Avidity enhancement effectively eradicates tumors in Acute Myeloid Leukemia (AML)
AML, the most common type of leukemia in adults, poses significant clinical challenges due to its resistance to therapies and its bleak prognosis. To date, little is known about the effectiveness of CAR T cell therapies on myeloid cancers such as AML, due to the lack of a suitable antigen and off-target killing. The lab of Dr. Marcela Maus identified an immunotherapy strategy for AML by increasing binding avidity between tumor and CAR T cells through the generation of a modified CAR T construct. This modified construct enabled T cells to escape surface CAR cleavage by evading proteases secreted by AML cells.
When the researchers examined the effects of their strategy on tumor cell killing effectiveness, cell avidity measurement proved to be the most accurate predictor of in vivo tumor control compared to classical assays used for characterization of CARs, such as in vitro cytotoxicity and cytokine secretion (Figure 1). These results confirm that cell avidity is a crucial biomarker in immune oncology.
Figure 1 Cell avidity measurement accurately predicts in vivo tumor control. Cell avidity measurement proved to be more accurate than other standard assays used to characterize CARs, such as in vitro cytotoxicity and cytokine secretion.
Building upon their previous research indicating avidity escape as an evasion mechanism against CAR T cell therapies, the results of this study show that improving cell avidity leads to a more effective eradication of tumors in mouse models of AML. Thus, avidity enhancement may be a promising strategy to improve the clinical success of CAR T therapies.
Cell avidity measurement with the z-Movi Cell Avidity Analyzer is a new and exciting method that can be used to accurately predict the in vivo efficacy of CAR T cells against tumor cells, enabling researchers to quickly identify and develop the most effective immunotherapies.
Solid tumors avoid CAR T cell killing through cell avidity escape
CAR T cell therapies have been transformative in battling blood-related cancers; however, the more prevalent solid tumors have been less responsive to this therapeutic approach. This study aimed to investigate the mechanisms involved in solid tumor escape of CAR T cell therapies. By screening several glioblastoma cell lines, including ones derived from patients, the team of Dr. Marcela V. Maus found that loss of genes in the interferon-γ receptor (IFNγR) signaling pathway (IFNGR1, JAK1 or JAK2) rendered them more resistant to CAR T cell killing. Similar results were observed in vivo in IFNγR knockout mouse models.
Transcriptional profiling of IFNγR knockout (KO) cells showed a lower upregulation of cell-adhesion molecules after exposure to CAR T cells, leading to reduced cell avidity (Figure 2A) and reduced CAR T cell killing. This, ultimately, resulted in solid tumor escape. The avidity escape resistance mechanism to CAR T cell killing was further established by showing that ICAM-1 overexpression in IFNγR1 KO cells restored cell avidity, which in turn led to restored tumor cell killing (Figure 2B and 2C). Therefore, enhancing cell avidity between T cells and solid tumors may be the key to improving responses to CAR T cell therapies.
Figure 2 Cell avidity accurately predicts CAR T cell killing ability in solid tumors. Reduced avidity between IFNγR1 KO and CAR T cells, as measured with the z-Movi Cell Avidity Analyzer (A), was restored in IFNγR1 KO cells overexpressing ICAM-1 (B). This led to increased CAR T cell killing (C), showing the power of cell avidity in accurately predicting CAR T cell functionality.
Cell avidity measurement with the z-Movi Cell Avidity Analyzer is a unique, exciting method that can predict the efficacy of CAR T cell therapies against solid tumors rapidly and reliably. This can lead to faster therapy development and optimization and, potentially, better clinical outcomes.
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 3A and 3B). In agreement with these results, the cells showed increased in vitro cytotoxicity against their target cells (Figure 3C), 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.
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 4). The cell killing results also demonstrated significantly higher toxicity induced by high functioning CAR T cells on low antigen-expressing healthy-like cells (Figure 5).
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.
Rapidly evaluate immunotherapeutic strategies with cell avidity
Here, researchers from the Memorial Sloan Kettering Cancer Center (MSKCC) 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 6 and 7). 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.
Figure 6 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. Figure 7 Bar graph representing fold increases of bound CAR T cells at the plateau force gated from the avidity curve in Figure 6.