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Phage display technology is a unique gene recombination expression technology, and it is also a simple and effective screening tool. Through panning, a protein or peptide with high affinity and selectivity to the target is obtained. Antibody phage display has become the first and most widely used in vitro screening technology. Phage display derivatives play an important role in the diagnosis and treatment of diseases. This article reviews the phage display system of phage display technology, the size and classification of antibody libraries and their applications, and discusses the application prospects and challenges of phage display technology. This thesis lays the foundation for the theoretical and experimental research of bacteriophages.

PURPOSE: The aberrant Hepatocyte growth factor (HGF)/ mesenchymal-epithelial transition factor (c-Met) signaling pathway in various malignancies and its correlation with tumor invasion and poor prognosis has validated c-Met as a compelling therapeutic target. Up to now, several monoclonal antibodies and small molecule inhibitors targeting c-Met have been introduced with different outcomes, none are yet clinically approved. Toward the generation of novel fully human anti-c-Met molecules, we generated a large naïve Fab antibody library using phage display technology, which subsequently screened for novel Fabs against c-Met. METHODS: A phage library, with a functional size of 5.5 × 10(10) individual antibody clones, was prepared using standard protocols and screened for c-Met-specific Fabs by successive rounds of panning. A panel of Fabs targeting c-Met were isolated, from which four clones were selected and further characterized by DNA sequencing. The c-Met binding ability of our selected Fabs was evaluated by c-Met ELISA assay and flow cytometry techniques. RESULTS: Among the confirmed anti-c-Met Fabs, clone C16, showed the highest affinity (K(aff): 0.3 × 10(9) M(−1)), and 63% binding to MKN45 cells (a human gastric adenocarcinoma cell-line) as compared to c-Met negative T47D cell-line (9.03%). CONCLUSION: Together, our study presents a single-pot antibody library, as a valuable source for finding a range of antigen-specific Fab antibodies, and also, a fully human, high affinity and specific anti c-Met Fab antibody, C16, which has the potential of developing as a therapeutic or chemotherapeutic delivery agent for killing c-Met-positive tumor cells. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s40199-020-00334-z) contains supplementary material, which is available to authorized users.

Despite great values in many applications, heavy chain-only antibodies (HcAbs) are naturally only produced in camelids and sharks, which are not easy to access and handle. Production of the type of antibodies in small laboratory animals would remarkably facilitate their applications. We previously reported a mouse line in which the CH1 exon of mouse γ1 was deleted that could express heavy chain-only IgG1 antibodies. However, these mice showed an extremely weak IgG1 response to specific antigens when immunized, and we could only achieve single VH domains with low affinity to antigens using these mice. One possibility is that the mouse germline VH repertoire was not sufficient to support the expression of functional heavy chain-only antibodies. In this study, we report the generation of a rat line in which the CH1 exon of the γ2a gene was removed and the γ1 and γ2b genes were silenced. Although the genetically modified rats expressed heavy chain-only IgG2a, they also exhibited a very weak IgG2a response to antigen immunization. Panning of a phage library constructed using IgG2a VH segments amplified from immunized rats identified antigen-specific single VH antibodies, which also exhibited much lower affinity than that of commercial mAbs. Together with our previous report, this study suggests that the simple genetic removal of the CH1 exon does not guarantee the successful expression of functional heavy chain-only antibodies.

This protocol is a comprehensive guide to phage display based selection of virus neutralizing VH antibody domains. It details three optimized parts including 1) Construction of large size (theoretically > 1011) naïve human antibody heavy chain domain library, 2) SARS-COV-2 antigen expression and stable cell line construction, and 3) Library panning for selection of SARS-CoV-2 specific antibody domains. Using this protocol, we identified a high-affinity neutralizing human VH antibody domain, VH ab8, which exhibits high prophylactic and therapeutic efficacy., Graphical Abstract

Antibody phage display has been pivotal in the quest to generate human monoclonal antibodies for biomedical and research applications. Target antigen preparation is a main bottleneck associated with the panning process. This includes production complexity, downstream purification, quality and yield. In many instances, purified antigens are preferred for panning but this may not be possible for certain difficult target antigens. Here, we describe an improved procedure of affinity selection against crude or non-purified antigen by saturation of non-binders with blocking agents to promote positive binder enrichment termed as Yin-Yang panning. A naïve human scFv library with kappa light chain repertoire with a library size of 109 was developed. The improved Yin-Yang biopanning process was able to enrich monoclonal antibodies specific to the MERS-CoV nucleoprotein. Three unique monoclonal antibodies were isolated in the process. The Yin-Yang biopanning method highlights the possibility of utilizing crude antigens for the isolation of monoclonal antibodies by phage display.

Phage display technology (PD) is a powerful technique for the generation of tumor‑targeting antibodies. However, there are a number of different selection methods established in different laboratories around the world. Cell‑based PD panning methods using primary tumor cells are particularly heterogeneous between laboratories, which can lead to inconsistent results. Therefore, the present study evaluated different cell‑based PD selection methods regarding their potential to generate acute myeloid leukemia (AML) blast‑binding antibodies. In addition to this evaluation, the present study improved the PD procedure by optimizing selection as well as depletion strategies. To the best of our knowledge, the current study demonstrated for the first time that antigen diversity during the depletion step is of importance for the enrichment of tumor‑targeting phage antibodies. It is demonstrated that medium levels of depletion antigen diversity led to the most promising antibody candidates. In addition, it was determined that purification of blast cells from patients with AML by immunomagnetic separation ameliorated the selection of AML‑binding phages during panning. Furthermore, suggesting a common design‑related mechanism using a 'single‑pot' PD library, such as the well‑known Tomlinson single‑chain fragment variable (scFv) library, the present study identified specific binding consensus phage particles in independent panning procedures. By means of these optimized strategies, four promising AML blast‑binding phage particles were isolated and soluble scFv‑Fc (scFv cloned to a fragment crystallizable of an IgG2a mouse antibody) fusion proteins were produced. These scFv‑Fc antibodies bound the surface of AML blasts and were successfully internalized into their cytoplasm, indicating that they are potential immunoconjugate candidates for AML immunotherapy.

Zearalenone (ZEN), a type of non-steroidal estrogenic mycotoxin, is mainly produced by several species of Fusarium molds. It is ubiquitous in contaminated grains and grain products all over the world, posing a serious threat to animal and human health. This study aims to screen the mimotopes of ZEN from a phage display random 12-mer peptide library and explore their immunogenicity. A monoclonal antibody (mAb) against ZEN was employed as the target for mimotope selection from a phage display random peptide library. After four rounds of panning, six mimotopes that could specifically bind to ZEN mAb were obtained. In order to explore the immunogenicity of these mimotopes, Balb/c mice were immunized with phages Z8, Z21, Z35, Z8:Z21:Z35(1:1:1) and the conjugate of ZEN-bovine serum albumin (ZEN-BSA), respectively. The titers of antibodies in the mice immunized with mimotopes were 1:3200 (Z8), 1:3200 (Z21), 1:6400 (Z35), 1:6400 (1:1:1 mixture of Z8, Z21 and Z35), and the binding between serum antibodies and ZEN-OVA could be blocked by ZEN standards. These results demonstrated that the mimotopes of ZEN could induce specific antibodies against ZEN, suggesting that these displayed peptides were immunogenic.

