Evaluation of backbone-cyclized HER2-binding 2-helix affibody molecule for in vivo molecular imaging.
BACKGROUND
Affibody molecules, small scaffold proteins, have demonstrated an appreciable potential as imaging probes. Affibody molecules are composed of three alpha-helices. Helices 1 and 2 are involved in molecular recognition, while helix 3 provides stability. The size of Affibody molecules can be reduced by omitting the third alpha-helix and cross-linking the two remaining, providing a smaller molecule with better extravasation and quicker clearance of unbound tracer. The goal of this study was to develop a novel 2-helix Affibody molecule based on backbone cyclization by native chemical ligation (NCL).


METHODS
The HER2-targeting NCL-cyclized Affibody molecule ZHER2:342min has been designed, synthesized and site-specifically conjugated with a DOTA chelator. DOTA-ZHER2:342min was labeled with (111)In and (68)Ga. The binding affinity of DOTA-ZHER2:342min was evaluated in vitro. The targeting properties of (111)In- and (68)Ga-DOTA-ZHER2:342min were evaluated in mice bearing SKOV-3 xenografts and compared with the properties of (111)In- and (68)Ga-labeled PEP09239, a DOTA-conjugated 2-helix Affibody analogue cyclized by a homocysteine disulfide bridge.


RESULTS
The dissociation constant (KD) for DOTA-ZHER2:342min binding to HER2 was 18nM according to SPR measurements. DOTA-ZHER2:342min was labeled with (111)In and (68)Ga. Both conjugates demonstrated bi-phasic binding kinetics to HER2-expressing cells, with KD1 in low nanomolar range. Both variants demonstrated specific uptake in HER2-expressing xenografts. Tumor-to-blood ratios at 2h p.i. were 6.1±1.3 for (111)In- DOTA-ZHER2:342min and 4.6±0.7 for (68)Ga-DOTA-ZHER2:342min. However, the uptake of DOTA-ZHER2:342min in lung, liver and spleen was appreciably higher than the uptake of PEP09239-based counterparts.


CONCLUSIONS
Native chemical ligation enables production of a backbone-cyclized HER2-binding 2-helix Affibody molecule (ZHER2:342min) with low nanomolar target affinity and specific tumor uptake.
Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, S-751 81 Uppsala, Sweden.
Hadis
H
Honarvar
Nima
N
Jokilaakso
Karl
K
Andersson
Jennie
J
Malmberg
Daniel
D
Rosik
Anna
A
Orlova
Amelie Eriksson
AE
Karlström
Vladimir
V
Tolmachev
Peter
P
Järver
D000818:Animals; D045744:Cell Line, Tumor; D003500:Cyclization; D005710:Gallium Radioisotopes; D006573:Heterocyclic Compounds, 1-Ring / Q000737:chemistry; D006801:Humans; D007205:Indium Radioisotopes; D051379:Mice; D057054:Molecular Imaging; D055550:Protein Stability; D017433:Protein Structure, Secondary; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
D005710:Gallium Radioisotopes; D006573:Heterocyclic Compounds, 1-Ring; D007205:Indium Radioisotopes; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; C533351:affibody (ZHER2-342)2; C071349:1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid