Characterization of INCB086550: A Potent and Novel Small-Molecule PD-L1 Inhibitor.

Blocking the activity of the programmed cell death protein 1 (PD-1) inhibitory receptor with therapeutic antibodies against either the ligand (PD-L1) or PD-1 itself has proven to be an effective treatment modality for multiple cancers. Contrasting with antibodies, small molecules could demonstrate increased tissue penetration, distinct pharmacology, and potentially enhanced antitumor activity. Here, we describe the identification and characterization of INCB086550, a novel, oral, small-molecule PD-L1 inhibitor. In vitro, INCB086550 selectively and potently blocked the PD-L1/PD-1 interaction, induced PD-L1 dimerization and internalization, and induced stimulation-dependent cytokine production in primary human immune cells. In vivo, INCB086550 reduced tumor growth in CD34+ humanized mice and induced T-cell activation gene signatures, consistent with PD-L1/PD-1 pathway blockade. Preliminary data from an ongoing phase I study confirmed PD-L1/PD-1 blockade in peripheral blood cells, with increased immune activation and tumor growth control. These data support continued clinical evaluation of INCB086550 as an alternative to antibody-based therapies. SIGNIFICANCE: We have identified a potent small-molecule inhibitor of PD-L1, INCB086550, which has biological properties similar to PD-L1/PD-1 monoclonal antibodies and may represent an alternative to antibody therapy. Preliminary clinical data in patients demonstrated increased immune activation and tumor growth control, which support continued clinical evaluation of this approach. See related commentary by Capparelli and Aplin, p. 1413. This article is highlighted in the In This Issue feature, p. 1397.

2020 White Paper on Recent Issues in Bioanalysis: BAV Guidance, CLSI H62, Biotherapeutics Stability, Parallelism Testing, CyTOF and Regulatory Feedback (Part 2A - Recommendations on Biotherapeutics Stability, PK LBA Regulated Bioanalysis, Biomarkers Assays, Cytometry Validation & Innovation Part 2B - Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine).

The 14th edition of the Workshop on Recent Issues in Bioanalysis (14th WRIB) was held virtually on June 15-29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14th WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by LCMS were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the Global Bioanalytical Community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication covers the recommendations on (Part 2A) BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation and (Part 2B) Regulatory Input. Part 1 (Innovation in Small Molecules, Hybrid LBA/LCMS & Regulated Bioanalysis), Part 3 (Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity) are published in volume 13 of Bioanalysis, issues 4, and 6 (2021), respectively.

A Potent and Selective Dual Inhibitor of AXL and MERTK Possesses Both Immunomodulatory and Tumor-Targeted Activity.

TYRO3, AXL, and MERTK constitute the TAM family of receptor tyrosine kinases, which play important roles in tumor growth, survival, cell adhesion, as well as innate immunity, phagocytosis, and immune-suppressive activity. Therefore, targeting both AXL and MERTK kinases may directly impact tumor growth and relieve immunosuppression. We describe here the discovery of INCB081776, a potent and selective dual inhibitor of AXL and MERTK that is currently in phase 1 clinical trials. In cellular assays, INCB081776 effectively blocked autophosphorylation of AXL or MERTK with low nanomolar half maximal inhibitory concentration values in tumor cells and Ba/F3 cells transfected with constitutively active AXL or MERTK. INCB081776 inhibited activation of MERTK in primary human macrophages and partially reversed M2 macrophage-mediated suppression of T-cell proliferation, which was associated with increased interferon-γ production. In vivo, the antitumor activity of INCB081776 was enhanced in combination with checkpoint blockade in syngeneic models, and resulted in increased proliferation of intratumoral CD4+ and CD8+ T cells. Finally, antitumor activity of INCB081776 was observed in a subset of sarcoma patient-derived xenograft models, which was linked with inhibition of phospho-AKT. These data support the potential therapeutic utility of INCB081776 as an immunotherapeutic agent capable of both enhancing tumor immune surveillance and blocking tumor cell survival mechanisms.