The pandemic caused by emerging Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) presents a global public health threat. Illustrating human antibody responding to viral antigen could potentially provide valuable information for basic research and clinical diagnosis. The antibody can be used as a complement to the viral detection for the rapid diagnosis of infected patients. Compared with spike protein (SP), nucleocapsid protein (NP) is normally conserved and highly immunogenic in many coronavirus members. As a major antigen, NP is a potential target for the diagnosis of SARS-CoV-2 infection. Here, we constructed a combinatorial fragment of antigen-binding (Fab)antibody phage library based on peripheral blood-derived from five coronavirus disease 2019 (COVID-19) infected donors. From the library, 159 Fab antibodies were obtained and identified by panning with NP. Among them, 16 antibodies were evaluated for their binding properties and epitopes recognition. Among these 16 antibodies, two well-paired antibodies were finally screened out for SARS-CoV-2 diagnosis by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) method. Our works may provide a potential resource for the clinical diagnosis of SARS-CoV-2 infection.

Antibody phage display is a powerful platform for discovery of clinically applicable high affinity monoclonal antibodies against a broad range of targets. Libraries generated from immunized animals offer the advantage of in vivo affinity-maturation of V regions prior to library generation. Despite advantages, few studies have described isolation of antibodies from rats using immune phage display. In our study, we describe a novel primer set, covering the full rat heavy chain variable and kappa light chain variable regions repertoire for the generation of an unbiased immune libraries. Since the immune repertoire of rats is poorly understood, we first performed a deep sequencing analysis of the V(D)J regions of VH and VLK genes, demonstrating the high abundance of IGVH2 and IGVH5 families for VH and IGVLK12 and IGVLK22 for VLK. The comparison of gene’s family usage in naïve rats have been used to validate the frequency’s distribution of the primer set, confirming the absence of PCR-based biases. The primers were used to generate and assemble a phage display library from human CD160-vaccinated rats. CD160 represents a valid therapeutic target as it has been shown to be expressed on chronic lymphocytic leukaemia cells and on the surface of newly formed vessels. We utilised a novel phage display panning strategy to isolate a high affinity pool (KD range: 0.399–233 nM) of CD160 targeting monoclonal antibodies. Subsequently, identified binders were tested for function as third generation Chimeric Antigen Receptors (CAR) T cells demonstrating specific cytolytic activity. Our novel primer set coupled with a streamlined strategy for phage display panning enable the rapid isolation and identification of high affinity antibodies from immunised rats. The therapeutic utility of these antibodies was demonstrated in CAR format.

Significance Effective therapies are urgently needed for COVID-19. We rapidly (within a week) identified a fully human monoclonal germline-like antibody (ab1) from phage-displayed libraries that potently inhibited mouse ACE2-adapted SARS-CoV-2 replication in wild-type BALB/c mice and native virus in transgenic mice expressing human ACE2 as well as in hamsters when administered before virus challenge. It was also effective when administered after virus infection of hamsters, although at lower efficacy than when used prophylactically. Ab1 was highly specific and did not bind to human cell membrane-associated proteins. It also exhibited good developability properties including complete lack of aggregation. Ab1 has potential for prophylaxis and therapy of COVID-19 alone or in combination with other agents., Effective therapies are urgently needed for the SARS-CoV-2/COVID-19 pandemic. We identified panels of fully human monoclonal antibodies (mAbs) from large phage-displayed Fab, scFv, and VH libraries by panning against the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) glycoprotein. A high-affinity Fab was selected from one of the libraries and converted to a full-size antibody, IgG1 ab1, which competed with human ACE2 for binding to RBD. It potently neutralized replication-competent SARS-CoV-2 but not SARS-CoV, as measured by two different tissue culture assays, as well as a replication-competent mouse ACE2-adapted SARS-CoV-2 in BALB/c mice and native virus in hACE2-expressing transgenic mice showing activity at the lowest tested dose of 2 mg/kg. IgG1 ab1 also exhibited high prophylactic and therapeutic efficacy in a hamster model of SARS-CoV-2 infection. The mechanism of neutralization is by competition with ACE2 but could involve antibody-dependent cellular cytotoxicity (ADCC) as IgG1 ab1 had ADCC activity in vitro. The ab1 sequence has a relatively low number of somatic mutations, indicating that ab1-like antibodies could be quickly elicited during natural SARS-CoV-2 infection or by RBD-based vaccines. IgG1 ab1 did not aggregate, did not exhibit other developability liabilities, and did not bind to any of the 5,300 human membrane-associated proteins tested. These results suggest that IgG1 ab1 has potential for therapy and prophylaxis of SARS-CoV-2 infections. The rapid identification (within 6 d of availability of antigen for panning) of potent mAbs shows the value of large antibody libraries for response to public health threats from emerging microbes.

Traditional antibody generation, using either phage display or animal immunization, relies on purified antigens. Many membrane proteins, such as G protein-coupled receptors, solute carriers, or ion channels, are important drug targets but very challenging for the formation of antibodies due to the difficulty of protein purification. Whole-cell panning is an alternative approach for generating antibodies without the need for antigen purification. However, it often suffers from background interference and therefore requires extensive screening with low success rates. Here, we develop a new phage selection method, dubbed affinity-tag-guided proximity selection (A-GPS), to efficiently isolate specific antibodies directly from the antigen-presenting cells. By engineering a genetically fused affinity tag for the target antigen, A-GPS confines the proximity labeling reaction near the target antigen and preferentially enriches the phage bound to the target antigen. Using surface-presented GFP on human cells as a model antigen, we demonstrated that A-GPS successfully enriched the antigen-specific clones in two rounds of selection. Among the 46 randomly picked clones, >95% of clones showed great affinity and specificity for GFP over the background of HEK293T surface proteins. One of the best clones expressed as a Fab fragment showed subnanomolar binding affinity for GFP. This clone was successfully applied to common biological applications, such as immunofluorescence and flow cytometry, reflecting the usefulness of A-GPS for generating commercial-grade antibodies.

