Proinflammatory activity of VEGF-targeted treatment through reversal of tumor endothelial cell anergy.

PURPOSE: Ongoing angiogenesis renders the tumor endothelium unresponsive to inflammatory cytokines and interferes with adhesion of leukocytes, resulting in escape from immunity. This process is referred to as tumor endothelial cell anergy. We aimed to investigate whether anti-angiogenic agents can overcome endothelial cell anergy and provide pro-inflammatory conditions. EXPERIMENTAL DESIGN: Tissues of renal cell carcinoma (RCC) patients treated with VEGF pathway-targeted drugs and control tissues were subject to RNAseq and immunohistochemical profiling of the leukocyte infiltrate. Analysis of adhesion molecule regulation in cultured endothelial cells, in a preclinical model and in human tissues was performed and correlated to leukocyte infiltration. RESULTS: It is shown that treatment of RCC patients with the drugs sunitinib or bevacizumab overcomes tumor endothelial cell anergy. This treatment resulted in an augmented inflammatory state of the tumor, characterized by enhanced infiltration of all major leukocyte subsets, including T cells, regulatory T cells, macrophages of both M1- and M2-like phenotypes and activated dendritic cells. In vitro, exposure of angiogenic endothelial cells to anti-angiogenic drugs normalized ICAM-1 expression. In addition, a panel of tyrosine kinase inhibitors was shown to increase transendothelial migration of both non-adherent and monocytic leukocytes. In primary tumors of RCC patients, ICAM-1 expression was found to be significantly increased in both the sunitinib and bevacizumab-treated groups. Genomic analysis confirmed the correlation between increased immune cell infiltration and ICAM-1 expression upon VEGF-targeted treatment. CONCLUSION: The results support the emerging concept that anti-angiogenic therapy can boost immunity and show how immunotherapy approaches can benefit from combination with anti-angiogenic compounds.

Are antiangiogenics a good 'partner' for immunotherapy in ovarian cancer?

Ovarian cancer (OC) is associated with poor survival because there are a limited number of effective therapies. Two processes key to OC progression, angiogenesis and immune evasion, act synergistically to promote tumor progression. Tumor-associated angiogenesis promotes immune evasion, and tumor-related immune responses in the peritoneal cavity and tumor microenvironment (TME) affect neovascular formation. Therefore, suppressing the angiogenic pathways could facilitate the arrival of immune effector cells and reduce the presence of myeloid cells involved in immune suppression. To date, clinical studies have shown significant benefits with antiangiogenic therapy as first-line therapy in OC, as well as in recurrent disease, and the vascular endothelial growth factor (VEGF) inhibitor bevacizumab is now an established therapy. Clinical data with immunomodulators in OC are more limited, but suggest that they could benefit some patients with recurrent disease. The preliminary results of two phase III trials have shown that the addition of immunomodulators to chemotherapy does not improve progression-free survival. For this reason, it could be interesting to look for synergistic effects between immunomodulators and other active drugs in OC. Since bevacizumab is approved for use in OC, and is tolerable when used in combination with immunotherapy in other indications, a number of clinical studies are underway to investigate the use of bevacizumab in combination with immunotherapeutic agents in OC. This strategy seeks to normalize the TME via the anti-VEGF actions of bevacizumab, while simultaneously stimulating the immune response via the immunotherapy. Results of these studies are awaited with interest.

Predictive markers of anti-VEGF and emerging role of angiogenesis inhibitors as immunotherapeutics.

The critical role of angiogenesis in promoting tumor growth and metastasis has been well established scientifically, and consequently blocking this pathway as a therapeutic strategy has demonstrated great clinical success for the treatment of cancer. The holy grail however, has been the identification of patients who derive significant survival benefit from this class of agents. Here we attempt to delineate the diverse mechanisms related to anti-VEGF including its role as an anti-vascular, anti-angiogenic or an anti-permeability factor and review the most promising predictive biomarkers interrogated in large clinical trials, that identify patients who may derive significant survival advantage with VEGF inhibition. Lastly, we describe the function of VEGF as an immunomodulator and illustrate the evidence for anti-VEGF in reprogramming the tumor milieu from an immunosuppressive to an immune permissive microenvironment in human cancers, thus elucidating the role of anti-VEGF as an optimal combination partner for immune checkpoint inhibitors.

