Tetrafluorophenolate of HBED-CC: a versatile conjugation agent for 68Ga-labeled small recombinant antibodies.
OBJECTIVE
The success of (68)Ga-labeled peptides for positron emission tomography of neuroendocrine tumors is mainly depending on the complex chemistry of this radioisotope. 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), the chelator of choice has however limitations if its application is expanded to heat-sensitive proteins. Recombinant antibodies like single chain Fv or diabodies belong to this class of proteins. They are suited to provide imaging contrast despite the short-lived (68)Ga because of their rapid blood clearances and nanomolar affinities. The heterobifunctional agent N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid (HBED-CC) was chosen as an alternative ligand because this agent is complexing [(68)Ga]Ga(3+) much faster than DOTA at ambient temperatures.


METHODS
A versatile technology for HBED-CC conjugation of proteins and (68)Ga-labeling has been developed. This included HBED-CC-tetrafluorophenol (TFP) ester synthesis, coupling to the antibody at various pH and complexation reactions performed in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer under different conditions.


RESULTS
The synthesis of the monoreactive 2,3,5,6-tetrafluorophenolate of HBED-CC at a carboxyl group not participating in complex formation used [Fe(HBED-CC)](-) for ester formation. The removal of Fe(3+) from purified (HBED-CC)TFP ester was achieved with RP(18) cartridge technology. The conjugation chemistry was performed with mAb425 which binds to the epidermal growth factor receptor (EGFR). This protein was used for optimizing purposes only. The influence of complexation parameters like temperature, pH, reaction time, and HBED-CC/antibody ratio on the biological activity of this model antibody was investigated. Furthermore, the outcome of this labeling procedure on the biological activity of a recombinant diabody (50 kDa) was studied.


CONCLUSIONS
It is known that small HBED-CC/antibody ratios are prerequisites for minimal interference of labels with antigen-binding domains. Here, the coupling of about one HBED-CC per antibody proved to be sufficient for efficient (68)Ga labeling, pointing to the successful application of (68)Ga for molecular imaging with small recombinant proteins.
Radiopharmaceutical Chemistry, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. m.eder@dkfz.de
Matthias
M
Eder
Björn
B
Wängler
Stefan
S
Knackmuss
Fabrice
F
LeGall
Melvyn
M
Little
Uwe
U
Haberkorn
Walter
W
Mier
Michael
M
Eisenhut
D000906:Antibodies / Q000737:chemistry* / Q000235:genetics; D004492:Edetic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D005710:Gallium Radioisotopes / Q000737:chemistry*; D006573:Heterocyclic Compounds, 1-Ring / Q000737:chemistry*; D007553:Isotope Labeling / Q000379:methods*; D010455:Peptides / Q000737:chemistry*; D019275:Radiopharmaceuticals / Q000138:chemical synthesis*; D011994:Recombinant Proteins / Q000737:chemistry*
D000906:Antibodies; D005710:Gallium Radioisotopes; D006573:Heterocyclic Compounds, 1-Ring; D010455:Peptides; D019275:Radiopharmaceuticals; D011994:Recombinant Proteins; C075313:N,N'-bis(2-hydroxy-5-(ethylene-beta-carboxy)benzyl)ethylenediamine N,N'-diacetic acid; C071349:1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid; D004492:Edetic Acid