Human T cells may be genetically modified to express targeted chimeric antigen receptors (CARs). We have previously demonstrated that T cells modified to express a CAR specific to the B cell tumor antigen CD19, termed 19-28z, successfully eradicate systemic human CD19+ tumors in SCID-Beige mice. While these results are encouraging, this xenogeneic tumor model fails to address potential limitations of this therapeutic approach in the clinical setting wherein these modified T cells encounter a hostile tumor microenvironment. Specifically, these models fail to address potential effector T cell inhibition mediated by endogenous regulatory T cells (Tregs). To investigate the role of inhibitory Tregs, we initially assessed the in vitro function of CAR-modified T cells in the context of Tregs. We found that CD19-targeted T cell proliferation and cytotoxicity were inhibited by purified natural Tregs. To further assess the role of these Tregs in vivo, we isolated and genetically modified Tregs to express the CD19-targeted 19z1 CAR. We verified specific trafficking of targeted Tregs to CD19+ tumors in vivo, and demonstrate that 19z1 Tregs wholly inhibit anti-tumor function of subsequently injected 19-28z effector T cells even at low Treg to effector T cell ratios (1:8). In order to overcome this limitation, we assessed whether the addition of a pro-inflammatory cytokine in vitro could overcome Treg inhibition. Indeed, the addition of exogenous IL-12 mediated resistance of 19-28z T cells to Treg inhibition. In light of this data we generated a bicistronic retroviral vector containing both the 19-28z CAR as well as the murine IL-12 fusion gene (19-28z IRES IL-12). Significantly, we found that 19-28z/IL-12+ T cells when compared to 19-28z+ T cells exhibited enhanced proliferation in vitro as well as resistance to Treg mediated inhibition. Finally, we demonstrate that 19-28z/IL-12+ T cells overcome Treg inhibition in vivo in our SCID-Beige Treg tumor model. In conclusion, tumor targeted T cells modified to express IL-12 demonstrate significantly enhanced in vivo anti-tumor efficacy in the presence of Tregs that are similarly targeted to the site of tumor. These results validate utilization of IL-12 secreting tumor targeted T cells in future clinical trials.
| Identifer | oai:union.ndltd.org:YALE_med/oai:ymtdl.med.yale.edu:etd-03052009-202819 |
| Date | 31 August 2009 |
| Creators | Lee, James C |
| Contributors | Dr. Thomas Duffy, Dr. Warren Shlomchik, Dr. Richard Edelson |
| Publisher | Yale University |
| Source Sets | Yale Medical student MD Thesis |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://ymtdl.med.yale.edu/theses/available/etd-03052009-202819/ |
| Rights | restricted, I hereby grant to the Yale School of Medicine the non-exclusive license to photocopy, archive and make accessible, under the conditions specified below, my print and electronic thesis, in whole or in part, in all forms of media. <p> I agree that the Yale School of Medicine may electronically store, copy or translate my thesis to any medium or format for the purpose of preservation and accessibility. The Yale School of Medicine is not under obligation to reproduce or display my thesis in the same format in which it was originally deposited. <p> I retain all ownership rights to the thesis, including but not limited to the right to use in future works (such as articles and books) all or part of this thesis. |
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