3501 YHCQGC(11) DYAVHP(2) YGCQGC(2) YFCOGC(1) YVCQGC(1) YXCQGC(1) YYCQGC(1) LWWYEW(1) LWWDCA(1) YAVHP 9 Phage display (common panning) 4 PMID:31434316 Anti-LDL (−) monoclonal antibody 1A3H2 Electronegative low-density lipoprotein, LDL (−) Not determined. Not determined. fUSE55-based X6 phage display library ELISA To prevent the selection of peptides binding to conserved domains of immunoglobulins,the anti-LDL(-) mAbs 1A3 and 2C7 were immobilized on microtiter plates and incubated in the presence of excess soluble, unrelated mouse immunoglobulin. 3502 CGEFLPORIC(3)[NA] CISTELLPSC(2)[NA] CIDFDLPGRC(2)[NA] CVSSEVLPSC(2)[NA] CMPSVILPSC(1)[0.818810±0.008400] CVSTVMLPSC(1)[NA] CISSVWLPSC(1)[NA] CYSSAMLPSC(1)[NA] CMSSEMLPSC(1)[NA] CVSSDVLPSC(1)[NA] CSSSAILPSC(1)[NA] CHPSAILPSC(1)[NA] CLSSFIIPSC(1)[NA] CSPSWLIPSC(1)[NA] CVPSVLLPSC(1)[NA] CLPSVFLPSC(1)[NA] CVPSYILPSC(1)[NA] CIPSMMLPSC(1)[NA] CLPSMILPSC(1)[NA] CWPSGVLPSC(1)[NA] CFTSVYLPSC(1)[NA] CDTSYLLPSC(1)[NA] CGDVLPSVSC(1)[NA] CGDTLPSVEC(1)[NA] CGSVLPSVVC(1)[NA] CGLYLPSVPC(1)[NA] CLDVFLPGRC(1)[NA] CFDVFLPGRC(1)[NA] CLDFELPGRC(1)[NA] CGESLPSRKC(1)[NA] CGVYLPSRLC(1)[NA] CVGKNFYPSC(1)[NA] CMWIPSGAMC(1)[NA] CLDIIPSGFC(1)[NA] CIQGDVIPSC(1)[NA] VLPS 35 Phage display (common panning) 4 PMID:31434316 Anti-LDL (−) monoclonal antibody 2C7D5F10 Electronegative low-density lipoprotein, LDL (−) Not determined. Not determined. fUSE55-based CX8C phage display library (CX8C) ELISA Absorbance at 450 nm was measured by spectrophotometry using a microplate reader (SynergyTM Mx, Biotek instruments Inc, Winooski, VT, USA). Data (OD450) shown were reproduced from Figure 1B in the reference. To prevent the selection of peptides binding to conserved domains of immunoglobulins,the anti-LDL(-) mAbs 1A3 and 2C7 were immobilized on microtiter plates and incubated in the presence of excess soluble, unrelated mouse immunoglobulin. P2C7 (CMPSVILPSC)) was quickly internalized by bone marrow-derived murine macrophages as shown by confocal microscopy. P2C7 increased the expression of TNFalpha, IL-1beta and iNOS as well as the secretion of TNFalpha, CCL2, and nitric oxide by murine macrophages, similar to the responses induced by LDL (-), although less intense. We identified a mimetic epitope associated with LDL (-), the P2C7 circular peptide, that activates macrophages. Our data suggest that this conformational epitope represents an important danger-associated molecular pattern of LDL (-) that triggers proinflammatory responses. 3503 CEVLPGRC(18) CMWLPGAC(4) GVDDGA(1) GRADIR(1) VLPS 4 Phage display (common panning) 3 PMID:31434316 Anti-LDL (−) monoclonal antibody 2C7D5F10 Electronegative low-density lipoprotein, LDL (−) Not determined. Not determined. ELISA To prevent the selection of peptides binding to conserved domains of immunoglobulins,the anti-LDL(-) mAbs 1A3 and 2C7 were immobilized on microtiter plates and incubated in the presence of excess soluble, unrelated mouse immunoglobulin. 3504 HFPFHHHKLRAH(9/20)[0.699] HPMHMLHKRQHG(5/20)[0.336] HPHWWQVFPKRT(4/20)[0.551] WPNLHHRFHTVR(2/20)[NA] 4 Phage display (common panning) 3 PMID:31733137 19-kDa fragment of Merozoite surface protein 1, MSP-I19 Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The binding affinity of each selected peptide clone to rPkMSP‐119 was validated using phage ELISA. The binding signals for each peptide were estimated using absorbance value at 405 nm towards rPkMSP‐119. Three rounds of biopanning were performed against purified PkMSP‐119 protein. After washing, bound phages were eluted by adding elution buffer 0.2 m Glycine–HCl (pH 2.2) supplemented with 1 mg/ml BSA into well, and the plate was gently rocked for 15 min at room temperature. The eluate was then neutralised with 1 m Tris‐HCl, pH 9.1. The second round of biopanning, 200 μl of 5e11 phage (eluates from first biopanning) was added into the PkMSP‐119 protein coated well and the same process was repeated as first biopanning. Four phage peptide variants, namely Pkd1 (HFPFHHHKLRAH), Pkd2 (HPMHMLHKRQHG), Pkd3 (HPHWWQVFPKRT) and Pkd4 (WPNLHHRFHTVR), that bound to PkMSP‐119 were identified from 20 randomly picked phage isolates in the third round of biopanning. Single Pro (P) and Arg (R) were detected among the three binding peptides (pkd1, pkd2 and pkd3). The sequences of both Pkd1 and Pkd2 consist of a large number of histidine residues. Pkd1 showed positive binding signal with 6.1× vs. BSA control. Docking results showed that Pkd1 and Pkd2 were ideal binding peptides for PkMSP‐119. 