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Vascular endothelial growth factor (VEGF) is an important regulating molecule of angiogenesis in tumor formation and progression. Cancer cells always secrete VEGF to stimulate angiogenesis that facilitate growth and invasion of the tumor. In this study, we established a VEGF164 overexpressing LL/2 lung cancer cell model and found that the postirradiated VEGF164-modified tumor cells protected the host against the challenge with LL/2 wild-type tumor cells. Histochemical assay showed that there were large areas of tumor necrosis with macrophage infiltration in the mice vaccinated with the VEGF164-modified tumor vaccine. T-cells isolated from the vaccinated mice showed cytotoxicity against the parental tumor cells in a dose-dependent manner. Meanwhile, sera from the mice vaccinated with LL/2-VEGF164 showed higher titers of antibodies against parental tumor cells compared with the nonvaccinated groups. Our results indicated that VEGF164-modified tumor vaccine could modulate host antitumor immune response and hold therapeutic potential for cancer.

Basic fibroblast growth factor (bFGF) is a mitogen for endothelial cells, which participates in tumor angiogenesis. Active immunity against bFGF could be a promising approach for the biotherapy of cancer. Because bFGF is abundant in normal and malignant tissues, it is presumably difficult for normal bFGF to induce immunity due to self-tolerance. In addition, previous studies have shown that a complex consisting of a cationic liposome and a non-coding plasmid DNA can be used to stimulate innate immunity. This stimulation initiates a potent cytokine response, which can inhibit tumor growth. To investigate the effects of immunity against bFGF on murine colon carcinomas, we employed an N-, C-terminally truncated basic fibroblast growth factor (tbFGF, of human origin) as an antigen and a liposome-DNA complex as an adjuvant. After six immunizations, a robust bFGF-specific immune response was elicited. Subsequently, inhibition of tumor growth and a significant reduction in tumor vasculature were observed. The antitumor effect was confirmed by adoptive therapy of activated spleen cells from the immunized mice. In vitro, a CTL assay revealed that bFGF-specific cytotoxic T lymphocytes (CTL) resulted in the lysis of mouse microvascular endothelial cells (MS1) rather than that of the CT26 colorectal cancer cells. These results suggest that anti-angiogenesis treatment induced by a bFGF-specific CTLs against microvascular endothelial cells may be a useful method for cancer therapy.

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 beta-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.

Matrix metalloproteinase-2 (MMP-2) has been used as a target for cancer immunotherapy. The activation of immunization by breaking immune tolerance to self-MMP-2 may be one of the promising approaches for the treatment of MMP-2-positive tumors. In this study, we constructed the xenogeneic tumor cell vaccine c-MMP-2 by transfecting CT26 and LLC cells with chicken MMP-2 cDNA constructs. MMP-2-specific autoantibodies in sera and tumor cells were found in mice immunized with c-MMP-2. Protection against tumor growth was evaluated in respect of the relative contributions of autoantibodies, CD4+, and CD8+ T cells. Treatment with this vaccine (c-MMP-2) also prolonged the survival time of mice bearing cancer. The specific cytotoxic T-cell responses suggested that the treatment increased CD8+ T-cell activity. The antitumor activity of c-MMP-2 was abrogated by in vivo depletion of CD4+ and CD8+ T-lymphocytes and improved by adoptive transfer of CD4+ and CD8+ T-lymphocytes from the mice treated with c-MMP-2. An alternative DNA vaccination strategy for cancer therapy was identified in this study by eliciting humoral and cellular immunoresponse with a crossreacting transfectant.

Active immunotherapy targeting epidermal growth factor receptor (EGFR) should be another attractive approach to the treatment of EGFR-positive tumors. To test this concept, the authors evaluated the potential immune responses and antitumor activities elicited by dendritic cells pulsed with recombinant ectodomain of mouse EGFR (DC-edMER). Spleen cells isolated from DC-edMER-vaccinated mice showed a high quantity of EGFR-specific antibody-producing cells. EGFR-reactive antibody in sera isolated from vaccinated mice was identified and shown to be effective against tumors in vitro and in vivo by adoptive transfer. DC-edMER vaccine also elicited cytotoxic T-lymphocyte responses that could mediate antitumor effects in vitro and adoptive transfer in vivo. In addition, EGFR-specific cytokines responses were elicited by DC-edMER vaccine. Immunization with DC-edMER resulted in tumor regression and prolonged survival in mice challenged with Lewis lung carcinomas and mammary cancer models. Depletion of CD4+ T lymphocytes could completely abrogate the antitumor activity and EGFR-specific antibody responses, whereas the depletion of CD8+ T lymphocytes showed partial abrogation of the antitumor activity but antibody was still detected. Furthermore, tumor-induced angiogenesis was suppressed in DC-edMER-vaccinated mice or mice treated with antibody adoptive transfer. Taken together, these findings suggest the antitumor immunity could be induced by DC-edMER, which may involve both humoral and cellular immunity, and may provide insight into the treatment of EGFR-positive tumors through the induction of active immunity against EGFR.

