A new scfv-based recombinant immunotoxin against EPHA2-overexpressing breast cancer cells; High in vitro anti-cancer potency.

Immunotoxin therapy is one of the immunotherapy strategies providing a new, effective and high potency treatment against various cancers. Breast cancer is the most common cancer among women in many countries. The EPH receptors are a large part of tyrosine kinase receptors family and play an effective role in tumor development and angiogenesis. Among EPH receptors, EPHA2 is more commonly well-known and widely expressed in many cancers like breast cancer. In this study, we evaluated the specification of a designed immunotoxin formed by EPHA2-specific scfv linked with PE38KDEL on EPHA2-overexpressing breast cancer cell line. This new scfv-based recombinant immunotoxin was studied in terms of features such as binding potency, cytotoxicity effects, apoptosis induction ability, and internalization. The flow cytometry results showed that the immunotoxin can significantly (approximately 99%) bind to EPHA2-overexpressing breast cancer cell line (MDA-MB-231) in a low concentration (2.5 ng/ul) while cannot significantly bind to the normal cell line (HEK-293) or even EPHA2-very low expressing cell line (MCF-7). Using the MTT assay and Annexin V/Propidium iodide (PI) double staining method by flow cytometry, we observed significant killing and apoptosis induction of the MDA-MB-231 cells at different concentrations. Immunotoxin tracking by confocal microscopy at 2 h and 6 h revealed a massive presence of immunotoxin in the cytoplasm. Finally, given the in vitro results, it seems that this immunotoxin is competent enough to serve as a good candidate for in vivo studies to further explore the possibility of breast cancer treatment.

Rezaie E ，Amani J ，Bidmeshki Pour A ，Mahmoodzadeh Hosseini H ... -  《-》

In vitro analysis of single chain variable fragment-based immunotoxins against Erythropoietin-producing hepatocellular A2 receptor overexpressed in breast cancer cells.

Breast cancer is one of the leading causes of cancer deaths worldwide. Thereafter, designing new treatments with higher specificity and efficacy is urgently required. In this regard, targeted immunotherapy using immunotoxins has shown great promise in treating cancer. To target a breast cancer cell, the authors used the antibody fragment against a receptor tyrosine kinase, EphA2, which is overexpressed in many cancers. This fragment was conjugated to a plant toxin, subunit A of ricin, in two different orientations from N to C-terminal (EphA2- C-Ricin and EphA2- N-Ricin). Then, these two immunotoxins were characterized using in vitro cell-based assays. Three different cell lines were treated, MDA-MB-231 (breast cancer) which has a high level of EphA2 expression, MCF-7 (breast cancer) which has a low level of EphA2 expression, and HEK293 (human embryonic kidney) which has a very low level of EphA2 expression. Moreover, binding ability, cytotoxicity, internalization, and apoptosis capacity of these two newly developed immunotoxins were investigated. The flow cytometry using Annexin V- Propidium iodide (PI) method indicated significant induction of apoptosis only in the MDA-MB-231 cells at different concentrations. It was also found that construct I, EphA2- C-Ricin immunotoxin, could bind, internalize, and induce apoptosis better than the EphA2- N-Ricin immunotoxin. In addition, the obtained data suggested that the N or C-terminal orientation conformation is of significant importance.

Faraz A ，Amani J ，Arbabian S ，Karizi SZ ，Torbati MB ... -  《-》

A novel EGFR-specific recombinant ricin-panitumumab (scFv) immunotoxin against breast and colorectal cancer cell lines; in silico and in vitro analyses.

The Epidermal Growth Factor Receptor (EGFR) has been of high importance as it is over expressed in a wide diversity of epithelial cancers, promoting cell proliferation and survival pathways. Recombinant immunotoxins (ITs) have emerged as a promising targeted therapy for cancer treatment. In this study, we aimed to investigate the antitumor activity of a novel recombinant immunotoxin designed against EGFR. Using an in silico approach, we confirmed the stability of the RTA-scFv fusion protein. The immunotoxin was successfully cloned and expressed in the pET32a vector, and the purified protein was analyzed by electrophoresis and western blotting. In vitro evaluations were conducted to assess the biological activities of the recombinant proteins (RTA-scFv, RTA, scFv). The novel immunotoxin demonstrated significant anti-proliferative and pro-apoptotic effects against cancer cell lines. The MTT cytotoxicity assay revealed a decrease in cell viability in the treated cancer cell lines. Additionally, Annexin V/Propidium iodide staining followed by flow cytometry analysis showed a significant induction of apoptosis in the cancer cell lines, with half maximal inhibitory concentration (IC50) values of 81.71 nM for MDA-MB-468 and 145.2 nM for HCT116 cells (P < 0.05). Furthermore, the EGFR-specific immunotoxin exhibited non-allergenic properties. The recombinant protein demonstrated high affinity binding to EGFR. Overall, this study presents a promising strategy for the development of recombinant immunotoxins as potential candidates for the treatment of EGFR-expressing cancers.

Naemi AA ，Salmanian AH ，Noormohammadi Z ，Amani J ... -  《-》

Soluble Cytoplasmic Expression and Purification of Immunotoxin HER2(scFv)-PE24B as a Maltose Binding Protein Fusion.

Park S ，Nguyen MQ ，Ta HKK ，Nguyen MT ，Lee G ，Kim CJ ，Jang YJ ，Choe H ... -  《INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES》

Design and construction of scFv-PE35KDEL as a novel immunotoxin against human epidermal growth factor receptor 2 for cancer therapy.

Human epidermal growth factor receptor 2 (HER2) is overexpressed on the surface of some kinds of cancer cells including breast cancer. In this study, we designed and produced a novel immunotoxin consisting anti-HER2 single-chain Fv (scFv) from pertuzumab and a modified form of Pseudomonas exotoxin (PE35KDEL). The three-dimensional (3D) structure of the fusion protein (anti-HER IT) was predicted by MODELLER 9.23 and its interaction with HER2 receptor was assessed using HADDOCK web server. Anti-HER2 IT, anti-HER2 scFv, and PE35KDEL proteins were expressed by Escherichia coli BL21 (DE3). After purification of the proteins using Ni2+ affinity chromatography and refolding through dialysis, the cytotoxicity of proteins against breast cancer cell lines was examined by MTT assay. In-silico studies showed that (EAAAK)2 linker can efficiently prevent the formation of salt bridges between two functional domains and the constructed fusion protein has a high affinity to HER2 receptor. The optimum condition of anti-HER2 IT expression was 25 °C and 1 mM IPTG. The protein was successfully purified and refolded by dialysis with a final yield of 45.7 mg per 1 L of bacterial culture. The cytotoxicity results showed that anti-HER2 IT was much more toxic on HER2-overexpressing cells, BT-474 (IC50 ~ 95 nM) compared with HER2-negative cells, MDA-MB-23 (IC50 ˃ 200 nM). This novel immunotoxin has the potential to be applied as a therapeutic candidate for HER2-targeted cancer therapy. However further in vitro and in vivo evaluations are still required to confirm the efficacy and safety of this protein.

Shariaty Vaziri Z ，Shafiee F ，Akbari V 《-》