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. Author manuscript; available in PMC: 2012 Jul 15.
Published in final edited form as: J Immunol. 2011 Jun 20;187(2):1015–1022. doi: 10.4049/jimmunol.1003780

FcRn blockade by Fc engineering ameliorates arthritis in a murine model

Dipesh A Patel *,2, Alberto Puig Cantó *,2, Dilip Kumar Challa *, Héctor Pérez Montoyo *,3, Raimund J Ober *,†, E Sally Ward *,4
PMCID: PMC3157913  NIHMSID: NIHMS298156  PMID: 21690327

Abstract

Multiple autoimmune diseases are characterized by the involvement of autoreactive antibodies in pathogenesis. Problems associated with existing therapeutics such as the delivery of intravenous immunoglobulin (IVIG) have led to interest in developing alternative approaches using recombinant or synthetic methods. Towards this aim, in the current study we demonstrate that the use of Fc-engineered antibodies (Abdegs, for antibodies that enhance IgG degradation) to block FcRn through high affinity, Fc region binding is an effective strategy for the treatment of antibody-mediated disease. Specifically, Abdegs can be used at low, single doses to treat disease in the K/BxN serum-transfer model of arthritis using BALB/c mice as recipients. 25–50 fold higher doses of IVIG are required to induce similar therapeutic effects. Importantly, we show that FcRn blockade is a primary contributing factor towards the observed reduction in disease severity. The levels of albumin, which is also recycled by FcRn, are not affected by Abdeg delivery. Consequently, Abdegs do not alter FcRn expression levels or subcellular trafficking behavior. The engineering of antibody constant regions to generate potent FcRn blockers therefore holds promise for the therapy of antibody-mediated autoimmunity.

INTRODUCTION

Although the focus of therapeutic approaches for autoimmunity has in the past been on targeting cellular immunity, considerable recent interest has been directed towards the humoral component for diseases in which antibodies play a role in pathogenesis (1, 2). In many cases current therapies for antibody-mediated diseases such as systemic lupus erythematosus and myasthenia gravis involve the use of immunosuppressive drugs or steroids which have undesirable side effects (3, 4). Although high doses of intravenous immunoglobulin (IVIG)5 can be effective in ameliorating inflammatory diseases (5, 6), the use of this reagent can result in adverse events such as immune complex-mediated damage (7, 8). Currently, there is also a worldwide shortage of IVIG (9). These shortcomings motivate the use of recombinant or synthetic approaches to develop new treatments.

A strategy that has recently been advocated to treat antibody-mediated autoimmunity is to design reagents that can lower antibody levels in vivo (10–12). This is of particular relevance since B cell depletion using antibodies to target CD20 does not lower the levels of autoreactive IgGs sufficiently to modulate autoantibody-mediated disease due to the lack of CD20 expression by long-lived plasma cells (1, 13). It is well established that the MHC Class I-related receptor, FcRn, regulates the levels and transport of antibodies throughout the body (14–16). The inhibition of this receptor therefore provides a possible target for the therapy of IgG-mediated autoimmune diseases (10–12, 17–19). However, the ability of relatively low and single doses of FcRn blockers to ameliorate such diseases is uncertain. In addition, whether FcRn blockade alone is effective in treating antibody-mediated disease is a major issue regarding the feasibility of this strategy. For example, it is unclear whether other anti-inflammatory pathways such as those involving FcγRIIB upregulation (20, 21) are necessary for therapeutic benefit. This question has been prompted by studies using high dose IVIG to treat inflammatory disease: some studies support a role for FcRn blockade in the reduction of disease activity (19, 22, 23), whereas others have reported that the beneficial effects of IVIG are solely due to upregulation of the inhibitory FcγR, FcγRIIB (20, 21), or to a combination of pathways involving both FcRn and FcγRIIB (24). Resolution of this issue is critical for the design of therapeutics for targeting antibody-mediated inflammatory disease.

Towards the goal of inhibiting FcRn function, we have recently described a class of antibodies called Abdegs (for antibodies that enhance IgG degradation) that are engineered to bind with increased affinity to FcRn through their Fc region at both acidic and near neutral pH (10, 25). To date, however, the activity of Abdegs in treating antibody-mediated disease is untested. Wild type antibodies bind with very low affinity to FcRn at near neutral pH and are dependent on fluid phase uptake for entry into cells, whereas Abdegs enter cells primarily by receptor-mediated processes (10, 26). Consequently, Abdegs compete very effectively with wild type IgGs for FcRn interactions since they not only bind more strongly to this receptor at endosomal pH, but also accumulate within cells to much higher concentrations (10, 25). As such, these engineered antibodies have a major competitive advantage for FcRn binding over existing lower affinity, wild type inhibitors such as IVIG. The binding properties of an antibody for FcRn also impact its in vivo half-life, with gain of binding at pH 7.4 resulting in lysosomal accumulation and lower in vivo persistence (25, 27, 28). Consequently, the competitive ability and in vivo half-life of an Abdeg can be tuned for specific applications (16).

