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Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.

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Molecular Imaging and Contrast Agent Database (MICAD) [Internet].

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Radioiodinated humanized monoclonal antibody A33

Radioiodinated-huA33 mAb
, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD 20894

Created: ; Last Update: October 22, 2007.

Chemical name:Radioiodinated humanized monoclonal antibody A33
Abbreviated name:Radioiodinated-huA33 mAb
Synonym:[125I/124I/131I]huA33
Agent Category:Humanized monoclonal antibody A33
Target:Glycoprotein A33
Target Category:Antibody-antigen binding
Method of detection:Single-photon emission computed tomography (SPECT) or gamma planar imaging
Source of signal / contrast:125I/131I
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
  • Checkbox Humans
Click here for protein and nucleotide sequence of human A33 antigen.

Background

[PubMed]

The A33 cell-surface differentiation antigen is a glycoprotein that belongs to the immunoglobulin superfamily (1). This antigen is expressed in >95% of primary and metastatic colon cancer cells, but is absent in most normal and tumor cell types (2, 3). Although the exact function of this glycoprotein is unknown, antibodies developed against the A33 antigen bind to tumor cells and exhibit prolonged retention in the tumor tissue (4). The A33 antigen was considered an important target of a mouse monoclonal antibody (mAb) developed for the treatment of colorectal cancers; the radiolabeled mAb was detectable in the tumor tissue up to 6 weeks after administration, but it was cleared from normal tissue within a week (4, 5). Radioimmunotherapy studies in a nude mouse model also showed that the antitumor effects of the A33 antigen mAb were enhanced by combination with chemotherapeutic agents (6, 7). However, patients treated with the radioiodinated murine mAb A33 showed minimal gut toxicity but developed a human anti-mouse antibody (HAMA) response after the first treatment (8). On re-treatment, the radiolabeled antibody was rapidly cleared from the blood circulation of these patients, and on imaging the label was observed to have accumulated in the liver, spleen, or adrenals, but not at the tumor sites (8).

To reduce immunogenicity of the murine mAb, a humanized version of the A33 antibody (huA33) was constructed and is under evaluation in various clinical trials (9-12). Recently huA33 was labeled with radioactive astatine (211At) or lutetium (177Lu) and studied for specificity, biodistribution, and tumor targeting in rodents (13, 14). From these studies it was concluded that the labeled antibody could be used for radioimmunotherapy of colorectal cancer.

Synthesis

[PubMed]

For some pre-clinical and clinical studies, a mouse mAb against glycoprotein A33 was produced as described by Welt et al. (5). Briefly, a mouse mAb A33 hybridoma cell line was derived by the fusion of spleen cells of BALB/c mice immunized with the AsPC-1 pancreatic adenocarcinoma cell line and SP2/0 myeloma cells (15). The mAb was used either as a supernatant from the hybridoma cultures or purified from A33 hybridoma ascites grown in pristine-treated and irradiated CByB6F1 mice as described elsewhere (4, 5, 8, 15). For clinical testing the antibody was tested according to standards set by the United States Food and Drug Administration (5).

The huA33 version of the antibody was produced in NSO cells as described by King et al. (9). A single plasmid was constructed to express the humanized heavy and light chains of mAb A33 and transfected into NSO cells to produce a stable cell line (9). The huA33 was purified from nitric oxide synthase supernatant by a three-step chromatographic procedure involving Q-Sepharose anion exchange, protein A affinity, and S-Sepharose cation exchange columns (12). The antibody was formulated in phosphate-buffered saline, sterile filtered, and stored at -70°C until required. The pure humanized mAb did not aggregate and had a binding specificity similar to the murine mAb (16).

Radioiodination of the murine mAb A33 was done with 125I/131I-labeled sodium iodide according to the chloramine-T method (17). The reaction was terminated with sodium metabisulphite, and the product was passed through a Sephadex G25 column to remove excess radioactivity. Fractions containing peak radioactivity were pooled, sterile filtered, and tested for binding and immunoreactivity (4). The purity was ≥90% as determined by thin-layer chromatography (TLC). The murine mAb had a specific activity up to 740 MBq/6.6 nmol (20 mCi/6.6 nmol) and an immunoreactivity of 50–80% (4).

