Generation and selection of immunized Fab phage display library against human B cell lymphoma

The approval of using monoclonal antibodies as a targeted therapy in the management of patients with B cell lymphoma has led to new treatment options for this group of patients. Production ofmonoclonal antibodies by the traditional hybridoma technology is costly, and the resulting murine antibodies often have the disadvantage of triggering human anti-mouse antibody （HAMA） response. Therefore recombinant Fab antibodies generated by the phage display technology can be a suitable alternative in managing B cell lymphoma. In this study, we extracted total RNA from spleen cells of BALB/c mice immunized with human B lymphoma cells, and used RT-PCR to amplify cDNAs coding for the κ light chains and Fd fragments of heavy chains. After appropriate restriction digests, these cDNA fragments were successively inserted into the phagemid vector pComb3H-SS to construct an immunized Fab phage display library. The diversity of the constructed library was approximately 1.94× 10^7. Following five rounds of biopanning, soluble Fab antibodies were produced from positive clones identified by ELISA. From eight positive clones, FabC06, FabC21, FabC43 and FabC59 were selected for sequence analysis. At the level of amino acid sequences, the variable heavy domains （VH） and variable light domains （VL） were found to share 88-92% and 89-94% homology with sequences coded by the corresponding murine germline genes respectively. Furthermore, reactivity with membrane proteins of the B cell lymphoma was demonstrated by immunohistochemistry and western blotting. These immunized Fab antibodies may provide a valuable tool for further study of B cell lymphoma and could also contribute to the improvement of disease therapy.

Construction of Human ScFv Phage Display Library against Ovarian Tumor

In order to construct a single chain fragment variable （ScFv） phage display library against ovarian tumor, by using RT-PCR, the human heavy chain variable region genes （VH） and light chain variable region genes （VL） were amplified from lymphocytes of ovarian tumor patients and subsequently assembled into ScFv genes by SOE. The resulting ScFv genes were electrotransformed into E. coli TG1 and amplified with the co-infection of helper phage M13KO7 to obtain phage display library. The capacity and titer of the resulting library were detected. The phage antibody library with a capacity of approximately 3 × 10^9 cfu/μg was obtained. After amplification with helper phage, the titer of antibody library reached 5 μ 10^12 cfu/mL. Human ScFv library against ovarian tumor was constructed successfully, which laid a foundation for the screening of ovarian tumor specific ScFv for the radioimmunoimaging diagnosis of ovarian tumor.

Construction of white spot syndrome virus (WSSV) whole genome phage display library

A rebuilt vector pCANTAB 5 EE was obtained by inserting a 34 bp double-stranded oligonucleotide which contained a EcoRV recognition sequence into pCANTAB 5 E. White spot syndrome virus （WSSV） genome DNA was fragmented by sonication to isolate fragments mainly in the range of 0.8 ～2.0 kb, then the fragments were blunt-ended with T4 DNA polymerase and cloned into the EcoRV site of pCANTAB 5 EE. The primary recombinant clone of the library was 3.0 × 10^5.Colony PCR of random selected recombinants showed that the size of the inserts was 0.12 ～ 1.77 kb. After the whole library recombinant phages infected Escherichia coli HB2151 cells, the extracellular and periplasmic extracts were dropped on PVDF membranes to perform dot blot, using polyclonal mouse anti-VP24 serum,anti-WSV026 serum,anti-WSV063 serum,anti-WSV069 serum,anti-WSV112 serum, anti WSV238 serum,anti-WSV303 serum and anti-VP26 serum as the primary antibody, respectively. The results showed that the display library could express the viral proteins.

Identification and Characterization of Peptides Binding AgEG1 from a Phage Display Library

Endoglucanases are the main cellulolytic enzymes digestion as well as its good kinetic properties make it an attractive of Anoplophora glabripennis. Their high activities in cellulose target for development of cellulase inhibitors. In this study, random pepfide phage display technology was employed to identify peptides that bound the AgEG1, a member of endoglucanase isozymes. Phage clones with peptide LPPNPTK and XPP （X is residue T, L, A or H） motif frequently occurred in the selected phage population and showed a higher phage recovery than other clones. Peptide LPPNPTK was chemically synthesized and characterized tor its binding activities to AgEG1. The synthetic peptide exhibited high specificity for AgEG1. The peptide LPPNPTK has the potential to be developed into inhibitors of the endoglucanase of A. glabripennis.

