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. 2014 Oct 30;124(18):2824-33.
doi: 10.1182/blood-2013-11-541235. Epub 2014 Jul 21.

"V体育官网入口" Invariant NKT cells with chimeric antigen receptor provide a novel platform for safe and effective cancer immunotherapy

Andras Heczey  1 , Daofeng Liu  1 , Gengwen Tian  2 , Amy N Courtney  1 , Jie Wei  1 , Ekaterina Marinova  1 , Xiuhua Gao  1 , Linjie Guo  1 , Eric Yvon  3 , John Hicks  2 , Hao Liu  4 , Gianpietro Dotti  5 , Leonid S Metelitsa  6
Affiliations

"V体育官网入口" Invariant NKT cells with chimeric antigen receptor provide a novel platform for safe and effective cancer immunotherapy

Andras Heczey et al. Blood. .

Abstract

Advances in the design of chimeric antigen receptors (CARs) have improved the antitumor efficacy of redirected T cells. However, functional heterogeneity of CAR T cells limits their therapeutic potential and is associated with toxicity. We proposed that CAR expression in Vα24-invariant natural killer T (NKT) cells can build on the natural antitumor properties of these cells while their restriction by monomorphic CD1d limits toxicity. Primary human NKT cells were engineered to express a CAR against the GD2 ganglioside (CAR. GD2), which is highly expressed by neuroblastoma (NB). We compared CAR. GD2 constructs that encoded the CD3ζ chain alone, with CD28, 4-1BB, or CD28 and 4-1BB costimulatory endodomains. CAR. GD2 expression rendered NKT cells highly cytotoxic against NB cells without affecting their CD1d-dependent reactivity. We observed a striking T helper 1-like polarization of NKT cells by 4-1BB-containing CARs. Importantly, expression of both CD28 and 4-1BB endodomains in the CAR. GD2 enhanced in vivo persistence of NKT cells. These CAR. GD2 NKT cells effectively localized to the tumor site had potent antitumor activity, and repeat injections significantly improved the long-term survival of mice with metastatic NB. Unlike T cells, CAR. GD2 NKT cells did not induce graft-versus-host disease. These results establish the potential of NKT cells to serve as a safe and effective platform for CAR-directed cancer immunotherapy. VSports手机版.

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Figures

Figure 1
Figure 1
Generation of CAR.GD2 NKT cells. (A) Schematic representation of recombinant retroviral vectors encoding CAR.GD2 constructs. (B) NKT cells were isolated from human PBMCs, stimulated with αGalCer, and transduced with the indicated CAR.GD2 constructs followed by expansion in culture with IL-2. The expanded cells were analyzed by FACS for the frequency of NKT cells using 6B11 and anti-CD3 mAbs (day 21 after transduction, upper panel) and for the frequency of CAR.GD2 NKT cells using anti-idiotype 1A7 mAb on days 7, 14, and 21. Results are from a representative of 5 experiments. (C) Absolute cell count of CAR.GD2-expressing NKT cells at day 21 of expansion. Data are mean ± standard deviation (SD) from 5 donors.
Figure 2
Figure 2
Dual-specific cytotoxicity of CAR.GD2 NKT cells. The cell-mediated cytotoxicity of parental and CAR.GD2 NKT cells with the indicated constructs was tested using a 4-hour 51Cr-release assay against the following targets: GD2+CD1dneg CHLA255 NB cell line (A); GD2negCD1d+ in vitro polarized M2 macrophages (B); and GD2negCD1dneg LA-N-6 NB cell line (C). (D) 7-AAD staining was performed on day 3 of NKT/NB cell culture. Histograms show percent 7-AAD positive cells after gating on CAR-positive cells with the indicated constructs. Results are from a representative of 3 independent experiments.
Figure 3
Figure 3
Cytokine release after CAR.GD2 stimulation. CAR.GD2 NKT cells were stimulated by CHLA255 NB cell line; supernatants were collected after 24 hours, and the concentrations of the indicated cytokines (A-F) were quantified using a Luminex assay. Plots represent combined data from 3 different donors. The maximal cytokine level in each experiment was used as 1, and data were normalized relative to the maximum in each experiment. Data are mean ± SD. *P < .05; **P < .01; ***P < .001, compared with Gz group, 1-way ANOVA with Bonferoni posttest analysis.
Figure 4
Figure 4
In vivo persistence of CAR.GD2 NKT cells in a metastatic NB model. Each mouse received IV injection of 106 CHLA-255 NB cells followed by (day 21) injection of 107 CAR.GD2 NKT cells with the indicated constructs. Cell persistence was monitored by bioluminescent imaging on the indicated days. (A) Bioluminescent images of 5 mice per group are shown from 1 of 2 independent experiments. (B) Bioluminescence photon count on indicated days. Data are mean ± standard error of the mean. Data were analyzed by Student t test at each time point after a logarithmic transformation to stabilize the variance, and relevant P values are described in the text. (C) Serum was collected from mice 24 hours after NKT-cell injection and analyzed by Luminex assay. Data are mean ± SD. *P < .05; **P < .01; ***P < .001, compared with Gz group, 1-way ANOVA with Bonferoni posttest analysis.
Figure 5
Figure 5
Antitumor activity of CAR.GD2 NKT cells in a metastatic NB model in hu-NSG mice. Three months after human cord blood stem cell transfer, each mouse received IV injection of 106 CHLA-255(ffluc+) NB cells followed by (day 7) injection of 107 CAR.GD2 NKT cells with the indicated constructs. For group “Repeat G28BBz,” additional injections were given on days 14 and 21. (A) Tumor growth was monitored using weekly bioluminescent imaging. Shown are 3 representative of 7 mice per group from 1 of 3 independent experiments. (B) Shown is a representative survival plot from 1 of 3 experiments. Data were analyzed by the Kaplan-Meier method. The differences in survival were then compared using the Gehan-Wilcoxon test.
Figure 6
Figure 6
CAR.GD2 NKT cells effectively localize to the tumor site and do not induce GVHD in hu-NSG mice. (A) Tumor xenografts were established after injection of CHLA-255 NB cells under the renal capsule for 3 weeks followed by IV injection of CFSE-labeled parental or G28BBz-transduced NKT or T cells from the same donor. Mice were euthanized after 48 hours, and tumor sections were analyzed by fluorescent microscopy. (B) Multiple random fields of tissue slides were scanned from each tumor, and the absolute numbers of CFSE-labeled cells were counted using the NIS Elements AR3.2 software. Whiskers cover 10 to 90 percentiles. Mean ± SD from 5 to 10 fields per mouse, 5 mice per group from 1 of 2 independent experiments. *P < .05; **P < .01; ***P < .001, 1-way ANOVA with Bonferoni posttest analysis. (C) Animals were euthanized after 4 to 5 weeks of receiving cell therapy. Macroscopic evaluation shows significant swelling and granular transformation of liver in mice treated with CAR.GD2 T cells, whereas CAR.GD2 NKT cells had no effect on liver. (D) Microscopic examination shows lymphocytic infiltrates and necrosis in both liver and lungs of mice treated with CAR.GD2 T cells, whereas mice treated with CAR.GD2 NKT cells have no detectable abnormalities in the same organs. Shown are representative images from 1 of 3 experiments with similar results.
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