Antibody C Region Influences TGN1412-like Functional Activity In Vitro

Abstract

The unexpected outcome of the clinical trial of the superagonistic CD28 mAb TGN1412 (IgG4κ) continues to stimulate interest. We show that TGN1412 binds similarly to human and cynomolgus macaque FcγR, eliminating the possibility that differences in Fc-mediated interactions with FcγR contributed to the failure of preclinical testing in macaques to predict toxicity in humans. The influence of the Fc domain and C region structure on the in vitro functional activity of TGN1412 was investigated using F(ab′)2 and Fab fragments derived from TGN1412 recovered from the trial and recombinant TGN1412 subclass variants and mutants. Superagonistic activity, as measured by cytokine release and proliferation, was assessed by exposing PBMCs to immobilized mAbs/fragments or to aqueous mAbs/fragments in the presence of HUVEC monolayers. Removing the Fc generally curtailed or abolished PBMC activation. However, eliminating detectable FcγR-binding of the IgG4 by mutation (L235E) did not abrogate activity. Stabilizing the “wild-type” IgG4 hinge (S228P) enhanced activity without increasing FcγR binding, which could only partially be explained by inhibition of Fab arm–exchange. Subclass switching the IgG4 mAb to IgG1 decreased activity, whereas switching to IgG2 markedly increased activity. We conclude that the C region strongly influences in vitro CD28-mediated superagonistic signaling. Superagonism requires an intact Fc, as shown by the absence of activity of TGN1412 Fab and F(ab′)2 fragments, but, notably, appears to be relatively independent of FcγR-binding properties. We propose that the Fc, potentially through restricting flexibility, maintains a favorable V region conformation to allow superagonistic activity. These findings have important implications for Ab design strategies.

Upregulation of FcγRIIb on monocytes is necessary to promote the superagonist activity of TGN1412

“…In our experiments, TGN1412 F(ab9) 2 failed to induce T-cell proliferation or cytokine release in accordance with findings by Ball et al 26 However, a recent study reported that TGN1412 F(ab9) 2 still induces residual intracellular TNF-a expression in T cells at 0.5 mg/mL. 10 The reason for this discrepancy is unclear.…”

Cell Contact–Dependent Priming and Fc Interaction with CD32+ Immune Cells Contribute to the TGN1412-Triggered Cytokine Response

“…Ball et al (40) recently described that removal of the Fc part of TGN1412 curtailed or fully abolished PBMC activation. Therefore we rigorously examined whether restored T cells responded to the F(ab9) 2 fragment of TGN1412.…”

A Monovalent Anti-Human CD28 Domain Antibody Antagonist: Preclinical Efficacy and Safety

“…finding, Poirier et al (22) reported no agonist activity with the monomeric Fab fragment, FR104. However, it was recently demonstrated that even a Fab fragment of TGN1412, dry-coated on plates could drive T cell proliferation (45). In this case the activity was attributed to the conformational impact of C-region on the binding epitope of the Fab, a region missing in the dAb but present in FR104, indicating that valency alone is not a guarantee that a molecule will lack agonist activity and suggested that cytokine release stimulated by TGN1412 is different from that of other therapeutic Abs.…”