3251 LPKQKRRQRRRM HASPPKNSHFAL LSNQSNPGIAKS LSKNRRNRRRQH WSFNWSMHSLGN KQRMQTSNRRRR NRRHRNLRRPPL QKRIRIQRRSLR QLRRRHRRIRTN RRHQSRILIQRS RQR 10 Phage display (common panning) 5 PMID:28860066 Mouse melanoma carcinoma cell line B16 Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) MT23 (LPKQKRRQRRRM) showed higher penetration efficiency based on fluorescence microcopy and quantitative assay, and it has capability for mediating functional Apoptin into cells in vitro or in vivo. 3252 HSVKPVVNLILR(34) HSIRLHTYPHMK(19) YSLRADSRWMPS(18) HSLRPEWRMPGP(15) HSIRTYWQSAQP(10) HSLREDWTLRMQ(2) HSLKPSLKQLAI(2) TMGFTAPRFPHY(2) APPGNWRNYLMP(2) HSLKPSWLLLGY(2) HSVKPDWAQMLR(1) HSIRSSHLHMFT(1) HSVKHDFRLLTK(1) SGHQLLLNKMPN(1) APRLPQSLLPQL(1) SHALPLTWSTAA(1) APPMSRQSFDGV(1) NFMESLPRLGMH(1) LLADTTHHRPWT(1) MEGQYKSNLLFT(1) NTELTSYGPPPA(1) TMHSLRPEWRMP(1) H-S-[ILV]-[KR], x-P-[KR], x(2)-P-[KR] 22 Phage display (subtractive panning) 4 PMID:28881621 Gap junction alpha-5 protein Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) A phage library was first pre-cleared in an uncoated well, and then presented to the bait protein. One of the retrieved peptides (HSLRPEWRMPGP) showed a 58.3% homology with amino acids 5-to-16 of IκBα, a member of the protein complex inhibiting NFκB activation. 3253 NKLLATW(18) NKLCCTK(1) NKLCCAK(1) GQLLCTM(1) GQLLATK(1) LL 5 Phage display (common panning) 4 PMID:28853252 Anti-VP2 monoclonal antibody 3G11 Outer capsid protein VP2 Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The results of sandwich ELISA test shown that MAb 3G11 can react positively with BTV-8, and with other 23,no positive reaction was found in the virus species. After the fourth round of panning, obtained positive sequence clones KLLAT, the 3 amino acids LL and T of the sequence are consistent with the amino acid residue LLST sequence of BTV-8 VP2. 3254 SAPSSKN(2) HAIYPRH(2) AMLPYTF(1) ANSTPPI(1) ANTTPRH(1) ATLTHPP(1) EDRANRQ(1) EPLQLKM(1) GKPMPPM(1) GKVQAQS(1) GRPHSAL(1) HTAPNFA(1) IPTLPSS(1) LHRPYST(1) LPTPPYA(1) MTSQHPK(1) NQLPLHA(1) QILAFNS(1) SHGNWWR(1) SLLNRMP(1) SLYKWTl(1) SPTQPKS(1) STPIQQP(1) TLHSLPP(1) TSLSMPS(1) TTVMGNL(1) VAHQLSR(1) WTITKHP(1) 28 Phage display (in vivo) 3 PMID:28916756 Human pancreatic ductal carcinoma cell Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) To conduct the phage screen, 10 µL of PhD-7 phage display library (New England Biolabs, Ipswich, MA, USA) was combined with 190 µL of Dulbecco’s phosphate buffered saline (DPBS) and injected intravenously into mice harboring orthotopic xenograft PDAC tumors. Phage circulated for 8–10min before the animals were killed and cardiac perfused with 30mL of DPBS. Tumors were minced into 1–2mm segments and Dounce homogenized in a solution consisting of DPBS+ 1% TritonX, ethylene diamine tetraacetic acid, and protease inhibitors (Thermo Scientific, Waltham, MA, USA). To assess the specificity of a few of the selected clones for blood vessels in vivo, fluorescently labeled phage were injected into animals bearing orthotopic tumors. Specificity of the phage for tumor vessels was assessed by immunofluorescence. We used three clones with similar homology, PRH, in our analysis. The “PRH” pool (HAIYPRH and LHRPYST) bound 73.4% (4.1-fold over normal pancreas vessels) of platelet endothelial cell adhesionmolecule1-positive tumor vessels. The immunofluorescent images indicate that the fluorescein isothiocyanate (FITC)-phage remained bound to the luminal surface of the blood vessels even after vigorous perfusion. 3255 DLTFTVNPLSKA(8)[0.134, 1.616] WHWSWWHPNQLT(7)[0.550, 1.949] TSVSYINNRHNL(1)[0.122, 0.377] 3 Phage display (common panning) 3 PMID:28929019 Refolded apical merozoite antigen 1 Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The binding signals for each peptide were estimated using absorbance value at 405 nm towards BSA (control) and rPvAMA1, respectively. The first column of the affinity values is the absorbance value towards BSA, the second is towards rPvAMA1. 