3451 SVEPSDSSNPIY HFGVDPNADFVS FRVDLKNDGMPE SPALHTTIPGAK YHAKNDGMVFPT TYNYDMPLRGRA TIPTRDPAMLHS TALPKVHTLLTH NPHDDVLWLHPS VPLDHAYGHSIP AYTVDALHELRH VGPLGQVMSGHS HPHDRQLISTYP 13 Phage display (common panning) 3 PMID:30187588 IgE from patients with allergy to Scylla paramamosain FLN c Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) Overnight, coating of the wells of microtiter plate (Nunc Maxisorp, Copenhagen, Denmark) at 4°C with goat anti-human IgE (ε‐chain specific) (Sigma‐Aldrich, St. Louis, MO, USA) was used to capture IgE from patients’ sera. The phage display peptide library was added after the blocking incubation with captured IgE and eluted after the washing steps. 3452 CLHQSPHIC(4/44) CPTNNPRSC(2/44) CAQIPRANC(2/44) CKLTHPQTC(2/44) CMNTDKRMC(2/44) CQPANSKMC(2/44) CDTRWSNLC(1/44) CEQRTSHKC(1/44) CFGSTTWKC(1/44) CGLAKETMC(1/44) CHTHPTHDC(1/44) CKSHAHQNC(1/44) CLHHNNAYC(1/44) CMGMGQAWC(1/44) CMTKMAPHC(1/44) CMHNGSWQC(1/44) CNKVHGKTC(1/44) CNSHQASAC(1/44) CNTKGPYQC(1/44) CPKPHSDTC(1/44) CPQNGKEVC(1/44) CPSNKLTQC(1/44) CRYGLEHKC(1/44) CRVNDQSQC(1/44) CSRSHHDHC(1/44) CSPALIGQC(1/44) CSKHRIHQC(1/44) CSQSYRHSC(1/44) CSSHPALRC(1/44) CSNTSLNAC(1/44) CSHSSMQKC(1/44) CTGKSAGSC(1/44) CTSANLRNC(1/44) CTSPSRRAC(1/44) CTTAKHTTC(1/44) CYTSPKGRC(1/44) 36 Phage display (in vivo) 3 PMID:31193742 Spinal Cord Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) The CX7C phage library was used for in vivo phage display for the screening of spinal cord homing peptides. The phage library was injected into C57BL/6 mice through the tail vein. At 5 min after injections, whole spinal cords were isolated after transcardiac removal of blood. Aliquots from 3–5 mice were combined and infected into Escherichia coli ER2738. The number of phages included in the spinal cord was titrated as PFU. After amplification to 1.0e11 PFU, the phages were again injected into other C57BL/6 mice. This biopanning procedure was repeated three times to select phages that specifically bound to the spinal cord. After the third round of biopanning, each phage genome was isolated from phage plaques and analyzed using DNA sequence-coding pIII protein. Analyses of the sequences of targeted phages yielded two candidate peptides targeting the spinal cord: SP1 (CLHQSPHIC) and SP2 (CPTNNPRSC). These peptides were synthesized and intravenously injected into mice to evaluate their tissue specificity and potential as gene delivery carriers. The complexes between SP1 or SP2 peptides and the plasmid vector expressing the reporter gene could induce gene transduction in the spinal cord through systemic injection without gene expression in the brain, liver, and kidney. In addition, intravenous injection of the complex between SP1 and the vectors induced interleukin-4 expression in the spinal cord, resulting in effective suppression of lipopolysaccharide-induced hyperalgesia. Therefore, intravenously administered spinal cord homing peptides complexed with a plasmid vector provided tissue-specific treatment featuring gene delivery to the CNS through systemic circulation. 3453 CLHQSPHIC(8/49) CPTNNPRSC(6/49) CFGQKASSC(2/49) CLTSTHMWC(2/49) CSKHGMPYC(2/49) CTSPSRRAC(2/49) CDLPWIQSC(1/49) CFGSTTWKC(1/49) CGLAKETMC(1/49) CGPHQFNLC(1/49) CHGPGHHSC(1/49) CHNPSIHNC(1/49) CKTYPSHLC(1/49) CKQTQTHFC(1/49) CLNPKTQYC(1/49) CMGMGQAWC(1/49) CMHNGSWQC(1/49) CMMETPGSC(1/49) CMMDHTLQC(1/49) CMTKMAPHC(1/49) CNHHSGLTC(1/49) CNLSSNRQC(1/49) CNNMPAKVC(1/49) CNPMGKLQC(1/49) CNTRAPSTC(1/49) CPGLCAHKC(1/49) CPTATPQLC(1/49) CRSANIYTC(1/49) CSQNQPKVC(1/49) CTHSSLPIC(1/49) CTSKPAHYC(1/49) CTYRPPHLC(1/49) CVPSRLPLC(1/49) 33 Phage display (in vivo) 4 PMID:31193742 Spinal Cord Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) The CX7C phage library was used for in vivo phage display for the screening of spinal cord homing peptides. The phage library was injected into C57BL/6 mice through the tail vein. At 5 min after injections, whole spinal cords were isolated after transcardiac removal of blood. Aliquots from 3–5 mice were combined and infected into Escherichia coli ER2738. The number of phages included in the spinal cord was titrated as PFU. After amplification to 1.0e11 PFU, the phages were again injected into other C57BL/6 mice. This biopanning procedure was repeated four times to select phages that specifically bound to the spinal cord. After the fourth round of biopanning, each phage genome was isolated from phage plaques and analyzed using DNA sequence-coding pIII protein. Analyses of the sequences of targeted phages yielded two candidate