Anti-VEGF agents confer survival advantages to tumor-bearing mice by improving cancer-associated systemic syndrome

The underlying mechanism by which anti-VEGF agents prolong cancer patient survival is poorly understood. We show that in a mouse tumor model, VEGF systemically impairs functions of multiple organs including those in the hematopoietic and endocrine systems, leading to early death. Anti-VEGF antibody, bevacizumab, and anti-VEGF receptor 2 (VEGFR-2), but not anti-VEGFR-1, reversed VEGF-induced cancer-associated systemic syndrome (CASS) and prevented death in tumor-bearing mice. Surprisingly, VEGFR2 blockage improved survival by rescuing mice from CASS without significantly compromising tumor growth, suggesting that “off-tumor” VEGF targets are more sensitive than the tumor vasculature to anti-VEGF drugs. Similarly, VEGF-induced CASS occurred in a spontaneous breast cancer mouse model overexpressing neu. Clinically, VEGF expression and CASS severity positively correlated in various human cancers. These findings define novel therapeutic targets of anti-VEGF agents and provide mechanistic insights into the action of this new class of clinically available anti-VEGF cancer drugs.     

Anti-VEGF– and anti-VEGF receptor–induced vascular alteration in mouse healthy tissues

Systemic therapy with anti-VEGF drugs such as bevacizumab is widely used for treatment of human patients with various solid tumors. However, systemic impacts of such drugs in host healthy vasculatures remain poorly understood. Here, we show that, in mice, systemic delivery of an anti-VEGF or an anti–VEGF receptor (VEGFR)-2 neutralizing antibody caused global vascular regression. Among all examined tissues, vasculatures in endocrine glands, intestinal villi, and uterus are the most affected in response to VEGF or VEGFR-2 blockades. Thyroid vascular fenestrations were virtually completely blocked by VEGF blockade, leading to marked accumulation of intraendothelial caveolae vesicles. VEGF blockade markedly increased thyroid endothelial cell apoptosis, and withdrawal of anti-VEGF resulted in full recovery of vascular density and architecture after 14 d. Prolonged anti-VEGF treatment resulted in a significant decrease of the circulating level of the predominant thyroid hormone free thyroxine, but not the minimal isoform of triiodothyronine, suggesting that chronic anti-VEGF treatment impairs thyroid functions. Conversely, VEGFR-1–specific blockade produced virtually no obvious phenotypes. These findings provide structural and functional bases of anti-VEGF–specific drug-induced side effects in relation to vascular changes in healthy tissues. Understanding anti-VEGF drug-induced vascular alterations in healthy tissues is crucial to minimize and even to avoid adverse effects produced by currently used anti-VEGF–specific drugs.     

Intravitreal anti-VEGF drugs as adjuvant therapy in diabetic retinopathy surgery

The use of intravitreal anti vascular endothelial growth factor (anti-VEGF) drugs such as pegaptanib, ranibizumab and bevacizumab has been widely reported to treat complications such as macular edema and rubeosis. During the past few years they have also been used as an adjuvant therapy to reduce intraocular bleeding during vitrectomy in eyes with proliferative diabetic retinopathy as well as to reduce the occurrence of vitreous haemorrhages in vitrectomized eyes and facilitate glaucoma surgery. In this paper we review the use of anti VEGF drugs in the surgical management of diabetic retinopathy related complications.     

Netzhautkomplikationen bei Diabetes

In September 2015 a revised version of the guideline “Prevention and therapy of diabetes-induced retinal complications” was released in Germany. It summarizes current recommendations for diagnosis and therapy of diabetic retinopathy with a special focus on practicability in daily routine. Newly included were the use of optical coherence tomography (OCT) for the diagnosis of diabetic macular edema and the evaluation of the therapeutic effect of anti-VEGF drugs (VEGF: vascular endothelial growth factor) or corticosteroids after intravitreal drug delivery. Screening intervals for diabetic retinopathy were extended to every 2 years for all diabetic patients with no retinopathy and limited systemic risk factors. Patients with known risk factors should be screened annually or in case of retinopathy, followed-up as recommended by the ophthalmologist.     

