alpha-Fetoprotein-specific tumor immunity induced by plasmid prime-adenovirus boost genetic vaccination.

alpha-Fetoprotein (AFP) is a potential target for immunotherapy in hepatocellular carcinoma; both the murine and human T-cell repertoires can recognize AFP-derived epitopes in the context of the MHC. Protective immunity can be generated with AFP-engineered dendritic cell-based vaccines. We now report a DNA-based immunization strategy using a prime-boost approach: coadministration of plasmid DNA encoding murine AFP and murine granulocyte-macrophage colony-stimulating factor followed by boosting with an AFP-expressing nonreplicating adenoviral vector. This immunization strategy can elicit a high frequency of Th1-type AFP-specific cells leading to tumor protective immunity in mice at levels comparable with AFP-engineered dendritic cells. This cell-free mode of immunization is better suited for large-scale vaccine efforts for patients with hepatocellular carcinoma.     

Specific antitumor immunity induced by cross-linking complex heat shock protein 72 and alpha-fetoprotein

Hepatocellular carcinoma (HCC) is one of the most common malignancies over the world. Alpha-fetoprotein (AFP) is an oncofetal protein during HCC development that could generate weaker and less reproducible antitumor protection, and it may serve as a target for immunotherapy. Therefore, it is imperative to enhance its immunogenicity and develop therapeutic vaccines to eliminate AFP-expressing tumors. In this study, by way of glutaraldehyde cross-linking, we constructed a potential therapeutic protein complex vaccine, heat shock protein 72 (HSP72)/AFP. Our results demonstrated that AFP and HSP72 synergistically exhibited significant increases in AFP-specific CD8(+) T cell responses and impressive antitumor effects against AFP-expressing tumors. Priming mice with the reconstructed vaccine, we elicited robust strong protective immunity. Our study suggests that a tumor vaccine by cross-linking tumor antigen and HSP72 is a promising approach for cancer therapy.     

Deoxyribonucleic acid (DNA) encoding a pan-major histocompatibility complex class II peptide analogue augmented antigen-specific cellular immunity and suppressive effects on tumor growth elicited by DNA vaccine immunotherapy

Vaccine immunotherapy must induce helper and cytotoxic cell-mediated immunity to generate the powerful antitumor immune responses needed to suppress cancer progression. We reported previously that a 16-amino acid peptide analogue derived from pigeon cytochrome c can bind broad ranges of MHC class II types and activate helper T cells in mice. To determine whether DNA encoding the Pan-MHC class II IA peptide (Pan-IA) can increase the efficacy of tumor suppression by DNA vaccine immunotherapy targeting tumor antigens, Pan-IA DNA was administered with ovalbumin (OVA) DNA to C57BL/6 mice bearing the OVA-expressing tumor cell line E.G7. Specific proliferative responses to and cytotoxic activities against OVA-expressing targets were induced in mice vaccinated with both OVA and Pan-IA DNA but not in those vaccinated with OVA DNA alone or control DNA plus Pan-IA DNA. Growth of E.G7 cells was suppressed only by combined vaccination with OVA and Pan-IA DNA, and tumors in five of the nine mice that received this combined vaccination were eradicated completely. In those mice, the frequency of CD8-positive T cells reactive with OVA(257-264) peptides in the context of H-2K(b) was significantly increased in the tumor site. Furthermore, immunofluorescent study of the inoculated tumors revealed increased accumulation of both CD4- and CD8-positive T cells producing IFN-gamma in the tumor only by this vaccine protocol. The data suggest that Pan-IA DNA can augment suppressive effects of DNA vaccines on tumor growth by increasing numbers of antigen-specific CTLs and helper T cells. This is the first study in which established tumors have been eradicated successfully by vaccination with DNA corresponding to CTL epitopes and helper T cell epitopes. Our animal model may contribute to the development of therapeutic DNA vaccines against cancer.     

