3276 YLWPDYPAPSLP(12)[6.14 ± 1.95] WVRVRSMLPVAP(11)[8.59 ± 1.81] WLHQECARMGAL(11)[23.19 ± 4.87] TCSRLAPWACVT(11)[8.61 ± 3.09] VFRGSPGYVEYV(10)[30.40 ± 2.86] PIPPLRWFEERV(10)[10.62 ± 2.00] WWTTKAFLSGPW(9)[31.12 ± 2.20] WSFLVPPSFRPI(9)[29.40 ± 5.94] GWYWTADLMDKM(9)[8.68 ± 3.57] TFHLMRPLMLPV(8)[17.57 ± 3.05] VPLRHSSLVWLV(7)[7.07 ± 2.65] WPDWTPRLPVPY(6)[37.74 ± 4.16] VRVAPSVMAPVL(6)[11.06 ± 4.93] LRPVVASKSWVI(6)[8.52 ± 5.38] YPAVPPRVVAAI(5)[23.98 ± 12.71] GFTWKGYCSELV(5)[12.96 ± 5.97] YPPVYRAEKTLA(3)[11.65 ± 0.33] WRGSLSYLKGPL(3)[13.32 ± 8.83] WRCIGSWVSASY(3)[19.03 ± 4.04] WLYDRVHSMWVL(3)[31.25 ± 2.87] WKSCGVWAGCPM(3)[10.89 ± 3.01] SVMFSWPAPMIP(3)[32.75 ± 1.83] RWYKPLPSLLLW(3)[28.92 ± 4.52] RCIYPAYTGCLF(3)[3.48 ± 0.41] MPARPTLPTGLI(3)[27.72 ± 3.83] HGWHCVYGESYV(3)[4.32 ± 1.08] YRTRALPSLVVR(2)[7.66 ± 2.95] WVYRGNLGIWVL(2)[35.54 ± 0.16] WSTGSYWGSLLW(2)[7.35 ± 1.26] WRGGPAYLKYIS(2)[5.72 ± 0.41] WRDSSGYVMGPW(2)[33.72 ± 1.11] VLLTHSQWSKLY(2)[14.49 ± 9.79] VFWRTALAIPGG(2)[23.43 ± 2.06] STRSQLPWHWSQ(2)[13.91 ± 0.34] LSRYGYAYEAYV(2)[4.88 ± 1.88] GWRWEYSYLVGP(2)[33.31 ± 1.02] GVVRGDAYWFYV(2)[5.28 ± 2.12] GRPAIRIPDIIG(2)[24.17 ± 1.58] GPPVSVKSWVSQ(2)[8.73 ± 5.69] GPIVPVPSWLGI(2)[40.11 ± 0.85] GLLDHRMWNELY(2)[24.30 ± 0.58] AAPVVPSVPLNW(2)[4.73 ± 0.40] YLVGTMAPWWPA(1)[6.18] YFWHDLTGTWSV(1)[4.04] YDPRGYEIPSWL(1)[41.05] YCWWVSFTDCPL(1)[14.10] WYREPMPTPVVM(1)[10.76] WWWPGYDAYMSV(1)[37.69] WWWDSWNGFELL(1)[4.58] WWMESTFLEGPS(1)[41.43] WWFLDSYVNGPL(1)[28.46] WRALPNPGVSYV(1)[24.50] WPDWTPTLQVPW(1)[36.99] WLTYPAPPTLPF(1)[4.32] WLHLHPPIWAPL(1)[9.72] WLFSETDQVWYL(1)[18.43] WGWWLLDDVITS(1)[4.39] WGRYLACYHHLD(1)[9.71] WGPVVAPRIESA(1)[6.43] WEFVRANLPVWG(1)[12.05] WCLLVPHSTCAV(1)[6.48] WARRQLPALPVV(1)[35.70] VSLAGRVAAVLP(1)[11.53] VRMLPTPPNYFD(1)[4.44] VRAPAVIPRVTY(1)[4.93] VPNFLPPAVKNL(1)[26.70] VPKVPSAPAYYL(1)[4.49] VPAVNRLAKFVE(1)[26.14] VLPRFQAPSPTV(1)[20.80] VFDHVRGQWVAI(1)[26.41] VAAKELWWWDVV(1)[20.99] TPWWRAAIPSSL(1)[14.46] TGPLTAPRSVLY(1)[11.41] TAPCLPTLPWWQ(1)[20.62] SVPVLPYRAVTL(1)[4.14] SRPAFPMPGIVS(1)[3.06] SRFSFWLPVPAG(1)[24.99] SLPGWAPARFVS(1)[5.87] SLAVKWAAYPGL(1)[3.56] SGRPVPPVRWVM(1)[9.82] SGPTVPPVMWMM(1)[10.59] SEVPLFRPILRV(1)[30.35] SAWMRNLPLVPQ(1)[30.03] RWWWGGVPVNLI(1)[8.19] RWSIWHTGYLLV(1)[13.89] RWSHGYLAYMLD(1)[8.26] RWGWSFDPWLSM(1)[3.25] RWGWCFDRSLLD(1)[3.80] RVVKDWARLAIP(1)[3.60] RVVAPYRIPDWL(1)[35.37] RVPPRIVAPLYG(1)[13.44] RRPVVASNSWVI(1)[6.59] RQTYSSYIAMAM(1)[3.87] RQKMPIPVGNLV(1)[20.55] RPPVVASNSWVI(1)[15.42] RPDKPVRFVVSG(1)[10.97] RPAIPSRPIQFQ(1)[17.16] RLQRPLPALPCK(1)[31.17] RLPLRPPLPHTS(1)[5.48] RIVYGGGRWFLM(1)[6.01] RIRPAVALPYLL(1)[3.06] RIPNFIPMGPMM(1)[24.72] RGSVGYLTYLEH(1)[8.08] RETFSTYWLRTH(1)[5.97] RAPMPVGQSWMV(1)[18.41] RACFFIQGAVRC(1)[8.67] QMLPSFSPAEVI(1)[26.64] QGCCFVRSLPLM(1)[22.51] PWSRALPPLPGD(1)[3.28] PWATWGILSPEL(1)[29.94] PRYRLLPLYPYL(1)[32.37] PRRPQLPVVAVS(1)[7.86] PPLTAPRFWAEI(1)[28.56] PPAIPSGFCIGF(1)[18.13] PLVDRHLKYMGV(1)[29.21] PKYSAGLPAVPQ(1)[5.35] PAVGSSYACPGL(1)[23.73] NRPKAQLPTCYL(1)[14.57] NLQLPKWSPAAI(1)[22.07] NIMFGRLAAVLP(1)[23.10] NIGRARFMVPFL(1)[3.49] NGWALFKQTYMV(1)[28.55] MPHRGPLSLPTA(1)[3.49] MGNRARMSIPFV(1)[14.14] MFPTFIPPLPGK(1)[6.12] LVPALPEYSWNW(1)[15.87] LSYVVRPLPELP(1)[11.41] LSQRARLELPWS(1)[12.19] LRYGSAYLRYVS(1)[10.61] LRTGEAYLRYVD(1)[10.12] LRGAYPFHSWMV(1)[6.61] LPLPPIPPSYIA(1)[14.85] LPFHSRANPHYL(1)[8.36] LIPVRPVLPMAM(1)[17.35] LIAVKPLRTAFI(1)[5.34] LFVVKPLRLSVL(1)[10.22] LAYRSRAFLALP(1)[8.93] LAVPTRPSLPWT(1)[27.27] LALRSLPLVGAQ(1)[3.82] KRYSALPRVTDT(1)[30.90] ISYIGPEVASRA(1)[10.65] IIWSPVSSWWDL(1)[3.06] IGRAQFPLPIIS(1)[24.68] ICFGSGVGWYEC(1)[4.88] HRLFRLPLPVPV(1)[20.67] HPAVPPRVVAAI(1)[32.89] GYWKWSPVLDIV(1)[4.54] GYFPVIPTSVAN(1)[14.78] GWYYFQGMMGTS(1)[3.04] GWYCNYQTSTVF(1)[4.41] GWYCEYVYQARQ(1)[3.59] GWWWVESPLKVI(1)[3.06] GWWFLEPPSGAP(1)[5.67] GWRVTAAYLTYG(1)[13.87] GWRGSYAFLQGP(1)[31.03] GVKWYRRAPLLT(1)[17.19] GVAPGLPLYGPW(1)[10.42] GRYPLVNWVLFP(1)[22.87] GRTYPA*SGCWL(1)[3.57] GRKPPLLMAPIY(1)[8.06] GPWFLDGYGVWW(1)[6.81] GPSVSERTFFYL(1)[3.63] GPSVPSLPLDFG(1)[29.98] GPPVWPRMPGLV(1)[4.44] GPPVNYATKYEV(1)[15.00] GPMVWPRQVVYV(1)[14.34] GPAVPRLTVCYT(1)[10.43] GNAPATMQRYFV(1)[4.23] GLKRYLPLLTWT(1)[28.74] GIRGRSFWLPVP(1)[27.42] GGACQYVQHWSC(1)[5.30] GFSHPEWPAPML(1)[10.63] GAPALPMRSWWL(1)[18.91] FPSWWGDGPVGV(1)[7.16] ESYIPAVGAVAF(1)[5.72] DIRNRDLPPLPL(1)[19.07] DIPQFWFRGVFY(1)[6.15] CYVYDSWHVCLP(1)[14.74] ATVRNRLPIPLL(1)[4.25] APPVPLRYLQRL(1)[3.25] APMVLPRSSVSL(1)[27.62] APIIGPKTWLVS(1)[8.88] APEIDRSYLFLF(1)[20.30] ALALPPFPHGYV(1)[12.66] AKRYGIAYEMYV(1)[6.83] 185 