Here we have used a murine model of rheumatoid arthritis (via transfer of K/BxN serum (29)) to both analyze the therapeutic activity of Abdegs and address the fundamental question as to whether FcRn blockade without other contributing anti-inflammatory effects can alleviate ongoing, IgG-mediated disease. In this model, arthritis develops in normal mice following the transfer of anti-glucose-6-phosphate isomerase (anti-GPI) antibodies which complex with endogenous GPI (30). These immune complexes infiltrate joints where they initiate an inflammatory cascade within minutes following transfer (31–33). This model is therefore instructive for the analysis of therapies that target the humoral response. We demonstrate that Abdegs can effectively treat arthritis through a mechanism involving FcRn inhibition. Collectively, our observations indicate that the use of Fc-engineered antibodies of the Abdeg class holds considerable promise for the treatment of arthritis, and by extension, other diseases in which antibodies play a role in pathology.

MATERIALS AND METHODS

Mice

K/BxN mice were generated by intercrossing KRN T cell receptor (TCR) transgenic mice (generously provided by Drs. C. Benoist and D. Mathis) with NOD mice (29). Offspring were typed by flow cytometric analysis of lymphocytes in the blood using an anti-Vβ6 antibody that recognizes the TCRβ chain of the transgene (29). Sera were harvested from 8–10 week old Vβ6+ K/BxN mice for use in transfer experiments. BALB/c and FcRn knockout mice (34) were purchased from The Jackson Laboratory (Bar Harbor, ME). All animal protocols have been approved by the Institutional Animal Care and Use Committee at the University of Texas Southwestern Medical Center (Dallas, TX).

Antibodies

Wild type human IgG1 and the mutant MST-HN (Met252 to Tyr, Ser254 to Thr, Thr256 to Glu, His433 to Lys and Asn434 to Phe) are both specific for hen egg lysozyme and were purified using lysozyme-Sepharose (10, 35). To generate MST-HN+N297A, an antibody heavy chain expression construct encoding the MST-HN mutant in combination with N297A (to express in aglycosylated form) was transfected into a light chain expressing NSO cell line (35). Antibody expressing clones were identified and expanded for protein expression as described (10, 35). Binding properties of purified human IgG1 and MST-HN for FcRn were confirmed using surface plasmon resonance (BIAcore) (36) prior to use in vivo. IVIG (Gammunex) was obtained at 100 mg/ml from Talecris. The mouse anti-lysozyme antibody, D1.3 (37), was purified using lysozyme-Sepharose.

Effect of MST-HN and IVIG on the clearance of radiolabeled IgG1

The effect of the MST-HN mutant on the clearance of 125I-labeled wild type mouse IgG1 (D1.3) was determined as described (10). Drinking water was supplemented with 0.1% Lugol 72 hours before radiolabeled mouse IgG1 was injected intraperitoneally into BALB/c mice and radioactivity monitored at the indicated times by whole-body counting (25) (Atom Lab 100 Dose Calibrator). 72 hours later, mice were intravenously injected with either PBS, 0.5, 1 or 2 mg MST-HN and whole body radioactivity was determined at the indicated times. Similarly, the effect of IVIG on the clearance of D1.3 was compared with that of MST-HN using an analogous approach, except that 72 hours post delivery of radiolabeled D1.3, mice were intravenously injected with either 1 mg wild type IgG1 or MST-HN, 25 or 50 mg IVIG and whole body counts were determined at the indicated times.

To analyze the effect of MST-HN on the clearance of mouse IgG1 in FcRn deficient mice (34), mice were treated as above except that a dose of 1 mg MST-HN or wild type IgG1 was injected into FcRn deficient mice 15–20 minutes following the intravenous delivery of radiolabeled mouse IgG1 (D1.3). β-phase half-lives of radiolabeled D1.3 were determined by fitting the data to a decaying biexponential model using MATLAB (Mathworks) and custom written software (38).

Appearance of intraperitoneally injected mouse IgG1 in the blood

Mouse IgG1 (D1.3) was radiolabeled as described (25). Following intraperitoneal injection of the radiolabeled antibody, counts in 10 μl of blood were determined at the indicated times by scintillation counting.

