Combining Mtor Inhibitors with Rapa-Resistant T Cells: a Two-Pronged Approach to Tumor Elimination
Authors: Huye Leslie E , Savoldo Barbara , Dotti Gianpietro , Wilson Matthew H , Rooney Cliona M 
Published: October 2019

Abstract
Abstract 2853
Although acute lymphoblastic leukemia and non-Hodgkin's lymphoma are curable in many cases, treatment-related complications remain a serious concern for survivors and the outcome is poor for patients with refractory/recurrent disease. Thus, new, less toxic therapies are necessary for improving the outcome and post-treatment quality of life for patients with these malignancies. Rapamycin and its rapalogs (rapa) are inhibitors of mTor, a central regulator of cell growth and survival, and have shown promise in both pre-clinical and clinical studies for the treatment of these tumors. However, despite clinical benefits in some lymphoma and leukemia subtypes as single agent therapy, rapalogs may be most beneficial when rationally combined with other therapeutic modalities, and thus it is important to understand the multiple effects that rapalogs have on tumors as well as host-tumor interactions including anti-tumor immune responses, which are an important factor in the efficacy of many chemotherapeutic agents. Rapalogs have direct anti-tumor activity, but also have paradoxical effects on anti-tumor immunity; rapalogs promote proinflammatory responses from myeloid dendritic cells and reverse multiple tumor-intrinsic immune evasion mechanisms, effects that should enhance the activity of tumor-specific T cells, but at the same time they directly inhibit effector T cells, which essentially negates their potential immune benefits. Therefore, to exploit the anti-tumor and untapped immune enhancing activities of rapa and create a two pronged approach to tumor therapy, we engineered T cells to express a rapa-resistant mutant of mTor (mTorRR) and directed them to B cell tumors by co-expressing a CD19 chimeric antigen receptor (CAR.CD19-28ζ). We stably expressed CAR.CD19-28ζ and/or mTorRR in T cells by transfecting PBMCs, isolated from 6 healthy donors, with transposon vectors encoding mTorRR-GFP, GFP only, or CAR.CD19-28ζ along with the PiggyBac transposase (on a separate plasmids). Transfected T cells were selected based on GFP expression and expanded by stimulation with OKT3 (50ng/ml) and IL-15 (5ng/ml) on irradiated allogeneic feeder cells. In the presence of rapa (25ng/ml), mTorRR-transfected but not control T cells maintained phosphorylation (73 ± 5% vs 24 ± 9%) of ribosomal S6 protein, a common readout of mTor activity, and expanded at the same rate as T cells without rapa (up to two logs in 14 days). The ability of mTorRR-expressing T cells to expand in rapa was attributable to their ability to resist the anti-proliferative effects of rapa. Furthermore, mTorRR-expressing CAR.CD19-28ζ T cells maintained their cytotoxic function and ability to secrete IFNγ after stimulation in the presence of rapa. Thus, the expression of mTorRR in human T cells provides resistance to rapa. To demonstrate the two pronged activity of rapa in the presence of rapa-resistant T cells, we assessed killing of the human CD19+ Burkitt's lymphoma line, Raji, in co-culture assays. We found that the combination of rapa and rapa-resistant-CAR.CD19-28ζ T cells produced greater anti-tumor activity against Raji cells compared with CAR.CD19-28ζ T cells (25±5% vs 47±6%, p=0.002, residual tumor cells after 4 days) or rapa alone (49±7%, p=0.02, residual tumor cells) We have also observed that this combination has superior anti-tumor activity against the pre-B ALL line, Nalm-6. These data correlate with the ability of rapa to reduce the levels of the anti-apoptotic factor, MCL-1, by 31%± 10%, p=0.002 as well as inhibit the production of the inhibitory cytokines, IL-10 (94pg/ml vs 630pg/ml for treated and untreated) and VEGF (82pg/ml vs 225pg/ml) by Raji cells, Together these data indicate that rapa-resistant CAR.CD19-28ζ T cells function in the presence of rapa and suggest that rapa may enhance the anti-tumor activity of rapa-resistant T cells, likely by inhibiting tumor-intrinsic immune evasion mechanisms. In conclusion, the combination of rapa and rapa-resistant, CAR.CD19-28ζ T cells may provide for a novel therapy for the treatment of B cell malignancies, an approach that can be extended to other cancers.

Disclosures:
No relevant conflicts of interest to declare.