Single-Chain Antigen-Binding Proteins

Bird, Robert E.; Hardman, Karl D.; Jacobson, James W.; Johnson, Syd; Kaufman, B.; Lee, Shwu Maan; Lee, Timothy J.; Pope, Sharon; Riordan, G S; Whitlow, Marc

doi:10.1126/science.3140379

Cited by 1,342 publications

(380 citation statements)

References 19 publications

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“…While the present study was being carried out, the x-ray structure of the wt GCN4 scFv (26) in complex with the antigenic peptide 4 as well as that of the framework donor, a hyperstable hybrid scFv of 4D5 and AB48 (29), was determined. 4 This allowed us to build a structural model of the -graft and the ⍀-graft with bound antigen (see under "Experimental Procedures").…”

Section: The In Vivo Performance Of An Intrabody Can Be Optimized By mentioning

confidence: 99%

“…Structural Modeling-The structural model of the ⍀-graft variant, a nickname of a triple point mutant of the -graft with the CDR-H3 sequence ALFDY, is based on the crystal structure of the originally selected (26) anti-GCN4 scFv in complex with the antigen 4 and the crystal structure 4 of the framework donor (29). Both domains of the -graft were individually superimposed with the domains of the anti-GCN4 scFv in complex with the antigen, as seen in the crystal structure.…”

Section: (Sigmaplot)mentioning

confidence: 99%

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Direct in Vivo Screening of Intrabody Libraries Constructed on a Highly Stable Single-chain Framework

Maur¹,

Zahnd²,

Fischer³

et al. 2002

Journal of Biological Chemistry

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Single-chain Fv antibody fragments (scFv) represent a convenient antibody format for intracellular expression in eukaryotic or prokaryotic cells. These so-called intrabodies have great potential in functional genomics as a tool to study the function of newly identified proteins in vivo, for example by binding-induced modulation of their activity or by blocking interactions with other proteins. However, the intracellular expression and activity of many single-chain Fvs are limited by their instability and folding efficiency in the reducing intracellular environment, where the highly conserved intrachain disulfide bonds do not form. In the present work, we used an in vivo selection system to isolate novel antigen-binding intrabodies. We screened two intrabody libraries carrying a randomized third hypervariable loop onto the heavy chain of a stable framework, which had been further optimized by random mutagenesis for better behavior in the selection system, and we biophysically characterized the selected variants to interpret the outcome of the selection. Our results show that singleframework intrabody libraries can be directly screened in vivo to rapidly select antigen-specific intrabodies.Antibodies are pivotal components of the vertebrate immune system that can bind to almost any molecule with a high degree of specificity and affinity. These characteristics have been exploited to turn the natural antibodies into powerful biotechnological tools in diagnostic and therapeutic applications. Advances in recombinant DNA technology have facilitated the manipulation, cloning, and expression of the antibody genes in a wide variety of hosts (1-3). Several forms of antibodies have been constructed to obtain derivatives that carry the binding site in a smaller assembly. One of the minimal forms still retaining the full binding site is the single-chain Fv fragment (scFv) 1 (4 -6). In the scFv form, the variable regions of the heavy and the light chains, which bear the hypervariable loops (or complementarity determining regions (CDR)), are connected by a flexible linker, allowing the expression of the protein from a single cDNA sequence.Natural antibodies, which are secreted by plasma cells, have evolved to function in an extracellular environment. In contrast to full-length, double-chain antibodies, the scFv antibody form can in principle be readily expressed also in the cytoplasm of eukaryotic cells and directed to any compartment to target intracellular proteins and thus evoke specific biological effects. Hence, intracellular scFvs, also known as "intrabodies," might have a great potential in functional genomics by blocking or modulating the activity of a growing number of newly identified proteins, thereby contributing to the understanding of their functions. In the long run, intrabodies might even be used in therapeutic applications, possibly in gene therapy settings.However, there are only few examples of successful applications (7-13), and the cytoplasmic expression of scFvs is generally confronted with the difficultie...

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Section: The In Vivo Performance Of An Intrabody Can Be Optimized By mentioning

confidence: 99%

Section: (Sigmaplot)mentioning

confidence: 99%

Direct in Vivo Screening of Intrabody Libraries Constructed on a Highly Stable Single-chain Framework

Maur¹,

Zahnd²,

Fischer³

et al. 2002

Journal of Biological Chemistry

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“…This type of recombinant polypeptide links the carboxyl terminus of the variable light chain (V L ) to the amino terminus of the variable heavy chain (V H ) by a peptide linker of sufficient length to bridge this distance (18). The use of scFv avoids the heterogeneous products of proteolysis of the whole antibody as well as problems of association between V H and V L domains in recombinant protein.…”

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confidence: 99%

Structure of an Anti-blood Group A Fv and Improvement of Its Binding Affinity without Loss of Specificity

