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

Abstract

Background: Immunotherapy has emerged as a promising strategy for multiple myeloma (MM), yet relapse remains frequent due to the immunosuppressive bone marrow (BM) microenvironment, characterized by T cell dysfunction and accumulation of immunosuppressive myeloid cells. The co-stimulatory receptor 4-1BB (CD137, TNFRSF9) can enhance T and NK cell effector functions, but its therapeutic utility in MM is not well established. Tasquinimod (TQ), a clinical-stage S100A9 inhibitor, offers a complementary approach by limiting the recruitment and activity of suppressive myeloid cells.

Methods: 4-1BB expression was assessed during disease progression in MM mice and in newly diagnosed MM patients using single-cell RNA sequencing and flow cytometry. Therapeutic potential was evaluated in 5TGM1 tumor-bearing mice treated with two 4-1BB agonists, LOB12.3 (IgG1κ) and 3H3 (IgG2a), using isotype controls. The lead agonist was subsequently combined with TQ to investigate dual targeting of the immunosuppressive tumor microenvironment. Tumor burden was quantified via BM and spleen plasmacytosis and serum M-protein levels. Immune modulation was analyzed using multi-parameter flow cytometry. Statistical significance was determined using the Mann-Whitney U test or one-way ANOVA (p < 0.05).

Results: 4-1BB expression progressively increased on T and NK cells during tumor development in mice. In primary MM patient BM samples, ex vivo 4-1BB stimulation with urelumab enhanced effector responses, increasing IFN-γ⁺ and Granzyme B⁺ CD3⁺ T cells, alongside trends toward increased CD56⁺ NK cells and elevated IFN-γ⁺ NK cell activity. In vivo, 4-1BB agonist treatment promoted expansion of T cell subsets, with clone-specific effects: the IgG2a clone 3H3 significantly reduced M-protein levels and BM plasmacytosis, whereas the IgG1 clone LOB12.3 induced NK cell depletion and demonstrated limited anti-tumor activity. Combining 3H3 with TQ provided superior anti-myeloma efficacy, reducing BM plasmacytosis from 62.5% in controls to 14.1% under combination treatment. Mechanistically, the combination enhanced Granzyme B expression, effector T cell differentiation, and dendritic cell maturation (CD86 upregulation), collectively overcoming BM immunosuppression.

Conclusions: These findings establish the isotype-specific efficacy of 4-1BB agonists and support 4-1BB stimulation combined with TQ as a promising strategy to enhance durable immunotherapeutic responses in MM.