Screening and Identification of a Novel Hepatocellular Carcinoma Cell Binding Peptide by Using a Phage Display Library

The purpose of this study was to screen peptides that can specifically bind to human hepatocellular carcinoma （hHCC） cells using phage display of random peptide library in order to develope a peptide-based carrier for the diagnosis or therapy of hHCC. A peptide 12-mer phage display library was employed and 4 rounds of subtractive panning were performed using the hHCC cell line HepG2 as the target. After panning, the phages that specifically bound to and internalized in hHCC cells were selected. The selected phages demonstrated highly specific affinity to HepG2 cells analyzed by ELISA and immunofluorescence analysis. 57.3% of the selected phage clones displayed repeated sequence FLLEPHLMDTSM, and 4 amino acid residues, FLEP were extremely conservative. Based on the sequencing results, a 16-mer peptide （WH-16） was synthesized. The competitive EL1SA showed that the binding of the phage clones displayed sequence FLLEPHLMDTSM to HepG2 cells was efficiently inhibited by WH-16. Our findings indicate that cellular binding of phage is mediated via its displayed peptide and the synthesized 16-mer peptide may have the potential to be a delivery carrier in target diagnosis or therapy for hHCC.

Identification and epitope mapping of anti-p72 single-chain antibody against African swine fever virus based on phage display antibody library

African swine fever virus(ASFV)is a lethal pathogen that causes severe threats to the global swine industry and it has already had catastrophic socio-economic effects.To date,no licensed prophylactic vaccine exists.Limited knowledge exists about the major immunogens of ASFV and the epitope mapping of the key antigens.As such,there is a considerable requirement to understand the functional monoclonal antibodies(mAbs)and the epitope mapping may be of utmost importance in our understanding of immune responses and designing improved vaccines,therapeutics,and diagnostics.In this study,we generated an ASFV antibody phage-display library from ASFV convalescent swine PBMCs,further screened a specific ASFV major capsid protein(p72)single-chain antibody and fused with an IgG Fc fragment(scFv-83-Fc),which is a specific recognition antibody against ASFV Pig/HLJ/2018 strain.Using the scFv-83-Fc mAb,we selected a conserved epitope peptide(221MTGYKH226)of p72 retrieved from a phage-displayed random peptide library.Moreover,flow cytometry and cell uptake experiments demonstrated that the epitope peptide can significantly promote BMDCs maturation in vitro and could be effectively uptaken by DCs,which indicated its potential application in vaccine and diagnostic reagent development.Overall,this study provided a valuable platform for identifying targets for ASFV vaccine development,as well as to facilitate the optimization design of subunit vaccine and diagnostic reagents.

PSMA mimotope isolated from phage displayed peptidelibrary can induce PSMA specific immune response

Prostate-specific membrane antigen (PSMA) is a cellsurface glycoprotein expressed predominantly in prostatesecretory acinar epithelium and prostate cancer cells aswell as in several extraprostatic tissues. Mouse monoclonal antibody 4G5 specific to the extracellular domainof PSMA was used to screen two phage displayed peptide libraries (9aa linear and 9aa cys library). Three 4G5reactive phagotopes were identified. Sequence analysis ofisolated clones demonstrated that the interaction motif'VDPA/SK' has high homology to 719-725aa on PSMA.Immunohistochemical staming of the prostate cancer sam ple with the PSMA-mimic phagotope (mimotope) immunized serum antibodies demonstrate that the mimotopeisolated from the phage displayed peptide libraries can induce PSMA specific immune response in vivo.

