Induction of an Altered CD40 Signaling Complex by an Antagonistic Human Monoclonal 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

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Supplementary http://www.jimmunol.org/content/suppl/2015/03/20/jimmunol.140290 Material 3.DCSupplemental http://www.jimmunol.org/

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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 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 (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 (University of Signaling assays Iowa). The Blood Center has University of Iowa Institutional Review Board approval to provide these deidentified samples to investigators. Cells A total of 1–2 3 106 cells was washed in RPMI 1640 medium, resuspended were cultured in RPMI 1640 medium with 10% heat-inactivated FCS in 1 ml BCM in 1.5-ml Eppendorf tubes, and rested for 45 min at 37˚C. (Atlanta Biologicals, Atlanta, GA), 10 mM 2-ME (Invitrogen, Grand Cells were stimulated for the time periods indicated in the figure legends Island, NY), 2 mM L-glutamine, penicillin, and streptomycin (B cell me- with anti-hCD40 or isotype-control mAbs at 10 mg/ml. Alternatively, cells dium [BCM]). Human primary monocyte-derived dendritic cells (DCs) were incubated with Hi5 insect cells, either wild-type or expressing

were obtained from the above-described human blood samples, as follows. hCD154, as described previously (20). Whole-cell lysates were prepared as Downloaded from Monocytes were isolated from these PBMCs by culturing for 30 min and described (21). washing away nonadherent cells. A cell scraper was used to dislodge monocytes, which were washed and resuspended in R10 (RPMI 1640 Immunoprecipitation and Western blotting medium with 10% FCS, 1% HEPES, 1% L-glutamine, 1% penicillin/ streptomycin, and 1% essential amino acids) at 1 3 106 cells/3ml with For immunoprecipitation of CD40 and associated proteins, Dynal protein G GM-CSF (100 ng/ml) and recombinant human IL-4 (10 ng/ml). Cells were magnetic beads (Invitrogen) were coated with anti-hCD40 mAb (10 mg/ cultured in six-well tissue culture plates at 3 ml/well. At days 2 and 5, 10 ml beads), as described previously (22). Abs were conjugated to beads 3 7 900 ml culture medium was removed and replenished with 1 ml fresh R10 according to the manufacturer’s protocols. A total of 2 10 cells was http://www.jimmunol.org/ containing 300 ng/ml GM-CSF and 30 ng/ml IL-4. To generate mature incubated in 1 ml BCM with mAb-coated beads for 10 min at 37˚C. Beads DCs, immature DCs were harvested on day 5, washed with PBS, seeded and cells were then pelleted and lysed, as described previously (22). Bead- 3 (5 3 105 cells/ml/well) in fresh R10 supplemented with GM-CSF (100 ng/ml) bound proteins were resuspended in 2 SDS-PAGE loading buffer and and recombinant human IL-4 (10 ng/ml), and stimulated with LPS (1 mg/ml) boiled for 3–5 min at 95˚C prior to loading samples for PAGE. For Western for 48 h. blotting, 10–15 ml sample was resolved on 10% SDS-PAGE. Proteins were transferred to Immobilon-P membranes (Millipore). Membranes were mAbs and CD154 blocked with 10% dry milk in TBST for 1 h, washed in TBST, incubated with primary Abs overnight, washed in 10% dry milk in TBST, and in- The agonistic anti-hCD40 mouse IgG1 mAb G28.5 (18) was produced by cubated with secondary Abs for 1–2 h or overnight. Protein bands were saturated AmSO4 precipitation of supernatant from a hybridoma obtained visualized using a chemiluminescent detection reagent (Pierce, Rockford, from the American Type Culture Collection (Manassas, VA). The antag- IL). Images of blots were recorded with a low-light imaging system by guest on September 27, 2021 onistic humanized anti-hCD40 mAb PG102 is a less immunogenic form of (LAS3000; Fujifilm Medical Systems Stamford, CT). Immunoblots were the humanized anti-hCD40 mAb ch5D12 (12) and was provided by Fast reprobed with appropriate Abs specific for control proteins, such as actin, Forward Pharmaceuticals. The humanized anti-hCD40 control mAb 4D11 to verify equal protein loading in each