Occupancy Assay Research Articles

P0929 Design and rationale for the phase 1c study evaluating the pharmacodynamic (PD) effects of vixarelimab in patients with Ulcerative Colitis (UC)

Abstract Background Vixarelimab, a selective oncostatin M receptor-beta (OSMRβ)-targeting, fully human monoclonal antibody, blocks OSM signaling and is hypothesized to target the inflammatory fibroblast-myeloid axis implicated in UC. An ongoing randomized, placebo-controlled Phase 2 MOONGLOW study (NCT06137183) is evaluating the efficacy and safety of vixarelimab in patients with UC. Here we report on the rationale and design of an open-label, single-arm Phase 1c study to provide deeper mechanistic insights regarding vixarelimab in patients with UC. The primary objective is to evaluate vixarelimab PD in the gut through assessment of the change in inflammatory fibroblast products from colonic tissue. Additional objectives include evaluation of receptor occupancy, pharmacokinetics, and safety. Methods The study will enroll ~24 patients to receive vixarelimab subcutaneously on two separate visits with a 6-week evaluation period and a subsequent safety follow-up (Figure 1). Patients will undergo endoscopy with biopsy collection from the colon and clinical assessments at screening/baseline and throughout the evaluation period. The timepoints of post-treatment assessments correspond to predicted steady state systemic drug exposures for the doses evaluated in the ongoing Phase 2 study. This enables PD evaluations in a Phase 1c population pharmacologically comparable to the Phase 2 study at timepoints earlier than possible in the Phase 2 study, whilst mitigating the confounding variables of disease resolution historically observed in UC studies after 12 weeks of placebo treatment.1 Results To facilitate mechanistic assessments of vixarelimab we have established A) a novel method for the assessment of OSMRβ occupancy using fresh colonic tissues and B) a sample collection approach for snRNA-seq aimed to identify and characterize inflammatory fibroblasts that are anticipated to decrease upon vixarelimab engagement of OSMRβ (Figure 2). Using bulk RNA-sequencing, we have also identified a reduction in inflammatory fibroblasts by weeks 4 to 6 in patients with UC who respond to biologic therapy (GSE73661). These data provide evidence that changes in inflammatory fibroblasts may be identifiable earlier than the typical week 12 evaluation in IBD induction trials providing rationale for the early time points chosen for the present study. Conclusion Integrating OSMRβ occupancy assay and snRNA-seq informs on assessment of inflammatory fibroblasts in patients with UC, enabling mechanistic insights for first-in-class therapeutic targeting of the inflammatory fibroblast-myeloid cell axis by vixarelimab in UC.

Abstract CT068: Interim results of the ongoing phase 1-2 clinical trial of KVA12123, an engineered IgG1 targeting VISTA, as monotherapy and in combination with pembrolizumab in patients with advanced solid tumors

Abstract V-domain Ig Suppressor of T-cell Activation (VISTA), a promising immuno-oncology target, is primarily expressed by immunosuppressive myeloid cells that infiltrate solid tumors. VISTA may contribute to resistance to anti-PD-(L)1 and anti-CTLA-4 therapies and correlates with a poor prognosis. KVA12123 is a human IgG1 monoclonal antibody that specifically binds to VISTA at neutral and acidic pHs. It was designed to improve pharmacokinetic (PK) characteristics as well as reduce the risk of cytokine release syndrome (CRS). KVA12123 exhibited strong anti-tumor activity alone and in combination with anti-PD1 in preclinical studies. It demonstrated an excellent safety profile in non-human primates. The VISTA-101 clinical trial is a first-in-human, Phase 1/2, multicenter, open-label, safety, PK and pharmacodynamic (PD) evaluation of KVA12123, both as monotherapy and in combination with pembrolizumab, in adult patients with advanced solid tumors (NCT05708950). METHODS: The phase 1 of VISTA-101 is an accelerated Bayesian Optimal Interval Design consisting of two arms: Part A) KVA12123 monotherapy dose escalation from 3 mg to 1000 mg including 6 cohorts and Part B) KVA12123 dose escalation from 30 mg to 1000 mg in combination with 400 mg fixed dosing of pembrolizumab including 4 cohorts. The primary objectives are safety, tolerability and to define a recommended phase 2 dose. Safety, PK and receptor occupancy data were considered during dose escalation. VISTA-101 is also assessing PD and predictive biomarkers of response. Patient tumor response is evaluated by iRECIST. RESULTS: Forty-two patients have been enrolled in the dose escalation monotherapy arm of the study. KVA12123 was administered at doses ranging from 3 to 1000 mg every 2 weeks over a 6-week cycle. KVA12123 was well tolerated at all dose levels and no DLTs were observed. The most frequently observed adverse events were grade 1 or 2 transient infusion-related reactions. No evidence of CRS-associated cytokines were detected. PK analysis demonstrated greater than dose-proportional exposure consistent with target-mediated metabolism. A VISTA receptor occupancy assay demonstrated full target engagement at doses of 30 mg and above. PD analysis indicated a dose-dependent induction of pro-inflammatory myeloid derived cytokines and chemokines involved in immune cell activation and recruitment to the tumor microenvironment including CCL2, CCL3, CCL4 and CXCL10. Increases in nonclassical monocytes, NK cells, CD4+ and CD8+ T cells were also demonstrated, consistent with observations made in preclinical models and indicative of VISTA target engagement. Patient tumor response evaluations in the monotherapy arm will be reported. The study continues to evaluate KVA12123 both as a monotherapy and in combination with pembrolizumab to define the recommended phase 2 dose. Citation Format: Evan Y. Yu, Jason Henry, Manish R. Patel, Paul Swiecicki, Ida Micaily, Benjamin Garmezy, Kalyan Banda, Vinny Hayreh, Kurt Lustig, Yulia Ovechkina, Shawn P. Iadonato, Thierry Guillaudeux, Lee Rosen. Interim results of the ongoing phase 1-2 clinical trial of KVA12123, an engineered IgG1 targeting VISTA, as monotherapy and in combination with pembrolizumab in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT068.

