Reduced Tumor Volume and Increased Necrosis of Human Breast Tumor Xenograft in Mice Pretreated by a Cocktail of Three Specific Anti-HER2 scFvs.

PURPOSE: We aimed to assess the effects of a cocktail comprising three specific antiHER2 scFvs on breast tumor formation in a xenograft mouse model and to evaluate quantitative changes in the tumor using stereological analysis. METHODS: Three specific anti-HER2 phage antibodies were produced from a scFv-library using phage display technology. The cell binding capacities of the antibodies were assessed via FACS analysis. Soluble forms of the antibodies were prepared by infecting HB2151-E. coli cells and purified using a centrifugal ultrafiltration method. The purification process was evaluated by SDSPAGE analysis. Two forms of scFv cocktails were prepared, soluble scFv and phage-scFv cocktail, which contained an equal amount/phage of the three specific anti-HER2 antibodies. Inbred female BALB/c mice were pretreated with 5 and 20 mg/kg of the soluble scFv cocktail and 1011 phagescFv cocktail/kg. The mice were then injected with 2×106 SKBR-3 human breast cancer cells. Total tumor, inflammatory and non-inflammatory volumes were estimated using the Cavalieri principle after preparing photomicrograph slides. RESULTS: The anti-HER2 scFvs showed significantly higher binding to SKBR-3 cells compared to the isotype control. SDS-PAGE analysis confirmed the high purification of the scFvs. Stereological analysis revealed that the group pretreated with 20 mg/kg of the soluble scFv cocktail exhibited the highest reductions in total tumor volume, non-inflammatory volume, and inflammatory volume, with reductions of 73%, 78%, and 72%, respectively, compared to PBS-pretreated mice (P-value < 0.0001). The volumetric ratio of necrotic tissue to total tumor volume increased by 2.2-fold and 2- fold in the 20mg/kg of soluble scFv cocktail and phage-scFv cocktail groups, respectively, compared to the PBS-treated mice (P-value < 0.05). CONCLUSION: Pre-treatment with a 20 mg/kg anti-HER2 scFv cocktail resulted in a significant reduction in tumor volume and increased necrotic area in a human breast cancer xenograft model, indicating the remarkable anti-tumor effect of the cocktail in vivo.

Development of Human Recombinant Antibodies Against ROR1 Tumor Antigen.

Background: Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncofetal antigen expressed on many types of cancer cells, but not normal adult cells. ROR1 antigen contributes to cancer development and progression by several signaling pathways. ROR1 expression has been associated with tumor growth, survival, and metastasis. In this study specific human recombinant antibodies were selected against ROR1 antigen for their use in cancer immunotherapy. Methods: Phage display technology was used to produce phage antibody from a human scFv library. Phage concentration was determined to confirm the phage rescue process. Panning procedure was performed to isolate specific scFv clones against ROR1 epitope. Phage ELISA was done to evaluate the reactivity of the selected scFvs. Results: Two specific human scFvs with frequencies of 20% and 25% were selected against ROR1 peptide. The antibodies showed specific reaction to the corresponding epitopes in phage ELISA. Discussion: Cancer targeted therapy using human specific antibodies is a new strategy, which is used in cancer therapy. The selected specific scFvs that target ROR1 epitope are human antibodies that originated from a human library and have the potential to be used in clinic in cancer immunotherapy of ROR1 positive tumors without induction of human anti mouse antibody (HAMA) response.

Single-chain antibody-decorated Au nanocages@liposomal layer nanoprobes for targeted SERS imaging and remote-controlled photothermal therapy of melanoma cancer cells.

