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Sachdev S Sidhu - One of the best experts on this subject based on the ideXlab platform.
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CellectSeq: In Silico Discovery of Antibodies Targeting Integral Membrane Proteins Combining In Situ Selections of Phage Displayed Synthetic Antibodies and Next-Generation Sequencing
2020Co-Authors: Abdellali Kelil, Jarrett Adams, Eugenio Gallo, Jason Moffat, Sachdev S SidhuAbstract:Abstract Synthetic Antibody (Ab) technologies are efficient and cost-effective platforms for the generation of monoclonal proteomic tools against human antigens. Yet, they typically depend on purified proteins, which exclude from interrogation integral membrane proteins that require the lipid bilayers to support their native form or function. Here, we present a novel Ab discovery strategy, termed CellectSeq, for targeting integral membrane proteins presented on native cells in complex environment. As proof of concept, we targeted the challenging tetraspanin receptor CD151, a target linked to cancer. First, we optimized in situ cell-based selections to enrich Ab pools for antigen-specific binders. Then, we designed novel NGS procedures to explore Ab pools diversities and abundances with enhanced accuracies. Finally, we developed novel motif-based scoring and error filtering algorithms for the comprehensive interrogation of NGS data to identify Abs with high diversities and specificities, even at extremely low abundances. We identified highly selective and diversified Abs against CD151 with abundance as low as 0.00009% for which manual sampling or identification using Abs abundances in NGS data would have been impossible. Here we show that CellectSeq enables the rapid discovery of diversified and selective antibodies against CD151, with implications for other integral membrane proteins and cell-surface receptors.
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inhibition of marburg virus rna synthesis by a Synthetic anti vp35 Antibody
ACS Infectious Diseases, 2019Co-Authors: Parmeshwar Amatya, Henry W Rohrs, Alevtina Pavlenco, Priya Luthra, Christopher F. Basler, Dominika Borek, Sachdev S Sidhu, Gang Chen, Nicole Wagner, Michael L. GrossAbstract:Marburg virus causes sporadic outbreaks of severe hemorrhagic fever with high case fatality rates. Approved, effective, and safe therapeutic or prophylactic countermeasures are lacking. To address this, we used phage display to engineer a Synthetic Antibody, sFab H3, which binds the Marburg virus VP35 protein (mVP35). mVP35 is a critical cofactor of the viral replication complex and a viral immune antagonist. sFab H3 displayed high specificity for mVP35 and not for the closely related Ebola virus VP35. sFab H3 inhibited viral-RNA synthesis in a minigenome assay, suggesting its potential use as an antiviral. We characterized sFab H3 by a combination of biophysical and biochemical methods, and a crystal structure of the complex solved to 1.7 A resolution defined the molecular interface between the sFab H3 and mVP35 interferon inhibitory domain. Our study identifies mVP35 as a therapeutic target using an approach that provides a framework for generating engineered Fabs targeting other viral proteins.
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functional genomic characterization of a Synthetic anti her3 Antibody reveals a role for ubiquitination by rnf41 in the anti proliferative response
Journal of Biological Chemistry, 2019Co-Authors: Jacob P Turowec, Frederic A Fellouse, James Pan, Jason Moffat, Esther Lau, Xiaowei Wang, Kevin R Brown, Kamaldeep K Jawanda, Sachdev S SidhuAbstract:Dysregulation of the ErbB family of receptor tyrosine kinases is involved in the progression of many cancers. Antibodies targeting the dimerization domains of family members EGFR and HER2 are approved cancer therapeutics, but efficacy is restricted to a subset of tumors and resistance often develops in response to treatment. A third family member, HER3, heterodimerizes with both EGFR and HER2 and has also been implicated in cancer. Consequently, there is strong interest in developing antibodies that target HER3, but to date, no therapeutics have been approved. To aid the development of anti-HER3 antibodies as cancer therapeutics, we combined Antibody engineering and functional genomics screens to identify putative mechanisms of resistance or Synthetic lethality with Antibody-mediated anti-proliferative effects. We developed a Synthetic Antibody called IgG 95, which binds to HER3 and promotes ubiquitination, internalization, and receptor down-regulation. Using an shRNA library targeting enzymes in the ubiquitin proteasome system, we screened for genes that effect response to IgG 95 and uncovered the E3 ubiquitin ligase RNF41 as a driver of IgG 95 anti-proliferative activity. RNF41 has been shown previously to regulate HER3 levels under normal conditions and we now show that it is also responsible for down-regulation of HER3 upon treatment with IgG 95. Moreover, our findings suggest that down-regulation of RNF41 itself may be a mechanism for acquired resistance to treatment with IgG 95 and perhaps other anti-HER3 antibodies. Our work deepens our understanding of HER3 signaling by uncovering the mechanistic basis for the anti-proliferative effects of potential anti-HER3 Antibody therapeutics.
