An Fc variant with two mutations confers prolonged serum half-life and enhanced effector functions on IgG antibodies.

The pH-selective interaction between the immunoglobulin G (IgG) fragment crystallizable region (Fc region) and the neonatal Fc receptor (FcRn) is critical for prolonging the circulating half-lives of IgG molecules through intracellular trafficking and recycling. By using directed evolution, we successfully identified Fc mutations that improve the pH-dependent binding of human FcRn and prolong the serum persistence of a model IgG antibody and an Fc-fusion protein. Strikingly, trastuzumab-PFc29 and aflibercept-PFc29, a model therapeutic IgG antibody and an Fc-fusion protein, respectively, when combined with our engineered Fc (Q311R/M428L), both exhibited significantly higher serum half-lives in human FcRn transgenic mice than their counterparts with wild-type Fc. Moreover, in a cynomolgus monkey model, trastuzumab-PFc29 displayed a superior pharmacokinetic profile to that of both trastuzumab-YTE and trastuzumab-LS, which contain the well-validated serum half-life extension Fcs YTE (M252Y/S254T/T256E) and LS (M428L/N434S), respectively. Furthermore, the introduction of two identified mutations of PFc29 (Q311R/M428L) into the model antibodies enhanced both complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity activity, which are triggered by the association between IgG Fc and Fc binding ligands and are critical for clearing cancer cells. In addition, the effector functions could be turned off by combining the two mutations of PFc29 with effector function-silencing mutations, but the antibodies maintained their excellent pH-dependent human FcRn binding profile. We expect our Fc variants to be an excellent tool for enhancing the pharmacokinetic profiles and potencies of various therapeutic antibodies and Fc-fusion proteins.

Selection and Characterization of YKL-40-Targeting Monoclonal Antibodies from Human Synthetic Fab Phage Display Libraries.

YKL-40, also known as chitinase-3-like 1 (CHI3L1), is a glycoprotein that is expressed and secreted by various cell types, including cancers and macrophages. Due to its implications for and upregulation in a variety of diseases, including inflammatory conditions, fibrotic disorders, and tumor growth, YKL-40 has been considered as a significant therapeutic biomarker. Here, we used a phage display to develop novel monoclonal antibodies (mAbs) targeting human YKL-40 (hYKL-40). Human synthetic antibody phage display libraries were panned against a recombinant hYKL-40 protein, yielding seven unique Fabs (Antigen-binding fragment), of which two Fabs (H1 and H2) were non-aggregating and thermally stable (75.5 °C and 76.5 °C, respectively) and had high apparent affinities (KD = 2.3 nM and 4.0 nM, respectively). Reformatting the Fabs into IgGs (Immunoglobulin Gs) increased their apparent affinities (notably, for H1 and H2, KD = 0.5 nM and 0.3 nM, respectively), presumably due to the effects of avidity, with little change to their non-aggregation property. The six anti-hYKL-40 IgGs were analyzed using a trans-well migration assay in vitro, revealing that three clones (H1, H2, and H4) were notably effective in reducing cell migration from both A549 and H460 lung cancer cell lines. The three clones were further analyzed in an in vivo animal test that assessed their anti-cancer activities, demonstrating that the tumor area and the number of tumor nodules were significantly reduced in the lung tissues treated with H1 (IgG). Given its high affinity and desirable properties, we expect that the H1 anti-hYKL-40 mAb will be a suitable candidate for developing anti-cancer therapeutics.

Generation of a Nebulizable CDR-Modified MERS-CoV Neutralizing Human Antibody.

Middle East respiratory syndrome coronavirus (MERS-CoV) induces severe aggravating respiratory failure in infected patients, frequently resulting in mechanical ventilation. As limited therapeutic antibody is accumulated in lung tissue following systemic administration, inhalation is newly recognized as an alternative, possibly better, route of therapeutic antibody for pulmonary diseases. The nebulization process, however, generates diverse physiological stresses, and thus, the therapeutic antibody must be resistant to these stresses, remain stable, and form minimal aggregates. We first isolated a MERS-CoV neutralizing antibody that is reactive to the receptor-binding domain (RBD) of spike (S) glycoprotein. To increase stability, we introduced mutations into the complementarity-determining regions (CDRs) of the antibody. In the HCDRs (excluding HCDR3) in this clone, two hydrophobic residues were replaced with Glu, two residues were replaced with Asp, and four residues were replaced with positively charged amino acids. In LCDRs, only two Leu residues were replaced with Val. These modifications successfully generated a clone with significantly greater stability and equivalent reactivity and neutralizing activity following nebulization compared to the original clone. In summary, we generated a MERS-CoV neutralizing human antibody that is reactive to recombinant MERS-CoV S RBD protein for delivery via a pulmonary route by introducing stabilizing mutations into five CDRs.