Vascular endothelial growth factor receptor-2 (VEGFR-2) has been shown to play a major role in inducing the full spectrum of VEGF biological response which is essential for tumor angiogenesis. We have demonstrated that immunotherapy of tumors with a vaccine based on quail homologous VEGFR-2 (qVEGFR) was effective in providing both protective and therapeutic antitumor immunity in several tumor models in mice. The purpose of this study was to determine whether the combination therapy of low-dose gemcitabine with qVEGFR as a vaccine could inhibit tumor growth to a greater extent. To test this concept, H22 hepatoma and Lewis lung carcinoma models were established in BALB/c mice and C57BL/6 mice, respectively. Mice were treated with either qVEGFR as a protein vaccine, gemcitabine, or both agents together. qVEGFR or low-dose chemotherapy treatment individually resulted in tumor inhibition to a certain extent.Remarkably, the combination therapy resulted in synergistic antitumor activity. Histological examination revealed that there was endothelial deposition of immunoglobulins within tumor tissues from mice treated with vaccine or combination therapy, especially intratumor angiogenesis was suppressed more significantly for the combination group. Also, ELISPOT analysis showed that mice treated with either qVEGFR alone or in combination with low-dose chemotherapy produced similar amount of anti-VEGFR antibody-producing B cells, which suggested that low-dose gemcitabine did not suppress the host's immune response, but potentiated the antitumor activity of the qVEGFR vaccine. Furthermore, TUNEL staining demonstrated a significant increase in the number of TUNEL-positive cells in the combination group compared with those of other groups. The observations may provide a new bio-chemotherapeutic approach for cancer.

Mouse colon cancer cells CT26 were transfected with constructed plasmid expressing mouse soluble B lymphocyte stimulator (msBlyS). Single cell clones were selected with 100 microg/ml Zeosin and subcloned by serial limiting dilution. Eight resistant transfectants were isolated and expanded, and five of them displayed the desirable msBlyS cDNA band amplified by semi-quantitative RT-PCR assay. Western blot analysis showed that only msBlyS molecules of the expected size were detected in the cell lysates from transfectants. The supernatant of transfectants could costimulate B cell proliferation in standard costimulation assay. Thus we have successfully screened the stable transfectants expressing high levels of msBlyS in CT26 cells, which could be used as cancer vaccines for further anti-tumor immunotherapy.

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.

Objective: To investigate the immunological mechanism for inhibiting melanoma growth in mouse by vaccination with xenogeneic melanocytes., Methods: Xenogeneic vaccine was prepared from pig eye melanocytes. By means of indirect ELISA the antibodies against pig melanocytes and B16 melanoma cells in immunized mice sera were detected and the immunoglobulin subclass were analyzed. Then after purification, the immunoglobulins were used for the inhibition of cell proliferation in vitro. Analyses of cross-reactive antigen in both pig melanocytes and B16 melanoma cells were performed by Western blot. Xenogeneic vaccine was used before B16 melanoma challenge in C57 BL/c mice and then the growth of tumor was monitored. Meanwhile, other mice immunized with xenogeneic vaccine were depleted of NK cells or CD4+ or CD8+ T lymphocytes., Results: The antibodies against pig melanocytes and B16 melanoma cells in mice sera were not detected by indirect ELISA until 2 weeks after first xenogeneic vaccination, and after the first finding, the antibody titers increased with the time of immunization. The anti-tumor activity and production of autoantibodies, conspicuously those of the elevated IgG, could be abrogated by the depletion of CD4+ T lymphocytes. The cross-reactive antigen with 180 kda protein in both pig melanocytes and B16 melanoma cells was confirmed. Xenogeneic vaccination resulted in inhibition of tumor growth in 90% of the immunized mice. The protective immune response elicited in this fashion was dispelled in the mice depleted of CD4+ T lymphocytes. However this response was found in 70% of the mice depleted of CD8+ T lymphocytes, and the depletion NK cells did not influence the anti-tumor effect of the vaccine., Conclusion: The anti-tumor immune response is capable of inhibiting melanoma growth; both humoral immunity and cellular immunity could be induced by xenogeneic melanocytes vaccination. This immune response is mainly mediated by CD4+ T lymphocytes.