Serum induced arthritis, therapy experiments and prophylactic treatment

Sera harvested from arthritic K/BxN mice were analyzed for anti-GPI titers using ELISAs. On day 0, arthritis was induced in BALB/c recipients by intraperitoneal delivery of 150 μl K/BxN serum. 6 hours following serum delivery, mice were treated with intravenous injections (200–250 μl per mouse) of MST-HN, wild type human IgG1, IVIG or PBS at the doses indicated. In cases where 50 mg IVIG was used, intravenous injections (250 μl each) were delivered twice, first at 6 hours post-serum transfer and then after an additional 3–4 hours. Mice were also treated either 72 hours following, or 12 hours prior to, K/BxN serum-transfer with intravenous injections of 1 mg/mouse of MST-HN, wild type IgG1 or PBS. Ankle and paw thicknesses were determined using an analog caliper (Dyer 313 series) by two independent observers.

Histopathology

Inflammatory cell infiltration into ankle joints was visualized in hindlimbs isolated from mice 6 days following serum delivery. BALB/c mice were injected intraperitoneally with 150 μl K/BxN serum and after 6 hours treated intravenously with either 1 mg/mouse of MST-HN, wild type human IgG1 or PBS. Mice were euthanized, hind limbs skinned and fixed in 10% neutral buffered formalin for about 60 hours. Following decalcification with Cal-Rite solution, tissue samples were embedded in paraffin wax, sagitally sectioned, deparaffinized and stained with hematoxylin and eosin (H&E). Slides were imaged and acquired using a Leica DM2000 upright photomicroscope (Leica Microsystems, Wetzlar, Germany) equipped with bright-field and epifluorescence optics and an Optronics Microfire color CCD camera (Optronics, Goleta, CA). A G4 Macintosh (OS 10.4.11) with Pictureframe 2.0 acquisition software was used to acquire and store images.

ELISAs to determine autoantibody, IgG and albumin levels

Serum samples of BALB/c mice prior to induction of arthritis and after treatment were obtained via retro-orbital bleeding at the indicated times. These samples were used to determine anti-GPI antibody levels, total IgG levels and albumin levels using sandwich ELISAs at serum dilutions of 1:500, 1:4000 and 1:50000, respectively. For measuring anti-GPI antibody levels, 1.25 μg/ml recombinant GPI (Roche) was used to coat 96 well plates, and bound antibody was detected using HRP-rabbit anti-mouse IgG (heavy and light chain specific; Dako). IgG and albumin levels were determined using ELISA quantitation kits for mouse IgG and albumin (Bethyl Laboratories).

Lectin immunoblotting

1 μg of each recombinant antibody or IVIG were electrophoresed on 12% polyacrylamide gels with BenchMark Prestained protein ladder (Invitrogen). Electrophoresed proteins were either transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore; Immobilon-P) or stained with Coomassie brilliant blue. The membrane was incubated with 1% Western blocking reagent (Roche) for 1 hour, followed by 1 μg/ml biotinylated Elderberry Bark Lectin (Sambucus nigra agglutinin, SNA; Vector Labs) in Western blocking reagent for 1 hour, and subsequently washed twice with TBS/0.1% Tween and once with TBS (pH 7.4). The membrane was then incubated with 2.25 μg/ml Extravidin-peroxidase conjugate (Sigma) in Western blocking reagent for 30 minutes prior to washing as above. Bound SNA was detected by incubation with 0.6 mg/ml 3,3- diaminobenzidine tetrahydrochloride (DAB) with 0.01% (v/v) hydrogen peroxide.

Statistical analyses

The n for each data set is indicated and statistical significance for each time point was determined by ANOVA using MATLAB (Mathworks). ANOVA was carried out by paired comparisons between different mouse groups using the Tukey-Kramer test at a 95% confidence interval (α = 0.05). Two-tailed Student's t test was used to compare two treatment groups. p values less than 0.05 were considered to be significant and actual values are indicated. Outliers due to experimental artifacts were removed from the analyses. Data shown are mean values of treatment groups with error bars indicating standard errors (s.e.m.).

RESULTS

Dose response analysis of the effects of the MST-HN Abdeg on IgG levels

The current study is directed towards analyzing the therapeutic efficacy of a mutated human IgG1 (MST-HN, Met252 to Tyr, Ser254 to Thr, Thr256 to Glu, His433 to Lys and Asn434 to Phe) (10) that is designed to inhibit FcRn function in a mouse model of antibody-mediated arthritis. We initially investigated how the dose of MST-HN Abdeg impacts the levels of competing wild type tracer IgG in vivo. BALB/c mice were used throughout these studies since this mouse strain is highly susceptible to arthritis following the transfer of arthritogenic serum derived from K/BxN mice (39). Mice were injected intraperitoneally with radiolabeled mouse IgG1 and, three days later, 0.5, 1 or 2 mg MST-HN per mouse delivered by intravenous injection. A dose of 0.5 mg per mouse (~25 mg/Kg) induces a lower decrease in tracer IgG1 levels relative to 1 mg (~50 mg/Kg) (Fig. 1). By contrast, comparison of doses of 1 and 2 mg MST-HN per mouse indicates no significant differences, demonstrating that for FcRn inhibition in BALB/c mice there is no advantage in using doses greater than 1 mg per mouse (Fig. 1).

