Phage versus phagemid libraries for generation of human monoclonal antibodies

Non-immune (na?ve) phage antibody libraries have become an important source of antibodies for reagent, diagnostic, and therapeutic use. To date, reported na?ve libraries have been constructed in phagemid vectors as fusions to pIII, yielding primarily single copy (monovalent) display of antibody fragments. For this work, we subcloned the single chain Fv (scFv) gene repertoire from a na?ve phagemid antibody library into a true phage vector to create a multivalently displayed scFv phage library. Compared to monovalently displayed scFv, multivalent phage display resulted in improved efficiency of display as well as antibody selection. A greater number of antibodies were obtained and at earlier rounds of selection. Such increased efficiency allows the screening for binding antibodies after a single round of selection, greatly facilitating automation. Expression levels of antigen-binding scFv were also higher than from the phagemid library. In contrast, the affinities of scFv from the phage library were lower than from the phagemid library. This could be overcome by utilizing the scFv in a multivalent format, by affinity maturation, or by converting the library to monovalent display after the first round of selection.     

Applying phage antibodies to proteomics: selecting single chain Fv antibodies to antigens blotted on nitrocellulose

Two-dimensional gel electrophoresis is a powerful tool for identification of proteins that differ between patients with qualitatively or quantitatively different disease states. Further characterization of these protein differences would be greatly facilitated by the availability of antibodies that could be used to detect and quantitate the temporo-spatial pattern and cellular and tissue location of the different proteins. To generate such antibodies, methods were developed which permit the successful selection of monoclonal phage antibodies from phage display libraries against antigens blotted from SDS-PAGE gels onto nitrocellulose. First, it was determined that nitrocellulose and PVDF membranes gave significantly lower levels of background phage binding than two other membranes studied. Next, it was determined that blocking with fish gelatin and binding in the presence of 0.5 M NaCl could reduce nonspecific binding 10,000-fold and result in enrichment ratios greater than 500-fold with antigen concentrations as low as 1 ng/mm(2). When optimized conditions were applied to phage antibody libraries, panels of monoclonal phage antibodies were generated against the proteins ErbB2 and bovine serum albumin electroblotted from SDS-PAGE gels onto nitrocellulose. Antibodies were obtained with as little as 10 to 1 ng of antigen, depending on whether the libraries displayed single or multiple copies of antibody per phage. The antibodies worked as reagents in both ELISA and Western blotting.     

Phage Peptide Libraries

Filamentous phage particles have been central in the construction of libraries displaying vast numbers of random peptides. These random peptides can be antigenically presented as fusions to coat proteins III and VIII of the phage. The isolation of ligate-reactive phage from an immense background of nonspecific phage is achieved by the biopanning process. Enrichment of reactive phage relative to unreactive phage occurs with alternate rounds of affinity selection to the desired molecular target and amplification of the specifically bound phage. This allows the isolation of rare binding species contained in the phage peptide libraries. Each phage particle contains the information in its genome pertaining to the type of random peptide insert displayed. Hence, the identification of binding motifs displayed on ligate-reactive phage is revealed by sequencing the relevant insert site in the phage genome. Phage peptide libraries have been used to isolate ligands to an array of protein ligates. The libraries have proved particularly effective in defining the binding sites of monoclonal antibodies and to some extent polyclonal sera. The analysis of the peptide insert sequences of a number of different clones of antibody binding phage can reveal a great deal about the nature and restriction of the amino acid residues critical for the antibody–antigen interaction.     

