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Recent advances in cancer immunotherapy had a positive impact on the life expectancy of patients for a large range of clinical indications. With new treatment strategies and druggable targets being identified at an increasing pace, the number of patients eligible for cancer immunotherapy is expected to expand steadily. However, promising therapeutic developments face hurdles in translating preclinical findings into therapy since conventional 2D cancer models hold low clinical predictive value. HUB developed an innovative alternative, build on the discovery that adult stem cells proliferate and organise into three-dimensional organotypic structures when they are embedded into extracellular matrix. Patient specific organoids are generated from healthy and malignant tissues and stored as biobanks with high quality and reproducibility. HUB Organoids recapitulate complex characteristics of the original parental tissue, including molecular heterogeneity, and morphological and functional traits. Since cancer progression and responses to immunotherapy are governed by immune cell interactions in the tumour microenvironment, we developed an assay in which various types of tumour organoids are co-cultured with T cells modified with chimeric antigen receptors (CAR T cells) to assess the cytotoxic potential. CAR-T cell therapy has been demonstrated to be efficacious against many hematological malignancies, however therapeutic application to treat solid cancers remains challenging. Our organoid-T cell co-culture assay offers a good platform to study the response of tumour organoids on the CAR T cells and will greatly contribute to our understanding of the critical factors that determine a successful CAR T cell therapy. In collaboration with GSK, HUB tested the cytotoxicity of GSK CAR T cells which target specific target antigen on human colorectal cancer (CRC) organoids. Six HUB CRC organoid models were selected based on differential expression levels of target antigen that was confirmed by FACS antibody staining. To evaluate GSK CAR T cell mediated organoid killing, caspase 3/7 expression was monitored at various timepoints and IFNγ secretion was measured by ELISA. For control, staurosporine and nonspecific-CAR T cells were included. Detected CRC-organoid killing demonstrated to be in line with antigen expression levels, indicating the specificity of the tested CAR T cells. Nevertheless, some CRC-organoid models demonstrated more resilient to CAR T cell mediated killing and further evaluation revealed upregulation of check point molecules such as PD-L1. This study reveals that tumour organoid-T cell co-culture assay hold value for preclinical development of CAR T-cell products and for evaluating tumour-evasion strategies. Moreover, this platform could potentially predict patient specific responses to CAR- T-cell therapy. Citation Format: Soura Mardpour, Mirella Georgouli, Ana Pereira Puri, Ravit Oren, Joshua Audu-Evans, Farzin Pourfarzad, Sara Brett, Pleun Hombrink, Sylvia F. Boj. Tumour organoid-t cell co-culture assay to evaluate CAR t cell cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 908.

Recent advances in cancer immunotherapy had a positive impact on the life expectancy of patients for a large range of clinical indications. With new treatment strategies and druggable targets being identified at an increasing pace, the number of patients eligible for cancer immunotherapy is expected to expand steadily. However, promising therapeutic developments face hurdles in translating preclinical findings into therapy since conventional 2D cancer models hold low clinical predictive value. We developed an innovative alternative, built on the discovery that adult stem cells proliferate and organise into three-dimensional organotypic structures when they are embedded into extracellular matrix. Patient specific organoids are generated from healthy and malignant tissues and stored as biobanks with high quality and reproducibility. HUB Organoids recapitulate complex characteristics of the original parental tissue, including molecular heterogeneity, and morphological and functional traits. Since cancer progression and responses to immunotherapy are governed by immune cell interactions in the tumor microenvironment, we developed an assay in which colorectal (CRC) and non-small cell lung cancer (NSCLC) tumor organoids are co-cultured with simultaneously expanded paired tumor infiltrating lymphocytes (TIL) as a source of tumor reactive cells. Our system allows robust expansion of TIL with preserved T-cell receptor repertoires and has an establishment rate of 75%. Paired resources allow for screening of immune-modulatory therapies under physiologic conditions not depending on peptide-pulsing and allo-reactivity. For this purpose, we developed a organoid-TIL co-culture system which quantifies and characterizes tumor organoid apoptosis by combining image-based analysis with flow cytometry and cytokine release assays as measurements of T-cell cytotoxicity against tumor (and normal-matched) organoids. Our system offers a powerful tool for the development and validation of cancer immunotherapy and may help to stratify cancer patients for susceptibility of various types of immunotherapies. Citation Format: Soura Mardpour, Claudia Beaurivage, Jane Sun, Lorenz Jahn, Rene Overmeer, Lars-Eric Fielmich, Pleun Hombrink, Farzin Pourfarzad, Sylvia F. Boj. Autologous organoids and T cell co-cultures as a powerful personalized platform for immunotherapy development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 188.