Preclinical studies in rats and squirrel monkeys for safety evaluation of the bivalent anti-human T cell immunotoxin, A-dmDT390-bisFv(UCHT1).

The bivalent anti-human T cell immunotoxin A-dmDT390-bisFv(UCHT1) for treatment of patients with T cell malignancies is a single chain fusion protein composed of the catalytic domain and translocation domains of diphtheria toxin fused to two tandem sFv molecules reactive with human CD3 epsilon. This immunotoxin selectively kills CD3 epsilon positive T cells. To determine the maximum tolerated dose (MTD), pharmacokinetics and immunogenicity of A-dmDT390-bisFv(UCHT1), rat and squirrel monkey studies were performed. In both animal studies, animals received either 0, 2.5 (low), 25 (medium), or 56.25 microg/kg (high) of A-dmDT390-bisFv(UCHT1) intravenously twice daily for four consecutive days. Although transient elevation of liver transaminases in the high groups was observed, the A-dmDT390-bisFv(UCHT1) administration did not affect liver function, renal function, the hemogram, or produce serious organ histopathology. Adverse events included transient lethargy, inappetence and weight loss in high groups. A-dmDT390-bisFv(UCHT1) plasma half life was 26.95 min in rats and 18.33 min in squirrel monkeys. Immune responses to A-dmDT390-bisFv(UCHT1) were minimal in squirrel monkeys and mild in rats. In vitro cytokine release, T cell activation and CD3 epsilon receptor occupancy assays using human PBMC were further performed since rat and squirrel monkey T cells do not react with A-dmDT390-bisFv(UCHT1). A-dmDT390-bisFv(UCHT1) did not induce cytokine release or T cell activation. The A-dmDT390-bisFv(UCHT1) concentration for 50% CD3 epsilon receptor occupancy was 7.4 nM. The MTD of 200 microg/kg total provides a dose level sufficient for anti-tumor activity in vitro and in a rodent model. Therefore, we propose that this agent is a promising drug for patients with surface CD3+ T cell malignancies.

Anthrax toxin-induced shock in rats is associated with pulmonary edema and hemorrhage.

Bacillus anthracis infections are frequently associated with severe and often irreversible hypotensive shock despite appropriate antibiotics and aggressive hemodynamic and pulmonary support. Based on the observations that the anthrax secreted proteins-protective antigen (PA), lethal factor (LF), and edema factor (EF) also produce shock and mortality in animal models, we chose to characterize further the clinical chemistries and microscopic pathology of toxin treated rats. Groups of three male Sprague Dawley rats received bolus intravenous infusions of PA/LF, PA/EF, LF, or EF alone and blood samples and tissues were collected and assayed for chemistries and tissue pathology. In PA/LF and PA/EF treated animals but not other groups, chemistries showed transaminasemia and elevated lactate dehydrogenase. PA/LF treated animals alone showed elevated hemoglobin and hematocrits; PA/EF treated animals alone showed lymphopenia. Pathology was remarkable for pulmonary edema in PA/LF treated rat lungs and pulmonary hemorrhage in PA/EF treated rat lungs. These results are consistent with our and others' previous findings that the morbidity and mortality associated with anthrax are not cytokine-mediated but due to a direct effect of the toxins on the cardiovascular system along with toxin-specific alterations in blood counts. PA/LF pathology matches that seen with acute cardiac failure, and PA/EF pathology coincides with direct vascular endothelial injury. These observations provide a rational basis for drug interventions to reduce the effect of these toxins on the heart and blood vessels.