Designing a HER2/neu promoter to drive alpha1,3galactosyltransferase expression for targeted anti-alphaGal antibody-mediated tumor cell killing.

Introduction: Our goal was to specifically render tumor cells susceptible to natural cytolytic anti-alphaGal antibodies by using a murine alpha1,3galactosyltransferase (malphaGalT) transgene driven by a designed form of HER2/neu promoter (pNeu), the transcription of which is frequently observed to be above basal in breast tumors. Indeed, the alphaGalT activity that promotes Galalpha1,3Galbeta1,4GlcNAc-R (alphaGal) epitope expression has been mutationally disrupted during the course of evolution, starting from Old World primates, and this has led to the counter-production of large amounts of cytotoxic anti-alphaGal antibodies in recent primates, including man.

Method: Expression of the endogenous c-erbB-2 gene was investigated in various cell lines by northern blotting. A malphaGalT cDNA was constructed into pcDNA3 vector downstream of the original CMV promoter (pCMV/malphaGalT) and various forms of pNeu were prepared by PCR amplification and inserted in the pCMV/malphaGalT construct upstream of the malphaGalT cDNA, in the place of the CMV promoter. These constructs were transferred into HEK-293 control and breast tumor cell lines. Stably transfected cells were analyzed by northern blotting for their expression of alphaGalT and c-erbB-2, and by flow cytometry for their binding with fluorescein isothiocyanate-conjugated Griffonia simplicifolia/isolectin B4.

Results: We show that expression of the malphaGalT was up- or down-modulated according to the level of endogenous pNeu activity and the particular form of constructed pNeu. Among several constructs, two particular forms of the promoter, pNeu250 containing the CCAAT box and the PEA3 motif adjacent to the TATAA box, and pNeu664, which has three additional PEA3 motifs upstream of the CCAAT box, were found to promote differential alphaGalT expression.

Conclusion: Our results strengthen current concepts about the crucial role played by the proximal PEA3 motif of pNeu, and may represent a novel therapeutic approach for the development of targeted transgene expression.