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1.
Theranostics ; 11(11): 5525-5538, 2021.
Article in English | MEDLINE | ID: mdl-33859761

ABSTRACT

Background: The non-homogenous distribution of antibody-drug conjugates (ADCs) within solid tumors is a major limiting factor for their wide clinical application. Nanobodies have been shown to rapidly penetrate into xenografts, achieving more homogeneous tumor targeting. However, their rapid renal clearance can hamper their application as nanobody drug conjugates (NDCs). Here, we evaluate whether half-life extension via non-covalent interaction with albumin can benefit the efficacy of a HER2-targeted NDC. Methods: HER2-targeted nanobody 11A4 and the irrelevant nanobody R2 were genetically fused to an albumin-binding domain (ABD) at their C-terminus. Binding to both albumin and tumor cells was determined by ELISA-based assays. The internalization potential as well as the in vitro efficacy of NDCs were tested on HER2 expressing cells. Serum half-life of iodinated R2 and R2-ABD was studied in tumor-free mice. The distribution of fluorescently labelled 11A4 and 11A4-ABD was assessed in vitro in 3D spheroids. Subsequently, the in vivo distribution was evaluated by optical molecular imaging and ex vivo by tissue biodistribution and tumor immunohistochemical analysis after intravenous injection of IRDye800-conjugated nanobodies in mice bearing HER2-positive subcutaneous xenografts. Finally, efficacy studies were performed in HER2-positive NCI-N87 xenograft-bearing mice intravenously injected with a single dose (250 nmol/kg) of nanobodies conjugated to auristatin F (AF) either via a maleimide or the organic Pt(II)­based linker, coined Lx®. Results: 11A4-ABD was able to bind albumin and HER2 and was internalized by HER2 expressing cells, irrespective of albumin presence. Interaction with albumin did not alter its distribution through 3D spheroids. Fusion to ABD resulted in a 14.8-fold increase in the serum half-life, as illustrated with the irrelevant nanobody. Furthermore, ABD fusion prolonged the accumulation of 11A4-ABD in HER2-expressing xenografts without affecting the expected homogenous intratumoral distribution. Next to that, reduced kidney retention of ABD-fused nanobodies was observed. Finally, a single dose administration of either 11A4-ABD-maleimide-AF or 11A4-ABD-Lx-AF led to long-lasting tumor remission in HER2-positive NCI-N87 xenograft-bearing mice. Conclusion: Our results demonstrate that genetic fusion of a nanobody to ABD can significantly extend serum half-life, resulting in prolonged and homogenous tumor accumulation. Most importantly, as supported by the impressive anti-tumor efficacy observed after a single dose administration of 11A4-ABD-AF, our data reveal that monovalent internalizing ABD-fused nanobodies have potential for the development of highly effective NDCs.


Subject(s)
Albumins/metabolism , Antineoplastic Agents/pharmacology , Immunoconjugates/pharmacology , Receptor, ErbB-2/metabolism , Single-Domain Antibodies/physiology , Aminobenzoates/pharmacology , Animals , Antineoplastic Agents/metabolism , Cell Line, Tumor , Female , Half-Life , Humans , Immunoconjugates/metabolism , Mice , Mice, Inbred BALB C , Mice, Nude , Oligopeptides/pharmacology , Single-Domain Antibodies/metabolism , Tissue Distribution/physiology
2.
Methods Mol Biol ; 2127: 167-184, 2020.
Article in English | MEDLINE | ID: mdl-32112322

ABSTRACT

Nanobodies, small recombinant binders derived from camelid single chain antibodies, have become widely used tools in a diversity of disciplines related to membrane proteins. They are applied as chaperones in crystallization and blockers or modifiers of protein activity among numerous other applications. Their simple architecture as a single polypeptide chain, in contrast to classical antibodies, enables straightforward cloning, library generation, and recombinant expression. The small diameter and the pointed wedge-like shape of the antigen-binding site underlies binding to hollows and crevices of membrane proteins and renders nanobodies often conformation specific making them a preferred type of chaperone. Here we describe a simple protocol for the recombinant production of nanobodies in E. coli and their purification. We expand the current repertoire of usage further by describing a procedure for enlarging nanobodies on their C-terminal end to generate "macrobodies," without interfering with their original characteristics. These enlarged nanobodies extend the application as a chaperone in crystallography and can serve to increase the mass for small targets in single particle electron cryo-microscopy, a field where nanobodies had so far only limited effect because of their small size.


