Abstract
Objective
To assess the role of active immunotherapy targeting VEGF with a peptide vaccine as a potential treatment for ovarian cancer.
Methods
A peptide vaccine targeting antigenic B-cell epitopes of VEGF were identified and linked to a promiscuous T-cell epitope. Elicited antibodies were assessed for their ability to recognize the VEGF protein, inhibit angiogenesis, inhibit the interaction of VEGF with its receptor, and inhibit cancer growth in mice.
Results
Following immunization, high-titered elicited antibodies were shown to be specific for the full-length VEGF protein by ELISA and Western blot. Anti-VEGF peptide antibodies inhibited cellular migration, proliferation, invasion, tube formation, and growth of aortic ring cultures. These antibodies inhibited the interaction between VEGF and its receptor (VEGFR2) in a concentration-dependent manner. Confirmation of this mechanism was demonstrated through inhibition of VEGFR2 phosphorylation following culture of human endothelial vein endothelial cells with anti-VEGF peptide antibodies. These antibodies were shown to inhibit ovarian cancer xenograft growth in a nude mouse model following intraperitoneal passive immunization. Active immunization with the VEGF peptide vaccine inhibited VEGF-dependent pancreatic islet cell tumor growth in RIP1-Tag2 transgenic mice and was associated with decreased vasculogenesis in these tumors compared with animals vaccinated with an irrelevant peptide. Active immunization also inhibited growth of tumors from a VEGF overexpressing ovarian cancer cell line, resulting in decreased tumor size and tumor vessel density compared with control mice.
Conclusions
Active immunization with VEGF peptides elicits antibodies that inhibit tumor growth by blocking VEGF-dependent angiogenesis.
Keywords: Ovarian cancer, Angiogenesis, VEGF, Active immunization, Vaccine
Introduction
Ovarian cancer is the most lethal gynecologic malignancy, with more than 14,600 women in the United States expected to die of the disease in 2009 [1]. Unfortunately, there is no effective means for detection of early ovarian cancer, and as such over 75% of cases are diagnosed when the disease has spread to the upper abdomen or lymph nodes. Despite intensive cytotoxic chemotherapy following radical surgery to reduce ovarian cancer volume, the median survival of women with advanced and large-volume ovarian cancer is under 40 months [2].
Recent studies have demonstrated the critical role of angiogenesis in tumor development and the formation of metastatic tumor deposits. The inhibition of tumor angiogenesis has emerged as a promising new therapeutic modality. A number of biologic activities have been identified as being involved in this complex process; however, vascular endothelial growth factor (VEGF) is now known to be one of the most potent and specific pro-angiogenic factors responsible for tumor-induced angiogenesis [3] and is the most promising target for inhibition of tumor-induced angiogenesis. VEGF is overexpressed in a number of human solid malignancies, including ovarian cancer [4,5]. VEGF overexpression has also been demonstrated in women with ovarian cancer and has been shown to be a poor prognostic factor [6–8]. Thus, VEGF is a rational target against which immunization may have a role in the treatment or prevention of ovarian cancer.
Various strategies have been used to inhibit the function of VEGF. These include targeting the VEGF receptor (VEGFR), using gene therapy techniques that deliver antisense oligonucleotides, use of soluble VEGFR, development of receptor tyrosine kinase (RTK) inhibitors, and monoclonal antibodies (Mab) directed against VEGF. The most promising approach appears to be a recombinant humanized version of a murine anti-human VEGF Mab (rhuMab VEGF, bevacizumab) [9]. This Mab has been tested in patients with metastatic cancer [10,11]. There are, however, several disadvantages to the use of antibody therapy. Importantly, passive immunization strategies involve the transfer of antibody to the patient, and immunity is short lived as the antibodies are cleared from the circulation. Likewise, Mabs are often immunogenic themselves, thereby limiting their long-term use. Also, large antibody volumes are necessary for effective sustained immunization. The use of vaccines to prevent or treat ovarian cancer is a highly attractive approach because of the expected minimal side effects of vaccine therapy. Many cancers express tumor-associated antigens (TAA) that serve as targets for cancer vaccines. Strategies for immunization have included whole cell vaccines, protein and DNA vaccines, as well as peptide vaccines; each type of antitumor vaccine has its advantages and limitations. Peptides are an attractive anticancer vaccine in that they are safe (free of pathogens and oncogenic potential), stable, easily constructed, and are a cost-effective vaccine system [12–14]. Importantly, peptide vaccines lead to sustained immune responses and memory, unlike that from passive immunization. Limitations of peptide vaccines include the fact that unmodified peptides are rarely immunogenic; thus, rational peptide design is imperative to the development of an effective antitumor vaccine. We describe the development, synthesis, and properties of a VEGF peptide that may serve as an effective method of prevention or treatment of women at risk for or with ovarian cancer.
