scholarly journals Antibody Fragments as Tools for Elucidating Structure-Toxicity Relationships and for Diagnostic/Therapeutic Targeting of Neurotoxic Amyloid Oligomers

2020 ◽  
Vol 21 (23) ◽  
pp. 8920
Author(s):  
André L. B. Bitencourt ◽  
Raquel M. Campos ◽  
Erika N. Cline ◽  
William L. Klein ◽  
Adriano Sebollela
Keyword(s):  
Structural Complexity ◽  
Antibody Fragments ◽  
Single Chain ◽  
Amyloidogenic Proteins ◽  
Amyloid Oligomers ◽  
The Central Nervous System ◽  
Single Domain Antibodies ◽  
Single Chain Variable Fragments ◽  
Fragment Antigen Binding

The accumulation of amyloid protein aggregates in tissues is the basis for the onset of diseases known as amyloidoses. Intriguingly, many amyloidoses impact the central nervous system (CNS) and usually are devastating diseases. It is increasingly apparent that neurotoxic soluble oligomers formed by amyloidogenic proteins are the primary molecular drivers of these diseases, making them lucrative diagnostic and therapeutic targets. One promising diagnostic/therapeutic strategy has been the development of antibody fragments against amyloid oligomers. Antibody fragments, such as fragment antigen-binding (Fab), scFv (single chain variable fragments), and VHH (heavy chain variable domain or single-domain antibodies) are an alternative to full-length IgGs as diagnostics and therapeutics for a variety of diseases, mainly because of their increased tissue penetration (lower MW compared to IgG), decreased inflammatory potential (lack of Fc domain), and facile production (low structural complexity). Furthermore, through the use of in vitro-based ligand selection, it has been possible to identify antibody fragments presenting marked conformational selectivity. In this review, we summarize significant reports on antibody fragments selective for oligomers associated with prevalent CNS amyloidoses. We discuss promising results obtained using antibody fragments as both diagnostic and therapeutic agents against these diseases. In addition, the use of antibody fragments, particularly scFv and VHH, in the isolation of unique oligomeric assemblies is discussed as a strategy to unravel conformational moieties responsible for neurotoxicity. We envision that advances in this field may lead to the development of novel oligomer-selective antibody fragments with superior selectivity and, hopefully, good clinical outcomes.

scholarly journals Central Nervous System Delivery of Antibodies and Their Single-Domain Antibodies and Variable Fragment Derivatives with Focus on Intranasal Nose to Brain Administration

Antibodies ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 47
Author(s):  
Arghavan Soleimanizadeh ◽  
Heiko Dinter ◽  
Katharina Schindowski
Keyword(s):  
Drug Delivery ◽  
Central Nervous System ◽  
Nervous System ◽  
Single Domain ◽  
Production Costs ◽  
Single Chain ◽  
The Central Nervous System ◽  
Single Domain Antibodies ◽  
Single Chain Variable Fragments ◽  
Specific Tissue

IgG antibodies are some of the most important biopharmaceutical molecules with a high market volume. In spite of the fact that clinical therapies with antibodies are broadly utilized in oncology, immunology and hematology, their delivery strategies and biodistribution need improvement, their limitations being due to their size and poor ability to penetrate into tissues. In view of their small size, there is a rising interest in derivatives, such as single-domain antibodies and single-chain variable fragments, for clinical diagnostic but also therapeutic applications. Smaller antibody formats combine several benefits for clinical applications and can be manufactured at reduced production costs compared with full-length IgGs. Moreover, such formats have a relevant potential for targeted drug delivery that directs drug cargo to a specific tissue or across the blood–brain barrier. In this review, we give an overview of the challenges for antibody drug delivery in general and focus on intranasal delivery to the central nervous system with antibody formats of different sizes.

scholarly journals Single Chain Variable Fragment Fused to Maltose Binding Protein: A Modular Nanocarrier Platform for the Targeted Delivery of Antitumorals

2021 ◽  
Author(s):  
Francisco J. Reche-Perez ◽  
Simona Plesselova ◽  
Eduardo De los Reyes-Berbel ◽  
Mariano Ortega-Muñoz ◽  
F. Javier Lopez-Jaramillo ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Targeted Delivery ◽  
Specific Binding ◽  
Protein A ◽  
Antibody Fragments ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Binding Properties ◽  
Single Chain Variable Fragments ◽  
Selective Delivery

The use of the specific binding properties of monoclonal antibody fragments such as single-chain variable fragments (ScFv) for the selective delivery of antitumor therapeutics for cancer cells is attractive due...

