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J. Biol. Chem., Vol. 282, Issue 13, 9834-9845, March 30, 2007

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Selection of Anthrax Toxin Protective Antigen Variants That Discriminate between the Cellular Receptors TEM8 and CMG2 and Achieve Targeting of Tumor Cells^*

Kuang-Hua Chen, Shihui Liu, Laurie A. Bankston, Robert C. Liddington, and Stephen H. Leppla¹

From the Laboratory of Bacterial Diseases, NIAID, National Institutes of Health, Bethesda, Maryland 20892-3202 and Infectious and Inflammatory Disease Center, Burnham Institute for Medical Research, La Jolla, California 92037

Anthrax toxin, a three-component protein toxin secreted by Bacillus anthracis, assembles into toxic complexes at the surface of receptor-bearing eukaryotic cells. The protective antigen (PA) protein binds to receptors, either tumor endothelial cell marker 8 (TEM8) or CMG2 (capillary morphogenesis protein 2), and orchestrates the delivery of the lethal and edema factors into the cytosol. TEM8 is reported to be overexpressed during tumor angiogenesis, whereas CMG2 is more widely expressed in normal tissues. To extend prior work on targeting of tumor with modified anthrax toxins, we used phage display to select PA variants that preferentially bind to TEM8 as compared with CMG2. Substitutions were randomly introduced into residues 605–729 of PA, within the C-terminal domain 4 of PA, which is the principal region that contacts receptor. Candidates were characterized in cellular cytotoxicity assays with Chinese hamster ovary (CHO) cells expressing either TEM8 or CMG2. A PA mutant having the substitutions R659S and M662R had enhanced specificity toward TEM8-overexpressing CHO cells. This PA variant also displayed broad and potent tumoricidal activity to various human tumor cells, especially to HeLa and A549/ATCC cells. By contrast, the substitution N657Q significantly reduced toxicity to TEM8 but not CMG2-overexpressing CHO cells. Our results indicate that certain amino acid substitutions within PA domain 4 create anthrax toxins that selectively kill human tumor cells. The PA R659S/M662R protein may be useful as a therapeutic agent for cancer treatment.

Received for publication, December 5, 2006 , and in revised form, January 22, 2007.

^* This work was supported by National Institutes of Health Grant AI055789 (to R. C. L.) and by intramural research funds of the NIAID, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

¹ To whom correspondence should be addressed: Laboratory of Bacterial Diseases, Bldg. 33, Rm. 1W20B, NIAID, National Institutes of Health, Bethesda, MD 20892-3202. Tel.: 301-594-2865; Fax: 301-480-0326; E-mail: sleppla{at}niaid.nih.gov.

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