The reciprocal function and regulation of tumor vessels and immune cells offers new therapeutic opportunities in cancer.

Tumor angiogenesis and escape of immunosurveillance are two cancer hallmarks that are tightly linked and reciprocally regulated by paracrine signaling cues of cell constituents from both compartments. Formation and remodeling of new blood vessels in tumors is abnormal and facilitates immune evasion. In turn, immune cells in the tumor, specifically in context with an acidic and hypoxic environment, can promote neovascularization. Immunotherapy has emerged as a major therapeutic modality in cancer but is often hampered by the low influx of activated cytotoxic T-cells. On the other hand, anti-angiogenic therapy has been shown to transiently normalize the tumor vasculature and enhance infiltration of T lymphocytes, providing a rationale for a combination of these two therapeutic approaches to sustain and improve therapeutic efficacy in cancer. In this review, we discuss how the tumor vasculature facilitates an immunosuppressive phenotype and vice versa how innate and adaptive immune cells regulate angiogenesis during tumor progression. We further highlight recent results of antiangiogenic immunotherapies in experimental models and the clinic to evaluate the concept that targeting both the tumor vessels and immune cells increases the effectiveness in cancer patients.

Targeted vaccination against the bevacizumab binding site on VEGF using 3D-structured peptides elicits efficient antitumor activity.

Therapeutic targeting of the VEGF signaling axis by the VEGF-neutralizing monoclonal antibody bevacizumab has clearly demonstrated clinical benefit in cancer patients. To improve this strategy using a polyclonal approach, we developed a vaccine targeting VEGF using 3D-structured peptides that mimic the bevacizumab binding site. An in-depth study on peptide optimization showed that the antigen's 3D structure is essential to achieve neutralizing antibody responses. Peptide 1 adopts a clear secondary, native-like structure, including the typical cysteine-knot fold, as evidenced by CD spectroscopy. Binding and competition studies with bevacizumab in ELISA and surface plasmon resonance analysis revealed that peptide 1 represents the complete bevacizumab binding site, including the hairpin loop (ß5-turn-ß6) and the structure-supporting ß2-α2-ß3 loop. Vaccination with peptide 1 elicited high titers of cross-reactive antibodies to VEGF, with potent neutralizing activity. Moreover, vaccination-induced antisera displayed strong angiostatic and tumor-growth-inhibiting properties in a preclinical mouse model for colorectal carcinoma, whereas antibodies raised with peptides exclusively encompassing the ß5-turn-ß6 loop (peptides 15 and 20) did not. Immunization with peptide 1 or 7 (murine analog of 1) in combination with the potent adjuvant raffinose fatty acid sulfate ester (RFASE) showed significant inhibition of tumor growth in the B16F10 murine melanoma model. Based on these data, we conclude that this vaccination technology, which is currently being investigated in a phase I clinical trial (NCT02237638), can potentially outperform currently applied anti-VEGF therapeutics.

Immunogenicity moderation effect of interleukin-24 on myelogenous leukemia cells.

Previous studies have shown that interleukin-24 (IL-24) has tumor-suppressing activity by multiple pathways. However, the immunogenicity moderation effect of IL-24 on malignant cells has not been explored extensively. In this study, we investigated the role of IL-24 in immunogenicity modulation of the myelogenous leukemia cells. Data show that myelogenous leukemia cells express low levels of immunogenicity molecules. Treatment with IL-24 could enhance leukemia cell immunogenicity, predominantly regulate leukemia cells to produce immune-associated cytokines, and improve the cytotoxic sensitivity of these cells to immune effector cells. IL-24 expression could retard transplanted leukemia cell tumor growth in vivo in athymic nude mice. Moreover, IL-24 had marked effects on downregulating the expression of angiogenesis-related proteins vascular endothelial growth factor, cluster of differentiation (CD) 31, CD34, collagen IV and metastasis-related factors CD147, membrane type-1 matrix metalloproteinase (MMP), and MMP-2 and MMP-9 in transplanted tumors. These findings indicated novel functions of this antitumor gene and characterized IL-24 as a promising agent for further clinical trial for hematologic malignancy immunotherapy.

Optimized Expression and Characterization of a Novel Fully Human Bispecific Single-Chain Diabody Targeting Vascular Endothelial Growth Factor165 and Programmed Death-1 in Pichia pastoris and Evaluation of Antitumor Activity In Vivo.