Decreased angiogenin concentration in vitreous and serum in proliferative diabetic retinopathy

Diabetic retinopathy is the most common cause of vision loss in young adults in developed countries. The disease therapy with anti-vascular endothelial growth factor (VEGF) agents gives some positive results, but is associated with retinal ischemia and vasoconstriction. Therefore, determination of factors involved in the physiological and pathological angiogenesis in the diabetic eye is of great importance for understanding of the pathogenesis of diabetic retinopathy and its effective treatment.Previously, we found that diabetic patients were characterized by increased serum concentration of VEGF, but decreased levels of other proangiogenic factor—angiogenin. The involvement of VEGF in pathogenesis of diabetic retinopathy is well established, but there is lack of data regarding angiogenin in retinopathy. Therefore, in the present study we measured angiogenin concentration in vitreous and serum samples of the patients with type 1 diabetes to determine its role in diabetic retinopathy. In addition, in each time, we compared the level of angiogenin with level of VEGF as a known factor involved in the pathogenesis of the disease.Angiogenin was found to be significantly more abundant in serum than in vitreous in both diabetic groups. In addition, patients with retinopathy had twofold lower vitreous angiogenin levels than diabetic individuals without complications. On the contrary, vitreous concentration of VEGF was dramatically increased only in participants with retinopathy. Patients without diabetic complications had significantly lower VEGF levels in vitreous than in serum and were characterized by high local and systemic concentration of angiogenin.These data suggest a local imbalance between two proangiogenic factors—VEGF and angiogenin in retinopathy. Low vitreous concentration of angiogenin in diabetic patients suggests that this factor is not responsible for pathological neovascularization in diabetic eye. Further studies will elucidate if angiogenin can be used to improve the insufficient angiogenesis in diabetes and prevent retinal ischemia after retinopathy treatment with anti-VEGF agents.     

Cystic Fibrosis Related Diabetes

Vascular endothelial growth factor (VEGF) plays a pivotal role in the development of diabetic macular edema (DME), the leading cause of vision loss among working-aged individuals. A decade of clinical trials demonstrated that drugs that bind soluble VEGF restore the integrity of the blood-retinal barrier, resolve macular edema, and improve vision in most patients with DME. Four drugs (pegaptanib, ranibizumab, bevacizumab, and aflibercept) effectively treat DME when administered by intravitreal injections. Only ranibizumab has received U.S. Food and Drug Administration (FDA) approval for DME, but bevacizumab is commonly used off-label, and an FDA application for aflibercept is pending. Effective treatment requires repeated injections, although recent data suggest that the treatment burden diminishes after 1 year. Intravitreal therapy is generally safe, although the incidence of systemic thromboembolic events varies among trials.     

Aflibercept exhibits VEGF binding stoichiometry distinct from bevacizumab and does not support formation of immune-like complexes

Anti-vascular endothelial growth factor (VEGF) therapies have improved clinical outcomes for patients with cancers and retinal vascular diseases. Three anti-VEGF agents, pegaptanib, ranibizumab, and aflibercept, are approved for ophthalmic indications, while bevacizumab is approved to treat colorectal, lung, and renal cancers, but is also used off-label to treat ocular vascular diseases. The efficacy of bevacizumab relative to ranibizumab in treating neovascular age-related macular degeneration has been assessed in several trials. However, questions persist regarding its safety, as bevacizumab can form large complexes with dimeric VEGF₁₆₅, resulting in multimerization of the Fc domain and platelet activation. Here, we compare binding stoichiometry, Fcγ receptor affinity, platelet activation, and binding to epithelial and endothelial cells in vitro for bevacizumab and aflibercept, in the absence or presence of VEGF. In contrast to bevacizumab, aflibercept forms a homogenous 1:1 complex with each VEGF dimer. Unlike multimeric bevacizumab:VEGF complexes, the monomeric aflibercept:VEGF complex does not exhibit increased affinity for low-affinity Fcγ receptors, does not activate platelets, nor does it bind to the surface of epithelial or endothelial cells to a greater degree than unbound aflibercept or control Fc. The latter finding reflects the fact that aflibercept binds VEGF in a unique manner, distinct from antibodies not only blocking the amino acids necessary for VEGFR1/R2 binding but also occluding the heparin-binding site on VEGF₁₆₅.     