The huA33 was also radioiodinated (124I/131I) as above (17) and had a purity of >98% as determined by TLC, a specific activity of 7.4 MBq/6.6 nmol (2 mCi/6.6 nmol), and an immunoreactivity of 78% (18). In another study, huA33 was radioiodinated with 131I, and the labeled antibody had a radiochemical purity of 99.6 ± 0.63% with an immunoreactivity of 63.8 ± 18.0%. The specific activity of this labeled mAb was not provided by the investigators (11).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

The cytotoxicity and subcellular distribution of radioiodinated murine mAb A33 was studied in a line of human colon cancer cells (19). The investigators showed that 125I/131I-labeled A33 was cytotoxic to the cells because the labeled A33 accumulated into the cytoplasmic vesicles and was transported close to the nucleus. Only 10% of the cells survived when they were exposed to 10 µCi/ml (370 kBq/ml) [125I]A33, but the survival increased to 90% when the cells were pretreated with a 100-fold excess of cold A33 and then exposed to 100 µCi/ml (3,700 kBq/ml) of the labeled antibody (19). Similar experiments performed with [131I]A33 showed it was toxic to the cells at either concentration given above.

In another study, Hoglund et al. investigated the in vitro cellular processing of [125I]huA33 in a human cancer cell line (20). The investigators observed that degradation of the intracellular antibody was slow, and an early loss of the label from the cells was caused by dissociation of the [125I]huA33 bound to the cell surface.

The in vitro properties of 124I-labeled huA33 were investigated with a human colorectal cell line, and the apparent association constant (Ka) of the antibody was determined to be 2.2 × 109 M-1 (18).

Animal Studies

Rodents

[PubMed]

With the use of positron emission tomography (PET), the biodistribution of [124I]huA33 was studied in xenografted mice (n = 5 animals/time point) bearing tumors from the human cancer cell line (18). The investigators observed uptake of the label (a total of six time points up to 240 h after the injection) mainly in the blood and various organs at 4 h, but by 24 h after injection the uptake was distinctly observed in the tumors. By 10 days after administration of the label, the radioactivity was localized primarily in the tumor and thyroid. The investigators did not perform blocking studies with cold huA33 (18).

The biodistribution of [131I]huA33 was investigated in nude mice bearing tumors from the human cancer cell line (21). Peak uptake of the label by the tumors was observed between 48 and 96 h with a mean uptake of 34% of the injected dose per gram of tissue. In these studies, specificity of antibody label uptake was determined by several techniques, including blocking by pretreatment of mice with unlabeled huA33. Using immunohistochemical techniques, the investigators demonstrated that a homogeneous distribution of the antibody in the tumors could be achieved in this model (21).

Other Non-Primate Mammals

[PubMed]

No publications are currently available.

Non-Human Primates

[PubMed]

No publications are currently available.

Human Studies

[PubMed]

The biodistribution and imaging characteristics of 131I-labeled murine mAb A33 were studied by Welt et al. in colorectal carcinoma patients with liver metastases (5). Imaging showed that the labeled antibody had a tumor tissue localization in 19 of 20 patients that correlated with surgical, pathological, and tissue radioactivity examination (5). One week after administration of the labeled antibody, the tumor/liver ratio ranged from 6.9/1 to 100/1 and the tumor/serum ratio ranged from 4.1/1 to 25.2/1. The isotope was visualized in the large bowel of some patients, but it was unclear if this represented specific antibody uptake or if it was gastric iodine secretion (5).

Only preliminary observations were obtained from a phase I/II study designed to determine the maximum tolerated dose (MTD) of murine [131I]A33 mAb (8). In this study, the localization of the labeled antibody in the metastized lesions of the patients was shown by single-photon emission computed tomography (SPECT) to last for at least 3 weeks after administration. The 131I-labeled antibody showed some hematological toxicity, but there was no toxicity in the bowel in spite of this being the only antigen-positive tissue in the body (8).

In another phase I/II study, also designed to determine the MTD of murine [125I]A33 mAb, SPECT analysis was performed on patients treated with the labeled antibody (4). Imaging revealed that the labeled antibody was located at all tumor sites identified by radiography. In this study, the MTD of the [125I]A33 mAb could not be established, and it was shown that the label was retained by the tumor tissue for up to 6 weeks after administration of the labeled antibody (4).

