3601 TWWNPRLVYFDY(32)[248.506±48.1295] GVWWTWGNYGQM(5)[99.1517±25.2107] YTPNWHFRWMPA(2)[82.7265±48.3205] APTTWFNSDSIT(2)[13.5881±14.3243] 4 Phage display (common panning) 3 PMID:34962367 Graphene paper, GP Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) To assess the binding affinity of GP-binding peptides, four selected GP-binding phages (with wild-type M13 phage as a control group) were amplified by infecting E. coli. After GP containers were UV treated and washed by TBS buffer, 200 μL of the corresponding phage solution (1010 pfu/ mL) was added into five containers separately and incubated for 60 min. The unbound phages were washed away with TBST. To get the GP-bound phages out of the GP substrates, 240 μL of the elution buffer was allowed to interact with the substrates for 8 min. Finally, 40 μL of neutralizer was added, and 10 μL of resultant was titrated to quantify the phages. First, GP was folded into a container and exposed to ultraviolet irradiation for an hour. Then, the container was washed using TBS. The phage library (2.0e11 pfu) was added to the GP container for incubation. After 1 h of incubation, the unbound phages were washed away with TBST. To elute bound phages, 240 μL of glycine-HCl (0.2 M, pH 2.2) was added and incubated for 8 min. Then the eluate was neutralized with 40 μL of Tris-HCl (1 M, pH 9.1). Then, 10 μL of the eluate was titrated. The rest of the eluate was amplified in ER2738 E. coli culture and treated as a sublibrary for the next round of biopanning. Output phages in round 3 were sequenced to identify the sequences of foreign peptides displayed at their protein 3 (p3) tips. We first screened a high-affinity graphene paper (GP) binding peptide (TWWNPRLVYFDY) by the phage display technique. Then we chemically conjugated the GP-binding peptide to the synthetic hydroxyapatite (HA) nanorods. The GP-binding peptide on the resultant HA nanorods enabled them to be bound and assembled onto the GP substrate with high affinity, forming a GP-peptide-HA composite with significantly improved hydrophilicity of GP. The composite promoted the attachment and proliferation of mesenchymal stem cells (MSCs), demonstrating its outstanding biocompatibility. Due to the unique compositions of the composite, it was also found to induce osteogenic differentiation of MSCs in vitro in the absence of other inducers in the medium, by verifying the expression of the osteogenic markers including collagen-1, bone morphogenetic proteins 2, runx-related transcription factor 2, osteocalcin, and alkaline phosphatase. Our work suggests that the GP-binding peptide can be used to link inorganic nanoparticles onto GP to facilitate the biomedical applications of GP. 3602 ACGTKPTKFC(6/26)[1.3898±0.0246] ACPTTSTQYC(5/26)[0.8509±0.0099] ACTDKASSSC(5/26)[0.7775±0.0246] ACTPRSANYC(3/26)[1.0217±0.0915] ACLKTYWYNC(2/26)[1.2293±0.0772] 5 Phage display (common panning) 4 PMID:37230729 Nucleoprotein, N Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) ELISA SARS-CoV-2 NP (1 μg/mL) was fixed in the wells of the microplates overnight. peptides are powerful biomolecular tools for SARS-CoV-2 detection, providing a new and inexpensive method of rapidly screening infections as well as rapidly diagnosing coronavirus disease 2019 patients 3603 SFYDFEMQGFFI(4)[2.6799±0.2464] SFYDEMGFI(4)[3.0158±0.2156] SYFMQFF(3)[3.1003±0.1193] 3 Phage display (common panning) 3 PMID:34170090 Anti-infectious bronchitis virus (IBV) M41 strain S1 protein monoclonal antibody 3D9 Spike protein S1 of Spike glycoprotein Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The absorbance at 450 nm was measured using a Bio-Rad Microplate Reader. A standard biopanning procedure was carried out according to the manufacturer's instructions. Briefly, one well of a 96-well microtiter plate was coated with 100 µL of mAb 3D9 with a final concentration of 100 µg/mL. After blocking, 100 µL of the phages (about 2.0e11 pfu/mL) from the Ph.D.-12 phage library was added to the wells and incubated for 1 h at room temperature. The unbound phages were removed, and the wells were washed with 0.1%TBST. An elution buffer was added to each well with gentle shaking for 1 h at room temperature. The eluent containing the phages was mixed with a neutralization buffer to determine the titer of the phages and for amplification. The coated mAb 3D9 concentration (100 µg/mL, 75 µg/mL, and 50 µg/mL) was reduced and the concentration of Tween-20 in TBST buffer (0.1%, 0.2%, and 0.3%) was increased according to the number of biopanning steps. Sequence analysis showed that the dominant sequence was SFYDFEMQGFFI. Indirect competitive enzyme-linked immunosorbent assay showed that SFYDFEMQGFFI is a mimotope of the S1 protein that was predicted by PepSurf. The mimotope may provide information for further structural and functional analyses of the S1 protein. 3604 NFWISPKLAFAL(6/24)[1.2595±0.0186] AEAWTGFSASGV(2/24)[0.1887±0.0186] NHHYTYHQYTVG(2/24)[0.4545±0.0165] HVNYGMHGYETT(4/24)[0.359±0.0304] NTACDMSGRHCH(3/24)[0.4685±0.0469] SSFGMSSQLISH(1/24)[0.1211±0.0068] WDMWPSMDWKAE(3/24)[0.1748±0.0068] QINPEFTPTMRW(1/24)[0.2306±0.0072] 8 4 PMID:35948070 Spike protein S1 of spike glycoprotein Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The optical density of each well at 452 nm (OD452nm) was measured. The bovine serum albumin (BSA) negative selection was added to rounds 2, 3, and 4 biopanning. That is, the phage was incubated with BSA for 4 h before incubating the phage with SARS-CoV-2 S1. After four rounds of biopanning from the pIII phage display library, a phage monoclone expressing the NFWISPKLAFAL peptide was identified, which had the best affinity and selectivity for SARS-CoV-2 S1 with a dissociation constant of 3.45 ± 0.58 nM.  3605 DGFIRPSGVRVA(19/78)[0.6748±0.4406] WDMWPSMDWKAE(8/78)[1.5887±0.1874] DGSMLNRMRGFS(3/78)[0.3231±0.0985] 3 Phage display (common panning) 4 PMID:36944950 Cathepsin B Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The absorbance was measured at 405 nm using a Multiskan FC microplate photometer (Thermo Scientific, Waltham, MA, USA). Data shown were reproduced from Figure 2A. Biotinylated cathepsin B (99 μL, 500 nM) was pre-reacted with the Ph.D.