Evaluate the avidity of T cells engaged by cell engagers
Cell engagers (CE) are molecules that can steer immune cells, such as T cells, NK cells, and macrophages, towards cancer cells. Most CEs are bispecific antibodies (BsAb), powerful tools adopted by immuno-oncology that can enhance the recruitment of T cells to specific cancer cells. They facilitate the formation of the immunological synapses (IS), which are key for a proper T-cell response against target cancer cells.
The z-Movi® Cell Avidity Analyzer, specialized in measuring avidity between immune cells and their targets, expands the set of parameters to select the best antibodies for optimal T-cell responses. Using avidity to screen and rank a panel of CEs against the same target, it is possible to find the most potent candidate within a few hours, with clear and reproducible results. Furthermore, the precise control of CE concentration offered by the z-Movi enables researchers to determine the impact of CE concentration on physiological IS formation. Overall, combining cell avidity analysis with the use of BsAbs provides a powerful approach for enhancing T-cell responses against cancer cells.
Quantifying IS formation in the presence of BsAbs as a predictor for in vivo functionality
In a study by Skokos et al. published in Science Translational Medicine (2020), the authors quantified IS formation in the presence or absence of BsAb to assess anti-tumor efficacy. In the presence of BsAbs, artificial IS formation was found to be enhanced, resulting in reduced tumor burden (Figure 1). In vivo functionality can thus be predicted by quantifying IS formation in the presence of BsAbs.
Although fruitful, the methods used in this study to quantify IS formation, such as confocal microscopy and immunofluorescent staining, were time-consuming, low throughput, laborious, and prone to “cherry picking” problems (i.e., bias selection of data sets to show a desired outcome). Measuring cell avidity can help overcome these problems and provide fast, robust, and reproducible results with just one simple assay. In one run, cell avidity measurements can quantify the binding of thousands of T cells to examine the influence of CEs on T-cell engagement on target cells.
Ranking cell engagers using cell avidity
Cell avidity is a crucial parameter that can be used to screen a panel of CEs against the same target, as it can detect clear and reproducible differences between them. Cell avidity measurement is a simple, robust, fast, and reproducible assay that provides results in just a few hours, in contrast to the lengthy in vitro assays traditionally used. Results obtained by measuring cell avidity have been found to correlate with in vitro cytotoxic assays performed by our collaborators.
By quantifying cell avidity, the impact of CEs on the initial cell-to-cell binding event can be determined, enabling researchers to rank CEs based on their avidity (Figure 2). Notably, increasing CE avidity has been shown to correlate with improved functionality.
Figure 2: Cell engagers can be successfully and reproducibly ranked using cell avidity
Workflow for establishing BsAb maximum activity (Emax) using cell avidity
Avidity is a crucial factor in the binding between target and effector cells in the development of CEs.
To precisely control CE concentrations and accurately represent physiological binding between target and effector cells in a microfluidic chip, we developed a co-injection workflow method (Figure 3). Through titration, we can establish a bell-shaped curve that identifies a window of maximum activity for each BsAb, as previously demonstrated by Betts and van der Graaf. Notably, using this method we demonstrated that excessive or inadequate CE concentrations can have a negative impact on the binding avidity between T cells and their target.
These findings emphasize the importance of cell avidity measurements in establishing the optimal CE concentration for better immunotherapy treatments.
Avidity data obtained from a nondisclosed biotech.
Betts and van der Graaf et al., 2020, Clin. Pharm. & Ter.
What are some obstacles faced when testing CEs and how do we overcome them?
Conventional Assay
Avidity Assay
Functional assays (e.g., killing, cytokine secretion, etc.) are time-consuming, laborious, and complex
Simple workflow, fast and robust
End-point assays mostly provide bulk information
Provides data at a single–cell resolution in real-time
Highly variable and inconsistent results obtained from traditional in vitro studies
Highly reproducible data
Legacy assays quantify the outcome of T-cell engagement and, therefore, neglect the initial cell-to-cell binding event formed by the bridging of CEs
Cell avidity quantifies the immunological synapse strength between a T cell and target cell in the presence of CEs. Measuring the initial cell-to-cell binding event provides more sensitive measurements, enabling researchers to distinguish subtle differences between CEs during preclinical studies
Difficult to identify lead CE candidates that demonstrate both potent and safe responses in vivo
Measures the avidity of live T cells to live healthy or cancerous cell targets in the presence of CEs. This application improves the ability to find optimal lead CE candidates by quantifying the physiological cell binding to find CEs that are potent but maintain safe profiles for their target. CE candidates can be ranked based on their avidity
CE concentration directly influences functionality, with a bell-shaped trend observed with increasing concentration vs. Emax (maximum functionality)
Assay enables precise control of bispecific concentration conditions within the microfluidic chip. This enables researchers to determine the impact of antibody concentration on physiological cell-to-cell interactions using different CEs and cell models
Conventional Assay
Functional assays (e.g., killing, cytokine secretion, etc.) are time-consuming, laborious, and complex
End-point assays mostly provide bulk information
Highly variable and inconsistent results obtained from traditional in vitro studies
Legacy assays quantify the outcome of T cell engagement and, therefore, neglect the initial cell-to-cell binding event formed by the bridging of CEs
Difficult to identify lead CE candidates that demonstrate both potent and safe responses in vivo
CE concentration directly influences functionality, with a bell-shaped trend observed with increasing concentration vs. Emax (maximum functionality)
Avidity assay
Simple workflow, fast and robust
Provides data at a single–cell resolution in real-time
Highly reproducible data
Cell avidity quantifies the immunological synapse strength between a T cell and target cell in the presence of CEs. Measuring the initial cell-to-cell binding event provides more sensitive measurements, enabling researchers to distinguish subtle differences between CEs during preclinical studies
Measures the avidity of live T cells to live healthy or cancerous cell targets in the presence of CEs. This application improves the ability to find optimal lead CE candidates by quantifying the physiological cell binding to find CEs that are potent but maintain safe profiles for their target. CE candidates can be ranked based on their avidity
Assay enables precise control of bispecific concentration conditions within the microfluidic chip. This enables researchers to determine the impact of antibody concentration on physiological cell-to-cell interactions using different CEs and cell models
Our solutions
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
