Phage Display Antibody Library Research Articles

Preparation of VCSM13 Helper Phage for Display Library Reamplification and Bio-Panning.

Phage-displayed antibody libraries can be constructed using any species that is easily immunized. The pComb3XSS phagemid vector is commonly used for library cloning and phage display. This phagemid encodes the origin of replication of the filamentous bacteriophage f1 but lacks all the genes required for replication and assembly of phage particles. The replication and the assembly of phage from these phagemids thus requires a "helper" phage that provides the genes essential for those steps during library production and bio-panning. One of those helper phages is VCSM13. In this protocol, we describe the preparation of VCSM13 helper phage. Users should prepare VCSM13 helper phage for library reamplification and for bio-panning.

Identification of monoclonal antibodies from naive antibody phage-display libraries for protein detection in formalin-fixed paraffin-embedded tissues

Most antibodies used in immunohistochemistry (IHC) have been developed by animal immunization. We wanted to explore naive antibody repertoires displayed on filamentous phages as a source of full-length antibodies for IHC on Formalin-Fixed and Paraffin-Embedded (FFPE) tissues. We used two isogenic mouse fibroblast cell lines that express or not human HER2 to generate positive and negative FFPE pseudo-tissue respectively. Using these pseudo-tissues and previously described approaches based on differential panning, we isolated very efficient antibody clones, but not against HER2. To optimize HER2 targeting and tissue specificity, we first performed 3–4 rounds of in vitro panning using recombinant HER2 extracellular domain (ECD) to enrich the phage library in HER2 binders, followed by one panning round using the two FFPE pseudo-tissues to retain binders for IHC conditions. We then analyzed the bound phages using next-generation sequencing to identify antibody sequences specifically associated with the HER2-positive pseudo-tissue. Using this approach, the top-ranked clone identified by sequencing was specific to the HER2-positive pseudo-tissue and showed a staining pattern similar to that of the antibody used for the clinical diagnosis of HER2-positive breast cancer. However, we could not optimize staining on other tissues, showing that specificity was restricted to the tissue used for selection and screening. Therefore, future optimized protocols must consider tissue diversity early during the selection by panning using a wide collection of tissue types.

Generation of a Naïve Human scFv Phage Display Library and Panning Selection.

Phage display antibody libraries have been successfully used as the essential tool to produce monoclonal antibodies against a plethora of targets ranging from diseases to native biologically important proteins as well as small molecules. It is well documented that diverse antibody genes are the major genetic source for the construction of a high-quality antibody library and selection of high-affinity antibodies. Naïve antibody libraries are derived using the IgM repertoire of healthy donors obtained from B-cells isolated from human peripheral blood mononuclear cell (PBMC). Single-chain fragment variable (scFv) is a routinely used format due to its smaller size and preference for phage display. The process involves the use of a two-step cloning method for library construction. The protocol also covers the biopanning process for target positive clone selection.

Generation of anti-idiotypic antibodies mimicking Cry2Aa toxin from an immunized mouse phage display library as potential insecticidal agents against Plutella xylostella

This study tried to generate anti-idiotypic antibodies (Ab2s) which mimic Cry2Aa toxin using a phage-display antibody library (2.8 × 107 CFU/mL). The latter was constructed from a mouse immunized with F (ab’)2 fragments digested from anti-Cry2Aa polyclonal antibodies. The F (ab’)2 fragments and Plutella xylostella (P. xylostella) brush border membrane vesicles (BBMV) were utilized as targets for selection. Eight mouse phage-display single-chain variable fragments (scFvs) were isolated and identified by enzyme-linked immunoassay (ELISA), PCR and DNA sequencing after four rounds of biopanning. Among them, M3 exhibited the highest binding affinity with F (ab’)2, while M4 bound the best with the toxin binding region of cadherin of P. xylostella (PxCad-TBR). Both of these two fragments were chosen for prokaryotic expression. The expressed M3 and M4 proteins with molecular weights of 30 kDa were purified. The M4 showed a binding affinity of 29.9 ± 2.4 nM with the PxCad-TBR and resulted in 27.8 ± 4.3 % larvae mortality against P. xylostella. Computer-assisted molecular modeling and docking analysis showed that mouse scFv M4 mimicked some Cry2Aa toxin binding sites when interacting with PxCad-TBR. Therefore, anti-idiotypic antibodies generated by BBMV-based screening could be useful for the development of new bio-insecticides as an alternative to Cry2Aa toxin for pest control.

