Defining New Therapeutics Using a More Immunocompetent Mouse Model of Antibody-Enhanced Dengue Virus Infection.

UNLABELLED: With over 3.5 billion people at risk and approximately 390 million human infections per year, dengue virus (DENV) disease strains health care resources worldwide. Previously, we and others established models for DENV pathogenesis in mice that completely lack subunits of the receptors (Ifnar and Ifngr) for type I and type II interferon (IFN) signaling; however, the utility of these models is limited by the pleotropic effect of these cytokines on innate and adaptive immune system development and function. Here, we demonstrate that the specific deletion of Ifnar expression on subsets of murine myeloid cells (LysM Cre(+) Ifnar(flox/flox) [denoted as Ifnar(f/f) herein]) resulted in enhanced DENV replication in vivo. The administration of subneutralizing amounts of cross-reactive anti-DENV monoclonal antibodies to LysM Cre(+) Ifnar(f/f) mice prior to infection with DENV serotype 2 or 3 resulted in antibody-dependent enhancement (ADE) of infection with many of the characteristics associated with severe DENV disease in humans, including plasma leakage, hypercytokinemia, liver injury, hemoconcentration, and thrombocytopenia. Notably, the pathogenesis of severe DENV-2 or DENV-3 infection in LysM Cre(+) Ifnar(f/f) mice was blocked by pre- or postexposure administration of a bispecific dual-affinity retargeting molecule (DART) or an optimized RIG-I receptor agonist that stimulates innate immune responses. Our findings establish a more immunocompetent animal model of ADE of infection with multiple DENV serotypes in which disease is inhibited by treatment with broad-spectrum antibody derivatives or innate immune stimulatory agents. IMPORTANCE: Although dengue virus (DENV) infects hundreds of millions of people annually and results in morbidity and mortality on a global scale, there are no approved antiviral treatments or vaccines. Part of the difficulty in evaluating therapeutic candidates is the lack of small animal models that are permissive to DENV and recapitulate the clinical features of severe human disease. Using animals lacking the type I interferon receptor only on myeloid cell subsets, we developed a more immunocompetent mouse model of severe DENV infection with characteristics of the human disease, including vascular leakage, hemoconcentration, thrombocytopenia, and liver injury. Using this model, we demonstrate that pathogenesis by two different DENV serotypes is inhibited by therapeutic administration of a genetically modified antibody or a RIG-I receptor agonist that stimulates innate immunity.

Nonclinical evaluation of GMA161--an antihuman CD16 (FcγRIII) monoclonal antibody for treatment of autoimmune disorders in CD16 transgenic mice.

Fc receptors are a critical component of the innate immune system responsible for the recognition of cross-linked antibodies and the subsequent clearance of pathogens. However, in autoimmune diseases, these receptors play a role in the deleterious action of self-directed antibodies and as such are candidate targets for treatment. GMA161 is an aglycosyl, humanized version of the murine antibody 3G8 that targets the human low-affinity Fcγ receptor III (CD16). As CD16 expression and sequence have high species specificity, preclinical assessments were conducted in mice transgenic for both isoforms of human CD16, CD16A, and CD16B. This transgenic mouse model was useful in transitioning into phase I clinical trials, as it generated positive efficacy data in a relevant disease model and an acceptable single-dose safety profile. However, when GMA161 or its murine parent 3G8 were dosed repeatedly in transgenic mice having both human CD16 isoforms, severe reactions were observed that were not associated with significant cytokine release, nor were they alleviated by antihistamine administration. Prophylactic dosing with an inhibitor of platelet-activating factor (PAF), however, completely eliminated all signs of hypersensitivity. These findings suggest that (1) GMA161 elicits a reaction that is target dependent, (2) immunogenicity and similar adverse reactions were observed with a murine version of the antibody, and (3) the reaction is driven by the atypical hypersensitivity pathway mediated by PAF.

Therapeutic control of B cell activation via recruitment of Fcgamma receptor IIb (CD32B) inhibitory function with a novel bispecific antibody scaffold.

OBJECTIVE: To exploit the physiologic Fcgamma receptor IIb (CD32B) inhibitory coupling mechanism to control B cell activation by constructing a novel bispecific diabody scaffold, termed a dual-affinity retargeting (DART) molecule, for therapeutic applications. METHODS: DART molecules were constructed by pairing an Fv region from a monoclonal antibody (mAb) directed against CD32B with an Fv region from a mAb directed against CD79B, the beta-chain of the invariant signal-transducing dimer of the B cell receptor complex. DART molecules were characterized physicochemically and for their ability to simultaneously bind the target receptors in vitro and in intact cells. The ability of the DART molecules to negatively control B cell activation was determined by calcium mobilization, by tyrosine phosphorylation of signaling molecules, and by proliferation and Ig secretion assays. A DART molecule specific for the mouse ortholog of CD32B and CD79B was also constructed and tested for its ability to inhibit B cell proliferation in vitro and to control disease severity in a collagen-induced arthritis (CIA) model. RESULTS: DART molecules were able to specifically bind and coligate their target molecules on the surface of B cells and demonstrated a preferential simultaneous binding to both receptors on the same cell. DART molecules triggered the CD32B-mediated inhibitory signaling pathway in activated B cells, which translated into inhibition of B cell proliferation and Ig secretion. A DART molecule directed against the mouse orthologs was effective in inhibiting the development of CIA in DBA/1 mice. CONCLUSION: This innovative bispecific antibody scaffold that simultaneously engages activating and inhibitory receptors enables novel therapeutic approaches for the treatment of rheumatoid arthritis and potentially other autoimmune and inflammatory diseases in humans.

Defining limits of treatment with humanized neutralizing monoclonal antibody for West Nile virus neurological infection in a hamster model.

A potent anti-West Nile virus (anti-WNV)-neutralizing humanized monoclonal antibody, hE16, was previously shown to improve the survival of WNV-infected hamsters when it was administered intraperitoneally (i.p.), even after the virus had infected neurons in the brain. In this study, we evaluated the therapeutic limit of hE16 for the treatment of WNV infection in hamsters by comparing single-dose peripheral (i.p.) therapy with direct administration into the pons through a convection-enhanced delivery (CED) system. At day 5 after infection, treatments with hE16 by the peripheral and the CED routes were equally effective at reducing morbidity and mortality. In contrast, at day 6 only the treatment by the CED route protected the hamsters from lethal infection. These experiments suggest that hE16 can directly control WNV infection in the central nervous system. In support of this, hE16 administered i.p. was detected in a time-dependent manner in the serum, cerebrospinal fluid (CSF), cerebral cortex, brain stem, and spinal cord in CSF. A linear relationship between the hE16 dose and the concentration in serum was observed, and maximal therapeutic activity occurred at doses of 0.32 mg/kg of body weight or higher, which produced serum hE16 concentrations of 1.3 microg/ml or higher. Overall, these data suggest that in hamsters hE16 can ameliorate neurological disease after significant viral replication has occurred, although there is a time window that limits therapeutic efficacy.