PD-1 Receptor Occupancy Research Articles

Multiparameter Flow Cytometry Assay for Quantification of Immune Cell Subsets, PD‐1 Expression Levels and PD‐1 Receptor Occupancy by Nivolumab and Pembrolizumab

We report the development and validation of a 12 parameter immunofluorescence flow cytometry method for the sensitive determination of cell concentrations, their expression of PD-1, and PD-1 receptor occupancy. Cell subsets include CD4+ and CD8+ -T-cells, B-cells, natural killer cells, classical-, intermediate- and non-classical monocytes, and myeloid- and plasmacytoid dendritic cells. Cells were isolated from peripheral blood by density gradient centrifugation. The validation parameters included specificity, linearity, limit of quantification, precision, biological within- and between subject variations. The lower limit of quantification was 5.0% of PD-1+ cells. Samples were stable for at least 153 days of storage at -80°C. The clinical applicability of the method was demonstrated in 11 advanced cancer patients by the successful determination of immune cell concentrations, relative number of PD-1+ immune cells, and number of PD-1 molecules per immune cell. Shortly after infusion of nivolumab, receptor occupancy on CD8+ -T-cells was 98%. Similar values were found predose cycle 2, suggesting receptor occupancy remained high throughout the entire cycle. © 2019 International Society for Advancement of Cytometry.

Abstract 4624: Profiling antitumor activity, immune cell infiltration, pharmokinetics, and receptor occupancy in a murine colon cancer model following immune checkpoint blockade

Abstract Clinical success of immune checkpoint blocking antibodies has accelerated the evaluation of immune-related targets for novel anti-cancer therapies. Preclinical testing of immune-targeted oncology agents requires preclinical models with functional immune systems. Utilization of murine syngeneic tumor models provides a robust system to evaluate anti-tumor activity and mechanism of action. This study evaluates the anti-tumor activity and immune response of different immune-oncology therapies in the CT26.WT murine colon cancer model. In addition, an anti-PD-1 pharmacokinetic profile was established and receptor occupancy evaluated. For this study, C57BL/6JOlaHsd mice were subcutaneously inoculated with CT26.WT cells. Tumor bearing mice were administered anti-CTLA-4, anti-PD-1, anti-PD-L1, anti-OX40, or anti-LAG3 monotherapy twice weekly for 3 weeks. Tumor volume was used to assess anti-tumor activity. Immune response to checkpoint blockade was monitored in a subset of tumors, and lymphocyte and myeloid populations were analyzed following two weeks of dosing. In addition, an anti-PD-1 pharmacokinetic profile experiment was performed using LC-MS/MS on mouse plasma from multiple time points. PD-1 receptor occupancy was also determined by flow cytometry utilizing a saturation/detection method. Following 3 weeks of monotherapy treatment all immune checkpoint blocking therapies, anti-CTLA4, anti-PD-1, anti-PD-L1, anti-OX40, and anti-LAG3, resulted in significant antitumor response in CT26.WT derived tumors. Phenotypes of tumor infiltrating lymphocytes (TILs) resident in tumors were profiled across check point inhibitor treatments: CD45+ lymphocytes were analyzed for populations of cytotoxic lymphocytes (CTLs, CD8+ cells), T-helper cells (CD4+ cells), regulatory T-cells (T-regs, CD4+/CD25+/FoxP3+ cells). The immune cell responses to checkpoint inhibitors were distinct and varied across immune checkpoint blocking antibodies. Furthermore, an empirical exposure-response analysis of anti-PD-1 in the CT26.WT colon model was demonstrated using a novel LC/MS-MS method of detection. Dose dependent occupancy of PD-1 receptors by anti-PD-1 in CT26.WT tumors was also observed. Our results demonstrate a significant anti-tumor response across five immune checkpoint blocking antibodies in the CT26.WT colon cancer model; however, immune response profiles are notably different across these blocking antibodies. Importantly, a novel LC/MS-MS method was developed specifically in mouse plasma to determine exposure response. Utilizing these multiple data sets will allow for PK/PD modeling to generate expectations for future dose response. This type of comprehensive analysis constitutes a highly-relevant tool to evaluate efficacy and mechanism of action for novel immune-targeted therapies for oncology. Citation Format: Patrick Allison, Hua-Chen Chang, Paul Trampont, Lindsey Standarski, Lauren Urisic, Jennifer Rusk, Jaydeep Mehta, Joshua Fohey, Eric Thomas, Karan Agrawal, Brandy Wilkinson. Profiling antitumor activity, immune cell infiltration, pharmokinetics, and receptor occupancy in a murine colon cancer model following immune checkpoint blockade [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 4624.