scholarly journals Llama-Derived Single-Chain Antibody Fragments Directed to Rotavirus VP6 Protein Possess Broad Neutralizing Activity In Vitro and Confer Protection against Diarrhea in Mice

2008 ◽  
Vol 82 (19) ◽  
pp. 9753-9764 ◽  
Author(s):  
Lorena Garaicoechea ◽  
Aurelien Olichon ◽  
Gisela Marcoppido ◽  
Andrés Wigdorovitz ◽  
Marina Mozgovoj ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Binding Capacity ◽  
Antibody Fragments ◽  
Target Antigen ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Group A Rotavirus ◽  
Group A

ABSTRACT Group A rotavirus is one of the most common causes of severe diarrhea in human infants and newborn animals. Rotavirus virions are triple-layered particles. The outer capsid proteins VP4 and VP7 are highly variable and represent the major neutralizing antigens. The inner capsid protein VP6 is conserved among group A rotaviruses, is highly immunogenic, and is the target antigen of most immunodiagnosis tests. Llama-derived single-chain antibody fragments (VHH) are the smallest molecules with antigen-binding capacity and can therefore be expected to have properties different from conventional antibodies. In this study a library containing the VHH genes of a llama immunized with recombinant inner capsid protein VP6 was generated. Binders directed to VP6, in its native conformation within the viral particle, were selected and characterized. Four selected VHH directed to conformational epitopes of VP6 recognized all human and animal rotavirus strains tested and could be engineered for their use in immunodiagnostic tests for group A rotavirus detection. Three of the four VHH neutralized rotavirus in vivo independently of the strain serotype. Furthermore, this result was confirmed by in vivo partial protection against rotavirus challenge in a neonatal mouse model. The present study demonstrates for the first time a broad neutralization activity of VP6 specific VHH in vitro and in vivo. Neutralizing VHH directed to VP6 promise to become an essential tool for the prevention and treatment of rotavirus diarrhea.

scholarly journals Multimeric humanized varicella-zoster virus antibody fragments to gH neutralize virus while monomeric fragments do not

2001 ◽  
Vol 82 (8) ◽  
pp. 1959-1963 ◽  
Author(s):  
P. D. Drew ◽  
M. T. Moss ◽  
T. J. Pasieka ◽  
C. Grose ◽  
W. J. Harris ◽  
...  
Keyword(s):  
Varicella Zoster Virus ◽  
Cultured Cells ◽  
Cross Reactivity ◽  
Antibody Fragments ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Neutralizing Activity ◽  
Varicella Zoster ◽  
Complementarity Determining Region

Murine monoclonal antibody 206 (MAb mu206) binds to gH, the varicella-zoster virus (VZV) fusogen, neutralizing the virus in vitro in the absence of complement and inhibiting cell-to-cell spread and egress of VZV in cultured cells. We have humanized this antibody to generate MAb hu206 by complementarity determining region grafting. MAb hu206 retained binding and in vitro neutralizing activity, as well as cross-reactivity with ten different VZV strains. Single-chain antibody fragments (scAb) derived from MAb hu206 were produced in Escherichia coli. These scAb retained the binding properties of the whole antibody. However, monomeric scAb exhibited markedly reduced neutralizing activity compared to the bivalent parental MAb hu206. Shortening the peptide linker joining the VH to the Vκ domain from 14 to 5 or even 0 residues encouraged multimerization and increased neutralizing efficacy. The fact that Fab fragments enzymatically generated from whole MAb hu206 lost their neutralizing potency lent support to the proposal that valency is important for VZV neutralization at this epitope.

scholarly journals Ribosome display and selection of single-chain variable fragments effectively inhibit growth and progression of microspheres in vitro and in vivo

2018 ◽  
Vol 109 (5) ◽  
pp. 1503-1512
Author(s):  
Shangke Huang ◽  
Lu Feng ◽  
Gaili An ◽  
Xiaojin Zhang ◽  
Zixuan Zhao ◽  
...  
Keyword(s):  
Single Chain ◽  
Ribosome Display ◽  
Single Chain Variable Fragments ◽  
Selection Of

scholarly journals Enhanced Bovine Herpesvirus Type 1 Neutralization by Multimerized Single-Chain Variable Antibody Fragments Regardless of Differential Glycosylation