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construction of Synthetic Antibody phage display libraries
Methods of Molecular Biology, 2018Co-Authors: Johan Nilvebrant, Sachdev S SidhuAbstract:Synthetic Antibody libraries provide a vast resource of renewable Antibody reagents that can rival or exceed those of natural antibodies and can be rapidly isolated through controlled in vitro selections. Use of highly optimized human frameworks enables the incorporation of defined diversity at positions that are most likely to contribute to antigen recognition. This protocol describes the construction of Synthetic Antibody libraries based on a single engineered human autonomous variable heavy domain scaffold with diversity in all three complementarity-determining regions. The resulting libraries can be used to generate recombinant domain antibodies for a wide range of protein antigens using phage display. Furthermore, analogous methods can be used to construct Antibody libraries based on larger Antibody fragments or second-generation libraries aimed to fine-tune Antibody characteristics including affinity, specificity, and manufacturability. The procedures rely on standard reagents and equipment available in most molecular biology laboratories.
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Synthetic Antibody Engineering: Concepts and Applications
Structural Biology in Immunology, 2018Co-Authors: Jonathan R. Lai, Gang Chen, Sachdev S SidhuAbstract:Abstract Monoclonal antibodies (mAbs) are widely used for diagnostic, research, and therapeutic applications. A critical performance feature of a mAb is its specificity toward a particular target. Here, we discuss the use of protein engineering methods (phage display and structure-based design) to develop novel mAbs, in some cases with exquisite specificity for particular target structural conformations, posttranslation modifications, or point mutations. The phage display libraries are designed and produced using Synthetic oligonucleotides (Synthetic antibodies), and the mAb identification procedure involves entirely in vitro screening methods. Therefore, the Synthetic Antibody approach allows stringent control over the state of the target during the Antibody selection process. Furthermore, the libraries encode amino acid side chains that are biased toward residues with favorable physicochemical attributes for intermolecular recognition. Thus, the method is not biased by immunodominance and allows for identification of mAbs against targets that are intractable by other methods.
Urpo Lamminmäki - One of the best experts on this subject based on the ideXlab platform.
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Phage Display Selection of an Anti-Idiotype-Antibody with Broad-Specificity to Deoxynivalenol Mycotoxins.
Toxins, 2020Co-Authors: Janne Leivo, Markus Vehniäinen, Urpo LamminmäkiAbstract:The use of Synthetic Antibody libraries and phage displays provides an efficient and robust method for the generation of antibodies against a wide range of targets with highly specific binding properties. As the in vitro selection conditions can be easily controlled, these methods enable the rapid generation of binders against difficult targets such as toxins and haptens. In this study, we used deoxynivalenol mycotoxin as a target to generate anti-idiotype-antibodies with unique binding properties from Synthetic Antibody libraries. The binding of the selected anti-idiotype antibodies can be efficiently inhibited with the addition of free isoforms of deoxynivalenol. The Antibody was consecutively used to develop deoxynivalenol-specific ELISA and TRF-immunoassays, which can detect deoxynivalenol and two of the most common metabolic isoforms in the range of 78-115 ng/mL.
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Development of anti-immunocomplex specific antibodies and non-competitive time-resolved fluorescence immunoassay for the detection of estradiol
Analytical and Bioanalytical Chemistry, 2019Co-Authors: Janne Leivo, Laura Kivimäki, Etvi Juntunen, Kim Pettersson, Urpo LamminmäkiAbstract:Detection of circulatory estradiol has widespread use in various clinical applications. Particularly, the use of estradiol-specific antibodies in immunoassays is routinely used, mainly due to the cost efficiency and simplicity of the sample handling process. However, the circulatory levels of estradiol can be extremely low in some conditions, and beyond the current detection limit of existing competitive immunoassays. We describe the generation of anti-immunocomplex specific antibodies derived from Synthetic Antibody repertoire and the development of high-performance non-competitive immunoassay for the detection of estradiol. Phage display selections were used to isolate new antibodies from Synthetic Antibody library with the use of existing estradiol specific Fab fragment. The found antibodies were consecutively used to set up a time-resolved fluorescence-based immunoassay (TRFIA), which can be used to detect estradiol with exceptional sensitivity and specificity. The limit of detection and EC50 were shown to be 3.0 pg mL−1 and 32.4 pg mL−1 respectively. Graphical abstract
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Development of anti-immunocomplex specific antibodies and non-competitive time-resolved fluorescence immunoassay for the detection of estradiol.
