Generation of Single Domain Antibody Fragments Derived from Camelids and Generation of Manifold Constructs

Vincke, Cécile; Gutiérrez, Carlos; Wernery, Ulrich; Devoogdt, Nick; Hassanzadeh-Ghassabeh, Gholamreza; Muyldermans, Serge

doi:10.1007/978-1-61779-974-7_8

Cited by 151 publications

(184 citation statements)

References 14 publications

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“…Anti-MMR sdAbs were isolated from an immune sdAb phagedisplay library after immunization of an alpaca (Vicugna pacos) according to a 6-wk alternating schedule of weekly injections of recombinant human or mouse monomeric fusion proteins (11,15). After production of sdAb-displaying bacteriophages in Escherichia coli, biopannings and enzyme-linked immunosorbent assay screenings were performed to select MMR binding sdAbs.…”

Section: Materials and Methods Sdab Generation And Selection Of Lead mentioning

confidence: 99%

“…After production of sdAb-displaying bacteriophages in Escherichia coli, biopannings and enzyme-linked immunosorbent assay screenings were performed to select MMR binding sdAbs. Purification of the hexahistidine-tagged sdAbs from the periplasm was performed as described previously (11,15). The binding characteristics of selected sdAbs were compared using surface plasmon resonance and flow cytometry.…”

Section: Materials and Methods Sdab Generation And Selection Of Lead mentioning

confidence: 99%

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PET Imaging of Macrophage Mannose Receptor–Expressing Macrophages in Tumor Stroma Using ¹⁸F-Radiolabeled Camelid Single-Domain Antibody Fragments

et al. 2015

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Tumor-associated macrophages constitute a major component of the stroma of solid tumors, encompassing distinct subpopulations with different characteristics and functions. We aimed to identify M2-oriented tumor-supporting macrophages within the tumor microenvironment as indicators of cancer progression and prognosis, using PET imaging. This can be realized by designing 18 F-labeled camelid single-domain antibody fragments (sdAbs) specifically targeting the macrophage mannose receptor (MMR), which has been identified as an important biomarker on this cell population. Methods: Crossreactive anti-MMR sdAbs were generated after immunization of an alpaca with the extracellular domains of both human and mouse MMR. The lead binder was chosen on the basis of comparisons of binding affinity and in vivo pharmacokinetics. The PET tracer 18 F-fluorobenzoate (FB)-anti-MMR sdAb was developed using the prosthetic group N-succinimidyl-4-18 F-fluorobenzoate ( 18 F-SFB), and its biodistribution, tumor-targeting potential, and specificity in terms of macrophage and MMR targeting were evaluated in mouse tumor models. Results: Four sdAbs were selected after affinity screening, but only 2 were found to be cross-reactive for human and mouse MMR. The lead anti-MMR 3.49 sdAb, bearing an affinity of 12 and 1.8 nM for mouse and human MMR, respectively, was chosen for its favorable in vivo biodistribution profile and tumor-targeting capacity. 18 F-FB-anti-MMR 3.49 sdAb was synthesized with a 5%-10% radiochemical yield using an automated and optimized protocol. In vivo biodistribution analyses showed fast clearance via the kidneys and retention in MMRexpressing organs and tumor. The kidney retention of the fluorinated sdAb was 20-fold lower than a 99m Tc-labeled counterpart. Compared with MMR-and C-C chemokine receptor 2-deficient mice, significantly higher uptake was observed in tumors grown in wild-type mice, demonstrating the specificity of the 18 F tracer for MMR and macrophages, respectively. Conclusion: Anti-MMR 3.49 was denoted as the lead cross-reactive MMR-targeting sdAb. 18 F radiosynthesis was optimized, providing an optimal probe for PET imaging of the tumor-promoting macrophage subpopulation in the tumor stroma. Dur ing tumor development, myeloid cells are attracted to the tumor stroma. These infiltrating immune cells are versatile, adopting different activation states in response to a changing microenvironment, leading to subsets of tumor-associated macrophages (TAMs) with specialized functions (1,2). Two main morphologically distinct TAM subsets can be distinguished on the basis of major histocompatibility complex (MHC) class II expression levels in multiple mouse tumor models. Tumor promotion has been linked with an accumulation of M2-oriented MHC II low TAMs in lung and breast carcinoma (3,4). Accordingly, MHC II low TAMs were found to reside primarily in less oxygenated zones, express hypoxia-regulated genes, and facilitate the angiogenic switch (5). Interestingly, the macrophage mannose receptor (MMR, CD206), a typical M2...

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Section: Materials and Methods Sdab Generation And Selection Of Lead mentioning

confidence: 99%

Section: Materials and Methods Sdab Generation And Selection Of Lead mentioning

confidence: 99%

PET Imaging of Macrophage Mannose Receptor–Expressing Macrophages in Tumor Stroma Using ¹⁸F-Radiolabeled Camelid Single-Domain Antibody Fragments

et al. 2015

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“…Generation of Anti-uPA Nanobodies-The immunization and construction of the nanobody phage library were conducted as described previously using single-chain zymogen uPA as antigen (38). Briefly, selection of anti-human uPA nanobodies was performed by immobilizing two-chain active uPA (ProSpec-Tany TechnoGene) (100 g/ml) in 96-well MaxiSorp immunoplates (Nunc).…”