Bispecific antibodies, which can bind to two different epitopes on the same or different antigens simultaneously, have recently emerged as attractive candidates for study in various diseases. Our present study successfully constructs and expresses a fully human, bispecific, single-chain diabody (BsDb) that can bind to vascular endothelial growth factor 165 (VEGF165) and programmed death-1 (PD-1) in Pichia pastoris. Under the optimal expression conditions (methanol concentration, 1%; pH, 4.0; inoculum density, OD600 = 4, and the induction time, 96 h), the maximum production level of this BsDb is achieved at approximately 20 mg/L. The recombinant BsDb is purified in one step using nickel-nitrilotriacetic acid (Ni-NTA) column chromatography with a purity of more than 95%. Indirect enzyme-linked immune sorbent assay (ELISA) and sandwich ELISA analyses show that purified BsDb can bind specifically to VEGF165 and PD-1 simultaneously with affinities of 124.78 nM and 25.07 nM, respectively. Additionally, the BsDb not only effectively inhibits VEGF165-stimulated proliferation, migration, and tube formation in primary human umbilical vein endothelial cells (HUVECs), but also significantly improves proliferation and INF-γ production of activated T cells by blocking PD-1/PD-L1 co-stimulation. Furthermore, the BsDb displays potent antitumor activity in mice bearing HT29 xenograft tumors by inhibiting tumor angiogenesis and activating immune responses in the tumor microenvironment. Based on these results, we have prepared a potential bispecific antibody drug that can co-target both VEGF165 and PD-1 for the first time. This work provides a stable foundation for the development of new strategies by the combination of an angiogenesis inhibition and immune checkpoint blockade for cancer therapy.

Meta-analysis of individual patient safety data from six randomized, placebo-controlled trials with the antiangiogenic VEGFR2-binding monoclonal antibody ramucirumab.

BACKGROUND: Ramucirumab, the human immunoglobulin G1 monoclonal antibody receptor antagonist of vascular endothelial growth factor receptor 2, has been approved for treating gastric/gastroesophageal junction, non-small-cell lung, and metastatic colorectal cancers. With the completion of six global, randomized, double-blind, placebo-controlled, phase III trials across multiple tumor types, an opportunity now exists to further establish the safety parameters of ramucirumab across a large patient population. MATERIALS AND METHODS: An individual patient meta-analysis across the six completed phase III trials was conducted and the relative risk (RR) and associated 95% confidence intervals (CIs) were derived using fixed-effects or mixed-effects models for all-grade and high-grade adverse events (AEs) possibly related to vascular endothelial growth factor pathway inhibition. The number needed to harm was also calculable due to the placebo-controlled nature of all six registration standard trials. RESULTS: A total of 4996 treated patients (N = 2748 in the ramucirumab arm and N = 2248 in the control, placebo arm) were included in this meta-analysis. Arterial thromboembolic events [ATE; all-grade, RR: 0.8, 95% CI 0.5-1.3; high-grade (grade ≥3), RR: 0.9, 95% CI 0.5-1.7], venous thromboembolic events (VTE; all-grade, RR: 0.7, 95% CI 0.5-1.1; high-grade, RR: 0.7, 95% CI 0.4-1.2), high-grade bleeding (RR: 1.1, 95% CI 0.8-1.5), and high-grade gastrointestinal (GI) bleeding (RR: 1.1, 95% CI 0.7-1.7) did not demonstrate a definite increased risk with ramucirumab. A higher percentage of hypertension, proteinuria, low-grade (grade 1-2) bleeding, GI perforation, infusion-related reaction, and wound-healing complications were observed in the ramucirumab arm compared with the control arm. CONCLUSIONS: Ramucirumab may be distinct among antiangiogenic agents in terms of ATE, VTE, high-grade bleeding, or high-grade GI bleeding by showing no clear evidence for an increased risk of these AEs in this meta-analysis of a large and diverse patient population. Ramucirumab is consistent with other angiogenic inhibitors in the risk of developing certain AEs. Clinical Trial Numbers: NCT00917384 (REGARD), NCT01170663 (RAINBOW), NCT01168973 (REVEL), NCT01183780 (RAISE), NCT01140347 (REACH), and NCT00703326 (ROSE).

The role of the prolactin/vasoinhibin axis in rheumatoid arthritis: an integrative overview.

