Establishment and validation of in-house cryopreserved CAR/TCR-T cell flow cytometry quality control

Abstract

Background Chimeric antigen receptor (CAR) or T-cell receptor (TCR) engineered T-cell therapy has recently emerged as a promising adoptive immunotherapy approach for the treatment of hematologic malignancies and solid tumors. Multiparametric flow cytometry-based assays play a critical role in monitoring cellular manufacturing steps. Since manufacturing CAR/TCR T-cell products must be in compliance with current good manufacturing practices (cGMP), a standard or quality control for flow cytometry assays should be used to ensure the accuracy of flow cytometry results, but none is currently commercially available. Therefore, we established a procedure to generate an in-house cryopreserved CAR/TCR T-cell products for use as a flow cytometry quality control and validated their use. Methods Two CAR T-cell products: CD19/CD22 bispecific CAR T-cells and FGFR4 CAR T-cells and one TCR-engineered T-cell product: KK-LC-1 TCR T-cells were manufactured in Center for Cellular Engineering (CCE), NIH Clinical Center. The products were divided in aliquots, cryopreserved and stored in the liquid nitrogen. The cryopreserved flow cytometry quality controls were tested in flow cytometry assays which measured post-thaw viability, CD3, CD4 and CD8 frequencies as well as the transduction efficiency and vector identity. The long-term stability and shelf-life of cryopreserved quality control cells were evaluated. In addition, the sensitivity as well as the precision assay were also assessed on the cryopreserved quality control cells. Results After thawing, the viability of the cryopreserved CAR/TCR T-cell controls was found to be greater than 50%. The expression of transduction efficiency and vector identity markers by the cryopreserved control cells were stable for at least 1 year; with post-thaw values falling within ± 20% range of the values measured at time of cryopreservation. After thawing and storage at room temperature, the stability of these cryopreserved cells lasted at least 6 h. In addition, our cryopreserved CAR/TCR-T cell quality controls showed a strong correlation between transduction efficiency expression and dilution factors. Furthermore, the results of flow cytometric analysis of the cryopreserved cells among different laboratory technicians and different flow cytometry instruments were comparable, highlighting the reproducibility and reliability of these quality control cells. Conclusion We developed and validated a feasible and reliable procedure to establish a bank of cryopreserved CAR/TCR T-cells for use as flow cytometry quality controls, which can serve as a quality control standard for in-process and lot-release testing of CAR/TCR T-cell products.

ZNF165 as a potential novel immunotherapeutic target for gastric cancer

“…Combined with GTEx dataset to evaluated the expression levels of 14 differentially expressed CTAs, we found that CT83 (KK-LC-1) and ZNF165 were expressed at low levels in normal human tissues (with the exception of testis tissue). To date, KK-LC-1 has already been used as immunotherapeutic target that has attempted to produce anti-tumor immune response by using TCR-T, CAR-T vaccines [32,33]. Moreover, we identified that the different mutation profiles of CTAs in both eastern and western GC populations, and found out that the TMB scores in the mutation type of the majority of mutated CTA genes were significantly higher than those in the wild type.…”

Post-thaw application of ROCK-inhibitors increases cryopreserved T-cell yield

“…38,39 This becomes even more relevant when discussing CAR-T cell therapy cryopreservation, as it is important to ensure that these act efficiently and conserve high viabilities. 40,41 Studies have shown lower cell viability and cytokine expression, 42 lower surface marker expression, 43 upregulation of apoptosis-associated gene expression 7 and lower cellular expansion after thawing compared to fresh CAR-T products. 44 Although results vary along studies, their cryopreservation is usually supported, as evidenced by the FDA approval of cryopreserved CAR-T products, which allow their centralised manufacturing.…”

Development of Robust Cell Therapy Manufacturing Processes Largely Depends on the Choice of Cell Counting Method Considering Cell Type, Precision, and Accuracy Requirements

“…Cell count and viability (CCV) measurement is perhaps the most fundamental analytical method used in development and execution of manufacturing processes for cell therapy and regenerative medicine. CCV is also a routine release specification for almost all cell therapy products and is used to determine populations to densities, potency assays, functional cell assays, ELISA and Gene modification assays [1][2][3]. Therefore, the choice and implementation of cell counting methodology can play a pivotal role in the development of manufacturing processes, and eventually commercialization of cell therapies.…”