2019 White Paper on Recent Issues in Bioanalysis: Chromatographic Assays (Part 1 - Innovation in Small Molecules and Oligonucleotides & Mass Spectrometric Method Development Strategies for Large Molecule Bioanalysis).

The 2019 13th Workshop on Recent Issues in Bioanalysis (WRIB) took place in New Orleans, LA, USA on April 1-5, 2019 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers, immunogenicity and gene therapy. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS, LBA cell-based/flow cytometry assays and qPCR approaches. This 2019 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2019 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1) covers the recommendations on Innovation in Small Molecules and Oligonucleotides & Mass Spec Method Development Strategies for Large Molecules Bioanalysis. Part 2 (2018 FDA BMV Guidance, 2019 ICH M10 BMV Draft Guideline and regulatory agencies' input on bioanalysis, biomarkers, immunogenicity and gene therapy) and Part 3 (New Insights in Biomarkers Assays Validation, Current & Effective Strategies for Critical Reagent Management, Flow Cytometry Validation in drug discovery & development & CLSI H62, Interpretation of the 2019 FDA Immunogenicity Guidance and The Gene Therapy Bioanalytical Challenges) are published in volume 11 of Bioanalysis, issues 23 and 24 (2019), respectively.

2018 White Paper on Recent Issues in Bioanalysis: focus on immunogenicity assays by hybrid LBA/LCMS and regulatory feedback (Part 2 - PK, PD & ADA assays by hybrid LBA/LCMS & regulatory agencies' inputs on bioanalysis, biomarkers and immunogenicity).

The 2018 12th Workshop on Recent Issues in Bioanalysis took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS and LBA/cell-based assays approaches. This 2018 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations for PK, PD and ADA assays by hybrid LBA/LCMS and regulatory agencies' input. Part 1 (LCMS for small molecules, peptides, oligonucleotides and small molecule biomarkers) and Part 3 (LBA/cell-based assays: immunogenicity, biomarkers and PK assays) are published in volume 10 of Bioanalysis, issues 22 and 24 (2018), respectively.

2016 White Paper on recent issues in bioanalysis: focus on biomarker assay validation (BAV): (Part 2 - Hybrid LBA/LCMS and input from regulatory agencies).

The 2016 10th Workshop on Recent Issues in Bioanalysis (10th WRIB) took place in Orlando, Florida with participation of close to 700 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. WRIB was once again a 5-day, weeklong event - A Full Immersion Week of Bioanalysis including Biomarkers and Immunogenicity. As usual, it is specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecules involving LCMS, hybrid LBA/LCMS, and LBA approaches, with the focus on biomarkers and immunogenicity. This 2016 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. This White Paper is published in 3 parts due to length. This part (Part 2) discusses the recommendations for Hybrid LBA/LCMS and regulatory inputs from major global health authorities. Parts 1 (small molecule bioanalysis using LCMS) and Part 3 (large molecule bioanalysis using LBA, biomarkers and immunogenicity) have been published in the Bioanalysis journal, issues 22 and 23, respectively.

2015 White Paper on recent issues in bioanalysis: focus on new technologies and biomarkers (Part 2 - hybrid LBA/LCMS and input from regulatory agencies).

The 2015 9th Workshop on Recent Issues in Bioanalysis (9th WRIB) took place in Miami, Florida with participation of over 600 professionals from pharmaceutical and biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. It is once again a 5-day week long event - a full immersion bioanalytical week - specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches including the focus on biomarkers and immunogenicity. This 2015 White Paper encompasses recommendations that emerged from the extensive discussions held during the workshop, and is aimed at providing the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to advance scientific excellence, improve quality and deliver better regulatory compliance. Due to its length, the 2015 edition of this comprehensive White Paper has been divided into three parts. Part 2 covers the recommendations for hybrid LBA/LCMS and regulatory agencies' inputs. Part 1 (small molecule bioanalysis using LCMS) and Part 3 (large molecule bioanalysis using LBA, biomarkers and immunogenicity) will be published in volume 7 of Bioanalysis, issues 22 and 24, respectively.

