Biochemical and Biophysical Research Communications 418 (2012) 748–753
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Chemically dimerized intravenous immunoglobulin has potent ameliorating activity in a mouse immune thrombocytopenic purpura model Yusuke Machino ⇑, Emiko Suzuki, Saki Higurashi, Hiroto Ohta, Mami Suzuki, Junya Kohroki 1, Yasuhiko Masuho Faculty of Pharmaceutical Sciences, Tokyo University of Science, Yamazaki 2641, Noda, Chiba 278-8510, Japan
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Article history: Received 17 January 2012 Available online 28 January 2012 Keywords: Intravenous immunoglobulin Immune thrombocytopenic purpura Fc gamma receptor Chemical dimerization Platelet
a b s t r a c t High-dose intravenous immunoglobulin (IVIG) preparations are currently used for the treatment of autoimmune diseases such as immune thrombocytopenic purpura (ITP). Although the mechanisms of IVIG efficacy remain enigmatic, some clinical and laboratory studies suggest that interaction of the Fc domain of IgG, especially the Fc domain of dimeric IgG, with its receptors (Fc gamma receptors; FccRs) plays an essential role. In this study, IVIG was dimerized with chemical crosslinkers to augment its therapeutic efficacy. Dimerized IVIG was found to have a much higher affinity for FccRs than monomeric IVIG. In a mouse ITP model, chemically dimerized IVIG abrogated the decrease in platelet numbers in the blood that was caused by an anti-platelet antibody at a dose that was one tenth of the required dose of IVIG. These results suggest that chemical dimerization of IVIG should greatly improve the efficacy of IVIG therapy of ITP. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Intravenous immunoglobulins (IVIG) are preparations of gammaglobulin (GG) that are treated so that they do not cause untoward reactions when they are administered intravenously. IVIG was originally used as an antibody substitution therapy for agammaglobulinemic patients. Currently, IVIG is also used for therapy of autoimmune diseases such as Guillain–Barré syndrome and inflammatory polyneuropathy [1,2]. IVIG was first used for autoimmune disease by Imbach et al. [3], who found that IVIG therapy was effective for the treatment of immune thrombocytopenic purpura (ITP). ITP is an autoimmune disease that is characterized by a platelet decrease, which is mediated by pathogenic anti-platelet antibodies [4]. This platelet decrease was suggested to be mediated by Fcc receptor (FccR)-bearing macrophages in the reticuloendothelial system (RES) [5]. The mechanisms of IVIG efficacy have been extensively studied but remain to be elucidated. Although some reports suggest that the Fab domain of the IgG in IVIG neutralizes pathogenic factors such as complement and cytokines [6–8], the Fc fragments of IVIG have been found to be as effective as IVIG itself, not only in the mouse ITP model, but also in clinical ITP therapy [9,10]. These findings suggest that interaction of the Fc domain with FccRs plays an essential role in IVIG effects. ⇑ Corresponding author. Address: Kyowa Hakko Kirin Co., Ltd., 3-6-6 Asahi-machi, Machida-shi, Tokyo 194-8533, Japan. Fax: +81 42 726 8330. E-mail address:
[email protected] (Y. Machino). 1 Present address: Osaka Research Center, Dainippon Sumitomo Pharma Co. Ltd., 3-1-98 Kasugade-naka, Konohana-ku, Osaka 554-00 22, Japan. 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2012.01.092
Three different types of FccRs are expressed on phagocytic cells [11]. FccRI is a high affinity receptor for the Fc domain of IgG and binds monomeric IgG, whereas FccRII and FccRIII are receptors that preferentially bind immune complexes (IC) or polymeric IgG, which they bind with low affinity. These receptors are functionally divided into activating receptors such as FccRI, FccRIIA and FccRIIIA, and an inhibitory receptor, FccRIIB [12]. Regarding Fc receptor-mediated mechanisms that mediate IVIG efficacy for ITP therapy, a simple explanation is that IVIG blocks activating Fc receptors, thereby preventing the binding of autoantibody-bearing platelets to these activating receptors [13]. In the other report, immunosuppression that was mediated by activating FccRs on dendritic cells was observed in an ITP model [14]. In addition, we recently found that cleavage of the interchain disulfide bonds of the IgG in IVIG decreased the binding of IVIG to FccRIIIA but not to either FccRIIA or FccRIIB and reduced the ability of IVIG to ameliorate ITP in a model mouse [15]. These data suggest that IVIG binding to Fc receptors, especially activation receptors, is essential for therapeutic activity in mouse ITP model. IVIG is clinically used at high dose (up to 2 g/kg) and its consumption is increasing. Materials of IVIG are prepared from blood donation, thus the supplied amount has limitations. In fact, product shortage occurred during the late 1990s [16]. Thus, further augmentation of therapeutic activity of IVIG is necessary to overcome these problems. Teeling et al. showed that a small amount of IgG dimers are present in IVIG preparations and that it is the IgG dimers and not the IgG monomers that are effective for amelioration of mouse ITP [17]. Actually, we showed IgG dimers in IVIG had potent binding activity to FccRs [15]. These results suggest that a
