SLAS Discovery The level of cytokine secretion was determined by cyto- Table 2. Summary of Phage Library Biopanning against kine-specific ELISAs with culture supernatants from lym- Glycoprotein-2 phocytes restimulated with the Ags for 24 h as described Input Phages previously. Cytokine concentrations were calculated from Round Phages(pfu) Enrichment" the plotted standard curves of serial dilutions of the recom- binant cytokine and expressed as mean t standard error First round ×10 2.3x10 2.3×10 (SE)(ng/mL)of each group. To measure the number of Second round 5x10% antigen-specific cytokine(interferon [INFIY, interleukin Third roun 2.0x [IL1, IL-5 and IL-6)secreting cells, enzyme-linked Fourth round 4x10 3.7×105 9.3×10 lospot(ELISPOT) assays were performed with lym- Enrichment is expressed as the relative percentage of the total number phocytes isolated from spleens and PPs as described of phages recovered compared to the input phages Table 3. Binding Affinity(K, values) of Selected Peptides Ag-Specific Lymphocyte Proliferation Ag-specific lymphocyte stimulation was determined using Sequence K, (HM) Dojindo CCK-8 reagent(Dojindo Laboratories, Kumamoto, HPNYDHDRMHTQGb-I16/50(32)0.068 Japan). Purified lymphocytes were plated in flat-bottom FRVIYTDMPGAH Gb-2 9/50(18) 0250 96-well plates at a density of 5 x 10- cells per well and SMQYADHPVNTG Gb-3 13/50(26) simulated with recombinant EGFP(1 mg/mL) or PBS (negative control) at 37C in a 5%Co, incubator. Plates were incubated for 72 h and pulsed with 10 uL CCK-8 obtained seven different amino acid sequences and named reagent(Dojindo Laboratories) per well for another 4 h. them GP-2 binding peptides(Gb-l to Gb-7), of which three Absorbance was measured at 450 nm, and the stimulation sequences appeared more frequently than the others and index(SI)was calculated as the ratio of the average ODsn were selected for further characterization. SAROTUP scan- value of wells containing antigen-stimulated cells to the ning results showed that none of these sequences have been average OD4so value of wells containing cells stimulated reported before for other targets or contain any known TUP with PBs. All assays were performed in triplicate motif or confer propagation advantages. Alignment analysis using Insight II showed that no conserved structural fea Statistical Analysis tures exist among the selected peptides The results are expressed as mean+ SE(SEM) using Microsoft Excel 2016(Microsoft Corp, Redmond, WA), Selected Peptides Bind to GP-2 with High Affinity and at least three independent experiments were performed The ability of the selected peptides ligands to bind to GP-2 unless otherwise stated. An unpaired Student t test(two- was measured in terms of dissociation constant(K)values nificant nonlinear regression analysis and Scatchard transformation using GraphPad Prism 6. The binding of peptides to GP-2 Results was specific and hyperbolic. All three peptides showed K values in the nanomolar range: Gb-1(K-68 nM), Gb-2 Biopanning of Phage Library (K,=250 nM), and Gb-3(K,=272 nM)as shown in Table To select peptide ligands against mucosal transcytotic 3. The binding pattern of these peptides to GP-2 measured receptor GP-2, we screened a 12-mer peptide phage display by saturation binding assay was consistent with the binding library against recombinant GP-2 as described in Materials pattern measured by phage-binding ELISA. Competition Ind Methods. Four rounds of biopanning were performed, ELISA results showed that the selected peptides do not bind and comparatively stringent conditions were applied in to any common or overlapping epitope on GP-2 each round to ensure enrichment in favor of GP-2. Phage titer increased more than 4000-fold from the first round to Binding of Ligand-Fused EGFPs to GP-2 and the fourth round (Table 2). The overall enrichment of the Transcytosis to M Cells library suggests that enrichment for selectively binding phage was achieved. After four rounds of biopanning, 3.7x To test the GP-2 targeting ability of selected peptides, we 10 phages were recovered and 50 plaques were randomly expressed peptide-fused EGFPs and analyzed their binding picked for sequence determination. From 50 plaques, we to M cells on PPs. We performed ligated gut loop assay and4 SLAS Discovery The level of cytokine secretion was determined by cyto￾kine-specific ELISAs with culture supernatants from lym￾phocytes restimulated with the Ags for 