Bispecific Designed Ankyrin Repeat Proteins (DARPins) Targeting Epidermal Growth Factor Receptor Inhibit A431 Cell Proliferation and Receptor Recycling

Boersma, Ykelien L.; Chao, Ginger; Steiner, Daniel; Wittrup, K. Dane; Plückthun, Andreas

doi:10.1074/jbc.m111.293266

Cited by 95 publications

(82 citation statements)

References 66 publications

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“…DARPins also have many favorable biophysical properties, such as exceptional stability and highyield prokaryotic production, which renders this type of molecule a promising candidate for use as a topical microbicide. Furthermore, the construction of bispecific binders is very straightforward, making biepitopic targeting and thus very high avidities achievable (72).…”

Section: Discussionmentioning

confidence: 99%

Conformation-Dependent Recognition of HIV gp120 by Designed Ankyrin Repeat Proteins Provides Access to Novel HIV Entry Inhibitors

Mann

Friedrich

Krarup

et al. 2013

J Virol

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Here, we applied the designed ankyrin repeat protein (DARPin) technology to develop novel gp120-directed binding molecules with HIV entry-inhibiting capacity. DARPins are interesting molecules for HIV envelope inhibitor design, as their high-affinity binding differs from that of antibodies. DARPins in general prefer epitopes with a defined folded structure. We probed whether this capacity favors the selection of novel gp120-reactive molecules with specificities in epitope recognition and inhibitory activity that differ from those found among neutralizing antibodies. The preference of DARPins for defined structures was notable in our selections, since of the four gp120 modifications probed as selection targets, gp120 arrested by CD4 ligation proved the most successful. Of note, all the gp120-specific DARPin clones with HIV-neutralizing activity isolated recognized their target domains in a conformation-dependent manner. This was particularly pronounced for the V3 loop-specific DARPin 5m3_D12. In stark contrast to V3-specific antibodies, 5m3_D12 preferentially recognized the V3 loop in a specific conformation, as probed by structurally arrested V3 mimetic peptides, but bound linear V3 peptides only very weakly. Most notably, this conformation-dependent V3 recognition allowed 5m3_D12 to bypass the V1V2 shielding of several tier 2 HIV isolates and to neutralize these viruses. These data provide a proof of concept that the DARPin technology holds promise for the development of HIV entry inhibitors with a unique mechanism of action.

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Section: Discussionmentioning

confidence: 99%

Conformation-Dependent Recognition of HIV gp120 by Designed Ankyrin Repeat Proteins Provides Access to Novel HIV Entry Inhibitors

Mann

Friedrich

Krarup

et al. 2013

J Virol

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“…3, 4, and 6). Because we can generate such retargeting DARPin modules to essentially any target using ribosome or phage display (37,44,50,51,(58)(59)(60)(61)(62)(63)(64), we can exploit these capabilities [e.g., in tumor therapy in animal models (63,(65)(66)(67)] to use Ad5 to deliver payloads with tumorcontrolling potential.…”

Section: Discussionmentioning

confidence: 99%

“…To construct adapters, the C terminus of the knob-binding DARPins (omitting the last three residues of the DARPin, which were disordered in the crystal structure) were fused to the N terminus of the phage capsid protein SHP, used as trimerization domain (43) (SHP, V13-P115), and was spaced by different linkers (SHP1, G; SHP2, GA; SHP3, GLKAGADVNA), introduced by a PCR-based strategy and cloned into pDST72. In a second step, the cDNA was inserted into pQIBi2_2 with either an N-or C-terminal fusion of the retargeting DARPins G3, 9.29, Ec4, Ac2, E01, or E69, reported earlier (44,45,50,51), spaced by a (Gly 4 Ser) 4 linker. These retargeting DARPins can be exchanged readily using either BamHI/HindIII (for N-terminal fusions) or BglII/BsaI (for C-terminal fusions).…”

Section: Methodsmentioning

confidence: 99%

“…These retargeting DARPins can be exchanged readily using either BamHI/HindIII (for N-terminal fusions) or BglII/BsaI (for C-terminal fusions). For competition, monomeric DARPins or bispecific DARPins with increased valency (LZ constructs) expressed and purified as previously described (37,50,51) were used. Expression of the MA(H) 6 mutant knobΔTAYT was performed as previously described (37).…”

