PMID- 24168270
OWN - NLM
STAT- MEDLINE
DCOM- 20141021
LR  - 20131122
IS  - 1520-4812 (Electronic)
IS  - 1043-1802 (Linking)
VI  - 24
IP  - 11
DP  - 2013 Nov 20
TI  - Orthogonal assembly of a designed ankyrin repeat protein-cytotoxin conjugate with 
      a clickable serum albumin module for half-life extension.
PG  - 1955-66
LID - 10.1021/bc4004102 [doi]
AB  - The generation of drug conjugates for safe and effective tumor targeting requires 
      binding proteins tolerant to functionalization by rational engineering. Here, we 
      show that Designed Ankyrin Repeat Proteins (DARPins), a novel class of binding 
      proteins not derived from antibodies, can be used as building blocks for facile 
      orthogonal assembly of bioconjugates for tumor targeting with tailored 
      properties. DARPin Ec1, which targets the Epithelial Cell Adhesion Molecule 
      (EpCAM), was genetically modified with a C-terminal cysteine for conjugation of 
      the small molecule cytotoxin monomethylauristatin F (MMAF). In addition, it was 
      N-terminally functionalized by metabolic introduction of the non-natural amino 
      acid azidohomoalanine to enable linkage of site-specifically 
      dibenzocyclooctyne-modified mouse serum albumin (MSA) for half-life extension 
      using Cu(I)-free click chemistry. The conjugate MSA-Ec1-MMAF was assembled to 
      obtain high yields of a pure and stable drug conjugate as confirmed by various 
      analytical methods and in functional assays. The orthogonality of the assembly 
      led to a defined reaction product and preserved the functional properties of all 
      modules, including EpCAM-specific binding and internalization, FcRn binding 
      mediated by MSA, and cytotoxic potency. Linkage of MMAF to the DARPin increased 
      receptor-specific uptake of the drug while decreasing nonspecific uptake, and 
      further coupling of the conjugate to MSA enhanced this effect. In mice, albumin 
      conjugation increased the serum half-life from 11 min to 17.4 h, resulting in a 
      more than 22-fold increase in the area-under-the-curve (AUC). Our data 
      demonstrate the promise of the DARPin format for facile modular assembly of drug 
      conjugates with improved pharmacokinetic performance for tumor targeting.
FAU - Simon, Manuel
AU  - Simon M
AD  - Department of Biochemistry, University of Zurich , Winterthurerstrasse 190, 
      CH-8057 Zurich, Switzerland.
FAU - Frey, Raphael
AU  - Frey R
FAU - Zangemeister-Wittke, Uwe
AU  - Zangemeister-Wittke U
FAU - Pluckthun, Andreas
AU  - Pluckthun A
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20131111
PL  - United States
TA  - Bioconjug Chem
JT  - Bioconjugate chemistry
JID - 9010319
RN  - 0 (Ankyrins)
RN  - 0 (Antineoplastic Agents)
RN  - 0 (Cytotoxins)
RN  - 0 (Oligopeptides)
RN  - 0 (Serum Albumin)
RN  - 0 (monomethylauristatin F)
SB  - IM
MH  - Animals
MH  - *Ankyrin Repeat
MH  - Ankyrins/*chemistry
MH  - Antineoplastic Agents/blood/*chemistry/*pharmacokinetics/pharmacology
MH  - Cell Proliferation/drug effects
MH  - *Click Chemistry
MH  - Cytotoxins/blood/*chemistry/*pharmacokinetics/pharmacology
MH  - Dose-Response Relationship, Drug
MH  - Drug Screening Assays, Antitumor
MH  - HEK293 Cells
MH  - HT29 Cells
MH  - Half-Life
MH  - Humans
MH  - Mice
MH  - Models, Molecular
MH  - Molecular Structure
MH  - Oligopeptides/blood/chemistry/pharmacokinetics/pharmacology
MH  - Serum Albumin/*chemistry
MH  - Structure-Activity Relationship
MH  - Tumor Cells, Cultured
EDAT- 2013/10/31 06:00
MHDA- 2014/10/22 06:00
CRDT- 2013/10/31 06:00
PHST- 2013/10/31 06:00 [entrez]
PHST- 2013/10/31 06:00 [pubmed]
PHST- 2014/10/22 06:00 [medline]
AID - 10.1021/bc4004102 [doi]
PST - ppublish
SO  - Bioconjug Chem. 2013 Nov 20;24(11):1955-66. doi: 10.1021/bc4004102. Epub 2013 Nov 
      11.