Anti-CD38-CAR IPS-Derived Cytotoxic T Lymphocytes Efficiently Eliminate Myeloma Cells Treated with Daratumumab and Isatuximab in Vitro and In Vivo
Authors: Hattori Yuji , Kubo Takanori , Nagano Seiji , Miyama Takahiko , Taniguchi Yoshitaka , Iba Yoshitaka , Ohshima Nobuko , Kanehara Rie , Kawase Takakazu , Takahashi Masahide , Kawamoto Hiroshi , Mihara Keichiro 
Published: December 2023

Patients with myeloma have improved prognosis with proteasome inhibitor (PI), immunomodulatory drugs (IMiDs), and antibodies, especially anti-CD38 antibodies such as daratumumab and isatuximab. However, even introduction of these novel therapeutics still gives dismal prognosis in relapsed and refractory myeloma patients. Recently, although anti-BCMA-CAR-T therapy is clinically available for these patients, this option has profoundly problems including long-time preparation of CAR-T cells and limited number of suitable patients. We previously developed anti-CD38-CAR-T cells and reported that these CAR-T cells efficiently eliminated myeloma cells. To use anti-CD38-CAR-T cells as off-the-shelf, we tried to develop anti-CD38-CAR iPS-derived cytotoxic T lymphocytes (iPS-rCTLs). Firstly, we investigated whether CD38 express on iPS-rCTLs because CD38 is induced or enhanced on T cells in activation for viral transduction. Interestingly, iPS-rCTLs never expressed CD38 even in the case of activation. Thus, we never needed addition of anti-CD38 antibody in the culture medium for blockade of interaction of CD38 and anti-CD38-CAR or knockdown/knockout of CD38 on T cells. Accordingly, we retrovirally transduced iPS-rCTLs with anti-CD38-CAR and then we obtained anti-CD38-CAR iPS-rCTLs with &gt;70% of transduction efficiency. Secondly, we performed co-culture experiments of anti-CD38-CAR iPS-rCTLs and myeloma cells in vitro. Using flow cytometric analysis, these cells efficiently abrogated myeloma cells in time-dependent and in cell number-dependent manner (48-hour incubation of both cells at E:T ratio of 1:2 showed &gt;90 % of cytotoxicity with anti-CD38-CAR iPS-rCTLs). xCELLigence cell analyzer also got results which were almost consistent with those of flow cytometry. Next, we tried to investigate the cytotoxicity of anti-CD38-CAR iPS-rCTLs in the xenograft model of NOG mice inoculated with RPMI8226 cells. Following inoculation of RPMI8226 cells (1x106), anti-CD38-CAR iPS-rCTLs were intravenously injected into mice (1 x 106) twice. Intriguingly, anti-CD38-CAR iPS-rCTLs clearly inhibited myeloma growth in xenografted mice until day 30. Next, we investigated their efficacy to myeloma cells treated with daratumumab or isatuximab. Surprisingly, anti-CD38-CAR iPS-rCTLs efficiently killed myeloma cells in the presence of daratumumab or isatuximab in vitro. Now that, we are investigating the cytotoxicity of anti-CD38-CAR iPS-rCTLs in xenografted mice model of myeloma in vivo. In conclusion, we obtained the cytotoxic results of anti-CD38-CAR iPS-rCTLs against myeloma cells in vitro in the presence of daratumumab or isatuximab. And anti-CD38-CAR iPS-rCTLs definitively inhibited the myeloma growth in vivo. These results suggest that the epitope recognizable by anti-CD38-CAR iPS-rCTLs was different from that of daratumumab and isatuximab. Anti-CD38-CAR iPS-rCTLs could shed light on myeloma patients as an off-the-shelf, leading to the increased number of the cutting edged therapeutic option.