IgG anti-hinge antibody against IgG4 F(ab’)2 generated by not matrix metalloproteinase-3 but pepsin is a useful diagnostic marker for rheumatoid arthritis, even seronegative rheumatoid arthritis.

Background The pepsin agglutinator, discovered over 50 years ago, has recently referred to be an anti-hinge antibody (AHA) because of main reacting with the IgG hinge epitope. AHA shows different reactivity for each hinge epitope generated by each protease that cleaves the hinge region at different sites. Moreover, AHA shows different reactivity against different hinge epitopes derived from each IgG subclass even when the same protease is used. Since production of matrix metalloproteinase-3 (MMP-3) is enhanced in rheumatoid arthritis (RA), the specic AHA might be increasingly produced. The purpose of this study is to determine whether AHA against IgG hinge epitopes produced by MMP-3 is specically elevated in RA.

IgG [7]. Furthermore, AHA usually targets a C terminus of amino-acid sequence in lower hinge region [8,9], implying it reacts speci cally to hinge neoepitope but not other epitopes existing in the IgG F(ab') 2 .
AHA reacting with human IgG1 or IgG4 hinge epitopes that appeared after cleavage with pepsin showed higher positivity rate and titer in RA patients than in healthy controls (HC) [10]. However, AHA against IgG1 or IgG4 F(ab') 2 generated by other protease such as IgG-degrading enzyme of Streptococcus pyogens (IdeS) did not show the difference, suggesting a certain AHA may be associated with a speci c disorder.
Matrix metalloproteinase-3 (MMP-3) is produced in synovial cells and has been pointed out the relation to RA disease activity [11,12]. It seems likely in RA to result in excessive production of IgG F(ab') 2 fragment by MMP-3 followed by overproduction of the speci c AHA, since activated MMP-3 generates human IgG1 F(ab') 2 fragment in vitro and in vivo [3,13].
The purpose of the present study is to measure IgG or IgA AHA against IgG1, IgG2, and IgG4 monoclonal therapeutic biologics cleaved by MMP-3 or pepsin, and to evaluate their characteristics in RA. Clinical study of serum AHA against IgG1-and IgG4 F(ab') 2 fragment generated by MMP-3 seems to be the rst time to our knowledge.

RA patients and healthy controls (HC)
In this cross-sectional and case-control study, serum samples were collected from 111 patients with RA who met the ACR classi cation criteria [14] and from 81 HC. All of the patient sera were leftovers and 5 sera were shortage for testing IgA class of anti-hinge antibodies. All the samples were stored at -80°C until use.
Characterizations of the RA patients and the HC were shown in Table 1. The RA was signi cantly older than the HC group, although no gender difference between the two groups. Many RA patients had long disease duration and treated with biologics. Positive for rheumatoid factor and anti-CCP2 antibodies due to routine laboratory examination were 67/111 (60.4%) and 77/111 (69.4%), respectively.
Although IFX is chimeric, the hinge region and CH2 domain were derived from human IgG1 according to the database from the national center for biotechnology information (NCBI, USA). We used pepsin (Sigma-Aldrich, USA) and human MMP-3 as proteases to cleave the biologics. The pro-MMP-3 was a kind gift from Daiichi-Fine Chemical, Toyama, Japan.
The biologics were dialyzed in the 0.1 M sodium citrate buffer (SCB, pH3.5), and then 100 μg of pepsin (Sigma-Aldrich, USA) in SCB was incubated with 10 mg of TCZ or NTZ overnight and of PAN for 2 hours. For stopping the digestion, 1M Tris was added to the IgG solution until the pH increased to 7.4. Proteolysis by MMP-3 was performed after activation of pro-MMP-3 by incubation at 55°C for 25 min [15]. The activated MMP-3, 50μg, in 50mM Tris-HCl (pH 7.5) containing 150 mM NaCl and 10 mM CaCl 2 was mixed with 5mg of each biologic at 37°C. After 2 hours incubation, each small amount of the reaction mixture (20 μL) was removed and the reaction was stopped by rapid freezing. The remaining reaction mixture was continued for 24 hours and stopped by adjustment to 20 mM ethylenediaminetetraacetic acid (EDTA).
Puri cation of IgG F(ab') 2 At the outset, human IgG digested by pepsin or MMP-3 was separated by gel ltration on Sephadex G-150 (Pharmacia Fine Chemicals AB, Uppsala, Sweden). Estimated IgG F(ab') 2 fractions were concentrated by Vivaspin 20 (Sartorius Stedium, Goettingen, Germany). Finally to remove IgG possessing Fc, the concentrated crude IgG F(ab') 2 were applied to a Protein G Mag Sepharose (GE Healthcare, Uppsala, Sweden) in 50 mM Tris, 150 mM NaCl pH 7.5. Puri cation of the IgG F(ab') 2 was con rmed by the SDS-PAGE described above. Each puri ed F(ab') 2 fragment was denoted by addition of an italic subscript meaning the protease responsible for the cleavage (e.g. IgG1 F(ab') 2MMP-3 ).
Levels of IgG AHA were calculated by a calibration curve using pooled human IgG puri ed by 40% ammonium sulfate and DEAE sephadex. We arbitrarily de ned 1 mg/mL of the pooled IgG as containing 800 arbitrary units (AU)/mL of IgG AHA to IgG F(ab') 2pepsin . We also used this calibration curve for measuring IgG or IgA AHA to other IgG F(ab') 2 fragments.
Inhibition study for speci cities of IgG AHA against IgG1-or IgG4 F(ab') 2pepsin Same volume of various concentrations of inhibitors (5,000, 1,000, 200, 40, 8, 1.6, 0.32, 0 μg/mL) and 1:200 diluted IgG AHA positive serum from RA patient was mixed well in the tube, followed by incubation for 2h at RT. The mixtures were added to ELISA plate (100 μL/well) coated with IgG1-or IgG4 F(ab') 2pepsin , and then allowed to react for 2h at RT. The subsequent procedure was the same as the measurement of AHA described above. The extents of inhibition by inhibitors were expressed as percent inhibition of the AHA responses, calculated as follows: Percent inhibition =1-(absorbance in the presence of inhibitors/absorbance in the absence of inhibitors) x 100