3505 CVRARTR(4.1%)[281±92,2.309867±0.004492] CLQKTPKQC(2.7%)[373±114,2.456670±0.006287] CVPRRGR(2.7%)[NA,2.163067±0.005392] CRPVRVR(2.7%)[NA,2.15684] CRIRDPRR(1.2%)[NA,2.28912] CTKRIR(1.2%)[NA,2.28912] CRRAR(1.2%)[NA,2.441107±0.009884] CKLRT(1.2%)[NA,2.750273±0.009884] 8 Phage display (subtractive panning, competitive panning) 3-5 PMID:32278214 Programmed cell death ligand-1 overexpressing HEK 293T cells Not determined. Not determined. Not determined. CX7C T7 phage display library Surface plasmon resonance (SPR) The binding affinity (KD, nM) of PD-L1-binding peptides was measured by the surface plasmon resonance (SPR) method using an SPR instrument (Reichert Technologies, Depew, NY) at 25 °C. The KD value was displayed in the first column of the affinity value. In addition, absorbance at 450 nm was measured by using a microplate reader (Tecan, Zurich, Switzerland). Data (OD450, the second column) shown were reproduced from Figure 1D in the reference. To subtract phage that non-specifically bind HEK 293T cells, the phage library, containing 1e9 plaque-forming units (pfu), was incubated with non-transfected HEK 293T cells at 4 °C for 1 h. Unbound phage were collected from the supernatant and incubated with PD-L1-transfected HEK 293T cells at 4 °C for 1 h. Bound phage was eluted by incubation with the Escherichia coli host BL21 for 10 min at 24 °C or room temperature (RT). The eluates were used for titration of pfu and for amplification in BL21. The biopanning procedure was repeated five times. Two selected peptides, CLQKTPKQC and CVRARTR (PD-L1Pep-1 and PD-L1Pep-2, respectively) that appeared to block PD-L1. PD-L1Pep-1 and PD-L1Pep-2 preferentially bound to high PD-L1-expressing cells over low PD-L1-expressing cells; binding was further enhanced by interferon-γ, an inducer of PD-L1 expression. Binding affinities of PD-L1Pep-1 and PD-L1Pep-2 were approximately 373 and 281 nM, respectively. Cellular binding of the PD-L1-binding peptides was reduced by silencing PD-L1 gene expression or competition with anti-PD-L1 antibody. PD-L1Pep-1 and PD-L1Pep-2 induced the internalization and downregulated cell surface levels of PD-L1. The PD-L1-binding peptides restored cytokine secretion and T-cell proliferation to cells inhibited by co-culture with tumor cells or culture on PD-L1-coated plates. Intravenously injected PD-L1Pep-1 and PD-L1Pep-2 efficiently homed to tumor tissues, inhibited tumor growth, and increased CD8+/FoxP3+ ratio in mice. The PD-L1-binding peptides in combination with doxorubicin or PD-L1-targeted liposomal doxorubicin inhibited tumor growth and increased CD8+/FoxP3+ ratio more efficiently than doxorubicin alone and untargeted liposomal doxorubicin, respectively. These results suggest that PD-L1Pep-1 and PD-L1Pep-2 block PD-L1 and reinvigorate T-cell activity, inhibiting tumor growth by enhancing anti-tumor immunity. 3506 KLWSIPTNFLLP(9/10) NAKYLPTILGRL(1/10) 2 Phage display (competitive panning) 3 PMID:32881706 Major allergen Can f 1 Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) Bound phages were eluted competitively in 100 μL solution of the respective free target (100 μg/mL Can f1 in TBS) on a rotating shaker at RT for 60 min. Panning procedure was repeated three times. The KLWSIPTNFLLP peptide corresponded to the antigen-binding site of a putative γδT-cell receptor. Additional biochemical investigations confirmed this interaction. 3507 YEHAMYRSAVLL[1.67] HDGTLLPRSSLH[2.16] TIGVVRDVATQL[2.06] ISTVYTGLTEKD[1.31] DSLTMKRLVLPY[1.58] RNVELHDALRRT[1.38] NSSTPMLSDVLR[1.70] NRVSDHLVRAVS[6.71] HAPMPYANWPRG[1.78] HVKAAYPMVTMS[3.42] 10 Phage display (common panning) PMID:31308251 Serum IgG of Systemic lupus erythematosus(SLE) patients Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA Optical density was read at 450 nm using a Behring EL311 ELISA microplate reader (Dade Behring Marburg Gmbh, Berlin, Germany). A typical four-parameter logistic nonlinear regression model was used for standard curve fitting for the ELISA, which was further used to estimate sample content (units/ml) from the absorbance measurement data. The concentration of IgG from the samples were measured with the commercial Human IgG ELISA Quantitation kits (eBioscience, Massachusetts) according to the instructions. Besides the serum samples, we also added 1 μg of an anti-6×His antibody (Millipore, California) and 10 μl PBS buffer to a 100 μl immunoprecipitation mix as positive control and negative control, respectively. About 1 × 109 M13-phage particles were added to 100 μl immunoprecipitation mix in a 96-well PCR plate. The plate was then carefully sealed with adhesive optical tape (Applied Biosystems, California) and rotated overnight at 4 °C. 10 μl of protein G Dynabeads (Invitrogen, Massachusetts) were then added to each well. The resealed plate was placed back