جستجوی مقالات مرتبط با کلیدواژه « Phage display » در نشریات گروه « پزشکی »

CD19 is an important antigen in manners of immunotherapy and B cell development. Studies showed that presence of CD19 is essential for B cell differentiations in various stages of a B lymphocyte. In most B cell associated malignancies, CD19 is expressed in normal to high levels making it a strong marker for targeting malignant B cells. Single chain antibodies are a derivative of antibody which only composed of variable regions of the antibody joined to each other by a polypeptide linker. They have been used for various purposes such as diagnostics, therapy and these act like a targeting part in binding to other molecules. Production of this binding molecules in E.coli expression systems have been challenging because of inability of these hosts to correctly fold the recombinant protein. Therefore, expression and purification condition that improve the solubility of scFvs in this expression system may enable us to obtain a higher yield of functional scFvs for in vitro and in vivo application.

In this study, we used a pET expression system induced by IPTG in BL21 (DE3) strain to express our phage display derived anti-CD19 scFvs. For this purpose, we cloned and expressed three clones of scFvs selected by soluble panning of a human scFv library against our target (CD19 extracellular domain) in phage display. After selection of the positive colonies, bacterial crude extracts of each colony were prepared and their affinity was checked with ELISA against CD19.

PZ7 was selected for cloning in pET28a vector, expression in BL21 and purification as it had highest affinity in crude extract ELISA. We observed the ~750 bp fragment of scFv after cloning in pET28a vector. Appropriate protein size was checked in SDS-PAGE before and after purification with NI-NTA. A protein band of ~27 kDa was confirmed in SDS-PAGE and western blotting. Furthermore, the sequence analysis showed that scFv PZ7 belonged to the human immunoglobulins family which our scFv library has derived from.

Successful implementation of the pET28a vector enabled efficient cloning and expression of a CD19-specific scFv antibody. The hybrid protein purification method employed demonstrates its potential for diverse protein types.

The phage display method is a technology that enables the expression of exogenous polypeptides on the surface of bacteriophage particles. Phage titration and ELISA are applied to measure helper phage particles or polypeptide bearing phages and also evaluation the interaction between polypeptide bearing phages and coated antigens, respectively. Although several procedures have been introduced to perform phage titration and ELISA, they face some limitations, such as being time-consuming, expensive, and low reproducibility. We developed a new system called EnzyPha by engineering the M13KO7 expressing Secreted Acid Phosphatase of Mycobacterium tuberculosis (SapM enzyme) on its pIX protein for applying in colorimetric phage titration and ELISA methods. To evaluate the idea, colorimetric phage titration and ELISA were performed and compared to the traditional methods. SapM enzyme was expressed on the pIX protein of M13KO7 properly. The colorimetric phage titration and phage ELISA showed better and comparable results against the traditional approaches. The results showed that the proposed model would titrate phages more sensitively than the plating titration method through a shorter timeframe. Moreover, it could be a better alternative to the routine phage ELISA due to time-saving, cost-effectiveness, and higher sensitivity.

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.

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.

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. 

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.

Fibroblast growth factors (FGFs) are involved in angiogenesis, wound healing and embryonic development. However, one of the causes of cancer cell growth in fibroblast-dependent cancers is FGF7 secreted by fibroblasts. Therefore, antibodies against FGF7 can be used for the treatment of these types of cancers.

In the previous studies, a phage displaying single domain antibody, D53, against human FGF7 has been identified using the phage display technique. In the present study, D53 was produced and purified in its isolated form. ELISA experiment was performed to evaluate the binding of D53 to FGF7. The mode of interaction of D53-FGF7 was explored using docking study and molecular dynamics (MD) simulations.

The expression and purification processes were verified using western blotting and SDS-PAGE analyses. ELISA experiment showed that D53 is able to recognize and bind FGF7. Docking study and MD simulations indicated that compared to dummy VH, D53 has more affinity towards FGF7.

The findings in the current study can be useful for the generation and the development of FGF7 inhibitors with a potential use in fibroblast-dependent cancers.

Production of functional recombinant antibody fragments in the periplasm of E. coli isa prerequisite step to achieve sufficient reagent for preclinical studies. Thus, the cost-effectiveand lab-scale production of antibody fragments demands the optimization of culture conditions.

The culture conditions such as temperature, optical density (OD600) at induction,induction time, and IPTG concentration were investigated to optimize the functional expressionof a phage-derived scFv molecule using a design of experiment (DoE). Additionally, the effectsof different culture media and osmolyte supplements on the expression yield of scFv wereexamined.