A Neutralizing Antibody Assay Based on a Reporter of Antibody-Dependent Cell-Mediated Cytotoxicity.

Benralizumab is a humanized anti-IL5 receptor α (IL5Rα) monoclonal antibody (mAb) with enhanced (afucosylation) antibody-dependent cell-mediated cytotoxicity (ADCC) function. An ADCC reporter cell-based neutralizing antibody (NAb) assay was developed and characterized to detect NAb against benralizumab in human serum to support the clinical development of benralizumab. The optimal ratio of target cells to effector cells was 3:1. Neither parental benralizumab (fucosylated) nor benralizumab Fab resulted in ADCC activity, confirming the requirement for ADCC activity in the NAb assay. The serum tolerance of the cells was determined to be 2.5%. The cut point derived from normal and asthma serum samples was comparable. The effective range of benralizumab was determined, and 35 ng/mL [80% maximal effective concentration (EC80)] was chosen as the standard concentration to run in the assessment of NAb. An affinity purified goat anti-benralizumab polyclonal idiotype antibody preparation was shown to have NAb since it inhibited ADCC activity in a dose-dependent fashion. The low endogenous concentrations of IL5 and soluble IL5 receptor (sIL5R) did not demonstrate to interfere with the assay. The estimated assay sensitivities at the cut point were 1.02 and 1.10 µg/mL as determined by the surrogate neutralizing goat polyclonal and mouse monoclonal anti-drug antibody (ADA) controls, respectively. The assay can detect NAb (at 2.5 µg/mL) in the presence of 0.78 µg/mL benralizumab. The assay was not susceptible to non-specific matrix effects. This study provides an approach and feasibility of developing an ADCC cell-based NAb assay to support biopharmaceuticals with an ADCC function.

Correlation of screening and confirmatory results in tiered immunogenicity testing by solution-phase bridging assays.

Biotherapeutic proteins induce undesired immune responses that can affect drug efficacy and safety. For this reason, immunogenicity assessment is an integral part of drug development and is mandated by the regulatory authorities. Immunogenicity is typically evaluated by a tiered approach consisting of a screening assay followed by a competitive inhibition with unlabeled drug serving as confirmatory assay and additional characterization of the immune response. The confirmatory assay is intended to reduce the number of false positive responses generated in the screening tier and ensure that all samples are correctly classified as positive or negative. The positive-negative sample decisions are based on screening and confirmatory assay cut points that are statistically derived through evaluation of drug-naive samples. In this paper, we describe the analysis of cut point data for the presence of statistical correlation between the screening and confirmatory results. Data were obtained from validations of solution-phase bridging assays for detection of anti-drug antibodies against monoclonal antibody therapeutics. All data sets showed moderate to strong positive correlation, indicating that the screening and confirmatory assays were not independent and were likely to generate similar information. We present theoretical evidence that correlated results may be a general feature of the tiered approach when the same test platform is used for both screening and confirmatory assays. The competitive inhibition test, therefore, may be of limited value beyond reduction of the overall false positive rate. Our results indicate that similar sample results could be obtained by using just the screening assay with the false positive rate set to 1%.

Validation and comparative analysis of a multiplexed assay for the simultaneous quantitative measurement of Th1/Th2 cytokines in human serum and human peripheral blood mononuclear cell culture supernatants.

There is increasing evidence suggesting a relationship between cytokine levels and disease pathogenesis, which has led to interest in analyzing multiple cytokines in biological fluids and culture supernatants for various research and clinical studies. The introduction of methodologies allowing simultaneous measurement of interrelated biomarkers/cytokines has further revolutionized this process. In contrast to tissue culture supernatant, the measurement of cytokines in serum has proven to be difficult to characterize in multiplexed formats because of the presence of large dynamic concentration ranges of proteins and other interfering factors that are present in this matrix. In the present study, we have used the microsphere-based multiplex method to simultaneously quantitate and compare six analytes, encompassing a representation of the Th1/Th2 cytokine panel (interleukin (IL)-2, IL-4, IL-5, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and IL-10), in both serum and culture supernatants from peripheral blood mononuclear cells (PBMCs). A detailed validation procedure for these determinations is described along with a comparative analysis of the performance of the multiplexed assay in serum and culture supernatant matrices. Our results indicate that precision of the multiplexed assay is comparable in both culture supernatant and serum. However, the accuracy of quantification of cytokines in the serum matrix but not in culture supernatant may be compromised depending upon the cytokine being analyzed. Therefore, one must use caution when interpreting data from such complex matrices. Nevertheless, this assay format is appropriate to profile cytokines in clinical trial samples.