Y. Machino et al. / Biochemical and Biophysical Research Communications 418 (2012) 748–753
dimeric IgG fraction could potentially be used instead of IVIG for autoimmune disease therapy. However, the IgG dimers and monomers in IVIG exist in a dynamic equilibrium that depends on the IgG concentration and the temperature [18]. It is therefore difficult to make an IVIG preparation that is composed only of dimeric IgG. In this study, we prepared IVIGs that were stably dimerized by chemical crosslinking and evaluated their binding to FccRs and their therapeutic activity in a mouse ITP model. 2. Materials and methods 2.1. IVIG In this study, gammaglobulin fractions (GG) were used instead of IVIG. GG were kindly provided by The Chemo-Sero-Therapeutic Research Institute (Kumamoto, Japan). 2.2. Gel filtration analysis A gel filtration HPLC was done by using a Protein Pak 300SW column (Waters, Milford, MA) at a flow rate of 1 ml/min in 0.1 M phosphate buffer (pH 6.6) containing 0.15 M NaCl at 25 °C. The protein concentration in the eluate was determined by measurement of the absorbance at 280 nm and the chromatographic results were analyzed using the software package HPLC ChromNAV (JASCO Corporation, Tokyo, Japan). 2.3. Preparation of chemically dimerized and polymerized GG We prepared highly purified chemically dimerized and polymerized IgG as described below. To separate monomeric IgG from GG, GG was applied to a Sephacryl S-200HR column (u2.5 cm 110 cm) at a flow rate of 0.2 ml/min in PBS containing 0.2% PEG4000. One half of the separated monomers were reacted with 2-iminothiolane (2-IT, Sigma–Aldrich Co.) in pH 7.5 and the other half were reacted with Sulfo-HMCS (Dojindo Lab., Kumamoto, Japan) in pH 7.0 at the ratio of IgG: crosslinker = 1:2.5 at 26 °C for 1 h. These modified IgGs were mixed in pH 7.0 at 26 °C for 1 h to form crosslinks. Free maleimide residues and SH groups were blocked by adding cysteine and iodoacetamide. The IgG was then purified by two gel-filtration chromatography steps and a highly purified chemically polymerized and dimerized IgG was obtained. For in vivo use, a dimer-rich fraction was prepared using the above chemical method with a slight modification. Briefly, GG was directly reacted with 2-IT or Sulfo-HMCS without separation of monomers, and the dimers and polymers were separated from the monomers by one gel-filtration step. 2.4. Evaluation of FccR-binding activities and antigen-binding activity by Enzyme-linked immunosorbent assay (ELISA) FccR-binding activities were measured by ELISA [15]. For measurement of antigen-binding activity, Escherichia coli (E. coli) DH5a was used as an antigen. Microtiter plates (Becton–Dickinson Co., NJ, USA) were coated with E. coli that was diluted to an OD600nm of 0.4 in 20 mM phosphate buffer (pH 7.0) containing 0.02% sodium azide at 4 °C overnight and subjected to the ELISA [15]. 2.5. Induction and treatment of an ITP model mouse This study was approved by the Animal Experiment Committee of Tokyo University of Science. Male BALB/c mice (5–6 weeks of age) were purchased from Sankyo Labo Service Corporation (Tokyo, Japan). All mice were kept at the animal facility at Tokyo University
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of Science. Mice were rendered thrombocytopenic by mAb MWReg30 (rat IgG1 antibody against mouse CD41; Santa Cruz Biotechnology Inc., CA, USA) and treated with GG or prepared dimers [15]. Blood samples were collected from the tail vein 6 h after MWReg30 administration. Groups of five mice were used for each experiment. The platelet counts of the five mice in each group are expressed as means ± SD. Statistical analysis was performed using Welch’s t-test. The criterion of significance was taken to be P < 0.05. 