Lipoprotein-associated phospholipase A2 (Lp-PLA2) as a therapeutic target to prevent retinal vasopermeability during diabetes

Lipoprotein-associated phospholipase A₂ (Lp-PLA₂) hydrolyses oxidized low-density lipoproteins into proinflammatory products, which can have detrimental effects on vascular function. As a specific inhibitor of Lp-PLA₂, darapladib has been shown to be protective against atherogenesis and vascular leakage in diabetic and hypercholesterolemic animal models. This study has investigated whether Lp-PLA₂ and its major enzymatic product, lysophosphatidylcholine (LPC), are involved in blood–retinal barrier (BRB) damage during diabetic retinopathy. We assessed BRB protection in diabetic rats through use of species-specific analogs of darapladib. Systemic Lp-PLA₂ inhibition using SB-435495 at 10 mg/kg (i.p.) effectively suppressed BRB breakdown in streptozotocin-diabetic Brown Norway rats. This inhibitory effect was comparable to intravitreal VEGF neutralization, and the protection against BRB dysfunction was additive when both targets were inhibited simultaneously. Mechanistic studies in primary brain and retinal microvascular endothelial cells, as well as occluded rat pial microvessels, showed that luminal but not abluminal LPC potently induced permeability, and that this required signaling by the VEGF receptor 2 (VEGFR2). Taken together, this study demonstrates that Lp-PLA₂ inhibition can effectively prevent diabetes-mediated BRB dysfunction and that LPC impacts on the retinal vascular endothelium to induce vasopermeability via VEGFR2. Thus, Lp-PLA₂ may be a useful therapeutic target for patients with diabetic macular edema (DME), perhaps in combination with currently administered anti-VEGF agents.Breakdown of the blood–retinal barrier (BRB) is a significant pathophysiological event during diabetic retinopathy (DR). Leakage of plasma proteins and lipids into the neuropile form exudates, whereas excessive vasopermeability can lead to diabetic macular edema (DME), which is a major cause of vision loss (1). Both DME in patients and BRB dysfunction in animal models have been linked to inflammatory processes, endothelial dysfunction, and compromise of normal neuroglial–vascular interactions (2). A number of therapeutic options are available to treat DME. Laser photocoagulation is effective in slowing the progression of DME; however, it carries risk of serious adverse side effects including visual field defects, retinal scarring, and foveal burns. Intravitreal injection of antiinflammatory corticosteroids has proven value, but this intervention also carries significant side effects including cataract formation and elevated intraocular pressure. More recently, neutralization of vascular endothelial growth factor (VEGF) in the retina has become a mainstream treatment of DME (3) although this is not effective for all DME patients (4). Moreover, repeated intravitreal injections can result in some patients becoming refractory to VEGF blockade (5), which suggests that other mechanisms also drive vascular permeability during DME. Concerns have also been raised that targeting the VEGF pathway could inadvertently compromise retinal neuroglial and microvascular survival (6) although, thus far, no evidence of these outcomes have been observed in clinical trials of anti-VEGF for DME. Nevertheless there is a need for additional therapeutic approaches to treat DME, either alone or adjunctively with currently available therapeutics.An alternative permeability-inducing agent in diabetic retina could be lysophosphatidylcholine (LPC), which is elevated in plasma of diabetic patients (7) and has demonstrated permeability-enhancing activity in cultured nonneural endothelial cells (ECs) (8). The principal enzyme responsible for the production of LPC is a calcium-independent phospholipase A₂ called lipoprotein-associated phospholipase A₂ (Lp-PLA₂) (also known as platelet-activating factor acetylhydrolase or type VIIA PLA₂). Lp-PLA₂ can elicit a broad range of proinflammatory and proapoptotic effects in blood vessels related to its hydrolysis of modified polyunsaturated fatty acids within oxidized low-density lipoprotein (oxLDL) into LPC and oxidized nonesterified fatty acids (OxNEFA) (9). Elevated Lp-PLA₂ has been proposed as a predictive biomarker in stroke (10), atherosclerosis (11), and coronary heart disease (12). Macrophages and other proinflammatory cells are a primary source of Lp-PLA₂ in the systemic circulation (13), although endothelial cells also express this enzyme (14).Darapladib, a specific inhibitor of Lp-PLA₂, has been shown to reduce atherosclerosis in both diabetic/hypercholesterolemic pigs (15) and ApoE-deficient mice (16). In diabetic/hypercholesterolemic pigs, darapladib protected against blood–brain barrier (BBB) dysfunction and vascular permeability (17). Darapladib has been studied in nearly 16,000 patients with coronary heart disease, and approximately one-third of this study population had diabetes mellitus (18). In a further study, a 3-month daily treatment with 160 mg of darapladib orally showed reduction of DME and an improvement in visual acuity in patients (19). These observations suggest that Lp-PLA₂ and its mechanism of action warrant further investigation in the context of DR. In this study, we have studied the inhibition of Lp-PLA₂ in diabetic rats with regards to BRB function and assessed the effect of LPC on neural microvascular endothelial cells. We show inhibition of Lp-PLA₂ was effective in reducing diabetes-induced retinal vasopermeability and that the effect of this inhibition was additive with VEGF neutralization, suggesting this enzyme could be an efficacious therapeutic target for DME, either alone or in combination with anti-VEGF therapeutics.     

