Investigating in vivo CAR-T cell biology requires methods to track and assess in vivo CAR-T cell expansion kinetics, persistence, biodistribution, and effector functions in patients and animal models. Developing CAR-T cell therapy for solid tumors and elucidating clinical non-response/relapse mechanisms in B cell cancers require methods to stain and sort CAR-T cells from clinical samples for downstream applications, such as multiparameter flow cytometry and next-generation sequencing. Currently, CARs that target human epidermal growth factor 2 (HER2) and epidermal growth factor receptor variant III (EGFRvIII) against glioblastoma, GD2 disialoganglioside against neuroblastoma, and mesothelin (MSLN) against mesothelioma, are being evaluated in clinical trials ( 4).Īlthough CAR-T cell therapy's preliminary clinical success in B cell cancers warrants optimism, there are several challenges in the CAR-T field that need to be addressed: (1) CAR-T cell therapy does not work on solid tumors (2) clinical non-response/relapse mechanisms in B cell cancers need elucidation (3) the in vivo biology of CAR-T cells in human subjects needs investigation and (4) the molecular designs of the CAR immunoreceptor need optimization.Īccurate and reproducible CAR detection methods are required to address these challenges. These preliminary successes ignited interest in extending CAR-T cell therapies from hematological malignancies to solid tumors ( 1). Both formulations yielded unprecedented 40% complete response rates against relapsed/refractory B-cell leukemia and lymphoma ( 2, 3). Since 2017, two formulations of anti-CD19 CAR-T cell therapy won FDA approval: Kymriah and Yescarta. The CAR directs T cells to recognize, activate, proliferate, and kill in response to scFv-driven recognition of tumor-associated antigens ( 1).
At the center of CAR-T cell therapy is the CAR, an engineered immunoreceptor consisting of an extracellular single-chain antibody fragment (scFv) and hinge, a transmembrane region, and intracellular signaling domains. Finally, we consider current scientific and clinical needs in order to provide future perspectives for improved CAR detection.Ĭhimeric antigen receptor-T (CAR-T) cell therapy is a breakthrough application of adoptive cell therapy (ACT), a novel immunoengineering field where T cells are genetically modified ex vivo and infused for anti-tumor, anti-viral, or immunomodulatory effects in vivo. For each method, we discuss: (1) what it measures (2) how it works (3) its scientific and clinical importance (4) relevant examples of its use (5) specific protocols for CAR detection and (6) its strengths and weaknesses. Here, we review the plethora of CAR detection methods, which can operate at the genomic, transcriptomic, proteomic, and organismal levels. In vivo tracking methods, including two-photon microscopy, bioluminescence imaging, and positron emission tomography scanning, have monitored CAR-T cell biodistribution across blood, tissue, and tumor. In vitro visualization methods, including confocal and total internal reflection fluorescence microscopy, have captured the molecular details underlying CAR immunological synapse formation, signaling, and cytotoxicity. Molecular assays, such as quantitative polymerase chain reaction, integration site analysis, and RNA-sequencing, have characterized CAR transduction, expression, and in vivo CAR-T cell expansion kinetics. For instance, CAR-staining/labeling agents have enabled flow cytometry analysis, imaging applications, cell sorting, and high-dimensional clinical profiling. Methods that detect, quantify, track, and visualize the CAR, have catalyzed the rapid advancement of CAR-T cell therapy from preclinical models to clinical adoption.
2Pritzker School of Medicine, University of Chicago, Chicago, IL, United StatesĬhimeric antigen receptor-T (CAR-T) cell therapy is a promising frontier of immunoengineering and cancer immunotherapy.1Pritzker School of Molecular Engineering, University of Chicago, Chicago, IL, United States.
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