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Background and purpose: SARS-CoV-2 causes a severe respiratory disease known as COVID-19 and is responsible for a global viral pandemic. The SARS-CoV-2 receptor binding domain (RBD) is located on the spike protein, which identifies and binds to the angiotensin-converting enzyme 2 (ACE2) receptor. The RBD is an important target for developing virus-neutralizing antibodies, vaccines, and inhibitors. Experimental approach: In this study, recombinant SARS-CoV-2 RBD was expressed in E. coli BL21 (DE3) and purified and its binding activity was determined. Purification was conducted using the Ni-NTA column. ELISA. flow cytometry assays were set to evaluate the binding ability of recombinant RBD to different anti-RBD antibodies and native ACE2 receptors on HEK293A cells, respectively. Findings/Results: The SDS-PAGE analysis revealed the corresponding band at 27 kDa in the culture after induction with 0.7 mM IPTG, while the corresponding band was not observed in the culture without IPTG induction. ELISA results showed that antibodies produced in the human sera could bind to the recombinant RBD protein and the commercial anti-RBD antibody. Also, flow cytometry analysis revealed that the recombinant RBD could bind to human ACE2 on the surface of HEK293A cells. Conclusion and implication: Our outcomes displayed that the recombinant RBD expressed in the E. coli strain has biological activity and can be used as an antigen for the development of diagnosis kits and vaccines as well as a tool for screening drugs against SASR-CoV-2.

Background and purpose: The global emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has prompted widespread concern. Bacteriophages have recently gained attention as a cost-effective and stable alternative for vaccine development due to their adjuvant properties. This study aimed to design and validate a poly epitope composed of viral proteins. Experimental approach: SARS-CoV-2 proteins (spike, nucleocapsid, membrane, envelope, papain-like protease, and RNA-dependent RNA polymerase) were selected for analysis. Immunoinformatic methods were employed to predict B and T cell epitopes, assessing their antigenicity, allergenicity, and toxicity. Epitopes meeting criteria for high antigenicity, non-allergenicity, and non-toxicity were linked to form poly epitopes. These sequences were synthesized and cloned into pHEN4 plasmids to generate Poly1 and Poly2 phagemid vectors. Recombinant Poly1 and Poly2 phages were produced by transforming M13ΔIII plasmids and phagemid vectors into E. coli TG1. Female Balb/c mice were immunized with a cocktail of Poly1 and Poly2 phages, and their serum was collected for ELISA testing. Interferon-gamma (IFN-γ) testing was performed on spleen-derived lymphocytes to evaluate immune system activation. Findings/Results: Recombinant Poly1 and Poly2 phages were produced, and their titer was determined as 1013 PFU/mL. Efficient humoral immune responses and cellular immunity activation in mice were achieved following phage administration. Conclusion and implication: Poly epitopes displayed on phages exhibit adjuvant properties, enhancing humoral and cellular immunity in mice. This suggests that phages could serve as adjuvants to bolster immunity against SARS-Cov-2. Recombinant phages could be applied as effective candidates for injectable and oral vaccine development strategies.

Borna disease virus (BDV) is a neurotropic, enveloped and ribonucleic acid (RNA) virus. BDV induces persistent neurologic disease in a wide host range included several vertebrate species as well as human. The BDV genome encodes 6 proteins but p24 protein was identified at higher rates than other proteins at BDV-infected tissues. In this study, BDV-p24 protein was constructed and subcloned into expression plasmid pET22. Confirmation of recombinant protein expression was performed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and western blotting. P24 protein was injected into rabbits with the aim of polyclonal antibody production and immunization. Compared to other diagnostic methods, ELISA is a fast method with cost effective and high sensitivity as well as lower probability of contamination. ELISA method was performed to evaluate the infection in laboratory rabbits and retrospective infection was examined in 50 rabbits. The obtained results in this study indicated that the ELISA method based on p24 protein has a high potential to detect BDV infection.

Abstract 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.

Abstract 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.

Objective: Despite of antiviral drugs and successful treatment, an effective vaccine against hepatitis C virus (HCV)infection is still required. Recently, bioinformatic methods same as prediction algorithms, have greatly contributed tothe use of peptides in the design of immunogenic vaccines. Therefore, finding more conserved sites on the surfaceglycoproteins (E1 and E2) of HCV, as major targets to design an effective vaccine against genetically different virusesin each genotype was the goal of the study. Materials and Methods: In this experimental study, 100 entire sequences of E1 and E2 were retrieved from the NCBIwebsite and analyzed in terms of mutations and critical sites by Bioedit 7.7.9, MEGA X software. Furthermore, HCV-1asamples were obtained from some infected people in Iran, and reverse transcriptase-polymerase chain reaction (RTPCR)assay was optimized to amplify their E1 and E2 genes. Moreover, all three-dimensional structures of E1 andE2 downloaded from the PDB database were analyzed by YASARA. In the next step, three interest areas of humoralimmunity in the E2 glycoprotein were evaluated. OSPREY3.0 protein design software was performed to increase theaffinity to neutralizing antibodies in these areas. Results: We found the effective in silico binding affinity of residues in three broadly neutralizing epitopes of E2glycoprotein. First, positions that have substitution capacity were detected in these epitopes. Furthermore, residuesthat have high stability for substitution in these situations were indicated. Then, the mutants with the strongest affinityto neutralize antibodies were predicted. I414M, T416S, I422V, I414M-T416S, and Q412N-I414M-T416S substitutionstheoretically were exhibited as mutants with the best affinity binding. Conclusion: Using an innovative filtration strategy, the residues of E2 epitopes which have the best in silico bindingaffinity to neutralizing antibodies were exhibited and a distinct peptide library platform was designed.