The development of theranostic platforms combining surface-enhanced Raman spectroscopy (SERS) imaging with NIR-stimulated photothermal therapy (PTT) is of utmost importance for the precise diagnosis and selective treatment of cancers, especially in superficial solid tumors. For this purpose, a versatile theranostic nanoprobe of liposomal layer-coated Au nanocages (AuNCs) was decorated with an anti-MUC18 single-chain antibody (scFv). 4-mercapto benzoic acid (p-MBA)-labeled AuNCs (p-AuNCs) were coated by a liposomal layer (p-AuNCs@lip), followed by conjugating anti-MUC18 scFv via post-insertion method to form immuno-liposomal layer-coated AuNCs (p-AuNCs@scFv-lip). Physicochemical characterizations of the p-AuNCs@scFv-lip were investigated by transmission electron microscopy (TEM) and UV-vis and Raman spectroscopy. Furthermore, the targeting ability and theranostic efficiency of the nanoprobe were evaluated for specific diagnosis and treatment of cancerous melanoma cells by flow cytometry, SERS mapping, and live/dead assay. The formation of lipid layer on p-AuNCs surface was confirmed by TEM imaging. After decorating the liposomal layer with scFv, a relevant red shift was observed in the UV-vis spectrum. Moreover, p-AuNCs@lip presented characteristic peaks in the Raman spectrum, which exhibited only a minor change after scFv conjugation (p-AuNCs@scFv-lip). Interestingly, the cellular uptake of AuNCs@scFv-lip by A375 cell line (MUC18+) showed a 24-fold enhancement compared with SKBR3 cells (MUC18-). AuNCs@scFv-lip specifically identified A375 cells from SKBR cells via SERS mapping and effectively killed A375 cells through the PTT mechanism. Taken together, this theranostic platform can provide a promising tool for both in situ diagnosis and remote-controlled thermal ablation of cancer cells.

Novel single-chain antibodies against highly conserved epitopes in the hemagglutinin of influenza A viruses: Promising agents for universal therapies.

OBJECTIVES: Development of new antibodies with broad activity would provide anti-influenza prophylaxis and treatment. Human single-chain variable fragments (scFvs) are considered effective agents against viruses. In this study specific human scFvs against highly conserved epitopes in the hemagglutinin (HA) of influenza A viruses were selected and their neutralizing activity was evaluated. MATERIALS AND METHODS: Bioinformatic methods were used to evaluate HA epitopes. The panning process selected specific clones from a scFv library. PCR and DNA fingerprinting differentiated the common patterns. Soluble forms of scFvs were produced and evaluated using Western blot analysis. The neutralizing effects of anti-HA scFvs were assessed by microneutralization assay using MDCK cells. Real-time PCR was done to determine the exact copy number of the virus following neutralization. RESULTS: Bioinformatic evaluation confirmed the antigenicity and accessibility of the epitopes. Four specific anti-HA scFvs, scFvs I, II, I', and II' were selected. The scFvs neutralized 2009 H1N1 pandemic and 83.34%, 79.17%, 75%, and 62.5% reduction in the virus titers were obtained following treatments with scFv-II', I, I', and II, respectively. Real-time PCR demonstrated 98.6%, 95.7%, 95.26%, and 91.19% reductions in virus numbers following neutralization with scFv-II', I, I', and II, respectively. CONCLUSION: Anti-HA scFvs selected against highly conserved HA of influenza A virus with high neutralizing effects, offer novel human antibodies for prophylaxis and treatment of a wide range of influenza viruses including different subtypes of H1N1, H3N2, and H5N1 influenza A virus. The antibodies have the potential to be used for universal therapy.

Anti-Proliferative Effect of Specific Anti-EGFR Single Chain Antibody on Triple Negative Breast Cancer Cells.

