H Wang1, X Du2, W-H Chen2, J Lou2, H-L Xiao1, Y-M Pan2, H Chen2, N An2, Q-X Zhang3. 1. Shenzhen Bone Marrow Transplantation Public Service Platform, Shenzhen Institute of Hematology, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen, China; Guangzhou Medical University, Guangzhou, China. 2. Shenzhen Bone Marrow Transplantation Public Service Platform, Shenzhen Institute of Hematology, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen, China. 3. Shenzhen Bone Marrow Transplantation Public Service Platform, Shenzhen Institute of Hematology, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen, China. Electronic address: qiaoxiazhang@163.com.
Abstract
BACKGROUND: The chimeric antigen receptor (CAR) consists of an antigen recognition moiety from a monoclonal antibody fused to an intracellular signalling domain capable of activating T cells. The specific structure of the CAR molecule has been used in various basic research and clinical settings to detect CAR expression, but it is necessary to develop more specific and simpler monitoring methods to observe real-time changes. MATERIALS AND METHODS: To develop a quantitative assay for the universal detection of DNA from anti-CD19 CAR-T cells, a TaqMan real-time quantitative polymerase chain reaction (qPCR) assay was developed using primers based on FMC63-28Z gene sequences. We identified the numbers of copies of CAR gene on T cells transduced with the CAR gene that were obtained from peripheral blood. RESULTS: The assay had a minimum detection limit of 10 copies/μL and a strong linear standard curve (y = -3.3682x + 38.594; R2 = 0.999) within the range of the input CAR gene (10-107 copies/μL). The reproducibility test showed a coefficient of variation ranging from 0.63%-1.65%. Real-time qPCR is a highly sensitive, specific, reproducible, and universal method that can be used to detect anti-CD19 CAR-T cells in peripheral blood.
BACKGROUND: The chimeric antigen receptor (CAR) consists of an antigen recognition moiety from a monoclonal antibody fused to an intracellular signalling domain capable of activating T cells. The specific structure of the CAR molecule has been used in various basic research and clinical settings to detect CAR expression, but it is necessary to develop more specific and simpler monitoring methods to observe real-time changes. MATERIALS AND METHODS: To develop a quantitative assay for the universal detection of DNA from anti-CD19 CAR-T cells, a TaqMan real-time quantitative polymerase chain reaction (qPCR) assay was developed using primers based on FMC63-28Z gene sequences. We identified the numbers of copies of CAR gene on T cells transduced with the CAR gene that were obtained from peripheral blood. RESULTS: The assay had a minimum detection limit of 10 copies/μL and a strong linear standard curve (y = -3.3682x + 38.594; R2 = 0.999) within the range of the input CAR gene (10-107 copies/μL). The reproducibility test showed a coefficient of variation ranging from 0.63%-1.65%. Real-time qPCR is a highly sensitive, specific, reproducible, and universal method that can be used to detect anti-CD19 CAR-T cells in peripheral blood.