99mTc-Hydrazinonicotinamide-anti-TAG-72 CC49 tetravalent single-chain Fv monoclonal antibody (2007)

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.