Improving in vivo folding and stability of a single-chain Fv antibody fragment by loop grafting

Jung, Sora; Plückthun, Andreas

doi:10.1093/protein/10.8.959

Cited by 145 publications

(100 citation statements)

References 30 publications

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“…The consensus mutations cluster strikingly around V H CDR3; it has been argued previously that the V H CDR3 loop exerts the greatest influence on antigen-binding specificity (26)(27)(28). The 4-4-20 CDR loops can be grafted onto a different scFv framework without loss of affinity (29), indicating that fluorescein recognition is dominated by the CDR loops. In fact, 9 of the 10 consensus mutations identified in the present work are located at 4-4-20 residues that were present in the loop-grafted 4D5Flu hybrid protein, indicating that they lie within the portion of the scFv largely responsible for binding specificity.…”

Section: Resultsmentioning

confidence: 99%

Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity

Boder

Midelfort

Wittrup

2000

Proc. Natl. Acad. Sci. U.S.A.

615

437

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Single-chain antibody mutants have been evolved in vitro with antigen-binding equilibrium dissociation constant Kd ‫؍‬ 48 fM and slower dissociation kinetics (half-time > 5 days) than those for the streptavidin-biotin complex. These mutants possess the highest monovalent ligand-binding affinity yet reported for an engineered protein by over two orders of magnitude. Optimal kinetic screening of randomly mutagenized libraries of 10 5 -10 7 yeast surfacedisplayed antibodies enabled a >1,000-fold decrease in the rate of dissociation after four cycles of affinity mutagenesis and screening. The consensus mutations are generally nonconservative by comparison with naturally occurring mouse Fv sequences and with residues that do not contact the fluorescein antigen in the wildtype complex. The existence of these mutants demonstrates that the antibody Fv architecture is not intrinsically responsible for an antigen-binding affinity ceiling during in vivo affinity maturation.

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Section: Resultsmentioning

confidence: 99%

Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity

Boder

Midelfort

Wittrup

2000

Proc. Natl. Acad. Sci. U.S.A.

615

437

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“…To efficiently exploit the high degree of antigen specificity and affinity of antibodies for intracellular applications, it will be necessary to use antibody frameworks that have been optimized for intracellular stability and solubility. In principle, there are two approaches to obtain a more stable framework, namely by protein engineering (15,43) or by directed evolution (42,44,45).…”

Section: Discussionmentioning

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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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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“…In contrast, un-fused scFvs that were recovered from iodoacetamide-treated cells were totally inactive (not shown), con®rming similar published observation. 8,52 Stability of MBP-scFvs…”

Section: Cytoplasmic Overexpression and Purification Of Mbp-scfv Fusionsmentioning

confidence: 99%

Escherichia coli maltose-binding protein as a molecular chaperone for recombinant intracellular cytoplasmic single-chain antibodies 1 1Edited by R. Huber

Bach

Mazor

Shaky

et al. 2001

Journal of Molecular Biology

166

119

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Recombinant single-chain antibodies (scFvs) that are expressed in the cytoplasm of cells are of considerable biotechnological and therapeutic potential. However, the reducing environment of the cytoplasm inhibits the formation of the intradomain disul®de bonds that are essential for correct folding and functionality of these antibody fragments. Thus, scFvs expressed in the cytoplasm are mostly insoluble and inactive.Here, we describe a general approach for stabilizing scFvs for ef®cient functional expression in the cell cytoplasm in a soluble, active form. The scFvs are expressed as C-terminal fusions with the Escherichia coli maltose-binding protein (MBP). We tested a large panel of scFvs that were derived from hybridomas and from murine and human scFv phage display and expression libraries by comparing their stability and functionality as un-fused versus MBP fused proteins. We found that MBP fused scFvs are expressed at high levels in the cytoplasm of E. coli as soluble and active proteins regardless of the redox state of the bacterial cytoplasm. In contrast, most un-fused scFvs can be produced (to much lower levels) in a functional form only when expressed in trxB À but not in trxB E. coli cells. We show that MBP-scFv fusions are more stable than the corresponding un-fused scFvs, and that they perform more ef®ciently in vivo as cytoplasmic intrabodies in E. coli. Thus, MBP seems to function as a molecular chaperone that promotes the solubility and stability of scFvs that are fused to it.

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Improving in vivo folding and stability of a single-chain Fv antibody fragment by loop grafting

Cited by 145 publications

References 30 publications

Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity

Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity

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

Escherichia coli maltose-binding protein as a molecular chaperone for recombinant intracellular cytoplasmic single-chain antibodies 1 1Edited by R. Huber

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