Evidence that thrombocytopenia observed in humans treated with orally bioavailable glycoprotein IIb/IIIa antagonists is immune mediated.

Glycoprotein (GP) IIb/IIIa antagonists are effective therapeutic agents, but elicit thrombocytopenia with a frequency that approaches 2%. Here, we provide evidence that thrombocytopenia in humans treated with the GP IIb/IIIa antagonist roxifiban is immune mediated. Two patients underwent conversion to a highly positive drug-dependent antibody (DDAB) status temporally associated with thrombocytopenia. Despite the continued presence of DDABs, the fall in platelet count was reversed by discontinuation of drug treatment, pointing to the exquisite drug dependency of the immune response. DDABs appear to bind to neoepitopes in GP IIb/IIIa elicited on antagonist binding. This information was used to develop an enzyme-linked immunosorbent assay (ELISA) for DDAB using solid-phase GP IIb/IIIa. A high level of specificity is indicated by the observation that DDAB binding is dependent on the chemical structure of the GP IIb/IIIa antagonist and that only 2% to 5% of human blood donors and 5% of chimpanzees present with pre-existing DDABs. Furthermore, none of 108 nonthrombocytopenic patients from the phase II roxifiban study showed an increase in antibody titer. Absorption of thrombocytopenia plasma with platelets reduced the DDAB ELISA signal, indicating that the test detects physiologically relevant antibodies. Screening patients for pre-existing or increasing DDAB titer during treatment with GP IIb/IIIa antagonists may reduce the incidence of drug-induced thrombocytopenia.

Effects of glycoprotein IIb/IIIa antagonists on platelet activation: development of a transfer method to mimic peak to trough receptor occupancy.

Several oral glycoprotein (GP) IIb/IIIa antagonists, Sibrafiban, Orbofiban and Lotrafiban, have been studied in large phase III trials; each has failed to provide efficacy and has been associated with increased mortality. Roxifiban has pharmacokinetic and pharmacodynamic properties believed to be more favorable than the earlier oral agents. Here, we revisit the controversial hypothesis of platelet activation liabilities of GP IIb/IIIa antagonists. The effects of site occupancy by four fibans (Roxifiban, Sibrafiban, Orbofiban and Lotrafiban) on platelet activation was assessed using P-selectin expression, fibrinogen binding and microaggregate formation. All four fibans inhibited ADP and TRAP-stimulated fibrinogen binding and microaggregate formation in a concentration-dependent manner, whereas P-selectin expression was relatively unaltered. To more vigorously test for activation liabilities, the effects of transition from peak to trough receptor occupancy upon platelet stimulation was analyzed. The high affinity of Roxifiban for resting platelets precluded reduction of site occupancy by dialysis or gel filtration. A method was developed that takes advantage of the rapid equilibrium of Roxifiban between platelets and soluble GPIIb/IIIa. The platelet occupancy is controlled by the ratio of platelet GPIIb/IIIa to soluble GPIIb/IIIa. This method allows in vitro investigation of peak/trough transitions on platelet activation. A decrease in site occupancy from peak to trough of Roxifiban or Sibrafiban did not result in increased activation of platelets. The loss of platelet-bound antagonist upon incubation with purified soluble GPIIb/IIIa returned fibrinogen binding/microaggregate formation to no drug levels. In conclusion, these studies do not provide evidence for an activation liability of GPIIb/IIIa antagonists in vitro.