Analytical and bioanalytical chemistry, 2019Co-Authors: Janne Leivo, Laura Kivimäki, Etvi Juntunen, Kim Pettersson, Urpo LamminmäkiAbstract:Detection of circulatory estradiol has widespread use in various clinical applications. Particularly, the use of estradiol-specific antibodies in immunoassays is routinely used, mainly due to the cost efficiency and simplicity of the sample handling process. However, the circulatory levels of estradiol can be extremely low in some conditions, and beyond the current detection limit of existing competitive immunoassays. We describe the generation of anti-immunocomplex specific antibodies derived from Synthetic Antibody repertoire and the development of high-performance non-competitive immunoassay for the detection of estradiol. Phage display selections were used to isolate new antibodies from Synthetic Antibody library with the use of existing estradiol specific Fab fragment. The found antibodies were consecutively used to set up a time-resolved fluorescence-based immunoassay (TRFIA), which can be used to detect estradiol with exceptional sensitivity and specificity. The limit of detection and EC50 were shown to be 3.0 pg mL−1 and 32.4 pg mL−1 respectively. Open in a separate window Graphical abstract
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Identification and analysis of anti-HDL scFv-antibodies obtained from phage display based Synthetic Antibody library
Clinical Biochemistry, 2016Co-Authors: Priyanka Negi, Päivi Malmi, Nina Sirkka, Tuomas Huovinen, Eeva-christine Brockmann, Jari Metso, Matti Jauhiainen, Janita Lovgren, Kim Pettersson, Urpo LamminmäkiAbstract:Objective: In epidemiological studies plasma high density lipoprotein cholesterol (HDL-C) levels are found to correlate inversely with atherosclerotic cardiovascular events. HDL consists of different subpopulations and they vary in their anti-atherogenic properties. The aim of this study is to isolate coronary artery disease (CAD) specific anti-HDL scFv-antibodies. Design and methods: To obtain CAD specific HDL binders, we used phage displayed Synthetic Antibody libraries to enrich specific antibodies against HDL isolated from CAD patients. The antibodies were affinity purified. Their capability to recognize apolipoproteins A-I and A-II, various HDL forms differing in lipid/protein ratios and plasma HDL, was studied using time-resolved fluorescence based immunoassay. Results: Using different selection strategies and immunoassay based screening we obtained altogether 1200 clones displaying HDL binding activity. By sequencing 337, we identified 264 unique antibodies against HDL. A set of 61 antibodies were selected for further analysis. We found a variety of antibodies with different binding profiles, including apoA-I binding antibodies either in lipid-dependent or lipid-independent manner and binders against apoA-II. Several antibodies were able to discriminate between HDL derived from CAD patients and healthy controls. A majority of the antibodies were immunoreactive with HDL in plasma. Conclusion: The novel HDL recognizing antibodies isolated from Synthetic Antibody phage library have displayed interesting HDL-binding characteristics suggesting that, in addition to use as research tools, a part of them might be useful for the development of diagnostic methods for CAD risk assessment.
An-suei Yang - One of the best experts on this subject based on the ideXlab platform.
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High throughput cytotoxicity screening of anti-HER2 immunotoxins conjugated with Antibody fragments from phage-displayed Synthetic Antibody libraries
Scientific Reports, 2016Co-Authors: Shin-chen Hou, Wei Ting Chao, Yao Sheng Chen, Chi Yu Fu, Hung-wei Lin, Chung Ming Yu, Kai-fa Huang, Andrew H.-j. Wang, Hong-sen Chen, An-suei YangAbstract:Immunotoxins are an important class of Antibody-based therapeutics. The potency of the immunotoxins depends on the Antibody fragments as the guiding modules targeting designated molecules on cell surfaces. Phage-displayed Synthetic Antibody scFv libraries provide abundant Antibody fragment candidates as targeting modules for the immunoconjugates, but the discovery of optimally functional immunoconjugates is limited by the scFv-payload conjugation procedure. In this work, cytotoxicity screening of non-covalently assembled immunotoxins was developed in high throughput format to discover highly functional Synthetic Antibody fragments for delivering toxin payloads. The principles governing the efficiency of the antibodies as targeting modules have been elucidated from large volume of cytotoxicity data: (a) epitope and paratope of the Antibody-based targeting module are major determinants for the potency of the immunotoxins; (b) immunotoxins with bivalent Antibody-based targeting modules are generally superior in cytotoxic potency to those with corresponding monovalent targeting module; and (c) the potency of the immunotoxins is positively correlated with the densities of the cell surface antigen. These findings suggest that screening against the target cells with a large pool of antibodies from Synthetic Antibody libraries without the limitations of natural Antibody responses can lead to optimal potency and minimal off-target toxicity of the immunoconjugates.
