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Anti-IgG1 Hinge Autoantibodies Reconstitute the Effector Functions of Proteolytically Inactivated IgGs

This information is current as Randall J. Brezski, Jennifer L. Luongo, Diane Petrone, Mary of September 29, 2021. H. Ryan, Degang Zhong, Susan H. Tam, Albert P. Schmidt, Marian Kruszynski, Brian P. Whitaker, David M. Knight and Robert E. Jordan J Immunol 2008; 181:3183-3192; ;

doi: 10.4049/jimmunol.181.5.3183 Downloaded from http://www.jimmunol.org/content/181/5/3183

References This article cites 58 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/181/5/3183.full#ref-list-1 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 © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Human Anti-IgG1 Hinge Autoantibodies Reconstitute the Effector Functions of Proteolytically Inactivated IgGs1

Randall J. Brezski,2 Jennifer L. Luongo, Diane Petrone, Mary H. Ryan, Degang Zhong, Susan H. Tam, Albert P. Schmidt, Marian Kruszynski, Brian P. Whitaker, David M. Knight, and Robert E. Jordan

A number of of potential importance to human physiology possess the ability to selectively degrade and inactivate ؅ Igs. Proteolytic cleavage within and near the hinge domain of human IgG1 yielded products including Fab and F(ab )2 possessing full Ag binding capability but absent several functions needed for immune destruction of cellular pathogens. In ؅ parallel experiments, we showed that the same proteolytically generated Fabs and F(ab )2s become self-Ags that were widely ؅ recognized by autoantibodies in the human population. Binding analyses using various Fab and F(ab )2, as well as single- chain analogues, indicated that the autoantibodies targeted the newly exposed sequences where proteases cleave the Downloaded from hinge. The point of cleavage may be less of a determinant for autoantibody binding than the exposure of an otherwise cryptic stretch of hinge sequence. It was noted that the autoantibodies possessed an unusually high proportion of the IgG3 isotype in contrast to Abs induced against foreign immunogens in the same human subjects. In light of the recognized potency of IgG3 effector mechanisms, we adopted a functional approach to determine whether human anti-hinge (HAH) autoantibodies ؅ could reconstitute the (missing) Fc region effector functions to Fab and F(ab )2. Indeed, in in vitro cellular assays, purified /؅ http://www.jimmunol.org HAH autoantibodies restored effector functions to F(ab )2 in both Ab-dependent cellular cytotoxicity and complement- dependent cytotoxicity assays. The results indicate that HAH autoantibodies selectively bind to proteolytically cleaved IgGs and can thereby provide a surrogate Fc domain to reconstitute lytic functions. The Journal of Immunology, 2008, 181: 3183–3192.

he Fab and Fc regions of Igs are attributed with resistance have been shown to possess strict selectivity for mammalian to under most physiological conditions. How- IgGs (8–10) The IgG-degrading of Streptococcus pyo- T ever, our group and others have shown that several pro- (IdeS) specifically cleaves human IgG1 between teases secreted or expressed in environments where Igs are present 236 and glycine 237 (EU numbering) (11) in the lower hinge (1, by guest on September 29, 2021 can cleave Igs in the hinge region, resulting in a number of cleav- 9, 12). IdeS-mediated cleavage of Igs bound to the surface of S. Ј age products, including Fab or F(ab )2 (1–3). These fragments re- pyogenes inhibited killing of the by (1, tain the ability to bind to their respective epitopes but lose Fc- 2). Numerous mucosal pathogens such as Streptococcus san- mediated effector functions that are dependent on Fc domain guis, Haemophilus influenza, and Neisseria meningitides ex- interactions with Fc receptors, complement, etc. Pathogenic cell- press proteases that cleave IgA1 in the hinge region, blocking associated proteolytic cleavage of Igs has thereby been proposed its effector functions. Microbial proteases that cleave IgM have as a potential mechanism for evasion of host immune surveillance also been identified (13, 14). Collectively, these investigations (2, 4, 5). suggested that extracellular proteolysis of host IgGs might rep- Proteases have been associated with tumor invasion, inflamma- resent a pathogenic tactic for avoidance of host humoral im- tion, and metastasis (6, 7), as well as with bacterial . The mune functions. Our group recently showed that a broad group matrix (MMPs)3 matrilysin (MMP-7) and of human proteases including MMP-3, G, , stromelysin-1 (MMP-3) were reported to cleave all subclasses of and human (HNE), as well as GluV8 from human IgG in a step-wise process (4). Certain bacterial proteases , and IdeS all catalyzed specific in vitro cleavages of the human IgG hinge (1). Proteases present at sites of inflammation, , and tu- Discovery Research, Centocor R&D Inc., Radnor, PA 19087 mor environments (15, 16) that are capable of cleaving Igs may Received for publication March 5, 2008. Accepted for publication June 20, 2008. expose epitopes in the hinge region that would be hidden in The costs of publication of this article were defrayed in part by the payment of page intact Igs. Such cryptic epitopes could allow for activation of charges. This article must therefore be hereby marked advertisement in accordance anti-hinge autoreactive B cells. Autoreactive B cells are thought with 18 U.S.C. Section 1734 solely to indicate this fact. to be eliminated from the repertoire by three processes, namely, 1 This work was supported by a post-doctoral fellowship grant from Johnson & John- anergy, editing, or deletion (17–19). Yet, healthy in- son Corporate Office of Science and Technology (to R.J.B.). dividuals have circulating B cells with specificity for autoanti- 2 Address correspondence and reprint requests to Centocor R&D Inc., 145 King of Prussia Road, Radnor, PA 19087. E-mail address: [email protected] gens (20, 21). Autoantibodies exist that recognize different do- 3 Abbreviations used in this paper: MMP, matrix ; IdeS, IgG-de- mains of Igs, such as the Fc domain-binding rheumatoid factor grading enzyme of ; HNE, human ; ADCC, (22). There is also evidence for autoantibodies that recognize Ab-dependent cellular cytotoxicity; CDC, complement-dependent cytotoxicity; HAH, proteolytically cleaved Igs in the form of Fab and F(abЈ) (23– human anti-hinge. 2 25). However, most earlier anti-fragment autoantibody studies Ј Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 used Fab and F(ab )2 generated with and (26, 27),

