Synthesis of alga-coated copper oxide nanoparticles with potential applications in shrimp farming.

Copper (Cu) is a crucial element that plays a vital role in facilitating proper biological activities in living organisms. In this study, copper oxide nanoparticles (CuO NPs) were synthesized using a straightforward precipitation chemical method from a copper nitrate precursor at a temperature of 85 °C. Subsequently, these NPs were coated with the aqueous extract of Sargassum angustifolium algae. The size, morphology, and coating of the NPs were analyzed through various methods, revealing dimensions of approximately 50 nm, a multidimensional shaped structure, and successful algae coating. The antibacterial activity of both coated and uncoated CuO NPs against Vibrio harveyi, a significant pathogen in Litopenaeus vannamei, was investigated. Results indicated that the minimum inhibitory concentration (MIC) for uncoated CuO NPs was 1000 µg/mL, whereas for coated CuO NPs, it was 500 µg/mL. Moreover, the antioxidant activity of the synthesized NPs was assessed. Interestingly, uncoated CuO NPs exhibited superior antioxidant activity (IC50 ≥ 16 µg/mL). The study also explored the cytotoxicity of different concentrations (10-100 µg/mL) of both coated and uncoated CuO NPs. Following 48 h of incubation, cell viability assays on shrimp hemocytes and human lymphocytes were conducted. The findings indicated that CuO NPs coated with alga extract at a concentration of 10 µg/mL increased shrimp hemocyte viability. In contrast, uncoated CuO NPs at a concentration of 25 µg/mL and higher, as well as CuO NPs at a concentration of 50 µg/mL and higher, led to a decrease in shrimp hemocyte survival. Notably, this study represents the first quantitative assessment of the toxicity of CuO NPs on shrimp cells, allowing for a comparative analysis with human cells.

Investigating the anti-streptococcal biofilm effect of ssDNA aptamer-silver nanoparticles complex on a titanium-based substrate.

Streptococcus mutans is a commensal and opportunistic pathogen that causes several diseases by forming a biofilm in humans and animals in many areas such as nasopharyngeal, cardiac valves, lungs, and oral cavity. Biofilms are very important in prosthetic infections associated with medical implants. The use of nanoparticles is one of the evolving fields in biofilm targeting. Silver nanoparticles can be used for biofilm targeting due to their inherent antimicrobial properties. Hybridization of nanoparticles with small molecules increases their biological properties and makes them multifunctional. The present investigation aimed to design an appropriate silver nanoparticles-aptamer complex that binds to the surface receptors of streptococcal strains. For this reason, silver nanoparticles with particle sizes in a range of 50 to 70 nm were synthesized and connected to a designed aptamer with a streptavidin-biotin linker. Then, the effect of the complex was investigated on the S. mutans biofilm formed on the surface of a medical-grade titanium substrate. The silver nanoparticles-aptamer complex at a concentration of 100 µg mL-1 after 48 h inhibited 43% of the biofilm formation and degraded 63% of the formed biofilm. Also, the cell availability reached 96% and the complex was stable in cell medium culture for 360 min. It was concluded that this complex could be a good candidate for removing the formed biofilms on the surface of titanium implants.

Co-delivery of STAT3 siRNA and methotrexate in breast cancer cells.

Co-delivery of anticancer drugs and biologics can provide synergetic effects and outperform single delivery therapies. Here, a nanoparticle (NP) system for co-delivery of methotrexate (MTX) and STAT3 siRNA has been developed and tested in vitro. Mesoporous silica nanoparticles (MSNs) were functionalized with chitosan (ch) by covalent grafting mediated by aminopropyl triethoxysilane (APTES) via glutaraldehyde as the linker. Co-delivery of MTX and STAT3 siRNA to MCF7 cells was demonstrated in cells by flow cytometric analysis and confocal laser scanning fluorescence microscopy for use in breast cancer treatment. MTX either competitively inhibits the dihydrofolate reductase (DHFR) receptor or suppresses the STAT3 metabolic pathway. STAT3 protein plays an essential role in cell division, proliferation and survival. Reduction of the protein by both MTX and STAT3 siRNA, achieved by chMSNs, significantly decreased the viability of breast cancer cells compared to single treatments alone. Cellular uptake of modified NPs was increased over time when additional free MTX was added implicating the DHFR receptor in uptake. In addition, protein corona compositions coated the NPs outer surface, were different between the NPs with and without drug potentially modulating cellular uptake. This study is the first report on co-delivery of MTX and STAT3 siRNA by chitosan modified MSNs.