3. Results 3.1. Preparation of chemically dimerized GG For preparation of chemically dimerized GG, we used a twostep method with two chemical crosslinkers to avoid intramolecular crosslinking and to control formation of dimeric IgG. One crosslinker was 2-IT which react to primary amines to introduce sulfhydryl (SH) groups. Introduced 2-IT has 8.1 Å ethylene group spacer arm and confer flexibility. The other was Sulfo-HMCS which also react to primary amines to introduce maleimide residues. Introduced Sulfo-HMCS has 11.5 Å ethylene group spacer arm and confer flexibility. SH groups introduced molecules should react only to molecule with maleimide residues. As the result, intramolecular crosslinking does not occur, and dimerized GG has long flexible spacer between molecules. In practice, one half of the monomeric GG was reacted with 2-IT to introduce SH groups and the other half of the GG was reacted with Sulfo-HMCS to introduce maleimide residues. The two modified GGs were then mixed to form crosslinks between the introduced SH and maleimide groups, resulting in GG dimers and polymers (Fig. 1A). The efficiency of introduction of SH groups and maleimide residues into IgG by 2-IT and Sulfo-HMCS depended on the IgG and crosslinker concentration, reaction time, temperature and the pH of the reaction buffer. Approximately 1 SH group and 1 maleimide residue were introduced per IgG molecule using the protocol described in the Methods section (data not shown). Since chemically linked IgG molecules contain not only dimers but also monomers or polymers (>trimer), the removal of polymers and monomers was required to obtain chemically linked dimers with high purity. Purification of the products of crosslinked IgG monomers by two sequential gel-filtration chromatography steps yielded highly purified chemically crosslinked dimers and polymers. Thus, gel-filtration HPLC analysis indicated that the highly purified crosslinked dimers did not contain polymers or monomers and that the crosslinked polymers were also a highly pure preparation (Fig. 1B). These crosslinked preparations were used for in vitro characterization. However, only a very small amount (about 2 mg) of highly purified dimers was obtained from an initial 250 mg GG using this procedure. Therefore, for in vivo use, a chemically crosslinked dimer-rich fraction of GG was obtained by modification of the protocol described in Section 2. The resulting chemically crosslinked dimer-rich fraction was composed of 36.1% polymer and 35.8% dimer (data not shown). 3.2. Receptor-binding and antigen-binding activities of chemically linked dimers The binding of crosslinked IgG dimers and polymers to FccRs was measured using ELISA. For comparative purposes, non-crosslinked polymer, dimer and monomer fractions that were obtained from GG by gel filtration were also assayed. The polymer, dimer and monomer composition of these non-crosslinked fractions were first analyzed by HPLC before their FccR binding activity was assayed. HPLC analysis of the composition of the GG fractions obtained by gel filtration indicated that the percent of IgG present as
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polymers, dimers and monomers in the polymer fraction was 56%, 17% and 27%, in the dimer fraction was <0.1%, 74% and 26%, and in the monomer fraction was <0.1%, <0.1% and 100%, respectively (Fig. 1C). The original GG was composed of 1.3% polymers and 13.5% dimers (data not shown). Since IgG polymers, unlike IgG monomers, bind to FccRs in a polyvalent manner, binding activity is generally in the order of polymers, dimers and monomers. In the ELISA assay, crosslinked polymers displayed much higher Fc receptor binding than GG, showing 140-, 90- and 30-fold higher binding to FccRIIA, FccRIIB and FccRIIIA, respectively, and crosslinked dimers showed 20-, 4- and 6-fold higher binding than GG, respectively, for these three receptors (Fig. 2A). The non-crosslinked polymer and dimer fractions from GG showed lower binding than the crosslinked polymers and dimers, respectively. The noncrosslinked polymer fraction from GG showed 25-, 70- and 10-fold higher binding, and the non-crosslinked dimer fraction from GG showed 1.6-, 2- and 2.5-fold higher binding than the parental GG
to FccRIIA, FccRIIB and FccRIIIA, respectively. GG displayed 25-, 30- and 4-fold higher binding than the monomer fraction to these three receptors, respectively (Fig. 2B). Antigen binding activities of GG, crosslinked polymers, crosslinked dimers and monomers were measured by ELISA using E. coli as an antigen. Crosslinked dimers displayed similar antigen binding as monomers and the parental GG, whereas crosslinked polymers showed slightly higher binding than the other three GG/IgG preparations (Fig. 3). 3.3. Assay of ITP amelioration by crosslinked IgG dimers The ability of the crosslinked IgG dimers to ameliorate ITP was assessed using a mouse ITP model in which the activity of crosslinked dimerized IgG was compared with that of GG, the GG monomer fraction and the non-crosslinked GG polymer plus dimer fraction. Crosslinked IgG dimers or GG were i.p. administered
