No abstract

The genetic transfer of antigen receptors provides a means to rapidly generate autologous tumor-reactive T lymphocytes. However, recognition of tumor antigens by cytotoxic T cells is only one step towards effective cancer immunotherapy. Other crucial biological prerequisites must be fulfilled to expand tumor-reactive T cells that retain a functional phenotype, including in vivo cytolytic activity and the ability to travel to tumor sites without prematurely succumbing to apoptosis. We show that these requirements are met by expanding peripheral blood T cells genetically targeted to the CD19 antigen in the presence of CD80 and interleukin-15 (IL-15). T cells expanded in the presence of IL-15 uniquely persist in tumor-bearing severe combined immunodeficiency (SCID)-Beige mice and eradicate disseminated intramedullary tumors. Their anti-tumor activity is further enhanced by in vivo co-stimulation. In addition, transduced T cells from patients with chronic lymphocytic leukemia (CLL) effectively lyse autologous tumor cells. These findings strongly support the clinical feasibility of this therapeutic strategy.

Purpose: HumanTcells targeted to the B cell^specific CD19 antigen through retroviral-mediated transfer of a chimeric antigen receptor (CAR), termed 19z1, have shown significant but partial in vivo antitumor efficacy in a severe combined immunodeficient (SCID)-Beige systemic human acute lymphoblastic leukemia (NALM-6) tumor model. Here, we investigate the etiologies of treatment failure in this model and design approaches to enhance the efficacy of this adoptive strategy. Experimental Design: A panel of modified CD19-targeted CARs designed to deliver combined activating and costimulatory signals to theT cell was generated and tested in vitro to identify an optimal second-generation CAR. Antitumor efficacy of Tcells expressing this optimal costimulatory CAR, 19-28z, was analyzed in mice bearing systemic costimulatory ligand-deficient NALM-6 tumors. Results: Expression of the 19-28z CAR, containing the signaling domain of the CD28 receptor, enhanced systemic T-cell antitumor activity when compared with 19z1 in treated mice. A treatment schedule of 4 weekly T-cell injections, designed to prolong in vivo T-cell function, further improved long-term survival. Bioluminescent imaging of tumor in treated mice failed to identify a conserved site of tumor relapse, consistent with successful homing by tumor-specific T cells to systemic sites of tumor involvement. Conclusions: Both in vivo costimulation and repeated administration enhance eradication of systemic tumor by genetically targeted T cells. The finding that modifications in CAR design as well asT-cell dosing allowed for the complete eradication of systemic disease affects the design of clinical trials using this treatment strategy.

Advanced human thyroid cancers, particularly those that are refractory to treatment with radioiodine (RAI), have a high prevalence of BRAF (v-raf murine sarcoma viral oncogene homolog B1) mutations. However, the degree to which these cancers are dependent on BRAF expression is still unclear. To address this question, we generated mice expressing one of the most commonly detected BRAF mutations in human papillary thyroid carcinomas (BRAF V600E ) in thyroid follicular cells in a doxycycline-inducible (dox-inducible) manner. Upon dox induction of BRAF V600E , the mice developed highly penetrant and poorly differentiated thyroid tumors. Discontinuation of dox extinguished BRAF V600E expression and reestablished thyroid follicular architecture and normal thyroid histology. Switching on BRAF V600E rapidly induced hypothyroidism and virtually abolished thyroid-specific gene expression and RAI incorporation, all of which were restored to near basal levels upon discontinuation of dox. Treatment of mice with these cancers with small molecule inhibitors of either MEK or mutant BRAF reduced their proliferative index and partially restored thyroid-specific gene expression. Strikingly, treatment with the MAPK pathway inhibitors rendered the tumor cells susceptible to a therapeutic dose of RAI. Our data show that thyroid tumors carrying BRAF V600E mutations are exquisitely dependent on the oncoprotein for viability and that genetic or pharmacological inhibition of its expression or activity is associated with tumor regression and restoration of RAI uptake in vivo in mice. These findings have potentially significant clinical ramifications.

We developed a novel approach to bioluminescent T cell imaging (BLI) using a membrane-anchored form of the Gaussia luciferase (GLuc) enzyme, termed extGLuc, which we could stably express in both mouse and human primary T cells. In vitro, extGLuc+ cells emitted significantly higher bioluminescent signal when compared to cells expressing GLuc, Renilla luciferase (RLuc), and membrane-anchored RLuc (extRLuc). In vivo, mouse extGLuc+ T cells exhibited higher bioluminescent signal when compared to GLuc+ and RLuc+ T cells. Application of this imaging approach to human T cells genetically modified to express tumor-specific chimeric antigen receptors (CARs) enabled us to demonstrate in vivo CAR-mediated T cell accumulation in tumor, T cell persistence over time, and concomitant imaging of T cells and tumor cells modified to express firefly luciferase (FFLuc). This sensitive imaging technology has application to many in vivo cell based studies in a wide array of mouse models.