on a rotator for 4 h at 4 °C. The beads were washed six times in 200 μl wash buffer (150 mm NaCl, 50 mm Tris-HCl, 0.1% NP-40 (pH 7.5)) by pipetting up and down eight times per wash. After removal of the wash buffer, the beads were resuspended in 30 μl ddH2O and heated at 95 °C for 10 min. To discover biomarkers for Systemic lupus erythematosus(SLE),a phage displayed random peptide library (Ph.D. 12) and deep sequencing were applied to screen specific autoantibodies in a total of 100 serum samples from 50 SLE patients and 50 healthy controls. Then, four peptides (YEHAMYRSAVLL, HDGTLLPRSSLH,TIGVVRDVATQL, and ISTVYTGLTEKD) were discovered with high diagnostic power to differentiate Systemic lupus erythematosus(SLE) patients from healthy controls. Among them, two peptides, i.e. YEHAMYRSAVLL and HDGTLLPRSSLH, were confirmed between SLE with other autoimmune patients. 3508 LTPMPNW(3)[1.448491±0.940644] MMSYPKH(2)[2.745750±0.669996] MMTLPNN(1)[3.233583±0.013649] MAREMSY(1)[3.147363±0.008307] VLAPPCW(1)[2.65617] 5 Phage display (common panning) 3 PMID:31255726 ArsS N-terminal (ArsS-N) acid-sensing domain Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) ELISA OD450nm was assessed using a microplate reader (T100, Bio-Rad, Hercules, CA, USA). Data (OD450) shown were reproduced from Figure 4 in the reference. To identify peptides that antagonize the acid-sensing domain of H. pylori ArsS. Phage clones were selected by solution-phase panning with protein G magnetic beads (NEB) that exhibited high affinity for purified recombinant Helicobacter pylori ArsS N-terminal acid-sensing protein (P-ArsS-A). The phages were reacted with the magnetic beads at room temperature for 2 h, which were then washed five times with 0.05% PBS with Tween 20 (PBST) for 5 min. To elute the bound phages, 100 μl 0.2M glycine-HCl (pH 2.2) was added for 8 min. Subsequently, 15 μl 1M Tris-HCl (pH 9.1) was used to neutralize the solution. Two additional rounds of panning were performed, with a phage load of 2e11 PFU/ml in each round of panning. Positive monoclonal phages were obtained after a total of three rounds of panning. Eight phage clones that could specifically bind to P-ArsS-A were obtained and five amino acid sequences were identified, including P03 (MMSYPKH) and P06 (LTPMPNW). An in vitro minimum inhibitory concentration (MIC) evaluation showed that P03 and P06 significantly inhibited the growth of H. pylori J99. The MIC of P03 was 8 μM, and the MIC of P06 was >16 μM, indicating that P03 is a stronger inhibitor compared to P06. This was confirmed by colony counting on blood agar plates after P03 and P06 administration. Using homology modeling and molecular docking analysis, it was shown that P03 and P06 could bind to the ArsS N-terminal domain, and there were four shared binding sites: TYR25, ASN39, ARG73, and GLU74. Additionally, one hydrogen bond was found between P03 and ArsS, which is more cohesive than other forms of bonding (van der Waals force, other non-covalent bonds). 3509 DWSSWVYRDPQT(10%) SGVYKVAYDWQH(5%) 2 Phage display (competitive panning) 6 PMID:31902944 Folate receptor alpha Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) Bound phages were eluted by addition of 1 mL of folic acid (Acros organics, Fisher Scientific, UK) in PBS (100 µg/mL) by competitive inhibition for 1 h at 4 °C. Two phage peptides(DWSSWVYRDPQT,SGVYKVAYDWQH) showed binding specificity to SKOV3 cells in vitro screened by Ph.D.-12 phage display library. WSSWVYRDPQT peptide identified targets colon cancer cells in vitro with in silico analysis suggesting the peptide targets glypican-3 (a heparin sulphate proteoglycan (HSPG)). 3510 CIGNSNTLC(9%) CTVRTSADC(7%) CTVRTSAEC(4%) 3 Phage display (competitive panning) 6 PMID:31902944 Folate receptor alpha Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) Bound phages were eluted by addition of 1 mL of folic acid (Acros organics, Fisher Scientific, UK) in PBS (100 µg/mL) by competitive inhibition for 1 h at 4 °C. Three phage peptides(CIGNSNTLC,CTVRTSADC,CTVRTSAEC) showed binding specificity to SKOV3 cells in vitro screened by Ph.D.-C7C phage display library. Peptides CIGNSNTLC and CTVRTSAEC bound FRα in the context of the phage particle. And synthesised lead peptide, CTVRTSAEC, bound specifically to FRα and could be competitively inhibited with folic acid. To assess the capacity of the elucidated FRα-binding oligopeptides to target OV to FRα, we genetically incorporated the peptides into the HAdV-C5 fiber-knob HI loop including in vectors genetically ablated for hCAR interactions. Unfortunately, the recombinant vectors failed to efficiently target transduction via FRα due to defective intracellular trafficking following entry via FRα, indicating that whilst the peptides identified may have potential for applications for targeted drug delivery, they require additional refinement for targeted virotherapy applications. 