The allogeneic fully human anti-CD38-CAR-γδ T cells enable off-the-shelf CAR-T cell therapy for myeloma

Abstract

Abstract Introduction The prognosis for myeloma patients has been improved by proteasome inhibitors (PIs), immunomodulatory drugs (IMiDs), and antibodies, particularly anti-CD38 antibodies such as daratumumab and isatuximab. However, even with the introduction of these novel therapeutic agents, the prognosis for patients with relapsed or refractory myeloma remains poor. Recently, anti-BCMA-CAR-T therapy has become clinically available for these patients, but serious limitations remain, including the long time required for CAR-T cell preparation and the limited number of eligible patients. Furthermore, repeated administration of CAR-T cells to the same patient seems to be ineffective, because rodent-derived CARs are immunogenic to host T cells. Therefore, there is a need for allogeneic T cells containing CARs with non-immunogenic scFvs that can be used as “off-the-shelf” products applicable to “all patients, anytime, anywhere.” CD38 is an appropriate target for myeloma cells. We previously demonstrated that anti-CD38 CAR-T cells containing mouse scFv are effective against multiple myeloma cells. Thus, we attempted to develop fully human anti-CD38-CAR γδ T cells for repetitive off-the-shelf use in myeloma patients. Methods γδ T cells were cultured using zoledronic acid and IL-2 and expanded from PBMCs of healthy donors. We obtained fully anti-CD38 antibodies from human derived antibody libraries. A retroviral construct for anti-CD38-CAR with a fully human scFv was created based on the genetic information obtained from the anti-CD38 antibody. We developed a fully human anti-CD38 CAR γδ T cells by retroviral transduction. CD38 expression and CAR transduction efficiency were measured by flow cytometry using anti-linker antibody. The efficacy of fully human anti-CD38 CAR γδ T cells against myeloma cells was examined in vitro by co-culture with two independent myeloma-derived cell lines (KMM1 and MOLP-8) by using flowcytometry. In vivo studies were performed on NOG mice inoculated with 1 x 106 luciferase-transfected KMM1 cells followed by intravenous injection of 5 x 106 fully human anti-CD38 CAR γδ T cells. In vivo luminescence was monitored over time to assess tumor suppression by in vivo imaging system. Results First, we investigated whether CD38 is expressed on γδ T cells, since CD38 is induced and enhanced on αβ T cells during activation for viral transduction. Interestingly, γδ T cells showed only a little increase in CD38 expression even after activation. Therefore, we concluded that there is no interaction between CD38 and anti-CD38-CAR and that it is not necessary to perform CD38 knockdown/knockout in T cells to eliminate potential fratricide. We retrovirally transduced γδ T cells with fully human anti-CD38 gene and obtained over 90% of its transduction efficiency. Next, we performed experiments in which fully human anti-CD38-CAR γδ T cells were cocultured with myeloma cell line cells and freshly isolated myeloma cells from the patients in vitro. Flow cytometry analysis showed that these CAR γδ T cells efficiently eliminated myeloma cells in a time- and cell number-dependent manner (incubation of both cells at an E:T ratio of 1:2 for 48 hours demonstrated over 90% of cytotoxic activity by fully human anti-CD38-CAR γδ T cells). Next, we examined the cytotoxic activity of fully human anti-CD38-CAR γδ T cells in a xenograft model of NOG mice inoculated with KMM1 cells transduced with the luciferase gene. Interestingly, fully human anti-CD38-CAR γδ T cells clearly inhibited myeloma growth in xenografted mice up to day 28. Finally, we also obtained the comparable cytotoxicity of cryopreserved fully human anti-CD38-CAR γδT cells to live counterparts. Conclusion We obtained cytotoxic results of fully human anti-CD38-CAR γδ T cells against myeloma cells in vitro. Furthermore, fully human anti-CD38-CAR γδ T cells significantly inhibited myeloma growth in vivo. These CAR-γδT cells have fully human scFv and MHC-unrestricted cytotoxicity, so they can be used multiple times, which is economically advantageous. Allogeneic fully human anti-CD38-CAR γδ T cells may shed new light on the treatment of myeloma patients as an off-the-shelf therapeutic agent, potentially leading to an increase in cutting-edge treatment options.