BackgroundWe conducted a phase I/Ib, open-label, single-arm trial to assess the safety, tolerability and optimal scheduling regimen of OTSGC-A24 cancer vaccine in patients with advanced gastric cancer.MethodsPatients with advanced gastric cancer with HLA-A*24:02 haplotype were included in this study. OTSGC-A24 was administered at 1 mg in 3-weekly (3w), 2-weekly (2w), and weekly (1w) cohorts to evaluate the safety, immunological response and schedule. Based on the highest specific cytotoxic T lymphocyte (CTL) induction rate at 4 weeks, using the ELISPOT test, cohorts were expanded to define the optimal dosing schedule for OTSGC-A24.ResultsIn this study, 24 advanced gastric cancer patients with HLA-A*24:02 haplotype were enrolled and treated in 3 cohorts (3w cohort: 3; 2w cohort: 11 and 1w cohort: 10 patients). The most common adverse events were decreased appetite (29%), diarrhea (21%), myalgia (25%). The most common treatment-related adverse event was injection site erythema (25%). No dose-limiting toxicities were observed in any cohort and OTSGC-A24 was well tolerated. Positive CTL responses after vaccination were observed in 15 patients (75%) at 4 weeks: 3w cohort (33%), 2w cohort (88%), 1w cohort (78%). At 12 weeks, 18 patients had responded (90%); 3w cohort (100%), 2w cohort (100%), 1w cohort (78%). The best radiological was stable disease (40%). Median progression free survival was 1.7 months (95% CI: 1.4 to 3.5) and median overall survival was 5.7 months (95% CI 3.8 to 8.6).ConclusionsOTSGC-A24 combined peptide cancer vaccine was well tolerated. Significant responses in CTL were observed and the recommended phase 2 dose is 1 mg OTSGC-A24 sub-cutaneous, every 2 weeks. Although no radiological response was observed, a respectable overall survival was achieved, consistent with other immunotherapy agents being investigated in gastric cancer.Trial registrationClinicalTrials.gov Identifier: NCT01227772, Date registered: 21 Oct 2010.

Background We evaluated the relevance of PD-1+CD8+ T-cells in gastric cancer (GC) including prognostic significance, association with chemotherapy and immunotherapy sensitivity and correlations with the tumor microenvironment (TME). Methods Discovery cohort: GC samples were evaluated for AE1/3, CD8, PD-1, Ki-67 and Granzyme-B expression with fluorescence-based multiplex immunohistochemistry (mIHC). Validation cohorts: we analyzed bulk RNAseq GC datasets from TCGA, the “3G” chemotherapy trial and an immunotherapy phase 2 trial. The cox proportional hazards model was used to identify factors that influenced overall survival (OS). To study the TME, we analyzed single-cell RNAseq performed on GCs. Results In the discovery cohort of 350 GCs, increased PD-1 expression of CD8 T-cells was prognostic for OS (HR 0.822, p = 0.042). PD-1 expression in CD8 T-cells highly correlated with cytolytic [Granzyme-B+] (r = 0.714, p < 0.001) and proliferative [Ki-67+] (r = 0.798, p < 0.001) activity. Analysis of bulk RNAseq datasets showed tumors with high PD-1 and CD8A expression levels had improved OS when treated with immunotherapy (HR 0.117, p = 0.036) and chemotherapy (HR 0.475, p = 0.017). Analysis of an scRNAseq dataset of 152,423 cells from 40 GCs revealed that T-cell and NK-cell proportions were higher (24% vs 18% and 19% vs 15%, p < 0.0001), while macrophage proportions were lower (7% vs 11%, p < 0.0001) in CD8PD-1high compared to CD8PD-1low tumors. Conclusion This is one of the largest GC cohorts of mIHC combined with analysis of multiple datasets providing orthogonal validation of the clinical relevance of PD-1+CD8+ T-cells being associated with improved OS. CD8PD-1high tumors have distinct features of an immunologically active, T-cell inflamed TME.

Natural killer cell-based immunotherapy is a potential treatment for esophageal cancer due to the cytotoxic effect of NK cell and minimal side effects. Natural Killer Activation and Expansion System (NKAES) is our unique system that capable to produce highly cytotoxic and highly proliferative NK cells. Previous studies showed that NKAES-generated NK cells exerted high cytotoxicity on various types of leukemic cell lines and soft tissue cancer cell lines. Our study focuses on the feasibility of NKAES-generated NK cell immunotherapy for esophageal cancer. To generate NKAES-generated NK, peripheral blood mononuclear cell (PBMC) is co-cultured with feeder cells (genetically modified K562 leukemic cell) in presence of interleukin 2. The feeder cells express 4-1BB ligands and membrane bound interleukin 15, which enhance NK responsiveness to interleukin 2, NK activation, NK proliferation and NK survival. Our NK cytotoxicity assay results indicate that seven esophageal cancer cell lines (TE1, TE2, TE3, TE4, TE5, KYSE110 and KYSE30) are highly sensitive to NKAES-generated NK cells. We further compared the cytotoxicity between resting NK, IL-2 activated NK, and NKAES-generated NK on these cell lines. We found that NKAES-generated NK produced the strongest cytotoxicity on these cancer cell lines. NK cytotoxicity is strongly depending on the expression of NK Group 2 Member D (NKG2D) ligands. During cancer metastasis, NKG2D ligands are shedded off by metalloproteinase, which contributes to immune evasion. Interestingly, a previous study showed that a colorectal cancer cell line was sensitive to NK cytotoxicity in early phase of EMT. We further investigated this phenomena in esophageal cancer. To study this, we successfully established an in vitro model, in which stimulated EMT event in esophageal cancer cell lines by using GSK3β inhibitor and epithelial growth factor (EGF). Astonishingly, beside EMT features, we observed that surface NKG2D ligand expression was higher in EMTed esophageal cancer cells compared with control. In addition, EMTed esophageal cancer cells were much sensitive to NKAES-generated NK cytotoxicity. The difference of NK sensitivity was insignificant in IL-2 activated NK. These findings suggest that early EMT event is a crucial checkpoint to prevent further progression that ultimately lead to cancer metastasis, and NK Immunosurveillance is important in this checkpoint. As a conclusion, NKAES-generated NK cells are capable to exert high cytotoxic effect on esophageal cancer cells as well as to establish a strong checkpoint to prevent the progression of EMT event. Citation Format: Kee Siang Lim, Ley Fang Kua, Kosaku Mimura, Kensuke Shiraishi, Wee Joo Chng, Wei Peng Yong, Dario Campana, Koji Kono. Implication of highly cytotoxic natural killer cells for esophageal cancer treatment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3148. doi:10.1158/1538-7445.AM2015-3148