A high affinity nanobody against endothelin receptor type B: a new approach to the treatment of melanoma.
抗内皮素 B 型受体的高亲和力纳米抗体: 治疗黑色素瘤的新方法。
- 作者列表："Ji L","Dong C","Fan R","Qi S
:The aim of the study was to produce a single-domain antibody (nanobody) specific for endothelin receptor type B (EDNRB) which has high expression in melanoma. Cultured human melanoma cells were used as antigens to immunize alpacas. After antibody generation was verified in alpaca serum, total RNA was extracted from alpaca lymphocytes and the target VHH fragment was amplified by two-step PCR, cloned in the pCANTAB5E phagemid vector, and used to transform Escherichia coli TG1 cells to obtain a phage-display nanobody library, which was enriched by panning. The results indicated successful construction of a phage-display anti-human melanoma A375 nanobodies library with a size of 1.2 × 108/ml and insertion rate of 80%. After screening, eight positive clones of anti-EDNRB nanobodies were used to infect E. coli HB2151 for production of soluble nanobodies, which were identified by ELISA. Finally, we obtained a high-affinity anti-EDNRB nanobody, which consisted of 119 amino acids (molecular weight: 12.97 kDa) with 22 amino acids in CDR3 and had good affinity in vitro. The results suggest that the nanobody may be potentially used for the treatment of human melanoma.
: 本研究的目的是产生一种在黑色素瘤中高表达的内皮素 B 受体 (EDNRB) 特异性单域抗体 (纳米抗体)。用培养的人黑色素瘤细胞作为抗原免疫羊驼。在羊驼血清中验证抗体生成后，从羊驼淋巴细胞中提取总 RNA，通过两步 PCR 扩增目标 VHH 片段，克隆到 pCANTAB5E phacemid 载体中, 并用于转化大肠杆菌 TG1 细胞，获得噬菌体展示纳米抗体文库，经淘选富集。结果表明，噬菌体展示抗人黑色素瘤 A375 纳米抗体文库构建成功，大小为 1.2 × 108/ml，插入率为 80%。筛选后，用 8 个抗 EDNRB 纳米抗体阳性克隆感染大肠杆菌 HB2151 生产可溶性纳米抗体，用 ELISA 法进行鉴定。最后，我们获得了一种高亲和力的抗 EDNRB 纳米抗体，它由 119 个氨基酸 (分子量: 12.97 kDa) 组成，CDR3 中有 22 个氨基酸，在体外具有良好的亲和力。结果表明，纳米抗体可能用于人类黑色素瘤的治疗。
METHODS::In colorectal cancer (CRC), hepatic arterial infusion (HAI) chemotherapy may convert primarily unresectable CRC liver metastases (CLM) into resectability, although the risk of metastatic recurrence remains high after CLM ablation. We investigated the role of antitumour immunity invoked by first-line oxaliplatin-HAI for long-term CLM outcome. In a prospective study cohort of primarily unresectable CLM, we assessed patients' fms-related tyrosine kinase 3 ligand (FLT3LG) in serum, reflecting opportune intratumoural immune activity, at baseline and following 1-3 sequences of oxaliplatin-HAI. The end points were CLM resectability and overall survival. Patients who presented an immediate twofold increment of circulating FLT3LG during the treatment and at its completion were scored as CLM resectable (16.4% with both features), were alive at final follow-up 8-12 years later. All patients experienced FLT3LG increase during the treatment course, but those who remained unresectable or had the disease converted but presented a slow and gradual FLT3LG accretion, later died of the metastatic disease. These data provide further support to our previous findings that tumour-directed immunity invoked by oxaliplatin-containing therapy predicts excellent outcome of early advanced CRC if macroscopic tumour ablation is rendered possible by the 'classic' tumour response to the cytotoxic treatment.
METHODS::Prostate cancer is one of the primary causes of death around the world. As an important drug, flutamide has been used in the clinical diagnosis of prostate cancer. However, the over dosage and improper discharge of flutamide could affect the living organism. Thus, it necessary to develop the sensor for detection of flutamide with highly sensitivity. In this paper, we report the synthesis of lanthanum cobaltite decorated halloysite nanotube (LCO/HNT) nanocomposite prepared by a facile method and evaluated for selective reduction of flutamide. The as-prepared LCO/HNT nanocomposite shows the best catalytic performance towards detection of flutamide, when compared to other bare and modified electrodes. The good electrochemical performance of the LCO/HNT nanocomposite modified electrode is ascribed to abundant active sites, large specific surface area and their synergetic effects. Furthermore, the LCO/HNT modified electrode exhibits low detection limit (0.002 μM), wide working range (0.009-145 μM) and excellent selectivity with remarkable stability. Meaningfully, the developed electrochemical sensor was applied in real environmental samples with an acceptable recovery range.
METHODS::Several studies have indicated that cancer-associated fibroblasts (CAFs) could promote cancer progression in many malignancies. However, the mechanism by which CAFs promote the growth and metastasis of lung cancer remains poorly defined. In the present study, CAFs and normal fibroblasts (NFs) were isolated from human lung cancer and adjacent tissue. The data showed that the conditional medium (CM) of CAFs could increase the proliferation, migration and invasion of lung cancer cells. Vascular cell adhesion molecule-1 (VCAM-1) showed a higher expression in CAF-CM than NF-CM, and blocking VCAM-1 in CAF-CM attenuated the proliferation and invasion of cancer cells. Further, the results showed that VCAM-1 secreted from CAFs activated AKT and MAPK signaling via receptor α4β1 integrin (very-late antigen (VLA)-4) in lung cancer cells. Moreover, CAFs promoted VCAM-1 expression and tumor growth in vivo. Additionally, bioinformatics analysis indicated a positive correlation on the CAF marker protein alpha-smooth muscle actin (α-SMA) and VCAM-1 expression, which was associated with a poor prognosis in lung cancer patients. These findings demonstrate that the VCAM-1 secreted from CAFs enhances growth and invasion by activating the AKT and MAPK signaling of lung cancer cells.