peptides targeting the spinal cord: SP1 (CLHQSPHIC) and SP2 (CPTNNPRSC). These peptides were synthesized and intravenously injected into mice to evaluate their tissue specificity and potential as gene delivery carriers. The complexes between SP1 or SP2 peptides and the plasmid vector expressing the reporter gene could induce gene transduction in the spinal cord through systemic injection without gene expression in the brain, liver, and kidney. In addition, intravenous injection of the complex between SP1 and the vectors induced interleukin-4 expression in the spinal cord, resulting in effective suppression of lipopolysaccharide-induced hyperalgesia. Therefore, intravenously administered spinal cord homing peptides complexed with a plasmid vector provided tissue-specific treatment featuring gene delivery to the CNS through systemic circulation. 3454 CLHQSPHIC(31/47) CPTNNPRSC(12/47) CNMRTLMQC(2/47) CDMHQGKTC(1/47) CSPDKNRSC(1/47) 5 Phage display (in vivo) 5 PMID:31193742 Spinal Cord Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) The CX7C phage library was used for in vivo phage display for the screening of spinal cord homing peptides. The phage library was injected into C57BL/6 mice through the tail vein. At 5 min after injections, whole spinal cords were isolated after transcardiac removal of blood. Aliquots from 3–5 mice were combined and infected into Escherichia coli ER2738. The number of phages included in the spinal cord was titrated as PFU. After amplification to 1.0e11 PFU, the phages were again injected into other C57BL/6 mice. This biopanning procedure was repeated five times to select phages that specifically bound to the spinal cord. After the fifth round of biopanning, each phage genome was isolated from phage plaques and analyzed using DNA sequence-coding pIII protein. Analyses of the sequences of targeted phages yielded two candidate peptides targeting the spinal cord: SP1 (CLHQSPHIC) and SP2 (CPTNNPRSC). These peptides were synthesized and intravenously injected into mice to evaluate their tissue specificity and potential as gene delivery carriers. The complexes between SP1 or SP2 peptides and the plasmid vector expressing the reporter gene could induce gene transduction in the spinal cord through systemic injection without gene expression in the brain, liver, and kidney. In addition, intravenous injection of the complex between SP1 and the vectors induced interleukin-4 expression in the spinal cord, resulting in effective suppression of lipopolysaccharide-induced hyperalgesia. Therefore, intravenously administered spinal cord homing peptides complexed with a plasmid vector provided tissue-specific treatment featuring gene delivery to the CNS through systemic circulation. 3455 CTTNPFSLC(5/12)[2913.0±311.0] CRLSMETVC(3/12)[NA] CQAPHKPWC(1/12)[NA] CHNSKSTTC(1/12)[NA] CPSSMRGTC(1/12)[NA] CILKKNVSC(1/12)[NA] 6 Phage display (common panning) 2 PMID:31173215 Bone mesenchymal stem cell (BMSC) Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) Flow Cytometry The bone mesenchymal stem cell (BMSC) affinity properties of FITC-labeled peptides were analyzed quantitively by flow cytometry at a wavelength of 488 nm, using FlowJo v10 (Tree Star, Inc). A specific cyclic peptide for Sprague-Dawley rat bone mesenchymal stem cells (BMSCs), CTTNPFSLC (known as C7), was used, which was identified via phage display technology. Its high affinity for BMSCs was demonstrated using flow cytometry and fluorescence staining. Subsequently, the cyclic peptide was placed on β-tricalcium phosphate (β-TCP) scaffolds using absorption and freeze-drying processes. Adhesion, expansion and proliferation of BMSCs was investigated in vitro on the C7-treated β-TCP scaffolds and compared with pure β-TCP scaffolds. The results revealed that C7 had a promoting effect on the adhesion, expansion and proliferation of BMSCs on β-TCP scaffolds. Therefore, C7 may be effective in future tissue engineering therapy for osteonecrosis of the femoral head (ONFH). 