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. 2010 Jun;24(6):1160-70.
doi: 10.1038/leu.2010.75. Epub 2010 Apr 29.

Engineering CD19-specific T lymphocytes with interleukin-15 and a suicide gene to enhance their anti-lymphoma/leukemia effects and safety

V Hoyos  1 , B Savoldo, C Quintarelli, A Mahendravada, M Zhang, J Vera, H E Heslop, C M Rooney, M K Brenner, G Dotti
Affiliations

Engineering CD19-specific T lymphocytes with interleukin-15 and a suicide gene to enhance their anti-lymphoma/leukemia effects and safety

V Hoyos et al. Leukemia. 2010 Jun.

Abstract

T lymphocytes expressing a chimeric antigen receptor (CAR) targeting the CD19 antigen (CAR.19) may be of value for the therapy of B-cell malignancies. Because the in vivo survival, expansion and anti-lymphoma activity of CAR.19(+) T cells remain suboptimal even when the CAR contains a CD28 costimulatory endodomain, we generated a novel construct that also incorporates the interleukin-15 (IL-15) gene and an inducible caspase-9-based suicide gene (iC9/CAR.19/IL-15). We found that compared with CAR.19(+) T cells, iC9/CAR.19/IL-15(+) T cells had: (1) greater numeric expansion upon antigen stimulation (10-fold greater expansion in vitro, and 3- to 15-fold greater expansion in vivo) and reduced cell death rate (Annexin-V(+)/7-AAD(+) cells 10+/-6% for iC9/CAR.19/IL-15(+) T cells and 32+/-19% for CAR.19(+) T cells); (2) reduced expression of the programmed death 1 (PD-1) receptor upon antigen stimulation (PD-1(+) cells <15% for iC9/CAR.19/IL-15(+) T cells versus >40% for CAR.19(+) T cells); and (3) improved antitumor effects in vivo (from 4.7- to 5.4-fold reduced tumor growth). In addition, iC9/CAR.19/IL-15(+) T cells were efficiently eliminated upon pharmacologic activation of the suicide gene. In summary, this strategy safely increases the anti-lymphoma/leukemia effects of CAR.19-redirected T lymphocytes and may be a useful approach for treatment of patients with B-cell malignancies.

