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.

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.

Anti-angiogenic biomolecules in neovascular age-related macular degeneration; therapeutics and drug delivery systems.

Blindness in the elderly is often caused by age-related macular degeneration (AMD). The advanced type of AMD known as neovascular AMD (nAMD) has been linked to being the predominant cause of visual impairment in these people. Multiple neovascular structures including choroidal neovascular (CNV) membranes, fluid exudation, hemorrhages, and subretinal fibrosis, are diagnostic of nAMD. These pathological alterations ultimately lead to anatomical and visual loss. It is known that vascular endothelial growth factor (VEGF), a type of proangiogenic factor, mediates the pathological process underlying nAMD. Therefore, various therapies have evolved to directly target the disease. In this review article, an attempt has been made to discuss general explanations about this disease, all common treatment methods based on anti-VEGF drugs, and the use of drug delivery systems in the treatment of AMD. Initially, the pathophysiology, angiogenesis, and different types of AMD were described. Then we described current treatments and future treatment prospects for AMD and outlined the advantages and disadvantages of each. In this context, we first examined the types of therapeutic biomolecules and anti-VEGF drugs that are used in the treatment of AMD. These biomolecules include aptamers, monoclonal antibodies, small interfering RNAs, microRNAs, peptides, fusion proteins, nanobodies, and other therapeutic biomolecules. Finally, we described drug delivery systems based on liposomes, nanomicelles, nanoemulsions, nanoparticles, cyclodextrin, dendrimers, and composite vehicles that are used in AMD therapy.

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.

Highly in vitro anti-cancer activity of melittin-loaded niosomes on non-small cell lung cancer cells.

BACKGROUND: Development of promising medicines from natural sources, specially venom, is of highly necessitated to combat against life-threatening cancers. Non-small cell lung cancer (NSCLC) has a significant percentage of mortalities. Melittin, from bee venom, is a potent anticancer peptide but its toxicity has limited its therapeutic applications. Accordingly, this study aims to synthesize niosomes with suitable stability and capacity for carrying melittin as a drug. Additionally, it seeks to evaluate the anti-cancer activity of melittin-loaded niosomes on non-small cell lung cancer. METHODS: The niosome was prepared by thin film hydration method. Cytotoxicity and apoptosis were assessed on A549, Calu-3, and MRC5 cells. Real-time PCR was used to determine expression of apoptotic and pro-apoptotic Bax, Bcl2, and Casp3 genes. Immunocytochemistry (ICC) was also used to confirm expression of the abovementioned genes. Furthermore, wound healing assay was performed to compare inhibition effects of melittin-loaded niosomes with free melittin on migration of cancer cells. RESULTS: IC50 values of melittin-loaded niosomes for A549, Calu-3, and MRC5 cells were respectively 0.69 µg/mL, 1.02 µg/mL, and 2.56 µg/mL after 72 h. Expression level of Bax and Casp3 increased '10 and 8' and '9 and 10.5' fold in A549 and Calu-3, whereas Bcl2 gene expression decreased 0.19 and 0.18 fold in the mentioned cell lines. The cell migration inhibited by melittin-loaded niosomes. CONCLUSIONS: Melittin-loaded niosomes had more anti-cancer effects and less toxicity on normal cells than free melittin. Furthermore, it induced apoptosis and inhibited cancer cells migration. Our results showed that melittin-loaded niosomes may be a drug lead and it has the potential to be future developed for lung cancer treatment.

Specific Targeting of Zinc Transporter LIV-1 with Immunocytokine Containing Anti-LIV-1 VHH and Human IL-2 and Evaluation of its In vitro Antitumor Activity.

