89Zr immuno-PET: comprehensive procedures for the production of 89Zr-labeled monoclonal antibodies.
The use of immuno-PET, the combination of PET with monoclonal antibodies (mAbs), is an attractive option to improve tumor detection and mAb quantification. The long-lived positron emitter (89)Zr has ideal physical characteristics for immuno-PET, such as a half-life of 3.27 d, which is compatible with the time needed for intact mAbs to achieve optimal tumor-to-nontumor ratios. Thus far, a major limitation in the use of (89)Zr has been the lack of suitable methods for its stable coupling to mAbs. In this article, practical protocols for reproducible isolation of highly pure (89)Zr and the production of optimal-quality mAb-(89)Zr conjugates are provided.


METHODS
(89)Zr was produced by a (p,n) reaction on natural yttrium ((89)Y), isolated with a hydroxamate column, and used for labeling of premodified mAbs. mAbs were premodified with a novel bifunctional derivative of the chelate desferrioxamine B (Df) via a new linker chemistry. To this end, Df was initially succinylated (N-sucDf), temporarily filled with Fe(III), esterified by use of tetrafluorophenol, and then directly coupled to mAbs. Chimeric mAb (cmAb) U36, directed against head and neck cancer, was used for in vitro and in vivo evaluation. The in vitro stability of cmAb U36-N-sucDf-(89)Zr was assessed in human serum, and its in vivo behavior was evaluated by biodistribution and PET imaging studies in tumor-bearing nude mice. A cmAb U36-Df-(89)Zr conjugate containing a previously described succinimide ring-thioether unit in the linker was used as a reference.


RESULTS
(89)Zr was produced in large batches (6.5-13.5 GBq) and isolated with improved radionuclidic purity (>99.99%) and high yield (>94%). The Df-premodified mAbs gave (89)Zr-labeling efficiencies of 80% within 30 min, resulting in conjugates with preserved integrity and immunoreactivity. With respect to stability, the novel cmAb U36-N-sucDf-(89)Zr conjugate appeared to be superior to the reference conjugate. In vivo, the novel conjugate demonstrated selective tumor targeting, and on PET images obtained at 24, 48, and 72 h after injection of this conjugate, small tumors in the range of 19-154 mg were readily visualized.


CONCLUSIONS
Methods were developed for improved purification of the long-lived positron emitter (89)Zr. Moreover, a novel bifunctional Df chelate was synthesized for the reproducible coupling of (89)Zr to mAbs. The suitability of such conjugates to detect millimeter-sized tumors in xenograft-bearing nude mice was demonstrated.
Verel
Iris
I
Department of Otolaryngology/Head and Neck Surgery, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands.
Visser
Gerard W M
GW
Boellaard
Ronald
R
Stigter-van Walsum
Marijke
M
Snow
Gordon B
GB
van Dongen
Guus A M S
GA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000493:pharmacokinetics; D002289:Carcinoma, Non-Small-Cell Lung / Q000000981:diagnostic imaging / Q000378:metabolism; D004355:Drug Stability; D005240:Feasibility Studies; D005260:Female; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D008175:Lung Neoplasms; D051379:Mice; D008819:Mice, Nude; D009928:Organ Specificity; D016719:Radioimmunodetection / Q000379:methods; D011868:Radioisotopes / Q000737:chemistry* / Q000302:isolation & purification / Q000493:pharmacokinetics*; D019275:Radiopharmaceuticals / Q000138:chemical synthesis / Q000302:isolation & purification / Q000493:pharmacokinetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014018:Tissue Distribution; D014055:Tomography, Emission-Computed / Q000379:methods; D015040:Zirconium / Q000737:chemistry* / Q000302:isolation & purification / Q000493:pharmacokinetics*
D000911:Antibodies, Monoclonal; D011868:Radioisotopes; D019275:Radiopharmaceuticals; D015040:Zirconium