-12 random phage library (1 μL, 1.0 × 1013 PFU/mL) at 100 rpm for 1 h. Subsequently, 100 µL of the complex mixture was added onto a pre-washed streptavidin-coated microplate, and allowed to react at a shaking speed of 100 rpm for 10 min to facilitate specific binding between avidin and biotin. Then, 0.1 mM biotin was added as a blocking agent and allowed to react for 5 min. After removing the unbound phages and residual biotin, the plate was washed 10 times with 0.1% PBST (0.1 M phosphate-buffered saline (PBS) with 0.1% Tween 20), and the bound phages were eluted using 100 µL of 0.2 M glycine–HCl (pH 2.2) with 1 mg/mL of bovine serum albumin (BSA) solution. Finally, the eluent was neutralized with 15 µL of Tris–HCl (pH 9.1) to prevent deactivation or destruction of the desired phages. Throughout this process, the desired phages were amplified using Escherichia coli ER2738 making sufficient copies for the next rounds. The amplified phages were then harvested by polyethylene glycol (PEG)/NaCl precipitation (20% (v/v) PEG-8000 with 2.5 M NaCl). After every biopanning round, the phages were tittered, and the displayed peptide sequences were analyzed using the − 96 gIII sequencing primer (5ʹ-HOCCC TCA TAG TTA GCG TAA CG-3ʹ). A phage-display library was biopanned against biotinylated cathepsin B to identify a high-affinity peptide with the sequence WDMWPSMDWKAE. The identified peptide-displaying phage clones and phage-free synthetic peptides were characterized using enzyme-linked immunosorbent assays (ELISAs) and electrochemical analyses (impedance spectroscopy, cyclic voltammetry, and square wave voltammetry). Feasibilities of phage-on-a-sensor, peptide-on-a-sensor, and peptide-on-a-AuNPs/MXene sensor were evaluated. 3606 AHNHTPIKQKYL(2/55)[512.103±48.2444] ANTELALANRKH(2/55)[352.5796±17.2553] NWGVMPWIGATT(2/55)[389.5553±13.3817] 3 Phage display (common panning) 4 PMID:32971483 Tyrosine-protein kinase receptor UFO Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) EIS was carried out with a DC potential of 0.2 V using an alternating voltage of 10 mV in a frequency range of 10 Hz to 10 kHz. The AXL protein was dissolved in PBS buffer and subsequently transferred to a microwell plate. Following overnight incubation at 4 °C, the wells were washed with PBS buffer to remove the unbound AXL protein and then incubated with blocking buffer at 4 °C for 1 h. After removing the residual blocking solution, the wells were washed six times with PBST buffer (0.1 M PBS buffer with 0.1% Tween 20). The Ph.D.-12 peptide library (1 × 1011 plaque forming units [PFUs]) was added to the wells containing immobilized AXL protein and binding was performed by incubating the plate at 25 °C with shaking. Following successful shaking, the plate was washed 10 times with PBST to remove the unbound phage particles or residual protein and the AXL-bound phage particles were finally eluted using an elution buffer (0.2 M glycine-HCl, pH 2.2). To minimize non-specific binding during the entire biopanning process, the Tween 20 concentration used in the washing step of round 1, 2, and 3–5 was increased to 0.1, 0.3, and 0.5%, respectively. The elution fraction containing the most AXL-bound phage particles was immediately neutralized using Tris-HCl (pH 9.1) to prevent their activity. The neutralized eluted phage fraction was used to infect E. coli ER2738 to amplify sufficient copies of the peptides beyond preparation of DNA sequencing and next round of panning. The phage titer was determined using Luria-Bertani broth containing isopropyl β-D-thiogalactopyranoside and X-gal, and PFU was calculated by counting single blue plaques after overnight incubation at 37 °C. Biopanning of M13 phage library successfully identified a high affinity peptide, with the sequence AHNHTPIKQKYL. To study the feasibility of using free peptides for molecular recognition, we synthesized a series of amino acid-substituted peptides and examined their binding affinity for a tyrosine kinase receptor (AXL) using electrochemical impedance spectroscopy and square wave voltammetry. Most synthetic peptides had non-identical random coil structures based on circular dichroism spectroscopy. Of the peptides tested, AXL BP1 (AHNHTPIKQKYL) exhibited nanomolar binding affinity for AXL. To verify whether AXL BP1 could be used as a peptide inhibitor at the cellular level, two functional tests were carried out: a WST assay for cell viability and qRT-PCR for quantification of RNA levels in Zika virus-infected Huh7 cells. The results showed that AXL BP1 had low cytotoxicity and could block Zika virus entry. These results indicate that newly identified affinity peptides could potentially be used for the development of Zika virus entry inhibitors. 3607 WSLGYTG(4/20) GTIYWNS(1/20) SPLSPRY(1/20) ETGITRQ(1/20) WIFTPLG(1/20) RNSWPVW(1/20) GSSGKPG(1/20) RGTGHYW(1/20) WWSTHDR(1/20) RNMRGYG(1/20) WTARPTG(1/20) GSWTTGQ(1/20) 12 3 PMID:33481966 T-cell surface glycoprotein CD3 zeta chain Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) Flow Cytometry All candidate phage clones harvested from the biopanning experiments and two random clones from the stock library were amplified, tittered, and plated for the determination of each concentration (PFU/ml). Streptavidin-coated iron-oxide microbeads (Dynabeads M280, Invitrogen) were decorated with biotinylated human CD3e (Avitag) at a saturation density (>10 μg of biotinylated protein per 1 mg of Dynabeads). CD3e-coated beads were washed with PBS buffer once and incubated with candidate phage clones (10^12 virions per 1 mg beads) in 200 μl TBST buffer (tris-buffered saline with 0.1% tween) for 30 minutes at room temperature on a shaker. After washing the beads with FACS buffer once, the beads were incubated with anti-M13 Major Coat Protein antibody (1 : 100 dilution) for 30 minutes at room temperature on a shaker. The bead samples were finally washed and resuspended in FACS buffer to be examined using flow cytometry. Screening of the PhD-7 phage display peptide library on human recombinant CD3ε protein as the target protein was carried out according to the manufacturer's protocol with some modifications. Briefly, 50 μl of a 50% aqueous suspension of Pierce protein A/G magnetic beads was washed with 1 ml of TBST washing buffer (tris-buffered saline with 0.1% tween) on a magnetic separator and blocked in blocking buffer (0.1 M NaHCO3 (pH 8.6) with 0.5% (w/v) BSA) for 1 h at 4 °C on a rotator. 