Phage display (common panning) 5 PMID:28890361 SH3 domain Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA They validated most of the non-canonical specificity motifs by isolating individual clones from the phage pools to test for positive interactions with the cognate SH3 domains by phage ELISAs. Clonal ElISA signal is the ratio between protein and GST. Qualitatively, everything above 3 is considered a positive binder at uM or lower range. Based on ~70,000 unique binding peptides, we obtained 154 specificity profiles for 115 SH3 domains, which reveal that roughly half of the SH3 domains exhibit non-canonical specificities and collectively recognize a wide variety of peptide motifs, most of which were previously unknown. Crystal structures of SH3 domains with two distinct non-canonical specificities revealed novel peptide-binding modes through an extended surface outside of the canonical proline-binding site. Our results constitute a significant contribution toward a complete understanding of the mechanisms underlying SH3-mediated cellular responses. 3277 CDAGRKQKC(952)[0.48,0.65] CAKRNGKSC CGRKNVKLC CGRKQSERC CQGKRQTEC CGGRKQTAC CAPAGKKQC CYMGKKNAC CGLRGRRQC CKDMGKRNC CDRGRRNPC CGGTARRNC CARRNTRAC CAKKNATTC CSARKQKDC CVSAGKRQC CRYAKKNEC CRGRRNDEC CVGNAKKQC CGKRNARSC CIPGRKNPC CGGRRNRTC CAGKRQPMC CGARRNKSC CVARRNVGC CGRRNC [AG]-B-B-[NQ] 26 Phage display (in vivo) 1 PMID:29123083 Connective tissue growth factor (CTGF) Not determined. Not determined. Not determined. T7 CX7C phage display library ELISA To demonstrate the specific binding of DAG peptide to its receptor, ELISA plate wells were coated with full-length recombinant human CTGF/CCN2 (R&D systems; cat#9190-CC) at a concentration of 5 μ/ml for 2–3 h at room temperature.The reaction was stopped with the addition of equal volume of 1 M sulphuric acid and absorbance measured at 450 nm.(20um,60um) A CX7C naïve phage library (1e10 pfu of, in 100 µL of PBS) was intravenously injected into hAPP-J20 mice and allowed to circulate for 30 minutes due to its short half life, after which mice were anesthetized with 2.5% avertin and perfused with PBS intracardially. The brain was removed and the hippocampus was extracted and homogenized in LB-NP 40 (1%). The phages in the lysate were rescued by amplification in E.coli. 3278 MSSRSRPHINSL(3) TAELYPDLQSSQ(2) EDARRPPTSTEH(2) PVNKQHTSLQNN(2) VNHEYKLHSIKY(1) DDTYPSRPVYLK(1) DLYLSHGAPPQH(1) HVTHNITNESNS(1) ARMTFSQMSPHT(1) TGSIRPKLHASP(1) QLARMSSLHVPM(1) SHEISRITAVSK(1) VDMVTKQLLEYP(1) ELQIGSWRMPPM(1) SERLMTPPKLFR(1) MTHKQMHKHHGL(1) LVSLTPPWINVD(1) SSAQMNLNTFLN(1) LGSHNIRLGEGS(1) YPHPIRQNFFAY(1) KSHTENSFTNVW(1) KLPPMNSDSMVW(1) HMNAHLTFQSAI(1) DAVKTHHLKHHS(1) 24 Phage display (competitive panning) 3 PMID:29119720 1-deoxy-D-xylulose-5-phosphate synthase Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) For the first two rounds, DXS functionalized with N-hydroxysuccinimide (NHS)–desthiobiotin and Dynabeads MyOne Streptavidin C1 (Invitrogen 65001) were used to capture DXS from the solution. For the third round, to avoid selection of phages against streptavidin and given that D. radiodurans DXS contains an N-terminal Histag, MagneHis Ni Particles (Promega V8560) were used as capturing system. After the incubation step, 0.1 mL of beads were added to the solution, which was mixed in a thermo shaker at 48Cfor15 min. The tube was placed in a magnetic rack to allow for adhesion of the magnetic beads on one side of the tube. Phages expressing DXS binders were retained on the bead surface, and the buffer containing unbound phages was gently discarded from the tube. To remove weakly bound phages further, the beads were washed with PBST (10V1 mLPBS with 0.05% Tween 20) whilst retaining them using a magnetic rack. The elution of strongly bound phages from the beads was achieved by suspending the beads in the elution buffer (1 mL, 1mM Biotin in PBS for rounds 1 and 2 and 500 mm imidazole in PBS for round 3). 3279 MAIPTRGKMPQY(8) HSSPVQTDWITV(4) KAIRTRGKRPQY(1) YSSTIYTPTAVG(1) GSSLLYSGSGPA(1) ALWPPNLHAWVP(1) SSSPVAWALAMR(1) HSSPPFPWLLVT(1) DSSSGLYLRPLS(1) VSSSIFPIALPD(1) THPSTKVPGTPA(1) ASSVISPRWLLW(1) ALWPPNLHAWVP(1) TSSAAAPYYSPP(1) VSSMKGPTLSTN(1) DSSTWLFLSSYR(1) 16 Phage display (competitive panning) 2 PMID:29119720 1-deoxy-D-xylulose-5-phosphate synthase Not determined. Not determined. Not determined. XS2X9, Ph.D.