Date of Award

2001

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Abstract

Objectives . The broad objective of this study was to develop and characterize plasmid DNA anti-tumor vaccines encoding the likely immunogenic form of a TAA, in this case the variable region (idiotype), of a B-cell lymphoma/hybridoma cell, 2C3. Methods . The tumor model used in this study is a BALB/c B-cell lymphoma, 2C3, expressing clonotypic immunoglobulin (Ig) that is unique and highly immunogenic. The plasmid vector construct used in the study encoded 2C3 Id in the form of scFv designed to be expressed cytosolically. Purified plasmids were used as vaccines, with or without a plasmid encoding murine GM-CSF, in BALB/c mice. Sera of immunized mice were assayed for antibodies by ELISA, and their spleens for cytotoxic T-lymphocytes (CTLs) by 51 Cr release assay. DNA-immunized mice were challenged intra-peritoneally with viable 2C3 cells to assess for protection. A P815 mouse mastocytoma cell line, which is capable of presenting antigens in the context of MHC class I molecules, was used as a tool to determine the expression of scFv in a manner recognizable by anti-2C3 CTLs. Results . The results indicate that scFv-immunized mice, were able to induce anti-Id antibodies, but no CTLs. Persistent expression of the plasmid was observed in the muscle as well as in popliteal lymph nodes post-injection. The survival times of the immunized hosts were moderately prolonged after a lethal tumor challenge. However, 20% of mice vaccinated i.m. with both plasmid and GM-CSF remained tumor free. scFv transfected P815 (P815A4) was shown to express 2C3 Id epitopes that were recognized by anti-2C3 CTLs from early tumor-bearing mice. Majority of the cytolytic activity was biased against the variable light chain regions of the Id, especially VL3. Conclusions . To our knowledge, this is the first direct evidence on the in vivo existence of Id-specific CTLs in early tumor-bearing hosts using Id-transfected antigen presenting cells, such as P815A4, that are MHC restricted. DNA vaccination appears to be effective in evoking anti-tumor immunity and GM-CSF seems to be a promising cytokine adjuvant that may be co-administered along with the scFv plasmid. Currently, investigations are underway to determine various forms/structures of the antigen required for optimum immunogenicity.

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