lane. was used in Fig. 1 (19). The isotype-control Abs used were mIgG1 (Southern Biotech, Birmingham, AL) and hIgG4k (Sigma-Aldrich, St. Louis, TRAF-degradation assay MO) for G28.5 and PG102, respectively. The source of human CD154 was 6 membranes of CD154-expressing Hi5 insect cells produced in the Bishop Cells were placed in a 24-well plate at 1 3 10 cells/ml/well. Anti-CD40 laboratory, as previously described (20). Hi5 cells grow at 25˚C; they die mAbs were added to appropriate wells at 10 mg/ml, and cells were incu- and form membrane fragments when cultured at 37˚C. bated at 37˚C for 5 h. Cells were then transferred to 1.5-ml Eppendorf The following primary Abs were used for Western blotting and are listed tubes and pelleted (7000 rpm, 1 min, 4˚C). Cells were resuspended in 23 by their species and recognition specificities: rabbit anti–phospho-JNK, SDS sample dye and sonicated (Branson Sonifier 450, 1.5 output, 90% rabbit anti–phospho-IkBa, rabbit anti-total IkBa, mouse anti–IkB kinase duty cycle), and samples were boiled at 95˚C for 3 min. (IKK)g, rabbit anti–phospho-p38, rabbit anti–phospho-ERK, rabbit anti- TRAF6 (all from Cell Signaling Technology, Danvers, MA), rabbit anti- Analysis of detergent-soluble and insoluble membrane total JNK and rabbit anti-TRAF3 (Santa Cruz Biotechnology, Santa Cruz, signaling complexes CA), mouse anti-actin (Millipore, Billerica, MA), rabbit anti-TRAF2 3 7 (Medical and Biological Laboratories, Nagoya, Japan), and mouse anti– Cells (1 10 /condition) were stimulated with mAbs or insect cell cellular inhibitor of apoptosis-1 (cIAP1; R&D Systems, Minneapolis, MN). membranes, as described above, or left unstimulated and then pelleted 3 g The rabbit anti–HOIL1-L interacting protein (HOIP) Ab was obtained from (400 , 1 min). The cell pellets were resuspended in lysis buffer (1% b Dr. B. Hostager (University of Iowa). The sheep anti-hCD40 Ab (21) was Brij, 150 mM NaCl, 20 mM Tris [pH 7.5], 50 mM -glycerophosphate, generated in the Bishop laboratory. The secondary Abs used included goat 2 mini complete EDTA-free tablets, and sodium vanadate) and incubated anti-mouse IgG, goat anti-rabbit IgG, goat anti-chicken IgG, donkey anti- for 30 min on ice. After incubation, cell lysates were centrifuged (30 min, 3 g sheep IgG (all from Jackson ImmunoResearch, West Grove, PA), and goat 4˚C, 400 ), and supernatants were transferred to a new tube. Lysis anti-hamster IgG (Santa Cruz Biotechnology). buffer + 0.5% SDS and 1% 2-ME were added. Pellets were sonicated (1.5 output, 90% duty cycle), and 23 SDS- PAGE buffer was added to both Simultaneous binding of PG102 and human CD154 to hCD40 pellet and supernatant. Both fractions were boiled (3 min, 95˚C). ELISA. Flat-bottom 96-well EIA/RIA plates (Corning Life Sciences, Generation of PG102 Fab fragments Amsterdam, the Netherlands) were coated with human recombinant CD40- Fc fusion protein (Bioceros) at 0.25 mg/ml in PBS (pH 7.2). After blocking Fab fragments from PG102-intact IgGs were generated using the Pierce Fab with 1% w/v BSA, saturating (2 mg/ml) human CD154 (hCD154)–mouse Preparation Kit (Thermo Fisher Scientific, Boston, MA), according to CD8 fusion protein (Ancell, Bayport, MN) was added, followed by satu- the manufacturer’s instructions. After this procedure, only PG102 Fab rating (1 mg/ml) PG102 or 4D11 (or vice versa). hCD154 or PG102/4D11 fragments were found by standard SDS-PAGE and Coomassie Blue was detected with a combination of biotinylated rat anti-mouse CD8 Ab staining (data not shown). Subsequently, the binding of these PG102 Fabs (Ancell) and HRP-labeled streptavidin (Jackson ImmunoResearch) or with on hCD40-expressing JY B cells was compared with PG102-intact IgGs HRP-labeled goat anti-human k L chain Ab (The Jackson Laboratory), using flow cytometry. Results are presented in Supplemental Fig. 1B and respectively. Optical densities were read at 450 nm after development confirm effective binding of Fabs to hCD40, indistinguishable from the with a ready-to-use tetramethylbenzidine solution, according to the binding of intact PG102. The Journal of Immunology 3

