A 3D image-based phenotypic screen of bi-specific antibodies targeting stem cells in a panel of patient derived colon carcinoma organoids.

e23174 Background: Organoid cultures grown in 3D increase the likelihood for accurately predicting drug responses in patients, discriminating different drug responses and flagging toxicity. We used a high content screening platform and a panel of 40 patient derived colorectal cancer (CRC) organoids to characterize the responses to a large panel of (545) bispecific antibodies. These antibodies comprised a HER3 or EGFR targeting arm combined with a LGR4, LGR5, ZNRF3 or RNF43 targeting arm to target CRC stem cells. Methods: Automated 3D imaging and analysis of CRC organoids was performed. Results: The broad mutational spectrum of the organoids was reflected in a broad heterogeneity of organoid phenotypes. Some CRC organoids formed well-differentiated spheroids with a single lumen that resembled wild type organoids, whereas others had multiple lumens or were poorly differentiated without a luminal cavity. A rich set of morphological features was extracted from 3D image data, including organoid size and shape, planar cell polarity, lumen formation as well as cell number and nucleus shape. Some features, such as those that described lumen formation, were more sensitive in detecting drug treatment than features associated with cell proliferation, improving the sensitivity of the assay to detect active molecules. A set of 10 features was selected to create a drug response profile. The bispecific antibody screen was performed in two stages: a primary screen in three different tumoroid models (18,908 wells) and a validation screen in 25 different tumoroid models (23,040 wells). These screens simultaneously measured morphological alterations associated with growth, differentiation and survival (e.g. apoptosis) and identified a panel of bispecific antibodies that potently inhibited the growth of a significant majority of colorectal cancer tumoroid models tested. Conclusions: These results demonstrate that high content screening of CRC organoids is an effective strategy to identify novel inhibitors of CRC tumor outgrowth and enable identification of bispecific antibodies that target colorectal cancer stem cells with different mutational backgrounds.