Imaging of anthrax intoxication in mice reveals shared and individual functions of surface receptors CMG-2 and TEM-8 in cellular toxin entry.

Bacillus anthracis lethal toxin and edema toxin are binary toxins that consist of a common cell-binding moiety, protective antigen (PA), and the enzymatic moieties, lethal factor (LF) and edema factor (EF). PA binds to either of two receptors, capillary morphogenesis protein-2 (CMG-2) or tumor endothelial marker-8 (TEM-8), which triggers the binding and cytoplasmic translocation of LF and EF. However, the distribution of functional TEM-8 and CMG-2 receptors during anthrax toxin intoxication in animals has not been fully elucidated. Herein, we describe an assay to image anthrax toxin intoxication in animals, and we use it to visualize TEM-8- and CMG-2-dependent intoxication in mice. Specifically, we generated a chimeric protein consisting of the N-terminal domain of LF fused to a nuclear localization signal-tagged Cre recombinase (LFn-NLS-Cre). When PA and LFn-NLS-Cre were coadministered to transgenic mice expressing a red fluorescent protein in the absence of Cre and a green fluorescent protein in the presence of Cre, intoxication could be visualized at single-cell resolution by confocal microscopy or flow cytometry. Using this assay, we found that: (a) CMG-2 is critical for intoxication in the liver and heart, (b) TEM-8 is required for intoxication in the kidney and spleen, (c) CMG-2 and TEM-8 are redundant for intoxication of some organs, (d) combined loss of CMG-2 and TEM-8 completely abolishes intoxication, and (e) CMG-2 is the dominant receptor on leukocytes. The novel assay will be useful for basic and clinical/translational studies of Bacillus anthracis infection and for clinical development of reengineered toxin variants for cancer treatment.

Bismaleimide cross-linked anthrax toxin forms functional octamers with high specificity in tumor targeting.

In recent years, anthrax toxin has been reengineered to act as a highly specific antiangiogenic cancer therapeutic, shown to kill tumors in animal models. This has been achieved by modifying protective antigen (PA) so that its activation and toxicity require the presence of two proteases, matrix metalloproteinase (MMP) and urokinase plasminogen activator (uPA), which are upregulated in tumor microenvironments. These therapeutics consist of intercomplementing PA variants, which are individually nontoxic, but form functional toxins upon complementary oligomerization. Here, we have created a dual-protease requiring PA targeting system which utilizes bismaleimide cross-linked PA (CLPA) rather than the intercomplementing PA variants. Three different CLPA agents were tested and, as expected, found to exclusively form octamers. Two of the CLPA agents have in vitro toxicities equal to those of previous intercomplementing agents, while the third CLPA agent had compromised in vitro cleavage and was significantly less cytotoxic. We hypothesize this difference was due to steric hindrance caused by cross-linking two PA monomers in close proximity to the PA cleavage site. Overall, this work advances the development and use of the PA and LF tumor-targeting system as a practical cancer therapeutic, as it provides a way to reduce the drug components of the anthrax toxin drug delivery system from three to two, which may lower the cost and simplify testing in clinical trials. HIGHLIGHT: Previously, anthrax toxin has been reengineered to act as a highly specific antiangiogenic cancer therapeutic. Here, we present a version, which utilizes bismaleimide cross-linked protective antigen (PA) rather than intercomplementing PA variants. This advances the development of anthrax toxin as a practical cancer therapeutic as it reduces the components of the drug delivery system to two, which may lower the cost and simplify testing in clinical trials.

Solid tumor therapy by selectively targeting stromal endothelial cells.

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.

Reductive methylation and mutation of an anthrax toxin fusion protein modulates its stability and cytotoxicity.

We characterized an anti-cancer fusion protein consisting of anthrax lethal factor (LF) and the catalytic domain of Pseudomonas exotoxin A by (i) mutating the N-terminal amino acids and by (ii) reductive methylation to dimethylate all lysines. Dimethylation of lysines was achieved quantitatively and specifically without affecting binding of the fusion protein to PA or decreasing the enzymatic activity of the catalytic moiety. Ubiquitination in vitro was drastically decreased for both the N-terminally mutated and dimethylated variants, and both appeared to be slightly more stable in the cytosol of treated cells. The dimethylated variant showed greatly reduced neutralization by antibodies to LF. The two described modifications offer unique advantages such as increased cytotoxic activity and diminished antibody recognition, and thus may be applicable to other therapeutic proteins that act in the cytosol of cells.

