Background

[PubMed]

Radioiodinated anti–TAG-72 CC49 (Fab’)₂ antibody fragment (¹³¹I-CC49 (Fab’)₂), which is formed by the conjugation of ¹²⁵I/¹³¹I with an anti–tumor-associated glycoprotein 72 (TAG-72) (Fab’)₂ antibody fragment, 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, and ¹³¹I has a t_½ of 8 days. Although both can be used for in vivo gamma imaging, ¹²⁵I has a gamma energy that is too low and ¹³¹I has a gamma energy that is too high. Another radioiodine, ¹²³I, is more ideal for single-photon emission tomography (SPECT) and planar gamma 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 (12, 13).

Radiolabeled MAbs have been developed for both the diagnosis and treatment of tumors (14). Radiolabeled B72.3 and CC49 exhibit excellent tumor localization capabilities with potential diagnostic and therapeutic applications in the clinical setting (15, 16). 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, 17-19). One possible approach to minimize these problems is reducing intact antibodies to smaller antibody fragments such as F(ab’)₂ and Fab’ (20). Another approach is the development of genetic engineering methods to obtain single-chain Fv constructs (scFv) and multivalent scFv constructs (11, 21, 22). The F(ab’)₂ and Fab’ fragments can generally be prepared by simple enzymatic cleavage. Pepsin digestion of the intact IgG removes the antibody constant region and produces the F(ab’)₂ fragment with bivalent antigen-binding capability and a molecular weight of 100,000 (14). Because of the smaller size, the F(ab’)₂ fragment has a faster blood clearance and a better tumor penetration than the intact IgG (2, 23). The removal of the Fc portion during the enzymatic cleavage also reduces nonspecific binding of F(ab’)₂ to Fc receptors. The in vitro and in vivo properties of radioiodinated CC49 (Fab’)₂ fragments have been studied and compared to those of the single-chain Fv constructs (1-4, 24).

Synthesis

[PubMed]

CC49 MAb IgG was developed by the immunization of mice with purified TAG-72 (4). CC49 IgG was purified from ascetic fluid obtained from immunized mice. CC49 (Fab’)₂ was prepared by 2% pepsin digestion of the purified CC49 IgG at 37ºC for 16 h (1, 2). The digested fragments were dialyzed and purified using ion-exchange chromatography. Radioiodination of CC49 (Fab’)₂ was performed with the use of 1,3,4,6-tetrachloro-3α,6α-diphenylglycoluril (IodoGen) as the oxidizing agent. Briefly, 100 μg of CC49 (Fab’)₂ in 0.1 M sodium phosphate buffer (pH 7.2) was added to a glass tube coated with 20 μg IodoGen. Approximately 18.5 MBq (0.5 mCi) ¹²⁵I-sodium iodide or ¹³¹I-sodium iodide was added and the mixture was incubated for 2 min at room temperature. Size-exclusion chromatography was performed immediately to purify the radiolabeled antibody. The labeling efficiency was 50%. SDS-PAGE analysis identified the ¹²⁵I/¹³¹I-CC49 (Fab’)₂ fragment molecular weight as ~100,000. The specific activity of ¹²⁵I/¹³¹I-CC49 (Fab’)₂ was ~111–185 kBq/μg (3–5 μCi/μg) or ~11.1–18.5 MBq/nmol (0.3–0.5 mCi/nmol) on the basis of the molecular weight of 100,000. Yokota et al. (2) reported a specific activity of 81.4–432.9 kBq/μg (2.2–11.7 μCi/μg) or 8.1–43.3 MBq/nmol (0.2–1.2 mCi/nmol). In another study, Yokota et al. (3) reported a radiochemical yield and purity of 30–50% with a specific activity of 0.34–0.4 MBq/μg (9.2–10.8 μCi/μg) or 34–40 MBq/nmol (0.9–1.1 mCi/nmol). Pavlinkova et al. (4) reported a specific activity of 111–333 kBq/μg (3–9 μCi/μg) or 11.1–33.3 MBq/nmol (0.3–0.9 mCi/nmol).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