Objective(s): In this study, Boltorn® H40-PEG-MTX-anti-VEGFR2 nanobody was fabricated in which nanobody was selected for blocking the receptor, H40 as a nanocarrier for delivery of methotrexate (MTX) to the tumor cells, and polyethylene glycol (PEG) moieties for improving the blood circulation time and safety. Materials and Methods: The synthesis process of the nanosystem has been characterized by different analytical methods. Results: The prepared nanoplatform exhibited high drug loading capacity, excellent colloidal stability, and an average particle size of around 105 nm. MTX was successfully conjugated through ester bonds and its release profile clearly showed that the ester bond is in favor of releasing the drug in acidic pH (5.5). The cytotoxicity of the developed nanoplatform exhibited great anti-cancer activity against MCF7 and KDR293 (cells with overexpressed anti-VEGFR2 NB receptors) cell lines while no deleterious toxicity was observed for nanocarrier against HEK293 normal cells. Furthermore, both hemolysis and LD50 assay results confirmed the hemocompatibility and biocompatibility of the developed nanoplatform. Conclusion: The most striking result to derive from the data is that the designed nanoplatform could potentially inhibit cell migration and invasion and the anti-angiogenesis properties of the developed nanoplatform may serve as a promising nanosystem to suppress the formation of blood vessels around tumor cells and consequently inhibit tumor progression.

Abstract Interleukin-2 (IL-2) is an important cytokine in survival, expansion, function of CD8+ T cells and natural killer cells in immunotherapy of melanoma and renal cell carcinomas. Its severe toxicity following binding to its high affinity IL-2 receptor alpha (IL-2Rα) has restricted its application in cancer patients. In the present study, we investigated the antitumor efficacy and cytotoxicity of a mutated human IL-2 previously designed by selective amino acid substitutions, and its reduced affinity towards high-affinity IL-2Rα (CD25) was approved compared to the wild type IL-2 (wtIL-2). Furthermore, their ability to induce PBMC cell proliferation, and interferon-gamma secretion was compared. The mutant IL-2 also represented higher antitumor activity and more efficient cytotoxicity than wild type hIL-2. The developed mutant IL-2 can be an alternative tool in IL-2 associated immunotherapy of various cancers.

Objective(s): One of the important interactions in controlling the human immune system is the reaction between checkpoint proteins such as programmed cell death-1 (PD-1) and its ligand, PD-L1. These are negative immunoregulatory molecules that promote immune evasion of tumor cells. PD-L1 expression is an immune-mediated mechanism used by various malignant cells in order to down-regulate the immune system. Checkpoint inhibitors (CPIs) are a new class of anti-cancer agents that stimulate immune cells to elicit an antitumor response by blocking the ligand and receptor interactions. Nanobody (Nb) as a new type of antibody fragment, has some potential as CPI.Materials and Methods: A female camel was immunized with recombinant PD-L1 protein, nanobody library was constructed and PD-L1 specific Nb was selected. The selected Nb was characterized in terms of affinity, specificity, and binding potency in ELISA, Western blotting, and flow cytometry. Results: Developed nanobody, A22 binds to its cognate target with high specificity and affinity. Western blot and flow cytometry techniques showed that nanobody A22 was able to specifically detect and attach to human PD-L1 protein on the cell surface and in the cell lysate. MTT assay showed the inhibitory effect of PD-L1 by specific Nb on A431 and HEK293 cells, with no cytotoxic effect on cell growth.Conclusion: The results highlighted the potential of anti-PD-L1 Nb as a novel therapeutic in cancer therapy without undesirable cytotoxicity.

Objective(s): A variety of signaling molecules have been identified that play a role in angiogenesis, of prime importance, vascular endothelial growth factor (VEGF) and its resceptor (VEGFR), which is highly expressed in most human solid tumors. Targeting VEGF or/and VEGFR with immunotoxin may be a promising approach to directly affect cancer cells. Immunotoxins are for targeted treatment comprising two functional moieties, an antibody that binds to target cells along with toxin that kills molecules. Materials and Methods: In this study, an immunotoxin comprising domain of diphtheria toxin subunit A (DT386) genetically fused to mouse VEGF (mVEGF-DT) was developed. The second construct, which contains the DT386 domain, was made to investigate the action of the DT386 domain on tumor cells. Both gene constructs were cloned, expressed, and were further purified. The biological activity of mVEGF-DT and DT386 proteins was assessed on the TC1 cell line bearing mouse model. Proteins were injected intra-tumoral in mice, in separate groups. Results: Tumors in the mVEGF-DT group started to dwindle after six injections, but tumor size in both control groups (DT386 and PBS), continued to grow. Conclusion: Successful targeting of solid tumor cells by mVEGF-DT immunotoxin demonstrates the therapeutic potential utility of these conjugates for tumor targeting.

