The development of anti-angiogenic therapies was highly anticipated. This therapeutic strategy was hypothesized to avoid the tumor resistance pathways of traditional anti-cancer drugs by targeting the vasculature as opposed to the genetically instable and highly mutagenic tumor cell population. The pre-clinical success of targeting the VEGF pathway using mAb-based therapy further bolstered this hypothesis. However, clinical studies of anti- VEGF strategies in cancer patients have not delivered the level of efficacy anticipated. To date, bevacizumab is the most developed anti-VEGF pathway mAb. Bevacizumab is currently indicated as a first- or second-line treatment in five different tumor types, and is being evaluated in many clinical trials. Therefore, experience with bevacizumab in the clinic provides a working model for the benefits and pitfalls of anti-angiogenic mAb therapies, as well as a benchmark for other anti-angiogenic mAb discussed in this article.
As discussed previously, the results of bevacizumab in Phase 2 and 3 clinical trials have been modest when compared to the success of anti-VEGF therapy in pre-clinical models.90
In the clinic, responses with bevacizumab as a single agent therapy (in glioblastoma) or in combination with standard chemotherapy (in nonsmall cell lung cancer, metastatic colorectal cancer, breast cancer and renal cell carcinoma) is measured in a few months correlating with small, albeit statistically significant increases in PFS, and rarely in OS.93
This differs from some anti-angiogenic small molecule tyrosine kinase inhibitors (TKIs) that do not display improved efficacy when combined with chemotherapy.137
The differences between anti-angiogenic TKIs and mAbs may be due to functional changes occurring within the tumor in response to the different drugs. Treating tumors with bevacizumab, or other anti-VEGF pathway mAbs counteracts the inherent disorganization and abnormalities of the tumor vasculature. This process has been termed “normalization.”138
The pruned, normalized tumor vasculature achieved with anti-angiogenic therapy has increased pericyte coverage and stability and a reduction in vessel leakiness and interstitial fluid pressure, which in combination improves the subsequent delivery of chemotherapy and other drugs. This process allows for cytostatic anti-angiogenic therapy (e.g., bevacizumab and other anti-VEGF pathway mAbs) combined with cytotoxic chemotherapy to have improved clinical response as compared to either agent alone. This absence of synergy with some VEGFR TKIs and chemotherapy is perhaps due to the targeting of other tyrosine kinase receptors such as PDGFR that can disrupt the normalization process.137
A better understanding of the normalization process among tumor types would allow for optimization of anti-angiogenic and chemotherapeutic drug delivery schedules, perhaps improving the overall efficacy of these drugs in the clinic.
Bevacizumab therapy is associated with several adverse effects. The most common toxicities are hypertension, proteinuria and bleeding events that result from a loss of homeostatic VEGF signaling and vascular maintenance.139
Certain histologies, such as squamous non-small cell lung cancer, were more prone to fatal bleeding events, leading to the exclusion of these patients from future studies.93
In addition, perforations were more frequent in patients with metastatic colorectal cancer, ovarian cancer or metastases within the gastrointestinal tract. Inhibition of VEGFR signaling with sorafenib and sunitinib therapy is also associated with hypertension, proteinuria and bleeding events similar to treatment with bevacizumab. However, there are a host of off-target adverse effects with sorafenib and sunitinib therapy, including skin reactions, hand-foot syndrome, fatigue and diarrhea resulting from TKI inhibition of targets other than the VEGFRs.139
Based on these patterns, specific blockade of VEGF, PlGF, VEGFR1 or VEGFR2 with r84, VEGF-Trap, IMC-18F1 or IMC-1121B might induce toxicities more similar to bevacizumab rather than sorafenib and sunitinib. It is also possible that VEGFR1 signaling is important for maintaining the homeostatic function of VEGF, and thus therapies allowing for continued VEGFR1 signaling such as r84 and IMC-1121B may provide a less severe toxicity profile than bevacizumab. In support of this, pre-clinical studies in our laboratory with extended (12 week) treatment of tumor-bearing and non-tumor bearing mice with r84 failed to induce toxicity (unpublished data). Results from IMC- 1121B on-going clinical trials and future studies with r84 in the clinic will ultimately answer these questions about differences in toxicities between the VEGF pathway antibodies. Alternatively, the severity or frequency of toxicities between mAbs targeting the VEGF pathway may depend more on the relative affinity of the drug for its target. In pre-clinical studies with mice engineered to express human VEGF, anti-VEGF antibodies of increasing affinity had a greater toxicity induction.111
Additionally, chemotherapy regimens such as carboplatin and paclitaxel, in combination with bevacizumab or VEGFR TKIs can exacerbate the toxicities of these targeted therapies.140
Therefore, carefully assessing both drug affinity for its target and chemotherapy doses and regimens is required to control toxicity in patients receiving drugs targeting the VEGF pathway. The distribution of toxicities within mAb strategy and patient groups should be taken into consideration as future anti-VEGF pathway therapies are introduced into the clinic to minimize adverse effects and to monitor for new patterns of toxicity.