Subject(s)
Biochemistry/methods , Membrane Proteins/immunology , Single-Domain Antibodies/biosynthesis , Single-Domain Antibodies/isolation & purification , Single-Domain Antibodies/physiology , Animals , Chromatography, Gel , Cloning, Molecular/methods , Escherichia coli , Eukaryotic Cells , Gene Expression Regulation, Bacterial , Humans , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Protein Binding , Protein Conformation , Transformation, Bacterial
3.
J Biotechnol ; 293: 17-23, 2019 Mar 10.
Article in English | MEDLINE | ID: mdl-30690101

ABSTRACT

Vaccination is as one of the most beneficial biopharmaceutical interventions against pathogens due to its ability to induce adaptive immunity through targeted activation of the immune system. Each vaccine needs a tailor-made set of tests in order to monitor its quality throughout the development and manufacturing. The analysis of the conformational state of protein nanoparticles is one of the key steps in vaccine quality control. The enzyme lumazine synthase from Brucella spp. (BLS) acts as a potent oral and systemic immunogen. BLS has been used as a carrier of foreign peptides, protein domains and whole proteins, serving as a versatile platform for vaccine engineering purposes. Here, we show the generation and characterization of four families of nanobodies (Nbs) which only recognize BLS in its native conformational state and that bind to its active site. The present results support the use of conformation-sensitive Nbs as molecular probes during the development and production of vaccines based on the BLS platform. Finally, we propose Nbs as useful molecular tools targeting other protein scaffolds with potential applications in nano-and biotechnology.


Subject(s)
Multienzyme Complexes , Single-Domain Antibodies , Bacterial Proteins/chemistry , Bacterial Proteins/physiology , Brucella/enzymology , Escherichia coli/genetics , HEK293 Cells , Humans , Multienzyme Complexes/chemistry , Multienzyme Complexes/physiology , Protein Conformation , Protein Folding , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/physiology , Vaccines, Subunit
4.
Cell ; 174(3): 649-658.e16, 2018 07 26.
Article in English | MEDLINE | ID: mdl-30033369

ABSTRACT

Synthetic multicellular systems hold promise as models for understanding natural development of biofilms and higher organisms and as tools for engineering complex multi-component metabolic pathways and materials. However, such efforts require tools to adhere cells into defined morphologies and patterns, and these tools are currently lacking. Here, we report a 100% genetically encoded synthetic platform for modular cell-cell adhesion in Escherichia coli, which provides control over multicellular self-assembly. Adhesive selectivity is provided by a library of outer membrane-displayed nanobodies and antigens with orthogonal intra-library specificities, while affinity is controlled by intrinsic adhesin affinity, competitive inhibition, and inducible expression. We demonstrate the resulting capabilities for quantitative rational design of well-defined morphologies and patterns through homophilic and heterophilic interactions, lattice-like self-assembly, phase separation, differential adhesion, and sequential layering. Compatible with synthetic biology standards, this adhesion toolbox will enable construction of high-level multicellular designs and shed light on the evolutionary transition to multicellularity.


