Anthrax Protective Antigen Retargeted with Single‐Chain Variable Fragments Delivers Enzymes to Pancreatic Cancer Cells

Loftis, Alexander R.; Santos, Michael; Truex, Nicholas L.; Biancucci, Marco; Satchell, K.J.F.; Pentelute, Bradley L.

doi:10.1002/cbic.202000201

Cited by 14 publications

(22 citation statements)

References 42 publications

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“…For delivery of RRSP into mouse cells, we used the anthrax toxin-based delivery system wherein the anthrax toxin lethal factor N- terminus was fused with RRSP (LF N RRSP) or LF N RRSP with a catalytically inactivating H4030A amino acid substitution (here after referred to as LF N RRSP*). Intracellular delivery of RRSP (previously known as DUF5) with this system has been previously been demonstrated in several mammalian and mouse cell lines (23, 27, 28).…”

Section: Resultsmentioning

confidence: 99%

RAS specific protease Induces Irreversible Growth Arrest via p27 in several KRAS Mutant Colorectal Cancer cell lines

Stubbs¹,

Biancucci²,

Vidimar³

et al. 2020

Preprint

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RAS is one of the most frequently mutated oncogenes in cancer with ~30% of all human tumors harboring a mutation in either HRAS, NRAS, or KRAS isoforms. Despite countless efforts for development of small molecule inhibitors for RAS, it remains an elusive target in the clinic. Here we demonstrated that the pan-RAS biological inhibitor RAS/RAP1-specific endopeptidase (RRSP) has proteolytic activity in ‘Ras-less’ mouse embryonic fibroblasts expressing human RAS isoforms (H/N/KRAS) or major oncogenic KRAS mutants (G12C, G12V, G12D, G13D, and Q61R). The cleavage of RAS inhibited phosphorylation of ERK and cell proliferation. To investigate how RAS processing affects colon cancer cells, we tested RRSP against KRAS-dependent (SW620 and GP5d) and KRAS-independent (HCT-116, SW1463, and HT-29) cell lines and found that RRSP inhibited growth. The cleavage of RAS was cytotoxic in some cell lines and induced either irreversible cell cycle arrest or uncharacterized growth inhibition in others. The G1 cell cycle arrest in some colon cancer cells was mediated through rescue of p27 (Kip1) protein expression resulting in reduced phosphorylation of retinoblastoma protein. Together, this work demonstrated that complete ablation of RAS in cells induces growth inhibition, but the mechanism of inhibition can vary in different tumor cell lines. This ability of RAS processing to halt cell proliferation by multiple strategies highlights RRSP both as a potential anti-tumor therapy and as a tool for studying RAS signaling across tumor types.

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Section: Resultsmentioning

confidence: 99%

RAS specific protease Induces Irreversible Growth Arrest via p27 in several KRAS Mutant Colorectal Cancer cell lines

Stubbs¹,

Biancucci²,

Vidimar³

et al. 2020

Preprint

Self Cite

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“…In addition, there are multiple protein engineering approaches aimed to enhance intracellular delivery and/or subsequent therapeutic effects, some of which are only recently coming to the fore for engineered proteins. The therapeutic potency can be increased without affecting delivery itself, through either delivery of (target protein-inactivating) enzymes [ 8 , 38 ], targeted protein degradation [ 39 ], or by enhancing the stability of the delivered agent against proteasomal degradation [ 40 , 41 ].…”

Section: Resultsmentioning

confidence: 99%

A Computational Investigation of In Vivo Cytosolic Protein Delivery for Cancer Therapy

et al. 2021

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The ability to specifically block or degrade cytosolic targets using therapeutic proteins would bring tremendous therapeutic opportunities in cancer therapy. Over the last few years, significant progress has been made with respect to tissue targeting, cytosolic delivery, and catalytic inactivation of targets, placing this aim within reach. Here, we developed a mathematical model specifically built for the evaluation of approaches towards cytosolic protein delivery, involving all steps from systemic administration to translocation into the cytosol and target engagement. Focusing on solid cancer tissues, we utilized the model to investigate the effects of microvascular permeability, receptor affinity, the cellular density of targeted receptors, as well as the mode of activity (blocking/degradation) on therapeutic potential. Our analyses provide guidance for the rational optimization of protein design for enhanced activity and highlight the importance of tuning the receptor affinity as a function of receptor density as well as the receptor internalization rate. Furthermore, we provide quantitative insights into how enzymatic cargoes can enhance the distribution, extent, and duration of therapeutic activity, already at very low catalytic rates. Our results illustrate that with current protein engineering approaches, the goal of delivery of cytosolic delivery of proteins for therapeutic effects is well within reach.

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“…Additionally, Loftis et al used an mPA fused with the single-chain variable fragment (scFv) of an antibody to internalize and deliver LF N -DTA through EGFR or carcinoembryonic antigen, which could kill pancreatic cancer cells overexpressing the two receptors at the plasma membrane. For additional specificity towards their pancreatic cancer cell line, they made an LF-RRSP fusion protein which targets the Ras–ERK signaling pathway, crucial for many pancreatic cancer cells [ 57 ]. Similarly, Becker et al used designed ankyrin repeat proteins (DARPins) fused to a PA-CMG2-based construct to specifically target transmembrane glycoprotein epithelial cell adhesion molecule (EpCAM) at the surface of cells.…”

Section: Anthrax Toxinmentioning

confidence: 99%

“…Targeting to non-native receptors using Affibodies, DARPins or receptors ligand for the cytosolic delivery of non-native cargos [ 55 , 56 , 57 , 58 ]…”

Section: Figurementioning

confidence: 99%

Harnessing the Membrane Translocation Properties of AB Toxins for Therapeutic Applications

Piot

Goot

Sergeeva

2021

Toxins

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Over the last few decades, proteins and peptides have become increasingly more common as FDA-approved drugs, despite their inefficient delivery due to their inability to cross the plasma membrane. In this context, bacterial two-component systems, termed AB toxins, use various protein-based membrane translocation mechanisms to deliver toxins into cells, and these mechanisms could provide new insights into the development of bio-based drug delivery systems. These toxins have great potential as therapies both because of their intrinsic properties as well as the modular characteristics of both subunits, which make them highly amenable to conjugation with various drug classes. This review focuses on the therapeutical approaches involving the internalization mechanisms of three representative AB toxins: botulinum toxin type A, anthrax toxin, and cholera toxin. We showcase several specific examples of the use of these toxins to develop new therapeutic strategies for numerous diseases and explain what makes these toxins promising tools in the development of drugs and drug delivery systems.

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Anthrax Protective Antigen Retargeted with Single‐Chain Variable Fragments Delivers Enzymes to Pancreatic Cancer Cells

Cited by 14 publications

References 42 publications

RAS specific protease Induces Irreversible Growth Arrest via p27 in several KRAS Mutant Colorectal Cancer cell lines

RAS specific protease Induces Irreversible Growth Arrest via p27 in several KRAS Mutant Colorectal Cancer cell lines

A Computational Investigation of In Vivo Cytosolic Protein Delivery for Cancer Therapy

Harnessing the Membrane Translocation Properties of AB Toxins for Therapeutic Applications

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