Monitored whole gene in vitro evolution of an anti-hRaf-1 affibody molecule towards increased binding affinity

Grimm, Sebastian; Salahshour, Samaneh; Nygren, Per‐Åke

doi:10.1016/j.nbt.2011.10.008

Cited by 10 publications

(11 citation statements)

References 22 publications

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“…We also investigated PA-mediated delivery of a binder based on affibody (ABRaf), which was evolved to bind to human Raf-1 (hRaf-1, K d =100 n m ),[ 2c ] a protein kinase of central importance in the MAPK/ERK signaling pathway. Although ABRaf has been shown to inhibit the Ras/Raf interaction in vitro, it has not been tested for inhibition of MAPK signaling pathway in cells.…”

Section: Resultsmentioning

confidence: 99%

Delivery of Antibody Mimics into Mammalian Cells via Anthrax Toxin Protective Antigen

2014

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Antibody mimics have significant scientific and therapeutic utility for the disruption of protein–protein interactions inside cells; however, their delivery to the cell cytosol remains a major challenge. Here we show that protective antigen (PA), a component of anthrax toxin, efficiently transports commonly used antibody mimics to the cytosol of mammalian cells when conjugated to the N-terminal domain of LF (LFN). In contrast, a cell-penetrating peptide (CPP) was not able to deliver any of these antibody mimics into the cell cytosol. The refolding and binding of a transported tandem monobody to Bcr-Abl (its protein target) in chronic myeloid leukemia cells were confirmed by co-immunoprecipitation. We also observed inhibition of Bcr-Abl kinase activity and induction of apoptosis caused by the monobody. In a separate case, we show disruption of key interactions in the MAPK signaling pathway after PA-mediated delivery of an affibody binder that targets hRaf-1. We show for the first time that PA can deliver bioactive antibody mimics to disrupt intracellular protein–protein interactions. This technology adds a useful tool to expand the applications of these modern agents to the intracellular milieu.

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Section: Resultsmentioning

confidence: 99%

Delivery of Antibody Mimics into Mammalian Cells via Anthrax Toxin Protective Antigen

2014

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“…Fourth, affinities of selected binders for specific targets can differ widely, depending on several parameters including binder class, selection methods, library size and target characteristics (Grimm et al . ). SHMR4 nanobody recognizes BoNT/E toxin with no cross‐reactivity with other antigens, especially with related BoNT toxins.…”

Section: Discussionmentioning

confidence: 97%

“…; Grimm et al . ), while in this research, the parental VHH was screened from immune library which was quite specific. Second, all of the random mutations occurring in FR and CDR regions remained unchanged, whereas CDR regions, in particular CDR3, have striking effect in antibody specificity and affinity.…”

Section: Discussionmentioning

confidence: 99%

“…This affinity increase of 10% could be attributed to several factors. First, most of the affinity maturation processes were performed in antibodies obtained from nonimmune libraries (Yau et al 2005;Groves et al 2006;Grimm et al 2012), while in this research, the parental VHH was screened from immune library which was quite specific. Second, all of the random mutations occurring in FR and CDR regions remained unchanged, whereas CDR regions, in particular CDR3, have striking effect in antibody specificity and BoNT/E BoNT/A BoNT/B BSA UreC Bap LPS.V Antigen(10 µg ml -1 ) Figure 5 (a) ELISA assay of SHMR4 with recombinant BoNT/E Hc.…”

Section: Selective Clonesmentioning

confidence: 99%

“…Third, great affinity enhancements by random mutagenesis have not yet been reported for VHHs, and most of the affinity improvements by random or site-directed mutagenesis have been performed for scFvs that are not comparable with nanobodies in overall structure and other features. Fourth, affinities of selected binders for specific targets can differ widely, depending on several parameters including binder class, selection methods, library size and target characteristics (Grimm et al 2012). SHMR4 nanobody recognizes BoNT/E toxin with no cross-reactivity with other antigens, especially with related BoNT toxins.…”

Section: Selective Clonesmentioning

confidence: 99%

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Random mutagenesis of BoNT/E Hc nanobody to construct a secondary phage-display library

et al. 2014

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Aims: To construct secondary mutant phage-display library of recombinant single variable domain (VHH) against botulinum neurotoxin E by error-prone PCR. Methods and Results: The gene coding for specific VHH derived from the camel immunized with binding domain of botulinum neurotoxin E (BoNT/E) was amplified by error-prone PCR. Several biopanning rounds were used to screen the phage-displaying BoNT/E Hc nanobodies. The final nanobody, SHMR4, with increased affinity recognized BoNT/E toxin with no crossreactivity with other antigens especially with related BoNT toxins. Conclusions: The constructed nanobody could be a suitable candidate for VHH-based biosensor production to detect the Clostridium botulinum type E. Significance and Impact of the Study: Diagnosis and treatment of botulinum neurotoxins are important. Generation of high-affinity antibodies based on the construction of secondary libraries using affinity maturation step leads to the development of reagents for precise diagnosis and therapy.

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Ribosome Display: A Potent Display Technology used for Selecting and Evolving Specific Binders with Desired Properties

Kang

et al. 2018

Mol Biotechnol

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Monitored whole gene in vitro evolution of an anti-hRaf-1 affibody molecule towards increased binding affinity

Cited by 10 publications

References 22 publications

Delivery of Antibody Mimics into Mammalian Cells via Anthrax Toxin Protective Antigen

Delivery of Antibody Mimics into Mammalian Cells via Anthrax Toxin Protective Antigen

Random mutagenesis of BoNT/E Hc nanobody to construct a secondary phage-display library

Ribosome Display: A Potent Display Technology used for Selecting and Evolving Specific Binders with Desired Properties

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