Anti-tumor effects of a human VEGFR-2-based DNA vaccine in mouse models

Background

Vascular endothelial growth factor (VEGF) and its receptor, VEGFR-2 (Flk-1/KDR), play a key role in tumor angiogenesis. Blocking the VEGF-VEGFR-2 pathway may inhibit tumor growth. Here, we used human VEGFR-2 as a model antigen to explore the feasibility of immunotherapy with a plasmid DNA vaccine based on a xenogeneic homologue of this receptor.

Methods

The protective effects and therapeutic anti-tumor immunity mediated by the DNA vaccine were investigated in mouse models. Anti-angiogenesis effects were detected by immunohistochemical staining and the alginate-encapsulate tumor cell assay. The mechanism of action of the DNA vaccine was primarily explored by detection of auto-antibodies and CTL activity.

Results

The DNA vaccine elicited a strong, protective and therapeutic anti-tumor immunity through an anti-angiogenesis mechanism in mouse models, mediated by the stimulation of an antigen-specific response against mFlk-1.

Conclusion

Our study shows that a DNA vaccine based on a xenogeneic homologue plasmid DNA induced autoimmunity against VEGFR-2, resulting in inhibition of tumor growth. Such vaccines may be clinically relevant for cancer immunotherapy.     

Vaccine-induced ErbB (EGFR/HER2)-specific immunity in spontaneous canine cancer

Epidermal Growth Factor Receptor (EGFR) is overexpressed on a number of human cancers, and often is indicative of a poor outcome. Treatment of EGFR/HER2 overexpressing cancers includes monoclonal antibody therapy (cetuximab/trastuzumab) either alone or in conjunction with other standard cancer therapies. While monoclonal antibody therapy has been proven to be efficacious in the treatment of EGFR/HER2 overexpressing tumors, drawbacks include the lack of long-lasting immunity and acquired resistance to monoclonal therapy. An alternative approach is to induce a polyclonal anti-EGFR/HER2 tumor antigen response by vaccine therapy. In this phase I/II open-label study, we examined anti-tumor immunity in companion dogs with spontaneous EGFR expressing tumors. Canine cancers represent an outbred population in which the initiation, progression of disease, mutations and growth factors closely resemble that of human cancers. Dogs with EGFR expressing tumors were immunized with a short peptide of the EGFR extracellular domain with sequence homology to HER2. Serial serum analyses demonstrated high titers of EGFR/HER2 binding antibodies with biological activity similar to that of cetuximab and trastuzumab. Canine antibodies bound both canine and human EGFR on tumor cell lines and tumor tissue. CD8 T cells and IgG deposition were evident in tumors from immunized dogs. The antibodies inhibited EGFR intracellular signaling and inhibited tumor growth in vitro. Additionally, we illustrate objective responses in reducing tumors at metastatic sites in host animals. The data support the approach of amplifying anti-tumor immunity that may be relevant in combination with other immune modifying therapies such as checkpoint inhibitors.     

The Polyclonal Antibodies Induced by VBP3 Complex Peptide Targeting Angiogenesis and Tumor Suppression

Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) play a critical role in tumor-associated angiogenesis and have become the targets of anti-tumor therapy. The BALB/c mice were immunized with VEGF/bFGF complex peptide (VBP3) constructed with different epitope peptides of human VEGF and bFGF. The results of the immunogenicity showed that the VBP3 could effectively stimulate immune response in mice and elicit the mice to produce high titer specific anti-VEGF and anti-bFGF antibodies (anti-VBP3 antibodies). The polyclonal anti-VBP3 antibodies separated from the mouse immune serum could effectively inhibit the proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) and block the proliferation and migration of lung cancer A549 cells. Besides, the anti-VBP3 antibodies could effectively inhibit tumor growth and tumor angiogenesis in BABL/c nude mice. The results demonstrated that the VBP3 complex peptide could elicit the body to produce the high titer anti-VEGF and anti-bFGF antibodies, which showed anti-tumor and anti-angiogenic effects in vitro and in vivo. The results revealed that the VBP3 complex peptide could be used as a potential peptide vaccine in tumor therapy.     