3256 YYWGPPV(2) HYIDFRW(2) WNEVKPI(1) IIIRNSF(1) NYTLTDI(1) AQNQPMA(1) SLLGQTP(1) GLFHNAQ(1) 8 Phage display (common panning) 3 PMID:28888997 Human prostate cancer cell line PC3 Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) PC-3 cells were seeded onto a well of a 96 well microplate (Nunc Maxisorb) for 24 hours to bring the culture to about 75% confluence. The cells were washed with PBS and then exposed to 1.0e9 phage particles from the parent Ph.D-7 library in 100µl PBS. After incubation, the cells were washed with PBS and then 100µl of 0.2M Glycine-HCl, pH. 2.2 were added to elute cell-bound phage. After 10 mins, 15µl of 1M Tris-HCl, pH 9.1 was added to neutralize the solution and the eluted phage were recovered. The cells were washed again with PBS and then 100µl 30mM Tris, pH. 8.0 was added. The cells were frozenthawed until they had ruptured as judged by light microscopy (three cycles). The ruptured cell mixture was collected and centrifuged at 2000rpm for 2 min; the supernatant containing the PC3 internalized phage was recovered. Phage were titrated, amplified and the exposure procedure was repeated twice more. 3257 TPARHIY(3) AYPSAHR(2) TPARHIY(1) THISKLI(1) NERALTL(1) YWHPHMV(1) LSNNNLR(1) 7 Phage display (subtractive panning) 3 PMID:28888997 Human prostate cancer cell line PC3 Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) The first (termed L1) was the parent Ph.D-7 library. The second library (L2) was derived from L1 and comprised phage that did not bind normal mouse tissues. For this purpose, 1.0e11 pfu of L1 (10µl) in 100µl PBS were injected intravenously into a Balb/c mouse and phage were recovered from the blood 24hrs later using the phage isolation procedure described in the kit. Then three rounds of biopanning were performed against the PC-3 cells, which were seeded onto a well of a 96 well microplate (Nunc Maxisorb). Phage from a libraries displaying 7mer peptides were exposed to PC-3 cells and only internalized phage were recovered. The ability of these phage to internalize into other prostate cancer cells (LNCaP, DU-145) was validated. The displayed peptides of selected phage clones were synthesized and their specificity for target cells was validated in vitro and in vivo. One peptide (P12, LSNNNLR) which specifically targeted PC-3 tumors in vivo was incorporated into mono-drug (Chlorambucil, Combretastatin or Camptothecin) and dual-drug (Chlorambucil/Combretastatin or Chlorambucil/Camptothecin) PDCs and the cytotoxic efficacy of these conjugates for target cells was tested. Conjugation of P12 into dual-drug PDCs allowed discovery of new drug combinations with synergistic effects. 3258 CPRIWADSC(12)[4.84] CTYLNSAKC(10)[4.63] 2 Phage display (competitive panning) 3 PMID:28783899 Anti-imidaclothiz monoclonal antibody 1E7 Imidaclothiz Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) ELISA The absorbance at 450 nm was measured. Data shown as affinity values were IC50 (ng/mL). The imidaclothiz (2 μg/mL in PBS) was added to elute the binding phage with shaking for 1 h. 3259 CLPPRMIYEC(25)[4.02] 1 Phage display (competitive panning) 3 PMID:28783899 Anti-imidaclothiz monoclonal antibody 1E7 Imidaclothiz Not determined. Not determined. CX8C M13 phage display library ELISA The absorbance at 450 nm was measured. Data shown as affinity values were IC50 (ng/mL). The imidaclothiz (2 μg/mL in PBS) was added to elute the binding phage with shaking for 1 h. 3260 TMHLPYCPTNIC(29)[2.97] SQPWCPPSICGD(8)[5.08] DYHDPSLPTLRK(1)[5.74] 3 Phage display (competitive panning) 3 PMID:28783899 Anti-imidaclothiz monoclonal antibody 1E7 Imidaclothiz Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The absorbance at 450 nm was measured. Data shown as affinity values were IC50 (ng/mL). The imidaclothiz (2 μg/mL in PBS) was added to elute the binding phage with shaking for 1 h. 3261 DRWVARDPASIF(22/60) FPSSPGVSRAIN(3/60) GLHTSATNLYLH(2/60) GVAMLQSGYASR(2/60)[0.36] VTVELQHLSPAK(2/60)[0.45] FEHSLYKEMTHL(1/60)[1.79] NYPATNTHRYTP(1/60) TPQSFWQKGSLV(1/60) AFDTFNLYMDEL(1/60) VPGLVFSKLTTG(1/60) VPAQSGQGIEHE(1/60) SPDLSTRPTLGY(1/60) IPAPLLVPNLWH(1/60) SSDSVVKQAILT(1/60) HMNTHDSLFPVG(1/60) QPNNILPAFGNY(1/60) APTLYWNVPTHV(1/60) LFAAASSNHIHR(1/60) SVLPLLCCDSTP(1/60) VERSFLNERVTP(1/60) DVTRLLSTHMWI(1/60) GIQNGLAFQGLQ(1/60) QLQLNNQVALMY(1/60) 23 Phage display (subtractive panning) 5 PMID:28823053 Macrophage-stimulating protein receptor, MSP receptor Hepatocyte growth factor-like protein 4QT8, Not determined. Ph.D.