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Engineering anti-vascular endothelial growth factor single chain disulfide-stabilized Antibody variable fragments (sc-dsFv) with phage-displayed sc-dsFv libraries
Journal of Biological Chemistry, 2010Co-Authors: Yi Jen Huang, Anna Tung Ching Ching, Hung-ju Chang, Hung-ju Hsu, Chung Ming Yu, Ing Chien Chen, Yu Ching Lee, An-suei YangAbstract:Phage display of Antibody fragments from natural or Synthetic Antibody libraries with the single chain constructs combining the variable fragments (scFv) has been one of the most prominent technologies in Antibody engineering. However, the nature of the artificial single chain constructs results in unstable proteins expressed on the phage surface or as soluble proteins secreted in the bacterial culture medium. The stability of the variable domain structures can be enhanced with interdomain disulfide bond, but the single chain disulfide-stabilized constructs (sc-dsFv) have yet to be established as a feasible format for bacterial phage display due to diminishing expression levels on the phage surface in known phage display systems. In this work, biological combinatorial searches were used to establish that the c-region of the signal sequence is critically responsible for effective expression and functional folding of the sc-dsFv on the phage surface. The optimum signal sequences increase the expression of functional sc-dsFv by 2 orders of magnitude compared with wild-type signal sequences, enabling the construction of phage-displayed Synthetic antivascular endothelial growth factor sc-dsFv libraries. Comparison of the scFv and sc-dsFv variants selected from the phage-displayed libraries for vascular endothelial growth factor binding revealed the sequence preference differences resulting from the interdomain disulfide bond. These results underlie a new phage display format for Antibody fragments with all the benefits from the scFv format but without the downside due to the instability of the dimeric interface in scFv.
Won J Yang - One of the best experts on this subject based on the ideXlab platform.
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A phosphorylation pattern-recognizing Antibody specifically reacts to RNA polymerase II bound to exons
Experimental & Molecular Medicine, 2016Co-Authors: Jungwon Han, Aerin Yoon, Do B Hwang, Hwa K Lee, Min S Kim, Jong Hyuk Lee, Yujean Lee, Soomin Yoon, Sunyoung Park, Won J YangAbstract:The C-terminal domain of RNA polymerase II is an unusual series of repeated residues appended to the C-terminus of the largest subunit and serves as a flexible binding scaffold for numerous nuclear factors. The binding of these factors is determined by the phosphorylation patterns on the repeats in the domain. In this study, we generated a Synthetic Antibody library by replacing the third heavy chain complementarity-determining region of an anti-HER2 (human epidermal growth factor receptor 2) Antibody (trastuzumab) with artificial sequences of 7–18 amino-acid residues. From this library, antibodies were selected that were specific to serine phosphopeptides that represent typical phosphorylation patterns on the functional unit (YSPTSPS)2 of the RNA polymerase II C-terminal domain (CTD). Antibody clones pCTD-1stS2 and pCTD-2ndS2 showed specificity for peptides with phosphoserine at the second residues of the first or second heptamer repeat, respectively. Additional clones specifically reacted to peptides with phosphoserine at the fifth serine of the first repeat (pCTD-1stS5), the seventh residue of the first repeat and fifth residue of the second repeat (pCTD-S7S5) or the seventh residue of either the first or second repeat (pCTD-S7). All of these Antibody clones successfully reacted to RNA polymerase II in immunoblot analysis. Interestingly, pCTD-2ndS2 precipitated predominately RNA polymerase II from the exonic regions of genes in genome-wide chromatin immunoprecipitation sequencing analysis, which suggests that the phosphoserine at the second residue of the second repeat of the functional unit (YSPTSPS)2 is a mediator of exon definition.Gene expression: Antibodies explore gene activityAntibodies that bind to an enzyme that allows genes to make the proteins they encode could prove a valuable research tool. The ‘RNA polymerase II’ enzyme copies a gene into messenger RNAs – molecules that carry genetic information from the cell nucleus to direct protein synthesis out in the cytoplasm. The enzyme has an unusual pattern of amino acids carrying phosphate groups at one end of its protein chain. Hyori Kim at the University of Ulsan in South Korea, with Junho Chung and co-workers at Seoul National University, created a series of antibodies that selectively bind to these ‘phosphorylated’ regions of the enzyme depending on the phosphorylation pattern. The antibodies are being used to explore how different phosphorylation patterns control the binding of this vital enzyme to molecules required for gene activity, and to specific sites on DNA like exon and intron.