BACKGROUND: Targeted therapy is an important treatment strategy that is widely used for cancer therapy. Epidermal growth factor receptor (EGFR) is overexpressed in a significant percentage of Triple-negative breast cancer (TNBC) patients. Although Cetuximab, which targets EGFR, has shown some inhibitory effects on TNBC cells, Cetuximab resistance cases due to ligand-independent activating mutations in the EGFR gene limit its application. Due to various benefits of single chain antibodies (scFvs), the use of these antibodies in cancer targeted therapy is increasing. In this study, a specific anti-EGFR antibody was isolated and evaluated. METHODS: Panning procedure was used against an immunodominant epitope of EGFR in its dimerization arm using a diverse phage library. Polymerase Chain Reaction (PCR) and fingerprinting were applied to identify the specific clones. The MTT tetrazolium assay was performed to evaluate the inhibitory effects of selected anti- EGFR scFv phage antibody on MDA-MB-468, a TNBC cell line. RESULTS: After four round of panning, one dominant pattern was observed in DNA fingerprinting with frequency of 85%. The growth of MDA-MB-468 cells was decreased dose-dependently after treatment with anti-EGFR scFv phage antibody. No significant inhibitory effect of M13KO7 helper phage as negative control on the cell growth of MDA-MB-468 was observed (p> 0.05). CONCLUSION: The selected anti-EGFR scFv with high anti proliferative effect on TNBC cells offers an effective alternative for TNBC targeted therapy. The antibody, which binds to the dimerization arm of EGFR and inhibits EGFR dimerization, could also overcome TNBC cases with Cetuximab resistance due to ligandindependent activating mutations.

Antibody-guided nanomedicines as novel breakthrough therapeutic, diagnostic and theranostic tools.

Recent advances in nanotechnology, such as the development of various types of nanoparticles and hybrid nanomaterials, have revolutionized nanomedicine. The small size, customizable surface, enhanced solubility, and multi-functionality endow the nanoparticles with an ability to interact with complex cellular and biological functions in new ways. Furthermore, these systems can deliver drugs to specific tissues and provide a targeted therapy. For this purpose, different categories of molecules, particularly antibodies, have been used as ligands. Antibody-conjugated nanomaterials can significantly enhance the efficiency of nanomedicines, especially in the field of cancer. This review is focused on three major medical applications of antibody-conjugated nanomaterials, namely, therapeutic, diagnostic and theranostic applications. To provide comprehensive information on the topic and an overview of these hybrid nanomaterials for biomedical applications, a brief summary of nanomaterials and antibodies is given. Moreover, the review has depicted the potential applications of antibody-conjugated nanomaterials in different fields and their capabilities to empower nanomedicine, particularly in relation to the treatment and detection of malignancies.

Generation and characterization of a specific single-chain antibody against DSPP as a prostate cancer biomarker: Involvement of bioinformatics-based design of novel epitopes.

Isolation of specific single chain antibodies (scFvs) against key epitopes of cancer markers are applied for cancer immunotherapy and diagnosis. In this study following the prediction of the 3D structure of the DSP part of Dentin sialophosphoprotein (DSPP), the epitope was chosen using in silico programs. Panning process was applied to isolate specific human scFv against the epitope. PCR and DNA fingerprinting differentiated the specific clones, which were evaluated by phage ELISA. Following DNA sequencing, the 3D structure of isolated scFv was modeled and Docked on DSP. Results demonstrated the selection of a specific anti-DSPP scFv with 40% frequency, which reacted significantly with the predicted epitope and PCa patients' urines in ELISA tests (P-value < 0.05). The VH and VL of the isolated scFv were from VH1 and VL3 gene families with several amino acid changes in CDRs and FRs domains. The scFv tightly bound to the DSP epitope with the lowest energy level by hydrogen bonds, cation-pi, hydrophobic and ionic interactions demonstrating the specificity of Ag-Ab interactions. The anti-DSPP scFv selected in this study with significant specificity to DSPP antigen offers a promising new agent for both PCa early detection and treatment of cancers with DSPP expression.

Isolation of Neutralizing Human Single Chain Antibodies Against Conserved Hemagglutinin Epitopes of Influenza a Virus H3N2 Strain.

