99mTc-Hydrazinonicotinamide-anti-TAG-72 CC49 divalent single-chain Fv monoclonal antibody

99mTc-HYNIC-CC49 sc(Fv)2 MAb

Cheng KT.

Publication Details



In vitro Rodents



99mTc-Hydrazinonicotinamide-anti-TAG-72 CC49 divalent single-chain Fv monoclonal antibody (99mTc-HYNIC-CC49 sc(Fv)2 MAb), which is formed by the conjugation of 99mTc with a bioengineered anti–tumor-associated glycoprotein 72 (TAG-72) antibody construct, has been developed for single-photon emission computed tomography (SPECT) imaging of cancers that express TAG-72 (1). 99mTc is a gamma emitter with a half-life (t½) of 6.02 h.

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 (2-5). 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 (6). The murine MAb B72.3 against TAG-72 was initially generated by immunization of mice with a membrane-enriched fraction of a human breast carcinoma (7). With use of affinity-purified TAG-72 from LS-174T as an immunogen, CC49 and other anti–TAG-2 MAbs with higher affinity constants (Ka) have been produced and characterized (1-3, 7).

Radiolabeled MAbs have been developed for both the diagnosis and treatment of tumors (8). Radiolabeled B72.3 and CC49 have shown excellent tumor localization capabilities with potential diagnostic and therapeutic applications in the clinical setting (9, 10). Because of their relatively large size, radiolabeled intact MAbs tend to have unfavorable imaging kinetics, poor tumor penetration, and high potential for human anti-mouse antibody response (1, 11-13). One approach to minimize these problems is reducing intact antibodies to antibody fragments such as F(ab’)2 and Fab’ (14). Another approach is the development of genetic engineering methods to obtain single-chain Fv constructs (scFv) and multivalent scFv constructs (1, 15, 16). These scFv constructs contain the variable regions of the light chain (VL) and heavy chain (VH) connected by a flexible linker. Colcher et al. (17) constructed the monomeric CC49 scFv MAb (~27 kDa), which selectively recognizes a unique sialyl-Tn epitope of TAG-72. The radioiodinated CC49 scFv appeared to clear rapidly from the blood with good tumor penetration (16, 18). To further improve the imaging kinetics, Pavlinkova et al. (18) constructed the high-affinity dimer CC49 sc(Fv)2 (~60 kDa). The radioiodinated CC49 sc(Fv)2 showed good stability and increased avidity in vivo compared with the radioiodinated CC49 scFv construct.

With direct or indirect labeling, MAbs can be labeled with 99mTc, a gamma emitter with ideal SPECT imaging properties. Direct labeling involves reduction of 99mTc-pertechnetate and nonspecific binding of the reduced 99mTc to donor atoms, namely thiol, amide, amino, and carboxylate (19). Indirect labeling uses a bifunctional chelating agent, which can be more binding site–specific on the MAb molecule. Goel et al. (1) used hydrazinonicotinamide (HYNIC) as a bifunctional coupling agent to label CC49 sc(Fv)2 with 99mTc. The 99mTc-HYNIC-CC49 sc(Fv)2 MAb showed good tumor targeting and in vivo biodistribution properties.



Goel et al. (1) reported the construction and radiolabeling of the 99mTc-HYNIC-CC49 sc(Fv)2 MAb. The CC49 scFv (VL-linker-VH) was derived from the murine MAb CC49 cloned in the yeast expression vector pPICZαA and constructed with the 205C linker. The bacterial scFv construct was used as the template DNA for the expression of the scFv in competent P. pastoris KM71 cells. The construction of the divalent sc(Fv)2 (VL-linker-VH-linker-VL-linker-VH-His6) was performed as described by Goel et al. (20) using the 205C linker in a P. pastoris expression system, and the final construct was purified from the secreted medium with immobilized metal affinity chromatography. The preparation was shown to be >95% pure by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Size-exclusion high-performance liquid chromatography (HPLC) showed the molecular mass to be 60 kDa. Competitive solid-phase competition enzyme-linked immunosorbent assay (ELISA) with bovine submaxillary gland mucin (BSM) confirmed the immunoreactivity of the divalent construct. To radiolabel the agent, the hydrazino-modification of CC49 sc(Fv)2 was achieved by reacting the construct with the N-hydroxysuccinimide ester of succinimidyl-6-hydrazinonicotinate hydrochloride (SHNH) at a molar ratio of 10:1 in sodium phosphate buffer (pH 7.8). It was estimated that there were ~2.3 SHNH groups per sc(Fv)2. In the radiolabeling procedure, sodium 99mTc-pertechnetate, tricine, and stannous chloride were first mixed, then SHNH-derivatized CC49 sc(Fv)2 was added, and the reaction mixture was incubated at room temperature for 45 min. The final 99mTc-HYNIC-CC49 sc(FV)2 MAb was purified on a Sephadex G-25 column. The specific activity was 74–111 MBq/mg (2–3 mCi/mg) or 4.44–6.66 MBq/nmol (0.12–0.18 mCi/nmol) on the basis of the estimated 60-kDa molecular weight) with a radiochemical purity ≥95% (HPLC analysis). On SDS-PAGE, >90% of the total radioactivity was associated with the protein band of 58 kDa.

