A novel helper phage that improves phage display selection efficiency by preventing the amplification of phages without recombinant protein

Phage display is a widely used technology for the isolation of peptides and proteins with specific binding properties from large libraries of these molecules. A drawback of the common phagemid/helper phage systems is the high infective background of phages that do not display the protein of interest, but are propagated due to non-specific binding to selection targets. This and the enhanced growth rates of bacteria harboring aberrant phagemids not expressing recombinant proteins leads to a serious decrease in selection efficiency. Here we describe a VCSM13-derived helper phage that circumvents this problem, because it lacks the genetic information for the infectivity domains of phage coat protein pIII. Rescue of a library with this novel CT helper phage yields phages that are only infectious when they contain a phagemid-encoded pIII-fusion protein, since phages without a displayed protein carry truncated pIII only and are lost upon re-infection. Importantly, the CT helper phage can be produced in quantities similar to the VCSM13 helper phage. The superiority of CT over VCSM13 during selection was demonstrated by a higher percentage of positive clones isolated from an antibody library after two selection rounds on a complex cellular target. We conclude that the CT helper phage considerably improves the efficiency of selections using phagemid-based protein libraries.     

Multivalent scFv display of phagemid repertoires for the selection of carbohydrate-specific antibodies and its application to the Thomsen-Friedenreich antigen

The Thomsen-Friedenreich disaccharide (TF) is a promising target antigen for tumor immunotherapy, since it is almost exclusively expressed in carcinoma tissues. The TF-specific antibodies generated so far are IgMs of mouse origin with limited therapeutic potential. Phage-displayed scFv repertoires are an established source for recombinant antibodies; however, we were unable to identify scFvs binding to TF when applying libraries in the standard monovalent display format of phagemid systems. Here, we report on the successful selection of TF-specific antibody fragments using a multivalent scFv phagemid library format based on shortened linkers (one amino acid residue). The libraries were constructed from mice immunized with asialoglycophorin and selected using TF displayed on two different carrier molecules in combination with the proteolytically cleavable helper phage KM13. All isolated clones encoded the same framework genes and the same complementarity-determining regions. After affinity maturation only scFv with the founder sequence were selected from secondary repertoires. This indicates a very narrow sequence window for TF-specific antibodies. Investigating other linker-length formats revealed a clear inverse correlation between linker length and binding activity both as soluble proteins and displayed on phages. The highest affinity was obtained with the tetrameric format. The selected scFv was specific for TF on various carrier molecules and tumor cells and performed well in ELISA and immunohistochemistry. We postulate that scFv phagemid library formats with short linkers (i.e. multimeric scFvs) may, in general, be advantageous in selections for the generation of scFvs against carbohydrate epitopes or other epitopes associated with low intrinsic affinity per binding site), and expect that they will be superior in applications for diagnosis or therapy.     

Selection of human antibody fragments by phage display

Here, we describe a protocol for the selection of human antibody fragments using repertoires displayed on filamentous bacteriophage. Antigen-specific clones are enriched by binding to immobilized antigen, followed by elution and repropagation of phage. After multiple rounds of binding selection, specific clones are identified by ELISA. This article provides an overview of phage display and antibody technology, as well as detailed protocols for the immobilization of antigen, the selection of repertoires on purified or complex antigens and the identification of binders.     

Identification of natural ligands for SH2 domains from a phage display cDNA library

The cytoplasmic domain of the Fc gamma receptor IIB (FcgammaRIIB) can be successfully displayed on the surface of filamentous phage, and after phosphorylation in vitro, can interact specifically with the SH2 domains of SHP-2, a cytoplasmic tyrosine phosphatase. When full-length FcgammaRIIB is expressed on phage, however, this interaction is greatly compromised, illustrating that characteristics of the full-length sequence are not well tolerated by the phage display system. Many associations in cell physiology are driven by similar interactions involving small modular binding domains or ligands, and so a fragmented cDNA library will facilitate display of such domains free of sequences which compromise their expression. A fragmented leukocyte cDNA display library of 10(8) clones was constructed. This library was phosphorylated in vitro with fyn kinase and was selected against the tandem SH2 domains of SHP-2 in the search for additional ligands. A depletion strategy to remove non-specific clones was employed, using SHP-2 Sepharose, prior to in vitro phosphorylation and selection. This permitted the emergence of clones encoding the cytoplasmic domain of PECAM-1, another natural ligand for SHP-2. The importance of dual phosphorylation of tyrosine residues at positions 663 and 686 was confirmed in competition ELISA experiments using phosphorylated phage and synthetic peptides. Thus, phage display of fragmented cDNA libraries permits the identification and characterisation of phosphorylated ligands of modular binding domains based on their functional interaction.     

