Synergistic and Persistent Effect of T-Cell Immunotherapy with Anti-CD19 or Anti-CD38 Chimeric Receptor on B-Cell Lymphoma in Conjunction with Rituximab
Authors: Mihara Keichiro , Yanagihara Kazuyoshi , Takigahira Misato , Takihara Yoshihiro , Kimura Akiro 
Published: April 2020

Abstract
Approximately half of the patients with B-cell non-Hodgkin’s lymphoma (B-NHL) are refractory to the standard chemotherapy. Immunotherapeutic approaches may help overcome cellular drug-resistance without damaging normal tissues. By using artificial receptors, it is possible to redirect the specificity of immune cells to tumor-associated antigens that may provide a useful strategy to the cancer immunotherapy. Since B-NHL cells invariably express CD19 and CD38, these antigens may be suitable molecular candidates for immunotherapy. Thus, we prepared for GFP-containing retroviral vectors for anti-CD19 and anti-CD38 chimeric receptors. We transduced human peripheral T cells or T cell lines with an anti-CD19-chimeric receptor (CR), or anti-CD38-CR containing anti-CD19, or anti-CD38 antibody-derived single-chain variable domain respectively. Retroviral transduction led to anti-CD19-CR or anti-CD38-CR expression in T cells with high efficiency (&gt;80%). T cell line Hut78 retrovirally transduced with anti-CD19-CR or anti-CD38-CR exerted powerful cytotoxicity against B-NHL cell lines, HT, RL and lymphoma cells freshly isolated from patients with B-NHL in vitro individually. This killing effect was dependent on dose and duration. To examine the synergistic effect of two chimeric receptors, we examined effect of Hut78 transduced with anti-CD19-CR and/or -anti-CD38-CR on HT, RL cells or lymphoma cells from patients. Interestingly, we found that two sets of chimeric receptors exerted additive cytotoxic effect on HT, RL cells and lymphoma cells in vitro. To confirm the mutual effect of T cells with these chimeric receptors on lymphoma cells in vivo, we used NOD/SCID mice transplanted with HT cells, which were labeled with luciferase. We monitored intensity and localization of the luciferase activity from the tumor by in vivo photon counting system. After mice were subcutaneously inoculated with HT-luciferase cells, human peripheral T cells expressing either anti-CD19-CR or anti-CD38-CR were injected into mice. These T cells with either anti-CD19-CR or anti-CD38-CR exerted synergistic suppressing effect on tumor formation in the inoculated mice. Next, we examined whether there is any synergistic effect of those T cells and rituximab on mice inoculated with HT-luciferase cells. Intriguingly, we could not detect the luciferase activity in any mice treated with both of the T cells and rituximab for more than 40 days. Thus, we concluded that the T cells with either anti-CD19-CR or anti-CD38-CR enhanced cytotoxicity against HT-luciferase cells in xenografted mice in conjunction with rituximab. Moreover, the synergistic tumor-suppressing action was persistent for over two months in vivo. Next, we examined whether these therapeutic strategy has any adverse effect on mice with these T cells in conjunction with rituximab. We confirmed that mice treated with the T cells bearing either of two different CR in the presence of rituximab had no adverse effect on peripheral blood cells and also on bone marrow cells for over two months. Here we demonstrated that the simultaneous immunotherapy against different antigens augmented tumor-suppressing effect on B-lymphoma cells in vitro and in vivo with few side effects. These results may provide a powerful rationale for clinical testing of autologous T cells with anti-CD19-CR or anti-CD38-CR and rituximab in patients with aggressive or relapsed B-NHLs, which are refractory to the conventional therapy.