Two-state selection of conformation-specific antibodies

Department of Pharmaceutical Chemistry, University of California, 1700 4th Street, San Francisco, CA 94143, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 03/2009; 106(9):3071-6. DOI: 10.1073/pnas.0812952106
Source: PubMed


We present a general strategy for identification of conformation-specific antibodies using phage display. Different covalent probes were used to trap caspase-1 into 2 alternative conformations, termed the on-form and the off-form. These conformation-trapped forms of the protease were used as antigens in alternating rounds of selection and antiselection for antibody antigen-binding fragments (Fabs) displayed on phage. After affinity maturation, 2 Fabs were isolated with K(D) values ranging from 2 to 5 nM, and each bound to their cognate conformer 20- to 500-fold more tightly than their noncognate conformer. Kinetic analysis of the Fabs indicated that binding was conformation dependent, and that the wild-type caspase-1 sits much closer to the off-form than the on-form. Bivalent IgG forms of the Fabs were used to localize the different states in cells and revealed the activated caspase-1 is concentrated in a central structure in the cytosol, similar to what has been described as the pyroptosome. These studies demonstrate a general strategy for producing conformation-selective antibodies and show their utility for probing the distribution of caspase-1 conformational states in vitro and in cells.

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    • "Synthetic antibody libraries displayed on the surface of M13 filamentous bacteriophage (phage display) have been used to select antibodies that bind to targets such as ubiquitin [76], histones [77], and hemoglobins [78], and to select against precise antigenic conformations [79]. Antibodies with exquisite specificity have been obtained from synthetic antibody libraries, such as antibodies that can distinguish between chicken and quail lysozyme [80], which differ by only four amino acids, and those that can differentiate between two conformations of the same enzyme (caspase) [81]. An additional advantage is that the initial libraries are typically constructed on human scaffolds, therefore the resulting antibodies do not require extensive engineering to ''humanize'' prior to therapeutic use [75]. "
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    ABSTRACT: Vaccines that elicit a protective broadly neutralizing antibody (bNAb) response and monoclonal antibody therapies are critical for the treatment and prevention of viral infections. However, isolation of protective neutralizing antibodies has been challenging for some viruses, notably those with high antigenic diversity or those that do not elicit a bNAb response in the course of natural infection. Here, we discuss recent work that employs protein engineering strategies to design immunogens that elicit bNAbs or engineer novel bNAbs. We highlight the use of rational, computational, and combinatorial strategies and assess the potential of these approaches for the development of new vaccines and immunotherapeutics.
    Preview · Article · Oct 2013 · FEBS letters
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    • "Recent efforts have focused on developing libraries containing restricted diversity segments within the CDRs of stable heavy and light chain variable domain (VH and VL, respectively) frameworks [16-21]. This diversity is encoded by designed, synthetic oligonucleotides (‘synthetic antibodies’) which, when used in combination with screening by a display method (e.g., phage display, yeast display, or mRNA display), allows for identification of antibodies or antibody fragments with specificities and affinities comparable to or better than antibodies obtained from natural sources [22-26]. Additionally, restricted diversity libraries permit high-throughput mutagenesis studies of combining site residues to determine which characteristics most accurately reflect the physicochemical attributes of functional antibodies [4,16-18]. "
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    • "As a result, synthetic antibody libraries are not subject to biases of natural immune repertoires and the resulting antibodies can have enhanced properties. For example, synthetic antibodies with exquisite conformational or structural specificity have been isolated against several protein targets (Brawley et al., 2010; Gao et al., 2009). In addition, synthetic antibodies have been used to target post-translational modifications in high specificity (Newton et al., 2008), as well as nucleic acids, a class of antigens that has resisted traditional antibody isolation methods (Ye et al., 2008). "
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