Objective(s): SLC39A6 (solute carrier family 39) or LIV-1, is a zinc-transporter protein associated with estrogen-positive breast cancer and its metastatic spread. Significantly there is a direct relation between high zinc intake and unregulated proliferation and cancers. Blocking SLC39A6 protein may result in reduced metastasis and proliferation in many malignant tumors. This study aimed to develop an anti-SLC39A6 nanobody that is able to detect and block the SLC39A6 protein on the surface of cancerous cells.Materials and Methods: The recombinant SLC39A6 was expressed and used for camel immunization. The VHH library was constructed and screened for SLC39A6-specific nanobody. Then, the strength of nanobody in SLC39A6 detection was evaluated by Western blotting and flow cytometry.Results: We showed the ability of nanobody to detect SLC39A6 by Western blotting and flow cytometry and the specificity of the C3 nanobody for the SLC39A6 antigen. Furthermore, the selected nanobody potently inhibits cell proliferation.Conclusion: These data show the potential of SLC39A6-specific nanobody for the blockade of zinc transportation and provide a basis for the development of novel cancer therapeutics.

For adenoviruses (Ads) to be optimally effective in cancer theranostics, they need to be retargeted toward target cells and lose their natural tropism. Typically, this is accomplished by either engineering fiber proteins and/or employing bispecific adapters, capable of bonding Ad fibers and tumor antigen receptors. This study aimed to present a simple and versatile method for generating Ad-based bionanoparticles specific to target cells, using the SpyTag-SpyCatcher system. The SpyTag peptide was inserted into the HI loop of fiber-knob protein, which could act as a covalent anchoring site for a targeting moiety fused to a truncated SpyCatcher (SpyCatcherΔ) pair. After confirming the presence and functionality of SpyTag on the Ad type-5 (Ad5) fiber knob, an adapter molecule, comprising of SpyCatcherΔ fused to an anti-vascular endothelial growth factor receptor 2 (VEGFR2) nanobody, was recombinantly expressed in Escherichia coli and purified before conjugation to fiber-modified Ad5 (fmAd5). After evaluating fmAd5 detargeting from its primary coxsackie and adenovirus receptor (CAR), the nanobody-decorated fmAd5 could be efficiently retargeted to VEGFR2-expressing 293/KDR and human umbilical vein endothelial (HUVEC) cell lines. In conclusion, a plug-and-play platform was described in this study for detargeting and retargeting Ad5 through the SpyTag-SpyCatcher system, which could be potentially applied to generate tailored bionanoparticles for a broad range of specific targets; therefore, it can be introduced as a promising approach in cancer nanotheranostics.

Objective(s): Cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) is the most important human immune checkpoint that modulates T cells activity and brings about immune-homeostasis. Accordingly, checkpoint inhibitor cancer therapy has been approved as a growing method to block over-expressed immune checkpoints, such as CTLA-4 receptors. Considering the competitive characteristics of single-domain antibodies with monoclonal antibodies, we tried to develop a camelid Nanobody against human CTLA-4. Materials and Methods: We have constructed the VHH gene library by using immunized-camel peripheral blood mononuclear cells and carrying out the Nested-PCR technique. VHH-library was screened by phage display technique and specific nanobodies against CTLA-4 protein were selected and amplified with bio-panning steps. Stronger binders were screened by Periplasmic Extract-ELISA, followed by estimating the complexity of the library. Specific anti-CTLA-4 Nanobody and 3hCTL55, with longer CDR3 and a higher binding rate, were selected for more assays. Results: Results revealed the existence of two different clones in the library with 108 binders. In comparison with seven different antigens, using the ELISA technique confirmed the specificity of Nanobody 3hCTL55 against human CTLA-4 antigen. We calculated Nanobody 3hCTL55 affinity for human CTLA-4 antigen at 50×10-9 M, approximately. Performing western blot and Flow-cytometry techniques showed that Nanobody 3hCTL55 was able to specifically detect and attach both commercial human CTLA-4 protein and human CTLA-4 antigen on the cell surface and in the cell lysate. Conclusion: Taken together, this developed camelid-specific anti-CTLA-4 Nanobody 3hCTL55, selected from a high-quality immune library by phage display technique, may be effective for further study about cancer diagnosis and cancer-therapy purposes.

Background: Hemiscorpius lepturus is one of the most dangerous scorpions in Iran and the world. Numerous studies have been conducted on phospholipases, especially phospholipase D, in this scorpion’s venom, and the results have shown this protein to be the main cause of death. Therefore, one of the most effective ways of preventing fatalities is to produce a toxoid vaccine from the deadly toxin of the venom. The present study was conducted to assess the non-toxicity of this toxoid and the safety of the vaccine candidate in BALB/c mice. Methods: The production of interferon-gamma and interleukin-4 cytokines in the spleen cells of the mice was measured using ELISpot assay 28 days following immunization with rPLD toxoid. Results: The unpaired t-test results showed a significant increase in the concentration of IFN-γ cytokine in the vaccinat­ed mice (P= 0.001), indicating that the immune system is directed toward the Th1 pattern, while no significant differ­ence was observed in the levels of IL-4 (P= 0.16) despite an increase in this cytokine. The in-vivo tests showed that the mice immunized with interval doses of 80µg of toxoid were completely protected against 10 × the LD100 of the venom. Moreover, the toxoid had no dermonecrotic effects and caused no necrotic and inflammatory complications in the rabbit skin. Conclusion: As a vaccine, the toxoid has the potential to increase the Th1 cytokine response and, subsequently, in­crease acquired cellular immunity. Thus, this toxoid appears to be able to provide an effective vaccine against the ven­om of Hemiscorpius lepturus.

