Elevated LAG-3 expression in plasma cells and lymphoid subpopulations at distinct stages of Multiple Myeloma disease progression

Introduction
Immunotherapy dramatically changed the survival and quality of life of Multiple Myeloma (MM) patients. Despite the potential for durable responses, the majority of patients are still confronted with disease relapse. Immune checkpoints (ICPs) such as PD-1/PD-L1 were previously found to be overexpressed in MM, however targeting of these ICPs did not result in any clinical success so far. More recent in vitro studies suggested that blockade of the ICP LAG-3 could increase effector T-cell responses in the tumor microenvironment of MM patients. In this study, we aimed to further elucidate the expression of LAG-3 in myeloid and lymphoid subsets during MM disease progression using non-invasive imaging and flow cytometry in the immunocompetent 5T33MM model.

Methods
Previous developed anti-LAG-3 nanobodies were used to non-invasively image the LAG-3 expression in vivo. Anti-LAG-3 nanobodies and a control nanobody (R3B23) were radiolabeled with 99mTechnetium and injected in naïve and tumor-bearing C57BL/KaLwRij mice at different time points post-tumor inoculation (7 days post-injection (DPI), 14 DPI and 21 DPI). SPECT/CT imaging and organ biodistribution studies using a γ-counter were performed. LAG-3 expression on different immune subtypes was further evaluated using multi-parameter flow cytometry. Statistical differences were assessed using a One-way ANOVA, with p<0.05 considered as statistically significant.

Results
While no clear differences could be observed on SPECT/CT images of naïve and MM mice, ex vivo biodistribution analysis showed a significant uptake and increase of anti-LAG-3 nanobodies in the bone and spleen at 14 DPI (20-40% tumor load) and 21 DPI (60-80% tumor load) compared to naïve mice. Within the CD45+ population in the spleen, we observed an increase in neutrophils (CD11b+Ly6G+) and monocytes (CD11b+Ly6G) during MM disease progression. In the bone marrow (BM), we observed a significant increase in CD4+ T-cells and CD8+ T-cells at 14 DPI and 21 DPI within the CD45+ cell fraction. Flow cytometry analysis revealed a significant higher LAG-3 expression on MM cells upon disease progression. Within the lymphoid population, LAG-3 expression was increased in BM-derived CD8+ T-cells, while this remained unaffected in splenic T-cells. LAG-3 expression generally decreased in almost all myeloid subsets (e.g. macrophages, dendritic cells (DCs)), except splenic monocytes and plasmacytoid DCs, during MM disease progression.

Conclusion
Ex vivo biodistribution data revealed increased LAG-3 expression and nanobody uptake in MM-infiltrating organs. Using flow cytometry, a high expression of LAG-3 was observed in MM cells and lymphoid cells, which significantly increased within BM-derived CD8+ T-cells at end-stage of disease. Altogether, these data illustrate increased expression of LAG-3 upon disease progression and fosters the evaluation of LAG-3 blocking therapies, particularly in the context of immunotherapeutic approaches in MM.