Statistical analysis internalization of the CD40 complex; after 2 h of incubation with The statistical significance of the results from multiple experiments was PG102 at 37˚C, no detectable differences in CD40 surface levels determined by the Student t test. The ranges of p values are indicated in the were observed (Supplemental Fig. 1A). figure legends. Lack of effective induction of early CD40 signaling by PG102 Engagement of CD40 by agonistic anti-CD40 Abs in B lympho- Results cytes and myeloid cells results in rapid induction of activation of Independent CD40 binding by PG102 and CD154 MAPKs, including JNK, ERK, and p38, as well as activation of the Most reagents designed to inhibit CD40 signaling for therapeutic canonical NF-kB1 transcriptional pathway (23, 24). Because the purposes block the binding of CD40 to CD154 (reviewed in Refs. 2, 5D12/PG102 mAb inhibits human B cell proliferation and Ig 5). Thus, it was of interest to determine whether the previously production induced by either activated T cells or agonistic anti- reported inhibitory impact of the hCD40-specific mAb PG102 on hCD40 Ab (10, 11), we investigated whether this corresponds CD40-mediated immune cell activation results from blocking to a lack of CD40-mediated early signals induced by PG102. of the CD40–CD154 interaction. This was addressed using two Supplemental Fig. 2A demonstrates that the agonistic anti-hCD40 complementary approaches, described in detail in Materials and mAb G28.5, as expected, stimulated robust activation of MAPK Methods. An ELISA approach used plates coated with soluble and NF-kB1 in human primary DCs. In contrast, PG102 stimu- hCD40, incubated with various combinations of soluble hCD154, lated detectable, but quite modest, MAPK activation and little to PG102, or control Abs. Fig. 1A shows that, regardless of whether no NF-kB1 activation. hCD154 or PG102 was added first, neither inhibited the binding Initial studies of PG102 examined CD40-mediated B cell ac- Downloaded from of the other. The same results were obtained using intact human tivation, so we focused upon this cell type for our subsequent B cells, analyzed by flow cytometry (Fig. 1B). Fig. 1B also presents, experiments. Fig. 2 shows that PG102 did not induce detectable for comparison, results with the 4D11 anti-hCD40 mAb, which phosphorylation of either JNK or IkBa in the human B cell line blocked hCD154 binding to CD40. Thus, PG102 does not im- T5-1, which was previously reported as a robust model of CD40 pair CD154 binding to CD40 and vice versa. We also found that signaling to human B cells (20, 25). Fig. 2A compares the phos-

PG102 binding to hCD40 on human B cells did not result in rapid phorylated forms of JNK and IkBa in a representative Western http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 1. Lack of CD154–PG102 binding competition. (A) PG102 and hCD154 binding to plate-bound CD40. Binding assays were performed as described in Materials and Methods, using binding of HRP-labeled secondary reagents as a detection method. Cells were incubated first with PG102 or control mAb, followed by CD154, and CD154 was detected (upper panel). The initial incubations were with CD154, followed by PG102 or control mAbs; PG102 binding was detected (lower panel). Results represent the mean 6 SD of triplicate cultures from a representative of three experiments. (B) PG102 or 4D11 mAb and hCD154 binding to cell-expressed hCD40. hCD40-expressing human B lymphoblastoid cells (JY) were incubated with FITC-labeled hCD154, followed by PE-labeled PG102 or vice versa, as described in Materials and Methods (left panel). Cells were incubated first with PE-4D11 mAb and then FITC-hCD154 (right panel). Flow cytometry of stained cells from a representative of three experiments is shown. BG, background staining. 4 ALTERED CD40 SIGNALING COMPLEX INDUCED BY AN ANTAGONIST mAb

FIGURE 2. Failure of PG102 to effectively activate early CD40-mediated signaling events. (A) Cells of the human B cell line T5-1 were incubated with the hCD40-specific mAbs G28.5 or PG102 for the indicated minutes (Min), as described in Materials and Methods. Cell lysates were subjected to SDS-PAGE, and Western blot- ting was performed for total or p-JNK, p-IkBa, and actin (as a loading control; total IkBa de- creases with activation so cannot serve this func- tion). A representative of three experiments is shown. (B) Relative quantification, as in Materials and Methods, of CD40-induced phosphorylation of JNK and IkBa, normalized to total JNK or actin, respectively. Data are mean 6 SD of three experiments. **p , 0.01. Iso, cells treated for 30 min with isotype-control Abs for PG102 or G28.5. Downloaded from

blot of lysates from B cells stimulated with G28.5 or PG102 anti- Inhibition of CD154-mediated B cell activation by PG102 hCD40 mAbs for the indicated minutes. Fig. 2B shows quantifi- PG102 did not bind to or block binding of CD154 (Fig. 1). Thus, cation of phospho-JNK relative to total JNK, as well as phospho- we wished to test the hypothesis that binding of PG102 induces an http://www.jimmunol.org/ IkBa relative to actin (because total IkBa is degraded following altered CD40 signaling complex that is refractory to agonistic activation), providing the mean values obtained from three indi- stimuli. Therefore, we treated human B cells with membrane-bound vidual experiments. CD154 alone, together with PG102, or subsequent to PG102 addition. by guest on September 27, 2021

FIGURE 3. PG102-mediated inhibition of CD154 signaling. (A) T5-1 human B cells were stimulated with hCD154 alone, CD154+PG102, or PG102 alone for 30 min, followed by CD154 for the indicated minutes. Phosphorylation of JNK and IkBa was assessed as in Fig. 2. A representative of three experiments is shown. (B) Quantification, as in Materials and Methods, for three replicates of the experiment shown in (A), expressed as fold increase over values at the zero time point. Data are mean 6 SD. (C) T5-1 human B cells were stimulated with hCD154 alone, CD154+PG102, PG102 for 30 min + CD154, or CD154 for 30 min + PG102 for the indicated number of minutes. Phosphorylation of JNK and IkBa was assessed as in (A); line graphs represent fold increase over values at the zero time point. **p , 0.01. Iso, cells treated for 30 min with isotype-control Ab for PG102. The Journal of Immunology 5