Statistical analysis
We used the Mann-Whitney U test and the Kruskal-Wallis test to compare the difference between 2 groups and among multiple groups, respectively. We also used Fisher's exact χ 2 test for nominal characteristic. To elucidate the independent variables associated with RA diagnosis, univariate logistic regression followed by multivariate logistic regression analysis was performed. Age and gender were forcedly entered into the model. Two-tailed P value <0.05 was considered signi cant. All of the data were analyzed on a personal computer using SPSS version 19 (IBM Japan, Tokyo, Japan) and StatFlex version 6 (Osaka, Japan).
As shown in Figure 2, the Kruskal-Wallis test revealed signi cant difference of AHA levels among the four groups in AHA1, AHA2 and AHA6. Moreover, the Mann-Whitney U test was used to compare the difference between the groups and the AHA levels of the DNRA in AHA, AHA2 and AHA6 were signi cantly lower than those of the DPRA (AHA1; P=0.004, AHA2; P=0.003, AHA6; P=0.03).
Speci city of IgG AHA responses against IgG1-or IgG4 F(ab') 2pepsin To elucidate whether IgG AHA responses against IgG1-or IgG4 F(ab') 2pepsin possess speci city for epitopes on each IgG subclass F(ab') 2pepsin , inhibition studies were implemented. Response of serum AHA from a patient with RA (S-47) against IgG1 TCZ F(ab') 2pepsin was inhibited by neither IgG2-nor IgG4 F(ab') 2pepsin , but inhibited dose-dependently by both of IgG1 F(ab') 2pepsin ( Figure 3A). Meanwhile in another RA patient (S-212), AHA response against IgG4 F(ab') 2pepsin was inhibited by not only IgG4 F(ab') 2pepsin , but IgG1 F(ab') 2pepsin to a certain extent ( Figure 3B). These results indicate possibilities that IgG AHAs against IgG1 F(ab') 2pepsin speci cally react with IgG1 F(ab') 2pepsin , but those against IgG4 F(ab') 2pepsin cross-react with IgG1 F(ab') 2pepsin . To further ascertain the possible properties, percent inhibitions at de nite concentration of inhibitors (1,000 μg/mL) were calculated in sera from 9 RA patients. IgG AHAs against IgG1 F(ab') 2pepsin exhibited a predisposition to have speci c reaction to pepsin-digested IgG1 hinge neoepitopes ( Figure 3C). IgG1 F(ab') 2pepsin , however, inhibited the all reactions of ve IgG AHAs against IgG4 F(ab') 2pepsin to the same extent as IgG4 F(ab') 2pepsin ( Figure 3D). These observations indicate that AHAs against pepsin-digested IgG4 show a tendency to cross-react with IgG1 F(ab') 2pepsin , meanwhile speci cities of AHAs against IgG1 F(ab') 2 pepsin are mostly restricted to IgG1  An expansion and diversi cation of the AHA response in RA We looked into how many positive AHAs were obtained within each HC or RA patient (Figure 4). The AHA exceeding each optimal cutoff value was accepted as positive, and the cumulative number of positive AHA in each HC and RA patient was counted. The IgG and IgA AHAs were classi ed into 5 grades (0-4) according to the cumulative number. The grade of IgG AHAs in RA showed a tendency to be higher than that in HC ( Figure 4A). Furthermore, the highest grade (cumulative number of recognized epitopes: 4) of IgA AHAs was over 50% in RA, indicating extremely extensive recognition pro le ( Figure 4B). These results suggest AHA repertoire in RA is expanded and diversi ed.
Potential of AHA as an independent diagnostic marker for RA To further assess the potential ability of AHA in diagnostic performance of RA, we accomplished logistic regression analysis. Univariate analysis using 106 patients with RA and 81 HC showed that AHAs except AHA2 were proper to be selected for multivariate analysis as shown in analysis 1 of Table 3.
Multivariate logistic regression analysis revealed that AHA3, IgG AHA against NTZ IgG4 F(ab') 2pepsin , and weakly AHA5, IgA AHA against TCZ IgG1 F(ab') 2pepsin , were selected as an independent variable contributing to RA diagnosis. Additionally, logistic regression analysis using AHAs of patients with DNRA (n=18) revealed that only AHA3 was once more selected as an independent variable [odds ratio 1.18 (95% CI 1.06-1.32); P=0.003] as shown in analysis 2 of Table 3.

