Bin Kan

Immunogene Therapy of Tumors with Vaccine Based on Xenogeneic Epidermal Growth Factor Receptor

The breaking of immune tolerance against self epidermal growth factor receptor (EGFr) should be a useful approach for the treatment of receptor-positive tumors with active immunization. To test this concept, we constructed a plasmid DNA encoding extracellular domain of xenogeneic (human) EGFr (hEe-p) or corresponding control mouse EGFr (mEe-p) and empty vector (c-p). Mice immunized with hEe-p showed both protective and therapeutic antitumor activity against EGFr-positive tumor. Sera isolated from the hEe-p-immunized mice exhibited positive staining for EGFr-positive tumor cells in flow cytometric analysis and recognized a single 170-kDa band in Western blot analysis. Ig subclasses responded to rEGFr proteins were elevated in IgG1, Ig2a, and Ig2b. There was the deposition of IgG on the tumor cells. Adoptive transfer of the purified Igs showed the antitumor activity. The increased killing activity of CTL against EGFr-positive tumor cells could be blocked by anti-CD8 or anti-MHC class I mAb. In vivo depletion of CD4+ T lymphocytes could completely abrogate the antitumor activity, whereas the depletion of CD8+ cells showed partial abrogation. The adoptive transfer of CD4-depleted (CD8+) or CD8-depleted (CD4+) T lymphocytes isolated from mice immunized with hEe-p vaccine showed the antitumor activity. In addition, the increase in level of both IFN-γ and IL-4 was found. Taken together, these findings may provide a new vaccine strategy for the treatment of EGFr-positive tumors through the induction of the autoimmune response against EGFr in a cross-reaction between the xenogeneic homologous and self EGFr.

Active immunotherapy of tumors with a recombinant xenogeneic endoglin as a model antigen

Angiogenesis play a critical role in tumor growth and metastasis. Increasing evidence suggests that endoglin is a powerful marker of angiogenesis in solid malignancies. Thus, breaking of immune tolerance of self‐endoglin‐associated angiogenesis is an attractive approach to cancer therapy. To test this concept, we recombined the extracellular domains of porcine endoglin, and used it as a xenogeneic vaccine. We found that immunotherapy with porcine endoglin was effective at both protective and therapeutic anti‐tumor immunity in several mouse tumor models. Autoantibodies against mouseendoglin were identified by Western blot and ELISA. IgG1 and IgG2b were substantially increased. Anti‐endoglin antibody‐producing B cells were detectable by ELISPOT assay. There was endothelial deposition of immunoglobulins within tumors. The anti‐tumor activity was also induced by the adoptive transfer of the purified immunoglobulins. Angiogenesis was apparently inhibited within the tumor tissues and on the alginate beads. The increased apoptotic cells were found within the tumor tissues from the mice treated with porcine endoglin. The anti‐tumor activity and production of autoantibodies againstmouse endoglin could be abrogated by depletion of CD4+ T lymphocytes. Remarkably, no marked toxicity was found in the immunized mice. These observations may provide an alternative rationalstrategy for active cancer immunotherapy.

Combination of low‐dose cisplatin and recombinant xenogeneic endoglin as a vaccine induces synergistic antitumor activities

Angiogenesis is critical to the growth and metastasis of solid tumors, and acquired drug resistance is one of the major hindrances to chemotherapy. Thus, we sought a rational strategy using the combination of antiangiogenic biotherapy and chemotherapy for cancer therapy. We explored the efficacy of a strategy combining low‐dose cisplatin and a recombinant xenogeneic endoglin as a protein vaccine, which we previously demonstrated to have effective antiangiogenic effects in several mouse models. We found that both low‐dosage cisplatin and xenogeneic endoglin vaccine individually resulted in effective suppression of tumor growth in 2 tumor models via inhibition of tumor angiogenesis. Remarkably, the combination therapy resulted in not only significant antiangiogenic effects but also additional promotion of tumor cell apoptosis and inhibition of tumor cell proliferation, without any ensuing increase in host toxicity during the course of treatment, which lasted for 6 months. In addition, the combination demonstrated a synergistic relationship, which was shown in all of the synergistic indexes, i.e., tumor volume, angiogenesis, apoptosis and proliferation. Both antibodies and antibody‐producing B cells against mouse self‐endoglin were observed in all mice immunized by the xenogeneic endoglin vaccine (alone and combination), which suggested that low‐dose cisplatin did not suppress the host immune response but potentiated the antitumor activity of the xenogeneic endoglin vaccine. These observations may provide the basis for an effective alternative strategy for cancer therapy in the near future.

