Abstract A21: Chimeric antigen receptor T cells targeting primary CNS lymphoma: Visualizing the anti-tumor response in vivo
Authors: Mulazzani Matthias , Fräßle Simon , Buchholz Veit , Straube Andreas , Busch Dirk , Baumgarten Louisa von 
Published: March 2017

Abstract
Introduction: Advances in the treatment of primary CNS lymphoma (PCNSL) have led to prolonged survival. However, long-term remission is achieved infrequently. Chimeric antigen receptor T cells (CAR T cells) have proven to be a promising therapeutic approach for systemic B cell malignancies. Even in patients with advanced chemotherapy-refractory disease, syngeneic or allograft-derived CAR T cells can achieve durable remission. The most successful CAR targets CD19, a pan-B cell antigen also expressed in PCNSL. Whether CAR T cells are able to control PCNSL growth, and if so, if allogeneic or syngeneic CAR T cells are equally effective is not known. Additionally, information on the exact cellular interactions leading to tumor regression remains scarce.
Methods: To analyze the therapeutic effect of CAR T cells on orthotopic PCNSL at single-cell resolution in vivo, we established a novel mouse model. After microsurgical preparation of a cranial window, human red fluorescent B-cell lymphoma cells were stereotactically implanted into the cortex of nude mice. After the tumour reached a threshold diameter, we injected green fluorescent CAR T cells adjacent to the tumour. To unravel the ensuing CAR T cell - tumor interactions, we performed in vivo imaging using dual photon microscopy over several weeks.
Results: After implantation, allogeneic as well as syngeneic CAR T cells migrate throughout both hemispheres and preferentially accumulate in the subarachnoid compartment. Over time, CAR T cells infiltrate the tumor in high numbers. Upon encountering their cognate antigen, CAR T cells proliferate, their migration velocity decreases and they arrest in close contact with tumour cells. Cell-cell interactions can be observed in real time and are accompanied by tumor regression. Control CAR T cells, lacking the extracellular domain of the CAR, infiltrate the tumor in lower numbers and barely arrest, proliferate or kill tumor cells. After several weeks, allogeneic CAR T cells disappear and persisting lymphoma cells lead to tumor regrowth. However, repeated therapy with CAR T cells remains successful. Syngeneic CAR T cells show longer persistence and improved efficacy compared to allogeneic CAR T cells.
Conclusions: For the first time, CAR T cell interactions with tumor cells have been visualized repeatedly over several weeks. In vivo microscopy reveals important aspects of CAR T cell biology. Long-term analysis indicates that CAR T cells are generally able to control PCNSL growth. Allogeneic CAR T cells show lower persistence than syngeneic T cells. These findings corroborate preclinical data suggesting that CAR expression does not protect against allogeneic T cell deletion. However, syngeneic CAR T cell therapy as well as repeated therapy with allogeneic CAR T cells reduces tumor growth, indicating persistence as a key factor limiting efficacy of CAR T cell therapy in PCNSL. These results emphasize the need for new strategies to enhance CAR T cell persistence.
Citation Format: Matthias Mulazzani, Simon Fräßle, Veit Buchholz, Andreas Straube, Dirk Busch, Louisa von Baumgarten. Chimeric antigen receptor T cells targeting primary CNS lymphoma: Visualizing the anti-tumor response in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A21.