- Email: [email protected]
Synergistic antitumor efficacy of combined DNA vaccines targeting tumor cells and angiogenesis
Accepted Manuscript Synergistic antitumor efficacy of combined DNA vaccines targeting tumor cells and angiogenesis Xiaotao Yin, Wei Wang, Xiaoming Zhu, Yu Wang, Shuai Wu, Zicheng Wang, Lin Wang, Zhiyan Du, Jiangping Gao, Jiyun Yu PII:
S0006-291X(15)30393-4
DOI:
10.1016/j.bbrc.2015.08.003
Reference:
YBBRC 34376
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 29 July 2015 Accepted Date: 1 August 2015
Please cite this article as: X. Yin, W. Wang, X. Zhu, Y. Wang, S. Wu, Z. Wang, L. Wang, Z. Du, J. Gao, J. Yu, Synergistic antitumor efficacy of combined DNA vaccines targeting tumor cells and angiogenesis, Biochemical and Biophysical Research Communications (2015), doi: 10.1016/j.bbrc.2015.08.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Synergistic antitumor efficacy of combined DNA vaccines targeting tumor cells and angiogenesis
†
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Xiaotao Yina, b , Wei Wangb, c†, Xiaoming Zhub†, Yu Wangb, Shuai Wub, Zicheng Wangb, Lin Wangd, Zhiyan Dub, Jiangping Gaoa*, Jiyun Yub*
Department of Urology, Chinese PLA General Hospital, 28 Fu Xing Road, Beijing 100853,
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a
b
Beijing Institute of Basic Medical Sciences, 27 Tai Ping Road, Beijing 100850, China
Department of Urology, No. 261 Hospital of PLA, Beijing, China
d
†
Department of Internal Medicine, No. 316 Hospital of PLA, Beijing, China
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c
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China
These authors contributed equally to this study
author:
Jiangping
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*Corresponding
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([email protected])
Gao
([email protected]),
Jiyun
Yu
ACCEPTED MANUSCRIPT Abstract To further enhance the antitumor efficacy of DNA vaccine, we proposed a synergistic strategy that targeted tumor cells and angiogenesis simultaneously. In this study, a Semliki
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Forest Virus (SFV) replicon DNA vaccine expressing 1-4 domains of murine VEGFR2 and IL12 was constructed, and was named pSVK-VEGFR2-GFc-IL12 (CAVE). The expression of VEGFR2 antigen and IL12 adjuvant molecule in 293T cells in vitro were verified by western
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blot and enzyme-linked immune sorbent assay (ELISA). Then CAVE was co-immunized with
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CAVA, a SFV replicon DNA vaccine targeting survivin and β-hCG antigens constructed previously. The antitumor efficacy of our combined replicon vaccines was evaluated in mice model and the possible mechanism was further investigated. The combined vaccines could elicit efficient humoral and cellular immune responses against survivin, β-hCG and VEGFR2
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simultaneously. Compared with CAVE or CAVA vaccine alone, the combined vaccines inhibited the tumor growth and improved the survival rate in B16 melanoma mice model more effectively. Furthermore, the intratumoral microvessel density was lowest in combined
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vaccines group than CAVE or CAVA alone group. Therefore, this synergistic strategy of DNA
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vaccines for tumor treatment results in an increased antitumor efficacy, and may be more suitable for translation to future research and clinic.
Keywords: DNA vaccine; angiogenesis; VEGFR2; tumor.
ACCEPTED MANUSCRIPT Introduction Despite of the development of medicine and the improvement of lifespan of people, cancer is still the most serious medical problem, which is a leading cause of death [1]. Many
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malignant tumors are diagnosed at the advanced stage, and patients don’t have the opportunity to receive radical treatment. The standard therapies such as chemotherapy and radiotherapy
development of novel treatment approach is required.
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have limited efficacy for advanced malignant tumors but with severe side effects. So the
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Over the past few years, tumor associated antigen (TAA) specific DNA vaccine has become a promising immunotherapy for cancer [2,3]. It has the potency to induce specifically humoral and cellular immune response in vivo [4], which could eradicate systemic tumor cells and control the potential metastases. Besides, DNA vaccines also have the advantages of safety,
cancer therapy.
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stability, low-costs, and ease of preparation. Therefore DNA vaccine is an ideal approach for
However, many DNA vaccines targeting TAA that exhibit efficient antitumor activity in
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mice, don’t exhibit correlated antitumor efficacy in large animals or humans [3]. Some tumor
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cells may avoid immune surveillance through malignant transformation or immune editing [5,6]. So single DNA vaccine targeting TAA alone may be not enough to eradicate the tumor cells. This challenge may be overcome by combining another antitumor approach. The angiogenesis plays an important role in tumor growth, invasion and metastasis [7].
Without angiogenesis, tumor would not growth more than 2 mm. As a result, anti-angiogenesis has been already a well-recognized strategy for tumor therapy. Vascular endothelial growth factor (VEGF) is the most potent angiogenic cytokine, which is induced in
ACCEPTED MANUSCRIPT tumor cells due to hypoxia [8]. And vascular endothelial growth factor receptor 2 (VEGFR2), which is a major tyrosine kinase receptor for VEGF, mediates most angiogenic effects of VEGF. Meanwhile, it is mainly expressed in newborn vascular endothelial cells in tumor
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tissue, which makes VEGFR2 be an ideal antitumor target [9]. Recently we developed a Semliki Forest Virus (SFV) replicon DNA vaccine that targeted surviving and β-hCG antigens [10], and we named it CAVA. This DNA vaccine exhibited a
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antitumor efficacy was found to be unsatisfactory.
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promising antitumor effect in mice model. However, in our subsequent experiment, the
To further improve the antitumor efficacy of DNA vaccine, we proposed a synergistic antitumor strategy, which consists of targeting tumor cells and anti-angiogenesis simultaneously. Therefore in this study, we constructed a recombinant SFV replicon DNA
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vaccine expressing 1-4 domains of murine VEGFR2, which was intended to be co-immunized with CAVA. Then the antitumor efficacy of our combined replicon vaccines was evaluated in
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mice model and the possible mechanism was further investigated.
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Material and Methods Mice and cell lines
Female C57BL/6 mice (6-8 weeks old) were purchased from Beijing Experimental Animal
Center (Beijing, China) and were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Institute of Health publication no.85-23, Revised 1996). Animal experiment procedure in this study were approved by the Animal Ethics Committee of Beijing Institute of Basic Medical Sciences.
