Rounds Of Panning Research Articles

A cell-based subtractive panning strategy for selection of conformation-specific single-chain variable-fragment (scFv) against dimerization domain of EGFR

Background and objectiveOverexpression of EGFR, a member of the ErbB receptor family, has been observed in several cancers and causes resistance to therapeutic antibodies, such as Herceptin. In this study, we produced a recombinant single-chain variable fragment (scFv) antibody against the EGFR dimerization domain. MethodsThe recombinant scFv was generated using a cell-based subtractive panning strategy. Subtractive panning was performed on a genetically engineered, VERO/EGFR, cells as well as a triple-negative breast cancer, MDA-MB-468, cells. Phage cell-ELISA was used to monitor the binding of the selected scFvs to the dimerization domain of EGFR. Inhibition of EGFR and HER2 dimerization by the produced scFvs were finally evaluated using the dimerization inhibition test and the expression of apoptosis-related genes were measured using the quantitative RT-PCR. ResultsPCR fingerprinting results showed a uniform digestion pattern following the third round of panning that confirmed the success of subtractive panning. Moreover, cell-ELISA validated the reactivity of the produced scFvs to EGFR following stimulation with EGF. Dimerization inhibition test showed the capacity of the scFvs to inhibit EGFR and HER2 dimerization. Investigation of apoptosis-related genes showed that treatment with the scFv antibody caused increased Bax and decreased Bcl2 expression. ConclusionsDirected HER2 targeting was shown to be effective enough to block the functional domain of the cell receptor and its intracellular signaling pathway. The subtractive panning strategy used in this study could control the process of directed selection of specific antibodies against the dimerization domain of EGFR. Selected antibodies might then be functionally tested for antitumor effects in both in vitro and in vivo studies.

Isolation of atrazine nanobodies enhanced by depletion of anti-carrier protein phages and performance comparison between the nanobody and monoclonal antibody derived from the same immunogen

Nanobody, a single domain antibody, has been shown a great promise for immunoassay (IA) applications. To improve the panning efficiency so as to obtain a valuable nanobody, anti-carrier protein phages in a phage display library were depleted to enhance the selection of nanobodies against the herbicide atrazine by using immunomagnetic beads conjugated with bovine serum albumin (IMB-BSA). The depletion of anti-carrier protein phages from the atrazine phage display library tripled the number of atrazine positive phage clones after four rounds of panning. One of the most sensitive phage clones Nb3 selected from the IMB-BSA depleted library was used to compare the performance with the monoclonal antibody (mAb 5D9) developed from the same immunogen. The Nb3-based IA exhibited similar specificity with the mAb 5D9-based IA, but greater thermostability and organic solvent tolerance. The half-maximum inhibition concentration (IC50) of the former was 3.5-fold greater than that of the latter (36.7 ng/mL versus 10.2 ng/mL). Because the Nb3-based IA was more robust than the mAb 5D9-based IA, the method detection limit of the two assays was 7.8 ng/mL of atrazine in river samples. The depletion strategy can increase the chance to acquire high quality nanobody and can be applicable for effective development of nanobodies against other small molecules.

Improved sensitivity of the anti-microcystin-LR ELISA using phage-displayed alpha-type anti-idiotypic nanobody

Anti-idiotypic antibodies (Ab2) are valuable tools that can be used for a better understanding of molecular mimicry and the immunological network. In this work, we showed a new application of a phage-displayed alpha-type Ab2 (Ab2α) to improve the sensitivity of an enzyme-linked immunosorbent assay (ELISA) detecting cyanobacterial toxin microcystin-LR (MC-LR). A monoclonal antibody (mAb) against MC-LR was used as an antigen to isolate binders in a camelid nanobody library. After three rounds of panning, three unique clones with strong binding against anti-MC-LR mAbs were isolated. These clones could specifically bind to anti-MC-LR mAbs without influencing mAbs binding with MC-LR, meaning these clones were Ab2αs. Based on the signal amplification effect of phage coat proteins and the non-competitive nature of Ab2α, a novel competitive ELISA method for MC-LR was established with a phage-displayed Ab2α. It showed that the phage-displayed Ab2α greatly enhanced the ELISA signal and sensitivity of the method was improved 3.5-fold to the conventional one. Combining with the optimization of pre-incubation time, the optimized ELISA decreased its limit of detection (LOD) from 4.5 ng/mL to 0.8 ng/mL (5.6-fold improvement). This new application of Ab2α may potentially be employed to improve the sensitivity of immunoassays for other environmental pollutants.

