Previous studies have shown that a single course of FcR non-binding anti-CD3 mAbs can attenuate the loss of insulin production. In this study, we did not find a statistically significant improvement in C-peptide responses and the responses after 24 months were not different. This result is most likely due to the small number of subjects, in this trial that created an imbalance of baseline C-peptide values (e.g. higher values in the drug treated group). In addition, the repeated dosing of the drug, as was originally intended, was not given. Nonetheless, data from the earlier time points suggest a trend for improved C-peptide responses and the ad hoc analysis using partially matched controls is consistent with the previous findings. Moreover, when subjects were followed for a longer period of time, we found persistent C-peptide responses. Insulin use was significantly reduced in the primary and ad hoc analysis. In addition, we found a significant inverse correlation between C-peptide responses and insulin requirements suggesting that insulin use may be a useful parameter for evaluating residual C-peptide responses. The baseline C-peptide AUC did predict the C-peptide AUC in follow-up but not predict response to drug therapy in this study or in our earlier trial [2
]. However, the sample size may be too small to detect a relationship between the baseline response and the effects of treatment.
Our experience herein suggests that AEs with modified anti-CD3 mAbs may be dose-related. Compared to our previous study, we found higher coating and modulation of CD3 on peripheral T cells, and higher trough drug levels, and the AEs were more frequent, of higher grade, and occurred at an earlier time point in this treatment course. Despite the increased rate and severity of AEs, there was no evidence for improved efficacy based on C-peptide responses. Using our prior criteria for a clinical response (i.e. a < 15% decrease in C-peptide response), the response rate at 1 year was not improved with the higher dose of drug (3/6) compared to responses with a lower dose (15/21). Even the improvement in C-peptide responses in the first 6 months after drug treatment was not greater. However, a larger number of subjects are needed to definitively establish the relationship between drug dose and clinical efficacy as well as the long term preservation of C-peptide responses, a possibility raised by the plateau of C-peptide AUC during longer term follow up as shown in .
The mechanism(s) of action of FcR non-binding anti-CD3 mAb remains unknown. Although initial studies with OKT3 and even FcR non-binding anti-CD3 mAbs suggested that anti-CD3 mAbs deplete effector T cells, other preclinical studies and human data were not consistent with this mechanism [15
]. In murine studies with thymectomized mice Hirsch et al suggested that depletion was not the mechanism by which the mAb induced immunosuppression, and that TCR modulation was associated with disappearance of T cells from the circulation [7
]. Indeed, in our clinical study, the appearance of lymphocytes in the periphery coincided with reduced levels of coating and modulation of the TCR. The coating of CD3 fell from a peak of 76% and 75% on CD4 and CD8+ T cells to 13% and 31% by day 30, when T cells reappeared in the circulation. The levels of TRECs, a reflection of new thymic emigrants, in the circulating cells were constant after mAb treatment at the time when the cell counts recovered. Moreover, in a previous study, we did not find an increase in the proportion of CD45RA+ cells after recovery of circulating T cells (60.5±8.8%) compared to before treatment with anti-CD3 mAb (79.1±4.5%, n=7). However, the interpretation of the TREC analysis has certain limitations. For example, homeostatic proliferation of new thymic emigrants possibly after deletion of lymphocytes could have resulted in a rapid decline in TREC levels even though thymic output had increased [10
]. Nonetheless, the kinetics of return of circulating lymphocytes was too rapid (2 weeks) to be explained by thymic output, because it has been shown that the absolute production rate of newly divided CD4+ T cells is 10.4±6.5 cells/μl/d and of CD8+ cells 5.9±7.6 cells/μl/d[18
]. Depending on the extent of depletion in the secondary lymphoid organs and sites, it would take several months to recover peripheral lymphocytes if new thymic cells were the primary source of new cells. These findings are consistent with the margination or trafficking of T cells after treatment with teplizumab. However, it is also possible that both mechanisms (i.e. recovery from marginated cells and homeostatic proliferation of new thymic emigrants) are operative.
Preclinical studies suggested that the non-FcR binding anti-CD3 mAb may induce regulatory T cells[5
]. We had previously reported that there was an increased proportion of CD8+ T cells following treatment with anti-CD3 mAb and increased expression of Foxp3 in CD8+ T cells. We found, in this trial, a relatively greater proportion of CD8+ T cells during drug administration but did not find a difference in recovery of cells at day 90 unlike our previous studies[19
]. Further studies will be needed to evaluate the functional changes in the CD8+ subpopulation after mAb treatment.
The duration of the effects on C-peptide responses with mAb treatment is of primary importance for any immune therapy of T1DM. In an extended follow-up of the drug-treated subjects we found detectable insulin production in drug-treated subjects followed for up to 60 months with an increase in insulin production over time in 3/4 subjects followed beyond month 24. In addition, the insulin use 3 ½ yrs after diagnosis was, on average, less than 0.5 U/kg which has been used by some investigators to characterize the “honeymoon” period[20
]. Because our sample size is small, we cannot determine whether the persistence of C-peptide responses was significantly different from what might occur in non-treated subjects with T1DM of similar duration. Of note, in the DCCT, only 3% and 8% of subjects < 18 yrs and ≥ 18 yrs respectively had detectable C-peptide levels after 5 years of T1DM: whereas in a more recent analysis, >85% of subjects with T1DM duration up to 4 years had detectable C-peptide. However these studies were cross-sectional and did not evaluate changes in insulin production over time. [21
In summary, this study supports previous investigations showing that treatment of patients with new onset T1DM with a brief course of teplizumab results in a reduced rate of decline in insulin production and a decrease in insulin requirements for more than 2 years after diagnosis. The rapid return of circulating T cells and the absence of increased levels of TRECs suggests that the drug affects lymphocyte trafficking rather than causes depletion. A higher dose of drug, approximately 40% higher than the dose used previously, resulted in increased AEs. However, the relationship between dose of anti-CD3 mAb the duration of response.merits further investigation.