Oral PD-L1 inhibitor GS-4224 selectively engages PD-L1 high cells and elicits pharmacodynamic responses in patients with advanced solid tumors

BackgroundCheckpoint inhibitors targeting the programmed cell death 1 (PD-1)/programmed cell death 1 ligand 1 (PD-L1) pathway are effective therapies in a range of immunogenic cancer types. Blocking this pathway with...

Novel covalent CDK7 inhibitor potently induces apoptosis in acute myeloid leukemia and synergizes with Venetoclax

IntroductionThe emergence of resistance to the highly successful BCL2-directed therapy is a major unmet need in acute myeloid leukemia (AML), an aggressive malignancy with poor survival rates. Towards identifying therapeutic options for AML patients who progress on BCL2-directed therapy, we studied a clinical-stage CDK7 inhibitor XL102, which is being evaluated in solid tumors (NCT04726332).Materials and methodsTo determine the anti-proliferative effects of XL102, we performed experiments including time-resolved fluorescence resonance energy transfer, target occupancy, cell cycle and apoptosis-based assays. We also included genetically characterized primary myeloid blasts from de novo and relapsed/refractory AML patients. For mechanistic studies, CRISPR/Cas9 mediated knockout of CDK7 and c-Myc and immunoblotting were performed. NOD/SCID orthotropic and subcutaneous AML xenografts were used to determine anti-leukemic effects. To assess the synergistic effects of XL102 with Venetoclax, we performed RNA sequencing and gene set enrichment analysis using Venetoclax sensitive and resistant model systems.ResultsXL102, a highly specific, orally bioavailable covalent inhibitor of CDK7. Inhibitory effect on CDK7 by XL102 in primary myeloid blasts (n = 54) was in nanomolar range (mean = 300 nM; range = 4.0-952 nM). XL102 treated AML cells showed a reduction in phosphorylation levels of Serine 2/5/7 at carboxy-terminal domain of RNA polymerase II. T-loop phosphorylation of CDK1(Thr161) and CDK2(Thr160) was inhibited by XL102 in dose-dependent manner leading to cell-cycle arrest. c-Myc downregulation and enhanced levels of p53 and p21 in XL102 treated cells were observed. Increased levels of p21 and activation of p53 by XL102 were mimicked by genetic ablation of CDK7, which supports that the observed effects of XL102 are due to CDK7 inhibition. XL102 treated AML xenografts showed remarkable reduction in hCD45 + marrow cells (mean = 0.60%; range = 0.04%-3.53%) compared to vehicle control (mean = 38.2%; range = 10.1%-78%), with corresponding increase in p53, p21 and decrease in c-Myc levels. The data suggests XL102 induces apoptosis in AML cells via CDK7/c-Myc/p53 axis. RNA-sequencing from paired Venetoclax-sensitive and Venetoclax-resistant cells treated with XL102 showed downregulation of genes involved in proliferation and apoptosis.ConclusionTaken together, XL102 with Venetoclax led to synergistic effects in overcoming resistance and provided a strong rationale for clinical evaluation of XL102 as a single agent and in combination with Venetoclax.

Abstract 5673: Characterization of AB598, a therapeutic anti-human CD39 antibody for the treatment of cancer