Key tissue targets responsible for anthrax-toxin-induced lethality.

Bacillus anthracis, the causative agent of anthrax disease, is lethal owing to the actions of two exotoxins: anthrax lethal toxin (LT) and oedema toxin (ET). The key tissue targets responsible for the lethal effects of these toxins are unknown. Here we generated cell-type-specific anthrax toxin receptor capillary morphogenesis protein-2 (CMG2)-null mice and cell-type-specific CMG2-expressing mice and challenged them with the toxins. Our results show that lethality induced by LT and ET occurs through damage to distinct cell types; whereas targeting cardiomyocytes and vascular smooth muscle cells is required for LT-induced mortality, ET-induced lethality occurs mainly through its action in hepatocytes. Notably, and in contradiction to what has been previously postulated, targeting of endothelial cells by either toxin does not seem to contribute significantly to lethality. Our findings demonstrate that B. anthracis has evolved to use LT and ET to induce host lethality by coordinately damaging two distinct vital systems.

Anthrax edema factor toxicity is strongly mediated by the N-end rule.

Anthrax edema factor (EF) is a calmodulin-dependent adenylate cyclase that converts adenosine triphosphate (ATP) into 3'-5'-cyclic adenosine monophosphate (cAMP), contributing to the establishment of Bacillus anthracis infections and the resulting pathophysiology. We show that EF adenylate cyclase toxin activity is strongly mediated by the N-end rule, and thus is dependent on the identity of the N-terminal amino acid. EF variants having different N-terminal residues varied by more than 100-fold in potency in cultured cells and mice. EF variants having unfavorable, destabilizing N-terminal residues showed much greater activity in cells when the E1 ubiquitin ligase was inactivated or when proteasome inhibitors were present. Taken together, these results show that EF is uniquely affected by ubiquitination and/or proteasomal degradation.

Anthrax toxin-mediated delivery of the Pseudomonas exotoxin A enzymatic domain to the cytosol of tumor cells via cleavable ubiquitin fusions.

UNLABELLED: Anthrax toxin proteins from Bacillus anthracis constitute a highly efficient system for delivering cytotoxic enzymes to the cytosol of tumor cells. However, exogenous proteins delivered to the cytosol of cells are subject to ubiquitination on lysines and proteasomal degradation, which limit their potency. We created fusion proteins containing modified ubiquitins with their C-terminal regions fused to the Pseudomonas exotoxin A catalytic domain (PEIII) in order to achieve delivery and release of PEIII to the cytosol. Fusion proteins in which all seven lysines of wild-type ubiquitin were retained while the site cleaved by cytosolic deubiquitinating enzymes (DUBs) was removed were nontoxic, apparently due to rapid ubiquitination and proteasomal degradation. Fusion proteins in which all lysines of wild-type ubiquitin were substituted by arginine had high potency, exceeding that of a simple fusion lacking ubiquitin. This variant was less toxic to nontumor tissues in mice than the fusion protein lacking ubiquitin and was very efficient for tumor treatment in mice. The potency of these proteins was highly dependent on the number of lysines retained in the ubiquitin domain and on retention of the C-terminal ubiquitin sequence cleaved by DUBs. It appears that rapid cytosolic release of a cytotoxic enzyme (e.g., PEIII) that is itself resistant to ubiquitination is an effective strategy for enhancing the potency of tumor-targeting toxins. IMPORTANCE: Bacterial toxins typically have highly efficient mechanisms for cellular delivery of their enzymatic components. Cytosolic delivery of therapeutic enzymes and drugs is an important topic in molecular medicine. We describe anthrax toxin fusion proteins containing ubiquitin as a cytosolic cleavable linker that improves the delivery of an enzyme to mammalian cells. The ubiquitin linker allowed modulation of potency in cells and in mice. This effective strategy for enhancing the intracellular potency of an enzyme may be useful for the cytosolic delivery and release of internalized drugs.