Figure 1.

Figure 1

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MST-HN shows a dose dependent effect on tracer IgG1 levels. BALB/c mice were injected intraperitoneally on day 0 with 125I-labeled mouse IgG1. After 72 hours (indicated by arrow), mice were injected intravenously with either 0.5, 1, 2 mg MST-HN or PBS (n = 3 mice per treatment group). Levels of remaining radiolabeled mouse IgG1 were determined by whole body counting at the indicated times. Radioactivity levels were significantly lower for all MST-HN treatment groups relative to the PBS treatment group from 85–166 hours (p < 2×10−5; ANOVA). Relative to the 0.5 mg MST-HN treatment group, radioactivity levels were significantly lower in mice treated with 1 mg MST-HN (p < 2×10−5, 85 hours; p < 3×10−6, 96–124 hours; ANOVA, +) or 2 mg MST-HN (p < 3×10−6, 96–124 hours; p < 1.5×10−6, 142–148 hours; ANOVA, *). Differences between the 1 and 2 mg MST-HN treatment groups were not significant. Error bars indicate s.e.m. Data are representative of two independent experiments.

Dynamics of appearance of antibodies in the circulation following intraperitoneal delivery

Anti-GPI antibodies, primarily of the IgG1 isotype, are known to be the mediators of disease in the K/BxN model (32). In the current experiments, we induced arthritis by transferring serum from arthritic K/BxN mice by intraperitoneal delivery into BALB/c recipient mice. Prior to the delivery of potential therapeutics, initial analyses were therefore carried out to determine the time at which intraperitoneally injected mouse IgG1 could be detected in the serum of BALB/c mice. The levels of radiolabeled IgG1 in the blood approach maximal levels at the earliest sampling point of two hours, but continue to increase slowly to peak levels until 6 hours post-delivery (Fig. 2). PET imaging has shown that anti-GPI antibodies in the serum localize to joints at detectable levels within several minutes of delivery (33), indicating that extravasation to the target site is very rapid.

Figure 2.

Figure 2

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The appearance of intraperitoneally (i.p.) delivered mouse IgG1 in the circulation of recipient mice peaks at 6 hours post-delivery. Mice were injected intraperitoneally with 125I-labeled mouse IgG1 and radioactivity in blood samples determined at the indicated times (n = 6 mice). Error bars indicate s.e.m. Data are representative of two independent experiments.

MST-HN is effective in ameliorating arthritis

The therapeutic effect of treatment with 0.5 or 1 mg MST-HN (per mouse) on arthritis was initially assessed by transferring arthritogenic serum into BALB/c mice and treating 6 hours later. Mice were treated with MST-HN (0.5 or 1 mg), or as controls with wild type IgG1 (0.5 or 1 mg; same binding specificity as MST-HN) or PBS vehicle. Disease severity was monitored by determining ankle and paw thickness. The data shown in Figure 3A,B clearly demonstrate that MST-HN treatment at a time point when substantial accumulation of anti-GPI:GPI immune complexes in joints is expected (Fig. 2 and ref. 33) results in significantly lower disease activity relative to controls (wild type IgG1 or PBS). Although both 0.5 mg (~25 mg/Kg) and 1 mg (~50 mg/Kg) MST-HN induce therapeutic effects, the higher dose is more effective in treating disease. In addition, there was no significant difference between wild type IgG1 and PBS treatment groups (Fig 3A,B). Similar results were observed when paw swelling was used to assess disease activity (data not shown). Delivery of MST-HN to mice following injection of control (non-arthritogenic) serum did not have any impact on joint size (data not shown), indicating the specificity of the effects on autoantibody-mediated disease.

Figure 3.