Background: SLC39A6 Protein (solute carrier family 39) or LIV-1 is a zinc transporter protein that is overexpressed in positive estrogen cancers such as breast cancer. The LIV-1 protein transfer zinc into the cytoplasm through the plasma membrane. Today it is known that just as a decrease in the concentration of zinc in the cell can cause cancer, an excessive increase in the concentration of zinc can also stimulate irregular cell division and caused cancer. Thus, inhibition of zinc transporter protein may play a role in preventing malignancies and metastasis. It can also be used as a diagnostic marker in the diagnosis of cancers in various laboratory methods. The present study was performed to prepare a polyclonal camel antibody for the detection of LIV-1 protein at the cell surface. Methods: This study was started in the Pasture Institute of Iran in 2018 September and finished in February 2020. An expression construct containing the human LIV-1 gene was prepared and transferred to the E.coli BL21 by chemical (CaCl2) and heat shock method. The expression of the protein was induced by IPTG and then protein was purified by affinity (Ni-NTA) chromatography. After preparing recombinant protein one female camel was immunized, 6 times at two weeks intervals with Freundchr('39')s adjuvant. After immunization, the isolated polyclonal antibody was evaluated by ELISA, western blotting and flow cytometry in the detection of LIV-1 protein. Results: The result showed that LIV-1 protein was well purified and also the camel polyclonal antibody was able to detect LIV-1 protein in ELISA, western blot and also it can detect LIV-1 on the cell surface as shown by flow cytometry test. Conclusion: In recent years, LIV-1 has been shown to be a good candidate as a marker in breast cancer, so polyclonal antibodies against LIV-1 can be used for early detection of breast cancer by various diagnostic methods. In this study, it has been shown that polyclonal camel antibodies can be used in laboratory methods and can be considered for immunological tests and therapeutic applications.

Objective(s): Emergence of resistant tumor cells to the current therapeutics is the main hindrance in cancer treatment. Combination therapy, which mixes two or more drugs, is a way to overcome resistant problems of cancer cells to current treatments. Nanobodies are promising tools in cancer therapy due to their high affinity as well as high penetration to tumor sites. Materials and Methods: Here, the inhibitory effect of mixtures of two nanobodies (anti-vascular endothelial growth factor (VEGF) and anti-neuropilin-1 (NRP-1) nanobodies) on tube formation of human endothelial cells in vitro and ex vivo were analyzed.Results: Results showed that combination of two drugs significantly inhibited proliferation and tube formation of human endothelial cells. In addition, mixtures of two nanobodies inhibited angiogenesis in chick chorioallantoic membrane (CAM) assay efficiently compared with each individual nanobody. Conclusion: Results highlight the efficacy of combination therapy of cancer compared with mono-therapy and promises development of novel anti-cancer therapeutics based on nanobodies targeting two or more targets of tumor cells.

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.

Although high-dose IL-2 has clear antitumor effects, severe side effects like severe toxicity and activation of Tregs by binding of IL-2 to high-affinity IL-2R, hypotension, and vascular leak syndrome limit its applications as a therapeutic antitumor agent. Here in this study, a rational computational approach was employed to develop and design novel triple-mutant IL-2 variants with the aim of improving IL-2-based immunotherapy. The affinity of the mutants towards IL-2Rα was further computed with the aid of molecular dynamic simulations and umbrella sampling techniques and the obtained results were compared to those of wild-type IL-2. In vitro experiments by flow cytometry showed that the anti-CD25 mAb was able to bind to PBMC cells even after mutant 2 preincubation, however, the binding strength of the mutant to α-subunit was less than of wtIL-2. Additionally, reduction of IL-2Rα subunit affinity did not significantly disturb IL-2/IL2Rβγc subunits interactions.

