Background

[PubMed]

The palmotylated glycoprotein A33 (gpA33) is a cell-surface differentiation antigen that belongs to the immunoglobulin superfamily (1, 2). This antigen is expressed primarily in the normal intestine and in >95% of colon tumors, and it has not been detected in any other tissue (1, 2). Because of its presence only in the intestine or in tumors in the intestine, the gpA33 has been evaluated as a target for the detection and radioimmunotherapy of colon cancer tumors (2). The biodistribution of a radioiodinated humanized monoclonal antibody (Ab) directed against the human gpA33 antigen (huA33) is discussed in a separate MICAD chapter (3). It was reported that the labeled Ab used to treat the colon cancers bound to the entire intestinal tissue, including the tumors; however, although the radioactivity persisted in the tumors for at least 6 weeks, the label was lost from the normal gut tissue within 1 week after administration (1-3). The more rapid clearance of a radioiodinated huA33 from the normal intestinal tissue was observed to correspond to the turnover period of basal colonocytes in this tissue (4).

Despite the extensive use of immunotherapy to treat cancers, the efficacy of this regimen is limited by the fact that not all individuals are responsive to this therapy. This is either due to the lack of or irresponsiveness of the receptors (or antigens) to which the Abs are directed. Also, because of its large size, the Ab may not be able to penetrate solid cancerous tumors completely, remain in blood circulation for extended periods, or may not be effective due to other factors as described by von Mehren et al. (5). As an alternative to Abs, investigators have developed Ab fragments such as the single chain variable fragments (scFv), which are much smaller (~30 kDa) than intact Abs (~150 kDa), contain the antigen-binding site, exhibit high tumor penetration, generate excellent tumor/normal tissue concentration ratios, and show rapid clearance through the kidneys (6, 7). Several scFv fragments targeted toward different antigens are under evaluation in clinical trials approved by the United States Food and Drug Administration and by similar regulatory agencies around the world (8).

To further enhance scFv efficacy for preclinical or clinical application, these protein chains have been linked, either by synthesis or recombinant methods, to a variety of molecules such as enzymes (for prodrug therapy), toxins (for treatment of cancer), etc., to generate bifunctional molecules (7, 9, 10). The scFv chain directs the enzyme or toxin to a predetermined cellular antigen where the enzyme converts a prodrug into a cytotoxic molecule or the toxin is activated and has a lethal effect on the cell. Therefore, an Ab fused to an enzyme could be used for an Ab-directed enzyme-prodrug therapy for the treatment of cancers or other diseases. On the basis of this principle, Coelho et al. fused a recombinant scFv directed against the A33 antigen (A33scFv) with the yeast cytosine deaminase (CDy) enzyme to generate A33scFv::CDy as a possible colon cancer treatment (6). The CDy part of the recombinant fusion protein deaminates 5-fluorocytosine (5-FC), the prodrug, to produce cytotoxic 5-fluorouracil after the A33scFv part binds to the A33 antigen on the cell surface. Therefore, the A33scFv::CDy fusion protein is selectively lethal to only those cells that overexpress the A33 antigen, i.e., colon cancer cells. Panjideh et al. investigated the binding specificity of ¹³¹I-labeled A33scFv::CDy under in vitro conditions and also studied its biodistribution in athymic mice bearing xenograft tumors derived either from LIM 1215 cells, a gpA33-positive human colon carcinoma cell line, or the negative control HT29 cells, a gpA33-negative human colon carcinoma cell line (11).

Synthesis

[PubMed]

The production and purification of A33scFv::CDy were described by Coelho et al. (6). Purity of the recombinant fusion protein was reported to be >95% as determined with sodium dodecylsulfate polyacrylamide gel electrophoresis. The ¹³¹I labeling of purified A33scFv::CDy, to obtain [¹³¹I]A33scFv::CDy, was done according to the chloramine-T method as detailed by Panjideh et al. (11). The specific activity of [¹³¹I]A33scFv::CDy was reported to be ~400 kBq/mg (~10.8 μCi/mg). The radiochemical yield, purity, and stability of the labeled fusion protein were not reported.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

The binding activity and specificity of A33scFv::CDy was investigated with the use of flow cytometry using LIM 1215 cells (6). Using a secondary polyclonal Ab directed toward the CDy part of the fusion protein, both the crude production supernatant and the purified fusion protein were shown to bind to the LIM 1215 cells. In another assay, the binding of A33scFv labeled with the green fluorescent protein (A33scFv::GF) to LIM 1215 cells was shown to be inhibited quantitatively by A33scFv::CDy, which suggested that the fusion protein bound specifically to the A33 antigen.