3511 SCATPFSPQVCS(78/80)[0.30566] SCRLQVXPGFCS(1/80)[NA] SCGERGXAECCS(1/80)[NA] 3 Phage display (common panning) 4 PMID:31422560 Copper (II) oxide nanoparticles, CuO-NPs Not determined. Not determined. Not determined. T7 SCX8CS phage display library (SCX8CS) ELISA The absorbance of each sample was measured at 450 nm using a microplate reader (Spectra Max Plus 384; Molecular Devices, San Jose, CA, USA). Data (OD450) shown were reproduced from Figure 1b in the reference. Prepared CuO-NPs (500 μg) were mixed with the constructed libraries and incubated in TBS-T containing 0.1% Tween 20. After 1 h at room temperature, CuO-NPs were washed five times with TBS-T. For the proliferation of T7 phages bound to the surface of CuO-NPs, E. coli BLT5403 (Merck Millipore) proliferated to the log phase was mixed with the nanoparticles and incubated at 37 °C. We evaluated the cytotoxicity of copper (II) oxide nanoparticles (CuO-NPs) by coating with a novel cyclic peptide, CuO binding peptide 1 (CuBP1), cyclic-SCATPFSPQVCS, which binds to the surface of CuO-NPs. CuBP1 was found to promote the aggregation of CuO-NPs under mild conditions. The treated CuO-NPs with CuBP1 caused the reduction of the cytotoxicity against Escherichia coli, Lactobacillus helveticus, and five other microorganisms, including bacteria and eukaryotes. Similar effects were also demonstrated against human embryonic kidney (HEK293) cells in vitro. 3512 CRQTKN(9/22)[9.92179] CRGTAEG(6/22)[4.812447±0.023385] CKSRKDGAC(3/22)[3.667137±0.009220] CMPKRPSSC(1/22)[5.607913±0.032665] 4 Phage display (in vivo) 5 PMID:31243044 K-ras(LA2) mutant mouse model of lung cancer Not determined. Not determined. Not determined. T7 CX7C phage display library (CX7C) Binding assay Ex vivo fluorescence intensities were measured using the eXplore Optix and Analysis Workstation software (ART Inc.). Fluorescence intensities were reproduced from Figure 1C in the reference and shown. Data are presented as mean +/- SE. K-ras(LA2) mutant mice bearing lung tumors (18–20 weeks old) were intravenously injected with a solution containing phages at a total of 1e11 pfu. After 15 minutes of circulation, mice were sacrificed and tumor nodules from lungs were excised and weighed. Cell suspension of tumor nodules was prepared in culture media by chopping with a knife, homogenizing using Medimachine (DAKO), and passing through a 70 μm cell strainer. The cell bound phages were eluted by lysing cells with 100 mL of 1% NP-40 for 10 minutes on ice and then by adding BL21 host bacteria. Phages were amplified in the host bacteria and used for next round of selection. Compared with other candidate peptides selected from 5 rounds of phage display, the CRQTKN peptide homed to tumor nodules in the lung of mutant mice at higher levels. Photoacoustic tomography of mutant mice detected lung tumors via tumor homing of the near-infrared fluorescence dye-labeled CRQTKN peptide. Ex vivo photoacoustic images of isolated organs further demonstrated tumor homing of the CRQTKN peptide, whereas minimal accumulation was observed in control organs, such as the liver. Compared with untargeted liposomes and doxorubicin, doxorubicin-loaded liposomes whose surface was modified with the CRQTKN peptide more efficiently delivered doxorubicin and reduced the number or size of tumor lesions in K-ras(LA2) mutant mice. Analysis of hematologic parameters and liver and kidney function showed no significant systemic side effects by the treatments. Affinity-based identification was used to detect TNF receptor superfamily member 19L (TNFRSF19L), which was upregulated in lung tumors of mutant mice, as the receptor for the CRQTKN peptide. In conclusion, these results suggest that the CRQTKN peptide is a promising targeting probe for photoacoustic-guided detection and drug delivery to lung cancer, and acts by binding to TNFRSF19L. 3513 CKSRKDGAC(4/27)[3.667137±0.009220] CMPKRPSSC(3/27)[5.607913±0.032665] CRGTAEG(3/27)[4.812447±0.023385] 3 Phage display (in vivo) 4 PMID:31243044 K-ras(LA2) mutant mouse model of lung cancer Not determined. Not determined. Not determined. T7 CX7C phage display library (CX7C) Binding assay Ex vivo fluorescence intensities were measured using the eXplore Optix and Analysis Workstation software (ART Inc.). Fluorescence intensities were reproduced from Figure 1C in the reference and shown. Data are presented as mean +/- SE. K-ras(LA2) mutant mice bearing lung