Rare antibody phage isolation and discrimination (RAPID) biopanning enables identification of high-affinity antibodies against challenging targets

In vitro biopanning platforms using synthetic phage display antibody libraries have enabled the identification of antibodies against antigens that were once thought to be beyond the scope of immunization. Applying these methods against challenging targets remains a critical challenge. Here, we present a new biopanning pipeline, RAPID (Rare Antibody Phage Isolation and Discrimination), for the identification of rare high-affinity antibodies against challenging targets. RAPID biopanning uses fluorescent labeled phage displayed fragment antigen-binding (Fab) antibody libraries for the isolation of high-affinity binders with fluorescent activated sorting. Subsequently, discriminatory hit screening is performed with a biolayer interferometry (BLI) method, BIAS (Biolayer Interferometry Antibody Screen), where candidate binders are ranked and prioritized according to their estimated kinetic off rates. Previously reported antibodies were used to develop the methodology, and the RAPID biopanning pipeline was applied to three challenging targets (CHIP, Gαq, and CS3D), enabling the identification of high-affinity antibodies.

Human antibody VH domains targeting uPAR as candidate therapeutics for cancers.

The high expression of uPAR has been linked to tumor progression, invasion, and metastasis in several types of cancer. Such overexpression of uPAR makes it a potential target for immunotherapies across common cancers such as breast, colorectal, lung, ovarian cancer, and melanoma. In our study, two high-affinity and specific human VH domain antibody candidates, designed as clones 3 and 115, were isolated from a phage-displayed human VH antibody library. Domain-based bispecific T- cell engagers (DbTE) based on these two antibodies exhibited potent killing of uPAR-positive cancer cells. Thus, these two anti-uPAR domain antibodies are promising candidates for treating uPAR positive cancers.

Single domain antibodies specific for HER2 dimerization domain effectively disrupts HER2 dimerization

Dimer-dependent phosphorylation of HER2 receptor is a key event for the signal transduction of HER family of receptors which correlates with tumor invasion and metastasis. New generation of therapies based on dimerization domain inhibition using monoclonal or fragment antibodies was introduced. A potent method for manufacturing antibodies and antibody fragments is the phage display antibody library method. A recombinant phage was generated using the phage display method from synthetic dAb library. Subtractive biopanning was performed on sepharose 4b resin. Evaluation of success of subtractive biopanning was confirmed by the PCR fingerprinting after the fourth round of biopanning. The fourth round of biopanning results in the isolation of several dimerization domain reactive clones based on the polyclonal phage ELISA results. Monoclonal phage cell ELISA was used to select the positive clones with the highest affinity, and they were subsequently employed for functional tests. Cell-ELISA, MTT assay and dimerization inhibition test revealed that the reactivity and specificity of the selected monoclonal phage to dimerization domain of HER2. Further, Annexin V/PI staining and gene expression analysis showed that increased apoptosis rates. Also, in silico binding of the selected clones to conformational structure of HER2 was applied, using protein–protein docking tool of the ICM-Pro software, and showed sdAbs were specifically interacted with dimerization domain of the receptor. In conclusion, we have identified a single domain targeting HER2 dimerization, which represents a promising therapeutic and diagnostic candidate for HER2-positive cancers. Purified sdAb needs to more research to evaluate it both in vivo and in vitro via functional tests to determine if it can be applied for treatment and diagnostics.

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.

A structure and knowledge-based combinatorial approach to engineering universal scFv antibodies against influenza M2 protein

BackgroundThe influenza virus enters the host via hemagglutinin protein binding to cell surface sialic acid. Receptor-mediated endocytosis is followed by viral nucleocapsid uncoating for replication aided by the transmembrane viral M2 proton ion channel. M2 ectodomain (M2e) is a potential universal candidate for monoclonal antibody therapy owing to its conserved nature across influenza virus subtypes and its importance in viral propagation.MethodsThe phage-displayed naive human antibody libraries were screened against the short stretch of the N-terminal 10-mer peptide (SLLTEVETPI) of the M2e. ELISA, BLI, and flow cytometry assays were used to examine scFv binding to M2e epitopes. The scFv crystal structures were determined to examine the nature of the interactions. The potencies of the scFvs against the influenza virus were demonstrated by real-time PCR and confocal microscopy imaging.ResultsThe four unique scFv clones were obtained from the scFv phage-display antibody libraries and shown to exhibit binding with the 10-mer conserved part of the M2e and with full-length M2 protein expressed on the HEK293T cells. The crystal structure of scFv AU1 with M2e peptide showed the peptide as a dimer in the parallel beta-sheet conformation bound at the interface of two scFv CDRs. The scFv AU1 significantly restricted the release of H1N1 virus progeny from the infected A549 cells.ConclusionThis structural and biochemical study showcased the binding of antibody scFv molecules with M2e peptide dimer, providing the structural insights for the function effect in terms of recognizing and restricting the release of new viral particles from an infected host cell.

