Page 7 - P. Molecular medicine and imaging
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Protein Nanobio Lab sat UNIST
Target-switchable Intracellular Bacterial Toxin Delivery Systems
with improved therapeutic efficacy against target tumor
Seong Guk Park, Bongseo Choi, Yoonji Bae, Sebyung Kang
Department of Biological Sciences, School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), Ulsan, 44919, Korea
Abstract Scheme
Targeted cancer therapies have been extensively developed to selectively
suppress tumor growth as well as to avoid harming healthy tissues. However,
failure to escape endosome upon receptor-mediated endocytosis is a major
obstacle limiting the efficacy of protein-based targeted cancer therapeutics, such
as recombinant immunotoxin. Here, novel target-switchable intracellular toxin
delivery systems (TiTDS) is presented, using catalytic and translocation domain of
diphtheria toxin (dtA-T) as an intracellular toxin delivery platform and affibody
molecules targeting HER2 or EGFR (HER2Afb or EGFRAfb) as target-specific
ligands. The intracellular toxin delivery platform and affibody molecules are
genetically fused with SpyCatcher (SC) protein and SpyTag (ST) peptide, Schematic illustration of the construction of target-switchable intracellular bacteria
respectively, to generate dtA-T-SC and ST-HER2Afb or ST-EGFRAfb modules. toxin delivery systems and TiTDS-mediated selective cancer treatments. Toxin
delivery module (dtA-T-SC) composed of diphtheria toxin A-T domain and SC can be
These modules can be individually purified and post-translationally ligated to form ligated with ST-fused targeting ligands (ST-HER2Afb and ST-EGFRAfb) in a mixing-
dtA-T/HER2Afb or dtA-T/EGFRAfb, through simple mixing-and-matching. and-matching manner, forming dtA-T/HER2Afb or dtA-T/EGFRAfb. dtA-T/HER2Afb
dtA-T /HER2Afb and dtA-T/EGFRAfb can selectively bind to their corresponding and dtA-T/EGFRAfb selectively bind to their corresponding target cancer cells, enter
the cells through receptor-mediated endocytosis, and successfully release active
target cancer cells, efficiently enter the cells through receptor-mediated toxins to cytosol, leading to death of target cancer cells.
endocytosis, successfully escape endosome, and release active toxins to the
cytosol. They exhibit high target-specific cytotoxicity in vitro and can reduce tumor Target-specific toxicity of TiTDS
masses significantly in vivo. The TiTDS will be promising targeted cancer
therapeutic platforms because of their high target specificity, effective intracellular
delivery of active toxins with improved therapeutic efficacy, and versatile target
switchability.
Characterization
Figure 3. Target cell-specific cytotoxicity tests with dtA-T/Afb. Time-dependent
morphological changes of (A) NIH3T6.7 and (B) A431 cells upon treatment with PBS,
Figure 1. Construction and characterization of intracellular toxin delivery 20 μM of doxorubicin, and 2 μM of corresponding dtA-T-SC, ST-Afb, and dtA-T/Afb
modules (dtA-T-SC), targeting ligands (ST-Afb), and their ligated forms (dtA- monitored by the real-time microscope, IncuCyte® . In vitro quantitative
T/Afb). (A) Schematic illustration of dtA-T-SC and ST-Afb (ST-HER2Afb and ST- measurement of dose-dependent cytotoxicity of dtA-T/HER2Afb to NIH3T6.7 cells
EGFRAfb) constructs. L represents a linker peptide. (B) The SDS-PAGE and dtA-T/EGFRAfb to A431 cells assayed by CCK-8.
analysis of dtA-T-SC, ST-Afb, and dtA-T/Afb. (C) Molecular mass analyses of
dtA-T-SC, ST-Afb, and dtA-T/Afb with mass spectrometry. Calculated (Calc.) and
observed (Obs.) masses are indicated.
Figure 2. Trafficking released
dta upon target-cell binding of
dta-T/Afb and subsequent
receptor-mediated endocytosis.
Confocal fluorescence
microscopic cell images of (A)
NIH3T6.7 cells treated with
dta-T/HER2Afb and (B) A431
cells with dta-T/EGFRAfb were
obtained at indicated times.
Inactive diphtheria toxin A
fragments (dta, green) are
probed with anti- diphtheria
toxin A antibodies, which
selectively bind to the released
form of A fragment. Nuclei of
cells (blue) and endosomes
(red) were stained with DAPI
and lysotracker, respectively. Figure 4. Improved in vivo therapeutic efficacy of albumin-binding domain (AlBD)-
Scale bar = 20 μm.
fused dtA-T/Afb. (A) Schematic illustration of AlBD-fused dtA-T/Afb constructs and
their expected forms after ligation with ST-Afb. (B) PBS, AlBD-fused dtA-
T/HER2Afbs, and dtA-T/HER2Afb (5 μg, respectively) were intravenously injected to
NIH3T6.7-allografted nude mice, and their tumor volumes were measured with a
caliper. (C) A431 cells were inoculated to nude mice. PBS, AlBD-fused dtA-
T/EGFRAfb, and dtA-T/EGFRAfb (5 μg, each) were intravenously injected. A total of
eight injections at 2- to 3-day intervals were administered. Pictures of biopsied
tumors from sacrificed mice are presented below (B) and (C).
Acknowledgement Conclusion
Laboratory Members
In conclusion, our target-switchable intracellular bacterial toxin delivery system
Ph.D. : Bongseo Choi (previous) provides a great potential for developing versatile anti-cancer platforms that can
MS/Ph.D. Candidate: Hansol Kim be applied to various target cancers by adopting multiple targeting ligands on
MS/Ph.D. Candidate: Hyukjun Choi
MS/Ph.D. Candidate: Yoonji Bae demand.