Screening of nanobody targeting alkaline phosphatase PhoX in Microcystis and detection of the PhoX in situ by fluorescence immunoassays

Cyanobacterial alkaline phosphatases (APases) play a key role in organophosphate utilization in freshwater. Tracking the distribution of APases can provide insights into the physiological response of phytoplankton to phosphorus nutrition. Extracellular APase PhoX, one of three prokaryotic APase families, is important for organophosphate utilization. Because the existing methods only give information on bulk APases activity, the specific contribution of the PhoX is little evaluated. To develop an immunoassay for PhoX detection, the phoX gene of Microcystis aeruginosa PCC7806 was expressed in Escherichia coli BL21 and the purified PhoX was used as a coated antigen for screening anti-PhoX nanobodies from an alpaca antibody library. After three rounds of panning, a nanobody 3H with the highest affinity was selected. Further tests showed 3H could specifically bind to Microcystis PhoX and be used for PhoX detection by immunoblotting analysis. Then we constructed two different fluorescent-labeled 3H, EGFP-3H (with an enhanced green fluorescent protein, EGFP) and FITC-3H (with fluorescein 5-isothiocyanate, FITC), as specific fluorescent dyes for PhoX. The fluorescence staining tests with laboratory strains and water bloom samples showed that both fluorescent-labeled nanobodies could visualize the distribution and evaluate relative expression levels of PhoX in Microcystis. The nanobody 3H could be a useful regent to develop fluorescence immunoassays for in situ analyses of Microcystis PhoX.Graphical Abstract

Screening, Expression, and Identification of Nanobody against SARS-CoV-2 Spike Protein.

Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an infectious disease that has become a serious burden on global public health. This study screened and yielded specific nanobodies (Nbs) against SARS-CoV-2 spike protein receptor binding domain (RBD), following testing its basic characteristics. A nanobody phage library was established by immunizing a camel with RBD protein. After three rounds of panning, the positive colonies were screened by enzyme-linked immunosorbent assay (ELISA). By sequencing, four different sequences of nanobody gene fragments were selected. The four nanobody fusion proteins were expressed and purified, respectively. The specificity and affinity of the four nanobodies were identified by ELISA. Our results showed that an immune phage display library against SARS-CoV-2 has been successfully constructed with a library capacity of which was 4.7 × 108 CFU. The four purified nanobodies showed specific high-affinity binding SARS-CoV-2 S-RBD. Among these, the antigen binding affinity of Nb61 was more comparable to that of commercial rabbit anti-SARS-CoV-2 S-RBD antibodies. In sum, our study has obtained four nanobody strains against SARS-CoV-2 S-RBD with significant affinity and specificity, therefore laying an essential foundation for further research as well as the applications of diagnostic and therapeutic tools of SARS-CoV-2.

Development of Anti-LRRC15 Small Fragments for Imaging Purposes Using a Phage-Display ScFv Approach.

The human leucine-rich repeat-containing protein 15 (LRRC15) is a membrane protein identified as a marker of CAF (cancer-associated fibroblast) cells whose overexpression is positively correlated with cancer grade and outcome. Nuclear molecular imaging (i.e., SPECT and PET) to track LRRC15 expression could be very useful in guiding further therapeutic strategies. In this study, we developed an ScFv mouse phage-display library to obtain small fragment antibodies against human LRRC15 for molecular imaging purposes. Mice were immunized with recombinant human LRRC15 (hLRRC15), and lymph node cells were harvested for ScFv (single-chain variable fragment) phage-display analysis. The built library was used for panning on cell lines with constitutive or induced expression after transfection. The choice of best candidates was performed by screening various other cell lines, using flow cytometry. The selected candidates were reformatted into Cys-ScFv or Cys-diabody by addition of cysteine, and cloned in mammalian expression vectors to obtain batches of small fragments that were further used in site-specific radiolabeling tests. The obtained library was 1.2 × 107 cfu/µg with an insertion rate >95%. The two panning rounds performed on cells permittedenrichment of 2 × 10−3. Screening with flow cytometry allowed us to identify 28 specific hLRRC15 candidates. Among these, two also recognized murine LRCC15 and were reformatted into Cys-ScFv and Cys-diabody. They were expressed transiently in a mammalian system to obtain 1.0 to 4.5 mg of Cys fragments ready for bioconjugation and radiolabeling. Thus, in this paper, we demonstrate the relevance of the phage-display ScFv library approach for the fast-track development of small antibodies for imaging and/or immunotherapy purposes.

