George L. Spitalny

A High-Affinity Monoclonal Antibody to Anthrax Protective Antigen Passively Protects Rabbits before and after Aerosolized Bacillus anthracis Spore Challenge

ABSTRACT We have developed a therapeutic for the treatment of anthrax using an affinity-enhanced monoclonal antibody (ETI-204) to protective antigen (PA), which is the central cell-binding component of the anthrax exotoxins. ETI-204 administered preexposure by a single intravenous injection of a dose of between 2.5 and 10 mg per animal significantly protected rabbits from a lethal aerosolized anthrax spore challenge (∼60 to 450 times the 50% lethal dose of Bacillus anthracis Ames). Against a similar challenge, ETI-204 administered intramuscularly at a 20-mg dose per animal completely protected rabbits from death (100% survival). In the postexposure setting, intravenous administration of ETI-204 provided protection 24 h (8 of 10) and 36 h (5 of 10) after spore challenge. Administration at 48 h postchallenge, when 3 of 10 animals had already succumbed to anthrax infection, resulted in the survival of 3 of 7 animals (43%) for the duration of the study (28 days). Importantly, surviving ETI-204-treated animals were free of bacteremia by day 10 and remained so until the end of the studies. Only 11 of 51 ETI-204-treated rabbits had positive lung cultures at the end of the studies. Also, rabbits that were protected from inhalational anthrax by administration of ETI-204 developed significant titers of PA-specific antibodies. Presently, the sole therapeutic regimen available to treat infection by inhalation of B. anthracis spores is a 60-day course of antibiotics that is effective only if administered prior to or shortly after exposure. Based upon results reported here, ETI-204 is an effective therapy for prevention and treatment of inhalational anthrax.

A sensitive immunochemical assay for biologically active MuIFN-γ

An immunochemical assay for the detection of mouse gamma interferon (MuIFN-gamma) has been established. Using a purified monoclonal antibody (MAb) that neutralizes the antiviral activity of MuIFN-gamma, and a polyclonal antibody (PAb) prepared against affinity-purified MuIFN-gamma, we have developed a double-sandwich enzyme-linked immunosorbent assay (ELISA). The assay is specific for both natural and recombinant MuIFN-gamma and displays only background activity against MuIFN-alpha + beta, recombinant TNF (rTNF), human gamma interferon (HuIFN-gamma) and crude rat spleen cell supernatants. The assay is more sensitive and reproducible than the measurement of hydrogen peroxide (H2O2) release by macrophages, and is capable of detecting both crude and purified MuIFN-gamma down to 0.03 U/ml of antiviral activity, making this assay 10-100 times more sensitive than the conventional antiviral assay. The ELISA detects only biologically active MuIFN-gamma since treatment of the MuIFN-gamma at high temperature or low pH conditions resulted in abolishment of biological activity, as determined by inhibition of cytopathic effect, coincident with a dramatic decrease in ELISA titer.

CD5-mediated specific delivery of DNA to T lymphocytes: compartmentalization augmented by adenovirus.

Specific DNA delivery has been achieved via interactions between an asialoorosomucoid-polylysine conjugate and the asialoglycoprotein receptor. We have now extended this technology to another cell type. In order to achieve DNA delivery uniquely to T cells, we have employed an antibody-polylysine conjugate which binds and is internalized via CD5. Binding analyses of the T101 monoclonal antibody to Jurkat cells and freshly isolated human peripheral T lymphocytes were performed and Scatchard plots revealed Kd values of 1.4 and 1.2 pM, respectively. To introduce DNA into the T cell, a complex of T101-polylysine and the luciferase plasmid was formed (T101-PL-DNA). 125I-labeled antibody alone or T101-PL-DNA complexes were both shown to internalize. Subcellular fractionation indicated that the complex remained in the endosomal compartment of the cell for up to 90 min. However, with the addition of adenovirus particles, there was a decrease of labeled complex in the endosomal fraction over time suggesting it was no longer tethered to the endosome vesicle. In vitro transfections confirmed this result showing the addition of adenovirus particles during incubation resulted in increased expression of the luciferase protein. Without adenovirus, there was limited expression of the transduced gene. These data revealed that T101 can deliver DNA via an antibody-PL conjugate. The addition of adenovirus allowed the DNA to escape the endosome enabling expression of the reporter gene.

