We show that defined lymphocytes can be rapidly purified by immunoaffinity chromatography starting directly from whole blood. The method relies on low-affinity Fab-fragments attached to a column-matrix combined with the reversible Strep-tag technology. Compared to established cell enrichment protocols, the Strep-tag affinity chromatography of cells is independent of erythrocyte lysis or centrifugation steps, allowing for simple cell-enrichment with good yields, high purities, and excellent functionality of purified cells.

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.

Adoptive transfer of primary (unmodified) or genetically engineered antigen-specific T cells has demonstrated astonishing clinical results in the treatment of infections and some malignancies. Besides the definition of optimal targets and antigen receptors, the differentiation status of transferred T cells is emerging as a crucial parameter for generating cell products with optimal efficacy and safety profiles. Long-living memory T cells subdivide into phenotypically as well as functionally different subsets (e.g. central memory, effector memory, tissue-resident memory T cells). This diversification process is crucial for effective immune protection, with probably distinct dependencies on the presence of individual subsets dependent on the disease to which the immune response is directed as well as its organ location. Adoptive T cell therapy intends to therapeutically transfer defined T cell immunity into patients. Efficacy of this approach often requires long-term maintenance of transferred cells, which depends on the presence and persistence of memory T cells. However, engraftment and survival of highly differentiated memory T cell subsets upon adoptive transfer is still difficult to achieve. Therefore, the recent observation that a distinct subset of weakly differentiated memory T cells shows all characteristics of adult tissue stem cells and can reconstitute all types of effector and memory T cell subsets, became highly relevant. We here review our current understanding of memory subset formation and T cell subset purification, and its implications for adoptive immunotherapy.