Conformation of the Human Immunoglobulin G2 Hinge Imparts Superagonistic Properties to Immunostimulatory Anticancer Antibodies

Abstract

SummaryMonoclonal antibody (mAb) drugs that stimulate antitumor immunity are transforming cancer treatment but require optimization for maximum clinical impact. Here, we show that, unlike other immunoglobulin isotypes, human IgG2 (h2) imparts FcγR-independent agonistic activity to immune-stimulatory mAbs such as anti-CD40, -4-1BB, and -CD28. Activity is provided by a subfraction of h2, h2B, that is structurally constrained due its unique arrangement of hinge region disulfide bonds. Agonistic activity can be transferred from h2 to h1 by swapping their hinge and CH1 domains, and substitution of key hinge and CH1 cysteines generates homogenous h2 variants with distinct agonistic properties. This provides the exciting opportunity to engineer clinical reagents with defined therapeutic activity regardless of FcγR expression levels in the local microenvironment.

Influence of immunoglobulin isotype on therapeutic antibody function

“…ChiLob 7/4, a CD40 mAb that has recently completed a phase 1 clinical trial, 66 is agonistic in human CD40 transgenic mice as IgG2 in the absence of FcgR expression and even as a F(ab9) 2 fragment. 60 FcgRindependent activity has also been demonstrated for another IgG2 CD40 mAb in a clinical trial: CP870-893. 67 The key to IgG2 agonistic activity lies in the unique configuration of its hinge region, 60 which can adopt alternative conformations through disulphide rearrangement.…”

“…60 FcgRindependent activity has also been demonstrated for another IgG2 CD40 mAb in a clinical trial: CP870-893. 67 The key to IgG2 agonistic activity lies in the unique configuration of its hinge region, 60 which can adopt alternative conformations through disulphide rearrangement. [68][69][70] IgG2 is believed to be synthesized with all 4 heavy chain (HC) hinge cysteines involved in parallel inter-HC disulphide bonds, a conformation designated as IgG2(A).…”

“…For example, a recent study from Dahan et al 42 demonstrated greater efficacy with blocking anti-PD1 mAbs engineered to prevent FcgR engagement ( Figure 1C). Moreover, human IgG2 agonistic anti-TNFR mAbs also do not require FcgR interaction for agonistic activity 60 ( Figure 1B). ChiLob 7/4, a CD40 mAb that has recently completed a phase 1 clinical trial, 66 is agonistic in human CD40 transgenic mice as IgG2 in the absence of FcgR expression and even as a F(ab9) 2 fragment.…”

“…54 In contrast to direct targeting agents, the agonistic activity of these mAbs is dependent on their ability to engage inhibitory FcgRIIB, [55][56][57][58][59] and mAbs with high A:I ratios (eg, mouse IgG2a, human IgG1) are largely inactive in preclinical models, whereas those with low A:I ratios (eg, mouse IgG1 and rat IgG2a) are highly agonistic. [55][56][57][58][59][60] Signaling through FcgRIIB is not required to confer activity; rather, it provides a crosslinking scaffold for the mAbs to facilitate TNFR clustering and activation 57,[61][62][63] ( Figure 1B). Activatory FcgR can also mediate crosslinking in vitro 57 or in vivo when sufficiently expressed at the target location 58,63 ; thus, the predominant role of FcgRIIB in vivo may, in part, reflect its bioavailability.…”

Immunostimulatory Monoclonal Antibodies and Immunomodulation: Harvesting the Crop

“…The molecular mechanisms required for making good agonistic antibodies were dissected in depth by Dr. Glennie (University of Southampton School of Medicine, Southampton, United Kingdom). He showed that agonistic antibodies targeting the family of TNFR need crosslinking by FcgRIIB, but the antibody can also be engineered to obtain superagonistic antibodies independent of cross-linking, tampering with the structure of the antibody to confer a more rigid conformation of the hinge (19).…”