The emergence of coronavirus disease 2019 (COVID-19) pandemic led to an urgent need to develop therapeutic interventions. Among them, neutralizing antibodies play crucial roles for preventing viral infections and contribute to resolution of infection. Here, we describe the generation of antibody libraries from 17 different COVID-19 recovered patients and screening of neutralizing antibodies to SARS-CoV-2. After 3 rounds of panning, 456 positive phage clones were obtained with high affinity to RBD (receptor binding domain). Then the positive clones were sequenced and reconstituted into whole human IgG for epitope binning assays. After that, all 19 IgG were classified into 6 different epitope groups or Bins. Although all these antibodies were shown to have ability to bind RBD, the antibodies in Bin2 have more superiority to inhibit the interaction between spike protein and angiotensin converting enzyme 2 receptor (ACE2). Most importantly, the antibodies from Bin2 can also strongly bind with mutant RBDs (W463R, R408I, N354D, V367F and N354D/D364Y) derived from SARS-CoV-2 strain with increased infectivity, suggesting the great potential of these antibodies in preventing infection of SARS-CoV-2 and its mutations. Furthermore, these neutralizing antibodies strongly restrict the binding of RBD to hACE2 overexpressed 293T cells. Consistently, these antibodies effectively neutralized pseudovirus entry into hACE2 overexpressed 293T cells. In Vero-E6 cells, these antibodies can even block the entry of live SARS-CoV-2 into cells at only 12.5 nM. These results suggest that these neutralizing human antibodies from the patient-derived antibody libraries have the potential to become therapeutic agents against SARS-CoV-2 and its mutants in this global pandemic.

The worldwide spread of COVID-19 highlights the need for an efficient approach to rapidly develop therapeutics and prophylactics against SARS-CoV-2. The SARS-CoV-2 spike protein, containing the receptor-binding domain (RBD) and S1 subunit involved in receptor engagement, is a potential therapeutic target. We describe the development of a phage-displayed single-domain antibody library by grafting naive complementarity-determining regions (CDRs) into framework regions of a human germline immunoglobulin heavy chain variable region (IGHV) allele. Panning this library against SARS-CoV-2 RBD and S1 subunit identified fully human single-domain antibodies targeting five distinct epitopes on SARS-CoV-2 RBD with subnanomolar to low nanomolar affinities. Some of these antibodies neutralize SARS-CoV-2 by targeting a cryptic epitope located in the spike trimeric interface. Collectively, this work presents a versatile platform for rapid antibody isolation and identifies promising therapeutic anti-SARS-CoV-2 antibodies as well as the diverse immogneic profile of the spike protein.

Antibodies-recognising peptides bound to the major histocompatibility complex (pMHC) represent potentially valuable and promising targets for chimeric antigen receptor (CAR) T cells to treat patients with cancer. Here, a human phage-Fab library has been selected using HLA-A2 complexed with a heteroclitic peptide variant from an epitope shared among multiple melanoma-associated antigens (MAGEs). DNA restriction analyses and phage ELISAs confirmed selection of unique antibody clones that specifically bind to HLA-A2 complexes or HLA-A2-positive target cells loaded with native or heteroclitic peptide. Antibodies selected against heteroclitic peptide, in contrast to native peptide, demonstrated significantly lower to even negligible binding towards native peptide or tumour cells that naturally expressed peptides. The binding to native peptide was not rescued by phage panning with antigen-positive tumour cells. Importantly, when antibodies directed against heteroclitic peptides were engineered into CARs and expressed by T cells, binding to native peptides and tumour cells was minimal to absent. In short, TCR-like antibodies, when isolated from a human Fab phage library using heteroclitic peptide, fail to recognise its native peptide. We therefore argue that peptide modifications to improve antibody selections should be performed with caution as resulting antibodies, either used directly or as CARs, may lose activity towards endogenously presented tumour epitopes.

The infection of the novel coronavirus SARS-CoV-2 have caused more than 150,000 deaths, but no vaccine or specific therapeutic antibody is currently available. SARS-CoV-2 relies on its spike protein, in particular the receptor binding domain (RBD), to bind human cell receptor angiotensin-converting enzyme 2 (ACE2) for viral entry, and thus targeting RBD holds the promise for preventing SARS-CoV-2 infection. In this work, a competitive biopanning strategy of a phage display antibody library was applied to screen blocking antibodies against RBD. High-affinity antibodies were enriched after the first round using a standard panning process in which RBD-His recombinant protein was immobilized as a bait. At the next two rounds, immobilized ACE2-Fc and free RBD-His proteins were mixed with the enriched phage antibodies. Antibodies binding to RBD at epitopes different from ACE2-binding site were captured by the immobilized ACE2-Fc, forming a “sandwich” complex. Only antibodies competed with ACE2 for recognizing RBD at the same or similar epitopes can bind to the free RBD-His in the supernatant and be subsequently separated by the Ni-NTA magnetic beads. Top 1 lead from the competitive biopanning of a synthetic antibody library, Lib AB1, was produced as the full-length IgG1 format. It was proved to competitively block the binding of RBD to ACE2 protein, and potently inhibit SARS-CoV-2 pseudovirus infection of ACE2-overexpressing Hela cells with IC50 values of 12nM. Nevertheless, top 1 lead from the standard biopanning of Lib AB1, can only bind to RBD in vitro but not have the blocking or neutralization activity. Our strategy can efficiently isolate the blocking antibodies of RBD, and it would speed up the discovery of neutralizing antibodies against SARS-CoV-2.

The COVID-19 pandemic is spreading rapidly, highlighting the urgent need for an efficient approach to rapidly develop therapeutics and prophylactics against SARS-CoV-2. We describe here the development of a phage-displayed single-domain antibody library by grafting naïve CDRs into framework regions of an identified human germline IGHV allele. This enabled the isolation of high-affinity single-domain antibodies of fully human origin. The panning using SARS-CoV-2 RBD and S1 as antigens resulted in the identification of antibodies targeting five types of neutralizing or non-neutralizing epitopes on SARS-CoV-2 RBD. These fully human single-domain antibodies bound specifically to SARS-CoV-2 RBD with subnanomolar to low nanomolar affinities. Some of them were found to potently neutralize pseudotyped and live virus, and therefore may represent promising candidates for prophylaxis and therapy of COVID-19. This study also reports unique immunogenic profile of SARS-CoV-2 RBD compared to that of SARS-CoV and MERS-CoV, which may have important implications for the development of effective vaccines against SARS-CoV-2.

Trans-2-nonenal is a fatty aldehyde with a long-chain, containing nine carbons. It was known that trans-2-nonenal is a cause of the odor associated with aging, and mainly found from the ages of 40 years. Phage display was a famous laboratory technique for proteins, peptides, and DNA interaction using bacteriophages to connect proteins with the genetic information. In this study, we have carried out random phage display screening to identify specific binding peptides for trans-2-nonenal. Trans-2-nonenal was directly coated on the plate. To select the phages against trans-2-nonenal, the random phages were attached to trans-2-nonenal and selected by 1–5 panning steps. Individual DNA of phage after 5 panning was analyzed to find the specific peptide sequence against trans-2-nonenal. The affinity of positive and negative binding phages was verified by ELISA assay using M13 phage-specific antibody. Among the phages, the phage with sequence of AHKSKLHQHVMFGGG (called as P4) in the end of tail, has shown the highest response. To explore a role of the peptide selected in sequence analysis and ELISA assay, the peptide was connected to magnetic beads. The peptide-coated beads were treated within trans-2-nonenal: treatment of P4 peptide shows significant decrease of trans-2-nonenal compared to negative peptide. Based on our results, it is suggested that the peptide, which is selected by phage display, could be used for the removal of trans-2-nonenal and odor associated with aging.

