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Abstract
Background CART-cell therapy has shown clinical success in treating hematologic cancers. However, more effective CAR T-cell therapies are needed for the treatment of solid tumors. T-cell activation is a formative event that directs cell fate and function in mature T cells. In the context of cellular product derivation, these decisions critically impact the phenotypic and functional quality of the T-cell drug product. We hypothesized that optimization of signaling parameters during T-cell activation can generate more potent CAR T cells.
Methods To control signaling during T-cell activation, we employed our platform, the Stim-R technology, a synthetic cell mimic that mediates precise signal molecule presentation (figure 1). We designed and fabricated Stim-R technology formulations to present T-cell activating signals engaging CD3 and CD28 at different densities and stoichiometries. Utilizing these formulation variants, we generated arrays of diverse ROR1-targeted CAR T-cell products which we profiled phenotypically and functionally. Based on these metrics, we compared Stim-R-generated CAR T cells to CAR T cells generated using a conventional bead-based activator to identify lead formulations showing superior in vitro function. We interrogated the mechanisms underlying this improved function by performing transcriptomic analysis of Stim-R CAR T cells following multiple rounds of tumor cell stimulation in vitro and validated the Stim-R CAR T cells in vivo in an H1975 lung tumor xenograft model in mice.
Results In vitro, Stim-R CAR T cells exhibited increased polyfunctionality in response to ROR1+ target-cell stimulation. Stim-R CAR T cells also demonstrated enhanced expansion and cytotoxicity in response to repeated ROR1+ target-cell stimulation. Transcriptomic analysis revealed that Stim-R CAR T cells retained a unique subset of stem-like cells with effector-associated gene signatures and also displayed down-regulation of exhaustion-associated gene sets compared to conventional CAR T cells, following repeat antigen stimulation. In vivo, Stim-R-generated CAR T cells exhibited 10-40x greater peak T-cell numbers in the blood, prolonged persistence, and improved tumor control over two months.
Conclusions Potent CAR T-cell therapies may be engineered by optimizing signaling events during T-cell activation to direct the phenotypic and functional qualities of the resultant cell product. Stim-R technology formulations mediating optimized signal presentation enabled the production of ROR1-targeted CAR T cells with improved polyfunctionality, persistence, and anti-tumor activity that retained a stem-like subpopulation with effector-associated gene signatures and showed reduced exhaustion, following repeated antigen stimulation. Such enhanced T-cell products may result in improved therapeutic benefit against solid tumors.
Acknowledgements We acknowledge Brianna Parish, Carson Harms, and Melissa DeFrancesco for their help with Stim-R technology process development. We also acknowledge Blythe Sather, Suman Vodnala, Gary Lee, Stan Riddell, Stephen Hill, and Nick Restifo for their scientific input and strategic guidance.
Ethics Approval Animal studies were conducted in Explora BioLabs vivariums and are approved by Explora’s IACUC under Core protocol amendment EB17-010.
The Stim-R technology is a programmable cell-signaling platform. The technology comprises biodegradable lipid-coated silica micro-rods that can combinatorially present signals in precise densities and stoichiometries. Soluble signals are released in a controlled manner while surface-anchored signals are presented on a synthetic lipid membrane, mimicking physiologic presentation. By using the Stim-R technology to control cell-signaling parameters during T-cell activation, T-cell products with targeted phenotypic and functional profiles may be engineered.