Background While a diverse and complex array of cell-based and cell-engaging immunotherapies are currently under development, only a small subset will become approved therapies. 7 in 8 immunotherapeutic candidates fail in the clinic, suggesting that current in vitro approaches (e.g., killing assays, cytokine secretion) for evaluating efficacy, identifying toxicity, and predicting in vivo performance are insufficient and novel in vitro approaches are required. Methods Measurement of cell avidity by acoustic force1 2 interrogates the overall strength of the interaction between an effector cell and its target to comprehensively evaluate CAR-T, CAR-NK, TCRtg, and cell engager performance. Results Here, we review recent publications in Nature Biotechnology,3 Cancer Cell,4 and Journal of Immunotherapy of Cancer5 highlighting how the in vitro cell avidity assay can be used to: Generate unique mechanistic insights, where conventional in vitro assays often fail to differentiate therapeutic candidates De-risk on-target, off-tumor toxicities by profiling the avidity of effectors for healthy vs tumor targets Stratify candidates within minutes, often with more robust correlation to in vivo compared to conventional assays Reduce the time and expense leading up to murine studies while increasing confidence in lead selection Conclusions Higher avidity has been significantly correlated with improved tumor control in murine models, thereby increasing confidence in candidate selection and shortening the time to IND filing. References Sitters, et al. Acoustic force spectroscopy. Nat Methods 2015;12(1):47–50 Kamsma, et al. Single-Cell Acoustic Force Spectroscopy: Resolving Kinetics and Strength of T Cell Adhesion to Fibronectin. Cell Rep. 2018;24(11):3008–3016 Chockley, et al. Synapse-tuned CARs enhance immune cell anti-tumor activity. Nat Biotechnol. 2023;doi:10.1038/s41587–022-01650–2 Leick, et al. Non-cleavable hinge enhances avidity and expansion of CAR-T cells for acute myeloid leukemia. Cancer Cell 2022;40(5):494–508 Lee, et al. Limited efficacy of APRIL CAR in patients with multiple myeloma indicate challenges in the use of natural ligands for CAR T cell therapy. J. Immunother. Cancer 2023;in press

Cellular immunotherapies are increasingly complex in design. Affinity readouts, i.e., the measurements of the strength of single receptor-ligand interactions, are frequently used to assess the binding of chimeric antigen receptors (CARs) or T cell receptors (TCRs) to their ligand. However, affinity is a poor predictor of effector function of the engineered downstream product. Therefore, new tools are needed to evaluate the binding strength of CAR and TCR-transgenic cells to their cognate targets in a more biologically relevant context. Recent studies demonstrated the utility of measuring cellular avidity as a novel biomarker for identifying and developing potent and safe immunotherapies. Unlike affinity, cellular avidity is driven by the overall strength of dynamic surface interactions between effector cells and their targets by integrating receptor density, the sum of individual affinities, and engagement of the multitude of co-receptors within the immunological synapse. Here, we show that increased specific avidity, i.e., TCRs with the strongest antigen binding and the lowest background, correlated with improved effector function both in vitro and in vivo. For CAR-T, higher cellular avidity was significantly correlated with improved tumor control in in vivo murine models, but also associated with toxicities in patients, suggesting fine-tuning of cell therapies to the desired avidity for ideal function is needed.

Background With the increasing complexity of cell therapy construct design, novel methodologies to accelerate screening and improve evaluation of lead candidates for clinical benefit are needed. Several methods are currently employed including receptor-ligand affinity measurements. However, affinity between T cell receptors (TCRs) and peptide-MHC complexes (pMHC) has shown to be a poor predictor of T cell functional capacity. Methods We have developed the z-Movi Cell Avidity Analyzer to directly measure the overall binding strength, or cellular avidity, of effector to their target. Unlike affinity, cellular avidity is driven by the overall strength of dynamic surface interactions between effector cells and their targets; this novel biomarker integrates receptor density, the sum of individual affinities, and engagement of the multitude of co-receptors within the immunological synapse to characterize the interaction in a more biologically relevant context. Results We will discuss how increased specific cellular avidity, i.e., TCRs with the strongest antigen binding and the lowest background binding, correlated with improved effector function in vitro and in vivo. Another application of measuring cell avidity explored the effect of changes to the glycosylation pattern on the cell surface of BMDC on T cell activation. Here, altered glycosylation patterns on BMDC increased avidity towards T cells leading to enhanced T cell function upon Ag recognition. These results demonstrated the benefits of understanding cell avidity to predict and select potent T cell candidates or bispecific antibodies. Conclusions These results demonstrated the benefits of understanding cell avidity to predict and select potent T cell candidates or bispecific antibodies.

Background Affinity between himeric antigen receptors (CARs) to their target has shown to be a poor predictor of functional capacity of T cells. Recent studies have revealed that the overall binding strength (or avidity) between T cells and tumor cells represents a crucial parameter for identifying and developing potent cancer immunotherapies. Compared with affinity, cell avidity provides a more complete and physiologically relevant picture that reflects the bona fide interaction between T cells and tumor cells. Therefore, this interaction could better predict cellular responses in vitro, drive better, more informed decisions and potentially improve clinical outcome. However, one of the main obstacles in the process of measuring avidity is the lack of fast, specific, and accurate technologies to assess cellular avidity. Methods The z-Movi® Cell Avidity Analyzer is a novel and unique instrument for direct measurement of cell–cell interaction strength using acoustic forces. This new technology provides predictive, reproducible, and fast high-throughput results at a single-cell level. Results In this poster we will demonstrate that data obtained with the z-Movi Cell Avidity Analyzer correlates strongly with standard in vitro assays, such as cytokine release and cytotoxicity measurements. We will review the simple principles behind the z-Movi, the workflow to set up an experiment consisting of multiple runs, and how the intuitive software package will assist in getting precise cell avidity information of hundreds of cells simultaneously. Finally, we will demonstrate the great potential of the z-Movi for accelerating the development of CAR T immunotherapy against cancer. Conclusions Finally, we will demonstrate the great potential of the z-Movi for accelerating the development of CAR T immunotherapy against cancer.