Tumor is a serious threat to human health. At present, surgical resection, chemoradiotherapy, targeted therapy and immunotherapy are the main therapeutic strategies. Monoclonal antibody has gradually become an indispensable drug type in the clinical treatment of cancer due to its high efficiency and low toxicity. Phage antibody library technology (PALT) is a novel monoclonal antibody preparation technique. The recombinant immunoglobulin variable region of heavy chain (VH)/variable region of light chain (VL) gene is integrated into the phage vector, and the antibody is expressed on the phage surface in the form of fusion protein to obtain a diverse antibody library. Through the process of adsorption-elution-amplification, the antibody library can be screened to obtain the antibody molecule with specific binding antigen as well as its gene sequence. PALT has the advantages of short antibody production cycle, strong plasticity of antibody structure, large antibody yield, high diversity and direct production of humanized antibodies. It has been used in screening tumor markers and preparation of antibody drugs for breast cancer, gastric cancer, lung cancer and liver cancer. This article reviews the recent progress and the application of PALT in tumor therapy.
Humans ; Bacteriophages/genetics* ; Immunoglobulin Variable Region/genetics* ; Gene Library ; Antibodies, Monoclonal/therapeutic use* ; Immunotherapy ; Peptide Library

Autoantigens ; blood ; Gene Library ; Hepatitis, Autoimmune ; genetics ; immunology ; Hepatocytes ; immunology ; Humans ; Peptide Library ; Polymerase Chain Reaction ; methods

OBJECTIVESTo construct and screen a primarily phage display library of HCV C and E1 genes evolved with an artificial pattern.

METHODSTwo genes of about 1 kb with different genotypes were evolved by DNA shuffling. The re-assembled HCV C and E1 genes were cloned into a phage vector. After being rescued with helper phage M13KO7, a phage display library was constructed. Then the library was screened with anti-C and E1 McAb. Double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was carried out on twenty individual phage clones selected randomly to detect their binding and reactive activity with high-titer HCV-positive sera. Normal sera were used as controls.

RESULTSThe phage display library of HCV C and E1 genes which evolved with an artificial pattern was constructed. Their capacity amounted to 1.64 x 10(6), and 86 percent of the clones contained C and E1 genes. After four rounds of panning, the phage library was specifically enriched. Twelve positive clones were successfully screened.

CONCLUSIONThe capacity and diversity of the constructed library are enough for screening. The results demonstrate the superiority of the specific binding and reactive activity and affinity of the 12 phage clones from the HCV positive sera.

DNA, Viral ; genetics ; Gene Library ; Hepacivirus ; genetics ; Peptide Library ; Viral Core Proteins ; genetics ; Viral Envelope Proteins ; genetics

The application of in vitro selection method to isolate nucleic acids, peptides and proteins according to their functions has been studied intensively in recent years. In vitro display technologies are not limited by cellular transformation efficiencies; thus, very large libraries of up to 10(13)-10(14) members can be built. The most popular in vitro display technologies are ribosome display and mRNA display; ribosome display achieves the mRNA-ribosome-nascent peptide complexes by stalling the translating ribosome in an in vitro translation reaction. In mRNA display, the mRNA-protein complex is achieved by binding the two macromolecules through a small adaptor molecule, typically puromycin; these mRNA-peptide fusions can then be purified and subjected to in vitro selection. In vitro display technologies provide a different approach to the in vitro selection and directed evolution of peptides and proteins. This review focuses on the principle and method of ribosome display and mRNA display technologies, and discusses their applications.
Animals ; Directed Molecular Evolution ; Gene Library ; Humans ; Peptide Library ; Protein Interaction Mapping ; methods ; RNA, Messenger ; chemistry ; genetics ; Ribosomes ; chemistry ; genetics