Results presented in Fig. 3 indicate that either simultaneous or pretreatment of B cells with PG102 abrogated CD154-mediated JNK and IkBa phosphorylation. Fig. 3A (representative Western blot) and Fig. 3B (quantification of results from three experi- ments) show that either PG102 pretreatment or simultaneous ad- dition of PG102 and hCD154 to B cells resulted in marked inhibition of early signaling events. Fig. 3C further demonstrates that this inhibition lasted throughout the course of JNK or NF-kB activation and could be mediated by PG102, even when this mAb was added to cells subsequent to an hCD154 stimulus. Similar findings were obtained in myeloid cells (M. Thewissen and M. de Boer, unpublished observations). These data suggest several po- tential mechanisms, which are not mutually exclusive. One ex- planation is that a distinct, negative signal delivered via PG102 to the CD40 signaling complex counteracted and prevented nor- FIGURE 4. CD40-mediated TRAF degradation. (A) T5-1 human B cells mal agonist-mediated activating signals. Another possibility is that were incubated for 5 h with mAbs G28.5 or PG102 or BCM alone. Cell PG102 induced formation of a defective CD40 signaling complex, lysates were prepared and separated by SDS-PAGE, followed by Western which competed with the functional signaling complex induced by blotting for TRAF2, TRAF3, and actin, as described in Materials and

CD154. We next examined the nature of CD40 signals induced by Methods. A representative of three experiments is shown. Arrows indicate Downloaded from PG102. size markers (kDa). (B) Quantification of TRAF band intensities, as in Materials and Methods, normalized to actin. Data are mean 6 SD of three CD40-mediated TRAF degradation induced by PG102 experiments. *p , 0.05, **p , 0.01. ns, not significant. CD40 signals to B cells rapidly induce recruitment of TRAF2 and TRAF3 to the CD40 cytoplasmic domain, followed by TRAF2- reduced TRAF2/3 binding was consistent, and Fig. 5C indicates dependent K48-linked polyubiquitination and proteasome-mediated that this is seen for PBMCs from multiple healthy human blood http://www.jimmunol.org/ degradation of both of these TRAFs (21, 26–28). Because PG102 donors. could not induce CD40-mediated activating signals, we examined Another feature of CD40-induced signaling complex formation whether the mAb induced this regulatory event. Data presented in is the localization of the complex to cholesterol-rich plasma Fig. 4 indicate that PG102 induced CD40-mediated degradation of membrane fractions (“rafts”), separated by their relative detergent both TRAF2 and TRAF3 in human B cells, to an extent that was insolubility in cell lysates (21, 32). Stimulation with either CD154 not detectably different from degradation induced by the ago- or PG102 induced movement of TRAF2 and TRAF3 to these nistic mAb G28.5. This was also true in primary human DCs membrane rafts; however, because their overall recruitment to (Supplemental Fig. 2B). Consistent with these findings, relative CD40 was decreased (Fig. 5), the amounts in the rafts were also levels of ubiquitination of CD40-associated TRAF2 induced by lower (data not shown). Fig. 5 shows that there was not a signifi- by guest on September 27, 2021 PG102 versus G28.5 in human B cells were indistinguishable cant decrease in total TRAF6–CD40 binding induced by PG102 (K.C. Bankert and G.A. Bishop, unpublished observations). versus CD154. However, although CD40 translocates to the detergent-insoluble fraction following PG102 stimulation, PG102, CD40 signaling complex formation induced by PG102 when used alone to stimulate B cells, was unable to induce TRAF6 Data shown in Fig. 4 indicate that PG102 showed selective in- localization to this fraction (Fig. 6). Notably, PG102 treatment for duction of CD40-mediated signaling events, with normal degra- 30 min or 2 h prior to the addition of hCD154 also inhibited dation of TRAF2 and TRAF3. We next examined recruitment of hCD154-induced TRAF6 translocation (Fig. 6). Thus, the CD40 signaling proteins known to play major roles in CD40-mediated signaling complex induced by PG102 displayed altered associa- signals to B cells (24, 29–31). Following stimulation with either tions of membrane CD40 with TRAF2, TRAF3, and TRAF6. G28.5 or PG102 mAbs, CD40 was immunoprecipitated from human B cells, and associated proteins were normalized to CD40 Requirement for CD40 cross-linking to induce to determine their relative amounts. Although amounts of TRAF6 PG102-mediated signaling associated with the signaling complex were not statistically dif- The results described above show that PG102-mediated CD40 ferent, CD40 from PG102-stimulated B cells had significantly less engagement induced normal degradation of TRAF2 and TRAF3 associated TRAF2 and TRAF3 (Fig. 5). Representative Western but inhibited CD154-mediated CD40 signaling, without interfering blots in Fig. 5A show that this was not due to the decreased ability with CD154 binding to CD40. Taken together, these data support of PG102 to bind or precipitate hCD40. In addition, the impor- the hypothesis that PG102-mediated inhibition of CD40 signaling tant TRAF2-associated signaling proteins cIAP1, IKKg,and is not passive, but instead induces formation of an abnormal sig- HOIP showed significantly reduced amounts in the PG102- naling complex that inhibits CD154-induced CD40 signaling. induced CD40 signaling complex. Normal CD40 signaling requires cross-linking of membrane CD40. Although T5-1 cells are a well-validated model for CD40 sig- To determine whether aggregation of CD40 by PG102 is required naling to human B cells, this is a transformed cell line, and CD40 is for PG102-induced effects, we prepared Fab fragments of PG102, also biologically important in a variety of immune cells, particu- which bound normally to CD40 (Supplemental Fig. 1B). Fig. 7 larly myeloid cells. Thus, we examined CD40 signaling complex demonstrates that, similar to intact mAb, PG102 Fabs did not formation in primary human PBMCs. Fig. 5C presents a repre- induce CD40-mediated JNK or NF-kB1 activation, but the Fabs sentative Western blot demonstrating that the marked decrease in were ineffective in impairing agonist-induced activation of these TRAF2 and with CD40 in PG102- versus G28.5-stimulated cells events. Additionally, in contrast to intact PG102, Fab fragments is also seen in primary human immune cells. The decrease in did not induce TRAF degradation in human B cells. Thus, PG102 TRAF3 was more modest and did not reach statistical signifi- must induce CD40 aggregation to inhibit CD40 signaling by al- cance, as it did in the T5-1 cells (Fig. 5B). However, the trend of teration of the signaling pathway. 6 ALTERED CD40 SIGNALING COMPLEX INDUCED BY AN ANTAGONIST mAb Downloaded from http://www.jimmunol.org/

