Background

[PubMed]

Tn antigen is a tumor-associated carbohydrate epitope (N-acetyl-galactosamine (GalNAc)-O-Ser/Thr (GalNAca-O-Ser/Thr)) (1-3). Three-step targeting with biotinylated MLS128 monoclonal antibody (Bt-MLS128 mAb), streptavidin (SA), and ¹¹¹In-diethylenetriamine pentaacetic acid (DTPA)-biotin (¹¹¹In-biotin) was developed for in vivo imaging of Tn antigen-expressing tumors (4). It was designed on the basis of avidin’s extraordinarily high binding affinity for biotin.

Avidin’s high affinity for biotin was first exploited in histochemical applications in the mid-1970s (5, 6). This affinity is more than one million times higher than that of most antibodies for most antigens. Avidin has four binding sites for biotin, and most proteins, including enzymes, can be conjugated with several molecules of biotin. The avidin-biotin binding is essentially irreversible. These properties allow molecular complexes to be formed between avidin and biotinylated antibodies. In addition, small molecular sizes of avidin and biotin allow improved tumor uptake and rapid intratumoral spatial distribution.

Altered glycosylation on the cell surface is a hallmark of malignant transformation and tumor progression. Incomplete synthesis of the carbohydrate chains and precursor accumulation result in loss of the normal carbohydrate antigens and high expression of the tumor-associated carbohydrate antigens (7-9). Lewis Y, TF, Globo H, GM2, polysialic acid, sialyl Lewis A, Tn, and sialyl Tn are some of the antigens investigated intensively as diagnostic markers or as vaccine antigens (8-11). Tn antigen was first reported as a tumor-associated antigen nearly 40 years ago (12). It is composed of a single GalNAc glycan residue attached via an α-linkage to either the serine (Ser) or the threonine (Thr) of a polypeptide chain (9, 11). In normal tissues, Tn antigen is masked by covalently bound terminal carbohydrate moieties, but in tumors it is unmasked because of defective O-glycosylation. Accordingly, Tn antigen is rarely expressed in normal tissues, but it is widely expressed in human carcinomas or hematological cancers. It has been reported that the Tn antigen is expressed in 70–90% of breast, colon, lung, bladder, cervical, ovarian, stomach, and prostate tumors (1, 3, 7). The expression levels of the Tn antigen are closely associated with tumor aggressiveness and poor survival of patients (8, 10). In addition, Tn antigen is recognized by the human immune system as a novel epitope, provoking immune responses in patients. There is a strong correlation among the expression of the Tn antigen, the development of the spontaneous antibodies against Tn, and the prognosis for patients with carcinomas. Clinical trials are under way to deliberately provoke or enhance human immune responses by injecting patients with synthetic peptide antigens bearing Tn structure (3, 8, 13-15). Tn antigen has attracted significant interest as a target for tumor diagnosis and immunotherapy. A number of anti-Tn IgG and IgM antibodies have been generated and investigated for their imaging feasibilities and anti-tumor activities (2, 4, 16-22). The results are generally inconsistent. There are still some issues to be resolved, such as immunogenicity, reduced effectiveness in vivo, and cross-reactivity against type-A blood antigen. In addition, directly radiolabeled antibodies usually show a slow and low uptake by tumors, and their blood clearance is also slow. Nakamoto et al. tested the imaging feasibility of three-step targeting with Bt-MLS128, SA, and ¹¹¹In-biotin in mice bearing LS180 human colon cancer xenografts (4).

Note: Investigators from the same research group as Nakamoto et al. also labeled the anti-Tn MLS128 mAb directly with ¹²⁵I/¹³¹I (¹²⁵I/¹³¹I-MLS128) and ¹¹¹In (¹¹¹In-MLS128), separately, and investigated their biodistribution and the feasibility of imaging tumors in mice bearing LS180 tumor xenografts. They also tested the imaging feasibility of two-step targeting with Bt-MLS128 and ¹²⁵I-SA (Bt-MLS128-¹²⁵I-SA) in mice with LS180 tumor xenografts (17, 19, 20, 23, 24).

Synthesis

[PubMed]

Imaging with three-step targeting included three agents: Bt-MLS128, SA, and ¹¹¹In-biotin (4, 20). The anti-Tn MLS128 is a mouse IgG3 antibody with κ light chain, and it was produced by immunizing mice with LS180 human colorectal cancer cells (21). The antibody was purified from the ascitic fluid of the hybridoma-bearing mice with protein A affinity chromatography. The murine OST6 mAb against an alkaline phosphatase-related substance was used as the control. The antibodies were biotinylated by mixing them with sulfosuccinimidyl-6-(biotinamido)hexanoate, and they were then purified with chromatography on PD-10 gel. The average numbers of biotin molecules coupled to the MLS128 and OST6 were 1.2 and 1.7, respectively. Bt-MLS128, Bt-OST6, and SA were also labeled with ¹²⁵I with the chloramine-T method. The labeling efficiencies of radioiodinated Bt-MLS128, Bt-OST6, and SA were 63.2, 58.3, and 81.3%, respectively, and their specific activities were 23.4, 21.6, and 30.1 GBq/µg (0.63, 0.58, and 0.81 kCi/µg), respectively. The ¹¹¹In-DTPA-biotin was developed by mixing DTPA-biotin with ¹¹¹In-chloride. The labeling efficiency of the ¹¹¹In-biotin was >99%, and its specific activity was 296.0 GBq/µg (8 kCi/µg).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Nakamoto et al. analyzed the binding abilities of ¹²⁵I-Bt-MLS128 and ¹²⁵I-SA to the immobilized avidin, biotin, and LS180 tumor cells by incubating them together for 1 h at 4ºC (4). More than 95% of ¹²⁵I-Bt-MLS128 bound to immobilized avidin. The immunoreactive fraction of ¹²⁵I-Bt-MLS128 that bound to the LS180 tumor cells was 47.2%, which was slightly lower than that of the unmodified MLS128 (data not shown). ¹²⁵I-SA showed >90% binding to the biotin-coated beads. There was no significant binding between the control ¹²⁵I-Bt-OST6 and the LS180 cells.

Animal Studies

Human Studies

[PubMed]

No references are currently available.

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