Selective B cell depletion upon intravenous infusion of replication-incompetent anti-CD19 CAR lentivirus

Abstract

Anti-CD19 CAR-T therapy for B cell malignancies has shown clinical success, but a major limitation is the logistical complexity and high cost of manufacturing autologous cell products. Direct infusion of viral gene transfer vectors to initiate in vivo CAR-T transduction, expansion and anti-tumor activity could provide an alternative, universal approach for CAR-T and related immune effector cell therapies that circumvents ex vivo cell manufacturing. To explore the potential of this approach we first evaluated human and mouse CD8 + T cells transduced with VSV-G pseudotyped lentivectors carrying an anti-CD19CAR-2A-GFP transgene comprising either an 1D3 or FMC63 anti-CD19 binding domain. To evaluate CD19 antigen-driven CAR-T proliferation in vitro we co-cultured transduced murine T cells with an excess of irradiated splenocytes. We observed markedly greater expansion over a 9 week period for CAR-transduced T cells compared to control T cells transduced with a GFP-only transgene cultured under the same conditions (mean fold expansion +/-SD above control: ID3-CD19CAR-GFP modified T cells, 12.2 +/-0.09 (p < 0.002); FMC63-CD19CAR-GFP modified T cells 8.8 +/-0.03 (p < 0.004)). CAR-T cells isolated at the end-point of these co-cultures showed potent B cell directed cytolytic activity in vitro. Next, we administered approximately 20 million replication-incompetent lentiviral particles carrying either ID3-CD19CAR-GFP, FMC63-CD19CAR-GFP, or GFP-only transgene to wild-type C57BL/6 mice by tail vein infusion and monitored the dynamics of immune cell subsets isolated from peripheral blood at weekly intervals. We saw emergence of a persistent CAR-transduced CD3 + T cell population beginning at week 3-4 that reached a maximum of 13.5 +/-0.58 % (mean +/-SD) and 7.8 +/-0.76% of the peripheral blood CD3 + T cell population in mice infused with ID3-CD19CAR-GFP lentivector or FMC63-CD19CAR-GFP lentivector, respectively. This was followed by a rapid decline in each group of the B cell content of peripheral blood. Complete B cell aplasia was apparent by week 5 and was sustained until the end of the protocol (week 8). None of these changes were observed in mice infused with GFP-only control lentivector, and significant CAR positive populations were not observed within other immune cell subsets, including macrophage, natural killer, or B cells. Modest weight loss of 5.5 +/-2.97 % (mean +/-SD) was observed in some animals receiving an anti-CD19CAR-GFP transgene during the protocol. These results indicate that direct IV injection of lentiviral particles carrying an anti-CD19 CAR transgene can transduce T cells that then fully ablate endogenous B cells in wild type mice. Based on these results it may be useful to further explore, using existing vectors, the feasibility of systemic gene therapy as a modality for CAR-T and related genetically-engineered Immune Effector Cell therapies.

Complete sequence verification of plasmid DNA using the Oxford Nanopore Technologies’ MinION device

“…Our novel approach was developed and validated using three unique plasmids previously generated in-house: BCRxV.TF.1, BCRxV.GagPolRev.1 and BCRxV.VSVG.1 [20] ( Additional file 1 ). The sequence identity of each plasmid had been previously confirmed by Sanger sequencing following the NIH Human Genome finishing standard (> Phred30, double read coverage of every base) [21].…”