Induction of an antigen specific humoral immune response by immunization with the aggregate‐free human TGFα‐P64k fusion protein

Transforming growth factor alpha (TGFα) is an important epidermal growth factor receptor (EGFR) ligand. Over-expression of both molecules in epithelial tumors has been correlated with poor prognosis and disease progression. Due to the importance of TGFα in tumorigenesis, this molecule has great potential for cancer immunotherapy. We previously designed a TGFα-based vaccine consisting of a fusion protein between human TGFα (hTGFα) and P64k protein from Neisseria meningitidis expressed in Escherichia coli. However, this protein was obtained highly aggregated, which hampered its introduction into clinical use. In this study, we demonstrate that this aggregation state is not a consequence of IMAC purification, but is formed after bacterial disruption. To obtain this protein as a monomer, we designed a procedure that included an unfolding/refolding step at the end of purification. We verified that hTGFα in the refolded fusion protein (hTGFα-P64k-r) is immunogenic in mice. The latter was capable of inducing a humoral immune response against hTGFα identical to that generated with the aggregated fusion protein, demonstrating that the aggregation level has no influence on hTGFα immunogenicity. We also showed that TGFα-directed antibodies induced apoptosis in A431 cells. The present results also validated the potential use of this vaccine in cancer patients with tumors overexpressing TGFα. Drug Dev Res 69 2008. © 2008 Wiley-Liss, Inc.