3456 CTTNPFSLC(6/12)[2913.0 ± 311.0] CQAPHKPWC(2/12)[NA] CRLSMETVC(1/12)[NA] CPSSMRGTC(1/12)[NA] CDLLESERC(1/12)[NA] CKMWNGSGC(1/12)[NA] 6 Phage display (common panning) 3 PMID:31173215 Bone mesenchymal stem cell (BMSC) Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) Flow Cytometry The bone mesenchymal stem cell (BMSC) affinity properties of FITC-labeled peptides were analyzed quantitively by flow cytometry at a wavelength of 488 nm, using FlowJo v10 (Tree Star, Inc). A specific cyclic peptide for Sprague-Dawley rat bone mesenchymal stem cells (BMSCs), CTTNPFSLC (known as C7), was used, which was identified via phage display technology. Its high affinity for BMSCs was demonstrated using flow cytometry and fluorescence staining. Subsequently, the cyclic peptide was placed on β-tricalcium phosphate (β-TCP) scaffolds using absorption and freeze-drying processes. Adhesion, expansion and proliferation of BMSCs was investigated in vitro on the C7-treated β-TCP scaffolds and compared with pure β-TCP scaffolds. The results revealed that C7 had a promoting effect on the adhesion, expansion and proliferation of BMSCs on β-TCP scaffolds. Therefore, C7 may be effective in future tissue engineering therapy for osteonecrosis of the femoral head (ONFH). 3457 CVPSKPGLC[60] 1 Phage display (competitive panning) 3 PMID:31152222 Anti-aflatoxin B1 (AFB1) monoclonal antibody (MAb) 2F5 Aflatoxin B1 Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) ELISA IC50 (ng/mL) was calculated in competitive phage ELISA. The competitive panning method was employed in this study. In brief, one well of a microtiter plate was coated with 100 μL of MAb 2F5 and incubated overnight at 4 °C. The next day, the phage library diluted with PBS was first added to the MAb 2F5-coated well and incubated for 1 h at 25 °C. After washing with PBST, the combined peptide was competitively eluted using 500 ng/mL AFB1 in 10% methanol PBS. Subsequently, the supernatants of the four wells were collected and infected into E. coli ER2738 for amplification by mixing with PEG solution. The entire panning procedure was repeated twice, except for the use of concentrations of AFB1, MAb 2F5, and Tween 20 in PBST in the second and third round of panning, respectively. After each round of panning, the eluted phage titer was determined according to the manufacturer’s protocol. The phage DNA was sequenced by Sangon Biotech using 96 gIII primer. 3458 LPSYPGYYQITI(7)[1.860 ± 0.056] CWTISLFGGVTQ(3)[1.576 ± 0.055] SKRSTGKDYTAT(1)[1.461 ± 0.078] RRVISARWTSDR(1)[1.461 ± 0.055] MQTSFLKHHSVT(1)[1.306 ± 0.066] 5 Phage display (common panning) 4 PMID:31149727 Apical membrane antigen-1 Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The optical density (OD) was measured at 490 nm using a microplate reader. The OD values were reproduced from Figure 2 and shown. Two corresponding specific Eimeria tenella apical membrane antigen-1 (EtAMA1) binding peptides (LPSYPGYYQITI (L) and CWTISLFGGVTQ (C)) showed significant effects on inhibiting sporozoite invasion of MDBK cells. 3459 ITMSVPAHNAKE(39/169)[34.2 ± 2.11] TNTSWDPQYNPD(11/169)[80.1 ± 1.13] NHFVPTSNRFNA(11/169)[120 ± 1.23] NFTINGKTHRLW(6/169)[298 ± 1.45] NAITLLSPPLHK(5/169)[57.1 ± 1.77] SSHNHDSYHGTK(2/169)[476 ± 2.73] LMNPATMKTSSG(1/169)[360 ± 1.87] TNTSWDPQYNPD(1/169)[47.6 ± 2.51] SNMKPSMEYSSR(1/169)[273 ± 1.79] IGNSWPLTSHSW(1/169)[144 ± 1.1] SYNTFMYERASK(1/169)[562 ± 1.69] MVHSKASMWPGK(1/169)[690 ± 2.27] KVYAINSWTNYY(1/169)[1200 ± 90.2] 13 Phage display (competitive panning) 5 PMID:31146360 Tubulin alpha chain Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The binding affinities of phages were determined by the enzyme-linked immunosorbent assay (ELISA) method with anti-M13 phage antibody. The optical densities were measured at 450 nm with a Biotrak multiwall plate reader (Amersham Bioscience). Kd (pM) was calculated and shown. In the final round, bound phages were eluted by addition of a 10 M excess of tubulin alpha chain. One peptide with the sequence of ITMSVPAHNAKEK demonstrated the high inhibitory effect on microtubule formation with a nanomolar range of IC50 values, which were much lower than a well-known chemical inhibitor—benomyl. Based on these results, this peptide can be employed to further develop promising candidates for novel antifungal agents against Phytophthora blight. 3460 TNPQARWHEYNF(61/112)[41.9 ± 1.05] NPIGDNYSGTGL(10/112)[37.1 ± 1.41] 2 Phage display (competitive panning) 5 PMID:31146360 Tubulin beta chain Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The binding affinities of phages were determined by the enzyme-linked immunosorbent assay (ELISA) method with anti-M13 phage antibody. The optical densities were measured at 450 nm with a Biotrak multiwall plate reader (Amersham Bioscience). Kd (pM) was calculated and shown. In the final round, bound phages were eluted by addition of a 10 M excess of tubulin beta chain. 