Assessment of Subcutaneous vs Intravenous Administration of Anti–PD-1 Antibody PF-06801591 in Patients With Advanced Solid Tumors

We assessed feasibility of monthly subcutaneous administration of PF-06801591, a humanized immunoglobulin G4 monoclonal antibody that binds to the programmed cell death (PD-1) receptor and blocks its interaction with PD-1 ligands. To evaluate the safety, efficacy, and pharmacokinetics of PF-06801591 administered intravenously vs subcutaneously. Ongoing phase 1, open-label, multicenter, dose-escalation study of 40 patients, 18 years or older, with locally advanced or metastatic solid tumors, enrolled between March 8, 2016, and March 5, 2018, from 4 US medical centers. An intravenous dose of 0.5, 1, 3, or 10 mg/kg of PF-06801591 was administered every 3 weeks or a subcutaneous dose of 300 mg was administered every 4 weeks. Dose escalation occurred after 2 to 4 patients were enrolled per dose level, with additional patients enrolled in each cohort for further assessment. The primary end points were dose-limiting toxic effects and safety. Secondary end points included pharmacokinetics, immunogenicity, PD-1 receptor occupancy, and efficacy. Of 40 enrolled patients (12 men and 28 women; mean [SD] age, 61 [13] years) in this phase 1 dose-escalation trial, 25 received PF-06801591 intravenously at escalating dose levels (0.5, 1, 3, or 10 mg/kg) and 15 patients received the monoclonal antibody subcutaneously at a single dose level. No dose-limiting toxic effects were observed. Grade 3 or higher treatment-related adverse events occurred in 4 (16%) patients treated intravenously and 1 (6.7%) patient treated subcutaneously. Immune-related adverse events occurred in 10 (40%) patients treated intravenously and 3 (20%) treated subcutaneously. No dose-adverse event associations were observed during intravenous dose escalation, and no serious skin toxic effects occurred with subcutaneous delivery. Responses were seen in 5 patients receiving PF-06801591 intravenously and in 2 patients treated subcutaneously for an overall objective response rate of 18.4%. Median overall survival was not reached with intravenous dosing vs 10.7 months with subcutaneous administration. Exposure to PF-06801591 increased in a dose-proportional manner over the range of intravenous doses. Median time to maximum observed serum concentration was 8 days after subcutaneous administration. Full PD-1 receptor occupancy was seen in all dose cohorts. Anti-PD-1 antibody PF-06801591 was tolerable and showed antitumor activity in a variety of tumor types across all dose levels of intravenous and subcutaneous administration. Monthly subcutaneous administration of PF-06801591 offers a convenient, effective alternative to currently available intravenously administered checkpoint inhibitors. ClinicalTrials.gov identifier: NCT02573259.

Preclinical evaluation of the efficacy, pharmacokinetics and immunogenicity of JS-001, a programmed cell death protein-1 (PD-1) monoclonal antibody

JS-001 is the first monoclonal antibody (mAb) against programmed cell death protein-1 (PD-1) approved by the China Food and Drug Administration (CFDA) into the clinical trails. To date, however, no pre-clinical pharmacological and pharmacokinetic (PK) data are available. In this study, we investigated the efficacy of JS-001 and conducted a preclinical PK study, including the monitoring of anti-drug antibodies (ADAs). We found that JS-001 specifically bound to PD-1 antigen with an EC50 of 21 nmol/L, and competently blocked the binding of PD-1 antigen to PD-L1 and PD-L2 with IC50 of 3.0 and 3.1 nmol/L, respectively. Furthermore, JS-001 displayed distinct species cross-reactivity: it could bind to the PD-1 antigen on the peripheral blood mononuclear cells (PBMCs) of humans and cynomolgus monkeys, but not to those of mice and woodchucks; the Kd values for the interaction between JS-001 and PD-1 antigens on CD8+ T cells of human and cynomolgus monkey were 2.1 nmol/L and 1.2 nmol/L, respectively. In vitro, treatment with JS-001 (0.01-10 μg/mL) dose-dependently stimulated human T cell proliferation, as well as IFN-γ and TNF-α secretion. In HBsAg-vaccinated cynomolgus monkeys, the expression of PD-1+/CD4+ and PD-1+/CD8+ was significantly elevated, intramuscular injection of JS-001 (1 and 10 mg/kg) resulted in dramatic decreases in PD-1+/CD4+ and PD-1+/CD8+ expression in a dose-dependent manner, which was supported by PD-1 receptor occupancy (RO) results. In the PK study, 18 cynomolgus monkeys treated with single, ascending doses of 1, 10, and 75 mg/kg, and another 6 cynomolgus monkeys received 10 mg/kg successive administration. The plasma clearance of JS-001 followed a linear PK profile with single administration in the 1 and 10 mg/kg groups and a non-linear PK profile in the 75 mg/kg group. In the successive 10 mg/kg administration group, no drug accumulation was observed. But the AUC from the last exposure was lower than that of the first administration, which was probably due to the production of ADAs, as demonstrated in immunogenicity study. These non-clinical data are encouraging and provide a basis for the efficacy and safety of JS-001 in clinical trials.

