Small Molecule Agents Targeting PD-1 Checkpoint Pathway for Cancer Immunotherapy: Mechanisms of Action and Other Considerations for Their Advanced Development.

Pioneering success of antibodies targeting immune checkpoints such as programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) has changed the outlook of cancer therapy. Although these antibodies show impressive durable clinical activity, low response rates and immune-related adverse events are becoming increasingly evident in antibody-based approaches. For further strides in cancer immunotherapy, novel treatment strategies including combination therapies and alternate therapeutic modalities are highly warranted. Towards this discovery and development of small molecule, checkpoint inhibitors are actively being pursued, and the efforts have culminated in the ongoing clinical testing of orally bioavailable checkpoint inhibitors. This review focuses on the small molecule agents targeting PD-1 checkpoint pathway for cancer immunotherapy and highlights various chemotypes/scaffolds and their characterization including binding and functionality along with reported mechanism of action. The learnings from the ongoing small molecule clinical trials and crucial points to be considered for their clinical development are also discussed.

PD-1 derived CA-170 is an oral immune checkpoint inhibitor that exhibits preclinical anti-tumor efficacy.

Small molecule immune checkpoint inhibitors targeting PD-1 and other pathways may offer advantages including ease of dosing, ability to manage immune-related adverse events (irAEs) due to their shorter pharmacokinetic exposure and opportunity to target more than one pathway for improving efficacy. Here we describe the identification and characterization of CA-170, an amino acid inspired small molecule inhibitor of PD-L1 and VISTA derived from the interface of PD-1 and PD-L1. CA-170 exhibited potent rescue of proliferation and effector functions of T cells inhibited by PD-L1/L2 and VISTA with selectivity over other immune checkpoint proteins as well as a broad panel of receptors and enzymes. Observed blocking of PD-L1 signaling and binding to PD-L1 in the cellular context without preventing the assembly of PD-1:PD-L1 complex support the formation of a defective ternary complex as the mechanism of action of CA-170. Oral administration of CA-170 resulted in increased proliferation and activation of T cells in the tumor, and significant anti-tumor efficacy in a number of immunocompetent mouse tumor models either as a single agent or in combination with approved therapeutics. These results prompted the advancement of CA-170 to human clinical trials.

A Rationally Designed Peptide Antagonist of the PD-1 Signaling Pathway as an Immunomodulatory Agent for Cancer Therapy.

Pioneering success of antibodies targeting immune checkpoints such as PD-1 and CTLA4 has opened novel avenues for cancer immunotherapy. Along with impressive clinical activity, severe immune-related adverse events (irAE) due to the breaking of immune self-tolerance are becoming increasingly evident in antibody-based approaches. As a strategy to better manage severe adverse effects, we set out to discover an antagonist targeting PD-1 signaling pathway with a shorter pharmacokinetic profile. Herein, we describe a peptide antagonist NP-12 that displays equipotent antagonism toward PD-L1 and PD-L2 in rescue of lymphocyte proliferation and effector functions. In preclinical models of melanoma, colon cancer, and kidney cancers, NP-12 showed significant efficacy comparable with commercially available PD-1-targeting antibodies in inhibiting primary tumor growth and metastasis. Interestingly, antitumor activity of NP-12 in a preestablished CT26 model correlated well with pharmacodynamic effects as indicated by intratumoral recruitment of CD4 and CD8 T cells, and a reduction in PD-1+ T cells (both CD4 and CD8) in tumor and blood. In addition, NP-12 also showed additive antitumor activity in preestablished tumor models when combined with tumor vaccination or a chemotherapeutic agent such as cyclophosphamide known to induce "immunologic cell death." In summary, NP-12 is the first rationally designed peptide therapeutic targeting PD-1 signaling pathways exhibiting immune activation, excellent antitumor activity, and potential for better management of irAEs.

Small-Molecule Immune Checkpoint Inhibitors Targeting PD-1/PD-L1 and Other Emerging Checkpoint Pathways.

Advances in harnessing the immune system for cancer treatment have been spectacular in recent years as witnessed by the approval of a number of antibodies targeting the PD-1/PD-L1 immune checkpoint pathway spanning an expanding list of indications. However, it is well recognized that while these antibodies show impressive clinical activity, they suffer from shortcomings including the failure to show response in a majority of patients, their need to be administered by intravenous injection, and immune-related adverse events due to the breaking of immune self-tolerance. Small-molecule-based therapeutic approaches offer the potential to address the shortcomings of these antibody-based checkpoint inhibitors. In the first part of this review, we discuss the rationale for small-molecule-based checkpoint therapy followed by efforts on the discovery of small-molecule-based approaches targeting the PD-1/PD-L1 axis and other immune checkpoint pathways. In the latter part of the article, we describe small-molecule inhibitors simultaneously targeting two non-redundant checkpoint inhibitor pathways as an approach to improve the response rate. A brief review of the progress of an oral small-molecule checkpoint inhibitor currently in clinical development is presented at the end.

Anti-PD-L1 peptide improves survival in sepsis.