Preparation of functional single-chain antibodies against bioactive gibberellins by utilizing randomly mutagenized phage-display libraries

Screening randomly mutagenized proteins displayed on a phage surface by biopanning is a powerful strategy to obtain evolved clones with improved properties such as higher stability and functionality. We utilized this method to overcome the problem that functional single-chain antibodies against active gibberellins, a class of plant hormones, can not be prepared by some of the conventional methods. Single-chain antibody libraries with random mutations were constructed from two independent anti-bioactive gibberellin monoclonal antibody lines in a phagemid vector, so that the mutagenized scFvs were expressed in a phage-displayed form upon helper phage infection. From both libraries, scFv clones with binding activity to GA(4) were successfully obtained by successive rounds of biopanning against BSA-GA(4), the original immunogen. The results are highly suggestive that this approach might be a general solution when a single-chain antibody does not show binding activity. We found further that a ribosomal frameshift to complement a nonsense mutation frequently occurred in an amber suppressor strain of E. coli TG1, resulting in the display of a functional antibody, while such a nonsense mutant failed to produce a soluble antibody in a non-amber suppressor strain. This result explains at least partly why single-chain antibodies are sometimes functional only in a phage-displayed form, not in a soluble form.     

工程抗体酶的研究进展

本文评述了工程抗体酶的研究现状和抗体酶在防化医学研究中的应用。抗体库技术的出现使抗体酶的研究呈现了新的生机。不仅使不具备细胞工程实验条件的实验室能够制备抗体酶,而且实验周期大大缩短,比杂交瘤技术提供多20倍的结合性抗体作为潜在的抗体酶,使抗体酶更易获得。抗体库技术能够容易地评价编码抗体酶的基因,因而可以在分子水平上认识和改造抗体酶。噬菌体抗体库可以不经免疫即可制备抗体的特点,使通过人源性噬菌体抗体库生产的抗体酶能够直接用于临床治疗。随着工程抗体酶研究的不断深入,新颖实用的抗体酶将会被应用于包括军事医学在内的多种领域中。     

Function blocking antibodies to neuropilin-1 generated from a designed human synthetic antibody phage library

Non-immune (na?ve) antibody phage libraries have become an important source of human antibodies. The synthetic phage antibody library described here utilizes a single human framework with a template containing human consensus complementarity-determining regions (CDRs). Diversity of the libraries was introduced at select CDR positions using tailored degenerate and trinucleotide codons that mimic natural human antibodies. Neuropilin-1 (NRP1), a cell-surface receptor for both vascular endothelial growth factor (VEGF) and class 3 semaphorins, is expressed on endothelial cells and neurons. NRP1 is required for vascular development and is expressed widely in the developing vasculature. To investigate the possibility of function blocking antibodies to NRP1 as potential therapeutics, and study the consequence of targeting NRP1 in murine tumor models, panels of antibodies that cross-react with human and murine NRP1 were generated from a designed antibody phage library. Antibody (YW64.3) binds to the CUB domains (a1a2) of NRP1 and completely blocks Sema3A induced neuron collapse; antibody (YW107.4.87) binds to the coagulation factor V/VIII domains (b1b2) of NRP1 and blocks VEGF binding and VEGF induced cell migration. YW107.4.87 inhibits tumor growth in animal xenograft models. These antibodies have provided valuable tools to study the roles of NRP1 in vascular and tumor biology.     

Surface presentation of protein epitopes using bacteriophage expression systems.

Vast libraries of filamentous phage expression vectors that display foreign (poly)peptides on the virion surface can be screened by affinity-purifying those phage whose displayed foreign peptide binds to an antibody or another binding protein. Present libraries display only short random peptides, but work is presently underway to create libraries displaying antibodies with a great diversity of binding specificities.     

A Sortase A Programmable Phage Display Format for Improved Panning of Fab Antibody Libraries

Phage display of combinatorial antibody libraries is a versatile tool in the field of antibody engineering, with diverse applications including monoclonal antibody (mAb) discovery, affinity maturation, and humanization. To improve the selection efficiency of antibody libraries, we developed a new phagemid display system that addresses the complication of bald phage propagation. The phagemid facilitates the biotinylation of fragment of antigen binding (Fab) antibody fragments displayed on phage via Sortase A catalysis and the subsequent enrichment of Fab-displaying phage during selections. In multiple contexts, this selection approach improved the enrichment of target-reactive mAbs by depleting background phage. Panels of cancer cell line-reactive mAbs with high diversity and specificity were isolated from a naïve chimeric rabbit/human Fab library using this approach, highlighting its potential to accelerate antibody engineering efforts and to empower concerted antibody drug and target discovery.     

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.     

Effects of oxidoreduction potential combined with acetic acid,NaCl and temperature on the growth,acidification, and membrane properties of Lactobacillus plantarum

Antibody phage display libraries (Griffin and Tomlinson I) displaying antibody genes and maintained and amplified in Escherichia coli were used to isolate antibodies to the hapten target microcystin LR (1000 Da) conjugated to either bovine serum albumin or keyhole limpet haemocyanin. In competition enzyme-linked immunosorbent assay, bacterially expressed antibodies selected via the Griffin library showed at least 300 times greater sensitivity than those isolated from the Tomlinson library, for free microcystin. Bacterially expressed phage antibody libraries provide a rapid and relatively easy route for the selection of monoclonal antibodies specific for even the most difficult of antigenic targets such as free haptens.     

The isolation of super-sensitive anti-hapten antibodies from combinatorial antibody libraries derived from sheep

The complexity and expense of producing anti-hapten monoclonals via the traditional hybridoma route and the preferential selection of antibodies that recognise the conjugated form of the hapten, over antibodies that specifically recognise free hapten, are two of the more important problems that have limited the development and application of anti-hapten antibodies. The advent of phage display technology allows the rapid isolation of monoclonal antibody fragments from libraries of different antibodies (>10⁸) displayed on the surface of filamentous bacteriophages. Much of the power of this new approach lies in the flexibility with which these libraries can be screened for suitable binders. Using an optimised selection procedure, we have isolated from a sheep antibody phage display library, super-sensitive anti-hapten antibodies specific for the herbicide and environmental pollutant, atrazine. In particular, two phage clones have been isolated that can be expressed cheaply and in quantity in Escherichia coli, demonstrate excellent stability in nonphysiological conditions and are exciting prospects for immunoassay applications including ELISA, dip-stick formats, on-line monitoring and biosensor technologies. In ELISA formats they show low levels of cross reactivity with related molecules and a limit of detection of a 1–2 parts per trillion (p.p.t.), well within the 100 p.p.t. required by EC legislation.     

De Novo Selection of High-Affinity Antibodies from Synthetic Fab Libraries Displayed on Phage as pIX Fusion Proteins

Filamentous phage was the first display platform employed to isolate antibodies in vitro and is still the most broadly used. The success of phage display is due to its robustness, ease of use, and comprehensive technology development, as well as a broad range of selection methods developed during the last two decades. We report here the first combinatorial synthetic Fab libraries displayed on pIX, a fusion partner different from the widely used pIII. The libraries were constructed on four V_L and three V_H domains encoded by IGV and IGJ germ-line genes frequently used in human antibodies, which were diversified to mirror the variability observed in the germ-line genes and antibodies isolated from natural sources. Two sets of libraries were built, one with diversity focused on V_H by keeping V_L in the germ-line gene configuration and the other with diversity in both V domains. After selection on a diverse panel of proteins, numerous specific Fabs with affinities ranging from 0.2 nM to 20 nM were isolated. V_H diversity was sufficient for isolating Fabs to most antigens, whereas variability in V_L was required for isolation of antibodies to some targets. After the application of an integrated maturation process consisting of reshuffling V_L diversity, the affinity of selected antibodies was improved up to 100-fold to the low picomolar range, suitable for in vivo studies. The results demonstrate the feasibility of displaying complex Fab libraries as pIX fusion proteins for antibody discovery and optimization and lay the foundation for studies on the structure-function relationships of antibodies.     

