Preparation, Characterization, and Radiolabeling of Anti-HER2 scFv With Technetium Tricarbonyl and Stability Studies.

Breast cancer is the most common diagnosed cancer, and the second cause of cancer death among women, worldwide. HER2 overexpression occurred in approximately 15% to 20% of breast cancers. Invasive biopsy method has been used for detection of HER2 overexpression. HER2-targeted imaging via an appropriate radionuclide is a promising method for sensitive and accurate identification of HER2+ primary and metastatic lesions. 99mTc-anti-HER2 scFv can specifically target malignancies and be used for diagnosis of the cancer type and metastasis as well as treatment of breast cancer. We radiolabeled anti-HER2 scFv that was expressed in Escherichia coli and purified through Ni-NTA resin under native condition with 99mTc-tricarbonyl formed from boranocarbonate. HER2-based ELISA, BCA, TLC, and HPLC were used in this study. In the current study, anti-HER2 scFv was lyophilized before radiolabeling. It was found that freeze-drying did not change the binding activity of anti-HER2 scFv to HER2. Results demonstrated direct anti-HER2 scFv radiolabeling by 99mTc-tricarbonyl to hexahistidine sequence (His-tag) without any changes in biological activity and radiochemical purity of around 98%. Stability analysis revealed that 99mTc-anti-HER2 scFv is stable for at least 24 h in PBS buffer, normal saline, human plasma proteins, and histidine solution.

Predictors of the rate of cognitive decline in older adults using machine learning.

BACKGROUND: The longitudinal rates of cognitive decline among aging populations are heterogeneous. Few studies have investigated the possibility of implementing prognostic models to predict cognitive changes with the combination of categorical and continuous data from multiple domains. OBJECTIVE: Implement a multivariate robust model to predict longitudinal cognitive changes over 12 years among older adults and to identify the most significant predictors of cognitive changes using machine learning techniques. METHOD: In total, data of 2733 participants aged 50-85 years from the English Longitudinal Study of Ageing are included. Two categories of cognitive changes were determined including minor cognitive decliners (2361 participants, 86.4%) and major cognitive decliners (372 participants, 13.6%) over 12 years from wave 2 (2004-2005) to wave 8 (2016-2017). Machine learning methods were used to implement the predictive models and to identify the predictors of cognitive decline using 43 baseline features from seven domains including sociodemographic, social engagement, health, physical functioning, psychological, health-related behaviors, and baseline cognitive tests. RESULTS: The model predicted future major cognitive decliners from those with the minor cognitive decline with a relatively high performance. The overall AUC, sensitivity, and specificity of prediction were 72.84%, 78.23%, and 67.41%, respectively. Furthermore, the top 7 ranked features with an important role in predicting major vs minor cognitive decliners included age, employment status, socioeconomic status, self-rated memory changes, immediate word recall, the feeling of loneliness, and vigorous physical activity. In contrast, the five least important baseline features consisted of smoking, instrumental activities of daily living, eye disease, life satisfaction, and cardiovascular disease. CONCLUSION: The present study indicated the possibility of identifying individuals at high risk of future major cognitive decline as well as potential risk/protective factors of cognitive decline among older adults. The findings could assist in improving the effective interventions to delay cognitive decline among aging populations.

Therapeutic potential of a novel IP-10-(anti-HER2 scFv) fusion protein for the treatment of HER2-positive breast cancer.

OBJECTIVES: Interferon-γ-inducible protein 10 (IP-10) is a potent antitumor agent and acts by its angiostatic and immunomodulatory properties. IP-10 can target to tumor site by linking with single chain variable fragment (scFv) that recognized specific tumor antigen. In this study, we evaluated biological activity of the fusion protein including IP-10 and anti-HER2 scFv (IP-10-(anti-HER2 scFv)). RESULTS: The HER2- and cell-based ELISA as well as the flow cytometry analysis demonstrated that the fusion protein specifically binds to HER2 antigen. In addition, competitive ELISA demonstrated that the fusion protein recognized the same epitope of HER2 antigen as trastuzumab. The results of MTT assay demonstrated that the growth of HER2-enriched SK-BR3 cells was inhibited in the presence of the fusion protein. Moreover, the cytotoxic effect of the fusion protein was not significantly different from that of trastuzumab. However, no significant cytotoxic effect compared to trastuzumab and anti-HER2 scFv was observed in HER2-low-expressing MDA-MB-231 cells. The obtained findings demonstrated that IP-10-(anti-HER2 scFv) can selectively reduce the cell viability in HER2+ cells. Moreover, similar inhibitory effect on growth of both SK-BR-3 and MDA-MB-231 cell lines was observed in the presence of anti-HER2 scFv protein even at high concentration after 72 h. The chemotaxis properties of the fusion protein were also analyzed by a chemotaxis assay. It was demonstrated that the fusion protein induced migration of activated T cell similar to recombinant IP-10 protein. CONCLUSIONS: Our findings suggested that IP-10-(anti-HER2 scFv) fusion protein can specifically direct IP-10 to the HER2-expressing tumor cells and may act as an adjuvant along with HER2-based vaccine to gather the elicited immune response at the site of HER2-overexpressimg tumors.

