Few alternative therapies exist for organ failure except organ transplantation, but immune response makes transplantation difficult (1). Rejection of organ grafts by immune response has become a serious problem. Using ABO blood group-identical or compatible grafts is one solution to this problem (2, 3). ABO-incompatible solid-organ transplantation is generally avoided: outcomes generally are unfavorable because of humoral rejection (4, 5). Humoral rejection associated with ABO-incompatible transplantation is triggered by antigen recognition of antidonor blood group A/B antibodies, which bind to blood group A/B-antigen on the endothelium of the graft. Subsequently, the complement and blood clotting cascades initiate (2, 5, 6). The classical pathway, which is a pathway of complement activation, is induced when the C1q binds to the Fc fragment of the antibody reacted with the antigen (1, 7, 8). In the graft organ, endothelial damage causes the formation of fibrin thrombi that destroy the microvasculature, leading to acute graft dysfunction and graft failure (4, 9, 10).
The preoperative anti-A/B antibody titer has been reported to be correlated with long-term graft survival in ABO-incompatible kidney transplantation (11). In ABO-incompatible liver transplantation, patients with high titers of antiblood group antibody are also at very high risk of graft failure (12, 13). Various strategies have been developed to overcome ABO-incompatible transplantation, such as plasmapheresis, splenectomy, intensive immunosuppression, anti-CD20 monoclonal antibody, intravenous immunoglobulin, and intraportal infusion therapy, which have improved transplantation outcomes (14–19). These strategies, however, present the risks of infection and thrombosis. Furthermore, despite these various therapies, outcomes of ABO-incompatible transplantation are worse than those of ABO-identical or compatible transplantation. Recently, the strategy for overcoming ABO-incompatible transplantation is mainly the reduction of antibodies in plasma or the suppression of antibody production. The outcome of ABO-incompatible transplantation might be more favorable if the blood group antigen on the graft organ was suppressed and the humoral rejection were controlled.
For use in this study, we produced a novel anti-A monoclonal antibody, designated as K7508, and performed A-antigen suppressing procedures. In vitro, A-antigen-expressing cells such as blood group A-red blood cells (A-RBCs) or A431 were coated with K7508 or its Fab fragment. Their reactivity to another anti-A antibody was measured using flow cytometry. In vivo, we immunized mice with A-antigen-expressing cells after coating them with K7508 or Fab fragment, and then measured the production of anti-A antibody in mice sera. This in vivo model using A-antigen-expressing cells might mimic solid organ transplantation.
MATERIALS AND METHODS
Female BALB/c mice were obtained (Charles River Japan, Inc., Kanagawa, Japan); they were used and maintained in a temperature-controlled room with alternating 12-hour light/dark cycles. The Animal Care and Use Committee of the National Institute of Advanced Industrial Science and Technology approved all mouse manipulations.
Cell Lines, Antibodies, and Tissue Microarrays
An epidermoid carcinoma cell line, A431, which is known to express blood group A-antigen, was obtained from Cell Resource Center for Biomedical Research, Tohoku University (Sendai, Japan) (20). The A431 cells were cultured at 37°C in a humidified atmosphere of 5% CO2 and 95% air in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sigma, St. Louis, MO), 2 mM l-glutamine (Invitrogen Corp., Carlsbad, CA), 100 units/mL of penicillin, and 100 μg/mL of streptomycin (Invitrogen Corp.). From healthy volunteers, RBCs of blood groups A, B, and O were extracted. An anti-A monoclonal antibody (IgM) and rabbit anti-A polyclonal antibody were purchased (Wako Pure Chemical Industries Ltd., Tokyo, Japan), as were normal liver tissue microarrays (Cybrdi Inc., Frederick, MD).
Female BALB/c mice (4 weeks old) were immunized by neck subcutaneous injections of the salivary mucin of the blood group A-individual with Adjuvant Complete Freund (Difco Laboratories, Detroit, MI) to obtain anti-A-antigen, which is attached to protein. One week later, secondary intraperitoneal immunization was performed. The booster injection was given intraperitoneally 2 days before spleen cells were recovered. The spleen cells were fused with mouse myeloma P3U1 cells using polyethylene glycol (Mr 4,000); the hybridomas were grown in RPMI 1640 medium with hypoxanthine, aminopterin and thymidine selection medium supplement (Sigma). The culture supernatants were screened using ELISA for binding to the synthetic A-antigen.
