Targeting of human fibroblast growth factor receptor 2 by a novel specific nanobody.

Inhibition of FGFR2 signaling is promising in targeted therapy of FGFR2-related tumors. In this study, anti-FGFR2 nanobodies (Nbs) were isolated through screening of an immune camelid phage display library. Four rounds of biopanning were carried out with commercial human FGFR2 antigen and enrichment was assessed by ELISA and phage titration. The gene of Nb was sub-cloned into the expression vector, and the recombinant vector was transformed into Escherichia coli WK6 cells. The recombinant protein was purified using Ni-NTA affinity chromatography. The anti-FGFR2 Nb (C13) was characterized by SDS-PAGE, western blotting, competitive inhibition ELISA, flow cytometry, MTT, and migration assay. C13 Nb recognized FGFR2 with high specificity and no cross-reactivity was observed with other tested antigens. The affinity of C13 Nb was calculated to be 1.5 × 10-9 M. Results of cytotoxicity showed that C13 Nb (10 µg/ml) inhibited 85% of the proliferation of T-47D cells (p < 0.001). In addition, C13 inhibited the migration of 68% of T-47D toward the source of the growth factor (p < 0.01). The flow cytometry showed that C13 Nb bound to the surface of FGFR2+ cells, T-47D cell line (96%). Results indicate the potential of anti-FGFR2 Nb for targeted therapy of FGFR2-overexpressing tumors after complementary investigations.

Investigation of the therapeutic potential of recombinant bispecific bivalent anti-PD-L1/VEGF nanobody in inhibition of angiogenesis.

OBJECTIVE: Immunotherapy using monoclonal antibodies targeting programmed death ligand-1 (PD-L1) on cancer cells as a biomarker of escape from response to immune checkpoint has demonstrated efficacy in treating many solid tumors. In addition, some of the signals, such as vascular endothelial growth factor (VEGF), bind to receptors on the surface of normal endothelial cells and encourage angiogenesis, or the formation and survival of new blood vessels. METHODS: Due to the special features of nanobodies with high specificity and affinity as a powerful new tool in cancer therapy, here, a recombinant bispecific bivalent anti-PD-L1/VEGF nanobody was constructed and its functionality in inhibition of angiogenesis in vitro was investigated. RESULTS: Results demonstrated that bivalent anti-PD-L1/VEGF nanobody efficiently inhibited HUVEC and A431 cells proliferation and tube formation. In addition, bivalent anti-PD-L1/VEGF nanobody efficiently inhibited angiogenesis in an ex ovo Chick Chorioallantoic Membrane assay. DISCUSSION: The results indicate for the potential of bivalent anti-PD-L1/VEGF nanobody as a novel promising tool for cancer therapy.

Effective blocking of neuropilin-1activity using oligoclonal nanobodies targeting different epitopes.

Neuropilin-1 (NRP-1) is a non-tyrosine kinase receptor and when overexpressed, leads to angiogenesis. High expression of NRP-1 has been observed in various cancers. Unique characteristic of nanobodies (small size, high affinity and stability, and ease production) make them potential therapeutic tools. Oligoclonal nanobodies which detect multiple functional epitopes on the target antigen could be potential tools for inhibition of cancer resistance problems due to escape variant of tumor cells. In this study, oligoclonal anti-NRP-1 nanobodies were selected from camel immune library and their binding activities as well as in vitro functionality were evaluated. Anti-NRP-1 nanobodies were expressed in an Escherichia coli host, and purified using nickel affinity chromatography. The effect of each individual and oligoclonal nanobodies on human endothelial cells were evaluated by MTT, Tube formation, and migration assay as well. Results showed that oligoclonal anti-NRP-1 nanobodies detected different epitopes of NRP-1 antigen and inhibited in vitro angiogenesis of human endothelial cells better than each individual nanobody. Results indicate promising oligoclonal anti-NRP-1 nanobodies for inhibition of angiogenesis.

Diabetes as one of the long-term COVID-19 complications: from the potential reason of more diabetic patients' susceptibility to COVID-19 to the possible caution of future global diabetes tsunami.