VEGF Trap complex formation measures production rates of VEGF, providing a biomarker for predicting efficacious angiogenic blockade

VEGF is the best characterized mediator of tumor angiogenesis. Anti-VEGF agents have recently demonstrated impressive efficacy in human cancer trials, but the optimal dosing of such agents must still be determined empirically, because biomarkers to guide dosing have yet to be established. The widely accepted (but unverified) assumption that VEGF production is quite low in normal adults led to the notion that increased systemic VEGF levels might quantitatively reflect tumor mass and angiogenic activity. We describe an approach to determine host and tumor production of VEGF, using a high-affinity and long-lived VEGF antagonist now in clinical trials, the VEGF Trap. Unlike antibody complexes that are usually rapidly cleared, the VEGF Trap forms inert complexes with tissue- and tumor-derived VEGF that remain stably in the systemic circulation, where they are readily assayable, providing unprecedented capability to accurately measure VEGF production. We report that VEGF production is surprisingly high in non-tumor-bearing rodents and humans, challenging the notion that systemic VEGF levels can serve as a sensitive surrogate for tumor load; tumor VEGF contribution becomes significant only with very large tumor loads. These findings have the important corollary that anti-VEGF therapies must be sufficiently dosed to avoid diversion by host-derived VEGF. We further show that our assay can indicate when VEGF is optimally blocked; such biomarkers to guide dosing do not exist for other anti-VEGF agents. Based on this assay, VEGF Trap doses currently being assessed in clinical trials are in the efficacious range.     

Failure of treatment with anti-VEGF monoclonal antibody for long-standing POEMS syndrome

We present the case of a 71-year-old woman with polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes (POEMS) syndrome. Overproduction of vascular endothelial growth factor (VEGF), secreted by plasmacytoma, is considered responsible for the characteristic symptoms, and therefore anti-VEGF monoclonal antibody (bevacizumab) could be a therapeutic option. The patient was treated with bevacizumab 7 years after onset. Despite a dramatic decrease in serum VEGF levels, there was no clinical improvement, possibly because aberrant angiogenesis had already developed systemically. We suggest that careful consideration should be taken for indication of bevacizumab therapy, and this agent may be used in selected patients with a short duration POEMS syndrome.     

Ramucirumab for gastric cancer

In recent years, various molecular target agents have been investigated for gastric cancer. VEGF is one of the most potent angiogenic factors and is a signaling molecule secreted by many solid tumors. High VEGF expression is one of the characteristic features of gastric carcinomas, thus targeting VEGF is considered a promising strategy for gastric cancer. Ramucirumab, an anti-VEGF receptor antibody, has proven to be effective for previously treated advanced gastric cancer. Details of ramucirumab, including two pivotal Phase III studies, will be discussed in this review. Ramucirumab, with or without chemotherapy, improved survival in gastric cancer after previous systemic chemotherapy, thus becoming the standard of care for this patient population. Optimal timing of ramucirumab use and adequate biomarkers for patient selection as well as mechanism of resistance should be explored in future research.     