ACCEPTED MANUSCRIPT B16F10-β-hCG melanoma cell line (C57BL/6), which stably expressed human β-hCG antigen, was established previously in our laboratory [10]. 293T cells were obtained from ATCC (Rockville, MD). All cells were cultured in RPMI 1640 medium (Gibco-BRL,
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Gaitherburg, MD, USA) supplemented with 10% fetal calf serum, penicillin (100 U/mL) and streptomycin (100 U/mL), and they were maintained at 37℃，5% CO2 in a humidified
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incubator.
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Construction of DNA replicon vaccine
The plasmid DNA replicon vaccine CAVA was constructed and researched in our previous study [10]. In this study we constructed the DNA replicon vaccine pSVK-VEGFR2-GFc-IL12, which was named CAVE. The schematic representation of CAVE vaccine was shown in
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Figure 1C. Briefly, the VEGFR2-GFc-IL12 gene fragment was obtainded by double enzyme digestion from plasmid pVAX1-VEGFR2-GFc-IL12, which was constructed in previous study. Then the DNA fragment was inserted into the replicon vector pSVK to create
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pSVK-VEGFR2-GFc-IL12 (CAVE). In this recombinant plasmid, the gene sequences of Fc
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fragment of human IgG1 and glycosyl phosphatidyl inositol (GPI) signal peptide were fused to VEGFR2. And internal ribosome entry site (IRES) and IL-12 were then fused to the end of GPI. Both CAVA and CAVE plasmids were prepared by Endotoxin free Giga kit (Qiagen, Shanghai, China). 293T cells were transfected with CAVE plasmids, and the expression of mVEGFR and IL-12 were identified by western blot and ELISA respectively.
Plasmids DNA vaccines immunization in C57BL/6 mice
ACCEPTED MANUSCRIPT Female C57BL/6 mice aged 6-8 weeks were randomly devided into 5 groups (5 mice per group). Plasmids dosage used for each vaccination was explored and optimized by preliminary experiments. The five groups of mice were vaccinated three times at 10-day
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intervals with 100 µL PBS, 100 µL (0.1 µg/µL) pSVK-vector, 100 µL (0.1 µg/µL) CAVA, 100 µL (0.1 µg/µL) CAVE and 100 µL (0.1 µg/µL CAVA plus 0.1 µg/µL CAVE) combined vaccines respectively via intramuscular injection followed by electroporation stimulation.
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Two weeks after the last vaccination, bloods and spleens of each group of mice were collected
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for the evaluation of humoral and cellular immune activity.
Antibody detection
Anti-suvivin, anti-β-hCG and anti-VEGFR2 antibodies were detected by enzyme-linked
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immune sorbent assay (ELISA). 96-well plates were coated with purified survivin, β-hCG and VEGFR2 protein (0.25 µg/well) respectively overnight at 4℃. The blood was collected at 2nd and 4th weeks after the last vaccination. The isolated serum was diluted at 1:100 and incubated
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in the precoated plates for 2 hours at 37℃. Then the plates were washed with PBST (0.05%
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Tween 20 in PBS) and blocked with 1% bovine serum albumin for 1 hour at 37℃. HRP-conjugated goat anti-mouse IgG (dilution 1:5000) was added into the plates (100 µL/well), and the plates were incubated for 1 hour at 37℃. Finally, the antibody titers were detected using TMB system and the OD values were measured at 492 nm by a Bio-Rad plate reader.
IFN-γ ELISPOT assay
ACCEPTED MANUSCRIPT To evaluate cellular immune responses to sepcific antigen in vaccinated mice, splenic lymphocytes were tested for IFN-γ secretion using enzyme-linked immunospot assay (ELISPOT). First, spleen cells were were havested two weeks after the last immunization.
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Then RBC were lysed and the spleenic lymphocytes were isolated by density gradient centrifugation method using lymphocyte isolation liquid (TIAN JIN HAO YANG Biological Manufactory Co. Ltd, China). The isolated lymphocytes were suspended in 10% FCS/RPMI
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1640 and were adjusted to the concentration of 5×106 cells/mL. 100 µL cell suspention (5×105
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cells) was added to each well of the 96-well pecoated IFN-γ Elispot plate (Dakewe bio tech Ltd, Shenzhen, China). The lymphocytes were stimulated with purified recombinant survivin, β-hCG, and VEGFR2 protein 1 µg/well, and were cultured for 48 h at 37℃，5% CO2 in a humidified incubator. After removal of cells and washing with PBST for three times,
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biotinylated rat anti-mouse INF-γ antibody was added and the plate was incubated for 1 h at 37℃. Then the plate was washed and 100 µL 3-amino-9-ethycarbazole (AEC) was added per well, followed by incubation for 45 min at 37℃. Finally, the plate was wash for six times and
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the IFN-γ spots were counted by the ImmunoSpot analyzer (CTL).
Tumor protection and survival analysis in C57BL/6 mice 6-8 weeks female C57BL/6 were divided into 5 groups (n=10) and were vaccinated three
times at a 10-days interval with PBS, pSVK-vector, CAVA, CAVE and combined vaccines as described above. Seven days after the last immunization, 7.5×104 B16F10-β-hCG cells which were suspended in 100 µL PBS, were injected subcutaneously into the left flank of the mice. The tumor protection efficacy was monitored by tumor volume. The length and width (mm)
ACCEPTED MANUSCRIPT of tumor was measured with a digital caliper every 2 days, and the tumor volume was calculated as the following formula: tumor volume (mm3) = 0.5 × length × width2. The mice were observed for 38 days after tumor challenge. Besides, in survival analysis study another 5
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groups of mice (n=5) were vaccinated as the above protocol. The mice were observed for 70 days after the tumor challenge, and the survival rates were calculated.
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Hematoxylin and eosin (H&E) staining and Immunohistochemistry
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To explore the antiangiogenesis of our combined vaccines, the microvessel density in tumor tissue was counted. Briefly, the tumors of mice were removed 38 days after tumor challenge. Then they were paraffin-embed and sectioned to 5 µm-thick slices. After dewaxed, the slides were incubated with 3% hydrogen peroxide for 10 min to block the endogenous
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peroxidase activity. Then the slides were immerged in blocking buffer for 45 min followed by incubation with rabbit anti-CD31 antibody (Abcam) and HRP-conjugated goat anti-rabbit IgG (Santa). After staining with diaminobezidin and conterstarining with hematoxylin, the
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microvessel density was captured and calculated by analyzing 10 random fields per section.