DNA vaccination against neu reduces breast cancer incidence and metastasis in mice

The gene for HER2/neu is overexpressed in 30-40% of breast and ovarian cancers, and this overexpression correlates with increased metastasis and poor prognosis. The HER2/neu gene product, a transmembrane protein kinase member of the EGF receptor family, has significant potential as a tumor antigen for vaccination. We inserted the sequence for neu into a novel plasmid called ELVIS containing a Sindbis virus replicon that reproduces multiple copies of mRNA. Mice vaccinated one time intramuscularly demonstrated a strong antibody response against A2L2, a murine breast cancer cell line transfected to express neu. Vaccinated mice challenged in the mammary fatpad with A2L2 had reduced tumor incidence and reduced tumor mass compared to mice challenged with tumor cells lacking the neu insert. Intradermal vaccination was also protective and required 80% less plasmid for a similar level of protection. Vaccination reduced the incidence of lung metastasis from mammary fatpad tumors and reduced the number of lung metastases resulting from intravenous injection of A2L2 cells. Cytotoxic T lymphocytes cultures of immune spleen cells with P815-neu cells produced high levels of interferon-gamma indicating an antigen-specific Th1-type immune response resulting from the vaccination. In a spontaneous breast tumor model using neu transgenic mice, vaccination with ELVIS-neu protected against development of spontaneous breast tumors. Our preclinical data indicate that therapeutic vaccination of patients with ELVIS-neu may reduce metastasis from HER2/neu-expressing breast and ovarian tumors.     

Th2-dominated antitumor immunity induced by DNA immunization with the genes coding for a basal core peptide PDTRP and GM-CSF

Our previous study showed that DNA vaccination with a plasmid vector encoding a core peptide of mucin1 (PDTRP) provided modest protection against challenge with tumor cells that expressed mucin1 protein. We report here that a DNA vaccine comprising a modified PDTRP plasmid and GM-CSF coding sequence at the C-terminus induced better protection against tumor challenge. The increased protection was directly correlated with a stronger PDTRP-specific immune response induced by the GM-CSF fusion plasmid. The plasmid encoding GM-CSF and the target PDTRP antigen induced a greater PDTRP-specific Th proliferation, antibodies, and cytotoxicity. Interestingly, the modified plasmid vaccine predominantly enhanced the type 2 immune responses manifested by an increased IgG1 to IgG2a antibody ratio and a greater induction of GATA-3 and IL-4 mRNA than that of T-bet and IFN-gamma mRNA in spleen cells from vaccinated mice. In addition, protection against tumor challenge in vaccinated mice showed that there was no significant change in mice survival after in vivo CD8+CTL depletion, indicating that antitumor immunity augmented by plasmid encoding GM-CSF and target PDTRP gene vaccine was dominated by Th2 immune response.     

An attenuated Salmonella oral DNA vaccine prevents the growth of hepatocellular carcinoma and colon cancer that express alpha-fetoprotein

Antitumor vaccination therapies using attenuated Salmonella typhimurium carrying plasmid DNA encoding tumor-associated antigens are currently under preclinical development. In the present study, we first established a useful method to facilitate in vivo monitoring of attenuated S. typhimurium uptake using a bioluminescent lux gene operon plasmid. Following transformation with the lux gene operon construct, mice were fed with various amounts of attenuated S. typhimurium-lux to monitor in vivo clearance over a period of 24 h. We found that the ingested attenuated S. typhimurium-lux cells were almost cleared out 9 h postfeeding, as judged by a significant decrease in bioluminescence. We further examined the therapeutic efficacy of vaccination using attenuated S. typhimurium carrying the mouse alpha-fetoprotein (AFP) gene against a cancer line CT26-murine alpha-feto protein (mAFP) that stably expresses AFP and mouse hepatocellular carcinoma (HCC) Hepa1-6. Attenuated S. typhimurium oral DNA vaccine was found to promote protective immunity against both CT26-mAFP and Hepa1-6 tumor cells growth. The oral DNA vaccine significantly increased the life span of tumor-challenged mice in both tumor models. Together, these results suggest that vaccination with the attenuated S. typhimurium oral DNA vaccine that carries the AFP gene could be a promising strategy to prevent HCC development.     