www.jimmunol.org 3184 ANTI-HINGE AUTOANTIBODIES AGAINST IgG1 FRAGMENTS proteases of questionable relevance for most human tissue and ELISA disease. Many of these studies did not indicate whether the ELISAs for human Ig binding to proteolytic fragments of mAbs were Ј anti-Fab and anti-F(ab )2 autoantibodies were specific for vari- adapted from the format previously described (1). In brief, mAb, Fab, and Ј ␮ able, hinge, or Fab constant regions. In one study, it was sug- F(ab )2 were coated on 96-well plates at 10 g/ml. Single donor serum and gested that these autoantibodies function to negatively regulate pooled healthy donor serum were incubated on Ag-coated wells beginning Ag-engaged B cells by binding to Fc␥RIIb and inducing B cell at 1/50 dilutions. Detection of bound human Abs was by isotype-specific, HRP-conjugated secondary Abs; anti-IgG1 mAb (Zymed; 1/3200), anti- apoptosis (28). IgG2 mAb (Zymed; 1/1000), anti-IgG3 mAb (Zymed; 1/3000), anti-IgG4 The presence of autoantibodies to proteolytically cleaved IgG1 mAb (Sanquin; 1/10,000), anti-IgM mAb (Zymed; 1/1200), anti-IgE mAb was also documented in human clinical trials or preclinical testing (Nordic Immunol.; 1/600), goat anti-IgA (Zymed; 1/1000), and anti-IgD using therapeutic monoclonal Fab or F(abЈ) , respectively (23, 29, mAb (Nordic Immunol.; 1/24,000). Isotype standards of IgG1, IgG2, IgG3, 2 IgG4, IgM, IgE, and IgD were from myeloma serum and were obtained 30). In five human subjects treated with a human/murine chimeric from Athens Research & Technology. Human serum-derived IgA was ob- Fab (papain-generated), serum anti-human hinge reactivity was tained from Jackson ImmunoResearch Laboratories. Isotype standards observed to increase soon after administration. The timing of the were directly coated in microtiter wells at 0.1 ␮g/ml to serve both as spec- increased immune reactivity was suggestive of a memory recall ificity controls and as reactivity standards against which to normalize hu- response (23), since immune responses to the fully murine version man serum IgG binding. The plates were developed using SIGMAFAST OPD (Sigma-Aldrich) and stopped by acidification with HCl. of the Ab in other subjects usually did not appear until 4–6 wk ELISAs for detection of IgG3 binding to peptide analogues of the hu- after administration. In a preclinical study with a different human- man IgG1 hinge were performed as follows. Microtiter plate wells were ized Ab to the same glycoprotein IIb/IIIa (␣IIb␤3) target, treat- precoated with 10 ␮g/ml streptavidin (Pierce) overnight at 4°C. With the ment of cynomolgus monkeys with a pepsin-generated F(abЈ) re- exception of the omission of from the blocking buffer, the washing Downloaded from 2 and detection steps were otherwise as previously described for the frag- sulted in an acute decrease in the platelet count that was attributed ment ELISA (1). were added to replicate wells at 1 ␮g/ml in a Ј to host anti-F(ab )2 (30). To our knowledge, clinical or animal volume of 50 ␮l. Pooled human serum was added to microtiter wells at a Ј studies have not been conducted using cell-targeting Fab or F(ab )2 dilution of 1/50 in PBS (pH 7.4) containing 3% BSA. Experiments with generated by more physiologically relevant proteases, such as IdeS IVIg were conducted at 2 mg/ml. Detection of bound Abs used an anti- or the MMPs. IgG3-specific secondary as described above.

Since multiple proteases involved with infection, inflammation, Peptides http://www.jimmunol.org/ Ј and tumor environments can generate Fab and F(ab )2 that lack Fc-mediated effector function, we hypothesize that human anti- The 14-mer hinge peptides were synthesized on a Rainin SMPS-110 using hinge (HAH) autoantibodies counteract proteolytic cleavage of standard Fmoc protocols for HBTU activation. Acetyl-Lys(Fmoc)-OH (Bachem) was coupled as the N-terminal residue and, after Fmoc depro- IgGs by providing a surrogate Fc domain. We document the ex- tection, biotinylated with N-(Biotinyloxy) succinimide (Bachem). Fmoc- istence of anti-hinge autoantibodies in healthy human donors that Glu(biotinyl-PEG3)-OH (a spacer of three ethylenoxy units (PEG3)in- are specific for the hinge region of IgG1. To study the effect of serted between the biotin and the ␥-carboxy group of glutamic ; these autoantibodies in vitro, we affinity purified HAH autoanti- NovaBiochem) was coupled to Rink resin. Peptides were deprotected and cleaved from resin by acidolysis using trifluoroacetic acid cleavage mixture bodies from pooled human IgG preparations (IVIg). HAH autoan- then purified by preparative reversed-phase HPLC as previously described tibodies were tested in both Ab-dependent cellular cytotoxicity (32). Two sets of peptides were prepared: Acetyl-Lys(biotin)-(hinge-14- by guest on September 29, 2021 (ADCC) and complement-dependent cytotoxicity (CDC) assays mer peptide)-␣-acid and ␣-amino-(hinge-14-mer peptide)-Glu(biotinyl- and were shown to restore both functions to cell-bound F(abЈ) at PEG3)-amide. Analytical reversed-phase HPLC and capillary electrophore- 2 Ͼ Ab concentrations that correspond to in vivo levels. sis showed a high degree ( 97%) of peptide homogeneity (data not shown). Surface-enhanced laser desorption-ionization and matrix-assisted laser desorption-ionization mass spectrometry gave the expected molecular masses. Materials and Methods Human materials Proteases and IgG digestions Ј Human donor blood cells for use in ADCC assays were collected from Proteolytic used to generate Fab and F(ab )2 of mAbs were pro- employee volunteers at Centocor with all necessary permissions and ap- duced or obtained as previously described (1). Proteolytic digestions of provals. The protocol and informed consent form, as well as any relevant mAbs were performed at physiological pH and ionic strength as previously study-related documentation, were approved by a third party Institutional described (1). IgG cleavage was assessed by Agilent Biosizing microcap- Review Board. Healthy human serum samples were commercially obtained illary electrophoresis in SDS denaturing solution without reduction of from Bioreclamation and used as individual test samples or as pools. disulfide bonds (31). Purification of mAb fragments used A ad- Ј sorption steps and yielded Fab and F(ab )2 without detectable levels of Fc-containing materials. Designations of individual purified fragments in- Isolation of HAH autoantibody clude a subscript to denote the responsible for the cleavage (e.g., Autoantibodies specific for the hinge region of IgG were affinity purified Ј F(ab )2 MMP-3). from IVIg (Octapharma AB). In brief, mAb3 was digested with either Ј 0.02% IdeS (Genovis) or GluV8, and the resultant F(ab )2 of mAb3 were purified from the Fc fragment over Mabselect Protein A (GE Healthcare). Ј Abs and protein reagents A total of 200 mg of each mAb3 F(ab )2 was coupled to NHS-activated Sepharose 4 Fast Flow (GE Healthcare) and 5g of IVIg was applied to the mAbs included fully human, recombinant humanized Abs or human/mu- Ј Sepharose-coupled mAb3 F(ab )2. Bound Ab was eluted with 0.1 M gly- rine chimeric Abs possessing human constant domains and hinge regions. cine (pH 2.5), and the pH was neutralized with 2 M Tris (pH 7.0) (1/10 The mAb1 (anti-viral Ag) is a human IgG1. The mAb2 (anti-human cy- volume). Cross-reactive anti-whole IgG Abs were removed by further tokine) and mAb3 (anti-human CD41/CD61) are human/murine chimeric chromatography on 40 mg of intact mAb3 coupled to NHS-activated IgG1s possessing human constant regions and hinge domains. The mAb4 Sepharose 4 Fast Flow. The unbound flow through on the IgG column was (anti-human CD142) is a CDR-grafted humanized IgG1. The mAb5 (anti- termed the HAH autoantibody. human CD20), a chimeric IgG1, is a product of Genentech. The mAbs contained ␬ L chains. The above mAbs were used as substrates for pro- ADCC assay Ј teolytic to Fab and F(ab )2. IgG concentrated from large pools of human plasma (IVIg) was a product of Octapharma AB. ADCC assays were performed as previously described (33). In brief, EBV Ag was obtained from BiosPacific. was from Af- PBMCs were purified from human blood and used as effector cells for finity BioReagents. Tetanus toxoid was a product of List Biological Lab- ADCC assays. MDA-MB-231 human breast carcinoma cells (ATCC) were oratories. was obtained from Sigma-Aldrich. used as target cells with a ratio of 1 target cell to 50 effector cells. Target The Journal of Immunology 3185

separate plates, and CDC was measured with LDH cytotoxicity detection A B kit (Roche). Absorbance was measured using a Spectra max Plus 384 150 (PerkinElmer). Data were normalized to maximal cytotoxicity with Triton 50 125 X-100 (Sigma-Aldrich) and minimal control containing only cells and complement alone. Data were fit to a sigmoidal dose-response model using 100 30 GraphPad Prism v5. EC50 values were calculated from the 4-parameter 75 regression analyses.

