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Purpose: To assess if pretargeting, using a combination of a recombinant bispecific antibody (bsMAb) that binds divalently to carcinoembryonic antigen (CEA) and monovalently to the hapten histamine-succinyl-glycine and a (90)Y-peptide, improves therapeutic efficacy in a human colon cancer-nude mouse xenograft compared with control animals given (90)Y-humanized anti-CEA immunoglobulin G (IgG)., Experimental Design: Clearance and biodistribution were monitored by whole-body readings and necropsy. Animals were monitored for 34 weeks with a determination of residual disease and renal pathology in survivors. Hematologic toxicity was assessed separately in non-tumor-bearing NIH Swiss mice., Results: Hematologic toxicity was severe at doses of 100 to 200 microCi of (90)Y-IgG, yet mild in the pretargeted animals given 500 or 700 microCi of the (90)Y-peptide. Evidence of end-stage renal disease was found at 900 microCi of the pretargeted (90)Y-peptide whereas animals given 700 microCi showed only mild renal pathology, similar to that seen in control animals given (90)Y-IgG. Biodistribution data indicated that the average amount of tumor radioactivity by a 700-microCi dose of the pretargeted peptide over a 96-hour period was increased 2.5-fold (48 microCi/g) compared with 150 microCi of (90)Y-IgG (18.9 microCi/g). At these doses, survival (i.e., time to progression to 2.5 cm(3)) was significantly improved (P < 0.04) compared with (90)Y-IgG, with ablation of about one third of the tumors, whereas viable tumor was present in all of the (90)Y-IgG-treated animals., Conclusion: Pretargeting increases the amount of radioactivity delivered to colorectal tumors sufficiently to improve the therapeutic index and responses as compared with conventional radioimmunotherapy.

Here we describe molecular imaging of cancer using signal amplification of a radiotracer in situ by pretargeting a multivalent, bispecific antibody to carcinoembryonic antigen (CEA), which subsequently also captures a radioactive hapten-peptide. Human colon cancer xenografts as small as approximately 0.15 g were disclosed in nude mice within 1 h of giving the radiotracer, with tumor/blood ratios increased by >or=40-fold (approximately 10:1 at 1 h, approximately 100:1 at 24 h), compared to a (99m)Tc-labeled CEA-specific F(ab') used clinically for colorectal cancer detection, while also increasing tumor uptake tenfold ( approximately 20% injected dose/g) under optimal conditions. This technology could be adapted to other antibodies and imaging modalities.

The article reviews the background and current status of pretargeting for cancer imaging and therapy with radionuclides. Pretargeting procedures were introduced approximately 20 years ago as an alternative to directly radiolabeled antibodies. Because they were multistep processes, they were met with resistance but have since progressed to simple and improved procedures that could become the next generation of imaging and therapy with radionuclides. The separation of the radiolabeled compound from the antibody-targeting agent affords pretargeting procedures considerable flexibility in the radiolabeling process, providing opportunities for molecular imaging using gamma- or positron-emitting radionuclides and a variety of beta- and alpha-emitting radionuclides of therapeutic applications. Pretargeting methods improve tumor/nontumor ratios, exceeding that achieved with directly radiolabeled Fab' fragments, particularly within just a few hours of the radionuclide injection. In addition, tumor uptake exceeds that of a Fab' fragment by as much as 10-fold, giving pretargeting a greatly enhanced sensitivity for imaging. Advances in molecular biology have led to the development of novel binding proteins that have further improved radionuclide delivery in these systems. Studies in a variety of hematologic and solid tumor models have shown advantages of pretargeting compared with directly radiolabeled IgG for therapy, and there are several clinical studies under way that are also showing promising results. Thus, the next generation of targeting agents will likely employ pretargeting approaches to optimize radionuclide delivery for a wide range of applications.