Enhancement of astaxanthin production in Haematococcus pluvialis using zinc oxide nanoparticles.

Today, there is a great interest in using astaxanthin due to its potential health advantages. Application of different types of nanoparticles (NPs) as stress agents to enhance astaxanthin production in Haematococcus pluvialis, a microalgae strain, has been reported in the literature. In this study, the effect of different concentrations of zinc oxide (ZnO) NPs on the enhancement of astaxanthin production in H. pluvialis was investigated. First, ZnO NPs were synthesized from zinc nitrate as the precursor and sodium hydroxide (chemical method), and peel extract of pomegranate (green method) as reducing agents. To study the cell viability and stimulate the astaxanthin production, H. pluvialis cells were exposed to the different concentrations (i.e. 50, 100, 200, and 400 µg.ml-1) of ZnO NPs. The synthesized powders were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), and dynamic light scattering (DLS) methods. The characterization results showed that the pure ZnO NPs were successfully synthesized via both methods with uniform particle size distribution. But, the average particle size of the green synthesized ZnO NPs (about 30 nm) was smaller than that of the chemically synthesized ones (about 80 nm). Maximum astaxanthin production (~ 20 mg.g-1 of dry biomass of H. pluvialis) was achieved at 100 µg.ml-1 of green synthesized ZnO NPs exposure to the H. pluvialis in comparison with the control culture after 15 days. However, ZnO NPs concentration above 200 µg.ml-1 was toxic to the microalgae. From these results, it can be concluded that a specific amount of ZnO NPs could be considered as a worthy candidate for the enhancement of astaxanthin production in H. pluvialis.

Biodegradable nanocarriers based on chitosan-modified mesoporous silica nanoparticles for delivery of methotrexate for application in breast cancer treatment.

Nanocarriers have demonstrated great promise in the delivery of hydrophobic drugs particularly to tumor spaces by enhanced permeability and retention (EPR) effects. Mesoporous silica nanoparticles (MSNs) are the attractive nanocarrier system to reduce the drug's toxic side effects, enable controlled drug release, prevent drug degradation and provide a biocompatible and biodegradable high surface area carrier. Surface-modified MSNs have been applied to increase drug loading and efficiency. In this study, functionalized MSNs loaded with methotrexate (MTX) were designed for use as a cytotoxic agent. The MSNs were first modified with 3-triethoxysilylpropylamine (APTES) and then with chitosan through covalent coupling mediated by glutaraldehyde. The physicochemical properties of the nanoparticles were optimized for each step. The loading percentage (12.2%) and release profile of MTX as an anti-breast cancer drug, loaded at amine-modified MSNs, were measured via high performance liquid chromatography (HPLC). Moreover, the uptake profiles of fluorescein isothiocyanate (FITC)-labeled MSN-APTES-chitosan with or without MTX were monitored on MCF7 cancer cells via confocal microscopy. Following exposure of nanoparticles to body fluids, they were surrounded by specific proteins that may affect their cellular uptake. Hence, the adsorption profiles of protein corona on the surface of MSN, amine-modified MSN and MTX-loaded MSN-APTES-chitosan were analyzed. The cytotoxic potential for killing breast cancer cells was also studied. The MTX loaded MSN-APTES-chitosan showed a positive effect at a low dose (0.5 µM MTX). In this study, we introduce a new method to synthesize biodegradable MSNs with small and uniform particle size, achieve high MTX loading via covalent amine and chitosan-functionalization, monitor the cellular uptake and demonstrate the potential to decrease the viability of breast cancer cells at low dose.

