Oncolytic Herpes Simplex Virus-Loaded CD19 CAR-T Cells Improves the Therapeutic Effect of Diffuse Large B-Cell Lymphoma

Background In the clinical practice of treating patients with diffuse large B-cell lymphoma, CD19 CAR-T cell therapy has failed to achieve the desired therapeutic effect and is highly prone to recurrence. In the field of solid tumors, the combined therapy of CAR-T cell therapy and oncolytic viruses has made breakthrough progress. Therefore, we introduce this combined therapeutic idea into the treatment of diffuse large B-cell lymphoma. In order to deal with the characteristic that diffuse large B-cell lymphoma is highly prone to systemic metastasis and to avoid the problem that oncolytic viruses are neutralized by antibodies in the blood,we abandon the two common administration methods of oncolytic viruses, intravenous infusion and intratumoral injection, and chose to load the oncolytic viruses onto CAR-T cells for combined treatment.

Method We used the CRISPR Cas9 technology to knockout the cytotoxic genes ICP34.5 and ICP47 on Herpes Simplex Virus (HSV), and added the GFP expression cassette to obtain the oncolytic herpes simplex virus-GFP（ oHSV-GFP）. By co-culturing oHSV-GFP (MOI = 100) with CD19 CAR-T cells in a 37°C, 5% CO2 incubator for 24 hours, CD19 CAR-T^oHSV cells were obtained, and evaluated using flow cytometry and qPCR techniques. We evaluated its anti-tumor effect and safety using the human diffuse large B-cell lymphoma cell line (U2932) and the human diffuse large B-cell lymphoma animal model.

Result Flow cytometry analysis demonstrated that this method could successfully prepare CD19 CAR-T^oHSV cells, and the proportion of CD19 CAR-T cells expressing fluorescence accounted for approximately 10% of the total. It was verified again by qPCR technology. Using CCK8 to evaluate the proliferation situation, we found that this preparation method had no effect on the proliferation of CAR-T cells. CD19 CAR-T cells and CD19 CAR-T^oHSV cells were co-cultured with diffuse large B-cell lymphoma cell lines respectively. We found that the anti-tumor effect of CD19 CAR-T^oHSV cells was significantly better than that of CD19 CAR-T cells. In the evaluation of cytokines, the secretion of IFN-γ by CD19 CAR-T^oHSV cells was significantly higher than that of CD19 CAR-T cells, which might be one of the reasons for its high tumor effect. In the animal model of diffuse large B-cell lymphoma, we found that the tumor volume of mice treated with CD19 CAR-T^oHSV cells was significantly smaller than that of the control group and the mice treated with CD19 CAR-T cells. Meanwhile, immunohistochemistry showed that the infiltration of CAR-T cells in the CD19 CAR-T^oHSV cell treatment group was significantly better than that in the CD19 CAR-T cell group. This proved the superior inhibitory effect on tumor growth and tumor infiltration ability of CD19 CAR-T^oHSV cell therapy. Through qPCR analysis of the main organs and tumors of mice, we found that only the tumor showed viral replication. Evaluation of HE staining sections of the liver, spleen and kidney of mice did not reveal obvious tissue damage. This preliminarily proved the safety of CD19 CAR-T^oHSV cell therapy.

Conclusion Oncolytic herpes simplex virus-loaded CD19 CAR-T cells is a relatively safe and effective method to enhance the therapeutic effect of diffuse large B-cell lymphoma.