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.

Drug delivery strategies in maximizing anti-angiogenesis and anti-tumor immunity.

Metronomic chemotherapy has been shown to elicit anti-tumor immune response and block tumor angiogenesis distinct from that observed with maximal tolerated dose (MTD) therapy. This review delves into the mechanisms behind anti-tumor immunity and seeks to identify the differential effect of dosing regimens, including daily low-dose and medium-dose intermittent chemotherapy (MEDIC), on both innate and adaptive immune populations involved in observed anti-tumor immune response. Given reports of VEGF/VEGFR blockade antagonizing anti-tumor immunity, drug choice, dose, and selective delivery determined by advanced formulations/vehicles are highlighted as potential sources of innovation for identifying anti-angiogenic modalities that may be combined with metronomic regimens without interrupting key immune players in the anti-tumor response. Engineered drug delivery mechanisms that exhibit extended and local release of anti-angiogenic agents both alone and in combination with chemotherapeutic treatments have also been demonstrated to elicit a potent and potentially systemic anti-tumor immune response, favoring tumor regression and stasis over progression. This review examines this interplay between various cancer models, the host immune response, and select anti-cancer agents depending on drug dosing, scheduling/regimen, and delivery modality.

Neoangiogenesis in Melanoma: An Issue in Biology and Systemic Treatment.

Neoangiogenesis is a recognized hallmark of cancer, granting tumor cells to dispose of metabolic substrates through a newly created vascular supply. Neoangiogenesis was also confirmed in melanoma, where vascular proliferation is associated with increased aggressiveness and poorer prognosis. Furthermore, melanoma cells show the so-called vascular mimicry, consisting in the assumption of endothelial-like features inducing the expression of pro-angiogenic receptors and ligands, which take part in the interplay with extracellular matrix (ECM) components and are potentiated by the ECM remodeling and the barrier molecule junction alterations that characterize the metastatic phase. Although neoangiogenesis was biologically proven and clinically associated with worse outcomes in melanoma patients, in the past anti-angiogenic therapies were employed with poor improvement of the already unsatisfactory results associated with chemotherapic agents. Among the novel therapies of melanoma, immunotherapy has led to previously unexpected outcomes of treatment, yet there is a still strong need for potentiating the results, possibly by new regimens of combination therapies. Molecular models in many cancer types showed mutual influences between immune responses and vascular normalization. Recently, clinical trials are investigating the efficacy of the association between anti-angiogenetic agents and immune-checkpoint inhibitors to treat advanced stage melanoma. This paper reviews the biological bases of angiogenesis in melanoma and summarizes the currently available clinical data on the use of anti-angiogenetic compounds in melanoma.

The Binding of CD93 to Multimerin-2 Promotes Choroidal Neovascularization.

Purpose: The purpose of this study was to investigate the involvement of CD93 and Multimerin-2 in three choroidal neovascularization (CNV) models and to evaluate their contribution in the neovascular progression of age-related macular degeneration (AMD). Methods: Choroidal neovascular membranes collected during surgery from AMD patients were analyzed by microscopy methods. Laser-induced CNV mouse models and choroid sprouting assays (CSAs) were carried out using the CD93 knockout mouse model. An original ex vivo CSA of vascular angiogenesis, employing choroid tissues isolated from human donors, was developed. Results: In contrast to healthy choroid endothelium, hyperproliferative choroidal endothelial cells (ECs) of AMD patients expressed high levels of CD93, and Multimerin-2 was abundantly deposited along the choroidal neovasculature. CD93 knockout mice showed a significant reduced neovascularization after laser photocoagulation, and their choroidal ECs displayed a decreased ability to produce sprouts in ex vivo angiogenesis assays. Moreover, the presence of an antibody able to hamper the CD93/Multimerin-2 interaction reduced vascular sprouting in the human CSA. Conclusions: Our results demonstrate that CD93 and its interaction with Multimerin-2 play an important role in pathological vascularization of the choroid, disclosing new possibilities for therapeutic intervention to neovascular AMD.

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.

In vivo tumor-suppressing and anti-angiogenic activities of a recombinant anti-CD3ε nanobody in breast cancer mice model.

Aim: Achievements in cancer immunotherapy require augmentation of a host's anti-tumor immune response for anti-cancer modality. Materials & methods: Different concentrations of recombinant anti-CD3 nanobody were administered at predetermined time intervals during a 24-day treatment period and then expression of angiogenic biomarkers including VEGFR2, MMP9 and CD31, as well as tumor cell proliferation marker ki67, was determined in tumor sections by immunohistochemistry. Furthermore, expression of cytokines was examined in peripheral blood of mice. Results: Based on our results, administration of nanobody could reduce biomarker expression in tumor sections. Tumor growth was also delayed and survival rate was increased in response to nanobody treatment. Moreover, expression of pro-inflammatory cytokines was reduced. Conclusion: In conclusion, we demonstrated that administration of nanobody could effectively suppress angiogenesis as well as tumor growth.

