3476 CPKSNNGVC[0.27 ± 0.04] CKTPNGHLC[0.17 ± 0.01] CQSISTAHC[0.18 ± 0.03] CNDDVPNKC[0.17 ± 0.02] CEIPGKVVC[0.17 ± 0.02] CLRTPANHC[0.20 ± 0.02] 6 Phage display (subtractive panning) 4 PMID:30850705 Human colorectal cancer cell line RKO Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) ELISA Assessment by cell-ELISA of the binding selectivity to RKO of six phage clones was performed. The plate was read at 562 nm on an automated ELISA plate reader (Biotech Synergy HT). The M13KE wild-type phage was used as a negative control. The OD562 values were reproduced from Figure 1. The OD562 value of the M13KE wild-type phage is 0.05 ± 0.01. 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, followed by a negative selection step against normal colon CCD-841-CoN cell line. 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. After four rounds of selection plus a negative step with normal colorectal cells, CCD-841-CoN, there was an obvious phage enrichment that specifically bound to RKO cells. Cell-based enzyme-linked immunosorbent assay (ELISA) was performed to assess the most specific peptides leading to the selection of the peptide sequence CPKSNNGVC. Through fluorescence microscopy and cytometry, the synthetic peptide RKOpep was shown to specifically bind to RKO cells, as well as to other human colorectal cancer cells including Caco-2, HCT 116 and HCT-15, but not to the normal non-cancer cells. Moreover, it was shown that RKOpep specifically targeted human colorectal cancer cell tissues. A bioinformatics analysis suggested that the RKOpep targets the monocarboxylate transporter 1, which has been implicated in colorectal cancer progression and prognosis, proven through gene knockdown approaches and shown by immunocytochemistry co-localization studies. The peptide herein identified can be a potential candidate for targeted therapies for colorectal cancer. 3477 QATHRSH 1 Phage display (subtractive panning, competitive panning) 3 PMID:30796964 Aromatic-L-amino-acid decarboxylase (EC:4.1.1.28), AADC Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) Two petri dishes were coated with 10 μg/ml of either MBP (maltose binding protein) or MBP-DDC in 0.1 Μ NaHCO3, pH 8.6, overnight at 4°C. After removal of the residual protein solution, non-specific binding sites of the petri dish were blocked with 5 mg/ml bovine serum albumin) and the petri dishes were incubated at 4°C for 1 hr. All remaining procedures were performed at room temperature. The blocking buffer was removed from each petri dish and subsequently the dishes were washed 6 times with TBST (Tris buffered saline, 0.1% Tween 20, pH 7.4). 2x1011 library phages were added to the petri dish coated with ΜΒΡ and incubated for 1 hr. The phages interacting with MBP or BSA were bound to the petri dish (non-specific interaction). This first incubation resulted in removing the phages that were bound to the fusion protein by interacting with MBP, thus allowing that in the next step only the phages interacting specifically with DDC would bind. The supernatant that contained the phages not bound to MBP or BSA was incubated for 1 hr with the petri dish coated with the fusion protein. Following incubation and removal of the supernatant, 10 washes with TBST were performed. Elution of the phages was performed with a 1 hr incubation with shaking with free MBP-DDC (~100 μg/ml in TBS) or with L-Dopa (~100 μg/ml in TBS). For the (His)6DDC fusion protein the above technique was used, but with agarose magnetic beads. Human phosphatidyl-inositol-3-kinase (GenBank accession number AAG61115.1) contains the peptide ATHRS at positions 127–131, showing high similarity with the heptapeptide QATHRSH, isolated by the phage display method ten times in total, eluted both with MBP-DDC (eight times) and L-Dopa (two times). 3478 HSVEDVS(14/50)[1.35] PGVAERS(10/50)[2.58] LPRSHPI(7/50)[5.25] SSIWYSP(6/50)[2.61] TFHSTFS(5/50)[10.23] VAVSNTH(4/50)[3.20] 6 Phage display (subtractive panning) 4 PMID:30986050 D2-MT1-MMP (MT-loop deleted MMP-14) Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) ELISA The binding affinities of the top six high ratio phage peptides were determined by ELISA. The resulting Kd value (nM) is a dissociation constant, representing the proportion of dissociated molecules to initial molecule number in unit time, indicating that the higher the Kd value, the lower the affinity, and vice versa. In each round of panning, one group of WT-MT1-MMP and three groups of D2-MT1-MMP (2 mL each) were coated onto a 35 mm sterile polystyrene dish to make sure that the surface is completely wet. The plates were then kept in a humidified incubator at 4 °C overnight and blocked using a blocking buffer (0.1 M NaHCO3, pH = 9.6, 5 mg/mL BSA) for 2 h at room temperature (RT) to avoid nonspecific