Subject(s)
Cell Adhesion/physiology , Metabolic Engineering/methods , Synthetic Biology/methods , Bacterial Physiological Phenomena , Biological Evolution , Cell Adhesion/genetics , Cell Differentiation/genetics , Cell Differentiation/physiology , Escherichia coli/genetics , Gene Library , Metabolic Networks and Pathways , Single-Domain Antibodies/genetics , Single-Domain Antibodies/immunology , Single-Domain Antibodies/physiology
5.
Toxins (Basel) ; 10(3)2018 03 01.
Article in English | MEDLINE | ID: mdl-29494518

ABSTRACT

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) are a subset of pathogens leading to illnesses such as diarrhea, hemolytic uremic syndrome and even death. The Shiga toxins are the main virulence factors and divided in two groups: Stx1 and Stx2, of which the latter is more frequently associated with severe pathologies in humans. RESULTS: An immune library of nanobodies (Nbs) was constructed after immunizing an alpaca with recombinant Shiga toxin-2a B subunit (rStx2aB), to retrieve multiple rStx2aB-specific Nbs. The specificity of five Nbs towards rStx2aB was confirmed in ELISA and Western blot. Nb113 had the highest affinity (9.6 nM) and its bivalent construct exhibited a 100-fold higher functional affinity. The structure of the Nb113 in complex with rStx2aB was determined via X-ray crystallography. The crystal structure of the Nb113-rStx2aB complex revealed that five copies of Nb113 bind to the rStx2aB pentamer and that the Nb113 epitope overlaps with the Gb3 binding site, thereby providing a structural basis for the neutralization of Stx2a by Nb113 that was observed on Vero cells. Finally, the tandem-repeated, bivalent Nb1132 exhibits a higher toxin neutralization capacity compared to monovalent Nb113. CONCLUSIONS: The Nb of highest affinity for rStx2aB is also the best Stx2a and Stx2c toxin neutralizing Nb, especially in a bivalent format. This lead Nb neutralizes Stx2a by competing for the Gb3 receptor. The fusion of the bivalent Nb1132 with a serum albumin specific Nb is expected to combine high toxin neutralization potential with prolonged blood circulation.


Subject(s)
Antibodies, Neutralizing , Recombinant Proteins , Shiga Toxin 2 , Single-Domain Antibodies , Animals , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/physiology , Camelids, New World/immunology , Chlorocebus aethiops , Protein Domains , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Shiga Toxin 2/chemistry , Shiga Toxin 2/genetics , Shiga Toxin 2/immunology , Shiga Toxin 2/metabolism , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/physiology , Vero Cells
6.
Proc Natl Acad Sci U S A ; 115(12): 3036-3041, 2018 03 20.
Article in English | MEDLINE | ID: mdl-29507218

ABSTRACT

Protein-protein binding is key in cellular signaling processes. Molecular dynamics (MD) simulations of protein-protein binding, however, are challenging due to limited timescales. In particular, binding of the medically important G-protein-coupled receptors (GPCRs) with intracellular signaling proteins has not been simulated with MD to date. Here, we report a successful simulation of the binding of a G-protein mimetic nanobody to the M2 muscarinic GPCR using the robust Gaussian accelerated MD (GaMD) method. Through long-timescale GaMD simulations over 4,500 ns, the nanobody was observed to bind the receptor intracellular G-protein-coupling site, with a minimum rmsd of 2.48 Å in the nanobody core domain compared with the X-ray structure. Binding of the nanobody allosterically closed the orthosteric ligand-binding pocket, being consistent with the recent experimental finding. In the absence of nanobody binding, the receptor orthosteric pocket sampled open and fully open conformations. The GaMD simulations revealed two low-energy intermediate states during nanobody binding to the M2 receptor. The flexible receptor intracellular loops contribute remarkable electrostatic, polar, and hydrophobic residue interactions in recognition and binding of the nanobody. These simulations provided important insights into the mechanism of GPCR-nanobody binding and demonstrated the applicability of GaMD in modeling dynamic protein-protein interactions.