Generation of bioluminescent enzyme immunoassay for ferritin by single-chain variable fragment and its NanoLuc luciferase fusion.

Ferritin, widely present in liver and spleen tissue, is considered as a serological biomarker for liver diseases and cancers. The detection of ferritin may be an important tool in health diagnosis. In this study, 14 non-immunized chicken spleens were utilized to construct a single-chain fragment (scFv) phage library. After 4 rounds of panning, 7 unique clones were obtained. The optimal clone was further screened and combined with NanoLuc luciferase (Nluc) as a dual functional immunoprobe to bioluminescent enzyme immunoassay (BLEIA), which was twice as sensitive as its parental scFv-based double-sandwich enzyme-linked immunoassay (ds-ELISA). The cross-reactivity analysis revealed that the proposed methods were highly selective and suitable for clinical detection. To further verify the performance of the immunoassays, serum samples were tested by the proposed methods and a commercial ELISA kit, and there was a good correlation between the results. These results suggested that scFv fused with Nluc might be a powerful dual functional tool for rapid, practically reliable, and highly sensitive ferritin detection.

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.

Isolation and characterization of phage display-derived scFv antibodies against human parechovirus 1 VP0 protein

Human parechoviruses (PeVs) are common viruses that are associated with a variety of diseases from mild gastrointestinal and respiratory symptoms to severe central nervous system infections. Until now there has not been antibodies for visualizing parechovirus infection. We used E. coli recombinant PeV-A1-VP0 protein as a target in phage display single chain variable fragment (scFv) antibody library panning. Three rounds of panning allowed identification and isolation of several candidate scFv clones, which tested positive in enzyme-linked immunosorbent assay (ELISA) against VP0. Three scFv clones (scFv-55, -59 and -71) with different CDR-3 sequences were further purified and tested in ELISA, Western blot and immunofluorescence microscopy (IFA) against a set of PeV-A1 isolates and a few isolates representing PeV types 2–6. In IFA, all three scFv binders recognized twenty PeV-A1 isolates. ScFv-55 and -71 also recognized clinical representatives of PeV types 1–6 both in IFA and in capture ELISA, while scFv-59 only recognized PeV-A1, -A2 and -A6. PeV-A1-VP0 (Harris strain) sequence was used to generate a peptide library, which allowed identification of a putative unique conformational antibody epitope with fully conserved flanking regions and a more variable core VVTYDSKL, shared between the scFv antibodies. Sequencing of the VP0 region of virus samples and sequence comparisons against parechoviral sequences in GenBank revealed 107 PeV-A1, -A3, -A8, -A17, -A (untyped) sequences with this exact epitope core sequence, which was most dominant among PeV-A1 isolates. These data suggest the first-time isolation of broad range phage display antibodies against human parechoviruses that may be used in diagnostic antibody development.

Trypanosoma evansi RoTat 1.2 variant surface antigen mimotopes selected by panning of the random peptide phage-display library against monoclonal antibodies.