This article describes a procedure for isolating T cell subpopulations using various methods including indirect panning and immunopanning by microarray. In these methods, cells are selected by their capacity to bind to antibody-coated plates (or slides) on the basis of particular cell-surface markers. Such methods can be superior to the antibody/complement lysis method (Alternate Protocol), as they can select additional cell population for analysis. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Isolation of T cell populations by indirect panning Basic Protocol 2: Immunopanning with microarray Alternate Protocol: Isolation of T cell populations by antibody/complement-mediated cytotoxicity.

Objective To isolate and characterize novel high-affinity llama single-domain antibodies against human HER2. Results We immunized a llama with human HER2, constructed a phage-displayed VHH library from the lymphocytes of the animal, and isolated six unique HER2-specific VHHs by panning. All six VHHs were unique at the amino acid level and were clonally unrelated, as reflected by their distinct CDR3 lengths. All six VHHs recognized recombinant human HER2 ectodomain with monovalent affinities ranging from 1 to 51 nM, had comparable affinities for cynomolgus monkey HER2, and bound HER2+ SKOV3 cells by flow cytometry. Three of the VHHs recognized recombinant murine HER2 with no loss of affinity compared with human and cynomolgus monkey HER2. The VHHs recognized three major epitopes on HER2 (including one conserved across the human, simian and murine orthologues), all of which were distinct from that of trastuzumab. These VHHs may be useful in the design of modular cancer immunotherapeutics. Electronic supplementary material The online version of this article (10.1186/s13104-018-3955-8) contains supplementary material, which is available to authorized users.

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.

Recombinant antibodies with well-characterized epitopes and known conformational specificities are critical reagents to support robust interpretation and reproducibility of immunoassays across biomedical research. For myocilin, a protein prone to misfolding that is associated with glaucoma and an emerging player in other human diseases, currently available antibodies are unable to differentiate among the numerous disease-associated protein states. This fundamentally constrains efforts to understand the connection between myocilin structure, function, and disease. To address this concern, we used protein engineering methods to develop new recombinant antibodies that detect the N-terminal leucine zipper structural domain of myocilin and that are cross-reactive for human and mouse myocilin. After harvesting spleens from immunized mice and in vitro library panning, we identified two antibodies, 2A4 and 1G12. 2A4 specifically recognizes a folded epitope while 1G12 recognizes a range of conformations. We matured antibody 2A4 for improved biophysical properties, resulting in variant 2H2. In a human IgG1 format, 2A4, 1G12, and 2H2 immunoprecipitate full-length folded myocilin present in the spent media of human trabecular meshwork (TM) cells, and 2H2 can visualize myocilin in fixed human TM cells using fluorescence microscopy. These new antibodies should find broad application in glaucoma and other research across multiple species platforms.

Antibodies cross-reacting with homologue antigens in different species would be essential reagents for the development of comparative oncology studies. In comparison with conventional immunoglobulin Gs, recombinant nanobodies (single-domain variable regions of heavy-chain only antibodies of Camelidae origin) can be easily isolated in vitro and engineered into a variety of reagents with optimized characteristics for different research and clinical applications. We recovered an anti-human epidermal growth factor receptor 2 (anti-HER2) nanobody from a naïve llama library by direct panning on whole cells and expressed it fused to Fc and green fluorescent protein. These immunoreagents were assessed by flow cytometry and immunofluorescence with both human and canine cells overexpressing HER2 and its canine homologue dog epidermal growth factor receptor 2. The reported data illustrate the potential of using this class of antibodies in comparative oncology and suggest some development perspectives enabled by in vitro panning of pre-immune nanobody libraries.

Chondroitin sulfate proteoglycan 4 (CSPG4) is a promising target for cancer immunotherapy due to its high level of expression in a number of malignant tumors, and its essential role in tumor growth and progression. Clinical application of CSPG4-targeting immunotherapies is hampered by the lack of fully human high-affinity CSPG4 antibodies or antibody fragments. To overcome this limitation, we performed affinity maturation on a novel human CSPG4 single-chain Fv fragment (scFv) using the random mutagenesis approach and screened for improved variants from a yeast display library using a modified whole-cell panning method followed by fluorescence-activated cell sorting. After six rounds of panning and sorting, the top seven mutant scFvs were isolated and their binding affinities were characterized by flow cytometry and surface plasmon resonance. These highly specific, affinity-matured variants displayed nanomolar to picomolar binding affinities to the CSPG4 antigen. While each of the mutants harbored only two to six amino acid substitutions, they represented ~270–3000-fold improvement in affinity compared to the parental clone. Our study has generated affinity-matured scFvs for the development of antibody-based clinical therapeutics targeting CSPG4-expressing tumors.

Recombinant antibodies are now very important in both therapeutics and diagnostics and offer significant advantages over conventional antibodies. The generation of a single-chain variable antibody fragment (scFv) (a common and important recombinant antibody format) is used to demonstrate the construction of a recombinant antibody library. An immunotube-based two-day panning approach, using Escherichia coli as an expression system, is utilised for antibody screening. The methods used for antibody selection and purification using immobilised metal affinity chromatography (IMAC) are described.

Matrix metalloproteinase (MMP)-14 is an important target for cancer treatment due to its critical roles in tumor invasion and metastasis. Previous failures of all compound-based broad-spectrum MMP inhibitors in clinical trials suggest that selectivity is the key for a successful therapy. With inherent high specificity, monoclonal antibodies (mAbs) therefore arise as attractive inhibitors able to target the particular MMP of interest. As a routine screening method, enzyme-linked immunosorbent assays (ELISA) have been applied to panned phage libraries for the isolation of mAbs inhibiting MMP-14. However, because of suboptimal growth conditions and insufficient antibody expression associated with monoclonal ELISA, a considerable number of potentially inhibitory clones might not be identified. Taking advantage of next-generation sequencing (NGS), we monitored enrichment profiles of millions of antibody clones along three rounds of phage panning, and identified 20 Fab inhibitors of MMP-14 with inhibition IC50 values of 10-4000 nM. Among these inhibitory Fabs, 15 were not found by monoclonal phage ELISA. Particularly, Fab R2C7 exhibited an inhibition potency of 100 nM with an excellent selectivity to MMP-14 over MMP-9. Inhibition kinetics and epitope mapping suggested that as a competitive inhibitor, R2C7 directly bound to the vicinity of the MMP-14 catalytic site. This study demonstrates that deep sequencing is a powerful tool to facilitate the systematic discovery of mAbs with protease inhibition functions. This article is protected by copyright. All rights reserved

Cell surface antigens represent the most common targets for antibody-based cancer therapy. Isolation of lead antibodies to these membrane targets from antibody repertoires, such as immunized or naive phage display libraries, has been a challenging task, which is an outstanding issue when soluble portion of the target(s) is not available, and/or a naive phage display library is used. Common cell-based panning methods often encounter numerous difficulties, including high background and loss of cells during repeated washes. Here we described a novel FACS sorter-based protocol to isolate single-chain Fv molecules specific for defined antigen MSIIR expressed on stably transformed mammalian cells, and screening of unique binders to the tumor target.