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A phosphorylation pattern-recognizing Antibody specifically reacts to RNA polymerase II bound to exons
Experimental & Molecular Medicine, 2016Co-Authors: Jungwon Han, Aerin Yoon, Do B Hwang, Hwa K Lee, Min S Kim, Jong Hyuk Lee, Yujean Lee, Soomin Yoon, Sunyoung Park, Won J YangAbstract:Antibodies that bind to an enzyme that allows genes to make the proteins they encode could prove a valuable research tool. The ‘RNA polymerase II’ enzyme copies a gene into messenger RNAs – molecules that carry genetic information from the cell nucleus to direct protein synthesis out in the cytoplasm. The enzyme has an unusual pattern of amino acids carrying phosphate groups at one end of its protein chain. Hyori Kim at the University of Ulsan in South Korea, with Junho Chung and co-workers at Seoul National University, created a series of antibodies that selectively bind to these ‘phosphorylated’ regions of the enzyme depending on the phosphorylation pattern. The antibodies are being used to explore how different phosphorylation patterns control the binding of this vital enzyme to molecules required for gene activity, and to specific sites on DNA like exon and intron. The C-terminal domain of RNA polymerase II is an unusual series of repeated residues appended to the C-terminus of the largest subunit and serves as a flexible binding scaffold for numerous nuclear factors. The binding of these factors is determined by the phosphorylation patterns on the repeats in the domain. In this study, we generated a Synthetic Antibody library by replacing the third heavy chain complementarity-determining region of an anti-HER2 (human epidermal growth factor receptor 2) Antibody (trastuzumab) with artificial sequences of 7–18 amino-acid residues. From this library, antibodies were selected that were specific to serine phosphopeptides that represent typical phosphorylation patterns on the functional unit (YSPTSPS)_2 of the RNA polymerase II C-terminal domain (CTD). Antibody clones pCTD-1stS2 and pCTD-2ndS2 showed specificity for peptides with phosphoserine at the second residues of the first or second heptamer repeat, respectively. Additional clones specifically reacted to peptides with phosphoserine at the fifth serine of the first repeat (pCTD-1stS5), the seventh residue of the first repeat and fifth residue of the second repeat (pCTD-S7S5) or the seventh residue of either the first or second repeat (pCTD-S7). All of these Antibody clones successfully reacted to RNA polymerase II in immunoblot analysis. Interestingly, pCTD-2ndS2 precipitated predominately RNA polymerase II from the exonic regions of genes in genome-wide chromatin immunoprecipitation sequencing analysis, which suggests that the phosphoserine at the second residue of the second repeat of the functional unit (YSPTSPS)_2 is a mediator of exon definition.
Yi Jen Huang - One of the best experts on this subject based on the ideXlab platform.
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A panel of anti-influenza virus nucleoprotein antibodies selected from phage-displayed Synthetic Antibody libraries with rapid diagnostic capability to distinguish diverse influenza virus subtypes.