Rheumatoid arthritis (RA) is a chronic, autoimmune, inflammatory disease destroying articular cartilage and bone. The female preponderance and the influence of reproductive states in RA have long linked this disease to sexually dimorphic, reproductive hormones such as prolactin (PRL). PRL has immune-enhancing properties and increases in the circulation of some patients with RA. However, PRL also suppresses the immune system, stimulates the formation and survival of joint tissues, acquires antiangiogenic properties upon its cleavage to vasoinhibins, and protects against joint destruction and inflammation in the adjuvant-induced model of RA. This review addresses risk factors for RA linked to PRL, the effects of PRL and vasoinhibins on joint tissues, blood vessels, and immune cells, and the clinical and experimental data associating PRL with RA. This information provides important insights into the pathophysiology of RA and highlights protective actions of the PRL/vasoinhibin axis that could lead to therapeutic benefits.

A Review of Anti-Angiogenic Targets for Monoclonal Antibody Cancer Therapy.

Tumor angiogenesis is a key event that governs tumor progression and metastasis. It is controlled by the complicated and coordinated actions of pro-angiogenic factors and their receptors that become upregulated during tumorigenesis. Over the past several decades, vascular endothelial growth factor (VEGF) signaling has been identified as a central axis in tumor angiogenesis. The remarkable advent of recombinant antibody technology has led to the development of bevacizumab, a humanized antibody that targets VEGF and is a leading clinical therapy to suppress tumor angiogenesis. However, despite the clinical efficacy of bevacizumab, its significant side effects and drug resistance have raised concerns necessitating the identification of novel drug targets and development of novel therapeutics to combat tumor angiogenesis. This review will highlight the role and relevance of VEGF and other potential therapeutic targets and their receptors in angiogenesis. Simultaneously, we will also cover the current status of monoclonal antibodies being developed to target these candidates for cancer therapy.

Vaccination approach to anti-angiogenic treatment of cancer.

Improvement of patient survival by anti-angiogenic therapy has proven limited. A vaccination approach inducing an immune response against the tumor vasculature combines the benefits of immunotherapy and anti-angiogenesis, and may overcome the limitations of current anti-angiogenic drugs. Strategies to use whole endothelial cell vaccines and DNA- or protein vaccines against key players in the VEGF signaling axis, as well as specific markers of tumor endothelial cells, have been tested in preclinical studies. Current clinical trials are now testing the promise of this specific anti-cancer vaccination approach. This review will highlight the state-of-the-art in this exciting field of cancer research.

Anti-bevacizumab idiotype antibody vaccination is effective in inducing vascular endothelial growth factor-binding response, impairing tumor outgrowth.

Tumors require blood supply and, to overcome this restriction, induce angiogenesis. Vascular endothelial growth factor (VEGF) plays an important role in this process, which explains the great number of antiangiogenic therapies targeting VEGF. The research and development of targeted therapy has led to the approval of bevacizumab, a humanized anti-VEGF monoclonal antibody (mAb), in clinical settings. However, side effects have been reported, usually as a consequence of bolus-dose administration of the antibody. This limitation could be circumvented through the use of anti-idiotype (Id) antibodies. In the present study, we evaluated the efficacy of an active VEGF-binding immune response generated by an anti-bevacizumab idiotype mAb, 10.D7. The 10.D7 anti-Id mAb vaccination led to detectable levels of VEGF-binding anti-anti-Id antibodies. In order to examine whether this humoral immune response could have implications for tumor development, 10.D7-immunized mice were challenged with B16-F10 tumor cells. Mice immunized with 10.D7 anti-Id mAb revealed reduced tumor growth when compared to control groups. Histological analyses of tumor sections from 10.D7-immunized mice showed increased necrotic areas, decreased CD31-positive vascular density and reduced CD68-positive cell infiltration. Our results encourage further therapeutic studies, particularly if one considers that the anti-Id therapeutic vaccination maintains stable levels of VEGF-binding antibodies, which might be useful in the control of tumor relapse.

Acute tumour response to a bispecific Ang-2-VEGF-A antibody: insights from multiparametric MRI and gene expression profiling.