Cloning allergens via phage display

Although an impressive list of allergenic structures has been elucidated during the last decade by classical cloning methods, the size of the repertoire of molecular structures able to elicit allergic reactions is still unknown. Selective enrichment of cDNA libraries displayed on phage surface with serum IgE from allergic individuals combined with robotic-based high-throughput screening technology has proved to be extremely successful for the rapid isolation of allergens. The basic concept of linking the phenotype, expressed as gene product displayed on the phage coat, to its genetic information integrated into the phage genome, creates fusion proteins covalently associated with the infectious particle itself. Therefore, cDNA libraries displayed on phage surface can be screened for the presence of specific clones using the discriminative power of affinity purification. The selection of IgE-binding clones involves the enrichment of phage binding to serum IgE immobilised to a solid phase during consecutive rounds of affinity selection. As a consequence of the physical linkage between genotype and phenotype, sequencing of the DNA of the integrated section of the phage genome can readily elucidate the amino acid sequence of the surface-displayed allergen. In spite of some biological limitations imposed by Escherichia coli as expression host, phage surface display technology has strongly contributed to the rapid isolation of a vast variety of IgE-binding structures.     

Mimotopes and proteome analyses using human genomic and cDNA epitope phage display

In the post-genomic era, validation of candidate gene targets frequently requires proteinbased strategies. Phage display is a powerful tool to define protein-protein interactions by generating peptide binders against target antigens. Epitope phage display libraries have the potential to enrich coding exon sequences from human genomic loci. We evaluated genomic and cDNA phage display strategies to identify genes in the 5q31 Interleukin gene cluster and to enrich cell surface receptor tyrosine kinase genes from a breast cancer cDNA library. A genomic display library containing 2 x 106 clones with exon-sized inserts was selected with antibodies specific for human Interleukin-4 (IL-4) and Interleukin-13. The library was enriched significantly after two selection rounds and DNA sequencing revealed unique clones. One clone matched a cognate IL-4 epitope; however, the majority of clone insert sequences corresponded to E. coli genomic DNA. These bacterial sequences act as 'mimotopes' (mimetic sequences of the true epitope), correspond to open reading frames, generate displayed peptides, and compete for binding during phage selection. The specificity of these mimotopes for IL-4 was confirmed by competition ELISA. Other E. coli mimotopes were generated using additional antibodies. Mimotopes for a receptor tyrosine kinase gene were also selected using a breast cancer SKBR-3 cDNA phage display library, screened against an anti-erbB2 monoclonal antibody. Identification of mimotopes in genomic and cDNA phage libraries is essential for phage display-based protein validation assays and two-hybrid phage approaches that examine protein-protein interactions. The predominance of E. coli mimotopes suggests that the E. coli genome may be useful to generate peptide diversity biased towards protein coding sequences.ABBREVIATIONS USED: IL, interleukin; ELISA, enzyme linked immunoabsorbant assay; PBS, phospho-buffered saline; cfu, colony forming units.     

An efficient method for variable region assembly in the construction of scFv phage display libraries using independent strand amplification

Phage display library technology is a common method to produce human antibodies. In this technique, the immunoglobulin variable regions are displayed in a bacteriophage in a way that each filamentous virus displays the product of a single antibody gene on its surface. From the collection of different phages, it is possible to isolate the virus that recognizes specific targets. The most common form in which to display antibody variable regions in the phage is the single chain variable fragment format (scFv), which requires assembly of the heavy and light immunoglobulin variable regions in a single gene.

In this work, we describe a simple and efficient method for the assembly of immunoglobulin heavy and light chain variable regions in a scFv format. This procedure involves a two-step reaction: (1) DNA amplification to produce the single strand form of the heavy or light chain gene required for the fusion; and (2) mixture of both single strand products followed by an assembly reaction to construct a complete scFv gene. Using this method, we produced 6-fold more scFv encoding DNA than the commonly used splicing by overlap extension PCR (SOE-PCR) approach. The scFv gene produced by this method also proved to be efficient in generating a diverse scFv phage display library. From this scFv library, we obtained phages that bound several non-related antigens, including recombinant proteins and rotavirus particles.     