Shark VNAR phage display libraries: An alternative source for therapeutic and diagnostic recombinant antibody fragments.

The development of recombinant antibody fragments as promising alternatives to full-length immunoglobulins offers vast opportunities for biomedicine. Antibody fragments have important advantages compared with conventional monoclonal antibodies that make them attractive for the biotech industry: superior stability and solubility, reduced immunogenicity, higher specificity and affinity, capacity to target the hidden epitope and cross the blood-brain barrier, the ability to refold after heat denaturation and inexpensive and easy large-scale production. Different antibody formats such as antigen-binding fragments (Fab), single-chain fragment variable (scFv) consisting of the antigen-binding domains of Ig heavy (VH) and light (VL) chain regions connected by a flexible peptide linker, single-domain antibody fragments (sdAbs) like camelid heavy-chain variable domains (VHHs) and shark variable new antigen receptor (VNARs), and bispecific antibodies (bsAbs) are currently being evaluated as diagnostics or therapeutics in preclinical studies and clinical trials. In the present review, we summarize and discuss studies on VNARs, the smallest recombinant antibody fragment, obtained after the screening of different types of phage display antibody libraries. Results published until March 2023 are discussed.

Phage display technology for fabricating a recombinant monoclonal ScFv antibody against tetanus toxin.

Tetanus is a specific infectious disease, often associated with lower immunization in developing countries and catastrophic events (such as earthquakes). Millions of people, especially children, die every year from tetanus disease. Therefore, it is necessary to devise a rapid and sensitive detection method for tetanus toxin to ensure an early diagnosis and clinical treatment of tetanus. The current study looks at developing a novel, high specific, low-cost, and sensitive ScFv antibody. It is capable of tetanus detection immunoassays in clinical diagnosis, suspicious foods, and water monitoring. For this regard, a high-quality phage display antibody library (8.7×107 PFU/ml) was constructed. Tetanus-specific antibodies with high affinity retrieved from libraries. After phage rescue and four rounds of biopanning, clone screening was performed by phage ELISA. Recombinant antibodies expressed from the AC8 clone showed the highest affinity for tetanus. SDS-PAGE and western blotting confirmed the presence of a high-quality, pure ScFv band at 32kDa. ELISA was used to determine the affinity value, estimated to be around 10-8M. The results suggest that the proposed detection method by ScFv antibodies is an alternative diagnostic tool enabling rapid and specific detection of the tetanus toxin.

Sequence enrichment profiles enable target-agnostic antibody generation for a broad range of antigens

Phenotypic drug discovery (PDD) enables the target-agnostic generation of therapeutic drugs with novel mechanisms of action. However, realizing its full potential for biologics discovery requires new technologies to produce antibodies to all, a priori unknown, disease-associated biomolecules. We present a methodology that helps achieve this by integrating computational modeling, differential antibody display selection, and massive parallel sequencing. The method uses the law of mass action-based computational modeling to optimize antibody display selection and, by matching computationally modeled and experimentally selected sequence enrichment profiles, predict which antibody sequences encode specificity for disease-associated biomolecules. Applied to a phage display antibody library and cell-based antibody selection, ∼105 antibody sequences encoding specificity for tumor cell surface receptors expressed at 103-106 receptors/cell were discovered. We anticipate that this approach will be broadly applicable to molecular libraries coupling genotype to phenotype and to the screening of complex antigen populations for identification of antibodies to unknown disease-associated targets.

Targeted innovative design of Bt Cry toxin insecticidal mimics

Bt Cry toxin is the mostly studied and widely used biological insect resistance protein, which plays a leading role in the green control of agricultural pests worldwide. However, with the wide application of its preparations and transgenic insecticidal crops, the resistance to target pests and potential ecological risks induced by the drive are increasingly prominent and attracting much attention. The researchers seek to explore new insecticidal protein materials that can simulate the insecticidal function of Bt Cry toxin. This will help to escort the sustainable and healthy production of crops, and relieve the pressure of target pests' resistance to Bt Cry toxin to a certain extent. In recent years, the author's team has proposed that Ab2β anti-idiotype antibody has the property of mimicking antigen structure and function based on the "Immune network theory" of antibody. With the help of phage display antibody library and specific antibody high-throughput screening and identification technology, Bt Cry toxin antibody was designed as the coating target antigen, and a series of Ab2β anti-idiotype antibodies (namely Bt Cry toxin insecticidal mimics) were screened from the phage antibody library. Among them, the lethality of Bt Cry toxin insecticidal mimics with the strongest activity was close to 80% of the corresponding original Bt Cry toxin, showing great promise for the targeted design of Bt Cry toxin insecticidal mimics. This paper systematically summarized the theoretical basis, technical conditions, research status, and discussed the development trend of relevant technologies and how to promote the application of existing achievements, aiming to facilitate the research and development of green insect-resistant materials.