Subject(s)
Receptor, Muscarinic M2/metabolism , Receptors, G-Protein-Coupled/metabolism , Single-Domain Antibodies/physiology , Computer Simulation , Models, Molecular , Protein Binding , Protein Conformation , Receptor, Muscarinic M2/chemistry , Receptors, G-Protein-Coupled/chemistry , Thermodynamics
7.
Biomed Pharmacother ; 102: 230-241, 2018 Jun.
Article in English | MEDLINE | ID: mdl-29567535

ABSTRACT

Cortactin is a multidomain actin binding protein that activates Arp2/3 mediated branched actin polymerization. This is essential for the formation of protrusive structures during cancer cell invasion. Invadopodia are cancer cell-specific membrane protrusions, specialized at extracellular matrix degradation and essential for invasion and tumor metastasis. Given the unequivocal role of cortactin at every stage of invadopodium formation, it is considered an invadopodium marker and potential drug target. We used cortactin nanobodies to examine the role of cortactin domain-specific function at endogenous protein level. Two cortactin nanobodies target the central region of cortactin with high specificity. One nanobody interacts with the actin binding repeats whereas the other targets the proline rich region and was found to reduce EGF-induced cortactin phosphorylation. After intracellular expression as an intrabody, they are both capable of tracing their target in the complex environment of the cytoplasm, and disturb cortactin functions during invadopodia formation and extracellular matrix degradation. These data illustrate the use of nanobodies as a research tool to dissect the role of cortactin in cancer cell motility. This information can contribute to the development of novel therapeutics for tumor cell migration and metastasis.


Subject(s)
Actins/metabolism , Cell Surface Extensions/metabolism , Cortactin/metabolism , Extracellular Matrix/pathology , Neoplasm Invasiveness , Proline/metabolism , Single-Domain Antibodies/physiology , Carrier Proteins/metabolism , Cell Line, Tumor , Extracellular Matrix/metabolism , Head and Neck Neoplasms/metabolism , Head and Neck Neoplasms/pathology , Humans , Microfilament Proteins/metabolism , Neoplasms, Squamous Cell/metabolism , Neoplasms, Squamous Cell/pathology , Protein Binding , Protein Domains
8.
Proc Natl Acad Sci U S A ; 115(9): 2126-2131, 2018 02 27.
Article in English | MEDLINE | ID: mdl-29440485

ABSTRACT

Sensitive and specific antibodies are essential for detecting molecules in cells and tissues. However, currently used polyclonal and monoclonal antibodies are often less specific than desired, difficult to produce, and available in limited quantities. A promising recent approach to circumvent these limitations is to employ chemically defined antigen-combining domains called "nanobodies," derived from single-chain camelid antibodies. Here, we used nanobodies to prepare sensitive unimolecular detection reagents by genetically fusing cDNAs encoding nanobodies to enzymatic or antigenic reporters. We call these fusions between a reporter and a nanobody "RANbodies." They can be used to localize epitopes and to amplify signals from fluorescent proteins. They can be generated and purified simply and in unlimited amounts and can be preserved safely and inexpensively in the form of DNA or digital sequence.


Subject(s)
DNA, Complementary/chemistry , Single-Domain Antibodies/physiology , Amino Acid Sequence , Animals , Antibody Specificity , Antigen-Antibody Reactions , Camelus/immunology , DNA, Complementary/metabolism , Enzyme-Linked Immunosorbent Assay , Green Fluorescent Proteins/metabolism , Immunohistochemistry , Sensitivity and Specificity , Single-Domain Antibodies/chemistry
9.
Plant Biotechnol J ; 16(2): 660-671, 2018 02.
Article in English | MEDLINE | ID: mdl-28796912

