Induction of an Altered CD40 Signaling Complex by an Antagonistic Human Monoclonal Antibody to CD40 This information is current as Katherine C. Bankert, Kyp L. Oxley, Sonja M. Smith, John of September 27, 2021. P. Graham, Mark de Boer, Marielle Thewissen, Peter J. Simons and Gail A. Bishop J Immunol published online 20 March 2015 http://www.jimmunol.org/content/early/2015/03/20/jimmun ol.1402903 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2015/03/20/jimmunol.140290 Material 3.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 27, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 20, 2015, doi:10.4049/jimmunol.1402903 The Journal of Immunology Induction of an Altered CD40 Signaling Complex by an Antagonistic Human Monoclonal Antibody to CD40 Katherine C. Bankert,*,1 Kyp L. Oxley,*,1,2 Sonja M. Smith,*,3 John P. Graham,†,2 Mark de Boer,‡ Marielle Thewissen,‡ Peter J. Simons,x and Gail A. Bishop*,†,{,‖ Blocking the interaction of CD40 with its ligand CD154 is a desirable goal of therapies for preventing and/or ameliorating auto- immune diseases and transplant rejection. CD154-blocking mAbs used in human clinical trials resulted in unanticipated vascular complications, leading to heightened interest in the therapeutic potential of antagonist mAbs specific for human CD40. Abs that do not require physical competition with CD154 to inhibit CD40 signaling have particular therapeutic promise. In this study, we dem- onstrate that the antagonist anti-human CD40 mAb PG102 fails to trigger CD40-mediated activation, as well as impairs CD154- mediated CD40 activation, via a distinct nonstimulatory CD40 signaling mechanism. PG102 did not induce early CD40-induced signaling events, and it inhibited early kinase and transcription factor activation by CD154 or agonist anti-CD40 mAbs. However, Downloaded from PG102 stimulated normal CD40-mediated TNFR-associated factor (TRAF)2 and TRAF3 degradation. PG102 induced the forma- tion of a CD40 signaling complex that contained decreased amounts of both TRAF2 and TRAF3 and TRAF2-associated signaling proteins. Additionally, PG102-induced CD40 signaling complexes failed to recruit TRAF6 to detergent-insoluble membrane frac- tions. Fab fragments of PG102, while retaining CD40 binding, did not induce TRAF degradation, nor could they inhibit CD154- stimulated B cell signaling, indicating that CD40 aggregation is required for the signaling inhibition induced by PG102. The an- tagonistic impact of PG102 on CD40 signaling reveals that the manner of CD40 ligation can determine sharply different outcomes http://www.jimmunol.org/ for CD40 signaling and suggests that such information can be used to therapeutically manipulate these outcomes. The Journal of Immunology, 2015, 194: 000–000. D40, a member of the TNFR superfamily, is expressed by promise in preclinical mouse models of arthritis (6) and allograft multiple immune and nonimmune cell types and plays key transplant (7). However, the first human clinical trial using anti- C roles in stimulating effective Ag presentation and hu- CD154–blocking mAb was halted as the result of several fatal moral immunity (1). CD40 signaling is implicated in the patho- thromboembolic events (8), which may have been due to the effect genesis of both autoimmunity (2, 3) and transplant rejection (4, 5), of immune complexes upon activated platelets expressing CD154. by guest on September 27, 2021 and disruption of CD40-mediated activation has been of consid- Although a variety of approaches to blocking CD40–CD154 in- erable therapeutic interest (reviewed in Ref. 5). The first inter- teractions have since been explored and may prove successful (5), ventions tested used mAbs specific for the CD40L, CD154, to an attractive alternative is direct inhibition of the CD40-mediated block the receptor–ligand interaction. This approach showed great activation pathway. The mAb PG102 was derived from the previously described anti- human CD40 (hCD40) mAb 5D12 (9), which inhibits the prolif- *Department of Microbiology, University of Iowa, Iowa City, IA 52242; †Interdisciplinary eration of human B cells stimulated with activated T cells or ag- Graduate Program in Immunology, University of Iowa, Iowa City, IA 52242; ‡Fast Forward Pharmaceuticals BV, Utrecht, the Netherlands; xBioceros Holding onistic CD40-specific mAbs (10, 11). Subsequently, a