Production of monoclonal antibodies for measuring Avastin and its biosimilar by Sandwich ELISA.

The development of Bevacizumab (Avastin) biosimilar products has grown rapidly over the last ten years as the original Avastin's patent will expire soon. The approval of Avastin biosimilars requires the demonstration of similarity between the biosimilar and the reference product. To support pre-clinical and clinical studies, pharmacokinetic (PK) assays are required to measure the biosimilar and Avastin with comparable precision and accuracy. The PK assay of Avastin employed by Genentech was a Sandwich ELISA which could detect the total drug concentration. However, it was developed in-house and not commercially available. Therefore, in most of the Avastin biosimilar pre-clinical studies, the antibody drug concentrations were measured using an indirect ELISA against coated VEGF, which could only measure the free instead of the total antibody drugs. It failed the essential requirement to develop the biosimilars. In this study, we reported the generation of mouse monoclonal antibodies (mAbs) that specifically recognize Avastin in a VEGF non-competitive manner. Using a pair of non-VEGF competing anti-Avastin mAbs, a Sandwich ELISA was developed with a lower limit of quantitation (LLOQ) at 400 ng/mL and upper limit of quantitation (ULOQ) at 12800 ng/mL. The assay validation was carried out with serum samples from monkey treated with Avastin biosimilar at seven different time points. Our data showed that the Sandwich ELISA kit we developed is sensitive, simple, reproducible and ready for use in human clinical trials.

Human Recombinant VEGFR2D4 Biochemical Characterization to Investigate Novel Anti-VEGFR2D4 Antibodies for Allosteric Targeting of VEGFR2.

VEGF-A/VEGFR2 complex is the major signaling pathway involved in angiogenesis and the inhibition of this axis retards tumor growth and inflammatory disorders progression, reducing vessel sprouting. Signaling by VEGFR2 requires receptor dimerization and a well-defined orientation of monomers in the active dimer. The extracellular portion of receptor is composed of seven Ig-like domains, of which D2-3 are the ligand binding domains, while D4 and D7, establishing homotypic contacts, allosterically regulate receptor activity. The allosteric targeting of VEGFR2 represents a promising alternative to study neovascular disorders overcoming drawbacks related to competition with VEGF. In this work, we expressed in bacterial host domain 4 of VEGFR2 (VEGFR2D4). After protein refolding, we characterized the purified domain and administered it in mice for monoclonal antibodies production. One of them, mAbD4, was tested in ELISA assays, showing a nanomolar affinity for VEGFR2D4. Finally, the methodology here described could contribute to the development of antibodies which can allosterically bind VEGFR2 and therefore to be used for imaging purposes or to modulate receptor signaling.

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.

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.

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.

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 Therapeutic Antibody LM609 Selectively Inhibits Ligand Binding to Human αVß3 Integrin via Steric Hindrance.

The LM609 antibody specifically recognizes αVß3 integrin and inhibits angiogenesis, bone resorption, and viral infections in an arginine-glycine-aspartate-independent manner. LM609 entered phase II clinical trials for the treatment of several cancers and was also used for αVß3-targeted radioimmunotherapy. To elucidate the mechanisms of recognition and inhibition of αVß3 integrin, we solved the structure of the LM609 antigen-binding fragment by X-ray crystallography and determined its binding affinity for αVß3. Using single-particle electron microscopy, we show that LM609 binds at the interface between the ß-propeller domain of the αV chain and the ßI domain of the ß3 chain, near the RGD-binding site, of all observed integrin conformational states. Integrating these data with fluorescence size-exclusion chromatography, we demonstrate that LM609 sterically hinders access of large ligands to the RGD-binding pocket, without obstructing it. This work provides a structural framework to expedite future efforts utilizing LM609 as a diagnostic or therapeutic tool.

