抗OX40/EGFR双特异性抗体的制备和活性鉴定

Construction and activity identification of anti-OX40/anti-EGFR bispecific antibody

摘要

目的  构建同时靶向表皮生长因子受体（epidermal growth factor receptor，EGFR）和OX40的双特异性抗体，并在体外初步验证其对T细胞的特异性激活作用。方法  构建抗OX40/EGFR双特异性抗体的真核表达载体，转染293F细胞进行表达和纯化。构建外源表达OX40和EGFR的HEK293T细胞，利用流式细胞术分析双抗与靶蛋白表达细胞的结合活性。并利用Jurkat-OX40-NF-κB-GFP报告细胞，验证双特异性抗体对报告细胞的激活活性。通过酶联免疫吸附法（enzyme-linked immunosorbent assay，ELISA）检测外周血单个核细胞（peripheral blood mononuclear cell,PBMC）的白细胞介素-2（interleukin-2，IL-2）和γ-干扰素(interferon-γ，IFN-γ）分泌，进一步验证抗OX40/EGFR双抗对原代T细胞的激活。结果  成功构建并且纯化了抗OX40/EGFR双特异性抗体，并且验证了该双抗可以特异性地结合表达EGFR和OX40的HEK293T细胞。在表达EGFR的A549肺癌细胞介导的交联作用下，该双特异性抗体较OX40单抗更强地激活OX40-NF-κB报告细胞，并且促进PBMC分泌IL-2和IFN-γ细胞因子。结论  抗OX40/EGFR双特异性抗体构建成功，可同时识别OX40和EGFR受体并激活肿瘤特异性T细胞。

Abstract

Objective  To construct a bispecific antibody targeting epidermal growth factor receptor（EGFR） and OX40 and evaluate the function for tumor-specific T cell activation. Methods  The gene of anti-OX40/anti-EGFR bispecific antibody was cloned into eukaryotic expression vector, and then the constructed vector were transfected to 293F cells for the bispecific antibody purification. The binding activity of anti-OX40/anti-EGFR bispecific antibody with the cells expressing target proteins  were detected by flow cytometry. To identify the activation of T cells mediated by anti-OX40/anti-EGFR bispecific antibody, the activation of NF-κB signal activation was evaluated by Jurkat-OX40-NF-κB-GFP reporter cells and the activation of primary T cells was detected by interleukin-2（IL-2） and interferon-γ（IFN-γ） secretion of peripheral blood mononuclear cell(PBMC). Results  Anti-OX40/anti-EGFR bispecific antibody was successfully constructed and purified, and its binding ability to HEK293 cells expressing OX40 and EGFR was verified. Jurkat-OX40-NF-κB-GFP reporter cells were activated by the bispecific antibody with the crosslinking of A549 cells. Further, the anti-OX40/anti-EGFR bispecific antibody promoted the secretion of IL-2 and IFN-γ of PBMC. Conclusion  Anti-OX40/anti-EGFR bispecific antibody was successfully constructed which could specifically recognize OX40 and EGFR and activate tumor specific T cells.

关键词

OX40 / 表皮生长因子受体 / 双特异性抗体 / 癌症 / T细胞激活

Key words

OX40 / epidermal growth factor receptor (EGFR) / bispecific antibody (BsAb) / cancer / T cell activation