Development of an allogeneic whole-cell tumor vaccine expressing xenogeneic gp100 and its implementation in a phase II clinical trial in canine patients with malignant melanoma

A xenogeneic melanoma-antigen-enhanced allogeneic tumor cell vaccine (ATCV) is an appealing strategy for anti-cancer immunotherapy due to its relative ease of production, and the theoretical possibility that presentation of a multiplex of antigens along with a xenogeneic antigen would result in cross-reaction between the xenogeneic homologs and self-molecules, breaking tolerance and ultimately resulting in a clinically relevant immune response. In this study, we evaluated the efficacy of such a strategy using a xenogeneic melanoma differentiation antigen, human glycoprotein 100 (hgp100) in the context of a phase II clinical trial utilizing spontaneously arising melanoma in pet dogs. Our results demonstrate that the approach was well tolerated and resulted in an overall response rate (complete and partial response) of 17% and a tumor control rate (complete and partial response and stable disease of >6 weeks duration) of 35%. Dogs that had evidence of tumor control had significantly longer survival times than dogs that did not experience control. Delayed type hypersensitivity (DTH) to 17CM98 canine melanoma cells used in the whole cell vaccine was enhanced by ATCV and correlated with clinical response. In vitro cytotoxicity was enhanced by ATCV, but did not correlate with clinical response. Additionally, anti-hgp100 antibodies were elicited in response to ATCV in the majority of patients tested; however, this also did not correlate with clinical response. This approach, along with further elucidation of the mechanisms of tumor protection after xenogeneic immunization, may allow the development of more rational vaccines. This trial also further demonstrates the utility of spontaneous tumors in companion animals as a valid translational model for the evaluation of novel vaccine therapies. E.G. MacEwen deceased     

Specific blockade of VEGF and HER2 pathways results in greater growth inhibition of breast cancer xenografts that overexpress HER2

We have previously reported that breast cancer cells which overexpress HER2 produce higher levels of VEGF than cells with low levels of HER2. This study tested the hypothesis that dual targeting of the VEGF (with VEGF-Trap) and HER2 (with trastuzumab) pathways would result in greater growth inhibition of HER2-overexpressing breast cancer xenografts than either agent alone. In this study we found that human and murine endothelial cells expressed high levels of VEGF receptors (VEGFR1, VEGFR2, & VEGFR3). VEGF-Trap decreased levels of secreted VEGF derived from both human and murine cells and effectively blocked VEGF-induced tyrosine phosphorylation of VEGFR2. VEGF-Trap as a single treatment inhibited tumor microvessel density (MVD), tumor vasculature, cell proliferation, and tumor growth of BT474 xenografts in a dose-dependent manner from 2.5 mg/kg to 25 mg/kg. VEGF-Trap decreased levels of both human VEGF and PlGF protein in vivo. Trastuzumab as a single agent effectively inhibited BT474 tumor growth in a dose-dependent manner, associated with a decrease in human VEGF, tumor MVD and tumor cell proliferation. Treatment with a combination of VEGF-Trap (2.5-10 mg/kg) and trastuzumab (1 mg/kg) produced significantly greater inhibition of BT474tumor growth than either individual agent, associated with greater inhibition of tumor MVD and tumor cell proliferation. Thus, VEGF-Trap in combination with trastuzumab produces superior growth inhibition of tumor xenografts which overexpress HER2, which may result from inhibition of both tumor angiogenesis and proliferation. Similar mechanisms may contribute to the clinical anti-tumor activity of trastuzumab in combination with inhibitors of VEGF signaling pathway in women with breast cancers which overexpress HER2.     

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-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.     

Vaccination against angiogenesis-associated antigens: a novel cancer immunotherapy strategy

Therapeutic vaccines represent an attractive approach to cancer treatment. Traditionally, cancer immunotherapy targets antigens expressed by the tumor cells. Although numerous clinical trials studying different cancer vaccines have been conducted during the past twenty years, very limited clinical responses have been observed. The inefficient anti-tumor immunity is thought to be due, in major part, to the escape mechanisms exerted by the genetically unstable tumor cells, e.g., emergence of antigen-loss mutants, downregulation of MHC molecules and lack of expression of costimulatory molecules. Recently, a novel vaccine strategy has been developed to circumvent these obstacles. Taking advantage of the importance of angiogenesis in tumor growth and the genetic stability of endothelial cells, this immunotherapy strategy targets antigens (e.g., angiogenic growth factor receptors) overexpressed by the tumor neo-vasculature rather than the tumor cells per se. For example, active immunization against vascular endothelial growth factor receptor-2 (VEGFR-2) has been shown to generate strong cellular and humoral immune responses, which lead to the inhibition of angiogenesis and tumor growth and metastasis. This review provides an outline of this emerging field and discusses the advantages and potential pitfalls of such a vaccine strategy.     