tumors (18–20 weeks old) were intravenously injected with a solution containing phages at a total of 1e11 pfu. After 15 minutes of circulation, mice were sacrificed and tumor nodules from lungs were excised and weighed. Cell suspension of tumor nodules was prepared in culture media by chopping with a knife, homogenizing using Medimachine (DAKO), and passing through a 70 μm cell strainer. The cell bound phages were eluted by lysing cells with 100 mL of 1% NP-40 for 10 minutes on ice and then by adding BL21 host bacteria. Phages were amplified in the host bacteria and used for next round of selection. Compared with other candidate peptides selected from 4 rounds of phage display, there peptides(CKSRKDGAC,CMPKRPSSC,CRGTAEG) were selected. 3514 WHWRLPS(2)[1.359865±0.157799] NPMHIYDTLPAR(2)[0.400335±0.030196] 2 Phage display (common panning) 3-5 PMID:32717427 Multiple Sclerosis recombinant antibodie A1, MS-A1 (ST-176) Not determined. Not determined. Not determined. Ph.D.-12 and Ph.D.-7 phage library pool ELISA Absorbance at 405 nm was determined spectrophotometrically with a Microplate Reader (Bio-Rad, Hercules, CA). Data (OD405) shown were reproduced from Figure 1A in the reference. The Phage Display Peptide Libraries (12mer + 7mer) (New England BioLabs, Beverly, MA) were used for affinity selection of specific peptides under five rounds of biopannning for the MS brain rAbs. We identified 4 high affinity phage peptides from which 2 peptides (NPMHIYDTLPAR,WHWRLPS) are unique. Database searches revealed that peptide A1-A1(NPMHIYDTLPAR) shared sequence homologies with TRPM8 channel-associated factor 2 and ORF4 polyprotein [Nora virus]. And peptide A1-C3(WHWRLPS) shared sequence homologies with Tyrosine-protein kinase-defective receptor EPH-6, Sodium leak channel non-selective protein and Major structural protein VP1 [Mesocricetus auratus polyomavirus 1]. 3515 AHIPYQGRDTSQ(4) 1 Phage display (common panning) 3-5 PMID:32717427 Multiple Sclerosis recombinant antibodie B1, MS-B1 (G2-160) Not determined. Not determined. Not determined. Ph.D.-12 and Ph.D.-7 phage library pool ELISA The Phage Display Peptide Libraries (12mer + 7mer) (New England BioLabs, Beverly, MA) were used for affinity selection of specific peptides under five rounds of biopannning for the MS brain rAbs. We identified 4 high affinity phage peptides from which 1 peptides(AHIPYQGRDTSQ) are unique. Database searches revealed that peptide B1-A3(AHIPYQGRDTSQ) shared sequence homologies with Cytospin-A and Epstein-Barr nuclear antigen 4. 3516 NISAWFQPHQAL(13)[0.270370±0.088490] WQWGPYMVGAGV(4)[NA] 2 Phage display (common panning) 3-5 PMID:32717427 Multiple Sclerosis recombinant antibodie A2, MS-A2 (ST-196) Not determined. Not determined. Not determined. Ph.D.-12 and Ph.D.-7 phage library pool ELISA Absorbance at 405 nm was determined spectrophotometrically with a Microplate Reader (Bio-Rad, Hercules, CA). Data (OD405) shown were reproduced from Figure 1B in the reference. The Phage Display Peptide Libraries (12mer + 7mer) (New England BioLabs, Beverly, MA) were used for affinity selection of specific peptides under five rounds of biopannning for the MS brain rAbs. We identified 17 high affinity phage peptides from which 2 peptides(NISAWFQPHQAL,WQWGPYMVGAGV) are unique. Database searches revealed that peptide A2-D2(WQWGPYMVGAGV) shared sequence homologies with G-protein coupled receptor 161,Myomesin-3 and RNA-directed RNA polymerase L [Oliveros mammarenavirus]. And peptide A2-F4(NISAWFQPHQAL) shared sequence homologies with Dual specificity protein phosphatase 2,Ubiquitin-like modifier-activating enzyme Epstein-Barr nuclear antigen 6 and Capsid assembly protein [Human herpesvirus 4 strain B95-8]. 3517 SCSSLTTLRPCG(8/20)[1.71407] SQRKLAAKLTSK(6/20)[1.939233±0.016779] VILTGPEAEYFW(3/20)[0.659633±0.004151] HAMSPVFLSKYA(1/20)[NA] QVNGLGERSQQM(1/20)[NA] DYHDPSLPTLRK(1/20)[NA] 6 Phage display (common panning) 4 PMID:31678742 P. aeruginosa H103 (PAO1 wild-type prototroph) Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA Azino-bis (3-ethylbenzothiazole sulfonic acid) diammonium salt (ABTS) peroxidase substrate (Sigma-Aldrich) was used to detect peptide binding and the colour development at 415 nm was recorded by using a Model 680 microplate reader (Bio-Rad, Hercules, CA, USA). Data (OD415) shown were reproduced from Figure 1A in the reference. Four rounds of biopanning were performed against whole cells of P. aeruginosa H103. After washing, the bound phages were eluted by the addition of 100 μL elution buffer (0.2 M glycine-HCl, pH 2.2, 1 mg/mL BSA) at room temperature for 10 min.After neutralization with 1 M Tris-HCl, pH 9.1, the eluted