Construction Of Functional Macrophages Expressing Transmembrane Affibody And Antitumor Study Of Targeting HER2~+ Breast Cancer Model

At present,tumor immunotherapy has made remarkable progress,but still faces many challenges.The poor in-depth delivery efficiency of chimeric antigen receptor-T(CAR-T)cells and immune checkpoint blockade therapy for solid tumors is one of the main limitations of its clinical application.Macrophages,as innate immune cells,have the recruitment tendency to solid tumor and can infiltrate into the solid tumor microenvironment(TME),which makes macrophages become a good cell therapy tool through the gene engineering.Therefore,Affibody is used as a model molecule to establish a transmembrane protein expression platform.Through gene engineering technology,Affibody domain of HER2 ligand is expressed in macrophages,so that it can not only directly target HER2-EGF signal pathway of HER2 positive breast cancer cells to inhibit tumor growth,but also be used as a drug carrier to deliver antitumor drugs to living cells synergistic antitumor.The specific research contents and results of this paper are as follows:(1)With the help of CAR-T’s idea of extracellular binding,benefiting from the infection and budding expression mechanism of enveloping virus,the lentiviral vector was used to construct functional protein transmembrane display technology guided by signal peptide.The protein transmembrane expression platform was successfully established with Affibody as the model molecule,Affibody was expressed on the surface of cell membrane and the Affibody-RAW264.7 stable cell line with high HER2 binding activity was screened.QPCR,WB and confocal results showed the successful expression of Affibody-GFP fusion protein,and flow cytometry,fluorescence microscopy and CO-IP results confirmed the HER2 binding activity of Affibody;(2)Transwell technique was used to verify the HER2 positive tumor cells trend of functional macrophage Affbody-RAW264.7.The results showed that,compared with unmodified macrophage RAW264.7,the modified functional macrophage Affibody-RAW264.7 showed stronger HER2 positive tumor targeting ability in vitro;the killing ability in vitro of Affibody-RAW264.7 was monitored by living cell monitoring and LDH technology at cell level,and the results showed that the functional macrophages expressing Affibody had a significant killing effect of HER2 positive breast cancer,andthis characteristic increased with the increase of effective target ratio;(3)The functional macrophages were used as drug delivery system,and the PLGA containing DOX were used as model molecules to develop a new drug delivery platform.In the same way,we used living cell monitoring and CCK8 technology to monitor and compare the cytotoxic effect in vitro with macrophage RAW264.7 group,functional macrophage Affibody-RAW264.7 group,drug loaded functional macrophage Affibody-RAW264.7/PLGA-DOX group.PBS was used as the control group without treatment.The results showed that Affibody-RAW264.7/PLGA-DOX group had the most significant antitumor effect in vitro,the second was Affibody-RAW264.7,the worst was RAW264.7,and the cytotoxic effect of which were also proportional to their effective target ratio;(4)A HER2 positive breast cancer model was established.The tumor delivery ability of Affibody-RAW264.7,Affibody-RAW264.7,Affibody-RAW264.7 and Affibody-RAW264.7/PLGA-DOX group were determined at the mouse level by using histological section technique.PBS was also used as a control group without treatment.The data of mouse tumor experiment showed the same conclusion as that of the cytotoxic effect in vitro,which further ensured the possibility of functional macrophages expressing Affibody molecules as a new generation of cancer treatment platform.