BACKGROUND: Immunotherapies using monoclonal antibodies against influenza A hemagglutinin (HA) has been an effective means for controlling Influenza spread. An alternative method for viral prophylaxis and treatment is the development of human single-chain variable fragment (scFv) antibodies with no human anti-mouse antibody (HAMA) response and high specificity. In the present study, two highly conserved sequences of HA were used to select specific neutralizing scFvs against H3N2 strain of influenza A virus. METHODS: Biopanning process was performed to isolate specific scFv antibodies against highly conserved HA sequences, aa173-181 and 227-239, of the influenza A H3N2 strain from a scFv library. The peptide-binding specificity of the selected clones was examined via phage ELISA. The soluble forms of the clones were prepared and assessed using western blot analysis and neutralization efficiency of the selected clones were examined by TCID50 neutralizing assay and real-time PCR. RESULTS: scFv 1 and scFv 2 were selected against HA of H3N2 influenza A virus with frequencies of 95% and 30% in the panning process, respectively. Western blot analysis confirmed the scFv band size. Significant neutralization in the presence of scFv 1 and scFv 2 were obtained. Real time PCR revealed significant decrease in viral copy number. CONCLUSION: Two specific neutralizing scFvs against two highly conserved neutralizing epitopes of the influenza A virus HA glycoprotein were selected. A strong neutralization effect of scFv1, showed the potential of this antibody for H3N2 influenza A controlling in the viral spread.

Isolation of Specific Human Recombinant Antibodies Against Glycoprotein 41 of HIV.

BACKGROUND: Blocking of gp41 of HIV virus, which is involved in the virus entry has been introduced as an effective strategy against HIV infection. In this study we used phage display technology to select specific single chain antibody (scFv) against gp41 HIV for its application in clinical use. METHODS: Single chain antibodies against an epitope located in C- terminal part of gp41 were selected using the panning process which enriched a phage antibody display library of scFv. Following panning, 20 clones were amplified by PCR and fingerprinted. To test the specificity of the selected antibodies phage ELISA was performed. RESULTS: PCR of the library clones demonstrated the presence of VH-linker-VL inserts. Fingerprinting of the clones showed a diverse library with different patterns. Fingerprinting of selected clones after panning revealed two specific single chain antibodies with frequency of 25% and 20%. These clones were preserved for further investigations. Phage ELISA results showed specificity of the two scFvs against the immunodominant epitope of gp41. The absorbance of the scFv1 and scFv2 were 0.72 and 0.63 while the absorbance of the no peptide were 0.18 and 0.12, respectively. CONCLUSION: In this study we successfully selected two specific recombinant antibodies against gp41. These libraries are human antibodies with high affinity and specificity and have the potential to be used for diagnosis and treatment. Further investigations are needed to show the effects of the antibodies in vitro and in vivo.

Selection and Evaluation of Specific Single Chain Antibodies against CD90, a Marker for Mesenchymal and Cancer Stem Cells.

BACKGROUND: CD90, a membrane-associated glycoprotein is a marker used to identify mesenchymal stem cells (MSCs). Recent studies have introduced CD90, which induces tumorigenic activity, as a cancer stem cell (CSC) marker in various malignancies. Blocking CD90 activity with anti-CD90 monoclonal antibodies enhanced anti-tumor effects. To date, highly specific antibody single-chain variable fragments (scFvs) have been isolated against various targets and showed promising results in cancer immunotherapy. METHODS: A phage antibody was produced from a scFv library using M13KO7 helper phage. The phage library was panned against a CD90 epitope. To select specific clones, PCR and DNA fingerprinting were performed and common patterns were identified. The panning results were confirmed by phage ELISA. RESULTS: Of 20 clones selected after panning, 16 shared identical fingerprints. One clone from this group reacted specifically with the epitope in phage ELISA. The average absorbance of wells coated with the CD90 peptide was significantly greater than that of wells containing no peptide (p=0.03). CONCLUSION: Currently, recombinant antibodies are used not only as highly specific detection tools, but due to their specific characteristics, are applied in targeted cancer therapies. The anti-CD90 scFv selected in this study has the potential to be used to detect MSCs and target CSCs and offers promising strategies for treatment of various cancers.

