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Engineering an IgG Scaffold Lacking Effector Function with Optimized Developability.

ABSTRACT: IgG isotypes can differentially bind to Fc? receptors and complement, making the selection of which isotype to pursue for development of a particular therapeutic antibody important in determining the safety and efficacy of the drug. IgG2 and IgG4 isotypes have significantly lower binding affinity to Fc? receptors. Recent evidence suggests that the IgG2 isotype is not completely devoid of effector function, whereas the IgG4 isotype can undergo in vivo Fab arm exchange leading to bispecific antibody and off-target effects. Here an attempt was made to engineer an IgG1-based scaffold lacking effector function but with stability equivalent to that of the parent IgG1. Care was taken to ensure that both stability and lack of effector function was achieved with a minimum number of mutations. Among the Asn297 mutants that result in lack of glycosylation and thus loss of effector function, we demonstrate that the N297G variant has better stability and developability compared with the N297Q or N297A variants. To further improve the stability of N297G, we introduced a novel engineered disulfide bond at a solvent inaccessible location in the CH2 domain. The resulting scaffold has stability greater than or equivalent to that of the parental IgG1 scaffold. Extensive biophysical analyses and pharmacokinetic (PK) studies in mouse, rat, and monkey further confirmed the developability of this unique scaffold, and suggest that it could be used for all Fc containing therapeutics (e.g. antibodies, bispecific antibodies, and Fc fusions) requiring lack of effector function or elimination of binding to Fc? receptors.

SUBMITTER: Jacobsen FW

PROVIDER: S-EPMC5290958 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Engineering an IgG Scaffold Lacking Effector Function with Optimized Developability.

Jacobsen Frederick W FW Stevenson Riki R Li Cynthia C Salimi-Moosavi Hossein H Liu Ling L Wen Jie J Luo Quanzhou Q Daris Kristine K Buck Lynette L Miller Sterling S Ho Shu-Yin SY Wang Wei W Chen Qing Q Walker Kenneth K Wypych Jette J Narhi Linda L Gunasekaran Kannan K

The Journal of biological chemistry 20161219 5

IgG isotypes can differentially bind to Fcγ receptors and complement, making the selection of which isotype to pursue for development of a particular therapeutic antibody important in determining the safety and efficacy of the drug. IgG2 and IgG4 isotypes have significantly lower binding affinity to Fcγ receptors. Recent evidence suggests that the IgG2 isotype is not completely devoid of effector function, whereas the IgG4 isotype can undergo in vivo Fab arm exchange leading to bispecific antibo ...[more]

PMID: 27994062

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Project description:Therapeutic performance of recombinant antibodies relies on two independent mechanisms: antigen recognition and Fc-mediated antibody effector functions. Interaction of Fc-fragment with different FcR triggers antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity and determines longevity of the antibody in serum. In context of therapeutic antibodies Fc?Rs play the most important role. It has been demonstrated that the Fc-attached sugar moiety is essential for IgG effector functionality, dictates its affinity to individual Fc?Rs and determines binding to different receptor classes: activating or inhibitory. In this study, we systematically analyze effector functions of monoclonal IgG1 and its eight enzymatically engineered glycosylation variants. The analysis of interaction of glycovariants with FcRs was performed for single, as well as for antigen-bound antibodies and IgGs in a form of immune complex. In addition to functional properties we addressed impact of glycosylation on the structural properties of the tested glycovariants. We demonstrate a clear impact of glycosylation pattern on antibody stability and interaction with different Fc?Rs. Consistent with previous reports, deglycosylated antibodies failed to bind all Fc?-receptors, with the exception of high affinity Fc?RI. The Fc?RII and Fc?RIIIa binding activity of IgG1 was observed to depend on the galactosylation level, and hypergalactosylated antibodies demonstrated increased receptor interaction. Sialylation did not decrease the Fc?R binding of the tested IgGs; in contrast, sialylation of antibodies improved binding to Fc?RIIa and IIb. We demonstrate that glycosylation influences to some extent IgG1 interaction with FcRn. However, independent of glycosylation pattern the interaction of IgG1 with a soluble monomeric target surprisingly resulted in an impaired receptor binding. Here, we demonstrate, that immune complexes (IC), induced by multimeric ligand, compensated for the decreased affinity of target bound antibody towards FcRs, showing the importance of the IC-formation for the FcR- mediated effector functions.

Dataset Information

Engineering an IgG Scaffold Lacking Effector Function with Optimized Developability.

Publications

Engineering an IgG Scaffold Lacking Effector Function with Optimized Developability.

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