Renal Effects of Anti-angiogenesis Therapy: Update for the Internist

Angiogenesis has become an innovative target in cancer therapy. Agents that inhibit vascular endothelial growth factor (VEGF), one of the most potent promoters of angiogenesis, and its receptor have significant implications for clinical practice. Bevacizumab, sorafenib, sunitinib and other anti-VEGF drugs are frequently complicated by mild proteinuria and hypertension. Other unique renal effects, such as high-grade proteinuria and acute kidney injury, have been described. The most common histopathologic kidney lesion is thrombotic microangiopathy, with other glomerular lesions and interstitial nephritis occurring less frequently. The mechanism for anti-VEGF therapy-induced hypertension is not well understood; however, nitric oxide pathway inhibition, rarefaction, and oxidative stress may be important in its pathogenesis. Glomerular injury may develop from loss of VEGF effect on maintaining the filtration barrier. Adverse effects of anti-VEGF class of drugs are manageable but require close attention and follow-up. Understanding the fundamentals of anti-VEGF drugs' mechanism of action and their clinical implications is crucial when caring for patients receiving anti-VEGF therapy.     

New therapies for ovarian cancer: cytotoxics and molecularly targeted agents

Ovarian cancer, although a chemo-sensitive disease, is associated with high morbidity and mortality due to its often-late presentation. Platinums and taxanes have improved the prognosis over recent years but median overall survival is still unacceptably low (24-60 months). Apart from the manipulation of doses, schedules, mode of delivery, and combinations of existing drugs, new cytotoxics and molecularly targeted agents with different mechanisms of action must be evaluated in this patient population. This article will review the most recent clinical trials data pertaining to these new cytotoxic drugs including patupilone, telcyta, and trabectedin, as well as those of small molecules and inhibitors of the EGFR and VEGF receptor families. It will also discuss other potential signal transduction targets worthy of further evaluation in future trials.     

Effect of weight‐reducing agents on glycaemic parameters and progression to Type 2 diabetes: a review

Weight loss is associated with improvements in glycaemic control and cardiovascular disease risk factors. However, in the diabetic population, weight management is more challenging, in part because of the weight‐promoting effects of the majority of glucose‐lowering therapies. This review summarizes evidence from 23 placebo‐controlled randomized trials, of at least 1 year duration, on the effects of drugs promoting weight loss (orlistat, sibutramine and rimonabant) on glycaemic variables, diabetes incidence and diabetes control. Fifteen studies of non‐diabetic subjects were found, eight of which included a longer treatment period. Eight studies in diabetic patients were reviewed. In non‐diabetic subjects, weight loss agents led to a significant improvement in fasting glucose, fasting insulin and insulin resistance. In the diabetic population, glycated haemoglobin decreased by 0.28–1.1% with orlistat and 0.6% with sibutramine and rimonabant. Orlistat reduces progression to diabetes in patients with glucose intolerance treated for 4 years (risk reduction of 45%). In summary, despite leading to only modest weight loss after 12 months, agents promoting weight loss have beneficial effects on glycaemic parameters, glycaemic control and progression to diabetes. These additional benefits of weight loss agents need to be highlighted in order to increase their judicious use in clinical practice, although this may be limited by their well‐known adverse side effects. The longer‐term safety of these agents beyond a few years is yet to be established.     

Heparin-binding VEGFR1 variants as long-acting VEGF inhibitors for treatment of intraocular neovascular disorders

Neovascularization is a key feature of ischemic retinal diseases and the wet form of age-related macular degeneration (AMD), all leading causes of severe vision loss. Vascular endothelial growth factor (VEGF) inhibitors have transformed the treatment of these disorders. Millions of patients have been treated with these drugs worldwide. However, in real-life clinical settings, many patients do not experience the same degree of benefit observed in clinical trials, in part because they receive fewer anti-VEGF injections. Therefore, there is an urgent need to discover and identify novel long-acting VEGF inhibitors. We hypothesized that binding to heparan-sulfate proteoglycans (HSPG) in the vitreous, and possibly other ocular structures, may be a strategy to promote intraocular retention, ultimately leading to a reduced burden of intravitreal injections. We designed a series of VEGF receptor 1 variants and identified some with strong heparin-binding characteristics and ability to bind to vitreous matrix. Our data indicate that some of our variants have longer duration and greater efficacy in animal models of intraocular neovascularization than current standard of care. Our study represents a systematic attempt to exploit the functional diversity associated with heparin affinity of a VEGF receptor.

