Multi-institutional team applies TIMING to explain differences in tumor rejection kinetics
Differences in dynamics explain the distinct cytolytic behavior of CAR T cell therapy and can guide engineering of more adaptive and effective cellular products
PUBLICATION ALERT
HOUSTON, Texas — January 28, 2025. CellChorus®, the leader in applying artificial intelligence to visually evaluate how cells perform and interact over time, announced a publication by an interdisciplinary multi-institutional team from three top research universities that extensively applied the CellChorus® TIMING™ platform (Time-lapse Imaging Microscopy In Nanowell Grids) to test how different types of CAR T cells eradicate tumors. The TIMING platform was used to map the dynamics of CAR T cell interactions with tumor cells as a function of the CAR being used in two different clinical products. The results of the study will help guide the design of CAR T cell therapies for more effective, personalized treatment. The publication, “Molecular dynamics at immune synapse lipid rafts influence the cytolytic behavior of CAR T cells,” was published in Science Advances. The research team included those from Baylor College of Medicine, the University of Houston, and the University of Virginia.
Figure 1. (Left) Filmstrip of the TIMING cooperative/additive killing experiment of HER2-CAR T cells and Raji.HER2 at 2:1. (Right) Percentage of events that lead to 0, 1, or 2 target cell deaths in wells that have one CD4 or CD8 HER2-CAR T cell that contacted two tumor targets.
The research team applied TIMING to study the interaction dynamics and potency of two different types of CAR T cells (HER2-CAR-TCD28ζ and HER2-CAR-T4-1BBζ). HER2 is a common marker of solid tumors like breast cancer. A major difference between these engineered cells is how the CAR T cells are activated after binding the tumor antigen they recognize, based on the CD28.ζ or 4-1BB.ζ domains in the CAR. HER2-CAR-T cells with CD28.ζ domains eliminate tumor cells quickly. In contrast, HER2-CAR-T cells with 4-1BBζ domains are slow and steady killers that exert lasting tumor control. The team observed (a) that CD28.ζ-CART cells engaged in brief, highly lethal CAR-T immune synapse (CARIS) and mastered serial killing and (b) that 4-1BB.ζ-CART cells formed lengthy CARIS and relied on robust CAR-T cell expansion and cooperative tumor cell killing. The result is important because the differences in molecular dynamics at the CARIS explain the distinct cytolytic behavior of CAR T cells in different CAR-T therapies (CD19 CAR T cells harboring either CD28.ζ or 4-1BB.ζ are clinically approved for lymphomas) and can guide engineering of more effective cellular products targeting solid tumors.
Figure 2. Kinetics of synapse formation and target cell killing. (A) Time needed by CAR T cells to seek target cells (Tseek). (B) The duration in contact (Tcontact). (C) The time to achieve target death (Tdeath) once effector-target conjugation is initiated. (D) Percentage of Raji.HER2 survival after CAR T cell contact in wells having CD4 versus CD8 of HER2-CAR T cells and Raji.HER2 at 1:1. n = 457. (*P < 0.05, **P < 0.01, ***P < 0.001).
The TIMING platform comprehensively studies cells to quantify how immune cells move, interact, kill, survive, and secrete biomolecules at single-cell resolution over time. In TIMING assays, immune cells like CAR T cells are constrained in thousands of individual nanowells and imaged over time to create time-lapse videos of cell-cell interactions in each nanowell (as shown in Figure 1, left). The videos are processed by AI/ML models to quantify cell migration, cell-cell contact dynamics, potency (target cell killing and serial killing) (Figure 1, right), immune cell survival, biomolecule secretion, and additional cellular functions that can only be understood by tracking individual cells over time. Data and insights from the TIMING platform enable the field to develop, manufacture and deliver novel therapies with higher efficacy faster, at less expense, and with higher rates of success to benefit patients in oncology, infectious diseases, and a wide range of other diseases and disorders. Example TIMING data that led to the insights in the paper include (A) the time CAR T cells spent to find and interact with target cells (Tseek), (B) the duration of cell-cell synapse (Tcontact), and (C) the time it took the CAR-T to kill the target (Tdeath) once effector-target conjugation is initiated (Fig. 2).
Source: Ahmed Z. Gad et al. Molecular dynamics at immune synapse lipid rafts influence the cytolytic behavior of CAR T cells. Sci. Adv. 11, eadq8114(2025). doi:10.1126/sciadv.adq8114
About CellChorus
CellChorus® is the leader in applying artificial intelligence to quantify the function and performance of cells over time to improve the development and delivery of novel therapies that improve patient care. The company applies Time-lapse Imaging Microscopy In Nanowell Grids™ (TIMING™) with neural network-based detection to identify how multiple types of cells move, activate, kill, proliferate, and survive at single-cell resolution. The patent-protected platform links TIMING data with other single-cell modalities to provide a comprehensive understanding of cellular function and therapeutic potential. Please visit cellchorus.com for more information.
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