-12 and wild-type phage library pool Two rounds of selection were performed by using a custom-made phage library. TBS (trisbuffered saline, 50 mm Tris·HCl pH 7.5, 150 mm NaCl) was used as incubation buffer,TBST (TBS with 0.05% Tween 20) was used as washing buffer, MagneHis Ni Particles (Promega V8560) were employed for protein recovery, and 1mm ThDP in TBS was used as elution buffer. ThDP was chosen as competitive eluent to elute peptides specifically interacting with the ThDP-binding site of DXS. A D-GAP-competitive peptidic inhibitor, P12 (MAIPTRGKMPQY), with a Ki value of (113 ± 4) μM, was identified by phage display targeting the thiamine diphosphate (ThDP)-binding pocket. 3280 CSPGAKVRC(322/143637) CGEKRTRGC(279/143637) CDSRSRKRC(226/143637) CPKGAGSKC(196/143637) CRKGTLGRC(149/143637) CVKGTVKTC(148/143637) CRGTIRGRC(143/143637) CLDRRKKPC(136/143637) CPARRSNRC(117/143637) CKDSAEMRC(113/143637) CERSNNVSC(113/143637) CGKRAPFRC(110/143637) CVSSSSGRC(108/143637) 13 Phage display (in vivo) 1 PMID:29116108 Peritoneal macrophage of mouse bearing breast cell line 4T1 Not determined. Not determined. Not determined. T7 CX7C phage display library Naïve phage library was injected intraperitoneally in 4T1 tumor-bearing mice, and allowed to circulate for 2 h. Peritoneal cells were collected, the accompanying phages were rescued and the peptide-encoding segment of phage DNA was sequenced. Tumor-associated macrophages (TAMs) expressing the multi-ligand endocytic receptor mannose receptor (CD206/MRC1) contribute to tumor immunosuppression, angiogenesis, metastasis, and relapse. Here, we describe a peptide that selectively targets MRC1-expressing TAMs (MEMs). Deep sequencing of the peptide-encoding inserts in the selected phage pool revealed enrichment of the peptide CSPGAKVRC (codenamed “UNO”). Intravenously injected FAM-labeled UNO (FAM-UNO) homed to tumor and sentinel lymph node MEMs in different cancer models: 4T1 and MCF-7 breast carcinoma, B16F10 melanoma, WT-GBM glioma and MKN45-P gastric carcinoma. Fluorescence anisotropy assay showed that FAM-UNO interacts with recombinant CD206 when subjected to reducing conditions. Interestingly, the GSPGAK motif is present in all CD206-binding collagens. FAM-UNO was able to transport drug-loaded nanoparticles into MEMs, whereas particles without the peptide were not taken up by MEMs. In ex vivo organ imaging, FAM-UNO showed significantly higher accumulation in sentinel lymph nodes than a control peptide. This study suggests applications for UNO peptide in diagnostic imaging and therapeutic targeting of MEMs in solid tumors. 3281 CTVAGSGPC(77/197903) CIGNSKKKC(66/197903) CVRLREKRC(63/197903) CRKKSRAQC(60/197903) CGKRAPFRC(59/197903) CAANDYSDC(57/197903) CLEDDSAKC(57/197903) CNDKS(55/197903) CVGRKVRGC(55/197903) CVDRRESRC(53/197903) CRQEEESRC(52/197903) CGKTRYSRC(50/197903) CSPGAKVRC(50/197903) 13 Phage display (in vivo) 1 PMID:29116108 Peritoneal macrophage of healthy mouse Not determined. Not determined. Not determined. T7 CX7C phage display library Naïve phage library was injected intraperitoneally in eight-week old Balb/c mice (Charles River, Wilmington, MA, USA), and allowed to circulate for 2 h. Peritoneal cells were collected, the accompanying phages were rescued and the peptide-encoding segment of phage DNA was sequenced. 3282 CRTLRSKAC(775) CRRTRQRSC(500) CIGNK(420) CVLNESGDC(216) CRDKRGSKC(194) CVGRKVRGC(193) CKRPNENVC(183) CNRRTKIGC(182) CSPKMRATC(180) CKRTRRREC(176) CLSSITPEC(169) CVDQDPL(163) CRGTRSNRC(157) CGPCQEGLC(153) CMTLRSRKC(147) CSTKTSLKC(146) CGDEA(146) CTTSTGADC(141) CSTLKRRVC(136) CRGVAKVRC(133) CSVGRLK(131) CHQDF(128) CSFDEANPC(124) CRNRA(123) CVSDRKVAC(123) CKRGRFAKL(120) CAQPNSR(115) CRPTRRVSC(114) CRNGLNKRC(113) CGFRSD(112) CRKTVGPRC(112) CEVMQRKRC(107) CVASVKRKC(106) CDANQ(106) CRRTAIKKC(106) CDSRSRKRC(105) CLSKRTPRC(105) CVDSEATDC(105) CPRTAKVLC(105) CQSRSPRNC(101) CNKNGTAPC(101) CTDRHSTNC(94) CDALAPNSC(93) CIDGRTDLC(92) CMNVESSPC(91) CREKNSQRC(91) CLVRPERKC(90) CRKRMNRTC(90) CVDITSPDC(89) CSYEKEPVC(86) CSPGAKVRC(10) 51 Phage display (common panning) 1 PMID:29116108 Mouse macrophage cell line Raw 267.4 Not determined. Not determined. Not determined. T7 CX7C phage display library 3283 CNDAPLTEC[0.105±0.015] CYSANASSC[0.121±0.012] CGPLPHRHC[0.095±0.045] CNPHFYKHC[0.059±0.006] CTESPNPLC[0.062±0.009] CSMPLMGHC[0.080±0.012] CTMREQHFC[0.021±0.007] CNTHEKPTC[0.060±0.015] CQNTHTKQC[0.195±0.013] CGGSMLSSC[0.133±0.002] CWASNVQSC[0.110±0.007] CTMSHENTC[0.098±0.006] CDSKHVPFC[0.045±0.003] CGSAPVRSC[0.048±0.003] CHMFTGSTC[0.052±0.004] CSKEATPFC[0.058±0.002] 16 Phage display (common panning) 1 PMID:28986145 bB2-crystallin fibrils Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) ELISA The absorbance of each well was measured at 450 nm using FlexStation® 3 multimode plate reader.Data were reproduced from Figure 6 and shown. 