Preparation of Fab Fragment
Papain digestion was performed to obtain the Fab fragment using Immobilized Papain (Pierce Biotechnology Inc., Rockford, IL). The 10 mg of anti-A antibody was added to a final concentration of 20 mM cystein·HCl and 10 mM EDTA. The 1.0 mL of sample was mixed with the 0.25 mL of Immobilized Papain and incubated overnight in a shaker at 37°C. After separating the immobilized enzyme from the product by 0.22-μm filter, the whole IgG that was not disassembled by the enzyme was removed using Amicon Ultra 100 K (Millipore Corp., Tokyo, Japan).
The RBCs or A431 cells were incubated with purified monoclonal antibodies or mice sera (diluted in phosphate-buffered saline [PBS]) for 30 min at 4°C. After washing with PBS, diluted secondary antibodies (1:1000): fluorescein-conjugated goat IgG fraction to mouse IgM; μ chain-specific (MP Biomedicals, Solon, OH) or Oregon Green 488 goat anti-mouse IgG(H+L) (Invitrogen Corp.) were reacted for 30 min at 4°C. Fluorescence data were collected using a flow cytometer (FACS Caliber; BD Biosciences, Braintree, MA).
Enzyme-Linked Immunosorbent Assay (ELISA)
The A-trisaccharides conjugated to bovine serum albumin (BSA) (Calbiochem Novabiochem Corp., San Diego, CA), B-trisaccharides conjugated to BSA (Dextra Laboratories, Reading, UK), and H-disaccharides conjugated to BSA (Dextra Laboratories) were independently coated onto Nunc-immuno plates (Nunc, Roskilde, Denmark) at 1 μg/mL for 1 hr. After blocking with 1% BSA, the plates were incubated with purified monoclonal antibodies or mice sera for 1 hr. After washing with 0.05% Tween 20 in PBS, the plates were incubated with HRP anti-mouse IgG (Dako, Glostrup, Denmark) or peroxidase-conjugated affinipure donkey anti-mouse IgM, μ chain specific (Jackson Immuno Research Laboratories Inc., Suffolk, UK) for 1 hr. After subsequent washing, the enzymatic reaction was conducted with 1-Step Ultra TMB-ELISA (Pierce). After the reaction was stopped with 1 M H2SO4, the optical density was measured at 450 nm using an auto-plate reader. These reactions were performed with a volume of 50 μL at room temperature.
Immunohistochemical staining was performed using the avidin-biotinylated immunoperoxidase method. In brief, 5-μm sections were deparaffinized and rehydrated. All tissues were then exposed to 3% hydrogen peroxidase for 5 min. Then, K7508, its Fab fragment (K7508Fab), or rabbit anti-A polyclonal antibody (Wako Pure Chemical Industries Ltd.) was added to the sections for 30 min at room temperature. Biotinylated secondary anti-mouse IgG (Dako) or anti-rabbit IgG (Dako) was incubated for 30 min at room temperature followed by the peroxidase-conjugated biotin-streptavidin complex (Vectastain ABC Kit; Vector Laboratories Inc., Burlingame, CA) for 30 min at room temperature. Color was developed using 3,3-diaminobenzidine tetrahydrochloride tablet sets (Dako) for 1 min. The sections were counterstained with Mayer's hematoxylin.
Immunization of A431 Cells
The A-antigen-expressing cells, A431, were coated with K7508 or its Fab fragment for 30 min at 4°C. Mice were immunized by intraperitoneal injections of A-antigen-expressing cells mixed with the Imject Alum (Pierce).
Results are expressed as the mean ± standard deviation. Student's t test was used to determine significance among the groups. A value of P<0.05 was considered significant.