Cell growth inhibition and apoptosis in breast cancer cells induced by anti-FZD7 scFvs: involvement of bioinformatics-based design of novel epitopes.

BACKGROUND: FZD7 has a critical role as a surface receptor of Wnt/ß-catenin signaling in cancer cells. Suppressing Wnt signaling through blocking FZD7 is shown to decrease cell viability, metastasis and invasion. Bioinformatic methods have been a powerful tool in epitope designing studies. Small size, high affinity and human origin of scFv antibodies have provided unique advantages for these recombinant antibodies. METHODS: Two epitopes from extracellular domain of FZD7 were designed using bioinformatic methods. Specific anti-FZD7 scFvs were selected against these epitopes through panning process. The specificity of the scFvs was assessed by phage ELISA and the ability to bind to FZD7 expressing cell line (MDA-MB-231) was determined by flowcytometry. Antiproliferative and apoptotic effects of the scFvs were evaluated by MTT and Annexin V/PI assays. The effects of selected scFvs on expression level of Surivin, c-Myc and Dvl genes were also evaluated by real-time PCR. RESULTS: Results demonstrated selection of two specific scFvs (scFv-I and scFv-II) with frequencies of 35 and 20%. Both antibodies bound to the corresponding peptides and cell surface receptors as shown by phage ELISA and flowcytometry, respectively. The scFvs inhibited cell growth of MDA-MB-231 cells significantly as compared to untreated cells. Growth inhibition of 58.6 and 53.1% were detected for scFv-I and scFv-II, respectively. No significant growth inhibition was detected for SKBR-3 negative control cells. The scFvs induced apoptotic effects in the MDA-MB-231 treated cells after 48 h, which were 81.6 and 74.9% for scFv-I and scFv-II, respectively. Downregulation of Surivin, c-Myc and Dvl genes were also shown after 48h treatment of cells with either of scFvs (59.3-93.8%). ScFv-I showed significant higher antiproliferative and apoptotic effects than scFv-II. CONCLUSIONS: Bioinformatic methods could effectively select potential epitopes of FZD7 protein and suggest that epitope designing by bioinformatic methods could contribute to the selection of key antigens for cancer immunotherapy. The selected scFvs, especially scFv-I, with high antiproliferative and apoptotic effects could be considered as effective agents for immunotherapy of cancers expressing FZD7 receptor including triple negative breast cancer.

Inhibition of Intercellular Communication between Prostate Cancer Cells by A Specific Anti-STEAP-1 Single Chain Antibody.

BACKGROUND: Six-Transmembrane epithelial antigen of the prostate-1 (STEAP-1) is present at the intercellular junctions of the secretory epithelium of prostate and is overexpressed in all steps of prostate cancer. STEAP-1 acts as a transporter protein or a putative channel between cancer cells while it has limited expression in normal human tissues. This protein has been suggested as an attractive target for prostate cancer immunotherapy. OBJECTIVE: This study aimed at the development of a specific single chain fragment variable (scFv) antibody against STEAP-1 epitope and testing the inhibitory effect of the selected scFv antibody in blocking gap junctions between tumor cells. METHOD: In the current study, a phage library was used and a specific scFv antibody was isolated against STEAP-1 epitope using panning process. RESULTS: PCR and DNA fingerprinting of the obtained clones demonstrated a dominant pattern of a specific clone. Binding of the selected scFv to the corresponding target on PC3 and LNCaP cell lines was tested using ELISA and flow cytometry techniques. The inhibitory effect of the selected scFv antibody in blocking gap junctions between the cells was tested using intercellular communication assay. The selected antibody reacted with the corresponding epitope in ELISA and bound to prostate cancer cells with an intensity of 44.6% (PC3 cells) and 73.4% (LNCap cells) as shown by FACS analysis. Intercellular communication assay indicated that dye transfer between the cells in PC3 and LNCaP cell lines treated with 1000 scFv/cell was significantly inhibited (80-90%). CONCLUSION: Our results suggested that the selected specific anti-STEAP1 scFv highly inhibited intercellular communication between prostate cancer cells and has the potential to be used as a new effective agent in prostate cancer immunotherapy.

