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

99mTc-HYNIC-CC49 [sc(Fv)2]2 MAb
, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD, vog.hin.mln.ibcn@dacim

Created: ; Last Update: December 28, 2007.

Chemical name:99mTc-Hydrazinonicotinamide-anti-TAG-72 CC49 tetravalent single-chain Fv monoclonal antibody
Abbreviated name:99mTc-HYNIC-CC49 [sc(Fv)2]2 MAb
Synonym:99mTc-CC49 [sc(Fv)2]2 MAb, 99mTc-CC49 MAb
Agent Category:Tetravalent single-chain Fv monoclonal antibody ([sc(Fv)2]2 MAb)
Target Category:Antibody to antigen binding
Method of detection:Single-photon emission computed tomography (SPECT), planar gamma imaging
Source of signal:99mTc
  • Checkbox In vitro
  • Checkbox Rodents
Click on protein, nucleotide (RefSeq), and gene for more information about TAG-72



99mTc-Hydrazinonicotinamide-anti-TAG-72 CC49 tetravalent single-chain Fv monoclonal antibody (99mTc-HYNIC-CC49 [sc(Fv)2]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 monoclonal antibody B72.3 (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 monoclonal antibodies 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 monoclonal antibodies 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. Goel et al. (19) formed the tetravalent [sc(Fv)2]2 construct (~120 kDa) that exhibited four potentially active antigen-binding sites and showed improved in vitro binding properties.

MAbs can be labeled with 99mTc, a gamma emitter with ideal SPECT imaging properties, by direct or indirect labeling. Direct labeling involves reduction of 99mTc-pertechnetate and nonspecific binding of the reduced 99mTc to donor atoms, namely thiol, amide, amino, and carboxylate (20). 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 divalent CC49 sc(Fv)2 and tetravalent CC49 [sc(Fv)2]2 with 99mTc. Both 99mTc-HYNIC-CC49 sc(Fv)2 MAb and 99mTc-HYNIC-CC49 [sc(Fv)2]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]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 with 25 amino acids. The bacterial scFv construct was used as the template DNA for the expression of the scFv in competent methylotrophic 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. (21) using the 205C linker in a P. pastoris expression system. On expression as a secreted protein by the P. pastoris, 20–30% of the divalent form was found to spontaneously associate through noncovalent interactions into the tetravalent [sc(Fv)2]2 or higher aggregates (>200 kDa) (19). The construct was purified from the secreted medium with immobilized metal affinity chromatography, and the [sc(Fv)2]2 was separated on a Superdex 200 column. The yield was reported to be 2.0–3.5 mg/liter. The preparation was shown to be >95% pure by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE study also confirmed that the two polypeptide chains of the tetramer were noncovalently linked. Size-exclusion high-performance liquid chromatography (HPLC) showed the molecular mass to be 120 kDa. Competitive solid-phase competition enzyme-linked immunosorbent assay (ELISA) with bovine submaxillary gland mucin (BSM) confirmed the immunoreactivity of the tetravalent construct with Ka = 1.0 × 108 M−1. 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) in the dark at 4ºC overnight. It was estimated that there were ~2.8 SHNH groups per [sc(Fv)2]2. In the radiolabeling procedure, sodium 99mTc-pertechnetate, tricine, and stannous chloride were first mixed, then SHNH-derivatized CC49 [sc(Fv)2]2 was added, and the reaction mixture was incubated at room temperature for 45 min. The final 99mTc-HYNIC-CC49 [sc(FV)2]2 MAb was purified on a Sephadex G-25 column. The final radiochemical yield was not reported. The specific activity was 74–111 MBq/mg (2–3 mCi/mg) or 8.88–13.32 MBq/μmol (0.24–0.36 mCi/μmol) on the basis of the estimated 120-kDa molecular mass with a radiochemical purity ≥95% (HPLC analysis).

In Vitro Studies: Testing in Cells and Tissues


The immunoreactivity of 99mTc-HYNIC-CC49 [sc(Fv)2]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]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, 65.2% of 99mTc-HYNIC-CC49 [sc(Fv)2]2 MAb remained intact, and 6.4% and 18.2% were associated with >130-kDa and <50-kDa proteins, respectively; ~10.2% of the 99mTc radioactivity was associated with a 60-kDa protein. This suggested the possibility of dissociation of the tetramers to the dimmers.

Using the Scatchard plot and surface plasmon resonance technique to measure the real-time interactions, Goel et al. (19) reported the Ka of unlabeled CC49 [sc(Fv)2]2 to be 1.02 × 108 M−1 in binding to the immobilized BSM. In comparison, the Ka for the intact CC49 MAb and CC49 sc(Fv)2 constructs were 1.14 × 108 M−1 and 2.75 × 107 M−1, respectively.

Animal Studies



Biodistribution studies of 99mTc-HYNIC-CC49 [sc(Fv)2]2 MAb were performed in nude mice bearing LS-174T 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 307 min, and the whole-body clearance t½ was 265 ± 39 min (n = 3). The radioactivity levels (n = 3 × 2) in percentage injected dose per gram (% ID/g) of the tumors were 13.3 ± 1.4 (0.5 h), 14.5 ± 0.9 (1 h), 15.0 ± 1.2 (4 h), 19.1 ± 1.1 (6 h), 5.6 ± 0.2 (16 h), and 2.5 ± 0.0 (24 h). At 16 h, the tumor/blood ratio was 12.7:1 with tumor localization approximately three-fold higher than that of 99mTc-sc(Fv2). At 0.5 h, the radioactivity levels (% ID/g) of major organs were 27.3 ± 1.5 (blood), 18.0 ± 1.3 (liver), 13.2 ± 0.8 (spleen), 17.2 ± 0.5 (kidneys), and 7.2 ± 1.1 (lungs). At 4 h, these levels changed to 7.5 ± 0.5 (blood), 20.1 ± 1.1 (liver), 10.9 ± 0.5 (spleen), 14.3 ± 1.9 (kidneys), and 2.7 ± 0.5 (lungs). By 24 h, these levels declined to 0.1 ± 0.0 (blood), 1.6 ± 0.0 (liver), 1.0 ± 0.0 (spleen), 1.1 ± 0.0 (kidneys), and 0.1 ± 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. The tumor remained positive with decreased background radioactivity at 16 h after injection. In comparison, the radioactivity level of 99mTc-HYNIC-CC49 [sc(Fv)2]2 MAb was about three-fold less than that of the divalent 99mTc-HYNIC-CC49 sc(Fv)2 MAb

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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