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 mouse endoglin 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 against mouse 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 rational strategy for active cancer immunotherapy.

Background & Objective: Chemokines are small molecule proteins that are the main mediators of cell migration. Studies indicated that chemokines could induce antitumor immune response strongly and steadily. At present, there was not antitumor therapy about chemokine BLC combined with chemotherapy. This study was designed to explore the anti-tumor effects and mechanisms of BLC-modified tumor cell vaccine combined with cisplatin., Methods: Firstly, we transfected pcDNA-BLC into murine tumor cell lines and established stable transfected tumor cell lines (Colon26 and LL/2) to express BLC. Null plasmid pcDNA3.1 (+) was also transfected into Colon 26 or LL/2 cells stably for comparison. We established mouse model with pcDNA-BLC stable transfected tumor cells (Tc), control mouse model with pcDNA3.1(+) stable transfected tumor cells (Pc), and mouse model with parental tumor cells (Nc). Then the 60 mice were inoculated subcutaneously (s.c.) in the right flanks with a total of 5x10(5) viable tumor cells (Tc to 20 mice, Pc to 20 mice, and Nc to 20 mice, respectively; 6-8 week BALB/c female mouse in colon 26 mouse model or 6-8 week C57BL/6 female mouse in LL/2 mouse model). The groups (Tc, Pc, and Nc) were randomly subdivided into Group A and Group B with 10 mice for each, resulting in Tc-A/Tc-B, Pc-A/Pc-B, and Nc-A/Nc-B, respectively. Group A (including Tc-A, Pc-A, and Nc-A) as chemotherapy group was injected (i.p.) with 0.1 ml cisplatin at the concentration of 2 mg/kg once a week for two weeks; Group B (including Tc-B, Pc-B, and Nc-B) as control with 0.1 normal saline. We further observed anti-tumor activity including the tumor growth, mice survival rate, side effects as well as tumor morphological analysis. Apoptotic cells were also determined in tumor tissues., Results: The combination therapy group Tc-A showed significant anticancer activities. The tumor growth was inhibited efficiently with 3/10 mice showing complete regression in LL/2 mouse model and 1/10 mice showing complete regression in Colon 26 mouse model. The combination therapy group Tc-A showed that the survival rate of mice was 100% in the two mouse models, compared with 68% in group Tc-B in C57BL/6 mouse model and 65% in group Tc-B in Colon 26 mouse model and those in other groups. In addition, the combination therapy group Tc-A induced tumor cell apoptosis significantly., Conclusions: The therapy of BLC-modified tumor cell vaccine combined with cisplatin had significant synergistic effect against tumor. It might develop a new approach for specific immunotherapy of tumors.

The breaking of immune tolerance of "self-antigens" associated with angiogenesis is an attractive approach to cancer therapy by active immunity. We used vascular endothelial growth factor receptor-2 (VEGFR-2) as a model antigen to explore the feasibility of the immunotherapy with a vaccine based on a xenogeneic homologous protein. To test this concept, we prepared a quail homologous VEGFR-2 protein vaccine (qVEGFR) based on quail VEGFR-2. At the same time, a protein vaccine based on the corresponding ligand-binding domain of mouse self-VEGFR-2 (mVEGFR) was also prepared and used as a control. We found that immunotherapy with qVEGFR was effective at protective and therapeutic antitumor immunity in several solid and hematopoietic tumor models in mice. Autoantibodies against mouse VEGFR-2 (Flk-1) were identified by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). Anti-VEGFR antibody-producing B cells were detectable by ELISPOT. Endothelial deposition of immunoglobulins developed within tumor. VEGF-mediated endothelial cell proliferation was inhibited in vitro by immunoglobulins from qVEGFR-immunized mice. Antitumor activity was caused by the adoptive transfer of the purified immunoglobulins. Antitumor activity and production of autoantibodies against Flk-1 could be abrogated by the depletion of CD4+ T lymphocytes. Angiogenesis was apparently inhibited within the tumors, and the vascularization of alginate beads was also reduced. No marked toxicity was found in the immunized mice. The observations may provide a vaccine strategy for cancer therapy through the induction of autoimmunity against the growth factor receptor associated with angiogenesis in a cross-reaction with single xenogeneic homologous protein.