Materials and methods
VEGF epitope selection
The selection of candidate VEGF B-cell epitopes was performed using computer-aided analysis using specific correlates for antigenicity such as flexibility, motility, hydropathy, protrusion indices, and antigenicity [12]. Sequences were given a score of 1 to 6 based on their respective index values and were ranked. The sequences receiving the highest scores were selected for further investigation. Measles virus fusion (MVF) protein sequence 288-302 was chosen as the promiscuous epitope to overcome the challenge of tolerance and MHC polymorphism [15]. The MVF epitope was linearly joined to the VEGF epitope by a four-residue linker (GPSL) on a peptide synthesizer. Peptides were purified by reverse-phase HPLC to ensure >95% purity. The identity of the peptides was performed by matrix-assisted LASER desorption ionization-time of flight spectrometry (MALDI-TOF). The sequence homology between human VEGF165 and mouse VEGF164 isoform is >90%, and the sequence homology against which the VEGF peptide was developed is 100% between the human and mouse VEGF sequences.
Vaccination and elicitation of VEGF peptide antibodies
Approval from the institutional Laboratory Animal Care and Use Committee was obtained prior to initiation of this study. To generate anti-VEGF peptide antibodies, New Zealand white rabbits (Charles River Laboratories, Inc., Wilmington, MA) and BALB/c mice (Harlan, Indianapolis, IN) were immunized subcutaneously at multiple sites with a total of 1 mg of each peptide emulsified in a Squaline/Arlacel vehicle containing nor-MDP (N-acetyl-glucosamine-3 yl-acetyl l alanyl-d-isoglutamine). Subsequent booster injections were given at 3 weeks (secondary immunization, 2 years) and 6 weeks (tertiary immunization, 3 years) after primary (1 year) immunization. Rabbit and mouse sera were collected weekly, and complement was inactivated by heating to 56 °C. High-titered sera were purified on a protein A/G-agarose column (Pierce, Rockford, IL), and eluted antibodies were concentrated and exchanged in PBS using Mr 100,000 cutoff centrifuge filter units (Millipore, Bedford, MA). Antibody concentration was quantified by ELISA.
Antibody characterization
Immunoprecipitation was undertaken to determine whether the VEGF peptide antibodies recognize the VEGF protein. Proteins (including rhVEGF) immunoprecipitated with VEGF peptide antibodies or a rabbit VEGF polyclonal antibody (R&D Systems, Minneapolis, MN) were resolved by 15% SDS–PAGE, transferred to nitrocellulose, and probed with a goat VEGF polyclonal antibody (Ab-4, R&D Systems, Minneapolis, MN) and detected by enhanced chemiluminescence. Confirmation of specificity and antibody concentrations were determined by direct ELISA against rhVEGF.
Characterization of the ability of anti-VEGF peptide antibodies to inhibit angiogenesis
The ability of anti-VEGF peptide antibodies to inhibit angiogenesis in assays of proliferation, migration, tube formation, and inhibition of outgrowths from aortic rings was assessed as described in the supplementary materials and methods.