binding. After discarding the blocking solution and washing six times with TBST (TBS containing 0.1% [v/v] Tween-20), 10 μL of Ph.D. 7-peptide library (2.0e11 pfu) was diluted in 1 mL of TBST and exposed to the No.1 D2-MT1-MMP plate for 1 h at RT with gentle shaking. Then, the unbound phages were transferred to No.2 D2-MT1-MMP plate for 1 h at RT under gentle rotation. This procedure was repeated for No.3 D2-MT1-MMP plate. After these three steps, phage peptide which did not combination with D2-MT1-MMP was obtained and placed in No.4 plate, which is WT-MT1-MMP. After 1 h incubation, the bound phages were collected by adding 1 mL of 0.2 M Glycine-HCl (pH = 2.2) containing 1 mg/mL BSA to the plate for 8 min and neutralized with 150 μL of 1 M Tris-HCl (pH = 9.1). Then, 1 μL of phages was collected for phage tittering, and the remaining phages were mixed with 200 μL of ER2738 culture (at early log stage) for further amplification. After bacteriolysis, by centrifugation and PEG precipitation, the phages were recovered from the culture supernatant, and then dissolved in TBS. The titer was determined on LB/IPTG/Xgal plates and used as input phages for the next cycle. After four cycles of panning, the phage clones that had higher binding affinity to MT-loop were obtained. Several clones were randomly selected and −96 gIII sequencing primer was used to obtain the peptide sequences. In this study, we considered the 3-dimensional (3-D) conformation of the MT-loop area in the MT1-MMP molecule and designed a novel strategy to screen the Ph.D. peptide library. The peptide HSVEDVS (HS7) we obtained showed a better binding affinity to WT-MT1-MMP than the peptide HWKHLHNTKTFL (AF7p) as observed through enzyme-linked immunosorbent assay (ELISA) and biolayer interferometry (BLI). The new peptide labeled and attached MT1-MMP expression cell lines HT1080 and did not show any toxicity to cells. Furthermore, for in vivo imaging, HT1080 tumor-bearing mice with higher MT1-MMP expression accumulated more Cy5.5-HS7 than mice with MT1-MMP low-expression cell lines A549 at tumor sites, and the half-life of HS7 was longer than that of AF7p, as confirmed by ex vivo imaging of the main organs. These results suggest the feasibility of using the subtraction biopanning strategy to screen the affinity peptide targeting MT-loop regions and HS7 is a superior probe for noninvasively imaging MT1-MMP expression in MT1-MMP-positive tumor models. It provides impetus for further studies to use HS7 in early diagnosis of tumors and in peptide-mediated drugs. 3479 CNAGHLSQC(2)[0.67 ± 0.03] CSLNHTVNC(1)[0.86] CSAKTTSAC(1)[0.63] 3 Phage display (subtractive panning, competitive panning) 3 PMID:30968602 Fibroblast growth factor receptor 1, FGFR-1 Fibroblast growth factor 1, FGF-1 1EVT, Not determined. Ph.D.-C7C phage display library (CX7C) ELISA ELISA was conducted after the third round of selection. The absorbance was measured at 450 nm and reproduced from Figure 1A. After the second and third rounds of biopanning, additional counterselection with Fc fragment was performed. Additionally after the last round of selection, binding phages were eluted with 100 molar excess of fibroblast growth factor 1 (FGF1) over applied phage library. We used the phage display technique to select cyclic peptides F8 (CSLNHTVNC) and G10 (CSAKTTSAC) as binders of the fibroblast growth factor 1 (FGF1)–FGFR1 interface. ELISA and in vitro cell assays were performed to reveal that cyclic peptide F8 is more effective in preventing the FGF1–FGFR1 interaction, and also decreases FGF1-induced proliferation of BA/F3 FGFR1c cells by over 40%. Such an effect was not observed for BA/F3 cells lacking FGFR1. Therefore, cyclic peptide F8 can act as a FGF1–FGFR1 interaction antagonist, and may be suitable for further development for potential use in therapies against FGFR1-expressing cancer cells. 3480 HPSALMKPTSHA(4)[6.12 ± 0.24] DRWVARDPASIF(4)[7.48 ± 0.35] NPHAPSSFYEAY(3)[5.47 ± 0.10] TVGLPMTYYMHT(3)[8.23 ± 0.30] TSSPLTRWSSSL(2)[5.51 ± 0.16] AWADQPVTAPNR(2)[4.54 ± 0.05] HLTTTHPEPPYG(2)[4.17 ± 0.16] IPLGRDGGSYQR(2)[3.57 ± 0.13] MDENVATNQLMI(2)[5.87 ± 0.48] ALGDSCRYCRLL(1)[NA] ATEERSRIWMFL(1)[NA] CVASARGAQIGM(1)[NA] DDFRVWWPNFPR(1)[NA] DPVGLGGWWAKV(1)[NA] DSSQWDKIYSWT(1)[NA] GFAVGARDSLMF(1)[NA] GSAPLLTVDTSK(1)[NA] HSKAFPVLYPLR(1)[NA] LGHSGGPTRPSW(1)[NA] MLDQRPMSSYAG(1)[NA] MSLDSFRVDRRA(1)[NA] QGMVAESYSPLS(1)[NA] SALKGLFPADHH(1)[NA] SLECDELIHSQI(1)[NA] STLGFNPPAILP(1)[NA] STPGCCAHDHFR(1)[NA] SVPMGSLASLES(1)[NA] TAHASLDDQGLR(1)[NA] TPQSFWQKGSLV(1)[NA] VSGQRSVGTPLS(1)[NA] WDFRQWWQPSGG(1)[NA] 31 Phage display (common panning) 4 PMID:30937184 Proheparin-binding EGF-like growth factor Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA Quantification of binding abilities of peptides was performed by using enzyme-linked immunosorbent assay (ELISA). Absorbance was measured at 405 nm. The relative binding abilities were calculated using the following equation: Absorbance when sHB-EGF coated/Absorbance when bovine serum albumin (BSA) coated. The relative binding values were reproduced from Figure 3B and shown. The relative binding value of the wild type M13 phage is 2.6079 ± 0.14361. Six hundred microliters of sHB-EGF (100 μg/ml in NaHCO3, pH 8.6) was coated on polystyrene 6-well cell culture plate overnight at 4 °C. Other steps were performed according to manual of Ph.D-12. In order to avoid nonspecific binding, the phage elusion in each round was incubated with a new plate for 20 min at room temperature. After 4 rounds of biopanning, the isolated colonies of E. coli ER2738 were cultured and the remained phages were sequenced. Two peptides, no. 7 (HLTTTHPEPPYG) and no. 29 (TPQSFWQKGSLV) were found mildly binding to HB-EGF. Then the effects of these peptides on HB-EGF functions were examined and both peptides no. 7 and no. 29 were found indeed inhibiting the functions of HB-EGF in promoting migration and invasion of SKOV3 and HO-8910 cells in vitro. Further mechanism investigation showed that peptides no. 7 and no. 29 inhibited HB-EGF-promoted cell migration and invasion through attenuating activation of the EGFR signaling pathway manifested by decreased p-Erk1/2 and Snail levels. More importantly, peptides no. 7 and no. 29 showed strong activities in inhibiting migration of SKOV3 cells in vivo. 3481 ERTYSPSTAVRS[1.458 ± 0.28] PSTAVR[NA] SPSTAVRS[NA] TYSPSTAVRSAA[NA] PSTETR[NA] LPSTETRHV[NA] YSPSTETRSA[NA] 7 Phage display (subtractive panning) 3 PMID:31132884 The catalytic domain of rho-associated protein kinase 1 Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) The strength of interactions between Peptide7 and ROCK1 and ROCK2 was then alyzed by performing an SPR study. The binding of Peptide7 to ROCK1 as compared to ROCK2 demonstrated small differences even though bith proteins have 92% similarity in sequence in the catalytic domain. Upon inspection, there are only 3 amino acid differences in the activation loop. The average of three experiments yielded KD values of 1.458 ± 0.28 µM for ROCK1 (1–553) and 5.153 ± 1.15 µM for ROCK2. Phage particles were first mixed with MBP protein to avoid the negative selection by elimiting phages that could bind to MBP. The supertant which contained only non-MBP-binding phages was then incubated with MBP-ROCK1 (5–348). Peptide7(ERTYSPSTAVRS), a promising ROCK inhibitory peptide for both ROCK isoforms, measured at 1.45 ± 0.28 µM for ROCK1 (1–553) and 5.15 ± 1.15 µM for ROCK2. Peptide7 reduced cellular migration in wound healing assays. The binding epitope on ROCK1 was mapped to the flexible activation loop within the catalytic domain. Peptide alanine scanning mutants helped identify critical amino acids to generate optimized Peptide22. This compact ROCK inhibitor facilitated vascular relaxation, blocked neovascularization of endothelial cells, and inhibited MLC phosphatase phosphorylation. 3482 CSLTQDQGC(328,760) CADAQADVC(140,578) CKASRLGRC(139,938) CRAKGRDAC(103,044) CVGRPDA*C(84,760) CSRTEGDVC(67,529) C*SSLVKTC(61,014) CKRSS*YFC(57,309) CRNGEGAQC(54,832) CGEL*CNDC(54,226) 10 1 PMID:32035108 Inflamed central nervous system (CNS) of mice with experimental autoimmune encephalomyelitis (EAE) Not determined. Not determined. Not determined. CX7C T7 phage display library A tissue homogete equivalent to 100 mg of tissue was incubated with phage library at 4 °C for 1 h. Thereafter, the tissue homogete was washed to remove the unbound phages, whereas the bound-phages were rescued using BLT5615 strain of E. coli. The phages were then amplified and phages were injected intravenously (i.v.) into EAE mice that had clinical score between 2 and 3. The phages were circulated for 15 min. Thereafter, animals were anesthetized, perfused with saline, and CNS tissue was collected and homogenized. MS-1 (amino acid sequence CRGGKRSSC), was shown to selectively recognize CNS tissue in EAE mice. Individually cloned phages with this insert preferentially homed to EAE CNS after an intravenous injection. Similarly, systemically-administered fluorescence-labeled synthetic MS-1 peptide showed selective accumulation in the spil cord of EAE mice. 3483 CSLTQDQGC CRAKGRDAC CAMGNGGDC CTTPAKNNC CTEQIEERC CKASRLGRC CVGRPDA*C C*SSLVKTC CKRSS*YFC SLTQDQG, RAKGRDA, AMGNGGD, RPGESS, PGTVKR, KRSS, GDRLV, TTPAKNN and TEQIEER 9 3 PMID:32035108 Inflamed central nervous system (CNS) of mice with experimental autoimmune encephalomyelitis (EAE) Not determined. Not determined. Not determined. CX7C T7 phage display library A tissue homogete equivalent to 100 mg of tissue was incubated with phage library at 4 °C