Production and Evaluation of Specific Single-Chain Antibodies against CTLA-4 for Cancer-Targeted Therapy.

BACKGROUND: Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) molecules are expressed on T-cells and inhibit their function by inhibiting activation of subsequent T-cell molecular pathways. Blocking of CTLA-4 inhibits the growth of malignant tumor cells. Anti-CTLA-4 monoclonal antibodies activate the immune system against cancer. Due to several advantages of single-chain antibodies (scFvs) compared to monoclonal antibodies in cancer immunotherapy, specific anti-CTLA-4 scFvs (single-chain variable fragment) were selected in this study. METHODS: A phage antibody display library of scFvs was analyzed and a panning process was performed against an immunodominant epitope of CTLA-4. PCR and DNA fingerprinting were used to differentiate the specific clones. The specificity of the selected clones was investigated by phage ELISA (Enzyme-linked immunosorbent assay). RESULTS: Two specific clones with frequencies of 35 and 20% were identified. The clones reacted with the corresponding epitope on ELISA, while no reactivity was observed with an unrelated peptide, M13KO7 helper phage, unrelated scFvs, or no peptide as negative controls. CONCLUSION: Targeted therapy against cancer markers is an ideal treatment strategy. Specific human anti-CTLA-4scFvs were selected in this study. These scFvs bound the related epitope. These antibodies have the potential to be used for targeted therapy, where the blocking of CTLA4 receptor is needed. The study suggests further evaluation of the selected scFvs to reveal the effects of the selected antibodies.

Avian gametologs as molecular tags for sex identification in birds of prey of Iran.

Global environmental change and rapid destruction of natural habitats necessitate the conservation of endangered and threatened birds of prey. Recently, molecular sex identification methods based on amplification of introns of chromodomain-helicase DNA binding protein1 (CHD1) have provided valuable tools for ecological study and conservation breeding programs of birds. These methods employ a primer pair flanking an intron which varies considerably in length between the avian gametologs CHD1Z and CHD1W. Herein, we test the applicability of CHD1Z and CHD1W as universal tags for molecular sex identification in birds of prey of Iran. We showed successful sex identification in 22 species of birds of prey using feathers as the source of DNA. The results suggest that the regions of CHD1W and CHD1Z amplified in this study are conserved among most of Falconiformes, enabling accurate sex identification in birds of prey.

Cell growth inhibition and apoptotic effects of a specific anti-RTFscFv antibody on prostate cancer, but not glioblastoma, cells.

Background: Single chain antibody (scFv) has shown interesting results in cancer immunotargeting approaches, due to its advantages over monoclonal antibodies. Regeneration and tolerance factor (RTF) is one of the most important regulators of extracellular and intracellular pH in eukaryotic cells. In this study, the inhibitory effects of a specific anti-RTF scFv were investigated and compared between three types of prostate cancer and two types of glioblastoma cells.  Methods: A phage antibody display library of scFv was used to select specific scFvs against RTF using panning process. The reactivity of a selected scFv was assessed by phage ELISA. The anti-proliferative and apoptotic effects of the antibody on prostate cancer (PC-3, Du-145 and LNCaP) and glioblastoma (U-87 MG and A-172) cell lines were investigated by MTT and Annexin V/PI assays.  Results: A specific scFv with frequency 35% was selected against RTF epitope. This significantly inhibited the proliferation of the prostate cells after 24 h. The percentages of cell viability (using 1000 scFv/cell) were 52, 61 and 73% for PC-3, Du-145 and LNCaP cells, respectively, compared to untreated cells. The antibody (1000 scFv/cell) induced apoptosis at 50, 40 and 25% in PC-3, Du-145 and LNCaP cells, respectively. No growth inhibition and apoptotic induction was detected for U-87 and A172 glioblastoma cells.  Conclusions: Anti-RTFscFv significantly reduced the proliferation of the prostate cancer cells. The inhibition of cell growth and apoptotic induction effects in PC-3 cells were greater than Du-145 and LNCaP cells. This might be due to higher expression of RTF antigen in PC-3 cells and/or better accessibility of RTF to scFv antibody. The resistance of glioblastoma cells to anti-RTF scFv offers the existence of mechanism(s) that abrogate the inhibitory effect(s) of the antibody to RTF. The results suggest that the selected anti-RTF scFv antibody could be an effective new alternative for prostate cancer immunotherapy.