The development of a neovascular supply or angiogenesis serves crucial homeostatic roles, since the blood vessels carry nutrients to tissues and organs and remove catabolic products (1). However, uncontrolled growth of blood vessels can promote or facilitate numerous disease processes, including tumors and intraocular vascular disorders (1). Although numerous angiogenic factors were initially identified and characterized (2), work performed in many laboratories has established vascular endothelial growth factor (VEGF) as a key regulator of normal and pathological angiogenesis as well as vascular permeability (3–5). Alternative exon splicing results in the generation of multiple isoforms that differ in their affinity for heparin, including VEGF₁₂₁, VEGF₁₆₅, and VEGF₁₈₉. VEGF₁₂₁ lacks significant heparin binding. While VEGF₁₆₅ has a single, exon-7-encoded, heparin-binding domain, VEGF₁₈₉ has two heparin-binding domains encoded respectively by exon-6 and exon-7 (6, 7). Much experimental evidence documents the key role of the heparin-binding VEGF isoforms in the establishment of biochemical gradients required for angiogenesis (8–10). VEGF is a member of a gene family that also includes PlGF, VEGF-B, VEGF-C, and VEGF-D. Three related receptor tyrosine kinases (RTKs) have been reported to bind VEGF ligands: VEGFR1 (11, 12), VEGFR2 (13), and VEGFR3 (14). VEGF binds both VEGFR1 and VEGFR2, while PlGF and VEGF-B interact selectively with VEGFR1. VEGFR3 and its two ligands, VEGF-C and VEGF-D, are primarily implicated in lymphangiogenesis (15, 16). Each member of this RTK class has seven immunoglobulin (Ig)-like domains in the extracellular portion (17). There is agreement that VEGFR2 is the main signaling receptor for VEGF (16), although VEGFR1 binds VEGF with substantially higher affinity than VEGFR2 (17).VEGF inhibitors have become a standard of therapy in multiple tumors and have transformed the treatment of intraocular neovascular disorders such as the neovascular form of age-related macular degeneration (AMD), proliferative diabetic retinopathy, and retinal vein occlusion, which are leading causes of severe vision loss and legal blindness (3, 5, 18). Currently, three anti-VEGF drugs are widely used in the United States for ophthalmological indications: bevacizumab, ranibizumab, and aflibercept (3). Bevacizumab is a full-length IgG antibody targeting VEGF (19). Even though bevacizumab was not developed for ophthalmological indications, it is widely used off-label due to its low cost. Ranibizumab is an affinity-matured anti-VEGF Fab (20). Aflibercept is an IgG-Fc fusion protein (21), with elements from VEGFR1 and VEGFR2, that binds VEGF, PIGF, and VEGF-B (22). Importantly, after a 5-y treatment with ranibizumab or bevacizumab, about half of neovascular AMD patients had good vision, i.e., visual acuity 20/40 or better, an outcome that would not have been possible before anti-VEGF agents were available (23). However, in real-life clinical settings, many patients receive fewer anti-VEGF injections than in clinical trials, and it has been hypothesized that this correlates with less satisfactory visual outcomes (24). Therefore, there is a need to develop agents with longer duration after intraocular injection, thus reducing the frequency of injections, and a number of approaches to this end have been attempted (25, 26). Aflibercept (Eylea) was approved based on clinical trials showing that every 8-wk administration of the dose of 2 mg could match the efficacy of monthly ranibizumab (0.5 mg). However, despite the prediction that a switch to aflibercept would reduce the number of intravitreal injections, recent studies suggest that it is not the case (27). Another approach aiming to reduce frequency of injections is the use of high doses of brolucizumab, a recently Food and Drug Administration (FDA)-approved high-affinity single-chain variable fragments (scFV) targeting VEGF (28). However, an increased incidence of retinal vasculitis and intraocular inflammation that adversely affected vision has been observed in a subset of patients treated with brolucizumab (29). A refillable slow-release device system implanted in the vitreous that enables continuous delivery of ranibizumab has been developed (30). The phase II data reported are promising, but this approach requires surgery and complications such as bleeding have been noted (30). Therefore, there is still an unmet medical need for intravitreal anti-VEGF agents with improved half-life.In 1996, in the course of structure–function studies aiming to identify VEGF-binding elements in VEGFR1, we found that deletion of Ig-like domain (D) 2, but not of other Ds, abolished VEGF or PIGF binding (31). Replacing D2 of VEGFR3 with D2 of VEGFR1 conferred on VEGFR3 the ligand specificity of VEGFR1 and abolished the ability of the mutant VEGFR3 to interact with VEGF-C (31). Subsequent studies documented the interaction between D2 and VEGF (or PlGF) by X-ray crystallography (32–34). However, D3 was important for optimal VEGF binding (31, 32). These initial studies led to the design of a construct comprising the first three lg-like Ds of VEGFR1, fused to an Fc-lgG (Flt-1-3-IgG) (31). Flt-1-3-lgG showed a potent ability to neutralize VEGF in vitro and in several in vivo models of physiological and pathological angiogenesis (35–40). However, the half-life of this molecule following systemic administration was relatively short due to the presence of clusters of basic residues in D3, which resulted in binding to heparan-sulfate proteoglycans (HSPG) and sequestration in the extracellular matrix of various tissues.In 2002, Holash et al. (22) described an IgG fusion construct comprising VEGFR1 D2 and VEGFR2 D3, which has much lower heparin affinity than VEGFR1 D3. This molecule, known today as aflibercept, ziv-aflibercept, or Eylea, was reported to have a significantly longer systemic half-life than Flt(1-3-lgG) (22). These pharmacokinetic characteristics, combined with high binding affinity for VEGF and the ability to bind PlGF and VEGF-B, led to the prediction that aflibercept would be a more effective antitumor agent than other VEGF inhibitors (22, 41). However, aflibercept has gained FDA approval only for second line treatment of colorectal cancer, while bevacizumab and the anti-VEGFR2 antibody ramucirumab received several FDA approvals in multiple cancer types (3, 5), suggesting that the above-mentioned characteristics did not provide a therapeutic advantage. Clearly, aflibercept has had its major clinical impact as an intravitreal treatment for ocular vascular disorders.We hypothesized that heparin-binding mediated by D3 (or other Ig-like domain) of VEGFR1 (12), while a disadvantage for systemic administration, might confer significant advantages for intravitreal and potentially other local administration.     

