rG17PE38，一种新型免疫毒素，靶向表达 CCK-2R 的胃癌。

rG17PE38, a novel immunotoxin target to gastric cancer with overexpressed CCK-2R.

机构信息

Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, Jilin University, Changchun, PR China.

出版信息

J Drug Target. 2013 May;21(4):375-82. doi: 10.3109/1061186X.2012.757770. Epub 2013 Jan 11.

DOI:10.3109/1061186X.2012.757770

PMID:23311704

Abstract

BACKGROUND

Gastrin/cholecystokinin subtype 2 receptor (CCK2R) is overexpressed in several types of tumors. Gastrin-17 (G17) peptide has a high affinity with CCK2R. These characters suggest that G17 may be useful for target cancer therapy.

PURPOSE

Construct a new immunotoxin (IT) targeting of CCK2R overexpressed gastric cancer.

METHODS

Two ITs were generated using forward and reverse G17 peptides fused with PE38. To get a high yield, codon optimized gene and optimized fermentation parameters were used in large-scale protein expression. An immunoaffinity technique was introduced into pseudomonas exotoxin (PE)-derived IT purification procedure. G17 competition, GST pull-down and indirect immunoflourescence assays were carried out to confirm the interaction between rG17 and CCK2R. Then, several cytotoxic assays were carried out on 18 cell lines, and an in vivo antitumor activity experiment was tested in nude mice.

RESULTS

The rG17PE38 showed specific cytotoxicity on three gastric cancer cells, while G17PE38 did not. After optimization, the expression level reached about 40% in medium deprived of NaCl. Next, 15-27.5 mg of pure rG17PE38 per 1 L of cultures was obtained. Results of G17 competition, GST pull-down and indirect immunoflourescence assays demonstrated that rG17 have a specific interact with CCK2R. Purified rG17PE38 showed high cytotoxicity on gastric cancer cell lines with the IC50 value of 0.6-4 ng·mL(-1). Treatment of nude mice inoculated with BGC-823 tumor xenografts with rG17PE38 efficiently inhibited tumor size.

CONCLUSIONS AND DISCUSSION

The present study demonstrates that reversed G17 could be used as target moiety of PE-derived IT and the rG17PE38 could be developed as a new immunotherapy agent. Codon optimized gene could increase the rG17PE38 expression level in E. coli and furthermore NaCl inhibits the rG17PE38 expression in large scale. Meanwhile, our present study inducts an immunoaffinity method in the IT purification procedure, which could purify the PE-derived ITs in native form.

摘要

背景

胃泌素/胆囊收缩素 2 型受体（CCK2R）在多种类型的肿瘤中过度表达。胃泌素-17（G17）肽与 CCK2R 具有高亲和力。这些特征表明 G17 可能对癌症的靶向治疗有用。

目的

构建一种针对 CCK2R 过表达胃癌的新型免疫毒素（IT）。

方法

使用正向和反向 G17 肽融合 PE38 构建了两种 IT。为了获得高产率，使用密码子优化基因和优化的发酵参数进行了大规模蛋白质表达。免疫亲和技术被引入到假单胞菌外毒素（PE）衍生的 IT 纯化过程中。进行了 G17 竞争、GST 下拉和间接免疫荧光测定，以确认 rG17 与 CCK2R 之间的相互作用。然后，在 18 种细胞系上进行了几种细胞毒性测定，并在裸鼠中进行了体内抗肿瘤活性实验。

结果

rG17PE38 对三种胃癌细胞具有特异性细胞毒性，而 G17PE38 则没有。经过优化，在不含 NaCl 的培养基中的表达水平达到约 40%。接下来，从每升培养物中获得了 15-27.5 毫克的纯 rG17PE38。G17 竞争、GST 下拉和间接免疫荧光测定的结果表明，rG17 与 CCK2R 具有特异性相互作用。纯化的 rG17PE38 对胃癌细胞系表现出高细胞毒性，IC50 值为 0.6-4ng·mL(-1)。用 rG17PE38 处理 BGC-823 肿瘤异种移植的裸鼠，有效地抑制了肿瘤的大小。

结论和讨论

本研究表明，反转的 G17 可以用作 PE 衍生的 IT 的靶位，rG17PE38 可以作为一种新的免疫治疗剂进行开发。密码子优化基因可以提高 E. coli 中 rG17PE38 的表达水平，并且 NaCl 可以在大规模培养中抑制 rG17PE38 的表达。同时，我们的研究在 IT 纯化过程中引入了免疫亲和方法，可以以天然形式纯化 PE 衍生的 IT。