Purpose: A disadvantage of conventionally radioiodinated monoclonal antibodies (mAb) for cancer therapy is the short retention time of the radionuclide within target cells. To address this issue, we recently developed a method in which radioiodine is introduced onto antibodies using an adduct consisting of a nonmetabolizable peptide attached to the aminopolycarboxylate diethylenetriaminepentaacetic acid, designated IMP-R4. This adduct causes the radioiodine to become trapped in lysosomes following antibody catabolism. Clinical-scale production of 131I-IMP-R4-labeled antibodies is possible using a recently developed facile method., Experimental Design: The properties of 131I-IMP-R4-labeled anticarcinoembryonic antigen (CEA) humanized mAb hMN-14 were compared with the directly radioiodinated hMN-14 (131I-hMN-14) in CEA-expressing human colon cancer cell lines, LoVo and LS174T, and in nude mice bearing established LoVo tumor xenografts., Results: 125I-IMP-R4-hMN-14 retention in the cell lines was significantly increased (61.5% after 3 days) compared with 125I-hMN-14. In vivo, a significant improvement in tumor accretion of radiolabel was obtained using 131I-IMP-R4-hMN-14, which led to a marked improvement in therapeutic efficacy. Eight weeks post-treatment, mean tumor volumes were 0.16 +/- 0.19 and 1.99 +/- 1.35 cm3 in mice treated with 131I-IMP-R4-hMN-14 and 131I-hMN-14, respectively, with complete remissions observed in 27% of mice treated with 131I-IMP-R4-hMN-14 and none using 131I-hMN-14., Conclusion: 131I-IMP-R4-hMN-14 provides a significant therapeutic advantage in comparison to the conventionally 131I-labeled antibody. The ability of this labeling method to lend itself to clinical-scale labeling, the broad applicability of a humanized anti-CEA mAb for CEA-expressing cancers, and the clinical benefits of radioimmunotherapy with anti-CEA mAb shown recently for small-volume and minimal residual disease combine to make 131I-IMP-R4-hMN-14 a promising new agent for radioimmunotherapy.

Unlabelled: Radiolabeling of monoclonal antibodies (mAbs) with an intracellularly trapped form of (131)I (residualizing (131)I) involves radioiodinating a small molecular entity, conjugating it to the mAb, and purification. Column purifications are impractical during procedures involving multi-gigabecquerel levels of radioactivity. The goal of this study was to develop a simple, remote, "1-pot" method of radiolabeling and purification for the scaled-up radioiodination of a humanized anti-carcinoembryonic antigen (CEA) mAb, humanized MN-14 (hMN-14; labetuzumab), with an optimized residualizing (131)I moiety, (131)I-IMP-R4. IMP-R4 is MCC-Lys(MCC)-Lys(X)-d-Tyr-d-Lys(X)-OH, where MCC is 4-(N-maleimidomethyl)-cyclohexane-1-carbonyl and X is 1-((4-thiocarbonylamino)benzyl)-diethylenetriaminepentaacetic acid., Methods: An IODO-GEN-based remote labeling system was used. IMP-R4 was radioiodinated (0.13 mumol per 3.7 GBq of (131)I) at a pH of 7.0-7.4 and conjugated to disulfide-reduced hMN-14 after quenching of unused reactive (131)I. The product was purified by stirring for 5 min with a 20% (w/v) suspension of an anion-exchange resin and sterilely filtered into a sealed vial. Human serum albumin was added at a final concentration of 1%-2.5%. Immunoreactivity was determined by mixing with CEA and determining the complexation level by size-exclusion high-pressure liquid chromatography. Two control radiolabelings, either with unreduced hMN-14 or with IMP-R4 omitted, also were performed., Results: In 18 radiolabelings with (131)I in the range of 2.04-4.81 GBq (55-130 mCi), yields of 59.9% +/- 7.9% (mean +/- SD) at specific activities of 200 +/- 26 MBq/mg (5.4 +/- 0.7 mCi/mg) were obtained, with > or =95% of the radioactivity being associated with hMN-14 and with < or =4% aggregation. Similar yields were obtained in a subset of radiolabelings (n = 7) with >3.7 GBq of (131)I. The immunoreactivities of the preparations were typically >95%. Nonspecific incorporation in the absence of IMP-R4 was 0.5%, whereas that obtained with unreduced IgG was approximately 8%, possibly because of conjugation of IMP-R4 at lysine sites. The process also removed >99% of the quenching reagent used. Radiolabelings performed with freshly prepared solutions or lyophilized preparations produced similar yields, a result that suggested the option for a single-use kit design., Conclusion: Efficient removal of (131)I-IMP-R4 and quenched (131)I by 5 min of stirring with anion-exchange resin renders a multi-gigabecquerel-level preparation of (131)I-IMP-R4-hMN-14 safe, convenient, and practical.