Mimotope peptides of native antigens are valuable for diverse applications such as diagnostics, therapeutics and modern vaccine design. Here, we report for the first time the selection and identification of peptide mimotopes of Trypanosoma evansi RoTat 1.2 variant surface glycoprotein (VSG) for their potential uses in surra diagnostics and multi-epitope vaccine research. First, we produced the mouse monoclonal antibodies (mAbs), designated as 2E11 (IgG1) and 1C2 (IgG1), against the antigens in T. evansi RoTat 1.2 lysates. We then used 2E11 mAb to immunoprecipitate the target antigen. The immunoprecipitated antigen was then identified to be the VSG by mass spectrometry. Both 2E11 and 1C2 mAbs reacted with the VSG in immunoblots. The surface plasmon resonance immunosensors developed with both the mAbs detected VSG in the parasite lysates as well as in the rodent sera. Further, the mAbs were biotinylated and used in three rounds of panning to select peptide mimotopes from the random peptide phage display library (PhD-12; New England Biolabs, USA). The phage clones selected against each mAb were amplified and tested by phage capture ELISA for specificity. The peptide coding regions of the selected phages were sequenced and the protein blast search of the deduced amino acid sequences was performed by accessing the non-redundant protein database at https://blast.ncbi.nlm.nih.gov/. The conformational B epitope prediction of the selected mimotope sequences was done by using 3D Pepitope algorithms accessed at: http://pepitope.tau.ac.il/. The potential applications of the selected mimotopes in surra diagnostics and research are being explored.

Identification of New Antibodies Targeting Malignant Plasma Cells for Immunotherapy by Next-Generation Sequencing-Assisted Phage Display.

To identify new antibodies for the treatment of plasma cell disorders including multiple myeloma (MM), a single-chain Fragment variable (scFv) antibody library was generated by immunizing mice with patient-derived malignant plasma cells. To enrich antibodies binding myeloma antigens, phage display with cellular panning was performed. After depleting the immune library with leukocytes of healthy donors, selection of antibodies was done with L-363 plasma cell line in two consecutive panning rounds. Monitoring the antibodies’ enrichment throughout the panning by next-generation sequencing (NGS) identified several promising candidates. Initially, 41 unique scFv antibodies evolving from different B cell clones were selected. Nine of these antibodies strongly binding to myeloma cells and weakly binding to peripheral blood mononuclear cells (PBMC) were characterized. Using stably transfected Chinese hamster ovary cells expressing individual myeloma-associated antigens revealed that two antibodies bind CD38 and intercellular adhesion molecule-1 (ICAM-1), respectively, and 7 antibodies target yet unknown antigens. To evaluate the therapeutic potential of our new antibodies, in a first proof-of-concept study the CD38 binding scFv phage antibody was converted into a chimeric IgG1. Further analyses revealed that #5-CD38-IgG1 shared an overlapping epitope with daratumumab and isatuximab and had potent anti-myeloma activity comparable to the two clinically approved CD38 antibodies. These results indicate that by phage display and deep sequencing, new antibodies with therapeutic potential for MM immunotherapy can be identified.

Abstract 5976: Engineering easy-to-implement [18F] nanobody-PET for emerging immuno-onco target, ICAM-1

