Identification Of Peptide Mimetics Of Xenoreactive α-Gal Antigenic Epitope By Phage Display

The prevention of interaction between the human natural anti-Gal antibody and the a-Gal antigen epitope expressed on pig cells is the key to success in xenotransplantation . Hyperacute rejection induced by the interaction of human natural anti-Gal antibody with the a-Gal antigen on the xenograft cells was a major immune barrier in xenotransplantation. The phage display technology offers an effective strategy for selecting peptides which can interact with the anti-Gal antibody to block a-Gal antigen binding site. Two peptide libraries, linear 7 peptide library and C7C library , were panned on the anti-B monoclonal antibody which have the characteristic of binding to the a-Gal antigen epitope. After four rounds of panning, 22 positive phage clones were selected. Highly homologous sequence PT and STL existed among these selected peptides. Stachyose competitive ELISA assays revealed that these peptides specifically bound to a-Gal antigen binding site. 8 peptide mimics of a-Gal epitope could inhibit the agglutination of pig Red blood cells mediated by human sera in a dose-dependent manner. These results demonstrated that the selected peptides can mimic the conformational structure of a-Gal antigen and have the therapeutic potential in xenotransplantation.1. Biopanning process of two librariesThe two peptide libraries, linear 7 peptide library and C7C peptide library, werecycled through four rounds of affinity selection. The immunosorbant plates were coated with Anti-B antibody in sodium carbonate buffer at 4 ℃ overnight, followed by blocking for 2 hour at 37 ℃ with bovine serum albumin(BSA) in Tris-buffered saline(TBS).The phage libraries(~1012 transducing uints) were added to the coated wells. After incubation for 1 hour at room temperature, the plates were washed 10 times with TBST(Tris-buffered saline containing 0.1% tween-20). The phages bound to the antibody were eluted with 0.1M Glycine-HCl buffer (PH2.2), then was neutralized by 1M Tris-HCl buffer (PH 9.1). The eluted phage was amplified by propagation in E.coli ER2738 for 4.5 hours and harvested by precipitation with PEG/NaCl. After four rounds of selection, approximate 17000-fold increase in the number of eluted phage for linear 7 peptide library and 20-fold for C7C peptide library were observed.2. Detection of the positive phage clones by ELISA assayThe 96-well immunsorbant plates were coated with Anti-B antibody at 4 ℃ overnight and blocked with 5% BSA for 2 hour at 37 ℃. -1012 phage particles of the selected clones were added to each well and incubated for 1 hour with shaking at room temperature. The plates were washed six times with TBST, then incubated with 100ul of horseradish peroxidase anti-M13 antibody conjugate diluted 1:5000 in TBS for 1 hour and were washed 6 times with TBST. The plates were developed with the substrate tetramethylbenlldine(TMB) and read at 450 nm. Assessing the different interaction of individual phage clones with antibodies and BSA, we determined the phage clones which specifically bind to antibodies, but not BSA. we isolated 16 phage clones from linear 7 peptide library and 6 clones from C7C peptide library which displayed anti-Gal antibody-binding specificity.3. The phage DNA sequencingThe positive phage clones selected by ELISA assay were sequenced to determine the amino acid sequence of peptides. The -96 primer 5'-CCCTCATAGTTA GCGTAACG-3' was used for antomated sequencing. Alignment of the sequences revealed a number of conserved residues existed in the selected peptides. There was a very clear consensus sequence PT among these petides from two libraries.4. Affinity measurement by stachyose competitive ELISA assayThe competitive ELISA assays were carried out on the 96-well immunsorbant plates coated with Anti-B antibody. 50ul of positive phage clones (-1012 transducing uints) and 50ul of serially diluted stachyose solution were added into each well, and incubated at room temperature for 1 hour. Then plates were washed and developed as the method described above. The inhibition rate was calculated by the formula:Inhibition rate= [A450(-s>A450(+s)]/ A450(-s)A450(-s) represent A450 without stachyose, A450(-i-s) is A450 with stachyose.The positive clones were examined by stachyose competitive ELISA assays in which phage-displayed peptides binding to the antibody was inhibited corresponding to the concentration of stachyose. This indicated the positive clones bound to antibody at the a-Gal antigen binding site.5. DTT reduction assay:The 96-well immunsorbant plates were coated with lOOul of Anti-B antibody at 4 1C overnight and blocked with 300ul of 5% BSA for 2 hour at 37X:. ~1012 phage particles of each positive clone incubated in TBS with 2mM DTT for 60 minutes. Negative controls were phage solution without DTT. Each clone was added to plates and incubated for 1 hour at room temperature. The plates were washed six times with TBST, incubated with lOOul of horseradish peroxidase anti-Mi3 antibody conjugate diluted 1:5000 in TBS for 1 hour, then were washed and developed as above. The absorbance was read at 450 nm. When the disulfide bond was disrupted by DTT, the binding capacity of the peptides was decreased to different levels, indicating that the suitable conformation of binding to the antibody were changed. In contrast, no obvious variation of OD450nm was observed with or without DTT for linear peptides.6. Peptide synthesisPeptides were synthesized using standard solid phase method. Purity and mass of each peptide were verified by liquid chromatography mass spectrometry(HPLC/MS).7. Inhibition assay of pig RBC agglutination.RBC agglutination assay was performed in U-shaped agglutination plates. Phage solutions were serially diluted in 40ul PBS. Each phage dilution was mixed with 40ul of 1% RBC which was washed 3 times and resuspend in PBS and 40ul of 1% human serum diluted in PBS. The mixtures were shook gently for 2 minutes and incubated for 1 hour. The agglutination of RBC was visible when RBC homologically distributed on the bottom of the wells to form blood dumps. In contrast, RBC precipitated as a small dot when no agglutination happened. The result indicated mat stachyose, positive phage clones and me synthetic peptides could inhibit RBC agglutination.In our experiment, eight peptide mimics of the a-Gal epitop were selected and identified from two peptide libraries. ELISA assays suggested mat these peptides bound specifically to anti-Gal antibody. It was further proved by stachyose competitive assay mat the phage-displayed peptides and stachyose competitively bound to the anti-Gal antibodies at the same binding site. The peptides displayed on the phage surface and the free synthetic peptides can inhibit the agglutination of pig RBC mediated by human natural anti-Gal antibody. These results revealed mat the peptides can mimic me structure of Gal-ol-3Gal at the molecular level and are able to replace a-Gal antigen to interact with the human natural anti-Gal antibody.