phage was amplified in E. coli ER2738 by an infection method. The amplified phages were titred and used for the second round of biopanning. After four rounds of biopanning, the eluted phage was used to prepare phage stocks to isolated phage genomic DNA for nucleotide sequencing. The DNA sequences were translated into amino acids by using the Snapgene viewer software. Eight clones encoded the sequence SCSSLTTLRPCG, referred to as PA1, six clones encoded the sequence SQRKLAAKLTSK, referred to as PA2, and three clones encoded the sequence VILTGPEAEYFW, referred to as PA3. The remaining three clones (PA4, 5, and 6) encoded unique peptide sequences. The targeting peptide (PA2) that binds specifically to OprF porin on P. aeruginosa and a hybrid peptide was constructed by addition of the targeting peptide to GNU7, a potent antimicrobial peptide. The resulting hybrid peptide PA2-GNU7 exhibited potent antimicrobial activity against P. aeruginosa without causing host toxicity. Confocal laser scanning microscopy analysis and time-kill experiments demonstrated that PA2-GNU7 exhibited a high degree of specificity for P. aeruginosa, and rapidly and selectively killed P. aeruginosa cells in mixed cultures. In addition, in vivo treatment efficacy of PA2-GNU7 was significantly greater than that of conventional antibiotics in a mouse model of MDR P. aeruginosa infection. Taken together, the data suggest that PA2-GNU7 may be a promising template for further development as a novel anti-MDR P. aeruginosa therapeutic agent. 3518 GSAPLLTVDTSK(22)[12.45 ± 7.40] RVAPDFSCAYPY(18)[12.62 ± 11.01] SGVYKVAYDWQH(13)[35.80 ± 18.22] QSHDQNNYNQRS(7)[NA] 4 Phage display (subtractive panning) 4 PMID:32828793 Amyloid beta 42, Aβ42 Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA Absorbance was measured at 450 nm (OD450) using a microplate reader (Molecular Devices). The Kd value (pM) was determined and shown. Negative selection was performed for reducing non-specific phages between the third and forth round of panning, adding the amplified phage to non-target coated plate for 1 h. Non-bound phages in the supernatant were used for the forth round of biopanning. The synthesized peptides were confirmed to have excellent binding affinity to Aβ aggregates, by immunohistochemical staining and western blotting using the brains of 3X transgenic (Tg) AD mice at different stages (5–7, 12–17 months old) of AD severity. In the present study, it was confirmed that newly developed amyloid-binding peptides could be used as novel probes for the detection of Aβ aggregates, which can be used for clinical diagnosis of AD in the future. 3519 SPAQHTHERAHT(21)[13.07 ± 5.88] SPHLHTSSPWER(17)[217.97 ± 27.01] MKAHHSQLYPRH(12)[1548.22 ± 835.51] NYPHPHLSNYHP(8)[637.02 ± 815.98] NAPKHAHRLPVH(2)[1282.66 ± 269.73] 5 Phage display (subtractive panning) 4 PMID:32828793 Amyloid beta 42, Aβ42 Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA Absorbance was measured at 450 nm (OD450) using a microplate reader (Molecular Devices). The Kd value (pM) was determined and shown. Two rounds of negative selection were performed for reducing non-specific phages, by adding the amplified phage to non-target-coated resin for 1 h. One was performed after the second round of positive selection, the other after the third round of positive selection. After centrifugation, non-bound phages in the supernatant were used for the following cycles. The synthesized peptides were confirmed to have excellent binding affinity to Aβ aggregates, by immunohistochemical staining and western blotting using the brains of 3X transgenic (Tg) AD mice at different stages (5–7, 12–17 months old) of AD severity. In the present study, it was confirmed that newly developed amyloid-binding peptides could be used as novel probes for the detection of Aβ aggregates, which can be used for clinical diagnosis of AD in the future. 3520 MKAHHSQLYPRH[14.269347±0.042793] HRPYLQSHHAKM[16.550423±0.042787] SGHFFMKDHWDV[2.154010±0.017321] VDWHDKMFFHGS[2.64163] HAPDTIKRSLAM[17.884483±0.042793] DKDVTHFLERTR[17.678607±0.042787] NHHMMPAWNVKH[10.816170±0.038036] NLGAEPGTPYLV[15.191657±0.042787] YQPAREHRVPAG[21.867440±0.038036] TNNNTPSQMGLS[13.025873±0.042787] 10 Phage display (common panning) 5 PMID:32375468 Ni–Ti Stents Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) Binding assay The stent or coil was observed by an upright fluorescence microscope (BX53-44-FL-1, Olympus, Tokyo, Japan).Fluorescence intensity of Ni−Ti stents surfaces after exposure to FITC-labeled peptides was determined. Fluorescence intensities were reproduced from Figure 2B in the reference and shown. To wash out nonbinding phages, the