The developed 2FI regression model indicated the significant linear effect of theincubation temperature, the induction time, and the induction OD600 on the expression yieldof functional scFv. Besides, the statistical analysis indicated that two significant interactions ofthe temperature/induction time and the temperature/induction OD600 significantly interplay toincrease the yield. Further optimization showed that the expression level of functional scFvwas the most optimal when the cultivation was undertaken either in the TB medium or in thepresence of media supplements of 0.5 M sorbitol or 100 mM glycine betaine.

In the present study, for the first time, we successfully implemented DoE tocomprehensively optimize the culture conditions for the expression of scFv molecules in aphage antibody display setting, where scFv molecules can be isolated from a tailor-made phageantibody library known as “Human Single Fold scFv Library I.”

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.

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.

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.

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.

Cancer is a disease advanced via</em> surplus angiogenesis. The development of new anti-angiogenic therapeutic agents with more efficacy and fewer side effects is still quite necessary. Conventional therapies saving the life of many cancer patients but due to drug resistance and lack of specificity utilizing these methods is faced with limits. Recently, new therapeutic agents have been developed and used to treat cancers such as scaffold proteins, monoclonal antibodies, tyrosine kinase inhibitors, and peptides. In antiangiogenic drug development, anti-angiogenic peptides design is a significant aim. Peptides have developed as substantial therapeutics that are being carefully investigated in angiogenesis-dependent diseases because of their high penetrating rate into the cancer cells, high specificity, and low toxicity. In this review, we focus on anti-angiogenic peptides in the field of cancer therapy that are designed, screened, or derived from nanobodies, mimotopes, phage displays, and natural resources.

Phage display technology provides a new approach for making human antibody fragments that could be applicable in passive immune therapy. We applied the use of this technology to make human single-chain variable fragments (scFvs)specific for tetanus toxin.Tetanus toxin is a neurotoxin constituted by the association of two subunits, mediates its lethal action by blocking neuromuscular vesicle docking.

We previously found that six Human scFvclones inhibit toxin binding to ganglioside GT1b. This is the final report of human tetanus scFvs (scFv 8 and scFv 13) isolated from an immunized library of more than 106 scFv clones with in vivo neutralizing activity.

Only scFv 13 can reduce the in vivo toxicity induced by tetanus toxin. Also, scFv 8 has a weak capability of reducing the in vivo toxicity of the toxin.

These selected ScFvscan be considered as a possible option to substitute the human tetanus immunoglobulin (HTIG) which is extensively current immunotherapy for tetanus patients. Taken together, our results suggest that the use of human tetanus scFvs may lead to a less aggressive passive immune therapy against tetanus.

Bacteriophages, viruses which infect the bacteria are the most abundant organisms on the earth. Among them, the best studied and the most-exploited group is the filamentous phages especially M13 phage. They have shown a lot of interesting applications because of their unique features. Therefore, to get maximum performance of bacteriophage like M13, techniques need to be engaged for proper concentrations. In this review, most of these methods were explored in PubMed, Scopus and Google Scholar, using keywords including M13 bacteriophage, phage concentration, phage purification, phage display. Accordingly, the most important research papers about this subject have been collected, categorized and discussed. As a conclusion, to select an appropriate method for the concentration of M13 bacteriophages different criteria should be considered, including cost, equipment, yield and purity of the product. In general, subsequent applications of M13 phage is the most important factor for the selection of the concentration method.

Generation of antibodies which potentially discriminate between malignant andhealthy cells is an important prerequisite for early diagnosis and treatment of gastric cancer(GC). Comparative analysis of cell surface protein landscape will provide an experimental basisfor biomarker discovery, which is essential for targeted molecular therapies. This study aimedto isolate phage-displayed antibody fragments recognizing cell surface proteins, which weredifferentially expressed between two closely related GC cell lines, namely AGS and MKN-45.

We selected and screened a semisynthetic phage-scFv library on AGS, MKN-45, andNIH-3T3 cell lines by utilizing a tailored selection scheme that was designed to isolate phagescFvsthat not only recognize the differentiated AGS cells but also distinguish them from NIH-3T3 fibroblasts and the poorly differentiated MKN-45 cells.

After four rounds of subtractive whole cell panning, 14 unique clones were identifiedby ELISA screening and nucleotide sequencing. For further characterization, we focused on fourphage-scFvs with strong signals in screening, and their specificity was confirmed by cell-basedELISA. Furthermore, the selected phage-scFvs were able to specifically stain AGS cells with38.74% (H1), 11.04% (D11), 76.93% (G11), and 69.03% (D1) in flow cytometry analysis whichsupported the ability of theses phage scFvs in distinguishing AGS from MKN-45 and NIH-3T3cells.