The biodistribution of [131I]huA33 was investigated in a phase I trial of patients with colorectal carcinomas (3). Imaging studies revealed that the label was distributed in the blood pool only during day 0 after administration, and there was distinct uptake in the colorectal carcinoma sites by up to the second day after the infusion. A similar uptake was also observed in the metastatic site of the liver and the lungs (3). A gradual clearance of the label through the bowel was observed in these patients.

A phase I dose escalation study of [131I]huA33 was performed in patients with pretreated metastatic colorectal carcinomas (10). Initially the label was distributed in the blood pool and then it was followed by tumor uptake and clearance through the bowel. In this study, whole-body planar images were acquired for all patients after infusion of the labeled antibody into the tumors, and persistent uptake of the antibody was observed up to 4 weeks after the infusion.

Sakamoto et al. investigated the in vivo characteristics of [131I]huA33 in patients with gastric carcinomas (11). Gamma imaging and ex vivo scintigraphy of the resected specimens showed a selective localization of the labeled antibody to the gastric cancer. From these observations the investigators concluded that the targeting characteristics of huA33 indicated it had a potential for the targeted treatment of advanced gastric carcinomas.

Supplemental Information

[Disclaimer]

No information is currently available.

References

1.
Heath J.K., White S.J., Johnstone C.N., Catimel B., Simpson R.J., Moritz R.L., Tu G.F., Ji H., Whitehead R.H., Groenen L.C., Scott A.M., Ritter G., Cohen L., Welt S., Old L.J., Nice E.C., Burgess A.W. The human A33 antigen is a transmembrane glycoprotein and a novel member of the immunoglobulin superfamily. Proc Natl Acad Sci U S A. 1997;94(2):469–74. [PMC free article: PMC19536] [PubMed: 9012807]
2.
Ritter G., Cohen L.S., Nice E.C., Catimel B., Burgess A.W., Moritz R.L., Ji H., Heath J.K., White S.J., Welt S., Old L.J., Simpson R.J. Characterization of posttranslational modifications of human A33 antigen, a novel palmitoylated surface glycoprotein of human gastrointestinal epithelium. Biochem Biophys Res Commun. 1997;236(3):682–6. [PubMed: 9245713]
3.
Scott A.M., Lee F.T., Jones R., Hopkins W., MacGregor D., Cebon J.S., Hannah A., Chong G. P. U, A. Papenfuss, A. Rigopoulos, S. Sturrock, R. Murphy, V. Wirth, C. Murone, F.E. Smyth, S. Knight, S. Welt, G. Ritter, E. Richards, E.C. Nice, A.W. Burgess, and L.J. Old, A phase I trial of humanized monoclonal antibody A33 in patients with colorectal carcinoma: biodistribution, pharmacokinetics, and quantitative tumor uptake. Clin Cancer Res. 2005;11(13):4810–7. [PubMed: 16000578]
4.
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5.
Welt S., Divgi C.R., Real F.X., Yeh S.D., Garin-Chesa P., Finstad C.L., Sakamoto J., Cohen A., Sigurdson E.R., Kemeny N. et al. Quantitative analysis of antibody localization in human metastatic colon cancer: a phase I study of monoclonal antibody A33. J Clin Oncol. 1990;8(11):1894–906. [PubMed: 2230877]
6.
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7.
Tschmelitsch J., Barendswaard E., Williams C. Jr, Yao T.J., Cohen A.M., Old L.J., Welt S. Enhanced antitumor activity of combination radioimmunotherapy (131I-labeled monoclonal antibody A33) with chemotherapy (fluorouracil). Cancer Res. 1997;57(11):2181–6. [PubMed: 9187118]
8.
Welt S., Divgi C.R., Kemeny N., Finn R.D., Scott A.M., Graham M., Germain J.S., Richards E.C., Larson S.M., Oettgen H.F. et al. Phase I/II study of iodine 131-labeled monoclonal antibody A33 in patients with advanced colon cancer. J Clin Oncol. 1994;12(8):1561–71. [PubMed: 8040668]
