Erratum: Overcome trastuzumab resistance of breast cancer using anti-HER2 chimeric antigen receptor T cells and PD1 blockade.

[This corrects the article on p. 688 in vol. 10, PMID: 32195036.].

The Third Generation Anti-HER2 Chimeric Antigen Receptor Mouse T Cells Alone or Together With Anti-PD1 Antibody Inhibits the Growth of Mouse Breast Tumor Cells Expressing HER2 in vitro and in Immune Competent Mice.

Chimeric Antigen Receptor (CAR)-T cells have great efficacy against CD19+ leukemia but little success for solid tumors. This study explored the effectiveness of third generation anti-HER2 CAR-T cells alone or in combination with anti-PD1 antibody on breast tumor cells expressing HER2 in vitro and in immune competent mouse model. The PDL1-positive mouse mammary tumor cell line 4T1 engineered to express luciferase and human HER2 was used as the target cell line (4T1-Luc-HER2). Anti-HER2 CAR-T cells were generated by transducing mouse spleen T cells with recombinant lentiviruses. ELISA analysis showed that IL-2 and IFN-γ secretion was increased in CAR-T cells co-cultured with the target cells, and the secretion of these two cytokines was increased further with the addition of anti-PD1 antibody. Lactate dehydrogenase assay revealed that CAR-T cells displayed a potent cytotoxicity against the target cells, and the addition of anti-PD1 antibody further enhanced the cytotoxicity. At the effector: target ratio of 16:1, cytotoxicity was 39.8% with CAR-T cells alone, and increased to 49.5% with the addition of anti-PD1 antibody. In immune competent syngeneic mouse model, CAR-T cells were found to be present in tumor stroma, inhibited tumor growth and increased tumor apoptosis significantly. Addition of anti-PD1 antibody further enhanced these anti-tumor activities. Twenty-one days after treatment, tumor weight was reduced by 50.0% and 73.3% in CAR-T group and CAR-T plus anti-PD1 group compared with blank T group. Our results indicate that anti-PD1 antibody can greatly increase the efficacy of anti-HER2 CAR-T against HER2-positive solid tumors.

Overcome trastuzumab resistance of breast cancer using anti-HER2 chimeric antigen receptor T cells and PD1 blockade.

Trastuzumab-resistance is still a major challenge in treating patients with HER2 positive breast cancer. In this study, we tried to overcome transtuzumab-resistance by examining the therapeutic efficacy of third generation anti-HER2 chimeric antigen receptor (CAR)-T cells alone and in combination with PD1 blockade against HER2 positive and trastuzumab-resistance breast cancer cells in vitro and xenograft model. The anti-HER2 CAR-T cells were generated by infecting CD3/CD28 activated peripheral blood mononuclear cells with lentivirus expressing third generation anti-HER2 CAR. Anti-HER2 CAR-T cells were specifically targeted to HER2 positive BT474 and trastuzumab resistant HCC1954 cells compared with HER2 negative breast cancer cells. Results from ELISA revealed that the secretion of IL-2 and IFN-γ was increased in anti-HER2 CAR-T cells after being co-cultured with HCC1954 cells, and was further increased with the addition of anti-PD1 antibody in the co-culture system. Furthermore, data from lactate dehydrogenase assay showed that anti-HER2 CAR-T cells displayed a potent cytotoxicity against HCC1954 and BT474 cells. Addition of anti-PD1 antibody further enhanced the cytotoxicity of anti-HER2 CAR-T cells against HCC1954 cells. Lastly, injection of anti-HER2 CAR-T cells significantly reduced the growth of HCC1954 xenograft tumors. Combining anti-HER2 CAR-T cells with anti-PD1 antibody further impaired the growth of HCC1954 tumors. The present results indicate that anti-HER2 CAR-T cells have therapeutic efficacy against trastuzumab resistant breast tumors and addition of the PD1 antibody can further enhance the therapeutic effect of anti-HER2 CAR-T cells. Thus, third generation anti-HER2 CAR-T cells along with PD1 blockade is a potential therapy to overcome trastuzumab resistance of breast cancer.