In Vitro Studies: Testing in Cells and Tissues


The immunoreactivity of 99mTc-HYNIC-CC49 sc(Fv)2 MAb by solid-phase ELISA with immobilized BSM was 85–95% with a nonspecific binding of 0.8–1.5% (1).

In vitro stability studies of 99mTc-HYNIC-CC49 sc(Fv)2 MAb were conducted by incubating the radiolabeled MAb construct in 1% bovine serum albumin (BSA) or 1% mouse serum at 37ºC for 24 h (1). By HPLC analysis, <20% loss of 99mTc label was detected in 1% BSA. In the 1% mouse serum, ~20% and 10% of the 99mTc were associated with low molecular weight proteins (<50 kDa) and high molecular weight proteins (>130 kDa), respectively. This latter protein fraction suggested the possibility of aggregation of the radiolabel with serum proteins.

Using the Scatchard plot and surface plasmon resonance technique to measure the real-time interactions, Goel et al. (21) reported the Ka of unlabeled CC49 sc(Fv)2 to be 2.75 × 107 M−1 in binding to the immobilized BSM. In comparison, the Ka for the intact CC49 MAb construct was 1.14 × 108 M−1.

Animal Studies



Biodistribution studies of 99mTc-HYNIC-CC49 sc(Fv)2 MAb were performed in nude mice bearing LS-147T s.c. human colon carcinomas (~250–300 mm3) (1). Each mouse received 0.37 MBq (10 μCi) of 99mTc-HYNIC-CC49 sc(Fv)2 mAb by i.v. administration. The blood elimination t½ was 144 min, and the whole-body clearance t½ was 184 ± 19 min (n = 3). The radioactivity levels (n = 3 × 2) in percentage injected dose per gram (% ID/g) of the tumors were 9.9 ± 0.7 (0.5 h), 10.9 ± 0.8 (1 h), 12.6 ± 0.4 (4 h), 7.2 ± 0.7 (8 h), 2.3 ± 0.1 (16 h), and 1.2 ± 0.0 (24 h). At 16 h, the tumor/blood ratio was 4.6:1. At 0.5 h, the radioactivity levels (% ID/g) of major organs were 13.9 ± 1.1 (blood), 18.9 ± 1.5 (liver), 13.4 ± 1.2 (spleen), 33.9 ± 1.8 (kidneys), and 5.4 ± 0.8 (lungs). At 4 h, these levels changed to 4.3 ± 0.2 (blood), 14.8 ± 1.3 (liver), 9.7 ± 0.7 (spleen), 27.3 ± 1.4 (kidneys), and 1.3 ± 0.0 (lungs). By 24 h, these levels declined to 0.1 ± 0.0 (blood), 1.2 ± 0.0 (liver), 1.0 ± 0.0 (spleen), 2.4 ± 0.3 (kidneys), and 0.0 ± 0.0 (lungs). Macroautoradiography studies performed in mice at 6 h and 16 h after radioactivity administration confirmed a high degree of tumor localization and negligible retention in the blood and normal organs (1). The exceptions were the liver and pancreas, but the tumor radioactivity levels were still two-fold higher at both 6 and 16 h in these two organs. The tumor remained positive at 16 h after injection. The study suggested that 99mTc-HYNIC-CC49 sc(Fv)2 MAb probably underwent hepatobiliary excretion.

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.


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