Objective(s): In this study, the neutralizing abilities of the equine and the recently introduced camelid antivenoms on the hemodynamic parameters (inotropism, chronotropism, and arrhythmogenicity) were assessed following envenomation by Hemiscorpius lepturus venom in rats. Materials and Methods: At first, the electrophoretic profiles of both products were obtained by using the SDS-PAGE method (12.5%) and stained with Coomassie blue and silver nitrate. Secondly, different doses of the camelid antivenom (10, 50, and 100 µl) were given intravenously in 10 min before venom injection (400 µg/rat). The neutralizing potencies of camelid and equine antivenoms were measured by preincubation (100 µl) with H. lepturus venom for 30 min at room temperature. Finally, equal amounts of the antivenoms were injected intravenously to observe the hemodynamic changes.Results: Based on the electrophoretic profile, it was evident that undesired proteins significantly decreased in equine antivenom, owing to impurities. Pretreatment with the camelid antivenom (100 µl), neutralized the elevation of the mean arterial pressure evoked with scorpion venom injection (88.15±4.56 versus 10.2±1.23 percent at the 8th min). The Incubation of the venom and the camelid antivenom counteracted the hemodynamic changes, but the equine product had no effect. The intravascular injection of the equine antivenom transiently increased the mean arterial pressure as compared to the control (108.67±8.63 mmHg versus 52.67±1.93 mmHg at the 10th min).Conclusion: The most obvious finding emerging from this study was that the camelid antivenom neutralized the hemodynamic changes in rats significantly, but in comparison, the equine antivenom had just a minor ability.

While monoclonal antibodies are efficient therapeutics for cancer treatment, nanobodies or variable heavy domain – due to their small size, high stability, and solubility – have many advantages in comparison. Oligoclonal nanobodies are a mixture of nanobodies against different epitopes of an antigen. Specific nanobodies against vascular endothelial growth factor (VEGF, which has an important role in cancer angiogenesis) were selected from an immune camel library using biopanning. Specific binding of the nanobodies to VEGF antigen was assessed by periplasmic extract enzyme-linked immunosorbent assay (ELISA). Bioinformatics analysis and molecular docking were performed on selected nanobodies against VEGF. The in vitro inhibitory effects of each single nanobody, as well as a pool of selected nanobodies (oligoclonal nanobodies), on proliferation and tube formation by/in human umbilical vein endothelial cells (HUVEC) cells was evaluated using MTT and Tube formation assays, respectively. Four nanobodies showed the highest signal intensity in the periplasmic extract ELISA. Sequencing revealed that four unique nanobodies with different CDR3 rejoin were selected. Oligoclonal nanobodies inhibited proliferation and tube formation of the HUVEC cells more potently than did each individual nanobody. Taken together, this data from this study suggests that in vitro use of nanobodies (in an oligoclonal mode) that target distinct epitopes on VEGF could be promising as a novel therapy to treat VEGF-dependent pathologies. However, this needs to be further tested in in vivo studies.

Objective(s): Filamentous bacteriophage M13 was genetically engineered to specifically target mammalian cells for gene delivery purpose. Materials and Methods: A vascular endothelial growth factor receptor 2 (VEGFR2)-specific nanobody was genetically fused to the capsid gene III of M13 bacteriophage (pHEN4/3VGR19). A mammalian expression construct containing Cop-green fluorescent protein (Cop-GFP), as a reporter gene, was amplified by PCR and then sub-cloned in the pHEN4/3VGR19 phagemid. The resulting construct was transfected into 293KDR cell. The recombinant phage was extracted and confirmed and then transduced into VEGFR2 expressing cell (293KDR). Results: Seventy-two hr after transfection, green fluorescence was detected in 30% of the cells. About 1% of the cells which transduced by recombinant phages were able to express GFP. Conclusion: It is hoped that the results from this study will help to find potential vectors to improve the efficiency of gene delivery. Taken together, we conclude that this newly-introduced vector can be used in cancer researches.

Streptococcus pneumoniae is a major pathogen in human respiratory tract which causes significant morbidity and mortality across from the world. Currently available vaccines are not completely effective and cannot cover all pathogenic strains so there is an important need to develop an alternative cost-effective vaccine, based on conserved protein antigens. Pneumococcal surface protein A (PspA) is one of interesting candidates for development of a serotype-independent vaccine against pneumococcal infections. PspA is grouped into two major families with five clades, and broad-reacting PspA-based vaccines should contain at least one functional fragment from each of the two families.In this study, we developed two immunogenic antigens based on recombinant PspA proteins that including the different antigenic regions of PspA from both two families. The cross-reactivity of antibodies elicited against two PspA proteins PspAB1-5 and PspA4ABC and their role in complement deposition with three strains of pneumococci were tested. The protective effects of developed anti-PspA antibodies in mice in intranasal and intraperitoneal challenges were evaluated using a strain from clade 2. Sera from immunized mice with PspAB1-5 in comparison with PspA4ABC was able to deposit more C3 complement component on surface of pneumococci bearing diverse PspA from both families 1 and 2, and immunized mice with the PspAB1-5 showed a higher protection than PspA4ABC in pneumococcal challenges.The obtained results from this study indicate that a PspA-based antigen composed of B region from all clades in addition to conserved domains, can provide a significant protection against multiple strains of S. pneumoniae and may overcome the limitation of polysaccharide vaccines. Keywords: Streptococcus pneumoniae, Pneumococcal surface protein A (PspA), Immune protection

Objective(s): Nanobodies, the single domain antigen binding fragments of heavy chain-only antibodies occurring naturally in camelid sera, are the smallest intact antigen binding entities. Their minimal size assists in reaching otherwise largely inaccessible regions of antigens. However, their camelid origin raises a possible concern of immunogenicity when used for human therapy. Humanization is a promising approach to overcome the problem. Materials and Methods: Here, we designed a humanized version of previously developed nanobody (anti vascular endothelial growth factor nanobody), evaluated and compared its predicted 3D structure, affinity and biological activity with its original wild type nanobody. Results: Our in silico results revealed an identical 3D structure of the humanized nanobody as compare to original nanobody. In vitro studies also demonstrated that the humanization had no significant visible effect on the nanobody affinity or on its biological activity. Conclusion: The humanized nanobody could be developed and proposed as a promising lead to target pathologic angiogenesis.