According to recent researches, people with diabetes mellitus (type 1 and 2) have a higher incidence of coronavirus disease 2019 (COVID-19), which is caused by a SARS-CoV-2 infection. In this regard, COVID-19 may make diabetic patients more sensitive to hyperglycemia by modifying the immunological and inflammatory responses and increasing reactive oxygen species (ROS) predisposing the patients to severe COVID-19 and potentially lethal results. Actually, in addition to COVID-19, diabetic patients have been demonstrated to have abnormally high levels of inflammatory cytokines, increased virus entrance, and decreased immune response. On the other hand, during the severe stage of COVID-19, the SARS-CoV-2-infected patients have lymphopenia and inflammatory cytokine storms that cause damage to several body organs such as ß cells of the pancreas which may make them as future diabetic candidates. In this line, the nuclear factor kappa B (NF-κB) pathway, which is activated by a number of mediators, plays a substantial part in cytokine storms through various pathways. In this pathway, some polymorphisms also make the individuals more competent to diabetes via infection with SARS-CoV-2. On the other hand, during hospitalization of SARS-CoV-2-infected patients, the use of some drugs may unintentionally lead to diabetes in the future via increasing inflammation and stress oxidative. Thus, in this review, we will first explain why diabetic patients are more susceptible to COVID-19. Second, we will warn about a future global diabetes tsunami via the SARS-CoV-2 as one of its long-term complications.

Inhibition of neovascularisation in human endothelial cells using anti NRP-1 nanobody fused to truncated form of diphtheria toxin as a novel immunotoxin.

Neuropilin-1 (NRP-1) regulates a range of physiological and pathological processes, including angiogenesis. Targeting of NRP1 is considered a significant approach in cancer therapy. In the present study, a novel antiNRP1 immunotoxin (αNRP1 IT) was developed by genetic fusion of a single domain (VHH) anti-NRP-1 antibody fragment to a truncated diphtheria toxin. The αNRP1 IT was expressed into bacterial cells as an inclusion body (IB). Expression of αNRP1 IT was confirmed by SDS-PAGE and western blotting. Recombinant αNRP1 IT was purified using nickel affinity chromatography. Toxicity and antiangiogenesis effect of αNRP1 IT was investigated both in vitro and in vivo. Results showed that αNRP1 IT significantly reduced the viability of human umbilical vein endothelial cell line (HUVEC) (p < .05). The αNRP1 IT significantly inhibited tube formation of HUVEC cells (p < .001). Furthermore, αNRP1 IT inhibited angiogenesis in Chick Chorioallantoic Membrane (CAM) Assay. These data suggest the potential of αNRP1 IT as a novel therapeutic in targeted cancer therapy.

A nanobody-derived mimotope against VEGF inhibits cancer angiogenesis.

Vascular Endothelial Growth Factor (VEGF) promotes angiogenesis in tumours of various cancers. Monoclonal antibodies and nanobodies are one of the potent agents in the treatment of cancer. Due to their high costs, researchers are considering to design and produce peptides as a substitute approach in recent years. The aim of the current study was designing a mimotope against VEGF and evaluate its effects on cell proliferation and tube formation in the HUVEC cell line. For this, a peptide was designed against VEGF and chemically produced. The effects of synthetic peptide and nanobody on the inhibition of proliferation of HUVEC cells were examined using MTT and tube formation assays. The data indicate that the peptide was able to significantly inhibit both HUVEC cell proliferation and tube formation through inhibition of VEGF, highlighting the potential of peptides as a 'novel' class of candidate drugs to inhibit angiogenesis.

The inhibitory effect of the combination of two new peptides on biofilm formation by Acinetobacter baumannii.

The emergence of extensively drug-resistant (XDR) Acinetobacter baumannii strains and the limited number of efficacious antibiotics demonstrate an urgent need to develop novel agents to treat infections caused by this dangerous pathogen. To find antimicrobial peptides against A. baumannii growing either in planktonic or in biofilm mode, biopanning was carried out with a peptide library on five XDR A. baumannii strains grown in the medium containing human blood (blood biopanning) and biofilms formed by these strains (biofilm biopanning). Two groups of peptides were identified, among which two peptides N10 (from blood biopanning) and NB2 (from biofilm biopanning) were selected and synthesized for more assessments. The selected peptides showed significant binding to A. baumannii rather than to the human cell line Caco-2. Both peptides were effective against A. baumannii and showed antibacterial activities (minimum inhibitory concentration (MIC) 500 µg/ml). In the biofilm inhibition assay, NB2 reduced biofilm more efficiently (75%) than N10 (50%). The combination of the two peptides could function better than each peptide alone to prevent biofilm formation by A. baumannii. Supplementation of conventional therapy with a mixture of peptides targeting A. baumannii or using peptides to deliver antibiotics specifically to the site of infection may be promising to control A. baumannii-related diseases.

An antibody fragment against human delta-like ligand-4 for inhibition of cell proliferation and neovascularization.