Characterization of the ability of anti-VEGF peptide antibodies to inhibit VEGF-VEGF receptor interaction
VEGF Fluorokine (R&D Systems, Minneapolis MN) was used to quantitatively determine the percentage of cells expressing the VEGF receptors and to estimate the receptor density for VEGF on the surface of HUVECs by flow cytometry, as described in the supplementary materials and methods. Also, the ability of anti-VEGF peptide antibodies to inhibit phosphorylation of the VEGFR2 was evaluated by immunoprecipitation, as described in the supplementary materials and methods.
Characterization of the ability of anti-VEGF peptide antibodies to inhibit tumorigenesis
Human ovarian cancer SKOV-3 cells were injected intraperitoneally in female nu/nu mice. Seven weeks later, 107 cells were harvested by peritoneal lavage and injected into a new set of recipients. Three weeks later, this was repeated, and the final passage of cells harvested and cultured for investigation. The n, 5×106 subcloned cells were mixed with matrigel and injected subcutaneously in 7-week-old athymic nude mice. Seven days later, mice were treated twice weekly with intraperitoneal PBS or 5μg/g antibody: normal rabbit IgG, mouse monoclonal anti-VEGF antibody, or anti-VEGF peptide antibodies. Tumor measurements were undertaken beginning 7 days after inoculation and twice weekly. Tumor volume was calculated according to the formula [volume=0.52×(width)2×length in mm3]. Mice were sacri-ficed 4 weeks after challenge, and tumors were imbedded in OTC and sections immunostained with rat anti-CD31 monoclonal antibody (1:1000, Pharmingen, San Diego, CA). Microvessel hot spots were identified under 10× power, and photographed at 100×. Microvessel density was expressed as the percentage of CD31 staining versus section image. Statistical difference between groups was analyzed by Student's t-test.
Characterization of the ability of VEGF peptides to inhibit VEGF-dependent tumor growth in a transgenic mouse model
RIP1-Tag2 mice, which develop endocrine pancreatic tumors under the influence of VEGF, were used to investigate the effect of active immunization with VEGF peptides. Subcutaneous immunization with 100 μg of MVF-VEGF epitope or an irrelevant MVF-peptide sequence from HTLV-1 was performed at 5 weeks of age, with booster immunizations at 7, 9, and 11 weeks. Serum was collected prior to every immunization and quantified for concentrations of anti-VEGF peptide antibodies and VEGF (VEGF ELISA kit, R&D Systems, Minneapolis, MN). Mice were sacrificed at 12.5 weeks; sera were collected and tumors were measured and harvested prior to embedding in OTC. Tumors were immunostained with rat anti-CD31 monoclonal antibody, 1:1000. Microvessel hot spots were identified at 10× and photographed at 100×. Microvessel density was expressed as the percentage of CD31 staining versus section image. Statistical difference between groups was analyzed by Student's t-test. In a parallel experiment, mice that were vaccinated with VEGF peptides were allowed to survive until they died spontaneously or demonstrated evidence of debility from disease. Special IACUC approval was obtained for the use of death as an endpoint in the transgenic mouse model. The survival of these mice was compared with either untreated mice or those treated with an irrelevant peptide from HTLV-1. Survival was calculated with the Kaplan–Meier method and compared using the log rank test.
Characterization of the ability of VEGF peptides to inhibit growth of VEGF-dependent transgenic cell lines
Female C57BL/6 mice were immunized subcutaneously with 100 μg of MVF-VEGF epitope or an irrelevant MVF-peptide sequence from HTLV-1 at 6 weeks of age, with booster immunizations at 9, 12, and 15 weeks. ID8 cells (derived from spontaneous malignant transformation of C57BL/6 mouse ovarian surface epithelial cells) transfected with overexpressing VEGF and GFP were kindly provided by George Coukos, M.D., Ph.D. (University of Pennsylvania). ID8-VEGF-GFP cells were cultured and 5×106 cells were mixed with growth factor reduced Matrigel. Mice were challenged with subcutaneously injected cells at 11 weeks. Sera were collected weekly and quantified by ELISA. Tumors were measured twice weekly. At 18 weeks, mice were sacrificed, tumors harvested and observed using a fluorescence stereomicroscope, embedded in OTC, and frozen sections stained for CD31 expression. Microvessel hot spots were identified under 10× power and photographed at 100×. Microvessel density was expressed as the percentage of CD31 staining versus section image. Statistical difference between groups was analyzed by Student's t-test.
