A low-dose immunotherapy targeting Fc-gamma Receptors and Heparan Sulfate Proteoglycan to impact myeloid cells and control tumor growth in cancers with varying immunosuppressive profiles

ABSTRACT Background Myeloid cells play a central role in cancer-associated immunosuppression. Indeed, their accumulation and functional reprogramming attenuate effective immune responses and may facilitate tumor progression. To modulate the activity of the myeloid subsets, we chose to target Fcγ receptors (FcγRs), since all myeloid cell populations variably express FcγRs. As FcγRIIb provides an inhibitory signaling that might alter efficacy, we developed an immunotherapy, active at a low dose to limit binding via this low-affinity receptor while still interacting with higher-affinity FcγRs. We engineered a Fc-based fusion protein whose activity is potentiated by its ability to engage both FcγRs and a coreceptor, Heparan Sulfate Proteoglycan (HSPGs). We designed and fused a HSPG-ligand, named T54, to a human IgG1-Fc molecule to produce the Fc-T54 fusion protein. Methods Fc-T54 was produced using recombinant technologies. Binding characteristics were assessed using ELISA and flow-cytometric assays. Immune activity was investigated using cell-culture assays. Ability to affect tumor growth was investigated using four syngeneic tumor models with deserted to inflamed characteristics that differ in their sensitivity to immune checkpoint inhibitors (ICIs). Tumor microenvironment (TME) was analysed by flow cytometry. Results Compared to Fc, Fc-T54 demonstrates superior binding to low-affinity FcγRs and interacts more effectively with human leukocytes, including neutrophils, and B lymphocytes, as well as with monocytes and dendritic cells (DCs) within peripheral blood mononuclear cells. In activation assays Fc-T54 increases the number of monocytes/macrophages and B-lymphocytes, decreases neutrophil abundance, and enhances DC activation. Fc-T54 also demonstrates enhanced interaction with murine DCs accompanied by increased activation. Subcutaneous administration of low dose Fc-T54 - or its murine surrogate - significantly inhibits tumor growth in immune-deserted and immune-excluded mouse models, and synergizes with anti-PD-1 therapy in the immune-inflamed model. TME analysis in the MB49 bladder cancer model reveals that the immunotherapy decreases the proportion of granulocytic myeloid-derived suppressor cells while increasing CD8+ T-cells and natural killer cells, promoting a microenvironment more prone to tumor control. Conclusions This new FcγR/HSPG-engaging immunotherapy, administered via subcutaneous route, offers a novel approach to modulate the myeloid compartment and expand therapeutic options for ICI-resistant, deserted/excluded tumors, and for inflamed tumors when used in combination regimens.