The immunoglobulin (Ig) CH2 domain is a promising scaffold for the development of candidate therapeutics. We have previously shown that the stability of isolated CH2 could be increased by the introduction of an additional disulfide bond and removal of seven N-terminal residues (m01s). However, both isolated CH2 and m01s aggregate, likely due to the existence of aggregation-prone regions (APRs) that we identified by using computational methods. This knowledge was used to generate a phage display library of mutants. The library was incubated at high temperature to remove aggregating CH2 domains, and then panned against a mouse anti-human CH2 monoclonal antibody targeting a conformational epitope to remove misfolded CH2s. After two rounds of panning, one clone, m01s5, with smaller APRs, was identified. After additional mutagenesis one clone, m01s5.4, which aggregated much less than m01s as measured by a turbidity assay and dynamic light scattering, was identified. m01s5.4 also exhibited much lower nonspecific binding than m01s. Engineering of a previously identified m01s-based tumor antigen-specific binder led to a dramatic reduction of its aggregation without affecting its binding. In summary, we describe a new approach for reducing aggregation based on a combination of computational and phage display methodologies, and show that aggregation of CH2-based scaffolds can be significantly reduced by the newly identified mutants, which can improve the developability of potential CH2-based therapeutics.

CAR-T cell therapy emerged in the last years as a great promise to cancer treatment. Nowadays, there is a run to improve the breadth of its use, and thus, new chimeric antigen receptors (CAR) are being proposed. The antigen-binding counterpart of CAR is an antibody fragment, scFv (single chain variable fragment), that recognizes a membrane protein associated to a cancer cell. In this chapter, the use of human scFv phage display libraries as a source of new mAbs against surface antigen is discussed. Protocols focusing in the use of extracellular domains of surface protein in biotinylated format are proposed as selection antigen. Elution with unlabeled peptide and selection in solution is described. The analysis of enriched scFvs throughout the selection using NGS is also outlined. Taken together these protocols allow for the isolation of new scFvs able to be useful in the construction of new chimeric antigen receptors for application in cancer therapy.

Single domain antibodies have certain advantages including their small size, high stability and excellent tissue penetration, making them attractive drug candidates. Rabbit antibodies can recognize diverse epitopes, including those that are poorly immunogenic in mice and humans. In the present study, we established a method to isolate rabbit VH single domain antibodies for potential cancer therapy. We immunized rabbits with recombinant human B7-H3 (CD276) protein, made a phage-displayed rabbit VH single domain library with a diversity of 7 × 109, and isolated two binders (A1 and B1; also called RFA1 and RFB1) from phage panning. Both rabbit VH single domains exhibited antigen-dependent binding to B7-H3-positive tumor cell lines but not B7-H3 knockout tumor cell lines. Our study shows that protein immunization followed by phage display screening can be used to isolate rabbit single domain antibodies. The two single domain antibodies reported here may have potential applications in cancer immunotherapy.

Antibody phage display is the most used in vitro technology to generate recombinant, mainly human, antibodies as tools for research, for diagnostic assays, and for therapeutics. Up to now (autumn 2018), eleven FDA/EMA-approved therapeutic antibodies were developed using phage display, including the world best-selling antibody adalimumab.A key to generate successfully human antibodies in vitro is the choice of the most appropriate antibody selection method, for our goal. In this book chapter, we describe the antibody selection process (panning) in solution and its advantages over panning on immobilized antigens. Detailed protocols on the panning procedure and the screening of monoclonal binders are given.

We selected epitope-specific antibodies by cross-linking hits to the site-specific photocrosslinker on the target antigen., Currently, there are no methods available offering solutions to select and identify antibodies binding to a specific conformational epitope of an antigen. Here, we developed a method to allow epitope-directed antibody selection from a phage display library by photocrosslinking bound antibodies to a site that specifically incorporates a noncanonical amino acid, p-benzoyl-l-phenylalanine (pBpa), on the target antigen epitope. By one or two rounds of panning against antibody phage display libraries, those hits that covalently bind to the proximity site of pBpa on specific epitopes of target antigens after ultraviolet irradiation are enriched and selected. This method was applied to specific epitopes on human interleukin-1β and complement 5a. In both cases, more than one-third of hits identified bind to the target epitopes, demonstrating the feasibility and versatility of this method.

Phage display antibody libraries have proven an invaluable resource for the isolation of diagnostic and potentially therapeutic antibodies, the latter usually being antibody fragments converted into IgG formats. Recent advances in the production of highly diverse and functional antibody libraries are considered here, including for Fabs, scFvs and nanobodies. These advances include codon optimisation during generation of CDR diversity, improved display levels using novel signal sequences, molecular chaperones and isomerases and the use of highly stable scaffolds with relatively high expression levels. In addition, novel strategies for the batch reformatting of scFv and Fab phagemid libraries, derived from phage panning, into IgG formats are described. These strategies allow the screening of antibodies in the end-use format, facilitating more efficient selection of potential therapeutics.

Panning approaches using antibody libraries often result in the isolation of antibodies that bind to cells through an unknown cellular receptor. Here, we describe a protocol that uses ligand-directed crosslinking with the aminooxy-sulfhydryl-biotin (ASB) trifunctional crosslinker followed by a proteomic analysis to identify the cellular receptors for orphan ligands. We describe the synthesis of the ASB crosslinker, labelling of the ligand with ASB, and cell binding of the labelled ligands. Next, biotin affinity purification and trypsin digestion of cell surface proteins that have been crosslinked by ASB are described. Lastly, several hints and tips to improve the proteomic analysis for these types of samples are provided., Series: Methods in Molecular Biology