-12 phage display library (X12) ELISA A subtractive biopanning was first performed by adding a peptide-displaying phage library to pre-cold NIH3T3 cells. Subsequently, the supernatant was transferred onto pre-cold NIH3T3-RON cells and incubated. Totally, five rounds of biopanning were carried out using NIH3T3-RON cell line. Then,the recovered phage from fifth round were used for subtractive biopanning using EBC-1-Dox+ (RON negative) in order to eliminate possible non-RON binding phage particles. Binding modes and affinities were also predicted by docking and molecular dynamics (MD) simulation. The results of ELISA experiment showed that P6 peptide (FEHSLYKEMTHL) displaying phage has higher affinity for RON compared to others and its binding site is located out of ligand binding site. Docking and MD simulation results also indicated higher affinity of P6 to RON as well as its exosite-binding feature. Taken together,our data suggest a capacity for P6 peptide to be utilized as RON binding agent,and hence be used for various purposes,including design of drug delivery systems for transferring cytotoxic agents to RON-positive cancer cells, interfering with RON signaling, peptidomimetics design, and diagnostic imaging. 3262 DRWVARDPASIF(39/60) FPSSPGVSRAIN(20/60) 2 Phage display (subtractive panning) 5 PMID:28823053 Macrophage-stimulating protein receptor, MSP receptor Hepatocyte growth factor-like protein 4QT8, Not determined. Ph.D.-12 phage display library (X12) A subtractive biopanning was first performed by adding a peptide-displaying phage library to pre-cold MDCK cells. Subsequently, the supernatant was transferred onto pre-cold MDCK-RON cells and incubated. Totally, five rounds of biopanning were carried out using MDCK-RON cell line. Then,the recovered phage from fifth round were used for subtractive biopanning using EBC-1-Dox+ (RON negative) in order to eliminate possible non-RON binding phage particles. 3263 NCWSSLRGICENLG[21.7] SCSWNVERIRGCSV[18.8] CWNLKRIGSQGC[30.7] CWNLKRIGSQGC[26.4] GCVKYRGSVSCQESKT[18.0] ACLNHFVSGNMIRC[26.1] CPFYDWRCSDF[25.0] NVRCYRDVPSCS[25.9] EDVVCYGRVDYMPLCV[25.2] DCNLWGDDGKYRLCFG[23.6] CVKVGLSWIGDCNS[23.9] CMEAWQCSLA[24.6] QCYPWCRL[25.1] CWTYLRGGCSVT[22.1] GDCGYHYMCSMT[25.0] NMCYLRGKMDCNIV[30.9] VCSDHYIGGKEVRC[27.4] CCSYGYEKCCNG[28.6] SPCRVGYTPC[27.7] NCARLYSHQRDYSQVC[24.4] CLDRSGGCYTREGYLS[29.0] CYPLCRVG[16.2] KCDSHYIRGVVACH[30.2] GCGYRYMCDMINGL[32.8] GDCYPHCRLV[28.9] DCLRYRVGSSCS[29.6] RCESKGWANTCVSE[20.7] CYPLCRVL[31.1] RCGLAWCVVLVDQ[20.8] 29 Phage display (common panning) 4 PMID:28825773 Proprotein convertase subtilisin/kexin type 9, PCSK9 Low-density lipoprotein receptor 2W2M,2W2N,2W2O,2W2P,2W2Q,3BPS,3GCW,3GCX,3M0C,3P5B,3P5C, Not determined. TAFTSWEEYLDWVGSG fusion M13 phage display library (TAFTSWEEYLDWVGSGX(8,10,12,14,16)) ELISA The s/n ratio is the signal:noise ratio, wherein “signal” and "noise" are the spot phage ELISA signals of phage binding to NeutrAvidin-captured PCSK9 and to NeutrAvidin, respectively. The s/n ratios were shown. 3264 KLWNLGRV[28.2] VLWNHSRI[24.9] GPWNLNRV[27.6] PLLWNLQKVH[23.5] KLWNLTRISS[28.1] VVLWNHSRIL[26.8] VPWNLARL[29.3] VPWNLAKI[22.5] GHPLWNLSRI[25.7] NELWNINRLRSS[12.0] SIPWNLERITPPR[28.9] VLWNHSRI[29.7] MPWNLARIER[27.8] ALWNMRRVESVAFR[23.9] GEYLWNLKRLES[28.7] TRYADRGVMVYSLS[23.0] MVYVDRGVRVFT[28.0] EHQFYSYRGVDVYR[22.3] KNNYAYLINMPRAPGI[24.7] RGGYELNIPR[26.1] NPTYWLTRIS[23.1] PHSYWLDRVQ[25.3] GVLGVGGMWIA[12.8] SYRGAWSVFGGSTD[19.5] PRTYLRGLVD[25.6] QTTYLRVSSS[27.0] QISYLRNA[22.9] RGEVYDKGGVIVHL[30.5] GREMRSVIQM[28.2] QELPNHRRLS[19.1] SETGLGTYYG[28.1] SSTWHFVGGVRV[24.4] TRYADRGVMVYSLS[30.3] KMTHMKAD[22.4] PTSGLTPS[27.5] QVTSLPQA[27.9] RGGYELNIPR[29.8] W-N-L-x-R-I 37 Phage display (common panning) 4 PMID:28825773 PCSK9ΔP' Not determined. Not determined. Not determined. WNLVRIGLLR fusion peptide library (X(2-5)(G/S)WNLVRIGLLR) ELISA The s/n ratio is the signal:noise ratio, wherein “signal” and "noise" are the spot phage ELISA signals of phage binding to NeutrAvidin-captured PCSK9 and to NeutrAvidin, respectively. The s/n ratios were shown. One cluster contained a motif, WNLxRI , homologous to the sequence of the native P' helix. 