2 picomoles of target protein was mixed with 1011 pfu (plaque-forming unit) of the phage library in 200 μl of TBST buffer and incubated for 15 minutes at room temperature on a rotator and then incubated with pre-washed and pre-blocked magnetic beads for 15 minutes at room temperature on a rotator. After the microbeads were washed vigorously 15 times with 1 ml TBST, the bound phage was eluted by 1 ml of glycine buffer (0.2 M glycine–HCl (pH 2.2) + 0.1% (w/v) BSA) and neutralized by the addition of 150 μl of 1 M Tris–HCl (pH 9.1). The harvested phages were amplified by infecting E. coli (ER2738) cells and precipitated using PEG8000/NaCl solution. After each round, a negative selection was performed by incubating the harvested phage clones from the previous round with the empty protein A/G magnetic beads in TBST buffer for 20 minutes at room temperature on a rotator. Only the unbound phages in this negative selection were used for the next rounds of panning. After the third and the fifth round of biopanning, the resulting phage clones tittered on LB/IPTG/Xgal plates, and 20 out of 100 blue plaques were randomly selected for sequencing. WSLGYTG demonstrated a superior binding behavior to other clones in the binding assays against recombinant T-cell surface glycoprotein CD3 zeta chain (CD3 zeta) on microbeads or Jurkat cells. The synthesized peptide also showed specific binding to Jurkat cells in a dose-dependent manner but not to B cell lymphoma line, 2PK3 cells. Molecular modeling and docking simulation confirmed that the selected peptide ligand in an energetically stable conformation binds to a pocket of CD3 zeta that is not hidden by either CD3 gamma (T-cell surface glycoprotein CD3 gamma chain) or CD3 delta (T-cell surface glycoprotein CD3 delta chain). Lastly, magnetic microbeads conjugated with the synthesized peptide ligands showed a weak but specific association with Jurkat cells and induced the calcium flux, a hallmark indication of proximal T cell receptor signaling, which gave rise to an enhancement of IL-2 section and cell proliferation. The novel peptide ligand and its various multivalent forms have a great potential in applications related to T cell biology and T cell immunotherapy. 3608 WSLGYTG(18/20) GSWTTGQ(1/20) YNHTMMY(1/20) 3 5 PMID:33481966 T-cell surface glycoprotein CD3 zeta chain Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) Flow Cytometry All candidate phage clones harvested from the biopanning experiments and two random clones from the stock library were amplified, tittered, and plated for the determination of each concentration (PFU/ml). Streptavidin-coated iron-oxide microbeads (Dynabeads M280, Invitrogen) were decorated with biotinylated human CD3e (Avitag) at a saturation density (>10 μg of biotinylated protein per 1 mg of Dynabeads). CD3e-coated beads were washed with PBS buffer once and incubated with candidate phage clones (10^12 virions per 1 mg beads) in 200 μl TBST buffer (tris-buffered saline with 0.1% tween) for 30 minutes at room temperature on a shaker. After washing the beads with FACS buffer once, the beads were incubated with anti-M13 Major Coat Protein antibody (1 : 100 dilution) for 30 minutes at room temperature on a shaker. The bead samples were finally washed and resuspended in FACS buffer to be examined using flow cytometry. Screening of the PhD-7 phage display peptide library on human recombinant CD3ε protein as the target protein was carried out according to the manufacturer's protocol with some modifications. Briefly, 50 μl of a 50% aqueous suspension of Pierce protein A/G magnetic beads was washed with 1 ml of TBST washing buffer (tris-buffered saline with 0.1% tween) on a magnetic separator and blocked in blocking buffer (0.1 M NaHCO3 (pH 8.6) with 0.5% (w/v) BSA) for 1 h at 4 °C on a rotator. 2 picomoles of target protein was mixed with 1011 pfu (plaque-forming unit) of the phage library in 200 μl of TBST buffer and incubated for 15 minutes at room temperature on a rotator and then incubated with pre-washed and pre-blocked magnetic beads for 15 minutes at room temperature on a rotator. After the microbeads were washed vigorously 15 times with 1 ml TBST, the bound phage was eluted by 1 ml of glycine buffer (0.2 M glycine–HCl (pH 2.2) + 0.1% (w/v) BSA) and neutralized by the addition of 150 μl of 1 M Tris–HCl (pH 9.1). The harvested phages were amplified by infecting E. coli (ER2738) cells and precipitated using PEG8000/NaCl solution. After each round, a negative selection was performed by incubating the harvested phage clones from the previous round with the empty protein A/G magnetic beads in TBST buffer for 20 minutes at room temperature on a rotator. Only the unbound phages in this negative selection were used for the next rounds of panning. After the third and the fifth round of biopanning, the resulting phage clones tittered on LB/IPTG/Xgal plates, and 20 out of 100 blue plaques were randomly selected for sequencing. WSLGYTG demonstrated a superior binding behavior to other clones in the binding assays against recombinant T-cell surface glycoprotein CD3 zeta chain (CD3 zeta) on microbeads or Jurkat cells. The synthesized peptide also showed specific binding to Jurkat cells in a dose-dependent manner but not to B cell lymphoma line, 2PK3 cells. Molecular modeling and docking simulation confirmed that the selected peptide ligand in an energetically stable conformation binds to a pocket of CD3 zeta that is not hidden by either CD3 gamma (T-cell surface glycoprotein CD3 gamma chain) or CD3 delta (T-cell surface glycoprotein CD3 delta chain). Lastly, magnetic microbeads conjugated with the synthesized peptide ligands showed a weak but specific association with Jurkat cells and induced the calcium flux, a hallmark indication of proximal T cell receptor signaling, which gave rise to an enhancement of IL-2 section and cell proliferation. The novel peptide ligand and its various multivalent forms have a great potential in applications related to T cell biology and T cell immunotherapy. 