Figure 3

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MST-HN is effective in treating arthritic mice. A, BALB/c mice were injected on day 0 with arthritogenic serum and 6 hours later (indicated by arrow) treated with 0.5 mg MST-HN, 0.5 mg wild type IgG1 or PBS (n = 6 mice per treatment group). Ankle swelling (both ankles analyzed for each mouse) was not significantly different between wild type IgG1 and PBS treated mice, whereas MST-HN treatment significantly reduced swelling with respect to wild type IgG1 or PBS treatment from day 4 onwards (all p < 2×10−7; ANOVA, *). B, Mice were treated and analyzed as described in Figure 3A, but 1 mg MST-HN, 1 mg wild type IgG1 or PBS (n = 6 mice per treatment group) was administered. Ankle swelling in MST-HN treated mice was significantly lower compared with that for either wild type IgG1 or PBS treated mice from day 3 onwards (p < 1×10−2, day 3; all p < 1×10−8, day 4 onwards; ANOVA, *). No significant difference was observed between wild type IgG1 and PBS treatment groups. C, Mice (n = 4 per treatment group) were treated as described in Figure 3B and hind limbs were harvested 6 days after K/BxN serum delivery. Limbs were subsequently skinned, fixed, decalcified and embedded in paraffin wax before 5 μm thick sections were cut. Ankle joint sections were stained with hematoxylin and eosin (H&E). Sections shown are at 4× and 10× magnification (black box in 4× images indicates region observed at 10× magnification). Images are representative of each treatment group. D, Mice were treated as described in Figure 3B and anti-GPI antibody levels in sera of mice (4–6 mice per treatment group; duplicate samples for each mouse) were determined by ELISA before and following treatment. Serum anti-GPI levels were significantly lower at the indicated times (*) in MST-HN treated mice compared with mice treated with PBS or wild type IgG1 (p = 6.7×10−7, 10 hours; p = 2.8×10−9, 24 hours; p = 1.4×10−9, 33 hours; p = 9×10−9, 48 hours; ANOVA). Error bars indicate s.e.m.

Histopathological analysis of ankle joints showed markedly less infiltration of immune cells in mice treated with MST-HN relative to mice in control treatment groups (Fig. 3C). As a result, no obvious bone damage was observed in MST-HN treated mice, whereas mice treated with either wild type IgG1 or PBS showed substantial inflammatory cell invasion and bone/tissue damage (Fig. 3C). We also determined the anti-GPI levels in sera of mice immediately prior to and following delivery of MST-HN (~50 mg/Kg), wild type IgG1 or PBS (Fig. 3D). Disease activity was found to correlate with anti-GPI antibody levels, with MST-HN treatment resulting in a rapid decrease in these antibody levels within several hours of delivery.

In order to further investigate the therapeutic effects of MST-HN, we analyzed whether MST-HN delivery during established disease (3 days post-serum transfer) ameliorated arthritis. Relative to control groups, MST-HN treatment resulted in a rapid attenuation of disease (Fig. 4A). In addition, we determined the effect of administration of MST-HN prior to K/BxN serum-transfer. Prophylactic treatment with MST-HN 12 hours prior to K/BxN serum-transfer resulted in less severe disease relative to that observed in wild type IgG1 treated mice (Fig. 4B). However, the reduction was not as pronounced compared with the effect of Abdeg delivery 6 hours post-transfer of arthritogenic serum.

Figure 4.

Figure 4

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MST-HN is effective in ameliorating arthritis both during ongoing disease and prophylactically. A, To treat established disease, BALB/c mice were injected on day 0 with arthritogenic serum and 72 hours later (indicated by arrow) treated with 1 mg MST-HN, 1 mg wild type IgG1 or PBS (n = 7–8 mice per treatment group). Ankle swelling was not significantly different between wild type IgG1 and PBS treated mice, whereas MST-HN treatment significantly reduced swelling with respect to wild type IgG1 or PBS treatment from day 5 onwards (p < 2×10−4, day 5; all p < 7×10−8, day 6 onwards; ANOVA, *). B, BALB/c mice were treated with 1 mg MST-HN or 1 mg wild type IgG1 (n = 6 mice per treatment group) 12 hours prior to K/BxN serum-transfer (day 0). Ankle swelling was significantly lower (*) in mice treated with MST-HN compared with wild type IgG1 treated mice from day 3 onwards (Student's t test: p < 3×10−2, day 3; p < 4×10−3, day 4; p < 8×10−5, day 5; p < 2×10−4, day 6; all p < 9×10−5, day 7 onwards).

The therapeutic effect of MST-HN on disease involves FcRn blockade

Earlier studies have indicated that sialylation through α2,6 linkage to the N-linked carbohydrate, present on 1–3% of IgG molecules in IVIG preparations, mediates anti-inflammatory effects by inducing the upregulation of FcγRIIB (20, 21, 40). It was therefore important to determine whether similar effects could contribute to disease amelioration induced by MST-HN. Immunoblotting with a lectin (Sambucus nigra agglutinin, SNA) specific for α2,6-linked sialic acid indicated that MST-HN, wild type IgG1 and IVIG all bear similar levels of this type of sialic acid (Fig. 5). A possible contribution of this sialic acid form to the anti-inflammatory effects of MST-HN was therefore directly assessed by comparing the activity of this antibody with its parent, wild type counterpart. Significantly, our data demonstrate that at a dose of 0.5 or 1 mg per mouse, wild type IgG1 has no therapeutic activity (Fig. 3A,B), suggesting that the disease amelioration induced by the same doses of MST-HN involves FcRn inhibition.

