RasIns: Genetically Encoded Intrabodies of Activated Ras Proteins

Cetin, Mehmet Sevket; Evenson, William E.; Gross, Garrett G.; Jalali-Yazdi, Farzad; Krieger, Daniel; Arnold, Don W.; Takahashi, Terry T.; Roberts, Richard W.

doi:10.1016/j.jmb.2016.11.008

Cited by 33 publications

(29 citation statements)

References 36 publications

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“…Also, important will be the creation of designer binding domains that can reliably differentiate between posttranslationally modified proteins and their unmodified counterparts, making it possible to deplete certain protein subpopulations while sparing others and bestowing proteome editing with an additional layer of specificity. Toward this objective, several groups including ours have described methods for isolating binders that specifically recognize particular protein states such as active versus inactive conformations, mutant versus wild‐type proteins, and phosphorylated versus non‐phosphorylated isoforms …”

Section: Resultsmentioning

confidence: 99%

Proteome editing using engineered proteins that hijack cellular quality control machinery

2019

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Protein silencing is an important aspect of both scientific investigation of native protein function and therapeutic targeting of aberrant protein activity. Many techniques for silencing proteins at the DNA or RNA level exist such as CRISPR, RNAi, or TALEN. Cellular proteins can also be selectively removed at the posttranslational level using proteome editing techniques, many of which employ engineered proteins to engage natural cellular quality control machinery for accelerating the removal of otherwise stable proteins. Here, we summarize recent progress in the development of such engineered proteins, comparing them to analogous microbial-, viral-, and small molecule-based strategies for selectively degrading proteins of interest. Finally, we highlight the advantages and limitations of proteome editing technologies and discuss how the application of directed evolution could alleviate current challenges and usher in a new wave of designer proteins for diagnostic and therapeutic application.

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Section: Resultsmentioning

confidence: 99%

Proteome editing using engineered proteins that hijack cellular quality control machinery

2019

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“…These advantages were validated as part of human safety studies with an 18 F loaded Adnectin, where imaging could be completed on the same day as injection and antibody-based reagents may take several days of radiation burden to clear non-specific binding [67,68]. This was further confirmed after loading the Adnectin with 64 Cu where the monobody was able to provide same-day PET visualisation of tumour cells, resulting in an increase in the monobody uptake in tumours over 24 h post-injection [62,69]. This concept has been taken even further with a Centyrin domain evolved to bind EGFR and conjugated with a fluorescent dye, which was then applied to guide surgical removal of tumour cells [70].…”

Section: Biosensorsmentioning

confidence: 86%

“…Tumour imaging with monobodies conjugated to (B) radioisotopes [62] and (C) microbubbles [52]. (D) Targeted degradation of endogenous intracellular proteins [63] and (E) targeted intracellular fluorescence reporters for endogenous proteins [64].…”

Section: Delivery Agentsmentioning

confidence: 99%

“…These intracellular monobodies were then applied to visualise dynamics in living cells without altering expression or location of the endogenous targets, and was able to follow the movement of an intracellular vesicle at 7 µm/s [29]. This work was taken even further with the development of state-selective intracellular binders of Ras, a commonly mutated oncoprotein in tumours with poor prognoses [64]. The anti-RAS monobodies specifically targeted K-and H-Ras, two variants that are the most commonly mutated genes in human tumours, and were demonstrated to be highly specific tools for monitoring the conformational or mutational state of RAS proteins while also potentially modulating their signalling pathways [64].…”

Section: Intracellular Applicationsmentioning

confidence: 99%

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Development and Differentiation in Monobodies Based on the Fibronectin Type 3 Domain

Chandler

Buckle

2020

Cells

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As a non-antibody scaffold, monobodies based on the fibronectin type III (FN3) domain overcome antibody size and complexity while maintaining analogous binding loops. However, antibodies and their derivatives remain the gold standard for the design of new therapeutics. In response, clinical-stage therapeutic proteins based on the FN3 domain are beginning to use native fibronectin function as a point of differentiation. The small and simple structure of monomeric monobodies confers increased tissue distribution and reduced half-life, whilst the absence of disulphide bonds improves stability in cytosolic environments. Where multi-specificity is challenging with an antibody format that is prone to mis-pairing between chains, multiple FN3 domains in the fibronectin assembly already interact with a large number of molecules. As such, multiple monobodies engineered for interaction with therapeutic targets are being combined in a similar beads-on-a-string assembly which improves both efficacy and pharmacokinetics. Furthermore, full length fibronectin is able to fold into multiple conformations as part of its natural function and a greater understanding of how mechanical forces allow for the transition between states will lead to advanced applications that truly differentiate the FN3 domain as a therapeutic scaffold.

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“…Tumour imaging with monobodies conjugated to (B) radioisotopes [61] and (C) microbubbles [51]. (D) Targeted degradation of endogenous intracellular proteins [62] and (E) targeted intracellular fluorescence reporters for endogenous proteins [63].…”

Section: Delivery Agentsmentioning

confidence: 99%

Development and Differentiation in Monobodies Based on the Fibronectin Type 3 Domain

Chandler

Buckle

2020

Preprint

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As a non-antibody scaffold, monobodies based on the fibronectin type III (FN3) domain overcome antibody size and complexity while maintaining analogous binding loops. However, antibodies and their derivatives remain the gold standard for design of new therapeutics. In response, clinical therapeutic proteins based on the FN3 domain are beginning to use native fibronectin function as a point of differentiation. The small and simple structure of monomeric monobodies confers increased tissue distribution and reduced half-life, whilst the absence of disulphide bonds improves stability in cytosolic environments. Where multi-specificity is challenging with an antibody format that is prone to mis-pairing of chains, FN3 domains in the fibronectin assembly already interact with a large number of molecules. As such, multiple monobodies engineered for interaction with therapeutic targets are being combined in a similar beads-on-a-string assembly which improves both efficacy and pharmacokinetics. Furthermore, full length fibronectin is able to fold into multiple conformations as part of its natural function and a greater understanding of how mechanical forces allow for the transition between states will lead to advanced applications that truly differentiate the FN3 domain as a therapeutic scaffold.

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RasIns: Genetically Encoded Intrabodies of Activated Ras Proteins

Cited by 33 publications

References 36 publications

Proteome editing using engineered proteins that hijack cellular quality control machinery

Proteome editing using engineered proteins that hijack cellular quality control machinery

Development and Differentiation in Monobodies Based on the Fibronectin Type 3 Domain

Development and Differentiation in Monobodies Based on the Fibronectin Type 3 Domain

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