3609 LTPHKHHKHLHA(35)[+] RLATYPQLSVTQ(NA)[+] 2 Phage display (common panning) 3 PMID:34111485 Anti-bovine viral diarrhea virus (BVDV) serum Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) Western blot BSA-conjugated peptides were separated by SDS-PAGE, transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). After blocking with 5% non-fat milk in PBST for 1 h at room temperature, the membranes were incubated with goat anti-BVDV specific antibody overnight at 4 ◦C, and donkey anti-goat IgG-HRP for 1 h at room temperature. After extensive washing with PBST, the reactive protein bands were developed using Clarity Western ECL substrate (BioRad, cat# 1705061). The phage library panning was performed following the protocol specified by the vendor with modification. In brief, 60 mm dishes were coated with commercial goat anti-BVDV serum (1:100 dilution) in 0.1 M NaHCO3 (pH 8.6) overnight at 4 °C. The coated dishes were blocked with 5 mg/ml BSA in 0.1 M NaHCO3 (pH 8.6) for 2 h at 4 °C. After rapid washing 6 times with TBST (TBS + 0.1% [v/v] Tween-20), the dishes were incubated with 1 × 1011 clones of the phage from Ph.D.-12 phage display library in 1 ml of TBST for 30 min at room temperature with gentle rocking. Unbound phages were washed away by 10 times of washing with TBST at room temperature. Bound phages were eluted with 1 ml of 0.2 M Glycine-HCl (pH 2.2) containing 1 mg/ml BSA with gentle rocking for 15 min, then neutralized with 150 μl of 1 M Tris-HCl, pH 9.1. Recovered phages were amplified by incubation with 20 ml of E. coli ER2738 (OD600 0.01–0.05) with vigorous shaking for 4.5 h at 37 °C. The amplified phages, were purified by precipitation with 1/6 volume of 20% PEG/2.5 M NaCl overnight at 4 °C, and centrifugation for 30 min at 10,000g. The purified phages were taken over additional binding/amplification/purification cycles to enrich the pool in favor of binding sequences. After 3 rounds of panning, 100 individual clones are characterized by DNA sequencing with primer 28gIII primer (5′-GTA TGG GAT TTT GCT AAA CAA C-3′) as described in the manufacturer's protocol.  We screened a 12-mer phage display peptide library using commercial goat anti-bovine viral diarrhea virus (BVDV) serum, and identified a mimotope “LTPHKHHKHLHA” referred to as P3. With sequence alignment, a putative B-cell epitope “77ESRKKLEKALLA88” termed as P3-BVDV1/2 residing in BVDV core protein was identified. The synthesized peptides of both P3 and P3-BVDV1/2 show strong reactivity with BVDV serum in immune blot assay. Immunization of mice with these individual peptides leads to the production of antibody that cannot neutralize virus infectivity. 3610 CLNTPLKSC(6)[1.038±0.1546] CQPGSLGSC(2)[0.7684±0.0491] CGPRAATSC(1)[0.6455±0.0903] CYKPNQTWC(1)[0.9324±0.0903] CQDPPAAPC(1)[1.0148±0.1886] CFNGIAAEC(1)[0.842±0.1728] CTKPAVKFC(1)[0.6375±0.0729] 7 Phage display (subtractive panning) 3 PMID:35408679 Insulinoma cell line MIN6 Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) ELISA The 96-well plates were then measured at 450 nm using an ELISA reader (Bio-Tek ELX 800, Vermont, NE, USA). After the first round of phage screening on mice insulinoma cell line MIN6, the eluted phage was added to the blocked murine pancreatic cancer cell line KPC cells to absorb the phages, which can combine with KPC cells. The remaining phages (not absorbed by KPC cells) were collected for amplification. Three rounds of phage screening were performed on MIN6 (positive selection) and KPC cells (negative selection). We used phage display libraries to screen a beta-cell-targeted peptide, LNTPLKS, which was tagged with fluorescein isothiocyanate (FITC). This peptide was validated for targeting beta-cell with in vitro and in vivo studies. Immunocytochemistry (ICC) and fluorescence-activated cell sorting (FACS) analysis were used to validate the target specificity of the peptide. FITC-LNTPLKS displayed much higher fluorescence in beta cells vs. control cells in ICC. This discrimination was consistently observed using primary rodent islet. FACS analysis showed right shift of peak point in beta cells compared to control cells. The specific bind to in situ islet was verified by in vitro experiments using rodent and human pancreatic slices. The peptide also showed high affinity of islet grafts under the renal capsule. In the insulinoma animal model, we could find FITC-LNTPLKS accumulated specifically to the tumor, thus indicating a potential clinical application of molecular imaging of insulinoma. 3611 DHAQRYGAGHSG(20/30) 1 Phage display (common panning) 2 PMID:36177466 Recombinant receptor-binding domain (RBD) of glycoprotein protein Angiotensin-converting enzyme 2 Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA Optical absorbance was measured at 450 nm in a microplate reader (Synergy H1, BioTek Instruments, Inc.,Winooski, VT, United States). Data shown were preproduced from Figure 1. In the competitive S-trimer-ACE2-binding experiments, synthetic C2 (DHAQRYGAGHSG) peptide inhibited S-trimer binding onto 293T-ACE2hR cells at high concentrations (50 mM) but not at lower concentrations (10 mM and below), neither for the settings of S-trimer binding onto recombinant ACE2 proteins. 3612 DHAQRYGAGHSG(16/25) 1 Phage display (common panning) 3 PMID:36177466 Recombinant receptor-binding domain (RBD) of glycoprotein protein Angiotensin-converting enzyme 2 Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA Optical absorbance was measured at 450 nm in a microplate reader (Synergy H1, BioTek Instruments, Inc.,Winooski, VT, United States). Data shown were preproduced from Figure 1. In the competitive S-trimer-ACE2-binding experiments, synthetic C2 (DHAQRYGAGHSG) peptide inhibited S-trimer binding onto 293T-ACE2hR cells at high concentrations (50 mM) but not at lower concentrations (10 mM and below), neither for the settings of S-trimer binding onto recombinant ACE2 proteins. 