TY - JOUR
T1 - Bioanalytical Methods for Characterization of CAR-T Cellular Kinetics
T2 - Comparison of PCR Assays and Matrices
AU - Masilamani, Madhan
AU - Jawa, Vibha
AU - Dai, Yanshan
AU - Das, Romita
AU - Park, Alice
AU - Lamba, Manisha
AU - Wu, Fan
AU - Zheng, Xirong
AU - Lu, Edwin
AU - Gleason, Carol
AU - Mack, Tim
AU - Mora, Johanna
AU - Surapaneni, Sekhar
N1 - Publisher Copyright:
© 2023 Bristol Myers Squibb. Clinical Pharmacology & Therapeutics © 2023 American Society for Clinical Pharmacology and Therapeutics.
PY - 2023/9
Y1 - 2023/9
N2 - Recently, multiple chimeric antigen receptor T-cell (CAR-T)-based therapies have been approved for treating hematological malignancies, targeting CD19 and B-cell maturation antigen. Unlike protein or antibody therapies, CAR-T therapies are “living cell” therapies whose pharmacokinetics are characterized by expansion, distribution, contraction, and persistence. Therefore, this unique modality requires a different approach for quantitation compared with conventional ligand binding assays implemented for most biologics. Cellular (flow cytometry) or molecular assays (polymerase chain reaction (PCR)) can be deployed with each having unique advantages and disadvantages. In this article, we describe the molecular assays utilized: quantitative PCR (qPCR), which was the initial platform used to estimate transgene copy numbers and more recently droplet digital PCR (ddPCR) which quantitates the absolute copy numbers of CAR transgene. The comparability of the two methods in patient samples and of each method across different matrices (isolated CD3+ T-cells or whole blood) was also performed. The results show a good correlation between qPCR and ddPCR for the amplification of same gene in clinical samples from a CAR-T therapy trial. In addition, our studies show that the qPCR-based amplification of transgene levels was well-correlated, independent of DNA sources (either CD3+ T-cells or whole blood). Our results also highlight that ddPCR can be a better platform for monitoring samples at the early phase of CAR-T dosing prior to expansion and during long-term monitoring as they can detect samples with very low copy numbers with high sensitivity, in addition to easier implementation and sample logistics.
AB - Recently, multiple chimeric antigen receptor T-cell (CAR-T)-based therapies have been approved for treating hematological malignancies, targeting CD19 and B-cell maturation antigen. Unlike protein or antibody therapies, CAR-T therapies are “living cell” therapies whose pharmacokinetics are characterized by expansion, distribution, contraction, and persistence. Therefore, this unique modality requires a different approach for quantitation compared with conventional ligand binding assays implemented for most biologics. Cellular (flow cytometry) or molecular assays (polymerase chain reaction (PCR)) can be deployed with each having unique advantages and disadvantages. In this article, we describe the molecular assays utilized: quantitative PCR (qPCR), which was the initial platform used to estimate transgene copy numbers and more recently droplet digital PCR (ddPCR) which quantitates the absolute copy numbers of CAR transgene. The comparability of the two methods in patient samples and of each method across different matrices (isolated CD3+ T-cells or whole blood) was also performed. The results show a good correlation between qPCR and ddPCR for the amplification of same gene in clinical samples from a CAR-T therapy trial. In addition, our studies show that the qPCR-based amplification of transgene levels was well-correlated, independent of DNA sources (either CD3+ T-cells or whole blood). Our results also highlight that ddPCR can be a better platform for monitoring samples at the early phase of CAR-T dosing prior to expansion and during long-term monitoring as they can detect samples with very low copy numbers with high sensitivity, in addition to easier implementation and sample logistics.
UR - http://www.scopus.com/inward/record.url?scp=85167397697&partnerID=8YFLogxK
U2 - 10.1002/cpt.2991
DO - 10.1002/cpt.2991
M3 - Article
C2 - 37422675
AN - SCOPUS:85167397697
SN - 0009-9236
VL - 114
SP - 664
EP - 672
JO - Clinical Pharmacology and Therapeutics
JF - Clinical Pharmacology and Therapeutics
IS - 3
ER -