Reagents and methods for PET using bispecific antibody pretargeting and 68Ga-radiolabeled bivalent hapten-peptide-chelate conjugates.
The aim of this work was to develop reagents and methods potentially useful in PET, using (68)Ga in a 2-step pretargeting protocol.


METHODS
We prepared bispecific antibodies (bsAbs) for disease-specific targeting of carcinoembryonic antigen-positive cells and recognition of later-administered bivalent hapten-peptide conjugates. The secondary antibody arm (antibody 679) recognizes a histaminyl-succinyl-glycine (HSG) structural subunit. The bsAbs were prepared as Fab' x Fab' conjugates using chemical cross-linking methods and as bispecific diabodies using recombinant DNA technologies. A HSG-bivalent hapten conjugate bearing the macrocyclic ring chelating agent 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) was designed to be readily radiolabeled with (68)Ga taken directly from a (68)Ge/(68)Ga generator system. Reagents were tested in vitro and, then, for their targeting properties in a preclinical animal model of human cancer.


RESULTS
A chemically cross-linked hMN-14 x 679 F(ab')(2) and a fully humanized bispecific diabody construct (BS1.5H), expressed in Escherichia coli, were prepared for this work. We synthesized the bivalent peptide termed IMP 241 [DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH(2)] and labeled it with (68)Ga and (67)Ga at temperatures from 45 degrees C to 100 degrees C, over times of 15 min to 1 h, establishing 15 min at 95 degrees C as a useful condition for (68)Ga labeling. When we formulated the IMP 241 bivalent hapten-peptide with ammonium acetate buffer at pH 4-5 and eluted the (68)Ga from the generator directly into the peptide solution, we achieved an almost quantitative incorporation of the (68)Ga into IMP 241, as analyzed by size-exclusion high-performance liquid chromatography, after mixing the complex with the 679 antibody. For in vivo studies we used (67)Ga-IMP 241 as a surrogate for (68)Ga-IMP 241, in view of the short, 68-min half-life of the (68)Ga nuclide. The (67)Ga-IMP 241 was successfully pretargeted to human colon tumor xenografts in athymic mice with both the chemical and the diabody bispecific proteins. High tumor-to-normal tissue ratios for (67)Ga uptake were found for all tissues at 1 to 6 h after injection of (67)Ga-IMP 241. When using the BS1.5H diabody for pretargeting, tumor-to-blood, tumor-to-liver, and tumor-to-lung ratios of (67)Ga-IMP 241 at 1 and 3 h after injection were 41:1 and 137:1, 51:1 and 106:1, and 16:1 and 46:1, respectively.


CONCLUSIONS
The general approach described, along with the new compositions and the labeling methods we have developed, may eventually allow for use of (68)Ga-labeled specific targeting agents in a routine clinical PET application.
Griffiths
Gary L
GL
Immunomedics, Inc., Morris Plains, New Jersey. Garden State Cancer Center, Belleville, New Jersey 07950, USA. ggriffiths@immunomedics.com
Chang
Chien-Hsing
CH
McBride
William J
WJ
Rossi
Edmund A
EA
Sheerin
Agatha
A
Tejada
German R
GR
Karacay
Habibe
H
Sharkey
Robert M
RM
Horak
Ivan D
ID
Hansen
Hans J
HJ
Goldenberg
David M
DM
D000818:Animals; D018033:Antibodies, Bispecific / Q000737:chemistry; D045744:Cell Line, Tumor; D002614:Chelating Agents / Q000737:chemistry; D003110:Colonic Neoplasms / Q000000981:diagnostic imaging* / Q000378:metabolism*; D006241:Haptens / Q000737:chemistry; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry; D007553:Isotope Labeling / Q000379:methods*; D008657:Metabolic Clearance Rate; D051379:Mice; D008819:Mice, Nude; D009842:Oligopeptides / Q000737:chemistry / Q000493:pharmacokinetics*; D009928:Organ Specificity; D009942:Organometallic Compounds / Q000737:chemistry / Q000493:pharmacokinetics*; D019275:Radiopharmaceuticals / Q000493:pharmacokinetics; D014018:Tissue Distribution; D014055:Tomography, Emission-Computed / Q000379:methods*
D018033:Antibodies, Bispecific; D002614:Chelating Agents; D006241:Haptens; D007202:Indicators and Reagents; D009842:Oligopeptides; D009942:Organometallic Compounds; D019275:Radiopharmaceuticals; C483470:gadolinium IMP241