FIGURE 5. CD40 signaling complex composition. (A) T5-1 human B cells were stimulated with magnetic beads coated with mAbs G28.5 or PG102; these beads were also used to immunoprecipitate the CD40 signaling complex, as described in Materials and Methods. CD40-associated proteins were by guest on September 27, 2021 subjected to SDS-PAGE, and Western blotting was performed for the indicated proteins. A representative of five experiments is shown. (B) Relative quantities of CD40-associating proteins in signaling complexes induced by G28.5 versus PG102. Amounts are normalized to the amount of hCD40 immunoprecipitated. The number of experiments averaged for each panel is represented by the “n” value. The p values were calculated as described in Materials and Methods.(C) CD40 signaling complex induced by G28.5 or PG102 in primary human PBMCs. PBMCs from normal human blood donors were stimulated, and hCD40 immunoprecipitates were analyzed as in (A). The “n” value refers to the number of human blood donors tested.

Discussion to CD154 showed efficacy in the suppression of transplant rejection Initial discovery of the CD40 receptor preceded that of its ligand. and graft-versus-host disease in both mouse and preclinical primate Although agonistic mAbs to CD40 initially suggested its role as an models (4, 7, 46–48). However, initial human clinical trials of the immune cell activator (18, 33), the discovery of its key physio- anti-hCD154 mAb hu5c8/BG9588/ruplizumab in systemic logical roles in immune responses followed identification of the erythematosus patients, based upon these promising preclinical ligand for CD40 (initially called gp39 or CD40L, now designated results, resulted in unexpected and serious thromboembolic com- CD154) (34–36). This, in turn, led to elucidation of the critical plications in some patients. This problem also emerged in trials of role for defective CD40 signaling in the human immunodeficiency other anti-CD154 mAbs (reviewed in Ref. 5). The cause of these disease X-linked hyper-IgM syndrome (37–40). Soon after the complications is not entirely clear, but it may be related to the appreciation of CD40’s important functions in host immune expression of CD154 on human platelets, together with FcRs that defenses, studies implicated an important contribution of CD40- can bind the therapeutic mAbs. mediated activation in pathogenic processes, including transplant Thus, alternative approaches to inhibit CD40 signaling are rejection, autoimmune diseases, and B cell malignancies (reviewed needed. This could include development of CD154-blocking Abs in Refs. 2–4, 41–43). Thus, CD40, and its activation by CD154 or Ab fragments that cannot bind FcgRs but retain high affinity for became attractive targets for these clinical problems. CD154 and show normal, unaccelerated serum clearance. How- The earliest and most frequent approach to inhibiting CD40- ever, if direct ligation of platelet CD154, not just FcR binding, is mediated signaling in vivo is to interfere with CD40–CD154 responsible for certain complications, this drawback will remain. interactions. Blocking Abs to CD154 interfere with the development Peptidomimetics can also provide a way to block CD40–CD154 of collagen-induced arthritis, a mouse model of human rheumatoid interactions (49). However, peptide-binding affinities can frequently arthritis (6), as well mouse models of human systemic lupus erythe- be modest, requiring large and potentially impractical quantities to matosus (44, 45). These findings were followed by many studies in effectively compete with CD154 for CD40 binding. various laboratory models, showing promise for blocking CD40– An approach that circumvents the limitations of both of the prior CD154 signaling in the alleviation of autoimmune and chronic in- strategies is the use of nonagonistic CD40 mAbs. A number of such flammatory diseases (reviewed in Ref. 5). Additionally, blocking Abs reagents act by competing directly with CD154 for CD40 binding The Journal of Immunology 7