Results
Chimeric VEGF B-cell epitope constructs
Computer-aided analysis of candidate B-cell epitopes of VEGF was used to select residues 127-144 (KCECRPKKDRARQENPCG) and residues 102-122 (ITMQIMRIKPHQGQHIGEMSF), which correlates with a secondary structure of turn–helix–turn, as being potentially immunogenic and antigenic. These epitopes were synthesized with the promiscuous T-helper cell epitope, MVF (measles virus fusion protein), at the COOH terminus. The amino acid sequences, predicted secondary structures, and the molecular weights of the MVF-conjugated VEGF peptide constructs are indicated in supplementary Table 1.
Immunogenicity of chimeric VEGF B-cell epitope peptides in outbred rabbits
The VEGF oligopeptides were highly immunogenic, as evidenced by antibody titers of over 1:300,000 following immunization (Fig. 1). Both MVF-VEGF(102-122) and MVF-VEGF(127-144) elicited immediate and high antibody titers 1 week after the first booster, with an eventual rise in antibody titers to maximal levels after the tertiary boost. The polyclonal IgG sera did not cross-react with the MVF T-cell sequence.
Fig. 1.
High-titer sera recognizing the B-cell epitope (VEGF, lighter bars) and immunogen (MVF-VEGF, darker bars) following active immunization with MVF-VEGF(127-144) peptides. ELISA of a single New Zealand white rabbit sera against VEGF, demonstrating titers >1:250,000 at 1 week following the second booster (3Y+1). High-titer sera persisted through repeated booster immunizations (at 3Y+3, 4Y+3, 5Y+3, and 6Y+3). Similar results were seen in all rabbits immunized with the VEGF(127-144) and VEGF(102-122) peptides. ELISA against MVF alone showed no immmunoreactivity, demonstrating specificity of the elicited antibodies towards the B-cell epitope and immunogen.
Binding of peptide antibodies to native human VEGF
Peptide-based vaccines will be effective only if the antibodies elicited by peptide immunogens bind native full-length VEGF. Two different methods were used to test the binding of antipeptide antibodies to the native protein. First, the capacity of VEGF peptide antibodies to immunoprecipitate VEGF protein from the tumor lysates of RIP1-Tag2 mouse pancreatic islet tumors overexpressing VEGF was assessed. Both of the peptide antibodies efficiently immunoprecipitated the native mouse VEGF from RIP1-Tag 2 tumors (Fig. 2). Second, binding of the peptide antibodies to rhVEGF was determined by ELISA. The pattern of reactivity of peptide antibodies with the protein paralleled the observation of the immunoprecipitation (data not shown).
Fig. 2.
Anti-human VEGF(102-122) and VEGF(127-144) antibodies recognize mVEGF164 by immunoprecipitation. Following lysis of mouse VEGF-expressing tumor, immunoprecipitation of mouse VEGF with anti-VEGF peptide antibodies and resolution of protein by Western blot with anti-mouse VEGF polyclonal antibody was undertaken. Resolution of mouse VEGF was seen in lanes immunoprecipitated with rabbit VEGF peptide antibodies with the appropriate positive (anti-mouse VEGF antibody right lane) and negative (normal IgG, left lane) controls.