Inhibition of macrophage-induced antigen-specific T-cell proliferation by interferon-γ

The effects of IFN-gamma on macrophage (M phi)-mediated antigen-specific T-cell proliferation was investigated. A well-defined assay system using purified resident populations of antigen-pulsed peritoneal M phi and immune T cells was used to measure M phi-induced antigen-specific T-cell proliferation. Antibody affinity purified or recombinant IFN-gamma inhibited M phi-induced T-cell proliferation when KLH-pulsed M phi from mice given IFN-gamma prior to KLH were cultured with KLH immune T cells from normal mice. Monoclonal rat anti-IFN-gamma antibody neutralized the inhibitory effect of IFN-gamma. This inhibition of T-cell proliferation occurred despite the fact that these M phi appeared to be activated by IFN-gamma treatment as measured by increased tumoricidal activity. The mechanism for the inhibition was unrelated to class II (Ia) expression, IL-1 secretion, and prostaglandin secretion. These results demonstrate the complex and sensitive role IFN-gamma has in regulating the immune response.

A DeImmunized chimeric anti-C3b/iC3b monoclonal antibody enhances rituximab-mediated killing in NHL and CLL cells via complement activation.

Complement-dependent cytotoxicity (CDC) is a key mechanism of Rituximab (RTX) action in killing non-Hodgkin’s lymphoma (NHL) cells both in vitro and probably in vivo. A DeImmunized, mouse/human chimeric monoclonal antibody (Mab), H17, specific for cell-associated complement C3 cleavage products, C3b and iC3b, was generated to enhance RTX-mediated killing of target cells by CDC. When NHL cell lines were treated with RTX and H17 in the presence of complement for 1xa0h, there was 40–70% more cell death than that observed with RTX alone. The enhancing effect of H17 was also seen over longer treatment periods. H17 was tested ex vivo against primary cells from NHL and chronic lymphocytic leukemia (CLL) patients. In RTX-resistant NHL samples, H17 enhanced RTX-mediated killing; in the remaining samples RTX + complement alone promoted more than 80% killing, and no significant enhancement was observed. The H17 antibody also increased RTX-mediated killing in four out of nine CLL samples. H17 may have therapeutic applications in NHL and CLL treatment as an adjunctive therapy to RTX. It might also enhance the activity of other therapeutic antibodies that work through CDC.

In Vivo Gene Therapy via Receptor-Mediated DNA Delivery

Human gene therapy has advanced during the past ten years from a theoretical concept to a rapidly emerging technology. Tremendous technological strides in recombinant DNA methodologies have fostered molecular studies in such fields as regulation of gene expression, human genetics and disease states, and gene transfer techniques. The confluence of these fields has led to the emergence of gene therapy as a reality, albeit limited at present to a small number of clinical studies (Anderson, 1992; Miller, 1992). As practiced today most gene therapy protocols lie within the realm of high-cost, technology-intensive, individualized treatments, akin to such procedures as organ and bone marrow transplantation (Mulligan, 1991). This type of procedure, though beneficial, is limited in usefulness since it is not readily accessible to much of the population. Even for those to whom this type of gene therapy strategy would be available, the current inability to achieve permanent transgene expression necessitates periodic retreatments, making these procedures highly cost-ineffective over the long term.

In vitro expression of secondary antitumor immunity by in vivo tumor-sensitized T cells: Inhibition by tumor-induced suppressor T cells

SummaryIn vitro cultivation of memory immune cells from P815- or P388-immune mice with corresponding irradiated tumor cells induced generation of cytolytic T cells (CTL). The induction of CTL generation, as well as the cytolytic activity itself, was tumor-specific. The in vitro generation of CTL from P815- or P388-immune cells was suppressed by spleen cells from mice bearing corresponding progressive tumors (tumor size 15 mm). The tumor-induced suppressor cells suppressed the in vitro generation of CTL, but did not affect their cytolytic function. The suppression was tumor-specific and was mediated by Ly1+2−L3T4+ T cells. Treatment of suppressor cell donors with cyclophosphamide or sublethal γ-radiation completely abolished the ability of their spleen cells to inhibit the in vitro CTL generation.

IFN-γ is not an essential mediator of murine antibody responses in vitro

Abstract We have used purified murine gamma-interferon (Mu IFN-γ) and anti-Mu IFN-γ monoclonal antibody to study the participation of gamma-interferon (IFN-γ) in in vitro plaque-forming cell (PFC) responses to antigen. We have studied several supernatants with T-cell replacing factor (TRF) activity as well as helper T-cell dependent anti-sheep red blood cells (SRBC) PFC responses. Our findings demonstrate that neither TRF-nor T-cell mediated responses are critically dependent upon IFN-γ.