Selection of anthrax toxin protective antigen variants that discriminate between the cellular receptors TEM8 and CMG2 and achieve targeting of tumor cells - PubMed (original) (raw)

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 et al. J Biol Chem. 2007.

Abstract

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.

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Figures

Fig. 1

Fig. 1

Amino acid sequences of PA domain 4 variants expressed in T7 bacteriophage and selected by panning. Mutations were produced by 8-oxo-dGTP used in equimolar amount (400 μM) with the four normal dNTPs in a PCR reaction, the product of which was introduced into the bacteriophage T7 phage display system. Bacteriophage were enriched for those that bind to monoclonal antibody 14B7 (a receptor mimic) but not to soluble CMG2. The PA variant sequences are sorted according to the more frequent substitutions into panels A-D. The summary results line shown at the bottom of each panel summarizes results for all panels and identifies positions substituted more than once (X) and those having codons containing only G and C and therefore not expected to change at significant frequencies (*).

Fig. 1

Fig. 1

Amino acid sequences of PA domain 4 variants expressed in T7 bacteriophage and selected by panning. Mutations were produced by 8-oxo-dGTP used in equimolar amount (400 μM) with the four normal dNTPs in a PCR reaction, the product of which was introduced into the bacteriophage T7 phage display system. Bacteriophage were enriched for those that bind to monoclonal antibody 14B7 (a receptor mimic) but not to soluble CMG2. The PA variant sequences are sorted according to the more frequent substitutions into panels A-D. The summary results line shown at the bottom of each panel summarizes results for all panels and identifies positions substituted more than once (X) and those having codons containing only G and C and therefore not expected to change at significant frequencies (*).

Fig. 1

Fig. 1

Amino acid sequences of PA domain 4 variants expressed in T7 bacteriophage and selected by panning. Mutations were produced by 8-oxo-dGTP used in equimolar amount (400 μM) with the four normal dNTPs in a PCR reaction, the product of which was introduced into the bacteriophage T7 phage display system. Bacteriophage were enriched for those that bind to monoclonal antibody 14B7 (a receptor mimic) but not to soluble CMG2. The PA variant sequences are sorted according to the more frequent substitutions into panels A-D. The summary results line shown at the bottom of each panel summarizes results for all panels and identifies positions substituted more than once (X) and those having codons containing only G and C and therefore not expected to change at significant frequencies (*).

Fig. 1

Fig. 1

Amino acid sequences of PA domain 4 variants expressed in T7 bacteriophage and selected by panning. Mutations were produced by 8-oxo-dGTP used in equimolar amount (400 μM) with the four normal dNTPs in a PCR reaction, the product of which was introduced into the bacteriophage T7 phage display system. Bacteriophage were enriched for those that bind to monoclonal antibody 14B7 (a receptor mimic) but not to soluble CMG2. The PA variant sequences are sorted according to the more frequent substitutions into panels A-D. The summary results line shown at the bottom of each panel summarizes results for all panels and identifies positions substituted more than once (X) and those having codons containing only G and C and therefore not expected to change at significant frequencies (*).

Fig. 2

Fig. 2

Close-up of the PA-CMG2 binding interface, with selected sidechains shown as ball-and-stick, and hydrogen bonds as dashed black lines. The Mg2+ ion (blue ball) coordinates D683 from PA, as well as residues from CMG2 (in red) at the MIDAS motif. The double mutant R659S/M662R is shown, modeled with side-chain conformations based on the wild-type complex. The two mutated side-chains are close together, and the R659S mutation prevents a clash of buried positive charges with the 662R side-chain. In the wild-type complex, the side-chain of CMG2 E117 (in red) turns away from M662, making a strong hydrogen bond to the main-chain of D683. In TEM8, the analogous residue is aspartic acid, shown in its most probable conformation (colored in gold), which allows it to form a salt-bridge to the M662R mutant side-chain. The side-chain of N657 is shown in its buried location, making hydrogen bonds (dashed lines) to the β-strand above it. The N657Q mutant, with its longer side-chain, would not fit well, and modeling suggests that the mutation would engender a downward motion of the loop (red arrow) towards the MIDAS motif, leading to a more intimate packing between PA and the receptor. Some mutants with smaller side-chains at the 657 position are tolerated, while large hydrophobic or charged residues are not.

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