VEGF peptide antibodies inhibit angiogenesis in vitro
Previous studies have demonstrated that the in vitro properties of migration, proliferation, and tube formation are valuable surrogate methods of testing anti-angiogenic compounds in the preclinical setting. The ability of rhVEGF to induce migration of HUVECs through a permeable membrane in a Boyden chamber was significantly inhibited by rabbit anti-VEGF peptide antibodies, with 20% of the HUVECs migrating through the membrane in the presence of peptide antibodies, compared with 40% with pre-immune sera (p<0.01, data not shown). Furthermore, inhibition of rhVEGF-induced proliferation of HUVECs was inhibited by these anti-VEGF peptide antibodies (>20% inhibition in the presence of peptide antibodies, compared with 2% with pre-immune sera (p<0.01, data not shown). The anti-VEGF peptide antibodies also significantly disrupted the ability of rhVEGF to induce endothelial cells to form tubes (>20% inhibition in the presence of peptide antibodies, compared with 2% with pre-immune sera (p<0.01) and inhibit endothelial cell growth into Matrigel under the influence of rhVEGF compared with normal rabbit IgG (p<0.01).
VEGF peptide antibodies inhibit interaction of VEGF with VEGF receptors
Peptide-based vaccines targeting angiogenesis will be effective only if the antibodies elicited by peptide immunogens can prevent mitogens of angiogenesis (VEGF) from interacting with their receptors. In a flow-cytometry-based system, the presence of VEGF peptide antibodies effectively prevented the interaction between VEGF and VEGF receptors in a concentration-dependent fashion (Fig. 3). The ability of VEGF peptide antibodies to modify the interaction between VEGF receptors and its ligand VEGF was confirmed through demonstrating the ability of VEGF peptide antibodies to prevent the activation through completely blocking phosphorylation of the VEGF receptor compared with treatment with an irrelevant peptide.
Fig. 3.
VEGF peptide antibodies recognize and bind VEGF, leading to a decrease in the interaction between the VEGF ligand and its receptors. Flow cytometry demonstrating a shift in population when rhVEGF-stimulated human umbilical vein endothelial cells (HUVEC) are pre-incubated with monoclonal anti-VEGF antibodies, anti-MVF-VEGF (102-122) antibodies, and anti-MVF-VEGF(127-144) antibodies. Compared with cells treated with rhVEGF alone (right population), those treated with VEGF blocking antibodies (middle population) are shifted towards the population of HUVECs not stimulated with rhVEGF (left population). This shift in population demonstrates a decrease in the interaction between the VEGF ligand and VEGF receptors. Further investigation demonstrates that this decreased VEGF-VEGFR interaction is partially a result of blocking phosphorylation of VEGFR2 (KDR).
MVF-VEGF peptide constructs inhibit angiogenesis-dependent tumor growth in RIP1-Tag2 transgenic mice
Following immunization with MVF-VEGF peptide constructs, RIP1-Tag2 mice (average n=16 per group) developed high-titer VEGF peptide antibodies that immunoprecipitated mouse VEGF. At 12.5 weeks, tumor growth of mice immunized with VEGF peptides were significantly smaller than those untreated or treated with an irrelevant peptide (85 mm3 vs. 210 mm3, p=0.01 for MVF-VEGF(102-122) vs. irrelevant MVF-HTLV-1peptide, Fig. 4A). The reduced tumor volume in mice immunized with VEGF peptide constructs was associated with a significant reduction in microvessel density (data not shown) and a reduction in serum VEGF levels (74.9 vs. 83.7 pg/mL, p=0.05) without a reduction in tumor VEGF levels. Vaccinated mice survived longer than control mice or those treated with an irrelevant HTLV-1 peptide (median survival 15 weeks for MVF-VEGF(127-144) and MVF-VEGF(102-122) vs. 13 weeks for those treated with irrelevant MVF-HTLV-1 peptide, p=0.0015, Fig. 4B).
Fig. 4.
(A) At the time of animal necropsy, the tumor volume of RIP1-Tag2 mice immunized with MVF-VEGF(102-122) or MVF-VEGF(127-144) is significantly smaller than that of mice immunized with a control peptide or PBS. Compared with control mice immunized with MVF-HTLV-1 peptides, tumors from the mice immunized with MVF-VEGF(102-122) were significantly smaller, p<0.01. There is no difference in tumor volume between mice immunized with irrelevant MVF-HTLV-1 peptide and a non-immunized control group (data not shown). (B) Vaccination with MVF-VEGF(127-144) and MVF-VEGF(102-122) led to significantly improved survival in RIP1-Tag2 mice compared with mice vaccinated with irrelevant MVF-HTLV-1 control peptide (median survival 13 versus 15 weeks, p=0.0015).