Does immune checkpoint inhibitor exhibit limited efficacy against non-viral hepatocellular carcinoma?: A review of clinical trials

Immunotherapy with immune checkpoint inhibitors has been shown to be beneficial for cancers originating from various organs. In May 2020, combination therapy with anti-programmed death-ligand 1 antibody atezolizumab and anti-vascular endothelial growth factor (VEGF) antibody bevacizumab was approved as a novel first-line systemic therapy for hepatocellular carcinoma (HCC). The number of patients with HCC not caused by hepatitis virus infection (non-viral HCC), including non-alcoholic steatohepatitis (NASH)-related HCC, has been increasing in recent years. Recently, Pfister and colleagues reported that immune checkpoint inhibitors may exhibit limited efficacy against NASH-related HCC, based on basic research and clinical data. This review will discuss the mechanism of impaired tumor immune surveillance in NASH and analyze the results of previously published clinical trials of immune checkpoint inhibitors to investigate whether patients with non-viral HCC are less likely to benefit from immunotherapy with immune checkpoint inhibitors. Furthermore, we also discuss the possibility of enhancing the therapeutic effect of immune checkpoint inhibitors for NASH-related HCC by combining anti-VEGF agents.     

TLR9 agonist acts by different mechanisms synergizing with bevacizumab in sensitive and cetuximab-resistant colon cancer xenografts