A cytotoxicity assay was performed to confirm the dual activity of A33scFv::CDy (6). LIM 1215 cells treated with A33scFv::CDy were exposed to different concentrations of 5-FC, and, with the use of appropriate controls, it was shown that 1 ng/ml of the fusion protein was sufficient for the maximum cytotoxic effect (25% cell survival) under these experimental conditions (6). Prior exposure of the cells to A33scFv::GF followed by treatment with A33scFv::CDy and 5-FC, as described above, was reported to eliminate the cytotoxic effects of the prodrug.

Using a saturation assay by exposing LIM 1215 cells to [¹³¹I] A33scFv::CDy, the equilibrium dissociation constant and the binding capacity of A33scFv::CDy were determined to be 15.8 nM and 5.02 nM/2 × 10⁶ cells, respectively (11).

Animal Studies

Human Studies

[PubMed]

No references are currently available.

Supplemental Information

[Disclaimer]

No information is currently available.

References

1.

Sakamoto J., Kojima H., Kato J., Hamashima H., Suzuki H. Organ-specific expression of the intestinal epithelium-related antigen A33, a cell surface target for antibody-based imaging and treatment in gastrointestinal cancer. Cancer Chemother Pharmacol. 2000;46 Suppl:S27–32. [PubMed: 10950144]

2.

Ackerman M.E., Chalouni C., Schmidt M.M., Raman V.V., Ritter G., Old L.J., Mellman I., Wittrup K.D. A33 antigen displays persistent surface expression. Cancer Immunol Immunother. 2008;57(7):1017–27. [PMC free article: PMC2836164] [PubMed: 18236042]

3.

Chopra, A., Radioiodinated humanized monoclonal antibody A33 [Radioiodinated-huA33 mAb]. Molecular Imaging and Contrast agent Database (MICAD) [database online]. National Library of Medicine, NCBI, Bethesda, MD, USA. Available from www​.micad.nih.gov, 2004 -2009.

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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]

5.

von Mehren M., Adams G.P., Weiner L.M. Monoclonal antibody therapy for cancer. Annu Rev Med. 2003;54:343–69. [PubMed: 12525678]

6.

Coelho V., Dernedde J., Petrausch U., Panjideh H., Fuchs H., Menzel C., Dubel S., Keilholz U., Thiel E., Deckert P.M. Design, construction, and in vitro analysis of A33scFv::CDy, a recombinant fusion protein for antibody-directed enzyme prodrug therapy in colon cancer. Int J Oncol. 2007;31(4):951–7. [PubMed: 17786329]

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Holliger P., Hudson P.J. Engineered antibody fragments and the rise of single domains. Nat Biotechnol. 2005;23(9):1126–36. [PubMed: 16151406]

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Hudson P.J., Souriau C. Engineered antibodies. Nat Med. 2003;9(1):129–34. [PubMed: 12514726]

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Delves P.J., Roitt I.M. The immune system. First of two parts. N Engl J Med. 2000;343(1):37–49. [PubMed: 10882768]

10.

Delves P.J., Roitt I.M. The immune system. Second of two parts. N Engl J Med. 2000;343(2):108–17. [PubMed: 10891520]

11.

Panjideh H., Da Silva Coelho V.C., Dernedde J., Bachran C., Forster G.J., Franke J., Fasold P., Fuchs H., Thiel E., Deckert P.M. Biodistribution and efficacy of [131I]A33scFv::CDy, a recombinant antibody-enzyme protein for colon cancer. Int J Oncol. 2008;32(4):925–30. [PubMed: 18360720]