for 1 h. Thereafter, the tissue homogete was washed to remove the unbound phages, whereas the bound-phages were rescued using BLT5615 strain of E. coli. The phages were then amplified and phages were injected intravenously (i.v.) into EAE mice that had clinical score between 2 and 3. The phages were circulated for 15 min. Thereafter, animals were anesthetized, perfused with saline, and CNS tissue was collected and homogenized. MS-1 (amino acid sequence CRGGKRSSC), was shown to selectively recognize CNS tissue in EAE mice. Individually cloned phages with this insert preferentially homed to EAE CNS after an intravenous injection. Similarly, systemically-administered fluorescence-labeled synthetic MS-1 peptide showed selective accumulation in the spil cord of EAE mice. 3484 YYVSWPPDMMHY(11/17)[500 ± 25 nM, 299.3 nM] GRTPLLDSFLAG(3/17)[~43 μM, NA] TQQDYAWLLDH(1/17)[NA] SYSNFPLLHSYW(1/17)[NA] HDTWPLLATKTS(1/17)[NA] [PL]LL[DAH] 5 Phage display (common panning) 3 PMID:32659860 Signal transducer and activator of transcription 3 Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The affinity (Kd) of the peptides to STAT3 was determined by isothermal titration calorimetry (ITC) using no ITC calorimeter (TA Instruments, DE, USA). The alysis of which shows that peptide 2 interacted with higher affinity (Kd = 500 ± 25 nM) while peptide 1 was a slightly weaker (Kd = 1.79 ± 0.12 μM). Peptide 3, however, showed nearly 80x weaker compared to peptide 2 (Kd ~ 43 μM). All the three peptides were found to bind with a release of heat. For peptide ELISA, the strength of the association was recorded by Cytation 3 Imaging reader (BioTek Instruments). The result shows that the peptides interacted with STAT3 in a dose‐dependent manner. Peptide 1 was shown to have an affinity of 299.3 nM while the value for peptide 2 is 232.9 nM. Peptide 3 did not reach saturation under the identical conditions, suggesting a weaker interaction with STAT3. The first column of the affinity values is the Kd value determined by ITC. The second column is the strength of the association determined by peptide ELISA. A 100‐fold dilution of the PhD‐12™ library (New England Biolabs) was then added and incubated for 1 hr.The precipitated phages were pelleted by centrifugation (60 min, 16,100 g, 4°C), and the tubes dried for 20 min at room temperature (RT). The pellets were then re‐suspended in 1 ml of TBST buffer containing 0.02% w/v N3 and 1% (w/v) BSA. After re‐precipitation and suspension, the top 80% of the supertant was used for the next round of phage amplifications and selection. The YYVSWPPDMMHY sequence was found to be enriched by 36% while another with a short consensus motif was displayed in 20% of the phages. Binding alysis by isothermal titration calorimetry shows the most displayed peptide interacted with a Kd of 1.79 μM, which on modification of its structure to mimic the tural binding partners of STAT3 brought the affinity to high nomolar range (Kd = 500 nM). Using a panel of tumor cell lines, we show that the peptides prevented the proliferation of triple‐negative breast cancer cells with a moderate activity (GI50 = 50 μM). 3485 AHLPTSMLKGQG(12)[0.0224] YHFNGCEDPLCR(2)[0.0712] KVWSLVNQGGQF(2)[3.13] GHIPTCLTPMCR(1)[0.137] HSIRDGFRSTPV(1)[0.466] TGQTVTGLSYIF(1)[2.32] KVVSLSALQSMT(1)[0.665] WTSQPHLQHVDD(1)[5.29] GHLAVNMPRASL(1)[0.257] HHTGCLSPLSCS(1)[0.00257] HWKVTTWNSSTV(1)[3.52] SGVYKVAYDWQH(1)[5.00] HPWCCGLRLDLR(1)[0.0873] GH,PT,QGGQ,KV 13 Phage display (common panning) 3 PMID:32664518 Extracellular domain of low-density lipoprotein (LDL) receptor Low-density lipoprotein Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The apparent dissociation constant (K*d, M) was determined by ELISA. Each clone was incubated with LDLR and BSA-coated wells at a concentration of 5x1011 phages in 100µl of buffer for 2 hours under stirring (350 rpm). The blank was incubated with the buffer. Then the HRP-conjugated monoclol anti-M13 antibody (Amersham Pharmacia Biotech Benelux, Roosendaal, The Netherlands) was incubated for 1 hour (dilution 1:5000 in TBSC with 0.5% BSA) to detect bound phages. The phage displayed random library of linear dodecapeptides (Ph.D.-12, New England Biolabs Inc., Bioké, Leiden, The Netherlands) was screened against the extracellular domain (ED) of LDLR.Three rounds of selection were performed to obtain a pool of phages with an increasing affinity to the target. Fifty clones were isolated from this 3rd pool of phages, and their binding to the ED-LDLR and the BSA employed as a control protein was evaluated at one concentration. The peptide (HPWCCGLRLDLR) was able to bind the ED-LDLR in the presence of tural ligands and dissociated at acidic pH and in the absence of calcium, in a similar manner as the LDL. In vitro, The peptide was endocytosed by endothelial cells through the caveolae-dependent pathway, proper to the LDLR route in BBB, suggesting the prevention of its lysosomal degradation. The in vivo studies performed by magnetic resonce imaging and fluorescent lifetime imaging suggested the brain penetration of this ED-LDLR-targeted peptide. 