Engineering Anthrax Toxin Variants That Exclusively Form Octamers and Their Application to Targeting Tumors

Phillips, Damilola D.; Fattah, Rasem J.; Crown, Devorah; Zhang, Yi; Liu, Shihui; Moayeri, Mahtab; Fischer, Elizabeth R.; Hansen, Bryan; Ghirlando, Rodolfo; Nestorovich, Ekaterina M.; Wein, Alexander N.; Simons, Lacy M.; Leppla, Stephen H.; Leysath, Clinton E.

doi:10.1074/jbc.m113.452110

Cited by 37 publications

(60 citation statements)

References 31 publications

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“…Thus, replacing the furin target sequence RKKR with MMP or uPA substrate sequences has yielded specific antitumor agents (20)(21)(22). To further increase tumor specificity, based on the fact that each LF-binding site on PA oligomers is located at the bridge region of adjacent PA63 monomers, we have previously generated intermolecular complementing PA variants dependent on the simultaneous presence of both MMPs and uPA for activation (23)(24)(25). Because both cancer cells and many tumor stromal cells overexpress MMPs and uPA (26)(27)(28), these PA variants are preferentially activated in solid tumors, thereby being able to selectively deliver effector proteins, such as LF or recombinant LF Significance Anthrax toxin proteins engineered to require activation by tumorassociated proteases show high specificity and potency in suppression of solid tumor growth through actions on tumor endothelial cells.…”

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confidence: 99%

Solid tumor therapy by selectively targeting stromal endothelial cells

Liu

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Engineered tumor-targeted anthrax lethal toxin proteins have been shown to strongly suppress growth of solid tumors in mice. These toxins work through the native toxin receptors tumor endothelium marker-8 and capillary morphogenesis protein-2 (CMG2), which, in other contexts, have been described as markers of tumor endothelium. We found that neither receptor is required for tumor growth. We further demonstrate that tumor cells, which are resistant to the toxin when grown in vitro, become highly sensitive when implanted in mice. Using a range of tissue-specific loss-of-function and gain-of-function genetic models, we determined that this in vivo toxin sensitivity requires CMG2 expression on host-derived tumor endothelial cells. Notably, engineered toxins were shown to suppress the proliferation of isolated tumor endothelial cells. Finally, we demonstrate that administering an immunosuppressive regimen allows animals to receive multiple toxin dosages and thereby produces a strong and durable antitumor effect. The ability to give repeated doses of toxins, coupled with the specific targeting of tumor endothelial cells, suggests that our strategy should be efficacious for a wide range of solid tumors.anthrax toxin | tumor targeting | angiogenesis | CMG2 | TEM8 R ecognition that aberrant activation of the RAS and PI3K pathways is often the mechanism of human tumorigenesis has inspired development of many small molecule inhibitors of these pathways and has led to improved treatments in certain cancers (1). However, these therapies are effective only in patients having defects in the specific targets of these drugs, and the therapies are rarely curative due to the development of resistance through acquisition of additional oncogenic mutations (2). Therefore, strategies are needed that are effective against a broad spectrum of cancers and that act through features that are not subject to development of resistance. This unmet need has fostered continued interest in strategies that target host-derived tumor vasculature.Anthrax toxin, a major virulence factor of Bacillus anthracis, consists of three individually nontoxic proteins: the cellular binding component, protective antigen (PA), and two enzymatic moieties, lethal factor (LF) and edema factor (EF) (3). PA binds to two host cell-surface integrin-like proteins: tumor endothelium marker-8 (TEM8) [also termed anthrax toxin receptor 1 (ANTXR1)] and capillary morphogenesis protein-2 (CMG2 or ANTXR2) (4, 5). Receptor-bound PA is processed by the ubiquitous cell-associated furin protease to a 63-kDa fragment (PA63), which then forms LFand EF-binding competent PA oligomers. Three or four molecules of LF or EF bind to the PA oligomers, and the complexes are then endocytosed (6-8). The acidic pH within the endosomes causes the PA oligomer to form a pore in the endosomal membrane, allowing translocation of LF or EF into the cytosol of cells to exert their cytotoxic actions (9). Thus, LF plus PA forms lethal toxin and EF plus PA forms edema toxin, with both toxins playing essenti...

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confidence: 99%

Solid tumor therapy by selectively targeting stromal endothelial cells

Liu

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…As a result, pore formation occurs only in cells expressing both uPA and MMP, as processing only by both proteases conditionally triggers the formation of a hetero-heptameric or octameric pore that mediates cell entry of the cytotoxic components. Using LF as effector, the engineered toxin system is conditionally toxic to lung tumor cells in vitro and in murine xenograft transplant models when the two variants are administered together (84). The cytotoxic potency of LF using conditionally bispecific ATx (∼10 −11 M in vitro) is within 10-fold of its wildtype counterpart.…”

Section: Role For the Prodrug Concept In Conditional Multi-specific Tmentioning

confidence: 95%

“…No microbiological agents, such as the anthrax bacterium or its spore, are involved. Surviving animals at the maximum tolerated dose show no acute nephro-or hepatotoxicity as judged by clinical biochemistry parameters (84). Conditionally bispecific prodrug conjugates.…”

Section: Role For the Prodrug Concept In Conditional Multi-specific Tmentioning

confidence: 99%

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Prodrug Applications for Targeted Cancer Therapy