With the expanding use of anti-angiogenic therapy in the clinic, it has become more apparent that not all tumors respond or will remain responsive to this treatment option. The inherent non-responsiveness of certain tumors and the development of acquired resistance to therapy following an initial response, has been termed intrinsic and evasive resistance, respectively.141
Pre-clinical research from a number of investigators has identified several mechanisms mediating resistance to anti-angiogenic therapy. This includes a switch from a primary reliance on VEGF to an alternative growth factor such as fibroblast growth factor, interleukin-8 and ephrins, increased stabilization of existing vessels by improved pericyte coverage, stimulating infiltration of pro-angiogenic immune cells and the co-option of normal vasculature through enhanced tumor invasiveness and metastasis.141
Patients with intrinsically resistant tumors would have no clinical benefit from anti-angiogenic therapy and therefore should be excluded from these treatments; however, we currently lack effective biomarkers that would allow for the stratification of intrinsically resistant or sensitive tumors, or for the monitoring of tumor response and the development of resistance to therapy.142
The identification of biomarkers of response to anti-angiogenic drugs is being actively pursued. In mBC, baseline levels of circulating VEGF have shown promise as a biomarker of response to bevacizumab therapy, but this has not translated to other tumor types such as mCRC or NSCLC.143
In addition, the utility of baseline VEGF levels as a biomarker of response varies depending on tumor type and therapy regimen (mAb or VEGFR TKI), bringing into question the universal applicability of this marker. Genetic polymorphisms of VEGF and interleukin-8 have been indicated as predictive biomarkers of response to bevacizumab in mBC and ovarian cancer, respectively; however these markers have yet to be validated in other tumor types or in relation to other anti-VEGF pathway therapies.143
In addition, there are increased levels of circulating PlGF following anti-VEGF therapy that have been positively correlated with improved outcome in bevacizumab-treated rectal cancer patients, although it remains to be seen if this increase in PlGF is predictive or prognostic of response to therapy.143
A systemic increase in blood pressure has potential as a pharmacodynamic biomarker of the effectiveness of VEGF blockade by bevacizumab and VEGFR TKIs. Recent studies have implicated the degree of hypertension in patients as a diagnostic biomarker of response to VEGF targeted therapy, although this has yet to be validated in large studies.143
Although there are several candidates awaiting validation as pharmacodynamic or prognostic biomarkers of anti-angiogenic therapy, including circulating levels of VEGF and PlGF, genetic polymorphisms of angiogenic factors and the degree of hypertension following therapy, we still lack biomarkers predictive of response to therapy that exist for other targeted drugs such as the overexpression of human epidermal growth factor receptor 2 (HER2/neu) in BC as a predictive marker of response to trastuzumab therapy. Similar to HER2/neu overexpression, predictive markers of response to anti-angiogenic therapy may be tumor-dependent. Our laboratory and others are currently assessing tumor-specific factors in xenograft models while others are evaluating patient samples for potential biomarkers of response. Further studies are required to identify and validate predictive versus prognostic biomarkers and may require carefully designed clinical trials and more frequent collection of patient tumor and serum samples throughout treatment. A better understanding of what dictates patient response and how to monitor resistance could lead to improved efficacy of mAbs targeting the VEGF pathway.
The opportunity to better understand the function of individual components of the VEGF pathway in the tumor microenvironment is afforded by evaluation of the efficacy and biology of the anti-VEGF strategies outlined in this review. As there are very few studies that directly compare anti-VEGF pathway mAbs, it is difficult to say with certainty that one strategy is best or will work for every patient. All of the mAbs discussed in this review target the VEGF pathway; however the specificity of the different mAbs affects the function of these therapies within the tumor microenvironment and provides clues to the potential advantages and disadvantages among the different therapies. It is of particular interest to compare the strategies that are specific to VEGF (bevacizumab and r84) to VEGF-Trap and those that inhibit VEGFR1 (MF1, IMC-18F1) or VEGFR2 (DC101, IMC-1C11, IMC-1121B) directly.
