Radiolabeling Of CART Cells And In Vivo Tracing Study

Purpose:To develop a new method for early in vivo distribution,migration,homing and prediction of CART cells,we labeled CART cells using ⁶⁸Ga and ⁸⁹Zr and compared the in vivo biological behavior of CART cells.In order to clarify the relationship between targeting and efficacy of CART cells in solid tumors,pharmacokinetic（PK）and pharmacodynamic（PD）studies of CD19 CART cells were performed in a mouse model of human leukemia xenograft tumors using a dual-modality imaging approach（PET and IVIS）.Methods:First,⁶⁸Ga/⁸⁹Zr were labeled with oxine,the labeling process was optimized and the quality control and stability were performed for the two labeled products respectively;second,⁶⁸Ga-oxine and ⁸⁹Zr-oxine were used to label CART cells and cell viability and labeling stability were evaluated;third,the biological behaviors of⁶⁸Ga/⁸⁹Zr-CART cells in KM mice and severe immunodeficiency model NCG mice were investigated using micro PET and the correlations were also studied.Finally,CD19-positive and negative K562 subcutaneous transplantation tumor models were constructed,respectively,and the pharmacokinetics（PK）and pharmacodynamics（PD）of CART cells in solid tumor models were investigated.For PK studies,⁸⁹Zr-CD19-CART cells（2*10⁶/3 MBq/per animal）were injected tail vein in CD19-positive and negative K562-luc animal models,respectively,and the in vivo biological behavior of CART cells was monitored using Micro PET scans at 2,4,6,24,48,72,90,112,140,168 and 260 h of CART cells in vivo biological behavior.For PD studies,optical imaging monitoring,tumor volume measurements,and DNA copy number assays were performed at days 2,4,6,8,10,and 13 after tail vein injection of CD19-CART cells.Results:Radiolabeling of CART cells was achieved using both ⁶⁸Ga and ⁸⁹Zr nuclides,and the cell viability after labeling was>90%and stable for two half-lives.PK results of Micro PET imaging after direct labeling of CART cells showed that ⁶⁸Ga and ⁸⁹Zr radiolabeled CART cells were mainly distributed in the lung within 10 min after injection,and gradually migrated to the spleen and liver within 6 h,followed by a plateau of radioactivity in the spleen and liver during the observation period.PET imaging of ⁶⁸Ga-labeled CART cells could be traced in vivo for at least 6 h.The in vivo distribution behavior of CART cells labeled with both ⁶⁸Ga and ⁸⁹Zr nuclides within 6h was basically the same,and the radioactive uptake values（%ID/g）in the same tissue were significantly correlated.The distribution behavior of CART cells in the early phase was highly correlated with the long-term biologic behavior in vivo was highly correlated.Moreover,the effective absorbed dose of ⁶⁸Ga-labeled CART cells was only one twenty-fourth of that of ⁸⁹Zr-labeled CART cells,with greater potential for clinical translation.Pharmacodynamic（PD）studies of CART cells using optical imaging combined with tumor volume monitoring showed that the fluorescence intensity of K562 tumor cells in the CD19-positive group was lower than that in the CD19-negative group after CART cell treatment,and in the early stage of treatment（day 4 of treatment）,the fluorescence intensity of K562 tumors in the CD19-positive group（1.19±0.20）was significantly lower than that in the CD19-negative group（3.28±1.03）,p=0.011,and maintained tumor suppression until the monitoring endpoint;at day 13 of treatment,the fluorescence intensity of CD19-positive tumors（3.85±1.64）was extremely significantly lower than that of CD19-negative tumors（8.18±1.68）,p=0.0005.The PD results with tumor volume monitoring were consistent with optical imaging results on day 10 after CART cells treatment,the relative tumor proliferation rate in the CD19-positive tumor CART cell treatment group was 450±142 T/C%,which was significantly lower than that in the CD19-negative tumor CART cell treatment group（853±360 T/C%）and the CD19-positive tumor lysis control group（978±177 T/C%）（p<0.05）.Further results of specificity analysis of CART cell uptake in CD19-positive and negative K562 tumors using the Patlak model revealed that tumor-specific uptake was higher in the CD19-positive model than in the CD19-negative model,which was consistent with the PD results,while in tumor tissue CART cell copy number assays using the conventional assay q PCR,the results were below the detection The valid data were not obtained.Conclusions:The dynamic distribution and migration of CART cells in the early phase in vivo correlated with the long-term fate of CART cells,and the safety of in vivo tracing using ⁶⁸Ga-labeled CART cells was higher than that of ⁸⁹Zr-labeled cells.The in vivo tracing of ⁶⁸Ga-labeled CART cells is safer than ⁸⁹Zr labeling and has greater potential for clinical translation.In vivo PK and PD studies of CD19 CART cells using PET scans and optical imaging showed that CD19-CART cells have specific targeted uptake and better tumor suppression efficiency in CD19-positive transplanted tumors.The preliminary method established in this study is expected to provide a basis for in vivo PK-PD studies of CART cells,and help the development and clinical translation of CART cell therapeutics.