In this study, we screened for a 7-mer BATP, RPRNPNK, for use in developing a novel therapeutic strategy for ABO-I KTx. It is well known that the major barrier to ABO-I KTx is humoral rejection triggered by antigen–antibody reactions (19–21). Serum antibody reduction and antibody production suppressive treatments have been used to overcome ABO-I KTx (22–24). However, blocking of ABO histo-blood group antigens has never been tested clinically because ABO histo-blood group antigen-blocking tests are very difficult to perform in vivo. To the best of our knowledge, this study is the first use of an ex vivo human model that mimics ABO-I KTx.
We performed ABO blood group antigen-neutralizing experiments with BATP using ELISA, HA assays, and human kidney tissues. ELISA and HA assays were performed in ex vivo experiments that mimic ABO-I KTx. Human kidney tissues were used for assessing possible clinical applications. A and B trisaccharide BSA on plates and A and B blood group antigens on RBCs were significantly blocked by BATP (Fig. 1F). BATP bound to peritubular and microvascular endothelia in type A and B kidney tissues but not in type O kidney tissues (Fig. 2), suggesting that BATP specifically recognizes both the A antigen [GalNAc(α–3)(Fuc(α1–2))Gal-] and the B antigen [Gal(α1–3)(Fuc(α1–2))Gal-] structures but not the H antigen [Fuc(α1–2)Gal] structure. Immunohistochemical analyses showed that A and B histo-blood group antigens are highly expressed in glomerular capillaries and peritubular and microvascular endothelia in type A and B kidney tissues. BATP significantly inhibited the reactivity of anti-blood group antibodies against ABO histo-blood group antigens expressed in glomerular capillaries as well as peritubular and microvascular endothelia (Fig. 3). Takasi et al. (25) reported that the ABO histo-blood group antigens in kidney tissues are different from those on RBCs because of differences in carrier proteins. Therefore, we speculate that BATP may recognize A and B trisaccharide epitopes and inhibit ABO histo-blood group antigen–antibody interactions by masking their trisaccharide structures.
Because in vivo experiments using human kidney tissues are ethically difficult, we performed an ex vivo study using normal tissue sections of kidneys excised from renal cell carcinoma patients. The results of ex vivo perfusion with BATP strongly suggested that BATP binds to ABO blood group antigens as an anti-blood group A/B antibody epitope mimetic that acts like an anti-A/B antibody and significantly suppressed IgM and IgG deposition in the glomerular capillaries. The A/B blood group antigens on RBC and in kidney tissues may be neutralized by BATP. These findings imply a lack of reaction between ABO histo-blood group antigens and de novo synthesized anti-A/B histo-blood group antibodies, which may contribute to long-term graft survival without rejection. There is evidence that this accommodation state is established within 2 weeks after transplantation (26–28). We therefore speculate that blocking of A and B antigens in donor organs by BATP administration during the first 2 weeks after transplantation (i.e., until accommodation has most likely occurred) is a reasonable and practical strategy. Ex vivo perfusion of the donor organ during cold storage and before transplantation is also possible. Neutralization of blood group antigens by BATP may represent one strategy for overcoming the challenges of ABO-I KTx. This alternative approach using a peptide may also be useful for minimizing antibody removal and anti–B-cell immunosuppression as adjuvant therapies in ABO-I KTx. We do not yet have data on in vivo peptide stability and systemic side effects. Further studies on ABO-I KTx using an animal model such as baboon (13, 14) are required to establish ABO antigen-blocking therapy using BATP.
MATERIALS AND METHODS
Construction of T7 Phage-Displayed 7-Mer Peptide (X7) Library
Seven-mer peptide (X7) libraries were constructed using the T7Select 415-1b vector as described by Krumpe et al. (29). In brief, random oligonucleotide insert DNA was synthesized as 5′-AACTGCAAGCTTTTA-(MNN)7-ACCACCACCAGAATTCGGATCCCCGAGCAT-3′, where N was a mixed equimolar ratio of each nucleotide and M was a mixed equimolar ratio of adenine and cytosine nucleotides. Amino acid translation of the complementary nucleotide sequence was MLGDPNSGGGX7. The insert DNA was incubated with a complementary extension primer (5′-ATGCTCGGGGATCCGAATTCTGGT-3′), Klenow enzyme (Takara Bio, Shiga, Japan), and deoxyribonucleotide triphosphates to form the complementary DNA strand. The mixed product was digested with EcoRI and HindIII (New England Biolabs, Ipswich, MA) followed by phenol/chloroform extraction and ethanol precipitation using standard techniques. The purified fragments were ligated into the predigested T7Select 415-1b vector using the DNA Ligation Kit “Mighty Mix” containing T4 DNA ligase (Takara Bio). This method inserts the randomized oligonucleotide library DNA in-frame following amino acid 348 of the capsid 10B gene. The ligation reaction mixture was incubated for 16 hr at 16°C and then subjected to in vitro packaging immediately followed by phage titration in a plaque assay. The remaining in vitro packaging solution was amplified once using BL21 cells until lysis. The lysate was centrifuged, titered, and frozen at −80°C in 0.5 M NaCl as a glycerol stock.