Ni–Ti stents were rinsed by PBS containing Tween 20 (0.1%) once and then by PBS four times at room temperature. The phages bound to the surface of the stent and coil were eluted with 37.5 μg/mL glycine/HCl solution (pH 2.2) containing 1 mg/mL BSA and 0.1 mg/mL phenol red for 15 min at room temperature. The eluted phage solution was neutralized with Tris-HCl (pH 9.1). Then, E. coli cells were infected with the eluted phages and cultured for the phage amplification. These amplified phages were used for the next round of panning. After the final panning at the fifth round, E. coli cells infected with the obtained phages were plated and incubated on LB-agar plates containing IPTG and X-gal (overnight at 37 °C). Phage display method was used to identify peptide ligands that had high affinity for the metallic surface of Ni–Ti stents. The binding assay using fluorescence labeling revealed that several synthetic peptides could bind onto those surfaces. 3521 AHNHTPIKQKYL[7.41469] LTPHKHHKHLHA[23.9808] SAVQWFELNTHA[5.38701] 3 Phage display (common panning) 5 PMID:32375468 Pt–W Coils Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) Binding assay The stent or coil was observed by an upright fluorescence microscope (BX53-44-FL-1, Olympus, Tokyo, Japan).Fluorescence intensity of Pt−W coils surfaces after exposure to FITC-labeled peptides was determined. Fluorescence intensities were reproduced from Figure 3B in the reference and shown. To wash out nonbinding phages, the Pt–W coils were rinsed by PBS containing Tween 20 (0.1%) once and then by PBS four times at room temperature. The phages bound to the surface of the stent and coil were eluted with 37.5 μg/mL glycine/HCl solution (pH 2.2) containing 1 mg/mL BSA and 0.1 mg/mL phenol red for 15 min at room temperature. The eluted phage solution was neutralized with Tris-HCl (pH 9.1). Then, E. coli cells were infected with the eluted phages and cultured for the phage amplification. These amplified phages were used for the next round of panning. After the final panning at the fifth round, E. coli cells infected with the obtained phages were plated and incubated on LB-agar plates containing IPTG and X-gal (overnight at 37 °C). Phage display method was used to identify peptide ligands that had high affinity for the metallic surface of Pt–W coils. The binding assay using fluorescence labeling revealed that several synthetic peptides could bind onto those surfaces. 3522 HSQSAEHHTSWP[12.77396] TLLPKNFGHRPL[118.495593±0.214277] TFLNSVPTYSYW[53.734663±0.242966] TSCFGDSRCQPP[23.902547±0.242966] LTKTNLDPMEFK[36.52743] TICWTYPKCTED[16.42114] TSNLWRYDRLTM[65.33084] STVSSLARILTG[61.356347±0.242966] 8 Phage display (common panning) 5 PMID:32375468 Co–Cr Stents Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) Binding assay The stent or coil was observed by an upright fluorescence microscope (BX53-44-FL-1, Olympus, Tokyo, Japan). Fluorescence intensity of Co−Cr stents after exposure to the FITC-peptide was determined. Fluorescence intensities were reproduced from Figure 4B in the reference and shown. To wash out nonbinding phages, the Co–Cr stents were rinsed by PBS containing Tween 20 (0.1%) once and then by PBS four times at room temperature. The phages bound to the surface of the stent and coil were eluted with 37.5 μg/mL glycine/HCl solution (pH 2.2) containing 1 mg/mL BSA and 0.1 mg/mL phenol red for 15 min at room temperature. The eluted phage solution was neutralized with Tris-HCl (pH 9.1). Then, E. coli cells were infected with the eluted phages and cultured for the phage amplification. These amplified phages were used for the next round of panning. After the final panning at the fifth round, E. coli cells infected with the obtained phages were plated and incubated on LB-agar plates containing IPTG and X-gal (overnight at 37 °C). Phage display method was used to identify peptide ligands that had high affinity for the metallic surface of Co–Cr stents. The binding assay using fluorescence labeling revealed that several synthetic peptides could bind onto those surfaces. 