Stochastic Reaction-Diffusion Model of the Binding of Monoclonal Antibodies to CD4 Receptors on the Surface of T Cells.

A stochastic reaction-diffusion model was developed to describe the binding of labeled monoclonal antibodies (mAbs) to CD4 receptors on the surface of T cells. The mAbs diffused to, adsorbed on, and underwent monovalent and bivalent binding to CD4 receptors on the cell surface. The model predicted the time-dependent nature of all populations involved in the labeling process. At large time, the populations reached equilibrium values, giving the number of antibodies bound to the T cell (ABC) defined as the sum of monovalently and bivalently bound mAbs. The predicted coefficient of variation (CV%) of the (ABC) values translated directly to a corresponding CV% of the measured mean fluorescence intensity (MFI). The predicted CV% was about 0.2% from the intrinsic fluctuations of the stochastic reaction process, about 5% after inclusion of the known fluctuations in the number of available CD4 receptors, and about 11% when fluctuations in bivalent binding affinity were included. The fluorescence detection process is expected to contribute approximately 7%. The abovementioned contributions to CV% sum up to approximately 13%. Work is underway to reconcile the predicted values and the measured values of 17% to 22%.

Evaluation protocol for CRISPR/Cas9-mediated CD19 knockout GM24385 cells by flow cytometry and Sanger sequencing.

Although several genome editing options are available, CRISPR/Cas9 is one of the most commonly used systems for protein and advanced therapies. There are some long-term data regarding genomic and phenotypic stability, however, information is sparse. Flow cytometry can offer a method to characterize these edited cells for longitudinal studies. The objective of this work is to describe a protocol for using flow cytometry to measure the edits from CRISPR/Cas9 on a well-characterized B-lymphoblast cell line, GM24385, with the goal of supporting safe and effective CRISPR/Cas9-engineered therapies.