BACKGROUND: To assess antivascular effects, and evaluate clinically translatable magnetic resonance imaging (MRI) biomarkers of tumour response in vivo, following treatment with vanucizumab, a bispecific human antibody against angiopoietin-2 (Ang-2) and vascular endothelial growth factor-A (VEGF-A). METHODS: Colo205 colon cancer xenografts were imaged before and 5 days after treatment with a single 10 mg kg(-1) dose of either vanucizumab, bevacizumab (anti-human VEGF-A), LC06 (anti-murine/human Ang-2) or omalizumab (anti-human IgE control). Volumetric response was assessed using T2-weighted MRI, and diffusion-weighted, dynamic contrast-enhanced (DCE) and susceptibility contrast MRI used to quantify tumour water diffusivity (apparent diffusion coefficient (ADC), × 10(6) mm(2) s(-1)), vascular perfusion/permeability (K(trans), min(-1)) and fractional blood volume (fBV, %) respectively. Pathological correlates were sought, and preliminary gene expression profiling performed. RESULTS: Treatment with vanucizumab, bevacizumab or LC06 induced a significant (P<0.01) cytolentic response compared with control. There was no significant change in tumour ADC in any treatment group. Uptake of Gd-DTPA was restricted to the tumour periphery in all post-treatment groups. A significant reduction in tumour K(trans) (P<0.05) and fBV (P<0.01) was determined 5 days after treatment with vanucizumab only. This was associated with a significant (P<0.05) reduction in Hoechst 33342 uptake compared with control. Gene expression profiling identified 20 human genes exclusively regulated by vanucizumab, 6 of which are known to be involved in vasculogenesis and angiogenesis. CONCLUSIONS: Vanucizumab is a promising antitumour and antiangiogenic treatment, whose antivascular activity can be monitored using DCE and susceptibility contrast MRI. Differential gene expression in vanucizumab-treated tumours is regulated by the combined effect of Ang-2 and VEGF-A inhibition.

Therapeutic antibody targeting of individual Notch receptors.

The four receptors of the Notch family are widely expressed transmembrane proteins that function as key conduits through which mammalian cells communicate to regulate cell fate and growth. Ligand binding triggers a conformational change in the receptor negative regulatory region (NRR) that enables ADAM protease cleavage at a juxtamembrane site that otherwise lies buried within the quiescent NRR. Subsequent intramembrane proteolysis catalysed by the gamma-secretase complex liberates the intracellular domain (ICD) to initiate the downstream Notch transcriptional program. Aberrant signalling through each receptor has been linked to numerous diseases, particularly cancer, making the Notch pathway a compelling target for new drugs. Although gamma-secretase inhibitors (GSIs) have progressed into the clinic, GSIs fail to distinguish individual Notch receptors, inhibit other signalling pathways and cause intestinal toxicity, attributed to dual inhibition of Notch1 and 2 (ref. 11). To elucidate the discrete functions of Notch1 and Notch2 and develop clinically relevant inhibitors that reduce intestinal toxicity, we used phage display technology to generate highly specialized antibodies that specifically antagonize each receptor paralogue and yet cross-react with the human and mouse sequences, enabling the discrimination of Notch1 versus Notch2 function in human patients and rodent models. Our co-crystal structure shows that the inhibitory mechanism relies on stabilizing NRR quiescence. Selective blocking of Notch1 inhibits tumour growth in pre-clinical models through two mechanisms: inhibition of cancer cell growth and deregulation of angiogenesis. Whereas inhibition of Notch1 plus Notch2 causes severe intestinal toxicity, inhibition of either receptor alone reduces or avoids this effect, demonstrating a clear advantage over pan-Notch inhibitors. Our studies emphasize the value of paralogue-specific antagonists in dissecting the contributions of distinct Notch receptors to differentiation and disease and reveal the therapeutic promise in targeting Notch1 and Notch2 independently.

Generation and characterization of a human nanobody against VEGFR-2.