Abstract Intercellular adhesion molecule-1 (ICAM-1) participates in many important processes, including leukocyte endothelial transmigration, cell signaling, cell-cell interaction, cell polarity and tissue stability. ICAM-1 is highly expressed in inflammatory conditions, chronic diseases and several cancers, like anaplastic thyroid cancer (ATC) and triple negative breast cancer (TNBC). This rationale has motivated the pursuit of ICAM-1 as an emerging Immuno-Onco target, with many groups developing adoptive cell therapies against ICAM-1. Visualizing the ICAM-1 expression in the patients noninvasively is critical for a successful treatment. Identifying the ideal candidate patient, track the therapeutic effect of these treatments, the changes implicated in these diseases, or even after therapy for efficacy. PET imaging would be ideal due to its quantifiability, but unfortunately so far there is no such PET tracer to image ICAM-1 in patients. Here, we developed a nanobody based PET platform to image ICAM-1. Nanobodies are the smallest known functional antibody fragment. They are derived from heavy-chain only antibodies (HcAbs) in camelids. They retain high affinity and specificity for their target antigens, with low off-target accumulation due to their hydrophilic properties. Their nanoscale dimensions enable deep penetration of tumors, tolerance of high temperatures, elevated pressures, non-physiological pH, and even strong chemical denaturants. We generated these nanobodies by immunizing alpaca with Fc-fused human ICAM-1, produced and purified from mammalian cell transfection media. The harvested B cells from these immunized alpaca were used to generate a cDNA library, containing transcripts of the VHH domain of the HcAbs. This cDNA library was subsequently cloned into a yeast-surface display platform. After three rounds of panning we screened 160+ ICAM-1 binding clones from which we identified 8 unique clones with high to moderate binding to ICAM-1. Among which, clone A7 was utilized for nanobody-PET due to its high affinity (~115nM) and specificity to ICAM-1. With the goal of having a nanobody-PET that would be easy to execute at a moment’s notice and translated into clinic, we decided to utilize NOTA/aluminum-fluoride chemistry to radio label our PET tracer. For consistency in labeling, we utilized sortase to label the nanobody with NOTA at a 1:1 ratio. We demonstrated the utility of this ICAM-1 nanobody-PET tracer in subcutaneous tumor mouse xenograft model. PET/CT imaging shows clear localization to ICAM-1-positive xenograft, with low background uptake apart from minor gallbladder and kidney clearance. The success of this tracer in conjunction with the ease of NOTA/aluminum-fluoride chemistry opens up the possibility to carry over nanobody-PET to other targets (e.g., MSLN, EpCAM). Citation Format: Yogindra Vedvyas, Juan Gonzalez-Valdivieso, Yago Alcaina, Yanping Yang, Moonsoo M. Jin. Engineering easy-to-implement [18F] nanobody-PET for emerging immuno-onco target, ICAM-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5976.

Identification of single domain antibodies with insect cytotoxicity using phage-display antibody library screening and Plutella xylostella ATP-binding cassette transporter subfamily C member 2 (ABCC2) -based insect cell expression system

It is extremely imminent to study a new strategy to manage agricultural pest like Plutella xylostella (P. xylostella) which is currently resistant to most of pesticides, including three domain-Cry toxins from Bacillus thuringiensis (Bt). In this study, we reported a phage displayed single domain antibody screening from human domain antibody (DAb) library targeted on Spodoptera frugiperda 9 (Sf9) cells expressed Cry1Ac toxin receptor, ATP-dependent binding cassette transporter C2 in P. xylostella (PxABCC2). After three rounds of panning, three cytotoxic antibodies (1D2, 2B7, 3C4) were obtained from thirty-eight antibodies and displayed high binding ability towards PxABCC2-expressed Sf9 cells. Through homology modeling and molecular docking, the interaction mode indicated that the most cytotoxic 1D2 of the three antibodies presented the lowest binding free energy required and had the most hydrogen bond formed with PxABCC2 in molecular docking analysis. Functional assay of key regions in 1D2 via Alanine replacement indicated that complementarity-determining region (CDR) 3 played a crucial role in antibody exerts binding activity and cytotoxicity. This study provides the first trial for discovering of potential cytotoxic antibodies from the human antibody library via specific receptor-expressed insect cell system biopanning.

Anti-SARS-CoV-2 Omicron Antibodies Isolated from a SARS-CoV-2 Delta Semi-Immune Phage Display Library.

This report describes the discovery and characterization of antibodies with potential broad SARS-CoV-2 neutralization profiles. The antibodies were obtained from a phage display library built with the VH repertoire of a convalescent COVID-19 patient who was infected with SARS-CoV-2 B.1.617.2 (Delta). The patient received a single dose of Ad5-nCoV vaccine (Convidecia™, CanSino Biologics Inc.) one month before developing COVID-19 symptoms. Four synthetic VL libraries were used as counterparts of the immune VH repertoire. After three rounds of panning with SARS-CoV-2 receptor-binding domain wildtype (RBD-WT) 34 unique scFvs, were identified, with 27 cross-reactive for the RBD-WT and RBD Delta (RBD-DT), and seven specifics for the RBD-WT. The cross-reactive scFvs were more diverse than the RBD-WT specific ones, being encoded by several IGHV genes from the IGHV1 and IGHV3 families combined with short HCDR3s. Six cross-reactive scFvs and one RBD-WT specific scFv were converted to human IgG1 (hIgG1). Out of the seven antibodies, six blocked the RBD-WT binding to angiotensin converting enzyme 2 (ACE2), suggesting these antibodies may neutralize the SARS-CoV-2 infection. Importantly, one of the antibodies also recognized the RBD from the B.1.1.529 (Omicron) isolate, implying that the VH repertoire of the convalescent patient would protect against SARS-CoV-2 Wildtype, Delta, and Omicron. From a practical viewpoint, the triple cross-reactive antibody provides the substrate for developing therapeutic antibodies with a broad SARS-CoV-2 neutralization profile.