OBJECTIVES: Angiogenesis targeting is an attractive approach for cancer treatment. Delta-like ligand 4 (DLL4) plays a pivotal role in neovascular development and its inhibitors have recently entered clinical trials for solid tumors. The aim of this study was to evaluate the possibilities of using anti-DLL4 antibody fragment as an angiogenesis maturation inhibitor. MATERIALS AND METHODS: In this study, a DLL4-specific Nanobody, named 3Nb3, was selected and assessed by western blotting and internalization assays. Functional assessments included MTT, apoptosis, and chicken chorioallantoic membrane (CAM) assays. RESULTS: Based on the results, 3Nb3 specifically binds to DLL4 and internalizes into MKN cell. Furthermore, 3Nb3 significantly inhibited the proliferation of cells and also neovascularization in the CAM. CONCLUSIONS: These data demonstrated the potential of Nanobody for application in targeting DLL4. Our findings may provide a basis for the development of novel therapeutic techniques to inhibit growth and neovascularization of tumors.

Neuropilin-1 Binding Peptide as Fusion to Diphtheria Toxin Induces Apoptosis in Non-small Cell Lung Cancer Cell Line.

BACKGROUND: Targeted cancer therapy can be considered as a new strategy to overcome the side effects of current cancer treatments. Neuropilin-1 (NRP-1) is a transmembrane glycoprotein that is expressed in endothelial cells and tumor vessels to stimulate angiogenesis progression. Targeted diphtheria toxin (DT)- based therapeutics are promising tools for cancer treatment. This study aimed to construct a novel NRP-1 binding peptide (as three repeats) (CRGDK) as a fusion to truncated DT (DTA) (DTA-triCRGDK) for targeted delivery of DT into NRP-1 expressing cells. METHODS: The concept of DTA-triCRGDK was designed, synthesized and cloned into the bacterial host. Expression of DTA-triCRGDK was induced by Isopropyl ß-D-1-thiogalactopyranoside (IPTG) and purification was performed using Ni-NTA chromatography. Biological activity of DTA-triCRGDK was evaluated using MTT, apoptosis, and wound healing assays. In addition, expression levels of apoptotic Bax, Bcl2, and Casp3 genes were determined by Real-time PCR. RESULTS: Cytotoxicity analysis showed the IC50 values of DTA-triCRGDK for A549 and MRC5 were 0.43 nM and 4.12 nM after 24 h, respectively. Bcl2 expression levels decreased 0.4 and 0.72 fold in A549 and MRC5, respectively. However, Bax and Casp3 expression level increased by 6.75 and 8.19 in A549 and 2.51 and 3.6 in MRC5 cells. CONCLUSION: Taken together, DTA-triCRGDK is a promising tool for targeted therapy of NRP-1 overexpressing cancer cells.

Targeted Delivery of Diphtheria Toxin into VEGFR1/VEGFR2 Overexpressing Cells Induces Anti-angiogenesis Activity.

BACKGROUND: Vascular Endothelial Growth Factor Receptors (VEGFR1 and VEGFR2) are tyrosine kinase receptors expressed on endothelial cells and tumor vessels and play an important role in angiogenesis. In this study, three repeats of VEGFR1 and VEGFR2 binding peptide (VGB3) were genetically fused to the truncated diphtheria toxin (TDT), and its in vitro activity was evaluated. METHODS: The recombinant construct (TDT-triVGB3) was expressed in bacteria cells and purified with nickel affinity chromatography. The binding capacity and affinity of TDT-triVGB3 were evaluated using the enzyme-linked immunosorbent assay. The inhibitory activity of TDT-triVGB3 on viability, migration, and tube formation of human endothelial cells was evaluated using MTT, migration, and tube formation assays. RESULTS: TDT-triVGB3 selectively detected VEGFR1 and VEGFR2 with high affinity in an enzyme- linked immunosorbent assay and significantly inhibited viability, migration, and tube formation of human endothelial cells. CONCLUSION: The developed TDT-triVGB3 is potentially a novel agent for targeting VEGFR1/ VEGFR2 over-expressing cancer cells.

Development and characterization of nanobody against envenomation by Naja naja oxiana.