Section: Methodsmentioning

confidence: 99%

A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior

Kromann-Hansen

Oldenburg

Yung

et al. 2016

Journal of Biological Chemistry

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A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30 -40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors.Serine proteases catalyze the hydrolysis of peptide bonds and are involved in numerous physiological processes, including digestion, blood clotting, fibrinolysis, complement activation, and turnover of the extracellular matrix (1). Neutralizing serine protease activity using orthosteric inhibitors, i.e. active site binding inhibitors, has been shown to be a successful therapeutic strategy for a number of pathological conditions, although the similar active site topology in all serine proteases increases the risk of off-target effects. Today, serine protease inhibitors are clinically used for therapy of several diseases, including thrombosis and bleeding disorders (2-4).All serine proteases catalyze the same type of hydrolytic reaction utilizing the same biochemical mechanism. Serine protease-catalyzed hydrolysis of a scissile bond proceeds through a highly conserved mechanism involving two tetrahedral intermediates and an acyl-enzyme complex. The polypeptide substrate is aligned in the active site of the protease interacting with the substrate specificity pockets denoted S1-Sn and S1Ј-SnЈ on the acyl and leaving group side of the scissile bond, respectively (5). The P1 residue of the substrate binds into the S1 pocket, and its carbonyl oxygen atom is inserted into the so-called oxyanion hole (backbone amides of chymotrypsinogen numbering). The catalytic triad (His-57, in the protease generates the required nucleophile for the attack of the hydroxyl group of Ser-195 on the carbonyl group of the P1-P1Ј scissile bond to form the first tetrahedral intermediate and later the acyl-enzyme. Following release of the P1Ј-leaving group, a water molecule performs a second nucleophilic attack, thereby completing the cycle (6).Peptide segments that bind the active site of serine proteases in a substrate-like manner may behave like an inhibitor or substrate. Ho...

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“…Three weeks later, the animal was subcutaneously boosted with 2 Â 10 7 HEK-293T cells that were transiently transfected with the pMet7 vector harboring the hCD20 gene. Four days later, anticoagulated blood was collected for sdAb library construction as described earlier (26). Next, sdAbs were phage displayed and biopannings (27) were performed on hCD20 pos CHO-K1 cells.…”

Section: Generation Of Anti-hcd20 Sdabsmentioning

confidence: 99%

Theranostic Radiolabeled Anti-CD20 sdAb for Targeted Radionuclide Therapy of Non-Hodgkin Lymphoma

Krasniqi

D’Huyvetter

Xavier

et al. 2017

Molecular Cancer Therapeutics

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Anti-CD20 radioimmunotherapy is an effective approach for therapy of relapsed or refractory CD20 pos lymphomas, but faces limitations due to poor tumor penetration and undesirable pharmacokinetics of full antibodies. Camelid single-domain Ab fragments (sdAb) might circumvent some of the limitations of radiolabeled full antibodies. In this study, a set of hCD20-targeting sdAbs was generated, and their capacity to bind hCD20 was evaluated in vitro and in vivo. A lead sdAb, sdAb 9079, was selected on the basis of its specific tumor targeting and significant lower kidney accumulation compared with other sdAbs. SdAb 9079 was then radiolabeled with 68 Ga and 177 Lu for PET imaging and targeted therapy. The therapeutic potential of 177 Lu-DTPA-sdAb was compared with that of 177 Lu-DTPA-rituximab and unlabeled rituximab in mice bearing hCD20 pos tumors. Radiolabeled with 68 Ga, sdAb 9079 showed specific tumor uptake, with very low accumulation in nontarget organs, except kidneys. The tumor uptake of 177 Lu-DTPA-sdAb 9079 after 1.5 h was 3.4 AE 1.3% ID/g, with T/B and T/M ratios of 13.3 AE 4.6 and 32.9 AE 15.6. Peak tumor accumulation of 177 Lu-DTPA-rituximab was about 9 times higher, but concomitantly with high accumulation in nontarget organs and very low T/B and T/M ratios (0.8 AE 0.1 and 7.1 AE 2.4). Treatment of mice with 177 Lu-DTPA-sdAb 9079 significantly prolonged median survival compared with control groups and was as effective as treatment with rituximab or its 177 Lu-labeled variant. Taken together, sdAb 9079 displays promising features as a theranostic drug to treat CD20 pos lymphomas.

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Generation of Single Domain Antibody Fragments Derived from Camelids and Generation of Manifold Constructs

Cited by 151 publications

References 14 publications

PET Imaging of Macrophage Mannose Receptor–Expressing Macrophages in Tumor Stroma Using ¹⁸F-Radiolabeled Camelid Single-Domain Antibody Fragments

PET Imaging of Macrophage Mannose Receptor–Expressing Macrophages in Tumor Stroma Using ¹⁸F-Radiolabeled Camelid Single-Domain Antibody Fragments

A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior

Theranostic Radiolabeled Anti-CD20 sdAb for Targeted Radionuclide Therapy of Non-Hodgkin Lymphoma

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Generation of Single Domain Antibody Fragments Derived from Camelids and Generation of Manifold Constructs

Cited by 151 publications

References 14 publications

PET Imaging of Macrophage Mannose Receptor–Expressing Macrophages in Tumor Stroma Using 18F-Radiolabeled Camelid Single-Domain Antibody Fragments

PET Imaging of Macrophage Mannose Receptor–Expressing Macrophages in Tumor Stroma Using 18F-Radiolabeled Camelid Single-Domain Antibody Fragments

A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior

Theranostic Radiolabeled Anti-CD20 sdAb for Targeted Radionuclide Therapy of Non-Hodgkin Lymphoma

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Product

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PET Imaging of Macrophage Mannose Receptor–Expressing Macrophages in Tumor Stroma Using ¹⁸F-Radiolabeled Camelid Single-Domain Antibody Fragments

PET Imaging of Macrophage Mannose Receptor–Expressing Macrophages in Tumor Stroma Using ¹⁸F-Radiolabeled Camelid Single-Domain Antibody Fragments