靶向EGFR的金葡菌肠毒素A的毒性与抗原性弱化及其治疗肺癌的实验研究

Lung cancer is the leading cause of cancer mortality worldwide and lung adenocarcinoma is the most common type, and there is no effective treatments so far. Epidermal growth factor receptor (EGFR) is overexpressed in a variety of human cancer cells. It has been considered as a rational target for drug delivery. Phage display has identified the dodecapeptide YHWYGYTPQNVI (GE11) as a ligand that binds to the epidermal growth factor receptor (EGFR) but does not activate the receptor. Staphylococcal enterotoxin A (SEA) has good anti-tumor effects in experimental studies, but it has not been used for cancer treatment owing to its unavoidable side-effects and antigenicity. In this study,the most probably MHC class II binding sites and B-cell epitopes of SEA were predicted by several bioinformatic softwares, and were subjected to site-directed mutagenesis,and we designed five SEA mutants with ten key amino acids.For specific targeting to EGFR over-expressing cancers, superantigen SEA was genetically fused to dodecapeptide GE11 which was identified using phage display screening to have high affinity for EGFR (Kd=22nM). In our previous studies, GE11-SEAm5 mutant could bind to EGFR, activate T lymphocytes, and display a reduced systemic toxicity. This project is intended to express five different GE11-SEA mutants. The toxicity of GE11-SEA mutants will be determined by MHC class II expressing Raji cells. The EGFR-binding activity of these mutants will be determined by cell-ELISA and immunofluorescence. The inhibitory effect of GE11-SEA mutants on EGFR over-expressing lung cancer cells was examined both in vitro and in vivo. The antigenicity of GE11-SEA mutants will be identified by detecting the anti-SEA mAb concentration in mice. Eventually, we will figure out the GE11-SEA mutant which can target EGFR over-expressing lung cancer cells, retain superantigen (SAg) characteristics and with low toxicity and low antigenicity. Our research will be widely used in development of safe and long-term effects superantigen in cancer treatments， and it will provide new ideas for other protein drugs with low toxicity and antigenicity.

金葡菌肠毒素SEA抗癌作用的实验效果显著，却因为其较强的抗原性和毒副作用，未能进入临床，本课题针对此问题展开研究。GE11是从噬菌体12肽库中筛选出的EGFR高亲和力多肽，本研究首次将GE11与SEA融合表达，靶向EGFR表达的肺癌细胞。同时在生物信息学软件预测的基础上，针对SEA的MHC-II结合位点和B细胞表位设计了毒性和抗原性弱化的5种突变体。前期研究从细胞水平初步证明其中一种突变体能靶向并杀伤EGFR表达的肺癌细胞，且毒副作用减弱。现申请课题拟从细胞水平和动物模型深入分析5种突变体对EGFR表达的肺癌细胞的靶向毒性、对MHC-II表达的Raji细胞的毒性、在动物体内产生抗体的能力及过敏原性，期望获得靶向并杀伤EGFR表达的肺癌细胞、低毒性及低抗原性的SEA，并确定其毒性、抗原性相关的氨基酸位点。不仅为SEA的临床应用提供实验基础，而且为其他蛋白质药物的毒性及抗原性改造提供新的思路。

金葡菌肠毒素SEA抗癌作用的实验效果显著，却因为其较强的抗原性和毒副作用，未能进入临床，本课题针对此问题展开研究。GE11是从噬菌体12肽库中筛选出的EGFR高亲和力多肽，本研究首次将GE11与SEA融合表达，ELISA和免疫荧光结果表明，GE11-SEA能靶向EGFR表达的肺癌细胞A549。在生物信息学软件预测的基础上，针对SEA的MHC-II结合位点和B细胞表位设计并原核表达了毒性和抗原性弱化的5种突变体。细胞增殖试验证明其中一种突变体GE11-SEAm5对MHC-II表达的Raji细胞的毒性减弱；MTS细胞增殖试验、细胞凋亡试验和细胞毒性试验证明GE11-SEAm5能杀伤肺癌A549细胞；GE11-SEAm5可以抑制小鼠Lewis肺癌细胞的移植瘤生长，降低小鼠血清中SEA特异性IgG抗体及总IgE抗体浓度，以上结果从细胞水平和在体水平充分证明改造后的SEA突变体的抗原性和毒性被弱化。我们还对SEA抑制肺癌的分子机制做了进一步的研究。ELISA结果表明SEA能够刺激PBMC分泌细胞因子，如IFN-γ、TNF-α、IL-2、IL-4、IL-6、IL-10、IL-17、CCL-2。荧光定量PCR结果表明SEA能够刺激后的PBMC的细胞因子IFN-γ、TNF-α、IL-2、IL-4、IL-6、IL-7、IL-9、IL-10、IL-12A、IL-13、IL-15、IL-17A 、IL-18、IL-24、IL-27、IL-32、IL-33及趋化因子CCL-2、CXCL9、CXCL10、CXCL11 mRNA的水平明显上调。WB结果表明，SEA可以激活ERK及STAT1，3，5，6，用MEK的抑制剂U0126可以抑制ERK的磷酸化，用JAK的广谱抑制剂P6可以抑制STAT1，3，5，6的激活。我们还发现U0126可以抑制STAT1，3，5，6的磷酸化，P6可以在早期部分的抑制ERK的磷酸化。U0126和P6可以抑制PBMC的激活及细胞因子IFN-γ、TNF-α、IL-2的分泌，并抑制SEA对肺癌细胞A549的杀伤作用。因此，SEA通过激活ERK和STATs，从而激活PBMC分泌细胞因子，发挥抗肿瘤作用。SEA诱导的ERK和STATs信号通路的激活有一定的crosstalk。该研究不仅为SEA的临床应用提供理论和实验基础，而且为其他蛋白质药物的毒性及抗原性改造提供新的思路。

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