Construction of Synthetic Antibody Phage Display Libraries.

Synthetic antibody libraries provide a vast resource of renewable antibody reagents that can rival natural antibodies and be rapidly isolated through controlled in vitro selections. Use of highly optimized human frameworks enables the incorporation of defined diversity at positions that are most likely to contribute to antigen recognition. This protocol describes the construction of synthetic antibody libraries based on a single engineered human autonomous variable heavy domain scaffold with diversity in all three complementarity-determining regions. The resulting libraries can be used to generate recombinant domain antibodies targeting a wide range of protein antigens using phage display. Furthermore, analogous methods can be used to construct antibody libraries based on larger antibody fragments or second-generation libraries aimed to fine-tune antibody characteristics including affinity, specificity, and manufacturability. The procedures rely on standard reagents and equipment available in most molecular biology laboratories.

Computational Analysis of Antibody Paratopes for Antibody Sequences in Antibody Libraries.

To ensure the functionalities of the antibodies in phage-displayed synthetic antibody libraries, we use computational method to evaluate the designs of the antibody libraries. The computational methodologies developed in our lab for designing antibody library provide rich information on the function of the designed antibody sequences-adequate antibody designs for a specific antigen type should have predicted paratopes for the antigen type. This computational assessment of the designed antibody sequences helps eliminate non-functional designs before proceeding to construct the library designs in the wet lab. As such, only reasonable antibody designs are constructed for antibody discoveries.

Construction of recombinant adenovirus expressing EGFRvIII extracellular domain gene and preparation of single domain antibody

The aim of this study was to construct a recombinant adenovirus expressing extracellular domain gene of human epidermal growth factor receptor variant Ⅲ (EGFRvIII ECD), and to prepare single domain antibody targeting EGFRvIII ECD by immunizing camels and constructing phage display antibody library. Total RNA was extracted from human prostate cancer cell line PC-3 cells and reversely transcribed into cDNA. EGFRvIII ECD gene was amplified using cDNA as template, and ligated into pAdTrack-CMV plasmid vector and transformed into E. coli BJ5183 competent cells containing pAdEasy-1 plasmid for homologous recombination. The recombinant adenovirus expressing EGFRvIII ECD was obtained through transfecting the plasmid into HEK293A cells. The recombinant adenovirus was used to immunize Bactrian camel to construct EGFRvIII ECD specific single domain antibody library. The single domain antibody was obtained by screening the library with EGFRvIII protein and the antibody was expressed, purified and identified. The results showed that recombinant adenovirus expressing EGFRvIII ECD was obtained. The capacity of EGFRvIII specific phage single domain antibody library was 1.4×109. After three rounds of enrichment and screening, thirty-one positive clones binding to EGFRvIII ECD were obtained by phage-ELISA, and the recombinant single domain antibody E14 with highest OD450 value was expressed and purified. The recombinant E14 antibody can react with EGFRvIII ECD with high affinity in ELISA assessment. The results indicated that the EGFRvIII specific single domain antibody library with high capacity and diversity was constructed and the single domain antibody with binding activity to EGFRvIII was obtained by screening the library. This study may facilitate the diagnosis and treatment of EGFRvIII targeted malignant tumors in the future.

Isolation of a human SARS-CoV-2 neutralizing antibody from a synthetic phage library and its conversion to fluorescent biosensors

Since late 2019, the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the resultant spread of COVID-19 have given rise to a worldwide health crisis that is posing great challenges to public health and clinical treatment, in addition to serving as a formidable threat to the global economy. To obtain an effective tool to prevent and diagnose viral infections, we attempted to obtain human antibody fragments that can effectively neutralize viral infection and be utilized for rapid virus detection. To this end, several human monoclonal antibodies were isolated by bio-panning a phage-displayed human antibody library, Tomlinson I. The selected clones were demonstrated to bind to the S1 domain of the spike glycoprotein of SARS-CoV-2. Moreover, clone A7 in Fab and IgG formats were found to effectively neutralize the binding of S protein to angiotensin-converting enzyme 2 in the low nM range. In addition, this clone was successfully converted to quench-based fluorescent immunosensors (Quenchbodies) that allowed antigen detection within a few minutes, with the help of a handy fluorometer.