10 50 25 Statistics % Isotype Standard % Isotype Standard -10 0 Statistics for Fig. 1A were determined as follows. Eight isotypes were A 2 IgE g IgG1 IgG2 IgG3 IgG4 IgM I IgD IgG1 IgG IgG3 IgG4 measured in four donors and compared with controls on 6 days. On each day, three replicates from each donor were tested. Repeated measures FIGURE 1. Ј ELISA detection of human Ig binding to mAb1 F(ab )2 gen- ANOVA was conducted on data expressed as percent of the isotype erated with IdeS. A, The detection of distinct isotypes from serum of four control. Three repeated effects were included in the model: isotype, day, healthy donors using specific secondary Abs. The assay signals were nor- and replicate within day. Pairwise comparisons between the isotypes malized to standard wells containing directly coated isotype standards and were evaluated. Adjustments for multiple comparisons were made using were calculated as the percent of isotype standard. Determinations were the Tukey-Kramer method. Adjusted p values of 0.05 or less were con- performed in triplicate in six independent experiments and the results ex- sidered significant. pressed as the mean Ϯ SEM. B, Binding of IgG isotypes from the pooled serum of 20 healthy donors (f) and from IVIg (Ⅺ). Bar heights correspond

to the mean percent of isotype standard Ϯ SEM from six replicate analyses. Results Downloaded from Human serum autoantibodies bind to the hinge region of proteolytically cleaved IgG1 cells were pre-labeled with BAPTA (PerkinElmer) for 20 min at 37°C, washed twice, and resuspended in DMEM, 5% heat-inactivated FBS, 0.1 The major goal of this study was to establish functional properties mM nonessential amino , 1 mM sodium pyruvate, penicillin (500 for the HAH autoantibodies that recognize proteolytically exposed L Ј U/ml), streptomycin (500 U/ml), and 2 mM -glutamine (all from Invitro- epitopes in IgG. We generated a panel of purified Fab and F(ab )2 4 6 gen). Target (1 ϫ 10 cells) and effector cells (0.5 ϫ 10 cells) were from mAbs as Ags for autoantibody binding. The mAbs included http://www.jimmunol.org/ combined, and 100 ␮l of cells were added to the wells of 96-well U-bottom plates. An additional 100 ␮l was added with or without Abs. All samples a human IgG1, as well as humanized and human/murine chimeric were performed in triplicate. The plates were centrifuged at 200 g for 3 Abs – each possessing the same human IgG1 hinge and constant Ј min, incubated at 37°C for 2 h, and then centrifuged again at 200 g for 3 region sequences. A F(ab )2 was generated from hu- min. A total of 20 ␮l of supernatant was removed per well, and cell man IgG1␬ mAb1, using IdeS as previously described (1). Fig. 1 ␮ was measured by the addition of 200 l of the DELPHIA Europium-based depicts the ELISA binding reactivity in diluted serum from a small reagent (PerkinElmer). Fluorescence was measured using an Envision 2101 ϭ Multilabel Reader (PerkinElmer). Data were normalized to maximal cyto- test group (n 4) of individual healthy donors to this mAb1 F(abЈ) . Fig. 1A shows that reactivity was found within the IgG1, toxicity with 0.67% Triton X-100 (Sigma-Aldrich) and minimal control 2 IdeS determined by spontaneous release of BAPTA from target cells in the IgG2, IgG3, IgM, and IgA isotypes, whereas no anti-fragment re- by guest on September 29, 2021 absence of any Ab. Data were fit to a sigmoidal dose-response model using activity was detected for IgG4, IgE, or IgD. IgA and IgM anti- GraphPad Prism v5. EC50 values were calculated from the 4-parameter regression analyses. fragment autoantibodies have been reported previously (34). The remaining studies focused on IgG isotypes since pooled human CDC assay IgG (e.g., IVIg) was used extensively for characterization and as a source of autoantibodies. WIL2-S cells (ATCC) were used as target cells for CDC assays. A total of Fig. 1B depicts the average binding of IgG isotypes to mAb1 50 ␮l of cells were added to the wells of 96-well plates for a final con- 4 F(abЈ) from pooled serum from 20 healthy donors, as well as centration of 8 ϫ 10 cells per well in RPMI, 5% heat-inactivated FBS, 0.1 2 IdeS mM nonessential amino acids, 1 mM sodium pyruvate, penicillin (500 from IVIg (IgG purified from a plasma pool of Ͼ3500 donors). U/ml), streptomycin (500 U/ml), and 2 mM L-glutamine. An additional 50 The binding results for these two pools of increased size confirmed ␮l was added to the wells with or without Abs and the plates were incu- that among the IgG isotypes, reactivity was detectable with IgG1, bated at room temperature for 2 h. A total of 50 ␮l of 10% rabbit com- plement (Invitrogen) was added to the wells, and the plates were incubated IgG2, and IgG3, but not IgG4. The serum pool and IVIg contained for 20 min at 37°C. All samples were performed in triplicate. The plates detectable IgG1 and IgG2 reactivity, but IgG3 was the major com- ␮ Ј were centrifuged at 200 g for 3 min, 50 l of supernatant was removed to ponent among autoantibody reactivity to this human F(ab )2.

Table I. Comparisons of human serum Ab isotypes binding in ELISA to IgG fragments and exogenous Agsa

Anti-IgG1b Anti-IgG2 Anti-IgG3 Anti-IgG4

mAb2 F(abЈ)2 MMP-3 ϩϩϩϩϪ mAb2 F(abЈ)2 MMP-12 ϪϪϩϩϪ mAb2 F(abЈ)2 Glu. Endopep. 1 ϩϩ Ϫ ϩϩ Ϫ mAb2 F(abЈ)2 Pepsin ϪϪϩϩϪ mAb2 F(abЈ)2 IdeS ϩϩ ϩϩ ϩϩϩϩ Ϫ Staphlokinase ϩϩ ϩ ϩ ϩ Streptokinase ϩϩϩϩϩϩ ϩϩϩϩϩϩ ϩ Ϫ EBV Capsid Ag ϩϩϩϩ ϩ ϩ Ϫ Tetanus Toxiod ϩϩϩϩϩϩ ϩϩϩ ϩ ϩϩϩϩϩ

a Pooled serum from 20 healthy donors was tested at serial 2-fold dilutions starting at 1/50. The IgG fragments, staphy- lokinase and EBV were coated at 10 ␮g/ml; streptokinase and tetanus toxoid were coated at 4 ␮g/ml. b The reactivity was assigned based on the highest dilution at which the serum pool resulted in 10% of the OD of the directly coated isotype control . —, No activity; ϩ, Assigned from one to six in correspondence with positive readings at 1/50, 1/100, 1/200, 1/400, 1/800, and 1/1600, respectively. 3186 ANTI-HINGE AUTOANTIBODIES AGAINST IgG1 FRAGMENTS

The content of IgG3 within HAH autoantibodies is A disproportionate compared with the isotype profiles of induced 3.0 immune responses against foreign Ags The isotype profile of HAH autoantibodies was compared with 2.5 human immune responses elicited to three commonly encountered exogenous immunogens; EBV capsid Ag, streptokinase, and 2.0 staphylokinase, as well as tetanus toxoid. The isotype distribution