24 h as described previously. Cytokine concentrations were calculated from the plotted standard curves of serial dilutions of the recom￾binant cytokine and expressed as mean ± standard error (SE) (ng/mL) of each group. To measure the number of antigen-specific cytokine (interferon [INF]–γ, interleukin [IL]–4, IL-5 and IL-6) secreting cells, enzyme-linked immunospot (ELISPOT) assays were performed with lym￾phocytes isolated from spleens and PPs as described previously.15 Ag-Specific Lymphocyte Proliferation Ag-specific lymphocyte stimulation was determined using Dojindo CCK-8 reagent (Dojindo Laboratories, Kumamoto, Japan).18 Purified lymphocytes were plated in flat-bottom 96-well plates at a density of 5 × 105 cells per well and stimulated with recombinant EGFP (1 mg/mL) or PBS (negative control) at 37 °C in a 5% CO2 incubator. Plates were incubated for 72 h and pulsed with 10 µL CCK-8 reagent (Dojindo Laboratories) per well for another 4 h. Absorbance was measured at 450 nm, and the stimulation index (SI) was calculated as the ratio of the average OD450 value of wells containing antigen-stimulated cells to the average OD450 value of wells containing cells stimulated with PBS. All assays were performed in triplicate. Statistical Analysis The results are expressed as mean ± SE (SEM) using Microsoft Excel 2016 (Microsoft Corp., Redmond, WA), and at least three independent experiments were performed unless otherwise stated. An unpaired Student t test (two￾tailed) was used to compare groups, and p values <0.05 were considered statistically significant. Results Biopanning of Phage Library To select peptide ligands against mucosal transcytotic receptor GP-2, we screened a 12-mer peptide phage display library against recombinant GP-2 as described in Materials and Methods. Four rounds of biopanning were performed, and comparatively stringent conditions were applied in each round to ensure enrichment in favor of GP-2. Phage titer increased more than 4000-fold from the first round to the fourth round (Table 2). The overall enrichment of the library suggests that enrichment for selectively binding phage was achieved. After four rounds of biopanning, 3.7 × 105 phages were recovered and 50 plaques were randomly picked for sequence determination. From 50 plaques, we obtained seven different amino acid sequences and named them GP-2 binding peptides (Gb-1 to Gb-7), of which three sequences appeared more frequently than the others and were selected for further characterization. SAROTUP scan￾ning results showed that none of these sequences have been reported before for other targets or contain any known TUP motif or confer propagation advantages. Alignment analysis using Insight II showed that no conserved structural fea￾tures exist among the selected peptides. Selected Peptides Bind to GP-2 with High Affinity The ability of the selected peptides ligands to bind to GP-2 was measured in terms of dissociation constant (Kd ) values of the synthetic peptides. Kd values were determined by nonlinear regression analysis and Scatchard transformation using GraphPad Prism 6. The binding of peptides to GP-2 was specific and hyperbolic. All three peptides showed Kd values in the nanomolar range: Gb-1 (Kd = 68 nM), Gb-2 (Kd = 250 nM), and Gb-3 (Kd = 272 nM) as shown in Table 3. The binding pattern of these peptides to GP-2 measured by saturation binding assay was consistent with the binding pattern measured by phage-binding ELISA. Competition ELISA results showed that the selected peptides do not bind to any common or overlapping epitope on GP-2. Binding of Ligand-Fused EGFPs to GP-2 and Transcytosis to M Cells To test the GP-2 targeting ability of selected peptides, we expressed peptide-fused EGFPs and analyzed their binding to M cells on PPs. We performed ligated gut loop assay and Table 2. Summary of Phage Library Biopanning against Glycoprotein-2. Round Input Phages (pfu) Recovered Phages (pfu) Enrichmenta First round 1 × 1011 2.3 × 105 2.3 × 10–6 Second round 5 × 109 5.3 × 105 1.1 × 10–4 Third round 1 × 107 2.0 × 104 2.0 × 10–3 Fourth round 4 × 107 3.7 × 105 9.3 × 10–3 a Enrichment is expressed as the relative percentage of the total number of phages recovered compared to the input phages. Table 3. Binding Affinity (Kd Values) of Selected Peptides. Sequence Name Frequency, No. (%) Kd (µM) HPNYDHDRMHTQ Gb-1 16/50 (32) 0.068 FRVIYTDMPGAH Gb-2 9/50 (18) 0.250 SMQYADHPVNTG Gb-3 13/50 (26) 0.272