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111In-(1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaaetic acid)-anti–TAG-72 humanized CH2 domain-deleted antibody

111In-(Mx-DTPA)-HuCC49ΔCH2 Ab
, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD, vog.hin.mln.ibcn@dacim

Created: ; Last Update: February 12, 2008.

Chemical name:111In-(1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid)-anti–TAG-72 humanized CH2 domain-deleted antibody
Abbreviated name:111In-(Mx-DTPA)-HuCC49ΔCH2 Ab
Synonym:111In-HuCC49ΔCH2 Ab, 111In-CC49 Ab
Agent Category:Humanized CH2 domain-deleted antibody variant
Target:(Sialyl-Tn) TAG-72
Target Category:Antibody to antigen binding
Method of detection:Single-photon emission computed tomography (SPECT), planar gamma imaging
Source of signal /contrast:111In
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
Click on protein, nucleotide (RefSeq), and gene for more information about TAG-72

Background

[PubMed]

111In-(1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid)-anti–TAG-72 humanized CH2 domain-deleted antibody (111In-(Mx-DTPA)-HuCC49ΔCH2 Ab), which is formed by the conjugation of 111In 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). 111In is a gamma emitter with a half-life (t½) of 2.8 d.

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 (2, 3, 7, 8). CC49 MAb appears to react with a unique disaccharide Sialyl-Tn (STn) epitope on the TAG-72 (9).

Radiolabeled MAbs have been developed for both the diagnosis and treatment of tumors (10). Radiolabeled B72.3 and CC49 have shown excellent tumor localization capabilities with potential diagnostic and therapeutic applications in the clinical setting (11, 12). Because of their relatively large sizes, radiolabeled intact MAbs tend to have unfavorable imaging kinetics, poor tumor penetration, and high potential for human anti-mouse antibody response (8, 13-15). One approach to minimize these problems is breaking up intact antibodies into antibody fragments such as F(ab’)2 and Fab’ (16). Another approach is the development of genetic engineering methods to obtain single-chain Fv constructs (scFv) and multivalent scFv constructs (8, 17, 18). The removal of the CH2 domain of IgG also appears to result in a significantly faster blood clearance and more rapid tumor uptake (1, 19, 20). Slavin-Chiorini et al. (21) bioengineered a complementary determining region-grafted humanized CC49 MAb (HuCC49) with a CH2 domain deletion (ΔCH2). Radiolabeled HuCC49ΔCH2 constructs have demonstrated faster blood clearance in mice (1, 21, 22). Clinical studies with radioiodinated HuCC49ΔCH2 Ab in patients did show faster blood clearance (23). Chinn et al. (1) prepared and evaluated 111In-(Mx-DTPA)-HuCC49ΔCH2 Ab as a potential SPECT imaging molecular probe for tumors expressing TAG-72.

Synthesis

[PubMed]

