6060

Therapeutic vaccines to treat cancer are designed to stimulate the ability of cytotoxic T cells and other effector cells to recognize class I antigens in a context that minimizes anergy and maximizes T cell activation. One powerful way to stimulate CTL activity is to present antigen the context of a particulate delivery system that stimulates uptake by professional antigen presenting cells and augments cross priming. We are testing the ability of non-pathogenic tobacco mosaic virus (TMV) to function as a vaccine antigen carrier by fusing peptide epitopes on the surface of the TMV coat protein. TMV coat forms a semi-crystalline array of >2100 subunits around a non-integrating RNA, thereby permitting the display of the target antigen at very high density. To test the ability of such fusions to stimulate T cell activation, we have displayed the Ova SIINFEKL peptide on the surface of TMV as either a genetic fusion or through a chemical linker. The SIINFEKL peptide has been shown previously to stimulate activated T cell immunity and protection from Ova EG.7 tumor challenge. The genetic TMV-peptide fusions were produced in tobacco plants by transient viral infection and the purified virus qualified for purity and epitope integrity. The chemical conjugate vaccines were produced by heterobifunctional linkage of synthetic peptides to a modified TMV, and the degree of conjugation determined by a combination of protein gel electrophoresis and MADLI-Tof mass spectrometry. These two distinct TMV vaccines were used to vaccinate C57b/6 mice at 10ug per dose. Interestingly, the chemical conjugate vaccines generated greatly superior T cell activation as measured by both IFNg Elispot, and protection from tumor challenge. The peptide sequence, amount delivered, and the TMV carrier were identical for both vaccines, indicating that the nature of the bond type between the peptide and the carrier was a critical factor in determining the efficiency of antigen delivery and cross priming. A second generation of vaccine compositions pair Ova antigen with a T-helper epitope, or a cellular fusion epitope, in order to augment antigen uptake, processing and/or epitope-specific immunogenicity. Multivalent vaccines are being produced by chemical conjugation, and are being qualified and tested by methods similar to the mono-valent vaccines previously described. Data will be presented regarding their ability to stimulate specific T cell responses, as well as to prevent tumor outgrowth. Ultimately, we hope to apply this technology to develop low cost and highly adaptable vaccines that can be used in therapeutic applications to induce CTL responses against human tumor antigens.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]