Characterisation of an Anti-Vaccinia Virus F13 Single Chain Fragment Variable from a Human Anti-Vaccinia Virus-Specific Recombinant Immunoglobulin Library.

Vaccinia virus (VACV) belongs to the genus Orthopoxvirus of the family Poxviridae. There are four different forms of infectious virus particles: intracellular mature virus (IMV), intracellular en-veloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV). The F13 protein occupies the inner side of the CEV- and EEV-membranes and the outer side of the IEV-membranes. It plays an important role in wrapping progress and EEV production. We constructed a human single-chain fragment variable (scFv) library with a diversity of ≥4 × 108 independent colonies using peripheral blood from four vaccinated donors. One anti-F13 scFv was isolated and characterised after three rounds of panning. In Western blotting assays, the scFv 3E2 reacted with the recombinant F13VACV protein with a reduction of binding under denatured and reduced conditions. Two antigenic binding sites (139-GSIHTIKTLGVYSDY-153 and 169-AFNSAKNSWLNL-188) of scFv 3E2 were mapped using a cellulose membrane encompassing 372 15-mere peptides with 12 overlaps covering the whole F13 protein. No neutralisation capa-bilities were observed either in the presence or absence of complement. In conclusion, the con-struction of recombinant immunoglobulin libraries is a promising strategy to isolate specific scFvs to enable the study of the host-pathogen interaction.

Selection and Identification of Single-Domain Antibodies Against Bartonella Invasion-Associated Locus B

At least 15 species or subspecies of Bartonella are zoonotic, each species appears to be highly adapted to one or a limited number of reservoir animals in which it is asymptomatic, while it can be transmitted to humans in which a variety of clinical manifestations can be caused. It was reported that Bartonella invasion associated locus B (IalB) played an important role in Bartonella pathogenesis. Our recent study showed that IalB had high sequence homology among different Bartonella species, and anti-IalB antibodies reacted strongly with Bartonella whole-cell lysates, which suggested that anti-IalB antibodies might be a good candidate for diagnosis of Bartonella infection. In the present study, we screened for single domain antibody fragment (sdAb) candidates against Bartonella birtlesii IalB from a sdAb phage display library. Following four rounds of panning, several positive phage clones were isolated. Among them, VH2 and VH7 were shown high enrichment. Their capable of recognizing IalB was verified by binding assays, phage ELISA and soluble fragment ELISA. Their CDRs are speculated to perform IalB binding function by molecular docking. And VH2 and VH7 had high specificity for Bartonella and did not cross-react with other bacteria. The VH2 and VH7 might be good candidates for specific diagnosis of Bartonella infection.

Cross-neutralization of RBD mutant strains of SARS-CoV-2 by convalescent patient derived antibodies.