3284 CNAGHLSQC[0.081±0.010] CDFRIRNAC[0.084±0.031] CNTQMHPKC[0.135±0.046] CKQEDILRC[0.131±0.009] CETSNTKSC[0.325±0.042] CGKHQFPHC[0.205±0.051] CGPTAKYIC[0.288±0.052] CSKEATPFC[0.295±0.032] CVNNADNSC[0.125±0.022] CPKGDENTC[0.198±0.020] CVGREPHAC[0.190±0.005] CNVWRAPGC[0.245±0.002] CIKHKAWKC[0.195±0.040] 13 Phage display (common panning) 1 PMID:28986145 bB2-crystallin fibrils Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) ELISA The absorbance of each well was measured at 450 nm using FlexStation® 3 multimode plate reader.Data were reproduced from Figure 6 and shown. 3285 CKQFKDTTC[0.695±0.001] CVPSKPGLC[0.585±0.121] CFTSTLSRC[0.562±0.112] CMLPTRIAC[0.571±0.041] CDAHYEKSC[0.312±0.052] CNKHNMLNC[0.173±0.031] CYFHYPQRC[0.758±0.110] CSLFSKNYC[0.605±0.035] 8 Phage display (common panning) 1 PMID:28986145 bB2-crystallin fibrils Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) ELISA The absorbance of each well was measured at 450 nm using FlexStation® 3 multimode plate reader.Data were reproduced from Figure 6 and shown. 3286 YKRGYK(50)[10 ± 2, 620 ± 60] WPQGPT(11)[NT, NT] ETWGYK(6)[NT, NT] TRSGPT(3)[NT, NT] RNSGWS(3)[NT, NT] ARAGTT(2)[NT, NT] 6 Phage display (common panning) 3 PMID:29050803 Platinum Not determined. Not determined. Not determined. T7 LARFH gene-based phage display library (X3GX2) Quartz crystal microbalance (QCM) The interaction of YKRGYK with a platinum surface was monitored and quantified by usinga hand-made flow-quartz crystal microbalance system (f-QCM) with a custom-made acryl flow cell equipped with 9-MHz Pt or Au quartz crystals (Kyocera, Japan). The dissociation constant (KD, nM) was shown. The majority of the selected variants contained the Tyr-Lys-Arg-Gly-Tyr-Lys (YKRGYK) sequence in their randomized segment. We characterized the platinum-binding properties of mutant LARFH by using quartz crystal microbalance analysis. Mutant LARFH seemed to interact with platinum through its loop containing the YKRGYK sequence, as judged by the estimated exclusive area occupied by a single molecule. Furthermore, a 10-residue peptide containing the YKRGYK sequence bound to platinum with reasonably high affinity and basic side chains in the peptide were crucial in mediating this interaction. In conclusion, we have identified an amino acid sequence, YKRGYK, in the loop of a helix-loop-helix motif that shows high platinum-binding affinity. This sequence could be grafted into loops of other polypeptides as an approach to immobilize proteins on platinum electrodes for use as biosensors among other applications. 3287 CSLFKNFRC CRADFYTTC CRENTNHTC CWNEDHTWC CLNSLFGSC CTYSPTEVC CQLFPLFRC CTLQDQATC CMQHSMRVC 9 Phage display (common panning) 1 PMID:29103911 Mouse heart Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) For in vitro evaluation of uptake by skeletal and cardiac cells, we synthesized the best 12 candidate peptides equipped with a fluorescein isothiocyanate (FITC) label we used the Ph.D.-C7C phage display peptide library. A schematic outline of the screening procedure is given in Figure 1. We performed a first-round in vitro screening using human control myotubes in which internalized and surface-bound phages were isolated and amplified. Subsequently, the naive library was also used for a first-round in vivo enriched internalized phage fraction, and the enriched surface-bound phage fraction was used for a second in vivo screening round in mdx mice, a mouse model for DMD.All enriched libraries from the biopanning selections, and the naive library with and without one round of bacterial amplification, were sequenced using a published NGS approach. 3288 VHWDFRQWWQPS(5)[1.04±0.08] GDGNSVLKPGNW(1)[0.49±0.07] HLSLPLWKWEKS(1)[0.61±0.09] VFAYHLRIPSGD(1)[0.43±0.13] 4 Phage display (common panning) 3 PMID:29163684 The second extracellular loop (107-130) of C-C chemokine receptor type 9 Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA Affinity was detected using ELISA. The OD450 values were reproduced from Figure 1 in the literature. P1 was (VHWDFRQWWQPS) able to bind specifically with MOLT4 cells which exhibit marked expression of CCR9. In addition, P1 promoted the apoptosis of MOLT4 cells induced by doxorubicin, and inhibited the migration of MOLT4 cells in the presence of chemokine (C-C motif) ligand 25. It was suggested that decreased activity in the phosphorylation of protein kinase B in MOLT4 cells may be responsible for the inhibition. In conclusion, the peptide P1 derived from a screened phage is able to specifically bind to CCR9 and inhibit the activity of CCR9. It has potential use as an antagonist in the treatment of CCR9-overexpressed carcinoma. 