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Comparing the cytosolic delivery efficiency of protein uptake systems
Dr. Wouter VerdurmenAssistant ProfessorDepartment of BiochemistryRadboud University Medical CenterNijmegen, the Netherlands
Antibody Engineering & Therapeutics, San Diego December 12, 2017
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Dec 12, 2017 Uptake of proteins to the cytosolPage 2
The rationale for intracellular protein delivery
▪ Advances in tumor profiling (e.g. genome, transcriptome) molecular understanding
▪ Genome-wide synthetic lethality studies quality of tumor vulnerability prediction
▪ Limited increase in available therapies, especially against intracellular proteins
Jerby-Arnon et al. Cell (2014)
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Dec 12, 2017 Uptake of proteins to the cytosolPage 3
The rationale for intracellular protein delivery
▪ Advances in tumor profiling (e.g. genome, transcriptome) molecular understanding
▪ Genome-wide synthetic lethality studies quality of tumor vulnerability prediction
▪ Limited increase in available therapies, especially against intracellular proteins
Tumor detection
Tumor profiling(e.g. transcriptome,
tumor genome)Treatment selection
Time
# o
f o
pti
on
s
Time
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dge
Schematic workflow of precision medicine
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Dec 12, 2017 Uptake of proteins to the cytosolPage 4
The rationale for intracellular protein delivery
▪ Advances in tumor profiling (e.g. genome, transcriptome) molecular understanding
▪ Genome-wide synthetic lethality studies quality of tumor vulnerability prediction
▪ Limited increase in available therapies, especially against intracellular proteins
Tumor detection
Tumor profiling(e.g. transcriptome,
tumor genome)Treatment selection
Time
# o
f o
pti
on
s
Time
kno
wle
dge
Schematic workflow of precision medicine
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Dec 12, 2017 Uptake of proteins to the cytosolPage 5
Easily achieved via endocytosis
Internalization into target cells
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Translocation from the endosome into the cytosol
Dec 12, 2017 Uptake of proteins to the cytosolPage 6
Difficult to achieve
Difficult to measure
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Objectives
Overall objective
▪ To speed up the advancement of cancer cell-selective intracellularprotein therapies
Specific aims
▪ To design an assay for quantifying cytosolic protein delivery
▪ To construct and assess receptor-specific protein delivery systems
▪ To compare the efficiencies of several prototypical protein deliverysystems with respect to cytosolic protein delivery
Dec 12, 2017 Uptake of proteins to the cytosolPage 7
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Model cargo: Designed ankyrin repeat proteins (DARPins)
Dec 12, 2017Page 8 Uptake of proteins to the cytosol
▪ Based on the regularity of the
ankyrin protein scaffold
▪ Disulfide-free
▪ Active in the cytosol
▪ Highly stable proteins
▪ Not prone to aggregation
Highly specific DARPins can be generated against virtually all targets:
▪ Members of large protein families (e.g. kinases)
▪ Protein-protein interactions
Binz et al. Nat. Biotech. (2004)
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Modular transport system design
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receptor
binding translocation payload
Possible via DARPins▪ Her2▪ EpCAM▪ EGFR
Inspired by nature's proteintranslocation mechanisms▪ anthrax toxin▪ Pseudomonas exotoxin A (ETA)
Possible via DARPins▪ Ras family▪ Bcl2 family ▪ ERK
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PA = protective antigenLF = lethal factor
Changetargetreceptor
Keep only binding domain
Add useful payload
Exploiting the anthrax toxin mechanism
Gao and Schulten. Biophys. J. (2006)
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Exploiting Pseudomonas exotoxin A (ETA)
Changetargetreceptor
Inactivate or remove catalytic domain and add useful payload
Becker et al. Antibodies (2012)
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Dec 12, 2017Page 12 Uptake of proteins to the cytosol
Covalent peptide modification via cytosolic biotin ligase (BirA)
Independent of:
▪ Cargo▪ Cellular characteristics▪ Observation
Quantifiable as:
▪ % Translocated molecules▪ Cytosolic concentration
A cytosolic delivery assay employing biotin ligase
Verdurmen et al. Methods Mol. Biol. (2017)
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Proof of concept
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Western blot shows:
▪ Total internalization (i.e. HA tag)
▪ Cytosolic delivery (i.e. a-biotin)
Transient co-expression of biotin ligase with avi-tagged DARPin and controls