Specificity Definition of a Novel Monoclonal Antibody to the Blood Group A-Antigen
After generating a monoclonal antibody, K7508, to the blood group A-antigen, we first characterized its specificity to the A-antigen by flow cytometric analysis. Both K7508 and its Fab fragment (K7508Fab) demonstrated specificity for the blood group A-antigen by strong binding with A-RBC (Figs. 1A and 1B) as well as A431, a cell line that expresses the blood group A-antigen (Figs. 1G and 1H). Specificity of K7508 or K7508Fab for the A-antigen was shown by the lack of reactivity with O-RBC (Figs. 1E and 1F) and weak reactivity with B-RBC (Figs. 1C and 1D). The specificities of K7508 and K7508Fab were also characterized by ELISA (Fig. 2). The data also show that specificity is directed to the blood group A-antigen (trisaccharide) and not to the B- or O-antigens (tri- or disaccharides, respectively). K7508 is of the IgG1 subclass (data not shown). Immunostaining studies revealed that K7508 reacted strongly with RBCs and endothelia of capillaries and arteries of liver from a blood group A individual but did not react with those tissues from B or O-individuals (Fig. 3).
Blood Group A-Antigen Neutralization by Anti-A Antibody (K7508) in Vitro
We next investigated whether A-antigen on cells were neutralized by K7508 in flow cytometric analyses or immunohistochemistry. Results of flow cytometric analyses indicated that A-RBCs coated with K7508 were not detected by another anti-A antibody (A-IgM): A-antigen on A-RBCs was masked by K7508 in vitro (Fig. 4A). In the same way, the reactivity of A-IgM was diminished after A431 cells were coated with K7508 (Fig. 4C). It was noteworthy that K7508Fab also inhibited A-IgM-binding to A-antigen on A-RBCs and A431 (Figs. 4B and 4D), indicating that K7508Fab had similar binding activity to that of K7508 and masked A-antigen on the cells. In immunohistochemistry, polyclonal anti-A antibodies recognized RBCs and endothelia of capillaries and arteries of blood group-A liver (Fig. 5A). By contrast, the liver tissues coated with K7508 or K7508Fab were not stained by polyclonal rabbit anti-A antibody (Fig. 5B and 5C), indicating that A-antigen on the endothelia of the liver tissues was masked by K7508 or K7508Fab. These results suggest that K7508 and K7508Fab masked A-antigen on tissues and might be useful for therapeutic use in vivo.
Suppression of Anti-A Antibody Production in Mice by Neutralization of A-Antigen With K7508 or K7508Fab
We next investigated whether K7508 or K7508Fab suppress the anti-A antibody production by neutralizing A-antigen in vivo. We first immunized mice with A431 cells after coating with K7508 by intraperitoneal injections. This in vivo model might mimic organ transplantation because the A-antigen of the donor graft induces the production of anti-A antibodies. Flow cytometry (Fig. 6A) showed that sera from mice immunized by A431 coated with K7508 (S-A431-K7508) reacted to A-RBCs more weakly than sera from mice immunized with non-coated A431 (S-A431), suggesting that S-A431-K7508 contained a lower concentration of anti-A antibodies. These results indicate that K7508 neutralizes A-antigen on A431 cells and that the production of mouse anti-A antibodies is inhibited in vivo. Furthermore, sera from mice immunized by A431 coated with K7508Fab (S-A431-K7508Fab) reacted weakly to A-RBCs (Fig. 6B), indicating that K7508Fab also possesses neutralizing activity against A-antigen.
We next investigated the concentrations of mouse anti-A antibodies in S-A431-K7508, S-A431-K7508Fab, and S-A431 using ELISA with A-trisaccharides (Fig. 6C). The concentration of anti-A antibodies increased gradually in S-A431 for three weeks. In contrast, that of S-A431-K7508 slightly increased for one week, and then stopped increasing from one week to three weeks. Similarly, the concentration of anti-A antibodies of S-A431-K7508Fab stopped increasing after one week. These results suggest that A-antigen on A431 cells was neutralized by K7508 or K7508Fab.