Discussion
There has been con icting for a long time as concerning the speci city of anti-F(ab') 2 antibodies in human sera [5,16,17]. Nowadays, studies using human monoclonal IgG1 F(ab') 2 and synthetic peptide analogues of IgG1 hinge region revealed that most of anti-IgG F(ab') 2 antibodies targeted the lower hinge epitopes, but neither idiotopes nor other epitopes in the IgG F(ab') 2 fragment [9, 10, 18].
Serum AHA exists in healthy persons and in patients with a variety of diseases [19][20][21][22][23]. Notably, higher positive incidence and level of serum AHA in RA compared to HC have been reported [5,6,24], and were in line with our results. Contrary to our expectations, IgG AHA against IgG1 F(ab') 2MMP-3 was not signi cantly higher in RA than HC, although IgA AHA against IgG1 F(ab') 2MMP-3 and moreover both IgG and IgA AHA against IgG4 F(ab') 2MMP-3 were signi cantly elevated in RA. Given those signi cantly elevated IgA AHA against IgG1 F(ab') 2MMP-3 and IgG/IgA AHA against IgG4 F(ab') 2MMP-3 in RA, it seems likely that up-regulated immune responses by IgG1/IgG4 F(ab') 2MMP-3 fragments are resulted in RA.
However, why did not the level of IgG AHA against IgG1 F(ab') 2MMP-3 show signi cant difference? Is there any up-regulated mechanism associated with increment of the IgG AHA in HC? MMP-12 that could cleave IgG1 at the same location in the lower hinge as do MMP-3 [7] could participate the increased AHA production in HC? Are these proteases involved in generation of IgG1 F(ab') 2MMP-3 that induce upregulated production of the IgG AHA throughout their life? In any case, we cannot clearly explain this issue in the present situation. Importantly, it was remarkable that all IgA AHAs were signi cantly higher in RA than HC, which might re ect abnormal conditions of mucosal immunity resulting from dysbiosis of respiratory, gut and oral mucosa in RA [25,26].
About the epitopes targeted by AHA in HC, Falkenburg et al reported that IgG AHA against IgG1 F(ab') 2pepsin was inhibited by IgG1 F(ab') 2pepsin but not IgG4 F(ab') 2pepsin , although IgG AHA against IgG4 F(ab') 2pepsin was inhibited by both IgG4 F(ab') 2pepsin and IgG1 F(ab') 2pepsin [10]. These ndings almost agree with the results of our inhibition studies using RA patients as shown in Figure 3. However, they also reported that the speci city of AHA against IgG4 F(ab') 2pepsin was clearly different between HC and RA, namely, the AHA in HC cross-reacted with IgG1 F(ab') 2pepsin , whereas the AHA in RA were inhibited only by IgG4 F(ab') 2pepsin . Unlike their ndings, AHA responses against IgG4 F(ab') 2pepsin showing cross-reactive with IgG1 F(ab') 2pepsin were recognized in RA. We suspect this discrepancy owing to different methods detecting AHAs, namely, our direct coating of IgG F(ab') 2 to ELISA plate vs. their indirect coating that results in stable and conformational hinge epitope (anti-biotin IgG F(ab') 2 bound to biotinylated human serum albumin).
We found a much more extended hinge epitope recognition pro le in RA compared to HC. This nding might indicate the possibility of the epitope-spreading phenomenon in which the immune response is extended to involve new intramolecular or intermolecular epitopes [27][28][29], although our study was crosssectional. The phenomenon has been revealed by observation of ACPA in RA, which occurs before clinical disease onset [30,31]. More study using preclinical and longitudinal RA patients is needed to con rm whether the same phenomenon as ACPA is observed.
