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A major roadblock prohibiting effective cellular immunotherapy of pancreatic ductal adenocarcinoma (PDAC) is the lack of suitable tumor-specific antigens. To address this challenge, here we combine flow cytometry screenings, bioinformatic expression analyses and a cyclic immunofluorescence platform. We identify CLA, CD66c, CD318 and TSPAN8 as target candidates among 371 antigens and generate 32 CARs specific for these molecules. CAR T cell activity is evaluated in vitro based on target cell lysis, T cell activation and cytokine release. Promising constructs are evaluated in vivo. CAR T cells specific for CD66c, CD318 and TSPAN8 demonstrate efficacies ranging from stabilized disease to complete tumor eradication with CD318 followed by TSPAN8 being the most promising candidates for clinical translation based on functionality and predicted safety profiles. This study reveals potential target candidates for CAR T cell based immunotherapy of PDAC together with a functional set of CAR constructs specific for these molecules., There is an unmet clinical need to identify therapeutic options for the treatment of pancreatic cancer (PDAC). Here the authors present a systematic screening approach for the identification of potential PDAC cell surface target candidates for CAR-T cell based immunotherapy, followed by their functional validation in preclinical models.

The in-depth characterization of tumor-infiltrating leukocytes (TILs) is crucial to further improve cancer immunotherapies. Since TILs generally constitute a small subpopulation of solid tumors, they can be lost in the background noise of downstream analyses such as flow cytometry or single cell sequencing. Therefore, improving pre-enrichment methods for TILs is necessary to increase the sensitivity of such studies and save resources spent on the analysis of contaminating cell populations. We have established a reliable workflow combining automated tissue dissociation with positive selection of CD45+ TILs from human tumors as well as humanized mouse models. Human tumor samples were stored and shipped in MACS® Tissue Storage Solution, maintaining cell viability and phenotype up to 48 hours after collection. Tumor dissociation was automated using the gentleMACS™Octo Dissociator and optimized for epitope conservation to overcome bias in immune-phenotyping caused by dissociation with aggressive or impure enzymes. The direct isolation of TILs by MACS® Technology was optimized for the characteristics of solid tumors, such as the presence of debris and dead cells, based on a CD45-specific enrichment reagent. As an example, TIL isolation from a human colorectal carcinoma led to purities of target cells >92% and yields >80%. To allow for the isolation of label free TILs, we developed an isolation strategy based on the REAlease™ Technology. Again, this method is independent of the tumor entity and even tumors with low frequencies of TIL infiltration, such as an ovarian carcinoma with a frequency of 4%, could be enriched to >90% purity. Upon Bead release, >99% of the cells showed no residual magnetic labeling, hence TILs could be subsequently used for a second round of magnetic isolation for the isolation of multiple subpopulations. Additionally, we demonstrated that the REAlease Biotin Complex was effectively removed from the target cells as well, making them non-distinguishable from non-labeled cells before separation. Due to the removal of all labels, epitopes used for isolation are completely available for re-labeling and can be further used for any downstream application. Taken together, we have developed a reliable workflow for the dissociation of solid tumors followed by the isolation of TILs in less than 90 minutes. We could show efficient separation performance using the standard MACS® as well as REAlease™ Technology for positive isolation of TILs. The REAlease™ Technology allows for the efficient removal of MicroBeads and the entire labeling complex, resulting in cells that are suited for a second magnetic labeling step or any kind of downstream analysis. Citation Format: Janina Brauner, Wa'el Al Rawashdeh, Andreas Bosio, Olaf Thorsten Hardt. A complete workflow for the isolation of tumor-infiltrating leukocytes from human tumors and humanized mouse models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1592.