3265 HLWGWLYAPSFQ(15)[0.95] YTFHFDIFQPHF(1)[25.66] 2 Phage display (subtractive panning) 6 PMID:28986753 Galectin-1, Gal-1 Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA To determine the apparent dissociation constant (K*d) of the selected clones, ten serial dilutions of phages (generally ranging from 5e12 to 1.0e7 phages/100 µL) were incubated with gal-1 or control proteins in the same conditions as for the first clones' screening. The optical density (OD) was measured (405 nm, differential filter: 630 nm) using a microplate reader (StatFax-2100, Awareness Technology, Fisher Bioblock Scientific, Tournai, Belgium). The affinity constants (Ka = 1/K*d) against gal-1 or control proteins were determined for one representative clone per peptide sequence. The phage clone has a better specific affinity when the affinity ratio is high. Coll = collagen. Affinity ratio = KaGal-1/((KaHSA + KaColl)/2). Data shown are affinity ratios. Based on this parameter, the best peptide clone is YTFHFDIFQPHF (P2) A linear 12-mer random peptide phage display library (PhD-12, New England Biolabs Inc. Bioké, Leiden, The Netherlands) was screened against human gal-1 (Peprotech, London, UK) for six rounds, which was immobilized on a 96-well ELISA plate at a concentration of 100 µg/mL. The preselection step was performed by incubating the phage library with bovine serum albumin (BSA, 1 µg/mL, Sigma-Aldrich, Diegem, Belgium). The time for the preselection step is modified along the rounds (80 – 100 – 120 – 140 – 160 –180 min). Peptide P1 (HLWGWLYAPSFQ) was selected according to its affinity toward gal-1. Its binding has been revealed by immunohistochemistry on human thyroid cancer biopsies, and it was co-localized with gal-1 by immunofluorescence on TPC-1 cell line. Peptide P1 induced a decrease in TPC-1 cells' adhesion to gal-1 immobilized on culture plates. After coupling to USPIO, the peptide preserved its affinity toward gal-1. Its specific binding has been corroborated by co-localization with gal-1 expressed by TPC-1 cells and by its ability to compete with anti-gal-1 antibody. The peptide and its USPIO derivatives produce no toxicity in HepaRG cells as determined by MTT assay. The vectorized contrast agent is a potential imaging probe for thyroid cancer diagnosis. Moreover, the gal-1-targeted peptide prevent cancer cell adhesion by interacting with the carbohydrate-recognition domain of gal-1. 3266 HLWGWLYAPSF(15)[0.95] HYSWSWIAYSPG(2)[1.14] VHWDFRQWWQPS(2)[1.47] YSWHIDIVAPRN(2)[Infinite ratio] YHHSGPYAGPMW(1)[2.50] SVYVEISWVRTM(1)[1.17] DWSSWVYRDPQT(1)[2.36] 7 Phage display (subtractive panning) 3 PMID:28976753 Galectin-1, Gal-1 Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA Peptide P7 (YSWHIDIVAPRN) was selected according to its affinity toward gal-1. Its binding has been revealed by immunohistochemistry on human thyroid cancer biopsies, and it was co-localized with gal-1 by immunofluorescence on TPC-1 cell line. Peptide P7 induced a decrease in TPC-1 cells' adhesion to gal-1 immobilized on culture plates. After coupling to USPIO, the peptide preserved its affinity toward gal-1. Its specific binding has been corroborated by co-localization with gal-1 expressed by TPC-1 cells and by its ability to compete with anti-gal-1 antibody. The peptide and its USPIO derivatives produce no toxicity in HepaRG cells as determined by MTT assay. The vectorized contrast agent is a potential imaging probe for thyroid cancer diagnosis. Moreover, the gal-1-targeted peptide prevent cancer cell adhesion by interacting with the carbohydrate-recognition domain of gal-1. A linear 12-mer random peptide phage display library (PhD-12, New England Biolabs Inc. Bioké, Leiden, The Netherlands) was screened against human gal-1 (Creative Biomart, New York, USA) for three rounds, which was immobilized on a 96-well ELISA plate at a concentration of 100 µg/mL. The preselection step was performed by incubating the phage library with a mix of human serum albumin (HSA) and collagen (both at 100 µg/mL; Sigma-Aldrich). The time for the preselection step is modified along the rounds (80 – 100 – 120 min). Peptide P7 (YSWHIDIVAPRN) was selected according to its affinity toward gal-1. Its binding has been revealed by immunohistochemistry on human thyroid cancer biopsies, and it was co-localized with gal-1 by immunofluorescence on TPC-1 cell line. Peptide P7 induced a decrease in TPC-1 cells' adhesion to gal-1 immobilized on culture plates. After coupling to USPIO, the peptide preserved its affinity toward gal-1. Its specific binding has been corroborated by co-localization with gal-1 expressed by TPC-1 cells and by its ability to compete with anti-gal-1 antibody. The peptide and its USPIO derivatives produce no toxicity in HepaRG cells as determined by MTT assay. The vectorized contrast agent is a potential imaging probe for thyroid cancer diagnosis. Moreover, the gal-1-targeted peptide prevent cancer cell adhesion by interacting with the carbohydrate-recognition domain of gal-1. 