Immunity against Tumor Angiogenesis Induced by a Fusion Vaccine with Murine β-Defensin 2 and mFlk-1

Purpose: Previous studies indicated that humoral or cellular immunity against murine vascular endothelial growth factor 2 (mFlk-1) was elicited to inhibit tumor growth. Here we describe a genetic fusion vaccine, pMBD2-mFlk-1, based on the targeting of a modified mFlk-1 to antigen-presenting cells by a murine β-defensin 2 (MBD2) protein to induce both humoral and cellular immunity against mFlk-1, with the targeting especially focused on immature dendritic cells. Experimental Design: The protective and therapeutic antitumor immunity of the fusion vaccine was investigated in mouse models. Antiangiogenesis effect was detected by immunohistochemical staining and alginate-encapsulate tumor cell assay. The mechanisms of the fusion vaccine were primarily explored by detection of autoantibodies and CTL activity and confirmed by the deletion of immune cell subsets. Results: The fusion vaccine elicited a strong protective and therapeutic antitumor immunity through antiangiogenesis in mouse models, and this worked through stimulation of an antigen-specific CD8+ T-cell response as well as a specific B-cell response against mFlk-1. The findings were confirmed by depletion of immune cell subsets and in knockout mice. Conclusion: Our study showed that a fusion vaccine based on self immune peptide (MBD2) and self antigen (mFlk-1) induced autoimmunity against endothelial cells, resulting in inhibition of tumor growth, and could be further exploited in clinical applications of cancer immunotherapy.

Identification of ATP synthase beta subunit (ATPB) on the cell surface as a non-small cell lung cancer (NSCLC) associated antigen

BackgroundAntibody-based immuneotherapy has achieved some success for cancer. But the main problem is that only a few tumor-associated antigens or therapeutic targets have been known to us so far. It is essential to identify more immunogenic antigens (especially cellular membrane markers) for tumor diagnosis and therapy.MethodsThe membrane proteins of lung adenocarcinoma cell line A549 were used to immunize the BALB/c mice. A monoclonal antibody 4E7 (McAb4E7) was produced with hybridoma technique. MTT cell proliferation assay was carried out to evaluate the inhibitory effect of McAb4E7 on A549 cells. Flow cytometric assay, immunohistochemistry, western blot and proteomic technologies based on 2-DE and mass spectrometry were employed to detect and identify the corresponding antigen of McAb4E7.ResultsThe monoclonal antibody 4E7 (McAb4E7) specific against A549 cells was produced, which exhibited inhibitory effect on the proliferation of A549 cells. By the proteomic technologies, we identified that ATP synthase beta subunit (ATPB) was the corresponding antigen of McAb4E7. Then, flow cytometric analysis demonstrated the localization of the targeting antigen of McAb4E7 was on the A549 cells surface. Furthermore, immunohistochemstry showed that the antigen of McAb4E7 mainly aberrantly expressed in tumor cellular membrane in non-small cell lung cancer (NSCLC), but not in small cell lung cancer (SCLC). The rate of ectopic expressed ATPB in the cellular membrane in lung adenocarcinoma, squamous carcinoma and their adjacent nontumourous lung tissues was 71.88%, 66.67% and 25.81% respectively.ConclusionIn the present study, we identified that the ectopic ATPB in tumor cellular membrane was the non-small cell lung cancer (NSCLC) associated antigen. ATPB may be a potential biomarker and therapeutic target for the immunotherapy of NSCLC.

