Human Programmed Death-1 Research Articles

A novel bispecific peptide targeting PD-1 and PD-L1 with combined antitumor activity of T-cells derived from the patients with TSCC

Programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1) are key immune checkpoints (ICs) that critically influence immunotherapy. Tumor resistance to single IC-targeting drugs has increased interest in dual-target drugs, which have shown feasibility for cancer treatment. In this study, we aimed to develop dual-target peptide drugs targeting the PD-1/PD-L1 pathway and to evaluate their efficacy compared to functional antibodies in enhancing the cytotoxicity of human T cells against tongue squamous carcinoma cell lines. Through sequence analysis and peptide truncation, we modified a pre-existing peptide named nABPD-1 targeting PD-1. Subsequently, we obtained two novel peptides named nABPD-2 and nABPD-3, with nABPD-2 showing an enhanced affinity for human PD-1 protein compared to nABPD-1. Importantly, nABPD-2 exhibited dual-targeting capability, possessing a high affinity for both PD-L1 and PD-1. Furthermore, nABPD-2 effectively promoted the cytotoxicity of human T cells against tongue squamous carcinoma cell lines, surpassing the efficacy of anti-PD-1 or anti-PD-L1 functional antibodies alone. Considering that nABPD-2 has lower production costs and dose requirements, it can potentially be used in therapeutic applications.

Antitumor Effect of Korean Red Ginseng through Blockade of PD-1/PD-L1 Interaction in a Humanized PD-L1 Knock-In MC38 Cancer Mouse Model

Blocking immune checkpoints, programmed death-1 (PD-1) and its ligand PD-L1, has proven a promising anticancer strategy for enhancing cytotoxic T cell activity. Although we previously demonstrated that ginsenoside Rg3, Rh2, and compound K block the interaction of PD-1 and PD-L1, the antitumor effect through blockade of this interaction by Korean Red Ginseng alone is unknown. Therefore, we determined the effects of Korean Red Ginseng extract (RGE) on the PD-1/PD-L1 interaction and its antitumor effects using a humanized PD-1/PD-L1-expressing colorectal cancer (CRC) mouse model. RGE significantly blocked the interaction between human PD-1 and PD-L1 in a competitive ELISA. The CD8+ T cell-mediated tumor cell killing effect of RGE was evaluated using murine hPD-L1-expressing MC38 cells and tumor-infiltrating hPD-1-expressing CD8+ T cells isolated from hPD-L1 MC38 tumor-bearing hPD-1 mice. RGE also reduced the survival of hPD-L1 MC38 cells in a cell co-culture system using tumor-infiltrating CD8+ T cells as effector cells combined with hPD-L1 MC38 target cells. RGE or Keytruda (positive control) treatment markedly suppressed the growth of hPD-L1 MC38 allograft tumors, increased CD8+ T cell infiltration into tumors, and enhanced the production of Granzyme B. RGE exhibits anticancer effects through the PD-1/PD-L1 blockade, which warrants its further development as an immunotherapy.

PD-1 licenses activated Treg for lymphatic migration.

Abstract Rather than serving as fluid transport conduits, the lymphatic vessels have been proved to be critical portals for immune cell migration. As non-immune cell, the lymphatic endothelial cell (LEC) constitutively express high level of programmed death-1 (PD-1) ligand (PD-L1), known as an immune checkpoint protein. We hypothesized that LECs use PD-L1 to regulate PD-1-expressing immune cells during transendothelial migration (TEM). Activated T cells expressed substantially elevated levels of both PD-1 and PD-L1. For human and mouse Tregs PD-1 was in excess of PD-L1, while effector CD4 T cells (Teffs) expressed PD-L1 in excess of PD-1. PD-1−/− but not PD-L1−/− Tregs had reduced migration and motility compared to WT. Engagement by PD-L1 Fc induced rapid activation of Akt but suppressed classical NFkB-P65 signaling in Tregs. WT LECs constitutively expressed high level of surface PD-L1. PD-L1−/− LEC or lymphatic vessels (LV) had increased adhesion and junction proteins VCAM-1 and zonulin-1, and had more zipper junctions. PD-1 triggered PD-L1 signaling in LEC result in strong PI3K/Akt and NFkB-p65 responses that regulated endothelial structure and enhanced TEM. Similar changes to LEC junctions were observed when PD-1high Tregs migrated across LEC layers, resulting in rapidly induced NFkB-p65 translocation into the LEC nucleus. PD-1-induced PD-L1 signaling decreased VE-cadherin expression, which was restored by blocking ERK and PI3K/Akt. Importantly, PD-1-blockade of intra-tumoral Tregs reduced tumor egress and the detained Tregs converted to IFN-g-producing Th1Tregs. These data demonstrate novel roles for Treg PD-1 and LEC PD-L1 in regulation of lymphatic migration and ultimately Treg suppressive function. Supported by grants from NIH (2R37AI062765-17A1)

Screening and characterization of aptamers for recombinant human programmed death-1 and recombinant extracellular domain of human programmed death ligand-1.

