One of the important strategies to understand the pharmacology of a new drug candidate is the use of biomarkers that can provide pharmacodynamic information from clinical samples regarding the interaction of the drug with its target. The effects of ATR-107, an antibody against the IL-21R, in suppressing IL-21 effects on immune cells were analyzed by measuring the effects of this antibody on the activation of STAT3. Other γc cytokines, such as IL-2, IL-7, and IL-15, predominantly phosphorylate STAT5 upon stimulation, while IL-21 predominantly phosphorylates STAT3. Additionally, STAT3 has a prolonged activation over other STATs and plays a critical role in the IL-21 signaling . pSTAT3 measurements resulted in a very good dynamic range for flow cytometry analysis allowing the generation of excellent dose response curves and a good IC50 determinaion. Thus, phospho-STAT3 was selected as a biomarker for IL-21. Using Western blot, we clearly demonstrated the phosphorylation of STAT3 by IL-21 in T cells. The specificity of the pSTAT3 signal and, the inhibitory effects of ATR-107, were demonstrated by the concentration-dependent inhibition of pSTAT3 using Western Blot. To further our analysis into specific cell types, flow cytometry was used. Phospho-specific flow cytometry measures the phosphorylation state of the interested kinase(s) at a single cell level in a heterogeneous cell population . Since the specific cell type can be identified by the simultaneous surface staining, there is no need to isolate the cells of interest. Another advantage is that the cells are kept in the whole blood environment during stimulation enabling the measurements in a more physiological condition.
Using flow cytometry analysis, pSTAT3 was detected in CD4+ T and CD19+ B cells. STAT3 signal transduction pathway is well known. After activation by phosphorylation, pSTAT3 translocates to the nucleus and transcription is initiated . Indeed, when IL-21 was added to whole blood, a concentration- and time-dependent increase in pSTAT3 signal was observed that coincided with the movement of the transcription factor to the cell nucleus. This was clearly visualized using ImageStream in a single cell. In contrast, in the presence of ATR-107, no pSTAT3 signal was observed in the nucleus by looking at the cells using ImageStream, agreeing with the lack of nuclear translocation.
Interestingly, ATR-107 inhibited the signal and the mechanism of inhibition was shown to be non-competitive. This mechanism of inhibition was demonstrated in both T and B cells. The data suggest that ATR-107 binding to the IL-21R is initially competitive with IL-21. However, once the antibody binds to the receptor, IL-21 cannot displace it from the receptor, thus, ATR-107 behaves like a functional non-competitive agent. This phenomenon is likely due to the long koff (2.91 x 104/s, t1/2: 39 min) of this antibody  which generates slow-reversible binding, an inhibitory feature of ATR-107. The non-competitive nature of inhibition demonstrated by ATR-107 may have important pharmacological consequences, since this mechanism could produce pharmacodynamic effects much longer that what is expected based on the pharmacokinetic characteristic of the molecule. Indeed, initial clinical data is suggestive of this effect where full receptor occupancy was observed for at least 42 days after a single administration of ATR-107 , an effect that was also demonstrated using pSTAT3 as a pharmacodynamic biomarker. These results in humans confirmed the previous observation of a 5–13 week pharmacodynamic efficacy observed in non-human primates using the IL-2R transcript as a PD biomarker .
Another important piece of information obtained from this study is that it is feasible to consider that the antibody binds the receptor initially in the cells that are circulating in the blood compartment and these cells may either not be able to infiltrate to the inflamed site or if they do, they may help transport the antibody to the inflamed tissues increasing its concentration at the site where it is needed. These possibilities will need to be demonstrated in further studies. Recent studies (Palandra et al. submitted Analytical Chemistry 2013) using a very specific mass spectrometric method to detect and quantify IL-21 demonstrated that IL-21 is not present in the circulation and rather is detected in inflamed tissues. This observation suggests that the antibody needs to inhibit the IL-21R at the inflamed site to be effective.
In summary, the results reported have two clinical applications. First, the functional pSTAT3 assay correlates well with the IL-21 receptor occupancy, suggesting these two assays could be used to support each other in assessing the pharmacology of ATR-107 in patients. Second, the pSTAT3 assay helped to discover the non-competitive feature of ATR-107, which provides important new information to predict ATR-107 effective dose selection. The data shows ATR-107 can completely block IL-21 induced STAT3 phosphorylation, regardless of the IL-21 concentration. Thus, for therapeutic purposes, the amount of IL-21R present on the cells at the inflamed site, but not the IL-21 levels is key to predicting the ATR-107 dose.
An important caveat from our studies is that all the assays reported here were done using blood from healthy donors. Levels of IL-21 and IL-21R could be quite different in patients with autoimmune diseases. For example, in lupus, the IL-21 producing T cells have been shown to be increased [21, 22], while the IL-21R expression on B cells appears to be decreased  or unchanged . Also, there may be changes in the sensitivity of the receptor activation as has been suggested for IL-21 in B cells in systemic lupus . Thus, it is conceivable that the responses of immune cells to IL-21 and to ATR-107 blockade could be different in patients than in healthy controls. Thus, to accurately choose the therapeutic doses, the assay will need to be validated using blood from patients with the specific disease being targeted.