3461 KALLHHLALLALHLA[0.46] WIALHHLLHLAAHWI[4.2] WALAHKALHALAHKP[5.3] KWLAKHAAGLALHAL[4.6] VLALHHALALAHKKA[9.0] PAALHHALALAHHLW[12.0] WMHKHQALAAMHAHR[0.12] RQAHTHALHHLALWC[0.13] WRLHHRHFLALALKR[0.21] TPHLHMFHAHKLAPR[0.16] TPHAHMWHAHKRNPK[0.56] PVHHVHLTAHHAVGC[1.5] 12 Phage display (common panning) 5 PMID:12849992 Lipid A Not determined. Not determined. Not determined. f5-15mer phage display library (X15) Surface plasmon resonance (SPR) Hybrid bilayers containing 20 mol% lipid A in DMPC on HPA chip were formed. The rate constants were determined by the nonlinear least squares fitting of the primary sensogram data using BiaEvaluation, Version 3.0 software. The association constant Ka (Unit × e6 1/M) was determined and shown. The lipid A solution was then sonicated for 5 min with a needle probe. This solution (2.0 ml) was then added to polystyrene petri dishes and incubated at 60 ℃. Subsequently, the dishes were washed five times with TBS (50 mM Tris–HCl, pH 7.5, and 150 mM NaCl) and blocked with 5 mg/ml of BSA for 2 h. Phages (2.0e11) were pipetted into each coated and blocked dish and were kept for 2 h at 4 ℃. Unbound phages were washed off with TBS and the bound phages were eluted with 0.1 M glycine HCl buffer (pH 2.2) containing 1 mg/ml BSA and neutralized with 0.1 M Tris–HCl, pH 9.0. The eluate was titered and amplified for use in subsequent round of panning, which were carried out in similarly coated wells of a 96-well microtitre plate. A comparison of the sequences revealed no consensus sequence between the 12 selected peptides suggesting that the lipid A binding motif is not sequence specific which is in accord with the sequence variation seen with the naturally occurring anti-microbial and/or endotoxin binding peptides. 3462 RLCSWISPCSA(4)[6] FGCSWLFPCPF(2)[8] RLCSWVSPCSA(1)[10] 3 Phage display (common panning) 3 PMID:15233528 B-domain deleted form of human factor VIII (BDDrFVIII) Not determined. Not determined. Not determined. TN7/1 phage display library ELISA Upon completion of the second and third rounds of selection, randomly-chosen individual phage isolates from pools generated by both elution conditions were screened for binding to BDDrFVIII by indirect phage ELISA. Background binding levels were established using wells coated with streptavidin alone or with BSA. Phage isolates exhibiting ELISA signals significantly above background (A630 > 0.25) were scored as positive for binding to BDDrFVIII. The ratio of the ELISA signal with target to the ELISA signal without target was shown. Biotinylated BDDrFVIII protein that was confirmed to be active after derivatization was immobilized onto streptavidin-coated magnetic beads and used as a target in six separate selection campaigns. Each campaign incorporated three rounds of selection from a single library, using one elution condition for phage bound to the immobilized BDDrFVIII. A round of selection consisted of a binding step in which the phage library was first incubated in suspension with the target, followed by a plate wash step to remove unbound or weakly-bound members of the phage library, and an elution step to recover a pool of phage. This results in a population of phage isolates enriched in members that bind the target under binding and wash conditions and release the target under elution conditions. Finally, elution pools from each round of selection were separately amplified by infection and growth in E. coli cultures. The purified phage preparations from this amplification step served as the input for subsequent rounds of selection. The consensus sequence is rlCSWѲsPCsa (uppercase letters stronger than lowercase, Ѳ = V, I, L, F). 3463 HPCGSWLRPCLH(10)[16] HSCGSWLFPCFA(7)[10] HLCGAWFRPCDA(6)[6] HPCGSWFNPCAH(4)[8] HPCGSWFRPCFH(3)[16] HACGSWFRPCHA(3)[6] HPCGAWLRPCYN(1)[20] HLCFAWFRPCDA(1)[8] HPCGSWLHPCAA(1)[6] HRCGSWLHPCLA(1)[6] 10 Phage display (common panning) 3 PMID:15233528 B-domain deleted form of human factor VIII (BDDrFVIII) Not determined. Not determined. Not determined. TN8/6 phage display library ELISA Upon completion of the second and third rounds of selection, randomly-chosen individual phage isolates from pools generated by both elution conditions were screened for binding to BDDrFVIII by indirect phage ELISA. Background binding levels were established using wells coated with streptavidin alone or with BSA. Phage isolates exhibiting ELISA signals significantly above background (A630 > 0.25) were scored as positive for binding to BDDrFVIII. The ratio of the ELISA signal with target to the ELISA signal without target was shown. Biotinylated BDDrFVIII protein that was confirmed to be active after derivatization was immobilized onto streptavidin-coated