Anti-Metastatic and Anti-Invasion Effects of a Specific Anti-MUC18 scFv Antibody on Breast Cancer Cells.

Breast cancer is the most common malignancy in women. Altered expression of MUC18, a cell surface receptor, and its interaction with Wnt-5a as its ligand, affects the motility and invasiveness of breast cancer cells. In this study, we explored the Wnt-5a binding site and designed an antigenic epitope on the MUC18 receptor using in silico methods. A specific single-chain variable fragment (scFv) was isolated against the epitope by several panning processes. The binding ability of the scFv to the related epitope was evaluated in ELISA and flow cytometry. The inhibitory effects of the selected scFv on MUC18 positive cell line, MDA-MB231, was assessed by migration and invasion assays. The results demonstrated isolation of specific scFv with frequency of 40 % which showed significant binding with the epitope in both ELISA and fluorescence-activated cell sorting (FACS) analyses. The antibody inhibited the migration (76 %) and invasion (67 %) of MUC18 positive cell line. The results suggest the specific anti-MUC18 scFv as an effective antibody for breast cancer immunotherapy.

Neutralizing human recombinant antibodies against herpes simplex virus type 1 glycoproteins B from a phage-displayed scFv antibody library.

The HSV-1 envelope glycoprotein B (gB) plays a critical role in virus entry into host cells. Neutralizing antibodies can therefore potentially prevent virus entry into target cells and cell-to-cell spread of infection. Our present study focused on the selection of neutralizing single-chain Fv (scFv) antibodies of a phage-displayed nonimmune human scFv antibody library against gB of HSV-1. To enrich specific scFvs, two phage antibodies were isolated against amino acid residues 31-43 derived from the N-terminal part of gB using panning technique. Two scFvs, scFv-gB1 and scFv-gB2, with frequencies of 45% and 20% were obtained from scFv clones after performing PCR and MvaI fingerprinting. In phage ELISA analysis, both gB1 and gB2 scFvs demonstrated high reactivity with the gB peptide. In the neutralization assay, scFv-gB1 and scFv-gB2 represented neutralizing effects of 55% and 59%, respectively. Upon further enhancement of the neutralizing effects of these antibodies, they can be considered as new potential alternatives in the treatment and prophylaxis of HSV-1 infections.

Single Chain Antibodies Against gp55 of Human Cytomegalovirus (HCMV) for Prophylaxis and Treatment of HCMV Infections.

