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Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.

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Anti-mouse monoclonal antibody immunoglobulin G conjugated to GRKKRRQRRRPPQGYG-diethylenetriamine pentaacetic acid-[111In]

[111In]mIgG-tat peptide
, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD 20894, vog.hin.mln.ibcn@dacim

Created: ; Last Update: February 20, 2008.

Chemical name:Anti-mouse monoclonal antibody immunoglobulin G conjugated to GRKKRRQRRRPPQGYG-diethylenetriamine pentaacetic acid -[111In]
Abbreviated name:[111In]mIgG-tat peptide
Agent Category:Mouse immunoglobulin G
Target:Cell nucleus
Target Category:Nuclear uptake
Method of detection:Single-photon emission computed tomography (SPECT), gamma planar imaging
Source of signal:111In
  • Checkbox In vitro
  • Checkbox Rodents



Monoclonal antibodies (mAb) are often directed toward cell-surface molecules and may be radiolabeled for the imaging or treatment of cancer and other ailments (1-3). The radiolabeled mAbs are not readily internalized by the cell except by receptor-mediated endocytosis, which limits the use of these macromolecules either for the imaging or treatment of neoplastic solid tumors (4). However, Hu et al. showed that linking the mAb to the tat peptide (17 amino acid residues in the following sequence: GRKKRRQRRRPPQGYG) derived from the human immunodeficiency virus 1 (HIV-1) transactivator of the transcription (TAT) protein enabled it to be internalized and to be directed to the cellular nucleus (5, 6). Several investigators have used the tat peptide to enable cell penetration by a variety of large molecules including proteins, oligodeoxynucleotides, liposomes, and even nanoparticles (7-9). Conjugation of the tat peptide to mouse immunoglobulins (mIgG) labeled with radioactive iodine (123I) was shown to enhance cellular internalization of [123I]mIgG under in vitro conditions in human breast cancer MDA-MB-468 cells and also in xenograft tumors derived from these cells in athymic mice (6). The investigators used a similar approach to construct and direct p21WAF-1/Cip-1, another 123I-labeled mAb against the intranuclear cyclin-dependent kinase inhibitor; p21WAF-1/Cip-1 was preferentially retained in xenograft tumors in athymic mice injected intratumorally with epidermal growth factor to induce the expression of p21WAF-1/Cip-1 (5). However, radiostability of the tat peptide (i.e., 123I-labeled mAb) is a major limitation of its use to target the intracellular or intranuclear epitopes because loss of the radiolabel can lead to the delivery of a suboptimal radioactivity dose to the cancerous lesions; in addition, 123I has a short half-life (13.3 h), so it is not possible to perform imaging studies for prolonged periods, especially when the tumor/background ratios are usually highest at 72 h after the administration of a radiolabeled mAb (10).

Press et al. showed that mAbs labeled with radioactive indium (111In; half-life = 2.8 days) and conjugated to the tat peptide were more stable in vivo compared with those labeled with 123I; 111In-labeled mAbs generate a higher tumor/background ratio, including internalization into the nucleus of human breast cancer BT-474 cells (11). In addition, the 111In-labeled and tat peptide–conjugated, anti-mIgG ([111In]anti-mIgG-tat) were retained two times longer by the cells compared to the same tat peptide–linked mAb labeled with 123I (11). In an effort to extend this work, Cornelissen et al. investigated the biodistribution and nuclear importation of [111In]anti-mIgG-tat in normal tissue and BT-474 cell xenograft tumors in athymic mice (4). These studies are briefly described in this chapter.



The synthesis of [111In]mIgG-tat was described by Cornelissen et al. (12). The tat peptide used for conjugation to the anti-mIgG was a synthetic peptide, but details of the chemistry used to prepare the peptide were not provided (12). Purity of the tat peptide was reported to be >97% as determined with high-performance liquid chromatography (HPLC).

Site-specific conjugation was used to link the [111In]tat to the anti-IgG mAb. The mIgG was obtained from commercial sources, and it was reacted with a four-fold molar excess of diethylenetriamine pentaacetic acid (DTPA) in anhydrous dimethyl sulfoxide. The reaction was allowed to proceed at room temperature for 45 min, and the DTPA-conjugated anti-mIgG was purified with size-exclusion HPLC (SE-HPLC) on a P-6 mini-column, with the use of phosphate-buffered saline (PBS) as the elution solvent. The anti-mIgG conjugate was determined to have 0.8 ± 0.3 mol DTPA/mol anti-mIgG. The purified DTPA-anti-mIgG was then mixed with an 800-fold molar excess of sodium periodide to generate aldehyde groups on the carbohydrates of the Fc domain of the mAb. After the oxidation reaction, a 40-fold molar excess of tat peptides (in PBS, pH 6.5) was added to the oxidized anti-mIgG, and the mixture was incubated in the dark at room temperature for 24 h. After this incubation, sodium cyanoborohydride (in PBS, pH 6.0) was added to the mixture to reduce the Schiff base between the tat peptide and the DTPA-anti-mIgG. The tat peptide–conjugated DTPA-anti-mIgG (anti-mIgG-tat) was then purified on a P-6 mini-column and eluted into citrate buffer (pH 5.0) (12). Under these conditions 1.2 ± 0.2 tat peptides were determined (n = 3 batches) to be substituted on each molecule of the anti-mIgG antibody.