3486 APTTHMWLNYWK(25/39) THWMXNY 1 Phage display (common panning) PMID:31778678 Anti-D-antigen monoclonal antibody 1G10 D-antigen Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA Three phage clones were selected because of their high frequency in bio-panning, good affinity and specificity for binding to mAb 1G10 in phage ELISA , as well as their similarity with the epitope of PV-I Sabin antigen performed in phage competitive ELISA. Phage clones were selected by solution-phase panning with protein G magnetic beads (NEB) that exhibited high affinity for IgG. 3487 LPFMPWI(3/17) XPF 1 Phage display (common panning) PMID:31778678 Anti-D-antigen monoclonal antibody 1G10 D-antigen Not determined. Not determined. Ph.D.-7 phage display library (X7) ELISA Three phage clones were selected because of their high frequency in bio-panning, good affinity and specificity for binding to mAb 1G11 in phage ELISA , as well as their similarity with the epitope of PV-I Sabin antigen performed in phage competitive ELISA_x0000_ Phage clones were selected by solution-phase panning with protein G magnetic beads (NEB) that exhibited high affinity for IgG. 3488 CSLTQYRYC(1/15) XTQ 1 Phage display (common panning) PMID:31778678 Anti-D-antigen monoclonal antibody 1G10 D-antigen Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) ELISA Three phage clones were selected because of their high frequency in bio-panning, good affinity and specificity for binding to mAb 1G12 in phage ELISA , as well as their similarity with the epitope of PV-I Sabin antigen performed in phage competitive ELISA_x0000_ Phage clones were selected by solution-phase panning with protein G magnetic beads (NEB) that exhibited high affinity for IgG. 3489 LTPHKHHKHLHA(19/33) GPHHMHHHRTHH(7/33) WPRHHWHTNYMR(1/33) GWHSPHAHWRVK(1/33) THYNPLRINPIT(1/33) KVHIMHFHHHSL(1/33) HSWSTIKRIETM(1/33) WPHLQHHKATSR(1/33) HDRMTKSSFSPP(1/33) 9 Phage display (common panning) 3 PMID:32568515 Se0 nanoparticle (SeNP) Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) Phage binding to SeNPs proceeded for 1 hour in selection rounds 1 and 2 but was reduced to 10 minutes in round 3. After each selection round, bound phage were acid-eluted with a Glycine-HCl (pH 2.2) based elution buffer for 20 minutes. A selenium noparticle binding peptide (LTPHKHHKHLHA) was genetically fused to a metalloid reductase that reduces selenite to a Se0 noparticle (SeNP) form. The fusion of the Se binding peptide to the metalloid reductase regulates the size of the resulting SeNP to ~35 nm average diameter, where without the peptide, SeNPs grow to micron sized polydisperse precipitates. The SeNP product remains associated with the enzyme/peptide fusion. 3490 SGVYKVAYDWQH(13/42) VHWDFRQWWQPS(10/42) HHWNSLWWPWKT(9/42) HFWHWPLWTRDH(5/42) HHYWWPWQITNS(5/42) TWWHWPRMYMGL(1/15) WHWWPHWDTGRA(1/15) WYPYNWTHWFTH(1/15) HLWYPWIWPMQQ(1/15) WHWNAWNWSSQQ(1/15) WHWSWLVHPHTL(1/15) LTNNLHKQVGPW(1/15) WHFTWWVDNRMT(1/15) HFMSYAGASTWA(1/15) GLHTSATNLYLH(1/15) GSAPLLTVDTSK(1/15) WEYSVVTPVDFL(1/15) GLTSQRLDGLPP(1/15) NPHIAHSLHSWV(1/15) HPHDYNDLTSPF(1/15) 20 Phage display (common panning) 3 PMID:32413370 Anti-HIV-1 monoclonal antibody Y498 Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA A phage ELISA was performed to determine the binding 12 mer-peptides. The results were measured at a wavelength of 450 nm. A random phage display peptide library (PhD-12) was purchased from New England Biolabs.After three rounds of selection, the selected phage clones were identified by single-strand D sequencing and phage ELISA, and the encoded peptide sequences were deduced. The concentration of washing buffer was increased gradually each round to a maximum of 0.5%TBST. To identify the key amino acid recognition site contacted with neutralizing antibody Y498, peptides were panned from the PhD-12 peptide library and predicted using online software. Then, four key amino acid sites, G367, D368, E370, and V372 located on the CD4 binding loop on gp120 of envelope of human immunodeficiency virus-1 (HIV-1), were found to determine the neutralization of antibody Y498. Residue E370 is in the deep part of the CD4 binding loop, which affects Y498-mediated neutralization. 