Tumor-Targeting Ability of Novel Anti-Prostate-Specific Membrane Antigen Antibodies.

Patients with prostate-specific membrane antigen (PSMA)-positive tumors can benefit from PSMA-targeted therapy; thus, we have constructed a phage-displayed synthetic antibody library for the production of novel PSMA antibodies with superior PSMA-targeting ability, favoring clinical management. The binding affinities of anti-PSMA antibodies were verified by an enzyme-linked immunosorbent assay (ELISA). Several in vitro and in vivo experiments, including cellular uptake, internalization, and cytotoxicity studies, micro single photon emission computed tomography (microSPECT)/CT, and biodistribution studies, were performed to select the most promising antibody among six different antibodies. The results showed the target affinities of our antibodies in the ELISA assays (7A, 8C, 8E, and 11A) were comparable to the existing antibodies (J591). The half-maximal effective concentrations of 7A, 8C, 8E, 11A, and J591 were 2.95, 6.64, 5.50, 2.08, and 4.79, respectively. The radiochemical yield of 111In-labeled antibodies ranged from 30% to 50% with high radiochemical purity (>90%). In the cellular uptake studies, the accumulated radioactivity of 111In-J591, 111In-7A, and 111In-11A increased over time. The internalized percentage of 111In-11A was the highest (32.14% ± 2.06%) at 48 h after incubation, whereas that of 111In-J591 peaked at 22.43% ± 4.38% at 24 h and dropped to 13.52% ± 3.03% at 48 h postincubation. Twenty-four hours after injection, radioactivity accumulation appeared in the LNCaP xenografts of the mice injected with 111In-11A, 111In-8E, 111In-7A, and 111In-J591 but not in the xenografts of the 111In-8C-injected group. Marked liver uptake was noticed in all groups except the 111In-11A-injected group. Moreover, the killing effect of 177Lu-11A was superior to that of 177Lu-J591 at low concentrations. In conclusion, we successfully demonstrated that 11A IgG owned the most optimal biological characteristics among several new anti-PSMA antibodies and it can be an excellent PSMA-targeting component for the clinical use.

Generation of Human Domain Antibody Fragments as Potential Insecticidal Agents against Helicoverpa armigera by Cadherin-Based Screening.

New insecticidal genes and approaches for pest control are a hot research area. In the present study, we explored a novel strategy for the generation of insecticidal proteins. The midgut cadherin of Helicoverpa armigera (H. armigera) was used as a target to screen materials that have insecticidal activity. After three rounds of panning, the phage-displayed human domain antibody B1F6, which not only binds to the H. armigera cadherin CR9-CR11 but also significantly inhibits Cry1Ac toxins from binding to CR9-CR11, was obtained from a phage-displayed human domain antibody (DAb) library. To better analyze the relevant activity of B1F6, soluble B1F6 protein was expressed by Escherichia coli BL21 (DE3). The cytotoxicity assays demonstrated that soluble B1F6 induced Sf9 cell death when expressing H. armigera cadherin on the cell membrane. The insect bioassay results showed that soluble B1F6 protein (90 μg/cm2) caused 49.5 ± 3.3% H. armigera larvae mortality. The midgut histological results showed that soluble B1F6 caused damage to the midgut epithelium of H. armigera larvae. The present study explored a new strategy and provided a basic material for the generation of new insecticidal materials.

Antibody CDR amino acids underlying the functionality of antibody repertoires in recognizing diverse protein antigens

Antibodies recognize protein antigens with exquisite specificity in a complex aqueous environment, where interfacial waters are an integral part of the antibody–protein complex interfaces. In this work, we elucidate, with computational analyses, the principles governing the antibodies’ specificity and affinity towards their cognate protein antigens in the presence of explicit interfacial waters. Experimentally, in four model antibody–protein complexes, we compared the contributions of the interaction types in antibody–protein antigen complex interfaces with the antibody variants selected from phage-displayed synthetic antibody libraries. Evidently, the specific interactions involving a subset of aromatic CDR (complementarity determining region) residues largely form the predominant determinant underlying the specificity of the antibody–protein complexes in nature. The interfacial direct/water-mediated hydrogen bonds accompanying the CDR aromatic interactions are optimized locally but contribute little in determining the epitope location. The results provide insights into the phenomenon that natural antibodies with limited sequence and structural variations in an antibody repertoire can recognize seemingly unlimited protein antigens. Our work suggests guidelines in designing functional artificial antibody repertoires with practical applications in developing novel antibody-based therapeutics and diagnostics for treating and preventing human diseases.