An efficient method for variable region assembly in the construction of scFv phage display libraries using independent strand amplification

Phage display library technology is a common method to produce human antibodies. In this technique, the immunoglobulin variable regions are displayed in a bacteriophage in a way that each filamentous virus displays the product of a single antibody gene on its surface. From the collection of different phages, it is possible to isolate the virus that recognizes specific targets. The most common form in which to display antibody variable regions in the phage is the single chain variable fragment format (scFv), which requires assembly of the heavy and light immunoglobulin variable regions in a single gene. In this work, we describe a simple and efficient method for the assembly of immunoglobulin heavy and light chain variable regions in a scFv format. This procedure involves a two-step reaction: (1) DNA amplification to produce the single strand form of the heavy or light chain gene required for the fusion; and (2) mixture of both single strand products followed by an assembly reaction to construct a complete scFv gene. Using this method, we produced 6-fold more scFv encoding DNA than the commonly used splicing by overlap extension PCR (SOE-PCR) approach. The scFv gene produced by this method also proved to be efficient in generating a diverse scFv phage display library. From this scFv library, we obtained phages that bound several non-related antigens, including recombinant proteins and rotavirus particles.     

An anti-leukemic single chain Fv antibody selected from a synthetic human phage antibody library

The display of human antibody repertoire on the cell surface of the filamentous bacteriophage has offered a novel strategy for selecting antibodies to a diverse range of purified targets. However, the selection of antibodies with biological functions has not yet been fully investigated. To select phage antibodies with therapeutic potential, a synthetic human single chain Fv (scFv) phage antibody library was panned on whole premyelocytic leukemia cell line (HL60). Phages binding to common receptors and undesirable phages were subtracted by incubating the library with human glioma cells. High affinity binding phages to HL60 cells were enriched by fluorescence-activated cell sorting. After the 6th round of selection, 50% of the selected phage antibodies showed significant binding to HL60 cells, whereas none of the analyzed phage antibodies bound to human pre-B cells (Nalm-6). In addition to binding, one scFv antibody inhibited HL60 cell proliferation by 90% compared to irrelevant scFv antibodies. Taken together the data demonstrate that specific scFv antibodies with biological functions can be isolated by using whole cells as affinity matrix.     

Biotin-tagged cDNA expression libraries displayed on lambda phage: a new tool for the selection of natural protein ligands

cDNA expression libraries displayed on lambda phage have been successfully employed to identify partners involved in antibody–antigen, protein– protein and DNA–protein interactions and represent a novel approach to functional genomics. However, as in all other cDNA expression libraries based on fusion to a carrier polypeptide, a major issue of this system is the absence of control over the translation frame of the cDNA. As a consequence, a large number of clones will contain lambda D/cDNA fusions, resulting in the foreign sequence being translated on alternative reading frames. Thus, many phage will not display natural proteins, but could be selected, as they mimic the binding properties of the real ligand, and will hence interfere with the selection outcome. Here we describe a novel lambda vector for display of exogenous peptides at the C-terminus of the capsid D protein. In this vector, translation of fusion peptides in the correct reading frame allows efficient in vivo biotinylation of the chimeric phage during amplification. Using this vector system we constructed three libraries from human hepatoma cells, mouse hepatocytic MMH cells and from human brain. Clones containing open reading frames (ORFs) were rapidly selected by streptavidin affinity chromatography, leading to biological repertoires highly enriched in natural polypeptides. We compared the selection outcome of two independent experiments performed using an anti-GAP-43 monoclonal antibody on the human brain cDNA library before and after ORF enrichment. A significant increase in the efficiency of identification of natural target peptides with very little background of false-positive clones was observed in the latter case.     

Highly efficient selection of phage antibodies mediated by display of antigen as Lpp-OmpA' fusions on live bacteria

Delayed infectivity panning (DIP) is a novel approach for the in vivo isolation of interacting protein pairs. DIP combines phage display and cell surface display of polypeptides as follows: an antigen is displayed in many copies on the surface of F(+) Escherichia coli cells by fusing it to a Lpp-OmpA' hybrid. To prevent premature, non-specific infection by phage, the cells are rendered functionally F(-) by growth at 16 degrees C. The antigen-displaying cells are used to capture antibody-displaying phage by virtue of the antibody-antigen interaction. Following removal of unbound phage, infection of the cells by bound phage is initiated by raising the temperature to 37 degrees C that facilitates F pilus expression. The phage then dissociate from the antigen and infect the bacteria through the F pilus. Using specific scFv antibodies and the human ErbB2 proto-oncogene and IL2-Ralpha chain as model antibody-antigen pairs, we demonstrate enrichment of those phage that display a specific antibody over phage that display an irrelevant antibody of over 1,000,000 in a single DIP cycle. We further show the successful isolation of anti-toxin, anti-receptor, anti-enzyme and anti-peptide antibodies from several immune phage libraries, a shuffled library and a large synthetic human library. The effectiveness of DIP makes it suitable for the isolation of rare clones present in large libraries.Since DIP can be applied for most of the phage libraries already existing, it could be a powerful tool for the rapid isolation and characterization of binders in numerous protein-protein interactions.     

