[99mTc]1,4,7,10-tetraazacyclododecane-N, N’, N’’, N’’’-tetraacetic acid-Phe-Lys-histamine-succinyl-Gly-D-Tyr-Lys- histamine-succinyl-Gly-thiosemicarbazonyl-glyoxyl-cysteine

[99mTc]IMP-245

Chopra A.

Publication Details

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Table

In vitro Rodents

Background

[PubMed]

Radioimmunotherapy is one of the choices available for the treatment of cancer, but this technique is not entirely successful because the antibodies (Abs) used to target the cancers are not very effective against solid tumors. This is primarily because the solid tumors have a low radiosensitivity and the Abs cannot be used to deliver a very high dose of radiation (1). The radionuclides are delivered to the tumors either by directly binding the radionuclide to the Ab that is targeted to a cellular antigen or the tumor cells are first pretargeted with an Ab and then exposed to a suitable ligand that carries the radionuclide for binding to the Ab. Use of Ab bound radioactivity usually results in hematological dose-limiting toxicity because the Ab is cleared very slowly from the blood circulation (2). Some investigators envisioned retargeting the tumors with an Ab followed by an infusion of radioactivity linked to a suitable ligand that rapidly clears from the blood and binds to the Ab would perhaps alleviate the hematological toxicity observed with radiolabeled Abs. Although there are several methods available to pretarget tumors, a common approach is to use bispecific Abs (bsAbs) - these Abs are usually genetically engineered and can bind to two distinct ligands with great specificity, inconjunction with a radiolabeled small molecule such as a hapten (3). Pretargeting with bsAbs for the treatment of tumors is under evaluation in several clinical trials approved by the United States Food and Drug Administration.

Sharkey et. al. have evaluated the use of a bispecific monoclonal antibody (bsMoAb) that is divalent for the carcinoembryonic antigen (CEA) and monovalent for anti-histamine-succinyl-glycine (HSG) for the molecular imaging of metastatic human colonic carcinoma in a mouse model (4). The structure of this bsMoAb (designated as TF2) was described by Goldenberg et. al (3). Briefly, the bsMoAb consists of three F(ab) fragments. Two of the fragments were specific for human CEA and the third fragment had binding specificity for HSG. A divalent HSG peptide labeled with radioactive 99m technetium (99m Tc) was used for the imaging of tumors that bound the bsMoAb. Data obtained from the pretargeting study was compared to results obtained with radioactive fluorine (18F) labeled fluorodeoxyglucose ([18F]FDG), the HSG peptide alone or an irrelevant anti-CD22 bsMoAb.

Synthesis

[PubMed]

Assembly of the TF2 anti-CEA/anti-HSG bsMoAb used for pretargeting the tumors was done by using the dock-and-lock platform technology (5) as described by Goldenberg et. al. (3). Employing recombinant DNA techniques, a cysteine modified dimerization and docking domain (called DDD2) was used to link two F(ab) fragments of the humanized MoAb hMN-14 to obtain C-DDD2-Fab-hMN-14and this was designated as the “A” component (6). The DDD2 consists of amino acids (aas) 1-44 of human tumor necrosis factor receptor RIIα and was linked to the carboxy-terminal of the Fd chain through a 14 aa flexible peptide linker. A F(ab) precursor fragment of the humanized MoAb h679 that has specificity for HSG was used to generate a second, recombinant, “B” component by linking the AD2 sequence (this is a 17 aa sequence derived from the A kinase anchor protein that is optimized for selectively binding with RII and has cysteine residues both at the N and the C terminals of the peptide) to the carboxy-terminus of the Fd chain through a 15-residue flexible peptide linker to obtain h679-AD2 (7).

Details of the production, purification and analysis of the two components are given elsewhere (5). A molar excess of component A was mixed with the B component in presence of a thiol reducing agent at room temperature for 1 h (5). The thiol reducing agent was removed by hydrophobic interaction chromatography and dimethyl sulphoxide was added to the solution for the formation of disulfide bonds. The disulfide-linked structure, designated TF2, was purified to homogeneity by affinity chromatography using HSG as the ligand (8). The stability of TF2 was determined by incubating it in pooled fresh human serum at 37oC under 5% CO2 for 7 days. The TF2 was reported to retain 98% of its bispecific binding activity after 7 days as determined by Biacore analysis (5).