Programmed death ligand-1 (PD-L1) is expressed on tumor cells and macrophages. The detection of PD-L1 expression in cancer and the treatment by targeting the PD-L1/programmed death-1 (PD-1) are of great clinical significance. This work aims to screen the aptamers with high affinity and specificity for recombinant human PD-1 (rhPD-1)/recombinant human PD-L1 extracellular domain (rhPD-L1). In this study, we have expressed, purified, prepared, and identified rhPD-1 and rhPD-L1. The rhPD-L1/rhPD-1 aptamers with high affinity and specificity were obtained by systematic evolution of ligands by exponential enrichment technique. Ten aptamers sequences to rhPD-L1 and 10 aptamers sequences to rhPD-1 were obtained by cloning and sequencing. The affinity and specificity of candidate aptamers were analyzed by gold nanoparticles-based colorimetric assay, dot blot assay, and electrophoretic mobility shift assay. The aptamers named A6 were picked out as the optimal aptamers that recognize PD-1, specifically with the Kd value of 47.84 ± 24.78 nM. The aptamers named B10 were picked out as the optimal aptamers that recognize PD-L1, specifically with the Kd value of 59.72 ± 15.87 nM. The study lays a foundation for the development of detection methods and therapeutic drugs targeting PD-L1/PD-1.

A Small Molecule Antagonist of PD-1/PD-L1 Interactions Acts as an Immune Checkpoint Inhibitor for NSCLC and Melanoma Immunotherapy

Immune checkpoint inhibitors, such as monoclonal antibodies targeting programmed death 1 (PD-1) and programmed death ligand-1 (PD-L1), have achieved enormous success in the treatment of several cancers. However, monoclonal antibodies are expensive to produce, have poor tumor penetration, and may induce autoimmune side effects, all of which limit their application. Here, we demonstrate that PDI-1 (also name PD1/PD-L1 inhibitor 1), a small molecule antagonist of PD-1/PD-L1 interactions, shows potent anti-tumor activity in vitro and in vivo and acts by relieving PD-1/PD-L1-induced T cell exhaustion. We show that PDI-1 binds with high affinity to purified human and mouse PD-1 and PD-L1 proteins and is a competitive inhibitor of human PD-1/PD-L1 binding in vitro. Incubation of ex vivo activated human T cells with PDI-1 enhanced their cytotoxicity towards human lung cancer and melanoma cells, and concomitantly increased the production of granzyme B, perforin, and inflammatory cytokines. Luciferase reporter assays showed that PDI-1 directly increases TCR-mediated activation of NFAT in a PD-1/PD-L1-dependent manner. In two syngeneic mouse tumor models, the intraperitoneal administration of PDI-1 reduced the growth of tumors derived from human PD-L1-transfected mouse lung cancer and melanoma cells; increased and decreased the abundance of tumor-infiltrating CD8+ and FoxP3+ CD4+ T cells, respectively; decreased the abundance of PD-L1-expressing tumor cells, and increased the production of inflammatory cytokines. The anti-tumor effect of PDI-1 in vivo was comparable to that of the anti-PD-L1 antibody atezolizumab. These results suggest that the small molecule inhibitors of PD-1/PD-L1 may be effective as an alternative or complementary immune checkpoint inhibitor to monoclonal antibodies.

Abstract 3367: Preclinical characterization of YBL-006, a fully human anti-PD-1 antibody being ready for clinical studies