Effective particle-mediated vaccination against mouse melanoma by coadministration of plasmid DNA encoding Gp100 and granulocyte-macrophage colony-stimulating factor.

Particle-mediated gene delivery was used to immunize mice against melanoma. Mice were immunized with a plasmid cDNA coding for the human melanoma-associated antigen, gp100. Murine B16 melanoma, stably transfected with human gp100 expression plasmid, was used as a tumor model. Particle-mediated delivery of gp100 plasmid into the skin of na?ve mice resulted in significant protection from a subsequent tumor challenge. Co-delivery of murine granulocyte-macrophage colony-stimulating factor (GM-CSF) expression plasmid together with the gp100 plasmid consistently resulted in a greater level of protection from tumor challenge. The inclusion of the GM-CSF plasmid with the gp100 DNA vaccine allowed a reduction in the gp100 plasmid dose required for antitumor efficacy. Protection from tumor challenge was achieved with as little as 62.5 ng of gp100 DNA per vaccination. Tumor protection induced by the gp100 + GM-CSF gene combination was T cell mediated, because it was abrogated in vaccinated mice treated with anti-CD4 and anti-CD8 monoclonal antibodies. In addition, administration of the gp100 + GM-CSF DNA vaccine to mice bearing established 7-day tumors resulted in significant suppression of tumor growth. These results indicate that inclusion of GM-CSF DNA augments the efficacy of particle-mediated vaccination with gp100 DNA, and this form of combined gp100 + GM-CSF DNA vaccine warrants clinical evaluation in melanoma patients.     

The effect of a therapeutic dendritic cell-based cancer vaccination depends on the blockage of CTLA-4 signaling

Dendritic cells (DCs) were pulsed with the H-2K(b) binding OVA(257-264)-peptide (SIINFEKL), and used as one single-injection vaccine in combination with anti-CTLA-4 monoclonal antibody (mAb) to treat mice inoculated 3 days previously with 3x10(5) E.G7-OVA lymphoma cells. Neither DC vaccination nor CTLA-4 blockage alone prevented tumor growth in tumor challenged mice. In contrast, the combination of one vaccination and injection of anti-CTLA-4 mAb lead to rejection or retarded tumor growth in more than 60% of the mice. The OVA-transgene or the SIINFEKL-epitope was not lost in the progressing tumors of vaccinated mice, however, the highest degree of anti-SIINFEKL reactivity of host CTLs in an IFN-gamma ELISPOT assay was found only in mice showing complete tumor rejection. Vaccinated mice having rejected E.G7-OVA tumors were capable of rejecting subsequent challenges with 1x10(6) E.G7-OVA tumor cells, and later on these mice even rejected wild-type EL-4 tumor cells indicating that tumor epitope spreading takes place during the process of vaccination-induced E.G7-OVA rejection. In agreement with these observations, mice having rejected E.G7-OVA tumors showed long lasting CTL memory in spleen and bone marrow towards both the SIINFEKL-peptide and other EL-4-derived tumor rejecting epitopes.     

