A new soluble CSF1R-dimeric mutein with enhanced trapping of both CSF-1 and IL-34 reduces suppressive tumor-associated macrophages in mesothelioma.
Background: Signalling through the colony stimulating factor-1 receptor (CSF-1R) upon binding of its ligands CSF-1 and IL-34 has tumorigenic effects in several cancers through both induction of suppressive macrophages, and survival/proliferation of tumor cells. In addition, IL-34 tumorigenic effect can also be mediated by signalling through its other receptors, protein-tyrosine phosphatase zeta (PTPzeta), Syndecan-1 (CD138) and triggering receptor expressed on myeloid cells 2 (TREM2). Inhibition of CSF-1R signalling with small tyrosine kinase inhibitors or neutralization of CSF-1 and/or IL-34 using antibodies have been proposed to be used in cancer but their effect is limited, they have drawbacks since they only partially inhibit CSF-1 or IL-34 activity and in the case of small tyrosine kinase inhibitors lead to secondary effects due to lack of specificity. Thus, there is a need for a more specific and yet integrative approach. Methods: A human mutated form of the extracellular portion of CSF-1R was in silico modelized to trap both IL-34 and CSF-1 with higher affinity than the wild-type CSF-1R by replacing the methionine residue at position 149 by a Lysine (M149K). The extracellular portion of the mutated CSF-1R M149K was dimerized using the immunoglobulin Fc sequence of a silent human IgG1 (sCSF-1RM149K-Fc). Signalling through CSF-1R, survival of monocytes and differentiation of suppressive macrophages were analyzed using mesothelioma patient's samples and mesothelioma/macrophage spheroids in the presence of sCSF-1RM149K-Fc or sCSF-1R-Fc wild type control (sCSF-1RWT-Fc). Results: We defined that the D1 to D5 domains of the extracellular portion of CSF-1R were required for efficient binding to IL-34 and CSF-1. The mutein sCSF-1RM149K-Fc trapped with higher affinity than sCSF-1RWT-Fc both recombinant CSF-1 and IL-34 added in culture and naturally produced in mesothelioma pleural effusions. This was shown by inhibition of CSF-1R signalling, survival and differentiation of human suppressive macrophage in vitro and in vivo induced by mesothelioma cells. Neutralization of IL-34 and CSF-1 resulted in higher killing of mesothelioma cells by a tumor-specific CD8+ T cell clone in mesothelioma/macrophage spheroids. Conclusions: sCSF-1RM149K-Fc efficiently inhibited CSF-1R signalling, monocyte survival and suppressive macrophage differentiation induced by mesothelioma cells producing CSF-1 and IL-34 as well as restored cytotoxic T cell responses. sCSF-1RM149K-Fc efficiently and simultaneously trap both CSF-1 and IL-34 with high therapeutic potential versus other therapies under development targeting single components of this complex cytokine pathway involved in cancer. Overall design: mRNA from Meso13 (n=3), Meso13 + monocytes (n=3), Meso34 (n=3) and Meso34 + monocytes (n=3) MCTS were analyzed using 3'RNA sequencing