Engineering anthrax toxin variants that exclusively form octamers and their application to targeting tumors.

Anthrax toxin protective antigen (PA) delivers its effector proteins into the host cell cytosol through formation of an oligomeric pore, which can assume heptameric or octameric states. By screening a highly directed library of PA mutants, we identified variants that complement each other to exclusively form octamers. These PA variants were individually nontoxic and demonstrated toxicity only when combined with their complementary partner. We then engineered requirements for activation by matrix metalloproteases and urokinase plasminogen activator into two of these variants. The resulting therapeutic toxin specifically targeted cells expressing both tumor associated proteases and completely stopped tumor growth in mice when used at a dose far below that which caused toxicity. This scheme for obtaining intercomplementing subunits can be employed with other oligomeric proteins and potentially has wide application.

Recombinant expression and purification of a tumor-targeted toxin in Bacillus anthracis.

Many recombinant therapeutic proteins are purified from Escherichia coli. While expression in E. coli is easily achieved, some disadvantages such as protein aggregation, formation of inclusion bodies, and contamination of purified proteins with the lipopolysaccharides arise. Lipopolysaccharides have to be removed to prevent inflammatory responses in patients. Use of the Gram-positive Bacillus anthracis as an expression host offers a solution to circumvent these problems. Using the multiple protease-deficient strain BH460, we expressed a fusion of the N-terminal 254 amino acids of anthrax lethal factor (LFn), the N-terminal 389 amino acids of diphtheria toxin (DT389) and human transforming growth factor alpha (TGFα). The resulting fusion protein was constitutively expressed and successfully secreted by B. anthracis into the culture supernatant. Purification was achieved by anion exchange chromatography and proteolytic cleavage removed LFn from the desired fusion protein (DT389 fused to TGFα). The fusion protein showed the intended specific cytotoxicity to epidermal growth factor receptor-expressing human head and neck cancer cells. Final analyses showed low levels of lipopolysaccharides, originating most likely from contamination during the purification process. Thus, the fusion to LFn for protein secretion and expression in B. anthracis BH460 provides an elegant tool to obtain high levels of lipopolysaccharide-free recombinant protein.

Role of N-terminal amino acids in the potency of anthrax lethal factor.

Anthrax lethal factor (LF) is a Zn(+2)-dependent metalloprotease that cleaves several MAPK kinases and is responsible for the lethality of anthrax lethal toxin (LT). We observed that a recombinant LF (LF-HMA) which differs from wild type LF (LF-A) by the addition of two residues (His-Met) to the native Ala (A) terminus as a result of cloning manipulations has 3-fold lower potency toward cultured cells and experimental animals. We hypothesized that the "N-end rule", which relates the half-life of proteins in cells to the identity of their N-terminal residue, might be operative in the case of LF, so that the N-terminal residue of LF would determine the cytosolic stability and thereby the potency of LF. Mutational studies that replaced the native N-terminal residue of LF with known N-end rule stabilizing or destabilizing residues confirmed that the N-terminal residue plays a significant role in determining the potency of LT for cultured cells and experimental animals. The fact that a commercially-available LF preparation (LF-HMA) that is widely used in basic research studies and for evaluation of vaccines and therapeutics is 3-fold less potent than native LF (LF-A) should be considered when comparing published studies and in the design of future experiments.

APRIL and BAFF promote increased viability of replicating human B2 cells via mechanism involving cyclooxygenase 2.

