The Tol2 transposon system mediates the genetic engineering of T-cells with CD19-specific chimeric antigen receptors for B-cell malignancies

Abstract

Engineered T-cell therapy using a CD19-specific chimeric antigen receptor (CD19-CAR) is a promising strategy for the treatment of advanced B-cell malignancies. Gene transfer of CARs to T-cells has widely relied on retroviral vectors, but transposon-based gene transfer has recently emerged as a suitable nonviral method to mediate stable transgene expression. The advantages of transposon vectors compared with viral vectors include their simplicity and cost-effectiveness. We used the Tol2 transposon system to stably transfer CD19-CAR into human T-cells. Normal human peripheral blood lymphocytes were co-nucleofected with the Tol2 transposon donor plasmid carrying CD19-CAR and the transposase expression plasmid and were selectively propagated on NIH3T3 cells expressing human CD19. Expanded CD3(+) T-cells with stable and high-level transgene expression (~95%) produced interferon-γ upon stimulation with CD19 and specifically lysed Raji cells, a CD19(+) human B-cell lymphoma cell line. Adoptive transfer of these T-cells suppressed tumor progression in Raji tumor-bearing Rag2(-/-)γc(-/-) immunodeficient mice compared with control mice. These results demonstrate that the Tol2 transposon system could be used to express CD19-CAR in genetically engineered T-cells for the treatment of refractory B-cell malignancies.

Figure 1

CD19-CAR and the Tol2 transposon system used in this study. VH, variable heavy chain; VL, variable light chain; hatched box, CD8α signal peptide; black box, (GGGGS)₃ linker; pTol2-CD19-CAR, Tol2 transposon plasmid carrying CD19-CAR; CAGp, hybrid cyto-megalovirus enhancer/chicken β-actin promoter; TIR, terminal inverted repeat; pCAGGS-mT2TP, Tol2 transposase (TPase) expression plasmid.

Figure 2

CD19-CAR expression and ex vivo expansion of Tol2-modified T-cells. (a) Surface expression of CD19-CAR on T-cells after nucleofection with pTol2-CD19-CAR with or without pCAGGS-mT2TP (TPase) plasmids was examined by flow cytometry. Values represent the percentages of CD3⁺ CD19-CAR⁺ and CD3⁻ CD19-CAR⁺ cells. Data are representative of one of the three independent experiments using different donors. (b) CD19-CAR expression of Tol2-tranduced T-cells in the presence or absence of TPase as detected by western blotting with an anti-CD3ζ antibody. β-Actin was used as a loading control. (c) Growth rates of transduced T-cells with or without TPase. 3T3/CD19 cells were added weekly. Viable cells were enumerated by trypan blue exclusion. Data represent mean ± s.d. from three different donors.

Figure 3

Antigen-specific effector functions of CD19-CAR T-cells in vitro. (a) IFN-γ secretion by CD19-CAR T-cells after co-culture with 3T3/CD19 or parental 3T3 cells. (b) Cytotoxicity of CD19-CAR T-cells against Raji and K562 cells in Calcein-AM release assays. K562 cells served as CD19⁻ targets. (c) Cytotoxic activities in CD4- and CD8-sorted T-cells. (d) Cytotoxicity against primary CD19⁺ diffuse large B-cell lymphoma (DLBCL) cells. Values show the mean ±s.d. of triplicate wells.

Figure 4

(a) Bioluminescent imaging of systemic Raji tumor progression in Rag2^−/−γc^−/− mice following T-cell infusion. Mice inoculated intravenously with Raji/Luc cells on day 0 were infused with CD19-CAR T-cells alone on day 3 (T-cell treatment group, n =3) or in combination with three intraperitoneal injections of an anti-PD-1 antibody (n =4), whereas mice in the control group did not receive T-cells (n =4). (b) Quantitative bioluminescence imaging on day 27 as shown in panel (a). (c) Raji tumor-bearing mice were infused with CD19-CAR T-cells alone (n =3) on day 3 or in combination with a single intramuscular injection of AAV-IL-7 (n =4). (d) Quantitative bioluminescence image on day 27 as shown in panel (c). Bioluminescent imaging was performed on days 13, 20 and 27. *P<0.05, **P<0.01.