Solid tumors consist of malignant and nonmalignant cells that together create the local tumor microenvironment (TME). Additionally, the TME is characterized by the expression of numerous soluble factors such as TGF-β. TGF-β plays an important role in the TME by suppressing T cell effector function and promoting tumor invasiveness. Up to now CAR T cells exclusively target tumor-associated antigens (TAA) located on the cell membrane. Thus, strategies to exploit soluble antigens as CAR targets within the TME are needed. This study demonstrates a novel approach using Adapter CAR (AdCAR) T cells for the detection of soluble latent TGF-β within the TME of a pancreatic tumor model. We show that AdCARs in combination with the respective adapter can be used to sense soluble tumor-derived latent TGF-β, both in vitro and in vivo . Sensing of the soluble antigen induced cellular activation and effector cytokine production in AdCAR T cells. Moreover, we evaluated AdCAR T cells for the combined targeting of soluble latent TGF-β and tumor cell killing by targeting CD66c as TAA in vivo . In sum, our study broadens the spectrum of targetable moieties for AdCAR T cells by soluble latent TGF-β.

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Adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TILs) has shown remarkable results in patients with metastatic melanoma. However, only a small fraction within the TIL population reacts against the tumor. Therefore, the pre-enrichment of tumor-specific T-cells and subsequent ex vivo expansion may improve the efficiency of ACT therapies. In addition, tumor-reactive T lymphocytes circulating in the blood (TRLs) have been found in low frequencies, which represents a challenge for their isolation, but also an advantage over TIL therapy since blood is a more reliable and accessible source than tumor excisions. Another impediment to the widespread application of ACT is the conventional rapid expansion protocol (REP) that constitutes a laborious and extensive process with frequent culture manipulations, and thus requires specialized personell and equipment. Our aim is to develop a fully automated large scale ex vivo T-cell isolation and expansion procedure in the CliniMACS Prodigy in order to simplify the manufacturing of tumor-reactive T-cells for ACT. The CliniMACS Prodigy instrument is a controlled system that integrates a series of cell processes, from magnetic cell separation and cell culture to final product formulation, under GMP conditions in a closed system. This process focuses on the optimization of the REP procedure on the CliniMACS Prodigy for TILs and TRLs. As a proof of concept, we used both cryopreserved outgrown TILs and magnetically isolated virus-specific T-cells from healthy donor leukapheresis via CD137 upregulation upon in vitro antigen stimulation. The first results show expansions ranging from 3,000-15,000 fold, both for TILs and CD137-expressing T-cells. From low cell numbers (2x10e5 - 1x10e6 cells) and after 14 days of cell culture in TexMACS medium and in the presence of high amounts of IL-2 and irradiated feeder cells, we were able to obtain around 3-4x10e9 cells. We also compared two different stimulation reagents, anti-CD3 antibody (OKT3) and TransAct (a soluble polymeric nanomatrix conjugated to humanized CD3 and CD28 agonist), which resulted in comparable expansion rates. Furthermore, small-scale experiments showed no differences between TexMACS and the conventional TIL culture medium (50% RPMI/50% AIM-V medium). The phenotype and reactivity of the expanded T-cells were also assessed by flow cytometry. Currently, higher starting cell numbers up to 1x10e7 cells are being assessed and first results are promising when shaking and media exchange are commenced earlier in the process. In summary, these data provide proof of concept for the expansion of low numbers of TILs and virus-specific T-cells from peripheral blood in a closed, automated manner in the CliniMACS Prodigy. In the future, this expansion process will be combined with a tumor-reactive T-cell enrichment process (e.g., via CD137-conjugated magnetic beads) to achieve the desired efficiency, simplicity and automated production of ACT therapies against cancer. Citation Format: Bianca Heemskerk, Christina Maeder, Elvira Criado-Moronati, Lisa Boettcher, Andrew Kaiser, Mario Assenmacher, Andrzej Dzionek. Automated ex vivo expansion of low numbers of tumor-reactive T-cells on the CliniMACS Prodigy® [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B016.

Immunotherapy has proven clinical efficacy and tremendous potential. But clinical benefit is experienced by only a subset of patients, such that additional research is necessary to improve outcomes. In particular, analyze steady-state anti-tumor immunity and monitor the effects of therapy on the tumor microenvironement. However, TIL numbers can be very low. Flow cytometry phenotyping different cell populations requires dividing limited tumor material into multiple samples, reducing the number of cells available for analysis. Therefore, innovative tools and workflows are needed to maximize the quality of data obtained from limited tumor patient samples. We established workflows combining tissue storage, dissociation, cell isolation and analysis. We developed new reagents for magnetic cell sorting, incorporating novel technology enabling the removal of magnetic beads and antibody fragments (REAlease®). Tumor-infiltrating (TI) T-, B-, Myeloid cells, cancer-associated fibroblasts and tumor cells were sequentially isolated and characterized by flow cytometry, multi-parameter imaging and transcriptomics. Importantly, purification of TI T cells with high viability was essential for high-resolution immune profiling by single cell transcriptomics. In conclusion, we optimized workflows for the comprehensive analysis of the tumor microenvironement. Sequential isolation of the different cell types maximized the number of cells available for analysis. These workflows reduce experimental time and allow the performance of more complex experimental setups. We believe the use of these innovative tools and workflows can significantly increase the quality of the data obtained in immunooncology and immunotherapy research.

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