Bacillus anthracis secretes two bipartite toxins thought to be involved in anthrax pathogenesis and resulting death of the host. The current model for intoxication is that protective antigen (PA) toxin subunits bind a single group of cell-surface anthrax toxin receptors (ATRs), encoded by the tumor endothelial marker 8 (TEM8) gene. The ATR͞TEM8-PA interaction is mediated by the receptor's extracellular domain related to von Willebrand factor type A or integrin inserted domains (VWA͞I domains). A metal ion-dependent adhesion site (MIDAS) located within this domain of the ATR͞TEM8 protein chelates a divalent cation critical for PA binding. In this report, we identify a second PA receptor encoded by capillary morphogenesis gene 2 (CMG2), which has 60% amino acid identity to ATR͞TEM8 within the VWA͞I domain, as well as a conserved MIDAS motif. A recombinant CMG2 protein bound PA and mediated toxin internalization when expressed on receptor-deficient cells. Binding between the CMG2 VWA͞I domain and PA was shown to be direct and metal-dependent, although the cation specificity of this interaction is different than that observed with ATR͞TEM8. Northern blot analysis revealed that CMG2 is widely expressed in human tissues, indicating that this receptor is likely to be relevant for disease pathogenesis. Finally, a soluble version of the CMG2 VWA͞I domain inhibited intoxication of cells expressing endogenous toxin receptors when it was added to PA at a 3:1 ratio. These studies distinguish CMG2 as a second anthrax toxin receptor and identify a potent antitoxin that may prove useful for the treatment of anthrax.

Widespread drug resistance due to empiric use of broad-spectrum antibiotics has stimulated development of bacteria-specific strategies for prophylaxis and therapy based on modern monoclonal antibody (mAb) technologies. However, single-mechanism mAb approaches have not provided adequate protective activity in the clinic. We constructed multifunctional bispecific antibodies, each conferring three mechanisms of action against the bacterial pathogen Pseudomonas aeruginosa by targeting the serotype-independent type III secretion system (injectisome) virulence factor PcrV and persistence factor Psl exopolysaccharide. A new bispecific antibody platform, BiS4, exhibited superior synergistic protection against P. aeruginosa-induced murine pneumonia compared to parent mAb combinations or other available bispecific antibody structures. BiS4αPa was protective in several mouse infection models against disparate P. aeruginosa strains and unexpectedly further synergized with multiple antibiotic classes even against drug-resistant clinical isolates. In addition to resulting in a multimechanistic clinical candidate (MEDI3902) for the prevention or treatment of P. aeruginosa infections, these antibody studies suggest that multifunctional antibody approaches may be a promising platform for targeting other antibiotic-resistant bacterial pathogens.

IntroductionTargeted mAb-based therapies provide effective and safe treatments for hematologic malignancies. Rituximab, which specifically targets the B-cell antigen CD20, has had the greatest success, revolutionizing the treatment of the 2 most common forms of nonHodgkin lymphoma: follicular and diffuse large B-cell lymphoma. In addition, mAb-based therapies targeting CD52 (alemtuzumab) and CD33 (gemtuzumab ozogamicin) have been approved for the treatment of chronic lymphocytic leukemia and acute myelogenous leukemia, respectively. Despite the progress of these strategies, they do have limitations. Only a fraction of patients respond to rituximab, and the majority of those who do respond will eventually relapse. Treatment with alemtuzumab and gemtuzumab are limited by safety concerns, and many additional hematologic malignancies do not respond to treatment with any of these targeted therapies. Various therapies based on alternate mAbs, including second-generation anti-CD20 mAbs and those targeting alternate cell-surface proteins such as CD19, CD22, CD30, CD37, CD40, and CD74, have been developed and are at different stages of clinical testing in the hopes of providing approaches to treating a broader spectrum of hematologic malignancies that are poorly served by existing therapies. 1,2 Whereas targeting of cell-surface antigens themselves can mediate antitumor activity through the induction of apoptosis, most mAb-based activity against hematologic malignancies is reliant on either Fc-mediated effector functions such as complementdependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity 3,4 or is engineered through the conjugation of an immunotoxin or radiolabeled isotope. 1 Considering the potential of naturally occurring CTLs to mediate cell lysis, various strategies have also been explored to recruit CTLs to mediate tumor cell killing. Tumor-specific CTLs exert extremely potent effects through recognition of the corresponding peptide/MHC complex recognized by their TCR, and are among the most potent cells that mediate antitumor effects. A major limitation in generating tumorspecific CTLs in vivo is that their induction requires the use of vaccine strategies, such as dendritic cell-based vaccines, 5 that are capable of breaking tolerance to cancer self-antigens. One alternative is ex vivo expansion and activation of rare, tumor-specific CTLs for reinfusion into cancer patients. 6 However, cancer cells can down-regulate MHC expression as an escape mechanism, thus preventing the ability of CTLs to recognize their antigenic peptide. The genetic manipulation of patients' T cells to express chimeric antigen receptors comprising a tumor-specific antigen and T cellactivating properties before their adoptive transfer provides a non-MHC-restricted approach to targeting cancer, as was shown recently in the treatment of lymphoma with T cells engineered to recognize CD19. 7 However, the patient-specific manipulation and risk associated with this procedure represent major limitations to its expanded use. Alt...