Different strategies for expression and purification of the CT26-poly-neoepitopes vaccine in Escherichia coli.

BACKGROUND: Due to the association of hypermutated colorectal cancer (CRC) with many neo-antigens, poly-neo-epitopes are attractive vaccines. The molecular features of murine CT26 are similar to those of aggressive human CRC. CT26 contains some antigenic mutations, which can provide specific immunotherapy targets. Herein, we aimed to express, and purify the previously designed hexatope containing CT26 neoepitopes, CT26-poly-neoepitopes. METHODS AND RESULTS: In the current study, expression of the CT26-poly-neoepitopes was optimized in three different Escherichia coli strains including BL21 (DE3), Origami (DE3), and SHuffle®. Furthermore, the effect of ethanol on the CT26-poly-neoepitopes expression was investigated. The highest amount of CT26-poly-neoepitopes, which included CT26-poly-neoepitopes with the uncleaved pelB signal sequence and the processed one, was achieved when BL21 containing pET-22 (CT26-poly-neoepitopes) was induced with 0.1 mM IPTG for 48 h at 22 ºC in the presence of 2% ethanol. However, 37 ºC was the optimized induction temperature for expression of the CT26-poly-neoepitopes in the absence of ethanol. To purify the CT26-poly-neoepitopes, Ni-NTA affinity chromatography under denaturing and hybrid conditions were applied. High and satisfactory CT26-poly-neoepitopes purity was achieved by the combined urea and imidazole method. CONCLUSION: The effect of ethanol on expression of the CT26-poly-neoepitopes was temperature-dependent. Furthermore, the pelB-mediated translocation of the CT26-poly-neoepitopes into the periplasm was inefficient. Moreover, higher concentration of imidazole in the washing buffer improved the CT26-poly-neoepitopes purification under hybrid condition. Overall, the immunogenicity of CT26-poly-neoepitopes expressed in BL21 under the optimum condition and purified under hybrid condition can be studied in our future in vivo study.

Evaluating the efficacy of immobilized metal affinity chromatography (IMAC) for host cell protein (HCP) removal from anti-HER2 scFv expressed in Escherichia coli.

Host cell proteins (HCPs) are process-related impurities that have influence on product safety and efficacy. HCPs should effectively be removed by chromatographic steps in downstream purification process. In this study, we aimed to evaluate the efficacy of immobilized-metal affinity chromatography (IMAC) for separation of HCPs from anti-HER2 single chain fragment variable (scFv) expressed in E. coli. This study explored how different purification conditions including native, denaturing and hybrid affect HCP level in purified anti-HER2 scFv. Furthermore, the effects of NaCl concentration in wash buffer as well as imidazole concentration in wash and elution buffer on purification yield and HCP level in purified anti-HER2 scFv were evaluated. It was found that increasing imidazole concentration in wash and elution buffers in native conditions reduced the yield of anti-HER2 scFv purification. However, enhancing NaCl concentration in wash buffer in purification under native conditions led to significant increase in the amount of anti-HER2 scFv without any change in protein purity. Herein, none of the IMAC purification methods conducted on soluble cytoplasmic proteins under native conditions could reduce the amount of HCP to acceptable level. HCP content was only lowered to ˂ 10 ppm when inclusion bodies were purified under hybrid conditions. Furthermore, increasing imidazole concentration in wash buffer in purification under hybrid conditions led to significant increase in eluted anti-HER2 scFv concentration, while HCP content was also increased in this condition. Overall, purification under hybrid conditions using wash buffer containing 40 mM imidazole resulted in the highest yield and acceptable level of HCP.