Since its first report in the Middle East in 2012, the Middle East respiratory syndrome-coronavirus (MERS-CoV) has become a global concern due to the high morbidity and mortality of individuals infected with the virus. Although the majority of MERS-CoV cases have been reported in Saudi Arabia, the overall risk in areas outside the Middle East remains significant as inside Saudi Arabia. Additional pandemics of MERS-CoV are expected, and thus novel tools and reagents for therapy and diagnosis are urgently needed. Here, we used phage display to develop novel monoclonal antibodies (mAbs) that target MERS-CoV. A human Fab phage display library was panned against the S2 subunit of the MERS-CoV spike protein (MERS-S2P), yielding three unique Fabs (S2A3, S2A6, and S2D5). The Fabs had moderate apparent affinities (Half maximal effective concentration (EC50 = 123&ndash, 421 nM) for MERS-S2P, showed no cross-reactivity to spike proteins from other CoVs, and were non-aggregating and thermostable (Tm = 61.5&ndash, 80.4 &deg, C). Reformatting the Fabs into IgGs (Immunoglobulin Gs) greatly increased their apparent affinities (KD = 0.17&ndash, 1.2 nM), presumably due to the effects of avidity. These apparent affinities were notably higher than that of a previously reported anti-MERS-CoV S2 reference mAb (KD = 8.7 nM). Furthermore, two of the three mAbs (S2A3 and S2D5) bound only MERS-CoV (Erasmus Medical Center (EMC)) and not other CoVs, reflecting their high binding specificity. However, the mAbs lacked MERS-CoV neutralizing activity. Given their high affinity, specificity, and desirable stabilities, we anticipate that these anti-MERS-CoV mAbs would be suitable reagents for developing antibody-based diagnostics in laboratory or hospital settings for point-of-care testing.

Phage display technology is a common approach for discovery of therapeutic antibodies. Drug candidates are typically isolated in two steps: First, a pool of antibodies is enriched through consecutive rounds of selection on a target antigen, and then individual clones are characterized in a screening procedure. When whole cells are used as targets, as in phenotypic discovery, the output phage pool typically contains thousands of antibodies, binding, in theory, hundreds of different cell surface receptors. Clonal expansion throughout the phage display enrichment process is affected by multiple factors resulting in extremely complex output phage pools where a few antibodies are highly abundant and the majority is very rare. This is a huge challenge in the screening where only a fraction of the antibodies can be tested using a conventional binding analysis, identifying mainly the most abundant clones typically binding only one or a few targets. As the expected number of antibodies and specificities in the pool is much higher, complementing methods, to reach deeper into the pool, are required, called deep mining methods. In this study, four deep mining methods were evaluated: 1) isolation of rare sub-pools of specific antibodies through selection on recombinant proteins predicted to be expressed on the target cells, 2) isolation of a sub-pool enriched for antibodies of unknown specificities through depletion of the primary phage pool on recombinant proteins corresponding to receptors known to generate many binders, 3) isolation of a sub-pool enriched for antibodies through selection on cells blocked with antibodies dominating the primary phage pool, and 4) next-generation sequencing-based analysis of isolated antibody pools followed by antibody gene synthesis and production of rare but enriched clones. We demonstrate that antibodies binding new targets and epitopes, not discovered through screening alone, can be discovered using described deep mining methods. Overall, we demonstrate the complexity of phage pools generated through selection on cells and show that a combination of conventional screening and deep mining methods are needed to fully utilize such pools. Deep mining will be important in future phenotypic antibody drug discovery efforts to increase the diversity of identified antibodies and targets.

Phage display technology is a widely used practical tool for isolating binding molecules against the desired targets in phage libraries. In the case of targeting the membrane protein with its natural conformation, conventional bio-panning has limitations on the efficient screening of the functionally relevant antibodies. To enrich the single-chain variable fragment (scFv) pools for recognizing the natural conformation of the membrane targets, the conventional bio-panning and screening process was modified to include the semi-automated cell panning protocol. Using FGFR3-overexpressing patient-derived cancer cells, biotin-X-DHPE was introduced and coupled to Streptavidin-coated magnetic beads for use in the solution-phage bio-panning procedure. The resulting clones of scFv were compared to the diversity of the binding region, especially on CDR-H3. The clones enriched further by cell-based panning procedure possessed a similar binding site and the CDR-H3 loop structure. The resulting antibodies inhibited cell growth and induced target degradation. This process may be a useful tool for screening biologically related antibodies that recognize natural conformational structure on cell membrane protein. Furthermore, cell-based panning has the potential to further expand to a high-throughput screening (HTS) system and automation process.

Law enforcement against illicit use of cannabis and related substances requires rapid, feasible, and reliable tools for on-site testing of cannabinoids. Notably, methods based on cannabinoid-specific antibodies enable efficient screening of multiple specimens. Antibody engineering may accelerate development of modern and robust testing systems. Here, we used in vitro affinity maturation to generate a single-chain Fv fragment (scFv) that recognizes with high affinity the psychoactive cannabinoid, Δ9-tetrahydrocannabinol (THC). A mouse monoclonal antibody against THC, Ab-THC#33, with Ka 6.2×107 M-1 (as Fab fragment) was established by the hybridoma technique. Then, a "wild-type" scFv (wt-scFv) with Ka, 1.1×107 M-1 was prepared by bacterial expression of a fusion gene combining the VH and VL genes for Ab-THC#33. Subsequently, random point mutations in VH and VL were generated separately, and the resulting products were assembled into mutant scFv genes, which were then phage-displayed. Repeated panning identified a mutant scFv (scFv#m1-36) with 10-fold enhanced affinity (Ka 1.1×108 M-1) for THC, in which only a single conservative substitution (Ser50Thr) was present at the N-terminus of the VH-complementarity-determining region 2 (CDR2) sequence. In competitive enzyme-linked immunosorbent assay (ELISA), the mutant scFv generated dose-response curves with midpoint 0.27 ng/assay THC, which was 3-fold lower than that of wt-scFv. Even higher reactivity with a major THC metabolite, 11-nor-9-carboxy-Δ9-tetrahydrocannabinol, indicated that the mutant scFv will be useful for testing not only THC in confiscated materials, but also the metabolite in urine. Indeed, the antibody fragment is potentially suitable for use in advanced on-site testing platforms for cannabinoids.

The HSV-1 envelope glycoprotein B (gB) plays a critical role in virus entry into host cells. Neutralizing antibodies can therefore potentially prevent virus entry into target cells and cell-to-cell spread of infection. Our present study focused on the selection of neutralizing single-chain Fv (scFv) antibodies of a phage-displayed nonimmune human scFv antibody library against gB of HSV-1. To enrich specific scFvs, two phage antibodies were isolated against amino acid residues 31–43 derived from the N-terminal part of gB using panning technique. Two scFvs, scFv-gB 1 and scFv-gB 2 , with frequencies of 45% and 20% were obtained from scFv clones after performing PCR and MvaI fingerprinting. In phage ELISA analysis, both gB 1 and gB 2 scFvs demonstrated high reactivity with the gB peptide. In the neutralization assay, scFv-gB 1 and scFv-gB 2 represented neutralizing effects of 55% and 59%, respectively. Upon further enhancement of the neutralizing effects of these antibodies, they can be considered as new potential alternatives in the treatment and prophylaxis of HSV-1 infections.

