Efficacy of intracellular immune checkpoint-silenced DC vaccine

Danhong Wang, Xue F Huang, Bangxing Hong, Xiao-Tong Song, Liangding Hu, Min Jiang, Bin Zhang, Hongmei Ning, Yuhang Li, Chen Xu, Xiao Lou, Botao Li, Zhiyong Yu, Jiangwei Hu, Jianlin Chen, Fan Yang, Haiyan Gao, Guoliang Ding, Lianming Liao, Lisa Rollins, Lindsey Jones, Si-Yi Chen, Hu Chen, Danhong Wang, Xue F Huang, Bangxing Hong, Xiao-Tong Song, Liangding Hu, Min Jiang, Bin Zhang, Hongmei Ning, Yuhang Li, Chen Xu, Xiao Lou, Botao Li, Zhiyong Yu, Jiangwei Hu, Jianlin Chen, Fan Yang, Haiyan Gao, Guoliang Ding, Lianming Liao, Lisa Rollins, Lindsey Jones, Si-Yi Chen, Hu Chen

Abstract

Background: DC-based tumor vaccines have had limited clinical success thus far. SOCS1, a key inhibitor of inflammatory cytokine signaling, is an immune checkpoint regulator that limits DC immunopotency.

Methods: We generated a genetically modified DC (gmDC) vaccine to perform immunotherapy. The adenovirus (Ad-siSSF) delivers two tumor-associated antigens (TAAs), survivin and MUC1; secretory bacterial flagellin for DC maturation; and an RNA interference moiety to suppress SOCS1. A 2-stage phase I trial was performed for patients with relapsed acute leukemia after allogenic hematopoietic stem cell transplantation: in stage 1, we compared the safety and efficacy between gmDC treatment (23 patients) and standard donor lymphocyte infusion (25 patients); in stage 2, we tested the efficacy of the gmDC vaccine for 12 acute myeloid leukemia (AML) patients with early molecular relapse.

Results: gmDCs elicited potent TAA-specific CTL responses in vitro, and the immunostimulatory activity of gmDC vaccination was demonstrated in rhesus monkeys. A stage 1 study established that this combinatory gmDC vaccine is safe in acute leukemia patients and yielded improved survival rate. In stage 2, we observed a complete remission rate of 83% in 12 relapsed AML patients. Overall, no grade 3 or grade 4 graft-versus-host disease incidence was detected in any of the 35 patients enrolled.

Conclusions: This study, with combinatory modifications in DCs, demonstrates the safety and efficacy of SOCS1-silenced DCs in treating relapsed acute leukemia.

Trial registration: ClinicalTrials.gov NCT01956630.

Funding: National Institute of Health (R01CA90427); the Key New Drug Development and Manufacturing Program of the "Twelfth Five-Year Plan" of China (2011ZX09102-001-29); and Clinical Application Research of Beijing (Z131107002213148).

Keywords: Dendritic cells; Hematology; Immunology; Immunotherapy; Leukemias.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. Enhanced maturation of Ad-siSSF–transduced hDCs.
Figure 1. Enhanced maturation of Ad-siSSF–transduced hDCs.
(A) Expression levels of a panel of surface markers on human PBMC DCs at 48 hours after transduction with Ad-siSSF, control Ad-GFP virus (MOI = 25,000 vp), or PBS-treated (mock) (isotype controls: brown line). With the exception of OX40L and TLR5, all surface markers are upregulated after Ad-siSSF transduction compared with mock or Ad-GFP controls. (B) The levels of representative proinflammatory cytokines and chemokines in the culture media of human PBMC DCs 48 hours after transduction with different adenoviruses at a MOI of 25,000 vp, as analyzed by ELISA. The hDCs have significantly enhanced levels of these cytokines and chemokines after Ad-siSSF transduction compared with controls. (C) Migration rates of transduced human PBMC DCs in response to recombinant CCL21 (100 ng/ml) show the enhanced migratory activity of Ad-siSSF–transduced DCs compared with controls. Results are presented from 1 of 3 repeated assays (mean ± SEM). *P < 0.05, Ad-siSSF vs. Ad-SM DCs, as determined by Student’s 2-tailed t test.
Figure 2. Enhanced priming of TAA-specific T…
Figure 2. Enhanced priming of TAA-specific T cells by Ad-siSSF-DCs and enhanced TAA-specific CTL cytolytic activity against human tumor cells.
Human autologous T cells were cocultured with Ad-transduced DCs or PBS-treated hDCs (mock) (20:1) for 2 weeks. (A) Intracellular IFNG staining of cocultured CD4+ and CD8+ T cells. FACS analysis shows an increase of IFNG expression after coculture with Ad-siSSF-DCs compared with controls. (B) Increased IFNG-producing T cells after coculture with Ad-siSSF-DCs compared with coculture with controls is shown by ELISpot. Representative data from 1 of 5 HLA-A2+ donors are shown. (C) Cytolytic activities of HLA-A2+ T cells against various human tumor cell lines after 2 weeks of in vitro sensitization with different adenovirus-transfected autologous DCs were determined by standard 51Cr release assays. T cells sensitized by Ad-siSSF-DCs exhibited increased killing compared with those sensitized by controls. Cytolytic percentages are presented from 1 of 3 repeated experiments. Error bars represent mean ± SEM. *P < 0.05, Ad-siSSF vs. Ad-SM; **P < 0.01, Ad-siSSF vs. Ad-GFP or mock, as determined by Student’s 2-tailed t test.
Figure 3. ELISpot assay of immune responses…
Figure 3. ELISpot assay of immune responses induced by a single injection of Ad-siSSF vector in monkeys.
Groups of monkeys (n = 3 per group) were injected with Ad-siSSF or control PBS, and toxicities and immune responses were measured 14 days later. Frequencies of antigen-specific T cells against MUC1 (hMUC1 20mer), survivin (hSur protein), and against the MUC1-survivin fusion protein (hMUC1+Sur) were significantly increased in monkeys given Ad-siSSF compared with the mock group (PBS). P < 0.00001 for responses against designated peptides in the Ad-siSSF group vs. mock group, as determined by Student’s 2-tailed t test.
Figure 4. Consolidated Standards of Reporting Trials…
Figure 4. Consolidated Standards of Reporting Trials diagram.
Figure 5. Kaplan-Meier estimates of overall survival.
Figure 5. Kaplan-Meier estimates of overall survival.
The 3-year probability of OS was significantly higher in the Ad-siSSF-DC group (n = 23) than in the DLI group (n = 25) (48.9 % vs. 27.5%, respectively). P = 0.028, as determined by log-rank test.
Figure 6. Comparing WT1 expression in AL…
Figure 6. Comparing WT1 expression in AL patients before and after treatment.
For the majority of the patients (n = 12), WT1 expression decreased drastically after infusion of Ad-siSSF-DCs. Across all patients, the pretreatment mean of WT1 copies was 1.92 per 1,000 ABL copies versus 0.18 WT1 copies detected after treatment. The complete remission rate was 83%. The GVHD rate was 50%, and no cases of grade 3 or grade 4 GVHD were observed.

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