The three proteins that constitute anthrax toxin self-assemble into toxic complexes after one of these proteins, protective antigen (PA), binds to tumor endothelial marker 8 (TEM8) or capillary morphogenesis protein 2 (CMG2) cellular receptors. The toxin receptor complexes are internalized, and acidic endosomal pH triggers pore formation by PA and translocation of the catalytic subunits into the cytosol. In this study we show that the pH threshold for conversion of the PA prepore to the pore and for translocation differs by approximately a pH unit, depending on whether the TEM8 or CMG2 receptor is used. For TEM8-associated toxin, these events can occur at close to neutral pH values, and they show relatively low sensitivity to ammonium chloride treatment in cells. In contrast, with CMG2-associated toxin, these events require more acidic conditions and are highly sensitive to ammonium chloride. We show, furthermore, that PA dissociates from TEM8 and CMG2 upon pore formation. Our results are consistent with a model in which translocation depends on pore formation and pore formation, in turn, depends on release of PA from its receptor. We propose that because PA binds to CMG2 with much higher affinity than it does to TEM8, a lower pH is needed to attenuate CMG2 binding to allow pore formation. Our results suggest that toxin can form pores at different points in the endocytic pathway, depending on which receptor is used for entry.capillary morphogenesis protein 2 ͉ tumor endothelial marker 8 ͉ toxin entry B acillus anthracis, the causative agent of anthrax, secretes a toxin that is believed to be instrumental in causing anthrax disease symptoms leading to death. Anthrax toxin consists of three proteins, protective antigen (PA), which is a receptorbinding and pore-forming subunit; lethal factor (LF), which is a protease that cleaves mitogen-activated protein kinase kinase family members; and edema factor (EF), which is an adenylate cyclase that raises cAMP levels in cells (1). PA is synthesized as an 83-kDa protein (PA 83 ) for which two cell surface receptors have been identified: tumor endothelial marker 8 (TEM8) (2, 3) and capillary morphogenesis protein 2 (CMG2) (4). Two splice variant mRNAs derived from the TEM8 gene (sv1 and sv2) encode functional anthrax toxin receptors (2, 5). TEM8 expression has been documented in epithelium of the lung, intestine, and skin, the three routes of entry in anthrax infection (6). The CMG2 gene, which has been shown to be broadly expressed in different tissues (4), encodes three protein isoforms, two of which, CMG2 488 and CMG2 489 , are anthrax toxin receptors (4) (H.M.S., unpublished data).Receptor-bound PA 83 is cleaved by a cellular protease to generate a 20-kDa PA 20 subunit and a 63-kDa subunit (PA 63 ). The larger subunit assembles into the heptameric (PA 63 ) 7 prepore in lipid rafts (7-9). EF and LF bind to the prepore, and the toxin-receptor complexes are internalized by clathrindependent endocytosis and by other endocytic mechanisms (9, 10). These complexes are then exp...