Introduction: 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. Materials and Methods: 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. Results: 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. Conclusion: 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.

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

Vascular endothelial growth factor receptor 2 (VEGFR-2) is known as one of the important antigens playing a vital role in angiogenesis. In this study, phage display technology (PDT) was used to produce a Single-chain variable fragment (scFv) antibody against a region of the domain 3 in VEGFR-2 called kinase insert domain receptor 3 (KDR3). After designing the KDR3 peptide and biopanning, a colony was chosen for scFv antibody expression. Following expression and purification; western blotting, dot blotting and immunofluorescence (IF) were used to evaluate the antibody function. Surface plasmon resonance (SPR) was also employed to measure affinity of produced antibody. Once a colony was selected and transferred to the expression host, the scFv antibody was expressed in the expected range of 28 kDa. Using a designed chromatography column, antibody purification was found to be about 95%. In this study, a novel scFv with the capability of binding to KDR3 was isolated and purified and its intracellular function was investigated and verified.

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). Conclusions: 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.

Background: CD20 is an important cell surface receptor that is used for target therapy of B cell lymphoma and some related blood diseases due to vital function of CD20.In previous studies, a Rituximab based humanized Single chain variable fragment (scFv) antibody showed good reactivity against B cell related cancer cells. But this recombinant protein produced Inclusion Bodies (IBs) inEscherichia coli (E. coli) cytoplasm.The aim of this study was to investigate the effect of coexpression with cytoplasmic chaperones on expression and solubility of humanized anti-CD20 scFv inE.coli.Methods: For this purpose, the fragment coding for anti-CD20 huscFv subcloned into the pET22b (+) and transformed into theE. coli BL21 (DE3) was evaluated. In order to inhibit the production of IBs, the effects of co-expression with cytoplasmic chaperones GroEL, DnaK, GroES, Tig, DnaJ and GrpE were investigated.Result: Coexpression with cytoplasmic chaperones led to increased soluble expression of anti-CD20 recombinant protein. Among investigated chaperones, pKJE7 chaperone plasmid containing DnaJ, GrpE, DnaK chaperone genes had significant effects with an expression yield of 325μg/ml soluble anti-CD20 scFv.Conclusion: The result of this study demonstrated remarkable effect of pKJE7 chaperone on enhancement of soluble expression of anti-CD20 huscFv antibody inE. coli.