3613 DHAQRYGAGHSG(7/30) HWKAVNWLKPWT(6/30) 2 Phage display (common panning) 2 PMID:36177466 Spike glycoprotein trimer, S-trimer Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA Optical absorbance was measured at 450 nm in a microplate reader (Synergy H1, BioTek Instruments, Inc.,Winooski, VT, United States). Data shown were preproduced from Figure 1. In the competitive S-trimer-ACE2-binding experiments, synthetic C2 (DHAQRYGAGHSG) and C6 (HWKAVNWLKPWT) peptides inhibited S-trimer binding onto 293T-ACE2hR cells at high concentrations (50 mM) but not at lower concentrations (10 mM and below), neither for the settings of S-trimer binding onto recombinant ACE2 proteins. 3614 DHAQRYGAGHSG(10/25) HWKAVNWLKPWT(10/25) 2 Phage display (common panning) 3 PMID:36177466 Spike glycoprotein trimer, S-trimer Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA Optical absorbance was measured at 450 nm in a microplate reader (Synergy H1, BioTek Instruments, Inc.,Winooski, VT, United States). Data shown were preproduced from Figure 1. In the competitive S-trimer-ACE2-binding experiments, synthetic C2 (DHAQRYGAGHSG) and C6 (HWKAVNWLKPWT) peptides inhibited S-trimer binding onto 293T-ACE2hR cells at high concentrations (50 mM) but not at lower concentrations (10 mM and below), neither for the settings of S-trimer binding onto recombinant ACE2 proteins. 3615 LTRIKW(20%)[0.86] LTRMRY(20%)[0.46] FAPRWR(6.66%)[1.22] FMLRRL(6.66%)[1.13] MYIRPR(6.66%)[0.85] FARVRQ(6.66%)[0.71] LSRMPK(3.33%)[1.15] FSRHPR(3.33%)[1.23] MMRWKS(3.33%)[1.17] MILRKT(3.33%)[0.71] FTRLRA(3.33%)[0.97] LTLRRL(3.33%)[0.75] FGRVPR(3.33%)[0.56] FAFRPK(3.33%)[0.3] MSRMTR(3.33%)[0.56] LTWRTY(3.33%)[0.4] 16 Phage display (common panning) 3 PMID:35306102 Avian influenza virus (H9N2) particles Not determined. Not determined. Not determined. X6 fdMED1 phage display library ELISA Absorbance was measured at 450 nm after subtraction of background and shown. Affinity selection of H9N2-binding peptides was performed by direct biopanning the virus with a phage library displaying linear hexapeptides (Houimel et al., 1999). Purified H9N2 avian influenza virus (15µg/ml in PBS pH 7.4) was coated overnight at 4°C on a NUNC Maxisorbant microplate (NUNC, Danemark), then wells were washed three times with PBS pH 7.4 and saturated 2h at room temperature with 200  μl of PBS-BSA 1%. Phage-peptides (1012 tu/ml in 200  μl PBS-BSA 1%) were added to each well and then incubated for 2h at room temperature. Unbound phages were removed by washing the wells ten times with PBS containing 0.05% Tween-20 (PBST), ten further times with PBS alone, and specifically bound phage was eluted with 100 µl of 100  mM triethylamine, pH 12, immediately buffered with 1 ml of 1 M Tris-HCl, pH 7.4. Eluated phages were propagated in exponentially growing E. coli TG1 and grown overnight at 30°C selectively with 15 µg/ml tetracycline. The produced phage particles were precipitated from the bacterial supernatant with 20% PEG 6000/2.5 M NaCl on ice for 2 h and pelleted by centrifugation for 20 min at 4000 x g. The number of recovered phage was calculated for each of three rounds of panning with decreasing numbers of H9N2 virus after each round. Sixteen different phage-peptides were able to bind specifically the H9N2 virus, among them, 13 phage-peptides interacted with the hemagglutinin H9. Two selected peptides, P1 (LSRMPK) and P2 (FAPRWR) have shown antiviral activity in ovo and P1 was more protective in vivo then P2 when co-administered with the H9N2 virus. Mechanistically, these peptides prevent infection by inhibiting the attachment of the H9N2 virus to the cellular receptor. Molecular docking revealed that the peptides LSRMPK and FAPRWR bind to hemagglutinin protein H9, but interact differently with the receptor binding site (RBS). The present study demonstrated that the peptide P1 (LSRMPK) could be used as a new inhibitory molecule directed against the H9N2 virus. 3616 NQILSLLGI[no binding, no binding] ACHAWAPTR[5.14, 6] AKKIILRLS[no binding, no binding] 3 Phage display (common panning) 3-4 PMID:37191335 Tumor necrosis factor-α(TNF-α) Not determined. Not determined. Not determined. X9 phage display library The values of the dissociation constant KD (μM) were fitted using Nanotemper Analysis software v.2.3 and shown. A lead peptide, pep2 (ACHAWAPTR, KD = 5.14 μM), could directly bind to TNF-α and block TNF-α-triggered signaling activation. Peptide pep2 inhibits TNF-α-induced cytotoxicity and attenuates the inflammation by decreasing NF-κB and MAPK signaling activities in a variety of cells. Furthermore, pep2 attenuated colitis induced by dextran sodium sulfate in mice in both prophylactic and therapeutic settings. Moreover, pep2 reduced the phosphorylation of p38, ERK1/2, JNK1/2, p65, and IκBα in colonic tissues as well as downregulated inflammatory genes. And HIS3, TRP5, and ARG9 may be the key amino acids in pep2 to bind TNF-α by molecular docking. Collectively, targeting TNF-α with pep2 can attenuate the inflammation in vivo and vitro by inhibiting NF-κB and MAPK signaling pathways. 3617 TLWPFDLWLKTR(9/16)[0.2638±0.1027] SIWPFFNSFMTM(3/16)[0.2347±0.0682] TFWNWLPISRAI(2/16)[0.2767±0.0998] TFSWWPIPLPWG(1/16)[0.2593±0.058] TFLPFHWNLSWW(1/16)[0.2825±0.0521] 5 Phage display (subtractive panning) 4 PMID:35066280 Synthetic standard tyrosine-nitrated peptide (sTNP, sequence: H2N-GGGGY*GGG-COOH) Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The binding was quantified by OD450 measurement. OD450 was reproduced from Figure 2B and shown. The whole screening procedure is illustrated in Fig. 1. The phage-displayed 12-mer randomized peptide library was resuspended with TBS supplemented with 1% BSA (w/v) to make a 100 μL phage solution with a tilter of 1 × 1011 pfu/mL, which was incubated with the sepharose 4B immobilized non-nitrated peptides GGGGYGGG at 37 °C for 1 h. The supernatant was collected and further incubated with sepharose 4B immobilized standard TNP (sTNP, sequence: H2N-GGGGY*GGG-COOH) at 37 °C for 1 h. Then, the collected beads were washed five times with 1 mL TBST (TBS supplemented with 