AB03. In vitro and in vivo study of individualized cancer immunotherapy and a pilot trial on patients with gastric cancer

Introduction: Cancer immunotherapy has been used in clinical trials and shown an exciting foreground. But if it is beneficial for the treatment of gastric cancer is still not well studied. Present study discussed the antitumor efficiency of individualized immunotherapy induced by dendritic cells transfected with total antigen of autologous gastric cancer cells and reported the initial results of our pilot clinical trial.

Methods: Autologous DCs were isolated from PBMC and proliferated in supplement with GM-CSF and IL-4. Resulting immature DCs were loaded with autologous tumor antigen (tumor lysate or total RNA) and matured in supplement with TNF-α (tu-DC). Autologous T lymphocytes were isolated from PBMC and cocultured with tu-DC to prepare antigen-specific CTLs. Autologous primary cancer cells were obtained from resected specimens of the patients with gastric cancer and cultured as target cells. Phenotypic characters of DCs were detected and evaluated by FCM. The efficiency of cytotoxicity and cytokine release was measured and estimated by using assays of Cytotoxicity and ELISA.

Methods: Humanized immune system was reconstructed on 4-week-old nude mice by intraperitoneal (i.p.) injection of human PBMC with cell number of 3×10⁷ each. Prophylactic assay and therapeutic assay were performed by subcutaneously challenge of primary cancer cells after or before autologous DC vaccination, respectively, and the tumor burden were observed.

Methods: Forty two patients with gastric cancer, who underwent surgery during 2009 to 2012 in Hebei General Hospital, were subjected to a pilot clinical trial. Twenty six patients underwent a radical D2 resection, 7 patients, a palliative resection, 5 patients, an open-and-close operation, and 4 patients had recurrent tumor. Autologous tumor lysate were prepared from fresh excised specimens during operation. DC vaccines and tumor specific CTLs were prepared by using the methods above mentioned. All the 42 patients were therapied with 6 cycles of normative chemotherapy with XELOX regimens initiated within a month postoperatively. Two to four cycles of immunotherapy with the adoptive cell transfer (ACT) with tumor-specific CTLs and DC vaccines were administered during the intermission of chemotherapies. To suppress Treg and to promote calreticulin (CRT) exposure, CTX 1,000 mg and ADM 60 mg were intravenously infused a day before the immunotherapy. Thymosin alpha 1 (Tα1) was injected once a day subcutaneously during the therapy. The levels of Treg were measured before and after chemotherapy by FCM, and the Karnofsky performance scale, Incidence of recurrence, tumor burden, and side effects were evaluated.

Results: (I) Modality of target cells: Primary cancer cells were well growing on 15 to 18 days of culturing, and most of the cells showed a typical modality of cancer cells with more than 80% of the cells being PAS positive histologically; (II) Modality and function of DCs: a typical modality of mature DC is presented on day 7 of culturing. HLA-I molecule was persistently high expressed by immature and mature DCs. The expression of HLA-II, CD83, CD80 and CD86 were significantly going higher when immature DCs were matured. T cell proliferation assay indicated that mature DCs and tumor-loaded DCs (tu-DCs) have a stronger capability to induce autologous and allogenic T cell proliferation (CD3⁺ >95%); (III) Cytokine release: the IL-12 and INF-γ production in the culture supernatant of tu-DCs and CTLs was significantly increased (P<0.05, respectively); (IV) Cytotoxicity of CTL to cancer cells: CTLs induced by tu-DC have a much stronger cytotoxicity to autologous tumor cells than the allogenic tumor cells (P<0.05); (V) Prophylactic and therapeutic efficiency: in the prophylactic panel, final tumor volume and tumor dry weight in tu-DC vaccinated mice was significantly less than controls (P<0.05, respectively). Tumor inhibition rate in tu-DC group (53.7%) was significantly higher than controls (25.1%; P<0.05). Production of IL-12 and IFN-γ in tu-DC group was also significantly higher than controls (P<0.05, respectively). In the therapeutic panel, final tumor volume and tumor dry weight in tu-DC vaccinated mice was significantly less than controls (P<0.01, respectively). Tumor inhibition rate in tu-DC group (38.8%) was significantly higher than controls (13.2%; P<0.05). No autoimmune change was found histologicaly in all the tissues harvested from the experimental mice; (VI) Results of the pilot clinical trial: The percentage of Treg in peripheral blood was significantly decreased in all the 42 patients 4 days after chemotherapy (12.56%) compared with that before chemotherapy (25.51%; P<0.05). Twenty six patients with radical surgery were tumor-free survived with 18-32 months. Five of seven cases with palliative surgery were steady with 8-22 months, and 2 of them suffered tumor progress 8 and 17 months postoperatively. Five of the 9 tumor-loaded patients who underwent an open-and-close surgery or tumor recurrence were durable disease stabilization for 5 to 16 months, and 4 of them suffered a tumor progression during or after chemotherapy and ACT immunotherapy, while Karnofsky performance scale was significantly improved in all the nine tumor-loaded patients. No severe side effects were found except low fever with 37.5-38.5 °C in most, high fever in 10, fatigue in 6, and miled rash in 4 patients.

Discussion and conclusions: Our ex vivo study indicated that individualized immunotherapy, by using autologous tumor associated total antigen, DCs and effector cells, can induce a strong antitumor responses. Our pilot clinical trial demonstrated the feasibility of chemotherapy plus individualized immunotherapy based on regulation of tumor microenvironment to induce immune responses against gastric cancer clinically and benefit the overall survival and alive with neoplasm of the patients. Interestingly, postoperative systemic treatment of normative chemotherapy combined with ACT immunotherapy significantly benefits the tumor-free survival of the patients with gastric cancer.

Keywords: Gastric cancer; immunotherapy; individual therapy; chemotherapy