9.
King D.J., Antoniw P., Owens R.J., Adair J.R., Haines A.M., Farnsworth A.P., Finney H., Lawson A.D., Lyons A., Baker T.S. et al. Preparation and preclinical evaluation of humanised A33 immunoconjugates for radioimmunotherapy. Br J Cancer. 1995;72(6):1364–72. [PMC free article: PMC2034099] [PubMed: 8519646]
10.
Chong G., Lee F.T., Hopkins W., Tebbutt N., Cebon J.S., Mountain A.J., Chappell B., Papenfuss A., Schleyer P. P. U, R. Murphy, V. Wirth, F.E. Smyth, N. Potasz, A. Poon, I.D. Davis, T. Saunder, J. O'Keefe G, A.W. Burgess, E.W. Hoffman, L.J. Old, and A.M. Scott, Phase I trial of 131I-huA33 in patients with advanced colorectal carcinoma. Clin Cancer Res. 2005;11(13):4818–26. [PubMed: 16000579]
11.
Sakamoto J., Oriuchi N., Mochiki E., Asao T., Scott A.M., Hoffman E.W., Jungbluth A.A., Matsui T., Lee F.T., Papenfuss A., Kuwano H., Takahashi T., Endo K., Old L.J. A phase I radioimmunolocalization trial of humanized monoclonal antibody huA33 in patients with gastric carcinoma. Cancer Sci. 2006;97(11):1248–54. [PubMed: 17034367]
12.
Welt S., Ritter G., Williams C. Jr, Cohen L.S., John M., Jungbluth A., Richards E.A., Old L.J., Kemeny N.E. Phase I study of anticolon cancer humanized antibody A33. Clin Cancer Res. 2003;9(4):1338–46. [PubMed: 12684402]
13.
Almqvist Y., Steffen A.C., Tolmachev V., Divgi C.R., Sundin A. In vitro and in vivo characterization of 177Lu-huA33: a radioimmunoconjugate against colorectal cancer. Nucl Med Biol. 2006;33(8):991–8. [PubMed: 17127172]
14.
Almqvist Y., Steffen A.C., Lundqvist H., Jensen H., Tolmachev V., Sundin A. Biodistribution of (211)at-labeled humanized monoclonal antibody a33. Cancer Biother Radiopharm. 2007;22(4):480–7. [PubMed: 17803442]
15.
Garin-Chesa P., Sakamoto J., Welt S., Real F.X., Rettig W.J., Old L.J. Organ-specific expression of the colon cancer antigen A33, a cell surface target for antibody-based therapy. Int J Oncol. 1996;9:465–471. [PubMed: 21541536]
16.
Catimel B., Nerrie M., Lee F.T., Scott A.M., Ritter G., Welt S., Old L.J., Burgess A.W., Nice E.C. Kinetic analysis of the interaction between the monoclonal antibody A33 and its colonic epithelial antigen by the use of an optical biosensor. A comparison of immobilisation strategies. J Chromatogr A. 1997;776(1):15–30. [PubMed: 9286074]
17.
Hunter W.M., Greenwood F.C. Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature. 1962;194:495–6. [PubMed: 14450081]
18.
Lee F.T., Hall C., Rigopoulos A., Zweit J., Pathmaraj K., O'Keefe G.J., Smyth F.E., Welt S., Old L.J., Scott A.M. Immuno-PET of human colon xenograft- bearing BALB/c nude mice using 124I-CDR-grafted humanized A33 monoclonal antibody. J Nucl Med. 2001;42(5):764–9. [PubMed: 11337573]
19.
Daghighian F., Barendswaard E., Welt S., Humm J., Scott A., Willingham M.C., McGuffie E., Old L.J., Larson S.M. Enhancement of radiation dose to the nucleus by vesicular internalization of iodine-125-labeled A33 monoclonal antibody. J Nucl Med. 1996;37(6):1052–7. [PubMed: 8683300]
20.
Hoglund J., Orlova A., Sundin A., Lundqvist H., Tolmachev V. Cellular processing in the SW1222 cell line of mAb A33 directly and indirectly radiohalogenated. Oncol Rep. 2006;16(1):159–63. [PubMed: 16786140]
21.
Barendswaard E.C., Scott A.M., Divgi C.R., Williams C. Jr, Coplan K., Riedel E., Yao T.J., Gansow O.A., Finn R.D., Larson S.M., Old L.J., Welt S. Rapid and specific targeting of monoclonal antibody A33 to a colon cancer xenograft in nude mice. International Journal of Oncology. 1998;12(1):45–53. [PubMed: 9454885]

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