Angiogenesis is important for the growth of solid tumors. The breaking of the immune tolerance against the molecule associated with angiogenesis should be a useful approach for cancer therapy. However, the immunity to self-molecules is difficult to elicit by a vaccine based on autologous or syngeneic molecules due to immune tolerance. Basic fibroblast growth factor (bFGF) is a specific and potent angiogenic factor implicated in tumor growth. The biological activity of bFGF is mediated through interaction with its high-affinity receptor, fibroblast growth factor receptor-1 (FGFR-1). In this study, we selected Xenopus FGFR-1 as a model antigen by the breaking of immune tolerance to explore the feasibility of cancer therapy in murine tumor models. We show here that vaccination with Xenopus FGFR-1 (pxFR1) is effective at antitumor immunity in three murine models. FGFR-1-specific autoantibodies in sera of pxFR1-immunized mice could be found in Western blotting analysis. The purified immunoglobulins were effective at the inhibition of endothelial cell proliferation in vitro and at the antitumor activity in vivo. The antitumor activity and production of FGFR-1-specific autoantibodies could be abrogated by depletion of CD4+ T lymphocytes. Histological examination revealed that the autoantibody was deposited on the endothelial cells within tumor tissues from pxFR1-immunized mice, and intratumoral angiogenesis was significantly suppressed. Furthermore, the inhibition of angiogenesis could also be found in alginate-encapsulate tumor cell assay. These observations may provide a new vaccine strategy for cancer therapy through the induction of autoimmunity against FGFR-1 associated with angiogenesis in a cross-reaction.

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-gamma 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.

Matrix metalloproteinase (MMP) family, in particular MMP-2, may play a key role in angiogenesis and tumor growth. It is conceivable that the breaking of immune tolerance of MMP-2 should be a useful approach to cancer therapy by active immunity. To test this concept, we constructed a plasmid DNA encoding chicken homologous MMP-2 (c-MMP-2) and control vectors. We found that the vaccine based on chicken homologous MMP-2 as a model antigen could induce both protective and therapeutic antitumor immunity. Autoantibodies against MMP-2 in sera of mice immunized with c-MMP-2 could be found by Western blotting analysis and ELISA assay. There was the deposition of autoantibodies within the tumor. IgG1 and IgG2b were substantially increased in response to c-MMP-2 immunization. The elevation of MMP-2 in the sera of tumor-bearing mice was abrogated with the vaccination of c-MMP-2. Transmigration of human endothelial cells and tumor cells through gelatin-coated filters was inhibited with immunoglobulins isolated from mice immunized with c-MMP-2. The gelatinase activity of MMP-2, including both latent MMP-2 (M(r) 72,000) and active MMP-2 (M(r) 66,000) derived from tumor tissues, was apparently inhibited by the vaccination with c-MMP-2. The antitumor activity and the inhibition of angiogenesis were acquired by the adoptive transfer of the purified immunoglobulins. The antitumor activity and production of autoantibodies against MMP-2 could be abrogated by the depletion of CD4(+) T lymphocytes. Angiogenesis was apparently inhibited within tumor, and chick CAMs angiogenesis was also inhibited. Thus, our findings may provide a vaccine strategy for cancer therapy through the induction of an autoimmune response against MMP-2 in a cross-reaction by the immunization with the single xenogeneic homologous MMP-2 gene and may be of importance in the additional exploration of the application of other xenogeneic homologous genes identified in human and other animal genome projects in cancer therapy.

Objective: To study the inhibition of melanoma growth in mice by vaccination with xenogeneic melanocytes., Methods: Xenogeneic vaccine was prepared from pig eye melanocytes. It was used before or after B16 melanoma challenge in C57 mice. The size of tumor was monitored. Cytotoxic T lymphocyte (CTL) activity of mouse spleen cells was measured by 51Cr release assay. Antibody response against pig melanocytes and B16 melanoma cells were detected by indirect ELISA., Results: Preventive vaccination resulted in inhibition of tumor growth in 90% of the immunized mice, while therapeutic vaccination inhibited tumor growth in 50% of the treated mice. Specific CTL activity and antibodies in the immunized mice were detected., Conclusion: Anti-tumor immune response capable of inhibiting melanoma growth can be induced by xenogeneic melanocyte vaccination.