Anti-CD40L Immune Complexes Potently Activate Platelets In Vitro and Cause Thrombosis in FCGR2A Transgenic Mice

Abstract

Anti-CD40L immunotherapy in systemic lupus erythematosus patients was associated with thromboembolism of unknown cause. We previously showed that monoclonal anti-CD40L immune complexes (ICs) activated platelets in vitro via the IgG receptor (FcγRIIa). In this study, we examined the prothrombotic effects of anti-CD40L ICs in vivo. Because mouse platelets lack FcγRIIa, we used FCGR2A transgenic mice. FCGR2A mice were injected i.v. with preformed ICs consisting of either anti-human CD40L mAb (M90) plus human CD40L, or a chimerized anti-mouse CD40L mAb (hMR1) plus mouse CD40L. ICs containing an aglycosylated form of hMR1, which does not bind FcγRIIa, were also injected. M90 IC caused shock and thrombocytopenia in FCGR2A but not in wild-type mice. Animals injected with hMR1 IC also experienced these effects, whereas those injected with aglycosylated-hMR1 IC did not, demonstrating that anti-CD40L IC-induced platelet activation in vivo is FcγRIIa-dependent. Sequential injections of individual IC components caused similar effects, suggesting that ICs were able to assemble in circulation. Analysis of IC-injected mice revealed pulmonary thrombi consisting of platelet aggregates and fibrin. Mice pretreated with a thrombin inhibitor became moderately thrombocytopenic in response to anti-CD40L ICs and had pulmonary platelet-thrombi devoid of fibrin. In conclusion, we have shown for the first time that anti-CD40L IC-induced thrombosis can be replicated in mice transgenic for FcγRIIa. This molecular mechanism may be important for understanding thrombosis associated with CD40L immunotherapy. The FCGR2A mouse model may also be useful for assessing the hemostatic safety of other therapeutic Abs.

Engineering of a Novel Anti-CD40L Domain Antibody for Treatment of Autoimmune Diseases

“…To this end, our results and those from Robles-Carillo et al (18) confirm that IgG1 binding to platelet CD32a (FcgRIIA) is likely the link to susceptibility to TE. Interestingly, other IgG1 Abs against TNF superfamily members (including numerous anti-TNF-a mAbs) have been in the clinic with no apparent incidents related to TE; hence, why the predisposition with targeting CD40L?…”

“…Subsequent to the clinical findings two major issues were raised: 1) what was the relationship between TE and anti-CD40L mAbs and 2) in addition to blocking CD40 binding, was the Fc-effector function a necessary component of the anti-CD40L mAb for efficacy? To this end, recent data suggested that binding of the Fc domain of anti-CD40L mAbs to the activating FcgRIIa (CD32a) receptor on platelets results in activation and aggregation of platelets (18). These data could explain the TE events with hu5c8 and IDEC-131, because both were of the IgG1 isotype that binds avidly to FcgRIIa.…”

CD32a antibodies induce thrombocytopenia and type II hypersensitivity reactions in FCGR2A mice

“…8 The following were purchased from commercial sources: soluble human CD40L (Peprotech), mouse CD32 mAbs IV.3 (StemCell Technologies) and mouse AT-10 (Serotec), F(ab9) 2 -goat antihuman IgG (minimal cross-reactivity with mouse IgG; Jackson Immunoresearch Laboratories), fluorescein isothiocyanate-labeled anti-mouse CD41, CD62P, and control flow cytometry mAbs (BD Biosciences), and Alexa 488 -labeled goat-antihuman IgG (Invitrogen).…”

“…Signs of apparent shock were assessed by observation of balance, mobility, and respiration, and were ranked as fatal, severe (complete immobility, apparent loss of consciousness), moderate (impaired mobility, irregular respiration), mild (lethargy, shallow respiration), or none, as described. 8,12,13 Platelets were counted 30 minutes postinjection. Lungs were harvested en bloc for assessing thrombosis by hematoxylin-and-eosin (H&E) microscopy.…”

T Cell Targeted Therapies in Lupus: Do They Make Sense?

“…A subsequent openlabel study of another monoclonal antibody to CD40L, BG9588 in 28 patients with proliferative lupus nephritis failed to provide further insights into therapeutic efficacy, and was stopped prematurely due to thromboembolic events [35]. This could be due to the fact that platelets of patients with SLE strongly express CD40L on their surface and can be cross-linked via binding of the anti-CD40L Fc to platelet FcγRIIα receptor, potentially leading to platelet aggregation and thromboembolism [36]. Anti-CD40L constructs that do not trigger FcγRIIα signaling, such as single IgG molecules and domain antibodies, could bypass platelet activation-related toxicities while maintaining their biological efficacy [31].…”