The effects of DNA formulation and administration route on cancer therapeutic efficacy with xenogenic EGFR DNA vaccine in a lung cancer animal model

Background

Tyrosine kinase inhibitor gefitinib is effective against lung cancer cells carrying mutant epidermal growth factor receptor (EGFR); however, it is not effective against lung cancer carrying normal EGFR. The breaking of immune tolerance against self epidermal growth factor receptor with active immunization may be a useful approach for the treatment of EGFR-positive lung tumors. Xenogeneic EGFR gene was demonstrated to induce antigen-specific immune response against EGFR-expressing tumor with intramuscular administration.

Methods

In order to enhance the therapeutic effect of xenogeneic EGFR DNA vaccine, the efficacy of altering routes of administration and formulation of plasmid DNA was evaluated on the mouse lung tumor (LL2) naturally overexpressing endogenous EGFR in C57B6 mice. Three different combination forms were studied, including (1) intramuscular administration of non-coating DNA vaccine, (2) gene gun administration of DNA vaccine coated on gold particles, and (3) gene gun administration of non-coating DNA vaccine. LL2-tumor bearing C57B6 mice were immunized four times at weekly intervals with EGFR DNA vaccine.

Results

The results indicated that gene gun administration of non-coating xenogenic EGFR DNA vaccine generated the strongest cytotoxicty T lymphocyte activity and best antitumor effects. CD8(+) T cells were essential for anti-tumor immunityas indicated by depletion of lymphocytes in vivo.

Conclusion

Thus, our data demonstrate that administration of non-coating xenogenic EGFR DNA vaccine by gene gun may be the preferred method for treating EGFR-positive lung tumor in the future.     

Kinetics of synthesis and immunologically active fraction of anti-tumor Immune RNA

The kinetics of synthesis of anti-tumor Immune RNA (I-RNA) in immunized rodents was determined and an immunologically active fraction of I-RNA isolated. As measured in a microcytotoxicity assay for cell mediated immunity, cytotoxic immune reactivity of syngeneic I-RNA was maximal when extracted from the spleens of Fischer rats 21–28 days following their inoculation with 10⁶ syngeneic MC3-R tumor cells. Maximum immunoreactivity of xenogeneic I-RNA extracted from the lymphoid organs of guinea pigs immunized with MC-1 mouse tumor cells was reached 14 days after immunization. Both syngeneic and xenogeneic anti-tumor I-RNA were fractionated in preparative sucrose density gradients. The highest cytotoxic immune reactivity was consistantly obtained from I-RNA fractions with sedimentation values of 12–16S. The immunologically active I-RNA comprised only 5–7% of the total RNA extracted from the lymphoid tissues of immunized animals.     

Potent CTL induction by a whole cell pertussis vaccine in anti-tumor peptide immunotherapy

Promising yet limited clinical responses have been reported for peptide based immunotherapy against tumors. In order to induce more potent cytolytic CD8 T cell responses, we investigated the use of Bordetella pertussis vaccine as an adjuvant for peptide immunization. A whole cell (Wc) vaccine has been known to induce a Th1 biased immune response while an acellular (Ac) vaccine tends to induce that of the Th2 type. Natural infection by B. pertussis helps to maintain a robust Th1 memory in the host population. To examine the adjuvant activity of the pertussis vaccine, we immunized mice with an ovalbumin peptide as a model tumor antigen, and monitored the development of anti-tumor activities. The addition of either the Ac or the Wc vaccine helped expand the specific CD8 T cells. However, there was a marked difference in the induced cytolytic activity where the Wc vaccine was superior to the Ac. The Wc vaccine was also more effective in inducing in vivo tumor rejection. The adjuvant activity was not only effective against ovalbumin, but was also evident when an endogenous tumor antigen, Wilms' tumor 1 gene product, was targeted. These results indicate that, although the Wc vaccine does not share the same antigen specificity with tumor cells, it can aid in the development of highly cytolytic CD8 T cells as an adjuvant at the site of peptide immunization.     