tive” signaling pathways that bacterial superantigens are reported to trigger when they bind TCR/MHC complexes (reviewed in Ref. 53). To gain further insight into how possible allosteric effects of PG102 binding may alter formation of the CD40 signaling com- plex, we examined two key features of this process. CD40 ligation results in immediate redistribution of CD40 into cholesterol-rich detergent-insoluble fractions of the plasma membrane (21, 32), to which it rapidly recruits TRAF2 and TRAF3, followed by a FIGURE 6. Altered TRAF6 recruitment to the detergent-insoluble somewhat slower recruitment of TRAF6 (21, 54). The TRAF2/3 fraction of the plasma membrane by PG102. T5-1 human B cells were in- recruitment facilitates TRAF2-dependent K48-mediated poly- cubated, as in Materials and Methods, with control or hCD154-expressing insect cell (Hi5) membranes or PG102, as indicated, for 30 min. Cell lysates ubiquitination of these TRAFs, leading to their subsequent protea- were separated into detergent-soluble (Sol) and insoluble (In) fractions prior somal degradation (26–28). This degradation limits and prevents to Western blotting, as in Fig. 5. Cell fractions were blotted for TRAF6 and prolonged CD40 signaling (31, 55, 56). hCD40. A representative of three total experiments is shown. Thus, we examined each of these events in response to PG102 compared with agonistic stimuli. The results revealed that PG102 induced levels of TRAF2 and TRAF3 degradation that were in- (reviewed in Ref. 5); thus, they must be delivered at sufficiently distinguishable from those induced by an agonistic CD40 stimulus high concentration to outcompete CD154 binding. However, the in both human B cells and DCs (Fig. 4, Supplemental Fig. 2). nonagonistic mAb to hCD40 described in this report, PG102, does However, the amounts of TRAF2 and TRAF3 recruited to total Downloaded from not require or act by blocking CD154 binding. Importantly, cellular CD40 were decreased significantly in both a human B cell binding of hCD40 by PG102, although it does not remove hCD40 line and freshly isolated PBMCs from normal donors. This resulted from the cell surface, renders B cells refractory to subsequent in a concomitant decrease in PG102-induced CD40 binding of stimulation with CD154. This raises the attractive possibility that TRAF2-associated signaling proteins, including cIAP1, IKKg,and smaller amounts of PG102 can be therapeutically effective in the ubiquitination regulator HOIP.

inhibiting CD40-mediated immune activation in vivo, while also The latter findings are complex and intriguing. Reduced HOIP http://www.jimmunol.org/ avoiding any complications arising from ligation of CD154. and IKKg recruitment is consistent with the inability of PG102 to There is precedent for the concept that ligation of different stimulate CD40-mediated NF-kB activation. However, although epitopes on the CD40 molecule can induce distinct signaling cas- cIAP1 was reported to participate in CD40-mediated ubiquitination cades (50, 51), and even agonistic anti-CD40 Abs are not equivalent and subsequent degradation of TRAF2 and TRAF3, we found no to CD154 stimulation in all regards (52). The results demonstrate detectable impairment of this regulatory event following PG102 that strong binding to CD40, as is the case with PG102, does not ligation. Thus, although PG102 effectively stimulated events in- necessarily predict agonistic activity. The striking inability of PG102- volved in normal restraint of CD40 signaling, this mAb did not mediated CD40 engagement to trigger early signaling pathways in stimulate activation events. This suggests that the reduced recruit- immune cells is consistent with its reported inhibition of CD40- ment of TRAF2, cIAP1, and HOIP was nonetheless sufficient to by guest on September 27, 2021 mediated B cell effector functions (10, 11). This also correlates mediate TRAF2 and TRAF3 degradation downstream of PG102 with PG102-mediated inhibition of in vivo CD40-dependent pro- ligation and/or that additional unidentified CD40-binding mole- motion of inflammation and transplant rejection in animal models cules can also mediate these events. Recruitment of CD40 and (12–14) and early clinical human studies (15). In some regards, associated TRAF molecules to detergent-insoluble membrane this PG102-induced altered CD40 signaling resembles the “alterna- fractions was also altered when PG102 was used to engage hCD40

