Directed Evolution of High-Affinity Antibody Mimics Using mRNA Display

Xu, Lihui; Aha, Patti; Gu, Ke; Kuimelis, Robert G.; Kurz, Markus; Lam, Terence; Lim, Ai Ching; Liu, Hongxiang; Lohse, Peter; Sun, Lin; Weng, Shawn; Wagner, Richard W.; Lipovšek, Daša

doi:10.1016/s1074-5521(02)00187-4

Cited by 225 publications

(144 citation statements)

References 67 publications

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“…Single-domain antibodies (37) and structurally analogous domains (38) are increasingly being exploited as alternatives to single-chain antibodies for molecular recognition. The approach demonstrated here may find application in engineering existing intrabodies for increased potency against other disease targets, including Parkinson's (39) and Alzheimer's diseases, HIV, and cancer.…”

Section: Discussionmentioning

confidence: 99%

Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody

Colby

Chu

Cassady

et al. 2004

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

169

145

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Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expansion in the number of polyglutamine-encoding CAG repeats in the gene that encodes the huntingtin (htt) protein. A property of the mutant protein that is intimately involved in the development of the disease is the propensity of the glutamine-expanded protein to misfold and generate an N-terminal proteolytic htt fragment that is toxic and prone to aggregation. Intracellular antibodies (intrabodies) against htt have been shown to reduce htt aggregation by binding to the toxic fragment and inactivating it or preventing its misfolding. Intrabodies may therefore be a useful gene-therapy approach to treatment of the disease. However, high levels of intrabody expression have been required to obtain even limited reductions in aggregation. We have engineered a single-domain intracellular antibody against htt for robust aggregation inhibition at low expression levels by increasing its affinity in the absence of a disulfide bond. Furthermore, the engineered intrabody variable light-chain (VL)12.3, rescued toxicity in a neuronal model of HD. We also found that VL12.3 inhibited aggregation and toxicity in a Saccharomyces cerevisiae model of HD. VL12.3 is significantly more potent than earlier anti-htt intrabodies and is a potential candidate for gene therapy treatment for HD. To our knowledge, this is the first attempt to improve affinity in the absence of a disulfide bond to improve intrabody function. The demonstrated importance of disulfide bond-independent binding for intrabody potency suggests a generally applicable approach to the development of effective intrabodies against other intracellular targets

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Section: Discussionmentioning

confidence: 99%

Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody

Colby

Chu

Cassady

et al. 2004

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

169

145

View full text Add to dashboard Cite

show abstract

“…[126] de Wildt et al developed a technique for high-throughput screening of recombinant antibodies, involving robotic picking and high-density gridding of bacteria containing the antibody gene followed by filter-based ELISA to identify clones that express binding antibody fragments, thereby allowing up to 18 342 different antibody clones to be screened at a time against bovine serum albumin (BSA), human serum albumin (HSA), and several recombinant human proteins. [127] In the near future, antibody fragments might compete with the following alternative scaffolds: [128,129] fibronectin, [130,131] lipocalin, [132] and ankyrin or leucine-rich repeat domains [133] although these systems still have not reached the maturity of phage-displayed antibody fragments.…”

Section: Phage-displayed Antibody-and Protein-fragment Microarraysmentioning

confidence: 99%

Protein‐Detecting Microarrays: Current Accomplishments and Requirements

2005

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The sequencing of the human genome has been successfully completed and offers the chance of obtaining a large amount of valuable information for understanding complex cellular events simply and rapidly in a single experiment. Interestingly, in addressing these proteomic studies, the importance of protein-detecting microarray technology is increasing. In the coming few years, microarray technology will become a significantly promising and indispensable research/diagnostic tool from just a speculative technology. It is clear that the protein-detecting microarray is supported by three independent but strongly related technologies (surface chemistry, detection methods, and capture agents). Firstly, a variety of surface-modification methodologies are now widely available and offer site-specific immobilization of capture agents onto surfaces in such a way as to keep the native conformation and activity. Secondly, sensitive and parallel detection apparatuses are being developed to provide highly engineered microarray platforms for simultaneous data acquisition. Lastly, in the development of capture agents, antibodies are now probably the most prominent capture agents for analyzing protein abundances. Alternative scaffolds, such as phage-displayed antibody and protein fragments, which provide the advantage of increasing diversity of proteinic capture agents, however, are under development. An approach involving recombinant proteins fused with affinity tag(s) and coupled with a highly engineered surface chemistry will provide simple production protocols and specific orientations of capture agents on the microarray formats. Peptides and other small molecules can be employed in screening highly potent ligands as well as in measuring enzymatic activities. Protein-detecting microarrays supported by the three key technologies should contribute in accelerating diagnostic/biological research and drug discovery.

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“…5). Translation reactions were incubated at 30℃ for 60 min and protein products were analyzed by SDS-PAGE and autoradiography as described in section 1. from engineered libraries of linear peptides [18] , constrained peptides [19] , and single-domain antibody mimics [20] . In addition, mRNA display has been used to identify cellular polypeptides that bind specific signaling proteins [21] and small-molecule drugs as polypeptide substrates of v-abl kinase [22] .…”

Section: Inhibition Of Selected Peptides On Ts Mrna In Vitro Translationmentioning

confidence: 99%

In vitro selected peptides bind with thymidylate synthase mRNA and inhibit its translation

et al. 2007

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Thymidylate synthase (TS), an essential enzyme for catalyzing the biosynthesis of thymidylate, is a critical therapeutic target in cancer therapy. Recent studies have shown that TS functions as an RNA-binding protein by interacting with two different sequences on its own mRNA, thus, repressing translational efficiency. In this study, peptides binding TS RNA with high affinity were isolated using mRNA display from a large peptide library (>10(13) different sequences). The randomized library was subjected up to twelve rounds of in vitro selection and amplification. Comparing the amino acid composition of the selected peptides (12th round, R12) with those from the initial random library (round zero, R0), the basic and aromatic residues in the selected peptides were enriched significantly, suggesting that these peptide regions might be important in the peptide-TS mRNA interaction. Categorizing the amino acids at each random position based on their physicochemical properties and comparing the distributions with those of the initial random pool, an obvious basic charge characteristic was found at positions 1, 12, 17 and 18, suggesting that basic side chains participate in RNA binding. Secondary structure prediction showed that the selected peptides of R12 pool represented a helical propensity compared with R0 pool, and the regions were rich in basic residues. The electrophoretic gel mobility shift and in vitro translation assays showed that the peptides selected using mRNA display could bind TS RNA specifically and inhibit the translation of TS mRNA. Our results suggested that the identified peptides could be used as new TS inhibitors and developed to a novel class of anticancer agents.

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Directed Evolution of High-Affinity Antibody Mimics Using mRNA Display

Cited by 225 publications

References 67 publications

Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody

Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody

Protein‐Detecting Microarrays: Current Accomplishments and Requirements

In vitro selected peptides bind with thymidylate synthase mRNA and inhibit its translation

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