Monday, May 19, 2025
5/19/2025
Combinatorial engineering of a three-cell synthetic immunotherapy system - Project Summary/Abstract: T cells, natural killer (NK) cells, and macrophages engineered to express chimeric antigen receptors (CARs) have shown promise as therapies for cancers, autoimmune diseases, heart disease, aging, and chronic viral infections. Current therapies use these CAR immune cells in isolation from one another. This contrasts the natural immune system, which deploys multiple cell types and coordinates their activities to mount immune responses against pathogens and cancers. A major challenge is to extend our engineering of immune cells to include co-engineering distinct cell types to function as a synthetic immune system. Such a synthetic immune system may overcome limitations of the individual CAR immune cell types, provided the cells are engineered to act synergistically rather than antagonistically. We reason that we can design a three-cell (T-NK- macrophage) immunotherapy by treating each cell type as a modular part, creating and testing many combinations of engineered cells. The functions and phenotypes of CAR immune cells can be modulated by changing the signaling domains of CARs or other receptors such as synthetic cytokine receptors (SCRs). In this work, we propose to screen CARs and SCR libraries in three-cell immunotherapy systems against cancer models and to use the screen data to fit dynamical models that describe the time-dependent behavior of such systems. Using this approach, we will identify CAR-SCR pairs with signaling domains that tune three-cell immunotherapy function, and create quantitative models to aid in development of new receptors. In Aim 1, we will construct libraries of CAR-SCR pairs and screen them to engineer a CAR T-NK-macrophage synthetic immune system with synergistic anti-tumor activity against a leukemia model. In Aim 2, we will engineer a tri-specific CAR-T-NK-macrophage synthetic immune system to overcome tumor heterogeneity and antigen escape in the context of a glioblastoma model. To carry out these aims, we will combine CAR T cells, NK cells, and macrophages and perform in vitro arrayed screens using live-cell imaging, flow cytometry, and RNAseq to measure immune cell function (proliferation, killing, cell state) and anti-tumor activity. Dynamical models fit to these data will help us quantify the time evolution of these systems, the activity of each cell type, and their combined synergy. This work will identify receptors that optimize key anti-tumor phenotypes of CAR immune cells. This approach will also give us valuable insights into how multiple cell types synergize or antagonize in the context of a multi-cell therapy. A framework to engineer three-cell synthetic immune system with receptors that tune the therapeutic functions will facilitate improved therapies for cancers and autoimmune diseases.
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