Combined with other proteomic techniques, these phage-scFvs can be applied tomembrane proteome analysis and, subsequently, identification of novel tumor-related antigensmediating proliferation and differentiation of cells. Furthermore, such antibody fragments canbe exploited for diagnostic purposes as well as targeted drug delivery of GC.

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.

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.

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.

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.

Tumor necrosis factor alpha (TNF-α) is an inflammatory cytokine which plays crucial roles in pathogenesis of inflammatory diseases. The current study aimed to investigate the binding abilities of I44 and I49 domain antibodies to TNF-α. The dAbs were expressed in bacterial expression system and purified by affinity chromatography using Ni-sepharose column. The expression and purity of the proteins were evaluated using western blotting and SDS-PAGE techniques, respectively. ELISA experiment showed that I44 and I49 dAbs bind to TNF-α with the binding constants (Kd) of 5.18 ± 1.41 and 2.42 ± 0.55 µM, respectively. The inhibitory effect of dAbs on TNF-α biological effect was determined in MTT assay in which I44 and I49 prevented TNF-α cell cytotoxicity with IC50 values of 6.61 and 3.64 µM, respectively. The identified anti-TNF-α dAbs could bind to and inhibit TNF-α activity. The dAbs activities can be attributed to their ability to establish hydrogen bonds as well as hydrophobic contacts with TNF-α. The results of the current study can pave the way for further structural studies in order to introduce new more potent anti-TNF-α antibodies.

The single-chain variable fragment (scFv) domain of antibodies is now considered as one of the therapeutic tools that can be produced by phage display technology (PDT). Antibody purification is one of the most important steps in antibodies production. The aim of study was purification and characterization of anti-VEGFR2 scFv antibody fragments. After the coating of vascular endothelial growth factor receptor 2 (VEGFR2) peptide in ELISA microplates, the phage display library of Tomlinson was used for antibody isolation. The targeted scFv was purified by chromatography using a zeolite-based column. The purity and functional assessment of purified scFv were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting techniques, respectively. Affinity binding was evaluated by surface plasmon resonance (SPR). The desired scFv was selected after four stages of biopanning. SDS-PAGE analysis showed a 28 kDa scFv with high purity (>90%). The western bloting analysis confirmed the binding of produced scFv antibody to the desired peptide. The affinity binding of scFv antibody analyzed by SPR was about 60 μM. In this study, the novel scFv antibody against VEGFR2 peptide was purified by chromatography column containing zeolite. Based on our findings the produced antibody may be applied for diagnosis or targeting of VEGFR2 in antibody-based therapy strategies.

Nanobody (Nb) or VHH is the smallest binding domain of camelid heavy chain antibody (HcAb). Light chains of HcAb naturally removed and because of some evolutionary changes, Nbs have unique properties rather than conventional antibodies. The size of Nb is about one-tenth (0.1) of whole antibodies and this size improved some problems of four chains antibodies such as high yield of expression in prokaryotic systems and penetration to tissues. Some other characterizes of Nb like close homology to human VH, high stability in the extended range of pH and temperature, the capability to the identification of unusual epitope is very attractive for research and development of new Nb candidates for diagnosis, research, and therapeutic applications. Discovery of Nb almost coincided with advancement in phage display technology that was used along with Hybridoma technology in monoclonal antibody development. Currently, many of research groups focused on high-quality Nbs development against different targets especially in cancers and fortunately there are many of Nbs in clinical trial stages for use in extended ranges of diseases such as cancers, autoimmune and inflammatory diseases and infectious diseases. Recently two of them were approved for clinical use. Big companies like Ablynx and Merck have been invested in this field and in future, further drugs base Nbs were approved in different areas of health science. In this review, we focused on production, features, and clinical application of Nbs and will be noted to Nbs in clinical trials.

Tumor necrosis factor alpha (TNF-α) expression amplifies to excess amounts in several disorders such as rheumatoid arthritis and psoriasis. Although, Anti-TNF biologics have revolutionized the treatment of these autoimmune diseases, formation of anti-drug antibodies (ADA) has dramatically affected their use. The next generation antibodies (e.g. Fab, scFv) have not only reduced resulted immunogenicity, but also proved several benefits including better tumor penetration and more rapid blood clearance. Using affinity selection procedures in this study, a scFv antibody clone was isolated from naïve Tomlinson I phage display library that specifically recognizes and binds to TNF-α. The TNF-α recombinant protein was expressed in genetically engineered Escherichia coli SHuffle® T7 Express, for the first time, which is able to express disulfide-bonded recombinant proteins into their correctly folded states. ELISA-based affinity characterization results indicated that the isolated novel 29.2 kDa scFv binds TNF-α with suitable affinity. In-silico homology modeling study using ‘ModWeb’ as well as molecular docking study using Hex program confirmed the scFv and TNF-α interactions with a scFv-TNF- α binding energy of around -593 kj/mol which is well in agreement with our ELSIA results. The cloned scFv antibody may be potentially useful for research and therapeutic applications in the future.

