Zahirović Abida, Plavec Tina Vida, Berlec Aleš
Department of Biotechnology, Jožef Stefan Institute, Ljubljana, Slovenia.
Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia.
Front Bioeng Biotechnol. 2022 Jan 26;10:822823. doi: 10.3389/fbioe.2022.822823. eCollection 2022.
Pro-inflammatory cytokines play an important role in the development and progression of colorectal cancer (CRC). Tumor-targeting bacteria that can capture pro-inflammatory cytokines in the tumor microenvironment and thus block their tumor-promoting effects might provide clinical benefits in inflammation-associated CRC. The aim of this study was to develop bacteria with dual functionality for selective delivery of cytokine-binding proteins to the tumor by targeting specific receptors on cancer cells. We engineered a model lactic acid bacterium, , to co-display on its surface a protein ligand for tumor antigens (EpCAM-binding affitin; HER2-binding affibody) and a ligand for pro-inflammatory cytokines (IL-8-binding evasin; IL-6-binding affibody). Genes that encoded protein binders were cloned into a lactococcal dual promoter plasmid, and protein co-expression was confirmed by Western blotting. To assess the removal of IL-8 and IL-6 by the engineered bacteria, we established inflammatory cell models by stimulating cytokine secretion in human colon adenocarcinoma cells (Caco-2; HT-29) and monocyte-like cells (THP-1; U-937). The engineered removed considerable amounts of IL-8 from the supernatant of Caco-2 and HT-29 cells, and depleted IL-6 from the supernatant of THP-1 and U-937 cells as determined by ELISA. The tumor targeting properties of the engineered bacteria were evaluated in human embryonic kidney epithelial cells HEK293 transfected to overexpress EpCAM or HER2 receptors. Fluorescence microscopy revealed that the engineered specifically adhered to transfected HEK293 cells, where the EpCAM-targeting bacteria exhibited greater adhesion efficiency than the HER2-targeting bacteria. These results confirm the concept that can be efficiently modified to display two proteins simultaneously on their surface: a tumor antigen binder and a cytokine binder. Both proteins remain biologically active and provide the bacteria with tumor antigen targeting and cytokine binding ability.
促炎细胞因子在结直肠癌（CRC）的发生和发展中起重要作用。能够在肿瘤微环境中捕获促炎细胞因子并从而阻断其促肿瘤作用的肿瘤靶向细菌可能会在炎症相关的CRC中带来临床益处。本研究的目的是开发具有双重功能的细菌，通过靶向癌细胞上的特定受体将细胞因子结合蛋白选择性递送至肿瘤。我们对一种模式乳酸菌进行工程改造，使其在表面共展示肿瘤抗原的蛋白配体（EpCAM结合亲和体；HER2结合亲和体）和促炎细胞因子的配体（IL-8结合躲避素；IL-6结合亲和体）。将编码蛋白结合剂的基因克隆到乳球菌双启动子质粒中，并通过蛋白质印迹法确认蛋白共表达。为了评估工程菌对IL-8和IL-6的清除情况，我们通过刺激人结肠腺癌细胞（Caco-2；HT-29）和单核细胞样细胞（THP-1；U-937）中的细胞因子分泌建立了炎症细胞模型。通过酶联免疫吸附测定法（ELISA）测定，工程菌从Caco-2和HT-29细胞的上清液中去除了大量的IL-8，并从THP-1和U-937细胞的上清液中消耗了IL-6。在转染以过表达EpCAM或HER2受体的人胚肾上皮细胞HEK293中评估工程菌的肿瘤靶向特性。荧光显微镜检查显示，工程菌特异性粘附于转染的HEK293细胞，其中靶向EpCAM的细菌比靶向HER2的细菌表现出更高的粘附效率。这些结果证实了这样一个概念，即可以有效地对该菌进行改造，使其在表面同时展示两种蛋白：一种肿瘤抗原结合剂和一种细胞因子结合剂。两种蛋白均保持生物活性，并赋予细菌肿瘤抗原靶向和细胞因子结合能力。