Of relevance to both protective and pathogenic responses to Ag is the recent finding that soluble molecules of the innate immune system, i.e., IL-4, B cell-activation factor of the TNF family (BAFF), and C3, exhibit significant synergy in promoting the clonal expansion of human B2 cells following low-level BCR ligation. Although IL-4, BAFF, and C3dg each contribute to early cell cycle entry and progression to S phase, only BAFF promotes later sustained viability of progeny needed for continued cycling. The present study sought to further clarify the mechanisms for BAFF's multiple functions. By comparing BAFF and a proliferation-inducing ligand (APRIL) efficacy at different stages in the response (only BAFF binds BR3; both bind transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and B cell maturation Ag, the early role was attributed to BR3, while the later role was attributed to TACI/B cell maturation Ag. Importantly, BAFF- and APRIL-promoted viability of cycling lymphoblasts was associated with sustained expression of cyclooxygenase 2 (COX-2), the rate-limiting enzyme for PGE2 synthesis, within replicating cells. Supernatants of cultures with BAFF and APRIL contained elevated PGE2. Although COX-2 inhibitors diminished daughter cell viability, exogenous PGE2 (1-1000 nM) increased the viability and recovery of lymphoblasts. Increased yield of viable progeny was associated with elevated Mcl-1, suggesting that a BAFF/APRIL --> TACI --> COX-2 --> PGE2--> Mcl-1 pathway reduces activation-related, mitochondrial apoptosis in replicating human B2 cell clones.

Innate immunity and human B cell clonal expansion: effects on the recirculating B2 subpopulation.

Foci of autoantigen-specific B lymphocytes in nonlymphoid tissues have been associated with development of autoimmune disease. To better understand the genesis of such ectopic lymphoid tissue, this study investigated whether several B cell-tropic innate immune system molecules, known to be elevated in response to inflammatory stimuli, can cooperate in fostering the T cell-independent clonal expansion of mature human B2 cells under conditions of limiting BCR engagement. Notable synergy was observed between BCR coligation with the C3dg-binding CD21/CD19 costimulatory complex, B cell-activating factor belonging to the TNF family (BAFF), and IL-4 in generating B cell progeny with sustained CD86 and DR expression. The synergy was observed over a wide range of BCR:ligand affinities and involved: 1) cooperative effects at promoting early cell cycle progression and viability; 2) BCR:CD21 coligation-promoted increases in BAFF receptors that were highly regulated by IL-4; 3) reciprocal effects of IL-4 and BAFF at dampening daughter cell apoptosis typical of stimulation by BCR:CD21 and either cytokine alone; and 4) BAFF-sustained expression of antiapoptotic Mcl-1 within replicating lymphoblasts. The results suggest that significant clonal proliferation of recirculating B2 cells occurs upon limited binding to C3dg-coated Ag in an inflammatory in vivo milieu containing both BAFF and IL-4. When rare autoantigen-presenting B cells undergo such expansions, both B cell and T cell autoimmunity may be promoted.

Optimization of unique, uncharged thioesters as inhibitors of HIV replication.

A combinatorial chemistry approach was employed to prepare a restricted library of N-substituted S-acyl-2-mercaptobenzamide thioesters. It was shown that many members of this chemotype display anti-HIV activity via their ability to interact with HIV-1, HIV-2, SIV-infected cells, cell-free virus, and chronically and latently infected cells in a manner consistent with targeting of the highly conserved HIV-1 NCp7 zinc fingers. Compounds were initially screened using two different in vitro antiviral assays and evaluated for stability in neutral buffer containing 10% pooled human serum using a spectrophotometric assay. These data revealed that there was no significant correlation between thioester stability and antiviral activity, however, a slight inverse correlation between serum stability and virucidal activity was noted. Based on the virucidal capability and the ability to select lead compounds to inhibit virus expression from latently infected TNFalpha-induced U1 cells, we next determined if these compounds could prevent HIV cell-to-cell transmission. Several thioesters demonstrated potent inhibition of HIV cell-to-cell transmission with EC50 values in the 80-100 nM range. Thus, we have optimized a series of restricted thioesters and provided evidence that serum stability is not required for antiviral activity. Moreover, selected compounds show potential for development as topical microbicides.

Role of complement-binding CD21/CD19/CD81 in enhancing human B cell protection from Fas-mediated apoptosis.