The radioimmunoreactivity of ¹²⁵I-CC49 (Fab’)₂ was determined by a solid-phase radioimmunoassay using a TAG-72–positive extract of a LS-174T human colon carcinoma xenograft (1). ¹²⁵I-CC49 was used as the competitor. The binding of ¹²⁵I-CC49 (Fab’)₂ was 39.1%, and the relative K_a was ~4 × 10⁸ M^–1, which was similar to that of ¹²⁵I-CC49 IgG. The binding of ¹²⁵I-CC49 (Fab’)₂ to purified TAG-72 was 22.1%. Yokota et al. (2) reported a binding of 35% to a TAG-72–positive extract. In another study, Yokota et al. (3) found >90% binding of purified ¹²⁵I-CC49 (Fab’)₂ to bovine submaxillary gland mucin (BSM)-coated beads. Pavlinkova et al. (4) used the surface plasmon resonance technique to measure the real-time interactions, and the investigators reported that the K_a of unlabeled CC49 (Fab’)₂ was 1.04 × 10⁸ M−¹ in binding to the immobilized BSM. In comparison, the K_a for the intact CC49 IgG was 1.14 × 10⁸ M−¹. In the same study, the binding of ¹²⁵I-CC49 (Fab’)₂ to BSM was 81%.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

References

1.

Milenic D.E. , Yokota T. , Filpula D.R. , Finkelman M.A. , Dodd S.W. , Wood J.F. , Whitlow M. , Snoy P. , Schlom J. Construction, binding properties, metabolism, and tumor targeting of a single-chain Fv derived from the pancarcinoma monoclonal antibody CC49. Cancer Res. 1991; 51 (23 Pt 1):6363–71. [PubMed: 1933899]

2.

Yokota T. , Milenic D.E. , Whitlow M. , Schlom J. Rapid tumor penetration of a single-chain Fv and comparison with other immunoglobulin forms. Cancer Res. 1992; 52 (12):3402–8. [PubMed: 1596900]

3.

Yokota T. , Milenic D.E. , Whitlow M. , Wood J.F. , Hubert S.L. , Schlom J. Microautoradiographic analysis of the normal organ distribution of radioiodinated single-chain Fv and other immunoglobulin forms. Cancer Res. 1993; 53 (16):3776–83. [PubMed: 8339291]

4.

Pavlinkova G. , Beresford G.W. , Booth B.J. , Batra S.K. , Colcher D. Pharmacokinetics and biodistribution of engineered single-chain antibody constructs of MAb CC49 in colon carcinoma xenografts. J Nucl Med. 1999; 40 (9):1536–46. [PubMed: 10492377]

5.

Muraro R. , Kuroki M. , Wunderlich D. , Poole D.J. , Colcher D. , Thor A. , Greiner J.W. , Simpson J.F. , Molinolo A. , Noguchi P. et al. Generation and characterization of B72.3 second generation monoclonal antibodies reactive with the tumor-associated glycoprotein 72 antigen. Cancer Res. 1988; 48 (16):4588–96. [PubMed: 3396010]

6.

Johnson V.G. , Schlom J. , Paterson A.J. , Bennett J. , Magnani J.L. , Colcher D. Analysis of a human tumor-associated glycoprotein (TAG-72) identified by monoclonal antibody B72.3. Cancer Res. 1986; 46 (2):850–7. [PubMed: 3940648]

7.

Katari R.S. , Fernsten P.D. , Schlom J. Characterization of the shed form of the human tumor-associated glycoprotein (TAG-72) from serous effusions of patients with different types of carcinomas. Cancer Res. 1990; 50 (16):4885–90. [PubMed: 2379152]

8.

Xiao J. , Horst S. , Hinkle G. , Cao X. , Kocak E. , Fang J. , Young D. , Khazaeli M. , Agnese D. , Sun D. , Martin E. Pharmacokinetics and clinical evaluation of 125I-radiolabeled humanized CC49 monoclonal antibody (HuCC49deltaC(H)2) in recurrent and metastatic colorectal cancer patients. Cancer Biother Radiopharm. 2005; 20 (1):16–26. [PubMed: 15778575]

9.

Paterson A.J. , Schlom J. , Sears H.F. , Bennett J. , Colcher D. A radioimmunoassay for the detection of a human tumor-associated glycoprotein (TAG-72) using monoclonal antibody B72.3. Int J Cancer. 1986; 37 (5):659–66. [PubMed: 3699929]

10.