0.2% Tween-20) to clean the unbound phages and the bound phages were eluted with 1 mL elution buffer (0.2 M Gly-HCl supplemented with 1% BSA, pH = 2.2). The eluent was immediately neutralized by adding 0.15 mL neutralization buffer (1 M Tris-HCl, pH = 9.1). Collected phages were amplified and titer determination was performed following the manufacturer's instructions. A total of four rounds of the aforementioned “screening-amplification-titer determination-screening” cycle were conducted. Phages from the last biopanning were randomly selected and amplified for ELISA analysis to confirm their binding with the target peptide. Note a 10 or 100 folds decreased amount of target peptide was used for every next round of biopanning to increase the specificity of the screening. SPR analysis suggested that NT-1 (H2N-TLWPFDLWLKTR-COOH) should bind specifically to the nitrated tyrosine. Most importantly, immobilized NT-1 efficiently captured various types of endogenous TNPs in the presence of 100–1000 folds excessive amount of trypsinized BSA fragments. Compared with the commercially used anti-nitrotyrosine antibody, NT-1 enriched more TNPs with less sequence preference in the same experimental conditions. In summary, this study provided NT-1 as a useful start point to design and develop high-affinity binding ligand for TNPs, which could be useful in identifying nitrated tyrosine in proteins in physiological and pathophysiological conditions. 3618 GAQTCMN(15) 1 Phage display (common panning) 4 PMID:36353454 Mycolic acid Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) ELISA Absorbance was measured at 450 nm and reproduced from Figure 1D and shown. The efficacy of APTX4870 (GAQTCMN) against mycolic acid was demonstrated by evaluating clinical samples and conducting in vitro and Vivo. APTX4870 inhibited apoptosis, increased autophagy to decrease inflammation, and reduced Mycobacterium-derived mycolic acid (M.tb-MA)-induced lung damage. These findings suggest that this heptapeptide, which selectively targets M.tb-MA, might be exploited as a potential novel M.tb therapeutic treatment. 3619 CRGAMWMYKRC(5/6) CEGLAIQVKQC(1/6) 2 Phage display (competitive panning) 3 PMID:36070465 Hyaluronan-binding protein 2 Not determined. Not determined. Not determined. CX9C phage display library Tubes and streptavidin-coated Dynabeads (Thermo Fischer Scientific, Oslo, Norway) were blocked with PBS with 4% (w/v) skim milk powder. Approximately 1 × 10^13 virions of library and 1−10 μg (R1, 10 μg; R2, 3:1 μg) of biotinylated pro-FSAP were incubated with the appropriate amount of Dynabeads for 60 min at room temperature (RT). The beads/ protein/phage complexes were separated from the supernatant using a magnetic rack and washed with PBS-T20 (0.1% (v/v) Tween-20 in phosphate-buffered saline (PBS)). The complexes were then incubated with an excess amount of unbiotinylated pro-FSAP for 60 min at RT to remove phages bound with low-affinity and retain only high-affinity binding clones. The beads/protein/phage complexes were then separated from the supernatant containing the soluble FSAP using a magnet rack and washed with PBS-T20 (0.1% (v/v) Tween-20 in PBS) and with PBS pH 7.4. This was applied to both libraries and in all rounds. The remaining phages were then eluted from the beads with 500 μL of 100 mM triethylamine (pH 11) and neutralized in Tris−HCl pH 7.4. Using a phage display approach, we have identified a Cys-constrained 11 amino acid peptide (CRGAMWMYKRC), NNKC9/41, that activates pro-factor VII Activating protease (FSAP) in plasma. The synthetic linear peptide has a propensity to cyclize through the terminal Cys groups, of which the antiparallel cyclic dimer, but not the monocyclic peptide, is the active component. Other commonly found zymogens in the plasma, related to the hemostasis system, were not activated. Binding studies with FSAP domain deletion mutants indicate that the N-terminus of FSAP is the key interaction site of this peptide. In a monoclonal antibody screen, we identified MA-FSAP-38C7 that prevented the activation of pro-FSAP by the peptide. This antibody bound to the LESLDP sequence (amino acids 30−35) in an intrinsically disordered stretch in the N-terminus of FSAP. The plasma clotting time was shortened by NNKC9/41, and this was reversed by MA-FSAP-38C7, demonstrating the utility of this peptide. Peptide NNKC9/41 will be useful as a tool to delineate the molecular mechanism of activation of pro-FSAP, elucidate its biological role, and provide a starting point for the pharmacological manipulation of FSAP activity. 3620 IDCLMQNAGSA(10/11) DLPWSMPRPCR(1/11) 2 Phage display (competitive panning) 3 PMID:36070465 Hyaluronan-binding protein 2 Not determined. Not determined. Not determined. X11 phage display library Tubes and streptavidin-coated Dynabeads (Thermo Fischer Scientific, Oslo, Norway) were blocked with PBS with 4% (w/v) skim milk powder. Approximately 1 × 10^13 virions of library and 1−10 μg (R1, 10 μg; R2, 3:1 μg) of biotinylated pro-FSAP were incubated with the appropriate amount of Dynabeads for 60 min at room temperature (RT). The beads/ protein/phage complexes were separated from the supernatant using a magnetic rack and washed with PBS-T20 (0.1% (v/v) Tween-20 in phosphate-buffered saline (PBS)). The complexes were then incubated with an excess amount of unbiotinylated pro-FSAP for 60 min at RT to remove phages bound with low-affinity and retain only high-affinity binding clones. The beads/protein/phage complexes were then separated from the supernatant containing the soluble FSAP using a magnet rack and washed with PBS-T20 (0.1% (v/v) Tween-20 in PBS) and with PBS pH 7.4. This was applied to both libraries and in all rounds. The remaining phages were then eluted from the beads with 500 μL of 100 mM triethylamine (pH 11) and neutralized in Tris−HCl pH 7.4. 