chimeric BV, Utrecht, the Netherlands; {Department of Internal Medicine, University of form of 5D12 (ch5D12) containing human IgG4 constant regions ‖ Iowa, Iowa City, IA 52242; and VA Medical Center, Iowa City, IA 52242 was produced. The ch5D12 mAb prevents development of ex- 1K.C.B. and K.L.O. contributed equally to this work. perimental autoimmune encephalomyelitis in marmosets (12), as 2Current address: The Jackson Laboratory, Bar Harbor, ME. well as prolongs the survival of kidney allografts and allows re- 3Current address: Department of Pediatrics, University of Iowa, Iowa City, IA. peated systemic administration of adenoviral gene therapy vectors Received for publication November 17, 2014. Accepted for publication February 20, in rhesus monkeys (13, 14). In an open-label dose-escalation 2015. phase I/IIa study of Crohn’s disease patients, ch5D12 was well This work was supported in part by funds from the Holden Chair in Cancer Biology tolerated and showed promising clinical benefit (15). Several (to G.A.B.), PanGenetics BV (Utrecht, the Netherlands), and Fast Forward Pharma- amino acids in both H and L chains of the ch5D12 mAb were ceuticals. G.A.B. is a Senior Research Career Scientist of the Veterans Administra- tion. This material is based upon work supported in part by the Office of Research subsequently changed to remove potential MHC-binding epitopes and Development, Veterans Health Administration, Department of Veterans Affairs. and thus reduce possible immunogenicity (M. de Boer, personal Address correspondence and reprint requests to Dr. Gail A. Bishop, University of communication); the resultant mAb is PG102. Previous studies Iowa, 2193B MERF, 375 Newton Road, Iowa City, IA 52242. E-mail address: showed that ch5D12 inhibits CD40-induced production of proin- [email protected] flammatory cytokines (16), and we observed this as well with The online version of this article contains supplemental material. PG102 in B cell cultures (K.L. Oxley and G.A. Bishop, unpublished Abbreviations used in this article: BCM, B cell medium; ch5D12, chimeric form of 5D12; cIAP1, cellular inhibitor of apoptosis-1; DC, dendritic cell; hCD40, human observations). CD40; hCD154, human CD154; HOIP, HOIL1-L interacting protein; IKK, IkBkinase; The present study was undertaken to test the hypothesis that R10, RPMI 1640 medium with 10% FCS, 1% HEPES, 1% L-glutamine, 1% penicillin/ PG102 inhibits CD40 signaling by inducing a signaling cascade streptomycin, and 1% essential amino acids; TRAF, TNFR-associated factor. that is distinct from that initiated by binding of CD154 or agonistic Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 anti-CD40 Abs to CD40. Results reveal that engagement of CD40 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402903 2 ALTERED CD40 SIGNALING COMPLEX INDUCED BY AN ANTAGONIST mAb by PG102 induced formation of a dysfunctional CD40 signaling manufacturer’s instructions (Invitrogen), using an ELISA reader (Bio- complex characterized by altered TNFR-associated factor (TRAF) Rad, Hercules, CA). All binding reactions were performed at room tem- recruitment and association, with resultant ineffective induction of perature in the presence of 1% w/v BSA (Roche) and 0.05% v/v Tween-20 detergent. kinase and transcription factor activation. Importantly for potential FACS. After blocking with 50 mg/ml human IgG (Sigma-Aldrich), hCD40- therapeutic applications, although PG102 did not inhibit CD154– expressing JY human B lymphoblastoid cells (American Type Culture CD40 binding, it inhibited and prevented CD154-induced CD40 Collection) were incubated with saturating (15 mg/ml) hCD154–mouse activation. CD8 fusion protein (Ancell), followed by saturating (10 mg/ml) biotinylated PG102 (or vice versa). Subsequently, hCD154 and PG102 were detected with FITC-labeled rat anti-mouse CD8 Ab (Ancell) and PE-labeled strep- Materials and Methods tavidin (Jackson ImmunoResearch), respectively. Fluorescent signals were Cells measured using a flow cytometer (Becton Dickinson, Mt. View, CA). All The human B cell line T5-1 was described previously (17). PBMCs from binding reactions were performed at 4˚C in the presence of 0.1% v/v BSA human blood donors were isolated from discarded Leukocyte Reduction (Sigma-Aldrich) and 0.05% w/v sodium azide. System cones obtained from the DeGowin Blood Center