Vesicular stomatitis virus matrix protein gene enhances the antitumor effects of radiation via induction of apoptosis

Vesicular stomatitis virus (VSV) matrix (M) protein can directly induce apoptosis by inhibiting host gene expression when it is expressed in the absence of other viral components. Previously, we found that the M protein gene complexed to DOTAP-cholesterol liposome (Lip-MP) can suppress malignant tumor growth in vitro and in vivo; however, little is known regarding the biological effect of Lip-MP combined with radiation. The present study was designed to determine whether Lip-MP could enhance the antitumor activity of radiation. LLC cells treated with a combination of Lip-MP and radiation displayed apparently increased apoptosis compared with those treated with Lip-MP or radiation alone. Mice bearing LLC or Meth A tumors were treated with intratumoral or intravenous injections of Lip-MP and radiation. The combined treatment significantly reduced mean tumor volumes compared with either treatment alone in both tumor models and prolonged the survival time in Meth A tumor models and the intravenous injection group of LLC tumor models. Moreover, the antitumor effects of Lip-MP combined with radiation were greater than their additive effects when compared with the expected effects of the combined treatment in vivo. This study suggests that Lip-MP enhanced the antitumor activity of radiation by increasing the induction of apoptosis.

A protective role of IL-30 via STAT and ERK signaling pathways in macrophage-mediated inflammation

IL-30, the p28 subunit of IL-27, interacts with the Epstein-Barr virus-induced gene 3 (EBI3) to form IL-27, which modulates the inflammatory responses in autoimmune and infectious diseases. Several previous studies have provided evidence for the role of IL-30 in the anti-inflammatory process. However, the effect of IL-30 in macrophage-mediated immune responses is not well understood. With the recent observation in our experiment, we found that IL-30 exerted potent anti-inflammatory effects in the RAW 264.7 macrophages and in a lipopolysaccharide/D-galactosamine (LPS/D-GalN)-induced mouse model. IL-30 decreased the production of tumor necrosis factor (TNF)-α and IL-6 in LPS-stimulated RAW 264.7 macrophages in a dose-dependent manner. In the macrophage-mediated GalN and LPS model of acute liver injury, IL-30 prevented liver injury by suppressing serum enzyme activity and down-regulating the pro-inflammatory cytokines TNF-α, IL-1β, IL-6, and interferon (IFN)-γ. IL-30 treatment decreased apoptosis in liver tissue and increased glutathione (GSH) levels. We postulated that IL-30 might function through gp130-mediated signaling pathways and then demonstrated that IL-30 affects LPS-induced inflammation through the STAT1, STAT3, and ERK signaling pathways. These data indicate that IL-30 can provide critical protection against macrophage-mediated liver inflammation through anti-apoptotic, anti-oxidant, and anti-inflammatory activities.

A novel strategy for tumour therapy combining cell apoptosis and active immunity induced by caspy2, a zebrafish caspase

Caspy2, a zebrafish protease, is an active caspase for inducing apoptosis in mammalian cells. To investigate whether caspy2‐mediated apoptosis could be used in cancer therapy, its cDNA was amplified and cloned into eukaryotic expression vector pcDNA3.1+. The recombinant plasmid was mixed with cationic liposome and introduced into various tumour cell lines in vitro. Our data showed that caspy2 induced remarkable apoptosis of cancer cells in vitro. Treatment of mice‐bearing CT26 colon carcinoma or MethA fibrosarcoma with intratumoral injection of liposome‐caspy2 plasmid complex resulted in substantial killing of neoplastic cells and long‐term survivors. Apoptotic cells were widely distributed in caspy2‐treated tumour tissue. Infiltration of CD8+ T lymphocyte was also observed apparently in the tumour tissue after the treatment with caspy2. Tumour‐specific major histocompatibility complex (MHC) class I‐dependent CD8+ cytotoxic T lymphocyte activity was found by means of 51Cr release assay. In MethA model, the mice acquired a long‐time protective immunity against the parental tumour cell re‐challenge. These results indicated that caspy2 can act as both apoptosis inducer and immune response initiator, which may account for its extraordinary antitumour effect. Furthermore, in vivo depletion of CD8+ T lymphocytes could completely abrogate the antitumour immune activity, whereas the depletion of CD4+ cells showed partial abrogation. In this study, we developed a novel anticancer strategy that combines both induction of apoptosis and immune response in mice‐bearing tumours with a single caspy2 gene. This approach may provide an important way for treatment of cancer.