ABSTRACT

Since their discovery, single-domain antigen-binding fragments of camelid-derived heavy-chain-only antibodies, also known as nanobodies (Nbs), have proven to be of outstanding interest as therapeutics against human diseases and pathogens including viruses, but their use against phytopathogens remains limited. Many plant viruses including Grapevine fanleaf virus (GFLV), a nematode-transmitted icosahedral virus and causal agent of fanleaf degenerative disease, have worldwide distribution and huge burden on crop yields representing billions of US dollars of losses annually, yet solutions to combat these viruses are often limited or inefficient. Here, we identified a Nb specific to GFLV that confers strong resistance to GFLV upon stable expression in the model plant Nicotiana benthamiana and also in grapevine rootstock, the natural host of the virus. We showed that resistance was effective against a broad range of GFLV isolates independently of the inoculation method including upon nematode transmission but not against its close relative, Arabis mosaic virus. We also demonstrated that virus neutralization occurs at an early step of the virus life cycle, prior to cell-to-cell movement. Our findings will not only be instrumental to confer resistance to GFLV in grapevine, but more generally they pave the way for the generation of novel antiviral strategies in plants based on Nbs.


Subject(s)
Plant Diseases/immunology , Plant Diseases/virology , Nepovirus/pathogenicity , Plant Viruses/genetics , Plant Viruses/physiology , Single-Domain Antibodies/genetics , Single-Domain Antibodies/physiology
10.
FASEB J ; 31(6): 2460-2476, 2017 06.
Article in English | MEDLINE | ID: mdl-28235780

ABSTRACT

Cancer cells exploit different strategies to escape from the primary tumor, gain access to the circulation, disseminate throughout the body, and form metastases, the leading cause of death by cancer. Invadopodia, proteolytically active plasma membrane extensions, are essential in this escape mechanism. Cortactin is involved in every phase of invadopodia formation, and its overexpression is associated with increased invadopodia formation, extracellular matrix degradation, and cancer cell invasion. To analyze endogenous cortactin domain function in these processes, we characterized the effects of nanobodies that are specific for the N-terminal acidic domain of cortactin and expected to target small epitopes within this domain. These nanobodies inhibit cortactin-mediated actin-related protein (Arp)2/3 activation, and, after their intracellular expression in cancer cells, decrease invadopodia formation, extracellular matrix degradation, and cancer cell invasion. In addition, one of the nanobodies affects Arp2/3 interaction and invadopodium stability, and a nanobody targeting the Src homology 3 domain of cortactin enabled comparison of 2 functional regions in invadopodium formation or stability. Given their common and distinct effects, we validate cortactin nanobodies as an instrument to selectively block and study distinct domains within a protein with unprecedented precision, aiding rational future generation of protein domain-selective therapeutic compounds.-Bertier, L., Boucherie, C., Zwaenepoel, O., Vanloo, B., Van Troys, M., Van Audenhove, I., Gettemans, J. Inhibitory cortactin nanobodies delineate the role of NTA- and SH3-domain-specific functions during invadopodium formation and cancer cell invasion.


Subject(s)
Cortactin/chemistry , Neoplasm Invasiveness , Podosomes/physiology , Single-Domain Antibodies/physiology , Cell Line, Tumor , Cloning, Molecular , Cortactin/metabolism , Epitopes , Gene Expression Regulation, Neoplastic , Humans , Indoles/pharmacology , Protein Domains
11.
Annu Rev Pharmacol Toxicol ; 57: 19-37, 2017 01 06.
Article in English | MEDLINE | ID: mdl-27959623

ABSTRACT

Ligand-induced activation of G protein-coupled receptors (GPCRs) is a key mechanism permitting communication between cells and organs. Enormous progress has recently elucidated the structural and dynamic features of GPCR transmembrane signaling. Nanobodies, the recombinant antigen-binding fragments of camelid heavy-chain-only antibodies, have emerged as important research tools to lock GPCRs in particular conformational states. Active-state stabilizing nanobodies have elucidated several agonist-bound structures of hormone-activated GPCRs and have provided insight into the dynamic character of receptors. Nanobodies have also been used to stabilize transient GPCR transmembrane signaling complexes, yielding the first structural insights into GPCR signal transduction across the cellular membrane. Beyond their in vitro uses, nanobodies have served as conformational biosensors in living systems and have provided novel ways to modulate GPCR function. Here, we highlight several examples of how nanobodies have enabled the study of GPCR function and give insights into potential future uses of these important tools.