Tyrosine kinase inhibitors reprogramming immunity in renal cell carcinoma: rethinking cancer immunotherapy

The immune system regulates angiogenesis in cancer by way of both pro- and antiangiogenic activities. A bidirectional link between angiogenesis and the immune system has been clearly demonstrated. Most antiangiogenic molecules do not inhibit only VEGF signaling pathways but also other pathways which may affect immune system. Understanding of the role of these pathways in the regulation of immunosuppressive mechanisms by way of specific inhibitors is growing. Renal cell carcinoma (RCC) is an immunogenic tumor in which angiogenesis and immunosuppression work hand in hand, and its growth is associated with impaired antitumor immunity. Given the antitumor activity of selected TKIs in metastatic RCC (mRCC), it seems relevant to assess their effect on the immune system. The confirmation that TKIs improve cell cytokine response in mRCC provides a basis for the rational combination and sequential treatment of TKIs and immunotherapy (AU)

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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).

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.

Multiple therapeutic peptide vaccines for patients with advanced gastric cancer.

We performed a clinical trial using HLA-A24-binding peptide vaccines containing a combination of novel cancer-testis antigens and anti-angiogenic peptides for advanced gastric cancer (GC). Thirty-five GC patients who had shown resistance to the standard therapy were enrolled in this clinical trial using vaccinations with a mixture of multiple peptides derived from DEPDC1, URLC10, FoxM1, Kif20A and VEGFR1. The safety, the overall survival (OS), and the immunological responses based on an ELISPOT assay were determined to assess differences in patients who were HLA-A24-positive [24(+)] and HLA-A24-negative [24(-)]. No severe adverse effects were observed except for severe skin reactions in 4 patients. The differences in OS were not significant between patients who were 24(+) and 24(-). In the 24(+) group, patients who showed T cell responses specific to antigen peptides had a tendency towards better survival than those who showed no response, especially to the DEPDC1 peptide. The patients with local skin reactions had significantly better OS than the others. Peptide vaccine therapy was found to be safe and is expected to induce specific T cell responses in patients with advanced GC. The survival benefit of peptide vaccine monotherapy may not have been shown and further trials are needed to confirm these results.

Immuno-oncology for renal cell carcinoma treatment: future perspectives for combinations and sequences with molecularly targeted agents.

INTRODUCTION: From a theoretical viewpoint, combining molecularly targeted agents endowed with antiangiogenic properties with immunotherapy makes sense in treatment of metastatic renal cell carcinoma (RCC); this neoplasm is highly angiogenesis-dependent, as well as potentially immunogenic. Areas covered: The authors performed a literature search looking for clinical trials aimed at evaluating efficacy and tolerability of combinations (or sequences) of molecularly targeted agents and different immunotherapeutic approaches in metastatic RCC. Expert opinion: Combinations of molecularly targeted agents with old immunotherapeutics (i.e., cytokines) seem to add little to the presently available treatment standards (mainly monotherapy with targeted agents). Newer combinations with immune checkpoint inhibitors are promising but cumulative toxicity is an important issue, although highly dependent on the different companion drugs. Combinations with vaccines are ongoing, but first available data are not encouraging. A more thorough comprehension of the complex effects of these combinations on the immune system is mandatory to develop less empiric treatments.

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.

Identification and characterization of a novel nanobody against human placental growth factor to modulate angiogenesis.

Placental growth factor (PlGF), a member of vascular endothelial growth factors (VEGF) family, is considered as an important antigen associated with pathological conditions such as cancer cell growth, and metastasis. PlGF-targeting via nanobody (Nb) therefore could be beneficial to modulate these pathologies. In this work, phage-display and computational approach was employed to develop a high affinity PlGF-specific Nb. An Nb library was constructed against human recombinant PlGF (rPlGF). After panning on immobilized rPlGF the periplasmic-extract (PE) of individual colonies were screened by ELISA (PE-ELISA). The 3D structures of selected Nbs were then homology modeled and energy minimized using the AMBER force field. Binding score calculations were also assessed to reveal possible Nb-PlGF interactions. Via ELISA-based affinity/specificity determinations, the best-qualified Nb was further evaluated by proliferation, migration, 3D capillary formation, invasion assays and on Chick chorioallantoic membrane (CAM) model. An immune library of 1.5×107 individual Nb clones was constructed. By PE-ELISA 12 clones with strong signals were selected. Three out of 12 sequenced Nbs (Nb-C13, Nb-C18 and Nb-C62) showed high binding scores ranging between -378.7 and -461kcal/mol. Compared to a control Nb, Nb-C18 significantly inhibited proliferation, migration and the 3D-capillary formation of HUVEC cells (p<0.05) with an EC50 of 35nM, 42nM and 24nM and invasion of MDA-MB231was significantly suppressed (p<0.05) with an EC50 of57nM. The result of the CAM assay shows that Nb-C18 could inhibit the vascular formation in the chicken chorioallantoic membrane. This Nb can be used as anti-angiogenesis agent in future.