Identify the goldilocks CAR using cell avidity — a reliable predictor of T-cell efficacy

Part of: CAR T cells

Cell avidity as a parameter for optimal CAR-antigen combinations

This study aimed to identify CAR T cells that bind high antigen-expressing targets but not low antigen-expressing cells.

Researchers validated low functioning and high functioning CAR T cells based on functional assays and used non-transduced T cells as a negative control (NC). T cell avidities were tested on adherent cells with low antigen expression, high antigen expression, or no antigen expression.

The cell avidity data correlated with a corresponding in vitro cell killing assay, showing antigen-dependent binding strengths and cell killing (Figure 1). The cell killing results also demonstrated significantly higher toxicity induced by high functioning CAR T cells on low antigen-expressing healthy-like cells (Figure 2).

The z-Movi can distinguish between T cell specificities to improve CAR T cell therapy. Cell avidity is a reliable readout for identifying optimal CARs that can minimize on-target/off-tumor activity by CAR T cells.

1 Avidity scores based on the average detaching forces of the CAR T-cell populations relative to the non-transduced T-cell control population.

2 Cytotoxic effect of high and low functionality CARs on target cells with different antigen expression levels.

Data courtesy of Dr. Steven Albelda at University of Pennsylvania

Rapidly evaluate immunotherapeutic strategies with cell avidity

Here, researchers from the Dana-Farber Cancer Institute and Hospital Clinic Barcelona employed the z-Movi to evaluate two immunotherapeutic strategies that simultaneously target two multiple myeloma antigens, BCMA and GRC5F. Immunotherapies targeting BCMA have yielded a high response rate in multiple myeloma patients, however, BCMA antigen escape has caused remissions due to absent or low expression of the antigen. Targeting GPRC5D as an alternative or in addition to BCMA is a strategy to tackle BCMA antigen escape in especially relapsed patients. The illustration at the right shows the four different CAR configurations that were assessed in this study.

Illustration of the CAR designs evaluated in the study.

The aim of this study is to evaluate which CAR design is the most clinically relevant by predicting the CAR T-cell functionality using the z-Movi. The cell avidity of different CAR approaches was measured and compared against each other and with non-transduced (NTD) T cells. Both dtCAR populations required significantly higher force to detach from their target cells, compared with both mtCAR and NTD populations (Figures 3 and 4). With other words, dtCARs show exceptionally stronger binding strength to BCMA- and GPRC5D expressing multiple myeloma cells.

Most notably, subsequent in vivo studies evaluating the survival of mtCAR- or dtCAR-treated xenograft mouse models injected with myeloma cells expressing both antigens, showed a lower tumor burden and improved overall survival for the mice injected with dtCAR treatment. The results support the cell-avidity measurements, indicating that dtCARs effectively bind and kill cells expressing both BCMA and GPRC5D and demonstrate that measurements with the z-Movi® Cell Avidity Analyzer correlate with treatment outcomes in mouse models.

Cell avidity acts as a unique and reliable parameter to predict CAR T-cell functionality and provides information about clinical relevance. Furthermore, the fast and simple workflow of the z-Movi allows researchers to rapidly assess immunotherapeutic strategies, producing high-throughput data from different CAR T-cell populations within 24 hours without compromising cell viability.

3 Avidity curve showing the average proportion of bound CAR T cells and NTD T cells to BCMA and GPRC5D expressing cells upon application of an acoustic force ramp. The dashed line at 200 pN indicates plateau force (the force required to detach NTD cells). rForce represents forces calibrated on 10 μm polystyrene beads. 4 Bar graph representing fold increases of bound CAR T cells at the plateau force gated from the avidity curve in figure 3.

Data courtesy of Dr. Eric Smith at Dana-Farber Cancer Institute and Prof. Carlos de Larrea at Hospital Clínic Barcelona

Increased cell avidity through CD38 co-receptor drives enhanced
BCMA CAR T-cell sensitivity and persistence

The research team led by Dr. Maria Themeli from Amsterdam UMC (VUMC), Cancer Center Amsterdam, used co-targeting of CD38 via a chimeric costimulatory receptor (CCR) to tackle tumor escape due to low antigen density in multiple myeloma patients treated with anti-BCMA CAR T-cells. Using cell avidity measurements with the z-Movi, the researchers quickly identified that co-targeting induced higher cell avidity and subsequently potent anti-tumor effector function, even against antigen-low tumor variants.

Illustration of the CAR designs evaluated in the study.

Tumor escape through target antigen downregulation is one of the known mechanisms that leads to cancer patient relapse. To counteract this effect, the researchers set out to investigate if they could increase the cell avidity of BCMA CAR T cells against target cells expressing low levels of BCMA by co-transfecting the T cells with a CD38 targeting CCR. Indeed, cell avidity of BCMA-CD38 CAR/CCR T cells for the target cells, as measured using the z-Movi Cell Avidity Analyzer, was increased compared to the parental BCMA targeting CAR T cells (Figure 5a & 5b). In agreement with these results, the cells showed increased in vitro cytotoxicity against their target cells (Figure 5c), indicating that enhancing cell avidity by co-targeting CD38 using a CCR enhances BCMA CAR T-cell functionality.

Cell avidity analysis provides a deeper understanding on the mechanism and binding requirements behind multivalent binding, and cell avidity is a crucial biomarker for immuno-oncology.

Data courtesy of Dr. Maria Themeli at Amsterdam UMC (VUMC), Cancer Center Amsterdam (CCA)

5 (a) Avidity curve and (b) bar graphs showing the average percentage of bound T cells at indicated force applied for indicated constructs against MM1.S cells. (c) Cells transduced with indicated constructs were co-incubated with MM1.S cells at indicated E:T ratios.

Solutions

z-Movi® High-throughput Label-free Cell Interaction Studies
z-Movi

The z-Movi® is a unique instrument that measures the avidity between immune cells and their targets, enabling you to identify the most potent immunotherapeutic effector cells. This new technology provides you with predictive, reproducible, and fast high-throughput results at a single-cell resolution without compromising cell viability. All within a compact little box that easily fits inside the flow hood for sterile and safe sample handling

    Download the brochure to learn more



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