Giang

Boland

Poon

2014

AAPS J

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Abstract. Prodrugs are widely used in the targeted delivery of cytotoxic compounds to cancer cells. To date, targeted prodrugs for cancer therapy have achieved great diversity in terms of target selection, activation chemistry, as well as size and physicochemical nature of the prodrug. Macromolecular prodrugs such as antibody-drug conjugates, targeted polymer-drug conjugates and other conjugates that self-assemble to form liposomal and micellar nanoparticles currently represent a major trend in prodrug development for cancer therapy. In this review, we explore a unified view of cancer-targeted prodrugs and highlight several examples from recombinant technology that exemplify the prodrug concept but are not identified as such. Recombinant "prodrugs" such as engineered anthrax toxin show promise in biological specificity through the conditionally targeting of multiple cellular markers. Conditional targeting is achieved by structural complementation, the spontaneous assembly of engineered inactive subunits or fragments to reconstitute functional activity. These complementing systems can be readily adapted to achieve conditionally bispecific targeting of enzymes that are used to activate low-molecular weight prodrugs. By leveraging strengths from medicinal chemistry, polymer science, and recombinant technology, prodrugs are poised to remain a core component of highly focused and tailored strategies aimed at conditionally attacking complex molecular phenotypes in clinically relevant cancer.

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“…This decreased toxicity toward TEM8 suggests that this CMG2-specific variant will have an improved therapeutic index compared with PA when used as a component of tumortargeting agents. In ongoing work, the I656Q substitution is being placed into the previously described IC2-PA and "octamer" drugs (13,16,24), with an expectation of improving their specificity and, therefore, their therapeutic indices.…”

Section: Discussionmentioning

confidence: 99%

Anthrax Toxin Protective Antigen Variants That Selectively Utilize either the CMG2 or TEM8 Receptors for Cellular Uptake and Tumor Targeting

Chen

Liu

Leysath

et al. 2016

Journal of Biological Chemistry

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The protective antigen (PA) moiety of anthrax toxin binds to cellular receptors and mediates the translocation of the two enzymatic moieties of the toxin to the cytosol. Two PA receptors are known, with capillary morphogenesis protein 2 (CMG2) being the more important for pathogenesis and tumor endothelial marker 8 (TEM8) playing a minor role. The C-terminal PA domain 4 (PAD4) has extensive interactions with the receptors and is required for binding. Our previous study identified PAD4 variants having enhanced TEM8 binding specificity. To obtain PA variants that selectively bind to CMG2, here we performed phage display selections using magnetic beads having bound CMG2. We found that PA residue isoleucine 656 plays a critical role in PA binding to TEM8 but has a much lesser effect on PA binding to CMG2. We further characterized the role of residue 656 in distinguishing PA binding to CMG2 versus TEM8 by substituting it with the other 19 amino acids. Of the resulting variants, PA I656Q and PA I656V had significantly reduced activity on TEM8-expressing CHO cells but maintained their activity on CMG2-expressing CHO cells. The preference of these PA mutants for CMG2 over TEM8 was further demonstrated using mouse embryonic fibroblast cells and mice deficient in the CMG2 and/or the TEM8 receptors. The structural basis of the alterations in the receptor binding activities of these mutants is also discussed.Anthrax toxin is composed of three nontoxic proteins: protective antigen (PA), 3 lethal factor (LF), and edema factor (EF), which are released from Bacillus anthracis as monomers and assemble into toxic complexes on the surface of animal cells (1-3). PA is the moiety which binds to cell surface receptors and facilitates entry of the enzymatic EF and LF moieties into the host cell cytosol. Two anthrax toxin receptors have been identified; capillary morphogenesis protein 2 (CMG2, or anthrax toxin receptor 2) is the physiologically relevant toxin receptor mediating most of the in vivo toxicity of the toxin, whereas tumor endothelial marker 8 (TEM8, or anthrax toxin receptor 1) functions only as a minor anthrax toxin receptor (4 -8). Upon binding to the toxin receptors, PA is cleaved at the sequence 164 RKKR 167 by cell surface furin or furin-like proteases (9, 10), yielding the C-terminal 63-kDa fragment (PA63), which assembles to form a ring-shaped oligomer. The PA63 oligomer then binds LF and/or EF, and the toxin complex is internalized by receptor-mediated endocytosis. LF and EF are translocated into the cytosol from early or late endosomes to exert their cytotoxic effects. Therefore, PA plays the crucial role in anthrax toxin pathogenesis, serving as the delivery vehicle for translocation of LF and EF into the cytosol of the cell.The absolute requirement for cell surface proteolytic activation of PA for the toxin's action prompted our group and others to reengineer PA to make it depend on tumor-associated proteases for activation, thereby achieving high specificity for tumors (11)(12)(13)(14)(15). In a recent example, ...

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Engineering Anthrax Toxin Variants That Exclusively Form Octamers and Their Application to Targeting Tumors

Cited by 37 publications

References 31 publications

Solid tumor therapy by selectively targeting stromal endothelial cells

Solid tumor therapy by selectively targeting stromal endothelial cells

Prodrug Applications for Targeted Cancer Therapy

Anthrax Toxin Protective Antigen Variants That Selectively Utilize either the CMG2 or TEM8 Receptors for Cellular Uptake and Tumor Targeting

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