Statistical analysis
The results in this study were analyzed using SPSS software (version 17.0, Chicago, USA).
The statistical differences between groups were determined by One-Way ANOVA method. And p<0.05 was considered statistically significant in all results.
Results
ACCEPTED MANUSCRIPT Expression of DNA vaccine plasmid CAVE in eukaryotic cells The expression of CAVE plamids in the 293T cells were verified. Two days after transfection with CAVE plasmids, the expression of VEGFR2 in 293T cells was verified by
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western blot using rabbit anti-mouse VEGFR2 antibody. The 72 kDa molecular weight band in line 2 confirmed to the theoretic molecular weight of VEGFR2-GPI-Fc fusion protein
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was also verified using IL12 ELISA kit (Figure 1C).
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(Figure 1B). The expression of IL12 in the supernatants of 293T cells transfected with CAVE
Antibody assays
After the third vaccination, anti-VEGFR2 antibody, anti-survivin antibody and anti-β-hCG antibody in serum of mice were detected by ELISA assay respectively. The anti-VEGFR2
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antibody in mice vaccinated with CAVE and combined vaccines were verified at 2 weeks after last immunization, and the titers were significantly higher than those in other groups of mice (P<0.05) (Figure 2A, B and C). Anti-β-hCG and anti-survivin antibodies were also
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detected in the sera of mice immunized with CAVA and combined vaccines. And their titers in
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these two groups were higher than other groups. Moreover, 4 weeks after last immunization, the three antibodies titers were still high in corresponding groups (Figure 2A, B and C). No positive OD values were detected in the PBS and pSVK-vector groups.
ELISPOT assays Two weeks after the last immunization, the IFN-γ ELISPOT assay of spleen lymphocytes was performed to detect the antigen-specific cellular immune response. As shown in Figure
ACCEPTED MANUSCRIPT 2F, VEGFR2 specific IFN-γ secretion were induced in the mice immunized with CAVE and combined vaccines respectively. The spots number in the two groups was significantly higher than that in other groups (P<0.05). Besides, β-hCG and survivin specific IFN-γ secretion were
Anti-tumor efficacy of combined vaccines
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than that in other groups (P<0.05) (Figure 2D and E).
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also verified in CAVA and combined vaccines groups, and the number of spots was higher
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One week after the last immunization, the mice were challenged with B16F10-β-hCG tumor cells and the tumor volume was measured every 2 days. The tumor growth in mice treated with CAVE alone or CAVA alone was significantly inhibited compared with PBS and pSVK-vector (P<0.05). In addition, the combined vaccines inhibited the tumor growth more
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effectively than CAVA and CAVE alone (P<0.05) (Figure 3A). We also observed the survival rate of different groups of mice after tumor challenge. The survival rate of tumor-bearing mice immunized with CAVA alone or CAVE alone was significantly higher compared with
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the mice immunized with PBS and pSVK-vector (P<0.05). Furthermore, the mice threated
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with combined vaccines had a significantly higher survival rate than the mice treated with CAVA alone or CAVE alone (P<0.05) (Figure 3B).
Anti-angiogenesis effect of combined vaccines The anti-angiogenesis effect of combined vaccines was verified by immunohistochemical staining for CD31 in tumor tissue. CAVE encoding VEGFR2 antigen decreased the microvessel density significantly compared with PBS, pSVK and CAVA (P<0.05). And more
ACCEPTED MANUSCRIPT importantly, compared with CAVE alone, the combined vaccines achieved a greater reduction in microvessel density (P<0.05) (Figure 4).
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Discussion Immunologic therapy plays an increasingly important role in the treatment of advanced malignant tumor, such as malignant melanoma, renal cell carcinoma and hepatocellular
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carcinoma. And anti-angiogenesis has been already an efficient approach for tumor therapy in
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research and clinic. Bevacizumab, the first FDA approved anti-angiogenic agent, is a humanized anti-VEGF monoclonal antibody. It has been used to treat some solid tumors such as colorectal cancer, lung cancer, and breast caner, which can increase the survival of tumor patients significantly [11]. However, the anti-angiogenesis agents only suppress tumor cell
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growth and do not induce complete tumor regression. Therefore, it is reasonable and necessary to combine active immunotherapy targeting tumor cells with anti-angiogenesis
previously [12].
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agents. The combination approach could elicit complete tumor regression, as suggested
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Therefore, we presumed that the co-immunization of DNA vaccines targeting tumor cells and angiogenesis may produce a more powerful antitumor efficacy. And as expected, the combined replicon vaccines exhibited the stronger antitumor efficacy than CAVA or CAVE alone. Besides, the mice in co-immunized group had a superior survival rate compared with other groups. These results have demonstrated that the synergistic strategy targeting tumor cells and anti-angiogenesis is a promising approach for DNA vaccine research. We employ several strategies in vaccine design to break the immune tolerance of host and
ACCEPTED MANUSCRIPT increase the antitumor activity of our combined replicon vaccines. First, in this study the combined vaccines are both based on pSVK vector, which is a DNA based Semliki Forest virus (SFV) replicon vector [10]. It has been proved in many research that replicon vector
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could induce higher levels of gene expression without the risk of integration into the host cell genome [13,14,15,16]. Second, the presentation of antigen to DC cells is enhanced. Glycosyl phosphatidyl inositol (GPI) signal peptide is a protein that anchored on the cell membrane.