Abstract Cancer immunotherapy with immune checkpoint inhibitors which enhances T-cell regulatory pathways has provided unprecedented benefit to cancer patients. Programmed death 1 ligand (PD-L1) being expressed by cancer cells binds to programmed death-1 (PD-1) to avoid anti-tumor activity of immune cells. Antibodies to disrupt the interaction have been developed for cancer therapeutics. YBL-006 is a new anti-PD-1 human IgG4 monoclonal antibody. Here, we described in vitro & in vivo studies to evaluate pharmacological effect of YBL-006 along with non-clinical studies of pharmacokinetics, toxicokinetics & tissue cross reactivity (TCR). YBL-006 was bound specifically to human PD-1 among receptors of B7 family by ELISA. When affinity to recombinant PD-1 of various species was measured using surface plasmon response system, KD of YBL-006 was 0.372 nM to human PD-1 & 0.070 nM to cynomolgus monkey PD-1 which is higher affinity than nivolumab (1.37 nM & 2.50 nM) & pembrolizumab (1.44 nM & 0.817 nM). It did bind to mouse PD-1 with less affinity than to human PD1, while nivolumab & pembrolizumab did not bind to mouse PD-1. YBL-006 was able to inhibit interaction between PD-1 & both PD-L1 & PD-L2 which was confirmed in competitive binding assay. Inhibition of PD-1 by YBL-006 increased the level of IFN-γ in a mixed lymphocyte reaction model. In a syngeneic mouse model of MC38 colon cancer, weight of tumors in mice treated with YBL-006 was less than that in mice with human IgG control by 25%. In a humanized mouse model (miXeno mouse) of HCC827 non-small cell lung cancer, volume of tumors in YBL-006-treated mice was smaller (35 % less comparing to control mice) than that in nivolumab-treated mice (24 % less). Pharmacokinetic analysis in monkey showed that exposures to YBL-006 increased dose-dependently & in a dose-proportional manner. Maximum mean serum concentrations (Cmax) of YBL-006 were reached (Tmax) between 0.5- & 1.0-hour post onset of infusion & YBL-006 mean serum concentrations slowly declined at a mean estimated t1/2 value of 70.8~110 hours. Clearance & mean volume of distribution suggest that YBL-006 was mainly distributed throughout the blood. In 1-month IV infusion toxicity study in monkey, there was no toxicity when they were infused up to 100 mg/kg/dose of YBL-006, but anti-drug antibody was observed (7 out of 22 monkeys). There was no potential toxicity in TCR study. YBL-006 is an anti-PD-1 antibody with high affinity, promising anti-tumor activity in animal models, & favorable safety profile. First-in-human phase I trial to investigate the safety & efficacy of YBL-006 in advanced solid cancer will be held in 2020. Citation Format: Jaebong Yoon, Hyunmi Lee, Eunyoung Jeon, Jinsil Lee, Eunyoung Shim, Hyunju Lee, Eunjin Lee, Younggyu Cho, Bumchan Park, Hanseung Lee, Youngwoo Park. Preclinical characterization of YBL-006, a fully human anti-PD-1 antibody being ready for clinical studies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3367.

Engineering Chimeric Antigen Receptor T Cells against Immune Checkpoint Inhibitors PD-1/PD-L1 for Treating Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a 5-year survival rate of 9%. Major obstacles to successful treatment of pancreatic cancer are the immunosuppressive tumor microenvironment (TME) and antigenic complexity or heterogeneity. Programmed death-ligand 1 (PD-L1) is expressed on PDAC and immunosuppressed cells within the TME, providing suitable immunotherapy targets. We applied a chimeric antigen receptor (CAR) strategy to target immune checkpoint programmed death-1 (PD-1)/PD-L1 interactions. Lentiviral vectors were used to express the extracellular domain of human PD-1 (PD-1-CD28-4-1BB activating chimeric receptor [PD1ACR]) or the single-chain variable fragment (scFv) region of anti-PD-L1 (PDL1CAR) that binds to PD-L1, and each was fused to intracellular signaling domains containing CD3 zeta, CD28, and 4-1BB (CD137). Both engineered CAR T cells recognized and eliminated PD-L1-overexpressing CFPAC1 cells efficiently at approximately 80% in vitro. Adoptive transfer of both CAR T cells enhanced T cell persistence and induced specific regression of established CFPAC1 cancer by >80% in both xenograft and orthotopic models. Ki67 expression in tumors decreased, whereas proinflammatory cytokines/chemokines increased in CAR T cell-treated mouse sera. PD1ACR and PDL1CAR obtained a similar therapeutic efficacy. Thus, these armed third-generation PD-L1-targeted CAR T cells confer antitumor activity and the ability to combat T cell exhaustion, providing a potentially new and innovative CAR T cell immunotherapy against pancreatic cancers.

A high-affinity human PD-1/PD-L2 complex informs avenues for small-molecule immune checkpoint drug discovery

Immune checkpoint blockade of programmed death-1 (PD-1) by monoclonal antibody drugs has delivered breakthroughs in the treatment of cancer. Nonetheless, small-molecule PD-1 inhibitors could lead to increases in treatment efficacy, safety, and global access. While the ligand-binding surface of apo-PD-1 is relatively flat, it harbors a striking pocket in the murine PD-1/PD-L2 structure. An analogous pocket in human PD-1 may serve as a small-molecule drug target, but the structure of the human complex is unknown. Because the CC' and FG loops in murine PD-1 adopt new conformations upon binding PD-L2, we hypothesized that mutations in these two loops could be coupled to pocket formation and alter PD-1's affinity for PD-L2. Here, we conducted deep mutational scanning in these loops and used yeast surface display to select for enhanced PD-L2 binding. A PD-1 variant with three substitutions binds PD-L2 with an affinity two orders of magnitude higher than that of the wild-type protein, permitting crystallization of the complex. We determined the X-ray crystal structures of the human triple-mutant PD-1/PD-L2 complex and the apo triple-mutant PD-1 variant at 2.0 Å and 1.2 Å resolution, respectively. Binding of PD-L2 is accompanied by formation of a prominent pocket in human PD-1, as well as substantial conformational changes in the CC' and FG loops. The structure of the apo triple-mutant PD-1 shows that the CC' loop adopts the ligand-bound conformation, providing support for allostery between the loop and pocket. This human PD-1/PD-L2 structure provide critical insights for the design and discovery of small-molecule PD-1 inhibitors.

