Abstract
Although intracellular antibodies (intrabodies) are being explored as putative therapeutic and research reagents, little is known about the principles that dictate the efficacy of these molecules. In our efforts to address this issue, we generated a panel of five intrabodies, directed against catalytically inactive murine caspase-3, by screening single-chain antibody (Fv) phage display libraries. Here we determined criteria that single-chain Fv fragments must fulfill to act as efficient intrabodies. The affinities of these intrabodies, as measured by surface plasmon resonance, varied approximately 5-fold (50-250 nm). Despite their substantial sequence similarity, only two of the five intrabodies were able to significantly accumulate intracellularly. These disparities in intracellular expression levels were reflected by differences in the stability of the purified protein species when analyzed by urea denaturation studies. We observed varied efficiencies in retargeting the antigen murine caspase-3, from the cytosol to the nucleus, mediated by intrabodies tagged with an SV40 nuclear localization signal. Our results demonstrate that the intrinsic stability of the intrabody, rather than its affinity for the antigen, dictates its intracellular efficacy.
Highlights
Among the myriad applications of these molecules, the use of scFv fragments as intrabodies has received some attention recently
We observed varied efficiencies in retargeting the antigen murine caspase-3, from the cytosol to the nucleus, mediated by intrabodies tagged with an SV40 nuclear localization signal
Expression and Characterization of mCASP3—A catalytically inactive mutant of murine caspase-3 was engineered to prevent any proteolytic degradation of phage display scFv fragments during the screening process
Summary
Among the myriad applications of these molecules, the use of scFv fragments as intrabodies (intracellularly expressed antibodies) has received some attention recently The aim of such attempts has been to neutralize the function of endogenous target proteins, using intrabodies, by several methods [5,6,7]. A panel of scFv fragments was isolated from a phage display library directed toward the antigen murine caspase-3 (mCASP3) These scFv fragments were tested for properties such as affinity for the antigen, protein stability, and intracellular accumulation to determine characteristics that define an efficient intrabody. The intracellular stability of an intrabody was found to correlate with the stability of the purified protein, as measured by urea denaturation under standard and reducing conditions To our knowledge, this is the largest panel of scFv fragments against a common antigen that have been tested for intracellular stability and efficacy as intrabodies far
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