Date of Award

Fall 12-1-2003

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Life Sciences

First Advisor

Gary Stuart

Second Advisor

Swapan Ghosh

Third Advisor

Bill Flurkey

Abstract

Tumor vaccines are a promising alternative to chemotherapy for the treatment of cancer. To be effective and safe, a cancer vaccine should specifically target antigens expressed only on tumor cells. The B-cell tumor-derived Ig receptor may be considered a model tumor antigen for cancer vaccine development. Specifically the immunoglobulin idiotype (ld) encoded by the variable region genes (VH and VL) provides a target antigen. Promising results have been obtained in clinical studies of ld vaccination using ld proteins. However, ld protein is laborious and time-consuming to produce. Gene-based vaccines, on the other hand, are inexpensive and offer ease of manipulation and flexible design to activate effective attack on cancer. Various studies are being carried out, including the ones in our lab, to assess the potency and effectiveness of DNA vaccines for B-cell lymphomas. Using PCR techniques we have cloned the VH and VL genes from our B-cell tumor model 2C3 into an appropriate expression vector to be used as an idiotype vaccine. Since the cellular location of antigen has quantitative and qualitative effects on immune responses induced by genetic immunization, we wanted to investigate its effect on our idiotype vaccine by cloning its membrane, secretory and cytoplasmic versions. Also a thorough study comparing the effects of monomeric with the trimeric conformation of an idiotype as a single chain variable fragment (svFv) in the form of a DNA vaccine has not yet ,been undertaken. IV Pursuant to this goal we generated variants of all the above constructs with a 15 amino acid spacer between VH and VL genes. The spacer allows the idiotype to assume a monomeric conformation while the non-spacer counterparts exist as trimers. We compared the antibody responses in mice to the above constructs along with mouse survival following tumor challenge. A moderate antibody response was detected to all, with the highest response observed with the secretory form with spacer which also gave the highest extension in survival time. It was observed that in the absence of the spacer, sub-cellular localization of the idiotype antigen does not affect the antibody response. However antibody responses from mice immunized with the secretory venlion of VHVL with spacer were significantly higher than those from mice immunized with the membrane and cytoplasmic VHVL versions with spacer. This work indicates that the secretory form with a spacer appears to be better suited for antigen presentation in the context of class II MHC. To determine the same in the context of class I MHC we have generated transfectoma' s using all the membrane and secretory constructs. The ability of these transfectoma's to be killed by the effector splenocytes from tumor bearing mice was compared to that of the original parent tumor. Our results indicate that the presence of a spacer in the above vaccine constructs does not effect antigen presentation for CTL recognition. However, transmembrane forms of VHVL are more effective for CTL recognition. Overall the current study provides information about the sequence requirements and mechanism of presentation of a tumor vaccine. In future, this information can be applied to the design of more effective DNA vaccines against our tumor model 2C3.

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