1.5 toward these Ags was investigated in pooled serum from 20 healthy donors using a serial dilution-based criterion to compare

Optical Density Optical 1.0 reactivity. ELISA results in Table I point to IgG1 and IgG2 as the primary isotypes directed to exogenous Ags, as well as IgG4 in the 0.5 case of tetanus toxoid. IgG3 was the weakest among the isotypes detected against these “non-self” Ags. In contrast, autoantibody 0.0 reactivity to various F(abЈ) s of mAb2, generated by cleavage with S G G 2 V8 sin de g luV8 lu I 5 IgG 2 b five different proteases, MMP-3, MMP-12, GluV8, pepsin, and 2 G G 2 Pep Plasmin b 4 b Ab3 IgGAb4 I A mAb3 HNE A mAb mAb3mAb5 IdeS IdeS mAb2m Ig m m m IdeS, indicated a bias toward the IgG3 isotype. The remaining mAb3 Papain mAb2mAb4 MMP-3 MMP-3mA mAb mAb3 mAb2 MMP-12 experiments focused exclusively on IgG3; a departure from earlier B studies that did not report a specific isotype (35, 36) or that con- Downloaded from 1.4 centrated on other isotypes such as serum IgA (34). 1.2 To further test whether IgG3 HAH autoantibodies were selec- tive for the cleaved IgG hinge rather than to other elements of Ab 1.0 Ј structure, we used a group of different mAb Fab and F(ab )2 frag- 0.8 ments as Ags, as well as their intact IgG1 parents. The fragments 0.6 were produced by proteases catalyzing cleavage at the peptide http://www.jimmunol.org/ 0.4 bonds indicated in Fig. 2C. The purity and molecular size of rep- Optical Density Optical resentative proteolytically generated mAb fragments for ELISA 0.2 were confirmed by bio-sizing as shown in Fig. 2D. Levels of IgG3 0.0 anti-fragment reactivity above background were evident in indi- G G IdeS Ig ϭ luV8 luV8 3 IdeS vidual healthy donor sera (n 20) toward many of the human G Ab5 mAb2 mAb3Ig mAb4IgG mAb5 IgG mAb3 HNE mAb2 mAIdeS m IgG1 fragments in the ELISA (Fig. 2A). Autoantibody recognition mAb2 mAb3 Papain mAb2 mAb4MMP-3 MMP-3mAb2mAb4 G mAb3 Plasmin mAb2 MMP-12 was evident against each of the three Fabs of mAb4 generated by C enzymatic cleavage in the upper hinge – by papain, HNE, and Upper Hinge Core Lower Hinge human plasmin. The highest incidence of IgG3 reactivity in the by guest on September 29, 2021

(Fab Region) Hinge (F(ab')2 Region) upper hinge was against the HNE-generated fragment. Ј S-C-D-K-T-H-T-C-P-P-C-P-A-P-E-L-L-G-G-P-S-V IgG3 HAH autoantibody reactivities to F(ab )2 of “human” 219 12 3 456 7 240 IgG1s were consistently elevated as also illustrated in Fig. 2A. 1 Plasmin Ј Anti-F(ab )2 reactivity was found against each of the mAbs, 2 Human Neutrophil Elastase (HNE) mAb2, mAb3, mAb4, and mAb5, and was independent of the pro- 3 Papain 4 MMP-3, MMP-12 teases used for digestion since MMP-3, MMP-12, GluV8, pepsin, 5 Glutamyl I (GluV8), and IdeS were each represented. The detection of IgG3 HAH au- 6 Pepsin, MMP-7 toantibody binding to intact versions of these four mAbs was at 7 Ides baseline for 19 of the 20 healthy donors, in distinct contrast to the D fragments. No comparable binding of IgG3 HAH autoantibodies was detected to the cleaved Fc domain of IgG1 mAbs (data not shown). These findings indicated that C-terminal cleavage sites of

proteolyzed IgG1 Abs were primary targets for binding by IgG3

Standard

mAb4 IdeS IdeS mAb4 mAb4 MMP-3 MMP-3 mAb4

mAb3 Papain mAb3

mAb4 IgG mAb4 mAb4 Plasmin Plasmin mAb4

mAb4 GluV8 GluV8 mAb4 Pepsin mAb2

mAb2 MMP-3 MMP-3 mAb2 mAb3 HNE HNE mAb3 mAb2 MMP-12 MMP-12 mAb2 240- HAH autoantibodies.

150- Large pools of human plasma-derived IgGs possess 95- anti-fragment autoantibody reactivity 63- In Fig. 2B, the investigation of IgG3 binding to mAbs and to pro- 28- teolytically generated fragments was extended to IVIg as the 15- source of HAH autoantibodies; the bar heights indicated the mean 4.5- reactivity in the pool of Ͼ3500 donors. The binding patterns to this FIGURE 2. Binding of IgG3 autoantibodies to four different intact mAb panel of IgG1 Ags were consistent with the results in the individ- Ј IgG1s as well as to Fab and F(ab )2 generated from them with various ual donors (Fig. 2A). This indicated that IgG3 HAH autoantibodies proteases. A, Binding of serum IgG3 from 20 individual healthy donors. Symbols represent the average ELISA signal obtained for each serum sam- ple in three replicate analyses, and the mean value for the 20 samples is indicated by the horizontal line. The specific fragments of IgGs with the spanning 219 to 240, and the positions of cleavage by indi- sites of proteolytic cleavage are indicated on the x-axis. B, Detection of vidual proteases are indicated (1). D, Agilent biosizing analysis of repre- Ј IgG3 autoantibody binding to the same group of IgGs and fragments from sentative proteolytically generated Fab and F(ab )2. Microcapillary elec- pooled human IVIg at 2 mg/ml. Bar heights correspond to the mean Ϯ trophoresis was performed under nonreducing conditions as previously SEM from three tests. C, Depiction of the sequence of the IgG1 H chain described (1). The Journal of Immunology 3187