抗VEGF/VEGFR靶向肿瘤血管药物的研究进展

研究表明,肿瘤的生长转移和新血管的生成有密切关系,其中血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)及其信号途径在肿瘤血管生成中起关键作用。阻断该途径的任何环节均可有效抑制肿瘤血管的生成,进而抑制肿瘤的生长和转移。近年来,已有多种以VEGF/VEGFR为靶点的抗肿瘤血管生成药物投入临床应用,其中bevacizumab为第一个获批上市的抗肿瘤血管生成药物。继bevacizumab后,一种以基因工程手段获得的人Fc融合蛋白Zaltrap也成功在美国上市,这种杂交分子的药代动力学明显优于单克隆抗体,能更好的遏制肿瘤血管的发生并消退已形成的肿瘤血管。在肿瘤的临床治疗中,Zaltrap比bevacizumab显示出更大的优势。此外,VEGFC/D Trap及小分子酪氨酸激酶抑制剂也能有效抑制肿瘤血管的生成。在此对以VEGF/VEGFR为靶点的抗肿瘤血管生成药物进行综述。     

HER-2/neu Cancer Vaccines: Present Status and Future Prospects

Immunotherapeutic approaches to cancer should focus on novel undertakings that modulate immune responses by synergistic enhancement of anti-tumor immunological parameters. Cancer vaccines should preferably be composed of multiple defined tumor antigen specific B- and T-cell epitopes. The main focus of this article is to briefly review the present status of Her-2/neu vaccine strategies and to describe the innovative strategies developed in my laboratory for a vaccine against HER-2/neu (ErbB-2) with emphasis on the humoral arm of the immune response. Elucidating the underlining mechanisms of anti-tumor effects elicited by peptide vaccines against a self-protein is a requirement for developing an immunotherapeutic strategy that might be effective in human cancer vaccines. Our approach entails the identification of biologically relevant epitopes, establishing relevant in vitro assays for monitoring vaccine efficacy, devising strategies to engineer conformationally dependent sequences, developing highly immunogenic vaccines for an outbred population and delivering the immunogen/vaccine in a safe and efficacious vehicle, utilizing transgenic animal models for assessing tumor development, and developing challenge models using transplantable tumors to study efficacy of vaccine constructs. We have developed a multi-HER-2/neu B-cell epitope approach and shown in preclinical studies that immunization with a combination of two B-cell epitope was more effective in preventing mammary tumors than a single epitope. We have translated that work to the clinic (OSU 0105) in an FDA approved, NCI sponsored “Phase 1 Active Immunotherapy trial with Chimeric and Multi-epitope based peptide vaccine targeting HER-2 oncoprotein and nor-MDP adjuvant in patients with metastatic and/or recurrent solid tumors” at the James Cancer Hospital at the Ohio State University. The correlation between overexpression of HER-2/neu and up-regulation of VEGF has been demonstrated in breast cancer patients. Thus, blocking angiogenesis is an attractive strategy to inhibit tumor growth, invasion, and metastasis. The hypothesis that combination of anti-angiogenic therapy and tumor immunotherapy of cancer may be synergistic is an important future goal. In this review, I will discuss insights into our preclinical studies that might aid in the design of the next generation of cancer vaccines and become an integrated component of prophylactic/preventive and therapeutic approach.     

