240 Presentation of DLL4 and VCAM on paramagnetic beads replicates thymic Notch signaling and enables differentiation of functional of iPSC derived TCR T-cells in vitro

• Published in: Journal for immunotherapy of cancer 2023-11, Vol.11 (Suppl 1), p.A276-A276
• Main Authors: Chandrasekaran, Siddarth, Kardel, Melanie, Martinez, Elisa, Zeidan, Nabil, Deyati, Avisek, Wall, Valerie, Lee, Dylan, Farahani, Negin, AuYeung, Amanda, Subedar, Omar, Cadell, Michael, Apelu, Sommer, Prochazka, Laura, Sahaf, Zahra, Duong, Angela, Zmurchok, Cole, Yoon, Boyoung, Kirouac, Dan, Belew, Muluken, Mann, Jasdeep, Rajesh, Deepika, Bond, Chris
• Format: Article
• Language: English
• Subjects: Antigens; Cloning; Cytotoxicity; Genome editing; Immunotherapy; Lymphocytes; Regular and Young Investigator Award Abstracts
• ISSN: 2051-1426
• DOI: 10.1136/jitc-2023-SITC2023.0240

BackgroundAdoptive TCR T-cell therapies from autologous donor are expensive, time-consuming and depends on quality of T-cells in patients. One of the key challenges for autologous TCR therapy is the requirement of cell numbers that are orders of magnitude higher than CAR-T cells. This has several implications including the inability to manufacture enough cells for a repeat dosing regimen. To this end, we have developed a scalable method by which TCR+ CD8+ lymphocytes can be generated from induced pluripotent stem cells (iPSCs) using 3D engineered thymic niche (ETN) comprised of DLL4- and VCAM-conjugated paramagnetic microbeads.MethodsWe have successfully developed a gene-editing workflow for generation and characterization of iPSC clones engineered to express an exogenous TCR. We utilized a defined scalable differentiation process to generate lymphoid competent CD34+ HPCs from multiple iPSC clones. Cryopreserved CD34 cells were differentiated to Pro T cells and CD8+TCR+ cells by leveraging stage specific control of Notch signalling using the 3D ETN. Flow cytometry and single-cell RNA sequencing was used to characterize the phenotypic and transcriptional state of iPSC derived effector cells. Incucyte-based cytotoxic killing assay was used to demonstrate antigen specific effector cell proliferation and cytotoxic function. We have developed a computational tool to quantify the average number of target cells killed by each input effector cell per unit time.ResultsMultiple iPSC engineered clones expressing a TCR were successfully differentiated to hematopoietic precursor cells with an average efficiency of ~2.5 lympho-competent HPCs per input iPSC without magnetic sorting. HPCs were further differentiated in the presence of proprietary ETN beads to generate mature single positive T cells expressing TCR. The end stage cells expressed T-cell markers mimicking mature peripheral blood T-cells with a 90% TCR expression detected by an antigen-loaded tetramer. Transcriptional profiling of iPSC-derived and peripheral blood-derived CD8+ T-cells revealed similar gene expression signatures. Cryopreserved end-of-process CD8+ cells displayed target-specific cytotoxic activity against antigen expressing tumor cell lines in vitro across 4 rounds of stimulation in a long-term serial killing assay. iPSC derived CD8 TCR T-cells secreted perforin, granzyme B, IFNg, TNFa and GM-CSF and expanded ~1000-fold in response to target antigen.ConclusionsWe have successfully generated CD8+