Site-Specific, Thiol-Mediated Conjugation of Fluorescent Probes to Cysteine-Modified Diabodies Targeting CD20 or HER2

Sirk, Shannon J.; Olafsen, Tove; Barat, Bhaswati; Bauer, Karl B.; Wu, Anna M.

doi:10.1021/bc800113v

Cited by 42 publications

(45 citation statements)

References 34 publications

(57 reference statements)

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“…There have been a number of other diabodies and minibodies developed for different targets that have been modified for use in bioluminescence (436) and fluorescence (390) imaging studies (see Refs. 113, 317 and TABLE 2 for some examples).…”

Section: Diabodies and Minibodiesmentioning

confidence: 99%

A Molecular Imaging Primer: Modalities, Imaging Agents, and Applications

James

Gambhir

2012

Physiological Reviews

943

954

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Molecular imaging is revolutionizing the way we study the inner workings of the human body, diagnose diseases, approach drug design, and assess therapies. The field as a whole is making possible the visualization of complex biochemical processes involved in normal physiology and disease states, in real time, in living cells, tissues, and intact subjects. In this review, we focus specifically on molecular imaging of intact living subjects. We provide a basic primer for those who are new to molecular imaging, and a resource for those involved in the field. We begin by describing classical molecular imaging techniques together with their key strengths and limitations, after which we introduce some of the latest emerging imaging modalities. We provide an overview of the main classes of molecular imaging agents (i.e., small molecules, peptides, aptamers, engineered proteins, and nanoparticles) and cite examples of how molecular imaging is being applied in oncology, neuroscience, cardiology, gene therapy, cell tracking, and theranostics (therapy combined with diagnostics). A step-by-step guide to answering biological and/or clinical questions using the tools of molecular imaging is also provided. We conclude by discussing the grand challenges of the field, its future directions, and enormous potential for further impacting how we approach research and medicine.

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Section: Diabodies and Minibodiesmentioning

confidence: 99%

A Molecular Imaging Primer: Modalities, Imaging Agents, and Applications

James

Gambhir

2012

Physiological Reviews

943

954

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“…DOTA conjugation to antibody fragments was performed according to established protocols (8) by using metalfree buffers. The diabody was reduced by using dithiothreitol and reacted with 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10-maleimidoethylacetamide (Maleimido-monoamide-DOTA; Macrocyclics, Dallas, Tex) as described previously (9).…”

Section: Dota Conjugation and Radiolabelingmentioning

confidence: 99%

Development and Validation of an Immuno-PET Tracer as a Companion Diagnostic Agent for Antibody-Drug Conjugate Therapy to Target the CA6 Epitope

et al. 2015

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Purpose:To develop and compare three copper 64 ( 64 Cu)-labeled antibody fragments derived from a CA6-targeting antibody (huDS6) as immuno-positron emission tomography (immuno-PET)-based companion diagnostic agents for an antibody-drug conjugate by using huDS6. Materials and Methods:Three antibody fragments derived from huDS6 were produced, purified, conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and evaluated in the following ways: (a) the affinity of the fragments and the DOTA conjugates was measured via flow cytometry, (b) the stability of the labeled fragments was determined ex vivo in human serum over 24 hours, and (c) comparison of the in vivo imaging potential of the fragments was evaluated in mice bearing subcutaneous CA6-positive and CA6-negative xenografts by using serial PET imaging and biodistribution. Isotype controls with antilysozyme and anti-DM4 B-Fabs and blocking experiments with an excess of either B-Fab or huDS6 were used to determine the extent of the antibody fragment 64 Cu-DOTA-B-Fab binding specificity. Immunoreactivity and tracer kinetics were evaluated by using cellular uptake and 48-hour imaging experiments, respectively. Statistical analyses were performed by using t tests, one-way analysis of variance, and Wilcoxon and Mann-Whitney tests. Results:The antibody fragment 64Cu-DOTA-B-Fab was more than 95% stable after 24 hours in human serum, had an immunoreactivity of more than 70%, and allowed differentiation between CA6-positive and CA6-negative tumors in vivo as early as 6 hours after injection, with a 1.7-fold uptake ratio between tumors. Isotype and blocking studies experiments showed tracer-specific uptake in antigen-positive tumors, despite some nonspecific uptake in both tumor models. Conclusion:Three antibody fragments were produced and examined as potential companion diagnostic agents. 64 Cu-DOTA-BFab is a stable and effective immuno-PET tracer for CA6 imaging in vivo.q RSNA, 2015

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“…Hence, the location and number of cysteines (1 per scFv subunit, 2 per diabody) bypasses the issues associated with randomly located, stoichiometrically variable conjugation techniques. The Cys-Db format was recently extended to two additional targets, CD20 (B-cell lymphomas) and Her2/neu (Sirk et al, 2008). In vitro characterization of these fragments showed that the C-terminal cysteines could be readily modified, demonstrating the versatility and reproducibility of the system.…”

Section: Introductionmentioning

confidence: 99%

ImmunoPET imaging of B-cell lymphoma using 124I-anti-CD20 scFv dimers (diabodies)

Olafsen

Sirk

Betting

et al. 2010

Protein Engineering Design and Selection

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Edited by Paul CarterRapid clearing engineered antibody fragments for immunoPET promise high sensitivity at early time points. Here, tumor targeting of anti-CD20 diabodies (scFv dimers) for detection of low-grade B-cell lymphomas were evaluated. In addition, the effect of linker length on oligomerization of the diabody was investigated. Four rituximab scFv variants in the V L -V H orientation with different linker lengths between the V domains (scFv-1, scFv-3, scFv-5, scFv-8), plus the scFv-5 with a C-terminal cysteine (Cys-Db) for site-specific modification were generated. The scFv-8 and Cys-Db were radioiodinated with 124 I for PET imaging, and biodistribution of 131 I-Cys-Db was carried out at 2, 4 10 and 20 h. The five anti-CD20 scFv variants were expressed as fully functional dimers. Shortening the linker to three or one residue did not produce higher order of multimers. Both 124 I-labeled scFv-8 and Cys-Db exhibited similar tumor targeting at 8 h post injection, with significantly higher uptakes than in control tumors (P < 0.05). At 20 h, less than 1% ID/g of 131 I-labeled Cys-Db was present in tumors and tissues. Specific tumor targeting and high contrast images were achieved with the anti-CD20 diabodies. These agents extend the repertoire of reagents that can potentially be used to improve detection of low-grade lymphomas.

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Site-Specific, Thiol-Mediated Conjugation of Fluorescent Probes to Cysteine-Modified Diabodies Targeting CD20 or HER2

Cited by 42 publications

References 34 publications

A Molecular Imaging Primer: Modalities, Imaging Agents, and Applications

A Molecular Imaging Primer: Modalities, Imaging Agents, and Applications

Development and Validation of an Immuno-PET Tracer as a Companion Diagnostic Agent for Antibody-Drug Conjugate Therapy to Target the CA6 Epitope

ImmunoPET imaging of B-cell lymphoma using 124I-anti-CD20 scFv dimers (diabodies)

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