In order to develop a method to overcome the immune tolerance of cancer, we have designed an adenoviral vector, designated Ad-sig-TAA/ecdCD40L, which carries a transcription unit encoding the extracellular domain (ecd) of the CD40 ligand (CD40L) linked to TAA. The TAA are in turn linked to a secretory signal peptide (sig). This work was based on earlier DNA vaccines reported by Ralph Reisfeld. The binding of the CD40L to the CD40 receptor on DCs activates them. The binding of the TAA/CD40L chimeric protein leads to the internalization of the TAA and its ultimate presentation of Class I MHC on the DCs. In vivo and in vitro analysis showed that the subcutaneous injection of the Ad-sig-TAA/ecdCD40L adenoviral vector led to the development of a CD8+ T cell lymphocyte systemic immune response against TAA for up to a year. One of the TAA tested is MUC-1, which is overexpressed in epithelial cancer cells. We have shown that the Ad-sig-ecdhMUC-1/ecdCD40L vector makes the hMUC-1.Tg mice (which otherwise are killed by the growth of hMUC-1 positive cancer cell lines) resistant to the engraftment and growth of a hMUC-1 positive cancer cell line. In contrast, the cancer cell line which lacks the hMUC-1 antigen engrafts and grows well in these transgenic mice. We have also shown that subcutaneous injections of the hMUC-1/ecdCD40L protein following the vector injection increase the level of the hMUC-1 specific T cells, and generates high levels of serum antibodies against the hMUC-1 antigen. Importantly, these antibodies bind to breast and prostate cancer cancer cells in biopsy specimens from human subjects. The target of these antibodies is a 20 amino acid repeat in the MUC-1 extracellular domain which can completely block the binding of the antibodies to the human breast cancer epithelial cells in biopsy specimens. The schedule of one subcutaneous (sc) injection of the Ad-sig-MUC-1/ecdCD40L vector followed in 7 and 14 days by a sc injection of the MUC-1/ecdCD40L protein is the best schedule among many other combinations and schedules tested. In addition to the MUC-1 self antigen, we have tested the Ad-sig-rH2N/ecdCD40L vector which contains the rat her-2-neu (rH2N) antigen. We tested this vaccine in rH2N.Tg mice which are anergic to the rH2Nantigen. We found that one sc injection of the Ad-sig-rH2N/ecdCD40L vector followed by 2 sc injections of the rH2N/ecdCD40L protein breaks tolerance which exists in the rH2N.Tg mice which are otherwise tolerant of the rH2N TAA. This Ad-sig-TAA/ecdCD40L vector strategy outperforms both corresponding DNA vaccines as well as cellular based cytokine modified tumor cell vaccines. We plan to complete the preclinical testing necessary to bring this new strategy into phase I toxicity testing in the near future.