magnetic beads and used as a target in six separate selection campaigns. Each campaign incorporated three rounds of selection from a single library, using one elution condition for phage bound to the immobilized BDDrFVIII. A round of selection consisted of a binding step in which the phage library was first incubated in suspension with the target, followed by a plate wash step to remove unbound or weakly-bound members of the phage library, and an elution step to recover a pool of phage. This results in a population of phage isolates enriched in members that bind the target under binding and wash conditions and release the target under elution conditions. Finally, elution pools from each round of selection were separately amplified by infection and growth in E. coli cultures. The purified phage preparations from this amplification step served as the input for subsequent rounds of selection. The consensus sequence is HpCGSWѲrPCxa/h (uppercase letters stronger than lowercase, Ѳ = V, I, L, F). 3464 FCWVFAFDHCH(14)[24] FCWVFPFNHCS(6)[18] FCWVFPFNHCD(6)[8] FCWVFNFSHCS(3)[24] FCHVFNFVHCS(3)[8] FCWVFPFQHCA(2)[24] FCWVFNWVHCD(1)[14] FCWVFQFRHCH(1)[8] FCWVFPFHHCF(1)[6] 9 Phage display (common panning) 3 PMID:15233528 B-domain deleted form of human factor VIII (BDDrFVIII) Not determined. Not determined. Not determined. TN9 phage display library ELISA Upon completion of the second and third rounds of selection, randomly-chosen individual phage isolates from pools generated by both elution conditions were screened for binding to BDDrFVIII by indirect phage ELISA. Background binding levels were established using wells coated with streptavidin alone or with BSA. Phage isolates exhibiting ELISA signals significantly above background (A630 > 0.25) were scored as positive for binding to BDDrFVIII. The ratio of the ELISA signal with target to the ELISA signal without target was shown. Biotinylated BDDrFVIII protein that was confirmed to be active after derivatization was immobilized onto streptavidin-coated magnetic beads and used as a target in six separate selection campaigns. Each campaign incorporated three rounds of selection from a single library, using one elution condition for phage bound to the immobilized BDDrFVIII. A round of selection consisted of a binding step in which the phage library was first incubated in suspension with the target, followed by a plate wash step to remove unbound or weakly-bound members of the phage library, and an elution step to recover a pool of phage. This results in a population of phage isolates enriched in members that bind the target under binding and wash conditions and release the target under elution conditions. Finally, elution pools from each round of selection were separately amplified by infection and growth in E. coli cultures. The purified phage preparations from this amplification step served as the input for subsequent rounds of selection. The consensus sequence is FCWVFpFxHCx (uppercase letters stronger than lowercase). 3465 SVFPFEVWESLR[1.272, 1.02] YSWHEWYIPQLS[1.185, 0.69] SMPYIAWLALRG[0.929, 0.82] HSTLLNHTTGVL[0.937, 1.06] 4 Phage display (common panning) 3 PMID:30625581 Aflatoxin nanobody Nb28 Aflatoxin B1 Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA Supernatants containing mimotope-phages were screened by indirect competitive magnetic beads-based ELISA (MB-icELISA) in the presence (10 ng/ mL) or absence of aflatoxin B1 (AFB1). The absorbance was recorded at 450 nm. The maximum absorbance (ODmax) and the 50% inhibitory concentration (IC50, ng/mL) values were determined and shown in the left and right columns of affinity values, respectively. A rapid magnetic beads-based directed competitive ELISA (MB-dcELISA) was developed utilizing Nb28 and its mimotope ME17 (YSWHEWYIPQLS). The 50% inhibitory concentration and the detection limit of the MB-dcELISA were 0.75 and 0.13 ng/mL, respectively, with a linear range of 0.24–2.21 ng/mL. Further validation study indicated good recovery (84.2–116.2%) with low coefficient of variable (2.2%–15.9%) in spiked corn, rice, peanut, feedstuff, corn germ oil and peanut oil samples. 