Antigen binding and stability properties of non‐covalently linked anti‐CD22 single‐chain Fv dimers

Arndt, Michaela A.E.; Krauß, Jürgen; Rybak, Susanna M.

doi:10.1016/j.febslet.2004.11.011

Cited by 16 publications

(15 citation statements)

References 28 publications

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“…This abrogated all binding in ELISA. This finding supports the notion that linker dependent oligomerisation of scFvs may be affected by the sequence of the variable domains [35]. Removal of the terminal serine residues might therefore have prevented the variable domains of the multibodies from forming functional antigen binding sites.…”

Section: Discussionsupporting

confidence: 79%

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Improving the characteristics of a mycobacterial 16 kDa-specific chicken scFv

et al. 2011

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a b s t r a c tRecombinant antibodies can be engineered to improve their binding or other characteristics. A chicken single chain variable fragment (scFv) phage display library was panned against the mycobacterial 16 kDa antigen. Three fusion phages which bound specifically to the antigen were selected, each of which produced low signals in ELISA when secreted as a soluble scFv. One scFv was therefore chosen to be modified in an attempt to improve its binding. Firstly, a mutant sublibrary was created by random mutagenesis. High stringency panning of this sublibrary yielded binders which produced ELISA signals up to eleven times higher than the parent scFv. An increase in the intrinsic affinity was confirmed by surface plasmon resonance. Secondly, the flexible linker between the heavy and light chains of the parent scFv was either shortened to one glycine residue or deleted entirely. No ELISA signal was obtained when the linker was absent, but the glycine-linked scFv showed enhanced binding. Size exclusion chromatography revealed that the enhanced binder had aggregated to form tetramers. This study confirms that the strategies used to improve the binding of human and mouse scFvs can also enhance chicken scFvs.

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Section: Discussionsupporting

confidence: 79%

“…In mouse scFvs, directly joining the last residue of the heavy chain V H S 113 to the first residue of the light chain V L D 1 resulted in a mixture of trimers and tetramers [35,36]. Another study reported the formation of trimers exclusively when V H S 112 was fused to V L D 1 and tetramers when V H S 113 was ligated to V L D 1 [34].…”

Section: Discussionmentioning

confidence: 98%

Improving the characteristics of a mycobacterial 16 kDa-specific chicken scFv

et al. 2011

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“…Both 2-and 1-residue linked NC10 scFv V L -V H formed a mixture of dimers, trimers and tetramers, whereas the linker-less and 3-residue versions formed almost exclusively tetramers and dimers, respectively. In other studies, a linker-less anti-CD22 scFv V L -V H formed an almost pure dimer population (Arndt et al, 2004), whereas a linker-less anti-Lewis Y sc Fv V L -V H formed a mixture of trimers and tetramers (Kelly et al, 2008). Hence, the pattern of oligomerization appears to be highly variable among different antibodies, and a linker-less anti-CD20 scFv V L -V H may still not have resulted in oligomerization.…”

Section: Anti-cd20 Diabodies Discussionmentioning

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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“…(33,41,85) Fv fragments obtained by cloning the variable domains of mAb from hybridoma cells or by direct selection from phage libraries can also exhibit low thermodynamic stability even if they retain monovalent binding affinity and specificity of the parental mAb. The grafting of the antigen binding loops of such scFv onto the framework of a different, more stable scFv, was used to ''repair'' scFv fragments with suboptimal stability and made it possible to enhance their biophysical and functional properties.…”

Section: Stabilitymentioning

confidence: 99%

Multivalency: the hallmark of antibodies used for optimization of tumor targeting by design

2008

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High-precision tumor targeting with conventional therapeutics is based on the concept of the ideal drug as a "magic bullet"; this became possible after techniques were developed for production of monoclonal antibodies (mAbs). Innovative DNA technologies have revolutionized this area and enhanced clinical efficiency of mAbs. The experience of applying small-size recombinant antibodies (monovalent binding fragments and their derivatives) to cancer targeting showed that even high-affinity monovalent interactions provide fast blood clearance but only modest retention time on the target antigen. Conversion of recombinant antibodies into multivalent format increases their functional affinity, decreases dissociation rates for cell-surface and optimizes biodistribution. In addition, it allows the creation of bispecific antibody molecules that can target two different antigens simultaneously and do not exist in nature. Different multimerization strategies used now in antibody engineering make it possible to optimize biodistribution and tumor targeting of recombinant antibody constructs for cancer diagnostics and therapy.

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Antigen binding and stability properties of non‐covalently linked anti‐CD22 single‐chain Fv dimers

Cited by 16 publications

References 28 publications

Improving the characteristics of a mycobacterial 16 kDa-specific chicken scFv

Improving the characteristics of a mycobacterial 16 kDa-specific chicken scFv

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

Multivalency: the hallmark of antibodies used for optimization of tumor targeting by design

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