Expression cloning of cDNA by phage display selection.

Expression cloning of a mouse kappa chain fragment has been achieved from a cDNA library by display of expressed proteins on filamentous phage and affinity selection for binding to anti-mouse Fab antibodies. Expressed proteins were anchored to the phage coat by a synthetic, anti-parallel leucine zipper, which had been selected from a semi-randomized zipper library for the ability to connect a test protein to phage. From a library of 4 x 10(6) transformants, two separate clones displaying different size cDNA inserts were recovered after four selection rounds. These results further demonstrate the utility of phage display for cDNA expression cloning.     

Corruption of phage display libraries by target-unrelated clones: Diagnosis and countermeasures

Phage display is used to discover peptides or proteins with a desired target property—most often, affinity for a target selector molecule. Libraries of phage clones displaying diverse surface peptides are subject to a selection process designed to enrich for the target behavior and subsequently propagated to restore phage numbers. A recurrent problem is enrichment of clones, called target-unrelated phages or peptides (TUPs), that lack the target behavior. Many TUPs are propagation related; they have mutations conferring a growth advantage and are enriched during the propagations accompanying selection. Unlike other filamentous phage libraries, fd-tet-based libraries are relatively resistant to propagation-related TUP corruption. Their minus-strand origin is disrupted by a large cassette that simultaneously confers resistance to tetracycline and imposes a rate-limiting growth defect that cannot be bypassed with simple mutations. Nonetheless, a new type of propagation-related TUP emerged in the output of in vivo selections from an fd-tet library. The founding clone had a complex rearrangement that restored the minus-strand origin while retaining tetracycline resistance. The rearrangement involved two recombination events, one with a contaminant having a wild-type minus-strand origin. The founder’s infectivity advantage spread by simple recombination to clones displaying different peptides. We propose measures for minimizing TUP corruption.     

Selection of tumor-specific internalizing human antibodies from phage libraries

Antibody internalization into the cell is required for many targeted therapeutics, such as immunotoxins, immunoliposomes, antibody-drug conjugates and for targeted delivery of genes or viral DNA into cells. To generate directly tumor-specific internalizing antibodies, a non-immune single chain Fv (scFv) phage antibody library was selected on the breast tumor cell line SKBR3. Internalized phage were recovered from within the cell and used for the next round of selection. After three rounds of selection, 40 % of clones analyzed bound SKBR3 and other tumor cells but did not bind normal human cells. Of the internalizing scFv identified, two (F5 and C1) were identified as binding to ErbB2, and one (H7) to the transferrin receptor. Both F5 and H7 scFv were efficiently endocytosed into SKBR3 cells, both as phage antibodies and as native monomeric scFv. Both antibodies were able to induce additional functional effects besides triggering endocytosis: F5 scFv induces downstream signaling through the ErbB2 receptor and H7 prevents transferrin binding to the transferrin receptor and inhibits cell growth. The results demonstrate the feasibility of selecting internalizing receptor-specific antibodies directly from phage libraries by panning on cells. Such antibodies can be used to target a variety of molecules into the cell to achieve a therapeutic effect. Furthermore, in some instances endocytosis serves as a surrogate marker for other therapeutic biologic effects, such as growth inhibition. Thus, a subset of selected antibodies will have a direct therapeutic effect.     

Ribosome display and selection of human anti-cD22 scFvs derived from an acute lymphocytic leukemia patient

Novel in vitro methods for the display of antibody libraries against disease-related antigens have led to the development of powerful protein-based biotherapeutics. Eukaryotic ternary ribosome complexes can be used to display human single chain antibodies (scFvs) to isolate specific binding reagents to these antigens. Here, we present the isolation of human scFv against the immunotherapeutic target antigen CD22 from a patient-derived human scFv library using ribosome display technology. The ribosome complexes were enriched against the extra-cellular domain of human CD22 conjugated to magnetic beads. Isolated constructs were further affinity-matured and specific binding activity was demonstrated by surface plasmon resonance and validated using in vitro cell assays. The isolated human anti-CD22 scFvs can provide a basis for the development of new immunotherapeutic strategies in CD22-expressing malignant diseases.     