Defective expression of Fas leads to B cell autoimmunity, indicating the importance of this apoptotic pathway in eliminating autoreactive B cells. However, B cells with anti-self specificities occasionally escape such regulation in individuals with intact Fas, suggesting ways of precluding this apoptosis. Here, we examine whether coligation of the B cell Ag receptor (BCR) with the complement (C3)-binding CD21/CD19/CD81 costimulatory complex can enhance the escape of human B cells from Fas-induced death. This was warranted given that BCR-initiated signals induce resistance to Fas apoptosis, some (albeit not all) BCR-triggered events are amplified by coligation of BCR and the co-stimulatory complex, and several self Ags targeted in autoimmune diseases effectively activate complement. Using a set of affinity-diverse surrogate Ags (receptor-specific mAb:dextran conjugates) with varying capacity to engage CD21, it was established that BCR:CD21 coligation lowers the BCR engagement necessary for inducing protection from Fas apoptosis. Enhanced protection was associated with altered expression of several molecules known to regulate Fas apoptosis, suggesting a unique molecular model for how BCR:CD21 coligation augments protection. BCR:CD21 coligation impairs the generation of active fragments of caspase-8 via dampened expression of membrane Fas and augmented expression of FLIP(L). This, in turn, diminishes the generation of cells that would be directly triggered to apoptosis via caspase-8 cleavage of caspase 3 (type I cells). Any attempt to use the mitochondrial apoptotic protease-activating factor 1 (Apaf-1)-dependent pathway for apoptosis (as type II cells) is further blocked because BCR:CD21 coligation promotes up-regulation of the mitochondrial antiapoptotic molecule, Bcl-2.

In vivo antiviral activity of novel human immunodeficiency virus type 1 nucleocapsid p7 zinc finger inhibitors in a transgenic murine model.

Control of human immunodeficiency virus through the use of inexpensive chemotherapeutics, with minimal side effects and decreased potential for engendering resistant virus, is a long-term therapeutic goal. In principle, this goal can be accomplished if viral replication in reservoirs of chronically and latently infected cells is addressed. As a first step, we have developed novel antiviral compounds based on a 2-mercaptobenzamide thioester chemotype, including the pyridinioalkanoyl thioesters, which specifically target the zinc fingers of the human immunodeficiency virus nucleocapsid protein (NCp7). Using these compounds in a murine transgenic model, in which infectious human immunodeficiency virus is induced from an integrated provirus, we show inhibition of transgenic spleen cell p24 expression with potencies comparable to acute infection assays using human peripheral blood lymphocytes. More importantly, transgenic mice treated in vivo with two 2-mercaptobenzamide thioesters expressed significantly lower plasma p24, and splenocytes from these animals produced fewer infectious virions. Thus, these thioesters may provide an effective means for inhibiting the expression of human immunodeficiency virus from integrated viral reservoirs.

Antigen receptor triggered upregulation of CD86 and CD80 in human B cells: augmenting role of the CD21/CD19 co-stimulatory complex and IL-4.

The impact of BCR:CD21 co-engagement on B cell expression of molecules critical for T cell activation was investigated with receptor-specific mAbs conjugated to high MW dextran as stimulatory ligands. In the absence of IL-4, BCR:CD21 co-ligation augmented BCR-triggered CD86 only under conditions of very low BCR ligand dose or affinity, and CD80 was minimally induced by BCR and/or CD21 crosslinking. In the presence of IL-4, BCR:CD21 co-ligation augmented CD86 and CD80 expression under conditions of greater BCR engagement. However, with very high level BCR engagement, no bonus effect of BCR:CD21 crosslinking was observed. Co-ligation-promoted CD86 and CD80 expression was associated with heightened B cell activation of resting allogeneic T cells. The data suggest that co-clustering of BCR and the CD21/CD19 co-stimulatory complex following B cell engagement with C3d-bound microbial or self-antigens will enhance B cell recruitment of T cell help only when IL-4 is present and/or BCR engagement is very limiting.

Benzamide-based thiolcarbamates: a new class of HIV-1 NCp7 inhibitors.

The HIV-1 nucleocapsid protein NCp7, which contains two highly conserved zinc fingers, is being used as a novel target for AIDS therapy due to its pivotal role in viral replication and its mutationally intolerant nature. Herein we report a new class of NCp7 inhibitors that possess good antiviral activity with low cellular toxicity.