Mohammad Reza Tohidkia

Angiogenesis, as a tumor hallmark, plays an important role in the growth and development of the tumor vasculature system. There is a huge amount of evidence suggesting that the vascular endothelial growth factor receptor (VEGFR-2)/VEGF-A axis is one of the main contributors to tumor angiogenesis and metastasis. Thus, inhibition of the VEGFR-2 signaling pathway by anti-VEGFR-2 mAb can retard tumor growth. In this study, we employ phage display technology and solution-phase biopanning (SPB) to isolate specific single-chain variable fragments (scFvs) against VEGFR-2 and report on the receptor binding characteristics of the candidate scFvs A semisynthetic phage antibody library to isolate anti-VEGFR-2 scFvs through an SPB performed with decreasing concentrations of the VEGFR-2-His tag and VEGFR-2-biotin. After successful expression and purification, the specificity of the selected scFv clones was further analyzed by enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunoblotting. The competition assay was undertaken to identify the VEGFR-2 receptor-blocking properties of the scFvs. Furthermore, the molecular binding characteristics of candidate scFvs were extensively studied by peptide-protein docking. Polyclonal ELISA analysis subsequent to four rounds of biopanning showed a significant enrichment of VEGFR-2-specific phage clones by increasing positive signals from the first round toward the fourth round of selection. The individual VEGFR-2-reactive scFv phage clones were identified by monoclonal phage ELISA. The sequence analysis and complementarity-determining region alignment identified the four unique anti-VEGFR-2-scFv clones. The soluble and purified scFvs displayed binding activity against soluble and cell-associated forms of VEGFR-2 protein in the ELISA and flow cytometry assays. Based on the inference from the molecular docking results, scFvs D3, E1, H1, and E9 recognized domains 2 and 3 on the VEGFR-2 protein and displayed competition with VEGF-A for binding to VEGFR-2. The competition assay confirmed that scFvs H1 and D3 can block the VEGFR-2/VEGF-A interaction. In conclusion, we identified novel VEGFR-2-blocking scFvs that perhaps exhibit the potential for angiogenesis inhibition in VEGFR-2-overexpressed tumor cells.

The current study intended to evaluate two types of biorecognition element (BRE), namely recombinant antibody fragments and M13 bacteriophage-displayed antibody fragments, where protein L and electrostatic interactions were used to respectively conjugated antibodies and bacteriophages on AuNPs. The functionalization process was examined by DLS to monitor the changes in the size and zeta potential. The formation of the BRE-G17-Gly immunological complexes was manifested by aggregation (confirmed by FE-SEM) and color change from red to dark blue visible to the naked eye. Local refractive index variations of functionalized AuNPs were monitored by a UV − vis spectrophotometer, showing increasing size and decreasing zeta potential in all stages. The calibration plot was developed in the concentration range 1–5 µg/mL and the limit of detection (LOD) was 1 µg/mL. The LSRP nanobiosensor in combination with the phage-based BRE was an affordable and simple approach, as it was able to eliminate the time-consuming and costly step of extracting antibodies. Contrary to the traditional immunoassays, this method does not require additional amplification, e.g., enzymatic, to read the result. The proposed LSPR nanobiosensor model can be adapted to detect a wide range of pathogens, viruses, and biomarkers in the shortest possible time. Graphical abstract

Purpose: 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.Methods: 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.Results: 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.Conclusion: 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."

Single-chain variable fragments (scFvs) have gained increased attention among researchers in both academic and industrial fields owing to simple production in E. coli. The E. coli periplasm has been the site of choice for the expression of scFv molecules due to its oxidizing milieu facilitating correctly formation of disulfide bonds. Hence, the recovery of high-yield and biologically active species from the periplasmic space is a critical step at beginning of downstream processing. TES (Tris/EDTA/Sucrose) as a simple and efficient extraction method has been frequently used but under varied extraction conditions, over literature. This study, for the first time, aimed to interrogate the effects of four independent variables (i.e., Tris–HCl concentration, buffer’s pH, EDTA concentration, and incubation time) and their potential interactions on the functional extraction yield of an scFv antibody from the periplasmic space of E. coli. The results indicated that the Tris–HCl concentration and pH are the most significant variables in the TES method and displayed a positive effect at their lower values on the functional extraction yield. Besides, the statistical analysis revealed 4 significant interactions between different variables. Here is the first report on the successful application of a design of experiment based on a central composite design to establish a generic and optimal TES extraction condition. Accordingly, an optimal condition for TES extraction of scFv molecules from the periplasm of HB2151 at the exponential phase was developed as follows: 50 mM Tris–HCl at pH 7.2, 0.53 mM EDTA, and an incubation time of 60 min.

Monoclonal antibodies (mAbs) are a swiftly growing class of targeted therapeutics for malignancies. After their first advent, the antibody (Ab) engineering trail has shown an evolutionary trajectory – from the rodent-derived Abs to the chimeric, humanized and fully human Abs with higher efficacy and lower/no immunotoxicity. Despite possessing great clinical potentials, several reports have highlighted that monospecific mAbs, even with high-affinity, often fail to induce sufficient immunologic responses. The full activation of the immune system demands cooperative interactions of immunotherapies with target antigen (Ag) towards functional avidity. Although the monospecific mAbs show affinity to a target Ag, they often fail to render sufficient avidity necessary for the activation of intracellular signaling mechanisms and the provocation of the immune system. Thus, various Ab/non-Ab scaffolds with much greater therapeutic impacts have been engineered based on the adjustment of their affinity and avidity balance. Novel multivalent Ab scaffolds (e.g., MDX-447, MT110, CD20Bi, TF2, and FBTA05) and mimetic Abs (e.g., Adnectin, DARPins, Ecallantide) offer improved pharmacokinetic and pharmacodynamic properties. Here, we discuss the avidity and multivalency and provide comprehensive insights into advanced Ab scaffolds used for immunotargeting and therapy of cancer.

In this research, four novel and sensitive immunosensors for electrochemical determination of G17-Gly were designed based on signal amplification and tailor-made recombinant antibody technology. Anti-G17-Gly antibody fragments (i.e. scFv and VL specific to the N- and C-terminal of G17-Gly) were immobilized onto a polymeric nanocomposite comprising poly cetyl trimethyl ammonium bromide (P(CTAB)) as the conductive matrix, chitosan (CS) as a biocompatible agent and gold nanoparticles (AuNPs) as the signal amplification element. The high surface area provided by AuNPs and the small size of scFv/VL establish the basis for immobilizing a high amount of the anti-G17-Gly on the surface of the electrode for detecting G17-Gly in human plasma samples. Under optimal conditions, the designed immunosensors provide an excellent analytical capability for detecting and determining G17-Gly in human plasma samples with a linear range from 0.5 mM to 0.05 pM and a LLOQ of 0.05 pM. The sensitivity order of the immunosensors was Ag/2-mercaptoethanol/phage displaying scFv/P(CTAB–CS)–AuNP/GE, Ag/2-mercaptoethanol/phage displaying VL/P(CTAB–CS)–AuNP/GE, Ag/BSA/scFv/P(CTAB–CS)–AuNP/GE, and Ag/BSA/VL/P(CTAB–CS)–AuNP/GE. The aforementioned characteristics demonstrate that the proposed immune-devices can be used in biological and clinical diagnosis as reliable tools for identifying different oncobiomarkers.

Purpose: 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.Methods: 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.Results: 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.Conclusion: 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.