TY - JOUR
T1 - Selection of an Anticalin® against the membrane form of Hsp70 via bacterial surface display and its theranostic application in tumour models
AU - Friedrich, Lars
AU - Kornberger, Petra
AU - Mendler, Claudia T.
AU - Multhoff, Gabriele
AU - Schwaiger, Markus
AU - Skerra, Arne
N1 - Publisher Copyright:
© 2018 Walter de Gruyter GmbH, Berlin/Boston.
PY - 2018/2/23
Y1 - 2018/2/23
N2 - We describe the selection of Anticalins against a common tumour surface antigen, human Hsp70, using functional display on live Escherichia coli cells as fusion with a truncated EspP autotransporter. While found intracellularly in normal cells, Hsp70 is frequently exposed in a membrane-bound state on the surface of tumour cells and, even more pronounced, in metastases or after radiochemotherapy. Employing a recombinant Hsp70 fragment comprising residues 383-548 as the target, Anticalins were selected from a naïve bacterial library. The Anticalin with the highest affinity (KD=13 nm), as determined towards recombinant full-length Hsp70 by real-time surface plasmon resonance analysis, was improved to KD=510 pm by doped random mutagenesis and another cycle of E. coli surface display, followed by rational combination of mutations. This Anticalin, which recognises a linear peptide epitope located in the interdomain linker of Hsp70, was demonstrated to specifically bind Hsp70 in its membrane-associated form in immunofluorescence microscopy and via flow cytometry using the FaDu cell line, which is positive for surface Hsp70. The radiolabelled and PASylated Anticalin revealed specific tumour accumulation in xenograft mice using positron emission tomography (PET) imaging. Furthermore, after enzymatic coupling to the protein toxin gelonin, the Anticalin showed potent cytotoxicity on FaDu cells in vitro.
AB - We describe the selection of Anticalins against a common tumour surface antigen, human Hsp70, using functional display on live Escherichia coli cells as fusion with a truncated EspP autotransporter. While found intracellularly in normal cells, Hsp70 is frequently exposed in a membrane-bound state on the surface of tumour cells and, even more pronounced, in metastases or after radiochemotherapy. Employing a recombinant Hsp70 fragment comprising residues 383-548 as the target, Anticalins were selected from a naïve bacterial library. The Anticalin with the highest affinity (KD=13 nm), as determined towards recombinant full-length Hsp70 by real-time surface plasmon resonance analysis, was improved to KD=510 pm by doped random mutagenesis and another cycle of E. coli surface display, followed by rational combination of mutations. This Anticalin, which recognises a linear peptide epitope located in the interdomain linker of Hsp70, was demonstrated to specifically bind Hsp70 in its membrane-associated form in immunofluorescence microscopy and via flow cytometry using the FaDu cell line, which is positive for surface Hsp70. The radiolabelled and PASylated Anticalin revealed specific tumour accumulation in xenograft mice using positron emission tomography (PET) imaging. Furthermore, after enzymatic coupling to the protein toxin gelonin, the Anticalin showed potent cytotoxicity on FaDu cells in vitro.
KW - PASylation
KW - heat shock protein
KW - medical imaging
KW - protein engineering
KW - tumour targeting
UR - http://www.scopus.com/inward/record.url?scp=85037693367&partnerID=8YFLogxK
U2 - 10.1515/hsz-2017-0207
DO - 10.1515/hsz-2017-0207
M3 - Article
C2 - 29140786
AN - SCOPUS:85037693367
SN - 1431-6730
VL - 399
SP - 235
EP - 252
JO - Biological Chemistry
JF - Biological Chemistry
IS - 3
ER -