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And the Fc fragment of human IgG1 has efficient DC cells binding capacity. Therefore we
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fuse the antigen mVEGFR2 with GPI signal peptide and Fc fragment, so that the antigen could be anchored on the cell membrane and be recognized by APCs more efficiently [17,18]. This strategy has been also proved in our previous research [10,19]. Third, several efficient molecular adjuvants are co-expressed via the internal ribosome entry site (IRES) element in
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our combined replicon vaccines. GM-CSF and B7 are co-expressed in CAVA. GM-CSF activates the maturation and proliferation of T cells [20,21]. B7 promotes proliferation of T cells as well as the secretion of chemokines [22,23]. In addition, IL-12 is co-expressed in
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CAVE as adjuvant. It modulates innate and adaptive immunity, and also induce an
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antiangiogenic effect [24]. The co-expression of antigen and adjuvants simultaneously may create a suitable microenvironment for antigen-presenting cells [25]. At last, the electroporation is applied for the delivery of vaccines. It has been reported that the electroporation could significantly increase the activity of DNA vaccines [25,26]. Then we investigate the potential antitumor mechanism of our combined replicon vaccines. ELISA assays of the serum demonstrate that the combined vaccines successfully induce the generation of antibodies against survivin, β-hCG and VEGFR2 simultaneously, which means
ACCEPTED MANUSCRIPT combined replicon vaccines induce humoral immune response in mice successfully. And titers of the three antibodies have no significant differences from CAVA group and CAVE group. Four weeks after the last immunization, the antibody levels in mice of combined vaccines
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group are still high. The anti-VEGFR2 antibody plays an important role in anti-angiogenesis. It could inhibit the phosphorylation of VEGFR-2 induced by VEGF, and therefore block the VEGF/VEGFR2 signal pathway in angiogenesis [27,28]. Thus VEGF-mediated endothelial
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cell proliferation, migration and tube formation in tumor tissue are all inhibited, which may
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be partially responsible for the enhanced anti-tumor efficacy of the combined vaccines. We also detect the specific IFN-γ secreting splenic lymphocytes by quantitative ELISPOT approach. It is manifested that the combined replicon vaccines induce significant amount of specific
IFN-γ
secreting lymphocytes
regarding survivin,
β-hCG and
VEGFR2
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simultaneously. IFN-γ, which is an important factor in cellular immune response and is mainly produced by Th1 lymphocytes, activates the NK cells and CTL cells, and upregulates MHC II and costimulatory molecules of APCs. So the results demonstrate that the combined
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replicon vaccines successfully activate cellular immune response against survivin, β-hCG and
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VEGFR2 antigen simultaneously. And the cellular immune response is also the most crucial for the antitumor effect of vaccines. The other mechanism of our combined vaccines is the anti-angiogenesis effect. The vessel
densities in tumor tissue of different groups of mice are counted by immunohistochemistry staining with anti-CD31 antibodies. In mice immunized against VEGFR2, the inhibition of angiogenesis in tumor tissue was observed. And in the combined replicon vaccines group, more powerful anti-angiogenesis effect than CAVE groups was exhibited. The mechanism
ACCEPTED MANUSCRIPT underlying this synergistic efficacy was not totally clear, but may be associated with a decrease in secretion of other pro-angiogenic factors from the tumor cells themselves. In conclusion, the combined replicon vaccines, which could elicit efficient humoral and
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cellular immune responses against survivin, β-hCG and VEGFR2 simultaneously, not only target tumor cells specifically but also suppress the angiogenesis in tumor tissue. As a result, the combined vaccines exhibit an enhanced synergistic antitumor effect in mice, which inhibit
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the tumor growth and prolong the survival of tumor-bearing mice more effectively. This
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synergistic strategy of DNA vaccines for tumor treatment results in increased antitumor efficacy, and may be more suitable for translation to future research and clinic.
Acknowledgement
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This work was financially supported by the National Science Foundation of China (contract no. 31100655) and National Science and Technology Major Project of the Ministry of Science and Technology of China (contract no. 2014ZX09304313-003). The funders had no role in
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manuscript.
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study design, data collection and analysis, decision to publish, and preparation of the
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Figure Legends
Figure 1. Construction and expression of DNA-based replicon vaccine pSVK-VEGFR2-GFc-IL12 (CAVE). (A) Schematic illustration of the vaccine CAVE. (B) The expression of mVEGFR2 gene in 293T cells transfected with CAVE plasmids was detected by western blot with anti-mVEGFR2
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antibody; 1: 293T cells transfected with pSVK-vector, 2: 293T cells transfected with CAVE plasmids. (C) The expression of IL-12 gene in 293T cells transfected with CAVE plasmids was detected by ELISA assay; 1: the supernatant of 293T cells transfected with pSVK-vector, 2: the supernatant of
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293T cells transfected with CAVE plasmids.
Figure 2. Analysis of immune response in mice treated with different vaccines. The sera were collected at 2 weeks and 4 weeks after the last vaccination. The antigen-specific antibody in the sera was detected at a 1:100 dilution by ELISA for survivin (A), β-hCG (B), and VEGFR2 (C). Splenic lymphocytes of each group were harvested and cultured with IL-2 for 48h. The IFN-γ positive T cells were then counted by ELISPOT assay stimulated with protein survivin (D), β-hCG (E), and VEGFR2 (C) respectively.
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Figure 3. Antitumor efficacy in mice vaccinated with different DNA vaccines. (A) Five groups of mice (n=10) were vaccinated with PBS, pSVK-vector, CAVA, CAVE and bivalent vaccines
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respectively at a 10-days interval. Seven days after the third vaccination, 7.5×104 B16F10-β-hCG cells were injected subcutaneously into the the left flank of the mice. Then the tumor growth of
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of vaccinated mice after B16 tumor challenge.
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five groups of mice was measured until the 38th day after tumor challenge. (B) The survival curve
Figure 4. Angiogenesis effect of different DNA vaccines on tumor. The microvessel structures were indicated by immunohistochemical staining with murine CD31 antibody. Tumor tissue sections from mice vaccinated with (A) combined vaccines; (B) CAVA; (C) CAVE; (D)
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compared.
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pSVK-vector; (E) PBS. (F) Tumor microvessel density of each group was quantified and
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ACCEPTED MANUSCRIPT Highlights We developed combined replicon vaccines that target tumor cells and angiogenesis simultaneously.
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The combined vaccines exhibited an enhanced antitumor efficacy in B16 melanoma mice model.
The combined vaccines elicited efficient humoral and cellular immune responses against
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survivin, β-hCG and VEGFR2.
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The anti-angiogenesis effect was also enhanced in mice immunized with combined
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vaccines.