Analysis and comparison of alkaline and acid phosphatases of Gram-negative bacteria by bioinformatic and colorimetric methods.

Alkaline phosphatase (ALP) and acid phosphatase (ACP) are two important phosphatase enzymes that play fundamental roles in Gram-negative bacteria. Additionally, they are useful for various biotechnological and industrial applications. In the present study, different aspects of bacterial ALPs and ACPs such as pseudo amino acid composition (PseAAC), amino acid composition, dipeptide composition, physicochemical properties, secondary structures and structural motifs were studied. The binding affinity of the phosphomonoesters to ALP and ACP enzymes was predicted by docking, and the activity of ALPs and ACPs were measured using colorimetric assay. ROC curve statistical analysis the machine learning algorithms were applied for classification of these two phosphatase protein groups. The results indicated that the physicochemical properties of ALPs and ACPs were not significantly different, although the aliphatic index and Extinction coefficient of motifs of these two enzymes were significantly different. Classification based on the concept of PseAAC and dipeptide composition also indicated high accuracy. The result of docking demonstrated that the binding free energy of ALPs was less than ACPs and the experimental results demonstrated that the activity of ACPs was more than ALPs. In conclusion, there is a relationship between efficiency and PseAAC and dipeptide compositions of these two enzymes.

Production and partial purification of thermostable bacteriocins from Bacillus pumilus ZED17 and DFAR8 strains with antifungal activity.

The bacteria which are members of the genus Bacillus are known to produce a wide variety of antimicrobial substances and bacteriocins. The main objective of this study was to investigate the effect of these bacteriocins on eukaryotic cells such as fungi, yeast and plant seeds. Several strains were screened for antifungal activities and identified by the means of polymerase chain reaction (PCR) of the 16s rRNA gene and sequencing. Our experiments showed that the Bacillus pumilus ZED17 and DFAR8 strains, had antifungal activities against Rhizoctonia solani and selected for further investigations. Extracellular peptides produced by these strains were purified by ammonium sulfate precipitation and dialysis. Addition of these peptides to Potato Dextrose Agar (PDA) medium inoculated with R. solani indicated significant inhibition of the fungal growth. The antifungal peptides were thermo-stable and remained active after boiling at 100˚C for 15 min. The molecular weight of the peptide with antifungal activity was estimated by electrophoresis on the Sodium Dodecyl Sulfate Poly Acrylamide Gel (SDS-PAGE) as about 5 KDa. Structural nature of this peptide was determined after gel extraction by Fourier-transform infrared spectroscopy (FTIR). Moreover, this peptide showed inhibiting effect on seeds germination of some herbs. This peptide could be applied to control herbal fungal disease induced by R. solani which is a broad host range plant pathogen fungus and its inhibition is very important. The peptide also prevents seed germination. Hence, it can be appropriate for inhibiting weeds growth. No significant effect against Saccharomyces cerevisiae and Candida albicans was observed.

Investigating the antibacterial effects of some Lactobacillus, Bifidobacterium and acetobacter strains killed by different methods on Streptococcus mutans and Escherichia coli.

Although there are many health advantages assigned to different live bacteria such as probiotics, some health threatening effects have also been reported. For example, live bacteria can transfer antibiotic resistance genes to other commensal and opportunistic bacteria of gastrointestinal tract. Recently, it was shown that using killed bacteria have some advantages over live ones. In this research, heat, paraformaldehyde and ozone killing methods were used to kill the bacteria. Acetobacter cerevisiae, Lactobacillus acidophilus, Bifidobacterium lactis and traditional vinegar and fermented dairy product (Kumeh) derived bacteria were killed and their antibacterial activity against Streptococcus mutans and Escherichia coli was investigated. To identify the bacteria isolated from the traditional products, 16S rDNA gene was partially sequenced. The gene analysis showed vinegar and Kumeh derived bacteria were Acetobacter pasteurianus and Lactobacillus crustorum (LcK) strains respectively. The S. mutans growth inhibition was detected in the all concentrations of all killed samples. However, generally, E. coli showed more resistant to the killed bacteria than S. mutans and the antibacterial effect of heat-killed bacteria against E. coli was not observed in the all concentrations for some killed bacteria. Among the pathogenic bacteria, S. mutans was the most sensitive one to the killed bacteria with 70% of reduction in its viability. In conclusion, this research showed that different killed bacteria had different effects on other bacteria and the killing method showed an impact on these effects. Overall, paraformaldehyde-killed L.crustorum (LcK) showed the best antibacterial activity against S. mutans; about 70% decrease in bacterial viability.

