title: Enhancement of antibody functions through Fc multiplications.
abstract: Antibodies carry out a plethora of functions through their crystallizable fragment (Fc) regions, which can be naturally tuned by the adoption of several isotypes and post-translational modifications. Protein engineering enables further Fc function modulations through modifications of the interactions between the Fc and its functional partners, including FcγR, FcRn, complement complex, and additions of auxiliary functional units. Due to the many functions embedded within the confinement of an Fc, a suitable balance must be maintained for a therapeutic antibody to be effective and safe. The outcome of any Fc engineering depends on the interplay among all the effector molecules involved. In this report, we assessed the effects of Fc multiplication (or tandem Fc) on antibody functions. Using IgG1 as a test case, we found that, depending on the specifically designed linker, Fc multiplication led to differentially folded, stable molecules with unique pharmacokinetic profiles. Interestingly, the variants with 3 copies of Fc improved in vitro opsonophagocytic killing activity and displayed significantly improved protective efficacies in a Klebsiella pneumoniae mouse therapeutic model despite faster clearance compared with its IgG1 counterpart. There was no adverse effect observed or pro-inflammatory cytokine release when the Fc variants were administered to animals. We further elucidated that enhanced binding to various effector molecules by IgG-3Fc created a "sink" leading to the rapid clearance of the 3Fc variants, and identified the increased FcRn binding as one strategy to facilitate "sink" escape. These findings reveal new opportunities for novel Fc engineering to further expand our abilities to manipulate and improve antibody therapeutics.
authors.affiliation: a Department of Infectious Diseases , MedImmune , Gaithersburg , MD , USA.
authors.forename: Qun
authors.initials: Q
authors.lastname: Wang
authors.affiliation: b Department of Antibody Discovery and Protein Engineering , MedImmune , Gaithersburg , MD , USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Chen
authors.affiliation: a Department of Infectious Diseases , MedImmune , Gaithersburg , MD , USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Pelletier
authors.affiliation: a Department of Infectious Diseases , MedImmune , Gaithersburg , MD , USA.
authors.forename: Romana
authors.initials: R
authors.lastname: Cvitkovic
authors.affiliation: a Department of Infectious Diseases , MedImmune , Gaithersburg , MD , USA.
authors.forename: Jessica
authors.initials: J
authors.lastname: Bonnell
authors.affiliation: b Department of Antibody Discovery and Protein Engineering , MedImmune , Gaithersburg , MD , USA.
authors.forename: Chien-Ying
authors.initials: CY
authors.lastname: Chang
authors.affiliation: b Department of Antibody Discovery and Protein Engineering , MedImmune , Gaithersburg , MD , USA.
authors.forename: Adem C
authors.initials: AC
authors.lastname: Koksal
authors.affiliation: c Department of Purification Process Sciences , MedImmune , Gaithersburg , MD , USA.
authors.forename: Ellen
authors.initials: E
authors.lastname: O'Connor
authors.affiliation: d Department of Clinical Pharmacology & DMPK , MedImmune , Gaithersburg , MD , USA.
authors.forename: Xizhe
authors.initials: X
authors.lastname: Gao
authors.affiliation: d Department of Clinical Pharmacology & DMPK , MedImmune , Gaithersburg , MD , USA.
authors.forename: Xiang-Qing
authors.initials: XQ
authors.lastname: Yu
authors.affiliation: b Department of Antibody Discovery and Protein Engineering , MedImmune , Gaithersburg , MD , USA.
authors.forename: Herren
authors.initials: H
authors.lastname: Wu
authors.affiliation: a Department of Infectious Diseases , MedImmune , Gaithersburg , MD , USA.
authors.forename: C Kendall
authors.initials: CK
authors.lastname: Stover
authors.affiliation: b Department of Antibody Discovery and Protein Engineering , MedImmune , Gaithersburg , MD , USA.
authors.forename: William F
authors.initials: WF
authors.lastname: Dall'Acqua
authors.affiliation: b Department of Antibody Discovery and Protein Engineering , MedImmune , Gaithersburg , MD , USA.
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Xiao
mesh_terms: D000818:Animals; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000276:immunology* / Q000494:pharmacology; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology* / Q000494:pharmacology; D007710:Klebsiella Infections / Q000276:immunology; D007711:Klebsiella pneumoniae; D051379:Mice; D008810:Mice, Inbred C57BL; D015202:Protein Engineering / Q000379:methods*
chemical_list: D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G
keywords: Effector function; opsonophagocytic killing (OPK); pharmacokinetics (PK); serum clearance; tandem Fc