The human PD mutant A L exhibiting increased affinity for

The human PD-1 mutant (A132L) exhibiting increased affinity for both PD-L1 (45-fold) and PD-L2 (30-fold) was selected for further studies and the generation of a soluble chimeric Ig fusion protein (HA PD-1 Ig). As expected, compared to wild type PD-1 Ig, the HA PD-1 Ig showed significantly enhanced binding to mature human dendritic Tasquinimod Supplier expressing PD-L1 and PD-L2. Although it is not feasible to define a quantitative Kd in this cell-based system, due to a number of factors (e.g. variations in PD-Ligand expression levels, bivalency of the Ig fusion proteins, and this is not an equilibrium experiment), appreciation of the binding responses (Fig. 3A–B) indicates that the HA PD-1 Ig binds with an apparent affinity at least two orders of magnitude greater than the wild type PD-1 Ig. These data suggest that the apparent cell-surface binding of the HA PD-1 Ig is in the low single digit nanomolar range (~2nM), similar to many antigen antibody complexes. The enhanced binding of HA PD-1 Ig was consistent with the results of an allogeneic in vitro T cell activation assay, in which the HA PD-1 Ig increased T cell proliferation by at least 50%, (Fig. 3C–D), and the production of the Th1 cytokines IFN-γ and IL-12p70 6-fold or more; IL-2, IL-10, TNF-α, IL-13 by 4–5 fold; and IL-5 and TNF-β 2–3 fold, compared to untreated control (no Ig). The increase in proliferation and cytokine secretion for HA PD-1 Ig was dose-dependent, starting at concentrations as low as 0.5μg/ml (Supplementary Fig. 7). Wild type PD-1 Ig did not induce significant T cell proliferation or cytokine production, except at concentrations in the 50μg/ml range, which is above the usual concentration for such in vitro assays. The effects of HA PD-1 Ig on proliferation and cytokine production suggests two possible mechanisms: the first involving competitive binding, which sterically blocks the endogenous cell surface resident ligands from engaging cell surface resident PD-1; and the second exploiting reverse signaling through PD-L1 and PD-L2 on APCs elicited by the soluble high-affinity PD-1. A recent study indicates that PD-L1 may signal in tumor cells, such as ovarian cancer and melanoma, through a cancer cell-intrinsic, non-immune mechanism (Clark et al., 2016). No such data are available for PD-L2, which has not been extensively investigated in lung tumor models. Relevant to the current study, 3LL cells express PD-L1 in vitro, but not PD-L2 (Supplementary Fig. 8). Flow cytometric analysis of tumor-associated (Supplementary Fig. 9A) and splenic DCs (not shown) did not show significant increase in surface activation markers after treatment, although there was a trend towards increased expression of PD-L1 (Supplementary Fig. 9B), likely induced by IFN-γ release following radiation therapy. Furthermore, the allogeneic MLR studies utilized irradiated DCs, indicating that the enhanced proliferation and cytokine production elicited by HA PD-1 Ig (Fig. 3.) was due to direct inhibitory effects on T cells and not through reverse signaling into the APCs. Importantly, the effects of the HA PD-1 Ig are comparable to that of a PD-L1 blocking monoclonal antibody used at the same concentration. Based on these observations, the in vitro behavior of our high affinity mutant PD-1 Ig suggests it possesses utility as a modulator of the PD-L/PD-1 pathway, with a potency that is in the range of functionally blocking antibodies. Recent clinical trials highlighted the induction of anti-tumor immunity against various solid tumors by blockade of T cell inhibition through PD-1 and PD-L1 (Brahmer et al., 2012). These treatments resulted in durable tumor response rates of 10–15%, the highest rate for any immunotherapy approach for the treatment of cancer in the last 30years (Ribas, 2012). Moreover, in addition to metastatic melanoma, promising results were obtained in patients with non-small cell lung cancer, which has been largely resistant to immunotherapy. Phase 3 clinical trials have recently shown unprecedented successes for the treatment of NSCLC using anti-PD-1 monotherapy compared to platinum-based chemotherapy (Killock, 2016). It is clear that not all patients respond to these treatments, and accumulating evidence indicates that tumors that are immunogenic may be more responsive to this form of immunotherapy. Hence, there exists a continued need to examine new immunomodulatory agents and explore new combination therapies that result in synergistic responses between immunotherapy and other modalities such as chemotherapy and radiotherapy (Shahabi et al., 2015).