PD-1 Is Expressed at Low Levels on All Peripheral Blood Natural Killer Cells but Is a Significant Suppressor of NK Function Against PD-1 Ligand Expressing Tumor Targets

PD-1 Is Expressed at Low Levels on All Peripheral Blood Natural Killer Cells but Is a Significant Suppressor of NK Function Against PD-1 Ligand Expressing Tumor Targets

Abstract 1495: Alternative humanized PD-1 mouse models provide more options for PD-1 antibody efficacy study in vivo

Abstract The Programmed Death-1 (PD-1) binding to its ligands PD-L1 and PD-L2 plays a critical role in suppression of T cell function and tumor immune evasion. Blocking antibodies against PD-1 have been shown to induce durable antitumor responses in multiple cancers in many clinical trials. So a well-characterized mouse model is essential for preclinical studies of antibodies that target human PD-1. Here we generated several humanized PD-1 knock-in mouse models, one expresses a hybrid PD1 protein by replacing the amino acids that involve in binding to PD-L1 (mostly IgV domain) with the human counterpart in both C57BL/6J and Balb/C genetic background, another one have both the IgV domain and signal peptide replaced by human counterpart. These mouse models were confirmed by PCR and southern blotting. And homozygous mouse models from these different strategies were also successfully generated and both of them could survive and the birth rate were very similar to heterozygous and wild type littermates. Staining for human and mouse PD-1 in stimulated CD8+ and CD4+ T cells indicate the up regulated expression of human PD-1 and absence of mouse PD-1 in these mouse model. Homozygous human PD-1 knock-in mice displayed normal development, and no obvious signs of autoimmunity for at least six months, whereas PD-1 knockout mice developed tissue-specific autoimmunity. With these human PD-1 knock-in mice engrafted with MC38 and treated with anti-human PD-1 antibodies, we confirmed the binding of anti-human antibody to chimeric PD-1 protein, and found anti-human PD-1 antibodies could significantly inhibit tumor growth in these models. Head to head comparison of these mice with current commercial available models for in vivo efficacy studies are undergoing and will reveal how the signal peptide effect the protein refolding or signaling transduction pathway for these ‘human-mouse’ chimeric proteins. In summary, these human PD-1 knock-in mouse models are functional and can be used to evaluate anti-human PD1 antibodies in vivo, we provide more options for antibodies which is targeting PD-1. Citation Format: Qing Lin, Huimin Ma, Xiaoyu Zhang, Hengbin Li, Wenjing Yang, Zhewei Yang, Xingchen Shen, Qunsheng Ji. Alternative humanized PD-1 mouse models provide more options for PD-1 antibody efficacy study in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1495.

Molecular cloning, expression and characterization of Pekin duck programmed death-1

Programmed death-1 (PD-1) has a pivotal role in the attenuation of adaptive immune responses and peripheral tolerance. Here we describe the identification of the Pekin duck programmed death-1 orthologue (duPD-1). The duPD-1 cDNA encodes a 283-amino acid polypeptide that has an amino acid identity of 70%, 32% and 31% with chicken, murine and human PD-1, respectively. The duck PD-1 gene shares five conserved exons with chicken, murine and human PD-1 genes. A cluster of putative regulatory elements within the conserved region B (CR-B) of the basal promotor is conserved. Homology modeling was most compatible with the two β-sheet IgV domain structure of murine PD-1. Contact residues, shown to be critical for binding of the respective human and murine PD-1 ligands are mostly conserved between avian and mammalian species, whereas residues that define the cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM) are highly conserved across higher vertebrates and frog. Constitutive expression of duPD-1 transcripts was predominantly found in lymphocyte-rich tissues, and mitogen-stimulation of duck peripheral blood mononuclear cells transiently increased duPD-1 mRNA expression. A soluble duPD-1 protein was expressed and shown to engage the identified duck PD-1 ligands. Our observations show considerable evolutionary conservation between mammalian and avian PD-1 orthologues. This work will facilitate further investigation of the role of PD-1 signaling in adaptive immunity in the Pekin duck, a non-mammalian vertebrate and pathogen host with relevance for human and animal health.

