Anti-CD38-CAR-γδ T Cells Show Preclinical Cytotoxicity in Myeloma

Anti-CD38-CAR-gamma-delta (γδ) T cells demonstrated strong cytotoxicity against myeloma cells both in vitro and in vivo and remained active in the presence of anti-CD38 antibody drugs, according to data from a preclinical study presented at the 2025 ESMO Targeted Anticancer Therapies Congress.

Of note, throughout the culture period—including the stimulation phase—CD38 expression on γδ T cells was weaker compared with alpha-beta T cells. The functionality of anti-CD38-CAR-γδ T cells maintained potency after continuous culture and thawing from liquid nitrogen. Specifically, following the incubation of KMM1 cells with daratumumab (Darzalex) and isatuximab (Sarclisa), KMM1 cells were reduced by γδ cells in the absence of the antibody drugs. This indicated thatanti-CD38-CAR-γδ effectively destroyed myeloma cells 4 weeks after blood collection.

“We successfully induced the specific expansion of γδ T cells from peripheral blood,” lead study author Yuji Hattori, PhD, of the International Center for Cell and Gene Therapy in Toyoake, Japan, said in an oral presentation of the findings. “These γδ T cells exhibited low CD38 expression, allowing for the introduction of anti-CD38 CAR with genetic editing. In vitro, these γδ T cells effectively targeted [several] multiple myeloma lines, and their functionality was maintained in the presence of antibody drugs.”

Study Rationale and Design

CAR T-cell therapy has made a significant change to the treatment paradigm of hematologic malignancies, including multiple myeloma. However, several unmet needs persist.

"Alongside [the] remarkable progress [of CAR T-cell therapy], several challenges remain to be addressed,” Hattori said. “The processes involved in cell correction from patients, gene transfer, and ex vivo expansion are time-consuming and costly.”

To address these needs, the preclinical study was aimed at developing an off-the-shelf CAR T-cell therapy in tandem with allogeneic transplantation by reducing cost and stabilizing quality.

The focus of the study was to utilize γδ T cells—a minor subset of T cells—that can recognize and attack target cells in a major histocompatibility complex (MHC)–independent manner, which differs from alpha-beta T cells. These characteristics could allow for γδ T cells to be applied to allogeneic transplantation in the management of multiple myeloma.

In the study, γδ T cells were isolated from peripheral blood before being expanded ex vivo, and the CAR gene was introduced. During the ex vivo expansion of γδ T cells, peripheral blood was collected on day 0, and zoledronate (Reclast) was added. On day 4, the medium changed and zoledronate was removed. Following day 4, the medium was changed every few days.

“As a result of this cultivation, by 1 week after blood collection, the γδ T-cells [were] predominate in the viable cell population,” Hattori explained.

Regarding the CAR gene transfer into γδ T cells, the CAR gene was introduced by retrovirus after 4 days of activation with zoledronate. To evaluate CAR expression, a cytometry was performed using an antibody targeting G4S linker.

Following the success of generating anti-CD38-CAR-γδ T cells, the functionality was evaluated in vitro. CAR-γδ T cells were co-cultured with multiple myeloma cell lines (KMM1) for 24 hours.

Furthermore, the KMM1 cells were then incubated with daratumumab and isatuximab for 2 hours, and, subsequently, the effector passage for 24 hours.

The primary objective of the study was to achieve optimal expansion of γδ T cells from the peripheral blood and CAR transduction, as well as evaluate the efficacy of anti-CD38-CAR-γδ T cells in multiple myeloma cell lines.

In Vivo Cytotoxicity

Findings were further validated through in vivo studies with immunodeficient mice models. In vivo imaging monitored tumor progression, which was captured on days 22 and 29. Notably, tumor progression was significantly reduced after the intravenous anti-CD38-CAR-γδ T cells.

“These results demonstrate that γδ T cells exerted potential antitumor effects in vivo, effectively suppressing tumor growth,” Hattori concluded.

Disclosures: Hattori declared no conflicts of interest.

Reference

Hattori Y, Ohshima N, Taniguchi Y, et al. Development of anti-CD38-CAR allogeneic γδt cells as an off-the-shelf use for myeloma. ESMO Open. 2025;10(suppl 2):104179. doi:10.1016/j.esmoop.2025.104179