Compared to ACPA, AHA did not seem to be useful for RA diagnosis because of LR+ values indicating less than 5 at optimal cutoff values, since LR+ of anti-CCP2 in RA, when used HC and non-RA patients as control, was calculated as 71.6 and 12.1, respectively [32]. Meanwhile, LR+ of RF was not high and reported to be 4.86 [33] and seemed to approximate to the LR+ of AHAs. At cutoff value for over 95% speci city, however, two AHAs (AHA3 and AHA6) revealed LR+ close to 10, which can lead to an increase in the probability of RA diagnosis. In any case, as limitation of this study, it is impossible to obtain convincing LR+ because we used HC as control, and study using non-RA patients with joint symptom is needed.
Three AHAs (AHA3, AHA5, AHA8) were selected by univariate analysis and then AHA3, IgG anti-IgG4 F(ab') 2pepsin , and AHA 5, IgA anti-IgG1 F(ab') 2pepsin , were selected as independent variable for RA diagnosis by multivariate logistic regression analysis as shown in Table 3. Additionally, only AHA3 was also selected as independent variable when the multivariate analysis was conducted on DNRA (RF and anti-CCP2 double negative RA patients). Increment of the speci c AHA reactivity against IgG4 F(ab') 2pepsin in RA reminds us that 1) IgG4 is produced in the context of prolonged antigenic stimulation and 2) the AHA must be generated in vivo by other physiological proteases except pepsin, since pepsin needs activation in acidic stomach conditions and do not reach circulation. Activated matrix metalloproteinase-7 (MMP-7) seems to be a candidate of proteases for cleavage of human IgG so that it cleaves IgG4 at the same lower hinge site between F234 and L235 (EU numbering) as pepsin does [10,13]. Interestingly, the increased MMP-7 seems to mainly originate from not articular but extra-articular lesions such as nodules and lung of RA [12,34,35].
What role do the AHAs play in RA? Although we can not clearly explain, it has been proposed that several biological functions of AHA such as B cell suppression due to cross-linking the B cell receptor and the FcγRIIb, complement ampli cation via capture of dimeric C3b due to immune-complex formation of antigen-binding IgG F(ab') 2 and AHA, and restoring function of cleaved IgGs without Fc [7]. One possible proposal in RA is IgG4 ACPA that is the leading IgG subclass following IgG1 [36]. The IgG4 ACPA themselves would enervate IgG1-mediated ACPA-associated pathogenic progression through complement activation and triggering Fcγ receptors. In this situation, it has been proposed that speci c AHAs bind to IgG4 F(ab') 2 with ACPA reactivity, form immune complexes and potentially progress in ammatory processes [10]. Further studies will be needed to evaluate the pathogenic or protective participation of AHA in addition to RF and ACPA in joints and/or lung of RA.

Conclusions
We have found an extended epitope recognition pro le of AHA in RA, suggesting maturation of AHAproducing immune cells. IgG AHA against IgG4 F(ab') 2 generated by pepsin as an alternate protease of MMP-7, but not MMP-3, seemed to be a potential diagnostic marker for RA, even for seronegative RA.  Tables   Table 1.

Funding
This study was nancially supported by Iizuka Hospital.

Authors' contributions
TO conceived the study, performed all of the experiments, data analysis and wrote the manuscript. SO conceived the study and done critical revision of the manuscript. AU and SN performed acquisition of data. All authors read, commented on and approved the nal manuscript.
We thank T Matsuura, A Imamura and N Akagane for assistance with collection of participant samples and clinical details for this study. Comparison of AHA levels between HC and RA patients. AHA1, AHA2, AHA3 and AHA4 represent IgG AHA against TCZ IgG1 F(ab')2pepsin, TCZ IgG1 F(ab')2MMP-3, NTZ IgG4 F(ab')2pepsin and NTZ IgG4