DNA vaccination controls Her-2+ tumors that are refractory to targeted therapies

Her-2/neu(+) tumor cells refractory to antibody or receptor tyrosine kinase inhibitors are emerging in treated patients. To investigate if drug resistant tumors can be controlled by active vaccination, gefitinib and antibody sensitivity of four neu(+) BALB/c mouse mammary tumor lines were compared. Significant differences in cell proliferation and Akt phosphorylation were observed. Treatment-induced drug resistance was associated with increased chromosomal aberrations as shown by spectral karyotyping analysis, suggesting changes beyond neu signaling pathways. When mice were immunized with pneuTM encoding the extracellular and transmembrane domains of neu, antibody and T-cell responses were induced, and both drug-sensitive and drug-resistant tumor cells were rejected. In T-cell-depleted mice, drug-sensitive tumors were still rejected by vaccination, but drug-refractory tumors survived in some mice, indicating their resistance to anti-neu antibodies. To further test if T cells alone can mediate tumor rejection, mice were immunized with pcytneu encoding full-length cytoplasmic neu that is rapidly degraded by the proteasome to activate CD8 T cells without inducing antibody response. All test tumors were rejected in pcytneu-immunized mice, regardless of their sensitivity to gefitinib or antibody. Therefore, cytotoxic T lymphocytes activated by the complete repertoire of neu epitopes were effective against all test tumors. These results warrant Her-2 vaccination whether tumor cells are sensitive or resistant to Her-2-targeted drugs or antibody therapy.     

Antibodies elicited by naked DNA vaccination against the complementary-determining region 3 hypervariable region of immunoglobulin heavy chain idiotypic determinants of B-lymphoproliferative disorders specifically react with patients' tumor cells

Several reports have suggested that the mechanism of protection induced by antiidiotypic vaccination against low-grade lymphoproliferative disorders is likely to be antibody mediated. Here we test the hypothesis that DNA vaccination with the short peptide encompassing the complementary-determining region 3 hypervariable region of immunoglobulin heavy chain (VH-CDR3) may elicit a specific antibody immune response able to recognize the native antigens in the form required for therapy. As a test system, we used the VH-CDR3 sequences derived from two patients with non-Hodgkin's B lymphomas (PA, AS) and one patient with hairy cell leukemia (BA) to immunize outbred Swiss mice. This experimental model could mimic a clinical setting in which different patients present distinct HLA haplotypes. Individual tumor-specific VH-CDR3 sequences were amplified by a two-step procedure and directly cloned into multigenic plasmid vectors (pRC100 and derived) with and without mouse interleukin 2 (mIL-2). Each tumor-specific sequence was characterized by sequencing. Female Swiss mice were vaccinated i.m. with plasmids expressing the tumor-specific VH-CDR3 sequence alone (pRC101-PA), mIL-2 plus the VH-CDR3 sequence (pRC111-PA), or a different unrelated antigen (NS3 of hepatitis C virus; pRC112), the sole mIL-2 (pRC110), and the empty plasmid (pRC100). Boost injections were performed at 3 and 16 weeks from the first vaccination, and sera were drawn before each vaccination and at 6, 9, and 19 weeks. Induction of anti-VH-CDR3s antibodies in the sera and their ability to recognize native antigens on patients' tumor cells were evaluated by FACS analysis. Up to 56% (n = 25) of mice vaccinated with pRC111-PA plasmid and 20% (n = 15) of mice vaccinated with pRC101-PA developed a specific immune response that was maintained throughout 19 weeks of observation in 40% of pRC111-PA-vaccinated mice. No response was detected in sera obtained from mice vaccinated with the other plasmids (n = 45). pRC111-PA injection s.c. was less effective (13%, n = 15) than i.m. injection (53%, n = 15). Indeed, we demonstrated that antibodies elicited by naked DNA vaccination against three different patient-derived VH-CDR3 peptides (pRC111-PA or BA or AS) readily reacted with binding epitopes on the idiotypic proteins expressed on the surface of tumor cells derived from each patient; 60, 40, and 40% of, respectively, PA-, BA-, and AS-vaccinated mice developed specific antibodies. No cross-reactivity was detected among the three different CDR3s against tumor cells derived from the other two patients. The outbred mouse strategy confirmed the significant matching potential of three different VH-CDR3 peptides to be efficaciously presented through different MHCs. We conclude that individual VH-CDR3 DNA vaccination can result in a potentially effective specific immune response against non-Hodgkin's B lymphoma cells by a rapid and low-cost therapeutic approach.     