The efficacy of third generation anti­HER2 chimeric antigen receptor T cells in combination with PD1 blockade against malignant glioblastoma cells.

Without effective treatment, glioblastoma is one of the deadliest cancers worldwide. The aim of the present study was to explore whether combinational immunotherapy is effective for treating malignant glioblastoma in vitro. The therapeutic efficacy of third generation anti­human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR)­T cells alone and in combination with PD1 blockade was investigated for the treatment of malignant glioblastoma cells in vitro. Anti­HER2 CAR­T cells were prepared by transducing activated primary human T cells with lentiviruses which expressed third generation anti­HER2 CAR. The CAR­positive cell ratio was detected using flow cytometry. The expression level of CAR was detected by western blot analysis. The binding of anti­HER2 CAR­T cells to HER2+ U251 glioblastoma cells was examined under a fluorescence microscope. The cytokine secretion of CAR­T cells induced by target cells was analyzed via ELISA. The cytotoxicity of anti­HER2 CAR­T cells alone or in combination with anti­programmed death­1 (PD1) antibody against HER2+/PDL1+ U251 cells was examined using an LDH assay. The CAR­positive cell ratio and expression level of CAR in prepared CAR­T cells were both high enough. Anti­HER2 CAR­T cells could specifically bind to U251 cells. The IL­2 and IFN­Î³ secretion of CAR­T cells increased after being co­cultured with U251 cells, and further increased in the presence of anti­PD1 antibody. Anti­HER2 CAR­T cells displayed a potent cytotoxicity against U251 cells. In addition, the presence of anti­PD1 antibody further enhanced the efficacy of anti­HER2 CAR­T cells against U251 cells. The present results indicated that blocking PD1 immuno­suppression can increase the activation of CAR­T cells after they are activated by a targeting antigen. Third generation anti­HER2 CAR­T cells along with PD1 blockade have a great therapeutic potential for combatting malignant glioblastoma.

Comparative analysis and optimization of protocols for producing recombinant lentivirus carrying the anti-Her2 chimeric antigen receptor gene.

BACKGROUND: The production of anti-Her2 chimeric antigen receptor (CAR) T cells needs to be optimized to make it a reliable therapy. METHODS: Three types of lentiviral vectors expressing anti-Her2 CAR together with packaging plasmids were co-transfected into 293 T-17 cells. The vector with the best packaging efficiency was selected, and the packaging cell culture system and packaging plasmid system were optimized. Centrifugation speed was optimized for the concentration of lentivirus stock. The various purification methods used included membrane filtration, centrifugation with a sucrose cushion and the novelly-designed instantaneous high-speed centrifugation. The recombinant lentiviruses were transduced into human peripheral T cells with an optimized multiplicity of infection (MOI). CAR expression levels by three vectors and the efficacy of CAR-T cells were compared. RESULTS: When co-transfected, packaging cells in suspension were better than the commonly used adherent culture condition, with the packaging system psPAX2/pMD2.G being better than pCMV-dR8.91/pVSV-G. The optimal centrifugation speed for concentration was 20 000 g, rather than the generally used ultra-speed. Importantly, adding instantaneous centrifugation for purification significantly increased human peripheral T cell viability (from 13.25% to 62.80%), which is a technical breakthrough for CAR-T cell preparation. The best MOI value for transducing human peripheral T cells was 40. pLVX-EF1a-CAR-IRES-ZsGreen1 expressed the highest level of CAR in human peripheral T cells and the cytotoxicity of CAR-T cells reached 63.56%. CONCLUSIONS: We optimized the preparation of recombinant lentivirus that can express third-generation anti-Her2 CAR in T cells, which should lay the foundation for improving the efficacy of CAR-T cells with respect to killing target cells.