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

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)

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

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Schematic workflow of precision medicine

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

Dec 12, 2017 Uptake of proteins to the cytosolPage 5

Easily achieved via endocytosis

Internalization into target cells

Translocation from the endosome into the cytosol

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Difficult to achieve

Difficult to measure

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

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Model cargo: Designed ankyrin repeat proteins (DARPins)

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▪ 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)

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

PA = protective antigenLF = lethal factor

Changetargetreceptor

Keep only binding domain

Add useful payload

Exploiting the anthrax toxin mechanism

Gao and Schulten. Biophys. J. (2006)

Exploiting Pseudomonas exotoxin A (ETA)

Changetargetreceptor

Inactivate or remove catalytic domain and add useful payload

Becker et al. Antibodies (2012)

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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)

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

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

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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)

▪ 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)

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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Verdurmen et al. Methods Mol. Biol. (2017)

The biotin ligase assay for quantifying uptake

Uptake comparison in Flp-In 293 cells – 20 h

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Verdurmen et al. Sci Rep. (2017)

Cell-Penetratingpeptides

Bacterialtoxins

Superchargedproteins

ENDOSOMES + CYTOSOL(TOTAL CELL)

CYTOSOL ONLY

Flp-In 293 cells – 4 hours + proteasome inhibition

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Cell-Penetratingpeptides

Bacterialtoxins

Superchargedproteins

Verdurmen et al. Sci Rep. (2017)

Uptake comparison in MCF7 cells

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Verdurmen et al. Sci Rep. (2017)

Cell-Penetratingpeptides

Bacterialtoxins

Superchargedproteins

Uptake comparison in SKBR3 cells

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Verdurmen et al. Sci Rep. (2017)

Cell-Penetratingpeptides

Bacterialtoxins

Superchargedproteins

Uptake comparison in HT29 cells

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Verdurmen et al. Sci Rep. (2017)

Cell-Penetratingpeptides

Bacterialtoxins

Superchargedproteins

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

▪ 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

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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