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Fig. 2. FccR binding activity of crosslinked polymeric and dimeric IgG and of the non-crosslinked dimer and polymer fractions from GG. The binding of crosslinked polymeric and dimeric IgG (A) and of polymer, dimer and monomer IgG fractions from GG that were separated by HPLC gel-filtration (B) to FccRs was measured using an ELISA. GG and IgG monomers were also assayed. These samples were reacted with the extracellular domain of FccRIIA, FccRIIB or FccRIIIA coated on microtiter plates. The amount of bound IgG was measured using HRP-conjugated anti-human IgG (H + L) antibodies. Each experiment was performed in triplicate. The symbols and error bars indicate means ± SD. GG (closed circles), crosslinked polymers or polymer fraction from GG (open circles), crosslinked dimers or dimer fraction from GG (open triangles) and monomers (open squares).
and, 24 h later, a rat IgG1 mAb that was specific for mouse CD41 was i.p. administered to the mice to induce thrombocytopenia. GG ameliorated ITP in a dose dependent manner, as assessed by platelet counting (Fig. 4A). The polymer plus dimer GG fraction, which was composed of 6.3% polymers and 40% dimers, showed significantly higher amelioration of ITP at a dose of 0.2 g/kg body weight than the same dose of GG (P < 0.05), whereas the monomer fraction of GG was significantly less effective than GG at the same dose (P < 0.05). Crosslinked dimers at a dose of 0.1 g/kg were significantly more effective than 0.2 g/kg of GG (P < 0.05) (Fig. 4B) and as effective as 1 g/kg of GG or 0.2 g/kg of the polymer plus dimer fraction. Thus, chemically dimerized IgG appears to be about 10-fold more effective than GG in ameliorating ITP.
4. Discussion In this study, we proposed a new concept in which IVIG with high therapeutic efficacy can be achieved by dimerizing IVIG using
E. coli.
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IgG concentration (μg/ml) Fig. 3. Antigen binding activity of chemically dimerized IgG. The binding of prepared crosslinked polymeric and dimeric IgG to a bacterial antigen was measured using an ELISA and was compared to the binding of GG and IgG monomers. These samples were reacted with E. coli. (DH5a) that was coated on microtiter plates. The amount of bound IgG was measured using HRP-conjugated anti-human IgG (H + L) antibodies. This experiment was performed in triplicate. Anti-E. coli specific binding was calculated by subtraction of the OD450nm of an uncoated plate. The symbols and error bars indicate the means ± SD. GG (closed circles), crosslinked polymers (open circles), crosslinked dimers (open triangles) and monomers (open squares).
a chemical crosslinker. The resulting dimerized IVIG potently bound all of the FccRs that we tested and the antigen binding activity was conserved following chemical modification. Most importantly, the therapeutic activity of chemically dimerized IVIG was much higher than that of original IVIG in a mouse ITP model. Two different kinds of chemical linkers were used to crosslink IgG molecules to avoid intramolecular crosslinkage and to control the dimerization reaction of IVIG (Fig. 1A). Chemically dimerized IVIG contained not only dimers but also polymers (>trimers) and monomers, and gel filtration chromatography was required to obtain highly purified crosslinked dimers (Fig. 1B). However, the yield of crosslinked dimers was very low and a method that can produce a high yield should be developed for clinical use. It had been a concern that chemical modification of IVIG might affect its FccR binding activity and/or its antigen-binding activity. As the results, the antigen-binding activity was not affected and the FccR (FccRIIA, FccRIIB and FccRIIIA) binding activities of the crosslinked polymers and dimers were increased compared with those of the non-crosslinked polymers, dimers and monomers (Figs. 2 and 3). In terms of FccR binding activity, non-crosslinked dimers and polymers generally had high FccR binding activity due to their avidity effect compared with monomers. Surprisingly, crosslinked polymers and dimers showed more potent FccR binding affinity than non-crosslinked and dimers. Non-crosslinked dimers are existing as equilibrium of monomer and dimer [18]. In contrast, chemically crosslinked dimers are formed by covalent bonds and consequently dissociation never occurs. In addition, since these dimerized IgG molecules are connected by relatively