S0006-291X(15)30393-4
DOI:
10.1016/j.bbrc.2015.08.003
Reference:
YBBRC 34376
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 29 July 2015 Accepted Date: 1 August 2015
Please cite this article as: X. Yin, W. Wang, X. Zhu, Y. Wang, S. Wu, Z. Wang, L. Wang, Z. Du, J. Gao, J. Yu, Synergistic antitumor efficacy of combined DNA vaccines targeting tumor cells and angiogenesis, Biochemical and Biophysical Research Communications (2015), doi: 10.1016/j.bbrc.2015.08.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Synergistic antitumor efficacy of combined DNA vaccines targeting tumor cells and angiogenesis
†
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Xiaotao Yina, b , Wei Wangb, c†, Xiaoming Zhub†, Yu Wangb, Shuai Wub, Zicheng Wangb, Lin Wangd, Zhiyan Dub, Jiangping Gaoa*, Jiyun Yub*
Department of Urology, Chinese PLA General Hospital, 28 Fu Xing Road, Beijing 100853,
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a
b
Beijing Institute of Basic Medical Sciences, 27 Tai Ping Road, Beijing 100850, China
Department of Urology, No. 261 Hospital of PLA, Beijing, China
d
†
Department of Internal Medicine, No. 316 Hospital of PLA, Beijing, China
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c
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China
These authors contributed equally to this study
author:
Jiangping
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*Corresponding
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([email protected])
Gao
([email protected]),
Jiyun
Yu
ACCEPTED MANUSCRIPT Abstract To further enhance the antitumor efficacy of DNA vaccine, we proposed a synergistic strategy that targeted tumor cells and angiogenesis simultaneously. In this study, a Semliki
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Forest Virus (SFV) replicon DNA vaccine expressing 1-4 domains of murine VEGFR2 and IL12 was constructed, and was named pSVK-VEGFR2-GFc-IL12 (CAVE). The expression of VEGFR2 antigen and IL12 adjuvant molecule in 293T cells in vitro were verified by western
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blot and enzyme-linked immune sorbent assay (ELISA). Then CAVE was co-immunized with
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CAVA, a SFV replicon DNA vaccine targeting survivin and β-hCG antigens constructed previously. The antitumor efficacy of our combined replicon vaccines was evaluated in mice model and the possible mechanism was further investigated. The combined vaccines could elicit efficient humoral and cellular immune responses against survivin, β-hCG and VEGFR2
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simultaneously. Compared with CAVE or CAVA vaccine alone, the combined vaccines inhibited the tumor growth and improved the survival rate in B16 melanoma mice model more effectively. Furthermore, the intratumoral microvessel density was lowest in combined
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vaccines group than CAVE or CAVA alone group. Therefore, this synergistic strategy of DNA
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vaccines for tumor treatment results in an increased antitumor efficacy, and may be more suitable for translation to future research and clinic.
Keywords: DNA vaccine; angiogenesis; VEGFR2; tumor.
ACCEPTED MANUSCRIPT Introduction Despite of the development of medicine and the improvement of lifespan of people, cancer is still the most serious medical problem, which is a leading cause of death [1]. Many
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malignant tumors are diagnosed at the advanced stage, and patients don’t have the opportunity to receive radical treatment. The standard therapies such as chemotherapy and radiotherapy
development of novel treatment approach is required.
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have limited efficacy for advanced malignant tumors but with severe side effects. So the
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Over the past few years, tumor associated antigen (TAA) specific DNA vaccine has become a promising immunotherapy for cancer [2,3]. It has the potency to induce specifically humoral and cellular immune response in vivo [4], which could eradicate systemic tumor cells and control the potential metastases. Besides, DNA vaccines also have the advantages of safety,
cancer therapy.
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stability, low-costs, and ease of preparation. Therefore DNA vaccine is an ideal approach for
However, many DNA vaccines targeting TAA that exhibit efficient antitumor activity in
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mice, don’t exhibit correlated antitumor efficacy in large animals or humans [3]. Some tumor
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cells may avoid immune surveillance through malignant transformation or immune editing [5,6]. So single DNA vaccine targeting TAA alone may be not enough to eradicate the tumor cells. This challenge may be overcome by combining another antitumor approach. The angiogenesis plays an important role in tumor growth, invasion and metastasis [7].
Without angiogenesis, tumor would not growth more than 2 mm. As a result, anti-angiogenesis has been already a well-recognized strategy for tumor therapy. Vascular endothelial growth factor (VEGF) is the most potent angiogenic cytokine, which is induced in
ACCEPTED MANUSCRIPT tumor cells due to hypoxia [8]. And vascular endothelial growth factor receptor 2 (VEGFR2), which is a major tyrosine kinase receptor for VEGF, mediates most angiogenic effects of VEGF. Meanwhile, it is mainly expressed in newborn vascular endothelial cells in tumor
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tissue, which makes VEGFR2 be an ideal antitumor target [9]. Recently we developed a Semliki Forest Virus (SFV) replicon DNA vaccine that targeted surviving and β-hCG antigens [10], and we named it CAVA. This DNA vaccine exhibited a
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antitumor efficacy was found to be unsatisfactory.
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promising antitumor effect in mice model. However, in our subsequent experiment, the
To further improve the antitumor efficacy of DNA vaccine, we proposed a synergistic antitumor strategy, which consists of targeting tumor cells and anti-angiogenesis simultaneously. Therefore in this study, we constructed a recombinant SFV replicon DNA
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vaccine expressing 1-4 domains of murine VEGFR2, which was intended to be co-immunized with CAVA. Then the antitumor efficacy of our combined replicon vaccines was evaluated in
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mice model and the possible mechanism was further investigated.
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Material and Methods Mice and cell lines
Female C57BL/6 mice (6-8 weeks old) were purchased from Beijing Experimental Animal
Center (Beijing, China) and were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Institute of Health publication no.85-23, Revised 1996). Animal experiment procedure in this study were approved by the Animal Ethics Committee of Beijing Institute of Basic Medical Sciences.
ACCEPTED MANUSCRIPT B16F10-β-hCG melanoma cell line (C57BL/6), which stably expressed human β-hCG antigen, was established previously in our laboratory [10]. 293T cells were obtained from ATCC (Rockville, MD). All cells were cultured in RPMI 1640 medium (Gibco-BRL,
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Gaitherburg, MD, USA) supplemented with 10% fetal calf serum, penicillin (100 U/mL) and streptomycin (100 U/mL), and they were maintained at 37℃，5% CO2 in a humidified
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incubator.