Rapid Selection of Phage Se-scFv with GPX Activity via Combination of Phage Display Antibody Library with Chemical Modification

Glutathione peroxidase（GPX） plays an important role in scavenging reactive oxygen species. A series of catalytic antibodies with GPX activity have been generated by the authors of＇ this study. To obtain humanized catalytic antibodies, the phage-displayed human antibody library was used to select novel antibodies by repetitive screening, Phage antibodies, scFv-B8 and scFv-H6 with the GSH-binding site, were obtained from the library by enzyme-linked immu- nosorbent assay（ELISA） analysis with 4 rounds of scelection against their respective haptens, S-2,4-dinitriphenyl t-butyl ester（GStI-s-DNP-Bu） and S-2,4-dinit,-iphenyl t-hexyl ester（GSH-s-I）NP-He）. Nevertheless, several studies need to be condueted to determine whether scFv-B8 and seFv-tI6 possess GPX activity. 1＇o enhance the speed of the selection, selenocysteine（Sec, the catalytic group of GPX） was incorporated directly into the phages, scFv-B8 and seFv-H6, by chemical mutation to form the phages Se-scFv-B8 and Se-scFv-H6. The GPX activities were found to be 3012 units/μmol and 2102 units/μmol, respectively. To improve the GPX activity of the phage Se-scFv-B8, DNA shuffling was used to construct a secondary library and another positive phage antibody scFv-B9 was screened out by another panning against GSH-s-DNP-Bu. When Sec was incorporated via chemical mutation into the phage antibody scFv-B9, its GPX activity reached 3560 units/μmol, which is 1.17-fold higher than the phage antibody Se-scFv-B8 and almost approached the order of magnitude of native GPX. The rapid selection is the prerequisite for generating humanized Se-seFv with GPX activity.

Screening and Identification of a Targeting Peptide to nGLP-1R from Phage Display Peptide Library

In order to provide the structure information for designing new exendin-4 analogues, a phage display peptide library was screened by targeting the N-terminal extracellular domain of GLP-1R（nGLP-1R）. After four rounds of selection, nine sequences were obtained, four of them have higher affinity for nGLP-1R than the others. We chose two of them named X and Y peptides. Islet β-cell proliferation assay suggested that X and Y peptides didn＇t have any activity to increase islet β-cell proliferation. In other words, X and Y peptides were not agonists to GLP-1R. However, the conservative motifs of X and Y peptides provided us useful information to design new exendin-4 analogues.

Construction and diversity analysis of a murine IgEphage surface display library

To make further investigation of the IgE antibodyrepertoire in Trichosanthin (TCS) allergic responses, amurine IgE phage surface display library was constructed(3.0×105 independent clones). We first constructed theVe cDNA library (4.6×105 independent clones) and VκcDNA library (3.0×105 independent clones). Then, theVε and Vκ gene segments were amplified from both libraries by PCR respectively, and assembled into Fab fragment by SOE PCR. The phage library containing Fabs wasthus constructed. The diversity of Vε from this library wasanalyzed and proved. Fab clones with high specificity toTCS have been screened out.

MAPPING EPITOPES OF HUMAN PAPILLOMAVIRUS TYPE 16 L1 PROTEIN WITH A PHAGE DISPLAY EPITOPE LIBRARY

The objective of this study is to map epitopes on HPMAPV16 L1 protein and provide information to the design of HPV16 prophylactic peptide vaccine. The epitopes on L1 protein were screenedby polyclonal and two monoclonal antibodies (BS and F4G3) against RPV16 L1 protin from a 6-merfd phage display epitope library with the method or immuuo-afrinity screening (Biopauuing). Aferthree rounds or Bio-Panning, the Positive phages were detected by L1 antibodies again with ELISA.The positive phages reacted strongly with L1 antibodies were then identified by DNA sequencing.Three mimotopes have been screened by polycloual and two monoclonal antibodies. The mimotope(LSLFSC) reacted with mouoclonal antibody B8 showed 50% pomology with the sequence 270275a. a (DSLFFY) of prototype HPV16 L1. Another mimotope (LTSSYS) reacted with polyclonalantibodies had 66% pomology with the L1 sequence 516~521a. A(TTSSTS), also a mimotope (DRWDRF) was found had the bomologic RF with the known L1 sequence 441 ~446a. a. The mimotopesLSLFSC and DRWDRF were adjacent to the epitopes at 267~269a. a and 422~441 a. a reported byother researchers Previously. Our results suggest that there might be a batch of epitopes on HPV16L1 ppotein, and the predominant epitopes of HPV16 L1 protein are located in the above two domains. These results will be helpful for design or HPV16 prophylactic peatide vaccines and HPVpolyvalent vaccines.