Snakebites are considered a significant health issue. Current antivenoms contain polyclonal antibodies, which vary in their specificity against different venom components. Development and characterization of next generation antivenoms including nanobodies against Naja naja oxiana was the main aim of this study. Crude venom was injected into the Sephadex G50 filtration gel chromatography column and then toxic fractions were obtained. Then the corresponding fraction was injected into the HPLC column and the toxic peaks were identified. N. naja oxiana venom was injected into a camel and specific nanobodies screening was performed against the toxic peak using phage display technique. The obtained results showed that among the 12 clones obtained, N24 nanobody was capable of neutralizing P1, the most toxic peak obtained from HPLC chromatography. The molecular weight of P1 was measured with a mass spectrometer and was found to be about seven kDa. The results of the neutralization test of crude N. naja oxiana venom with N24 nanobody showed that 250 µg of recombinant nanobody could neutralize the toxic effects of 20 µg equivalent to LD50 × 10 of crude venom in mice. The findings indicate the potential of the developed nanobody to serve as a novel antivenom therapy.

CTXP, The Major Cobra Toxin Peptide from Naja Naja Oxiana Venom; A Promising Target for Antivenom Agent Development.

BACKGROUND AND OBJECTIVE: Snakebite envenoming is a serious public health issue causing more than 135,000 annual deaths worldwide. Naja Naja Oxiana is one of the most clinically important venomous snakes in Iran and Central Asia. Conventional animal-derived polyclonal antibodies are the major treatment of snakebite envenoming. Characterization of venom components helps to pinpoint the toxic protein responsible for clinical manifestations in victims, which aids us in developing efficient antivenoms with minimal side effects. Therefore, the present study aimed to identify the major lethal protein of Naja Naja Oxiana by top-down proteomics. METHODS: Venom proteomic profiling was performed using gel filtration (GF), reversed-phase (RP) chromatography, and intact mass spectrometry. The toxicity of GF-, and RP-eluted fractions was analyzed in BALB/c mice. The rabbit polyclonal antisera were produced against crude venom, GF fraction V (FV), and RP peak 1 (CTXP) and applied in neutralization assays. RESULTS: Toxicity studies in BALB/c identified FV as the major toxic fraction of venom. Subsequently, RP separation of FV resulted in eight peaks, of which peak 1, referred to as "CTXP" (cobra toxin peptide), was identified as the major lethal protein. In vivo neutralization assays using rabbit antisera showed that polyclonal antibodies raised against FV and CTXP are capable of neutralizing at least 2-LD50s of crude venom, FV, and CTXP in all tested mice. CONCLUSION: Surprisingly, the Anti-CTXP antibody could neutralize 8-LD50 of the CTXP peptide. These results identified CTXP (a 7 kDa peptide) as a potential target for the development of novel efficient antivenom agents.

Efficient Inhibition of Pathologic Angiogenesis using Combination Therapy of Anti-Epcam and Anti-VEGFR2 Nanobodies.

BACKGROUND: EpCAM and VEGFR2 play an important role in angiogenesis and tumorigenesis. It is currently of paramount importance to produce new drugs that can inhibit the angiogenesis and proliferation of tumor cells. Nanobodies are potential drug candidates for cancer therapy due to their unique properties. OBJECTIVE: This study aimed to investigate the combined inhibitory effect of anti-EpCAM and anti-VEGFR2 nanobodies in cancer cell lines. METHODS: Inhibitory activity of anti-EpCAM and anti-VEGFR2 nanobodies on MDA-MB231, MCF7, and HUVEC cells was investigated using both in vitro (MTT, migration, and tube formation assays) and in vivo assays. RESULTS: Results showed that the combination of anti-EpCAM and anti-VEGFR2 nanobodies efficiently inhibited proliferation, migration, and tube formation of MDA-MB-231 cells compared to each individual nanobodies (p < 0.05). In addition, the combination of anti-EpCAM and anti-VEGFR2 nanobodies efficiently inhibited tumor growth and volume of Nude mice bearing MDA-MB-231 cells (p < 0.05). CONCLUSION: Taken together, the results indicate the potential of combination therapy as an efficient approach to cancer therapy.

Use of Albumin for Drug Delivery as a Diagnostic and Therapeutic Tool.

Drug delivery is an important topic that has attracted the attention of researchers in recent years. Albumin nanoparticles play a significant role in drug delivery as a carrier due to their unique characteristics. Albumin is non-toxic, biocompatible, and biodegradable. Its structure is such that it can interact with different drugs, which makes the treatment of the disease faster and also reduces the side effects of the drug. Albumin nanoparticles can be used in the diagnosis and treatment of many diseases, including cancer, diabetes, Alzheimer's, etc. These nanoparticles can connect to some compounds, such as metal nanoparticles, antibodies, folate, etc. and create a powerful nanostructure for drug delivery. In this paper, we aim to investigate albumin nanoparticles in carrier format for drug delivery application. In the beginning, different types of albumin and their preparation methods were discussed, and then albumin nanoparticles were discussed in detail in diagnosing and treating various diseases.