3621 IMPIQMRRMQML(24)[0.6163±0.0085] IKRIMRPIRQSI(2)[0.4193±0.0125] MRISRPIMRQIT(2)[0.4088±0.0146] RRSHSPMRMPRK(1)[0.4172±0.0105] 4 Phage display (common panning) 3 PMID:33410516 Anti-hemagglutinin (HA) monoclonal antibody PR8-23 Hemagglutinin, HA Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The absorbance at 450 nm was measured. Data shown were reproduced from Figure 3B. Biopanning was performed according to the instructions of the manufacturer of the 12-mer peptide phage display library (New England Biolabs), with modifications. Three rounds of biopanning were conducted for antibody PR8-23. The biopanning in the three rounds was identical, except for the increased concentration of Tween-20 (0.5% [vol/vol]) used in the buffer solution in the second and third rounds and the reduction of the concentration of antibody PR8-23 by half in the third round. Ninety-six-well plates were coated with antibody PR8-23 (100 μg/ml) overnight at 4°C. The next day, the plates were blocked with 5% bovine serum albumin (BSA; Sigma-Aldrich) for 1 h at 4°C and then subjected to six washes with buffer solution (50 mM Tris-HCl, 150 mM NaCl, pH 7.5; containing 0.1% [vol/vol] Tween-20). Then, peptide phage library was added (1.62 × 10^11 in 100 μl/well) and incubated at room temperature for 30 min, which was followed by 10 washes with the same buffer solution. Subsequently, the elution buffer (0.2M glycine-HCl, pH 2.2) was used to wash the bound phages at room temperature for 30 min, and then the solution was neutralized to pH 7.8 with Tris-HCl (1M, pH 9.1). Finally, the Escherichia coli strain ER2738 was used to amplify the bound phages, and polyethylene glycol/NaCl (20% [wt/vol] polyethylene glycol-8000, 2.5M NaCl) was used for extraction. Clone 3 showed the highest reactivity among the four phages compared with the control antibody in phage ELISA. And Clone 3 displayed peptides with IMPIQMRRMQML, which interacted with 63‐IAPLQLGKCNIA‐74 (63aa‐74aa) on the HA protein of the A/PuertoRico/8/34(H1N1). 3622 CNLNTIDTC(1/70)[253 ± 79] CNEWQLKSC(1/70)[85 ± 18] CAQRPMKRC(1/70)[NT] CMDVNTFSC(1/70)[NT] CHELSHESC(1/70)[NT] CNDTTFTMC(1/70)[NT] CSKLTKSKC(1/70)[NT] CYNAYMNTC(1/70)[NT] CRGVKTNRC(1/70)[NT] CLRNVTQKC(1/70)[NT] CKLMMPADC(1/70)[NT] CVNMGGIPC(1/70)[NT] CEEHCINVC(1/70)[NT] CSDMMPIDC(1/70)[NT] CIQKSITKC(1/70)[NT] CIAPRKSKC(1/70)[NT] CKLSKKTTC(1/70)[NT] 17 Phage display (subtractive panning) 3-5 PMID:33391537 CD44 variant 6 (CD44v6) overexpressing HEK 293 cells Not determined. Not determined. Not determined. Byungheon Lee (Department of Biochemistry and Cell Biology, School Medicine, Kyungpook National University, Republic of Korea) Surface plasmon resonance (SPR) The binding affinity (KD) of CD44v6-binding peptides to the CD44v6 protein was measured using an SPR instrument (Reichert Technologies, Depew, NY). KD values (nM) were calculated by analyzing the steady-state binding data by fitting the curve of binding level against concentration with a 1:1 binding model (GraphPad Prism 7.0 software). The T7 415-1b phage vector was purchased from Novagen (Madison, WI) and used to construct a phage library displaying CX7C (C, cysteine; X7, seven random amino acids in which hydrophobic amino acids were enriched to occur at a frequency of at least one per every seven residues). The library had a diversity of approximately 1 × 10^9 plaque-forming units (pfu)/mL. Phages (1 × 10^9 pfu) were then incubated with CD44v6 expression vector-transfected HEK 293 cells at 4 °C for 1 h. Phages bound to the cells were eluted by incubation with 500 µL of a suspension of BL21 host bacteria for 10 min at room temperature. To remove phages that had bound non-specifically to cells, the eluted phages were then incubated with non-transfected parental HEK 293T cells at 4 °C for 30 min. Unbound phages in the supernatant were collected, diluted in 10 mL of LB broth, and amplified by culture with BL21 host bacteria. The amplified phages were used in the next round of the screening cycle. A total of 60 phage clones were selected after the third, fourth, and fifth rounds of screening with the transfected cells, and 10 phage clones were selected after the fifth round of screening with non-transfected cells. DNA inserts in the selected phage clones were subjected to a sequencing analysis by Macrogen Inc. (Seoul, Korea). The corresponding amino acid sequences were aligned and analyzed using the Clustal W program to identify shared amino acid sequences or consensus motifs. CNLNTIDTC (NLN) and CNEWQLKSC (NEW) peptides bound preferentially to CD44v6-high cells than to CD44v6-low cells. The binding affinities of NLN and NEW to CD44v6 protein were 253 ± 79 and 85 ± 18 nM, respectively. Peptide binding to CD44v6-high cells was inhibited by the knockdown of CD44v6 gene expression and competition with an anti-CD44v6 antibody. A pull-down assay with biotin-labeled peptides enriched CD44v6 from cell lysates. NLN and NEW induced CD44v6 internalization and inhibited hepatocyte growth factor-induced c-Met internalization, c-Met and Erk phosphorylation, and cell migration and invasion. In mice harboring tumors, intravenously administered NLN and NEW homed to the tumors and inhibited metastasis to the lungs. When combined with crizotinib, a c-Met inhibitor, treatment with each peptide inhibited metastatic growth more efficiently than each peptide or crizotinib alone. In addition, KLAKLAKKLAKLAK pro-apoptotic peptide guided by NLN (NLN-KLA) or NEW (NEW-KLA) killed tumor cells and inhibited tumor growth and metastasis. No significant systemic side effects were observed after treatments. 3623 CWTMVPWGW(4) CSEWWNWWTC(4) CEWYFGWWDC(2) CGPIWGLAG(2) CWWHWNSLW(2) CVWSFWGMY(2) CEIWWGVWLC(2) CWMQWWSPWC(2) CSGWFWAPW(2) CDYWWSNWYC(1) CYWWLPWSAC(1) CWPIMYWWEC(1) CSIYWWEWWC(1) CTYWQYFVWC(1) CKLFFRWIDC(1) CFPFFSQLVC(1) CEWFFGPLVC(1) CAGFFGWVVC(1) CSDFWRWLWC(1) CVHSAFGPWC(1) CWGSPFGWWC(1) CRWAWRWMWC(1) CAWYWGWVEC(1) CVRTWISMVC(1) CEWSWVWDWC(1) CRGVIWSWVC(1) CRGFWPSWVC(1) CGMWWPWVVC(1) CRCFGLRFC(1) CPWCFFGGRC(1) CPWWLFGYPC(1) CAFWIGGLWC(1) CNHAWAAGSC(1) CAWWLTVGLC(1) CHAWWLGGDC(1) CHSWWLGMWC(1) CLWWWEMGGC(1) CSWPVLWWYC(1) CQWLLFSSGC(1) CIDGWFMSAC(1) CLVLWMSAGC(1) CGLFLWRHSC(1) CWKLWRGSRC(1) CVRATSLMGC(1) CVKGRSLFPC(1) CLGLSWFGNC(1) CLSGIAVFLC(1) CGVDVFLWGC(1) CSLLGDVFFC(1) CGWWQWPYLC(1) CHWSSLLRAC(1) CGVGIMWWL(1) CVYWPDWPW(1) CYFWPWEDE(1) CATWGWPWWC(1) CLWPWWFNDC(1) CVWLWWEPYC(1) CMWTWWGSFC(1) CFWSWWGADC(1) CMQQWMGFPC(1) CDLIWWWSDC(1) CGRLAGGLTC(1) CWAIGYGPWC(1) CMLGFLMPGC(1) CWMEWFRWDC(1) CWWWDAWFLC(1) CSGLYKWWLC(1) CLFATGWFLC(1) CAWPWAGWWC(1) CGWFWGGWWC(1) CWLAGWKVLC(1) CNYGWSILC(1) CRVWPGLLMC(1) CPGWLPRLC(1) CEWWYWALGC(1) 75 Phage display (subtractive panning) 4 PMID:34643765 Chemically synthesized crotamine, SCRO Not determined. Not determined. Not determined. CX7C and CX8C phage display library pool ELISA For peptide binding validation, SCRO-coated plates were blocked with 3% casein in PBS and then incubated with phage diluted in Tris-buffered saline (TBS). After washing, rabbit anti-Fd bacteriophage antibody (Sigma, St. Louis, MO, USA Cat# B7786) was added, followed by anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz, Dallas, TX, USA). The ELISA was developed with 3,3′,5,5′-tetramethylbenzidine.  