2012 ◽  
Vol 19 (8) ◽  
pp. 1150-1157 ◽  
Author(s):  
Yfke Pasman ◽  
Eva Nagy ◽  
Azad K. Kaushik
Keyword(s):  
Amino Acid ◽  
Bovine Herpesvirus ◽  
Antibody Fragments ◽  
Virus Neutralization ◽  
High Avidity ◽  
Single Chain ◽  
Herpesvirus Type ◽  
Bovine Herpesvirus Type 1

ABSTRACTSingle-chain variable antibody fragments (scFvs) with a 2-amino-acid linker capable of multimerization as di-, tri-, or tetrabodies that neutralize bovine herpesvirus type 1 (BoHV-1)in vitrowere constructed and expressed inPichia pastoris. In contrast to the monomeric form, multimeric scFvs had a higher virus neutralization potency, as evidenced by a 2-fold increase in their ability to neutralize BoHV-1 due to avidity effects. Mass spectrum (quadrupole time of flight [Q-TOF]) analyses of multimeric scFv demonstrated extensive heterogeneity due to differential cleavage, variable glycosylation (1 to 9 mannose residues), and the incorporation of minor unidentified adducts. Regardless of the differential glycosylation patterns, the scFvs recognized non-gB or -gE target viral epitopes in the BoHV-1 envelope fraction in a Western blot and also neutralized BoHV-1 in infected Madin-Darby kidney (MDBK) cellsin vitro. Indirect evidence for the noncovalent multimerization of scFv was the presence of a major peak of multimerized scFv without a His tag (due to differential cleavage) in the Q-TOF profile, unlike monomeric scFv, which copurified with normally His-tagged scFv and recognized the target antigen. Overall, differentially glycosylated recombinant scFvs against BoHV-1 with a short linker (2 amino acids) are capable of assembly into functional multimers that confer high avidity, resulting in increased virus neutralizationin vitrocompared to that of monovalent scFv with a long (18-amino-acid) flexible linker. Overall, recombinant multimerized scFv5-2L potentially provides a high-potency therapeutic and immunodiagnostic reagent against BoHV-1, which is suitable for passive immunization and topical application.

Functional Analysis of Phage Display Selected Single-Chain Variable Antibody Fragments (scFvs) Specific for Anti-FVIII Antibodies

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1499-1499
Author(s):  
Kerstin Brettschneider ◽  
Anja Naumann ◽  
Sonja Neimanis ◽  
Joerg Kahle ◽  
Christine Heller ◽  
...  
Keyword(s):  
Phage Display ◽  
Hemophilia A ◽  
Antibody Fragments ◽  
Dependent Manner ◽  
Single Chain ◽  
Concentration Dependent Manner ◽  
Fviii Activity ◽  
Inhibitory Antibodies

Abstract The development of inhibitory antibodies against coagulation factor VIII (FVIII) is currently the most serious complication for hemophilia A patients that undergo FVIII replacement therapy. In addition, non-hemophilia A patients can spontaneously develop inhibitory auto-antibodies to FVIII, which results in acquired haemophilia A. The control of the allo- or autoimmune response to FVIII apparently includes the elicitation of anti-idiotypic antibodies. In this study the capacity of anti-idiotypic single-chain variable antibody fragments (scFvs) for neutralization of inhibitory anti-FVIII antibodies (FVIII inhibitors) was evaluated in vitro and in vivo. Anti-idiotypic scFvs were selected from phage-displayed libraries against murine monoclonal FVIII-specific inhibitors. As the majority of inhibitory antibodies is directed against the A2 or C2 domain of FVIII, strongly inhibitory A2- and C2-specific antibodies served as targets. Selected scFvs were expressed as scFv-Fc fusion proteins. Analysis of the scFv-Fcs by ELISA confirmed the specific binding to the cognate targets and binding studies via surface plasmon resonance revealed high affinities within the nanomolar range. Further characterization showed that binding of inhibitors to immobilized FVIII was blocked by specific scFv-Fcs in vitro. The ability of scFv-Fcs to neutralize their corresponding inhibitors was analyzed in a functional clotting assay. By adding scFv-Fcs to plasma spiked with inhibitors, FVIII activity was restored to 80% in a concentration dependent manner. FVIII knockout mice served as model organism for testing the capacity of scFv-Fcs to restore coagulation in vivo. Subsequent injection of FVIII following the injection of the inhibitors resulted in a largely reduced FVIII activity. However, FVIII activity was recovered in a concentration dependent manner by adding cognate anti-idiotypes. The scFv-Fcs were either preincubated with the corresponding inhibitor or added to the FVIII mixture without preincubation. The latter represents an adaption to a therapeutic setting. In conclusion, phage display selected anti-idiotypic scFvs are able to bind and effectively neutralize their target inhibitors in vitro and in vivo. Based on these promising results the potential of anti-idiotypic scFvs for the development of specific cell based immunotherapies for hemophilia A patients with inhibitors is currently under investigation. Disclosures No relevant conflicts of interest to declare.

