1 Long chain fatty acids have recently been identified as agonists for the G protein-coupled receptors GPR40 and GPR120. Here, we present the first description of GW9508, a small-molecule agonist of the fatty acid receptors GPR40 and GPR120. In addition, we also describe the pharmacology of GW1100, a selective GPR40 antagonist. These molecules were used to further investigate the role of GPR40 in glucose-stimulated insulin secretion in the MIN6 mouse pancreatic b-cell line. 2 GW9508 and linoleic acid both stimulated intracellular Ca 2 þ mobilization in human embryonic kidney (HEK)293 cells expressing GPR40 (pEC 50 values of 7.3270.03 and 5.6570.06, respectively) or GPR120 (pEC 50 values of 5.4670.09 and 5.8970.04, respectively), but not in the parent HEK-293 cell line. 3 GW1100 dose dependently inhibited GPR40-mediated Ca 2 þ elevations stimulated by GW9508 and linoleic acid (pIC 50 values of 5.9970.03 and 5.9970.06, respectively). GW1100 had no effect on the GPR120-mediated stimulation of intracellular Ca 2 þ release produced by either GW9508 or linoleic acid. 4 GW9508 dose dependently potentiated glucose-stimulated insulin secretion in MIN6 cells, but not in primary rat or mouse islets. Furthermore, GW9508 was able to potentiate the KCl-mediated increase in insulin secretion in MIN6 cells. The effects of GW9508 on insulin secretion were reversed by GW1100, while linoleic acid-stimulated insulin secretion was partially attenuated by GW1100. 5 These results add further evidence to a link between GPR40 and the ability of fatty acids to acutely potentiate insulin secretion and demonstrate that small-molecule GPR40 agonists are glucose-sensitive insulin secretagogues.

Programmed cell-death 1 ligand 1 (PD-L1) is a member of the B7/CD28 family of proteins that control T-cell activation. Many tumors can upregulate expression of PD-L1, inhibiting antitumor T-cell responses and avoiding immune surveillance and elimination. We have identified and characterized MEDI4736, a human IgG1 monoclonal antibody that binds with high affinity and specificity to PD-L1 and is uniquely engineered to prevent antibody-dependent cell-mediated cytotoxicity. In vitro assays demonstrate that MEDI4736 is a potent antagonist of PD-L1 function, blocking interaction with PD-1 and CD80 to overcome inhibition of primary human T-cell activation. In vivo MEDI4736 significantly inhibits the growth of human tumors in a novel xenograft model containing coimplanted human T cells. This activity is entirely dependent on the presence of transplanted T cells, supporting the immunological mechanism of action for MEDI4736. To further determine the utility of PD-L1 blockade, an anti-mouse PD-L1 antibody was investigated in immunocompetent mice. Here, anti-mouse PD-L1 significantly improved survival of mice implanted with CT26 colorectal cancer cells. The antitumor activity of anti-PD-L1 was enhanced by combination with oxaliplatin, which resulted in increased release of HMGB1 within CT26 tumors. Taken together, our results demonstrate that inhibition of PD-L1 function can have potent antitumor activity when used as monotherapy or in combination in preclinical models, and suggest it may be a promising therapeutic approach for the treatment of cancer. MEDI4736 is currently in several clinical trials both alone and in combination with other agents, including anti-CTLA-4, anti-PD-1, and inhibitors of IDO, MEK, BRAF, and EGFR.

Two series of CRF antagonists with N alpha- and C alpha-methylated alanine and leucines were evaluated for their biological activities in vitro and in vivo in several systems. The poly-N-methylated analogue of alpha-helical-CRF9-41, [N alpha MeLeu10,15,27,37,N alpha MeAla22,32,41]-alpha-Hel-CRF9-41, was found to be considerably less potent than the parent non-N-methylated analogue. This result was expected on the basis that alpha-helicity was thought to be required for biological activity and the prediction that backbone substitutions on the nitrogen have a tendency to break alpha-helices (a hypothesis that was confirmed by circular dichroism). Next, a series of constrained analogues of the potent CRF antagonist, [DPhe12,Nle21,38]h/rCRF12-41, was synthesized that contained C alpha-methylleucine and/or C alpha-methylalanine (Aib) residues at selected positions. Because C alpha-methylation is recognized to increase alpha-helicity, and because there is now strong NMR data suggesting that residues 6-36 assume a well-defined alpha-helix, it was expected that these analogues would be more potent. Although usual solid-phase peptide synthesis procedures were followed, success in coupling the C alpha-methyl amino acids was obtained only with a 1:1 mixture of BOP/HOBt. In vitro potencies of the synthesized compounds were measured in a collagenase-dispersed anterior pituitary cell culture bioassay. Monosubstituted analogues were shown to be twice to one fourth as potent as the parent compound; while the pluri-substituted peptides were slightly less potent. This decrease in potency might be correlated to an unexpected lower helical content of the pluri-substituted compounds (as determined by CD spectroscopy), as it was suggested that the bioactive conformation of the CRF was predominantly alpha-helical. Interestingly, one analogue, [DPhe12,Nle21,38,C alpha-MeLeu37]h/rCRF12-41, was found to be more potent and longer acting than the parent compound in two in vivo assays measuring ACTH release after intravenous administration to adrenalectomized rats and reversal of stress-induced delay in gastric emptying in the rat after intracisternal administration. The molecular basis for this increased duration of action and potency is being investigated.

To generate and characterize a murine GITR ligand fusion protein (mGITRL-FP) designed to maximize valency and the potential to agonize the GITR receptor for cancer immunotherapy. The EC value of the mGITRL-FP was compared with an anti-GITR antibody in an agonistic cell-based reporter assay. We assessed the impact of dose, schedule, and Fc isotype on antitumor activity and T-cell modulation in the CT26 tumor model. The activity of the mGITRL-FP was compared with an agonistic murine OX40L-FP targeting OX40, in CT26 and B16F10-Luc2 tumor models. Combination of the mGITRL-FP with antibodies targeting PD-L1, PD-1, or CTLA-4 was analyzed in mice bearing CT26 tumors. The mGITRL-FP had an almost 50-fold higher EC value compared with an anti-murine GITR antibody. Treatment of CT26 tumor-bearing mice with mGITRL-FP-mediated significant antitumor activity that was dependent on isotype, dose, and duration of exposure. The antitumor activity could be correlated with the increased proliferation of peripheral CD8 and CD4 T cells and a significant decrease in the frequency of intratumoral Tregs. The combination of mGITRL-FP with mOX40L-FP or checkpoint inhibitor antagonists enhanced antitumor immunity above that of monotherapy treatment. These results suggest that therapeutically targeting GITR represents a unique approach to cancer immunotherapy and suggests that a multimeric fusion protein may provide increased agonistic potential versus an antibody. In addition, these data provide, for the first time, early proof of concept for the potential combination of GITR targeting agents with OX40 agonists and PD-L1 antagonists. .