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Construction of DNA replicon vaccine
The plasmid DNA replicon vaccine CAVA was constructed and researched in our previous study [10]. In this study we constructed the DNA replicon vaccine pSVK-VEGFR2-GFc-IL12, which was named CAVE. The schematic representation of CAVE vaccine was shown in
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Figure 1C. Briefly, the VEGFR2-GFc-IL12 gene fragment was obtainded by double enzyme digestion from plasmid pVAX1-VEGFR2-GFc-IL12, which was constructed in previous study. Then the DNA fragment was inserted into the replicon vector pSVK to create
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pSVK-VEGFR2-GFc-IL12 (CAVE). In this recombinant plasmid, the gene sequences of Fc
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fragment of human IgG1 and glycosyl phosphatidyl inositol (GPI) signal peptide were fused to VEGFR2. And internal ribosome entry site (IRES) and IL-12 were then fused to the end of GPI. Both CAVA and CAVE plasmids were prepared by Endotoxin free Giga kit (Qiagen, Shanghai, China). 293T cells were transfected with CAVE plasmids, and the expression of mVEGFR and IL-12 were identified by western blot and ELISA respectively.
Plasmids DNA vaccines immunization in C57BL/6 mice
ACCEPTED MANUSCRIPT Female C57BL/6 mice aged 6-8 weeks were randomly devided into 5 groups (5 mice per group). Plasmids dosage used for each vaccination was explored and optimized by preliminary experiments. The five groups of mice were vaccinated three times at 10-day
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intervals with 100 µL PBS, 100 µL (0.1 µg/µL) pSVK-vector, 100 µL (0.1 µg/µL) CAVA, 100 µL (0.1 µg/µL) CAVE and 100 µL (0.1 µg/µL CAVA plus 0.1 µg/µL CAVE) combined vaccines respectively via intramuscular injection followed by electroporation stimulation.
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Two weeks after the last vaccination, bloods and spleens of each group of mice were collected
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for the evaluation of humoral and cellular immune activity.
Antibody detection
Anti-suvivin, anti-β-hCG and anti-VEGFR2 antibodies were detected by enzyme-linked
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immune sorbent assay (ELISA). 96-well plates were coated with purified survivin, β-hCG and VEGFR2 protein (0.25 µg/well) respectively overnight at 4℃. The blood was collected at 2nd and 4th weeks after the last vaccination. The isolated serum was diluted at 1:100 and incubated
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in the precoated plates for 2 hours at 37℃. Then the plates were washed with PBST (0.05%
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Tween 20 in PBS) and blocked with 1% bovine serum albumin for 1 hour at 37℃. HRP-conjugated goat anti-mouse IgG (dilution 1:5000) was added into the plates (100 µL/well), and the plates were incubated for 1 hour at 37℃. Finally, the antibody titers were detected using TMB system and the OD values were measured at 492 nm by a Bio-Rad plate reader.
IFN-γ ELISPOT assay
ACCEPTED MANUSCRIPT To evaluate cellular immune responses to sepcific antigen in vaccinated mice, splenic lymphocytes were tested for IFN-γ secretion using enzyme-linked immunospot assay (ELISPOT). First, spleen cells were were havested two weeks after the last immunization.
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Then RBC were lysed and the spleenic lymphocytes were isolated by density gradient centrifugation method using lymphocyte isolation liquid (TIAN JIN HAO YANG Biological Manufactory Co. Ltd, China). The isolated lymphocytes were suspended in 10% FCS/RPMI
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1640 and were adjusted to the concentration of 5×106 cells/mL. 100 µL cell suspention (5×105
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cells) was added to each well of the 96-well pecoated IFN-γ Elispot plate (Dakewe bio tech Ltd, Shenzhen, China). The lymphocytes were stimulated with purified recombinant survivin, β-hCG, and VEGFR2 protein 1 µg/well, and were cultured for 48 h at 37℃，5% CO2 in a humidified incubator. After removal of cells and washing with PBST for three times,
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biotinylated rat anti-mouse INF-γ antibody was added and the plate was incubated for 1 h at 37℃. Then the plate was washed and 100 µL 3-amino-9-ethycarbazole (AEC) was added per well, followed by incubation for 45 min at 37℃. Finally, the plate was wash for six times and
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the IFN-γ spots were counted by the ImmunoSpot analyzer (CTL).
Tumor protection and survival analysis in C57BL/6 mice 6-8 weeks female C57BL/6 were divided into 5 groups (n=10) and were vaccinated three
times at a 10-days interval with PBS, pSVK-vector, CAVA, CAVE and combined vaccines as described above. Seven days after the last immunization, 7.5×104 B16F10-β-hCG cells which were suspended in 100 µL PBS, were injected subcutaneously into the left flank of the mice. The tumor protection efficacy was monitored by tumor volume. The length and width (mm)
ACCEPTED MANUSCRIPT of tumor was measured with a digital caliper every 2 days, and the tumor volume was calculated as the following formula: tumor volume (mm3) = 0.5 × length × width2. The mice were observed for 38 days after tumor challenge. Besides, in survival analysis study another 5
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groups of mice (n=5) were vaccinated as the above protocol. The mice were observed for 70 days after the tumor challenge, and the survival rates were calculated.
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Hematoxylin and eosin (H&E) staining and Immunohistochemistry
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To explore the antiangiogenesis of our combined vaccines, the microvessel density in tumor tissue was counted. Briefly, the tumors of mice were removed 38 days after tumor challenge. Then they were paraffin-embed and sectioned to 5 µm-thick slices. After dewaxed, the slides were incubated with 3% hydrogen peroxide for 10 min to block the endogenous
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peroxidase activity. Then the slides were immerged in blocking buffer for 45 min followed by incubation with rabbit anti-CD31 antibody (Abcam) and HRP-conjugated goat anti-rabbit IgG (Santa). After staining with diaminobezidin and conterstarining with hematoxylin, the
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microvessel density was captured and calculated by analyzing 10 random fields per section.
Statistical analysis
The results in this study were analyzed using SPSS software (version 17.0, Chicago, USA).
The statistical differences between groups were determined by One-Way ANOVA method. And p<0.05 was considered statistically significant in all results.
Results
ACCEPTED MANUSCRIPT Expression of DNA vaccine plasmid CAVE in eukaryotic cells The expression of CAVE plamids in the 293T cells were verified. Two days after transfection with CAVE plasmids, the expression of VEGFR2 in 293T cells was verified by
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western blot using rabbit anti-mouse VEGFR2 antibody. The 72 kDa molecular weight band in line 2 confirmed to the theoretic molecular weight of VEGFR2-GPI-Fc fusion protein
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was also verified using IL12 ELISA kit (Figure 1C).