Enhancement of DNA vaccine potency by sandwiching antigen-coding gene between secondary lymphoid tissue chemokine (SLC) and IgG Fc fragment genes

DNA vaccine has become an attractive approach for generating antigen-specific immunity. Targeting antigens to FcRs for IgG (FcgammaRs) on dendritic cells (DCs) has been demonstrated to enhance antigen presentation. Secondary lymphoid tissue chemokine (SLC) has been shown to increase immune responses not only by promoting coclustering of T cells and DCs in the lymph nodes and spleen but also by regulating their immunogenic potential for the induction of T cell responses. In this study, using HPV 16 E7 as a model antigen, we constructed a chemotactic-antigen plasmid DNA vaccine (pSLC-E7-Fc) by linking SLC and Fc gene sequences to each end of E7 and evaluated its potency of eliciting specific immune response. We found that immunization with pSLC-E7-Fc generated much stronger E7-specific lymphocyte proliferative and cytotoxic T lymphocyte (CTL) responses than control DNA. All the mice receiving pSLC-E7-Fc prophylactic vaccination remained tumor free upon subcutaneous inoculation of TC-1 cells, while those given control DNA all developed tumors. These tumor-free mice were also protected against TC-1 rechallenge. Complete tumor regression with long-term survival occurred in 72% of mice given pSLC-E7-Fc as therapeutic vaccination. In experimental lung metastasis model wherein TC-1 cells were intravenously injected, therapeutic vaccination with pSLC-E7-Fc significantly reduced the number of tumor nodules in the lung. In vivo depletion with antibodies against CD4+or CD8+ T cells both resulted in complete abrogation of the pSLC-E7-Fc-induced immunotherapeutic effect. Our data indicate that the DNA vaccine constructed by the fusion of SLC and IgG Fc fragment genes to antigen-coding gene is an effective approach to induce potent anti-tumor immune response via both CD4+ and CD8+ T cells dependent pathways.     

Novel vaccination protocol consisting of injecting MUC1 DNA and nonprimed dendritic cells at the same region greatly enhanced MUC1-specific antitumor immunity in a murine model

In order to induce specific antitumor immunity in mice, we attempted to immunize C57BL/6 mice with DNA vaccine encoding MUC1 polypeptide. When the mice immunized with MUC1 DNA were challenged with EL4-muc, MUC1-transfected syngeneic lymphoma cells, they completely rejected tumors. When DNA vaccine was given to the EL4-muc tumor-bearing mice, this vaccination was insufficient to suppress tumor growth in the mice. However, activated, but nonprimed dendritic cells (DCs) obtained from syngeneic mice and MUC1 DNA vaccine were given simultaneously to the same site of EL4-muc tumor-bearing mice, tumor growth was markedly suppressed accompanying prolongation of survival time. MUC1 antigen was detected on the DCs at the vaccination site and in regional nodes in the mice which received MUC1 DNA vaccine and DCs. These mice showed markedly enhanced cellular immune responses specific for MUC1 compared to those in mice vaccinated with MUC1 DNA alone. No significant difference in titers of antibodies to MUC1 between the two groups was observed. These results suggest that nonprimed DCs inoculated at the DNA vaccine site are essential for eliciting strong antitumor cellular immunity to suppress tumor growth efficiently in DNA-vaccinated mice. This animal model is useful for developing DNA vaccine for anti-cancer immunotherapy.     