Anti-mIgG-tat was labeled by exposing it to labeled indium (111In) chloride for 45 min at room temperature (12). The final radiochemical purity of the product was >95% as determined with instant thin-layer chromatography developed in sodium citrate buffer (pH 5.0); the Rf values for the various reaction components or the product were not provided. The specific activity of the labeled tat-anti-mIgG ([111In]anti-mIgG-tat) was 11,000 TBq/mmol (297.3 Ci/mmol).

The anti-mIgG conjugated to DTPA and modified with the tat peptide was reported to have the ability to recognize the same epitopes and to have a binding affinity that was similar to the unmodified parent mAb (12).

In another publication, the stability of [111In]anti-mIgG-tat was determined by incubating it in mouse plasma for 24–72 h, after which the amount of 111In associated with the anti-IgG or transferrin was determined with SE-HPLC (4). The labeled mAb was reported to be stable up to 24 h, but at 48 and 72 h, 2.9 ± 0.2% and 4.7 ± 0.3% of the label, respectively, was determined to be transchelated from [111In]anti-mIgG-tat to transferrin (n = 3 determinations). The investigators cautioned that, because the [111In]anti-mIgG-tat and transferrin peaks were not separated completely by SE-HPLC, the actual extent of transchelation could be higher than reported in the publication (4).

In Vitro Studies: Testing in Cells and Tissues


Cornelissen et al. studied the cellular and nuclear penetration properties of [111In]anti-mIgG-tat with the use of BT-474 cells (12). The kinetics of [111In]anti-mIgG-tat uptake by the cells, cytoplasm, and nuclei was studied in the BT-474 cells. These investigators also performed competition studies with excess tat peptide or ammonium chloride (NH4Cl), an endosome acidification inhibitor(12). These results were compared with those obtained with 111In-labeled anti-IgG alone, which served as a control. The uptake and nuclear penetration of [111In]anti-mIgG-tat was reported to be two times greater compared with [111In]anti-mIgG, and the uptake was inhibited by the presence of either the tat peptide or NH4Cl. Of the total radioactivity taken up by the cells (127 ± 3.6 fmol) under these conditions, roughly half was detected in the cytoplasm (61.0 ± 2.5 fmol), and the other half was found in the nucleus (71.2 ± 3.0 fmol).

The efflux of incorporated radioactivity from the BT-474 cells after exposure to the labeled anti-mIgG was also studied (12). The cells were initially allowed to take up [111In]anti-mIgG-tat for 1 h at 37°C, and then the cells were washed and resuspended in growth medium for up to 24 h. During this time the amount of radioactivity remaining in the cell as free, cytoplasmic, and nuclear fractions was determined at various time points. The investigators reported that the elimination of radioactivity from the cell and the different compartments followed a biphasic model. Approximately 50% of the radioactivity was lost within 5 h, after which the amount of radioactivity remained constant within the cells, the cytoplasm and the nucleus (12).

Animal Studies



Cornelissen et al. investigated the biodistribution of [111In]anti-mIgG-tat in the tumors and normal tissue in athymic mice bearing subcutaneous BT-474 tumor xenografts (4). The uptake was compared after either an intravenous (IV) injection or intratumoral administration of [111In]anti-mIgG-tat (n = 3 animals) and compared with that of [111In]anti-mIgG (n = 3 animals) administered in the same manner. On the basis of the percent injected dose per gram tissue (% ID/g) and the area under the curve (AUC) calculations, after an IV injection of either radiochemical, [111In]anti-mIgG-tat (2.8 ± 0.2% ID/g) was reported to clear from blood two to three times faster than [111In]anti-mIgG (6.3 ± 1.5% ID/g) under these conditions at 72 h after injection (P = 0.0004). With the IV injection, at 72 h after administration, a two- to three-fold lower uptake of radioactivity was reported with [111In]anti-mIgG-tat compared to the control [111In]anti-mIgG, in the liver, lungs, and the small and large intestines of the animals (4). With either IV injected radiochemical, the accumulation of radioactivity was not significantly different in the kidneys or the xenograft tumors.

