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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    • "Thusly, intricate macromolecular cross-domain binding might be hypothetically achieved through this technology, an ideal scenario for an ADAM inhibitor [11]. In fact, antibodies have been produced through phage display, due to recent technical advances, capable of recognizing multiple distinct antigens [23] and different conformations of the same antigen [24, 25]. D1(A12) is a monoclonal antibody developed through antibody phage-display, which targets the TACE ectodomain. "
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    • "In addition, synthetic libraries, such as the recently published CDR-H3 library [72] are likely to provide a future source of interesting variable regions for this application. Covalent probes have been used to trap the caspase protease in 'on' and 'off' forms to enable alternating rounds of selection and anti-selection for Fab fragments displayed on phage [73]. Many of the most informative antibody fragments are very rare in immune repertoires or libraries, and methods with proven capability to mine these immune repertoires efficiently offer an advantage. "
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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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