Bispecific antibody therapy in multiple myeloma is impaired by tumor-associated macrophages through dual immunosuppressive and antigen-dependent mechanisms

Abstract Multiple myeloma (MM) is a malignant plasma cell disorder primarily affecting the bone marrow. Advances such as proteasome inhibitors and immunomodulatory drugs have improved outcomes. Recently, bispecific antibodies (BsMAbs) like GPRC5D/CD3 (talquetamab) have shown promise by redirecting T cells to MM cells and boosting cytotoxicity. However, resistance due to antigen loss or immune evasion remains a challenge. Tumor-associated macrophages (TAMs), abundant in the MM microenvironment, may promote tumor growth or suppress T-cell function. Their role in BsMAb therapy is not fully understood. TAMs may impair efficacy by blocking T-cell access or contributing to toxicities like cytokine release syndrome. Understanding TAM–BsMAb–T-cell interactions is key to improving treatment response in MM. In this study, we demonstrate that macrophages significantly impair the efficacy of BsMAbs in MM. Co-culture experiments using MM cell lines, T cells, and M2-like macrophages showed that talquetamab-induced MM cell lysis was strongly reduced in the presence of macrophages. Furthermore, we observed reduced T cell activation and decreased IFN-γ secretion, alongside a significant increase in IL-6 and IL-1β secretion by macrophages. These findings were confirmed using live-cell imaging and confocal microscopy. To elucidate the underlying mechanisms, we investigated the expression profile of macrophages and TAMs via Nanopore sequencing. Transcriptomic analysis revealed that TAMs express elevated levels of immunosuppressive molecules such as CD276 and CD200, compared to healthy bone marrow macrophages. Notably, TAMs and M2-like macrophages also expressed the BsMAb target antigen GPRC5D at both the RNA and protein levels, while M1-like macrophages did not. To determine whether talquetamab binds not only to MM cells but also to macrophages, we conjugated the BsMAb with a fluorochrome and incubated it with different cell populations. Interestingly, talquetamab labeled both MM targets and macrophages. Spatial transcriptomics further revealed that TAMs in proximity to MM cells express tight junction proteins (e.g., CLDN5, OCLN) and spatially separate T cells from MM cells, suggesting a potential barrier function.Taken together, our results suggest that TAMs may influence the efficacy of BsMAbs by forming an immunosuppressive barrier. Moreover, TAMs and certain macrophage phenotypes appear to express BsMAb target antigens, which could lead to off-target responses.