Abstract 577: Control of solid tumors by alphav beta3 CAR T cells is accompanied by profound tumor penetration and prevention of metastasis in pre-clinical models
Authors: Cobb Dustin A. , Liu Lixia , Dziegielewska Barbara , Mollica Philip , Lee Maria , Lee Daniel W. 
Published: June 2022

Abstract
Purpose: Using in vitro and xenograft models we aimed to determine the efficacy of αvβ3 CAR T cells deployed against melanoma and breast cancer tumors, two malignancies previously recognized for harnessing the αvβ3 pathway for angiogenic and invasion purposes.
Procedures: CAR T cells expressing an anti-αvβ3 scFv containing either a CD28 or 4-1BB co-stimulatory domain and CD3zeta were generated by retroviral transduction. In vitro cytotoxicity of αvβ3 CAR T cells was assessed by co-culture with melanoma or breast tumor cells assayed with bioluminescent- and impedance-based methods and effector cytokine production by ELISA. Xenograft studies, including melanoma (SK-MEL-28) and orthotopic breast tumors (MD-AMB-231), were carried out in NSG mice to evaluate in vivo efficacy of systemically administered αvβ3 CAR T cells. Immunohistochemistry was performed to evaluate T cell infiltration of melanoma tumors and changes within the tumor microenvironment. NSG mice harboring orthotopic breast tumors were monitored for disease progression, development of metastases using bioluminescent imaging and flow cytometry analysis of lung tissue, and overall survival.
Results: αvβ3 CAR T cells exhibited robust cytotoxicity and cytokine production against several melanoma and triple-negative breast tumor cell lines. Systemic administration of αvβ3 CAR T cells potently inhibited growth of SK-MEL-28 melanoma xenografts as demonstrated by significant differences in tumor volume relative to CD19 CAR treatment. Immunohistochemical analysis of tumors at the experimental endpoint revealed striking infiltration of residual tumors by human T cells in mice administered αvβ3.28z CARs, but to a lesser extent in αvβ3.BBz CAR-treated xenografts. This T cell infiltration was accompanied by marked expression of PD-L1 that was mostly absent in tumors of CD19 CAR-treated controls, suggesting treatment-induced up-regulation of PD-L1. In orthotopic xenografts of MD-AMB-231 breast tumors, αvβ3 CAR T cells were able to control tumor growth, prevent the formation of lung metastases, and resulted in enhanced overall survival.
Conclusions: These pre-clinical studies highlight a renewed potential for targeting integrins, specifically αvβ3, for the treatment of solid tumors. Data from this study suggests that αvβ3 CAR T cell therapy for melanoma may be further improved by combination therapy with checkpoint inhibition. Together with our prior work demonstrating robust efficacy of αvβ3 CAR T cells to treat glioblastoma and DIPG xenografts, these results highlight the broad applicability of utilizing CAR T cells targeting αvβ3 for treatment of multiple cancer types with reduced risk of on-target, off-tumor toxicity due to the restricted expression of αvβ3 in normal tissues. Results of these studies warrant further development of αvβ3 CAR T cells for clinical use.
Citation Format: Dustin A. Cobb, Lixia Liu, Barbara Dziegielewska, Philip Mollica, Maria Lee, Daniel W. Lee. Control of solid tumors by alphav beta3 CAR T cells is accompanied by profound tumor penetration and prevention of metastasis in pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 577.