A comparative study of stability, antioxidant, DNA cleavage and antibacterial activities of green and chemically synthesized silver nanoparticles.

Silver nanoparticles have a wide range of research, industrial and biomedical applications that make it essential to develop a low cost and eco-friendly approach with scaling up potential. Green synthesis of nanoparticles through bio-reactions leads to a reduction of silver ions to particles could be an acceptable selection using no additional reducing chemicals. Moreover, the simplicity of scale-up processes of the method makes it more efficient than chemical and physical synthesis methods. In this study, Datura stramonium leaf extract and sodium citrate were used as biological and chemical reducing and stabilizing agents to make silver nanoparticles. The main goal is to comprise properties and evaluate antibacterial activity of nanoparticles synthesized through two approaches. Size and morphology compared between the two types of the synthesized nanoparticle by UV-Visible spectroscopy, DLS, AFM, TEM and their antibacterial effects were evaluated through growth inhibition MIC and MBC methods. The results showed narrow size range, spherical shape, high anti-oxidant, antibacterial and DNA cleavage activities of green synthesized silver nanoparticles comparing to less average size, wider range of nanoparticle size, no anti-oxidant activity and less antibacterial and DNA cleavage activities of chemically synthesized nanoparticles. The green synthesized silver nanoparticles had more desirable characteristics and biological activities compared to chemically synthesized nanoparticles. For instance, the green nanoparticles showed narrow size range, spherical shape, high anti-oxidant, antibacterial and DNA cleavage activities versus the chemically synthesized which had less average size, higher range of nanoparticles size, no anti-oxidant activity and less antibacterial and DNA cleavage activities.

A novel method for sensitive, low-cost and portable detection of hepatitis B surface antigen using a personal glucose meter.

Hepatitis B virus (HBV) infection is the major public health problem leading cause of death worldwide. The most important diagnostic marker for this infection is hepatitis B surface antigen (HBsAg). In this study, a novel, inexpensive, portable and sensitive ELISA method was designed and investigated for diagnosis of HBsAg based on the functionalized Fe3O4 and Al2O3 nanoparticles, with the strategy for detecting the concentration of glucose using a cheap and accessible personal glucose meter (PGM). The ELISA system was constructed using hepatitis B antibody against HBsAg immobilized on streptavidin coated magnetic iron oxide particles (S-Fe3O4) as the capture antibody (Ab1). In addition, another hepatitis B antibody against different epitope of HBsAg (Ab2) and glucoamylase both were immobilized on Al2O3 nanoparticles. After formation of the sandwich immune complex between Ab1 and Ab2 immobilized on S-Fe3O4 and Al2O3 NPs, respectively, through HBsAg, starch was converted into glucose using glucoamylase. Then, the glucose concentration was measured using PGM. The concentration of HBsAg was calculated based on the linear relation between the concentrations of HBsAg and glucose. Under optimal conditions, this assay showed detection limit values of 0.3 to 0.4 ng ml-1 for "ay" and "ad" subtypes of HBsAg, respectively. The results indicate that the designed assay is comparable to the commercial kits in terms of sensitivity, on-site, specificity, cost, simplicity, portability and reproducibility. The presented method can be used in disadvantaged areas of the world and blood transfusion centers. To the best of our knowledge, this is the first report of using PGMs for HBSAg detection.

Biotic elicitation for scopolamine production by hairy root cultures of Datura metel.