Antibodies are immunoglobulins specifically introduced by immunity response of high animals, with the responsibility for recognising and cleaning out specific antigens. Antibody is not only a powerful weapon against pathogen invasion in the organism, but also a tool for specific molecular recognition used in basic scientific research. The diversity of antibody molecules resulted in the concept of antibody library; each individual animal is a natural antibody library. In the post-genome era, in order to fit various "omics", especially for proteomics requirement of high throughput technology, some gene engineering antibody libraries and antibody alternative libraries have been constructed based on phage display technology. Yet, more and more in vitro display systems such as ribosome display, mRNA display have been used for antibody library study, and that present more advantages than phage display. This mini review outlines the genesis, development and application prospect of antibody libraries according to the published reviews and research articles, and offers up to date development and application prospect of antibody library technology.
Animals ; Antibodies ; genetics ; physiology ; Antibody Diversity ; genetics ; Antibody Specificity ; Combinatorial Chemistry Techniques ; Gene Library ; Humans ; Peptide Library

OBJECTIVETo screen and identify proteins that interact with hepatitis C virus NS3 by means of T7-phage display system.

METHODSHepatitis C virus NS3 was expressed by prokaryotic expression and used as a selected molecule to biopan the T7 select human liver cDNA library; the selected positive clones were identified using DNA sequencing and analyzed with BLAST program in GenBank.

RESULTSAfter BLAST analysis in all the positive clones, the proteins which interacted with the hepatitis C virus NS3 were found to be serpin peptidase inhibitor, clade A, member 1 (SERPINA1) and cyclophilin-LC.

CONCLUSIONT7-phage display system is a convenient, rapid and effective method for screening interacting proteins. The proteins thus selected will provide an important means for studying the pathogenesis and carcinogenesis of HCV.

Cell Line ; Gene Library ; Hepacivirus ; metabolism ; Humans ; Peptide Library ; Protein Interaction Mapping ; methods ; Viral Fusion Proteins ; genetics ; isolation & purification ; metabolism ; Viral Nonstructural Proteins ; genetics ; metabolism

OBJECTIVESTo screen the hALR-interacting protein by phage-displayed technique and identify its biological activities.

METHODSThe specific phage clones that interacted with target protein hALR from a cDNA library of hepatocarcinoma cells were selected using the T7 phage-displayed technique. The acquired cDNA inserts were sequenced and analyzed by bioinformatic tools. The biological activities of the phage-displayed peptide affecting QGY hepatocarcinoma cells were studied using 3H-TdR method.

RESULTSThe cDNA inserts with 212 bp were acquired after 4 rounds of biopanning. They showed 100% homology with citron kinase. The phage-displayed peptide and the peptide combined with hALR affected QGY cells proliferation.

CONCLUSIONShALR-interacting peptide can be specifically screened by phage-displayed technique. Citron kinase that interacted with hALR potentially plays an important role in the proliferation of hepatocarcinoma cells.

Carcinoma, Hepatocellular ; metabolism ; pathology ; Gene Library ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Liver Regeneration ; Peptide Library ; Peptides ; metabolism ; Protein Binding ; Proteins ; genetics ; isolation & purification ; metabolism ; Tumor Cells, Cultured

OBJECTIVETo construct full-length human bladder cancer-specific antibody libraries for efficient display of full-length antibodies on the surface of mammalian cells.

METHODSThe total RNA was isolated from peripheral blood mononuclear cells from patients with bladder cancer. The repertoires of IgG1 heavy chain variable region (VH) and Kappa light chain were amplified by RT-PCR using specific primers. The antibody genes were inserted into the vector pDGB-HC-TM to construct the bladder-cancer-specific antibody libraries of heavy chains and light chains. Ten clones from each library were randomly picked for gene sequencing and transient transfection into FCHO cells to analyze antibody display on mammalian cell surface by flow cytometry after staining with corresponding fluorescent labeled antibodies.

RESULTSThe libraries of bladder-cancer-specific antibody heavy chain (IgG1) and light chain (LCk) were successfully constructed. Seven out of the 10 clones randomly selected from the heavy chain library and 9 out of the 10 clones from the light chain library showed correct open reading frame, coding for 7 unique VH and 9 unique LCk. The combinatory library size reached 3.32×10(11).

CONCLUSIONWe have successfully constructed a full-length human bladder-cancer-specific antibody library with a combinatory diversity of 3.32×10(11) based on mammalian display technology, which can be used for screening monoclonal antibodies against bladder-cancer-associated antigens.