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Figures

Figure 1
Figure 1. T cells transduced with the iC9/CAR.19/IL15 vector produce IL15 and expand in response to antigen stimulation
Panel A illustrates the kinetics of IL15 release by control NT, CAR.19+ and iC9/CAR.19/IL15+ T cells with or without antigen stimulation (CD19+ B-CLL cells). Panel B illustrates the release of IL15 by iC9/CAR.19/IL15+ T cells when these cells were maintained in culture for 4 weeks and stimulated once a week with the antigen (CD19+ B-CLL cells). Panel C illustrates the release of IL2 by control NT, CAR.19+ and iC9/CAR.19/IL15+ T cells with or without antigen stimulation (CD19+ B-CLL cells). Panels D, E. illustrate the expansion of control NT, CAR.19+ and iC9/CAR.19/IL15+ T cells upon weekly stimulation with CD19+ B-CLL cells. Viable cells were counted by trypan blue exclusion once a week. Data in these panels represent the mean ± SD of 4 T-cell lines.
Figure 2
Figure 2. T cells transduced with the iC9/CAR.19/IL15 vector have enhanced viability and higher expression of Bcl-2
Panel A. Control NT, CAR.19+ and iC9/CAR.19/IL15+ T cells were labeled with CFSE and stimulated with CD19+ B-CLL cells. CFSE dilution was measured by FACS analysis after 5 days of culture on live cells. Data are representative of 4 T-cell lines. CFSE negative cells of the top histogram (NT) represent residual tumor cells that are not expected to be eliminated by control T cells. Panel B illustrates Annexin-V/7-AAD staining of CAR.19+ or iC9/CAR.19/IL15+ T cells measured 5 days after the stimulation with B-CLL cells. Data are representative of 4 T-cell lines. Panel D shows BCL-2 expression as detected by FACS analysis in CAR.19+ or iC9/CAR.19/IL15+ T cells 5 days after the stimulation with B-CLL cells.
Figure 3
Figure 3. In vivo localization and expansion of T cells transduced either with CAR.19 or iC9/CAR.19/IL15 vectors
Panel A and D. SCID mice were infused i.v either with FFLuc labeled Daudi or Raji cells, respectively. Tumor cell bioluminescence was measured 10 or 15 days after infusion. Then SCID mice engrafted either with unlabeled Daudi or Raji cells, respectively, were injected either with CAR.19+ or iC9/CAR.19/IL15+ T cells labelled with eGFP-FFLuc (Panels B, E). T-cell signal intensity increased in mice receiving iC9/CAR.19/IL15+ T cells compared to CAR.19+ T cells. Panels C and F illustrate the maximum increase in T-cell bioluminescence obtained in 5 and 10 mice per group, respectively.
Figure 4
Figure 4. iC/CAR.19/IL15+ T cells have enhanced anti-tumor effects and lower expression of PD-1 as compared to CAR.19+ T cells
Panel A illustrates the cytotoxic activity of control NT, CAR.19+ and iC9/CAR.19/IL15+ T cells. Targets were CD19+ B-cell Lymphoma cell line (Daudi), CD19− lymphoma cell line (HDLM-2) and K562 cell line. Both CAR.19+ and iC9/CAR.19/IL15+ T cells retained specific cytotoxic activity. Data illustrate the mean ± SD of 4 T-cell lines. Panel B illustrates the release of IFNγ by control, CAR.19+ and iC9/CAR.19/IL15+ T cells with or without stimulation with the antigen (CD19+ B-CLL cells). Data represents the mean ± SD of 4 T-cell lines. Panel C illustrates the antitumor effects of CAR.19+ and iC9/CAR.19/IL15+ cells kept in culture for 4 weeks. iC9/CAR.19/IL15+ T cells had enhanced capacity to eliminate tumor cells (Karpas CD30+/CD19+) as compared to CAR.19+ cells. Results are representative of 4 T-cell lines. Panel D. PD-1 was significantly overexpressed in CAR.19+ T cells as compared to iC9/CAR.19/IL15+ T cells two days upon stimulation with B-CLL leukemic cells.
Figure 5
Figure 5. iC9/CAR.19/IL15+ T cells have enhanced anti-tumor effects in vivo as compared with CAR.19+ T cells
To evaluate anti-tumor effects, SCID mice were engrafted in the peritoneum (Panels A, B) or subcute (Panels C, D) with Daudi cells labeled with FFLuc, and then treated either with control NT, CAR.19+ or iC9/CAR.19/IL15+ T cells 7–10 days later. Tumor growth was monitored using an in vivo imaging system. Panels A, B illustrate tumor growth in representative mice. Enhanced control of tumor growth was observed in mice receiving iC9/CAR.19/IL15+ T cells. Panels B, D summarize the bioluminescence signal as a measurement of tumor growth by day 38 and 24 after T-cell infusion. Enhanced control of tumor growth was observed in mice treated with iC9/CAR.19/IL15+ T cells. Data represent mean ± SD of 12 mice per group.
Figure 6
Figure 6. Activation of the inducible caspase-9 suicide gene significantly eliminates iC9/CAR.19/IL15+ T cells
iC9/CAR.19/IL15+ T cells undergo apoptosis upon incubation with CID AP20187 at 50 nM(27). Results are representative of 4 T-cell lines. Panel B. SCID mice engrafted i.v. with Raji cells, infused with iC9/CAR.19/IL15+ T cells expressing eGFP-FFLuc were then treated by day 14 with 2 doses of the CID AP20187 (50 µg) i.p. two days apart(27). T-cell bioluminescence reduced upon CID administration. Panel C shows the kinetics of bioluminescence in 5 mice before and after treatment with CID.
Figure 7
Figure 7. T cells isolated from B-CLL patients and expressing iC9/CAR.19/IL15 produce IL15, expand in response to autologous B-CLL and provided enhanced anti-leukemia effect
Panels A, B. illustrate the expansion of control NT, CAR.19+ and iC9/CAR.19/IL15+ T cells obtained from B-CLL patients upon stimulation once a week with autologous B-CLL cells. Cells were counted by trypan blue exclusion once a week. Data in these panels represent the mean ± SD of 3 T-cell lines. Panel C illustrates the production of IL2 and IL15 by control, CAR.19+ and iC9/CAR.19/IL15+ T cells with or without weekly stimulation with autologous CD19+ B-CLL cells. Data in these panels represent the mean ± SD of 3 T-cell lines Panel D illustrates that IL15 protected iC9/CAR.19/IL15+ T cells from apoptosis after the stimulation with B-CLL cells. Data are representative of 3 T-cell lines. Panel E. iC9/CAR.19/IL15+ T cells retained enhanced capacity to eliminate autologous CD19+ B-CLL cells labeled with CFSE by week 4 of culture as compared to CAR.19+ T cells. Data are representative of 3 T-cell lines.
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