The (-)-hyoscyamine, atropine and scopolamine (hyoscine) are three valuable tropane alkaloids while scopolamine is the most important member of this group for the pharmaceutical industry due to its higher demand compared to hyoscyamine and atropine. Scopolamine is an anticholinergic reagent with several therapeutic applications. In the current study, the hairy roots culture of Daturametel was used as an advantageous method for production of scopolamine. The hairy roots are formed by Agrobacterium rhizogenes and have genetic stability, high growth rate and lateral branching. In this study, the effect of Bacillus cereus and Staphylococcus aureus as biotic elicitors on the production of scopolamine in D.metel hairy roots was investigated. The amount of scopolamine in the hairy roots was detected by HPLC analysis and compared with control samples after 0, 12 and 24 hours. Results showed that, B. cereus and S. aureus enhanced scopolamine production in the culture while the atropine content was decreased. Although in the control samples with no bacterial elicitation no scopolamine was detected, elicitation by B. cereus caused production of scopolamine and about 0.03 gram and 0.017 gram of it was detected in 100 gram dried D.metel hairy roots after 12 and 24 hours respectively. In S. aureus elicited hairy roots, scopolamine was not produced after 12 hours. However, about 0.025 gram of this tropane alkaloid was detected in 100 gram dried hairy roots after 24 hours. In conclusion, S. areus and B. cereus induced the scopolamine production in D. metel hairy roots.

In silico prediction of B cell epitopes of the extracellular domain of insulin-like growth factor-1 receptor.

The insulin-like growth factor-1 receptor (IGF-1R) is a transmembrane receptor with tyrosine kinase activity. The receptor plays a critical role in cancer. Using monoclonal antibodies (MAbs) against the IGF-1R, typically blocks ligand binding and enhances down-regulation of the cell-surface IGF-1R. Some MAbs such as cixutumumab are under clinical trial investigation. Targeting multiple distinct epitopes on IGF-1R, might be an effective strategy to inhibit IGF-1R pathway in cancer. In this study, new linear B cell epitopes for the extracellular domains of IGF-1R were predicted by in silico methods using a combination of linear B cell epitope prediction web servers such as ABCpred, Bepired, BCPREDs, Bcepred and Elliprro. Moreover, Discotope, B- pred and PEPOP web server tools were employed to predict new conformational B cell epitopes. In contrast to previously reported epitopes from extracellular region of the IGF-1R, we predicted new linear P8: (RQPQDGYLYRHNYCSK) and conformational Pc4: (HYYYAGVCVPACPPNTYRFE), Ppc6: (KMCPSTGKRENNESAPDNDT) and Ppc20: (ANILSAESSDSEFMQEPSGFI) epitopes. These epitopes are useful for further study as peptide antigens to actively immune host animals to develop new MAbs. Furthermore, the epitopes can be used in peptide-based cancer vaccines design.

Production and Characterization of New Anti-HER2 Monoclonal Antibodies.

Breast cancer is a major public health problem worldwide. Although in Iran cancer is the third cause of death after coronary heart disease and accidents, mortality from cancer has been on the rise during recent decades. About 15% to 20% of patients with invasive breast cancer have abnormally high levels of HER2 protein. HER2 is a specialized protein found on breast cancer cells that controls cancer growth and spread. This study describes the generation and characterization of new anti-HER2 MAbs towards HER2 protein using a chimeric peptide immunogen containing discontinuous B-cell epitope peptide (peptide 626) and promiscuous T-helper epitope (MVF). The specificity of these MAbs was confirmed in various immunoassays, including ELISA, Western blotting, and immunofluorescence. Moreover, the MTT assay results indicated that 5H5 and 5H11 MAbs could reduce the growth of SKBR3 cells by approximately 50% (p<0.05). These MAbs that can reduce cancer cell proliferation would be useful for cancer therapy. Furthermore, the synthetic peptide used in the current work was able to induce the immune system to generate antibodies, especially IgG isotype. Therefore, it could be further used as a peptide cancer vaccine that targets different epitopes or structural domains of HER2 ECD.

Immunization with a novel chimeric peptide representing B and T cell epitopes from HER2 extracellular domain (HER2 ECD) for breast cancer.