AIM: Nanobody is an antibody fragment consisting of a single monomeric variable antibody domain, which can be used for a variety of biotechnological and therapeutic purposes. The aim of this work was to isolate and characterize a human signal domain antibody against VEGFR-2 domain3 (VEGFR D3) from a phage display library. METHODS: To produce antigen-specific recombinant nanobodies with high affinity to VEGFR2 D3, a liquid phase panning strategy was used for all rounds of panning. For nanobody expression and purification, four VEGFR2 D3-blocking clones were subcloned into a pETduet-biotin-MBP expression vector. The recombinant proteins carried an MBP tag to facilitate purification by affinity chromatography. Recombinant NTV(1-4) was obtained after an additional gel filtration chromatography step. The interactions between VEGFR2 D3 and NTV(1-4) were assessed with luminescence-based AlphaScreen assay and SPR assay. Anti-angiogenesis effects were examined in human umbilical vein endothelial cells (HUVECs). RESULTS: In the AlphaScreen assay, NTV1 (100 and 200 nmol/L) elicited the highest binding signal with VEGFR2 D3; NTV2 showed moderate interactions with VEGFR2 D3; NTV3 and NTV4 exhibited little or no interaction with VEGFR2 D3. In the SPR assay, NTV1 displayed a high affinity for VEGFR2 D3 with an equilibrium dissociation constant (KD) of 49±1.8 nmol/L. NTV1 (1-1000 nmol/L) dose-dependently inhibited the proliferation of HUVECs and the endothelial tube formation by the HUVECs. CONCLUSION: The nanobody NTV1 is a potential therapeutic candidate for blocking VEGFR2. This study provides a novel and promising strategy for development of VEGFR2-targeted nanobody-based cancer therapeutics.

Detection of antiranibizumab antibodies among patients with exudative age-related macular degeneration.

PURPOSE: The aim of this study was to detect immune responses induced by intravitreal injection (IVT) of ranibizumab in patients with exudative age-related macular degeneration (AMD) in real life conditions. METHODS: An ELISA protocol from blood samples, following 2 different steps, was used to detect antibodies directed against the variable regions of ranibizumab. RESULTS: Among 91 patients included, 46 received more than 10 IVTs, 36 had received 10 IVTs or fewer, and 9 were treatment naïve. Specific antiranibizumab immunoglobulins G were detected in 14/82 treated patients (17.1%). No immunization was detected among naïve patients. For patients with 10 or fewer previous IVTs, immunization against ranibizumab was detected in 4/36 patients (11.1%) whereas immunization was observed in 10/46 patients (21.7%) with more IVTs (p = 0.20). CONCLUSIONS: Immunization against ranibizumab can be detected in 17% of treated patients. Further clinical studies are needed to investigate the relationship between specific immunization to anti-vascular endothelial growth factor antibodies and response or resistance to ranibizumab treatments.

Pharmacokinetics of a long-lasting anti-VEGF fusion protein in rabbit.

Conbercept(KH902), a recombinant fusion protein in clinical trial II/III, shows good potential to treat the neovascular age-related macular degeneration (AMD). This investigation evaluated its ocular pharmacokinetics and pharmacodynamic profile in rabbits following intravitreal administration (IVT). Rabbits (n = 120) received single bilateral conbercept IVT administration or single IV administration. Conbercept concentrations in ocular tissues and serum were measured after dosing. VEGF concentration was also measured simultaneously. The results showed that conbercept rapidly distributed from vitreous into targeted tissues and lasted over 81 days. Clearance in ocular tissues was parallel and exhibited a terminal half of 2.5-4.2 days. The drug exposure in the retina was 1/4 to 1/5 of that in vitreous. Serum conbercept concentrations after IVT dosing were low and bioavailability was approximately 44%. And single intravitreal injection induced that ocular VEGF concentration declined over 60 days and serum VEGF concentration decreased for a short time but rebounded to higher level than baseline later. All these indicated conbercept good pharmacokinetic profile in rabbits, with good ocular tropism and systemic tolerance. Combined with the efficacy data from our earlier in vitro and in vivo studies, it should have a promising clinical application for AMD treatment.

Anti-vascular endothelial growth factor antibody bevacizumab reduced the risk of anemia associated with chemotherapy-A meta-analysis.