Figure 5.

Figure 5

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Immunoblotting analyses of human IgGs using SNA. 1 μg MST-HN+N297A (aglycosylated MST-HN), wild type human IgG1, MST-HN and IVIG were electrophoresed on 12% SDS polyacrylamide gels and either stained with Coomassie brilliant blue or transferred onto a PVDF membrane for immunoblotting. Sizes of molecular weight standards (MW) are indicated in kDa on the left margin.

To further investigate the FcRn-dependence of MST-HN activity, the impact of MST-HN delivery on the clearance of 125I-radiolabeled mouse IgG1 in FcRn knockout mice (34) was examined. No significant difference in the half-life of radiolabeled IgG1 was observed in mice treated with either 1 mg MST-HN (30.4 hours ± 1.15, s.e.m, n = 5 mice) or 1 mg wild type IgG1 (32.2 hours ± 1.05, s.e.m., n = 5 mice). These results are therefore consistent with the concept that a primary mechanism of Abdeg action is through FcRn inhibition.

MST-HN treatment does not downregulate FcRn

In addition to the well established role of FcRn in regulating IgG levels (14, 15), it also functions to transport and recycle albumin (41). The lack of overlap between the IgG and albumin binding sites on FcRn (42) has enabled us to use albumin levels to assess whether the MST-HN mutant impacts FcRn expression and/or downregulation. For example, it is conceivable that through high affinity, bivalent binding to FcRn, MST-HN might promote lysosomal trafficking of this receptor. Importantly, albumin levels did not vary significantly between different treatment groups, whereas total IgG levels decrease (Fig. 6). These data demonstrate that the MST-HN mutant acts through competition with endogenous IgGs for binding to FcRn in the absence of significant effects on FcRn downregulation/degradation.

Figure 6.

Figure 6

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MST-HN does not affect serum albumin levels and lowers total IgG in mice. BALB/c mice were treated with 1 mg MST-HN or 1 mg wild type IgG1 6 hours following K/BxN serum-transfer, using the protocol described in Figure 3B. A, Serum albumin levels were determined by ELISA and were not significantly different (Student's t test) between mice treated with MST-HN or wild type IgG1 (n = 5 and 6 mice per treatment group, respectively; duplicate samples for each mouse). B, Total serum IgG levels were determined by ELISA for the same treatment groups as in Figure 6A and were significantly lower (*) in mice treated with MST-HN relative to those treated with wild type IgG1 (Student's t test: p = 6.8×10−3, 24 hours; p = 3.3×10−3, 33 hours; p = 1×10−4, 48 hours). Error bars indicate s.e.m. Data are representative of at least two independent experiments.

MST-HN is as effective as 25–50 fold higher doses of IVIG in treating arthritis

Comparison of the impact of 1 mg MST-HN and 1 mg IVIG on arthritis indicated that, by contrast with MST-HN, 1 mg IVIG had no significant therapeutic effect (data not shown). This result is not unexpected, since IVIG comprises primarily human IgG1 which at this low dose also does not alter disease incidence and severity (Fig. 3B). We therefore determined the dose of IVIG necessary to induce similar therapeutic effects as 1 mg MST-HN. Mice were treated with 1 mg MST-HN, 25 mg IVIG, 50 mg IVIG or as control, 1 mg wild type IgG1, and disease activity monitored (Fig. 7A). The reduction in ankle swelling in mice treated with 1 mg MST-HN was between that induced by 25 and 50 mg IVIG (~1,250 and 2,500 mg/Kg, respectively), although differences between MST-HN and IVIG treatment groups were not significant. Similar results were observed for paw swelling (data not shown). Consequently, approximately 25–50 fold more IVIG is needed to induce the therapeutic benefit that is achievable with 1 mg MST-HN. Consistent with the observations in Figure 3B,D, disease severity correlated with the amount of anti-GPI antibodies present (Fig. 7B). In addition, MST-HN and IVIG lowered levels of competing IgGs under non-inflammatory conditions in vivo (Fig. 7C).

Figure 7.