Abstract PR05: Combination therapy with anti-CTLA4 and anti-PD1 leads to distinct immunologic changes in-vivo

Abstract Therapies targeting T cell immune checkpoints such as CTLA4 and PD1/PDL1 axis have shown considerable promise in the therapy of human cancer. Combination therapy with dual immune checkpoint blockade (ICB) was recently shown to be highly active in melanoma. While signaling via both PD1 and CTLA4 is known to converge downstream and dampen T cell function, data comparing in vivo effects of blockade of these immune checkpoints either alone or in combination in vivo in humans are limited. Here we have analyzed paired pre/post therapy samples from patients treated either anti-CTLA4 (n=5) or anti-PD1 (n=6) alone, or a combination of anti-CTLA4 and anti-PD1 (n=8), using several methodologies including multi-parameter flow cytometry, single-cell mass-cytometry (CyTOF), Luminex and analysis of transcriptome of purified immune cells with exon-level arrays. We show that blockade of CTLA4, PD1 or combination blockade leads to distinct immunologic, genomic and cytokine signatures in vivo. CTLA4 blockade leads to a prominent proliferation signature in vivo, manifest as an increase in Ki-67 expression in a subset of T cells with transitional memory phenotype. PD1 blockade does not induce this phenotype and instead leads to marked changes in T cells expressing NK and cytolysis associated genes, as exemplified by Granzyme+ T cells. Combination blockade leads to non-overlapping changes in gene expression including proliferation-associated and chemokine genes and leads to an increase in both Ki67+and Granzyme+ T cells. Overall, therapy-induced changes are more prominent in T cells than in monocytes include also involve non-overlapping changes in several alternatively-spliced transcripts, and non-coding RNAs. Each of the ICB therapies also leads to a distinct cytokine profile with differential effects on systemic levels of sIL2R and IL1a. Changes seen in the peripheral blood T cells can also be seen in the tumor infiltrating lymphocytes. Combination therapy leads to an increase in interferon-gamma; producing T cells in both circulation as well as tumor bed. PD1 expression is higher on tumor infiltrating T cells when compared to T cells in circulation. Importantly, PD1 receptor occupancy following anti-PD1 therapy may be incomplete in the tumor infiltrating T cells even in the setting of complete receptor occupancy in circulating T cells. These data demonstrate that blockade of PD1, CTLA4 alone or in combination have distinct immunologic effects in vivo and each strategy serves as a unique immune-therapeutic. Improved understanding of the in vivo effects of ICB is needed for rational development of future immune-based combinations against cancer. This abstract is also being presented as Poster B06. Citation Format: Rituparna Das, Rakesh Verma, Mario Sznol, Chadra Sekhar Boddupalli, Scott Gettinger, Harriet Kluger, Madhav Dhodapkar, Kavita Dhodapkar. Combination therapy with anti-CTLA4 and anti-PD1 leads to distinct immunologic changes in-vivo. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr PR05.

Combination therapy with anti-CTLA-4 and anti-PD-1 leads to distinct immunologic changes in vivo.

Combination therapy concurrently targeting PD-1 and CTLA-4 immune checkpoints leads to remarkable antitumor effects. Although both PD-1 and CTLA-4 dampen the T cell activation, the in vivo effects of these drugs in humans remain to be clearly defined. To better understand biologic effects of therapy, we analyzed blood/tumor tissue from 45 patients undergoing single or combination immune checkpoint blockade. We show that blockade of CTLA-4, PD-1, or combination of the two leads to distinct genomic and functional signatures in vivo in purified human T cells and monocytes. Therapy-induced changes are more prominent in T cells than in monocytes and involve largely nonoverlapping changes in coding genes, including alternatively spliced transcripts and noncoding RNAs. Pathway analysis revealed that CTLA-4 blockade induces a proliferative signature predominantly in a subset of transitional memory T cells, whereas PD-1 blockade instead leads to changes in genes implicated in cytolysis and NK cell function. Combination blockade leads to nonoverlapping changes in gene expression, including proliferation-associated and chemokine genes. These therapies also have differential effects on plasma levels of CXCL10, soluble IL-2R, and IL-1α. Importantly, PD-1 receptor occupancy following anti-PD-1 therapy may be incomplete in the tumor T cells even in the setting of complete receptor occupancy in circulating T cells. These data demonstrate that, despite shared property of checkpoint blockade, Abs against PD-1, CTLA-4 alone, or in combination have distinct immunologic effects in vivo. Improved understanding of pharmacodynamic effects of these agents in patients will support rational development of immune-based combinations against cancer.