A method for development of murine Fab fragments towards extracellular domains of a surface receptor is presented. The GluA4 ionotropic glutamate receptor is used as a model system. Recombinant GluA4 ectodomain comprising both the N-terminal domain (NTD) and the ligand-binding domain (LBD) in one molecule was used for immunization. A Fab-phage library was constructed and a parallel panning approach enabled selection of murine Fab fragments towards either intact ectodomain or the isolated LBD of the GluA4 receptor. One LBD-Fab (FabL9) showed exclusive selectivity for the GluA4 LBD, over a panel of LBDs from GluA2, GluK1, GluK2 and GluD2. Soluble FabL9 was produced in amounts suitable for characterization. Competitive ELISA and rat-brain immunoprecipitation experiments confirmed that the FabL9 epitope is conserved in the LBD and in the intact native receptor. By an alignment of GluA2 and GluA4, the likely binding epitope for FabL9 was predicted. This study demonstrates a simple approach for development of antibody fragments towards specific sub-domains of a large ligand-gated ion channel, and this method could be utilized for all multi-domain surface receptors where antibody domain-selectivity may be desirable. Furthermore, we present for the first time a GluA4 subtype-specific murine Fab fragment targeting the LBD of the receptor.

The purpose of this study was to construct a single-chain variable fragment (scFv) antibody from chicken egg yolk immunoglobulin (IgY) by means of genetic engineering and subsequent panning for a specific antibody against Staphylococcus aureus. Methods and results We amplified the scFv using blood and spleen obtained from 100-day-old Roman chickens immunized with inactivated S. aureus and subsequently constructed a T7 phage display antibody library using phage display technology. Four non-repeated blood scFv and 6 spleen scFv were obtained following 3 rounds of panning of the T7 phage display antibody library, enzyme-linked immunosorbent assay and sequencing. These 10 scFv were cloned into the prokaryotic expression vector pCold I with expression induced at a low temperature. Four soluble proteins were obtained. Among them, soluble protein SFV6 derived from the spleen showed good reactivity against S. aureus using indirect ELISA and produced a particularly strong antibacterial effect in vitro. Conclusion We were successful in isolating a highly specific scFv antibody against S. aureus from the spleen phage display library. This study provides a simple and rapid method for the quick preparation of a large number of antibodies against S. aureus and provides the foundation for the positioning of antibodies in the organism and the study of the antibacterial mechanism through which the antibody functions.

In this manuscript, a method for the immunization of alpaca and the use of molecular biology methods to produce antigen-specific single domain antibodies is described and demonstrated. Camelids, such as alpacas and llamas, have become a valuable resource for biomedical research since they produce a novel type of heavy chain-only antibody which can be used to produce single domain antibodies. Because the immune system is highly flexible, single domain antibodies can be made to many different protein antigens, and even different conformations of the antigen, with a very high degree of specificity. These features, among others, make single domain antibodies an invaluable tool for biomedical research. A method for the production of single domain antibodies from alpacas is reported. A protocol for immunization, blood collection, and B-cell isolation is described. The B-cells are used for the construction of an immunized library, which is used in the selection of specific single domain antibodies via panning. Putative specific single domain antibodies obtained via panning are confirmed by pull-down, ELISA, or gel-shift assays. The resulting single domain antibodies can then be used either directly or as a part of an engineered reagent. The uses of single domain antibody and single domain antibody-based regents include structural, biochemical, cellular, in vivo, and therapeutic applications. Single domain antibodies can be produced in large quantities as recombinant proteins in prokaryotic expression systems, purified, and used directly or can be engineered to contain specific markers or tags that can be used as reporters in cellular studies or in diagnostics.

Antibody phage display is a key technology to generate recombinant, mainly human, antibodies for diagnostic and therapy, but also as tools for basic research. After antibody selection by "panning," a crucial step is the screening of monoclonal binders to isolate those which show antigen specificity. For this screening procedure, a highly parallelized approach to produce soluble antibody fragments in microtiter plates is essential. In this chapter, we give the protocol for the parallelized microscale production of scFvs for the screening procedure or further assays.

c-Met is a promising target in cancer therapy for its intrinsic oncogenic properties. However, there are currently no c-Met-specific inhibitors available in the clinic. Antibodies blocking the interaction with its only known ligand, hepatocyte growth factor, and/or inducing receptor internalization have been clinically tested. To explore other therapeutic antibody mechanisms like Fc-mediated effector function, bispecific T cell engagement, and chimeric antigen T cell receptors, a diverse panel of antibodies is essential. We prepared a chicken immune scFv library, performed four rounds of bio-panning, obtained 641 clones using a high-throughput clonal retrieval system (TrueRepertoireTM, TR), and found 149 antigen-reactive scFv clones. We also prepared phagemid DNA before the start of bio-panning (round 0) and, after each round of bio-panning (round 1−4), performed next-generation sequencing of these five sets of phagemid DNA, and identified 860,207 HCDR3 clonotypes and 443,292 LCDR3 clonotypes along with their clonal abundance data. We then established a TR data set consisting of antigen reactivity for scFv clones found in TR analysis and the clonal abundance of their HCDR3 and LCDR3 clonotypes in five sets of phagemid DNA. Using the TR data set, a random forest machine learning algorithm was trained to predict the binding properties of in silico HCDR3 and LCDR3 clonotypes. Subsequently, we synthesized 40 HCDR3 and 40 LCDR3 clonotypes predicted to be antigen reactive (AR) and constructed a phage-displayed scFv library called the AR library. In parallel, we also prepared an antigen non-reactive (NR) library using 10 HCDR3 and 10 LCDR3 clonotypes predicted to be NR. After a single round of bio-panning, we screened 96 randomly-selected phage clones from the AR library and found out 14 AR scFv clones consisting of 5 HCDR3 and 11 LCDR3 AR clonotypes. We also screened 96 randomly-selected phage clones from the NR library, but did not identify any AR clones. In summary, machine learning algorithms can provide a method for identifying AR antibodies, which allows for the characterization of diverse antibody libraries inaccessible by traditional methods.

Phage display is a powerful technique for drug discovery in biomedical research in particular for antibody libraries. But, several technical challenges are associated with the selection process. For instance, during the panning step, the successful elution of the phages bound to the antigen is critical in order to avoid losing the most promising binders. Here, we present an efficient protocol to establish, screen and select synthetic libraries of domain antibodies using phage display. We do not only present suitable solutions to the above-mentioned challenges to improve elution by 50-fold, but we also present a step by step in-depth protocol with miniaturized volumes and optimized procedures to save material, costs and time for a successful phage display with domain antibodies. Hence, this protocol improves the selection process for an efficient handling process. The here presented library is based on the variable domain (vNAR) of the naturally occurring novel antibody receptor (IgNAR) from cartilage fishes. Diversity was introduced in the Complementarity-Determining Region 3 (CDR3) of the antigen-binding site with different composition and length., publishedVersion