Plants can be exploited for the production of various recombinant proteins as valuable bioreactors because of many reasons. The use of plant viral vectors for the transient expression offers as a useful strategy for the large-scale production of proteins with pharmaceutical importance, such as antibodies within a very short time period. Today, cancer is the second cause of death in human society. Compelling evidence suggests that vascular endothelial growth factor (VEGF), due to its essential role in angiogenesis, is a critical target for cancer treatment. In This study, Anti-VEGF (Nb42) nanobody different fragments were transiently expressed under the control of a viral strong promoter in cucurbit plant using ZYMV-based viral vector. These fragments included natural nanobody sequence (Nb42I) and optimized nanobody sequence (Nb42II) for expression in plant. After inoculation of plants, the presence of transcripts of the Nb42 gene fragments in cucurbit inoculated plants was detected by using RT-PCR. Also, the recombinant protein expression was confirmed by Dot blotting, SDS-PAGE, Western blotting and ELISA. Based on the ELISA results, the expression level of Nb42I and Nb42II nanobody fragments was 2 and 6.8 μg/g of leaf tissue respectively. Taken together, recombinant Nb42 nanobody could be efficiently expressed in cucurbit plant and ZYMV-based expression system would be an appropriate platform for production of recombinant proteins in cucurbit plants

Phage display is very strong technique in drug discovery and development. Phage display has many applications in improving the immunological studies. Development of monoclonal 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 isolated. 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.

Objective(s): Lung cancer is the main leading cause of cancer death worldwide. Angiogenesis is the main step in proliferation and spreading of tumor cells. Targeting vascular endothelial growth factor (VEGF) is an effective approach for inhibition of cancer angiogenesis. Nanobodies (NBs) are a novel class of antibodies derived from the camel. Unique characteristics of Nbs like their small size and good penetration to tumor tissues makes them promising tools in drug development. Development of NBs targeting both human and mouse VEGF is required for understanding their in vivo functions. Therefore, development of cross-species reactive anti-VEGF Nbs for immunotherapy of lung cancer was the main aim of the current study. Materials and Methods: Here we developed NBs from Camelus dromedarius library with high specificity and binding affinity to both human and mouse VEGF. In vitro and In vivo function of developed NB was evaluated on human endothelial cells and lung epithelial tumor cells (TC-1). Results: A nanobody showed the highest affinity to human and mouse VEGF and potently inhibited VEGF in the ELISA experiment. Anti-VEGF NBs significantly inhibited in vitro human endothelial cell migration through blockade of VEGF (P=0.045). Anti-VEGF NBs also significantly inhibited in vivo TC-1 growth in a dose-dependent manner (P=0.001) and resulted in higher survival rate in the nanobody treated group Conclusion: These findings demonstrate the potential of anti-VEGF NBsin tumor growth inhibition and are promising as novel cancer therapeutic candidate.

Acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL) are common acute leukemia in adults and children, respectively. In these malignancies, chemotherapy is the main treatment strategy that fails in many cases and is usually associated with adverse effects on healthy cells. In this regard, the development of new therapies is essential. Monoclonal antibodies directed to the cell surface markers of leukemic blasts may have promising consequences with minimal toxic effects on normal cells. Since cluster of differentiation 45Ra (CD45Ra) and CD123 antigens, two considered surface markers of leukemic blasts in AML and ALL respectively, are overexpressed on AML and ALL blasts, CD34+ leukemic progenitors, and AML-LSCs in comparison with normal hematopoietic stem cells (HSCs), they were selected to be targeted; using specific monoclonal antibodies. In this project, CD45Ra+ cells and CD123+ cells were targeted by anti-CD45Ra and/or anti-CD123 monoclonal antibodies. Cytotoxicity effect and cell death induction was determined by 3-(4,5-dimethylthiazol-2-yl)-2–5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. Changes in the expression profile of MCL1, cMyc, Survivin, Id1, and PIM1 genes were assessed by real-time PCR. Statistical analysis of the results showed effective antibody-mediated cytotoxicity and induction of apoptosis in KG1α (CD45Ra+) and Nalm6 (CD123+) cell lines. Also, a significant change in the expression level of some of the apoptosis-related genes was observed. According to the results of this study, it can be concluded that an effective targeting of AML and ALL cancerous cell lines can be performed by anti-CD45Ra and anti-CD123 monoclonal antibodies through their effector functions and apoptosis induction.