A single heteroclitic epitope determines cancer immunity after xenogeneic DNA immunization against a tumor differentiation antigen

Successful active immunization against cancer requires induction of immunity against self or mutated self Ags. However, immunization against self Ags is difficult. Xenogeneic immunization with orthologous Ags induces cancer immunity. The present study evaluated the basis for immunity induced by active immunization against a melanoma differentiation Ag, gp100. Tumor rejection of melanoma was assessed after immunization with human gp100 (hgp100) DNA compared with mouse gp100 (mgp100). C57BL/6 mice immunized with xenogeneic full-length hgp100 DNA were protected against syngeneic melanoma challenge. In contrast, mice immunized with hgp100 DNA and given i.p. tolerizing doses of the hgp100 D(b)-restricted peptide, hgp100(25-33), were incapable of rejecting tumors. Furthermore, mice immunized with DNA constructs of hgp100 in which the hgp100(25-27) epitope was substituted with the weaker D(b)-binding epitope from mgp100 (mgp100(25-27)) or a mutated epitope unable to bind D(b) did not reject B16 melanoma. Mice immunized with a minigene construct of hgp100(25-33) rejected B16 melanoma, whereas mice immunized with the mgp100(25-33) minigene did not develop protective tumor immunity. In this model of xenogeneic DNA immunization, the presence of an hgp100 heteroclitic epitope with a higher affinity for MHC created by three amino acid (25 to 27) substitutions at predicted minor anchor residues was necessary and sufficient to induce protective tumor immunity in H-2(b) mice with melanoma.     

The role of angiostatin in the spontaneous bone and prostate cancers of pet dogs

Angiostatin is a potent inhibitor of angiogenesis generated in cancer-bearing hosts by tumor-derived proteases. Because the naturally occurring bone and prostate cancers of pet dogs provide unique model systems to study factors that regulate cancer progression and tumor dormancy, we investigated the capacity of these tumors to generate angiostatin. We determined that angiostatin fragments are present in urine of dogs with bone cancer. The identity of these fragments was confirmed by comparison of the experimentally determined protein sequence to that of a clone of canine angiostatin. Importantly, these fragments were absent in urine collected from the same dogs after complete surgical removal of the primary tumor. We also demonstrate that canine prostate cancer cells are capable of processing plasminogen to angiostatin in vitro. These findings provide rationale for using spontaneous canine tumor models to isolate endogenous angiogenesis inhibitors and to investigate their therapeutic use against cancer.     

VEGF, MVD 在胃癌预后及上消化道出血风险的研究进展

胃癌是危害人类健康的常见恶性肿瘤,也是上消化道出血常见的病因之一。其发生发展是多因素、多基因改变参与的长期、协同的病理过程。血管生成(angiogenesis)是这一发展过程中的关键步骤,血管内皮生长因子(vascular endothelial growth factor,VEGF)和微血管密度(micro vessel density,MVD)作为调控血管生成的主要因子和微血管的主要评价指标,在胃癌患者预后、临床病理因素中的作用被广泛研究。上消化道出血(upper gastrointestinal hemorrhage)是胃癌患者的常见临床症状威胁和影响患者的生存质量。新近有研究者提出,VEGF及MVD与胃癌患者的上消化道出血风险正相关,本文就相关研究进展进行介绍。     

NF—κB、VEGF在非小细胞肺癌中的表达及其与肿瘤血管形成的研究

目的探讨核因子-κB（NF-κB）和血管内皮生长因子（VEGF）在非小细胞肺癌（NSCLC）中的表达及其与肿瘤血管形成的关系。方法应用免疫组织化学方法,检测56例非小细胞肺癌及20例癌旁肺组织中的NF-κBP65、VEGF的表达,并用抗CD34测定肿瘤血管的密度（MVD）。结果（1）在非小细胞肺癌中NF-κBP65、VEGF的表达阳性率分别为83．9％（47／56）、69．6％（39／56）,明显高于癌旁组织（P〈O．05）;（2）NF-κBP65的表达在不同的TNM分期、淋巴结及胸腔积液、吸烟的分组之间差异有统计学意义,VEGF的表达在淋巴结及胸膜转移之间差异有统计学意义;（3）NF-κBP65、VEGF、MVD三者间存在明显相关性。结论NF-κB、VEGF异常表达与NSCLC的发生、发展及肿瘤血管的形成有着密切的关系。     

Targeting tumor vasculature with novel <Emphasis Type="Italic">Listeria</Emphasis>-based vaccines directed against CD105