Figure 7

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MST-HN has the same therapeutic effects as 25–50 fold higher doses of IVIG. A, BALB/c mice were injected on day 0 with arthritogenic serum and 6 hours later (indicated by arrow) treated with 1 mg wild type IgG1 (n = 7 mice), 1 mg MST-HN (n = 7 mice), 25 mg IVIG (n = 6 mice) or 50 mg IVIG (n = 5 mice) and disease monitored as in Figure 3A,B. Relative to mice treated with 1 mg wild type IgG1, disease activity was significantly lower in mice treated with 1 mg MST-HN (all p < 5×10−4, day 5 onwards; ANOVA, *), 25 mg IVIG (all p < 4×10−4, day 7 onwards; ANOVA) and 50 mg IVIG (p < 4×10−3, day 4; all p < 5×10−4, day 5 onwards; ANOVA). No significant differences were observed between mice treated with MST-HN and either 25 or 50 mg IVIG. Disease activity was significantly different between the 25 and 50 mg IVIG treatment groups from days 4–6 (p < 4×10−3, ANOVA). B, Mice were treated as described in Figure 7A and anti-GPI antibody levels in sera of mice (n = 4 mice per treatment group; triplicate samples for each mouse) were determined by ELISA. Serum anti-GPI levels were significantly lower in mice treated with 1 mg MST-HN or 50 mg IVIG (*) and 25 mg IVIG (+) compared with mice treated with wild type IgG1 (p = 2.5×10−3, 10 hours; p = 1×10−4, 24 hours; p = 2.3×10−3, 33 hours; ANOVA). C, BALB/c mice were injected with 125I-labeled mouse IgG1 as described in Figure 1. After 72 hours (indicated by arrow), mice were injected intravenously with either 1 mg wild type IgG1 or MST-HN, 25 or 50 mg IVIG (n = 4 mice per treatment group). Levels of remaining radiolabeled mouse IgG1 were determined by whole body counting at the indicated times. Radioactivity levels were significantly lower for all treatment groups relative to wild type IgG1 from 90–166 hours (p < 2×10−4, 90 hours; p < 7×10−6, 96–166 hours; ANOVA). Relative to the 1 mg MST-HN treatment group, radioactivity levels were significantly higher in mice treated with 25 mg IVIG (p < 2×10−4, 90 hours; p < 7×10−6, 96–118 hours; ANOVA, +) or 50 mg IVIG (p < 2×10−4, 90 hours; p < 7×10−6, 96–106 hours; ANOVA, *). No significant difference was observed between the 25 and 50 mg IVIG treatment groups. Error bars indicate s.e.m. Data are representative of two independent experiments.

DISCUSSION

Recent interest has focused on targeting the humoral arm of the immune response for the treatment of many autoimmune diseases (1, 2). In the current study, we demonstrate that recombinant antibodies of the Abdeg class can be used as an effective therapeutic to treat ongoing disease in the K/BxN serum-transfer model of antibody-mediated arthritis. Consistent with the ability of Abdegs to inhibit FcRn, disease blockade is accompanied by a reduction in serum anti-GPI levels. Abdegs therefore have considerable promise as therapeutic reagents for IgG-mediated autoimmunity.

Although IVIG is currently used to treat a number of autoimmune diseases, high doses (~1–2 g/Kg) are needed for efficacy (6, 43). This poses a challenge for use in the clinic that is exacerbated by the worldwide shortage of IVIG (9). In addition, the delivery of IVIG can result in adverse side effects due to the polyclonal nature of this therapeutic and immune complex-mediated damage (7, 8). These shortcomings have prompted the development of alternative approaches, such as the use of Fc-engineered antibodies that bind with increased affinity to FcRn at endosomal pH and have longer in vivo half-lives (11). Delivery of these antibodies alleviates autoimmunity in a murine model involving the passive transfer of human antibodies from arthritic patients (11). Antibodies of this class compete poorly for FcRn due to the need for fluid phase uptake into cells, and consequently they require high and repeated dosing to show efficacy. Another strategy which has shown beneficial effects in a rat model of myasthenia gravis is to use antibodies that block FcRn by binding through their variable domains (12). However, such antibodies bind to FcRn with similarly high affinity at both pH 6.0 and 7.4, resulting in short in vivo half-lives and the need for repeat and frequent dosing (12).

An alternative approach that is described here is to use Fc engineering to generate Abdegs that bind through their Fc region with high affinity to FcRn in the pH range 6.0–7.4 (10). Consequently, relative to their wild type counterparts Abdegs accumulate in cells to higher concentrations through receptor-mediated uptake, where they outcompete wild type IgGs for FcRn binding in endosomes (10, 25, 28). Nevertheless, due to the intrinsic pH dependence of FcRn-IgG interactions (27, 44), antibodies of the Abdeg class such as MST-HN retain significantly lower affinity at pH 7.4 relative to pH 6.0 (10). Consequently, MST-HN persists with a half-life in mice of around 40 hours (38), providing a significant advantage over the therapeutic use of shorter-lived antibodies that bind to FcRn through their variable regions (12, 17). Indeed, the available knowledge concerning how FcRn-IgG interaction properties impact the half-life and FcRn blocking activity of an Abdeg enables the optimization of Abdeg behavior for different applications (16, 25).