The human combinatorial antibody library HuCAL GOLD combines diversification of all six CDRs according to the natural immune system with a novel display method for efficient selection of high-affinity antibodies

This article describes the generation of the Human Combinatorial Antibody Library HuCAL GOLD. HuCAL GOLD is a synthetic human Fab library based on the HuCAL concept with all six complementarity-determining regions (CDRs) diversified according to the sequence and length variability of naturally rearranged human antibodies. The human antibody repertoire was analyzed in-depth, and individual CDR libraries were designed and generated for each CDR and each antibody family. Trinucleotide mixtures were used to synthesize the CDR libraries in order to ensure a high quality within HuCAL GOLD, and a β-lactamase selection system was employed to eliminate frame-shifted clones after successive cloning of the CDR libraries. With these methods, a large, high-quality library with more than 10 billion functional Fab fragments was achieved. By using CysDisplay, the antibody fragments are displayed on the tip of the phage via a disulfide bridge between the phage coat protein pIII and the heavy chain of the antibody fragment. Efficient elution of specific phages is possible by adding reducing agents. HuCAL GOLD was challenged with a variety of different antigens and proved to be a reliable source of high-affinity human antibodies with best affinities in the picomolar range, thus functioning as an excellent source of antibodies for research, diagnostic, and therapeutic applications. Furthermore, the data presented in this article demonstrate that CysDisplay is a robust and broadly applicable display technology even for high-throughput applications.     

Effective use of nitrocellulose-blotted antigens for phage display monoclonal antibody selection

The combinatorial phage display library approach to antibody repertoire cloning offers a powerful tool for the isolation of specific antibodies to defined target antigens. Panning strategy is often a very critical point for selecting antibody displayed on the surface of bacteriophages. Most selection strategies described to date have relied on the availability of purified and often recombinant antigen, providing the possibility to perform selections on a well defined antigen source. However, when the antigen is difficult to purify by means of laborious and time-consuming chromatography procedures, panning of phage antibody libraries has to be performed on complex antigen sources such as cell surfaces or tissue sections, or even by in vivo selection methods. This provides a series of technical and experimental complications. In the present work, we successfully generated a mouse monoclonal antibody fragment from a phage display library directed against protein E7 of HPV18 avoiding antigen purification as for immunizing mice as for antibody library selection. Our work demonstrates the feasibility of phage antibody selections on antigens transferred to a nitrocellulose membrane as solid support, using one-dimensional polyacrylamide gel electrophoresis system as the only practice to separate a given antigen present in bacterial crude cell lysate.     

Multiplexed fluid array screening of phage displayed anti-ricin single domain antibodies for rapid assessment of specificity

Phage display is a well-known technique that facilitates the selection of peptides or proteins that bind to a desired target. Using this tool, binding elements contained in the natural immune repertoires of the source animal or from a synthetically generated collection may be selected. The unpaired variable domain of the llama's heavy-chain-only classes of immunoglobulins represents an ideal source of genetic material to create phage display libraries. Initial panning of a semi-synthetic llama library yielded only one binder to the toxin ricin. In an effort to increase the number of monoclonal phage binders selected, the Luminex xMAP technology (Luminex, Austin, TX, USA) was used in addition to the enzyme-linked immunosorbent assay (ELISA) to screen clonal populations of phage after three rounds of selection. The xMAP technology detected phage displayed single domain antibody (sdAb) bound to ricin immobilized on the surface of microspheres under equilibrium conditions. This enhanced capability led directly to the identification of additional single domain antibodies of interest. The selected sdAbs were expressed, purified, and then evaluated for their specificity as well as enhanced thermal stability in comparison to conventional immunoglobulin G (IgG). We determined equilibrium dissociation constants and demonstrated their use as effective capture molecules in sandwich immunoassays.     

Kinetic analysis of engineered antibody-antigen interactions

Molecular engineering antibodies has made it possible to produce specific domains of the antibody molecule and combine them with other protein domains to achieve new properties. Using site directed mutagenesis, amino acid residues can be exchanged within the binding site; and, by analysis of crystal structures, the positions of these amino acids can be determined in three dimensions at atomic resolution. In addition, gene libraries and phage selection technology can be used to generate new antibody fragments directly from a gene pool. Both mutagenesis and selection from libraries offer opportunities to identify antibody-derived molecules with altered and useful antigen recognition properties. The detailed analysis both kinetic and equilibrium binding affinity are therefore essential to understand the activity of the molecules resulting from antibody engineering and to guide the progress of their further design. The paper reviews recently evolving techniques for the binging analysis of antibodies, their functional domains and antibody chimerae.     