Surface display of antibodies

To screen antibody libraries that contain many millions of different clones, a selection system is required with an efficiency comparable to that of the immune system. This can be achieved by displaying antibodies on the surface of microorganisms containing the antibody's gene, analogous to the expression of the IgM antigen receptor on the surface of unactivated B-lymphocytes. Specific clones can then be selected using immobilized antigens. The minor coat protein of filamentous phages, pIII, which initiates the infection of E.coli by binding to their F-pili, and the major coat protein, pVIII, have been used as carriers for displaying antibodies on the phage surface. Recombinant antibodies have also been targeted to the cell surface of bacteria by fusing them with outer membrane components derived from lipoproteins, OmpA and an IgA protease. However, only the pIII system has been routinely used for screening antibody libraries. Here we describe the various antibody surface display systems and the screening of antibody libraries generated from the gene repertoire of lymphocytes and by gene synthesis. Finally, we have made a short comparison of the bacterial production of Fabs versus single chain antibodies (scFv).     

A practical kinetic model for efficient isolation of useful antibodies from phage display libraries

To isolate phages displaying a practical and useful antibody with a high k_on value and/or a low k_off value from phage display antibody libraries, we developed a rational strategy based on a kinetic model. In the model, the recovery of a phage displaying an antibody after a round of biopanning is expressed as a function of five parameters, the apparent association rate constant of the phage antibody to the immobilized antigen (k_on′), the apparent dissociation rate constant of the phage antibody from the immobilized antigen (k_off′), the effective antigen concentration (C), the time for the binding process (t_b) and the time for the washing process (t_w). An optimum set of operating parameters (C, t_b and t_w) for isolating phages displaying an antibody with a high k_on value was designed based on the model. Three rounds of biopanning were carried out under the designed conditions, against a phage library in which the hypervariable regions of an original antibody were randomized. All isolated phages displayed an antibody with a higher k_on value and one displayed an antibody with a 30-fold greater k_on value than that of the original antibody. Experimental conditions which improve the efficiency of conventional off-rate selections are also described.     

Construction of high-complexity combinatorial phage display peptide libraries

Random peptide libraries displayed on the surface of filamentous bacteriophage are widely used as tools for the discovery of ligands for biologically relevant macromolecules, including antibodies, enzymes, and cell surface receptors. Phage display results in linkage of an affinity-selectable function (the displayed peptide) to the DNA encoding that function, allowing selection of individual binding clones by iterative cycles of in vitro panning and in vivo amplification. Critical to the success of a panning experiment is the complexity of the library: the greater the diversity of clones within the library, the more likely the library contains sequences that will bind a given target with useful affinity. A method for construction of high-complexity (> or = 10(9) independent clones) random peptide libraries is presented. The key steps are highly efficient binary ligation under conditions where the vector is relatively dilute, with only a modest molar excess of insert, followed by efficient electrotransformation into Escherichia coli. Library design strategies and a protocol for rapid sequence characterization are also presented.     

噬菌体展示技术筛选bFGF模拟短肽

目的：通过噬菌体展示技术筛选得到与FGFR结合的bFGF模拟短肽,为bFGF肽类抑制剂的研发提供实验基础。方法：以Balb/c 3T3细胞为靶标,以COS-7细胞作消减,对噬菌体随机七肽库进行4轮生物淘洗,再采用ELISA检测单克隆噬菌体对Balb/c 3T3亲和性和特异性,选取阳性克隆进行DNA测序分析。结果：从富集的噬菌体中获得12个阳性克隆,获得一组疏水性七肽及共同基序PR。结论：利用肽类新药开发的重要工具--噬菌体展示技术,得到2段bFGF的受体结合模拟肽,可望作为bFGF抑制剂的先导肽。     

Isolation of anti-MISIIR scFv molecules from a phage display library by cell sorter biopanning

While cell surface antigens represent the most common targets for antibody-based cancer therapy, isolation of new antibodies specific for these targets from single-chain Fv phage display libraries has been hindered by limitations associated with traditional selection techniques. Solid phase panning is often associated with conformational changes to the target protein due to its immobilization on plastic tubes that can limit the ability of the isolated scFv to bind to conformational epitopes and solution panning methods require the use of secondary tags that often mask desired sequences and create unintended epitopes. Commonly utilized cell-based panning methods typically yield a panel of single-chain Fv (scFv) molecules that are specific for numerous cell surface antigens, often obscuring the desired clones. Here, we describe a novel cell sorter-based system to isolate single-chain Fv molecules specific for defined antigen targets expressed on stably-transformed mammalian cells. We employed these methods to isolate promising scFv clones that bind specifically to the Müllerian inhibiting substance type II receptor, a cell surface ovarian cancer antigen that has proven to be a difficult target for selection strategies.