Abstract A174: The anti-LAG-3 antibody REGN3767 promotes immune activation in the tumor microenvironment and enhances antitumor activity of anti-PD-1 antibody REGN2810 in PD-1/LAG-3 humanized mice

Abstract The immune checkpoint receptor lymphocyte activation gene 3 (LAG-3) inhibits proliferation and cytokine production by activated CD4 and CD8 T-cells upon binding to major histocompatibility complex class II (MHC II). Another inhibitory receptor, programmed death 1 (PD-1), and its ligands PD-L1 and PD-L2, are critical for the establishment and maintenance of peripheral T-cell tolerance. Because PD-1 and LAG-3 signaling in the tumor microenvironment enables tumors to escape immune surveillance, combined targeting of PD-1 and LAG-3 has emerged as a promising therapeutic strategy. REGN3767 is a human monoclonal antibody that binds LAG-3 with high affinity and rescues T-cell function by blocking its interaction with MHC II. Similarly, the human monoclonal antibody REGN2810 blocks PD-1 interaction with PD-L1 and PD-L2. To test the in vivo activity of REGN3767 alone or in combination with REGN2810, we generated humanized PD-1/LAG-3 knock-in mice, in which the extracellular domains of mouse Pdcd1 and Lag3 genes were replaced with the corresponding regions of human PD-1 and human LAG-3, respectively. In these mice, combined REGN2810 and REGN3767 therapy reduced MC38.Ova tumor growth and prolonged survival, compared to REGN2810 and REGN3767 monotherapies. To explore the underlying molecular mechanisms, we conducted RNA sequencing and RT PCR analyses of MC38.Ova tumors in PD-1/LAG-3 humanized mice following administration of one or two doses of REGN2810, REGN3767, or the combination. Both REGN3767 and REGN2810 promoted robust transcriptional changes in tumors, and combined PD-1 and LAG-3 blockade resulted in enhanced immune activation signatures. Analysis of differentially expressed genes revealed T-cell expansion and activation induced by either antibody monotherapy. While tumor immune responses to REGN3767 were delayed compared to REGN2810, both agents induced similar gene changes via shared pathways. In addition to T-cell activation, REGN2810 or REGN3767 treatments engaged other tumor-infiltrating leukocytes, including neutrophils, myeloid and NK cells. Stronger tumor growth inhibition in response to REGN2810 or REGN3767 therapy was associated with more robust immune activation signatures. Combination of REGN2810 and REGN3767 enhanced antitumor efficacy, resulting in gene expression changes not seen with either monotherapy. The combination therapy also enhanced immune responses promoted by either antibody alone, including genes associated with T-cell activation and effector function. REGN2810 and REGN3767 combination also increased the expression of other checkpoint and costimulatory molecules, including CTLA-4, CD40 and VISTA. The robust intratumoral immune activation associated with antitumor efficacy in preclinical setting supports the clinical development of REGN2810 and REGN3767 as a combination cancer immunotherapy. Citation Format: Elena Burova, Gabor Halls, Omaira Allbritton, Wen Zhang, William Olson, Markus Mohrs, Gavin Thurston, Ella Ioffe. The anti-LAG-3 antibody REGN3767 promotes immune activation in the tumor microenvironment and enhances antitumor activity of anti-PD-1 antibody REGN2810 in PD-1/LAG-3 humanized mice [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A174.

Abstract 4561: Preclinical characterization of GLS-010 (AB122): A fully humanized clinical-stage anti-PD-1 antibody