3267 CIGNSNTLC(38.4%)[6.29] CTVRTSADC(23.2%)[15.4] CSNNGNALC(15.4%)[47.3] CISNGNQPC(15.4%)[64.2] CRVNTAALC(7.6%)[77.7] 5 Phage display (common panning) 3 PMID:29029605 Immunoglobulins (Igs) of murine 5T33MM cells Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) ELISA The specific binding of peptides to the 5T33MM sIgG was analyzed by ELISA. Streptavidin coated 96 well plates were washed extensively and supplemented with biotin-conjugated peptides by 1 h-incubation at 37 °C; after washing and blocking with blocking solution (1X PBS, 0.05% Tween-20, 5% milk), aliquots of 5T33MM sIgG were added overnight at 4 °C. Wells were extensively washed and coated with the anti-mouse IgG (Fc-specific) alkaline phosphatase-conjugated [Sigma Aldrich] for 1 h at 37 °C, incubated with the alkaline phosphatase substrate, and analyzed by an ELISA reader at 405 nm [Labsystems multiscan MS]. KD values (nM) of identified synthetic peptides are shown. The screening of phage displayed library was performed using the bait 5T33MM Igs. Briefly, the streptavidin-conjugated beads were coated with 5T33MM Igs and incubated with 1.0e11 phages overnight at 4 °C. 5T33MM Igs-interacting phages were eluted with 0.2 M glycine-HCl (pH 2.2, 1 mg/mL BSA) followed by the addition of neutralizing solution (1 M Tris-HCl pH 9.1). Ultimately, 3 cycles of panning were performed. Plaques of lysis from isolated phages were transferred to nitrocellulose filters, and membranes were blocked with PBS 1X, 0.1% NP-40; 5% milk; 0.02% NaN3 and then incubated for 2 h at RT with 100 μg of purified 5T33MM Igs. After washing, membranes were hybridized with an alkaline phosphatase-conjugated anti-mouse IgG (dilution 1:5000) and then washed 6 times. Immunoreactive phage clones were detected by BCIP/NBT premixed substrate. Based on the highest affinity binding to 5T33MM Igs, the insert amino acid sequence of phage clone 5 (CIGNSNTLC) was used for large-scale synthesis of the p5 peptide. By FACS, the FITC-conjugated P5 detected the MM-released exosomes in the serum of 5T33MM-engrafted mice, levels of which are correlated with tumor progression at an earlier time point compared to serum paraprotein. 3268 DWSSWVYRDPQT(3)[37933.89] LPKTVSSDMSLN(1)[NT] 2 Phage display (subtractive panning) 4 PMID:29065111 Human Colon cancer cell line COLO320HSR Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) Isothermal titration calorimetry (ITC) COLO320HSR was grown on coverslips overnight and fixed with 4% paraformaldehyde at room temperature for 30 min. Samples were blocked in 3% BSA (w/v) inPBS for 30 min at room temperature. Cells were then incubated with 10 mM FITC-labelled peptides for 15 min at room temperature and washed with PBS for three times. This was followed by nuclear staining with 4,6-diamidino-2-phenylindole (DAPI). A laser scanning confocal microscope was used to visualise the slides. The cumulative values of the integrated optical density (IOD) of green fluorescence (FITC) in each photo were analysed using the Image-Pro Plus 6.0 software. The IOD value was reproduced from Figure 3D in the literature and shown. Four rounds of reiterative biopanning were performed against a human colon cancer cell line (COLO320HSR). Then, the selected phages were applied to normal human intestinal epithelial cell line NCM460 in the same way, for subtractive screening. The peptides that specifically binds to human colon cancer cell line COLO320HSR but not to a normal human intestinal epithelial cell line NCM460 were selected finally. A peptide termed as CBP-DWS with the sequence of DWSSWVYRDPQT, which was demonstrated to be capable of binding to a panel of human colon cancer cell lines and tissues, was identified; it had virtually no binding to normal human intestinal epithelial cell line NCM460 and normal surrounding colon tissues. Bioinformatics analyses suggest that CBP-DWS targets human Glypican-3, which may be involved in important cellular functions in multiple cancer types. 