Single-chain Fv fragments of anti-neuraminidase antibody NC10 containing five- and ten-residue linkers form dimers and with zero- residue linker a trimer

Kortt, Alexander A.; Lah, M; Oddie, Geoffrey W.; Gruen, Charli; Burns, J E; Pearce, Lesley A.; Atwell, J. L.; McCoy, Airlie J.; Howlett, Geoffrey J.; Metzger, Dennis W.; Webster, Robert G.; Hudson, Peter J.

doi:10.1093/protein/10.4.423

Cited by 116 publications

(88 citation statements)

References 23 publications

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“…Holliger and colleagues pioneered the construction of a bivalent molecule by shortening the linker peptide to five residues, which prevents pairing of the variable domains on the same polypeptide chain but promotes association of two molecules to a bivalent diabody [8]. Further shortening the linker peptide below three residues was shown to result in the formation of trimeric, tetrameric, or larger aggregate species [9][10][11][12]. More recently, the formation of V H -V L oriented scFv antibodies with <3 residue linkers to predominantly dimeric molecules has been described [13,14].…”

Section: Introductionmentioning

confidence: 99%

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

Arndt¹,

Krauß²,

Rybak³

2004

FEBS Letters

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By varying linker length and domain orientation three multivalent derivatives of a monovalent anti-CD22 single-chain fragment variable (scFv) antibody were generated. Shortening the linker of the V H -V L oriented scFv to 5 or 0 residues resulted in the formation of diabodies or a mixture of tetramers and trimers, respectively. Unexpectedly, a V L -0-V H scFv assembled to homogenous dimers, remained substantially more stable than the V H -5-V L diabody when incubated in human serum at 37°C, and retained its dimeric state when concentrated up to 4 mg/ml. These properties suggest the V L -0-V H scFv could become an attractive vehicle for the selective delivery of multiple effector molecules to CD22 + tumor cells.

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Section: Introductionmentioning

confidence: 99%

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

Arndt¹,

Krauß²,

Rybak³

2004

FEBS Letters

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“…This in turn makes it possible to standardise assays with reproducible reagents and the antibodies can even be recovered by constructing a synthetic gene if necessary. A further advantage of recombinant antibodies is that their physical and chemical properties can be changed using standard recombinant DNA methods such as mutagenesis [7e13], multimerisation [14,15], chain and DNA fragment shuffling [16e18]. Affinity maturation by random mutagenesis, followed by increased selection pressure mimics somatic mutation in vitro, a process which often allows antibodies with higher affinity and specificity to be derived [7].…”

Section: Introductionmentioning

confidence: 99%

“…The sequence and the length of this linker can influence the properties of the scFv [15,23]. For example, mouse scFvs with linkers between 12 and 15 amino acids occur mostly as monomers while those joined with between 5 and 11 residues occur as dimers [14,24,25].…”

Section: Introductionmentioning

confidence: 99%

“…For example, mouse scFvs with linkers between 12 and 15 amino acids occur mostly as monomers while those joined with between 5 and 11 residues occur as dimers [14,24,25]. Variable domains joined either directly to each other or with linkers of up to four residues occur as trimers and tetramers [15]. This multimerisation results in an increase in cooperative binding and hence the avidity of the scFv.…”

Section: Introductionmentioning

confidence: 99%

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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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“…Non-covalent scFv dimers or trimers of MOC-31 and MOC-161 may easily be made by shortening the linker sequence separating the VH and VK domains to fewer than ten residues to yield diabodies (Holliger et al, 1993), or by deleting the linker completely to generate trimeric molecules (Kortt et al, 1997).…”

mentioning

confidence: 99%

High-affinity recombinant phage antibodies to the pan-carcinoma marker epithelial glycoprotein-2 for tumour targeting

Roovers

Henderikx²,

Helfrich³

et al. 1998

Br J Cancer

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Summary The tumour-associated antigen epithelial glycoprotein-2 (EGP-2) is a promising target for detection and treatment of a variety of human carcinomas. Antibodies to this antigen have been successfully used in patients for imaging of small-cell lung cancer and for adjuvant treatment of minimal residual disease of colon cancer. We describe here the isolation and complete characterization of high-affinity singlechain variable fragments (scFv) to the EGP-2 antigen. First, the binding kinetics of four murine whole antibodies directed to EGP-2 (17-1A, 323/A3, MOC-31 and MOC-1 61) were determined using surface plasmon resonance (SPR). The MOC-31 antibody has the lowest apparent off-rate, followed by MOC-161 and 323/A3. The V-genes of the two MOC hybridomas were cloned as scFv in a phage display vector and antigen-binding phage were selected by panning on recombinant antigen. The scFvs compete with the original hybridoma antibodies for binding to antigen and specifically bind to human carcinomas in immunohistochemistry. MOC-31 scFv has an off-rate which is better than those of the bivalent 17-1A and 323/A3 whole antibodies, providing it with an essential characteristic for tumour retention in vivo. The availability of these high-affinity anti-EGP-2 antibody fragments and of their encoding V-genes creates a variety of possibilities for their future use as tumour-targeting vehicles.

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Single-chain Fv fragments of anti-neuraminidase antibody NC10 containing five- and ten-residue linkers form dimers and with zero- residue linker a trimer

Cited by 116 publications

References 23 publications

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

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

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

High-affinity recombinant phage antibodies to the pan-carcinoma marker epithelial glycoprotein-2 for tumour targeting

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