A Novel Immunotoxin Targeting Epithelial Cell Adhesion Molecule Using Single Domain Antibody Fused to Diphtheria Toxin.

Epithelial Cell Adhesion Molecule (EpCAM) is overexpressed in a variety of cancers such as colon, stomach, pancreas, and prostate adenocarcinomas. Inhibition of EpCAM is considered as a potential target for cancer therapy. In current study, anti-EpCAM immunotoxin (α-EpCAM IT) was developed using genetic fusion of α-EpCAM single domain antibody (nanobody) (α-EpCAM Nb) to truncated form of diphtheria toxin. The expression of recombinant α-EpCAM IT was induced by Isopropyl ß-d-1-thiogalactopyranoside (IPTG) and confirmed by SDS-PAGE and western blot. Recombinant α-EpCAM IT was purified from the inclusion bodies and refolded using urea gradient procedure. The cytotoxicity and apoptosis activity of α-EpCAM IT on EpCAM over-expressing (MCF7), low-expressing (HEK293), and no-expressing (HUVEC) cells were evaluated by 3-4,5-Dimethylthiazol-2-yl (MTT) assay and annexin V-FITC-PI assay as well. In addition, anti-tumor activity of α-EpCAM IT was evaluated on nude mice bearing MCF7 tumor cells. Results showed success expression and purification of α-EpCAM IT. The α-EpCAM IT showed time and dose-dependent anti-proliferative activity on MCF-7 cells. However, α-EpCAM IT did not show any anti-proliferative activity on HEK293 and HUVEC cells as well. In addition, the annexin V-FITC-PI assay results showed that α-EpCAM IT significantly increased apoptotic rate in MCF-7 cells with no effect on HEK293 and HUVEC as well. Moreover, α-EpCAM IT significantly reduced tumor size in vivo study. The achieved results indicate the potential of designing α-EpCAM IT as a novel therapeutic for cancer therapy.

Positive effect of acellular amniotic membrane dressing with immobilized growth factors in skin wound healing.

The human amniotic membrane dressing has been shown to accelerate the wound healing process in the clinic. In this study, heparin was conjugated to a human Acellular Amniotic Membrane (hAAM) to provide affinity binding sites for immobilizing growth factors. To study the acceleration of the wound healing process, we bound epidermal growth factor and fibroblast growth factor 1 to heparinized hAAMs (GF-Hep-hAAMs). The heparinized hAAMs (Hep-hAAMs) were characterized by toluidine blue staining and infrared spectroscopy. The quality control of hAAM was performed by hematoxylin staining, swelling capacity test and biomechanical evaluation. The cytotoxicity, adhesion, and migration in vitro assays of GF-Hep-hAAMs on L-929 fibroblast cells were also studied by MTT assay, scanning electron microscopy, and scratch assay, respectively. Finally, in vivo skin wound healing study was performed to investigate the wound closure rate, re-epithelization, collagen deposition, and formation of new blood vessels. The results showed that GF-Hep-hAAMs enhance the rate of wound closure and epidermal regeneration in BALB/c mice. In conclusion, GF-Hep-hAAMs could accelerate the wound healing process, significantly in the first week.

Development of a Functional Nanobody Targeting Programmed Cell Death Protein-1 as Immune Checkpoint Inhibitor.

BACKGROUND: Programmed cell death protein 1 (PD-1) is a membrane receptor that is expressed on the surface of various immune cells, such as T cells, B cells, monocytes, natural killer T cells, and dendritic cells. In cancer, the interaction between PD-1 and its ligand PD-L1 suppresses the activation and function of T lymphocytes, leading to the impairment and apoptosis of tumor-specific T cells. This mechanism allows cancer cells to evade the immune response and promotes tumor progression. METHODS: Recombinant PD-1 protein was produced and used to immunize a camel. A nanobody library was generated from the camel's peripheral blood lymphocytes and screened for PD-1 binding. A specific nanobody (3PD9) was selected and characterized by affinity measurement, western blotting, and flow cytometry analysis. The ability of the selected nanobody to block the inhibitory signal of PD-1 in peripheral blood mononuclear cells (PBMCs) was evaluated by measuring the level of interleukin-2 (IL-2). RESULTS: The selected nanobody showed high specificity and affinity for human PD-1. Western blot and flow cytometry analysis confirmed that 3PD9 could recognize and bind to human PD-1 on the cell surface. It was demonstrated that the level of IL-2 was significantly increased in PBMCs treated with 3PD9 compared to the control group, indicating that the nanobody could enhance the T cell response by disrupting the PD-1/PD-L1 interaction. CONCLUSION: The results suggested that the anti-PD-1 nanobody could be a promising candidate for cancer immunotherapy.