3269 TLLVIRGLPGAC(38)[1.54] NLLTMARPSWLH(20)[0.99] ANPKVRVGHAAS(20)[0.80] VKKRTLCAAACC(6)[0.28] VKVTTASCCSMW(4)[0.31] HLMDHPIRSNNQ(1)[0.30] TTRSSAFLIGGP(1)[0.29] 7 Phage display (common panning) 5 PMID:23031494 Gold Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA To verify specificity of selected phages, two different and independent assays were conduced. Wild-type (Wt-M13) phages with no displayed peptide on their surface were used as negative control. Results were analyzed by an ELISA reader (LabSystem Multiscan MS) at 405 nm. The intensity of absorbance at 405 nm from selected phages adsorbed on gold surfaces was reproduced from Figure S6 in the literature. The intensity of absorbance of the Wt-M13 phage was 0.27. In the phage context, we demonstrated the adhesive motif was capable to adsorb on gold in a preferential way with a morphological and viscoelastic signature of the adsorbed layer as evidenced by QCM-D and AFM investigations. Out of the phage context, the linear dodecapeptide (TLLVIRGLPGAC) is reproducibly found to adhere to the gold surface, and by quantitative SPR measurements, high affinity constants (Keq ~ 1.0e6 M-1, binding energy ~ -8kcal/mol) were determined. We proved that the interactions occurring at gold interface were mainly hydrophobic as a consequence of high frequency of hydrophobic residues in the peptide sequence. Moreover, by CD, molecular dynamics and steered molecular dynamics, we demonstrated that the molecular flexibility only played a minor role in the peptide adsorption. 3270 CKKNSPTLC(15/24) CIGNSNTLC(7/24) CAKATCPAC(2/24) 3 Phage display (subtractive panning) 4 PMID:28029647 G-protein coupled receptor 55 Lysophosphatidylinositol, LPI Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) Briefly, phages of approximately 1.0e10 plaque-forming units (PFU) were pre-incubated for 1 h with 5.0e5 wild-type HEK293 cells. The supernatant containing unbound phage particles was subsequently transferred to 35-mm Petri dishes to be incubated with 5 ×105 ev-HEK293 cells for an additional 2 h, to further eliminate phage particles with a specific binding. The ultimate pre-cleaned supernatant was incubated with GPR55-HEK293 cells for 2 h, and unbound phage particles were removed by washing with 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween-20. The extracellular bound phages were eluted with 100 mM glycine-HCl (pH 2.2) for 10 min, and neutralized with 1 M Tris-HCl (pH 9.0). The plasma-membrane-bound phages were collected. Sequencing of selected phagotopes dictated the primary structure for the synthetic FITC-labeled peptide P1 (CKKNSPTLC), which was analyzed for binding specificity to immunoprecipitated HA-GPR55, and to endogenously expressed the lysophosphatidylinositol receptor GPR55, using cells interfered or not for GPR55, as well as for co-localization imaging with HA-GPR55 at the membrane level. The peptide P1 stimulated GPR55 endocytosis and inhibited GPR55-dependent proliferation of EHEB and DeFew cells, two human B-lymphoblastoid cell lines. Our data support the potential therapeutic application of peptide ligands of GPR55 for targeting and inhibiting growth of neoplastic cells, which overexpress GPR55 and are dependent on GPR55 signaling for their proliferation. 3271 CKKNSPTLC(14/18) CIGNSNTLC(4/18) 2 Phage display (subtractive panning) 4 PMID:28029647 G-protein coupled receptor 55 Lysophosphatidylinositol, LPI Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) Briefly, phages of approximately 1.0e10 plaque-forming units (PFU) were pre-incubated for 1 h with 5.0e5 wild-type HEK293 cells. The supernatant containing unbound phage particles was subsequently transferred to 35-mm Petri dishes to be incubated with 5 ×105 ev-HEK293 cells for an additional 2 h, to further eliminate phage particles with a specific binding. The ultimate pre-cleaned supernatant was incubated with GPR55-HEK293 cells for 2 h, and unbound phage particles were removed by washing with 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween-20. The extracellular bound phages were eluted with 100 mM glycine-HCl (pH 2.2) for 10 min, and neutralized with 1 M Tris-HCl (pH 9.0). An additional step of