Anti-HER2 scFv Expression in Escherichia coli SHuffle®T7 Express Cells: Effects on Solubility and Biological Activity.

Breast cancer is the second most commonly diagnosed cancer, worldwide. Human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer is correlated with poor prognosis. HER2-targeting monoclonal antibodies resulted in longer survival of HER2+ breast cancer. Single-chain variable fragment (scFv) demonstrates improved penetrability into tumors. Due to the presence of two disulfide bond, scFv expression in reducing bacterial cytoplasm may cause formation of inclusion bodies. Disulfide bond can be formed properly in cytoplasm of SHuffle® strain as it is trxB-, gor-, and overexpresses cytoplasmic DsbC chaperone. In this study, the anti-HER2 scFv was successfully expressed and purified in BL21 (DE3) and SHuffle® cells. Here, significant higher soluble anti-HER2 scFv was produced in SHuffle® than in BL21 strain. The specific binding of anti-HER2 scFv to HER2 was shown by flow cytometry analysis and ELISA. Moreover, it was demonstrated that the anti-HER2 scFv produced in SHuffle® binds to HER2 at higher level as compared to that expressed in BL21 cells. Furthermore, competitive ELISA-based study suggested that anti-HER2 scFv recognizes the same epitope of HER2 receptor as the trastuzumab antibody. Our findings indicated that correct disulfide bond formation in SHuffle® strain can result in enhanced solubility and higher biological activity level of anti-HER2 scFv.

The improvement of anti-HER2 scFv soluble expression in Escherichia coli

The relationship between the expression of HER2 and malignity of breast tumors has led to the generation of antibodies targeting HER2+ tumors. In addition, the expression of scFvs, as the smallest antigen-binding region of antibody containing two disulfide bonds in Escherichia coli often results in accumulating non-functional protein in the cytoplasm. A redox-modified strain of E. coli such as Origami (DE3) may facilitate the formation of proper disulfide bond in cytoplasm. The present study aimed to optimize the expression of anti-HER2 scFv in Origami and evaluate the influence of induction temperature, and host strain on the solubility of the protein. To this aim, chemicallysynthesized anti-HER2 scFv of Trastuzumab was cloned in pET-22b (+). The results demonstrated that anti-HER2 scFv is expressed in Origami, purified by using Ni-NTA column, and detected by anti-His antibody in Western blot analysis. The highest anti-HER2 scFv expression in Origami was achieved 24 h after IPTG induction (1 mM) at 37 ºC. Further, the total anti-HER2 scFv expression level was higher in BL21, compared to Origami strain. However, the ratio of soluble/insoluble forms of anti-HER2 scFv increased in Origami strain. Furthermore, higher soluble expression was achieved when the culture of recombinant Origami was conducted at lower temperature (25 ºC).

Preparation of Immunotoxin Herceptin-Botulinum and Killing Effects on Two Breast Cancer Cell Lines.

BACKGROUND: Worldwide, breast cancer is the most common cancer diagnosed among women and a leading cause of cancer deaths. The age of onset in Iran has become reduced by a decade for unknown reasons. Herceptin, a humanized monoclonal antibody, is a target therapy for breast cancer cells with over expression of HER2- neu receptors, but it is an expensive drug with only 20% beneficial rate of survival. This study introduces a novel approach to enhance the efficacy of this drug through immunoconjugation of the antibody to botulinum toxin. Decreasing the cost and adverse effects of the antibody were secondary goals of this study. MATERIALS AND METHODS: Botulinum toxin was conjugated with Herceptin using heterobifunctional cross linkers, succinimidyl acetylthiopropionate (SATP) and sulfo-succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) according to the supplier's guidelines and tested on two breast cancer cell lines: SK-BR-3 and BT-474. Toxin and Herceptin were also used separately as controls. The cytotoxicity assay was also performed using the new bioconjugate on cultured cells with Alamar blue and a fluorescence plate reader. RESULTS: Herceptin-Toxin bioconjugation significantly improved Herceptin efficacy on both breast cancer cell lines when compared to the control group. CONCLUSIONS: Toxin-Herceptin bioconjugation can be a potential candidate with increased efficiency for treating breast cancer patients with over expression of the HER2 receptor.