3289 CQVRSNTTC CSLFKNFRC CWNEDHTWC CLNSLFGSC CLLGHTNNC CFSHTYRVC CQLFPLFRC CMQHSMRVC 8 Phage display (common panning) 1 PMID:29103911 Mouse muscle Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) we used the Ph.D.-C7C phage display peptide library. A schematic outline of the screening procedure is given in Figure 1. We performed a first-round in vitro screening using human control myotubes in which internalized and surface-bound phages were isolated and amplified. Subsequently, the naive library was also used for a first-round in vivo enriched internalized phage fraction, and the enriched surface-bound phage fraction was used for a second in vivo screening round in mdx mice, a mouse model for DMD.All enriched libraries from the biopanning selections, and the naive library with and without one round of bacterial amplification, were sequenced using a published NGS approach. 3290 SRRPASFRTARE(15)[68] GLHTSATNLYLH(4)[NT] 2 Phage display (common panning) 4 PMID:29152066 Extracellular domain of Fibroblast growth factor receptor 2 [1-377] Not determined. 1EV2,1II4,1IIL, Not determined. Ph.D.-12 phage display library (X12) Flow Cytometry The Cy5.5-labeled peptide was serially diluted in PBS at concentrations ranging from 0 to 200 nM in 25 nM increments. QhTERT/FGFR2c cells (~1.0e5) were incubated with peptide at 4°C for 1 hour, washed with cold PBS, and the mean fluorescence intensities were measured using flow cytometry. The equilibrium dissociation constant kd=1/ka was calculated by performing a least squares fit of the data to the non-linear equation I=(I0+Imaxka[X])/(I0+ka[X]). I0 and Imax are the initial and maximum fluorescence intensities, corresponding to no peptide and at saturation, respectively, and [X] represents the concentration of the bound peptide. Prism 5.0 software (GraphPad Inc) was used to calculate kd (nM). Four rounds of biopanning were performed using a decreasing quantity (100, 80, 60, and 40 μg) of FGFR2-ECD in successive rounds to increase binding specificity. We used phage display to identify the peptide SRRPASFRTARE that binds specifically to the extracellular domain of FGFR2. We labeled this peptide with a nearinfrared fluorophore Cy5.5, and validated the specific binding to FGFR2 overexpressed in cells in vitro. We found high affinity kd = 68 nM and rapid binding k = 0.16 min-1 (6.2 min). In human esophageal specimens, we found significantly greater peptide binding to high-grade dysplasia (HGD) versus either Barrett’s esophagus (BE) or normal squamous epithelium, and good correlation with anti-FGFR2 antibody. We also observed significantly greater peptide binding to excised specimens of esophageal squamous cell carcinoma and gastric cancer compared to normal mucosa. These results demonstrate potential for this FGFR2 peptide to be used as a clinical imaging agent to guide tissue biopsy and improve methods for early detection of esophageal adenocarcinoma (EAC) and potentially other epithelial-derived cancers. 3291 TWPKHFDKHTFYSILKLGKH[45] 1 Phage display (common panning) 6 PMID:29090274 Domain 3332-3779 of prolow-density lipoprotein receptor-related protein 1 Alpha-2-macroglobulin receptor-associated protein, Alpha-2-MRAP 2FYL, Not determined. T7 X12, X16 and X20 phage display library pool ELISA Binding activity and selectivity of biotinylated TWPKHFDKHTFYSILKLGKH (L57) to LRP1(3332-3379)-Fc were evaluated by plate ELISA. Binding activity was detected with HRP-conjugated streptavidin and was measured by determining the absorbance at 450 nm. LRP1(3332-3379)-Fc and Act RIIB-Fc are counter proteins. The absorbance at 450 nm of biotinylated L57 was expressed as the mean ± SD (n = 2). The EC50 (nM) value of L57 for binding to LRP1(3332-3379)-Fc was shown. For the screening of T7 phage libraries, 20 μg LRP1-Fc was immobilized on Dynabeads Protein A (200 μL; Invitrogen, Carlsbad, CA, USA) in PBS (045–29795; Wako, Osaka, Japan) containing 0.5% BSA. After washing of the beads with PBS containing 0.1% Tween-20 (PBST), they were incubated with 1.0e12 pfu of phage libraries (mixture of X12, X16, and X20) for 1h at room temperature, and then washed with PBST (three times for the first round, five times for the second round, and 10 times for the third to sixth rounds). Bound phages were eluted with 200 μL of 0.5% SDS and then transfected into 80 mL of E. coli BLT5615 cells (Merck Millipore) in the log phase of growth for amplification. After bacteriolysis, the phages were recovered from the culture supernatant by centrifugation and PEG precipitation, according to the T7 Select manual. The recovered phages were dissolved in 1mL of PBS and 0.5 mL of the phages was used for the next round of biopanning. Using phage display technology, we obtained a novel peptide, L57 (TWPKHFDKHTFYSILKLGKH-OH), with an EC50 value of 45 nM for binding to cluster 4 (Ser3332–Asp3779) of LRP1. L57 was stable in mouse plasma for up to 20 min. In situ brain perfusion assay in mice revealed the significantly high BBB permeability of L57 3292 SVDLSSFIPPL(1)[2.97 ± 0.09] AAASLRDFISRW(1)[3.53 ± 0.14] DYGIWGRWIPLI(1)[3.19 ± 0.02] GYNLRAWLPPPY(1)[3.24 ± 0.07] VPVHLGSWLPPP(1)[3.34 ± 0.07] WSPLSLTRFIPP(1)[3.41 ± 0.01] FDPWLTKYIPYP(1)[3.54 ± 0.07] ELPRLNQYIPPP(1)[3.68 ± 0.07] GAMHLPWHMGTL(10)[1.64 ± 0.11] GAMHLPWHMGTP(1)[1.68 ± 0.07] TSMKLDRWIPPL(1)[3.37 ± 0.01] WNPMVNFIEPPH(1)[2.75 ± 0.08] GPFVYNQWIPIP(1)[3.36 ± 0.02] FLTDYIPTPSRK(1)[3.10 ± 0.07] VNMTQFVPPPAR(1)[3.50 ± 0.15] YSTNYTAFIPGP(1)[3.50 ± 0.13] EVNLSRFIQLPS(1)[2.70 ± .018] WEGRFLHFLNYP(1)[2.65 ± 0.24] 18 Phage display (common panning) 3 PMID:29310289 Pesticidal crystal protein Cry1Ab Anti-Cry1Ab monoclonal antibody Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA 100 μL of Cry1Ab protein was coated in microtiter plate wells and incubated overnight at 4 °C. After blocking with 3% skimmed milk at 37 °C for 2 h and washing 3 times with PBST, 100 μL of each serial concentration of selected phage stock was added to the Cry1Ab coated wells and incubated at 37 °C for 1 h. Following washing 3 times with PBST, 100 μL of HRP-conjugated anti-M13 phage antibody (1:5000, diluted in PBS) was added to the wells and incubated at 37 °C for 1 h. Then, 100 μL 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution was added to each well and incubated for 10 min at room temperature with gentle agitation. The optical density at 450 nm was detected on microplate reader (Thermo Scientific). Variety of phage display peptides capable of binding to Cry1Ab were obtained by phage display peptide library panning, in which Ab-39(TSMKLDRWIPPL) not only had good binding activity to Cry1Ab but also highly specific for Cry1Ab. After modification of electrode with gold nanoparticles, selected phage displayed peptide was applied to electrochemical immunosensor for Cry1Ab. 3293 ATRSANGM[6.18 ± 0.48] EQVPYPGE[0.29 ± 0.07] ASRSPTDK[0.27] ASRGTIAG[3.09] 4 Phage display (common panning) 3 PMID:29414074 Prostate-specific antigen Anti-human total PSA monoclonal antibody Not determined. Not determined. f8-8mer phage display library (X8) ELISA The phage was loaded into wells of a 96-well microtiter plate, and 50 μL of t-PSA solution was added to the wells, and the plate was incubated at 37 °C. Then the wells were washed six times with PBST, anti-PSA mAb was added. The plate was washed again, and then 50 μL of IgG-HRP was added and the plate was incubated for 1 h at room temperature. For the detection of t-PSA, 100 μL of 0.04% OPD was added. The absorbance at 492 nm was recorded on a microplate reader. ~10e11 phage virions from f8/8 landscape phage library in 50 μL library diluents were added to the wells as the input for the first round of biopanning. After incubation overnight at 4 °C, the wells were washed six times with TBST to remove unbound phages. After three rounds of selection, individual phage clones were picked randomly and amplified. Peptide ATRSANGM showed highest affinity and specificity against t-PSA. 3294 GTQYYAYSTTQKS GMIVDHLPIQVNT GEYDYACGVVGYE GTQAIRVHTISSQ GSYPKASLALLAP GGLNQVLRIPSFI GSPKHNLDMVKMM 7 PMID:29493687 MgF2 nanoparticles (NPs) Not determined. Not determined. Not determined. GX12 phage display library Biopanning on MgF2 nanoparticles (NPs) in water provided recently seven different 12mer peptides and one of which was selected to be investigated as peptide–poly-(ethylene glycol) (pep–PEG) conjugate with a PEG-block of Mn = 3000 (pep-I-PEG). 