cryogenic lysis of GPR55-HEK293 cells allowed the recovery of the intracellular bound phages. The internalized phages were collected. Sequencing of selected phagotopes dictated the primary structure for the synthetic FITC-labeled peptide P1 (CKKNSPTLC), which was analyzed for binding specificity to immunoprecipitated HA-GPR55, and to endogenously expressed the lysophosphatidylinositol receptor GPR55, using cells interfered or not for GPR55, as well as for co-localization imaging with HA-GPR55 at the membrane level. The peptide P1 stimulated GPR55 endocytosis and inhibited GPR55-dependent proliferation of EHEB and DeFew cells, two human B-lymphoblastoid cell lines. Our data support the potential therapeutic application of peptide ligands of GPR55 for targeting and inhibiting growth of neoplastic cells, which overexpress GPR55 and are dependent on GPR55 signaling for their proliferation. 3272 LMSTCTWLDECFRPQ(6)[1.025] PTPPCHRGDECQPLW(5)[0.703] SRPHCYPMDDCHPLW(4)[1.137] WSGMCESQWCKDFA(4)[1.039] TPHTCKLLDECVPLW(3)[2.925] TPTVCTWLDECPPWS(1)[1.052] HTPWCSIADPCLWEL(1)[1.597] YHEPCLWATACPTTP(1)[0.464] MQDMCNDDSCPLWS (1)[0.481] CTHPCEPPPLWPIAP(1)[2.082] PGPQCPYPAELWCTQ(1)[0.336] 11 Phage display (competitive panning) 2 PMID:24356816 Anti-CD43 monoclonal antibody UN1 Leukosialin Not determined. Not determined. f88-Cys5 phage display library (X4CX5CX4) ELISA Optical density (OD) at 405 nm (OD405) and 620 nm (OD620) were measured by an ELISA reader (Tecan), and values were expressed and shown as difference between OD405 and OD620. The UN1 mAb (10 μg) was linked to streptavidin-conjugated magnetic beads (Promega), which had been coated with 10 μg of goat anti-mouse IgG (Fc-specific) biotin-conjugated Ab (Sigma-Aldrich) in 200 μL of beads suspension. A total of 3.0e10 transducing units of phages from the library were added and incubated for 16 hours at 4 °C. After extensive washing, bound phages were eluted with 0.1 mol/L HCl/glycine buffer, pH 2.2, 1 mg/mL bovine serum albumin (BSA), and neutralized with 1 mol/L Tris, pH9.1. Eluted phages were amplified by infection of K91BK bacteria, and purified from plaques for a second round of affinity selection. Phage colonies were transferred according to an ordered grid on a lawn of K91BK cells on Luria-Bertani (LB) agar plates supplemented with 1 mmol/L isopropyl b-D-1-thiogalactopyranoside. Nitrocellulose filters were layered onto these plates and incubated overnight at 37 °C. Filters were blocked for 2 hours with blocking buffer(1 × PBS, 5% non-fat dry milk, 0.1% NP40, 0.01% NaN3) at room temperature and incubated O/N at 4 °C with the UN1 mAb (1 μg/mL) in blocking buffer. Then, filters were washed with washing buffer (1 × PBS, 0.1% NP40) and incubated with alkaline phosphatase-conjugated anti-mouse IgG (Fc specific) secondary antibody (SigmaAldrich) at the dilution of 1:5000 for 4 hours at 4 °C. After extensive washing, filters were incubated in developing solution (1-Step NBT/BCIP, Pierce, Thermo Scientific). Collected phage supernatants from positive clones were further analyzed by ELISA. By screening a phage-displayed random peptide library, we identified the phagotope 2/165 with the sequence of TPHTCKLLDECVPLW as a mimotope of the UN1 antigen, as it harbored a peptide sequence that was specifically recognized by the UN1 mAb and inhibited the binding of the UN1 mAb to UN1-positive tumor cells. On the basis of sequence homology with the extracellular region of CD43 (amino acids 64 to 83), the 2/165 peptide sequence was likely mimicking the protein core of the UN1/CD43 epitope. When used as vaccine in mice, the 2/165 phagotope raised antibodies against the UN1/CD43 antigen, indicating that the 2/165 phagotope mimicked the UN1 antigen structure, and could represent a novel immunogen for cancer immunotherapy. 