Structural analysis and molecular modeling of twoantitrichosanthin IgE clones from phage antibody library

Recently we constructed a murine IgE phage surfacedisplay library and screened out two IgE (Fab) cloneswith specific binding activity to Trichosanthin (TCS). Inthis work, the Vε and Vκ genes of the two clones weresequenced and their putative germline gene usages werestudied. On the basis of the known 3D structure of Trichosanthin and antibody, molecular modeling was carriedout to study the antigen-antibody interaction. The possible antigenic determinant sites on the surface of TCSrecognized by both the clones were analyzed, and the reaction forces between TCS and two Fab fragments werealso analyzed respectively.

Selection of the specific coalescent peptide of human CD59 by phage display techniques

To screen and identify the short peptides with specific binding activity to human CD59 and to design the short-peptide clamp against tumor escape, the phage display peptide library containing 12 peptides was used to select the highly expressed specific coalescent peptide of human CD59 in CHO cells. Positive phage clones obtained after 5 rounds of biopanning and detected with ELISA were obtained, in which 8 of them with high binding activity to human CD59 were sequenced. The 3 sequences thus obtained showed high homology with each and certain homology with sequence with human CD2 （PubMed 339HGAAENSLSPSS）, and all contained primary structure , of which this sequence may be the mimic confonnational epitope binding to human CD59. These results in the present study may be helpful to design the short-peptide clamp against the active sites of CD59 on tumor escape.

Transforming growth factor-β1 phage model peptides isolated from a phage display 7-mer peptide library can inhibit the activity of keloid fibroblasts

Background Transforming growth factor-β1 （TGF-β1） is known to have a role in keloid formation through the activation of fibroblasts and the acceleration of collagen deposition. The objective of this current study was to isolate TGF-β1 phage model peptides from a phage display 7-mer peptide library to evaluate their therapeutic effect on inhibiting the activity of keloid fibroblasts.Methods A phage display 7-mer peptide library was screened using monoclonal anti-human TGF-β1 as the target to obtain specific phages containing ectogenous model peptides similar to TGF-β1. Enzyme-linked immunosorbent assay （ELISA） was performed to select monoclonal phages with good binding activity, which underwent DNA sequencing. MTT assay and apoptosis assessment were used to evaluate the biological effects of the phage model peptides on keloid fibroblasts. Immunofluorescence assay was employed to show the binding affinity of the model peptides on phages causing keloid fibroblasts. Quantitative real-time PCR analysis was carried out to detect the expressions of nuclear factor κB （NF-κB） mRNA, connective tissue growth factor （CTGF） mRNA and TGF-β receptor Ⅱ （TβRII） mRNA in keloid fibroblasts.Results Specific phages with good results of ELISA were beneficiated. Four phage model peptides were obtained. The data of MTT showed that TGF-β1 and one phage model peptide （No. 4） could promote keloid fibroblasts proliferation,however, three phage model peptides （No. 1-3） could inhibit keloid fibroblasts proliferation. The results of apoptosis assessment showed that the three phage model peptides could slightly induce the apoptosis in keloid fibroblasts. The data of immunofluorescence assay revealed that the model peptides on phages rather than phages could bind to keloid fibroblasts. The findings of quantitative real-time PCR analysis suggested that the expressions of NF-κB mRNA and CTGF mRNA in the three phage model peptide groups decreased, while the expression of TβRII mRNA slightly increased.Conclusions Three phage model peptides isolated from a phage display 7-mer peptide library can inhibit keloid fibroblasts proliferation and induce the apoptosis in keloid fibroblasts. They can inhibit the activity of keloid fibroblasts by blocking TGF-β1 binding to its receptor and then regulating the expressions of NF-κB, CTGF and TβRII.

