Cellular Immunotherapy Monitoring
Monitoring persistence and pharmacokinetics of off-the-shelf or allogeneic cellular immunotherapies, such as chimeric antigen receptor T-cells (CAR-T) and engineered natural killer (NK) cells, is challenging due to the limited accuracy and sensitivity of testing methodologies. Traditional techniques developed for chimerism monitoring in bone marrow and solid organ transplantation simply do not have the sensitivity for immunotherapy monitoring.
The extraordinary sensitivity of TwinStrand Duplex Sequencing™ technology makes it possible to detect allogeneic cells below one-in-one-million frequency.
Analytical Sensitivity Below One-in-One-Million
As a measure of technical performance, allogeneic CAR-T DNA was serially diluted into control DNA at ratios from 1/300 to 1/1.1 million. Deep Duplex Sequencing was carried out on all samples in triplicate using a panel of 277 polymorphic SNP sites. Measured frequencies vs. expected frequencies are shown below with 95% confidence intervals.
Measured versus Expected CAR-T Fraction
Allogeneic CAR-T DNA was detected at the expected frequencies in all spike-ins down to below one-in-one-million. No allogeneic CAR-T DNA was detected in the negative control demonstrating unprecedented sensitivity and specificity.
TwinStrand Duplex Sequencing is the Most Sensitive Chimerism Detection Method
The accuracy of TwinStrand Duplex Sequencing technology provides the highest level of sensitivity for the detection of chimerism of any existing technology. In addition, because of the multi-locus nature of the Duplex Sequencing assay, it requires less DNA input than ddPCR at lower levels of sensitivity.
Figure reference: Limits of detection shown in the figure are based on published ranges for current chimerism detection methods. Abbreviations: STR CE – Short Tandem Repeat Capillary Electrophoresis, NGS – Next-Generation Sequencing, SNP – Single Nucleotide Polymorphism, qPCR – quantitative polymerase chain reaction, ddPCR – Droplet Digital polymerase chain reaction.
Monitoring CAR-T Cells In Vivo
To demonstrate clinical utility, the same Duplex Sequencing assay was applied to a series of longitudinally collected peripheral blood samples from two patients treated with an allogeneic CAR-T cell therapy. The fraction of CAR-T cells present in blood over time is shown as the blue and green lines. The dashed line indicates the sensitivity threshold of other available technologies.
Duplex Sequencing reveals complex cell therapy dynamics in these patients over time. The CAR-T abundance in both subjects fell below the detection limit of all other methods within one week of treatment. Subject 1 saw CAR-T levels drop to nearly 1/1,000,000 before rebounding thirty-fold at the 1-month time point, where it persisted for at least six months. Subject 2 saw drug-product levels drop below 1/100,000 within 2 weeks of treatment, where they remained for the duration of the 2-month sampling window.
Variation in the kinetics and absolute level of drug product likely correlates with clinical outcome, yet even being able to study this association has been impossible until the introduction of Duplex Sequencing methods.