3523 WALRVKAG[0.85168, 8.18e-4] SWGKMAKG[0.84287, NA] PAMARKMG[1.10182, NA] 3 Phage display (subtractive panning) 5 PMID:30903640 Interleukin-13 receptor subunit alpha-2 Interleukin-13, IL-13 3LB6, Not determined. T7 X7 phage display library (X7) ELISA,Surface plasmon resonance (SPR) The binding affinity of IL13Rα2 protein binding peptides was measured by ELISA, absorbance at 450 nm was measured by using a microplate reader (GE Healthcare Biosciences). Data (OD450, the first column) shown were reproduced from Supplementary Figure S1a in the reference. In addition, the surface plasmon resonance (SPR) method using an SPR instrument (Reichert Technologies, Depew, NY) at 25 °C. The Kd (M) value was displayed in the second column of the affinity value. For biopanning against IL‐13Rα2, a solution of the constructed T7 phage display library (3.6 × 109 pfu) was added to a well coated with BSA and incubated for 1 hr at room temperature. After incubation, the unbound T7 phages were removed and added to a well coated with IL‐13Rα1/Fc protein, incubated for 1 hr at room temperature, and the unbound T7 phages were removed and added to a well coated with IL‐13Rα2/Fc protein. The well was washed at least 10 times with PBS containing 0.2% Tween‐20 (PBST) after incubation for 10 min at room temperature, and E. coli BL21 cells were added for culturing and phage propagation. The A2b11 peptide (WALRVKAG), which was one of the positive clones, was shown to bind to IL‐13Rα2 protein by Biacore analysis and a binding assay using glioblastoma (GB) cell lines. This peptide was linked with a lytic peptide containing a linker sequence to form the IL‐13Rα2–lytic hybrid peptide. The IL‐13Rα2–lytic hybrid peptide showed cytotoxic activity against GB cell lines in vitro. The IL‐13Rα2–lytic hybrid peptide also affected Akt and Erk1/2 activation following treatment with interleukin‐13 and induced rapid ATP dynamics in GB cells. Anti‐tumor activity of the IL‐13Rα2–lytic hybrid peptide was observed in vivo after intratumoral injection in a mouse xenograft model of human GB cells. These results suggest that the IL‐13Rα2–lytic hybrid peptide might be a potent therapeutic option for patients with GB. 3524 ANLNLWTDYIRW[2.61538] 1 Phage display (common panning) 3 PMID:32454443 Human colon cancer cell line SW480 Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The binding ability of the selected phage clones was evaluated on colon cancer cells (SW480, HT29, HCT116, LoVo, and DLD1) as well as on a normal cell line (CCD-18co) by cell phage ELISA. Absorbance was measured at 450 nm (OD450) using a plate reader. The binding affinities (relative OD values) showed more than two- to five-fold higher selectivity for colorectal cells than the negative controls. The binding affinity (relative fold) was reproduced from the Supplementary Figure 1 and shown. The Ph.D.-12 Phage Display Peptide Library(New England BioLabs, Beverly, MA) were used for affinity selection of specific peptides under three rounds of biopannning for the human colon cancer cells SW480. The phage clone with the highest optical density (450 nm) compared to the negative control cells was selected. This peptide(ANLNLWTDYIRW) probe maintained binding affinity even after serum incubation. For therapeutic applications, this peptide probe was conjugated to hematoporphyrin, a photosensitizer, which showed a significantly enhanced cellular uptake and high photodynamic effect to kill tumor cells. As another application, we made a nanoparticle modified from the peptide probe. It efficiently delivered SN-38, an anticancer drug, into tumor cells, and its tumor-targeting ability was observed in vivo after intravenous injection to the same xenograft model. 3525 ANLNLWTDYIRW[2.25641] 1 Phage display (common panning) 3 PMID:32454443 Human colon cancer cell line HCT116 Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The binding ability of the selected phage clones was evaluated on colon cancer cells (SW480, HT29, HCT116, LoVo, and DLD1) as well as on a normal cell line (CCD-18co) by cell phage ELISA. Absorbance was measured at 450 nm (OD450) using a plate reader. The binding affinities (relative OD values) showed more than two- to five-fold higher selectivity for colorectal cells than the negative controls. The binding affinity (relative fold) was reproduced from the Supplementary Figure 1 and shown. The Ph.D.-12 Phage Display Peptide Library(New England BioLabs, Beverly, MA) were used for affinity selection of specific peptides under three rounds of biopannning for the human colon cancer cells HCT116. The phage clone with the highest optical density (450 nm) compared to the negative control cells was selected. This peptide(ANLNLWTDYIRW) probe maintained binding affinity even after serum incubation. For therapeutic applications, this peptide probe was conjugated to hematoporphyrin, a photosensitizer, which showed a significantly enhanced cellular uptake and high photodynamic effect to kill tumor cells. As another application, we made a nanoparticle modified from the peptide probe. It efficiently delivered SN-38, an anticancer drug, into tumor cells, and its tumor-targeting ability was observed in vivo after intravenous injection to the same xenograft model.