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Abstract
Background Adoptive cellular therapy with chimeric antigen receptor (CAR)-T cells has demonstrated remarkable clinical activity in a number of hematologic malignancies, but product chain of custody, individualized manufacturing, preparative chemotherapy, and patient management present technical and logistical hurdles to broader implementation.
Methods Lentiviral constructs for CARs (either CD19- or CD22-directed) co-expressed with a synthetic driver domain were identified from a >6 × 10 diversity combinatorial library of proliferative elements, transmembrane domains, leucine zippers, and an EGFR epitope screened for cellular expansion in a lymphoreplete model. Modified serum-free-lentiviral manufacturing process was developed to reduce complexity of CAR-T and to introduce CD3-activating elements into the viral envelope allowing activation and transduction of resting lymphocytes from peripheral blood.
Results Four-hour exposure of as little as 1 ml of blood to the CD3-directed CD19-targeted CAR encoding lentivirus followed by subcutaneous injection in NSG mice bearing CD19+/CD22+ Raji cells resulted in tumor regression (figure 1) and robust CAR-T cell expansion as determined by flow cytometry (figure 2) and qPCR (table 1), with peak levels >10,000 CAR-T cells/µl and less than three CAR copies per genome. In contrast, administration of the same products intravenously failed to support significant CAR-T expansion or control tumor growth (figure 3). Regression of established Raji tumors was also observed in NSG-(KbDb) (IA) animals following SC administration of CD19 or CD22 CARs with driver domains. CAR-T cells contracted in peripheral blood following tumor regression.
Tumor Regression
Regression of Raji tumor from the initial median volume of 151 mm3 throughout 40 days post subcutaneous administration of the LV transduced (at MOI 1 or 5) CD19-directed CAR T product (1M or 5M cells) in the NSG mice
CAR-T Cells expansion in Vivo Post SC injectionExpansion of CAR-T cells throughout 35 days post subcutaneous administration of the LV transduced (at MOI 1 or 5) CD19-directed CAR T product (1M or 5M cells) in the NSG mice bearing Raji tumor (groups G1-G4) or w/o tumor (G5-7G)
CAR-T Cells expansion in vivo post IV injectionExpansion of CAR-T cells throughout 35 days post intravenous administration of the LV transduced (at MOI 1 or 5) CD19-directed CAR T product (1M or 5M cells) in the NSG mice bearing Raji tumor (groups G1-G7).
qPCR Analysis of LV IntegrationNumber of LV integrations (LV copies) in the PBMC genome (RNase P copies) post transduction at MOI 1 or 5.
Conclusions We conclude that through a synthetic subcutaneous lymph node approach with modified lentiviruses and driver domains, rPOC SC may enable CAR-T generation with reduced complexity, while maintaining the ability of CAR-T cells to expand, persist and exert anti-tumor activity.
Ethics Approval All animal studies were IACUC approved.
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