Library Biopanning Procedure
Aliquots (100 μL) of Tris-buffered saline (TBS) containing 10 μg blood group A/B trisaccharide BSA (V-Labs, Covington, LA) were added to wells with high-binding capacity (BD Falcon, Franklin Lakes, NJ) and incubated at 4°C overnight. The wells were washed three times with 300 μL TBS and blocked for 1 hr at 4°C with 5% BSA and 5% normal goat serum (NGS) in TBS. The BSA/NGS-blocked blood group A/B trisaccharide BSA-coated wells were washed three times with 200 μL TBS containing 0.5% Tween 20 (TBST); then, 100 μL library phage was applied to each well. The plates were incubated at room temperature (RT) for 30 min with orbital shaking at 250g followed by washing 10 times with 200 μL TBST. Bound phages were eluted by incubation with 100 μL TBS containing 100 mM blood group A/B trisaccharide per well at RT for 20 min, with orbital shaking at 250g. The eluate was collected, and 10 μL were removed for titering by a plaque assay. The remaining eluted phages were amplified in 20 mL freshly prepared BL21 cells in baffled shaker flasks by incubating at 37°C for 3 hr. After centrifuging the inoculated phages at 3400g at 4°C for 15 min, 100 μL of the supernatant were subjected to the next round of biopanning. The second, third, and fourth rounds of biopanning were conducted in the same manner. For each round, the plaque-forming units (PFU) input was held constant (as determined by the plaque-forming assay) to keep the ratio of phage particles to target molecules approximately constant at 3.0×1010 PFU throughout biopanning.
Clone Isolation and DNA Sequence Analysis
In the plaque-forming assay, the eluted clones were plated at a concentration of approximately 75 PFU per 100 mm plate to ensure well-isolated plaques. Each plaque was lifted with an inoculation needle and placed into 500 μL BL21 cells in a plastic test tube. The tubes were incubated at 37°C with orbital shaking at 250g for 3 hr. The NaCl concentration of the lysate was adjusted to 0.5 M followed by centrifugation at 3400g at 4°C for 15 min. A 450 μL aliquot of clarified lysate was transferred to a 1.5 mL tube and stored at 4°C. The following components were used for PCR: Phire Hot Start DNA Polymerase (Finnzymes, Vantaa, Finland), sterile molecular biology grade water, T7 Up primer (10 μM in TE buffer, pH 8.0), and T7 Down primer (10 μM in TE buffer, pH 8.0). The primers were synthesized on a 1 μM scale and cartridge purified. The sequences were as follows: T7 Up, 5′-AGCGGACCAGATTATCGCTAA-3′ and T7 Down, 5′-AACCCCTCAAGACCCGTTTA-3′. Clarified phage lysate (1 μL) was added to each tube, and the T7 insert was amplified in a 30-cycle PCR. The PCR product was electrophoresed and purified using a QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) and sequenced using the T7 Up primer at the Sigma Genosys facility. The DNA and translated amino acid sequences were analyzed using Geneious Pro version 5.0.3 (Biomatters, Auckland, New Zealand).
Inhibitory Effect of Blood Group Antigen-Targeting Peptides on Hemagglutination of Human Red Blood Cells
RBCs of A and B blood groups were isolated from healthy volunteers. Anti-A (Z2A), anti-B (Z5H-2), and anti-H (87-N) mAbs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). HA assays were performed by the following microtitration method. Anti-A/B antibodies (100 μg/mL, 35 μL) were diluted in two serial steps and mixed with an equal volume of 0.5% human RBCs preincubated for 30 min at RT with 500 μg/mL control peptide or BATPs (ARARKTG, ASNKRPR, RPRNPNK, SPARRPR, RMSRKLP, GGKTRSK, or SRSSTRK). After incubation at RT for 2 hr, the reciprocal HA titer was determined as 2n.