The FDA approval of bevacizumab (Avastin^®, Genentech/Roche), a monoclonal antibody raised against human VEGF-A, as second-line therapy for colon and lung carcinoma validated the approach of targeting human tumors with angiogenesis inhibitors. While the VEGF/VEGFR pathway is a viable target for anti-angiogenesis tumor therapy, additional targets involved in tumor neovascularization have been identified. One promising target present specifically on tumor vasculature is endoglin (CD105), a member of the TGF-β receptor complex expressed on vascular endothelium and believed to play a role in angiogenesis. Monoclonal antibody therapy and preventive vaccination against CD105 has met with some success in controlling tumor growth. This report describes the in vivo proof-of-concept studies for two novel therapeutic vaccines, Lm-LLO-CD105A and Lm-LLO-CD105B, directed against CD105 as a strategy to target neovascularization of established tumors. Listeria-based vaccines directed against CD105 lead to therapeutic responses against primary and metastatic tumors in the 4T1-Luc and NT-2 mouse models of breast cancer. In a mouse model for autochthonous Her-2/neu-driven breast cancer, Lm-LLO-CD105A vaccination prevented tumor incidence in 20% of mice by week 58 after birth while all control mice developed tumors by week 40. In comparison with previous Listeria-based vaccines targeting tumor vasculature, Lm-LLO-CD105A and Lm-LLO-CD105B demonstrated equivalent or superior efficacy against two transplantable mouse models of breast cancer. Support is provided for epitope spreading to endogenous tumor antigens and reduction in tumor vascularity after vaccination with Listeria-based CD105 vaccines. Reported here, these CD105 therapeutic vaccines are highly effective in stimulating anti-angiogenesis and anti-tumor immune responses leading to therapeutic efficacy against primary and metastatic breast cancer.     

非小细胞肺癌中NF-κ B、VEGF 的表达及其与肿瘤血管形成的研究

目的:探讨核因子-κB(NF-κB)和血管内皮生长因子(VEGF)在非小细胞肺癌中的表达及其与肿瘤血管形成的关系。方法:应用免疫组织化学方法检测56例非小细胞肺癌及20例癌旁肺组织中的NF-κB P65、VEGF的表达,并用抗CD34测定肿瘤血管的密度(MVD)。结果:(1)在非小细胞肺癌中NF-κB P65、VEGF的表达阳性率分别为83.9%(47/56)、69.6%(39/56),明显高于癌旁组织(P<0.05);(2)NF-κB P65的表达在不同的TNM分期、淋巴结及胸腔积液、吸烟的分组之间差异有统计学意义,VEGF的表达在淋巴结及胸膜转移之间差异有统计学意义;(3)NF-κB P65、VEGF、MVD三者间存在明显相关性。结论:NF-κB、VEGF异常表达与NSCLC的发生、发展及肿瘤血管的形成密切的关系。     

An autologous dendritic cell canine mammary tumor hybrid-cell fusion vaccine

Mammary cancer is among the most prevalent canine tumors and frequently resulting in death due to metastatic disease that is highly homologous to human breast cancer. Most canine tumors fail to raise effective immune reactions yet, some spontaneous remissions do occur. Hybrid canine dendritic cell–tumor cell fusion vaccines were designed to enhance antigen presentation and tumor immune recognition. Peripheral blood-derived autologous dendritic cell enriched populations were isolated from dogs based on CD11c⁺ expression and fused with canine mammary tumor (CMT) cells for vaccination of laboratory Beagles. These hybrid cells were injected into popliteal lymph nodes of normal dogs, guided by ultrasound, and included CpG-oligonucleotide adjuvants. Three rounds of vaccination were delivered. Significant IgG responses were observed in all vaccinated dogs compared to vehicle-injected controls. Canine IgG antibodies recognized shared CMT antigens as was demonstrated by IgG-recognition of three unrelated/independently derived CMT cell lines, and recognition of freshly isolated, unrelated, primary biopsy-derived CMT cells. A bias toward an IgG2 isotype response was observed after two vaccinations in most dogs. Neither significant cytotoxic T cell responses were detected, nor adverse or side-effects due to vaccination or due to the induced immune responses noted. These data provide proof-of-principle for this cancer vaccine strategy and demonstrate the presence of shared CMT antigens that promote immune recognition of mammary cancer.