1.0 tern of binding of IgG3 HAH autoantibodies to hinge peptide analogues with free C-termini was not paralleled in peptides 0.8 that encompassed the same range of amino acid sequence, but 0.6 constructed with free N-terminal amino acids. The latter series 0.4 was conjugated with biotin on the C terminus of the 14-mer peptide and was assayed in ELISA in similar fashion to the 0.2 Optical Density Optical previous group. IgG3 HAH autoantibody binding was minimal 0.0 or undetectable to the latter series of hinge analogues (A. D K T H T C P P C P A P E L L G G P S V F L F P P K P K D T L 221 251 Schmidt, unpublished data). Thus, the results indicated substan- C-Terminal Amino Acid in 14-mer linear sythetic biotinylated peptide tial IgG3 HAH autoantibody binding to the C-terminal ana- FIGURE 3. ELISA binding of IgG3 autoantibodies from pooled serum logues of IgG1 Fab and F(abЈ) , but no comparable reactivity to from 20 healthy donors to peptide analogues of the human IgG1 hinge. 2 the cleaved end analogues of free Fcs. Peptides were biotinylated 14-mers with free C-termini corresponding to the amino acid positions indicated on the axis. Determinations were per- formed in triplicate wells in three independent experiments. Bar heights HAH autoantibodies were isolated by affinity adsorption on Ј correspond to the mean of the three experiments Ϯ SEM. proteolytically generated F(ab )2 IVIg was used as the source for the affinity purification of HAH Ј Ј autoantibodies. F(ab )2 IdeS or F(ab )2 GluV8 of mAb3 were sepa- against F(abЈ) , but not to intact IgG, were comparable in a much rately attached to affinity matrices, and 5 grams of human IVIg 2 Downloaded from larger healthy population. The results supported that the IgG3 re- Abs were applied to the respective columns. In each case, only a activity was specific for the cleavage-related epitopes in these small fraction of Abs was bound and then eluted under acidic mAbs and not to other nonhinge targets such as idiotypic deter- conditions (0.1M glycine (pH 2.5)). The pH-neutralized eluates Ј minants or constant region structures. Thus, proteolysis of IgG by from the F(ab )2 columns were further absorbed on a column of human and bacterial extracellular proteases exposed antigenic de- intact mAb3 IgG1 to minimize the presence of Abs to nonhinge terminants that were recognized by IgG3 HAH autoantibodies. determinants. In two replicate affinity chromatographies on the Ј http://www.jimmunol.org/ F(ab )2 IdeS matrix, the protein yields of HAH autoantibody The HAH autoantibody specificity to the IgG1 hinge was further IgGs were 0.037 and 0.032% of the total IgG applied to the defined using peptide analogues columns. The purified HAH autoantibody preparations were several hundred-fold enriched in IgG3 anti-hinge binding com- Because only seven positions were identified in the hinge as pro- pared with the starting IVIg based on ELISA reactivity (per tease cleavage sites, we wanted to determine whether other C- milligram total IgG; data not shown). This result supported the terminal amino acids within the hinge could be bound by autoan- previous findings that HAH Abs are enriched in IgG3 isotype. tibodies. In an attempt to address the positional gaps, a peptide analog approach was adopted. A series of advancing 14-mer pep-

Ј by guest on September 29, 2021 tide hinge analogues were synthesized that encompassed C-termi- HAH autoantibodies reconstituted ADCC to mAb F(ab )2 nal positions from 221 to leucine 251 (EU number- When tested in several cell-based assay systems, unfractionated ing) (11). Peptides were synthesized with an N-terminal Ac- IVIg contributed directly to cell killing independent of cell-bound ␧ Ј (N- -biotin) coupling to facilitate immobilization on streptavidin- F(ab )2. Thus, affinity-isolated HAH autoantibodies were tested for coated microtiter wells and to expose the C terminus of the pep- their ability to reconstitute Fc␥-mediated immune functions to Ј tide. Each consecutive C-terminal position within the hinge and cell-bound, effector-impaired F(ab )2. The mAb4 binds to CD142 adjoining constant region was represented in this single-chain pep- present on the plasma membrane of MDA-MB-231 cells (37). In- tide series. cubation of MDA-MB-231 cells with the intact IgG of mAb4 and Fig. 3 presents the ELISA results of pooled serum from 20 isolated human PBMCs resulted in mAb concentration-dependent healthy donors binding to the peptide series. A relatively low cell killing of the target cells in an ADCC assay (Fig. 4A). Nearly level of IgG3 HAH autoantibody reactivity was detected to the full cell killing was achieved at 200 ng/ml IgG, whereas no specific Ј upper hinge region encompassing aspartic acid 221 to cell lysis was evident with F(ab )2 of mAb4 generated with IdeS, 224; “DKTH.” There was no detectable HAH autoantibody GluV8, or MMP-3 at any concentration up to 36 ␮g/ml. A control binding to peptides with C-termini in and immediately flanking human IgG (mAb1) that was not specific for either MDA-MB-231 the core hinge (threonine 225 through 231, “TCP- cells, intact IgG, or any fragment of IgG did not restore ADCC Ј PCPA.” Substantial IgG3 anti-peptide reactivity was found for either alone or in combination with any of the mAb4 F(ab )2 (Fig. hinge analogues with C-termini at positions of the lower hinge 4B). However, a dose-dependent restoration of ADCC function from proline 232 to glycine 236 and the adjoining constant do- was observed when serial dilutions of HAH autoantibodies, puri- Ј main from glycine 237 to 241, “PELLGGPSVF.” fied by affinity chromatography on the F(ab )2 IdeS, were added to Ј Within this latter sequence were the C-terminal positions cor- a fixed 200 ng/ml of any of the three F(ab )2s (Fig. 4C). Maximum responding to protease cleavage sites in IgG1 by MMP-3 and restoration was observed at HAH autoantibody concentrations 1 ␮ MMP-12 (proline 232), by cathepsin G and GluV8 (glutamic g/ml and higher, and an EC50 was calculated for each reconsti- acid 233), by MMP-7 and pepsin (leucine 234), and IdeS (gly- tuted curve. A similar concentration-dependent pattern of restored cine 236). Similar “maps” of anti-IgG1-hinge-peptide-analog ADCC function was observed with the addition of the preparation Ј reactivity were established for individual donor serum and IVIg of HAH that had been affinity purified on F(ab )2 GluV8 to each of Ј (data not shown). Peptide analogues of IgG1 exhibited a peak the three F(ab )2 in contact with MDA-MB-231 cells (Fig. 4D). zone of Ag reactivity centered at the hinge/CH2 junction (also Additionally, using the opposite titration strategy, serial dilution of Ј the IdeS cleavage point) between glycine 236 and glycine 237. the respective F(ab )2 into a fixed HAH autoantibody concentra- However, some of the highly targeted peptides possessed C- tion of 5 ␮g/ml (the estimated physiological concentration), Ј Ј termini corresponding to putative F(ab )2s where no extracel- yielded EC50 values for F(ab )2 plus HAH autoantibodies that lular proteases are known to directly fragment IgG1. The pat- were similar to intact mAb4 IgG (Fig. 4, E and F). Addition of 3188 ANTI-HINGE AUTOANTIBODIES AGAINST IgG1 FRAGMENTS

A B 120 mAb4 IgG 100 mAb1

100 EC50 = 0.0029 µg/ml mAb4 F(ab')2 IdeS + mAb1 75 mAb4 F(ab')2 GluV8 + mAb1 80 mAb4 F(ab')2 IdeS 50 mAb4 F(ab')2 MMP-3 + mAb1 60 mAb4 F(ab')2 GluV8

40 mAb4 F(ab')2 MMP-3 25 20 % Maximal Lysis

% Maximal Lysis % Maximal 0 0

-20 -25 0.0001 0.001 0.01 0.1 1 10 100 0.001 0.01 0.1 1 10 100 [mAb4] (µg/ml) [mAb1] (µg/ml) C D 100 100 HAHIdeS HAH GluV8 mAb4 F(ab') + HAH 75 mAb4 F(ab')2 IdeS + HAHIdeS 75 2 IdeS GluV8 EC50 = 0.82 µg/ml EC50 = 0.24 µg/ml 50 50 mAb4 F(ab')2 GluV8 + HAH GluV8 mAb4 F(ab') + HAH 2 GluV8 IdeS EC50 = 0.88 µg/ml 25 EC50 = 0.28 µg/ml 25 mAb4 F(ab')2 MMP-3 + HAH GluV8 mAb4 F(ab') + HAH Downloaded from 2 MMP-3 IdeS EC50 = 1.2 µg/ml % Maximal Lysis 0 EC50 = 0.32 µg/ml Lysis % Maximal 0

-25 -25 0.001 0.01 0.1 1 10 100 0.001 0.01 0.1 1 10 100 µ µ [HAH IdeS ] ( g/ml) [HAH GluV8] ( g/ml) E F