Scientific reports, 2020Co-Authors: Yu Chung-ming, Hung Pin Peng, Jhih Wei Jian, Yueh-liang Tsou, Ing Chien Chen, Yi Jen Huang, Hong-sen Chen, Tung Chao-ping, Chiu Yi-kai, Wesley Wei-wen HsiaoAbstract:Immunoassays based on sandwich immuno-complexes of capture and detection antibodies simultaneously binding to the target analytes have been powerful technologies in molecular analyses. Recent developments in single molecule detection technologies enable the detection limit of the sandwich immunoassays approaching femtomolar (10-15 M), driving the needs of developing sensitive and specific antibodies for ever-increasingly broad applications in detecting and quantifying biomarkers. The key components underlying the sandwich immunoassays are Antibody-based affinity reagents, for which the conventional sources are mono- or poly-clonal antibodies from immunized animals. The downsides of the animal-based antibodies as affinity reagents arise from the requirement of months of development timespan and limited choices of Antibody candidates due to immunodominance of humoral immune responses in animals. Hence, developing animal antibodies capable of distinguishing highly related antigens could be challenging. To overcome the limitation imposed by the animal immune systems, we developed an in vitro methodology based on phage-displayed Synthetic Antibody libraries for diverse antibodies as affinity reagents against closely related influenza virus nucleoprotein (NP) subtypes, aiming to differentiating avian influenza virus (H5N1) from seasonal influenza viruses (H1N1 and H3N2), for which the NPs are closely related by 90-94% in terms of pairwise amino acid sequence identity. We applied the methodology to attain, within four weeks, a panel of IgGs with distinguishable specificities against a group of representative NPs with pairwise amino acid sequence identities up to more than 90%, and the antibodies derived from the Antibody libraries without further affinity refinement had comparable affinity of mouse antibodies to the NPs with the detection limit less than 1 nM of viral NP from lysed virus with sandwich ELISA. The panel of IgGs were capable of rapidly distinguishing infections due to virulent avian influenza virus from infections of seasonal flu, in responding to a probable emergency scenario where avian influenza virus would be transmissible among humans overlapping with the seasonal influenza infections. The results indicate that the in vitro Antibody development methodology enables developing diagnostic antibodies that would not otherwise be available from animal-based Antibody technologies.
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High throughput discovery of influenza virus neutralizing antibodies from phage-displayed Synthetic Antibody libraries
Scientific Reports, 2017Co-Authors: Ing Chien Chen, Cheng-chung Lee, Yi-kai Chiu, Chao-ping Tung, Yueh-liang Tsou, Chia-lung Lin, Chung Ming Yu, Yi Jen Huang, Hong-sen Chen, Andrew H.-j. WangAbstract:Pandemic and epidemic outbreaks of influenza A virus (IAV) infection pose severe challenges to human society. Passive immunotherapy with recombinant neutralizing antibodies can potentially mitigate the threats of IAV infection. With a high throughput neutralizing Antibody discovery platform, we produced artificial anti-hemagglutinin (HA) IAV-neutralizing IgGs from phage-displayed Synthetic scFv libraries without necessitating prior memory of Antibody-antigen interactions or relying on affinity maturation essential for in vivo immune systems to generate highly specific neutralizing antibodies. At least two thirds of the epitope groups of the artificial anti-HA antibodies resemble those of natural protective anti-HA antibodies, providing alternatives to neutralizing antibodies from natural Antibody repertoires. With continuing advancement in designing and constructing Synthetic scFv libraries, this technological platform is useful in mitigating not only the threats of IAV pandemics but also those from other newly emerging viral infections.
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Engineering anti-vascular endothelial growth factor single chain disulfide-stabilized Antibody variable fragments (sc-dsFv) with phage-displayed sc-dsFv libraries
Journal of Biological Chemistry, 2010Co-Authors: Yi Jen Huang, Anna Tung Ching Ching, Hung-ju Chang, Hung-ju Hsu, Chung Ming Yu, Ing Chien Chen, Yu Ching Lee, An-suei YangAbstract:Phage display of Antibody fragments from natural or Synthetic Antibody libraries with the single chain constructs combining the variable fragments (scFv) has been one of the most prominent technologies in Antibody engineering. However, the nature of the artificial single chain constructs results in unstable proteins expressed on the phage surface or as soluble proteins secreted in the bacterial culture medium. The stability of the variable domain structures can be enhanced with interdomain disulfide bond, but the single chain disulfide-stabilized constructs (sc-dsFv) have yet to be established as a feasible format for bacterial phage display due to diminishing expression levels on the phage surface in known phage display systems. In this work, biological combinatorial searches were used to establish that the c-region of the signal sequence is critically responsible for effective expression and functional folding of the sc-dsFv on the phage surface. The optimum signal sequences increase the expression of functional sc-dsFv by 2 orders of magnitude compared with wild-type signal sequences, enabling the construction of phage-displayed Synthetic antivascular endothelial growth factor sc-dsFv libraries. Comparison of the scFv and sc-dsFv variants selected from the phage-displayed libraries for vascular endothelial growth factor binding revealed the sequence preference differences resulting from the interdomain disulfide bond. These results underlie a new phage display format for Antibody fragments with all the benefits from the scFv format but without the downside due to the instability of the dimeric interface in scFv.
