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Monomeric IgG1 Fc molecules displaying unique Fc receptor interactions that are exploitable to treat inflammation-mediated diseases.

ABSTRACT: The IgG1 Fc is a dimeric protein that mediates important antibody effector functions by interacting with Fc? receptors (Fc?Rs) and the neonatal Fc receptor (FcRn). Here, we report the discovery of a monomeric IgG1 Fc (mFc) that bound to Fc?RI with very high affinity, but not to Fc?RIIIa, in contrast to wild-type (dimeric) Fc. The binding of mFc to FcRn was the same as that of dimeric Fc. To test whether the high-affinity binding to Fc?RI can be used for targeting of toxins, a fusion protein of mFc with a 38 kDa Pseudomonas exotoxin A fragment (PE38), was generated. This fusion protein killed Fc?RI-positive macrophage-like U937 cells but not Fc?RI-negative cells, and mFc or PE38 alone had no killing activity. The lack of binding to Fc?RIIIa resulted in the absence of Fc-mediated cytotoxicity of a scFv-mFc fusion protein targeting mesothelin. The pharmacokinetics of mFc in mice was very similar to that of dimeric Fc. The mFc's unique Fc?Rs binding pattern and related functionality, combined with its small size, monovalency and the preservation of FcRn binding which results in relatively long half-life in vivo, suggests that mFc has great potential as a component of therapeutics targeting inflammation mediated by activated macrophages overexpressing Fc?RI and related diseases, including cancer.

SUBMITTER: Ying T

PROVIDER: S-EPMC4622432 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Monomeric IgG1 Fc molecules displaying unique Fc receptor interactions that are exploitable to treat inflammation-mediated diseases.

Ying Tianlei T Feng Yang Y Wang Yanping Y Chen Weizao W Dimitrov Dimiter S DS

mAbs 20140101 5

The IgG1 Fc is a dimeric protein that mediates important antibody effector functions by interacting with Fcγ receptors (FcγRs) and the neonatal Fc receptor (FcRn). Here, we report the discovery of a monomeric IgG1 Fc (mFc) that bound to FcγRI with very high affinity, but not to FcγRIIIa, in contrast to wild-type (dimeric) Fc. The binding of mFc to FcRn was the same as that of dimeric Fc. To test whether the high-affinity binding to FcγRI can be used for targeting of toxins, a fusion protein of m ...[more]

PMID: 25517305

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Project description:The inhibition of Fcγ receptors (FcγR) is an attractive strategy for treating diseases driven by IgG immune complexes (IC). Previously, we demonstrated that an engineered tri-valent arrangement of IgG1 Fc domains (SIF1) potently inhibited FcγR activation by IC, whereas a penta-valent Fc molecule (PentX) activated FcγR, potentially mimicking ICs and leading to Syk phosphorylation. Thus, a precise balance exists between the number of engaged FcγRs for inhibition versus activation. Here, we demonstrate that Fc valency differentially controls FcγR activation and inhibition within distinct subcellular compartments. Large Fc multimer clusters consisting of 5-50 Fc domains predominately recruited Syk-mScarlet to patches on the plasma membrane, whereas PentX exclusively recruited Syk-mScarlet to endosomes in human monocytic cell line (THP-1 cells). In contrast, SIF1, similar to monomeric Fc, spent longer periods docked to FcγRs on the plasma membrane and did not accumulate and recruit Syk-mScarlet within large endosomes. Single particle tracking (SPT) of fluorescent engineered Fc molecules and Syk-mScarlet at the plasma membrane imaged by total internal reflection fluorescence microscopy (SPT-TIRF), revealed that Syk-mScarlet sampled the plasma membrane was not recruited to FcγR docked with any of the engineered Fc molecules at the plasma membrane. Furthermore, the motions of FcγRs docked with recombinant Fc (rFc), SIF1 or PentX, displayed similar motions with D ~ 0.15 μm2/s, indicating that SIF1 and PentX did not induce reorganization or microclustering of FcγRs beyond the ligating valency. Multicolor SPT-TIRF and brightness analysis of docked rFc, SIF1 and PentX also indicated that FcγRs were not pre-assembled into clusters. Taken together, activation on the plasma membrane requires assembly of more than 5 FcγRs. Unlike rFc or SIF1, PentX accumulated Syk-mScarlet on endosomes indicating that the threshold for FcγR activation on endosomes is lower than on the plasma membrane. We conclude that the inhibitory effects of SIF1 are mediated by stabilizing a ligated and inactive FcγR on the plasma membrane. Thus, FcγR inhibition can be achieved by low valency ligation with SIF1 that behaves similarly to FcγR docked with monomeric IgG.

Dataset Information

Monomeric IgG1 Fc molecules displaying unique Fc receptor interactions that are exploitable to treat inflammation-mediated diseases.

Publications

Monomeric IgG1 Fc molecules displaying unique Fc receptor interactions that are exploitable to treat inflammation-mediated diseases.

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