Four miniature swine, aged two months, were brought to our AAALAC accredited animal facility and were allowed to acclimate to their new surroundings for one week. Three days prior to the start of treatment they were bled for baseline blood values: complete blood counts, serum chemistry values and for flow cytometry analysis of CD3+ T cell populations. They were then injected through a 23g butterfly catheter, through a peripheral vessel, with an intravenous (IV) bolus of undiluted A-dmDT390biscFv (2-6-15) at a dose of 50 μg/kg, twice daily for 4 days. Prior to each day’s injections, morning blood samples were taken to monitor the aforementioned blood values. Each day’s values were then compared to the pre-treatment values. The animals were also injected with 4mg/kg diphenhydramine IV bolus immediately prior to immunotoxin infusion to prevent any unexpected anaphylactic reactions, of which none were observed. To ensure the full volume of immunotoxin was administered to the animals, each dose was flushed with 10 mL of phosphate buffered saline (pH of between 6.5 and 7.5). The animals were closely monitored for signs of change in clinical condition such as lethargy and significant weight loss and no signs of toxicity were seen in any animal. The animals were bled again on days 7 and 14 following the start of injections to monitor their T cell recovery and any possible toxicity.
The percentage of CD3+ T cells in the peripheral blood was determined by flow cytometry of heparinized whole blood samples after staining with FITC conjugated swine specific CD3 antibody (898H2-6-15, mouse IgGaK)13 using BD FACS lysing solution whole blood staining procedure according to the manufacturer (BD Biosciences, San Jose, Ca). The absolute T-cell count each day was calculated by multiplying the percentage of CD3+ cells in the peripheral blood by the white blood cell count. All experiments were approved by the Massachusetts General Hospital Institutional Animal Care and Use Committee.