Colcher D. , Hand P.H. , Nuti M. , Schlom J. A spectrum of monoclonal antibodies reactive with human mammary tumor cells. Proc Natl Acad Sci U S A. 1981; 78 (5):3199–203. [PMC free article: PMC319528] [PubMed: 6789331]

11.

Goel A. , Baranowska-Kortylewicz J. , Hinrichs S.H. , Wisecarver J. , Pavlinkova G. , Augustine S. , Colcher D. , Booth B.J. , Batra S.K. 99mTc-labeled divalent and tetravalent CC49 single-chain Fv's: novel imaging agents for rapid in vivo localization of human colon carcinoma. J Nucl Med. 2001; 42 (10):1519–27. [PubMed: 11585867]

12.

Beresford G.W. , Pavlinkova G. , Booth B.J. , Batra S.K. , Colcher D. Binding characteristics and tumor targeting of a covalently linked divalent CC49 single-chain antibody. Int J Cancer. 1999; 81 (6):911–7. [PubMed: 10362138]

13.

Colcher D. , Pavlinkova G. , Beresford G. , Booth B.J. , Batra S.K. Single-chain antibodies in pancreatic cancer. Ann N Y Acad Sci. 1999; 880 :263–80. [PubMed: 10415872]

14.

Kowalsky R.J. , Falen S.W. and Radiopharmaceuticals in nuclear pharmacy and nuclear medicine, American Pharmacists Association: Washington, D.C. p. 733-752. 2004

15.

Colcher D. , Minelli M.F. , Roselli M. , Muraro R. , Simpson-Milenic D. , Schlom J. Radioimmunolocalization of human carcinoma xenografts with B72.3 second generation monoclonal antibodies. Cancer Res. 1988; 48 (16):4597–603. [PubMed: 3396011]

16.

Colcher D. , Esteban J. , Carrasquillo J.A. , Sugarbaker P. , Reynolds J.C. , Bryant G. , Larson S.M. , Schlom J. Complementation of intracavitary and intravenous administration of a monoclonal antibody (B72.3) in patients with carcinoma. Cancer Res. 1987; 47 (15):4218–24. [PubMed: 3607761]

17.

Britton K.E. The development of new radiopharmaceuticals. Eur J Nucl Med. 1990; 16 (4-6):373–85. [PubMed: 2190837]

18.

Jain R.K. Transport of molecules across tumor vasculature. Cancer Metastasis Rev. 1987; 6 (4):559–93. [PubMed: 3327633]

19.

Primus F.J. , Bennett S.J. , Kim E.E. , DeLand F.H. , Zahn M.C. , Goldenberg D.M. Circulating immune complexes in cancer patients receiving goat radiolocalizing antibodies to carcinoembryonic antigen. Cancer Res. 1980; 40 (3):497–501. [PubMed: 7008935]

20.

Behr T. , Becker W. , Hannappel E. , Goldenberg D.M. , Wolf F. Targeting of liver metastases of colorectal cancer with IgG, F(ab')2, and Fab' anti-carcinoembryonic antigen antibodies labeled with 99mTc: the role of metabolism and kinetics Cancer Res 199555Suppl235777s–5785s. [PubMed: 7493346]

21.

Bird R.E. , Hardman K.D. , Jacobson J.W. , Johnson S. , Kaufman B.M. , Lee S.M. , Lee T. , Pope S.H. , Riordan G.S. , Whitlow M. Single-chain antigen-binding proteins. Science. 1988; 242 (4877):423–6. [PubMed: 3140379]

22.

Colcher D. , Bird R. , Roselli M. , Hardman K.D. , Johnson S. , Pope S. , Dodd S.W. , Pantoliano M.W. , Milenic D.E. , Schlom J. In vivo tumor targeting of a recombinant single-chain antigen-binding protein. J Natl Cancer Inst. 1990; 82 (14):1191–7. [PubMed: 2362290]

23.

Goldenberg D.M. Monoclonal antibodies in cancer detection and therapy. Am J Med. 1993; 94 (3):297–312. [PubMed: 8452154]

24.

Schott M.E. , Frazier K.A. , Pollock D.K. , Verbanac K.M. Preparation, characterization, and in vivo biodistribution properties of synthetically cross-linked multivalent antitumor antibody fragments. Bioconjug Chem. 1993; 4 (2):153–65. [PubMed: 7873647]