Amino Acid Sequence ; Animals ; Antibodies ; genetics ; Cell Surface Display Techniques ; Gene Library ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin kappa-Chains ; genetics ; Peptide Library ; Urinary Bladder Neoplasms ; genetics ; immunology

BACKGROUND: Hepatitis C virus(HCV), a family of Flaviviridae, has a host cell-derived envelope containing a positive-stranded RNA genome, and has been known as the maj or etiological agent for chronic hepatitis, hepatic cirrhosis, and hepatocellular carcinoma. There remains a need to dissect a molecular mechanism of pathogenesis for the development of therapeutic and effective preventive measure for HCV. Identification of cellular receptor is of central importance not only to understand the viral pathogenesis, but also to exploit strategies for prevention of HCV. This study was aimed at identifying peptide mimotopes inhibiting the binding of E2 protein of HCV to MOLT-4 cell . METHODS: In this study, phage peptide library displaying a random peptides consisting of 7 or 12 random peptides was employed in order to pan against E2 protein. Free HCV particles were separated from the immune complex forms by immunoprecipitation using anti-human IgG antibody, and used for HCV-capture ELISA. To identify the peptides inhibiting E2-binding to MOLT-4 cells, E2 protein was subj ect to bind to MOLT-4 cells under the competition with phage peptides. RESULTS: Several phage peptides were selected for their specific binding to E2 protein, which showed the conserved sequence of SHFWRAP from 3 different peptide sequences. They were also able to recognize the HCV particles in the sera of HCV patient s captured by monoclonal antibody against E2 protein. Two of them, showing peptide sequence of HLGPWMSHWFQR and WAPPLERSSLFY respectively, were revealed to inhibit the binding of E2 protein to MOLT-4 cell efficiently in dose dependent mode. However, few membrane-associated receptor candidates were seen using Fasta3 programe for homology search with these peptides. CONCLUSION: Phage peptides containing HLGPWMSHWFQR and WAPPLERSSLFY respectively, showed the inhibition of E2-binding to MOLT-4 cells. However, they did not reveal any homologues to cellular receptors from GenBank database. In further study, cellular receptor could be identified through the screening of cDNA library from MOLT-4 or hepatocytes using antibodies against these peptide mimotopes.
Antibodies ; Antigen-Antibody Complex ; Bacteriophages ; Carcinoma, Hepatocellular ; Conserved Sequence ; Databases, Nucleic Acid ; Enzyme-Linked Immunosorbent Assay ; Flaviviridae ; Gene Library ; Genome ; Hepacivirus* ; Hepatitis C* ; Hepatitis* ; Hepatitis, Chronic ; Hepatocytes ; Humans ; Immunoglobulin G ; Immunoprecipitation ; Liver Cirrhosis ; Mass Screening ; Peptide Library* ; Peptides ; RNA

OBJECTIVETo screen anti-c-Met Fab from a phage antibody library and identify its binding activity.

METHODSThe expression of c-Met of HCC lines was identified by Western blot and immunofluorescence. Antibodies against c-Met were screened with immobilized antigen. After five rounds of panning, 30 randomly selected clones were identified by phage ELISA to select specific clones with high affinity. The positive clones were selected for Fab soluble expression in TOP10F and the binding activities were analysed in HCC lines.

RESULTSc-Met expressed in HCC membrane was confirmed by Western blot and immunofluorescence. A Fab fragment named AM2-26 with fine activity to c-Met was selected. AM2-26 binding specificity was confirmed by IP, FACS and immunofluorescence.

CONCLUSIONThe anti-c-Met Fab binding to c-Met in HCC provides a promising candidate for the biotherapy of hepatoma.

Antibodies ; immunology ; isolation & purification ; Cell Line, Tumor ; Cloning, Molecular ; Gene Library ; Humans ; Immunoglobulin Fab Fragments ; immunology ; Immunoglobulin Variable Region ; immunology ; Peptide Library ; Proto-Oncogene Proteins c-met ; immunology ; Recombinant Fusion Proteins ; immunology