VEGF peptide antibodies inhibit growth of tumors from VEGF-expressing SKOV-3 ovarian cancer cells in female nu/nu mice
Intraperitoneal treatment with VEGF peptide antibodies inhibited tumor growth beginning 10 days after tumor engraftment. Inhibition of tumor growth persisted until the mice were sacrificed at 28 days following engraftment (Fig. 5A). The decreased tumor growth in the mice passively immunized (with either the VEGF peptide antibodies or commercially available VEGF mAb) was associated with a significant decrease in tumor microvessel growth (Fig. 5B), with microvessel density measurements of 1.94 after treatment with negative control (IgG) being reduced to 1.07 after treatment with anti-MVF-VEGF(127-144) antibodies, p<0.01, Fig. 5B).
Fig. 5.
(A) The tumor volume resulting from subcutaneous inoculation of SKOV-3 cells in athymic female nu/nu mice treated with mouse monoclonal anti-hVEGF antibody, rabbit polyclonal anti-MVF-VEGF(102-122) antibodies or anti-MVF-VEGF(127-144) antibodies was significantly smaller compared to PBS control mice from 11 days after inoculation, p<0.01). There is no difference in tumor volume between mice treated with normal rabbit IgG and mice of PBS control (p>0.05). (B) Immunostaining for endothelial cells (CD31) demonstrated reduced microvessel density in tumors from mice treated with mouse monoclonal anti-hVEGF antibody, rabbit polyclonal anti-MVFVEGF(102-122) antibodies or anti-MVF-VEGF(127-144) antibodies is significantly lower than that of mice treated with PBS (p<0.01). There is no difference in tumor microvessel density between mice treated with normal rabbit IgG and mice treated with PBS (p>0.05).
MVF-VEGF peptide constructs inhibit VEGF-overexpressing ovarian cancer tumor growth
When immunocompetent mice were immunized with MVF-VEGF peptide constructs (or an irrelevant MVF-HTLV-1 peptide control) and then challenged with ID8-VEGF-GFP transgenic cells, a significant reduction in the size of subcutaneous tumor explants was noted beginning on day 14, which persisted through the end of the experiment (mean irrelevant peptide 885±162mm3 vs. mean MVFVEGF(102-122) 466±100mm3, p=0.001, Fig. 6A). The reduced tumor volume in mice immunized with VEGF peptide constructs was associated with a significant reduction in microvessel density (Fig. 6B). Fluorescence microscopy of whole tumors demonstrated a gross decrease in tumor vascularity compared to control animals (data not shown).
Fig. 6.
(A) ID8 tumor volumes of mice vaccinated with MVF-VEGF(102-122) was significantly smaller than those vaccinated with irrelevant MVF-HTLV-1 peptide control beginning 7 days following tumor inoculation (p<0.01), and similar to tumor volumes in mice vaccinated with MVF-VEGF(127-144). Mice vaccinated with irrelevant MVFHTLV-1 peptide control demonstrated similar tumor volumes to those mice inoculated with PBS (p>0.05). (B) Immunostaining for endothelial cells (CD31) demonstrated reduced microvessel density in tumors from mice vaccinated with MVF-VEGF(102-122) or MVF-VEGF(127-144) compared to mice vaccinated with irrelevant MVF-HTLV-1 peptide control or PBS, p<0.01). There is no difference in tumor microvessel density between mice vaccinated with irrelevant HTLV-1 peptide and control mice without peptide treatment (p>0.05).