▪ Signal requires avi tag and biotin ligase ▪ No unspecific signal▪ Very sensitive detection ▪ Linear correlation between sample and signal
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Generating cell lines stably expressing biotin ligase
Uptake of proteins to the cytosol
▪ Verification through transient expression of avi-tagged DARPin
Full gel-shift 100 % of cytosolic protein is biotinylated
Available stable cell lines▪ Flp-In 293▪ RD▪ SKBR3▪ HT29▪ MCF7
Dec 12, 2017Page 14
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Dec 12, 2017 Uptake of proteins to the cytosolPage 15
Unprocessed protein
Furin-processed protein
biotin
avi-tag
+
cargo
HA-tag
Total cellular internalization: endosomes + cytosol▪ Flp-In 293 cells▪ Ac2 / Ec1: EpCAM-binding DARPins
Tm 60°C
Tm 90°C
Tm > 100°C
Verdurmen et al. J. Control. Rel. (2015)
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Cytosolic delivery
Furin-processed protein
Furin-processed protein
biotin
Verdurmen et al. J. Control. Rel. (2015)
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Endosomal vs cytosolic delivery
Cytosolic delivery
Furin-processed protein
Furin-processed protein
Total cellular internalization
Verdurmen et al. J. Control. Rel. (2015)
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▪ Introduction of Leu-Ala or Leu-Gly mutations mildly destabilize DARPins
▪ Destabilization rescues ability to be transported via anthrax toxin
Role of cargo stability for anthrax toxin system
Verdurmen et al. J. Control. Rel. (2015)
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cell-penetrating peptide fusions (e.g. tat / penetratin)
+ + ++
++++
Pseudomonas exotoxin A (ETA) fusions
anthrax toxin fusions (binary; PA + LF)
supercharged GFP (scGFP) fusions
+++
+
+
+++
++
+
Lawrence et al., J Am Chem Soc (2007) diphtheria toxin (DT) fusions
Comparing different protein transporters
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Dec 12, 2017 Uptake of proteins to the cytosolPage 20
Verdurmen et al. Methods Mol. Biol. (2017)
The biotin ligase assay for quantifying uptake
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Uptake comparison in Flp-In 293 cells – 20 h
Dec 12, 2017 Uptake of proteins to the cytosolPage 21
Verdurmen et al. Sci Rep. (2017)
Cell-Penetratingpeptides
Bacterialtoxins
Superchargedproteins
ENDOSOMES + CYTOSOL(TOTAL CELL)
CYTOSOL ONLY
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Flp-In 293 cells – 4 hours + proteasome inhibition
Dec 12, 2017 Uptake of proteins to the cytosolPage 22
Cell-Penetratingpeptides
Bacterialtoxins
Superchargedproteins
Verdurmen et al. Sci Rep. (2017)
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Uptake comparison in MCF7 cells
Dec 12, 2017 Uptake of proteins to the cytosolPage 23
Verdurmen et al. Sci Rep. (2017)
Cell-Penetratingpeptides
Bacterialtoxins
Superchargedproteins
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Uptake comparison in SKBR3 cells
Dec 12, 2017 Uptake of proteins to the cytosolPage 24
Verdurmen et al. Sci Rep. (2017)
Cell-Penetratingpeptides
Bacterialtoxins
Superchargedproteins
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Uptake comparison in HT29 cells
Dec 12, 2017 Uptake of proteins to the cytosolPage 25
Verdurmen et al. Sci Rep. (2017)
Cell-Penetratingpeptides
Bacterialtoxins
Superchargedproteins
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Dec 12, 2017 Uptake of proteins to the cytosolPage 26
Jerby-Arnon et al. Cell (2014) De Godoy et al. Nature (2008)
▪ Target prioritization should combine information from network prediction (synthetic
lethality) and quantitative proteomics (molecules/cell)
A perspective on achieved levels
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▪ The biotin ligase assay is a robust assay for the quantification of cytosolic delivery
▪ Cell-penetrating peptides: moderate total cell uptake, but low cytosolic uptake
▪ Supercharged proteins: massive total cell uptake, but poor cytosolic uptake
▪ Receptor-targeted bacterial toxins: moderate total cell uptake and highly efficient
cytosolic delivery
o Anthrax pores have a cargo ‘thermodynamic’ stability limit
o Cell-type preferences incompletely understood
▪ Current challenges
o Better understanding of the trafficking needed for a broader applicability
o To understand and improve delivery in controlled 3D tumor microenvironments
Conclusions
Dec 12, 2017Page 27 Uptake of proteins to the cytosol
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• Marigona Mazlami• Manuel Luginbühl• Damir Petkovic• Dr. Annemarie Honegger• Gabriela Nagy-Davidescu• Prof. Dr. Andreas Plückthun
Acknowledgements
• Ozan Topaloglu• Valentina Palacio Castañeda• Prof. Dr. Roland Brock
Radboud Institute for Molecular Life Sciences Today's molecules for tomorrow's medicine