Anti-HER-2 DNA vaccine protects Syrian hamsters against squamous cell carcinomas

This paper illustrates the efficacy of DNA vaccination through electroporation in the prevention of oral transplantable carcinoma in Syrian hamsters. At 21 and 7 days before tumour challenge, 19 hamsters were vaccinated with plasmids coding for the extracellular and transmembrane domains of rat HER-2 receptor (EC-TM plasmids), whereas 19 control hamsters were injected intramuscularly with the empty plasmid. Immediately following plasmid injection, hamsters of both groups received two square-wave 25 ms, 375 V cm(-1) electric pulses via two electrodes placed on the skin of the injection area. At day 0, all hamsters were challenged in the submucosa of the right cheek pouch with HER-2-positive HCPC I cells established in vitro from an 7,12-dimethylbenz[a]anthracene-induced oral carcinoma. This challenge gave rise to HER-2-positive buccal neoplastic lesions in 14 controls (73.37%), compared with only seven (36.8%, P<0.0027) vaccinated hamsters. In addition, the vaccinated hamsters displayed both a stronger proliferative and cytotoxic response than the controls and a significant anti-HER-2 antibody response. Most of the hamsters that rejected the challenge displayed the highest antibody titres. These findings suggest that DNA vaccination may have a future in the prevention of HER-2-positive human oral cancer.     

异位hCGβ基因免疫诱导的特异性抗肿瘤免疫应答

目的　观察异位hCGβ基因免疫诱导的肿瘤特异性免疫应答及其抗肿瘤作用 ,为肿瘤的免疫生物治疗寻求新途径。方法　构建含hCGβ编码基因的质粒TR4 2 1 hCGβ ,对BALB/c小鼠实施基因免疫 ,并以空质粒为对照。采用ELISA法和3 H TdR掺入法分别检测免疫小鼠血清中特异性抗hCGβ IgG抗体及其对肿瘤细胞体外生长的抑制作用 ;特异抗原体外刺激免疫小鼠脾细胞后 ,用3 H TdR掺入法和3 H TdR释放法分别检测其特异性增殖活性和细胞毒活性 ;皮下接种SP2 /0 hCGβ细胞攻击免疫小鼠 ,以成瘤率及实体瘤重量评估体内抗瘤效果。结果　全部TR4 2 1 hCGβ质粒免疫小鼠均产生高水平的抗hCGβ IgG抗体 ,该抗体可抑制肿瘤细胞的体外生长 ,与对照血清相比 ,差异有显著差性 (P <0 .0 5 ) ;hCGβ蛋白、灭活SP2 /0 hCGβ细胞以及两者混合均能刺激TR4 2 1 hCGβ质粒免疫小鼠脾细胞的体外增殖 (SI值分别为 1.5 3、1.81和 2 .0 5 ) ,与空质粒免疫小鼠相比 ,差异有显著性 (P <0 .0 1) ;特异抗原刺激的TR4 2 1 hCGβ质粒免疫小鼠脾细胞对SP2 /0 hCGβ细胞的细胞毒活性明显高于SP2 /0细胞(P <0 .0 1) ;空质粒免疫小鼠接种SP2 /0 hCGβ细胞后成瘤率为 10 0 % ,瘤重达 3.37g ,而TR4 2 1 hCGβ质粒免疫小鼠的成瘤率为 16 .6 6 % ,瘤重为     

Vaccination with liposome--DNA complexes elicits enhanced antitumor immunity

Cationic liposomes have been shown to potentiate markedly the ability of plasmid DNA to activate innate immune responses. We reasoned therefore that liposome-DNA complexes (LDC) could be used to produce more effective plasmid DNA vaccines for cancer. To test this hypothesis, tumor-bearing mice were vaccinated with conventional plasmid DNA vaccines or with LDC vaccines encoding model tumor antigens and CD8(+) T-cell responses and antitumor activity were assessed. We found that although plasmid DNA vaccines generated large increases in antigen-specific CD8(+) T cells, they failed to elicit significant antitumor immunity. In contrast, LDC vaccines elicited large numbers of antigen-specific CD8(+) T cells and also generated significant antitumor activity against established tumors. The antitumor activity elicited by immunization with LDC vaccines was mediated primarily by CD8(+) T cells. Studies of the interaction of LDC with antigen-presenting cells found that LDC triggered dendritic cell production of interleukin-12 and interferon (IFN)-gamma production by natural killer cells in vivo. Activation by LDC was also accompanied by upregulation of costimulatory molecule expression. These findings suggest that by concurrently activating strong systemic innate immune responses and generating cytotoxic T-lymphocyte responses, LDC may be used to increase the effectiveness of therapeutic plasmid DNA vaccination for cancer.     