3491 CHMRQGMAC(33/50)[1.40139] CSYPDTHRC(2/50)[0.3324] CQHPGSTMC(2/50)[0.31154] CTATESTVC(2/50)[0.38348] CNDWLSNSC(2/50)[0.49799] CQQGLSSQC(1/50)[0.79257] CHGPRASQC(1/50)[0.39923] CKQTTSSSC(1/50)[0.78065] CPLAYPHTC(1/50)[0.77129] CKETWWPHC(1/50)[1.19017] CMHTQTPWC(1/50)[0.34602] CTTHTYFGC(1/50)[0.63293] CGLKHTLKC(1/50)[0.48309] CLARNATWC(1/50)[0.50906] 14 Phage display (subtractive panning) 4 PMID:31247207 Plasma from 10 Alzheimer's disease patients Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) ELISA The absorbance value at 450 nm was determined with a microplate reader. Data (OD450) shown were reproduced from Figure 2C in the reference. In the first round of selection, phage library was selected against the pooled Alzheimer's disease (AD) plasma with albumin/IgG depletion. The second round was negative selection against pooled plasma from 10 Controls (5 male and 5 female) without albumin/IgG depletion. The third and fourth rounds of selection were against the same pooled AD plasma with albumin/IgG depletion. We further characterized one AD-specific peptide (AD#1 peptide, CHMRQGMAC) and one control-specific peptide (Con#1 peptide, CDGARHGRC), and evaluated their diagnostic performance in independent validation set (35 AD patients, 45 MCI, 45 controls and 20 PD patients). Our results show that both AD#1 peptide and Con#1 peptide could distinguish AD/MCI patients from controls and combition of these two peptides could greatly improve the diagnostic performance (AUC is above 0.80 in ROC curve alysis). In addition, we found that AD#1 peptide stained Aβ-treated primary astrocyte and bound to recombint human YKL-40 protein in in-vitro assay. It supports that AD#1 peptide detects AD inflammation related cytokine. 3492 CDGARHGRC(30/50)[1.63760] CQSPKTPFC(3/50)[1.08744] CSNGSDSSC(2/50)[1.13536] CTSVPHRYC(2/50)[0.40443] CTSTGRSNC(2/50)[0.74506] CSVSQLKLC(2/50)[0.54469] CTPLSYKPC(2/50)[0.40094] CLGQYHQRC(1/50)[1.18241] CLPSGNNRC(1/50)[0.53075] CKHTTSTSC(1/50)[0.36385] CWKHGNFNC(1/50)[0.43492] CWKAVTWLC(1/50)[0.43425] CPLGFVGDC(1/50)[0.26286] CNTSAYPDC(1/50)[0.22191] 14 Phage display (subtractive panning) 4 PMID:31247207 Plasma from 10 healthy persons Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) ELISA The absorbance value at 450 nm was determined with a microplate reader. Data (OD450) shown were reproduced from Figure 2D in the reference. In the first round of selection, phage library was selected against the pooled control plasma with albumin/IgG depletion. The second round was negative selection against pooled plasma from 10 Alzheimer's disease (AD) patients (5 male and 5 female) without albumin/IgG depletion. The third and fourth rounds of selection were against the same pooled control plasma without albumin/IgG. We further characterized one AD-specific peptide (AD#1 peptide, CHMRQGMAC) and one control-specific peptide (Con#1 peptide, CDGARHGRC), and evaluated their diagnostic performance in independent validation set (35 AD patients, 45 MCI, 45 controls and 20 PD patients). Our results show that both AD#1 peptide and Con#1 peptide could distinguish AD/MCI patients from controls and combition of these two peptides could greatly improve the diagnostic performance (AUC is above 0.80 in ROC curve alysis). 3493 NHRKVSRHATHF(4)[0.08643] PRHGKKPTNKRK(3)[0.79724] TVDSASLLQSRT(1)[0.08422] WGFHWPVYPPSR(1)[0.07299] ADARPWWKSQGF(1)[0.08063] FPYPMNKQTNGT(1)[0.08574] 6 Phage display (competitive panning) 3 PMID:31823885 Lectin α2,6-sialyllactose, 6'-SL Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA Absorbance was determined at 415 nm in a microtiter plate reader (Tecan Infinite® M1000 Pro, Tecan, Switzerland). Data (OD415) shown were reproduced from Figure 1 in the reference. Bound phages were eluted using 100 μL of 1 M α2,6-sialyllactose (6′-SL) (Cat# 35890-39-2, Dextra Laboratories, Reading, England, UK) in each round of panning. Mimetic peptide (PRHGKKPTNKRK), reverse peptide (KRKNTPKKGHRP) and scrambled peptide (RPGHKKTPKNKR) were tested for inhibition of 6′-SL binding to the lectin. Indeed, lectin binding to 6′-SL was inhibited by the mimetic peptide , but not by the reverse or scrambled peptides, showing that this peptide mimics 6′-SL. Functiolly, mimetic peptide, but not the reverse or scrambled peptides, increased viability and expression of neural cell adhesion molecule L1 in SK-N-SH human neuroblastoma cells, and promoted survival and neurite outgrowth of cultured mouse cerebellar granule neurons challenged by H2O2-induced oxidative stress. The combined results indicate that the 6′-SL mimetic peptide promotes neurol survival and neuritogenesis, thus raising hopes for the treatment of neurodegenerative diseases. 