FIGURE 7. CD40 cross-linking require- ment for PG102 signaling. (A) CD40-medi- ated degradation of TRAF2 and TRAF3 was assessed in T5-1 human B cells, as in Fig. 4, comparing intact mAbs with equimolar amounts of PG102 Fab fragments, prepared as described in Materials and Methods. Relative quantification of amounts of TRAFs in cell lysates was determined from three ex- periments, as in Fig. 4. (B) PG102 inhibition of CD154-mediated JNK and IkBa phos- phorylation was measured in T51 human B cells, as in Fig. 3, comparing intact PG102 with Fab fragments, as in (A). Values rep- resent the mean 6 SD of three experiments, quantified as in Fig. 3. *p , 0.05. ns, not significant. 8 ALTERED CD40 SIGNALING COMPLEX INDUCED BY AN ANTAGONIST mAb compared with CD154. Membrane subdomain redistribution of all 7. Larsen, C. P., E. T. Elwood, D. Z. Alexander, S. C. Ritchie, R. Hendrix, C. Tucker-Burden, H. R. Cho, A. Aruffo, D. Hollenbaugh, P. S. Linsley, et al. of these molecules was reduced in PG102-stimulated B cells, 1996. Long-term acceptance of skin and cardiac allografts after blocking CD40 despite robust binding of PG102 to CD40, as demonstrated by and CD28 pathways. Nature 381: 434–438. CD40 staining and immunoprecipitation by PG102. This suggests 8. Kawai, T., D. Andrews, R. B. Colvin, D. H. Sachs, and A. B. Cosimi. 2000. Thromboembolic complications after treatment with mAb against CD40L. Nat. that membrane recruitment to lipid-rich subdomains is of partic- Med. 6: 114–120. ular importance to initiate CD40-mediated activation signals. It is 9. de Boer, M., L. Conroy, H. Y. Min, and J. Kwekkeboom. 1992. Generation of again possible that this involves facilitation of access of CD40 to mAbs to human lymphocyte cell surface using insect cells expressing recombinant proteins. J. Immunol. Methods 152: 15–23. signaling proteins localized in these subdomains that have not yet 10. Kwekkeboom, J., M. De Boer, J. M. Tager, and C. De Groot. 1993. CD40 plays been identified. Although it was shown previously that CD40- an essential role in the activation of human B cells by murine EL4B5 cells. mediated TRAF6 recruitment to detergent-insoluble membrane Immunology 79: 439–444. 11. Kwekkeboom, J., D. de Rijk, A. Kasran, S. Barcy, C. de Groot, and M. de Boer. fractions of fibroblast cell lines impacts TRAF2-dependent CD40 1994. Helper effector function of human T cells stimulated by anti-CD3 mAb signals to these cells (57), in B cells, CD40 molecules that cannot can be enhanced by co-stimulatory signals and is partially dependent on CD40- bind TRAF6 still induce TRAF2-dependent signals (30), sug- CD40 ligand interaction. Eur. J. Immunol. 24: 508–517. 12. Boon, L., H. P. M. Brok, J. Bauer, A. Ortiz-Buijsse, M. M. Schellekens, gesting cell-type specificity in some aspects of CD40 signaling. S. Ramdien-Murli, E. Blezer, M. van Meurs, J. L. Ceuppens, M. de Boer, et al. PG102-induced TRAF2 degradation was not greater than that 2001. Prevention of EAE in the common marmoset (Callithrix jacchus) using a chimeric antagonist mAb against human CD40 is associated with altered B cell induced by agonistic CD40 signals (Fig. 4), also indicating that responses. J. Immunol. 167: 2942–2949. reduced PG102-mediated TRAF6 recruitment to lipid rafts was 13. Haanstra, K. G., J. Ringers, E. A. Sick, S. Ramdien-Murli, E.-M. Kuhn, L. Boon, not due to increased TRAF2 degradation. and M. Jonker. 2003. Prevention of kidney allograft rejection using anti-CD40 and anti-CD86 in primates. Transplantation 75: 637–643. The present study identified key characteristics of the mecha- 14. Haegel-Kronenberger, H., K. Haanstra, C. Ziller-Remy, A. P. Ortiz Buijsse, Downloaded from nism by which PG102 interacts with CD40 and its signaling J. Vermeiren, F. Stoeckel, S. W. Van Gool, J. L. Ceuppens, M. Mehtali, M. De complex. PG102 did not exert its inhibitory functions by competing Boer, et al. 2004. Inhibition of costimulation allows for repeated systemic ad- ministration of adenoviral vector in rhesus monkeys. Gene Ther. 11: 241–252. with CD154; instead, it induced a CD154-refractory state of early 15. Kasran, A., L. Boon, C. H. Wortel, R. A. Hogezand, S. Schreiber, E. Goldin, signaling pathways in B cells, features that enhance its potential to M. Boer, K. Geboes, P. Rutgeerts, and J. L. Ceuppens. 