100 100 http://www.jimmunol.org/ HAH IdeS HAH GluV8 mAb4 F(ab')2 IdeS + HAH IdeS F(ab')2 IdeS +HAH GluV8 75 75 EC50 = 0.0010 µg/ml EC50 = 0.0024 µg/ml mAb4 F(ab') + HAH F(ab') + HAH 50 2 GluV8 IdeS 50 2 GluV8 GluV8 EC50 = 0.0010 µg/ml EC50 = 0.0018 µg/ml

25 mAb4 F(ab')2 MMP-3 + HAH IdeS 25 F(ab')2 MMP-3 + HAH GluV8 % Maximal Lysis % Maximal % Maximal Lysis EC50 = 0.0008 µg/ml EC50 = 0.0008 µg/ml 0 0 0.0001 0.001 0.01 0.1 1 10 0.0001 0.001 0.01 0.1 1 10 µ µ [mAb4] ( g/ml) [mAb4] ( g/ml) by guest on September 29, 2021 Ј Ј FIGURE 4. Cell based ADCC assays of HAH affinity purified on either F(ab )2 IdeS (designated as HAHIdeS) or on F(ab )2 GluV8 (designated as

HAHGluV8). All assays were performed with human PBMCs from the blood of healthy donors and with the cell line MDA-MB-231 at a ratio of 50 PBMCs f Ј ‚ to 1 target cell. A, ADCC activity against MDA-MB-231 cells using intact mAb4 ( ) and F(ab )2 of mAb4 (anti-CD142) generated with IdeS ( ), GluV8 (ƒ), or MMP-3 (E). B, ADCC activity using an intact IgG (mAb1) nonspecific for MDA-MB-231 cells (f) or intact IgG of mAb1 added to a constant ␮ Ј ‚ ƒ E f concentration of 0.2 g/ml mAb4 F(ab )2 generated with IdeS ( ), GluV8 ( ), or MMP-3 ( ). C, ADCC activity using intact HAHIdeS alone ( )or ␮ Ј ‚ ƒ E HAHIdeS added to a constant concentration of 0.2 g/ml mAb4 F(ab )2 generated with IdeS ( ), GluV8 ( ), or MMP-3 ( ). D, ADCC activity using f ␮ Ј ‚ ƒ E HAHGluV8 alone ( )orHAHGluV8 added to a constant concentration of 0.2 g/ml mAb4 F(ab )2 generated with IdeS ( ), GluV8 ( ), or MMP-3 ( ). E, f ␮ Ј ‚ ADCC activity using HAHIdeS alone ( ) or a constant concentration of 5 g/ml HAHIdeS alone added to mAb4 F(ab )2 generated with IdeS ( ), GluV8 ƒ E f ␮ Ј ( ), or MMP-3 ( ). F, ADCC activity using HAHGluV8 alone ( ) or a constant concentration of 5 g/ml HAHGluV8 alone added to mAb4 F(ab )2 generated with IdeS (‚), GluV8 (ƒ), or MMP-3 (E). Bar heights correspond to the mean of triplicate wells Ϯ SD. The results are a representative of three independent experiments.

Ј Ј either HAH autoantibody preparation, in the absence of mAb4 sorption on mAb3 F(ab )2 IdeS or mAb3 F(ab )2 GluV8 reconsti- Ј F(ab )2 IdeS, was without effect. The results indicated that both tuted complement-mediated cell lysis to the three different Ј preparations of affinity-purified HAH autoantibodies reconstituted F(ab )2 of mAb5 with one exception (Fig. 5, C and D). HAH Ј Ј ADCC activity to low concentrations of F(ab )2 generated with isolated on mAb3 F(ab )2 GluV8 did not restore CDC to mAb5 IdeS, GluV8, and MMP-3. F(abЈ) . Higher concentrations of HAH were required for 2 IdeS CDC restoration compared with ADCC, and the reason for this Ј HAH autoantibodies reconstitute CDC to mAb F(ab )2 is uncertain. When the opposite titration strategy was performed Ј The affinity-isolated, HAH autoantibodies were tested for their abili- with serial dilution of the respective F(ab )2 into a fixed 5 ties to restore CDC function to F(abЈ) and F(abЈ) of ␮g/ml HAH autoantibody (purified on the IdeS-digested 2 IdeS 2 GluV8 Ј mAb5. The mAb5 IgG1 Ab binds to CD20 expressed on the human mAb3), restorations of CDC occurred at F(ab )2 concentrations B cell line WIL2-S and induces cell lysis in the presence of comple- that were within 2- to 3-fold of mAb5 IgG (Fig. 5E). Using Ј ment. Near maximum lysis of WIL2-S cells was obtained at ϳ1 HAH autoantibodies purified on mAb3 F(ab )2 GluV8, restora- ␮ Ј Ј g/ml mAb5 IgG. whereas none of the mAb5 F(ab )2 (generated tion was only seen with the mAb5 F(ab )2 GluV8 (Fig. 5F). De- with IdeS, GluV8, or MMP-3) demonstrated an effect at up to spite minor differences in concentration dependency and spec- 27 ␮g/ml (Fig. 5A). The nonspecific control, mAb1, had no ificity, ADCC and CDC independently provided supporting Ј activity alone or in combination with any of the F(ab )2 (Fig. evidence for HAH autoantibody-mediated restoration of effec- 5B). Addition of HAH autoantibodies isolated by affinity ad- tor functions to proteolytically cleaved IgGs. The Journal of Immunology 3189

A B 100 100 mAb5 IgG mAb1 80 mAb5 F(ab') + mAb1 EC50 = 0.029 µg/ml 80 2 IdeS mAb5 F(ab')2 GluV8 + mAb1 60 mAb5 F(ab')2 IdeS 60 mAb5 F(ab')2 MMP-3 + mAb1 mAb5 F(ab')2 GluV8 40 40 mAb5 F(ab')2 MMP-3 20 20 % Maximal Lysis % Maximal % Maximal Lysis 0 0

0.001 0.01 0.1 1 10 100 0.001 0.01 0.1 1 10 100 [mAb5] (µg/ml) [mAb1] (µg/ml) C D 100 100 HAH IdeS HAH GluV8 mAb5 F(ab') + 80 mAb5 F(ab')2 IdeS + HAH IdeS 80 2 IdeS HAH GluV8 mAb5 F(ab')2 GluV8 + HAH GluV8 60 EC50 = 2.5 µg/ml 60 EC50 = 6.7 µg/ml mAb5 F(ab')2 GluV8 + HAH IdeS 40 40 EC50 = 1.5 µg/ml mAb5 F(ab')2 MMP-3 + HAH GluV8 EC50 = 20.2 µg/ml

20 20 Downloaded from mAb5 F(ab')2 MMP-3 + HAH IdeS % Maximal Lysis % Maximal Lysis 0 EC50 = 4.8 µg/ml 0

0.001 0.01 0.1 1 10 100 0.001 0.01 0.1 1 10 100 µ µ [HAH IdeS ] ( g/ml) [HAH GluV8] ( g/ml) E F 100 100 HAH IdeS HAH GluV8 http://www.jimmunol.org/ 80 mAb5 F(ab')2 IdeS + HAH IdeS 80 mAb5 F(ab')2 IdeS + HAH GluV8 mAb5 F(ab')2 GluV8 + HAH GluV8 60 EC50 = 0.025 µg/ml 60 EC50 = 0.045 µg/ml mAb5 F(ab')2 GluV8 + HAH IdeS 40 40 EC50 = 0.052 µg/ml mAb5 F(ab')2 MMP-3 + HAHGluV8 20 20 IdeS % Maximal Lysis % Maximal mAb5 F(ab')2 MMP-3 + HAH % Maximal Lysis % Maximal 0 EC50 = 0.074 µg/ml 0

