IgG2a-formatted 4-1BB agonism combined with S100A9 inhibition enhances T cell activation and tumor control in a preclinical model of multiple myeloma

Abstract Introduction Immunotherapy has shown clinical promise in multiple myeloma (MM), but relapse remains common, largely due to the immunosuppressive bone marrow (BM) microenvironment, characterized by T cell exhaustion and the accumulation of myeloid-derived suppressor cells (MDSCs). Recent advances highlight the therapeutic promise of 4-1BB (CD137) agonist antibodies in enhancing T cell–mediated anti-tumor responses, though their efficacy in MM remains poorly defined. Investigating 4-1BB expression dynamics in a preclinical MM model informed our rationale for targeting this pathway. Tasquinimod (TasQ), a small-molecule immunomodulatory agent, currently evaluated in a phase Ib/IIa clinical trial in MM patients (NCT04405167), offers a complementary strategy. By inhibiting the S100A9 signaling pathway, TasQ interferes with the recruitment and function of MDSCs, resulting in a less suppressive tumor microenvironment (TME) which reestablishes the anti -tumor immunity. In this study, we assessed the therapeutic potential of two distinct 4-1BB agonist antibody clones in an immunocompetent preclinical MM model. Building on these findings, we further explored the combination of the most effective 4-1BB agonist with TasQ, aiming to overcome the immunosuppressive tumor microenvironment, and strengthen anti-myeloma immune activity. Methods 4-1BB expression was analyzed during disease progression in 5T33MM mice using single-cell RNA sequencing of spleen and BM, 2 myeloma-infiltrating organs. These findings were validated by flow cytometry in both 5T33MM and 5TGM1 models. Therapeutic potential was assessed by treating 5TGM1 tumor-bearing mice with two 4-1BB agonist clones—LOB12.3 (IgG1κ; n=7/group) and 3H3 (IgG2a; n=5/group), using clone-specific isotype controls. Beginning on day 3 post-tumor inoculation, mice received 100µg of antibody intraperitoneally twice weekly until end stage. In a follow-up experiment, the lead 4-1BB agonist was combined with TasQ, administered at 30mg/kg in drinking water, to evaluate the impact of dual targeting the immunosuppressive TME (n=11/group). Tumor burden was assessed by determining the percentage of plasmacytosis in BM and spleen through cytospin stainings, together with M-protein measurement by serum electrophoresis. Immunomodulating effects were investigated using multi-parameter flow cytometry. Statistical significance was determined using the Mann–Whitney U test or one-way ANOVA, with p<0.05 considered significant. Results 4-1BB was predominantly detected on T cells and natural killer (NK) cells, with its expression further increasing as the disease progressed. Treatment of 5TGM1 mice with 4-1BB agonists significantly increased the percentage of CD4+ and CD8+ T cells in the BM and spleen. Interestingly, treatment with clone LOB12.3 resulted in a significant reduction in NK cell percentages in both the BM and spleen, while clone 3H3 selectively reduced splenic NK cells. Therapeutically, clone 3H3 significantly decreased M-protein levels and BM plasmacytosis (p<0.01), while no significant effects were observed for clone LOB12.3. Treatment with the IgG2a-formatted 4-1BB agonist combined with TasQ led to a significant reduction in M-protein levels and BM plasmacytosis (p<0001). Plasmacytosis was 62.50% in the isotype control group, decreased to 36.18% with 4-1BB agonist, 37.64% with TasQ, and further reduced to 14.09% in the combination therapy group, highlighting the enhanced efficacy of dual treatment. These effects were mediated by increased granzyme B–mediated T and NK-cell activation and enhanced differentiation of effector T cells (CD44⁺CD62L⁻). While this therapy did not alter the frequency of dendritic cells (DCs) in the BM, it enhanced their maturation, as evidenced by increased CD86 expression—particularly on type 1 and type 2 conventional DC subsets. Conclusion In conclusion, our findings demonstrate that 4-1BB activation can enhance anti-tumor immunity in MM; however, the therapeutic efficacy is dependent on the specific agonist used. Notably, the IgG2a-formatted 4-1BB agonist showed superior activity, underscoring the importance of isotype selection in achieving optimal immunotherapeutic outcomes. Moreover, co-administration with TasQ enhanced therapeutic efficacy, supporting the potential benefit of a combinatorial approach. Further investigation is warranted to elucidate the mechanisms driving these responses and to refine 4-1BB-targeted strategies for effective clinical translation.