Abstract INTRODUCTION: Exhausted T cells express high levels of several immune checkpoint proteins, including the programmed death-1 (PD-1) receptor. Preclinical and clinical data support the role of the PD-1/PD-L1 axis in promoting tumor evasion by curtailing immune responses. We present here the preclinical characterization of GLS-010 (AB122), a novel fully human anti-PD-1 monoclonal antibody currently in Phase 1. METHODS: The affinity of GLS-010 (AB122) for human and cynomolgus monkey PD-1, its specificity for PD-1, and its ability to block the PD-1 interaction with PD-L1 and PD-L2 were measured by ELISA, flow cytometry and TCR-activated reporter gene assays. Functional assessment of GLS-010 (AB122) on immune responses (IFN-g, IL-2, and proliferation) was performed using mixed lymphocyte reaction (MLR) and re-stimulation with cytomegalovirus (CMV) pp665 peptide. The anti-tumor efficacy of GLS-010 (AB122) was evaluated using a mouse MC-38 tumor model grown in mice transgenic for human PD-1. For PK analysis, GLS-010 (AB122) was given in a single i.v. bolus to male and female cynomolgus monkeys at doses of 2, 6 and 18 mg/kg. RESULTS: GLS-010 (AB122) is a fully human IgG4 monoclonal antibody that binds to human PD-1 (EC50 ~ 210 pM, ELISA; 770 pM, flow cytometry), cyno PD-1 (EC50 ~ 150 pM, ELISA), but not rat or mouse PD-1. Lack of GLS-010 (AB122) binding to other related members of the CD28 family, such as ICOS, CD28 and CTLA-4, confirms the specificity of the interaction. Functional studies showed that binding of GLS-010 (AB122) to cell-expressed hPD-1 inhibits the interaction with both hPD-L1 and hPD-L2 with an IC50 of 580 pM and 670 pM, respectively (by flow cytometry) and 2.2 nM and 5.8 nM, respectively (in reporter gene assay). Using allogeneic monocyte-derived dendritic cells, we showed a dose-dependent enhancement of IFN-g production and proliferation by CD4+ T cells, saturating at concentrations below 100 pM. Similar results were obtained in an antigen-specific T cell recall response assay using CMV-infected donors. GLS-010 (AB122) was very effective at blocking MC-38 tumor growth in hPD-1 transgenic mice. PK profiles following a single i.v. dose of GLS-010 (AB122) administered to male and female cynomolgus monkeys were dose-proportional and the rate of clearance was dose-independent. CONCLUSIONS: GLS-010 (AB122) is a novel and selective antagonistic anti-hPD-1 antibody that potently blocks the interaction of human PD-1 with both PD-L1 and PD-L2. This blockade translates into potent enhancement of T cell activation in a variety of cell culture studies, which combined with its in vivo profile (in mice and monkeys) supports its ongoing clinical development in oncology. Citation Format: Joanne B. Tan, Chris Chen, Kai Chen, Guochun Li, Jing Li, Jieying Liu, Hema Singh, Guoyong Wang, Baotian Yang, Kristin Zhang, Xiaoning Zhao, Yong Zheng. Preclinical characterization of GLS-010 (AB122): A fully humanized clinical-stage anti-PD-1 antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4561.

PO-409 Signal peptide of PD-1 inhibits cancer cell growth

IntroductionCancer cells escape immune system surveillance by activating molecules called ‘immune checkpoints’, of which programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1) are known representative molecules. Immune cells do not become active when the PD-1 present on their cell surface recognises the PD-L1 present on a cancer cell’s surface, allowing cancer cells to evade the immune cell’s attack. Immune checkpoint-blocking antibodies developed to target PD-1/PD-L1 have remarkable anti-cancer activity. However, the biological reasons why cancer cells activate PD-L1 remain unclear. In addition, there is an issue with cells, such as myocardial cells, with PD-L1 present on their cell surfaces also being affected by immune checkpoint-blocking antibodies. We focused on a signal peptide of PD-1 expressed on the immune cell surface. Our hypothesis was that the signal peptide produced as part of the production process that leads to mature PD-1 has an anti-tumour function. We evaluated whether an artificial peptide designed based on this PD-1 signal peptide had cell growth inhibition activity against human melanoma A2058 and non-small cell lung cancer (NSCLC) HCC-827.Material and methodsWe chemically synthesised a conjugation peptide of the human PD-1 signal peptide (MQIPQAPWPVVWAVLQLGWR) and a cell penetrating peptide TAT (RRKKRRQRRR) and named it ‘PD1SP-TAT’ (MQIPQAPWPVVWAVLQLGWRRRKKRRQRRR). PD1SP-TAT was evaluated for growth inhibitory activity against A2058 and HCC-827 by WST assay.Results and discussionsThe results were 95% of human melanoma A2058 growth and NSCLC HCC-827 growth were inhibited in cells treated with a concentration of 25 µM PD1SP-TAT for 48 hours.ConclusionFrom these surprising results, PD1SP-TAT, based on the signal peptide of PD-1, shows remarkable anti-cancer activity against human melanoma and NSCLC, and suggests that PD1SP-TAT could be a candidate for a novel anti-cancer agent.

A phase I study of JS001, a humanized IgG4 mAb against programmed death-1 (PD-1) in patients with advanced solid tumors.

3067 Background: JS001 blocks the interaction between PD-1 and its ligands and eradicates established tumor in human PD-1 Knock-in mouse model. Methods: A Phase I open-label study is designed to evaluate the safety and tolerability of JS001 in advanced solid tumor pts who are refractory to standard therapy. The study has a 3+3 dose escalation design with planned cohorts at 1, 3, and 10 mg/kg followed by a dose expansion. (Clinical Trial ID: NCT02836795). Results: As of January 27, 2017, pts enrollment has been completed with 36 pts from 3 indications (22 Melanoma; 9 Urothelial Carcinoma; 5 Renal Cell Carcinoma). The majority of melanomas are acral and mucosal origin. No DLT was observed and no MTD was reached in the study. The most common treatment-related AEs were grade 1/2, including hyper- or hypo-thyroidism (42%), rash (39%), fever (28%), leukopenia (22%), elevation of liver enzymes (19%), anorexia (17%), and fatigue (14%). Treatment–related grade 3 AEs include proteinuria (n = 1), and lipase increase (n = 2). The emergence of AEs is not dose related. JS001 PK shows dose-dependent exposure with the elimination half-life of 6 to 12 days. Among 32 evaluable pts, 1 pt have complete response (melanoma), 6 pts have partial response (3 melanoma, 2 RCC and 1 UC), and 10 pts achieve stable disease, for an ORR of 22% and a DCR of 53%. 6 out of 7 CR/PR pts still have ongoing response. Two groups of pts benefited most from JS001 treatment, pts with high tumor-infiltrating lymphocytes (TIL) (50% ORR) and pts with > 1% PD-L1 expression in tumor biopsy (46% ORR). Conclusions: JS001 exhibited a favorable safety profile in human. Treatment related AEs are in line with those from approved drugs in the same class. JS001 has demonstrated promising anti-tumor activity, especially in previously under-evaluated acral (20% ORR, 53% DCR) and mucosal (25% ORR, 50% DCR) melanomas. Clinical trial information: NCT02836795.

