Abstract A102: Assessment of CIT-drugs MoA with in vivo and 3D in vitro imaging

Abstract Cancer Immunotherapies (CITs) have shown unprecedented results in improving tumor control. However, many patients are still refractory to treatment. A deeper understanding of CIT-drugs mode of action (MoA) may help improve therapeutic approaches to reach better anti-tumor response. To this end, we developed a skinfold chamber model in humanized mice that allows, by multi-photon intra-vital microscopy, the in vivo study of the tumor microenvironment. Analysis at the single cell level upon treatment with a CD20-targeting T-cell bispecific antibody, CD20-TCB (Glofitamab), showed that CD20-TCB localizes in the tumor (diffuse large B cell lymphoma, DLBCL) and acts on tumor-resident T cell motility within 1 hour post-treatment, causing a sharp reduction in their speed and an increase in tumor/T cell interaction time; those changes last up to 72h post-treatment. In addition, we show how the initial tumor/T cell interaction mediated by CD20-TCB leads to peripheral T cell recruitment into the tumor. This mechanism is dependent on the presence of tumor-resident T cells and on IFNg-CXCL10 pathway. Inhibiting any of these two pathways resulted in reduced T cell infiltration from the periphery and reduced anti-tumor efficacy. In order to expand and complement in vivo imaging for CIT-drug discovery e.g. for mid-throughput screening purposes, we further established a fully human mid-throughput 3D in vitro screening platform to image and quantify immune cell dynamics, trafficking and infiltration to tumors in response to CIT. To this aim, we have adopted the microfluidic OrganoPlate 3-lane 40 by MIMETAS. By combining endothelial tubules, peripheral immune cells, solid or liquid tumors, extracellular matrix and stromal components, we can faithfully recapitulate the tumor microenvironment organization and physiological in vivo T cell dynamics. Indeed, the 3D in vitro platform allows faithful reproduction of T cell recruitment and immune synapses formation upon CD20-TCB as observed in vivo. Furthermore, we tested CD20-TCB MoA in the context of three diverse cell lines of DLBCL. We show that treatment of diffuse large B-cell lymphomas leads to the formation of synapses between T cells and tumor cells, the stability of which correlates with tumor cell killing. Such findings offer fundamental observations for a better understanding of CD20-TCB MoA and how immunological synapses formation impacts T cell effector functions and tumor regression. Citation Format: Floriana Cremasco, Katerina Apostolopoulou, Elena Menietti, Leo Kunz, Sara Colombetti, Mario Perro. Assessment of CIT-drugs MoA with in vivo and 3D in vitro imaging [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A102.