Schistosoma japonicum: construction of phage display antibody library and its application in the immunodiagnosis of infection

Background A monoclonal antibody would be an effective tool for the detection of circulating antigens in the serum of patients with schistosomiasis, but the traditional way of producing monoclonal antibodies is not cost-effective. The objective of this study was to find a new method for the large-scale production of monoclonal antibodies against Schistosoma japonicum (Sj).Methods A phage display antibody library for Sj was constructed. To obtain a single-chain variable fragment antibody (scFv) against Sj, the library was screened with metabolic antigens from adult Sj worms (Sj-MAg) using enzyme-linked immunosorbent assay. The soluble scFvs select ed were used to detect Sj antigens in the serum of acute and chronic schistosomiasis patients.Results Six positive clones with good reactivity to Sj-MAg were obtained from the phage display antibody library of about 1.07×10 6 individual clones. Only two of these six clones bound specifically to Sj-MAg and were chosen for further analysis. Specific soluble anti-Sj-MAg scFvs were produced by inducing the 2 clones with isopropyl-D-thiogalactopyranoside. The characteristics of the scFvs were then determined. The results of Western blot showed that these scFvs could bind to Sj-MAg specifically an d had a molecular weight of about 31 kD. When testing serum from schistosomiasis patients with one of the two specific scFvs, its sensitivity was found to be 60% and 37% in acute and chronic patients, respectively, with a specificity of 90%. When the two specific scFvs were combined, their sensitivity was found to be 75% and 57% in acute and chronic patients, respectively, with a specificity of 85%.Conclusions The results indicate that the scFvs are potentially useful for the diagnosis of schistosomiasis. The library construct ion also provides a useful tool for the further screening of other antibodies for both diagnostic and immunotherapeutic applications and for epitope analysis and vaccine design.

Screening of Peptide Inhibitors of TACE from a Phage Display Random 15-Peptide Library by Recombinant TACE Ectodomain

Tumor necrosis factor(TNF)-α-converting enzyme(TACE)is the major protease responsible for processing pro-TNF-αfrom membrane-anchored precursors to secreted TNF-α.In the present study,a 15-peptide library was used to identify potential TACE antagonists.To obtain the recombinant TACE ectodomain and to use it as a selective molecule for the screening of peptide inhibitors of TACE,cDNA coding for the catalytic domain(T800)and full-length ectodomain(T1300)of TACE were amplified by reverse transcription–polymerase chain reaction.The expression plasmid were constructed by inserting T800/T1300 into plasmid pET-28a/c respectively and were transformed into Escherichia coli BL21(DE3).Sodium dodecyl sulfate–polyacrylamide gel electrophoresis(SDSPAGE)andWestern blot analysis revealed that T800/T1300 were highly expressed in the form of an inclusion body induced by isopropylthiogalactoside.After Ni2+–NTA resin affinity chromatography,the purity of the recombinant T800/T1300 protein was more than 90%.T800 and T1300 proteins were used in the screening of T800/T1300-binding peptides from a phage display random 15-peptide library.After four rounds of biopanning,the positive phage clones were analyzed by enzyme-linked immunosorbent assay,competitive inhibition assay(ELESA),and DNA sequencing.A common amino acid sequence(TRWLVYFS RPYLVAT)was confirmed and synthesized.A synthetic peptide was shown to bind to TACE and to inhibit TNF-αrelease from lipopolysaccharide(LPS)-stimulated human peripheral blood mononuclear cells(PBMC)by up to 60.3%.Fluorescence-activated cell sorter(FACS)analysis revealed that the peptide mediated the accumulation of TNF-αon an LPS-stimulated PBMC surface.These results demonstrate that the TACE-binding peptide is an effective antagonist of TACE and that the deduced motif might be applied to the molecular design of anti-inflammatory drugs.

