title: Reagents and methods for PET using bispecific antibody pretargeting and 68Ga-radiolabeled bivalent hapten-peptide-chelate conjugates.
abstract: 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.
authors.affiliation: Immunomedics, Inc., Morris Plains, New Jersey. Garden State Cancer Center, Belleville, New Jersey 07950, USA. ggriffiths@immunomedics.com
authors.forename: Gary L
authors.initials: GL
authors.lastname: Griffiths
authors.forename: Chien-Hsing
authors.initials: CH
authors.lastname: Chang
authors.forename: William J
authors.initials: WJ
authors.lastname: McBride
authors.forename: Edmund A
authors.initials: EA
authors.lastname: Rossi
authors.forename: Agatha
authors.initials: A
authors.lastname: Sheerin
authors.forename: German R
authors.initials: GR
authors.lastname: Tejada
authors.forename: Habibe
authors.initials: H
authors.lastname: Karacay
authors.forename: Robert M
authors.initials: RM
authors.lastname: Sharkey
authors.forename: Ivan D
authors.initials: ID
authors.lastname: Horak
authors.forename: Hans J
authors.initials: HJ
authors.lastname: Hansen
authors.forename: David M
authors.initials: DM
authors.lastname: Goldenberg
mesh_terms: 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*
chemical_list: D018033:Antibodies, Bispecific; D002614:Chelating Agents; D006241:Haptens; D007202:Indicators and Reagents; D009842:Oligopeptides; D009942:Organometallic Compounds; D019275:Radiopharmaceuticals; C483470:gadolinium IMP241