Artificial Anti-Tumor Opsonizing Proteins with Fibronectin Scaffolds Engineered for Specificity to Each of the Murine FcγR Types

Abstract

We have engineered a panel of novel Fn3 scaffold-based proteins that bind with high specificity and affinity to each of the individual mouse Fcγ receptors (mFcγR). These binders were expressed as fusions to anti-tumor antigen single-chain antibodies and mouse serum albumin, creating opsonizing agents that invoke only a single mFcγR response rather than the broader activity of natural Fc isotypes, as well as all previously reported Fc mutants. This panel isolated the capability of each of the four mFcγRs to contribute to macrophage phagocytosis of opsonized tumor cells and in vivo tumor growth control with these monospecific opsonizing fusion proteins. All activating receptors (mFcγRI, mFcγRIII, and mFcγRIV) were capable of driving specific tumor cell phagocytosis to an equivalent extent, while mFcγRII, the inhibitory receptor, did not drive phagocytosis. Monospecific opsonizing fusion proteins that bound mFcγRI alone controlled tumor growth to an extent similar to the most active IgG2a murine isotype. As expected, binding to the inhibitory mFcγRII did not delay tumor growth, but unexpectedly, mFcγRIII also failed to control tumor growth. mFcγRIV exhibited detectable but lesser tumor-growth control leading to less overall survival compared to mFcγRI. Interestingly, in vivo macrophage depletion demonstrates their importance in tumor control with mFcγRIV engagement, but not with mFcγRI. This panel of monospecific mFcγR-binding proteins provides a toolkit for isolating the functional effects of each mFcγR in the context of an intact immune system.

Selective Engagement of FcγRIV by a M2e-Specific Single Domain Antibody Construct Protects Against Influenza A Virus Infection

“…To our knowledge, this is the first report of an FcγRIV-specific VHH. A fibronectin scaffold protein that could specifically bind to FcγRIV with high affinity has been reported (63). In addition, this scaffold protein was able to delay tumor growth in a mouse model when linked to an anti-tumor antigen-specific single-chain antibody and mouse serum albumin (to extend the half-life of the fusion construct).…”

Anti-Tumor Strategies by Harnessing the Phagocytosis of Macrophages

“…Tumor-specific mAbs can help to bypass the anti-phagocytic signals by targeting tumor cells and interacting with macrophage FcγR to cause ADCP [158] (Figure 4a). The potential of four mFcγRs to support macrophage phagocytosis of opsonized tumor cells and tumor growth suppression in vivo was proven by Chen et al The results showed that while activating receptors (mFcγRI, mFcγRIII, and mFcγRIV) were equally capable of triggering specific tumor cell phagocytosis, the inhibitory receptor mFcγRII was unable to do so [159]. Some "don't eat me" signal blockades, such as RF231(a fully human anti-CD47), can not only promote CD47-mediated death signaling in tumor cells but also triggers FcγR-mediated phagocytosis of tumor cells [98].…”

DAKS1, a Kunitz Scaffold Peptide from the Venom Gland of Deinagkistrodon acutus Prevents Carotid-Artery and Middle-Cerebral-Artery Thrombosis via Targeting Factor XIa

“…Protein scaffolds are a wide research focus because they can be used as antibody replacement therapy [32][33][34][35]. Scaffold-based specific peptides are usually developed by using display technologies, such as phage, yeast, or ribosome/mRNA displays [36].…”