3494 TMANEAPYPGTQ[1.41642] HWDPPYPGSGTQ[1.32673] TEAPYPGSSVTN[1.20625] SALIDAAYPGTQ[1.14333] SEAPYPGAGVYN[1.1942] TFEAPYPGTMLV[1.12995] STEAPYPGIAVQ[1.31646] EAPYPG 7 Phage display (common panning) 3 PMID:31500272 Anti-σA monoclonal antibody 4E2 σA protein Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA Reactivity of phage clones to MAb 4E2 was determined by enzyme-linked immunosorbent assay. Data (OD450) were reproduced from Figure 3a in the reference. Purified MAb 4E2 was selected for epitope mapping under three rounds of biopanning with the Ph.D-12 TM Phage Display Peptide Library Kit (New England BioLabs, Cambridge, MA, USA). Sequencing of the phage clones with high OD values revealed the consensus sequence EAPYPG, which was identical to the 56EAPYPG61 (aa 217 to 223) of the DRV σA protein. Residues in epitope 56EAPYPG61 were completely homologous in DRV, GRV, ARV, and TRV. 3495 AFWSVPGTVSMT[1.21845] NWVVGGSVAAVT[1.31435] YNFVMAGLVATT[1.44127] IWVMAGAISLSM[1.07978] YVMAGLILSIPN[1.36512] CFTMASLITITA[1.25935] GAWVVAGLIMTV[1.23397] QWVVAGLVSVLG[1.27345] WV[V/M]AGL[I/V] 8 Phage display (common panning) 3 PMID:31500272 Anti-σA monoclonal antibody 1A7 σA protein Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA Reactivity of phage clones to MAb 1A7 binding was determined by enzyme-linked immunosorbent assay. Data (OD450) were reproduced from Figure 3b in the reference. Purified MAb 1A7 was selected for epitope mapping under three rounds of biopanning with the Ph.D-12 TM Phage Display Peptide Library Kit (New England BioLabs, Cambridge, MA, USA). Sequencing of the phage clones with high OD values revealed the consensus sequence WVV/MAGLI/V, which was identical to the 341WVVAGLI347 (aa 341–347) sequences of the DRV σA protein. Residues in the 341WVVAGLI347 were homologous in DRV and GRV, but divergent at residues 343V/M or/and 347I/V between DRV/GRV and ARV/TRV. 3496 GDGNSVLKPGNW[1.38809] DRWVARDPASIF[2.9589] 2 Phage display (common panning) 5 PMID:31279172 Neutrophil gelatinase-associated lipocalin Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The ELISA sigl was measured at 405 nm using a microplate spectrophotometer (Multiskan FC, Thermo Scientific, Waltham, MA, USA). Data (OD415) shown were reproduced from Figure S1a in the reference. Three rounds of biopanning were performed against recombint NGAL proteins. After washing, the bound phages were eluted using 100 μL of 0.2 M glycine-HCl (pH 2.2), and the eluent was immediately neutralized with 15 μL of Tris-HCl (pH 9.1) to prevent destruction of the phages.The eluted phages were amplified using E. coli ER2738 to make sufficient copies for subsequent rounds of biopanning. The amplified phages were harvested by Cl/polyethylene glycol precipitation. The neutrophil gelatise-associated lipocalin (NGAL) BP1 (DRWVARDPASIF) was selected as most promising recognition receptor, and its binding affinity was monitored by SWV and EIS. Using EIS, the limit of detection (LOD) was 1.74 ng/mL, while SWV had a LOD of 3.93 ng/mL. The detection performance of the peptide-incorporated sensor was comparable to commercially available ELISA NGAL detection kits. In addition, the validation of the peptide sensor was also confirmed with plasma from patients, and it was observed that the sensitivity of the peptide sensor showed a statistically significant difference. Our results show that the phage and peptide sensor system could detect NGAL with high sensitivity and selectivity, and this suggests its potential use as a biosensing platform for monitoring NGAL in a miniaturized electrochemical biosensor. 3497 HSFKWLDSPRLR(5)[2.580765±0.716041] GAYTSWRTSTNA(3)[1.788102±0.130236] DLVSWAGSGKKH(3)[1.855382±0.037913] VHWDFRQWWQPS(1)[2.125573±0.006749] AHAHTNWTSWWE(1)[2.594787±0.006743] ADWYHWRSHSSS(1)[2.043153±0.005618] IPNYSMQSREYR(1)[2.080793±0.006749] HYRPFTQEHRVT(1)[3.67144] HSFKGWDWPRLR(1)[2.795317±0.006743] GWKSHEPKGHGS(1)[2.694727±0.005618] EHLHASWNFSSG(1)[1.808223±0.006743] YDVPNKSWRTSW(1)[4.17569] 12 Phage display (subtractive panning) 3 PMID:31888393 AdipoR-12C Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The OD405 was measured using a microplate reader (StatFax-2100, Awarness Technology, Fisher Bioblock Scientific, Tournai, Belgium). Data (OD405) shown were reproduced from Figure 1G in the reference. Bovine serum albumin (BSA) immobilized on Dynabeads was used as control protein during the preselection steps of the 3 panning rounds to exclude non-specific phages. The selected peptide P17 (IPNYSMQSREYR) recognizes AdipoR1/R2 expressed by skeletal muscle, liver and pancreatic islets. In HepaRG and C2C12 cells, P17 induced the activation of AMPK (AMPKα-pT172) and the expression of succinate dehydrogenase and glucokinase; no cytotoxic effects were observed on HepaRG cells. In db/db mice, P17 promoted body weight and glycemia stabilization, decreased plasma triglycerides to the range of healthy mice and increased adiponectin (in high fat-fed mice) and insulin (in chow-fed mice) levels. It restored to the range of healthy mice the tissue levels and subcellular distribution of AdipoR1/R2, AMPKα-pT172 and PPARα-pS12. In liver, P17 reduced steatosis and apoptosis. The docking of P17 to AdipoR is reminiscent of the binding mechanism of adiponectin. To conclude, we have developed an AdipoR1/AdipoR2-targeted peptide that modulates adiponectin signalling pathways and has therapeutic relevance for T2D and obesity associated pathologies. 