Conversion Of Cellular Kinetic Data For Chimeric Antigen Receptor T-Cell Therapy (Car-T) Into Interpretable Units.

Abstract Objectives: Cellular kinetic (CK) measurement of CAR-T cell expansion in-vivo by quantitative polymerase chain reaction (qPCR) has been measured in units of transgene copy number/μg of DNA. We propose a formula to convert the qPCR, and flow cytometry (FC) measurements to interpretable numbers of CAR T-cells/μL blood. Further, after the conversion CK parameters were correlated with efficacy/safety endpoints. Methods: CK data measured using both qPCR and FC assays were utilized from the ELIANA trial in pediatric and young adult patients with relapsed and refractory acute lymphoblastic leukemia (pALL). qPCR measures the presence of CAR transgene in cells with units of copies of CAR-DNA/μg of genomic DNA, while FC quantifies surface expression of CAR T-cells as the % of either T-cells or white blood cells (WBCs) that express CAR. Neither measurement accounts for the typical significant increase in WBCs following CAR-T infusion. We propose equations for converting FC (Eq1) or qPCR (Eq2) into concentration of CAR-cells/μL of blood, using WBC counts from the complete blood count. For qPCR, the equation relies on 3 additional parameters M·F/N: amount of DNA/WBC (M), average number of copies of CAR-DNA/CTL019 cell (N), and fraction of cells with CAR-DNA that express CAR receptor (F). To estimate M·F/N, we performed regression of the cells/μL estimate from FC vs qPCR. We fit the CK model [1] to obtain the model parameters. Finally, CK parameters with safety/efficacy were correlated to compare the converted estimates to the native CK units. • (CD3+CAR+cells)/μl blood= WBC/μl blood × (CD3+CAR+cells)/WBC (1) • (CAR+cells)/μl blood=WBC/μl blood × CAR DNA copies/μg DNA × M μg DNA/WBC × 1 CAR cell/N CAR DNA copies×F (2) Results: There was high correlation between FC and qPCR estimates of CAR-T in the blood of pALL patients (r2=0.775). The M·F/N value derived based on FC and qPCR results was estimated as 2.68e-6μg DNA/CAR copies. Assuming M=6.6e-6μg DNA/WBC [2] and F=1, this predicts that N=2.46 CAR copies/CAR-T cell. There was also high correlation between the copies/μg and cells/μL estimates using qPCR (r2=0.752). The relationship between CK parameters and safety/efficacy endpoints was not improved when cells/μL was used. This can be attributed to high correlation between these metrics. Using the CK model, 11x greater fold expansion was predicted using the cells/μL estimate compared to copies/μg. This is because cells/μL estimate accounts the expansion of both CAR-T and WBC numbers following lymphodepletion. Conclusions: The conversion of CK into cells/μL allows for a physiological interpretation of CK data with a high correlation between the cells/μL and copies/μg indicating either metric can be used to predict safety/efficacy. References: 1 Stein M et al. CPT: Pharmacometrics & Systems Pharmacology(2019) 2 Gillooly JF et al. Cold Spring Harbor Perspectives in Biology vol.7,7 a019091(2015) Citation Format: Anwesha Chaudhury, Andrew Stein, Stephan Grupp, John Levine, Michael Pulsipher, G Doug Myers, Edward Waldron, Xu Zhu, Fraser McBlane, Rakesh Awasthi, Edmund K. Waller. Conversion of cellular kinetic data for chimeric antigen receptor T-cell therapy (CAR-T) into interpretable units [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 509.