Chinn et al. (1) reported the preparation and radiolabeling of HuCC49ΔCH2 . Kashmiri et al. (24) prepared the humanized CC49 MAb by grafting the CC49 MAb hypervariable regions onto the variable light and variable heavy frameworks of the human MAbs LEN and 21/28’ CL, respectively. Slavin-Chiorini et al. (21) described the development of the HuCC49ΔCH2 Ab from the humanized CC49 MAb. Before conjugation to Mx-DTPA, the HuCC49ΔCH2 Ab was diafiltrated at 2–8º into metal-free 0.9% sodium chloride. The pH of the solution was adjusted to 8.6 in sodium borate–buffered saline. This HuCC49ΔCH2 Ab solution was reacted with Mx-DTPA in a chelator/protein molar ratio of 3:1. The reaction mixture was incubated overnight for 16–20 h at ambient temperature. The unreacted Mx-DTPA was removed by diafiltration. This resulted in 1.1 mol Mx-DTPA per mol of HuCC49ΔCH2 Ab. Radiolabeling was conducted by using 111In chloride (111InCl3) solution in a specific activity of 74–148 MBq/mg (2–4 mCi/mg) or 8.97–17.76 MBq/nmol (0.24–0.49 mCi/nmol) based on a molecular weight of 121 kDa. The 111InCl3 solution was first adjusted to pH 6 with sodium citrate. (Mx-DTPA)-HuCC49ΔCH2 was added to the 111InCl3 solution and incubated for 15 min at ambient temperature. The reaction was quenched by adding human serum albumin and diethylenetriaminepentaacetic acid (DTPA). The radiochemical yield and radiochemical purity of 111In-(Mx-DTPA)-HuCC49ΔCH2 Ab was 97.1%. The final specific activity was not reported.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Chinn et al. (1) conducted in vitro solid-phase competition immunoassays of (Mx-DTPA)-HuCC49ΔCH2 with bovine submaxillary mucin (BSM) as the TAG-72 antigen source and Eu-labeled HuCC49ΔCH2 Ab as a tracer. The 50% inhibitory concentration value (IC50) and apparent affinity constant (Ka) of unlabeled (Mx-DTPA)-HuCC49ΔCH2 Ab were 15 nM and 4.20 nM, respectively. In comparison, the IC50 and Ka of Mx-DTPA-chimeric CC49 Ab (cCC49 Ab) were 5.8 nM and 0.62 nM, respectively. For 111In-(Mx-DTPA)-HuCC49ΔCH2 Ab, immunoreactivity assays were performed with BSM-coated resin. The immunoreactivity of HuCC49ΔCH2 Ab was 83.8%. In comparison, the immunoreactivity of 111In-(Mx-DTPA)-cCC49 was 80.5% of binding.

Animal Studies

Rodents

[PubMed]

Pharmacokinetics studies of 111In-(Mx-DTPA)-HuCC49ΔCH2 Ab were performed in normal nude mice (1). Each mouse received 74 kBq/1.3 μg (2 μCi/1.3 μg) of 111In-(Mx-DTPA)-HuCC49ΔCH2 Ab by i.v. administration. The blood clearance appeared to be monoexponential, and the biological t½ in the blood was 5.4 h. The radioactivity levels (n = 3–5) in percentage injected dose per gram (% ID/g) in the blood were 43.3 at 1 h and then fell to <1 at 24 h. Tumor localization of 111In-(Mx-DTPA)-HuCC49ΔCH2 Ab was evaluated in nude mice bearing LS-174T s.c. tumors expressing TAG-72 (~10 mm diameter) (1). Each mouse received 0.37 MBq (10 μCi) 111In-(Mx-DTPA)-HuCC49ΔCH2 Ab. The tumor radioactivity levels (n = 3) were 7.9 ± 0.7% ID/g and 14.4 ± 2.3 at 1 h and 24 h, respectively. The blood radioactivity levels were 25.5 ± 0.4% ID/g and 0.9 ± 0.1% ID/g at 1 h and 24 h, respectively. The tumor/blood ratios were 0.31 and 16.0 at 1 h and 24 h, respectively. In comparison, the tumor/blood ratios for 111In-Mx-DTPA-cCC49 were 0.35 and 3.7, respectively.

Other Non-Primate Mammals

[PubMed]

No publication is currently available.

Non-Human Primates

[PubMed]

No publication is currently available.

Human Studies

[PubMed]

No publication is currently available.

NIH Support

NIH Intramural Support.

References

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Chinn P.C., Morena R.A., Santoro D.A., Kazules T., Kashmiri S.V., Schlom J., Hanna N., Braslawsky G. Pharmacokinetics and tumor localization of (111)in-labeled HuCC49DeltaC(H)2 in BALB/c mice and athymic murine colon carcinoma xenograft. Cancer Biother Radiopharm. 2006;21(2):106–16. [PubMed: 16706631]
2.
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]
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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]
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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]
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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]
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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]
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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]
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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]
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Mueller B.M., Reisfeld R.A., Gillies S.D. Serum half-life and tumor localization of a chimeric antibody deleted of the CH2 domain and directed against the disialoganglioside GD2. Proc Natl Acad Sci U S A. 1990;87(15):5702–5. [PMC free article: PMC54395] [PubMed: 2198570]
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22.
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24.
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