3295 CFNLALHTC CISHGSPQC CLSTASNSC 3 Phage display (subtractive panning) 4 PMID:29516383 EAE-brain capillary endothelial cell Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) Brain endothelial cells derived from four deeply anesthetized rats with acute EAE (score 3) and four healthy rats were isolated. Briefly, brain pieces were gently homogenized, mixed with 30% dextran. The vascular fraction was filtered and digested in DMEM. Endothelial cells were pelleted and incubated with the phages for 2 h at 20 °C. The counter-selection step was performed by incubating 5e5 healthy brain capillary endothelial cells (BCECs) with 1e11 phages in 1 mL RPMI. After centrifugation of cells, the unbound phages were transferred on 4e5 EAE-BCEC for 2 h to obtain phages of the EAE-BCEC selection. The phages that remained bound to cells were recovered, amplified in E. coli ER 2738 bacteria. Four selection rounds were performed with isolated healthy or EAEBCEC. These peptides were respectively predicted to mimic the discointeracting protein 2 homolog A (DIP2A), the carboxypeptidase N, polypeptide 2 (CPN2), a plasma zinc metallocarboxypeptidase important in regulating vasoactive peptide hormones, growth factors and cytokines by specifically cleaving their C-terminal basic residues (Ph37), the intracellular protein huntingtin (Ph38), and the laminin subunit-1, a component of the extracellular matrix, in a region of epidermal growth factor (EGF)-like domain (Ph48). 3296 CSYKARVTC 1 Phage display (subtractive panning) 5 PMID:29516383 EAE-CNS brain endothelial cell Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) CNS cells were prepared from rat CNS. A rat with acute EAE (score 3) was deeply anesthetized and perfused with 50 mL ice-cold PBS 1×. The whole CNS tissues from both rats were recovered separately (about 2.3 g), cut into small pieces, and incubated for 50 min at 37 °C in 10 mL of PBS 1×. The recovered cells were concentrated and frozen. The Bex vivo total EAE selection^ was performed in two steps: (i) a counter-selection with CNS cells from a healthy rat realized to absorb healthy tissuespecific phages and (ii) the selection against cells from the EAE CNS. Thus, 1011 phages were incubated with 4.0 × 106 CNS cells from a healthy rat before incubation of the unbound phages with CNS cells from a EAE rat (4.0 × 106). After incubation, the cells were pelleted by centrifugation (1200 rpm/5 min/RT) and washed with 1 mL complemented RMPI. The phages that remained associated with cells were amplified. At each selection round, one additional washing step of cells associated with phages was added to increase the selection stringency, so that five washes were performed during the fifth round. This peptide mimics the discointeracting protein 2 homolog A (DIP2A), receptor for Follistatin 1, a glycoprotein involved in inflammatory reactions (Ph36). 3297 ACNTWNPWCPWDAPLCA[2.3 ± 2.7][0.40 ± 0.24] ACNNFPFRCVYYPDICA[0.44 ± 0.07][NT] 2 Phage display (common panning) PMID:29517911 Plasma kallikrein (EC:3.4.21.34) Not determined. Not determined. Not determined. CX6CX6C phage display library Inhibition assay NaCl, 1.5 μM bovine serum albumin, pH 7.5. PKal (typical nominal concentration 1−2 nM) was preincubated in 96-well plates with different concentrations of the tested peptide (or of aprotinin, used as a control molecule) for 15 min. The maximum final concentration of a given peptide was chosen based on its potency and was followed by a 1.25- to 3-fold serial dilution, plus a control with no peptide. The reactions were initiated by the addition of H-Pro-Phe-Arg-AMC at a final concentration of 10 to 25 μM, and initial rates of substrate hydrolysis were determined by linear fit of the raw fluorescence versus time traces. In the case of H-Pro-Phe-Arg-AMC however, the Km for PKal is large, and all experiments were performed under conditions where [S]≪ Km. The affinity of peptide binding to human and rat PKal are showed respectively. Bicyclic peptides with inhibitory activity against PKal were identified using Bicycle Therapeutics proprietary phage display platform. Briefly, linear peptide libraries containing randomized AA between 3 cysteine residues were displayed on the surface of filamentous phage and cyclized on the phage with a thiol-reactive molecular scaffold (TBMB). The libraries formats used were 5 × 5 and 6 × 6, such that either 5 or 6 randomized AA were between the first and second and second and third cysteine, respectively. Following cyclization of the phage libraries, repeated selections were performed against human and rat PKal. Selections and subsequent affinity maturation selections were performed against PKal using methodology described previously. Rat and human PKal orthologous enzymes were alternated as baits during each selection round in order to identify bicyclic peptides with good cross-reactivity. peptides were chemically synthesized for characterization. 