Because of direct stimulating immune system against disease, vaccination or active immunotherapy is preferable compared to passive immunotherapy. For this purpose, a newly designed chimeric peptide containing epitopes for both B and T cells from HER2 ECD subdomain III was proposed. To evaluate the effects of the active immunization, a discontinuous B cell epitope peptide was selected based on average antigenicity by bioinformatics analysis. The selected peptide was collinearly synthesized as a chimera with a T helper epitope from the protein sequence of measles virus fusion (208-302) using the GPSL linker. Three mice were immunized with the chimeric peptide. Reactive antibodies with HER2 protein in ELISA and immunofluorescence assays with no cross-reactivity were generated. The 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay indicated that the anti-peptide sera had inhibitory effects on proliferation of SK-BR-3 cells. Hence, the newly designed, discontinuous chimeric peptide representing B and T cell epitopes from subdomain III of HER2-ECD can form the basis for future vaccines design, where these data can be applied for monoclonal antibody production targeting the distinct epitope of HER2 receptor compared to the two broadly used anti-HER2 monoclonal antibodies, Herceptin and pertuzumab.

Binding analysis for interaction of diacetylcurcumin with ß-casein nanoparticles by using fluorescence spectroscopy and molecular docking calculations.

The interaction of diacetylcurcumin (DAC), as a novel synthetic derivative of curcumin, with bovine ß-casein (an abundant milk protein that is highly amphiphilic and self assembles into stable micellar nanoparticles in aqueous solution) was investigated using fluorescence quenching experiments, Forster energy transfer measurements and molecular docking calculations. The fluorescence quenching measurements revealed the presence of a single binding site on ß-casein for DAC with the binding constant value equals to (4.40±0.03)×10(4)M(-1). Forster energy transfer measurements suggested that the distance between bound DAC and Trp143 residue is higher than the respective critical distance, hence, the static quenching is more likely responsible for fluorescence quenching other than the mechanism of non-radiative energy transfer. Our results from molecular docking calculations indicated that binding of DAC to ß-casein predominantly occurred through hydrophobic contacts in the hydrophobic core of protein. Additionally, in vitro investigation of the cytotoxicity of free DAC and DAC-ß-casein complex in human breast cancer cell line MCF7 revealed the higher cytotoxic effect of DAC-ß-casein complex.

Protein bioinformatics applied to virology.

Scientists have united in a common search to sequence, store and analyze genes and proteins. In this regard, rapidly evolving bioinformatics methods are providing valuable information on these newly-discovered molecules. Understanding what has been done and what we can do in silico is essential in designing new experiments. The unbalanced situation between sequence-known proteins and attribute-known proteins, has called for developing computational methods or high-throughput automated tools for fast and reliably predicting or identifying various characteristics of uncharacterized proteins. Taking into consideration the role of viruses in causing diseases and their use in biotechnology, the present review describes the application of protein bioinformatics in virology. Therefore, a number of important features of viral proteins like epitope prediction, protein docking, subcellular localization, viral protease cleavage sites and computer based comparison of their aspects have been discussed. This paper also describes several tools, principally developed for viral bioinformatics. Prediction of viral protein features and learning the advances in this field can help basic understanding of the relationship between a virus and its host.

Linear and conformational B cell epitope prediction of the HER 2 ECD-subdomain III by in silico methods.

Human epidermal growth factor receptor 2 (HER2) is a member of the epidermal growth factor receptor family of receptor tyrosine kinases that play important roles in all processes of cell development. Their overexpression is related to many cancers, including examples in the breast, ovaries and stomach. Anticancer therapies targeting the HER2 receptor have shown promise, and monoclonal antibodies against subdomains II and IV of the HER2 extra-cellular domain (ECD), Pertuzumab and Herceptin, are currently used in treatments for some types of breast cancers. Since anti HER2 antibodies targeting distinct epitopes have different biological effects on cancer cells; in this research linear and conformational B cell epitopes of HER2 ECD, subdomain III, were identified by bioinformatics analyses using a combination of linear B cell epitope prediction web servers such as ABCpred, BCPREDs, Bepired, Bcepred and Elliprro. Then, Discotope, CBtope and SUPERFICIAL software tools were employed for conformational B cell epitope prediction. In contrast to previously reported epitopes of HER2 ECD we predicted conformational B cell epitopes P1C: 378-393 (PESFDGDPASNTAPLQ) and P2C: 500-510 (PEDECVGEGLA) by the integrated strategy and and P4: PESFDGD-X-TAPLQ; P5: PESFDGDP X TAPLQ; P6: ESFDGDP X NTAPLQP; P7: PESFDGDP-X-NTAPLQ; P8: ESFDG-XX-TAPLQPEQL and P9: ESFDGDP- X-NTAPLQP by SUPERFICIAL software. These epitopes could be further used as peptide antigens to actively immune mice for development of new monoclonal antibodies and peptide cancer vaccines that target different epitopes or structural domains of HER2 ECD.

