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

Yuan, Weiping; Chen, Jie; Cao, Ying; Yang, Leixiang; Shen, Luxi; B, Qi; Bin, Shufang; Li, Panyuan; Cao, Jianhua; Fang, Hezhi; Gu, Haihua; Li, Hongzhi

doi:10.1002/jgm.3027

Cited by 10 publications

(11 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The third generation anti-HER2 CAR was generated according to our published work ( 33 ). The recombinant lentivirus expressing the anti-HER2 CAR was produced by co-transfecting 293T-17 cells with recombinant lentiviral vector pLVX-EF1α-CAR-IRES-ZsGreen1 together with the packaging plasmids pSPAX2 and pMD2.G.…”

Section: Methodsmentioning

confidence: 99%

“…The recombinant lentivirus expressing the anti-HER2 CAR was produced by co-transfecting 293T-17 cells with recombinant lentiviral vector pLVX-EF1α-CAR-IRES-ZsGreen1 together with the packaging plasmids pSPAX2 and pMD2.G. The protocol for packaging, concentration and purification of the recombinant lentivirus was essentially as we described previously ( 33 ).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

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

Yang

et al. 2020

Front. Oncol.

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Yang

et al. 2020

Front. Oncol.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Lentiviruses can transduce not only dividing cells (without nuclear membranes) but also nondividing cells (with nuclear membranes), whereas retroviruses can only transduce dividing cells [113]. Lentiviral preintegration complexes can enter the nucleus via the ATP-dependent nuclear pore complex.…”

Section: Using An LV Can Increase the Persistence And Expansion Of Camentioning

confidence: 99%

Mechanisms underlying CD19-positive ALL relapse after anti-CD19 CAR T cell therapy and associated strategies

Nie

Chen

et al. 2020

Biomark Res

View full text Add to dashboard Cite

Chimeric antigen receptor (CAR) T cell therapy, especially anti-CD19 CAR T cell therapy, has shown remarkable anticancer activity in patients with relapsed/refractory acute lymphoblastic leukemia, demonstrating an inspiring complete remission rate. However, with extension of the follow-up period, the limitations of this therapy have gradually emerged. Patients are at a high risk of early relapse after achieving complete remission. Although there are many studies with a primary focus on the mechanisms underlying CD19relapse related to immune escape, early CD19 + relapse owing to poor in vivo persistence and impaired efficacy accounts for a larger proportion of the high relapse rate. However, the mechanisms underlying CD19 + relapse are still poorly understood. Herein, we discuss factors that could become obstacles to improved persistence and efficacy of CAR T cells during production, preinfusion processing, and in vivo interactions in detail. Furthermore, we propose potential strategies to overcome these barriers to achieve a reduced CD19 + relapse rate and produce prolonged survival in patients after CAR T cell therapy.

show abstract

“…The recombinant lentivirus was produced using 293T-17 cells co-transfected with recombinant lentiviral vector pLVX-EF1α-CAR-IRES-ZsGreen1 as well as packaging plasmids psPAX2 and pMD2.G. Packaging, concentration and purification of the recombinant lentivirus were performed following protocols we had optimized (28).…”

Section: Construction Of Anti-her2 Car and Production Of Recombinant mentioning

confidence: 99%

“…Preparation of human peripheral T lymphocytes. Preparation of human peripheral T lymphocytes, including isolation, activation, and purification, was performed following the protocols we have previously described (28,29) but with some further optimization. The present study was approved by the Ethical Committee of Wenzhou Medical University (Wenzhou, China).…”

Section: Construction Of Anti-her2 Car and Production Of Recombinant mentioning

confidence: 99%

The efficacy of third generation anti‑HER2 chimeric antigen receptor T�cells in combination with PD1 blockade against malignant glioblastoma cells

Shen

Bin

et al. 2019

Oncol Rep

Self Cite

View full text Add to dashboard Cite

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 anti-human 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. Anti-HER2 CART cells were prepared by transducing activated primary human T cells with lentiviruses which expressed third generation anti-HER2 CAR. The CAR-positive cell ratio was detected using flow cytometry. The expression level of CAR was detected by western blot analysis. The binding of anti-HER2 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 anti-HER2 CART cells alone or in combination with anti-programmed death-1 (PD1) antibody against HER2 + /PDL1 + U251 cells was examined using an LDH assay. The CAR-positive cell ratio and expression level of CAR in prepared CART cells were both high enough. Anti-HER2 CART cells could specifically bind to U251 cells. The IL-2 and IFN-γ secretion of CART cells increased after being co-cultured with U251 cells, and further increased in the presence of anti-PD1 antibody. Anti-HER2 CART cells displayed a potent cytotoxicity against U251 cells. In addition, the presence of anti-PD1 antibody further enhanced the efficacy of anti-HER2 CART cells against U251 cells. The present results indicated that blocking PD1 immuno-suppression can increase the activation of CART cells after they are activated by a targeting antigen. Third generation anti-HER2 CART cells along with PD1 blockade have a great therapeutic potential for combatting malignant glioblastoma.

show abstract

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

Abstract: 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.

Cited by 10 publications

References 44 publications

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

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

Mechanisms underlying CD19-positive ALL relapse after anti-CD19 CAR T cell therapy and associated strategies

The efficacy of third generation anti‑HER2 chimeric antigen receptor T�cells in combination with PD1 blockade against malignant glioblastoma cells

Contact Info

Product

Resources

About