Background

[PubMed]

The tumor-associated glycoprotein 72 (TAG-72) is a high molecular weight mucigenous protein that is found mainly in the extracellular matrix of neoplastic tumors (1). This glycoprotein is usually not expressed in normal tissues but is overexpressed in the tumors of several cancers, such as those of the colon and rectum (colorectal cancer), stomach, pancreas, ovaries, prostate, lung, breast, etc. Therefore, TAG-72 is considered to have much theranostic value (useful for the diagnosis and/or therapy of a disease) because it can be used for antigen-directed surgical resection of cancerous tumors with radiolabeled anti-TAG-72 monoclonal antibodies (mAb) (1). In addition, the detection or absence of TAG-72 expression in colorectal cancer tumors was shown to have prognostic value because individuals who had complete removal of tumors with surgery guided by mouse anti-Tag-72 mAb were shown to have a long-term survival advantage compared to patients who had an incomplete removal of the lesions (2). However, the main limitation of using radiolabeled antibodies (Ab) or their fragments to detect and treat malignant tumors is the long circulation half-life of these molecules and the development of human anti-mouse antibodies in the patients (3). In comparison, synthetic anti-tumor peptides that can be used for the noninvasive imaging and therapy of cancers show rapid clearance from circulation and non-target tissue, have more access and deeper penetration into the lesions, and may not be immunogenic (3-5).

Recently, the peptides GGVSCMQTSPVCENNL (A2-6) and NPGTCKDKWEICLLNGG (A3-10) were identified from a phase-display peptide library (f88-4/cys6) against TAG-72 and labeled with ^99mTc to generate [^99mTc]-A2-6 and [^99mTc]-A3-10 (4). The labeled peptides were then evaluated in mice for the detection of LS-174T cell xenograft tumors that overexpress the TAG-72 antigen in mice. In another study, a modified procedure (from that used to purify the A2-6 and A3-10 peptides) was used to purify peptides from the f88-4/cys6 phage library, and two new peptides, FRERCDKHPQKCTKFL (G3-15) and DPRHCQKRVLPCPAWL (T3-15), that had a high affinity for the TAG-72 antigen were identified (3). These peptides were then labeled with ^99mTc to obtain [^99mTc]-G3-15 and [^99mTc]-T3-15, and the labeled compounds were tested for the detection of LS-174T cell xenograft tumors in mice. This chapter describes the results obtained with [^99mTc]-A2-6 and [^99mTc]-A3-10. Studies performed with [^99mTc]-G3-15 and [^99mTc]-T3-15 are described in a separate chapter of MICAD (www.micad.nih.gov) (6).

Synthesis

[PubMed]

Peptides A3-6 and A3-10 were obtained in their native L-configuration from a commercial source, biotinylated, conjugated with N-hydroxysuccinimidyl-S-acetyl-mercaptoacetyltriglycine, and labeled with ^99mTc as described by Chen et al. (4). The peptides were purified on a Biogel P2 size-exclusion column and had a radiochemical purity of >90% as determined with instant thin-layer chromatography, paper chromatography, and reversed-phase high-performance liquid chromatography (4). The radiochemical yields of the labeled peptides were not reported. The specific activity of the peptides was reported to be 1.68 MBq/nmol (45.36 μCi/nmol).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Cell binding of the biotinylated peptides was determined with flow cytometry analysis as described by Chen et al. (4). Briefly, LS-174T cells (overexpress TAG-72) and HT-29 cells (controls; do not express TAG-72) were respectively incubated with increasing concentrations of the peptides, washed to remove the excess peptides, and treated with a mouse anti-biotin Ab. Subsequently, Phycoerythrin anti-mouse Ab was used to detect the cell-bound peptides with flow cytometry. Only the LS-174T cells were observed to bind the peptides, indicating a cell-binding specificity of the compounds.

The IC₅₀ values of the two ^99mTc-labeled peptides were determined in an in vitro competitive binding assay (4). The LS-174T cells were incubated with the labeled peptides in the presence of increasing concentrations (10−¹² to 10−⁴ mol/L) of the respective non-labeled peptides, and the IC₅₀ values were calculated as described by Chen et al. (4). The IC₅₀ values of A2-6 and A3-10 were determined to be 46.5 nmol/L and 420 nmol/L, respectively.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

Supplemental Information

[Disclaimers]

No supplemental information is currently available.

NIH Support

Studies reported in this chapter were supported by a National Institutes of Health grant CA111606.

References

1.

Povoski S.P., Hatzaras I.S., Mojzisik C.M., Martin E.W. Oncologic theranostics: recognition of this concept in antigen-directed cancer therapy for colorectal cancer with anti-TAG-72 monoclonal antibodies. Expert Rev Mol Diagn. 2011;11(7):667–70. [PubMed: 21902525]

2.

Povoski, S.P., I.S. Hatzaras, C.M. Mojzisik, M.W. Arnold, G.H. Hinkle, C.L. Hitchcock, D.C. Young, and E.W. Martin, Jr., Antigen-Directed Cancer Surgery for Primary Colorectal Cancer: 15-Year Survival Analysis. Ann Surg Oncol, 2011. [PubMed: 21732140]

3.

Xiao N., Cheng D., Wang Y., Chen L., Liu X., Dou S., Liu G., Liang M., Hnatowich D.J., Rusckowski M. Identification of a high affinity TAG-72 binding peptide by phage display selection. Cancer Biol Ther. 2011;11(1):22–31. [PMC free article: PMC3047098] [PubMed: 20980835]

4.

Chen L., Wang Y., Cheng D., Dou S., Liu X., Liu G., Hnatowich D.J., Rusckowski M. Comparing two TAG-72 binding peptides previously identified by phage display as potential imaging agents. Nucl Med Commun. 2011;32(10):920–4. [PMC free article: PMC3164986] [PubMed: 21876403]

5.

Rusckowski M., Gupta S., Liu G., Dou S., Hnatowich D.J. Evidence of specificity of radiolabeled phage display peptides for the TAG-72 antigen. Cancer Biother Radiopharm. 2007;22(4):564–72. [PubMed: 17803452]

6.

Chopra, A., 99mTc-Labeled tumor-associated glycoprotein 72 binding peptides, G3-15 and T3-15. Molecular Imaging and Contrast agent Database (MICAD) [database online]. National Library of Medicine, NCBI, Bethesda, MD, USA. Available from www​.micad.nih.gov, 2004 -to current. [PubMed: 22049575]