In the present study, rainbow trout weighting 3±0.3 g were vaccinated with an oral DNA vaccine encoding VP2 gene of a prevalent isolate of IPNV in Iranian trout farms encapsulated in sodium alginate microspheres and Chitosan tripolyphosphate (CS/TPP) nanoparticles. The vaccinated fish were then challenged with a virulent isolate of IPNV at 30 days post-vaccination. The transcriptional changes of adaptive- immune genes (IgM and IgT), as well as the VP4 gene of IPNV, as an indicators of viral replication were studied 45 days post-challenge. Analysis of RT-qPCR data showed lower levels of VP4 gene expression in the oral DNA vaccinated trout after IPNV challenge compared with the control one. Moreover, the constructed DNA vaccine did not enhance the expression of IgM and IgT genes above the levels observed in the carrier control group but it showed a mimic of viral activity and contributes to maintaining them at appreciable levels in vaccinated group.

Objective(s): As T-cell immunoglobulin and mucin domain 3 (TIM-3) is an immune regulatory molecule; its blocking or stimulating could alter the pattern of immune response towards a desired condition. Based on the unique features of nanobodies, we aimed to construct an anti-TIM-3 nanobody as an appropriate tool for manipulating immune responses for future therapeutic purposes. Materials and Methods:We immunized a camel with TIM-3 antigen and then, synthesized a VHH phagemid library from its B cell’s transcriptome using nested PCR. Library selection against TIM-3antigen was performed in three rounds of panning. Using phage-ELISA, the most reactive colonies were selected for sub-cloning in soluble protein expression vectors. The Nanobody was purified and confirmed with a nickel-nitrilotriacetic acid (Ni-NTA) column, SDS-PAGE and Western blotting. A flowcytometric analysis was performed to analyze the binding and biologic activities of theTIM-3 specific nanobody with TIM-3 expressing HL-60 and HEK cell lines. Results:Specific 15kD band representing for nanobody was observed on the gel and confirmed with Western blotting. The nanobody showed significant specific immune-reactivity against TIM-3 with a relatively high binding affinity. The nanobody significantly suppressed the proliferation of TIM-3 expressing HL-60 cell line. Conclusion: Finally, we successfully prepared a functional anti-humanTIM-3 specific nanobody with a high affinity and an anti-proliferative activity on an AML cell line in vitro.

Current therapies for acute myeloid leukemia (AML), are associated with high relapse rates. Hence, development of new therapeutic strategies is crucial to circumvent this problem. Bivalent antibody technology has been used to engineer novel antibody fragments with increased avidity, by assembling two scFv in a single molecule. Here, we present accompanying data from construction and characterization experiments of a biscFv antibody targeting CD123, the most important biomarker of leukemic cancer stem cells which play a key role in relapsed AML after chemotherapy. Data in this article are related to the research paper “Development of a novel engineered antibody targeting human CD123” Moradi-Kalbolandi S. et al. (2016) [1].

Integrons play an important role in multidrug resistance. The integron platform codes for integrase (intI) that is required for gene cassette integration through site-specific recombination. The recombination crossover occurs between the G and TT nucleotides in non-palindromic attI and palindromic attC sites. The aim of this study was to establish an efficient in vitro assay for integrase purification and activity detection. To this end, the intI gene was cloned into the pET-22b plasmid. Then, the resulting recombinant plasmid was transformed into Escherichia coli Origami™ strain. The recombinant protein expression was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot assays. The recombinant intI protein was purified by nickel–nitrilotriacetic acid (Ni–NTA) affinity chromatography, and its activity was measured by a newly introduced assay. Briefly, specific primers for each side of attI and attC were used, thereby, a polymerase chain reaction would be performed, if a fused plasmid containing both attI and attC sites was created upon recombination. SDS-PAGE and western blotting confirmed the presence of a 38-kDa recombinant protein. Optimum conditions were established for the measurement of the integrase activity and a new model assay was conducted to analyse the recombination activity in vitro. Although the electrophoretic mobility shift assay is an efficient and reliable method, the newly introduced assay provided new or enhanced capability to determine the integrase activity, suggesting that there is no need for expensive and advanced equipment.

The aim of this study was to produce a recombinant protein consisting of the catalytic and translocation domains of diphtheria toxin for its later application as a vaccine candidate against Corynebacterium diphtheria. To achieve this goal, at first, the amino acid sequence of DT386 was used for prediction of T and B cell epitopes using on-line servers. The DT386 coding sequence was synthesized and subcloned into the NcoI and XhoI sites of pET28a plasmid and recombinant pET28a plasmid was used to transform Escherichia coli BL21 (DE3) host cells. Afterwards, recombinant cells were selected and subjected to induction of expression by 1 mM isopropyl β-D-1-thiogalactopyranoside, (IPTG). Expression of the desired protein was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, and finally, the recombinant protein was purified using nickel affinity chromatography. The results of epitope prediction using on-line servers established the ability of DT386 for stimulation of immune system against diphtheria toxin. Restriction digestion of the recombinant plasmids using NcoI and XhoI enzymes confirmed the fidelity of cloning by producing a band of about 1200 bp. SDS-PAGE analysis following induction of expression and also purification step confirmed the expression of the desired protein by showing a band of about 45 kDa. In addition, Western blot analysis using anti-6X-His antibody confirmed the identity of the expected protein. In conclusion, in the present study we amplified and cloned the coding sequence of DT386 fragment, followed by its expression by E. coli BL21 (DE3) cells. Then, the expressed protein was purified and will be used for later studies of evaluation of its immunogenic properties.

