Abstract 2563: Macrophage Toll-like receptor-chimeric antigen receptors (MOTO-CARs) as a novel adoptive cell therapy for the treatment of solid malignancies

Abstract

Recent clinical trials using chimeric antigen receptors (CAR) T cells have demonstrated tremendous success in eradicating hematologic malignancies. Notwithstanding the excitement generated by CAR T cell therapy, its clinical efficacy has not been effectively translated to the context of solid tumors; the physical barriers of solid malignancies and the immunosuppressive conditions at the tumor site hinder the efficacy of CAR T cells. Macrophages have the ability to infiltrate almost every tissue and frequently are recruited into tumors. Therefore, macrophages are an attractive vehicle for CAR therapy and could help solve current challenges that CAR T cells face in the treatment of solid tumors. MOTO-CAR cells are monocyte-derived human macrophages that are genetically modified by a lentiviral or adenoviral approach to express a synthetic tumor-targeting receptor and to secrete cytokines, ligands or chemokine receptors. MOTO-CAR receptors are composed of a single-chain variable fragment (ScFv) that binds to a specific tumor target, a hinge to link it to a transmembrane domain, and an engineered Toll/Interleukin-1 receptor (TIR) signaling domain. When the ScFv binds to the tumor cell via its tumor target, an activation signal is transmitted. Myd88 dependent and independent signaling cascades are elicited, activating the macrophage and polarizing it towards a proinflammatory phenotype to eliminate cancer cells in a selective way. We previously reported the expression of Thymidine Kinase 1 (TK1) on the cell membrane of the non-small cell lung carcinoma NCI-H460 and A549 cell lines. The in vitro function of TK1 MOTO-CAR cells was evaluated against these cancer cell lines, using GFP-based phagocytosis and killing assays. Additionally, cell migration and interaction was recorded using time-lapse video with a confocal microscope. Upon co-culturing, with its target TK1 specific MOTO-CARs showed a nearly 4-fold increase in killing activity when compared with the controls (p<0.01). MOTO-CAR cells were produced through a lentiviral approach with around 30% of the cells expressing MOTO-CARs and with an adenoviral approach using the Ad5f35 vector with an efficiency of 70-80% of cells being transduced. Furthermore, after transduction MOTO-CAR cells showed a consistent M1 phenotype expressing high levels of CD14, CD80, CD206 and low levels of CD163. Time-lapse videos showed migration and clustering of MOTO-CAR cells around H460 GFP + cells. Moreover, cell death was observed upon contact of MOTO-CAR cells with target cells as well as phagocytic activity. In vivo testing using an orthotopic NOD scid gamma mice model is in progress. Our preclinical data show evidence that human macrophages are a suitable vehicle for CAR therapy and have the potential to successfully extrapolate the clinical efficacy of CAR therapy to the context of solid tumors. Citation Format: Edwin J. Velazquez, John E. Lattin, Taylor D. Brindley, Zachary Z. Reinstein, Roger Chu, Lu Liu, Evita G. Weagel, Michelle H. Townsend, Kiara V. Whitley, Eliza L. Lawrence, Brandon T. Garcia, Scott Weber, Richard A. Robison, Kim L. O'Neill. Macrophage Toll-like receptor-chimeric antigen receptors (MOTO-CARs) as a novel adoptive cell therapy for the treatment of solid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2563.

Toll-Like Receptor-Based Strategies for Cancer Immunotherapy

“…Another example of the application of TLR-derived domains for genetic engineering of the immune cells comes from CARmodified macrophages. Macrophages genetically modified to express CAR molecule consisting of an extracellular scFV domain and a cytosolic TIR domain are reported to show antigen-specific cytotoxicity and expansion both in vitro and in vivo [129,130]. These instances suggest that genetically engineered receptors mimicking TLR stimulation may be used to modify immune cells which naturally express and get stimulated by TLRs.…”

The Metabolic Features of Tumor-Associated Macrophages: Opportunities for Immunotherapy?

“…The in vitro analysis indicates that TK1 MOTO-CAR cells exhibit an M1-skewed phenotype and phagocytic activity. Moreover, these CAR cells specifically induce cell death and form clusters around the TK1-positive non-small cell lung carcinoma NCI-H460 cell line [ 94 , 95 ].…”

CARs: Beyond T Cells and T Cell-Derived Signaling Domains

“…The high prevalence of macrophages in solid tumors makes them a promising vehicle for CAR therapy, provided that the suppressive influence of the TME can be overcome [89]. CAR macrophages could contribute to shifting the TME towards tumor rejection by releasing inflammatory cytokines such as TNF, IL-6, IFNγ, and IL-12 [89,98,99], by promoting T cell recruitment and function, and tumor growth suppression [99]. Tumor cell lysis was achieved via CAR-directed phago-and trogo-cytosis [71].…”

“…A down-point of macrophages as CAR vehicles is their endogenous resistance to viral infection, resulting in low transduction rates [100]. Adenoviruses yielded the best transduction efficacy but are associated with high immunogenicity and an inflammatory phenotype in target cells, which is a risk factor for clinical applications [89,98,100]. Electroporation was also shown to be inefficient and resulted in low viability [100].…”

“…Another group worked on macrophage Toll-like receptor-chimeric antigen receptors (MOTO-CARs), which have a Toll Interleukin receptor (TIR) signaling domain ( Figure 1) [98]. Transduced into monocyte-derived human macrophages using Ad5f35, these receptors triggered phagocytosis of non-small cell lung carcinoma cell lines [98].…”