The screen for crotamine-binding peptides was performed using a 50:50 mixture of phage cyclic peptide libraries CX7C and CX8C (C: cysteine; X: any amino acid residue) provided by Erkki Koivunen. Maxisorp plates (Nunc, Thermo, Rockford, IL, USA) coated with SCRO were exposed to the phage library that had previously been depleted of non-specific phages by exposure to bovine serum albumin (BSA)–treated plates. After incubation, plates were washed and selected phages were amplified through K91 E. coli infection and quantified by colony counting. The viruses purified from these bacteria were named “enriched round 1.” Phage panning was repeated with the enriched library for another three rounds. Round 4 bacterial colonies were used for sequencing. One of the peptides (CVWSFWGMYC), synthesized chemically, was shown to bind both synthetic and natural crotamine and to block crotamine-DNA binding. 3624 VLGREEWSTSYW[10.6105] LEKGNTLSTSTV[NT] NPIVRSAEDGQL[NT] NESGITRIALQD[NT] YDADMFYMKTNM[NT] 5 Phage display (common panning) 3 PMID:36456600 Extracellular domain of epidermal growth factor tyrosine kinase receptor mutation variant III (EGFRvIII) Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) Flow Cytometry Ph.D.™-12 Phage Display Peptide Library Kit was purchased from New England Biolabs Inc. (Beverly, MA, USA). Biopanning procedures were done according to the manufacturer’s instruction with certain modifications. Briefly, a 96-well plate was coated with 150 µL hEGFRvIII (Sino Biological #29662-H08B) (in the first two rounds at 100 μg/mL, the third round at 10 μg/mL) overnight at 4 °C. Wells were washed with PBS, blocked with blocking buffer (3% BSA in PBS), washed six times with cold PBST (PBS + 0.1% [v/v] Tween-20), then incubated with 1010 pfu phage peptide library Ph.D.™-12 for 2 h at RT. Unbound phages were removed by washing 10 times with cold PBST (PBS + 0.1% [v/v] Tween-20 in the first round and 0.5% [v/v] in the other two rounds). Bound phages were eluted with 100 μL of 0.2 M Glycine–HCl (pH 2.2), 1 mg/mL BSA and neutralized with 15 μL of 1 M Tris–HCl, pH 9.1. The elution procedure was repeated three times and the final eluate was used for amplification in Escherichia coli ER2738 culture. Recovered phages were subjected for two more rounds of biopanning with hEGFRvIII proteins (Sino Biological #29662-H08B). The eluates of each round were titrated by qPCR to enumerate phages and eluate from the third round of screening for NGS sequencing. Streptavidin was used as a biopanning control with the same condition as the target except for elution. The unbound phages were eluted with 100 μL of 0.1 M Biotin in PBS for 30 min. The enriched peptides were characterized and their binding capacity towards stable cell lines expressing cancer-specific epidermal growth factor tyrosine kinase receptor mutation variant III (EGFRvIII), EGFR wild type (EGFR WT), or a low endogenous level of EGFR WT was confirmed by flow cytometry analysis. The best peptide candidate, VLGREEWSTSYW, was synthesized, and its binding specificity towards EGFRvIII was validated in vitro. Additionally, computational docking analysis suggested that the identified peptide binds selectively to EGFRvIII. The novel VLGREEWSTSYW peptide is thus a promising EGFRvIII-targeting agent for future applications in cancer diagnosis and therapy. 3625 CAINSLSRKC(21%) CAKSMGDIVC(11%) CGRKQVESSC(10%) CRGKSAEGTC(5%) 4 Phage display (in vivo) 4 PMID:33870243 Integrin alpha-3 Not determined. Not determined. Not determined. fUSE5-based CX8C phage display library Phage was 10^9 TU of the phage library per mouse administered via the intratracheal route with 50 μL of PBS with a MicroSprayer® Aerosolizer coupled to a high-pressure syringe (Penn-Century) and a small animal laryngoscope (Penn-Century). The devices were used to administer air-free liquid aerosol directly into the trachea of animals deeply anesthetized with 1% isoflurane. The four rounds of selection were performed as described. In round one (R1), animals received 109 TU of the CX8C library via aerosol (see scheme in Figure 1B). After 60 min, phage particles were recovered from the bloodstream, amplified and pooled. In round two (R2), R1-pooled phage particles (i.e., inhaled phage sub-library 1) were administered and recovered 30 min post-administration. The subsequent R2-pooled phage particles (i.e., inhaled phage sub-library 2) were amplified for administration in round three (R3). After 10 min, the R3-pooled phage particles (i.e., inhaled phage sub-library 3) were recovered and processed for aerosol administration in the final round four (R4). After 5 min, phage particles were recovered from the bloodstream, amplified, and the corresponding peptide-encoding genomes were analyzed by DNA sequencing. We screened a phage display random peptide library in vivo to select, identify, and validate a ligand (CAKSMGDIVC) that specifically targets and is internalized through its receptor, α3β1 integrin, on the surface of cells lining the lung airways and alveoli and mediates CAKSMGDIVC-displaying phage binding and systemic delivery without compromising lung homeostasis. As a proof-of-concept, we show that the pulmonary delivery of targeted CAKSMGDIVC-displaying phage particles in mice and non-human primates elicit a systemic and specific humoral response.