3466 NCKFSGCSVSVCH[5.13] NCRFSGCLQTMCV[8.77] NCRFSGCGTVACV[10.85] NCRFSGCGFKVCV[5.75] NCKFSGCPWELCI[0.78] NCRFSGCVWAKCS[16.90] NCKFSGCQESMCS[1.74] NCRFSGCQWGDCA[23.41] NCKFSGCQFLGCE[2.96] SCKFSGCHRKPCT[1.53] SCRFTFCYYQPCA[111.10] NCRFTFCNLNLCG[29.41] NCRFTFCEFGRCE[>200] NCRFSLCESMACL[71.18] NCKFSACYLTTCY[>200] QCWDRGCENRKCN[1.72] SCLFTFCYFVPCN[>200] TCRQSMCTARTCP[11.56] DCRWSSCTARTCA[8.65] TCGVQACLSARCY[12.9] GCSDQACWSARCV[10.50] NCKFSGCAQLRCY[NA] NCRYSGCFDRPCI[NA] NCRFTLCGLAPCG[NA] NCRYSGCFDRPCI[NA] NCYFSNCGAHSAE[NA] RCLLSACTARACN[NA] GCAQSVCTARYCE[NA] GCSPSRCTARFCP[NA] GCQTSQCTARTCL[NA] RCLPSWCSARTCF[NA] SCPPSRCTARTCP[NA] GCRESSCSARTCP[NA] ACRVSSCTARTCS[NA] ECRASDCSARVCW[NA] WCQASDCTARACI[NA] VCRQSDCSRRTCI[NA] VCRSSDCTKRSCE[NA] PCGEQACYTARCR[NA] LCLSQACWSARCA[NA] GCSAQACASARCL[NA] GCSVQACFSGRCT[NA] GCSQDACYSARCV[NA] ICSVLGCLSARCG[NA] GCGTLACQTARCR[NA] VCVERGCSTARCR[NA] LCADVSCATARCR[NA] GCVRPSCFSARCS[NA] ECQDRSCPFTMCS[NA] 49 Phage display (common panning) 2/3 PMID:24453110 Urokinase-type plasminogen activator Not determined. Not determined. Not determined. XCX4CX4CX phage display library Inhibition assay The inhibition constants Ki (μM) were determined by incubating different concentrations of bicyclic peptide with fixed concentration of human urokinase-type plasminogen activator. The indicated Ki values are averages of at least two measurements. The peptide library was cyclized with the thiol-reactive compound 1,3,5-tris(bromomethyl)benzene (TBMB). The library was then subjected to two or three iterative rounds of phage selection against human urokinase-type plasminogen activator. 3467 RCSGPSCPWQQCS[2.12] RCSGPACVWTQCT[6.88] RCAGPACITQFCT[4.61] RCAGPGCWWAVCG[2.06] RCAGPVCPWTRCG[0.80] RCAGPQCPWSICV[5.39] RCAGPVCPDDPCR[17.73] RCAGPRCPVDMCS[17.73] RCAGAKCPWAVCS[3.76] VCSERGCENRGCG[12.27] LCSDRGCENRWCK[17.73] WCHDRGCENRSCM[14.19] ICLGRGCENRYCG[6.48] TCRQSMCTARTCP[84.92] GCAPTACQSARCG[47.81] CCLGRGCENHRCL[NA] TCQNRGCENRECC[NA] CCRERGCENMVCP[NA] CCGERGCENRACT[NA] QCSINACLSSRCS[NA] GCSVQACLSGRCG[NA] TCGVQACLSARCY[NA] ACRLEACLSARCG[NA] GCTSSACQSARCR[NA] GCGTLACQTARCR[NA] GCGFQACQSARCL[NA] RCASQACYTARCG[NA] SCGVAACHTARCR[NA] VCVRQSCMTARCQ[NA] TCSRASCWTARCL[NA] YCAQASCWTARCG[NA] FCPQVSCLTARCM[NA] SCSQVSCYTARCR[NA] NCAVASCFSARCR[NA] YCVQQSCFSARCV[NA] SCSLQGCLSARCV[NA] RCSIAGCQTARCY[NA] ICRASVCTARTCW[NA] ECGWRACLAARCT[NA] SCSSQACAAARCR[NA] WCASVTCRLYGCM[NA] LCGNYRFSGSMCA[NA] 42 Phage display (common panning) 2/3 PMID:24453110 Urokinase-type plasminogen activator Not determined. Not determined. Not determined. XCX4CX4CX phage display library Inhibition assay The inhibition constants Ki (μM) were determined by incubating different concentrations of bicyclic peptide with fixed concentration of human urokinase-type plasminogen activator. The indicated Ki values are averages of at least two measurements. The peptide library was cyclized with the hydrophilic small molecule 1,3,5-triacryloyl-1,3,5-triazinane (TATA). The library was then subjected to two or three iterative rounds of phage selection against human urokinase-type plasminogen activator. 3468 GCVPQYCPPPLCG[7.33] KCQGSYCPPVACL[3.24] GCQVNYCPPVPCL[1.96] QCQVSYCPPVRCD[8.80] GCAVSYCPPQFCN[41.76] MCHVSYCPWQGCT[12.43] RCMVSYCPEIGCT[10.99] QCSVSYCPQVPCQ[3.54] GCFPSYCPQVACQ[0.78] GCGPSYCPQVACQ[1.77] RCGPSYCPDDFCF[35.90] FCGPSYCPYTGCA[2.98] GCGPSYCSYVPCG[13.09] VCQTSYCPYVPCL[3.68] YCQASYCPGVPCR[9.69] DCASYCPFVECV[84.91] WCQAEYCAFVPCI[4.39] QCQAEYCPRVRCT[1.41] RCQGEYCPPVRCL[6.28] SCQGEYCPFVVCE[6.77] WCQPEYCPQSGCD[103.60] GCQVEYCPPVPCL[15.76] SCQAQYCPEVQCR[4.38] NCQPQYCPRVTCI[1.57] ECQPQYCPSVGCK[20.74] VCQGQYCPPVECS[4.19] GCQVQYCPPVPCL[3.51] ACEVSYCPQVKCQ[NA] SCGPSYCPLVACQ[NA] ECVASYCPQVECL[NA] DCWGSYCQGSYCP[NA] FCDWSYCVLDRCT[NA] GCGSGRCGLTFCM[NA] WCWSGRCGSWGCS[NA] LCPSARCGWQNCE[NA] QCGPLACATARCA[NA] WCGPWGCGTGRCF[NA] WCTRDPCQTGRCY[NA] QCGLDPCQTGRCV[NA] QCRLSACTARSCF[NA] GCFPQYCPMMPCV[NA] DCNRPLCQNAPCL[NA] LCFDQECFWGLCF[NA] WCDWGFCKEQMCN[NA] LFQGACDTGRCA[NA] 45 Phage display (common panning) 2/3 PMID:24453110 Urokinase-type plasminogen activator Not determined. Not determined. Not determined. XCX4CX4CX phage display library Inhibition assay The inhibition constants Ki (μM) were determined by incubating different concentrations of bicyclic peptide with fixed concentration of human urokinase-type plasminogen activator. The indicated Ki values are averages of at least two measurements. The peptide library was cyclized with the hydrophilic small molecule N,N’,N’’-(benzene-1,3,5-triyl)-tris(2-bromoacetamide) (TBAB). The library was then subjected to two or three iterative rounds of phage selection against human urokinase-type plasminogen activator. 