Bevacizumab and r84 are both highly specific for VEGF-A and do not directly interfere with other VEGF family members. r84 is even more selective than bevacizumab due to the fact that it only inhibits VEGFR2 activation, leaving intact VEGFR1 signaling. In endothelial cells, VEGFR1 primarily functions as a negative inhibitor of VEGFR2 signaling by acting as a decoy receptor for VEGF and preventing VEGF from binding to and inducing angiogenesis through VEGFR2. This idea is supported by previously mentioned genetic experiments where loss of VEGFR1 leads to embryonic death due to too many endothelial cells, but mice expressing only the extracellular domain of VEGFR1 are viable.44,45
Additionally, VEGF binding to VEGFR1 can stimulate SHP-1 phosphatase to actively reduce levels of VEGFR2 phosphorylation.51
Further, whereas VEGF activation of VEGFR1 does not alter gene expression, PlGF binding to VEGFR1 in vitro changes the gene expression of more than 50 genes.144
PlGF may also provide an escape mechanism for anti-VEGF targeted therapy and blocking PlGF directly has been demonstrated to have anti-tumor effects.145
Treatment with r84 would allow for regulatory signaling through VEGFR1 and could reduce PlGF activation of VEGFR1 as a result of competition with PlGF for VEGFR1 binding. Therefore, VEGF binding to VEGFR1 may be an important negative regulator of tumor angiogenesis that could be harnessed with r84, but not with bevacizumab. VEGFTrap blocks VEGF from binding to VEGFR1 and VEGFR2 and blocks PlGF from binding to VEGFR1, thereby preventing negative regulation of VEGFR2 activity by VEGFR1 similar to bevacizumab and uniquely controlling VEGFR1 activation by PlGF. Thus the in vivo mechanisms of action of VEGF-Trap may fall somewhere between that of r84 and bevacizumab.
Alternatively, blocking VEGFR1 activity with mAbs has been very effective in VEGFR1 expressing tumors.122
VEGFR1 activity has also been linked to tumor metastasis and blocking VEGFR1 with MF1/IMC-18F1 reduces tumor growth, demonstrating the potential importance of this receptor in tumor progression and the need to inhibit its function in patients. Despite these data, the overall functions of VEGFR1 remain unclear and the full effects of VEGFR1 blockade are uncertain. However, there is still a strong possibility that VEGFR1 functions as a negative regulator of VEGFR2 and direct targeting of VEGFR1 with MF1/IMC-18F1 may be in effect, inhibiting an inhibitor of angiogenesis, which may be therapeutically counterproductive. Therefore, the results of on-going IMC-18F1 and IMC-1121B clinical trials and entry of r84 into the clinical arena are highly anticipated to elucidate the importance of VEGFR1 signaling in tumor angiogenesis and progression.
As previously mentioned, VEGFR2 is the predominant mediator of VEGF-induced angiogenesis and consequently, blocking functional signaling of this receptor with neutralizing antibodies such as DC101, IMC-1C11 and IMC-1121B is effective at reducing angiogenesis and tumor growth. There is increased expression of VEGFR2 in the tumor microenvironment, which in turn increases the potential of anti-VEGFR2 therapies to specifically target the tumor and not normal tissues. Directly targeting VEGFR2 also prevents receptor activation by other VEGF family members (e.g., VEGF-C, -D), which are not blocked by bevacizumab, r84, or VEGF-Trap. Additionally, the anti-VEGFR2 mAbs do not inhibit VEGFR1 negative regulation of VEGFR2, potentially enhancing blockade of VEGFR2 function. However, the anti-VEGFR2 mAbs will also block soluble VEGFR2, a natural inhibitor of lymphangiogenesis.59
This effect is a potential disadvantage of directly targeting VEGFR2, given the importance of the lymphatic vasculature in metastasis.146
Infiltration of immune cells such as macrophages within tumors can promote tumor survival and progression.147
Macrophages in tumor bearing animals express VEGFR2, and blockade of this receptor has been demonstrated to reduce macrophage migration and infiltration in tumors.60,109
Therefore, targeting VEGFR2 directly can negatively affect tumor growth and metastasis by reducing the population of tumor-promoting immune cells within the tumor microenvironment.
The anti-VEGFR antibodies have a broader specificity profile given that these agents interfere with signaling pathways stimulated by multiple members of the VEGF family. Thus far, it is unclear if a broader specificity anti-VEGF agent is more effective than bevacizumab or r84. In fact, a recent direct comparison of mouse chimeric r84 to sunitinib and a peptoid that binds and inhibits VEGFR1 and VEGFR2 demonstrated that r84 was as or more effective in controlling tumor growth in two models of breast cancer in immunocompetent mice.109
This study also evaluated the immunological phenotype of tumors under therapy and found that in general, treatment of r84 resulted in fewer immunosuppressive cells (e.g., myeloid-derived suppressor cells, Treg
, immature dendritic cells) in the tumor microenvironment. These cell based changes are likely the result of an altered cytokine profile after therapy.109
To our knowledge similar studies have not been performed with the other anti-VEGF agents discussed here. Species specificity issues and other inherent challenges with pharmaceutical-based novel therapies preclude a head-to-head test of these leading anti-VEGF strategies in pre-clinical models. Thus we are forced to make assumptions regarding the potential superiority of one agent over another based on the efficacy observed in similar models and the biology of the therapy employed.
In reality, arguing the benefits and shortcomings of the individual mAb-based strategies available for targeting the VEGF pathway in cancer may be short-sighted. Selectively targeting angiogenesis in patients will most likely require an arsenal of therapeutics and the best strategy may very well depend upon the tumor type, stage, histology or may be entirely patient specific. This again highlights the need for biomarkers that can predict a patient’s response to anti-angiogenic therapy, as well as the need for an array of selective therapies to improve patient survival by inhibiting tumor angiogenesis.