A resistin binding peptide selected by phage display inhibits 3T3-L1 preadipocyte differentiation

Background Resistin, a newly discovered cysteine-rich hormone secreted mainly by adipose tissues, has been proposed to form a biochemical link between obesity and type 2 diabetes. However, the resistin receptor has not yet been identified. This study aimed to identify resistin binding proteins/receptor. Methods Three cDNA fragments with the same 11 bp 5＇ sequence were found by screening a cDNA phage display library of rat multiple tissues. As the reading frames of the same 11 bp 5＇ sequence were interrupted by a TGA stop codon, plaque lift assay was consequently used to prove the readthrough phenomenon. The stop codon in the same 11 bp 5＇ sequence was replaced by tryptophan, and the binding activity of the coded peptide [AWIL, which was designated as resistin binding peptide （RBP）] with resistin was identified by the confocal microscopy technique and the affinity chromatography experiment, pDual GC-resistin and pDual GC-resistin binding peptide were co-transfected into 3T3-L1 cells to confirm the function of resistin binding peptide. Results Three cDNA fragments with the same 11 bp 5＇ sequence were found. The TGA stop codon in reading frames of the same 11 bp 5＇ sequence was proved to be readthroughed. The binding activity of RBP with resistin was consequently identified. The expression of the resistin binding peptide in 3T3-L1 preadipocytes expressing pDual G-C-resistin significantly inhibited the adipogenic differentiation. Conclusion RBP could effectively rescue the promoted differentiation of resistin overxepressed 3T3-L1 preadipocyte.

Keratinocyte growth factor phage model peptides can promote epidermal cell proliferation without tumorigenic effect

Background Keratinocyte growth factor （KGF） significantly influences epithelial wound healing. The aim of this study was to isolate KGF phage model peptides from a phage display 7-mer peptide library to evaluate their effect on promoting epidermal cell proliferation. Methods A phage display 7-mer peptide library was screened using monoclonal anti-human KGF antibody as the target. Enzyme linked immunosorbent assay （ELISA） was performed to select monoclonal phages with good binding activity. DNA sequencing was done to find the similarities of model peptides. Three-（4,5-dimethylthiazol-2-yl） -2,5-diphenyl tetrazolium bromide （MTT） assay, immunofluorescence assay and quantitative real-time PCR analysis were employed to evaluate the effect of the phage model peptides on epidermal cells. Results Thirty-three out of fifty-eight （56.9%） of the isolated monoclonal phages exhibited high binding activity by ELISA. Ten of fifteen obtained phage model peptides were similar to KGF or epidermal growth factor （EGF）. MTT assay data showed that four （No. 1-4） of the ten phage model peptides could promote epidermal cell proliferation. The expression of keratinocyte growth factor receptor （KGFR） mRNA in the KGF control group and the two phage model peptide groups （No. 1 and No. 2） increased. Expression of c-Fos mRNA and c-Jun mRNA in the KGF control group increased, but did not increase in the four phage model peptide groups （No.1-4）. Conclusion Four phage model peptides isolated from the phage display 7-mer peptide library can safely promote epidermal cell proliferation without tumorigenic effect.

Identification and Characterization of Peptides Mimicking the Epitopes of Metalloprotease of Schistosoma Japonicum

In an attempt to isolate and characterize peptides mimicking epitopes of metalloprotease and explore their immunological protection against Schistosoma japonicum （S. japonicum）, polyclonal anti-metalloprotease sera was prepared to screen a 12-mer random peptide library to isolate phages binding specially to antisera IgG. Then, phage ELISA, animal immunization, DNA sequencing, Western blotting and enzymatic activity neutralizing analysis were used to characterize the selected phage clones. All of ten randomly picked clones were shown to be positive. Five peptides of different amino acid sequences deduced from DNA sequences were obtained and two of them （peptides 2 and 3） could induce significant reduction （31.0% and 31.8%, respectively） in worm burden and high reduction （52.6% and 54.9%, respectively） in liver eggs per gram （LEPG）, while, unexpectedly, others （peptides 1, 4 and 5） could not elicit enough protection against infection of S. japonicum. Peptides 2 and 3 could be recognized by S. japonicum infected mouse sera （IMS） and could elicit neutralizing Abs. The results show that peptides 2 and 3 are antigenic and immunogenic. They are true mimics of epitopes of metalloprotease and useful as novel vaccine candidates against S.japonicum. Cellular ＆ Molecular Immunology. 2005;2（3）：219-223.