Toxicity of vaccination with MVF-VEGF constructs
Necropsy of BALB/c mice who had been vaccinated with the MVFVEGF(127-144) peptide constructs (primary vaccination with booster every 3 weeks for 4 vaccinations) was performed to determine the toxic effects. Compared with non-vaccinated mice, those treated with VEGF peptide constructs demonstrated mild serositis of the mesentery of the small bowel. Specifically, there was no evidence of large or small blood vessel changes.
Discussion
We demonstrate that with rational design, VEGF peptides can be developed that are specific, antigenic, immunogenic, and possess properties that inhibit angiogenesis and tumor growth in vitro and in vivo. This fact is contrary to the belief that peptide immunogens are considered to be weak and thus represent a poor method of cancer therapy. In general, immunization of animals with synthetic peptides has been shown to lead to elicitation of antibodies with low affinity to the native protein, partly because antibody recognition sites are usually of the conformational type, and the peptide immunogens lacked defined structure in solution. Peptides must mimic the native conformation of the protein for their respective antibodies to bind target antigens with an affinity high enough to be biologically significant. The genetically restricted stimulatory activity of peptides is also a major obstacle to developing vaccine approaches for use in an outbred human population. Covalent conjugation of B-cell epitope peptides to large carrier molecules is sometimes used to address this problem but often results in hypersensitivity, conformational changes, appearance of undefined structures, loss of epitopes, inappropriate presentation of epitopes, and batch-to-batch conjugate variability. We have addressed several of these issues in our approach to subunit peptide vaccine design. Our strategy involved de novo design of topographic determinants that focused on preserving the native protein sequence while facilitating folding of the peptide into a stable conformation that mimics the native protein structure. Our previous work in a variety of model systems has demonstrated that this approach can elicit high-titered antibodies that recognize native protein in an outbred population.
The progress of inhibiting angiogenesis as cancer therapy has progressed rapidly from the identification of VEGF as a mitogen for cancer-related blood vessel growth to the FDA-approval of these agents for cancer treatment with dozens more in development. Approaches to inhibiting angiogenesis include targeting the ligand, the receptor, or the cancer supporting vasculature. Each of these methods has its individual advantages and disadvantages; however, one common problem among each of these strategies includes the need to frequent repeated treatment, and the risk of tolerance or resistance [16]. It is possible that the strategy of active immunotherapy in an immunocompetent host may bypass these limitations. Specifically, given the fact that the FDA-approved monoclonal anti-VEGF antibody bevacizumab demonstrates only partial antitumor efficacy that is cytostatic in nature, prolonged treatment is necessary, including in the treatment of ovarian cancer [17,18]. Importantly, subunit peptide vaccines can focus immune responses to biologically active epitopes. The need for epitope-based vaccines stems from the fact that tolerance to self-antigens, such as VEGF, may limit a functional immune response to whole protein-based vaccines due to activation of suppressor T cells that maintain tolerance to host antigens or alternate regulatory mechanisms [19]. The capacity to narrowly focus the immune response is of particular relevance to VEGF, where interaction of the antibody with specific sites has the potential of inhibiting growth. In contrast to passive therapy, the continuous availability of tumor-targeting antibodies can be ensured at low cost.
Previous investigators have developed similar strategies of anti-VEGF cancer therapy. Interest in VEGF as a model antigen to explore therapy has been demonstrated through the construction of a plasmid DNA encoding Xenopus homologous VEGF [20]. This group determined that immunogene tumor therapy with this vaccine led to the development of VEGF-specific antibodies that were anti-angiogenic and inhibited tumor formation. Importantly, treatment of mice with the immunogene led to no significant toxic effects. In other work, vaccination with dendritic cells transfected with VEGF mRNA has been demonstrated to lead to cytotoxic T lymphocyte (CTL) responses, to the disruption of angiogenesis, and to antitumor efficacy without significant morbidity or mortality in vivo in a murine model [21]. Thus, previous work has demonstrated the feasibility of active immunization using VEGF as a TAA.
