The clinical success of gene-engineered T cells expressing a chimeric antigen receptor (CAR), as manifested in several clinical trials for the treatment of B cell malignancies, warrants the development of a simple and robust manufacturing procedure capable of reducing to a minimum the challenges associated with its complexity. Conventional protocols comprise many open handling steps, are labor intensive, and are difficult to upscale for large numbers of patients. Furthermore, extensive training of personnel is required to avoid operator variations. An automated current Good Manufacturing Practice-compliant process has therefore been developed for the generation of gene-engineered T cells. Upon installation of the closed, single-use tubing set on the CliniMACS Prodigy™, sterile welding of the starting cell product, and sterile connection of the required reagents, T cells are magnetically enriched, stimulated, transduced using lentiviral vectors, expanded, and formulated. Starting from healthy donor (HD) or lymphoma or melanoma patient material (PM), the robustness and reproducibility of the manufacturing of anti-CD20 specific CAR T cells were verified. Independent of the starting material, operator, or device, the process consistently yielded a therapeutic dose of highly viable CAR T cells. Interestingly, the formulated product obtained with PM was comparable to that of HD with respect to cell composition, phenotype, and function, even though the starting material differed significantly. Potent antitumor reactivity of the produced anti-CD20 CAR T cells was shown in vitro as well as in vivo. In summary, the automated T cell transduction process meets the requirements for clinical manufacturing that the authors intend to use in two separate clinical trials for the treatment of melanoma and B cell lymphoma.

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.

No abstract

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.

Introduction Metastasis to different organs or tissues may require distinct sets of regulators which may influence the homing and growth of tumour cells to specific secondary sites. Under the hypothesis that the immune microenvironment of the different niches may play an important role in this process, we have categorised metastatic samples from different primary tumours based on their immune profile. Material and methods Gene expression data from metastatic samples with different primary tumour origin (n=342) were downloaded from open repository GEO. Samples were scored using different gene expression profiles and characterised on the basis of their immune and stromal infiltration and activation of immune response pathways (Immunophenoscore, MCPcounter, ESTIMATE; among others). Resultant scores were analysed for statistical differences with ANOVA test. Multivariate analysis was used for clustering the samples based in their immune-features. Results and discussions As expected, significant differences were found between the immune profiles of samples metastasizing in distinct organs. For instance, breast cancer metastasis in lung showed a much higher immunogenic score than breast metastasis in brain (p=5e-4), suggesting a different immune microenvironment modulation. Also in breast, significant differences have been found in cell lineages infiltration, lung metastasis being the ones with the highest T cell component (p=0.002) and liver metastasis the ones with the lowest infiltration of endothelial cells (p=0.005). Moreover, in other cancer types like melanoma, samples showed differences among different metastatic locations. Interestingly, when comparing metastatic samples originating from different primary tumour, a high concordance among secondary tumours in immune scores were found; specifically in brain metastasis. These results suggest that cells needs to share similar molecular profiles to evade the immune surveillance and growth in a specific niche, independently of their origin. Conclusion Metastases from the same primary tumour growing in different organs show differences in their immune profile. However, those samples from different primary origin but growing in the same secondary organ shared a characteristic immune profile. These results suggest that immune system plays a role in determining the organ-specific homing of metastasis. 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 spe...