2B4 costimulatory domain enhancing cytotoxic ability of anti-CD5 chimeric antigen receptor engineered natural killer cells against T cell malignancies

Abstract

Background: Chimeric antigen receptor engineered T cells (CAR-T) have demonstrated extraordinary efficacy in B cell malignancy therapy and have been approved by the US Food and Drug Administration for diffuse large B cell lymphoma and acute B lymphocytic leukemia treatment. However, treatment of T cell malignancies using CART cells remains limited due to the shared antigens between malignant T cells and normal T cells. CD5 is considered one of the important characteristic markers of malignant T cells and is expressed on almost all normal T cells but not on NK-92 cells. Recently, NK-92 cells have been utilized as CAR-modified immune cells. However, in preclinical models, CART cells seem to be superior to CAR-NK-92 cells. Therefore, we speculate that in addition to the short lifespan of NK-92 cells in mice, the costimulatory domain used in CAR constructs might not be suitable for CAR-NK-92 cell engineering. Methods: Two second-generation anti-CD5 CAR plasmids with different costimulatory domains were constructed, one using the T-cell-associated activating receptor-4-1BB (BB.z) and the other using a NK-cell-associated activating receptor-2B4 (2B4.z). Subsequently, BB.z-NK and 2B4.z-NK were generated. Specific cytotoxicity against CD5 + malignant cell lines, primary CD5 + malignant cells, and normal T cells was evaluated in vitro. Moreover, a CD5 + T cell acute lymphoblastic leukemia (T-ALL) mouse model was established and used to assess the efficacy of CD5-CAR NK immunotherapy in vivo. Results: Both BB.z-NK and 2B4.z-NK exhibited specific cytotoxicity against CD5 + malignant cells in vitro and prolonged the survival of TALL xenograft mice. Encouragingly, 2B4.z-NK cells displayed greater anti-CD5 + malignancy capacity than that of BB.z-NK, accompanied by a greater direct lytic side effect versus BB.z-NK. Conclusions: Anti-CD5 CAR-NK cells, particularly those constructed with the intracellular domain of NK-cellassociated activating receptor 2B4, may be a promising strategy for T cell malignancy treatment.

Targeting T cell malignancies using CAR-based immunotherapy: challenges and potential solutions

“…This data confirms previously published data illustrating significantly improved survival and enhanced tumor reduction in irradiated T-ALL mouse models treated with CD5-CARmodified NK-92 cells compared to that of mice treated with control NK-92 cells [53]. Recently, another group tested CD5-CAR-modified NK-92 cells, using a NKspecific costimulatory domain 2B4 in their CAR constructs [82]. Interestingly, the CD5-2B4-CAR NK-92 cells displayed superiority to CD5-41BB-CAR NK-92 cells, in both in vitro and in vivo experiments [82].…”

“…Recently, another group tested CD5-CAR-modified NK-92 cells, using a NKspecific costimulatory domain 2B4 in their CAR constructs [82]. Interestingly, the CD5-2B4-CAR NK-92 cells displayed superiority to CD5-41BB-CAR NK-92 cells, in both in vitro and in vivo experiments [82].…”

You Have Got a Fast CAR: Chimeric Antigen Receptor NK Cells in Cancer Therapy

“…These deficiencies have led to NK-92 cells being genetically engineered to express a high-affinity CD16 receptor, with the rational of Cancers 2020, 12, 706 7 of 23 combining CD16-expressing NK-92 cells together with anti-tumor monoclonal antibodies to facilitate tumor elimination via ADCC [69]. Furthermore, numerous studies have overserved increased IFNγ release in CAR-expressing NK-92 cells compared to relatively low production in unmodified NK-92 cells in response to target exposure [70][71][72][73][74][75].…”

“…Only three constructs resulted in an increased cytolytic response, with the construct containing an NKG2D TM domain and 2B4 co-stimulatory domain being chosen for further analysis in iNK cells. Another comparison of 2B4 versus 4-1BB as co-stimulatory domains in NK-92 cells favored the 2B4 containing CAR construct, which induced rapid proliferation, increased cytokine production and degranulation, and decreased apoptosis in the transduced cells [70]. 2B4 is a member of the signaling lymphocytic activation molecule (SLAM)-family receptors and binds CD48, commonly expressed by hematopoietic cells.…”

Engineering of chimeric natural killer cell receptors to develop precision adoptive immunotherapies for cancer

“…Additionally, 2B4 co-stimulation resulted in superior anti-tumour efficacy of CD5-targeted CAR NK cells against T cell leukaemia compared to 4-1BB co-stimulated cells. This strategy was used to circumvent fratricide observed using CD5-targeted CAR T cells [148].…”