3273 CIIGSYVC CNLSVPAC CLSPIGEC CLGKGSVC CGRDSKQC CAAAPIRC CAGGGAYC CMRGKGLC CASGSENC CVPMRLQC CGRAKVRC CLPISSSC CMARQARC CRECGERC CAGKSSNC CIRREKRC CNIRRQGC CPQPRPLC CLANLQTC CAVVFSQC CSIRRESC CWRSVEVC CADVLRPC CEGESASC 24 Phage display (subtractive panning) 3 PMID:11689892 Human endothelial cells stimulated with vascular endothelial growth factor (VEGF) Not determined. Not determined. Not determined. CX6C phage display library A method termed biopanning and rapid analysis of selective interactive ligands (BRASIL) was used. BRASIL allows separation of phage-cell complexes from the remaining unbound phage; this is accomplished by a differential centrifugation that drives the cells from a hydrophilic environment into a non-miscible organic phase. Because the organic phase is hydrophobic, it excludes water-soluble materials surrounding cell surfaces. Bound phage are recovered from the cell pellet whereas the unbound phage remain soluble in the upper aqueous phase, eliminating the need for repeated washes. A two-step panning strategy to isolate phage that bind to angiogenic ECs was used. First, to decrease non-specific binding, we pre-cleared the phage library on starved ECs (before panning on the same cell line stimulated with recombinant VEGF165); starved human umbilical vein ECs (HUVECs) were incubated with the phage library and centrifuged through the organic phase. Second, the unbound phage pool left in the aqueous phase was transferred to a fresh tube and incubated with VEGF165-stimulated HUVECs. After centrifugation through the organic phase, phage bound to the VEGF165-stimulated HUVEC pellet were recovered by bacterial infection, amplified and subjected to two more rounds of selection. The motif PQPRPL is a novel chimeric ligand mimic that binds specifically to VEGF receptor-1 and to neuropilin-1. 3274 PFTVSVPFVWNFTAD(28/60)[0.59 ± 0.01] WAGPRVSSVYYAGAR(6/60)[0.72 ± 0.01] 2 Phage display (common panning) 3 PMID:29156833 Anti-leptospira monoclonal antibody P17 (8C1E11) Not determined. Not determined. Not determined. f5-15mer phage display library (X15) ELISA Eleven phage clones with the highest selection frequency and unique sequences were chosen for analysis of their ability to bind the v6 region peptide using phage ELISA. The optical densities were measured with a μQuant Universal Microplate Spectrophotometer (Bio-Tek Instruments, Winooski, VT) at 490 nm. The data shown were reproduced from Figure 1C. Libraries were selected against a v6 region peptide derivative, KEQWFGNRWHEGYR, which encompassed a 14 amino acid sequence of v6 domain of human CD44v6 protein. After three cycles of affinity selection, sixty individual phage clones from the elution pool of the fUSE5 library were randomly chosen for DNA sequencing. A 15 amino acid peptide (PFTVSVPFVWNFTAD, PFT) was identified by affinity selection against a peptide derived from the v6 region of CD44 (CD44v6). Synthesized PFT exhibited specific binding to CD44v6 with an equilibrium dissociation constant (Kd) of 743.4 nM. PFT also bound CD44v6 highly expressed on human PCa cell lines. Further, an aggressive form of PCa cells (v6A3) was isolated and tagged by a novel CSC reporter vector. The v6A3 cells had a CSC-like phenotype including enriched CD44v6 expression, enhanced clonogenicity, resistance to chemotherapeutics, and generation of heterogeneous offspring. PFT exhibited preferential binding to v6A3 cells compared to parental cells. Immunohistofluorescence studies with human PCa tissue microarrays (TMA) indicated that PFT was highly accurate in detecting CD44v6 positive aggressive PCa cells, and staining positivity was significantly higher in late stage, metastatic and higher-grade samples. Taken together, this study provides for the first time phage display selected peptides that target CD44v6 overexpressed on PCa cells. Peptide PFT may be explored as an aid in the diagnosis and therapy of advanced PCa disease. 3275 CSAYDRPLC(12/60)[0.74 ± 0.01] CWSNKGYDC(2/60)[0.79 ± 0.01] 2 Phage display (common panning) 3 PMID:29156833 Peptide KEQWFGNRWHEGYR Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) ELISA Eleven phage clones with the highest selection frequency and unique sequences were chosen for analysis of their ability to bind the v6 region peptide using phage ELISA. The optical densities were measured with a μQuant Universal Microplate Spectrophotometer (Bio-Tek Instruments, Winooski, VT) at 490 nm. The data shown were reproduced from Figure 1C. Libraries were selected against a v6 region peptide derivative, KEQWFGNRWHEGYR, which encompassed a 14 amino acid sequence of v6 domain of human CD44v6 protein. After three cycles of affinity selection, sixty individual phage clones from the elution pool of the M13C7C library were randomly chosen for DNA sequencing.