scholarly journals A microbial expression system for high-level production of scFv HIV-neutralizing antibody fragments in Escherichia coli

2019 ◽  
Vol 103 (21-22) ◽  
pp. 8875-8888 ◽  
Author(s):  
Marloes L. C. Petrus ◽  
Lukas A. Kiefer ◽  
Pranav Puri ◽  
Evert Heemskerk ◽  
Michael S. Seaman ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Neutralizing Antibodies ◽  
Expression System ◽  
Antibody Fragment ◽  
Green Fluorescence Protein ◽  
Antibody Fragments ◽  
Single Chain ◽  
Mammalian Expression ◽  
Anti Hiv

Abstract Monoclonal antibodies (mABs) are of great biopharmaceutical importance for the diagnosis and treatment of diseases. However, their production in mammalian expression hosts usually requires extensive production times and is expensive. Escherichia coli has become a new platform for production of functional small antibody fragment variants. In this study, we have used a rhamnose-inducible expression system that allows precise control of protein expression levels. The system was first evaluated for the cytoplasmic production of super folder green fluorescence protein (sfGFP) in various production platforms and then for the periplasmic production of the anti-HIV single-chain variable antibody fragment (scFv) of PGT135. Anti-HIV broadly neutralizing antibodies, like PGT135, have potential for clinical use to prevent HIV transmission, to promote immune responses and to eradicate infected cells. Different concentrations of L-rhamnose resulted in the controlled production of both sfGFP and scFv PGT135 antibody. In addition, by optimizing the culture conditions, the amount of scFv PGT135 antibody that was expressed soluble or as inclusions bodies could be modulated. The proteins were produced in batch bioreactors, with yields of 4.9 g/L for sfGFP and 0.8 g/L for scFv. The functionality of the purified antibodies was demonstrated by their ability to neutralize a panel of different HIV variants in vitro. We expect that this expression system will prove very useful for the development of a more cost-effective production process for proteins and antibody fragments in microbial cells.

Comparative study of inhibitory antibody derivatives towards thrombin activatable fibrinolysis inhibitor

2009 ◽  
Vol 102 (07) ◽  
pp. 69-75 ◽  
Author(s):  
Jan Develter ◽  
Maarten Dewilde ◽  
Ann Gils ◽  
Paul J. Declerck
Keyword(s):  
Antibody Fragments ◽  
Clot Lysis ◽  
Single Chain ◽  
Thrombin Activatable Fibrinolysis Inhibitor ◽  
Inhibitory Antibody ◽  
In Vivo Models ◽  
Fibrinolysis Inhibitor ◽  
Antibody Derivatives

SummaryThrombin activatable fibrinolysis inhibitor (TAFI) attenuates fibrinolysis and is considered as an attractive drug target. We generated two different antibody fragments, an antigen-binding fragment (Fab) and a single-chain variable fragment (scFv), derived from three distinct monoclonal antibodies (MAs) that inhibit the activation of TAFI by the thrombin/thrombomodulin complex (T/TM) and plasmin (MA-T1C10 and MA-T94H3) or by T/TM alone (MA-T12D11). The Fabs were obtained by papain digestion of the purified Mas, whereas the scFvs were cloned and subsequently expressed in bacteria. All antibody fragments revealed similar or slightly decreased affinities compared to those of the respective Mas, except scFv-T94H3. In the presence of a 16-fold molar excess of all antibody fragments, activation of TAFI by T/TM was completely blocked. Furthermore, Fab and scFv-derivatives from MA-T1C10 and MA-T94H3 were capable of interfering with the plasmin-mediated activation of TAFI. Addition of 850 nM of MA, Fab or scFv to an in-vitro clot lysis assay caused a significant reduction of clot lysis time (except for scFv-T94H3) and this effect was comparable to that of potato tuber carboxypeptidase inhibitor, a well-known TAFIa inhibitor. Dose-response experiments with the antibody (derivatives) in clot lysis and chromogenic assay revealed that the inhibitory capacity of the Fabs was comparable to that of the Mas, whereas the scFvs had a more reduced potency. In conclusion, these highly specific TAFI inhibitors are interesting tools to further evaluate the concept of TAFI inhibition in various in-vitro and in-vivo models.

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