2005. Safety and toler- inhibit CD40 signaling effectively without requiring high con- ability of antagonist anti-human CD40 Mab ch5D12 in patients with moderate to severe Crohn’s disease. Aliment. Pharmacol. Ther. 22: 111–122. centrations and without the risk for inducing complications related 16. Boon, L., J. D. Laman, A. Ortiz-Buijsse, M. T. den Hartog, S. Hoffenberg, P. Liu, http://www.jimmunol.org/ to binding to CD154. The altered signaling complex formation and F. Shiau, and M. de Boer. 2002. Preclinical assessment of anti-CD40 Mab 5D12 in cynomolgus monkeys. Toxicology 174: 53–65. distribution induced by PG102 can provide clues and predictive 17. Krangel, M. S., H. T. Orr, and J. L. Strominger. 1979. Assembly and maturation value as biomarkers for further development of effective thera- of HLA-A and HLA-B antigens in vivo. Cell 18: 979–991. peutic reagents to block CD40 signaling. 18. Clark, E. A., and J. A. Ledbetter. 1986. Activation of human B cells mediated through two distinct cell surface differentiation antigens, Bp35 and Bp50. Proc. There are numerous clinical conditions for which inhibiting Natl. Acad. Sci. USA 83: 4494–4498. CD40 signaling has considerable potential benefit, as discussed 19. Imai, A., T. Suzuki, A. Sugitani, T. Itoh, S. Ueki, T. Aoyagi, K. Yamashita, above. In addition, agonist binding to CD40 inhibits the survival M. Taniguchi, N. Takahashi, T. Miura, et al. 2007. A novel fully human anti- CD40 mAb, 4D11, for kidney transplantation in cynomolgus monkeys. Trans- and growth of malignant cells of various types (reviewed in Ref. plant. 84: 1020–1028. 43). Recently, evidence was presented for an important role for 20. Peters, A. L., R. M. Plenge, R. R. Graham, D. M. Altshuler, K. L. Moser, by guest on September 27, 2021 CD40 as an inhibitor of pathogenic T cell function in vis- P. M. Gaffney, and G. A. Bishop. 2008. A novel polymorphism of the human CD40 receptor with enhanced function. Blood 112: 1863–1871. ceral adipose tissue in diet-induced obesity (58). The ability of 21. Hostager, B. S., I. M. Catlett, and G. A. Bishop. 2000. Recruitment of CD40, PG102 to induce negative regulatory events downstream of CD40, TRAF2 and TRAF3 to membrane microdomains during CD40 signaling. J. Biol. Chem. 275: 15392–15398. such as TRAF degradation, may allow this reagent to trigger 22. Rowland, S. R., M. L. Tremblay, J. M. Ellison, L. L. Stunz, G. A. Bishop, and clinically desirable inhibitory pathways without the danger of B. S. Hostager. 2007. A novel mechanism for TRAF6-dependent CD40 signal- unintended immune activation. This will be an exciting possibility ing. J. Immunol. 179: 4645–4653. 23. Kehry, M. R. 1996. CD40-mediated signaling in B cells. Balancing cell survival, for future investigation. growth, and death. J. Immunol. 156: 2345–2348. 24. Bishop, G. A., and B. S. Hostager. 2001. Molecular mechanisms of CD40 sig- naling. Arch. Immunol. Ther. Exp. (Warsz.) 49: 129–137. Acknowledgments 25. Peters, A. L., and G. A. Bishop. 2010. Differential TRAF3 utilization by a variant We thank Dr. Bruce Hostager for valuable discussion and critical review human CD40 receptor with enhanced signaling. J. Immunol. 185: 6555–6562. of the manuscript. 26. Brown, K. D., B. S. Hostager, and G. A. Bishop. 2001. Differential signaling and receptor-associated factor (TRAF) degradation mediated by CD40 and the Epstein-Barr virus oncoprotein latent membrane protein 1 Disclosures (LMP1). J. Exp. Med. 193: 943–954. 27. Brown, K. D., B. S. Hostager, and G. A. Bishop. 2002. Regulation of TRAF2 M.d.B. and M.T. are employees of Fast Forward Pharmaceuticals, which signaling by self-induced degradation. J. Biol. Chem. 277: 19433–19438. owns the PG102 mAb. G.A.B. has consultant arrangements with, and has 28. Moore, C. R., and G. A. Bishop. 2005. Differential regulation of CD40-mediated received payments for travel expenses from, Fast Forward Pharmaceuticals. TRAF degradation in B lymphocytes. J. Immunol. 175: 3780–3789. The other authors have no financial conflicts of interest. 29. Bishop, G. A., C. R. Moore, P. Xie, L. L. Stunz, and Z. J. Kraus. 2007. TRAF proteins in CD40 signaling. Adv. Exp. Med. Biol. 597: 131–151. 30. Hostager, B. S. 2007. Roles of TRAF6 in CD40 signaling. Immunol. Res. 39: 105–114. 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46. Larsen, C. P., D. Z. Alexander, D. Hollenbaugh, E. T. Elwood, S. C. Ritchie, 58. Yi, Z., L. L. Stunz, and G. A. Bishop. 2014. CD40-mediated maintenance of immune http://www.jimmunol.org/ A. Aruffo, R. Hendrix, and T. C. Pearson. 1996. CD40-gp39 interactions play homeostasis in the adipose tissue microenvironment. Diabetes 63: 2751–2760. by guest on September 27, 2021