Medical school, Kunming University of Science and Technology, Kunming, 650500, P.R. China.
Department of Pathology, 920th Hospital of the Joint Logistics Support Force of PLA, 212 Daguan Rd, Kunming, 650032, P.R. China.
BMC Cancer. 2024 Jan 12;24(1):71. doi: 10.1186/s12885-023-11686-5.
Ras gene mutation and/or overexpression are drivers in the progression of cancers, including colorectal cancer. Blocking the Ras signaling has become a significant strategy for cancer therapy. Previously, we constructed a recombinant scFv, RGD-p21Ras-scFv by linking RGD membrane-penetrating peptide gene with the anti-p21Ras scFv gene. Here, we expressed prokaryotically RGD-p21Ras-scFv on a pilot scale, then investigated the anti-tumor effect and the mechanism of blocking Ras signaling.
The E. coli bacteria which could highly express RGD-p21Ras-scFv was screened and grown in 100 L fermentation tank to produce RGD-p21Ras-scFv on optimized induced expression conditions. The scFv was purified from E. coli bacteria using His Ni-NTA column. ELISA was adopted to test the immunoreactivity of RGD-p21Ras-scFv against p21Ras proteins, and the IC50 of RGD-p21Ras-scFv was analyzed by CCK-8. Immunofluorescence colocalization and pull-down assays were used to determine the localization and binding between RGD-p21Ras-scFv and p21Ras. The interaction forces between RGD-p21Ras-scFv and p21Ras after binding were analyzed by molecular docking, and the stability after binding was determined by molecular dynamics simulations. p21Ras-GTP interaction was detected by Ras pull-down. Changes in the MEK-ERK /PI3K-AKT signaling paths downstream of Ras were detected by WB assays. The anti-tumor activity of RGD-p21Ras-scFv was investigated by nude mouse xenograft models.
The technique of RGD-p21Ras-scFv expression on a pilot scale was established. The wet weight of the harvested bacteria was 31.064 g/L, and 31.6 mg RGD-p21Ras-scFv was obtained from 1 L of bacterial medium. The purity of the recombinant antibody was above 85%, we found that the prepared on a pilot scale RGD-p21Ras-scFv could penetrate the cell membrane of colon cancer cells and bind to p21Ras, then led to reduce of p21Ras-GTP (active p21Ras). The phosphorylation of downstream effectors MEK-ERK /PI3K-AKT was downregulated. In vivo antitumor activity assays showed that the RGD-p21Ras-scFv inhibited the proliferation of colorectal cancer cell lines.
RGD-p21Ras-scFv prokaryotic expressed on pilot-scale could inhibited Ras-driven colorectal cancer growth by partially blocking p21Ras-GTP and might be able to be a hidden therapeutic antibody for treating RAS-driven tumors.
Ras 基因突变和/或过表达是包括结直肠癌在内的癌症进展的驱动因素。阻断 Ras 信号已成为癌症治疗的重要策略。此前，我们通过将 RGD 膜穿透肽基因与抗 p21Ras scFv 基因连接，构建了重组 scFv，RGD-p21Ras-scFv。在这里，我们在试点规模上原核表达了 RGD-p21Ras-scFv，然后研究了其抗肿瘤作用和阻断 Ras 信号的机制。
筛选能够高表达 RGD-p21Ras-scFv 的大肠杆菌，在 100 L 发酵罐中培养，在优化的诱导表达条件下生产 RGD-p21Ras-scFv。使用 His Ni-NTA 柱从大肠杆菌中纯化 scFv。采用 ELISA 法检测 RGD-p21Ras-scFv 对 p21Ras 蛋白的免疫反应性，并通过 CCK-8 分析 RGD-p21Ras-scFv 的 IC50。免疫荧光共定位和下拉实验用于确定 RGD-p21Ras-scFv 与 p21Ras 之间的定位和结合。通过分子对接分析 RGD-p21Ras-scFv 与 p21Ras 结合后的相互作用力，并通过分子动力学模拟确定结合后的稳定性。通过 Ras 下拉检测 p21Ras-GTP 的相互作用。通过 WB 检测 Ras 下游 MEK-ERK/PI3K-AKT 信号通路的变化。通过裸鼠异种移植模型研究 RGD-p21Ras-scFv 的抗肿瘤活性。
建立了 RGD-p21Ras-scFv 试点规模表达技术。收获细菌的湿重为 31.064 g/L，从 1 L 细菌培养基中获得 31.6 mg RGD-p21Ras-scFv。重组抗体的纯度在 85%以上，我们发现制备的试点规模 RGD-p21Ras-scFv 可以穿透结肠癌细胞的细胞膜并与 p21Ras 结合，从而导致 p21Ras-GTP（活性 p21Ras）减少。下游效应物 MEK-ERK/PI3K-AKT 的磷酸化水平下调。体内抗肿瘤活性试验表明，RGD-p21Ras-scFv 抑制结直肠癌细胞系的增殖。
在试点规模上原核表达的 RGD-p21Ras-scFv 通过部分阻断 p21Ras-GTP 抑制 Ras 驱动的结直肠癌生长，可能成为治疗 RAS 驱动肿瘤的潜在治疗性抗体。
