双特异性抗体mAb04-MICA对人白血病细胞K562的体内外抗肿瘤活性  

Antitumor efficacy of bispecific antibody mAb04-MICA to human leukemia cell K562 in vitro and in vivo

作　　者：杜晓典 孙福谋[1] 袁敏讷 王斐 刘雅利 尚鹏钊 王旻[1] 张娟[1] DU Xiaodian;SUN Fumou;YUAN Minne;WANG Fei;LIU Yali;SHANG Pengzhao;WANG Min;ZHANG Juan(Antibody Engineering Laboratory,School of Life Science and Technology,China Pharmaceutical University,Nanjing 210009,China)

机构地区：[1]中国药科大学生命科学与技术学院抗体工程实验室,南京210009

出　　处：《中国药科大学学报》2018年第1期117-124,共8页Journal of China Pharmaceutical University

基　　金：国家自然科学基金资助项目(No.81473125);江苏省自然科学基金资助项目(No.BK20161459);江苏高校"青蓝工程"资助项目(2014);大学生创新创业训练计划资助项目(No.201710316032X)~~

摘　　要：探讨双特异性抗体m Ab04-MICA对人白血病细胞K562的体内外抗肿瘤活性。前期研究显示,m Ab04-MICA具有抗血管生成和重塑肿瘤微环境中的免疫监视作用,体外对K562细胞具有良好的抗肿瘤作用。通过ELISA鉴定m Ab04-MICA对抗原VEFGR2和受体NKG2D的亲和力;CCK8法分析m Ab04-MICA对K562的体外增殖抑制作用;采用流式细胞术分析m Ab04-MICA对鼠源VEGFR2的交叉反应能力;建立K562皮下荷瘤BALB/c裸鼠模型,通过测定瘤体积、称量瘤重、观测荷瘤鼠生存期等方法检测目的抗体的体内抗肿瘤活性;免疫组化方法测定其对肿瘤组织本身及组织内新生血管生成的影响。结果显示:m Ab04-MICA对VEGFR2和NKG2D都具有良好的亲和力,并且在体外能够特异性地抑制VEGF诱导的K562细胞的增殖;m Ab04-MICA与鼠源VEGFR2具有较高的结合能力;相同给药剂量下,双特异性抗体抑瘤作用显著优于母体单抗,且能延长荷瘤裸鼠生存期,其中Ki-67、p-VEGFR2、VEGF及CD34的表达量显著降低,提示m Ab04-MICA可通过特异性地阻碍瘤组织VEGFR2的磷酸化而控制瘤体新生血管的生成,在治疗白血病方面具有潜在的应用价值。This study aimed to investigate the efficacy of a bispecific antibody mAb04-MICA on human leukemia cell K562both in vitro and vivo.mAb04-MICA was previously found to posses excellent anti-angiogenic activity,and have the ability to recruit immune surveillance in tumor microenvironment.In this study,the affinity of mAb04-MICA to VEGFR2and NKG2D was identified by ELISA.CCK8was used to detect the effect of mAb04-MICA on K562proliferation.The cross reactivity of mAb04-MICA to murine VEGFR2was determined by flow cytometry assay.To evaluate the antitumor activity of mAb04-MICA,tumor volume,tumor weight and the survival of K562tumor-bearing nude mice were analyzed.The anti-angiogenic activity was determined by immunohistochemistry.The results indicated that mAb04-MICA could target to VEGFR2and NKG2D,and inhibit K562proliferation specifically.Besides,mAb04-MICA showed high binding capacity to murine VEGFR2.The bispecific antibody exhibited superior antitumor efficacy to the maternal monoclonal antibody and prolonged the survival of tumor-bearing mice.The expression of Ki-67,p-VEGFR2,VEGF and CD34in mAb04-MICA treated group was significantly reduced.The results indicated that mAb04-MICA could attenuate the phosphorylation of VEGFR2and impair angiogenesis of the tumor microenviroment.Therefore,mAb04-MICA could be further developed as a potential tumor targeted immunotherapeutic agent for leukemia.

关 键 词：双特异性抗体 血管内皮生长因子受体2 MHC-I相关抗原分子A 免疫监视 白血病 

分 类 号：Q511[生物学—生物化学] R965[医药卫生—药理学]