Targeting CD5 chimeric antigen receptor-engineered natural killer cells against T-cell malignancies.

Abstract

Background: Chimeric antigen receptor engineered T cells (CAR-T) have demonstrated promising clinical efficacy in B-cell malignancies, and the approach has been extended to T-cell malignancies. However, the use of allogeneic T cells in CAR therapy poses a challenge due to the risk of graft-versus-host disease. Recently, natural killer (NK) cells have exhibited "off‑the‑shelf" availability. The nanobody-based CAR structures have attracted much attention for their therapeutic potential owing to the advantages of nanobody, including small size, optimal stability, high affinity and manufacturing feasibility. CD5, a common surface marker of malignant T cells, has three scavenger receptor cysteine-rich domains (D1-D3) in the extracellular region. The present study aims to construct "off‑the‑shelf" CAR-NK cells targeting the membrane-proximal domain of CD5 derived from nanobody against T-cell malignancies.

Methods: Anti-CD5-D3 nanobody was screened by phage display technology, followed by constructing fourth-generation CAR plasmids ectopically producing IL-15 to generate CD5 CAR-NK cells derived from peripheral blood. And the second-generation CD5 CAR-T cells based on nanobody were generated, referred to as 5D.b CAR-T and 12 C.b CAR-T. Furthermore, CAR-NK cells without IL-15 (IL-15^△ CAR-NK) were generated to assess the impact on cytotoxicity of CAR-NK cells. Cytotoxic activity against CD5⁺ hematologic malignant cell lines and normal T cells was exerted in vitro and NOD/ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22/Gpt mouse model transplanted with Jurkat-Luc cells was used to evaluate the antitumor efficacy of CD5 CAR-NK cells in vivo.

Results: Two nanobodies (5D and 12 C) competed for binding to the epitope of CD5-D3. 12 C CAR-NK cells were superior to 5D CAR-NK cells in antitumor potential and 12 C.b CAR-T cells exhibited superior cytotoxic activity than 5D CAR-T cells ex vivo. So, 12 C was regarded as the optimal nanobody. 12 C CAR-NK cells and IL-15^△ CAR-NK cells exhibited robust cytotoxicity against CD5⁺ malignant cell lines and controlled disease progression in xenograft mouse model. 12 C CAR-NK cells demonstrated greater antitumor activity compared to that of IL-15^△ CAR-NK cells in vitro and in vivo.

Conclusions: Taken together, the fourth-generation nanobody-derived anti-CD5 CAR-NK cells may be a promising therapeutic against T-cell malignancies.