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(Figure 1B). The expression of IL12 in the supernatants of 293T cells transfected with CAVE
Antibody assays
After the third vaccination, anti-VEGFR2 antibody, anti-survivin antibody and anti-β-hCG antibody in serum of mice were detected by ELISA assay respectively. The anti-VEGFR2
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antibody in mice vaccinated with CAVE and combined vaccines were verified at 2 weeks after last immunization, and the titers were significantly higher than those in other groups of mice (P<0.05) (Figure 2A, B and C). Anti-β-hCG and anti-survivin antibodies were also
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detected in the sera of mice immunized with CAVA and combined vaccines. And their titers in
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these two groups were higher than other groups. Moreover, 4 weeks after last immunization, the three antibodies titers were still high in corresponding groups (Figure 2A, B and C). No positive OD values were detected in the PBS and pSVK-vector groups.
ELISPOT assays Two weeks after the last immunization, the IFN-γ ELISPOT assay of spleen lymphocytes was performed to detect the antigen-specific cellular immune response. As shown in Figure
ACCEPTED MANUSCRIPT 2F, VEGFR2 specific IFN-γ secretion were induced in the mice immunized with CAVE and combined vaccines respectively. The spots number in the two groups was significantly higher than that in other groups (P<0.05). Besides, β-hCG and survivin specific IFN-γ secretion were
Anti-tumor efficacy of combined vaccines
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than that in other groups (P<0.05) (Figure 2D and E).
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also verified in CAVA and combined vaccines groups, and the number of spots was higher
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One week after the last immunization, the mice were challenged with B16F10-β-hCG tumor cells and the tumor volume was measured every 2 days. The tumor growth in mice treated with CAVE alone or CAVA alone was significantly inhibited compared with PBS and pSVK-vector (P<0.05). In addition, the combined vaccines inhibited the tumor growth more
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effectively than CAVA and CAVE alone (P<0.05) (Figure 3A). We also observed the survival rate of different groups of mice after tumor challenge. The survival rate of tumor-bearing mice immunized with CAVA alone or CAVE alone was significantly higher compared with
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the mice immunized with PBS and pSVK-vector (P<0.05). Furthermore, the mice threated
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with combined vaccines had a significantly higher survival rate than the mice treated with CAVA alone or CAVE alone (P<0.05) (Figure 3B).
Anti-angiogenesis effect of combined vaccines The anti-angiogenesis effect of combined vaccines was verified by immunohistochemical staining for CD31 in tumor tissue. CAVE encoding VEGFR2 antigen decreased the microvessel density significantly compared with PBS, pSVK and CAVA (P<0.05). And more
ACCEPTED MANUSCRIPT importantly, compared with CAVE alone, the combined vaccines achieved a greater reduction in microvessel density (P<0.05) (Figure 4).
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Discussion Immunologic therapy plays an increasingly important role in the treatment of advanced malignant tumor, such as malignant melanoma, renal cell carcinoma and hepatocellular
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carcinoma. And anti-angiogenesis has been already an efficient approach for tumor therapy in
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research and clinic. Bevacizumab, the first FDA approved anti-angiogenic agent, is a humanized anti-VEGF monoclonal antibody. It has been used to treat some solid tumors such as colorectal cancer, lung cancer, and breast caner, which can increase the survival of tumor patients significantly [11]. However, the anti-angiogenesis agents only suppress tumor cell
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growth and do not induce complete tumor regression. Therefore, it is reasonable and necessary to combine active immunotherapy targeting tumor cells with anti-angiogenesis
previously [12].
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agents. The combination approach could elicit complete tumor regression, as suggested
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Therefore, we presumed that the co-immunization of DNA vaccines targeting tumor cells and angiogenesis may produce a more powerful antitumor efficacy. And as expected, the combined replicon vaccines exhibited the stronger antitumor efficacy than CAVA or CAVE alone. Besides, the mice in co-immunized group had a superior survival rate compared with other groups. These results have demonstrated that the synergistic strategy targeting tumor cells and anti-angiogenesis is a promising approach for DNA vaccine research. We employ several strategies in vaccine design to break the immune tolerance of host and
ACCEPTED MANUSCRIPT increase the antitumor activity of our combined replicon vaccines. First, in this study the combined vaccines are both based on pSVK vector, which is a DNA based Semliki Forest virus (SFV) replicon vector [10]. It has been proved in many research that replicon vector
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could induce higher levels of gene expression without the risk of integration into the host cell genome [13,14,15,16]. Second, the presentation of antigen to DC cells is enhanced. Glycosyl phosphatidyl inositol (GPI) signal peptide is a protein that anchored on the cell membrane.
