The binding of human IgG1 to human Fc gamma receptors (hFcγR) is highly sensitive to the presence of a single N-linked glycosylation site at asparagine 297 of the Fc, with deglycosylation resulting in a complete loss of hFcγR binding. Previously, we demonstrated that aglycosylated human IgG1 Fc variants can engage the human FcγRII class of the low-affinity hFcγRs, demonstrating that N-linked glycosylation of the Fc is not a strict requirement for hFcγR engagement. In the present study, we demonstrate that aglycosylated IgG variants can be engineered to productively engage with FcγRIIIA, as well as the human Fc gamma RII subset. We also assess the biophysical properties and serum half-life of the aglycosylated IgG variants to measure stability. Aglycosylated constructs DTT-IYG and DAT-IYG optimally drove tumor cell phagocytosis. A mathematical model of phagocytosis suggests that hFcγRI and hFcγRIIIA dimers were the main drivers of phagocytosis. In vivo tumor control of B16F10 lung metastases further confirmed the variant DTT-IYG to be the best at restoring WT-like properties in prevention of lung metastases. While deuterium incorporation was similar across most of the protein, several peptides within the CH2 domain of DTT-IYG showed differential deuterium uptake in the peptide region of the FG loop as compared to the aglycosylated N297Q. Thus in this study, we have found an aglycosylated variant that may effectively substitute for wild-type Fc. These aglycosylated variants have the potential to allow therapeutic antibodies to be produced in virtually any expression system and still maintain effector function.

Microbial type II polyketides serve as powerful medicinally relevant agents. These molecules are biosynthesized by polyketide synthases (PKSs) comprised of a core ketosynthase-chain length factor (KS-CLF) and phosphopantetheinylated acyl carrier protein (holo-ACP). While engineering type II PKSs holds potential to unlock sustainable access to diverse bioactive molecules, the inability to obtain cognate type II KS-CLFs andholo-ACPs forin vitrostudies represents a longstanding barrier. Herein, we share how the sequence and structural analysis of theGloeocapsa sp.PCC 7428 ACP allowed us to tune to a requisite weak yet specific interaction with a phosphopantetheinyl transferase to afford theholo-ACP. This, coupled with our ability to heterologously express the cognate KS-CLF in high quantities, unlocked access to polyketide products viain vitromultienzyme assembly. We hope this work inspires future studies of type II PKSs that have previously evaded heterologous expression or have yet to be explored.Abstract Figure

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.