BACKGROUND: Interleukin 2 (IL-2) is a vital cytokine in the induction of T and NK cell responses, the proliferation of CD8+ T cells, and the effective treatment of human cancers such as melanoma and renal cell carcinoma. However, widespread use of this cytokine is limited due to its short half-life, severe toxicity, lack of specific tumor targeting, and activation of Treg cells mediated by high-affinity interleukin-2 receptors. OBJECTIVE: In this study, a tumor-targeting LIV-1 VHH-mutIL2 immunocytokine with reduced CD25 (α chain of the high-affinity IL-2 receptor) binding activity was developed to improve IL-2 half-life by decreasing its renal infiltration in comparison with wild and mutant IL-2 molecules. METHODS: The recombinant immunocytokine was designed and expressed. The biological activity of the purified fusion protein was investigated in in vitro and in vivo experiments. RESULTS: The fusion protein represented specific binding to MCF7 (the breast cancer cell line) and more efficient cytotoxicity than wild-type IL-2 and mutant IL-2. The PK parameters of the recombinant immunocytokine were also improved in comparison to the IL-2 molecules. CONCLUSION: The observed results showed that LIV1-mIL2 immunocytokine could be considered as an effective agent in the LIV-1-targeted treatment of cancers due to its longer half-life and stronger cytotoxicity.

A novel nanobody-based immunocytokine of a mutant interleukin-2 as a potential cancer therapeutic.

The immunotherapeutic application of interleukin-2 (IL-2) in cancer treatment is limited by its off-target effects on different cell populations and insufficient activation of anti-tumor effector cells at the site of the tumor upon tolerated doses. Targeting IL-2 to the tumor microenvironment by generating antibody-cytokine fusion proteins (immunocytokine) would be a promising approach to increase efficacy without associated toxicity. In this study, a novel nanobody-based immunocytokine is developed by the fusion of a mutant (m) IL-2 with a decreased affinity toward CD25 to an anti-vascular endothelial growth factor receptor-2 (VEGFR2) specific nanobody, denoted as VGRmIL2-IC. The antigen binding, cell proliferation, IFN-γ-secretion, and cytotoxicity of this new immunocytokine are evaluated and compared to mIL-2 alone. Furthermore, the pharmacokinetic properties are analyzed. Flow cytometry analysis shows that the VGRmIL2-IC molecule can selectively target VEGFR2-positive cells. The results reveal that the immunocytokine is comparable to mIL-2 alone in the stimulation of Primary Peripheral Blood Mononuclear Cells (PBMCs) and cytotoxicity in in vitro conditions. In vivo studies demonstrate improved pharmacokinetic properties of VGRmIL2-IC in comparison to the wild or mutant IL-2 proteins. The results presented here suggest VGRmIL2-IC could be considered a candidate for the treatment of VEGFR2-positive tumors.

Tuning spacer length improves the functionality of the nanobody-based VEGFR2 CAR T cell.

BACKGROUND: The chimeric antigen receptor-expressing T (CAR-T) cells for cancer immunotherapy have obtained considerable clinical importance. CAR T cells need an optimized intracellular signaling domain to get appropriately activated and also for the proper antigen recognition, the length and composition of the extracellular spacer are critical factors. RESULTS: We constructed two third-generation nanobody-based VEGFR2-CARs containing either IgG1 hinge-CH2-CH3 region or hinge-only as long or short extracellular spacers, respectively. Both CARs also contained intracellular activating domains of CD28, OX40, and CD3ζ. The T cells from healthy individuals were transduced efficiently with the two CARs, and showed increased secretion of IL-2 and IFN-γ cytokines, and also CD69 and CD25 activation markers along with cytolytic activity after encountering VEGFR2+ cells. The VEGFR2-CAR T cells harboring the long spacer showed higher cytokine release and CD69 and CD25 expression in addition to a more efficient cytolytic effect on VEGFR2+ target cells. CONCLUSIONS: The results demonstrated that the third-generation anti-VEGFR2 nanobody-based CAR T cell with a long spacer had a superior function and potentially could be a better candidate for solid tumor treatment.

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.

An overview on application of phage display technique in immunological studies

Phage display is very strong technique in drug discovery and development. Phage display has many applications in improving the immunological studies. Development of mono-clonal antibody, peptides, peptidomimetics and epitope mapping are main application of phage display. Selection of monoclonal antibody or peptides that are displayed on the surface of the phages can be occurred through biopanning process. In biopanning process phage library is incubated with antigen and particular phages can be identified and iso-lated. Increasing the stringency in the biopanning rounds can be help to select phages with high affinity and specificity. Here, we describe an overview of phage display application with focusing on monoclonal antibody production and epitope mapping.