Background

[PubMed]

Radioiodinated anti–TAG-72 non-covalently linked CC49 divalent single-chain Fv antibody (¹²⁵I/¹³¹I-CC49 (scFv)₂ Ab), which is formed by the conjugation of ¹²⁵I/¹³¹I with a bioengineered recombinant anti–tumor-associated glycoprotein 72 (TAG-72) antibody construct, has been developed for gamma imaging of cancers that express TAG-72 (1-4). ¹²⁵I has a physical half-life (t_½) of 60 days with a gamma energy that is not ideal for in vivo imaging. ¹³¹I has a physical half-life (t_½) of 8.02 days with a gamma energy that is high but acceptable for in vivo imaging.

The TAG-72 antigen was isolated from the LS-174T human colon cancer xenograft as a high molecular weight glycoprotein (molecular mass of 10⁶ Da) with mucin-like characteristics (5-8). It is expressed on a variety of human adenocarcinomas such as pancreatic, breast, colorectal, prostate, endometrial, and ovarian cancers. This antigen has also been shown to be shed into the serum of cancer patients (9). The murine monoclonal antibody B72.3 (MAb B72.3) against TAG-72 mucin was initially generated by immunization of mice with a membrane-enriched fraction of a human breast carcinoma (10). With the use of affinity-purified TAG-72 from LS-174T as an immunogen, CC49 and other anti–TAG-72 MAbs with higher affinity constants (K_a) have been produced and characterized (5, 6, 10, 11). CC49 MAb appears to react with a unique disaccharide sialyl-Tn (STn) epitope on TAG-72 (2, 4).

Radiolabeled MAbs have been developed for both the diagnosis and treatment of tumors (12). Radiolabeled B72.3 and CC49 exhibit excellent tumor localization capabilities with potential diagnostic and therapeutic applications in the clinical setting (13, 14). Because of their relatively large size, intact radiolabeled MAbs tend to have unfavorable imaging kinetics, poor tumor penetration, and high potential for human anti-mouse antibody response (11, 15-17). One approach to minimize these problems is reducing intact antibodies to antibody fragments such as F(ab’)₂ and Fab’ (18). Another approach is the development of genetic engineering methods to obtain single-chain Fv constructs (scFv) and multivalent scFv constructs (11, 19, 20). These scFv constructs contain the variable regions of the light chain (V_L) and heavy chain (V_H) connected by a flexible linker. Colcher et al. (21) constructed the monomeric CC49 scFv Ab (~27 kDa), which selectively recognizes a unique STn epitope of TAG-72. The radioiodinated CC49 scFv appeared to clear rapidly from the blood with good tumor penetration (1, 20). To further improve the imaging kinetics by use of multivalency as a means of increasing the functional affinity, a non-covalently linked dimeric scFv was isolated on the basis of the spontaneous formation of the dimer from the monomeric CC49 scFv (1, 2, 4). This dimeric scFv was designated (scFv)₂ to distinguish it from the covalently-linked dimer sc(Fv)₂, in which two repeating chains of V_L and V_H are covalently linked in tandem (4). The radioiodinated CC49 (scFv)₂ showed good stability and improved in vivo pharmacokinetics compared with the intact CC49 IgG.

Synthesis

[PubMed]

Beresford et al. (4) and Pavlinkova et al. (1, 3) reported the construction and radiolabeling of the¹²⁵I/¹³¹I-CC49 (scFv)₂ Ab. The covalent CC49 scFv (V_L-linker-V_H) was derived from the murine MAb CC49 and constructed with the 205C linker with 25 amino acids (LSADDAKKDAAKKDDAKKDDAKKDL) (22). The sequence was confirmed by deoxyribonucleic acid sequencing. The fragment was cloned into the pRW83 vector, which contained a chloramphenicol resistance gene for recombination selection, the pen P gene with a penP promoter and terminator, and a pe1B signal peptide that directed the recombinant protein to the periplasmic space to produce the biologically active protein (4). The expression plasmid was transformed into Escherichia coli strain AG1. When expressed, this construct spontaneously formed the monomeric CC49 scFv and dimeric CC49 (scFv)₂. These soluble proteins were purified by ion-exchange chromatography, and CC49 (scFv)₂ was separated from CC49 scFv by gel filtration chromatography on a Superdex 75 column. The preparation was shown to be >90% pure by sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and the protein migrated to its theoretical molecular weight of 60,000 (3).

Radioiodination of CC49 (scFv)₂ Ab was performed with the use of 1,3,4,6-tetrachloro-3α,6α-diphenylglycoluril (IodoGen) as the oxidizing agent (1, 3, 4). Briefly, 20–100 μg of CC49 (scFv)₂ Ab in 0.1 M sodium phosphate buffer (pH 7.2) was added to a glass tube coated with 20 μg IodoGen. Approximately 0.014–0.027 MBq (0.5–1.0 mCi) ¹³¹I-labeled sodium iodide ([¹³¹I]NaI) or ¹²⁵I-NaI was added and the mixture was incubated for 3 min. Size-exclusion chromatography on a Sephadex G-25 column was performed immediately to purify the radiolabeled antibody. The labeling efficiency was ~90% by high-performance liquid chromatography (HPLC) (4). The specific activity of ¹²⁵I/¹³¹I-CC49 (scFv)₂ Ab was ~111–333 MBq/mg (3–9 mCi/mg) or 6.66–19.98 MBq/nmol (0.18–0.54 mCi/nmol) on the basis of the molecular mass of 60 kDa. The SDS-PAGE analysis showed that the non-covalently linked ¹³¹I-CC49 (scFv)₂ was dissociated by the SDS and appeared only as its 30-kDa form. The radiochemical purity was >90% (3).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

The immunoreactivity of radioiodinated CC49 (scFv)₂ Ab was assessed by solid-phase radioimmunoassay (RIA), with bovine submaxillary gland mucin (BSM) as the protein antigen attached to a solid-phase matrix (1, 2, 4). The specific binding for ¹²⁵I/¹³¹I-CC49 (scFv)₂ Ab was 88–94%. The binding kinetics of ¹²⁵I/¹³¹I-CC49 (scFv)₂ Ab was assessed by the real-time kinetic analysis with use of a surface plasmon resonance detector (4). The study reported the association constant (K_a) of unlabeled CC49 (scFv)₂ to be 4.46 × 10⁷ M−¹ (1-4). In comparison, the K_a for the intact CC49 MAb was 1.14 × 10⁸ M−¹. The relative affinity constant of¹²⁵I/¹³¹I-CC49 (scFv)₂ calculated from the Scatchard plot of the data obtained from the competition enzyme-linked immunosorbent assay was 1.70 × 10⁹ M−¹ (1).

In vitro stability studies of ¹²⁵I/¹³¹I-CC49 (scFv)₂ Ab showed that it was stable for at least 35 days when stored at 4ºC (1, 4). No significant protein degradation or radioactive iodine release was determined by HPLC and radioimmunoassay. Only ~2–3% loss in immunoreactivity was exhibited (1). Unlabeled (scFv)₂ was stable at −70ºC for >10 months without loss of immunoreactivity (1). When ¹²⁵I/¹³¹I-CC49 (scFv)₂ Ab was incubated in murine serum or 1% bovine serum albumin and incubated at 37ºC for up to 4 days, HPLC analysis showed that radioiodinated CC49 (scFv)₂ maintained its full integrity (3).

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

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