Complement Is Activated by IgG Hexamers Assembled at the Cell Surface

Diebolder, Christoph A.; Beurskens, Frank J.; Jong, Rob N. de; Koning, Roman I.; Strumane, Kristin; Lindorfer, Margaret A.; Voorhorst, Marleen; Ugurlar, Deniz; Rosati, Sara; Heck, Albert J. R.; Winkel, Jan G. J. van de; Wilson, Ian A.; Koster, Abraham J.; Taylor, Ronald P.; Saphire, Erica Ollmann; Burton, Dennis R.; Schuurman, Janine; Gros, Piet; Parren, Paul W.H.I.

doi:10.1126/science.1248943

Cited by 657 publications

(934 citation statements)

References 62 publications

(41 reference statements)

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“…For example, a hexameric assembly of Fc is required to elicit CDC activity. 42 The Fc hexameric assembly involves formation of an Fc-Fc interface, and residues involved in the formation of this interface partially overlap with the FcRn binding site. Therefore, it is critical to evaluate the capacity of our designs to not only bind to Fc receptors, but also mediate effector functions.…”

Section: Resultsmentioning

confidence: 99%

Extending human IgG half-life using structure-guided design

Booth

Ramakrishnan

Narayan

et al. 2018

mAbs

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Engineering of antibodies for improved pharmacokinetics through enhanced binding to the neonatal Fc receptor (FcRn) has been demonstrated in transgenic mice, non-human primates and humans. Traditionally, such approaches have largely relied on random mutagenesis and display formats, which fail to address related critical attributes of the antibody, such as effector functions or biophysical stability. We have developed a structure- and network-based framework to interrogate the engagement of IgG with multiple Fc receptors (FcRn, C1q, TRIM21, FcγRI, FcγRIIa/b, FcγRIIIa) simultaneously. Using this framework, we identified features that govern Fc-FcRn interactions and identified multiple distinct pathways for enhancing FcRn binding in a pH-specific manner. Network analysis provided a novel lens to study the allosteric impact of half-life-enhancing Fc mutations on FcγR engagement, which occurs distal to the FcRn binding site. Applying these principles, we engineered a panel of unique Fc variants that enhance FcRn binding while maintaining robust biophysical properties and wild type-like binding to activating receptors. An antibody harboring representative Fc designs demonstrates a half-life improvement of > 9 fold in transgenic mice and > 3.5 fold in cynomolgus monkeys, and maintains robust effector functions such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity.

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Section: Resultsmentioning

confidence: 99%

Extending human IgG half-life using structure-guided design

Booth

Ramakrishnan

Narayan

et al. 2018

mAbs

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“…Formation of ordered IgG hexamers as observed in the crystal packing of the structure of IgG gp120 increases affinity to C1 and thereby potentiates complement activation when viral or cellular surface targets are recognized. 40 Superposition of the CODV-Ig IL4 x IL13 / IL4/IL13 model on the immune-competent IgG gp120 hexamer reveals that no steric hindrance for hexamerization by Fc2-Fc3 is introduced by the CODV architecture. Furthermore, the CDR regions remain accessible to antigens (Fig.…”

Section: Codv-igs Retain Intrinsic Igg Functionsmentioning

confidence: 99%

“…36,40,[49][50][51][52][53] To determine the effects of CODV architecture on Fc functionality, we quantified the affinity of CODV-Ig IL4 x IL13 for FcRn and FcgR1 by SPR. The affinity for FcgR1 is very similar to isotype control IgG1 IL4 while the affinities for FcRn receptors are slightly decreased by »2.5-fold (Table S3).…”

Section: Codv-igs Retain Intrinsic Igg Functionsmentioning

confidence: 99%

“…3,[36][37][38][39] Fully functional Fc domains may also confer the potential to trigger the classical pathway of complementdependent humoral response, and thereby elicit complementdependent cytotoxicity (CDC). 40,41 To overcome major limitations of existing bispecific biotherapeutics and to create a universally applicable format that can be fine-tuned in multiparametric drug optimization, we designed the CODV format to serve as a versatile platform for the development of bispecific agents.…”

Section: Introductionmentioning

confidence: 99%

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CODV-Ig, a universal bispecific tetravalent and multifunctional immunoglobulin format for medical applications