0.0001 0.001 0.01 0.1 1 10 0.0001 0.001 0.01 0.1 1 10 [mAb5] µg/ml [mAb5] µg/ml by guest on September 29, 2021 Ј Ј FIGURE 5. Cell based CDC assays of HAH affinity purified on either F(ab )2 IdeS (designated as HAHIdeS) or on F(ab )2 GluV8 (designated as HAHGluV8). All assays were performed with the WIL2- line as described in Materials and Methods. A, CDC activity against WIL2-S cells using intact mAb5 (f) Ј ‚ ƒ E and F(ab )2 of mAb5 (anti-CD20) generated with IdeS ( ), GluV8 ( ), or MMP-3 ( ). B, CDC activity using an intact IgG (mAb1) nonspecific for WIL2-S f ␮ Ј ‚ ƒ E cells ( ) or intact IgG of mAb1 added to a constant concentration of 1 g/ml mAb5 F(ab )2 generated with IdeS ( ), GluV8 ( ), or MMP-3 ( ). C, CDC f ␮ Ј ‚ ƒ activity using HAHIdeS alone ( )orHAHIdeS added to a constant concentration of 1 g/ml mAb5 F(ab )2 generated with IdeS ( ), GluV8 ( ), or MMP-3 E f ␮ Ј ‚ ( ). D, CDC activity using HAHGluV8 alone ( )orHAHGluV8 added to a constant concentration of 1 g/ml mAb5 F(ab )2 generated with IdeS ( ), GluV8 ƒ E f ␮ Ј ( ), or MMP-3 ( ). E, ADCC activity using HAHIdeS alone ( ) or a constant concentration of 5 g/ml HAHIdeS alone added to mAb5 F(ab )2 generated ‚ ƒ E f ␮ with IdeS ( ), GluV8 ( ), or MMP-3 ( ). F, ADCC activity using HAHGluV8 alone ( ) or a constant concentration of 5 g/ml HAHGluV8 alone added Ј ‚ ƒ E Ϯ to mAb5 F(ab )2 generated with IdeS ( ), GluV8 ( ), or MMP-3 ( ). Bar heights correspond to the mean of triplicate wells SD. The results are a representative of three independent experiments.

Discussion and pathogical conditions such as systemic lupus erythematosus We recently investigated a number of human and bacterial pro- (38), cold agglutination (39), HIV (40), and rheumatoid teases that catalyze specific cleavages of human IgGs (1). These (25). One group also suggested a potential immunoregulatory role physiologically relevant proteases targeted both the upper and for anti-hinge autoantibodies via induction of B cell apoptosis (28). Ј In any case, the high incidence of anti-fragment reactivity in var- lower IgG1 hinge to generate fragments including Fab and F(ab )2 and the separated Fc domain. Moreover, the in vitro fragmentation ious populations is consistent with sensitization to these “self” patterns in purified systems were analogous to ex vivo patterns of Ags, and we were prompted to explore their potential inter-rela- endogenous IgG breakdown in synovial fluid samples from human tionship with IgG proteolysis. subjects with rheumatoid arthritis. Related observations also led to We focused on autoantibody binding to mAb IgG1 fragments speculations that widespread, targeted fragmentation of host Igs in with a view toward characterizing functional consequences of au- pathologic, protease-rich environments could cause localized im- toantibody/fragment interactions. Specifically, the studies concen- Ј mune dysfunction (2, 4, 5). In the current study, we investigated a trated on autoantibody association with Fab and F(ab )2 since these potential mechanism by which the host might combat proteolytic fragments retain their abilities to bind to the specific Ags on cell challenges to IgG1-mediated effector functions. surfaces. By the use of human and “humanized” mAb substrates, Independent investigations identified the presence of human au- as well as peptide analogues, the present approach departed from toantibodies in many healthy individuals directed against cleavage the majority of earlier investigations that focused on polyclonal, Ј site epitopes in Fab and F(ab )2 (23, 24). A functional role for serum-derived human IgGs as substrates for papain (vegetable- anti-hinge autoantibodies remains uncertain although several in- derived) or pepsin (from the digestive tract). Since the latter pro- vestigations showed correlations between serum titers of the Abs teases are not normally present in human vasculature or tissue, 3190 ANTI-HINGE AUTOANTIBODIES AGAINST IgG1 FRAGMENTS

Ј they may be less relevant to pathways leading to immune com- potential-terminal cleavage positions on Fabs and F(ab )2s promise in pathological (proteolytic) environments. Additionally, (whether or not these are known to be directly generated by pro- the use of mAbs facilitated the identification of enzymatic cleav- teases). Peptides terminating in the upper hinge, analogues of Fabs, age points (1) and minimized potential autoantibody interactions were bound to a lower extent than might have been suggested by with diverse idiotypic determinants in polyclonal preparations. the actual Fab with the same termini. The reasons are uncertain but Fab of IgG were generated by different proteases at three posi- could include a different conformational presentation of the tions in the upper hinge (1). When Fabs of the same parent IgG1 epitope in Fab related to orientation, flexibility, or the association were derived with plasmin, HNE, and papain (C-termini at lysine of the L chain. The most antigenic peptides possessed C-termini in 222, threonine 223, and histidine 224, respectively) and tested as the lower hinge “PELLGG” sequence within which several pro- Ags, autoantibody binding from healthy donor serum was most teases have been identified to cleave IgG1. Also within this se- pronounced toward the fragment produced by HNE. HNE is a quence resides the “LL” motif that is central to the recognition of potent, that is released from neutrophil granules, is IgG1 by Fc␥Rs on immune effector cells (48–51) and “LLG” present in extracellular inflammatory environments (16), and pos- found important for complement (52). This extended lower hinge sesses broad substrate reactivity including generation of Fab from sequence is highly conserved among several human IgG isotypes Ј IgG1 (Fig. 2D) and F(ab )2 from IgG2 and IgG4 (41). A relatively as well as IgGs of mice and many other mammals (53). Ј diminished level of autoantibody binding was seen toward papain- Pepsin-generated F(ab )2s also terminate within the antigenic generated Fab even though the two fragments differed in length by zone identified above at leucine 237, and one preclinical study Ј only a single amino acid (depicted in Fig. 2C). The heightened involving administration of such a F(ab )2 was informative regard- Downloaded from autoantibody reactivity to mAb3 FabHNE compared with Mab3 ing anti-hinge autoantibodies. When a humanized anti-platelet ␣ ␤ Ј Fabpapain implies that HAH autoantibodies may preferentially rec- IIb 3 monoclonal F(ab )2 was injected into 18 nonhuman pri- ognize Ags generated by physiologically relevant proteases. Nev- mates (30), an acute clearance of circulating platelets was noted in ertheless, the reactivity to mAb3 Fabpapain was higher than the 5 of the treated animals. The platelet clearance was in many cases reactivity to intact mAb3 IgG1 (Fig. 2A). profound (Ͼ75% from baseline) and rapid (within 24 h). The au- One human/murine chimeric Fab has been studied with regard thors showed that the monkeys exhibiting platelet clearance each

