Selection and characterization of camelid nanobodies towards urokinase-type plasminogen activator

Kaczmarek, Jakub Zbigniew; Skottrup, Peter Durand

doi:10.1016/j.molimm.2015.02.011

Cited by 11 publications

(8 citation statements)

References 42 publications

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“…Recently, new potential antitumor nanobodies have been developed against other membrane protein targets such as the DRs DR5 ( 63 , 80 ) and survivin ( 81 ), the chemokine receptors CXCR4 ( 82 ) and CXCR7 ( 83 ), the ion channel P2X7 ( 84 ), and the ecto-enzyme CD38 ( 24 , 85 ). Alternatively, antitumor nanobodies can be directed against receptor ligands, such as HGF ( 68 ), VEGF ( 86 , 87 ), urokinase-type plasminogen activator ( 88 ), or CXCL11/12 ( 89 ). In a number of in vivo xenograft studies, treatment with bispecific or multivalent nanobodies resulted in delay of tumor growth ( 64 , 68 ) and/or inhibition of angiogenesis ( 83 ).…”

Section: “Naked” Monomeric and Multimeric Antitumor Nanobodiesmentioning

confidence: 99%

Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics

Bannas¹,

2017

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Monoclonal antibodies have revolutionized cancer therapy. However, delivery to tumor cells in vivo is hampered by the large size (150 kDa) of conventional antibodies. The minimal target recognition module of a conventional antibody is composed of two non-covalently associated variable domains (VH and VL). The proper orientation of these domains is mediated by their hydrophobic interface and is stabilized by their linkage to disulfide-linked constant domains (CH1 and CL). VH and VL domains can be fused via a genetic linker into a single-chain variable fragment (scFv). scFv modules in turn can be fused to one another, e.g., to generate a bispecific T-cell engager, or they can be fused in various orientations to antibody hinge and Fc domains to generate bi- and multispecific antibodies. However, the inherent hydrophobic interaction of VH and VL domains limits the stability and solubility of engineered antibodies, often causing aggregation and/or mispairing of V-domains. Nanobodies (15 kDa) and nanobody-based human heavy chain antibodies (75 kDa) can overcome these limitations. Camelids naturally produce antibodies composed only of heavy chains in which the target recognition module is composed of a single variable domain (VHH or Nb). Advantageous features of nanobodies include their small size, high solubility, high stability, and excellent tissue penetration in vivo. Nanobodies can readily be linked genetically to Fc-domains, other nanobodies, peptide tags, or toxins and can be conjugated chemically at a specific site to drugs, radionuclides, photosensitizers, and nanoparticles. These properties make them particularly suited for specific and efficient targeting of tumors in vivo. Chimeric nanobody-heavy chain antibodies combine advantageous features of nanobodies and human Fc domains in about half the size of a conventional antibody. In this review, we discuss recent developments and perspectives for applications of nanobodies and nanobody-based human heavy chain antibodies as antitumor therapeutics.

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Section: “Naked” Monomeric and Multimeric Antitumor Nanobodiesmentioning

confidence: 99%

Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics

Bannas¹,

2017

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“…This interaction between scu-PA and plasminogen plays an important role in the initiation and acceleration of fibrinolysis. S-2444 is a specific substrate of tcu-PA that can catalyze its hydrolysis to p-nitroaniline [ 45 , 51 ]. Therefore, in our study, the uPA activity was monitored using S-2444 to effectively reflect the role of FGFC1 in the reaction system.…”

Section: Discussionmentioning

confidence: 99%

A Novel Marine Pyran-Isoindolone Compound Enhances Fibrin Lysis Mediated by Single-Chain Urokinase-Type Plasminogen Activator

et al. 2022

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Fungi fibrinolytic compound 1 (FGFC1) is a rare pyran-isoindolone derivative with fibrinolytic activity. The aim of this study was to further determine the effect of FGFC1 on fibrin clots lysis in vitro. We constructed a fibrinolytic system containing single-chain urokinase-type plasminogen activator (scu-PA) and plasminogen to measure the fibrinolytic activity of FGFC1 using the chromogenic substrate method. After FITC-fibrin was incubated with increasing concentrations of FGFC1, the changes in the fluorescence intensity and D-dimer in the lysate were measured using a fluorescence microplate reader. The fibrin clot structure induced by FGFC1 was observed and analyzed using a scanning electron microscope and laser confocal microscope. We found that the chromogenic reaction rate of the mixture system increased from (15.9 ± 1.51) × 10−3 min−1 in the control group to (29.7 ± 1.25) × 10−3 min−1 for 12.8 μM FGFC1(p < 0.01). FGFC1 also significantly increased the fluorescence intensity and d-dimer concentration in FITC fibrin lysate. Image analysis showed that FGFC1 significantly reduced the fiber density and increased the fiber diameter and the distance between protofibrils. These results show that FGFC1 can effectively promote fibrin lysis in vitro and may represent a novel candidate agent for thrombolytic therapy.

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“…Early evaluation of nanobodies raised against carbonic anhydrase and amylase demonstrated inhibitory activity for several of them (136), encouraging further experiments to deploy nanobodies to modulate enzyme activity. Nanobodies that bind to and inhibit the protease urokinase-type plasminogen activator (uPA) (137)(138)(139), which can contribute to cancer metastasis, may find clinical application. Crystallization of complexes between uPA and nanobodies shows how substrate binds and reveals the conformational equilibria that contribute (137,139).…”

Section: Targeting Extracellular Proteinsmentioning

confidence: 99%

Exploring cellular biochemistry with nanobodies

Cheloha

Harmand

Wijne

et al. 2020

Journal of Biological Chemistry

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Reagents that bind tightly and specifically to biomolecules of interest remain essential in the exploration of biology and in their ultimate application to medicine. Besides ligands for receptors of known specificity, agents commonly used for this purpose are monoclonal antibodies derived from mice, rabbits, and other animals. However, such antibodies can be expensive to produce, challenging to engineer, and are not necessarily stable in the context of the cellular cytoplasm, a reducing environment. Heavy chain-only antibodies, discovered in camelids, have been truncated to yield single domain antibody fragments (VHHs or nanobodies) that overcome many of these shortcomings. While known as crystallization chaperones for membrane proteins or as simple alternatives to conventional antibodies, nanobodies have been applied in settings where the use of standard antibodies or their derivatives would be impractical or impossible. We review recent examples in which the unique properties of nanobodies have been combined with complementary methods, such as chemical functionalization, to provide tools with unique and useful properties.

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Selection and characterization of camelid nanobodies towards urokinase-type plasminogen activator

Cited by 11 publications

References 42 publications

Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics

Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics

A Novel Marine Pyran-Isoindolone Compound Enhances Fibrin Lysis Mediated by Single-Chain Urokinase-Type Plasminogen Activator

Exploring cellular biochemistry with nanobodies

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