3298 ACSWPARCLHQDLCA[0.07 ± 0.02][0.90 ± 0.33] 1 Phage display (common panning) PMID:29517911 Plasma kallikrein (EC:3.4.21.34) Not determined. Not determined. Not determined. CX5CX5C phage display library Inhibition assay NaCl, 1.5 μM bovine serum albumin, pH 7.5. PKal (typical nominal concentration 1−2 nM) was preincubated in 96-well plates with different concentrations of the tested peptide (or of aprotinin, used as a control molecule) for 15 min. The maximum final concentration of a given peptide was chosen based on its potency and was followed by a 1.25- to 3-fold serial dilution, plus a control with no peptide. The reactions were initiated by the addition of H-Pro-Phe-Arg-AMC at a final concentration of 10 to 25 μM, and initial rates of substrate hydrolysis were determined by linear fit of the raw fluorescence versus time traces. In the case of H-Pro-Phe-Arg-AMC however, the Km for PKal is large, and all experiments were performed under conditions where [S]≪ Km.The affinity of peptide binding to human and rat PKal are showed respectively. Bicyclic peptides with inhibitory activity against PKal were identified using Bicycle Therapeutics proprietary phage display platform. Briefly, linear peptide libraries containing randomized AA between 3 cysteine residues were displayed on the surface of filamentous phage and cyclized on the phage with a thiol-reactive molecular scaffold (TBMB). The libraries formats used were 5 × 5 and 6 × 6, such that either 5 or 6 randomized AA were between the first and second and second and third cysteine, respectively. Following cyclization of the phage libraries, repeated selections were performed against human and rat PKal. Selections and subsequent affinity maturation selections were performed against PKal using methodology described previously. Rat and human PKal orthologous enzymes were alternated as baits during each selection round in order to identify bicyclic peptides with good cross-reactivity. peptides were chemically synthesized for characterization. 3299 PGATAGPSS[1.47 ± 0.04] STRRIGWLTD[1.50 ± 0.03] TDQTHRLA[1.46 ± 0.05] PGATAGPSS[1.55 ± 0.04] PGATAGPSS[1.38 ± 0.04] TDQTHRLA[1.42 ± 0.07] STRRIGWLTD[1.46 ± 0.03] SWVHEWVTSN[1.56 ± 0.01] TDQASRLA[1.46 ± 0.03] RSGCTSGLHR[1.51 ± 0.04] TDQASRLA[1.45 ± 0.03] RSGCTSGLHR[1.43 ± 0.03] 12 Phage display (common panning) 3 PMID:29522802 Anti-S.aureus GapC monoclonal antibody(mAb2A9) Glyceraldehyde-3-phosphate dehydrogenase Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA Detection of the binding activities of 12 strongly positive phage clones to mAb2A9 that were selected by a sandwich ELISA and the OD450 value of each well was read using a microplate reader (Bio-Rad, Hercules, CA, USA). Wild-type M13 phage was used as the negative control and an E. coli ER2738 culture supernatant was used as the blank control. Bovine serum albumen-coated wells were used to exclude cross-activity. The consensus motif PVATGSLT of the 12 clones is located at amino acids 236–243 of the GapC protein. These results suggest that the motif 236PVATGSLT243 is an epitope of the S. aureus GapC protein and it might be a potential component of an epitope-based vaccine against S. aureus. 3300 VQTVQIGSD[0.34±0.02] VSSVSTGES[0.15±0.02] VSTLESNQD[0.14±0.01] 3 Phage display (common panning) 3 PMID:29362753 Vibrio parahaemolyticus Not determined. Not determined. Not determined. f8-9mer phage display library (X9) Dot blot The specificity and affinity of the phages binding to V. parahaemolyticus were determined by the phage capture assay. A V. parahaemolyticus specific fusion phage ligand was discovered from the f8/9 landscape phage library through a biopanning procedure. The phages from the f8/9 landscape library were added to a well of a 96-well plate with immobilized V. parahaemolyticus. After washing to remove the unbound phages, the V. parahaemolyticus binding phages were eluted, propagated, and purified for use as the input in the next two rounds of biopanning. After 3 rounds of biopanning, we randomly picked up 5 phage clones for PCR amplification and sequencing. The frequency of the phage VQTVQIGSD was higher than that of others.