In this study, we produced a novel antiblood group A antibody, K7508, for developing a novel therapeutic strategy for the ABO-incompatible organ transplantation. The major barrier to ABO-incompatible organ transplantation is humoral rejection, which is known to be triggered by antigen-antibody complex reaction (2, 5, 6). For that reason, the reduction of antibodies in plasma or the suppression of antibody production has been performed to overcome ABO-incompatible transplantation (15–17). However, to date, the suppression of blood group antigen has not been performed clinically, probably because in vivo study about suppression of blood group antigen is very difficult. To the best of our knowledge, this study is the first one to use in vivo model which mimics ABO-incompatible transplantation.
In vitro study, we performed neutralizing experiments by K7508 using both A-antigen-expressing cells such as A-RBC or A431 and human liver tissues. A-antigen-expressing cells are used for application to in vivo study that mimics ABO-incompatible transplantation; on the other hand, human liver tissues were used for discussing the clinical application. A-antigen on A-RBC or A431 were completely masked by anti-A antibody, K7508 (Fig. 4), suggesting that K7508 is also applicable to in vivo study: whether antibodies against A-antigen-possessing graft are suppressed or not. K7508 reacted with A-antigen stronger than other anti-A antibodies in ELISA (data not shown). Furthermore, we speculate that these anti-A antibodies recognize the similar epitope of A-antigen; therefore other anti-A antibodies did not react with A-antigen after epitope of A-antigen was blocked by K7508. Immunohistochemical analyses showed that A-antigen is highly expressed in endothelia of capillaries and arteries of blood group-A liver (Fig. 3). Importantly, K7508 completely blocked the reactivity of sequentially applied other anti-A antibodies against endothelia of capillaries and arteries (Fig. 5). An in vivo study with the use of human liver tissues is ethically difficult to perform; therefore, we moved on to in vivo study using A-antigen-possessing cells.
The Fc portion of IgG is involved in antibody-dependent cell-mediated cytotoxicity and complement-dependent cell cytotoxicity. The classical pathway, which is one of complement activation, is triggered by the binding of the complement component C1q to the antigen-antibody complex. In the previous study, mouse RBCs coated in vitro with F(ab′)2 fragment of mouse RBC-specific antibody, when transfused into mice, survived longer in circulation than RBCs coated with whole IgG of mouse RBC-specific antibody (21). Therefore, we prepared the Fab fragment of K7508 (K7508Fab) to prevent the antibody-dependent cell-mediated cytotoxicity or complement-dependent cell cytotoxicity by K7508 for in vivo study. As shown in Figures 1–3, K7508Fab was also useful for flow cytometry, ELISA, and immunohistochemistry. As shown in Figure 6, when A-antigen-expressing cells were coated with K7508Fab, the production of anti-A antibody was also reduced in vivo. These findings suggest that A-antigen was neutralized by K7508Fab, and that A-antigen lost its antigenecity. In vitro study, K7508Fab completely blocked the reactivity of sequentially applied another anti-A antibodies against endothelia of capillaries and arteries (Fig. 5). Taken together, K7508Fab may be clinically applicable in ABO-incompatible organ transplantation.
Using A2 donors, who have less A-antigen than A1 donors, has brought success in ABO-incompatible liver transplantation and kidney transplantation (22–24). Fishbein et al. reported that transplantation of blood group A2 livers into blood group O recipients is safe and can be performed without graft loss and without regard to anti-A titer level (24). They concluded that the transplantation of A2 livers into O recipients can partially compensate for the more frequent use of O livers in recipients from other blood groups. Consequently, it is not necessary to neutralize blood group antigen completely, and masking the part of blood group antigen might turn around the outcome of ABO-incompatible transplantation. Moreover, the feature of this method is the specific treatment against blood group antigen. Therefore, broad and intensive immunosuppression, which causes severe infection and posttransplant lymphoproliferative disorder, might not be necessary (25, 26). In this study, we used only anti-A-antigen antibodies; however, we are also planning to produce anti-B-antigen antibodies, which may lead to a clinical use.
In conclusion, we produced a novel anti-A antibody: K7508. Production of anti-A antibody was suppressed significantly in vivo when blood group A-antigen-expressing cells were coated with K7508Fab. In the future, this neutralizing procedure might become an effective strategy to control humoral rejection related to ABO-incompatible solid organ transplantation.
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