Many critical advances in research utilize techniques that combine high-resolution with high-content characterization at the single cell level. We introduce the MICS (MACSima Imaging Cyclic Staining) technology, which enables the immunofluorescent imaging of hundreds of protein targets across a single specimen at subcellular resolution. MICS is based on cycles of staining, imaging, and erasure, using photobleaching of fluorescent labels of recombinant antibodies (REAfinity Antibodies), or release of antibodies (REAlease Antibodies) or their labels (REAdye_lease Antibodies). Multimarker analysis can identify potential targets for immune therapy against solid tumors. With MICS we analysed human glioblastoma, ovarian and pancreatic carcinoma, and 16 healthy tissues, identifying the pair EPCAM/THY1 as a potential target for chimeric antigen receptor (CAR) T cell therapy for ovarian carcinoma. Using an Adapter CAR T cell approach, we show selective killing of cells only if both markers are expressed. MICS represents a new high-content microscopy methodology widely applicable for personalized medicine.

Major advances have been achieved in targeted immunotherapy with significant clinical benefits for patients. However, on-target/off-tumor toxicity is a major concern. This issue highlights the clinical need for better targets to improve the safety profile of immunotherapies. On-target/off-tumor toxicity is mainly based on the expression of tumor-associated antigens (TAA) in healthy tissues under physiological conditions. Thus, new approaches have to be developed to restrict specificity of targeted immunotherapy selectively towards cancerous cells. One way to improve target specificity is multi-targeting of cancer cells, i.e. targeting more than one TAA per cancer cell. We developed a workflow to identify new tumor markers employing an unbiased high throughput flow cytometry-based screen on primary ovarian cancer samples. Co-expression of THY1, a marker of fibroblasts and hematopoietic stem cells, was revealed on cancer cells characterized by EPCAM expression, a marker of epithelial cells. We confirmed our findings by high content imaging analyzing several ovarian carcinoma samples. Next we assessed the safety profile of the target combination THY1-EPCAM by analyzing the expression across a multitude of healthy human tissue samples using again high content imaging. Cluster analysis showed correlation patterns within the datasets confirming our previous findings. In order to investigate the functionality of THY1-EPCAM as therapeutic t we combined this target pair with the adaptor CAR technology, a modular system composed of a CAR recognizing biotin and biotinylated antibodies which are specific for a certain antigen. This technology combines the flexibility and controllability of antibodies with the efficacy of CAR T cell-dependent killing of target cells. The functional in vitro characterization of adaptor CAR T cells targeting the THY1-EPCAM pair shows high specificity and efficacy. Moreover, we are going to conduct in vivo studies with adaptor CAR T cells to investigate the efficacy and safety of targeting THY1-EPCAM in a solid tumor model. In conclusion, we successfully identified a novel target combination in ovarian cancer utilizing flow cytometry-based screening complemented by high content imaging. The new target pair combination shows promising results in vitro in combination with adaptor CAR T cells indicating its potential use as future immunotherapy. Citation Format: Christoph Herbel, Vera Dittmer, Manuel Martinez-Osuna, Sandy Reiß, Peter Mallmann, Dominik Ratiu, Michael Mallmann, Paurush Praveen, Werner Müller, Dominik Eckardt, Andreas Bosio. Identification of a novel tumor marker combination THY1-EPCAM for adaptor CAR T cell therapy in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2813.

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Here we report the use of the MACSima™ imaging platform to perform high-content imaging for the pre-clinical validation of tumor target expression on tumor and healthy human tissues indicative for potential on-target/off-tumor toxicity in vivo. Major advances have been achieved in cancer therapy in the past decade. In particular, targeted-immunotherapy has progressed and clinical benefits for patients have been achieved. However, on-target/off-tumor toxicity is a potential threat which has been shown to be more pronounced in solid than in liquid tumors. These findings highlight the need for better pre-clinical assays to improve the safety profile of immunotherapies. On-target/off-tumor toxicity is mainly based on the expression of tumor-associated antigens (TAA) in healthy tissues under physiological conditions. Currently, most prediction methods for on-target/off-tumor expression are based on bulk mRNA expression data of healthy tissue. These prediction models, however, have limitations, mainly poor predictable relation between RNA and protein level. Moreover, it is frequently unclear which cell types are affected by on-target/off-tumor effects. To overcome these limitations we employ multi-parameter imaging to analyze the expression of TAAs directly at the protein level. Additionally, we gain spatial information about tissue and cellular distribution of TAAs. Notably, our novel high-content imaging technology potentially allows for the analysis of hundreds of markers in a single experiment, paving the way for high-dimensional characterization of cells within complex solid tissues. We performed high-content imaging with the MACSima™ platform to validate the expression of known TAAs across tumor and healthy tissue samples. Subsequently, we employed unbiased cluster analysis revealing correlation patterns within the datasets to identify cell types at risk. In detail, we analyzed several high-grade serous ovarian carcinoma and pancreatic ductal adenocarcinoma for the expression of known TAAs, described tumor markers, and tissue lineage markers. Additionally, we evaluated the expression of these TAAs across several healthy human tissues. Next we performed pixel- and object-based data analysis for unbiased cluster analysis. Thereby we identified cell clusters that express TAAs in ovarian and pancreatic cancers, as well as in healthy tissues. We found that primarily epithelial cells express the analyzed TAAs in different tissues and that TAA expression shows inter- and intra-tumor heterogeneity. Taken together, we established a novel workflow for multi-parameter characterization of tissues employing the MACSima™ imaging platform. Our work enables efficient identification of potential tissues for on-target/off-tumor toxicity, paving the way for improved pre-clinical evaluation of TAAs as new targets. Citation Format: Christoph Herbel, Vera Dittmer, Manuel Martinez-Osuna, Laura Nadine Kuester, Daniel Schaefer, Jan Drewes, Jutta Kollet, Werner Mueller, Michael Mallmann, Peter Mallmann, Philipp Stroebel, Olaf Hardt, Dominik Eckardt, Andreas Bosio. Evaluation of tumor-associated antigen expression with the MACSimaTM high-content imaging platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4694.

Many critical advances in research utilize techniques that combine high-resolution with high-content characterization at the single cell level. We introduce the MICS (MACSimaTM Imaging Cyclic Staining) technology, which enables the immunofluorescent imaging of hundreds of protein targets across a single specimen at sub-cellular resolution. MICS is based on cycles of staining, imaging, and erasure, using photobleaching of fluorescent labels of recombinant antibodies (REAfinityTM), release of antibodies (REAleaseTM) or their labels (REAdyeleaseTM). Multimarker analysis can identify potential targets for immune therapy against solid tumors. With MICS we analysed human glioblastoma, ovarian and pancreatic carcinoma, and 16 normal tissues. One potential target pair for chimeric antigen receptor (CAR) T-cell therapies identified for ovarian carcinoma is EPCAM/THY1. Using an adapter CAR T cell approach, we show selective killing of cells only in presence of both markers. MICS represents a new high content microscopy methodology to be widely used for personalized medicine.