Purpose: Angiogenesis or formation of new blood vessels is an essential process for tumor growth, invasion and metastasis. Vascular Endothelial Growth Factor (VEGF) and its receptors play an important role in angiogenesis-dependent tumors. VEGF-A is the most important factor in angiogenesis process. Human VEGF-A gene consists of eight exons that undergoes alternative exon splicing and produce five different proteins consisting of 121, 145, 165, 189 and 206 amino acids (named VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206). Methods: In this study, VEGF121 gene synthesized and cloned into the pET-26b plasmid. The recombinant plasmid was transferred into appropriate expression strain of BL-21. Expression of VEGF121 induced by IPTG (Isopropyl β-D-1-thiogalactopyranoside) and confirmed by SDS-PAGE and Western-Blotting. Recombinant VEGF121 was purified by nickel affinity chromatography. HUVECs (Human Umbilical Vein Endothelia Cells) cells were isolated from umbilical vein and the effect of VEGF121 on tube formation of endothelial cells was investigated. Results: SDS-PAGE and Western-Blotting results verified the purification of VEGF121. The final yield of recombinant protein was about 5mg per liter. Endothelial cell tube formation assay results showed that VEGF121 leads to tube formation of endothelial cell on matrix and induces angiogenesis in vitro. Conclusion: Recombinant VEGF121 is important factor in tube formation of endothelial cell, so it could be used in different cancer researches and angiogenesis assay.

Hemoscorpius lepturus is the most medically important scorpion in Iran. The clinical signs of H. lepturus envenomation are remarkably similar to those reported for brown spiders, including dermonecrosis, hematuria, renal failure and even death. The lethality and toxicity of brown spiders’ venom have been attributed to its phospholipase D activity. This study aims to identify a phospholipase D with possible lethality and dermonecrotic activity in H. lepturus venom. In this study, a cDNA library of the venom glands was generated by Illumina RNA sequencing. Phospholipase D (PLD) from H. lepturus was characterized according to its significant similarity with PLDs from brown spiders. The main chain designated as Hl‐RecPLD1 (the first recombinant isoform of H. lepturus PLD) was cloned, expressed and purified. Sphingomyelinase, dermonecrotic and lethal activities were examined. Hl‐PLD1 showed remarkable sequence similarity and structural homology with PLDs of brown spiders. The conformation of Hl‐PLD1 was predicted as a “TIM beta/alpha‐barrel”. The lethal dose 50 (LD50) and dermonecrotic activities of Hl‐RecPLD1 were determined as 3.1 μg/mouse and 0.7 cm2 at 1 μg respectively. It is the first report indicating that a similar molecular evolutionary mechanism has occurred in both American brown spiders and this Iranian scorpion. In conclusion, Hl‐RecPLD1 is a highly active phospholipase D, which would be considered as the lethal dermonecrotic toxin in H. lepturus venom.

Background: Angiogenesis which occurs mandatory in solid tumors, is a critical step in malignancy progression. Vascular endothelial growth factor (VEGF) is mainly responsible for angiogenesis process and facilitates the formation of new vessels. Distribution of monoclonal antibodies against VEGF or VEGF receptor (VEGFR) into the solid tumors is limited because of their huge dimensions. Moreover, many investigations have demonstrated the usefulness of immunotoxins to halt angiogenesis in solid tumors. Materials and Methods: We designed, expressed and evaluated the cytotoxicity of a novel nano-immunotoxin composed of VEGF splice variant containing 121 amino acids (VEGF121) and truncated the exotoxin A of Pseudomonas aeruginosa (PE38-KDEL). The fusion protein VEGF121-PE38 was successfully cloned and expressed in Escherichia coli, purified by Ni+ 2 affinity chromatography. The fusion protein was subsequently subjected to refolding using the reduced and oxidized glutathione. Results: The expression level of the fusion protein reached to 1 mg/ml. The VEGF121-PE38 immunotoxin showed a 59 KDa MW which had cytotoxic effect on HUVEC and 293/KDR cells as low and high expressing VEGFR2 cells, respectively. But the cytotoxicity on 293/KDR was 100 folds more than that of VEGFR2 low expressing cell HUVEC. Conclusion: The designed immunotoxin showed more selectivity for higher VEGFR2 expressing cells in vitro.

Antibodies and their derivative fragments have long been used as tools in a variety of applications, in fundamental research work, biotechnology, diagnosis, and therapy. Camels produce single heavy-chain antibodies (VHH) in addition to usual antibodies. These minimal-sized binders are very robust and bind the antigen with high affinity in a monomeric state. Vascular endothelial growth factor recepror-2 (VEGFR2) is an important tumor-associated receptor that blockade of its signaling can lead to the inhibition of neovascularization and tumor metastasis. Here, we describe the construction, expression, and purification VEGFR2-specific Diabody. Two variable fragments of a same camel anti-VEGFR2 antibody were linked together by the upper hinge segment of antibody to make a diabody. We showed the ability of diabody to recognition of VEGFR2 on the cell surface by FACS. Diabodies can be produced in the low-cost prokaryotic expression system, so they are suitable molecules for diagnostic and therapeutic issues.