Characterization of 11-dehydro-thromboxane B2 recombinant antibody obtained by phage display technology

Recombinant monoclonal antibodies specific for 11-dehydro-thromboxane B(2) (11D-TX) were isolated from the combinatorial libraries on a pComb3 phage-display vector using a magnetic cell sorting (MACS) system. The libraries were constructed from repertories of light and heavy-chains derived from the total RNA of 11D-TX conjugated keyhole limpet haemocyanin-immunized mice. Biotinylation of 11D-TX conjugated bovine serum albumin (BSA) was performed through free thiol groups on BSA using 1-biotinamido-4-[4'-(maleimidomethyl) cyclohexanecarboxamido] butane (Biotin-BMCC). Affinity bio-panning was performed to enrich the phage display libraries against biotinylated 11D-TX conjugated BSA with the MACS system. Results indicated that the selected anti-11D-TX Fab fragments expressed by E. coli exhibited a five-fold higher affinity for BSA conjugated 11D-TX compared to BSA alone and little specificity to other related compounds as determined by the binding assay and inhibition enzyme-linked immunosorbent assay (ELISA). This is the first report of an antibody against prostaglandin produced by phage display technology and also determination of the DNA sequence of this antibody. The MACS system was shown to be a simpler and more efficient method of panning than the conventional ELISA procedure. According to our results, we concluded that the phage display technique combined with the MACS system allowed the selection of the antibody with high affinity and some specificity.     

Engineering recombinant antibodies for immunotherapy

Recombinant antibody fragments binding with high affinity to their target can be obtained either from hybridomas or directly from antibody libraries on filamentous phage. These fragments are devoid of any activity other than antigen binding, and have to be processed and functionalized in order to be suitable for clinical applications. This article presents the authors’ view on the procedures and the features that are important for effective transformation of recombinant antibodies into useful immunotherapeutic agents. The topics presented include phage display methodologies, engineering of high-affinity binding, purification, and functionalization strategies of recombinant antibodies.     

PSMA mimotope isolated from phage displayed peptide library can induce PSMA specific immune response

Prostate-specific membrane antigen (PSMA) is a cell surface glycoprotein expressed predominantly in prostate secretory acinar epithelium and prostate cancer cells as well as in several extraprostatic tissues.Mouse monoclonal antibody 4G5 specific to the extracellular domain of PSMA was used to screen two phage displayed peptide libraries (9aa linear and 9aa cys library).Three 4G5-reactive phagotopes were identified.Sequence analysis of isolated clones demonstrated that the interaction motif“VDPA/SK” has high homology to 719-725aa on PSMA.Immunohistochemical staining of the prostate cancer sample with the psma-mimic phagotope(mimotope) immunized serum antibodies demonstrate that the mimotope isolated from the phage displayed peptide libraries can induce PSMA specific immune response in vivo.     

Peptide epitope identification by affinity selection on bacteriophage MS2 virus-like particles

Filamentous phages are now the most widely used vehicles for phage display and provide efficient means for epitope identification. However, the peptides they display are not very immunogenic because they normally fail to present foreign epitopes at the very high densities required for efficient B-cell activation. Meanwhile, systems based on virus-like particles (VLPs) permit the engineered high-density display of specific epitopes but are incapable of peptide library display and affinity selection. We developed a new peptide display platform based on VLPs of the RNA bacteriophage MS2. It combines the high immunogenicity of MS2 VLPs with the affinity selection capabilities of other phage display systems. Here, we describe plasmid vectors that facilitate the construction of high-complexity random sequence peptide libraries on MS2 VLPs and that allow control of the stringency of affinity selection through the manipulation of display valency. We used the system to identify epitopes for several previously characterized monoclonal antibody targets and showed that the VLPs thus obtained elicit antibodies in mice whose activities mimic those of the selecting antibodies.