The HSG containing peptide (IMP 245) was synthesized with a solid-phase, Fmoc-based method using Rink amide resin (9). The peptide was purified by reverse phase high performance liquid chromatography (RP-HPLC). The peptide was formulated into a lyophilized, single vial kit containing IMP 245, tin chloride, ascorbic acid and a 6-fold excess of unlabeled indium-to-peptide for binding to diethylenetriaminepentaacetic acid used during the synthesis and that may have co-eluted with the peptide. For labeling with radioactive technetium (99mTc) the vials were reconstituted with 99mTc pertechnetate in saline and incubated at room temperature for 10 min and transferred to a boiling water bath for 15 min. Purity of the 99mTc labeled peptide was determined by RP-HPLC and instant thin layer chromatography. To investigate the ability of the radiolabeled peptide to bind to CEA-bound bsMAb, it was mixed with a preformed mixture of CEA and the bsMAb. The specific activity of [99mTc]IMP 245 was between 1500 and 1600 Ci/mmol (5.5-5.9 TBq/mmol) with a radiochemical purity of >98% (10). A 100% of the radiolabeled peptide was reported to bind to the bsMAb (4).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

No references are currently available.

Animal Studies

Rodents

[PubMed]

The biodistribution of [99mTc]IMP 245 was investigated in NCr nude mice implanted with human colon cancer cell line, GW-39, tumors (10). The mice were injected with the radiochemical, sacrificed 30 min and 3 h later (n=5 animals per time point), and the radioactivity associated with the various organs determined. Results from this study indicated the labeled peptide was cleared primarily through the kidneys (8.63±2.42% of injected dose/gram tissue (%ID/g) at 30 min). During the same period only a small fraction of the radioactivity was observed in the liver (0.63±0.10%ID/g), small intestine (1.19±0.70%ID/g) and the large intestines (0.25±0.05%ID/g). The investigators also studied the biodistribution of [99mTc]IMP 245 in mice after pretargeting the tumors with the bsMAb (10). The mice were injected with the labeled peptide 24 h after treatment with the bsMAb and the animals were sacrificed at different time points (n=5 mice per time point), and accumulated radioactivity in the various organs was determined. A high accumulation of the radioactivity was observed in the tumors (14.2±5.27%ID/g) at 3 h compared to the other organs (kidneys, 2.68±0.50%ID/g; liver, 0.71±0.13%ID/g; blood, 2.23±0.39%ID/g) and a low uptake was noted in the gastrointestinal tract during the same period.

In another study with bsMAb targeted mice bearing GW-39 cell xenograft tumors that were injected with the IMP 245 radiotracer 24 h after treatment with the bsMAb, xenografts as small as ~0.15 g were visible within 1 h after the administration of [99mTc]IMP 245 (11). During this time the tumor/blood ratios were reported to increase ≥40-fold compared to a 99mTc-labeled CEA-specific F(ab”) used clinically for colorectal cancer detection. Based on these results the investigators suggested this technology could probably be used with other antibodies and imaging modalities also (11).

The tumor specificity of [99mTc]IMP 245 was clearly evident from the data obtained by Rossi et. al. in a study performed on TF2 pretargeted nude mice bearing LS-174T cell xenograft tumors (5). The tumor/nontumor ratios obtained from this study are given in the table below (ratio ± standard deviation):

Sharkey et. al. investigated the retargeting approach to detect small (<0.3 mm diameter) microdessimated human colon cancer GW-39 cell colonies in the lungs of nude mice (4). The investigators reported that by retargeting with the TF2 bsMAb the tumors were detected in lungs of the mice (n=32) within 1.5 h after administration of the [99mTc]IMP 245 peptide. In another set of mice that were treated with the peptide alone (n=20 animals), an irrelevant bsMAb (anti-CD22) (n=12 animals) and retargeted peptide and those treated with [18F]FDG (n=15 animals) did not detect the tumors. It was also reported that uptake of the labeled peptide in the lungs tumors of the TF2 pretargeted animals was nine fold higher than in lungs of the non-pretargeted mice (4). By comparison, the uptake was only 1.5 fold higher in the tumor bearing lungs with [18F]FDG or the labeled peptide alone. No blocking studies were performed.