In the current study, treatment of mice 6 hours following K/BxN serum-transfer with a single dose of either 0.5 or 1 mg Abdeg ameliorates arthritis, with the higher dose resulting in maximal competitive effects and almost complete ablation of disease symptoms. By comparison with Abdeg, our studies show that 25–50 fold higher concentrations of IVIG are required to induce a comparable reduction in disease activity. Thus, the binding properties of Abdegs translate into potent therapeutic effects. Importantly, MST-HN delivery 72 hours post-serum transfer reduces disease activity when joints are visibly swollen, demonstrating therapeutic benefit in established disease. The potency of the Abdeg in reducing arthritis, however, was not as pronounced when administered prophylactically (12 hours prior to K/BxN serum-transfer) compared with delivery at 6 hours post-serum transfer. This observation is consistent with the relatively short half-life of MST-HN (38), which is expected to render the prophylactically-delivered Abdeg less effective in competing with anti-GPI antibodies for FcRn binding.

Although in earlier studies the stoichiometry of FcRn binding to Fc (IgG) was uncertain, it is now established that the interaction comprises an asymmetric 2:1 FcRn:Fc (or IgG) complex (45–47). Relative to their homodimeric counterparts, heterodimeric Fc fragments with only one functional FcRn interaction site have shorter in vivo half-lives and are recycled less efficiently (48, 49). This, combined with our recent observations demonstrating that Abdegs enter the lysosomal pathway in cells (28), raises the question as to whether the presence of two relatively high affinity binding sites on an Abdeg for FcRn could decrease recycling activity by diverting this receptor into lysosomes. In addition to transporting IgG, FcRn recycles serum albumin (41), a protein that is critical in maintaining oncotic pressure, blood pH and transporting a range of small molecules. Importantly, the binding sites for albumin and IgG on FcRn are distinct (42). We therefore reasoned that by monitoring albumin levels, we could determine whether FcRn expression and/or intracellular trafficking were affected by Abdeg treatment. Our studies demonstrate that Abdegs function specifically through blocking FcRn-IgG interactions, and do not alter FcRn expression or distribution to impact albumin levels. The specificity of Abdeg effects for IgG indicate that the application of these reagents in the clinic will not result in the undesirable side effect of altering albumin homeostasis.

A question of direct relevance to the design of therapeutics is whether FcRn blockade alone is sufficient to treat antibody-mediated disease. For example, sialylation (α2,6-linked sialic acid) present on 1–3% of IgG molecules in IVIG has been shown to induce anti-inflammatory effects by upregulating FcγRIIB (21, 40). This upregulation has been reported to be either an important, or the sole, contributing factor to the therapeutic benefit of IVIG in the K/BxN serum-transfer model (20, 21, 24). Our immunoblotting data indicate that the levels of α2,6-linked sialic acid on MST-HN or wild type IgG1 are analogous to those present on IVIG, although the possibility of cross-reactivity of SNA with different forms of sialic/neuraminic acid on the recombinant antibodies cannot be excluded (50). We therefore reasoned that comparison of the effects of the delivery of MST-HN and wild type IgG1 on arthritis would enable us to assess the role of FcRn inhibition versus other possible anti-inflammatory pathways on the therapeutic effects of the Abdeg. Significantly, at the low doses (~25–50 mg/Kg) of recombinant antibodies used in this study, wild type IgG1 delivery does not modulate arthritis, suggesting that a primary mode of action of the MST-HN mutant is through FcRn blockade. This is also consistent with the inactivity of MST-HN in further reducing the half-life of IgG in FcRn knockout mice, combined with the relationship between therapeutic efficacy and the ability of MST-HN or relatively high doses of IVIG to decrease antibody levels in vivo.

In the current study we show that the use of antibodies of the Abdeg class offers advantages over alternative approaches, including the ability of relatively low, single doses to treat disease when delivered following the initiation of immune complex-mediated inflammation. Collectively, our observations demonstrate that Abdegs represent highly efficacious and specific therapeutics that could have broad applicability in the management of IgG-mediated autoimmunity.

ACKNOWLEGMENTS

We would like to thank Drs. Christophe Benoist and Diane Mathis for generously providing KRN transgenic mice. We are indebted to Dr. James Richardson and John Shelton (Molecular Pathology Core, University of Texas Southwestern Medical Center) for histologic preparations. We are grateful to Dr. Sripad Ram for expert advice on statistical analysis, Dr. Wentao Mi for advice on mouse husbandry and Tom Lo for assistance.

This study was supported in part by grants from the National Institutes of Health, RO1 AR 56478 and R01 AI 39167 (E.S.W.).

Footnotes

5

Abbreviations used in this paper: IVIG, intravenous immunoglobulin; FcRn, neonatal Fc receptor; K/BxN, a transgenic mouse strain that spontaneously develops arthritis; GPI, glucose-6-phosphate isomerase.

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