Section: Methodsmentioning

confidence: 99%

“…The specificity and efficiency of EGFR-mediated transduction was investigated using the previously described human epidermal carcinoma tumor cell line A431 overexpressing EGFR (Fig. 6A) (50). Ad5luc was coated with the adapters at an adapter-to-knob ratio of 1:1, and cells were infected at a multiplicity of 100 vp per cell.…”

Section: Shp As N-terminal Fusionsmentioning

confidence: 99%

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Development of a generic adenovirus delivery system based on structure-guided design of bispecific trimeric DARPin adapters

Dreier

Honegger

Hess

et al. 2013

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

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Adenoviruses (Ads) have shown promise as vectors for gene delivery in clinical trials. Efficient viral targeting to a tissue of choice requires both ablation of the virus' original tropism and engineering of an efficient receptor-mediated uptake by a specific cell population. We have developed a series of adapters binding to the virus with such high affinity that they remain fully bound for >10 d, block its natural receptor binding site and mediate interaction with a surface receptor of choice. The adapter contains two fused modules, both consisting of designed ankyrin repeat proteins (DARPins), one binding to the fiber knob of adenovirus serotype 5 and the other binding to various tumor markers. By solving the crystal structure of the complex of the trimeric knob with three bound DARPins at 1.95-Å resolution, we could use computer modeling to design a link to a trimeric protein of extraordinary kinetic stability, the capsid protein SHP from the lambdoid phage 21. We arrived at a module which binds the knob like a trimeric clamp. When this clamp was fused with DARPins of varying specificities, it enabled adenovirus serotype 5-mediated delivery of a transgene in a human epidermal growth factor receptor 2-, epidermal growth factor receptor-, or epithelial cell adhesion molecule-dependent manner with transduction efficiencies comparable to or even exceeding those of Ad itself. With these adapters, efficiently produced in Escherichia coli, Ad can be converted rapidly to new receptor specificities using any ligand as the receptor-binding moiety. Prefabricated Ads with different payloads thus can be retargeted readily to many cell types of choice.protein design | tumor targeting | viral retargeting | X-ray crystallography | protein engineering

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Anti‐EGFR scFv tetramer (tetrabody) with a stable monodisperse structure, strong anticancer effect, and a long in vivo half‐life

et al. 2016

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The development of single‐chain variable fragments (scFvs) as therapeutic agents has the potential to reduce the high cost of antibody production, but the development process often impairs scFv functions such as binding affinity and pharmacokinetics. Multimerization is one strategy for recovering or enhancing these lost functions. Previously, we constructed several antiepidermal growth factor receptor ( EGFR ) scFv multimers by modifying linker length and domain order. Antitumor effects comparable with those of the currently approved anti‐ EGFR therapeutic antibodies were observed for scFv trimers. In the present study, we fractionated an anti‐ EGFR scFv tetramer from the intracellular soluble fraction of an Escherichia coli transformant. Compared with the trimer, the tetramer showed higher affinity, greater cancer cell growth inhibition, and prolonged blood retention time. Furthermore, the tetramer did not dissociate into the trimer or other smaller species during long‐term storage (up to 33 weeks). Thus, our developed scFv tetramer is an attractive candidate next‐generation anti‐ EGFR therapeutic antibody that can be produced via a low‐cost bacterial expression system.

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Bispecific Designed Ankyrin Repeat Proteins (DARPins) Targeting Epidermal Growth Factor Receptor Inhibit A431 Cell Proliferation and Receptor Recycling

Cited by 95 publications

References 66 publications

Conformation-Dependent Recognition of HIV gp120 by Designed Ankyrin Repeat Proteins Provides Access to Novel HIV Entry Inhibitors

Conformation-Dependent Recognition of HIV gp120 by Designed Ankyrin Repeat Proteins Provides Access to Novel HIV Entry Inhibitors

Development of a generic adenovirus delivery system based on structure-guided design of bispecific trimeric DARPin adapters

Anti‐EGFR scFv tetramer (tetrabody) with a stable monodisperse structure, strong anticancer effect, and a long in vivo half‐life

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