BACKGROUND: Sepsis remains a leading cause of death in most intensive care units. Many deaths in sepsis are due to nosocomial infections in patients who have entered the immunosuppressive phase of the disorder. One cause of immunosuppression in sepsis is T-cell exhaustion mediated by programmed cell death-1 (PD-1) interaction with its ligand (PD-L1). Studies demonstrated that blocking the interaction of PD-1 with PD-L1 with knockout mice or inhibitory antibodies reversed T-cell dysfunction and improved sepsis survival. This study assessed the efficacy of a novel short-acting peptide (compound 8) that inhibits PD-1:PD-L1 signaling in a clinically relevant second-hit fungal sepsis model. METHODS: Mice underwent cecal ligation and puncture to induce peritonitis. Three days later, mice received intravenous injection of Candida albicans. Forty-eight hours after Candida infection, mice were treated with compound 8 or inactive peptide. The effect of Candida infection on expression of coinhibitory molecules, PD-1, and PD-L1 were quantitated by flow cytometry on CD4+ cells, CD8+ cells, natural killer (NK) cells, and natural killer T-cells (NKT). The effect of compound 8 on survival was also examined. RESULTS: Four days after fungal infection, PD-1 and PD-L1 expressions were markedly increased on CD4+, NK, and NKT cells in septic versus sham-operated mice (%PD-1 on CD4+, 11.9% versus 2.8%; and %PD-L1 on NKT, 14.8% versus 0.5%). Compared with control, compound 8 caused a 2-fold increase in survival from 30% to 60%, P < 0.05. CONCLUSIONS: Compound 8 significantly improved survival in a clinically relevant immunosuppressive model of sepsis. These results support immunoadjuvant therapy targeting T-cell exhaustion in this lethal disease.

Peptide hormone isoforms: N-terminally branched PYY3-36 isoforms give improved lipid and fat-cell metabolism in diet-induced obese mice.

The prevalence of obesity is increasing with an alarming rate worldwide and there is a need for efficacious satiety drugs. PYY3-36 has been shown to play a role in hypothalamic appetite regulation and novel analogs targeting the Y2 receptor have potential as drugs for the treatment of obesity. We have designed a series of novel PYY3-36 isoforms, by first adding the dipeptide Ile-Lys N-terminal to the N(α) of Ser-13 in PYY13-36 and then anchoring the N-terminal segment, e.g. PYY3-12, to the new Lys N(ε)-amine. We hypothesized that such modifications would alter the folding of PYY, due to changes in the turn motif, which could change the binding mode to the Y receptor sub-types and possibly also alter metabolic stability. In structure-affinity/activity relationship experiments, one series of PYY isoforms displayed equipotency towards the Y receptors. However, an increased Y2 receptor potency for the second series of PYY isoforms resulted in enhanced Y receptor selectivity compared to PYY3-36. Additionally, acute as well as chronic mice studies showed body-weight-lowering effects for one of the PYY isoforms, which was also reflected in a reduction of circulating leptin levels. Interestingly, while the stability and pharmacokinetic profile of PYY3-36 and the N-terminally modified PYY3-36 analogue were identical, only mice treated with the branched analogue showed marked increases in adiponectin levels as well as reductions in non-esterified free fatty acids and triglycerides.

Peptide architecture: adding an alpha-helix to the PYY lysine side chain provides nanomolar binding and body-weight-lowering effects.

The gut hormone PYY3-36 influences food intake and body weight via interaction with hypothalamic presynaptic Y2 receptors (Y2R). Novel Y2R-selective analogues of PYY3-36 are therefore potential drug candidates for the treatment of obesity. It has been hypothesized that PYY3-36 and possibly also the related PP-fold peptides, NPY and PP, bind to the membrane via their amphipathic alpha-helix prior to receptor interaction. The PYY3-36 amphipathic alpha-helix causes the peptide to associate with the membrane, making it essential for Y receptor potency as it potentially guides the C-terminal pentapeptide into the correct conformation for receptor activation. Based on this hypothesis, the importance of the amphipathic nature of PYY3-36, as well as the ability of amphipathic alpha-helices to interact in solution to form di- and tetramers, we redesigned the peptide architecture by addition of an amphipathic alpha-helix via the Lys 4 side chain of PYY3-36. Two different amphipathic sequences were introduced; first, PYY17-31, the native alpha-helix of PYY, and secondly, its retro counterpart, PYY31-17, which is also predicted to form an alpha-helix. Moreover, several different turn motifs between the branching point and the additional alpha-helix were tested. Several novel peptides with nanomolar Y2R binding affinities, as well as increased Y receptor selectivity, were identified. CD experiments showed the modifications to be well accepted, and an increase in mean ellipticity (ME) signifying an increased degree of alpha-helicity was observed. Receptor binding experiments indicated that the direction of the additional alpha-helix is less important, in contrast to the turn motifs, which greatly affect the Y1R binding and thus determine the Y1R activity. Conversely, the structure-activity relationships from in vivo data showed that the peptide containing the retro-sequence was inactive, even though the binding data demonstrated high affinity and selectivity. This demonstrates that radical redesign of peptide architecture can provide nanomolar binding with improved subtype selectivity and with in vivo efficacy.