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Abstract
Background The immune cells in the tumor microenvironment (TME) are not only powerful regulators of immunosuppression and tumorigenesis, but also represent a dominant cell type, with tumor-associated macrophages (TAMs) comprising up to 50% of total cell mass in solid tumors. Immunotherapies such as immune checkpoint inhibitors (ICIs) derive their efficacy from this cancer-immune cell interface, however, auto-immune related adverse events resulting from systemic administration remain a significant challenge.
Methods To address this need for potent, yet highly tumor-specific immunotherapies, we developed Tumor-Immune Cell Targeting Chimeras (TICTACs), antibody conjugates that are capable of selectively inhibiting immune checkpoint (ICP) molecules on TAMs. They consist of a novel synthetic ligand which recognizes CD206, a well-established TAM marker, and a non-blocking antibody that binds but does not inhibit the ICP (figure 1). By engaging CD206, which constitutively recycles between the plasma membrane and endosomes, we envisioned that TICTACs could facilitate removal of the ICP from the surface of CD206high TAMs, while having no effect on healthy CD206low macrophages. TICTACs against signal regulatory protein alpha (SIRPa) and CD54 were constructed through non-specific conjugation of the CD206 ligand to commercially available anti-SIRPa and CD54 antibodies. Macrophage cell lines RAW264.7, J774A.1, and BMA3.1A7 were first dosed with IL-4 to induced CD206 expression, treated with TICTACs, and the levels of SIRPa and CD54 were assessed by flow cytometry and confocal microscopy. CRISPR/Cas9 knockout of CD206 was performed to validate the involvement of the receptor in the mechanism.
Results Anti-SIRPa and CD54 TICTACs enabled robust downregulation of these proteins on the surface of CD206high, but not CD206low populations of macrophages in a dose-dependent manner (figure 2). Significant decrease of both SIRPa and CD54 were observed on CD206high macrophages at doses as low as 1 nM. Knocking out CD206 abrogated both uptake mediated by the CD206-targeting ligand and downregulation of the target ICP. Due to the dependence of CD206 expression on IL-4, the efficacy of TICTACs is also cytokine-dependent, and downregulation occurs only in the presence of IL-4.
Conclusions TICTACs are selective binders for CD206 and mediate significant downregulation of ICPs on the surface of tumor-associated, CD206high cells. By decoupling antibody selectivity from its blocking function, this presents a new paradigm for developing highly tumor-specific immunotherapies.
Acknowledgements The authors gratefully acknowledge support from the National Institute of Health (Award no. GM058867), Howard Hughes Medical Institute, and Stanford University.
Ethics Approval No human subjects or human data were used in this study.
In comparison to canonical immune checkpoint inhibitors, TICTAC activity is mediated by CD206, which is highly expressed on tumor-associated macrophages (TAMs)
Anti-SlRPa TICTACs induce robust downregulation of SIRPa on CD206high but not CD206low RAW264.7 cells
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