Immunotherapy approaches that engage T cells to attack tumors have proven clinical efficacy and tremendous potential in multiple cancers. Syngeneic mouse tumor models represent the gold standard to develop and analyze effects of immunotherapy, as they possess a fully competent immune repertoire. However, the phenotypic and functional analysis of tumor-infiltrating leukocytes (TILs) is technically challenging and labor intensive. The number of tumor infiltrating T cells can be very low and small subpopulations might escape analysis as they get lost in the background noise. Importantly, tumor infiltrating T cells are embedded in a cellular environment where antigen is abundant and surrounding cells express highly immunomodulatory molecules, such that unbiased cell-intrinsic functional characterization is hindered. When working with large cohort sizes, even immunophenotyping of TILs by flow cytometry is usually time consuming and data processing can be laborious. Therefore, it is fundamental to use effective innovative tools to streamline the workflow and to generate reliable data. We established complete workflows combining tissue storage, dissociation, T cell isolation and flow cytometric phenotyping. These workflows were validated in four different syngeneic mouse tumor models (B16-F10, B16-OVA, 4T1 and CT26.WT). Tissues were either processed immediately or stored in a solution that was shown to maintain cell viability and phenotype up to 48h after collection (Tissue Storage Solution™). Tumor dissociation was automated and optimized for epitope preservation using a tissue dissociator (gentleMACS™ Octo). Phenotypic analysis revealed that optimal enzymatic dissociation was essential for analysis of critical tumor-specific sub-populations, such as PD1hiTim3+Lag3+CD39+CD8+ T cells present in B16-F10 tumors. We developed new T cell-specific enrichment reagents for manual and (semi-) automated magnetic cell sorting, based on MACS® Technology, which enrich for rare tumor infiltrating T cells by up to 500-fold, while maintaining activation status and phenotype. Importantly, we compared labeling of TILs using conventional hybridoma-derived antibodies with recombinant antibodies engineered to eliminate Fc receptor-mediated background (REAfinity™). Use of REAfinity antibodies significantly diminished non-specific labeling of cells present in the tumor microenvironment. Finally, flow cytometric analysis was performed using an automated analyzer (MACSQuant X™). This instrument decreased hands-on as well as total acquisition time by facilitating fast and fully automated sample processing, including sample mixing and absolute cell counting. In conclusion, we have optimized workflows that include standardized processing of tumor samples, newly developed tools for (semi-) automated magnetic isolation of tumor infiltrating T cells and automated flow cytometric analysis. These workflows greatly 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 immuno-oncology and immunotherapy research. Citation Format: Anne Richter, Ramona Siemer, Carolin Steinbrueck, Manuela Herber, David Agorku, Janina Brauner, Olaf Hardt, Christian Dose, Cesar Evaristo. Tumor infiltrating T cells: complete workflows allow faster and improved flow cytometric analysis of syngeneic mouse tumors [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 LB-351.

Introduction Syngeneic mouse tumour models are widely used to analyse tumour immunology due to their fully competent immune-repertoire and have paved the way for novel immunotherapy agents in multiple tumour entities. However, the amount and composition of tumour infiltrating leukocytes (TIL) is highly variable. This complicates targeted analysis, in particular for small leukocyte subpopulations that may not be analysed properly or lost in the background noise. When working with large cohort sizes, immune-phenotyping by flow cytometry is time consuming and highly work intensive. We have developed improved workflows combining automated tissue dissociation with novel TIL specific isolation reagents to allow for more accurate and faster analysis of TILs and TIL subpopulations. Material and methods We have developed novel TIL specific enrichment reagents for the magnetic cell sorting (MACS) based isolation directly from dissociated tumour tissue. The composition of these populations before and after separation was analysed by flow cytometry. The developed tools were combined with optimised tissue dissociation and flow panels to establish comprehensive workflows. Results and discussions We have established workflows combining optimised and automated tissue dissociation using the gentleMACS platform with TIL specific isolation to improve and accelerate downstream analysis. Isolation of TIL was improved by developing new CD45, CD4+, CD8+, and pan T cell enrichment reagents for MACS-based isolation directly from dissociated tumour tissue. Applying this workflow, CD45 +TIL were enriched to purities above 80%–90% with high yields (>70%) from divers syngeneic mouse tumours. This allowed for rapid and reliable analysis even of small TIL subpopulations. Moreover, by using the newly developed T cell reagents, T cells were enriched to purities higher than 90%, allowing for a reliable analysis of rare T cell subpopulations. Importantly, while TIL or T cell enrichment significantly reduced analysis time and reagent costs in immune subset analysis, the composition of infiltrating cells was not affected, excluding the risk of introducing a bias by this method. Conclusion We have developed improved workflows for the isolation of generic TIL and T cells from mouse tumours reducing time and costs of downstream analysis while standardising and enhancing the detection and quantification of immune cell subpopulations. CD45 +TILs, pan-, CD4 + - or CD8 + -T cells can directly be isolated from dissociated mouse tumours and analysed for subpopulations.