Designing inducible chemotactic beacons for enhanced trafficking of CAR T cells to solid tumors

ABSTRACT CAR T cell immunotherapy has proven to be a real tour de force in treating refractory hematologic malignancies, and is being explored for treatment of solid tumors. However, solid tumors pose some obvious and unique challenges. One of the key differences between leukemia and solid tumors is that, leukemias are systemic, whereas solid tumors are highly localized. Hence, in the case of leukemia, access of intravenously infused CAR T cells to their cancerous targets is never an issue with both the killers and the targets being present by-default in the same location. However, the scenario is quite different in the case of solid tumors. Data from our and others’ labs show that the majority (80%-90%) of the adoptively transferred therapeutic CAR T cells sit on the sidelines in a resting state, and do not localize to the solid tumor to participate in the anti-tumor battle. Our studies further show that the CAR T cells that sit unengaged in the periphery – by virtue of default rest from antigenic encounter – exhibit phenotypic properties (TCF1Hi, CD127Hi, CD62LHi, PD-1Lo) and functional potential (IFN-gHi, TNF-aHi, IL-2Hi, GzmBHi) of classical memory T cells when benchmarked against the gold-standard acute viral infection-induced memory T cells. Hence, we propose that mobilization of this reservoir of highly functional resting memory-like CAR T cells from the periphery into the solid tumors would provide fresh reinforcements for enhanced tumor control, and increased synergy with checkpoint blockade immunotherapy. To address this hypothesis, in Aim 1, we will engineer tumor-inducible production of chemokines by intratumoral CAR T cells to serve as a localized beacon for relocating functionally potent CAR-Ts from periphery and thereby enhancing tumor control. Intratumoral injection or ectopic expression of chemokines by genetic modification of tumor cells has been shown to increase T cell infiltration, tumor control, and overall survival rates in preclinical studies. However, intratumoral injection is not feasible for inaccessible tumors, and the short half-life of chemokines necessitates impractically frequent injections. Overcoming these challenges, we propose a unique CAR bioengineering strategy. In solid tumors, where antigen is restricted to the tumor microenvironment (TME), utilization of NR4A response element (NRE) cassettes – activated by CAR-T cell signaling – will promote a localized, highly specific mechanism for increasing chemokine levels specifically in solid tumors. In Aim 2, we will query the impact of localized intratumoral chemokine expression by CAR T cells on the responsiveness to PD-1 checkpoint blockade immunotherapy. The working hypothesis in this aim is that chemokine-mediated continued recruitment of functional memory-like CAR T cells from the periphery into the tumors will build the intratumoral pool of less exhausted CAR T cells that are readily responsive to checkpoint blockade, and also provide a more conducive immune environment. Successful completion of these preclinical proof-of-concept studies will pave way for easy adaptation of inducible chemokine expression casettes into existing CAR-T therapies to transform clinical outcomes for a variety of solid tumors.