3498 GLKIWSLPPHHG NFMESLPRLGMH FDLNPRSLYGSL NPNGTTGPLDSV SMVYGNRLPSAL NLSNRLNLSPGI KVWTLDFQPPVL SLTVPFLPLYVP QPHKVFFPNLPR GHHSMTPGTAPH FATQQPPTAHIP LLADTTHHRPWT IPWTQHMAMSPM YSDQPTQSSQRP QVYAEFKTSFRS AVLDELRRRIFGA 16 Phage display (subtractive panning) 3 PMID:32840755 Soluble proteins differentially expressed between the control and dimethylhydrazine treated groups Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) For the second and third selection cycles, a pre-selection was carried out first using wells containing immobilized proteins for the control groups. The non-binding peptides (F1) were collected, amplified as previously described, and then used for selection against extracts from the test groups. Two peptides identified by phage display (GLKIWSLPPHHG denoted as peptide-1 and NFMESLPRLGMH peptide-2) were synthesized and exhibited purity higher than 84%. Poly(lactic acid)-block-polyethylene glycol nanospheres were prepared by nanoprecipitation and double emulsion methods in order to load the two peptides. Nanoparticles ranged in size from 114 to 150 nm and peptide encapsulation efficiency varied from 16 to 32%, depending on the methodology. No cytotoxic activity was observed towards Caco-2 tumor cell line, either free or loaded peptides in concentrations up to 3 μM at incubation times of 6 and 24 h, indicating safety as biomarkers. Fluorescein isothiocyanate–labeled peptides allowed evaluating selective interactions with Caco-2 cells, where peptide-1 entrapped in nanospheres showed greater intensity of co-localized cell fluorescence, in comparison to peptide-2. Peptide-1 loaded in nanospheres revealed promising to be investigated in further studies of selectivity with other human colon rectal cells as a potential biomarker. 3499 HSLWMASPMPGY(8/13)[0.12] QIFTSSPMPAMV(5/13)[0.09] 2 3 PMID:32222498 Anti-imidacloprid monoclonal antibody 3D11 Imidacloprid Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The half-maximum inhibition concentration (IC50, ng/mL) was calculated using the calibration curve and shown. For the first round of panning, the bound phages were eluted by adding 100 μL of 1 mg L−1 imidacloprid. To eliminate nonspecifically bound phages, the eluate was transferred into wells that were coated with 5% milk in PBS alone, and then the phages in the supernatants were amplified by infection of Escherichia coli ER2738. The amplified phages were subjected to two further panning procedures, which were carried out using mAb concentrations of 50 and 25 mg L−1, imidacloprid concentrations of 0.1 and 0.01 mg L−1, and PBS containing 0.3% Tween 20 and 0.5% Tween 20, respectively. We isolated two phage-borne peptides that compete with imidacloprid to bind the monoclonal antibody (mAb) 3D11 from phage display peptide libraries. A phage-enzyme-linked immunosorbent assay (P-ELISA) and two phage time-resolved fluoroimmunoassays (P-TRFIAs) for the detection of imidacloprid were developed using the phage-borne peptides as substitutes for chemically synthesized antigens. After systematic optimization, the half-maximum inhibition concentrations (IC50) of the P-ELISA, P-TRFIA-1, and P-TRFIA-2 were 0.067 ng mL−1, 0.085 ng mL−1, and 0.056 ng mL−1, respectively. Based on their IC50 values, the sensitivities of the P-ELISA and P-TRFIAs were more than four times greater than those of previous immunoassays. 3500 CWSYAVHPEC(4)[1.286620±0.009336] CDEYAVHMEC(4)[NA] CEGYAIHPEC(3)[NA] CFWYASHPEC(2)[NA] CIMYAIHPEC(1)[NA] CVLYALHDEC(1)[NA] CQFYVLHPEC(1)[NA] CAFYVMHPEC(1)[NA] CVWYVVHPEC(1)[NA] CILYAVHPEC(1)[NA] CDVYAVHPEC(1)[NA] CGDYAVHAMC(1)[NA] CEFYAVHALC(1)[NA] CVDYAVHVEC(1)[NA] YAVHP 14 Phage display (common panning) 4 PMID:31434316 Anti-LDL (−) monoclonal antibody 1A3H2 Electronegative low-density lipoprotein, LDL (−) Not determined. Not determined. fUSE55-based CX8C phage display library (CX8C) ELISA Absorbance at 450 nm was measured by spectrophotometry using a microplate reader (SynergyTM Mx, Biotek instruments Inc, Winooski, VT, USA). Data (OD450) shown were reproduced from Figure 1B in the reference. To prevent the selection of peptides binding to conserved domains of immunoglobulins,the anti-LDL(-) mAbs 1A3 and 2C7 were immobilized on microtiter plates and incubated in the presence of excess soluble, unrelated mouse immunoglobulin. P1A3 (CWSYAVHPEC) was quickly internalized by bone marrow-derived murine macrophages as shown by confocal microscopy. However, P1A3 did not show pro-inflammatory effects.