long, flexible linkers, chemically crosslinked dimers are expected to be more flexible than the non-crosslinked dimers and easy to bind FccR conformationally to maximize avidity effect. These are the reason why crosslinked dimers show higher FccR binding activity. As mentioned above, antigen binding activity was conserved in chemically crosslinked dimers (Fig. 3). IVIG is polyclonal antibody with reactivity against various antigens. Thus the proportion of antibodies that are reactive against a single antigen is very low i.e., the probability of dimerization of two antibodies against a single antigen is quite low. This is the reason why crosslinked dimeric IVIG did not show increased binding activity against E. coli. Some previous reports have shown the importance of the antigen
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Fig. 4. Therapeutic activity of chemically dimerized IgG in a mouse ITP model. A single dose of GG (0.5, 1 or 2 g/kg) (A) or of 0.2 g/kg of each of GG, of the dimer plus polymer fraction of GG, or of the monomer fraction of GG, or 0.1 g/kg of the crosslinked dimer fraction of GG (B) was administered intraperitoneally to mice (5 mice/group). After 24 h, 2 lg of anti-CD41 antibody was administered intraperitoneally. Blood samples were obtained 6 h after administration of the anti-CD41 mAb. Platelets were counted using an electronic cell counter. The x axis denotes the therapeutic reagent and the y axis denotes platelet counts 6 h after injection with the anti-platelet antibody. The white horizontal bar indicates the mean platelet count ± SD of non-treated mice. Data are presented as means ± SD. Treatment with 0.1 g/kg of the crosslinked dimer or with 0.2 g/ kg of the dimer plus polymer fraction of GG resulted in a significantly higher platelet count vs. the 0.2 g/kg GG group (P < 0.05), and 0.2 g/kg of the monomer fraction resulted in a significantly lower platelet count vs. the 0.2 g/kg GG group (P < 0.05).
binding activity of IVIG for therapeutic activity against autoimmune disease [6–8]. In addition, IVIG is also used for therapy of severe infection. IVIG has been shown to bind to various pathogenic bacteria and bacterial phagocytosis mediated by IVIG is at least partially mediated by FccRs [19,20]. These data suggest that the possibility of chemically dimerized IVIG will have superior therapeutic activity than conventional IVIG. As we expected, chemically dimerized IVIG showed a 10-fold increase in therapeutic activity over parental GG in a mouse ITP model (Fig. 4). In a comparison of FccR binding activity, the binding of the crosslinked dimer to FccRIIA, FccRIIB and FccRIIIA was 20, 4 and 6 times higher, respectively, than that of the parental GG (Fig. 2). We previously suggested the importance of FccRIIIA binding activity of IVIG for treatment of an ITP model mouse [15]. In support of this idea, the experiments reported here showed that the high binding of the crosslinked dimer to the FccRIIIA reflected its high in vivo therapeutic activity. Similar to the concept that multivalent Fc has high avidity for FccRs and high therapeutic activity, Bazin et al. reported that a complex of IgG with an anti-Fc antibody (tetra molecular immune complex) had augmented therapeutic activity in an ITP model [21]. Furthermore, co-administration of a soluble antigen with the counter part antibody (e.g., OVA with an anti-OVA antibody) was shown to be an effective therapy in this mouse model [22]. These therapeutic strategies will be more effective against ITP. However, in the clinical aspect, administration of exogenous protein antigen will trigger an immune response, which can be difficult to control. In contrast, chemically dimerized IVIG consists mainly of selfprotein, and therefore theoretically a major immune response will not occur. In terms of technological aspect, dimerization of protein is frequently used to augment biological activity. In this study, we used two steps, two linkers method for dimerization. Xiao et al. also reported that dimerized protein by crosslinking SH group introduced recombinant protein with crosslinker, monoethylene glycol dithioester [23]. While, in their case, only 1.2- to 1.4-fold increase in in vitro activity was reported, there were great improvements in our IVIG case. These results clearly show that binding independency and avidity of Fc to the FccR is important for the activity of IVIG because our linker, made by two crosslinkers has long and flexible property. In conclusion, chemically dimerized IVIG, in which IgG was stably conjugated by crosslinkers, showed increased binding to FccRs,
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