Precise mapping of an IGF-I-binding site on the IGF-1R.

The IGF-1R [type 1 IGF (insulin-like growth factor) receptor] is activated upon binding to IGF-I and IGF-II leading to cell growth, survival and migration of both normal and cancerous cells. We have characterized the binding interaction between the IGF-1R and its ligands using two high-affinity mouse anti-IGF-1R mAbs (monoclonal antibodies), 7C2 and 9E11. These mAbs both block IGF-I binding to the IGF-1R but have no effect on IGF-II binding. Epitope mapping using chimaeras of the IGF-1R and insulin receptor revealed that the mAbs bind to the CR (cysteine-rich) domain of IGF-1R. The epitope was finely mapped using single point mutations in the IGF-1R. Mutation of Phe241, Phe251 or Phe266 completely abolished 7C2 and 9E11 binding. The three-dimensional structure showed that these residues cluster on the surface of the CR-domain. BIAcore analyses revealed that IGF-I and a chimaeric IGF-II with the IGF-I C-domain competed for the binding of both mAbs with the IGF-1R, whereas neither IGF-II nor a chimaeric IGF-I with the IGF-II C-domain affected antibody binding. We therefore conclude the IGF-I C-domain interacts with the CR (cysteine-rich) domain of the receptor at the cluster of residues Phe241, Phe251 and Phe266. These results allow precise orientation of IGF-I within the IGF-I-IGF-1R complex involving the IGF-I C-domain binding to the IGF-1R CR domain. In addition, mAbs 7C2 and 9E11 inhibited both IGF-I- and IGF-II-induced cancer cell proliferation, migration and IGF-1R down-regulation, demonstrating that targeting the IGF-1R is an effective strategy for inhibition of cancer cell growth.

Production and characterization of monoclonal antibodies against insulin-like growth factor type 1 receptor.

The type 1 insulin-like growth factor receptor (IGF-1R) has been extensively reported to play an important role in cancer. Activation of the IGF-1R by IGF-I and IGF-II binding to the extracellular domains of the receptor induces mitogenic and anti-apoptotic effects, which are important events in tumor growth and survival. Several cancer cell types overexpress IGF-1R, suggesting a possible use of monoclonal antibodies (MAbs) against IGF-1R as diagnostic reagents. Here, we report the production and characterization of two independent MAbs, namely 7C2 and 9E11, generated by immunizing mice with the soluble extracellular part of this receptor (amino acids 1-906). Both MAbs bind to membrane bound IGF-1R and do not cross-react with insulin receptor isoforms, IR-A and IR-B expressed on IGF-1R() cells. MAbs 7C2 and 9E11 stained the IGF- 1R on frozen or paraffin-embedded tissue sections or frozen cells. The MAbs 7C2 and 9E11 immunoprecipitated the IGF-1R from P6 cell lysates (cells overexpressing human IGF-1R) and could detect non-reduced intact IGF-1R on immunoblots. However, the MAbs were not able to detect reduced and denatured receptor alpha and beta chains. Sequencing of the heavy- and light-chain variable regions revealed that the 7C2 and 9E11 CDR amino acid sequences are different but result in antibodies with similar properties. MAbs 7C2 and 9E11 are therefore potentially useful diagnostic tools and could be of therapeutic use for humans in the future.