Access Type

Open Access Dissertation

Date of Award

1-1-1998

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Molecular Biology and Genetics

First Advisor

Katrina T. Trevor, Ph.D.

Abstract

Autologous activated T cells (ATC) are being utilized in clinical trials as potential mediators of anti-tumor cytotoxicity in refractory cancer patients who suffer from hematologic malignancies or solid tumors. ATC are readily generated from peripheral blood and possess non-major histocompatibility complex (non-MHC) restricted killing properties. ATC are also powerful targets for genetic modification and hold promise for treatments of cancer, AIDS and T cell disorders. Retrovirus-mediated gene transfer and the fate of proviral gene expression were evaluated in human T cells using 1) immobilized anti-CD3 monoclonal antibody (mAb) plus interleukin-2 (IL-2), or 2) cis costimulation using beads carrying co-immobilized anti-CD3 and anti-CD28 mAbs. By cross-linking the CD3 and CD28 receptors, these mAbs mimic in vivo signaling events, leading to cytokine production and proliferation. A modified human interleukin-1β (IL-Iβ) cDNA inserted into the MFG retroviral vector served as an indicator gene in these studies. Optimized methodologies resulted in T cell transduction frequencies of approximately 50-75%. An important consideration for the success of T lymphocyte-based gene therapy is the relationship between T cell activity and the level of proviral gene expression. Early after mAb stimulation and virus exposure, proviral gene expression was greater at the RNA and protein levels in optimized anti-CD3/anti-CD28 bead-activated cultures, corresponding with augmented endogenous cytokine responses and mitogenesis. Proviral gene expression was not regulated by extrinsic cellular factors present in activated T cell supernatants. Regardless of the mAb stimulation method, proviral IL-lβ expression declined in long-term cultures concomitant with a decrease in cellular cytokines. Restimulation of transduced T cells by either mAb method reinduced both T cell activity and vector expression. The finding that proviral gene regulation is downmodulated in the absence of T cell signaling events suggests that in vivo, high-level proviral gene expression will not be maintained. To overcome the loss of activity in gene-modified T lymphocytes, we have applied a bispecific antibody (BsAb) to redirect cytotoxicity to tumor and enhance T cell activity. For successful cancer adoptive immunotherapy, a coordinated approach of BsAb arming of retrovirus-gene-modified T cells can be utilized for delivery of beneficial, tumoricidal molecules to the tumor site.

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