64Cu-p-NH2-Bn-DOTA-hu14.18K322A, a PET radiotracer targeting neuroblastoma and melanoma.
The hu14.18K322A variant of the GD2-targeting antibody hu14.18 has been shown to elicit a level of antibody-dependent cell-mediated cytotoxicity toward human neuroblastoma cells similar to that of the parent antibody. However, hu14.18K322A exhibited a decreased complement activation and associated pain, the dose-limiting toxicity in neuroblastoma immunotherapy. PET with a radiolabeled analog of the same antibody used in treatment will provide insight into the ability of hu14.18K322A to reach its target, as well as nontarget uptake that may cause side effects. Such antibody radiotracers might also provide a method for measuring GD2 expression in tumors, thus enabling the prediction of response to anti-GD2 therapy for individual patients.


METHODS
The conjugation of hu14.18K322A with p-NH(2)-Bn-DOTA was accomplished using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide with subsequent (64)Cu radiolabeling at 37°C for 30 min. Immunoreactivity of the conjugate was assessed by a dose-escalation blocking experiment measuring binding to purified GD2 versus GD1b as a negative control. Cell uptake and biodistribution studies in M21 (GD2-positive) and PC-3 (GD2-negative) tumor models were performed, as was small-animal PET/CT of M21 and PC-3 tumor-bearing mice.


RESULTS
The labeling of (64)Cu-p-NH(2)-Bn-DOTA-hu14.18K322A was achieved at more than 95% radiochemical purity and a specific activity of 127-370 MBq/mg (3.4-10 mCi/mg) after chromatographic purification. Preliminary in vitro data demonstrated a greater than 6-fold selectivity of binding to GD2 versus GD1b and dose-dependent inhibition of binding by unmodified hu14.8K322A. In vivo data, including small-animal PET/CT, showed significant GD2-positive tumor-targeting ability, with a persistent 2-fold-higher uptake of radiotracer than in GD2-negative tumors.


CONCLUSIONS
(64)Cu-p-NH(2)-Bn-DOTA-hu14.18K322A represents a novel PET radiotracer to facilitate clinical investigations of anti-GD2 immunotherapies and to complement other imaging modalities in the staging and treatment of neuroblastoma.
Division of Nuclear Medicine, Department of Radiological Sciences, St. Jude Children's Research Hospital, Memphis, TN, USA.
Amy L
AL
Vavere
Elizabeth R
ER
Butch
Jason L J
JL
Dearling
Alan B
AB
Packard
Fariba
F
Navid
Barry L
BL
Shulkin
Raymond C
RC
Barfield
Scott E
SE
Snyder
D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D045744:Cell Line, Tumor; D003301:Copper Radioisotopes; D005260:Female; D005732:Gangliosides / Q000276:immunology; D006207:Half-Life; D006573:Heterocyclic Compounds, 1-Ring / Q000737:chemistry*; D006801:Humans; D008545:Melanoma / Q000000981:diagnostic imaging* / Q000473:pathology; D051379:Mice; D009447:Neuroblastoma / Q000000981:diagnostic imaging* / Q000473:pathology; D049268:Positron-Emission Tomography / Q000379:methods*; D011849:Radioactive Tracers; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology / Q000493:pharmacokinetics
D000906:Antibodies; D003301:Copper Radioisotopes; D005732:Gangliosides; D006573:Heterocyclic Compounds, 1-Ring; D011849:Radioactive Tracers; D011993:Recombinant Fusion Proteins; C025447:sialogangliosides; C071349:1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid