1b). The 5 and 25 μg (4×/72 hr) dose regimens
produced ‘saw-tooth’ patterns, where expression of the CD3–TCR complex was quickly down-regulated after each dose but returned to near predose values before the subsequent dose. With each successive dose, the level of modulation of the CD3–TCR complex increased. In the 2 and 1 μg (4×/72 hr) dose regimens, the extent of modulation was considerably less than in other dose regimens and was clearly discernable only after the fourth dose (Fig. 1b). After the fourth dose, the difference in the percentage of modulation of the CD3–TCR complex between the 2 and 1 μg Metformin (4×/72 hr) dose regimens was significant (30·3% versus 19·7% modulation, P < 0·01). Furthermore, in all dose regimens, there was a transient decrease in lymphocyte numbers in the peripheral blood during and shortly after dosing (Fig. 2), consistent with what has been observed in Proteasome inhibitor the spleens of both NOD and non-autoimmune mice administered monoclonal anti-CD3 F(ab′)2.9,10,19 This observation of lymphopenia during dosing could be the result of either depletion of a subset of lymphocytes or retrafficking of monoclonal anti-CD3 F(ab′)2-bound lymphocytes from the peripheral blood. In Study B, the effectiveness of the various dose regimens in inducing remission of diabetes was investigated in new-onset diabetic NOD mice. In order to evaluate whether a shorter duration of modulation of
the CD3–TCR complex or a lower cumulative dose affects efficacy, Study B also included groups given only three doses. Animals were randomly enrolled into one of five monoclonal anti-CD3 F(ab′)2 dose regimens – 50 μg (5×/24 hr), 25 μg (4×/72 hr), 25 μg (3×/72 hr), 5 μg (4×/72 hr), or 5 μg (3×/72 hr) – or placebo. The 25 and 5 μg doses were chosen based
on the results of Study A, in which expression of the CD3/TCR complex, 24 hr after dose 4, was approximately 12% and 50% of baseline, respectively. No animals in the placebo group entered Amino acid remission during the 12-week observation of blood glucose levels. In all dose regimens, approximately half of the mice (44–60%) had long-term remission (Table 1). There was no statistically significant difference in remission rates between the various dose regimens. The well-established 50 μg (5×/24 hr) dose regimen resulted in 56% of the mice being in remission for 12 weeks, which is similar to the originally published 67% remission rate.10 There was no apparent relationship between the dose and the rate of remission. As in previous studies,10 the majority of mice in all dose regimens that entered remission did so 1–2 weeks after treatment and all remained in remission for the 12 weeks of follow-up. Study B demonstrated that a total dose as low as 15 μg resulted in long-term remission of diabetes in NOD mice. In Study C, lower doses were examined to determine the minimum effective dose with the 72 hr dose regimen.