Mosunetuzumab and lenalidomide combination therapy augments CD8+ T cells with enhanced T cell-dependent cytotoxicity against follicular lymphoma cells

Abstract

Abstract Introduction Follicular lymphoma (FL) represents most common form of indolent non-Hodgkin lymphoma. Despite significant improvements in five-year survival rates due to therapeutic advancements, relapsed/refractory (R/R) FL continues to present clinical challenges, necessitating innovative treatment approaches. Mosunetuzumab (Mosun), a CD20×CD3 bispecific antibody, functions by redirecting T cells to eliminate malignant B cells. Mosun monotherapy has been approved in the US, EU, and Japan for treating R/R FL patients who have received at least two prior lines of therapy. Currently, Phase III trials are evaluating the efficacy and safety of Mosun combined with lenalidomide (Len) in both R/R FL and first-line FL settings. Len, an immunomodulatory agent, exhibits multiple regulatory effects on immune cell functions, including T-cell activity (Griben JG et al., J Clin Oncol. (2015)). Previous research suggests that Len potentiates T-cell cytotoxicity when combined with CD3-engaging bispecific antibodies (Li J et al., Mol Cancer Ther. (2023), Olszewski AJ et al., ASH 2024). However, the precise mechanisms by which Len influences T-cell function during Mosun + Len combination therapy remain incompletely characterized (Bishton M et al., ASH 2022, Morschhauser F et al., ASH 2023). Therefore, in this study, we focused on examining the effects of Len on T cell function and its enhancement of Mosun efficacy in FL, while elucidating the underlying mechanisms of the Mosun + Len combination therapy. Methods T cells isolated from human peripheral blood mononuclear cells were co-cultured with FL cell lines (Minami-1 cells or SC-1 cells) at a 10:1 Effector: Target ratio and treated with Mosun (10 ng/mL) either alone or in combination with Len (1 μM). After 7 days, CD8+ T cells were isolated and their T cell-dependent cellular cytotoxicity (TDCC) was assessed using an xCELLigence immunotherapy assay. For this evaluation, identical numbers of CD8+ T cells were re-cocultured with fresh FL cell lines and exposed to multiple concentrations of Mosun (0-1,000 ng/ml). Flow cytometry was employed to analyze cytotoxic granule expression and CD8+ T cell number. Additionally, a multiplex immunoassay was utilized to quantify cytokine levels in co-culture supernatants. Results CD8+ T cells derived from co-cultures treated with the Mosun + Len combination demonstrated significantly enhanced cytotoxic activity against both Minami-1 and SC-1 target cells compared to those from co-cultures treated with Mosun alone, with significant differences observed at Mosun concentrations of 100 and 1000 ng/ml. To further investigate the mechanisms by which Len in combination with Mosun enhances TDCC activity, we conducted additional studies examining T cell functional characteristics after 7 days of co-culture between Minami-1 cells and T cells treated with Mosun alone or in combination with Len. Flow cytometric analysis revealed significant differences in T-cell number and character between treatment conditions. The combination of Mosun + Len resulted in a marked increase in CD8+ T-cell numbers compared to Mosun alone, Len alone, or vehicle conditions. Also, the proportion of CD8+ T cells expressing cytotoxic granules (granzyme B, perforin) was significantly increased in the Mosun + Len combination group compared to other groups. In a representative experiment, the mean percentages of granzyme B positive cells in CD8+ T cells were: vehicle (11.9%), Len (16.5%), Mosun (23.3%), and Mosun + Len (52.8%). Similarly, the mean percentages of perforin positive cells in CD8+ T cells were: vehicle (8.4%), Len (13.0%), Mosun (18.4%), and Mosun + Len (50.0%). In addition, the combination of Mosun + Len induced significantly higher levels of pro-inflammatory cytokines, such as IL-2, IFNγ, and TNFα, compared to Mosun monotherapy. Conclusion This investigation suggests that combining Mosun with Len results in expanded populations of CD8+ T cells exhibiting enhanced TDCC activity and increased pro-inflammatory cytokine production compared to Mosun monotherapy. These findings suggest a potential mechanistic basis supporting Mosun + Len combination for FL.