A Comprehensive Cellular Kinetics Strategy to Inform T-cell Therapy Programs

Adoptive T cell therapy is an innovative cancer treatment that involves the extraction and genetic modification of a patient’s T cells to enhance their ability to recognize and attack cancer cells. This case study describes a detailed cellular kinetics (CK) strategy for an autologous TCR-T cell program that targets a neoantigen expressed in certain solid tumors. This comprehensive clinical CK strategy combines 2 PCR assays and a flow cytometry assay to enable a clear analysis of the expansion and persistence of the engineered T cells in circulation. This presentation will highlight the challenges in the development of these 3 complementary assays and show qualification and validation data to support a first-in-human study.
The CK measurement using a ddPCR assay with reportable units of transgene copies per µg of blood DNA will support a secondary objective. The challenges of the ddPCR assay, including a high sensitivity requirement of 50 copies/µg blood DNA and the implemented solutions, will be discussed. Since lymphodepletion is an essential pre-treatment in T cell therapy clinical development and may impact the copies/µg DNA readout across patients, another exploratory ddPCR assay was developed to enable data reporting in copies/µL blood, a readout that is independent of the patients’ lymphodepletion status. For this exploratory assay, an exogenous DNA was incorporated into the assay workflow to enable the calculation of copies/µL blood. The consistency of this readout across samples with different levels of lymphodepletion was confirmed using CD45-depleted blood as well as blood from patients undergoing chemotherapy regimens.
A third assay uses multicolor flow cytometry to quantify the T cell product in blood, while also providing immunophenotyping information. The choice of critical reagents, assay challenges, solutions and validation data will be discussed.