Antibodies are widely used in a large variety of research applications, for diagnostics and therapy of numerous diseases, primarily cancer and autoimmune diseases. Antibodies are binding specifically to target structures (antigens). The antigen-binding properties are not only dependent on the antibody sequence, but also on the discrete antigen region recognized by the antibody (epitope). Knowing the epitope is valuable information for the improvement of diagnostic assays or therapeutic antibodies, as well as to understand the immune response of a vaccine. While huge progress has been made in the pipelines for the generation and functional characterization of antibodies, the available technologies for epitope mapping are still lacking effectiveness in terms of time and effort. Also, no technique available offers the absolute guarantee of succeeding. Thus, research to develop and improve epitope mapping techniques is still an active field. Phage display from random peptide libraries or single-gene libraries are currently among the most exploited methods for epitope mapping. The first is based on the generation of mimotopes and it is fastened to the need of high-throughput sequencing and complex bioinformatic analysis. The second provides original epitope sequences without requiring complex analysis or expensive techniques, but depends on further investigation to define the functional amino acids within the epitope. In this book chapter, we describe how to perform epitope mapping by antigen fragment phage display from single-gene antigen libraries and how to construct these types of libraries. Thus, we also provide figures and analysis to demonstrate the actual potential of this technique and to prove the necessity of certain procedural steps.

Antibody (Ab) repertoire sequencing using high-throughput massively parallel technologies has contributed substantially to the understanding of Ab responses following infection, vaccination and autoimmunity. Because individual B-cell receptors are recombined and diversified somatically, genomic comparisons are limited, and distinguishing rare variants from sequencing errors is a major challenge. Oxford Nanopore Technologies’ MinION is a highly portable and cost-effective third-generation sequencing instrument, but has not been used for Ab repertoire sequencing due to its high error rate (approximately 1/10 bases). Here, we applied nanopore sequencing to single-domain Ab (sdAb) repertoires and phage-displayed sdAb libraries. We show that despite low overall data fidelity, sdAb sequences could be reconstructed above a frequency threshold (∼100 copies); however, distinguishing clonal sdAb variants was not always possible. The data quality was sufficient to enable rapid identification of antigen-specific sdAb sequences enriched during panning of phage display libraries, obviating the need for screening single clones.

Isolation of specific single chain antibodies (scFvs) against key epitopes of cancer markers are applied for cancer immunotherapy and diagnosis. In this study following the prediction of the 3D structure of the DSP part of Dentin sialophosphoprotein (DSPP), the epitope was chosen using in silico programs. Panning process was applied to isolate specific human scFv against the epitope. PCR and DNA fingerprinting differentiated the specific clones, which were evaluated by phage ELISA. Following DNA sequencing, the 3D structure of isolated scFv was modeled and Docked on DSP. Results demonstrated the selection of a specific anti-DSPP scFv with 40% frequency, which reacted significantly with the predicted epitope and PCa patients' urines in ELISA tests (P-value < 0.05). The VH and VL of the isolated scFv were from VH1 and VL3 gene families with several amino acid changes in CDRs and FRs domains. The scFv tightly bound to the DSP epitope with the lowest energy level by hydrogen bonds, cation-pi, hydrophobic and ionic interactions demonstrating the specificity of Ag-Ab interactions. The anti-DSPP scFv selected in this study with significant specificity to DSPP antigen offers a promising new agent for both PCa early detection and treatment of cancers with DSPP expression.

Background Zika virus (ZIKV) is an emerging pathogen with no approved therapeutics and only limited diagnostics available. To address this gap, six mouse single-chain antibodies (scFvs) to ZIKV envelope (E) protein were isolated rapidly and efficiently from a ribosome-displayed antibody library constructed from the spleens of five immunized mice. Methodology/results In this report, we have generated a panel of mouse scFvs to ZIKV E protein using ribosome display. The six scFvs demonstrated no cross-reactivity with DENV2 NGC envelope protein, suggesting specificity for ZIKV E protein. These scFvs showed differences in their affinity: two (scFv45-3, scFv63-1) of them were dominant after four rounds of panning, and showed higher affinity (an apparent Kd values from 19 to 27 nM) than the other four (scFv5-1, scFv7-2, scFv38-1, and scFv51-2). All six scFvs showed ZIKV-neutralizing activity in the plaque reduction neutralization test (PRNT) assay and their neutralizing activity was positively correlated with their affinities. Conclusions/significance The scFvs (45-3 and 63-1) with highest affinity may have dual utility as diagnostics capable of recognizing ZIKV E subtypes and may be further developed to treat ZIKV infection. Our approach has the added advantage of generating Fc receptor-deficient antibodies, minimizing concern of antibody-dependent enhancement (ADE) of infection.

To understand the mechanism for inhibition of hepatitis B virus (HBV) infection is important. In this study, single-chain variable fragment (scFv) antibodies were generated and directed to the pre-S2 epitope of HBV surface antigen (HBsAg). These human scFvs were isolated from a person with history of HBV infection by phage display technology. An evaluation of panning efficiency revealed that the eluted phage titer was increased, indicating that specific clones were enriched after panning. Selected scFvs were characterized with the recombinant HBsAg through Western blotting and enzyme-linked immunosorbent assay to confirm the binding ability. Flow cytometry analysis and immunocytochemical staining revealed that one scFv, S17, could recognize endogenous HBsAg expressed on the HepG2215 cell membrane. Moreover, the binding affinity of scFv S17 to the pre-S2 epitope was determined to be 4.2 × 10-8 M. Two ion interactions were observed as the major driving forces for scFv S17 interacting with pre-S2 by performing a rational molecular docking analysis. This study provides insights into the structural basis to understand the interactions between an antibody and the pre-S2 epitope. The functional scFv format can potentially be used in future immunotherapeutic applications.

VEGFR1 is a receptor tyrosine kinase that has been implicated in cancer pathogenesis. It is upregulated in angiogenic endothelial cells and expressed on human tumor cells as well. VEGFR1 positive hematopoietic progenitor cells home to sites of distant metastases prior to the arrival of the tumor cells thus establishing a pre-metastatic niche. To discover high affinity human antibodies selective for VEGFR1 molecular imaging or for molecularly targeted therapy, a novel phage display scFv library was assembled and characterized. The library was constructed from the humanized 4D5 framework that was mostly comprised tyrosine and serine residues in four complimentarity determining regions (CDRs). The library produced diverse and functional antibodies against a panel of proteins, some of which are of biomedical interest including, CD44, VEGFA, and VEGFR1. After panning, these antibodies had affinity strong enough for molecular imaging or targeted drug delivery without the need for affinity maturation. One of the anti-VEGFR1 scFvs recognized its cognate receptor and was selective for the VEGFR1., Highlights • VEGFR1 contributes to the pathogenesis cancer. • To obtain VEGFR1 specific antibodies, a phage displayed scFv library was constructed. • Four complimentarity determining regions were principally comprised of tyrosine and serine. • High affinity antibody fragments were isolated and characterized. • This is the first human antibody fragment specific for VEGFR1 from a phage displayed library.