Nanoparticles as Powerful Tools for Crossing the Blood-brain Barrier.

The blood-brain barrier (BBB) is considered an important protective barrier in the central nervous system (CNS). The barrier is mainly formed by endothelial cells (ECs) interconnected by various junctions such as tight junctions (TJs), gap junctions, and adherent junctions. They collectively constitute an intensive barrier to the transit of different substances into the brain, selectively permitting small molecules to pass through by passive movement but holding off large ones such as peptides and proteins to cross the brain. Hence some molecules selectively transfer across the BBB by active routes via transcytosis. The BBB also forms a barrier against neurotoxins as well as pathogenic agents. Although various CNS disorders like Alzheimer's disease (AD) and Parkinson's disease (PD) could hamper the integrity of the border. Nevertheless, the BBB acts as a barrier for CNS disorders treatment because it prevents the drugs from reaching their target in the CNS. In recent years, different strategies, including osmotic disruption of BBB or chemical modification of drugs, have been used to transfer the chemotherapeutic agents into brain substances. Nowadays, nanoparticles (NPs) have been used as an effective and non-invasive tool for drug delivery and diagnosis of CNS disorders. In this review, we discuss the structural characteristic of BBB, safe passageways to cross the BBB, and the relation of barrier lesions with different CNS disorders. In the end, we explore the progress in drug delivery, diagnosis, imaging, and treatment of CNS disorders using nanoparticles.

Development of polyclonal heavy chain antibodies targeting programmed death ligand-1.

Programmed death ligand-1 (PD-L1, CD274 and B7-H1) has been described as a ligand for immune inhibitory receptor programmed death protein 1 (PD-1). With binding to PD-1 on activated T cells, PD-L1 can prevent T cell responses via motivating apoptosis. Consequently, it causes cancers immune evasion and helps the tumor growth; hence, PD-L1 is regarded as a therapeutic target for malignant cancers. The anti-PD-L1 monoclonal antibody targeting PD-1/PD-L1 immune checkpoint has attained remarkable outcomes in clinical application and has turned to one of the most prevalent anti-cancer drugs. The present study aimed to develop polyclonal heavy chain antibodies targeting PD-L1via Camelus dromedarius immunization. The extra-cellular domain of human PD-L1 (hPD-L1) protein was cloned, expressed, and purified. Afterwards, this recombinant protein was utilized as an antigen for camel immunization to acquire polyclonal camelid sera versus this protein. Our outcomes showed that hPD-L1 protein was effectively expressed in the prokaryotic system. The antibody-based techniques, such as enzyme-linked immunosorbent assay, western blotting, and flow cytometry displayed that the hPD-L1 protein was detected by generated polyclonal antibody. Due to the advantages of multi-epitope-binding ability, our study exhibited that camelid antibody is effective to be applied significantly for detection of PD-L1 protein in essential antibody-based studies.

Targeted therapy of angiogenesis using anti-VEGFR2 and anti-NRP-1 nanobodies.

PURPOSE: Targeted therapy in cancer researches is a promising approach that can resolve drawbacks of systematic therapeutics. Nanobodies are potent therapeutics due to their high specificity and affinity to the target. METHODS: In this study, we evaluated the effect of the combination of anti-vascular endothelial growth factor receptor 2 (anti-VEGFR2) and anti-neuropilin-1 (anti-NRP1) nanobodies both in vitro (MTT, and tube formation assay) and in vivo (chick chorioallantoic membrane (CAM), and Nude mice treatment assay). RESULTS: Our results showed that the combination of two nanobodies (anti-VEGFR2/NRP-1 nanobodies) significantly inhibited proliferation as well as tube formation of human endothelial cells effective than a single nanobody. In addition, the mixture of both nanobodies inhibited vascularization of chick chorioallantoic membrane ex ovo CAM assay as compared to a single nanobody. Moreover, the mixture of both nanobodies significantly inhibited tumor growth of the mice (tumor volume and weight) higher than individual nanobodies (P < 0.05). CONCLUSION: Our results offer a promising role of combination therapies in cancer therapy as well as angiogenesis.