Inhibitory Effect of Blood Group Antigen-Targeting Peptides on the Binding of Anti-A/B Antibodies to A/B Antigens Measured by Enzyme-Linked Immunosorbent Assay
Blood group A/B trisaccharide BSA-coated wells (5 μg/well) were washed three times with 300 μL TBS and blocked overnight at 4°C with 5% BSA/NGS in TBS. The wells were then treated with 100 μL of 0 to 500 μg/mL BATP (RPRNPNK) and incubated at RT for 1 hr. The wells were washed with 300 μL TBST followed by the addition of 100 μL anti-A (Z2A) or anti-B (Z5H-2) antibodies (1:1000 dilution in TBST) and incubated at RT for 1 hr. The wells were washed with 300 μL TBST, and 100 μL horseradish peroxidase (HRP)–labeled goat anti-mouse IgM antibody (1:10,000 dilution in TBST) was added followed by incubation at RT for 30 min. After the secondary antibody reaction, 100 μL tetramethylbenzidine peroxidase substrate (Funakoshi) was added followed by incubation at RT for approximately 3 min. The reaction was stopped with 100 μL of 1 N HCl, and optical density was measured at 450 nm.
Cytotoxicity of Blood Group Antigen-Targeting Peptides With Human Kidney Glomerular Capillary Microvascular Endothelial Cells
The blood type O HGMEC was obtained from the Applied Cell Biology Research Institute (Kirkland, WA) and grown in CSC complete serum-free medium with penicillin, streptomycin, and culture boost R at 37°C with 5% CO2. Blood type O HGMEC was cultured until near confluent and then cells were incubated with 1 mg/mL control peptide or BATP at 37°C with 5% CO2 for 24 hr. After treatment with BATP, cells were labeled with 5 μM CellEvent caspase-3/7 green detection reagent (Life Technologies, Carlsbad, CA) and with Hoechst 33342 (1 μg/mL in phosphate-buffered saline) in complete medium at 37°C. Stained cells were observed under fluorescence microscopy (floid cell imaging station, Life Technologies) at each time point.
Human Kidney Tissues
Normal human kidney tissues were obtained from renal tumor patients who underwent radical nephrectomy at the Department of Urology, Hirosaki University Hospital (Hirosaki, Japan). After routine radical nephrectomy for renal tumors, small sections of normal kidneys were removed and subjected to ex vivo perfusion for the BATP experiment. Informed consent was obtained from all patients before the initiation of the study. This study was approved by the Ethics Committee of the Faculty of Medicine, Hirosaki University. The study was performed in accordance with the Guidelines of the Declaration of Helsinki.
The normal kidney sections were fixed in formalin for hematoxylin–eosin and immunohistochemical staining. The deparaffinized sections were then exposed to 3% hydrogen peroxidase for 5 min. After washing with phosphate-buffered saline, expression of ABO histo-blood group antigens was examined using anti-A (Z2A), anti-B (Z5H-2), or anti-H (87-N) mAbs and HRP-labeled anti-mouse IgM antibody or Alexa 488–labeled anti-mouse IgM antibody. Binding of BATP (RPRNPNK) was examined using biotin-labeled BATP (200 μg/mL). Anti-blood group antigen antibodies and BATP were added to the kidney sections and incubated overnight at RT. The sections were then counterstained with hematoxylin and appropriately mounted. To examine the blood group antigen-blocking effect, 200 μg/mL control peptide or BATP (RPRNPNK) was added to the kidney sections followed by incubation for 30 min at 25°C before the first antibody staining. Stained tissues were observed and images collected by fluorescence microscopy (EZ-9000, Keyence, Osaka, Japan).
Ex vivo Perfusion With Blood Group Antigen-Targeting Peptides
The normal excised type A and B kidney tissues were perfused by gravity flow with normal saline and subsequently with 200 μg/mL control peptide or BATP and washed with normal saline. Next, the type A and B kidney tissues were perfused with 1 mL type B and A blood, respectively, followed by perfusion with normal saline. Ex vivo blood reperfusion performed within 1 min after blood withdrawal. Blood clotting formation in the glomeruli after ABO-I blood reperfusion was analyzed by immunohistochemistry and immunofluorescence. Ex vivo perfusion experiments using both blood type A and B kidneys were repeated in triplicate.
Results are expressed as mean±standard deviation. Student’s t test was used to determine the significance of differences between the groups. P<0.05 was considered statistically significant.
The authors thank Drs. Kazuyuki Mori and Shigeru Tsuboi for useful suggestions and comments.
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Keywords:© 2013 Lippincott Williams & Wilkins, Inc.
ABO-incompatible kidney transplantation; Blood group antigen; Blood group antigen-targeting peptide; Random peptide library-displaying T7 phage