Synthetic agonists of Toll-like receptor 9 (TLR9), a class of agents that induce specific immune response, exhibit antitumor activity and are currently being investigated in cancer patients. Intriguingly, their mechanisms of action on tumor growth and angiogenesis are still incompletely understood. We recently discovered that a synthetic agonist of TLR9, immune modulatory oligonucleotide (IMO), acts by impairing epidermal growth factor receptor (EGFR) signaling and potently synergizes with anti-EGFR antibody cetuximab in GEO human colon cancer xenografts, whereas it is ineffective in VEGF-overexpressing cetuximab-resistant GEO cetuximab-resistant (GEO-CR) tumors. VEGF is activated by EGFR, and its overexpression causes resistance to EGFR inhibitors. Therefore, we used IMO and the anti-VEGF antibody bevacizumab as tools to study IMO's role on EGFR and angiogenesis and to explore its therapeutic potential in GEO, LS174T, and GEO-CR cancer xenografts. We found that IMO enhances the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of cetuximab, that bevacizumab has no ADCC, and IMO is unable to enhance it. Nevertheless, the IMO-plus-bevacizumab combination synergistically inhibits the growth of GEO and LS174T as well as of GEO-CR tumors, preceded by inhibition of signaling protein expression, microvessel formation, and human, but not murine, VEGF secretion. Moreover, IMO inhibited the growth, adhesion, migration, and capillary formation of VEGF-stimulated endothelial cells. The antitumor activity was irrespective of the TLR9 expression on tumor cells. These studies demonstrate that synthetic agonists of TLR9 interfere with growth and angiogenesis also by EGFR- and ADCC-independent mechanisms affecting endothelial cell functions and provide a strong rationale to combine IMO with bevacizumab and EGFR inhibitory drugs in colon cancer patients.     

Anti-vascular endothelial growth factor drug treatment of diabetic macular edema: the evolution continues

Diabetic mellitus is the leading cause of blindness in working aged patients in developing nations. Due to the buildup of abnormal metabolites from several overactive biochemical pathways, chronic hyperglycemia causes oxidative stress in the retina which upregulates vascular endothelial growth factor (VEGF). Together with other growth factors and metabolites, VEGF causes endothelial cell proliferation, vasodilation, recruitment of inflammatory cells, and increased vascular permeability, leading to breakdown of the blood-retinal barrier. This allows trans-cellular exudation into the interstitial space resulting in diabetic macular edema (DME). For over 3 decades the standard treatment for DME has been laser photocoagulation. Though laser reduces the incidence of vision loss by 50%, few eyes with diffuse edema experience improved vision. This has led physicians to use the VEGF-binding drugs pegaptanib, ranibizumab, and aflibercept, each of which has been approved for the treatment of exudative macular degeneration, and bevacizumab which is commonly used off-label for a variety of chorioretinal disorders. Intravitreal administration of each drug frequently causes rapid improvement of DME with sustained improvement in vision through 2 years. Though these drugs significantly outperform laser photocoagulation over treatment periods of 1 year of less, the advantages appear to lessen when trials approach 2 years. Further studies to better determine relative efficacies of anti-VEGF drugs and laser photocoagulation are continuing.     

Drug insight: antiangiogenic therapies for gastrointestinal cancers--focus on monoclonal antibodies

Tumor angiogenesis is strongly induced by vascular endothelial growth factor (VEGF), which is overexpressed in most human gastrointestinal cancers. VEGF overexpression is known to be associated with poor prognosis and survival in patients with various solid tumors. The humanized monoclonal anti-VEGF antibody bevacizumab (Avastin, Genentech Inc., South San Francisco, CA) is a prototypic antiangiogenic compound, and has proven therapeutic benefit combined with conventional chemotherapy-namely, significantly improved progression-free survival in patients with metastatic colorectal cancer. Bevacizumab is the only anti-VEGF antibody that has been approved by the FDA and the European Medicines Agency for the treatment of metastatic colorectal cancer. Several ongoing clinical studies are evaluating the potential of bevacizumab therapy for other gastrointestinal cancers, in combination with chemotherapy, other targeted therapies and/or radiation. Soluble chimeric receptors, tyrosine kinase inhibitors, and monoclonal antibodies against VEGF and molecular targets in the integrin and Delta-like protein 4-Notch pathways are being developed. As tumors acquire resistance to anti-VEGF therapy, further development of antiangiogenic and vascular targets and therapy is warranted.     

No nephropathy in Type 2 diabetic patient with POEMS syndrome with an elevated plasma VEGF.

Vascular endothelial growth factor (VEGF) is considered to have a role in the pathogenesis of diabetic retinopathy. Recent experimental observations that anti-VEGF neutralizing antibody fully abolished the hyperfiltration and the increase in urinary albumin excretion suggested the contribution of VEGF to the development of diabetic nephropathy, as well. Here, we present a case of POEMS (Crow-Fukase) syndrome with Type 2 diabetes, which was associated with elevated plasma VEGF level, but no sign of diabetic nephropathy. The findings obtained from this case did not support the hypothesis that VEGF may enhance the development of diabetic nephropathy.