Revealing the Synergistic Potential of Mosunetuzumab Plus Polatuzumab Vedotin through CD20 Upregulation

Background

Despite the development of effective therapies for aggressive non-Hodgkin lymphoma, such as polatuzumab vedotin (Pola) in combination with rituximab, cyclophosphamide, doxorubicin, and prednisone (Pola-R-CHP), approximately 20–40% of patients experience relapsed or refractory disease after first-line treatment. Mosunetuzumab (Mosun) is a CD20×CD3 T cell–engaging bispecific antibody that engages and redirects T cells to eliminate malignant B cells. Pola is an antibody–drug conjugate that targets CD79b and delivers monomethyl auristatin E. Although clinical data supporting the safety and efficacy of the combination therapy of Mosun plus Pola in relapsed or refractory aggressive large B cell lymphoma has been reported (Budde et al., Nat Med., 2023), the molecular mechanism underlying the combination therapy remains to be elucidated. We previously demonstrated that Pola enhances CD20 expression through AKT and ERK signaling (Kawasaki et al., Br J Haematol., 2022), and a previous report indicated that loss of CD20 expression is associated with resistance to Mosun (Schuster et al., Blood, 2024). However, the impact of CD20 upregulation by Pola on Mosun sensitivity is unclear. Therefore, in this study, we investigated the involvement of Pola-induced CD20 upregulation in the efficacy of Mosun.

Methods

The in vitro efficacy of Mosun plus Pola in human diffuse large B-cell lymphoma (DLBCL) cell lines (SU-DHL-8, and HT) was evaluated by T cell–dependent cellular cytotoxicity (TDCC) assay, where T cells purified from human peripheral blood mononuclear cells (PBMCs) were co-cultured with the tumor cells at an effector-to-target ratio of 5:1. Cytotoxicity was assessed by flow cytometry. The synergy of the combination was assessed by the Bliss independence model. Control or CD20-stable-knockdown SU-DHL-8 cells were established using lentiviral short hairpin RNA (shRNA). Expression levels of surface proteins were determined by flow cytometry. For an in vivo study, SU-DHL-8 cells were subcutaneously inoculated into female NOD/ShiJic-scidJcl mice. T cells expanded from human PBMCs using Dynabeads Human T-Activator CD3/CD28 and IL-2 were intraperitoneally injected. Mousn (0.25 mg/kg) was intravenously administrated on Day 1, 8, and 15. Pola (2 mg/kg) was intravenously administrated on Day 1.

Results

We investigated the combination effect of Mosun plus Pola by TDCC assay in SU-DHL-8 cells, in which Pola is reported to enhance CD20 expression. The combination of Mosun plus Pola showed a synergistic effect in SU-DHL-8 cells. A similar synergistic effect was also demonstrated in the other DLBCL cell line, HT, in which CD20 upregulation by Pola has been also reported. To examine whether CD20 expression levels affected the efficacy of Mosun, CD20 knockdown cells were established by shRNA transduction. In CD20-downregulated SU-DHL-8 cells, Mosun sensitivity was decreased compared to the control cells. In SU-DHL-8 cells, Pola has been reported to enhance CD20 expression through AKT and ERK phosphorylation. Treatment of AKT and MEK inhibitors (MK-2206 [0.5 µM] and U0126 [10 µM]) reduced the synergistic combination effect of Mosun plus Pola in these cells. Subsequently, in the T cell–injection mouse model, we confirmed that Pola administration also enhanced the amount of CD20 expression compared to the control IgG group in SU-DHL-8 tumors on Day 5. The group administered a combination of Mosun plus Pola showed a significantly enhanced antitumor effect compared to each single-drug group on Day 15. Furthermore, to analyze the mechanism underlying the synergistic effect of Mosun plus Pola, we investigated the effect of Pola-induced CD20 upregulation on the activation status of T cells in vitro. Compared to Mosun alone, the combination of Pola and Mosun enhanced the levels of CD69, an early T cell activation marker, in both CD4⁺ and CD8⁺ T cells under the condition of the TDCC reaction against SU-DHL-8 cells.

Conclusions

In this study, we demonstrated that Pola combined with Mosun activated T cells in the Mosun-induced TDCC reaction and exhibited a synergistic efficacy against DLBCL cells. Our data suggest the possibility that Pola-induced upregulation of CD20 expression contributes to the synergy of this combination. From these findings, we identified a potential molecular rationale for the combined use of Mosun plus Pola, providing further supporting data for the effectiveness of this regimen.