Limitations of this investigation are the fact that the antigen chosen for investigation, VEGF, is ubiquitously expressed in normal and pathologic conditions, and its inhibition may lead to potentially serious biologic consequences. Although fetal development is strongly controlled by angiogenesis, only reproduction, wound healing, and cancer are controlled by angiogenesis in the adult host. As such, we believe that the relative control and VEGF overexpression in malignancy would lead to an acceptable therapeutic ratio in the treatment of solid tumors. This is supported by previous investigation of other methods of decreasing the effects of VEGF (i.e. through DNA vaccines or inhibition of VEGFR) that failed to demonstrate significant toxicity. We are also currently investigating other factors that may specifically control ovarian angiogenesis to circumvent the possibility of global disruption of VEGF-induced angiogenesis. Furthermore, the utility of active immunotherapy, which requires a relatively immunocompetent host, may not be feasible in patients undergoing conventional chemotherapy and radiation, both of which are inherently immunosuppressive.
Most women with ovarian cancer are diagnosed with advanced disease, and despite the majority obtaining a complete clinical response following induction chemotherapy, 80% will recur and succumb to their disease. This scenario suggests that microscopic residual disease after initial therapy is responsible for disease recurrence. For this reason, current clinical research in ovarian cancer focuses on the treatment of ovarian cancer with maintenance chemotherapy. Here, following initial treatment, patients achieving a complete clinical response have been demonstrated to have a better disease-free survival when a prolonged course of treatment is initiated immediately [22]. Interestingly, investigation of the role of active immunization with the anti-idiotype antibody ACA125 (which imitates the tumor-associated antigen CA125 in ovarian cancer) as a maintenance chemotherapy in ovarian cancer has demonstrated a positive impact on overall survival [23]. Whether the active immunization approach to inhibition of VEGF may lead to anti-idiotype antibodies, however, is uncertain. Given the recent finding that maintenance bevacizumab following primary treatment with cytotoxic chemotherapy and bevacizumab leads to prolonged disease-free survival (Burger RA, Brady MF, Bookman MA, et al.: A phase III trial of bevacizumab in the primary treatment of advanced epithelial ovarian cancer, primary peritoneal cancer and fallopian tube cancer. A Gynecologic Oncology Group study. J Clin Oncol 2010:28 (suppl: abstract LBA1)) is intriguing and suggests that targeting VEGF with strategies such as active immunotherapy to prevent symptomatic recurrence of ovarian cancer may be an attractive concept.
Angiogenesis has been demonstrated to influence cancer growth variably at different stages of malignant proliferation. Importantly, premalignant neoplastic conditions and small malignant tumors are thought to grow under the direct influence of endothelial mitogens such as VEGF, whereas larger malignant tumors may grow and metastasize independent of angiogenic factors [24]. The concept that angiogenic factors control early tumor growth has been applied to the clinical management of ovarian cancer. Current research efforts are directed at investigating chemotherapy agents that may act as anti-angiogenic, cytostatic agents. These compounds, such as tamoxifen and thalidomide, have been evaluated in women with early recurrent, asymptomatic ovarian cancer to determine if anti-angiogenic therapy may prevent the development of clinically significant, symptomatic disease. As such, anti-angiogenic therapy with active immunization using VEGF epitopes could serve as a rational maintenance therapy that could significantly impact the course of ovarian cancer.
From this investigation, we demonstrate that rational design of peptide vaccines against VEGF leads to elicitation of high-titered VEGF peptide antibodies that are specific and anti-angiogenic. Thus, further research is warranted into the potential for the application of active immunotherapy with VEGF in the treatment or prevention of ovarian cancer.
Supplementary Material
Footnotes
This work was supported by funding from the Gynecologic Cancer Foundation and the Liz Tilberis Scholars Program, The Ovarian Cancer Research Foundation (to D.E.C.). Peptides were synthesized in the Peptide Engineering Laboratory at Ohio State University for a fee.
Supplementary data to this article can be found online at doi:10.1016/j.ygyno.2010.07.037.
Conflict of interest
None of the authors report a conflict of interest relevant to the content of the manuscript.
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