Other Non-Primate Mammals

[PubMed]

No publications are currently available.

Non-Human Primates

[PubMed]

No publications are currently available.

Human Studies

[PubMed]

No publications are currently available.

Supplemental Information

[Disclaimers]

NIH Support

NIH Grant #

References

1.
Goldenberg D.M. Targeted therapy of cancer with radiolabeled antibodies. J Nucl Med. 2002;43(5):693–713. [PubMed: 11994535]
2.
Goldenberg D.M., Chatal J.F., Barbet J., Boerman O., Sharkey R.M. Cancer Imaging and Therapy with Bispecific Antibody Pretargeting. Update Cancer Ther. 2007;2(1):19–31. [PMC free article: PMC2034280] [PubMed: 18311322]
3.
Goldenberg D.M., Rossi E.A., Sharkey R.M., McBride W.J., Chang C.H. Multifunctional antibodies by the Dock-and-Lock method for improved cancer imaging and therapy by pretargeting. J Nucl Med. 2008;49(1):158–63. [PubMed: 18077530]
4.
Sharkey R.M., Karacay H., Vallabhajosula S., McBride W.J., Rossi E.A., Chang C.H., Goldsmith S.J., Goldenberg D.M. Metastatic human colonic carcinoma: molecular imaging with pretargeted SPECT and PET in a mouse model. Radiology. 2008;246(2):497–507. [PubMed: 18227543]
5.
Rossi E.A., Goldenberg D.M., Cardillo T.M., McBride W.J., Sharkey R.M., Chang C.H. Stably tethered multifunctional structures of defined composition made by the dock and lock method for use in cancer targeting. Proc Natl Acad Sci U S A. 2006;103(18):6841–6. [PMC free article: PMC1447525] [PubMed: 16636283]
6.
Sharkey R.M., Juweid M., Shevitz J., Behr T., Dunn R., Swayne L.C., Wong G.Y., Blumenthal R.D., Griffiths G.L., Siegel J.A. et al. Evaluation of a complementarity-determining region-grafted (humanized) anti-carcinoembryonic antigen monoclonal antibody in preclinical and clinical studies Cancer Res 199555Suppl235935s–5945s. [PubMed: 7493374]
7.
Rossi E.A., Sharkey R.M., McBride W., Karacay H., Zeng L., Hansen H.J., Goldenberg D.M., Chang C.H. Development of new multivalent-bispecific agents for pretargeting tumor localization and therapy. Clin Cancer Res. 2003;9(10 Pt 2):3886S–96S. [PubMed: 14506187]
8.
Rossi E.A., Chang C.H., Losman M.J., Sharkey R.M., Karacay H., McBride W., Cardillo T.M., Hansen H.J., Qu Z., Horak I.D., Goldenberg D.M. Pretargeting of carcinoembryonic antigen-expressing cancers with a trivalent bispecific fusion protein produced in myeloma cells. Clin Cancer Res. 2005;11(19 Pt 2):7122s–7129s. [PubMed: 16203811]
9.
Karacay H., McBride W.J., Griffiths G.L., Sharkey R.M., Barbet J., Hansen H.J., Goldenberg D.M. Experimental pretargeting studies of cancer with a humanized anti-CEA x murine anti-[In-DTPA] bispecific antibody construct and a (99m)Tc-/(188)Re-labeled peptide. Bioconjug Chem. 2000;11(6):842–54. [PubMed: 11087333]
10.
Sharkey R.M., McBride W.J., Karacay H., Chang K., Griffiths G.L., Hansen H.J., Goldenberg D.M. A universal pretargeting system for cancer detection and therapy using bispecific antibody. Cancer Res. 2003;63(2):354–63. [PubMed: 12543788]
11.
Sharkey R.M., Cardillo T.M., Rossi E.A., Chang C.H., Karacay H., McBride W.J., Hansen H.J., Horak I.D., Goldenberg D.M. Signal amplification in molecular imaging by pretargeting a multivalent, bispecific antibody. Nat Med. 2005;11(11):1250–5. [PubMed: 16258537]