BACKGROUND: Immunotherapy is a promising prospective new treatment for cytomegalovirus (CMV) infections. Neutralizing effects have been reported using monoclonal antibodies. Recombinant single chain antibodies (scFvs) due to their advantages over monoclonal antibodies are potential alternatives and provide valuable clinical agents. OBJECTIVES: The aim of this study was to select specific single chain antibodies against gp55 of CMV and to evaluate their neutralizing effects. In the present study, we selected specific single chain antibodies against glycoprotein 55 (gp55) of CMV for their use in treatment and diagnosis. MATERIALS AND METHODS: Single chain antibodies specific against an epitope located in the C-terminal part of gp55 were selected from a phage antibody display library. After four rounds of panning, twenty clones were amplified by the polymerase chain reaction (PCR) and fingerprinted by MvaI restriction enzyme. The reactivities of the specific clones were tested by the enzyme-linked immunosorbent assay (ELISA) and the neutralizing effects were evaluated by the plaque reduction assay. RESULTS: Fingerprinting of selected clones revealed three specific single chain antibodies (scFv1, scFv2 and scFv3) with frequencies 25%, 20 and 20%. The clones produced positive ELISA with the corresponding peptide. The percentages of plaque reduction for scFv1, scFv2 and scFv3 were 23.7, 68.8 and 11.6, respectively. CONCLUSIONS: Gp55 of human CMV is considered as an important candidate for immunotherapy. In this study, we selected three specific clones against gp55. The scFvs reacted only with the corresponding peptide in a positive ELISA. The scFv2 with 68.8% neutralizing effect showed the potential to be considered for prophylaxis and treatment of CMV infections, especially in solid organ transplant recipients, for whom treatment of CMV is urgently needed. The scFv2 with neutralizing effect of 68.8%, has the potential to be considered for treatment of these patients. The specific scFv1 and scFv3 with lower neutralizing effects can be used for diagnostic purposes.

Insilico analysis of three different tag polypeptides with dual roles in scFv antibodies.

Single chain fragment variable (scFv) antibodies are composed of variable heavy (VH) and variable light (VL) domains that are joined by a polypeptide linker. Typically, [(Gly4Ser) n] sequence is used as a linker to retain the integrity of the antigen-binding domain. Due to its low immunogenicity, this sequence cannot be used as a tag for scFv detection and purification. Several evidences have shown that the addition of an N or C-terminal tag for scFv detection and purification will result in the decreased expression and binding capacity of this antibody fragment. In this study, we substituted the traditional linker (GGGGS) with His-tag, C-myc or E-tag sequences through molecular modeling. Stability and integrity of all models were assessed by molecular dynamic (MD) simulation. Based on MD simulation analysis, the model containing E-tag sequence as a linker indicated more stability compared to other molecules. The results suggest that E-tag not only can be substituted for the traditional linker, also eliminates the necessity of using additional tag for scFv detection and purification.

Expression of Vascular Endothelial Growth Factor (VEGF) and Epidermal Growth Factor Receptor (EGFR) in Patients With Serous Ovarian Carcinoma and Their Clinical Significance.

BACKGROUND: Vascular endothelial growth factor (VEGF) has an essential role in tumor metastasis by inducing the construction of abnormal blood vessels. Epidermal growth factor receptor (EGFR) is involved in different parts of cancer growth such as tumor initiation, angiogenesis and metastasis. OBJECTIVES: The aim of this study was to evaluate the expression of VEGF and EGFR in ovarian cancer in southern Iran and to assess the correlation between expression of these two markers and patients' age, tumor stage, and grade. PATIENTS AND METHODS: In this cross-sectional study, 50 paraffin blocks of serous ovarian adenocarcinomas and 50 paraffin-embedded specimens from control individuals operated for reasons other than malignancy were immunohistochemically stained using anti-human VEGF and EGFR antibodies. RESULTS: A significant difference in the frequency of positive expression of VEGF was observed in ovarian cancer patients (25.0%) compared with the control group (8.0%) (P = 0.023). A significant difference between EGFR expression in patients (56.8%) and controls (24.0%) was also obtained (P = 0.001). No significant correlation between VEGF and EGFR expression and patients' age, tumor grade and stage were detected (P > 0.05). CONCLUSIONS: The significant increase in both VEGF and EGFR in the patients with ovarian cancer compared to healthy individuals could have prognostic value. Identifying these markers may be useful for chemopreventive and chemotherapeutic strategies for patients with serous ovarian cancer.