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And the Fc fragment of human IgG1 has efficient DC cells binding capacity. Therefore we
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fuse the antigen mVEGFR2 with GPI signal peptide and Fc fragment, so that the antigen could be anchored on the cell membrane and be recognized by APCs more efficiently [17,18]. This strategy has been also proved in our previous research [10,19]. Third, several efficient molecular adjuvants are co-expressed via the internal ribosome entry site (IRES) element in
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our combined replicon vaccines. GM-CSF and B7 are co-expressed in CAVA. GM-CSF activates the maturation and proliferation of T cells [20,21]. B7 promotes proliferation of T cells as well as the secretion of chemokines [22,23]. In addition, IL-12 is co-expressed in
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CAVE as adjuvant. It modulates innate and adaptive immunity, and also induce an
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antiangiogenic effect [24]. The co-expression of antigen and adjuvants simultaneously may create a suitable microenvironment for antigen-presenting cells [25]. At last, the electroporation is applied for the delivery of vaccines. It has been reported that the electroporation could significantly increase the activity of DNA vaccines [25,26]. Then we investigate the potential antitumor mechanism of our combined replicon vaccines. ELISA assays of the serum demonstrate that the combined vaccines successfully induce the generation of antibodies against survivin, β-hCG and VEGFR2 simultaneously, which means
ACCEPTED MANUSCRIPT combined replicon vaccines induce humoral immune response in mice successfully. And titers of the three antibodies have no significant differences from CAVA group and CAVE group. Four weeks after the last immunization, the antibody levels in mice of combined vaccines
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group are still high. The anti-VEGFR2 antibody plays an important role in anti-angiogenesis. It could inhibit the phosphorylation of VEGFR-2 induced by VEGF, and therefore block the VEGF/VEGFR2 signal pathway in angiogenesis [27,28]. Thus VEGF-mediated endothelial
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cell proliferation, migration and tube formation in tumor tissue are all inhibited, which may
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be partially responsible for the enhanced anti-tumor efficacy of the combined vaccines. We also detect the specific IFN-γ secreting splenic lymphocytes by quantitative ELISPOT approach. It is manifested that the combined replicon vaccines induce significant amount of specific
IFN-γ
secreting lymphocytes
regarding survivin,
β-hCG and
VEGFR2
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simultaneously. IFN-γ, which is an important factor in cellular immune response and is mainly produced by Th1 lymphocytes, activates the NK cells and CTL cells, and upregulates MHC II and costimulatory molecules of APCs. So the results demonstrate that the combined
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replicon vaccines successfully activate cellular immune response against survivin, β-hCG and
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VEGFR2 antigen simultaneously. And the cellular immune response is also the most crucial for the antitumor effect of vaccines. The other mechanism of our combined vaccines is the anti-angiogenesis effect. The vessel
densities in tumor tissue of different groups of mice are counted by immunohistochemistry staining with anti-CD31 antibodies. In mice immunized against VEGFR2, the inhibition of angiogenesis in tumor tissue was observed. And in the combined replicon vaccines group, more powerful anti-angiogenesis effect than CAVE groups was exhibited. The mechanism
ACCEPTED MANUSCRIPT underlying this synergistic efficacy was not totally clear, but may be associated with a decrease in secretion of other pro-angiogenic factors from the tumor cells themselves. In conclusion, the combined replicon vaccines, which could elicit efficient humoral and
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cellular immune responses against survivin, β-hCG and VEGFR2 simultaneously, not only target tumor cells specifically but also suppress the angiogenesis in tumor tissue. As a result, the combined vaccines exhibit an enhanced synergistic antitumor effect in mice, which inhibit
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the tumor growth and prolong the survival of tumor-bearing mice more effectively. This
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synergistic strategy of DNA vaccines for tumor treatment results in increased antitumor efficacy, and may be more suitable for translation to future research and clinic.
Acknowledgement
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This work was financially supported by the National Science Foundation of China (contract no. 31100655) and National Science and Technology Major Project of the Ministry of Science and Technology of China (contract no. 2014ZX09304313-003). The funders had no role in
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manuscript.
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study design, data collection and analysis, decision to publish, and preparation of the
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[25] S. Flingai, M. Czerwonko, J. Goodman, S.B. Kudchodkar, K. Muthumani, D.B. Weiner, Synthetic DNA vaccines: improved vaccine potency by electroporation and co-delivered genetic adjuvants, Front Immunol 4 (2013) 354. [26] N.Y. Sardesai, D.B. Weiner, Electroporation delivery of DNA vaccines: prospects for success, Curr Opin Immunol 23 (2011) 421-429.
ACCEPTED MANUSCRIPT [27] W.S. Lee, B.J. Pyun, S.W. Kim, S.R. Shim, J.R. Nam, J.Y. Yoo, Y. Jin, J. Jin, Y.G. Kwon, C.O. Yun, D.H. Nam, K. Oh, D.S. Lee, S.H. Lee, J.S. Yoo, TTAC-0001, a human monoclonal antibody targeting VEGFR-2/KDR, blocks tumor angiogenesis, MAbs (2015) 0.
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[28] Y. Yu, P. Lee, Y. Ke, Y. Zhang, J. Chen, J. Dai, M. Li, W. Zhu, G.L. Yu, Development of humanized rabbit monoclonal antibodies against vascular endothelial growth factor receptor 2
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with potential antitumor effects, Biochem Biophys Res Commun 436 (2013) 543-550.
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Figure Legends
Figure 1. Construction and expression of DNA-based replicon vaccine pSVK-VEGFR2-GFc-IL12 (CAVE). (A) Schematic illustration of the vaccine CAVE. (B) The expression of mVEGFR2 gene in 293T cells transfected with CAVE plasmids was detected by western blot with anti-mVEGFR2
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antibody; 1: 293T cells transfected with pSVK-vector, 2: 293T cells transfected with CAVE plasmids. (C) The expression of IL-12 gene in 293T cells transfected with CAVE plasmids was detected by ELISA assay; 1: the supernatant of 293T cells transfected with pSVK-vector, 2: the supernatant of
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293T cells transfected with CAVE plasmids.
Figure 2. Analysis of immune response in mice treated with different vaccines. The sera were collected at 2 weeks and 4 weeks after the last vaccination. The antigen-specific antibody in the sera was detected at a 1:100 dilution by ELISA for survivin (A), β-hCG (B), and VEGFR2 (C). Splenic lymphocytes of each group were harvested and cultured with IL-2 for 48h. The IFN-γ positive T cells were then counted by ELISPOT assay stimulated with protein survivin (D), β-hCG (E), and VEGFR2 (C) respectively.
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Figure 3. Antitumor efficacy in mice vaccinated with different DNA vaccines. (A) Five groups of mice (n=10) were vaccinated with PBS, pSVK-vector, CAVA, CAVE and bivalent vaccines
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respectively at a 10-days interval. Seven days after the third vaccination, 7.5×104 B16F10-β-hCG cells were injected subcutaneously into the the left flank of the mice. Then the tumor growth of
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of vaccinated mice after B16 tumor challenge.
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five groups of mice was measured until the 38th day after tumor challenge. (B) The survival curve
Figure 4. Angiogenesis effect of different DNA vaccines on tumor. The microvessel structures were indicated by immunohistochemical staining with murine CD31 antibody. Tumor tissue sections from mice vaccinated with (A) combined vaccines; (B) CAVA; (C) CAVE; (D)
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compared.
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pSVK-vector; (E) PBS. (F) Tumor microvessel density of each group was quantified and
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ACCEPTED MANUSCRIPT Highlights We developed combined replicon vaccines that target tumor cells and angiogenesis simultaneously.
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The combined vaccines exhibited an enhanced antitumor efficacy in B16 melanoma mice model.
The combined vaccines elicited efficient humoral and cellular immune responses against
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survivin, β-hCG and VEGFR2.
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The anti-angiogenesis effect was also enhanced in mice immunized with combined
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vaccines.