INTRODUCTION: Vascular endothelial growth factor (VEGF) may play a role in erythropoiesis. We performed a meta-analysis of randomized controlled trials (RCT) to determine the effect of the anti-VEGF antibody bevacizumab on anemia in cancer patients treated with chemotherapy. METHODS: Databases from PUBMED, the Web of Science, Embase, the Cochrane Library, and abstracts presented at the American Society of Clinical Oncology (ASCO) conferences until May 2010 were searched to identify relevant studies. Eligible studies included prospective RCTs in which the combination of bevacizumab and chemotherapy was compared with chemotherapy alone. Summary incidence rate, relative risk (RR), and 95% confidence interval (CI) were calculated. RESULTS: A total of 6439 patients with a variety of solid tumors were included for analysis from 11 RCTs. Among those patients receiving bevacizumab and chemotherapy, the incidences of all-grade and high-grade (grade 3 and above) anemia were 17.8% (95% CI: 11.1-27.1%) and 2.8% (95% CI: 1.6-5.0%) respectively. In comparison with chemotherapy alone, bevacizumab significantly reduced all-grade (RR, 0.79; 95% CI: 0.66-1.0, p = 0.007) and high-grade anemia (RR, 0.72; 95% CI: 0.57-0.90, p = 0.005). The effect did not vary significantly among bevacizumab doses (p = 0.88), tumor types (p = 0.75) or chemotherapy regimens (p = 0.98). DISCUSSION: Bevacizumab may significantly reduce the risk of anemia with chemotherapy in cancer patients.

An anti-vascular endothelial growth factor receptor 2/fetal liver kinase-1 Listeria monocytogenes anti-angiogenesis cancer vaccine for the treatment of primary and metastatic Her-2/neu+ breast tumors in a mouse model.

Thirty years after angiogenesis was shown to play an enabling role in cancer, modern medicine is still trying to develop novel compounds and therapeutics to target the tumor vasculature. However, most therapeutics require multiple rounds of administration and can have toxic side effects. In this study, we use anti-angiogenesis immunotherapy to target cells actively involved in forming new blood vessels that support the growth and spread of breast cancer. Targeting a central cell type involved in angiogenesis, endothelial cells, we immunized against host vascular endothelial growth factor receptor 2 to fight the growth of Her-2/neu(+) breast tumors. Using the bacterial vector, Listeria monocytogenes (Lm), we fused polypeptides from the mouse vascular endothelial growth factor receptor 2 molecule (fetal liver kinase-1) to the microbial adjuvant, listeriolysin-O, and used Lm to deliver the Ags and elicit potent antitumor CTL responses. Lm-listeriolysin-O-fetal liver kinase-1 was able to eradicate some established breast tumors, reduce microvascular density in the remaining tumors, protect against tumor rechallenge and experimental metastases, and induce epitope spreading to various regions of the tumor-associated Ag Her-2/neu. Tumor eradication was found to be dependent on epitope spreading to HER-2/neu and was not solely due to the reduction of tumor vasculature. However, vaccine efficacy did not affect normal wound healing nor have toxic side effects on pregnancy. We show that an anti-angiogenesis vaccine can overcome tolerance to the host vasculature driving epitope spreading to an endogenous tumor protein and drive active tumor regression.

Identification of novel neutralizing single-chain antibodies against vascular endothelial growth factor receptor 2.

Human vascular endothelial growth factor (VEGF) and its receptor (VEGFR-2/kinase domain receptor [KDR]) play a crucial role in angiogenesis, which makes the VEGFR-2 signaling pathway a major target for therapeutic applications. In this study, a single-chain antibody phage display library was constructed from spleen cells of mice immunized with recombinant human soluble extracellular VEGFR-2/KDR consisting of all seven extracellular domains (sKDR D1-7) to obtain antibodies that block VEGF binding to VEGFR-2. Two specific single-chain antibodies (KDR1.3 and KDR2.6) that recognized human VEGFR-2 were selected; diversity analysis of the clones was performed by BstNI fingerprinting and nucleotide sequencing. The single-chain variable fragments (scFvs) were expressed in soluble form and specificity of interactions between affinity purified scFvs and VEGFR-2 was confirmed by ELISA. Binding of the recombinant antibodies for VEGFR-2 receptors was investigated by surface plasmon resonance spectroscopy. In vitro cell culture assays showed that KDR1.3 and KDR2.6 scFvs significantly suppressed the mitogenic response of human umbilical vein endothelial cells to recombinant human VEGF(165) in a dose-dependent manner, and reduced VEGF-dependent cell proliferation by 60% and 40%, respectively. In vivo analysis of these recombinant antibodies in a rat cornea angiogenesis model revealed that both antibodies suppressed the development of new corneal vessels (p < 0.05). Overall, in vitro and in vivo results disclose strong interactions of KDR1.3 and KDR2.6 scFvs with VEGFR-2. These findings indicate that KDR1.3 and KDR2.6 scFvs are promising antiangiogenic therapeutic agents.