Cellular Kinetics by Flow Cytometry for Existing and Next Generation CAR-Ts

Distinct from the case of conventional antibody-based protein or small molecule therapeutics, in which PK refers to the quantitation of the levels of the drug in relevant preclinical or clinical matrices, the definition and conceptualization of pharmacokinetics for cell and gene therapies are more challenging. In the case of a vector-delivered transgene-based therapy, for example, quantitative measurements that may be made include levels of the capsid, levels of the vector genome, levels of transgene mRNA, and levels of transgene protein. Such measurements may necessitate the development of assays using technologies including PCR, LC-MS, ligand-binding modalities, or RT-PCR. In addition, quantitative measurements such as enzymatic activity resulting from the expression of a transgene protein are often made as a part of the pharmacodynamic assessment of the gene therapy, as well as downstream measurements of the level of pharmacodynamic and pathway-related biomarkers.

In the case of cell therapies like CAR-T, pharmacokinetic measurements typically consider circulating cell levels, although cells also distribute to other tissue sites. Potential technologies for CAR-T quantitation include qPCR for measurement of transgene levels, flow cytometry for the expression of CAR-expressing cells, and cellular labeling and imaging approaches, each with strengths and limitations.

In this panel, presenters will discuss the suite of quantitative methods involved in gene therapy programs, and then explore the development and implementation of methods for PK and biodistribution measurements for the AAV vector (including genome levels) as well as the PK of the transgene product. Important considerations for biodistribution studies, as well as options for assessment of levels of transgene products, will be examined. Similarly, techniques and considerations for CAR-T cellular quantitation will be explored. Finally, the implications of the definition of different types of assays to the performance requirements of these assays will be discussed.