3469 RCVQICPSWCG[>200] RCLALCPNWCT[>200] RCVALCPRWCY[>200] RCVKLCPLWCT[NA] RCVDLCPLWCV[NA] RCLAMCPSWCV[NA] 6 Phage display (common panning) 2/3 PMID:24453110 Urokinase-type plasminogen activator Not determined. Not determined. Not determined. XCX3CX3CX phage display library Inhibition assay The inhibition constants Ki (μM) were determined by incubating different concentrations of bicyclic peptide with fixed concentration of human urokinase-type plasminogen activator. The indicated Ki values are averages of at least two measurements. The peptide library was cyclized with the thiol-reactive compound 1,3,5-tris(bromomethyl)benzene (TBMB). The library was then subjected to two or three iterative rounds of phage selection against human urokinase-type plasminogen activator. 3470 RCLVPCRYLCW[>200] RCLLPCRYWCE[>200] RCVRPCYFPCA[>200] GCSWWCNRACP[>200] VCLMICHYPCL[>200] RCLVPCRLHCW[NA] RCLLPCRYWCP[NA] RCLVPCKYWCY[NA] RCLLPCNLWCF[NA] RCLGPCWIVCP[NA] RCVRPCFIPCA[NA] RCVRPCPWTCS[NA] RCVAPCWWGCN[NA] RCQYWCNRACL[NA] TCSWWCNRSCL[NA] GCSWWCNRACL[NA] LCYQICHYPCS[NA] TCFFICHYPCL[NA] 18 Phage display (common panning) 2/3 PMID:24453110 Urokinase-type plasminogen activator Not determined. Not determined. Not determined. XCX3CX3CX phage display library Inhibition assay The inhibition constants Ki (μM) were determined by incubating different concentrations of bicyclic peptide with fixed concentration of human urokinase-type plasminogen activator. The indicated Ki values are averages of at least two measurements. The peptide library was cyclized with the hydrophilic small molecule 1,3,5-triacryloyl-1,3,5-triazinane (TATA). The library was then subjected to two or three iterative rounds of phage selection against human urokinase-type plasminogen activator. 3471 VCNQICGRLCE[0.30] VCNQVCGRQCD[0.36] RCNYICGRDCS[0.37] SCNDVCGRDCT[1.48] SCNQICGRWCF[NA] TCNQICGRSCP[NA] TCNQICGRICV[NA] NCNQVCGRECG[NA] VCNQVCGRLCP[NA] LCNQVCGRQCL[NA] KCNQVCGRLCA[NA] LCNSLCGRICD[NA] WCNSICGRSCF[NA] KCNSICGRNCS[NA] VCNSVCGRLCS[NA] RCNDICGRHCL[NA] RCNAVCGRDCN[NA] SCNAICGRACD[NA] GCNRICGRLCE[NA] 19 Phage display (common panning) 2/3 PMID:24453110 Urokinase-type plasminogen activator Not determined. Not determined. Not determined. XCX3CX3CX phage display library Inhibition assay The inhibition constants Ki (μM) were determined by incubating different concentrations of bicyclic peptide with fixed concentration of human urokinase-type plasminogen activator. The indicated Ki values are averages of at least two measurements. The peptide library was cyclized with the hydrophilic small molecule N,N’,N’’-(benzene-1,3,5-triyl)-tris(2-bromoacetamide) (TBAB). The library was then subjected to two or three iterative rounds of phage selection against human urokinase-type plasminogen activator. 3472 CTGTPARQC(4/47)[15.66 ± 2.82] CKNSMFATC(4/47)[10.56 ± 1.09] CTNKHSPKC(3/47)[10.61 ± 0.73] CSPKNILHC(3/47)[NA] CFPSPTRTC(2/47)[NA] CNAGTLGRC(2/47)[NA] CNKEFASQC(2/47)[NA] CNSANNRIC(2/47)[NA] CSTQSTTSC(2/47)[NA] CAPPGKSEC(1/47)[NA] CAPKNILHC(1/47)[NA] CAPSSSATC(1/47)[NA] CESKTPKNC(1/47)[NA] CFDHHTNSC(1/47)[NA] CGTRETLSC(1/47)[NA] CHAALNRSC(1/47)[NA] CHHSNQRQC(1/47)[NA] CHRPDTRSC(1/47)[NA] CNRESPHLC(1/47)[NA] CNSRSHAIC(1/47)[NA] CPMPSTSYC(1/47)[NA] CPVTSRSDC(1/47)[NA] CRAWNEAPC(1/47)[NA] CSDRGLPSC(1/47)[NA] CSSKSDHSC(1/47)[NA] CTAYPAKAC(1/47)[NA] CTGAPARWC(1/47)[NA] CTKTGLHIC(1/47)[NA] CTSTAPLKC(1/47)[NA] CVTSPFHNC(1/47)[NA] CYSPRGGSC(1/47)[NA] CYTNPDNVC(1/47)[NA] 32 Phage display (subtractive panning, in vivo) 4 PMID:30788426 Human prostate cancer cell line LNCaP Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) Binding assay LN1 (CTGTPARQC), LN2 (CKNSMFATC), and LN3 (CTNKHSPKC) peptides modified with a biotin molecule were incubated with LNCaP cells for 24 h at a concentration of 1 mg/mL and the binding affinities of the respective peptides were compared using fluorescence with Avidin D-conjugated TRITC (biotinylated peptides are stained red). The fluorescence intensity of each target peptide bound to LNCaP cells (n = 6 per group) was reporduced from Figure 2C and shown. The phage library was injected into C57BL/6 mice through the tail vein at a titer of 1.0e9 plaque-forming units (PFU)/mL in 100 mL Tris-buffered saline (50 mM Tris-HCl [pH7.5]). Five minutes after the injection, blood was recovered from the heart in order to remove non-specifically bound phages. The collected blood was dissolved in DMEM (Thermo Fisher Scientific) containing protease inhibitor (Sigma-Aldrich, St Louis, MO, USA). The solution was introduced into Escherichia coli ER2738 (New England Biolabs), and the number of phages in the blood was titrated by plaque-forming units. After amplification to 1.0e11 PFU, the phages were injected into new C57BL/6 mice. After three rounds of in vivo panning with mice, the phages were injected into the femoral