to human immune responses following therapeutic administration possessed pre-existing anti-hinge autoantibodies to the humanized http://www.jimmunol.org/ Ј (23). In subjects treated with a papain-generated Fab, five individ- F(ab )2, and that the autoantibodies were similar in specificity to uals were observed to develop early increased anti-hinge autoan- HAH autoantibodies. No long-term follow-up of the animals was tibodies titers. The boost in autoantibody levels was evident at the reported to allow an assessment of potential recall responses to this earliest determination for three subjects (2 wk) and one subject (10 Ag, and isotype characterizations of the autoantibodies were not days). The early onset of specific anti-hinge autoantibodies were presented. Nevertheless, in addition to raising issues regarding fu- Ј temporally different from anti-murine variable region responses to ture development of F(ab )2 for human therapy, this primate study Ј the same Ab or to the fully murine Fab version of the Ab that provided insight into the potential action of anti-F(ab )2 autoanti- Ј occurred at 4–6 wk (14 of 15 subjects). This difference in timing bodies in association with a cell-bound F(ab )2. suggested a memory recall response to the hinge epitope in con- It proved important to separate HAH autoantibodies from the by guest on September 29, 2021 trast to the likely primary response to the murine variable region large excess of nonspecific Abs in IVIg since the latter were found components. Normal (baseline) anti-hinge autoantibody levels to to contain various anti-cell-specific Abs that interfered with our papain-generated Fab appear to be among the lowest for any IgG cell-based functional assays. To this end, purifications of HAH fragment (Fig. 2), and few autoimmune consequences have been autoantibodies from IVIg were separately performed by affinity associated with initial treatments with mAb3 Fab against the adsorption on two strongly antigenic fragments: immobilized ␣ ␤ Ј Ј IIb 3 receptor on platelets (42, 43). A relatively low level of mAb3 F(ab )2 IdeS or on mAb3 F(ab )2 GluV8. The procedures typ- HAH autoantibody binding to papain-generated Fab compared ically yielded HAH-autoantibody preparations containing less than Ј with pepsin-generated F(ab )2 had previously been observed (26). 0.04% of the total starting IgG, with several hundred-fold in- Ј Clinical investigations with other therapeutic Fabs possessing dif- creased binding to F(ab )2 (compared with starting IVIg), and with Ј ferent C-termini with higher reactivity for human autoantibodies an isotype profile enriched in IgG3. Binding to purified F(ab )2 and (e.g., the HNE cleavage site) have not been reported. to hinge peptide analogues confirmed that the reactivity of these Without question, the most antigenic sites were those exposed in preparations displayed some overlapping reactivity to fragments Ј or adjacent to the lower hinge by proteases that generated F(ab )2s. generated with different physiologically relevant proteases. The It also became apparent that IgG3 was highly represented among exact point of cleavage may be less important for autoantibody Ј the human anti-F(ab )2 autoantibodies, similar to reports of IgG3 binding than the exposure of an otherwise cryptic stretch of hinge anti-Fab autoantibodies in two diseases (27, 40). It is not clear sequence. what factors might bias anti-hinge autoantibodies toward IgG3. Although, our main emphasis was on HAH autoantibodies Ј The persistence and high incidence of IgG3 anti-hinge autoanti- against Fab and F(ab )2 that retain reactivity for target cells, we did bodies is indicative of a class-switching event and suggests that attempt to find related IgG3 autoantibody binding to purified Fc autoreactive B cells were selected for by exposure to cleaved hinge domains from proteolytically digested IgG1s. However, minimal self Ags. Although IgG3s are known to possess potent ADCC- and binding to purified, protease-generated Fcs was observed, and this CDC-mediated effector functions (44–47), it is uncertain whether was confirmed by near background IgG3 binding against single- this is related to a selection of IgG3 for the targeting of these peptide analogues of the hinge containing exposed N-termini. The self-Ags (in contrast to the use of other IgG isotypes vs foreign preferential autoantibody binding of IgG3 to peptides oriented challenges; Table I). This prompted an investigation of IgG3-en- with free C-termini differed from a previous and detailed epitope Ј riched HAH autoantibody binding to F(ab )2s on cells for recon- analysis (54). That work showed that two recombinant, human, stitution of the absent effector functions of the fragments (see fur- single-chain Fvs with anti-hinge specificity were bound to multiple ther below). synthetic double-chain peptides (and variants) containing the core The binding of HAH autoantibodies to a series of peptide ana- C-P-P-C hinge sequence with a free N terminus corresponding to logues of the IgG1 hinge provided a profile of reactivity to all threonine 223 in the upper IgG1 hinge (54, 55). Nevertheless, The Journal of Immunology 3191 when tested on actual fragments of IgG, those same two scFvs Anti-hinge autoantibodies could provide an additional layer of hu- Ј exhibited primarily anti-F(ab )2 reactivity with little or no anti-Fc moral immune protection to combat lost effector functions when binding (in accord with our own findings with serum IgG3) (55). endogenous Abs encounter localized, extracellular protease The authors concluded through additional peptide binding ap- activity. proaches that human serum IgG and IgA anti-hinge autoantibodies In summary, human circulation contains autoimmune reactivity most likely bound to conformational epitopes in the hinge (26, 34). directed to cryptic epitopes in the IgG1 hinge exposed by proteo- Despite the diversity of strategies and previous findings on the lytic enzymes; particularly by enzymes that are relevant to patho- specificity of anti-hinge autoantibodies, we chose to focus on au- logical conditions. The serum anti-hinge autoantibodies, in large Ј toantibodies that directly bind to the exposed hinge in Fab and part of IgG3 isotype, were shown to bind to cell-bound F(ab )2 and Ј F(ab )2 and their peptide analogues, because these fragments retain to restore complement and ADCC cell killing functions in vitro to Ј cellular Ag-binding reactivity and could thereby persist in local otherwise inactive F(ab )2. We propose that anti-hinge autoanti- Ј environments. Functional HAH autoantibody activities to F(ab )2 bodies could represent an immune countermeasure against the pro- were testable in in vitro cellular assays, whereas it was difficult to teolytic inactivation of host IgGs. envision a similar functional test for anti-Fc autoantibodies. As the final component of the present investigation, the affinity- Acknowledgments purified and enriched HAH autoantibody preparations were shown We thank Dr. Lars Bjo¨rk (University of Lund, Sweden) and Genovis AB Ј for the gift of IdeS protease used in the initial studies. We recognize the to restore in vitro ADCC and CDC lysis to cell-bound F(ab )2sin concentration-dependent manners. The reconstitution of in vitro insights of Dr. Carrie Wagner, Allen Schantz, David Hendricks, and Mary effector functions occurred with HAH autoantibodies in the ϳ1–10 Frances McAleer who performed earlier studies with hinge peptide ana- Downloaded from ␮ Ј logues and autoantibodies. We thank Paul Marsters for conducting the g/ml range when titrated against a fixed 200 ng/ml F(ab )2. When ␮ Ј statistical analyses. Finally, we are grateful to Dr. George Gunn and Dr. HAH autoantibody was present at a fixed 5 g/ml and F(ab )2 was Omid Vafa for their time in reviewing the manuscript. varied, partial restoration was seen at as low as 1 and 10 ng/ml of the fragment in ADCC and CDC, respectively. These concentra- Disclosures tions are compatible with the estimates of anti-hinge autoantibod- All authors are employees of Centocor R&D Inc. ies of ϳ5 ␮g/ml (Յ0.04% of total IgG in circulation) as calculated http://www.jimmunol.org/ Ј from the recoveries on F(ab )2 affinity columns. The two prepara- References tions of HAH autoantibodies, separately purified from IVIg on 1. Ryan, M. H., D. Petrone, J. F. Nemeth, E. Barnathan, L. Bjorck, and R. E. Jordan. Ј Ј 2008. 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