Thomas¹,

Patenaude²,

MacKenzie³

et al. 2002

Journal of Biological Chemistry

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The specificity of antibody recognition of the ABO blood group trisaccharide antigens has been explored by crystal structure analysis and mutation methods. The crystal structure of the Fv corresponding to the antiblood group A antibody AC1001 has been determined to 2.2-Å resolution and reveals a binding pocket that is complementary to the blood group A-trisaccharide antigen. The effect of mutating specific residues lining this pocket on binding to the A and B blood group oligosaccharide antigens was investigated through a panel of single point mutations and through a phage library of mutations in complementarity determining region H3. Both approaches gave several mutants with improved affinity for antigen. Surface plasmon resonance indicated up to 8-fold enhancement in affinity for the Apentasaccharide with no observable binding to the blood group B antigen. This is the first example of single point mutations in a carbohydrate-binding antibody resulting in significant increases in binding affinity without loss of specificity.The affinity of anti-carbohydrate antibodies for their antigens is commonly observed to be 3-5 orders of magnitude lower than affinities of anti-protein or anti-peptide antibodies for their antigens, yet there is no clear mechanism to explain this phenomenon. One means of exploring this question is to attempt to generate mutant anti-carbohydrate antibodies with higher affinities, either by design of site-directed mutants or by randomizing selected codons in a phage library. However, the production of antibodies with improved affinities that maintain antigen specificity has proven challenging (1-4). For example, in an exhaustive site-directed mutagenesis study of CDR 1 H3 of an anti-Salmonella Fab, Brummell et al. (1) found that all of the 90 mutant Fabs produced showed similar or decreased binding affinity for the O-antigen. Mutants of the anti-Lewis Y antibody BR96 were obtained as phage-displayed scFv; although the mutant scFv binding affinity had increased by up to 6-fold versus the native antibody, its specificity was altered (2, 3). More recently, higher affinity antibodies against levan, a model for the polysaccharide capsules of bacteria, were obtained by mutational analysis though their fine specificity varied from that of the parent antibody (5). In contrast, significant enhancements have been achieved with antibodies to protein antigens. For example, a mutant scFv of an anti-c-erbB-2 tumor antigen obtained by chain shuffling and phage display had a 5-6-fold increase in affinity (4). In a later study by the same group (6) sequential mutation of light and heavy chains of the scFv yielded 16-and 9-fold increases in affinity for the protein antigen. Yang et al. (7) obtained mutant anti-HIV-1 Fab by phage-display techniques with a 96-fold increase in binding, even though the native antibody fragments already possessed high affinity for the protein antigen. Though phage-display techniques have been reported to yield higher binding mutant scFvs, the resulting antibodies often contain mult...

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“…The interaction of the domains is known to be important for the stability and functionality of the antibody fragments of which they form part (Hochmann et al, 1976;Searle et al, 1995). The two variable domains may be expressed as heterodimeric, noncovalently linked Fv fragments (Skerra & Plückthun, 1988;Riechman et al, 1988) or as single-chain Fv 1 fragments (scFv's; Bird et al, 1988;Huston et al, 1988), where the two domains are connected by a flexible peptide linker. A simple two-state model can be used to fit the thermodynamic data from reversible solvent denaturation studies of Fv and scFv fragments in many cases (Pantoliano et al, 1991;Knappik & Plückthun, 1995), suggesting a coupling of the two variable domains into a cooperative folding unit.…”

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Folding Nuclei of the scFv Fragment of an Antibody

et al. 1996

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The folding kinetics of the variable domains of the phosphorylcholine-binding antibody McPC603, combined into a scFv fragment [V H -(Gly 4 Ser) 3 -V L ], were investigated by the use of fluorescence spectroscopy, nuclear magnetic resonance (NMR), and mass spectrometry (MS). All three methods gave evidence for the occurrence of a major kinetic intermediate during the refolding of the denatured, oxidized scFv fragment. This intermediate is formed within the first 30 s of folding and comprises exchangeprotected amide protons of hydrophobic and aromatic amino acids, most of which are localized within the inner -sheet of the V L domain. In the subsequent slow step, most of the amide protons become protected with rate constants that are very similar for residues of both domains. These data are in agreement with the MS results, which indicate a cooperative folding event from the intermediate to the native state of the scFv fragment.While antibody variable domains have become one of the structurally best characterized groups of proteins because of their central role in molecular recognition in the immune system (Alzari et al., 1988;Davies & Padlan, 1990), little is known about the structure formation during their folding in Vitro. The interaction of the domains is known to be important for the stability and functionality of the antibody fragments of which they form part (Hochmann et al., 1976;Searle et al., 1995). The two variable domains may be expressed as heterodimeric, noncovalently linked Fv fragments (Skerra & Plückthun, 1988; Riechman et al., 1988) or as single-chain Fv 1 fragments (scFv's; Bird et al., 1988;Huston et al., 1988), where the two domains are connected by a flexible peptide linker. A simple two-state model can be used to fit the thermodynamic data from reversible solvent denaturation studies of Fv and scFv fragments in many cases (Pantoliano et al., 1991;Knappik & Plückthun, 1995), suggesting a coupling of the two variable domains into a cooperative folding unit.Kinetic data from different antibody fragments reveal a more complex behavior. The rate-limiting step for the folding of a C L domain (Goto & Hamaguchi, 1982), an immunoglobulin light chain (Lang & Schmid, 1988), or a Fab fragment (Lilie et al., 1993) of an antibody has been shown to be proline isomerization. Isolated V L domains were shown to refold rapidly (Goto et al., 1979;Tsunenaga et al., 1987) and were suggested to undergo a retarded folding in the presence of the C L domain. Complete folding of denatured, oxidized Fv (Knappik & Plückthun, 1995) and scFv fragments is known to be slow (Pantoliano et al., 1991), but the mode of interaction of the two domains during the folding reaction remains to be elucidated.In order to investigate the nature of possible structured intermediates of antibody variable domains and to address the question of cooperativity at the level of individual amino acids, we made use of the recently obtained NMR assignments of most of the 15 N and 1 H resonances of the Fv and scFv fragments of the antibod...

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Single-Chain Antigen-Binding Proteins

Cited by 1,342 publications

References 19 publications

Direct in Vivo Screening of Intrabody Libraries Constructed on a Highly Stable Single-chain Framework

Direct in Vivo Screening of Intrabody Libraries Constructed on a Highly Stable Single-chain Framework

Structure of an Anti-blood Group A Fv and Improvement of Its Binding Affinity without Loss of Specificity

Folding Nuclei of the scFv Fragment of an Antibody

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