The overall objective is to understand mechanisms of action of scFv plasmid DNA vaccines that encode the idiotype (Id) ,i.e., the variable regions of heavy (VH) and light chains (VL) of the clonotypic immunoglobulin (Ig) of a murine B-cell lymphoma 2C3, and evaluate efficacy in terms of protection against the 2C3 tumor and the nature of immune responses evoked. Vaccination with Ig protein or scFv plasmid DNA usually induces humoral responses with limited prophylactic efficacy. With the 2C3 tumor, repeated immunizations using irradiated cells evoke both CTLs (cytotoxic T lymphocytes) and protective immunity. In contrast, similar immunization with purified secreted Ig is less effective, and results in indolent tumors. Furthermore, 2C3-Id- specific CTLs also occur at the early stages, but decline at late stages of tumor growth. The scFv plasmids are expected to overcome problems associated with protein immunogens by consistently producing only Id determinants of an Ig and thereby provoking memory immune cells. The question is whether scFv plasmids, to be effective, express cytosolic, secreted or membrane forms of the idiotype. To facilitate this study, we developed: (1) a prototype scFv construct encoding cytosolic VH- VL of 2C3 Ig based on the nucleotide sequences of both heavy and light chains; (2) a permanent transfectant P815A4, that expresses both intact scFv as well as CTL-recognized idiopeptides; (3) PCR, ELISA and cellular methodologies; (4) Id-specific CTL lines; and (5) anti-Id antibody reagents. With these tools, we will address the following: Design and construct a series of scFv producing plasmids that express distinct variants of the 2C3 idiotype. These variants will differ in the structure of the scFv itself, as well as in the subcellular localization of the scFv molecule (cytoplasmic, membrane-bound, or secreted); (2) Characterize the expression and mechanism of presentation of the above scFv variants after in vitro transfection into two different antigen-presenting cells: P815, and A20; and (3) Determine the in vivo effects of these scFv variants on the nature, magnitude and specificity of humoral and cellular (CTL) immune responses and on host survival rates against tumor challenge. We expect that these studies will provide understanding of molecular features of immunoglobulin idiotype that can be successfully exploited to design CTL- inducing antitumor responses.
| Lim, So-Yon; Ghosh, Swapan K (2005) Autoreactive responses to environmental factors: 3. Mouse strain-specific differences in induction and regulation of anti-DNA antibody responses due to phthalate-isomers. J Autoimmun 25:33-45 |
| Laxmanan, S; Stuart, G W; Ghosh, S K (2001) A stable single-chain variable fragment expressing transfectoma demonstrates induction of idiotype-specific cytotoxic T-cells during early growth stages of a murine B-lymphoma. Cancer Immunol Immunother 50:437-44 |