BackgroundThe emergence of COVID‐19 pandemic resulted in an urgent need for the development of therapeutic interventions. Of which, neutralizing antibodies play a crucial role in the prevention and resolution of viral infection.MethodsWe generated antibody libraries from 18 different COVID‐19 recovered patients and screened neutralizing antibodies to SARS‐CoV‐2 and its mutants. After 3 rounds of panning, 456 positive phage clones were obtained with high affinity to RBD (receptor binding domain). Clones were then reconstituted into whole human IgG for epitope binning assay and all 19 IgG were classified into 6 different epitope groups or Bins.ResultsAlthough all antibodies were found to bind RBD, the antibodies in Bin2 had superior inhibitory ability of the interaction between spike protein and angiotensin converting enzyme 2 receptor (ACE2). Most importantly, the antibodies from Bin2 showed stronger binding affinity or ability to mutant RBDs (N501Y, W463R, R408I, N354D, V367F, and N354D/D364Y) derived from different SARS‐CoV‐2 strains as well, suggesting the great potential of these antibodies in preventing infection of SARS‐CoV‐2 and its mutations. Furthermore, such neutralizing antibodies strongly restricted the binding of RBD to hACE2 overexpressed 293T cells. Consistently, these antibodies effectively neutralized wildtype and more transmissible mutant pseudovirus entry into hACE2 overexpressed 293T cells. In Vero‐E6 cells, one of these antibodies can even block the entry of live SARS‐CoV‐2 into cells at 12.5 nM.ConclusionsThese results indicate that the neutralizing human antibodies from the patient‐derived antibody libraries have the potential to fight SARS‐CoV‐2 and its mutants in this global pandemic.

Isolation and characterization of a novel GRP78-specific single-chain variable fragment (scFv) using ribosome display method.

Glucose-regulated protein 78 (GRP78) is a well-characterized endoplasmic reticulum (ER) chaperon frequently overexpressed at the surface of tumor cells and associated with tumor survival, metastasis, and chemoresistance. Hence, potential GRP78 binders emerge as promising candidates for cancer therapy and diagnosis. We applied ribosome display to isolate a single-chain variable domain (scFv) specific for the C-terminal domain of a recombinant human GRP78 (CGRP). Six female BALB/c mice were immunized and then splenocyte mRNA was extracted. An scFv-ribosome display library was established by joining the amplified VH/Vκ fragments through a 72-bp linker using overlap extension PCR. Then, selection was performed by applying two rounds of eukaryotic ribosome display panning with stepwise decreased amount of CGRP. Ultimately, the selected scFv was characterized using the indirect-ELISA assay, competitive-ELISA assay, Western blotting, Surface Plasmon Resonance (SPR), and in-silico analyses. The constructed library had a length of ~ 1100bp and the high-affinity scFvs were isolated using the outputs of the final panning round. Among 60 positive clones, GSF3 was selected and its expression, purification, and binding capacity was confirmed by SDS-PAGE and Western blotting. GSF3 exhibited an affinity of 13 × 107M-1 to CGRP as assessed by SPR. Moreover, the in-silico analyses indicated that GSF3 binds the C-terminal domain of GRP78 through key residues engaged in antibody-antigen interactions. We found that ribosome display is a swift and reliable technique for specific and high-affinity scFv isolation. Moreover, our results suggest that GSF3 might be applied as a potential cancer immunotherapeutic and diagnostic tool if this approach is carefully followed by successful preclinical and clinical evaluations to validate the findings for further confirmation.

Induction of anti-Zearalenone immune response with mimotopes identified from a phage display peptide library

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.

Screening of anti-idiotypic domain antibody from phage library for development of Bt Cry1A simulants

Anti-idiotypic antibody technique is a new approach for the rapid development of insecticidal protein. In this study, anti-Cry1A polyclonal antibodies were used as antigen to screen the anti-idiotypic antibody that can simulate Cry1A toxins from a phage display human domain antibody (DAB) library. After four rounds of panning, five positive clones that have binding activities with anti-Cry1A polyclonal antibodies were obtained. Indirect competitive ELISA (IC-ELISA) results showed that the positive clone D6 showed significant inhibition for the binding of Cry1A toxins with anti-Cry1A polyclonal antibodies, and the inhibition ratio increased with the increase of D6 content. While, B3, F4, G5, C7 and the controls showed no obvious inhibition to Cry1A toxins. The results suggest that D6 is the “β” subtype anti-idiotypic antibody, which can simulate Cry1A toxins and competitive binding with anti-Cry1A polyclonal antibodies. Meanwhile, D6 had certain binding activity with the brush border membrane vesicles (BBMV) of p. xylostella, which was the receptor of Cry1A toxins. The results of bioassay showed that D6 had certain insecticidal activity, and the lethal concentration of 50% (LC50) was 976 ng/cm2. This study provides basic materials and experience for the development of Cry toxin simulants.