Cornelissen et al. also studied the uptake of [111In]anti-mIgG-tat and [111In]anti-mIgG in the normal and the tumor tissue after an intratumoral injection of the radiochemicals in the mice bearing a xenograft tumor (n = 3 animals/group and time point) (4). Uptake of radioactivity was determined at various time points up to 144 h after the intratumoral injection. The radioactivity was initially very high (146–154% ID/g) after an intratumoral injection that declined 12- to 14-fold by 144 h after administration. When administered intratumorally, the accumulation of [111In]anti-mIgG-tat in the tumors was higher than when it was administered intravenously to the animals. Also, the AUC was ~three-fold (610 ± 157% ID•h) for the intratumorally injected [111In]anti-mIgG-tat compared to an AUC of 200 ± 37% ID h for the intratumorally injected [111In]anti-mIgG. An intratumoral injection of [111In]anti-mIgG-tat resulted in an encased nuclear localization of radioactivity only in the tumor cell nuclei compared with an increased localization of the radioactivity in the nuclei of the tumor, liver, and kidneys after IV administration. Although the tumors were not imaged after an IV injection, the investigators reported that when they were imaged after an intratumoral injection, the radioactivity was located primarily in the tumors with both [111In]anti-mIgG-tat and [111In]anti-mIgG (4). From these results the investigators concluded that radiolabeled immunoconjugates of the tat peptide have a good potential use for imaging intracellular epitopes or for localized radiotherapy of tumors.

Other Non-Primate Mammals


No references are currently available.

Non-Human Primates


No references are currently available.

Human Studies


No references are currently available.

Supplemental Information



1. Koyama, Y., T. Barrett, Y. Hama, G. Ravizzini, P.L. Choyke, and H. Kobayashi, In vivo molecular imaging to diagnose and subtype tumors through receptor-targeted optically labeled monoclonal antibodies. Neoplasia, 2007. 9(12): p. 1021-9.

2. Stillebroer, A.B., E. Oosterwijk, W.J. Oyen, P.F. Mulders, and O.C. Boerman, Radiolabeled antibodies in renal cell carcinoma. Cancer Imaging, 2007. 7: p. 179-88.

3. Dadachova, E. and A. Casadevall, Treatment of infection with radiolabeled antibodies. Q J Nucl Med Mol Imaging, 2006. 50(3): p. 193-204.

4. Cornelissen, B., K. McLarty, V. Kersemans, D.A. Scollard, and R.M. Reilly, Properties of [(111)In]-labeled HIV-1 tat peptide radioimmunoconjugates in tumor-bearing mice following intravenous or intratumoral injection. Nucl Med Biol, 2008. 35(1): p. 101-10.

5. Hu, M., P. Chen, J. Wang, D.A. Scollard, K.A. Vallis, and R.M. Reilly, 123I-labeled HIV-1 tat peptide radioimmunoconjugates are imported into the nucleus of human breast cancer cells and functionally interact in vitro and in vivo with the cyclin-dependent kinase inhibitor, p21(WAF-1/Cip-1). Eur J Nucl Med Mol Imaging, 2007. 34(3): p. 368-77.

6. Hu, M., P. Chen, J. Wang, C. Chan, D.A. Scollard, and R.M. Reilly, Site-specific conjugation of HIV-1 tat peptides to IgG: a potential route to construct radioimmunoconjugates for targeting intracellular and nuclear epitopes in cancer. Eur J Nucl Med Mol Imaging, 2006. 33(3): p. 301-10.

7. Bullok, K.E., S.T. Gammon, S. Violini, A.M. Prantner, V.M. Villalobos, V. Sharma, and D. Piwnica-Worms, Permeation peptide conjugates for in vivo molecular imaging applications. Mol Imaging, 2006. 5(1): p. 1-15.

8. Torchilin, V.P., R. Rammohan, V. Weissig, and T.S. Levchenko, TAT peptide on the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors. Proc Natl Acad Sci U S A, 2001. 98(15): p. 8786-91.

9. Zhang, Y.M., C.H. Tung, J. He, N. Liu, I. Yanachkov, G. Liu, M. Rusckowski, and J.L. Vanderheyden, Construction of a novel chimera consisting of a chelator-containing Tat peptide conjugated to a morpholino antisense oligomer for technetium-99m labeling and accelerating cellular kinetics. Nucl Med Biol, 2006. 33(2): p. 263-9.

10. Reilly, R.M., J. Sandhu, T.M. Alvarez-Diez, S. Gallinger, J. Kirsh, and H. Stern, Problems of delivery of monoclonal antibodies. Pharmaceutical and pharmacokinetic solutions. Clin Pharmacokinet, 1995. 28(2): p. 126-42.

11. Press, O.W., D. Shan, J. Howell-Clark, J. Eary, F.R. Appelbaum, D. Matthews, D.J. King, A.M. Haines, P. Hamann, L. Hinman, D. Shochat, and I.D. Bernstein, Comparative metabolism and retention of iodine-125, yttrium-90, and indium-111 radioimmunoconjugates by cancer cells. Cancer Res, 1996. 56(9): p. 2123-9.

12. Cornelissen, B., M. Hu, K. McLarty, D. Costantini, and R.M. Reilly, Cellular penetration and nuclear importation properties of 111In-labeled and 123I-labeled HIV-1 tat peptide immunoconjugates in BT-474 human breast cancer cells. Nucl Med Biol, 2007. 34(1): p. 37-46.

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