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Identification of human IgG1 variant with enhanced FcRn binding and without increased binding to rheumatoid factor autoantibody.

ABSTRACT: Various studies have demonstrated that Fc engineering to enhance neonatal Fc receptor (FcRn) binding is effective for elongating half-life or increasing cellular uptake of IgG. A previous study has shown that a N434H mutation to enhance FcRn binding resulted in increased binding to rheumatoid factor (RF) autoantibody, which is not desirable for therapeutic use in autoimmune disease. In this study, we first showed that all the existing Fc variants with enhanced FcRn binding also show increased RF binding, and then identified specific mutations that could be introduced to those Fc variants to reduce the RF binding. Furthermore, we generated novel Fc variants that do not increase RF binding and show half-lives of 45 d in cynomolgus monkey, which is longer than those of previously reported Fc variants. In addition, we generated novel Fc variants with antigen sweeping activity that do not increase RF binding. We expect that these novel Fc variants will be useful as antibody therapeutics against autoimmune diseases.

SUBMITTER: Maeda A

PROVIDER: S-EPMC5524163 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Identification of human IgG1 variant with enhanced FcRn binding and without increased binding to rheumatoid factor autoantibody.

Maeda Atsuhiko A Iwayanagi Yuki Y Haraya Kenta K Tachibana Tatsuhiko T Nakamura Genki G Nambu Takeru T Esaki Keiko K Hattori Kunihiro K Igawa Tomoyuki T

mAbs 20170407 5

Various studies have demonstrated that Fc engineering to enhance neonatal Fc receptor (FcRn) binding is effective for elongating half-life or increasing cellular uptake of IgG. A previous study has shown that a N434H mutation to enhance FcRn binding resulted in increased binding to rheumatoid factor (RF) autoantibody, which is not desirable for therapeutic use in autoimmune disease. In this study, we first showed that all the existing Fc variants with enhanced FcRn binding also show increased RF ...[more]

PMID: 28387635

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Project description:Despite the reasonably long half-life of immunoglogulin G (IgGs), market pressure for higher patient convenience while conserving efficacy continues to drive IgG half-life improvement. IgG half-life is dependent on the neonatal Fc receptor (FcRn), which among other functions, protects IgG from catabolism. FcRn binds the Fc domain of IgG at an acidic pH ensuring that endocytosed IgG will not be degraded in lysosomal compartments and will then be released into the bloodstream. Consistent with this mechanism of action, several Fc-engineered IgG with increased FcRn affinity and conserved pH dependency were designed and resulted in longer half-life in vivo in human FcRn-transgenic mice (hFcRn), cynomolgus monkeys, and recently in healthy humans. These IgG variants were usually obtained by in silico approaches or directed mutagenesis in the FcRn-binding site. Using random mutagenesis, combined with a pH-dependent phage display selection process, we isolated IgG variants with improved FcRn-binding, which exhibited longer in vivo half-life in hFcRn mice. Interestingly, many mutations enhancing Fc/FcRn interaction were located at a distance from the FcRn-binding site validating our random molecular approach. Directed mutagenesis was then applied to generate new variants to further characterize our IgG variants and the effect of the mutations selected. Since these mutations are distributed over the whole Fc sequence, binding to other Fc effectors, such as complement C1q and FcγRs, was dramatically modified, even by mutations distant from these effectors' binding sites. Hence, we obtained numerous IgG variants with increased FcRn-binding and different binding patterns to other Fc effectors, including variants without any effector function, providing distinct "fit-for-purpose" Fc molecules. We therefore provide evidence that half-life and effector functions should be optimized simultaneously as mutations can have unexpected effects on all Fc receptors that are critical for IgG therapeutic efficacy.

Project description:BackgroundRheumatoid arthritis (RA) is an inflammatory debilitating disease that affects the joints in the early and productive phases of an individual's life. Several cytokines have been linked to the disease pathogenesis and are known to contribute to the inflammatory state characteristic of RA. The participation of type I interferon (IFN) in the pathogenesis of the disease has been already described as well as the identity of the genes that are regulated by this molecule, which are collectively known as the type I IFN signature. These genes have several functions associated with apoptosis, transcriptional regulation, protein degradation, Th2 cell induction, B cell proliferation, etc. This article evaluated the expression of several genes of the IFN signature in different stages of disease and their correlation with the levels of anticitrullinated protein antibodies (ACPA) anticarbamylated protein (Anti-CarP) antibodies.MethodsSamples from individuals with early and established RA, high-risk individuals (ACPA+ and ACPA-), and healthy controls were recruited at "Unidad de Artritis y Rheumatismo" (Rheumatism and Arthritis Unit) in Guadalajara Jalisco Mexico. Determinations of ACPA were made with Eurodiagnostica ACPA plus kit. Anti-CarP determinations were made according to previously described protocols. RNA was isolated, and purity and integrity were determined according to RNA integrity number >6. Gene expression analysis was made by RT-qPCR using specific primers for mRNAs of the type I IFN signature. Relative gene expression was calculated according to Livak and Schmitgen.ResultsSignificant differences in gene expression were identified when comparing the different groups for MXA and MXB (P < 0.05), also when comparing established RA and ACPA- in both IFIT 1 and G15. An increased expression of ISG15 was identified (P < 0.05), and a clear tendency toward increase was identified for HERC5. EPSTRI1, IFI6, and IFI35 were found to be elevated in the chronic/established RA and early RA (P < 0.05). Significant correlations were identified for the IFN signature genes with the levels of ACPA and anti-CarP (P < 0.05).ConclusionOur data confirm previous observations in the role of IFN signature and the pathogenesis of RA. Also, we provide evidence of an association between several genes of the IFN signature (that regulate Th2 cells and B cell proliferation) with the levels of anti-CarP antibodies and ACPA.

Dataset Information

Identification of human IgG1 variant with enhanced FcRn binding and without increased binding to rheumatoid factor autoantibody.

Publications

Identification of human IgG1 variant with enhanced FcRn binding and without increased binding to rheumatoid factor autoantibody.

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