271. Helper-Dependent, Capsid-Modified Adenoviral Vectors for Immunotherapy of Breast Cancer

Abstract

Top of pageAbstract Introduction: Her2/neu is a membrane-spanning type I receptor tyrosine kinase. It has an extracellular domain (ECD), a single transmembrane region, and an intracellular domain (ICD). This oncogene is frequently overexpressed in breast and ovarian cancer. Even when it is a “self” antigen, patients have elevated Her2/neu antibody titers, and CTLs against this protein have been described. Immunotherapy strategies have been mostly aimed towards boosting the preexisting cytotoxic T-cell activity in order to break tolerance to Her2. Ex vivo transduction of autologous dendritic cells with adenoviral (Ad) vectors to mediate expression of tumor associated antigens and subsequent re-transplantation of these modified DCs, is considered to stimulate a potent anti-tumor immune response. Objective: to develop a strategy that will allow DCs to very efficiently present Her2/neu ECD epitopes by both MHC-I and -II molecules in order to generate CD4+ and CD8+ T cell immunity specific for the ECD of the human Her2/neu protein. Results: we have developed Ad vectors that have a number of new features over commonly used first-generation Ad.5 based vectors: i) Are devoid of all viral genes and grow in the presence of helper vectors (helper dependent (HD) vectors). HD vectors avoid toxicity or interference with DC functions associated with the expression of viral genes. ii) Because Ad5 based vectors transduce DCs very poorly (due to low levels of CAR, the Ad5 receptor), our HD-vectors are chimeric with Ad35 fibers, which allows them to transduce DCs more efficiently (using CD46 as a receptor). iii) The HD Ad5/35 vectors express the ECD of Her2/neu as a secreted protein fused to the Fc portion of human IgG (ECD.Fc). The intracellular expression of the protein should allow MHC class I presentation, and we expect that Fc receptor/s will efficiently enable the ECD antigen to be internalized through a pathway that will allow for efficient MHC class II presentation (and potentially also MHC class I cross-presentation). The following vectors were generated: HD.5/35.Fc, HD.5/35.ECD.Fc, and HD.5/35.GFP (expressing the green fluorescent protein). HD vector titers and helper contamination was assessed by RT-PCR. Transgene expression was detected by Western blotting (ECD.Fc and Fc), or flow cytometry (GFP). DCs were generated by ex vivo differentiation of peripheral blood CD14+ human monocytes in the presence of GM-CSF and IL-4. DC phenotype was analyzed by flow cytometry for MHC-II, CD14, CD80, CD83, and CD86 expression. Transduction with HD.5/35.GFP vectors at an MOI of 10 genomes per cell conferred transgene expression in 95% of DCs as measured by flow cytometry. Moreover, morphological observations indicate that HD.Ad5/35 infection activated DCs. We are currently analyzing the kinetics of ECD.Fc expression and assessing whether HD-Ad5/35.ECD.Fc transduced DCs are able to potently activate autologous T-cells from normal donors. T-cell activation studies will be done in comparison with ECD-peptide pulsed DCs.