vein of a 15-year-old female cynomolgus monkey. Five minutes after the injection, blood was taken from the femoral vein and dissolved in DMEM with protease inhibitor. The solution was centrifuged to collect the serum, from which phages were amplified to 1.0e11 PFU. These phages were injected into the tail vein of a C.B-17 SCID mouse with tumor xenografts (human prostate cancer cell line LNCaP (RCB2144)), and xenografted prostate cancer tissue was isolated after trans-cardiac removal of blood. Phages were recovered from the homogenized tissue and amplified as described above. Three additional cycles of in vivo phage panning were performed. After four cycles of in vivo panning in C.B-17 SCID mice, phage DNA was isolated from selected phage plaques with high affinity to prostate cancer tissue. LN1 (CTGTPARQC), LN2 (CKNSMFATC), and LN3 (CTNKHSPKC) were synthesized and evaluated for binding and biological activity. LN1 showed the highest avidity for LNCaP prostate cancer cells in vitro and was thus administered to tumor-bearing mice to evaluate in vivo binding. Strikingly, LN1 specifically bound to the tumor tissue and exhibited very low reactivity with normal liver and kidney tissues. To demonstrate that LN1 could specifically deliver drugs to prostate cancer tissue, a therapeutic peptide, LN1-KLA (C-TGTPARQ-CGGG-D[KLAKLAK]2), was prepared and used to treat LNCaP cells in vitro and was also administered to tumor-bearing mice. The therapeutic peptide significantly suppressed growth of the cells both in vitro and in vivo. 3473 ANTELALANRKH(2/110) NYLPHQSSSPSR(2/110) SLPNLPPTYAKP(2/110) ASNHSIPTFPLK(2/110) NPMNNVAQNPGP(2/110) TLGLRPVPVATT(2/110) GSWNTFRAQPTI(2/110) SQALSTSRQDLR(2/110) HSACLGPSNLQC(2/110) RVQPAHFNVMGQ(1/110) MVGTADGTLLDP(1/110) TMHHAAIAHPPH(1/110) GIVTNQHDSNAN(1/110) GLTFQVPWHANM(1/110) GHPMMPPKSEIR(1/110) TMAQGVAQRYGN(1/110) SHQPGDQSPANN(1/110) DLINIDRNHSFR(1/110) LPKQCSLLTSAC(1/110) NFTLQAHPHKYP(1/110) STDHGSWQKSRA(1/110) VPQLHHLMPHFD(1/110) TSMSQHFHVHRL(1/110) SPLTPPHAPETH(1/110) CPTDVRSGCMGT(1/110) IEMTRTNLNDVN(1/110) HTQHIQSDDHLA(1/110) NDLQRHRLTAGP(1/110) DDTQNSQNMDTL(1/110) 29 Phage display (common panning) 3 PMID:31040030 Gallium ion Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) Target preparation was carried out by immobilization of gallium ions on small monolithic ion exchange columns (CIM Disk Monolithic Column, BIA Separations d.o.o., Ajdovscina, Slovenia) for Äkta avant 25 FPLC system (GE Healthcare Europe GmbH, Freiburg,Germany). Original phage library or subsequently enriched phage pools were diluted in the respective buffer to a final volume of 1 ml and applied in a repetitive recycling loop of 15 ml. Unbound and rather unspecific binding phage were removed by washing with column volumes (CV) of the buffer at a flow rate of 1 ml/min. Good binding phage were eluted by applying 40 CV of the eluent at a flow rate of 1 ml/min. The eluate was collected in fractions, 1 ml each. In a final step gallium together with the remaining tight bound phage were stripped by applying 40 CV 1 M HCl and fractionated as well. Phage clones expressing the peptide sequences TMHHAAIAHPPH, SQALSTSRQDLR and HTQHIQSDDHLA were characterized to bind >10 fold better to a target that presents immobilized gallium ions than control phage, displaying no peptide sequence. 3474 CIAVPSNLC CKTPNGHLC CKXPKGHLC 3 Phage display (common panning) 3 PMID:30850705 Human colorectal cancer cell line RKO Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) An adaptation of the BRASIL method using RKO cells as target was used. A total of three rounds of selection with human epithelial CRC cell line RKO (ATCC CRL 2577) were performed through in vitro biopanning. In each round, the phages that specifically bound to target cells were recovered and used for the next round of selection. In the three rounds of selection, the obtained phage pool was not amplified between rounds. 3475 CRPTYSPSC CKIHSSETC CPKSNNGVC CIGNSNTLC 4 Phage display (common panning) 4 PMID:30850705 Human colorectal cancer cell line RKO Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) An adaptation of the BRASIL method using RKO cells as target was used. A total of four rounds of selection with human epithelial CRC cell line RKO (ATCC CRL 2577) were performed through in vitro biopanning. In each round, the phages that specifically bound to target cells were recovered and used for the next round of selection. In the three initial rounds of selection, the obtained phage pool was not amplified between rounds.