Subject(s)
Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/physiology , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/physiology , Animals , Dose-Response Relationship, Drug , Humans , Isoproterenol/chemistry , Isoproterenol/pharmacology , Ligands , Protein Binding/physiology , Protein Conformation/drug effects , Protein Structure, Secondary , Protein Structure, Tertiary
12.
Biochem J ; 459(1): 59-69, 2014 Apr 01.
Article in English | MEDLINE | ID: mdl-24438162

ABSTRACT

GAK (cyclin G-associated kinase) is a key regulator of clathrin-coated vesicle trafficking and plays a central role during development. Additionally, due to the unusually high plasticity of its catalytic domain, it is a frequent 'off-target' of clinical kinase inhibitors associated with respiratory side effects of these drugs. In the present paper, we determined the crystal structure of the GAK catalytic domain alone and in complex with specific single-chain antibodies (nanobodies). GAK is constitutively active and weakly associates in solution. The GAK apo structure revealed a dimeric inactive state of the catalytic domain mediated by an unusual activation segment interaction. Co-crystallization with the nanobody NbGAK_4 trapped GAK in a dimeric arrangement similar to the one observed in the apo structure, whereas NbGAK_1 captured the activation segment of monomeric GAK in a well-ordered conformation, representing features of the active kinase. The presented structural and biochemical data provide insight into the domain plasticity of GAK and demonstrate the utility of nanobodies to gain insight into conformational changes of dynamic molecules. In addition, we present structural data on the binding mode of ATP mimetic inhibitors and enzyme kinetic data, which will support rational inhibitor design of inhibitors to reduce the off-target effect on GAK.


Subject(s)
Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/physiology , Protein Multimerization/physiology , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/physiology , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/physiology , Animals , Apoproteins/chemistry , Apoproteins/physiology , Camelus , Catalytic Domain/physiology , Crystallization/methods , Enzyme Activation/physiology , Humans , Protein Conformation
13.
J Thromb Haemost ; 12(2): 229-36, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24354544

ABSTRACT

BACKGROUND: Down-regulation of fibrinolysis due to cleavage of C-terminal lysine residues from partially degraded fibrin is mainly exerted by the carboxypeptidase activity of activated thrombin-activatable fibrinolysis inhibitor (TAFIa). Recently, some intrinsic carboxypeptidase activity (i.e. zymogen activity) was reported for the proenzyme (TAFI); however, there is some discussion about its ability to cleave high molecular weight substrates. OBJECTIVE: We aimed to identify and characterize nanobodies toward mouse TAFI (mTAFI) that stimulate the zymogen activity and to test their effect in an in vitro clot lysis assay and an in vivo mouse thromboembolism model. METHODS AND RESULTS: Screening of a library of nanobodies toward mTAFI revealed one nanobody (VHH-mTAFI-i49) that significantly stimulates the zymogen activity of mTAFI from undetectable (< 0.35 U mg⁻¹) to 4.4 U mg⁻¹ (at a 16-fold molar ratio over mTAFI). The generated carboxypeptidase activity is unstable at 37 °C. Incubation of mTAFI with VHH-mTAFI-i49 revealed a time-dependent reduced activatability of mTAFI. Epitope mapping revealed that Arg227 and Lys212 are important for the nanobody/mTAFI interaction and suggest destabilization of mTAFI by disrupting the stabilizing interaction between the activation peptide and the dynamic flap region. In vitro clot lysis experiments revealed an enhanced clot lysis due to a reduced activation of mTAFI during clot formation. In vivo application of VHH-mTAFI-i49 in a mouse thromboembolism model decreased dose-dependently the fibrin deposition in the lungs of thromboembolism-induced mice. CONCLUSION: The novel, nanobody-induced, reduced activatability of mTAFI demonstrates to be a very potent approach to enhance clot lysis.


Subject(s)
Carboxypeptidase B2/antagonists & inhibitors , Single-Domain Antibodies/physiology , Animals , Mice
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