Murine liver metastasis model using L5178Y-ML lymphoma and the effect of antitumor agents on the metastasis

A reproducible tumor model for liver metastasis has been developed from murine L5178Y lymphoma line by sequential cycles of subcutaneous inoculation of liver tumor cells, that were originally generated in livers of female (BALB/c x DBA/2)F1 mice by injecting the parental cells into the tail vein. This variant (L5178Y-ML) metastasized predominantly to the liver after intravenous or subcutaneous injection. The livers of the animals killed 9 days after intravenous implantation of 5 x 10(5) tumor cells were about 3 times the weight of control livers. All tumor-bearing mice died 10 to 12 days after inoculation. Subcutaneous implantation of L5178Y-ML in the side flank of mice induced metastatic nodules spontaneously in the livers. The tumor cells proliferated more in livers than in the implanted sites, compared with the parental L5178Y cells. The effects of 5-fluorouracil, mitomycin C, cis-platinum and doxorubicin on the liver metastasis of L5178Y-ML were examined at subtoxic doses; 5-fluorouracil was the most effective in both inhibiting the tumor growth in livers and prolonging the survival period of mice. This model provides a useful tool for the experimental therapy of hepatic tumors in mice.     

Anti-angiogenic therapy subsequent to adeno-associated-virus-mediated immunotherapy eradicates lymphomas that disseminate to the liver

Liver cancer has a very poor prognosis and lacks effective therapy. We have previously demonstrated that intraportal injection of adeno-associated-viral (AAV) particles that express angiostatin lead to long-term expression of angiostatin capable of suppressing the outgrowth of EL-4 tumors in the liver. Here we combine AAV-mediated angiostatin therapy with immunotherapy by employing an AAV vector encoding the T-cell costimulator B7.1. Incubation of EL-4 cells with AAV-B7.1 viruses resulted in the rapid expression of B7.1 on the surface of 80% of EL-4 cells. Mice that were vaccinated with B7.1-engineered tumor cells rejected the tumor cells and resisted a secondary challenge with unmodified parental cells. Splenocytes from the vaccinated mice were highly cytotoxic towards parental EL-4 cells in vitro. However, the vaccinated mice failed to resist the challenge of a heavy burden of EL-4 cells. Intraportal injection of AAV particles that express angiostatin into mice that had been vaccinated 1 month earlier with B7.1-engineered tumor cells protected mice against the challenge of a heavy burden of EL-4 cells and eradicated tumors that had disseminated to the liver. The combinational therapy increased the survival rate of mice with advanced liver cancer. These encouraging results warrant investigation of the employment of anti-angiogenic therapy subsequent to cancer immunotherapy for targeting unresectable disseminated liver metastases.     

Vaccination against strontium-90-induced bone tumors

The thesis was tested that immunization against a murine osteosarcoma virus can reduce the incidence of bone tumors induced by 90Sr. C57BL/6J female mice (190) were divided into three sets of 2 groups. Each set consisted of a control group and an experimental group treated ip with 1.0 muCi 90Sr at 66 days of age. The three sets of groups received the following additional treatments: none (controls), 6 injections of Formalin-inactivated FBJ osteosarcoma virus (vaccinated group), or 6 injections of active FBJ virus (active virus controls). Only 1 bone tumor developed in a mouse not treated with 90Sr in the active virus controls. In 90Sr-treated mice, vaccination reduced bone tumor deaths during the first 600 days from 9 of 36 in controls to 1 of 33 in vaccinated mice (P less than .01), but bone tumor deaths during the entire life-span, 10 of 36 and 5 of 33, respectively, were not significantly different (P = .07). Thus the vaccination procedure delayed the development of bone tumors. In contrast, injection of active virus into 90Sr-treated mice increased the lifetime incidence of bone tumors from 10 of 36 in controls to 19 of 32 (P = .01).