Steinmetz

Vallée

Beil

et al. 2016

mAbs

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Bispecific immunoglobulins (Igs) typically contain at least two distinct variable domains (Fv) that bind to two different target proteins. They are conceived to facilitate clinical development of biotherapeutic agents for diseases where improved clinical outcome is obtained or expected by combination therapy compared to treatment by single agents. Almost all existing formats are linear in their concept and differ widely in drug-like and manufacture-related properties. To overcome their major limitations, we designed cross-over dual variable Ig-like proteins (CODV-Ig). Their design is akin to the design of circularly closed repeat architectures. Indeed, initial results showed that the traditional approach of utilizing (G4S) x linkers for biotherapeutics design does not identify functional CODV-Igs. Therefore, we applied an unprecedented molecular modeling strategy for linker design that consistently results in CODV-Igs with excellent biochemical and biophysical properties. CODV architecture results in a circular self-contained structure functioning as a self-supporting truss that maintains the parental antibody affinities for both antigens without positional effects. The format is universally suitable for therapeutic applications targeting both circulating and membrane-localized proteins. Due to the full functionality of the Fc domains, serum half-life extension as well as antibody-or complement-dependent cytotoxicity may support biological efficiency of CODV-Igs. We show that judicious choice in combination of epitopes and paratope orientations of bispecific biotherapeutics is anticipated to be critical for clinical outcome. Uniting the major advantages of alternative bispecific biotherapeutics, CODV-Igs are applicable in a wide range of disease areas for fast-track multi-parametric drug optimization.

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“…These findings are in keeping with data suggesting that different degrees of Fc multimerization are required to elicit FcgR and complement-mediated effects. For example, recent studies revealed the dimeric fraction of IVIG has increased ability of blocking Fc-FcgR interaction over IVIG monomers in macrophage phagocytosis, and a separate report suggested that C1q activation required the formation IgG hexamers (40,41). Consistent with these findings, whereas both lower-and higher-order fractions of GB4542 demonstrated decreased ADCP effect at higher concentrations, decreased CDC activity was observed only in the presence of high concentration of higher m.w.…”

Section: Discussionmentioning

confidence: 71%

Anti-CD20 Antibody with Multimerized Fc Domains: A Novel Strategy To Deplete B Cells and Augment Treatment of Autoimmune Disease

Zhang

Olsen

Chen

et al. 2016

The Journal of Immunology

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We developed a fully recombinant anti-CD20 protein derived from cDNA encoding one Fab domain, two IgG1 Fc regions, the IgG2 hinge, and an isoleucine zipper. This protein, called GB4542, contained both the homodimer and higher-order multimers. Binding studies revealed that GB4542 preferentially bound CD20+ cells yet also recognized CD20−FcγR+ PBMC. In contrast, a control mAb containing the identical Fab region, GB4500, failed to bind CD20−FcγR+ PBMC. Consistent with these findings, interactions between GB4542 and the canonical FcγRs had substantially lower KD values than correlate interfaces between GB4500 and these receptors. At low concentrations, GB4542 showed enhanced Ab-dependent cellular cytotoxicity, Ab-dependent cellular phagocytosis, and complement-dependent cytotoxicity compared with GB4500. However, at higher concentrations, an Fc analog of GB4542 inhibited anti-CD20 mAb–mediated B cell clearance through direct blocking of both Fc–FcγR interactions and C1q deposition on target cells. Furthermore, the higher-order multimer fraction of GB4542 demonstrated greater binding avidity with the canonical FcγRs and was associated with inhibitory effects observed in Ab-dependent cellular phagocytosis and complement-dependent cytotoxicity assays. These data suggest that GB4542 might have utility in the treatment of autoimmune diseases by combining both mAb-mediated B cell depletion and multimerized Fc-mediated tolerogenic effects.

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Complement Is Activated by IgG Hexamers Assembled at the Cell Surface

Cited by 657 publications

References 62 publications

Extending human IgG half-life using structure-guided design

Extending human IgG half-life using structure-guided design

CODV-Ig, a universal bispecific tetravalent and multifunctional immunoglobulin format for medical applications

Anti-CD20 Antibody with Multimerized Fc Domains: A Novel Strategy To Deplete B Cells and Augment Treatment of Autoimmune Disease

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