796 Enhanced in vitro T cell activation and cytotoxicity induced by a TROP2 ADC conjugated to the novel topoisomerase payload AZ14170133 in lung adenocarcinoma cells

<h3>Background</h3> Understanding of the temporal effects of antibody-drug conjugates (ADC) on the tumor immune microenvironment is limited. To better understand the dynamic changes of tumor-immune cell interactions and the effectiveness and potential combinatorial properties of ADC and immuno-oncology (IO) therapies, we developed a fluorescence image-based, in vitro tumor-T cell co-culture assay to elucidate temporal immunomodulatory aspects following ADC treatment. <h3>Methods</h3> The PC9 human non-small cell lung cancer (NSCLC) cell line was transduced to stably express the melanoma antigen, Melan-A/MART-1 (aa 27-35), and Green Fluorescent Protein (GFP) for a direct measure of tumor viability. Co-cultures of primary MART-1-specific CD8 T cells and tumor cells with a tool TROP2 ADC (hRS7) conjugated with a novel topoisomerase 1 inhibitor payload AZ14170133, volrustomig (PD1/CTLA4 monovalent bispecific antibody), or in combination. The fluorescence intensity of GFP expressed in tumor cells was measured continuously for up to 180 hours in an IncuCyte imaging system. As a measure of T cell activation in the co-culture system, we utilized a Jurkat T cell-based fluorescent reporter cell line and flow cytometry to assess the activity of NF-κB, NFAT and AP-1 in response to ADC treatment. <h3>Results</h3> In the real-time automated image analysis, we observed additive cytotoxic activities when combining T cells and ADC. Combining the ADC with volrustomig induced additional cytotoxic activities. During the course of experiments, we identified an early temporal pattern where the initial 72 hours of the cytotoxic activity of ADC or T cells against tumor does not induce loss of tumor viability but an apparent loss of tumor viability was observed when combining the ADC and T cells, suggesting ADC was enhancing the cytotoxic activities of T cells. To investigate the effects of ADC on T cell activation, we used the Jurkat T cell-based triple parameter reporter system in tumor-T cell co-culture experiments. We observed a greater than 3-fold increase in activation of NFAT and AP-1 in Jurkat T cells when co-cultured with ADC treated tumor cells compared to untreated tumor cells. <h3>Conclusions</h3> We uncovered novel temporal dynamics of ADC-IO monotherapy- and combination-induced cytotoxic responses in a lung adenocarcinoma model. The ADC induced activation of NFAT and AP-1 in Jurkat T cells during tumor-T cell co-culture indicates direct immunomodulatory properties of this ADC. Our study highlights an innovative approach to understanding the complex interplay between ADCs, immune cells, and tumor cells, with potential implications for the development of more effective ADC/IO combination treatment strategies. <h3>Acknowledgements</h3> Abigail Lara for the primary T cells. <h3>Ethics Approval</h3> Human materials used in the study were acquired from approved commercial vendors from donors who gave informed consent in accordance with AstraZeneca guidelines as determined by AstraZeneca human biological samples governance.