Expression of TAM-R in Human Immune Cells and Unique Regulatory Function of MerTK in IL-10 Production by Tolerogenic DC.

Tumor-infiltrating myeloid cells are a key component of the immune infiltrate often correlated with a poor prognosis due to their capacities to sustain an immunosuppressive environment. Among membrane receptors implicated in myeloid cell functions, Tyro3, Axl, and MerTK, which are a family of tyrosine kinase receptors (TAM-R), have been described in the regulation of innate cell functions. Here, we have identified MerTK among TAM-R as the major marker of both human M2 macrophages and tolerogenic dendritic cells (DC). In situ, MerTK expression was found within the immune infiltrate in multiple solid tumors, highlighting its potential role in cancer immunity. TAM-R ligands Gas6 and PROS1 were found to be constitutively produced by myeloid cells in vitro. Importantly, we describe a novel function of MerTK/PROS1 axis in the regulation of IL-10 production by tolerogenic DC. Finally, the analysis of TAM-R expression within the lymphoid compartment following activation revealed that MerTK, but not Axl or Tyro3, is expressed on activated B lymphocytes and regulatory T cells, as well as CD4+ and CD8+ T cells. Thus, our findings deepen the implication of MerTK in the regulation of myeloid cell-mediated immunosuppression and identified new cellular targets expressing MerTK that could participate in the antitumor immune response.

Single-domain antibody-based and linker-free bispecific antibodies targeting FcγRIII induce potent antitumor activity without recruiting regulatory T cells.

Antibody-dependent cell-mediated cytotoxicity, one of the most prominent modes of action of antitumor antibodies, suffers from important limitations due to the need for optimal interactions with Fcγ receptors. In this work, we report the design of a new bispecific antibody format, compact and linker-free, based on the use of llama single-domain antibodies that are capable of circumventing most of these limitations. This bispecific antibody format was created by fusing single-domain antibodies directed against the carcinoembryonic antigen and the activating FcγRIIIa receptor to human Cκ and CH1 immunoglobulin G1 domains, acting as a natural dimerization motif. In vitro and in vivo characterization of these Fab-like bispecific molecules revealed favorable features for further development as a therapeutic molecule. They are easy to produce in Escherichia coli, very stable, and elicit potent lysis of tumor cells by human natural killer cells at picomolar concentrations. Unlike conventional antibodies, they do not engage inhibitory FcγRIIb receptor, do not compete with serum immunoglobulins G for receptor binding, and their cytotoxic activity is independent of Fc glycosylation and FcγRIIIa polymorphism. As opposed to anti-CD3 bispecific antitumor antibodies, they do not engage regulatory T cells as these latter cells do not express FcγRIII. Studies in nonobese diabetic/severe combined immunodeficient gamma mice xenografted with carcinoembryonic antigen-positive tumor cells showed that Fab-like bispecific molecules in the presence of human peripheral blood mononuclear cells significantly slow down tumor growth. This new compact, linker-free bispecific antibody format offers a promising approach for optimizing antibody-based therapies.