Characterization of the Anti-PD-1 Antibody REGN2810 and Its Antitumor Activity in Human PD-1 Knock-In Mice.

The Programmed Death-1 (PD-1) receptor delivers inhibitory checkpoint signals to activated T cells upon binding to its ligands PD-L1 and PD-L2 expressed on antigen-presenting cells and cancer cells, resulting in suppression of T-cell effector function and tumor immune evasion. Clinical antibodies blocking the interaction between PD-1 and PD-L1 restore the cytotoxic function of tumor antigen-specific T cells, yielding durable objective responses in multiple cancers. This report describes the preclinical characterization of REGN2810, a fully human hinge-stabilized IgG4(S228P) high-affinity anti-PD-1 antibody that potently blocks PD-1 interactions with PD-L1 and PD-L2. REGN2810 was characterized in a series of binding, blocking, and functional cell-based assays, and preclinical in vivo studies in mice and monkeys. In cell-based assays, REGN2810 reverses PD-1-dependent attenuation of T-cell receptor signaling in engineered T cells and enhances responses of human primary T cells. To test the in vivo activity of REGN2810, which does not cross-react with murine PD-1, knock-in mice were generated to express a hybrid protein containing the extracellular domain of human PD-1, and transmembrane and intracellular domains of mouse PD-1. In these mice, REGN2810 binds the humanized PD-1 receptor and inhibits growth of MC38 murine tumors. As REGN2810 binds to cynomolgus monkey PD-1 with high affinity, pharmacokinetic and toxicologic assessment of REGN2810 was performed in cynomolgus monkeys. High doses of REGN2810 were well tolerated, without adverse immune-related effects. These preclinical studies validate REGN2810 as a potent and promising candidate for cancer immunotherapy. Mol Cancer Ther; 16(5); 861-70. ©2017 AACR.

Activity of CBT-501, a novel humanized anti-PD-1 antibody, in a humanized genetically engineered mouse model of colon carcinoma.

108 Background: CBT-501 (Genolimzumab, GB-226) is a novel humanized mAb (IgG4k) against human Programmed Death-1 (PD-1). CBT-501 demonstrated highly specific binding to PD-1 of human (KD= 505 pM) and cynomolgus (KD= 7.2 nM). It did not completely block the binding of nivolumab or pembrolizumab, suggesting a novel epitope. CBT-501 efficiently inhibited the binding of PD-L1/L2 to PD-1 through a competitive action for both human and monkey. It enhanced human T-cell activation in the Mix Lymphocyte Reaction (MLR), as shown by increased release of IL-2 and INF-γ. The Fc-fragment bound to FcRn dose-dependently without any effector function. The PK/PD studies in cynomolgus showed a linear dose exposure, with a T1/2of 115-142 h for single and repeat dosing. The PD-1 receptor occupancy after a single dose was dose-dependent reaching 100% at peak, maintained after multiple dosing and continued for 6 wk after last dose. Single and repeat dose toxicology studies in cynomolgus showed weight gain of the thyroid and mild expansion of the thyroid follicles at the highest dose of 100 mg/kg which were reversible. No abnormal drug-related toxicity was found. In rhesus vaccinated with adenovirus-mediated Simian Immunodeficiency Virus, biweekly IV CBT-501 promoted the antigen-specific T-cell response, as shown by ELISPOT and intracellular staining of IL-2, INF-γ and TNF-α in PBMC and T-cell subpopulation. Due to lack of immunoselectivity to mouse PD-1, investigation of the in vivo anti-tumor activity in rodent model is challenging. Methods: Crown Bioscience has developed humanized genetically engineered mouse model expressing human PD-1 extracellular domain with a fully functional murine immune system. Subcutaneous colon adenocarcinoma MC38 syngeneic tumor draft was established in mice. Results: CBT-501 injected IP q2w×3wk demonstrated significant anti-tumor activity. The tumor growth reduction was dose dependent with comparable or improved activity over nivolumab, and with the highest inhibition rate at 83%. Conclusions: Based on the strong nonclinical data and differentiation, and an acceptable safety profile, a first in human Phase 1 trial is planned in the Asia-Pacific region.