CD19 is a transmembrane glycoprotein of immunoglobulin superfamily. In order to treat lymphoma, monoclonal antibodies (mAb) can target different antigens, including CD19, CD20 and CD22 on the surface of B-cells. Along with biotechnology progress, a new generation of antibodies is introduced, with the purpose of eliminating the defects of the previous generation. Among the most developed one are nanobodies (Nb). Nbs are a unique kind of camelid single domain antibody fragments with a broad range of medical applications. Unique physicochemical properties of Nbs have made them ideal candidates for therapeutic and diagnostic applications.
An immune gene library was created, and several CD19 specific Nbs were selected through antigen panning process, and their molecular properties as well as specificity, sensitivity, affinity and immunoreactivity against CD19 positive and negative cells were evaluated.
The Nb library was prepared with 7.2 x107 members. We managed to isolate a panel of CD19-specific Nbs after the last round of selection with the affinity of isolated Nbs being estimated at the standard range of 15-35 nM. Sequence analysis of positive clones was indicative of the fact that 12 variable sequences were confirmed. Of all these 12 clones, 2 clones with the greatest level signal in ELISA underwent subsequent analysis. Our sequencing results indicated high sequence homology (approximately 90%) between the Nb and Homa variable immunoglobulin domains.
Specific Nbs possess the potential to be used as novel therapeutic approaches in order to treat autoimmune diseases and B-cell lymphoma.

Tumor-targeted therapies are playing growing roles in cancer research. The exploitation of these powerful therapeutic modalities largely depends on the discovery of tumor-targeting ligands. Phage display has proven a promising high throughput screening tool for the identification of novel specific peptides with high binding affinity to cancer cells. In the present study, we describe the use of phage display to isolate peptide ligands binding specifically to human lung cancer cells. Towards this goal, we screened a phage display library of 7-mer random peptides in-vitro on non-small cell lung carcinoma (A549) as the target cell. Following selection rounds, there was a highly considerable enrichment of lung cancer-binding phages and a significant increase – 170 fold - of the phage recovery efficiency. After three rounds of in-vitro panning, a group of peptides with different frequencies were obtained. The binding efficiency and selectivity of these peptides for target and control cells were studied. The results of cellular binding assay and cell ELISA (enzyme-linked immunosorbent assay) revealed that LCP1 (Lung Cancer Peptide1) with the displayed sequence AWRTHTP is the most effective peptide in binding to lung cancer cells compared with normal lung epithelial cells and different non-lung tumor cells. In conclusion, our findings suggest that LCP1 may represent a novel peptide that binds specifically to lung cancer cells and further studies can pave the way for its application as a potential targeting moiety in the targeted delivery of diagnostic and therapeutic agents into lung malignant cells.

EGFRvIII as the most common mutant variant of the epidermal growth factor receptor is resulting from deletion of exons 2–7 in the coding sequence and junction of exons 1 and 8 through a novel glycine residue. EGFRvIII is highly expressed in glioblastoma, carcinoma of the breast, ovary, and lung but not in normal cells. The aim of the present study was identification of a novel single chain antibody against EGFRvIII as a promising target for cancer therapy.
In this study, a synthetic peptide corresponding to EGFRvIII protein was used for screening a naive human scFv phage library. A novel five-round selection strategy was used for enrichment of rare specific clones.
After five rounds of screening, six positive scFv clones against EGFRvIII were selected using monoclonal phage ELISA, among them, only three clones had expected size in PCR reaction. The specific interaction of two of the scFv clones with EGFRvIII was confirmed by indirect ELISA. One phage clone with higher affinity in scFv ELISA was purified for further analysis. The purity of the produced scFv antibody was confirmed using SDS-PAGE and Western blotting analyses.
In the present study, a human anti- EGFRvIII scFv with high affinity was first identified from a scFv phage library. This study can be the groundwork for developing more effective diagnostic and therapeutic agents against EGFRvIII expressing cancers.

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.
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.
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.
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.