Antitumor Activity Of HER3-targeted HRG1?-CAR-T Against Breast Cancer

BackgroundChimeric antigen receptor modified T cells(CAR-T)therapy has become one of the hotspots for tumor immunotherapy due to its amazing clinical efficacy.The basic structure of CARs is composed of an extracellular antigen recognition domain,typically a single-chain variable fragment(scFv),linked to an intracellular signaling module that includes CD3? chain to induce T-cell activation upon antigen binding.To enhance efcacy and longevity of CAR-T cells,the construct has been continuously optimized,and the second,third and fourth generation CARs have been developed.Unlike other human epidermal growth factor receptor(HER)family members,HER3 has no receptor tyrosine kinases(RTKs)activity.However,it can still heterodimerize with other parters.In many tumors,HER3 acts as the main linker between RTK and PI3 K activation and is involved in tumor growth and solid tumor phenotype maintenance.In addition,as a co-receptor for HER2,HER3 is involved in tumor amplification and associated with resistance to HER2 targeted treatment in breast cancer.In view of its overexpression in tumors and compensatory effects in HER signaling,HER3 has gradually become an interesting target in the field of tumor immunotherapy.As a natural ligand for HER3/HER4,heregulin(HRG)contains an epidermal growth factor(EGF)-like sub-domain,which binds to the HER3/4 receptors and induces conformational changes,and then heterodimerizes with members of the HER tyrosine kinase receptor family.The follow-up signaling leads to cell proliferation and invasion of malignant tissues.Due to its higher receptor affinity,the ? isoform of HRG1 is considered to be the main factor for growth and proliferation in HER3/4 positive tumors.ObjectivesEndogenous ligands for tumor antigen may outperform single-chain variable fragments to serve as a component of CARs with the most appropriate affinity and minimal immunogenicity.In this study,we aim to engineer T cells with a second-generation CAR consisting of the extracellular domain of HRG1? and evaluate the specific cytotoxicity of these CAR-T cells in cultured HER3 positive breast cancer cells and xenograft tumors,thus providing a novel strategy to suppress breast cancer driven by HER family receptors and overcome cancer resistance to HER2-targeted therapy.MethodsThe lentiviral expression vector carrying the CAR gene was constructed by molecular cloning technique.The expression of CAR gene was examined by flow cytometry and western blot.T cells were obtained by magnetic beads separation and HRG1?-CAR-T cells were prepared upon lentivirus infection.Flow cytometry was used to detect the expression of target molecules,CFSE/PI double staining was used to analyze the specifc lysis by HRG1?-CAR-T cells in vitro,and ELISA was used to observe the secretion levels of cytokines IL-2 and IFN-?.Tumor cells were transfected with siRNAs targeting HER3 and the knockdown effect was analyzed by qRT-PCR and western blot.Nude mice were injected subcutaneously with HER3-positive SK-BR-3 cells to form xenograft tumors,followed by tail vein administration of control or HRG1?-CAR-T cells for treatment.Tumor volume and survival of nude mice were monitored and plotted.To detect the infiltration of T cells in tumor tissues,tumors were excised,sectioned and subject to immunohistochemical staining.ResultsThe lentiviral expression vector carrying the HRG1?-CAR gene was successfully constructed and the CAR gene expression was confirmed.T cells with high purity and certain activation levels were acquired and HRG1?-CAR-T cells were obtained after lentivirus infection.In vitro experiments showed that HRG1?-CAR-T cells can specifcally recognize and kill HER3-positive breast cancer cell lines SK-BR-3 and BT-474,and secrete high levels of cytokines IL-2 and IFN-?.Meanwhile,these CAR-T cells exert no obvious toxic effect on HER3 knockdown target cells,and HER3-negative tumor cells and normal breast epithelial cells.The results of in vivo experiments showed that HRG1?-CAR-T cells can significantly suppress tumor growth and effectively prolong the survival of nude mice.Immunohistochemical analysis revealed apparent infltration of CD3-positive T cells in the tumor tissues of mice receiving treatment with CAR-T cells,suggesting that CAR-T cells can infiltrate into the tumor site and to attack HER3-positive neoplastic cells.ConclusionsIn this study,we successfully constructed HRG1?-CAR and obtained HRG1?-CAR-T cells.The CAR-T cells specifically recognized and killed HER3-overexpressing breast cancer SK-BR-3 and BT-474 cells in vitro,and displayed potent tumoricidal effect on SK-BR-3 xenograft tumor models.Our results suggest that HRG1?-based CAR-T cells effectively suppress HER3-positive breast cancer,and may provide a novel strategy to overcome cancer resistance to HER2-targeted therapy.