Nivolumab as Programmed Death-1 (PD-1) Inhibitor for Targeted Immunotherapy in Tumor.

Targeted immunotherapy has become the most promising approach for tumor patients. Programmed death-1 (PD-1), an inhibitory receptor expressed on activated T cells, can reverse immune suppression and release T cell activation. Nivolumab, a fully human immunoglobulin G4 PD-1 immune checkpoint inhibitor antibody, blocks PD-1 and promotes antitumor immunity, and it is effective for treating non-small-cell lung cancer (NSCLC), melanoma, renal cell carcinoma (RCC) and other cancers. The present review summarizes the efficacy and current status of clinical trials of nivolumab and that enabled nivolumab to be investigated in patients.

Co-expression of PD-L1 and p-AKT is associated with poor prognosis in diffuse large B-cell lymphoma via PD-1/PD-L1 axis activating intracellular AKT/mTOR pathway in tumor cells

Programmed death-1 (PD-1) /programmed death-ligand 1 (PD-L1) engagement usually leads to diminished antitumor T-cell responses, which mediates the immune escape of tumor cells. However, little is known whether PD-1/PD-L1 could directly activates intracellular oncogenic signaling pathways in tumor cells. The purpose of this study is to investigate whether intracellular AKT/mTOR signaling could be directly activated by PD-1/PD-L1 during the malignant progression in diffuse large B-cell lymphoma (DLBCL). Detection of the expression of PD-L1 and p-AKT by immunohistochemistry (IHC) showed that both proteins were overexpressed in 54% and 48% DLBCL cases, respectively. Spearman test showed that PD-L1 expression was correlated with p-AKT expression (R=0.244, χ2=5.962; P=0.017) and the expression of PD-L1 and p-AKT were also correlated with clinic-pathological characteristics. In addition, survival analysis showed that DLBCL patients who co-expressed PD-L1 and p-AKT had significantly poorer outcome than patients with single positive or both negative expression (P<0.05). In vitro, total PD-L1 and membrane PD-L1 (mPD-L1) proteins were overexpressed in five DLBCL cell lines by western blot and flow cytometry. We observed that AKT/mTOR pathway was activated in DLBCL cells after stimulated with human recombination PD-1/Fc. Taken together, these results suggested that the combination of PD-1/PD-L1 antibodies and AKT/mTOR inhibitor might be a promising and novel therapeutic approach for DLBCL in the future.

Abstract B113: In vivo characterization of anti-PD-1 antibody REGN2810 in human PD-1 knock-in mice

Abstract The Programmed Death-1 (PD-1) receptor is an immunologic inhibitory checkpoint, expressed by activated T cells. PD-1 signals upon binding to its ligands PD-L1 and PD-L2 expressed on antigen presenting cells and some types of cancer cells, and delivers inhibitory signals to T cells, resulting in suppression of T-cell effector function. In cancer patients, PD-1 is expressed on tumor-infiltrating lymphocytes and inhibits antitumor immune responses through ligand binding. Clinically, it has been shown that antibodies (Abs) that block the interaction between PD-1 and PD-L1 can release the cytotoxic function of tumor-specific T cells, yielding durable objective responses in multiple cancer types. This study describes preclinical characterization of REGN2810, a fully human hinge-stabilized IgG4 monoclonal Ab that binds to the extracellular domain of human PD-1 with high affinity and specificity and inhibits interaction of PD-1 with its ligands. Taking advantage of the ability of human PD-1 to interact with murine PD-L1, we generated a mouse with human PD-1 gene knock-in allowing direct testing of our anti-human PD-1 Ab. Human PD-1 knock-in mice express a hybrid protein containing the extracellular portion of human PD-1, and transmembrane and intracellular domains of mouse PD-1. We demonstrated functional PD-1/PD-L1 signaling and immune responses in this model, and confirmed REGN2810 binding to hybrid PD-1 receptor on mouse T cells in vivo following REGN2810 injections. Prophylactic and therapeutic treatments of subcutaneous syngeneic tumors with REGN2810 in human PD-1 knock-in mice resulted in a dose-dependent suppression of tumor growth. Currently REGN2810 anti-PD-1 Ab is in phase 1 clinical trials for oncology indications. Citation Format: Elena Burova, Omaira Allbritton, William Poueymirou, Venus Lai, Janelle Waite, Dimitris Skokos, Nicholas Papadopoulos, Drew Murphy, Israel Lowy, Ella Ioffe, Gavin Thurston. In vivo characterization of anti-PD-1 antibody REGN2810 in human PD-1 knock-in mice. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B113.