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Supplementary Figure 4 NP-12 increased the levels of IFN-γ in the Cytomegalovirus (CMV) antigen recall assay

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.

Table 1A: Rescue of proliferation and IFN- γ release in the presence of PD-L1 or PD-L2 in mouse splenocytes Table 1B: Rescue of proliferation and IFN- γ release in the presence of PD-L1 or PD-L2 in human PBMCs

Supplementary Figure 5 Pharmacokinetic profile of NP-12

Supplementary Figure 3 NP-12 increased the levels of IFN-γ in the Tetanus toxoid recall assay

Supplementary methods

Supplementary Figure 1 NP-12 labeled with FITC shows higher binding to CHOK1 cells overexpressing PD-L1 as compared to WT CHOK1 cells

We studied the nucleation and growth of TiO2 nanoflowers and carbon nanotubes-TiO2 nanocomposite (n-CNT-TiO2) by the hydrothermal method in a single step at 150 °C and tested them for the photocatalytic degradation of MB. The microstructural analysis confirmed the transformation of TiO2 nanoflowers to CNT-TiO2 spheres due to the role of surface energy of CNT. The XRD peak profile analysis of TiO2 and n-CNT-TiO2 confirmed the formation of pure rutile phase of Titania sintered at 400 °C. A red-shift in the bandgap of n-CNT-TiO2 nanocomposite (2.7eV) was observed due to the donor states towards the conduction band edge. The core-level of XPS showed the strong interaction between carbon and titanium atoms. The CNT facilitate the interfacial charge transfer or avoid the formation of charge recombination due to the variation of Ti4+ to Ti3+ sites. The highest photocatalytic degradation was achieved by using the photocatalyst of n-CNT-TiO2 nanocomposite. The incorporation of CNT provides the interfacial charge transfer facility that creates new interbands energy states for the delay of electron-hole recombination, which cater the environmental remediation.

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.

We report a novel method for synthesizing monodisperse mesoporous TiO(2) spheres (sizes = 0.6-3.1 μm) by hydrolysis of titanium isopropoxide (TIP) in a mixture of C(8)-C(16)n-alkylamine, water, and ethanol. The size increases with decreasing temperature, TIP concentration, and water concentration, and upon purifying TIP. n-Dodecylamine gives the highest monodispersity.