Macrophages expressing chimeric cytokine receptors have an inflammatory phenotype and antitumoral activity upon IL-10 or TGFβ stimulation

Background: Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer that lacks hormone receptors and human epidermal growth factor receptor 2 (HER2) amplification, making it unresponsive to standard hormone or HER2-targeted therapies. Although immune checkpoint inhibitors (ICIs) have shown promise in tumors with high lymphocyte infiltration, their efficacy remains limited in tumors with minimal lymphocyte infiltration. Inflammatory macrophages are associated with better prognosis and response to ICIs. The tumor microenvironment (TME), however, is rich in interleukin (IL)-10 and transforming growth factor β (TGF β) inducing immunosuppressive, protumoral macrophages. Therefore, we engineer macrophages with a chimeric cytokine receptor (ChCR), designed to bind IL-10 or TGFβ and promote the generation of inflammatory and antitumoral active macrophages. Such genetically modified macrophages could be used as adoptive cell therapy (ACT). Methods: The ChCRs consist of the extracellular domains of the IL-10 or TGFβ receptor fused to the intracellular domains of interferon (IFN)-γ receptor. Upon binding IL-10 or TGFβ, the ChCRs shall trigger STAT1 signaling, eventually rendering the macrophages pro-inflammatory. We transduced human primary macrophages with a lentiviral vector expressing the ChCR and analyzed their phenotype, secretome, and transcriptome following stimulation. Moreover, we assessed their antitumoral activity in a three-dimensional (3D) co-culture assay with TNBC spheroids. Results: ChCR macrophages showed robust STAT1 activation in response to IL-10 or TGFβ stimulation, resulting in an inflammatory phenotype similar to IFN-γ-activated macrophages, as confirmed by phenotypic markers, and transcriptomic profiling. These stimulated ChCR macrophages demonstrated significant antitumoral effects in 3D TNBC spheroids. Moreover, ChCR stimulation led to the upregulation of and , chemokines essential for lymphocyte recruitment, and genes associated with good response to ICIs. Conclusion: We successfully engineered macrophages to express ChCRs that induce macrophages with an inflammatory phenotype and antitumoral activity within an IL-10-rich and TGFβ-rich environment. The induction of and expression via ChCRs stimulation could further support lymphocyte recruitment, potentially facilitating lymphocyte infiltration and disruption of the immunosuppressive TME. A future application of ChCR macrophages as an ACT might improve outcomes in patients with TNBC, particularly those with low immune cell infiltration, thereby addressing a critical unmet clinical need.