Abstract Background: CD39 catalyzes the conversion of extracellular adenosine triphosphate (ATP) into adenosine monophosphate (AMP), resulting in decreased levels of immunostimulatory ATP and increased levels of immunosuppressive adenosine in the tumor microenvironment (TME). By blocking CD39 in the TME, particularly in combination with immunogenic cell death (ICD)-inducing chemotherapies, local levels of ATP can increase, leading to myeloid cell activation and improved tumor control. AB598 potently binds and inhibits enzymatic activity of human and cynomolgus CD39 but not murine CD39. Human CD39 knock-in (hCD39KI) mice have been employed to determine anti-tumor efficacy of AB598 in animals with competent immune systems. Methods: Binding affinity and enzymatic inhibition of AB598 were determined in primary human immune cells. ATP-mediated activation of monocyte-derived dendritic cells (moDCs) and macrophages were assayed in vitro with and without AB598. Whole blood and tissue-based receptor occupancy (RO) assays were developed using AB598-competitive and non-competitive anti-CD39 reagents. In vitro binding of AB598 was determined in samples from human and cynomolgus donors. CD39 protein expression, target engagement, and enzymatic inhibition were assessed in whole blood and tissue samples collected from AB598-dosed animals. CD39 expression patterns in peripheral immune subsets were confirmed in samples from healthy human donors. CD39 enzymatic activity was evaluated in blood-derived cells and serum from healthy human donors and cancer patients. Results: AB598 binds to CD39-expressing monocytes, B cells, and other immune cell populations in human and cynomolgus whole blood with sub-nanomolar affinity. Following AB598 dosing in preclinical species, decreases in CD39 surface expression on peripheral immune cells were observed, consistent with other anti-CD39 therapeutic antibodies. Target engagement and enzymatic inhibition were evident in tissues collected from AB598-dosed animals. Treatment with AB598/chemotherapy combinations resulted in significant syngeneic tumor control in hCD39KI mouse models. Conclusion: When used in combination with an ICD-inducing chemotherapy agent, AB598 can promote anti-tumor immunity by activation of myeloid cells due to increased local ATP levels. Preclinical characterization of the pharmacodynamic effects of AB598 support our therapeutic rationale and demonstrate target engagement and inhibition both peripherally and intratumorally. Citation Format: Amy E. Anderson, Angelo Kaplan, Urvi Vani, Enzo Stagnaro, Kaustubh Parashar, Julie Clor, Nigel P. Walker, Steve W. Young, Matthew J. Walters, Ester Fernandez-Salas, Christine E. Bowman, Lisa Seitz. Characterization of AB598, a therapeutic anti-human CD39 antibody for the treatment of cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5673.

Abstract 1976: Development of a CD137 receptor occupancy assay to support the phase I/II study of BT7480, a Bicycle tumor-targeted immune cell agonist™ (Bicycle TICA®)

Abstract Bicycles are fully synthetic constrained peptides with antibody-like affinities that target selectively, readily penetrate tumor tissue, have relatively short half-lives, and can be chemically linked together to generate multifunctional molecules. BT7480 is a Bicycle TICA® being developed as a first-in-class CD137 therapeutic for the treatment of human cancers associated with Nectin-4 expression which is currently being investigated in an ongoing phase I/II clinical trial. Monitoring target engagement for a given therapeutic can be a key factor in recommending the phase II dose. While flow cytometry-based receptor occupancy (RO) assays are commonly used to monitor target engagement in the clinic, a CD137-specific RO assay presents several important challenges that have historically hampered monitoring RO in the clinic including the dynamic expression of CD137 on unstimulated and stimulated T cells, the low frequency of CD137+ cells in human blood and limited reagents to confidently detect CD137+ cells in the presence of CD137-targeting drugs. To address these challenges, a fit-for-purpose 14-plex flow cytometry panel was developed that incorporates a fluorescently labelled CD137-specific binding Bicycle®. This CD137 Bicycle® was shown to directly compete with BT7480 for binding to CD137, but not with fluorescently labelled anti-CD137 antibody, thereby enabling simultaneous detection of various CD137+ immune cell types as well as receptor occupancy by BT7480 in a single blood sample. Panel performance was tested across blood-based sample matrices routinely used in the clinic including EDTA and Cyto-Chex® blood collection tubes and Cell Preparation Tubes (CPT) (n=3 each). CPT were selected as the optimal sample matrix based on sample viability and highest detection of CD137 antibody+ and CD137 Bicycle® + cells. Ex vivo RO assessments in anti-CD3 stimulated and unstimulated healthy human blood, demonstrated dose-dependent detection of CD137 RO by BT7480 and the detection of >1000 CD137+ cells with sample viability >70% (n=5 each). The optimized method and dose-dependent detection of CD137+ cells and RO by BT7480 was further verified in unstimulated lung cancer patient whole blood samples (n=5). Results from this study represent the first report of a clinic-ready CD137 RO assay and the first flow cytometry assay using fluorescently labelled Bicycle® reagents and demonstrate the utility of the Bicycle® CD137 RO assay to monitor target engagement in the BT7480 first-in-human clinical trial. Citation Format: Heather Cohen, Drasti Kanakia, Johanna Lahdenranta, Punit Upadhyaya, Kristen Hurov, Julia Kristensson, Chintan Jobaliya, Greg Bannish, Adam Cotty, Kevin McDonnell, Sandra Hirschberg, Phil Brandish, Sebastien Hazard, Dominic Smethurst, Nicholas Keen, Stephen J. Blakemore. Development of a CD137 receptor occupancy assay to support the phase I/II study of BT7480, a Bicycle tumor-targeted immune cell agonist™ (Bicycle TICA®) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1976.

Abstract 1870: A simple and quantitative immunoassay for evaluation of receptor occupancy and antibody binding capacity in fixed cells or tissue samples for immune-directing antibody development

Abstract Receptor occupancy (RO) assays are used to quantify the binding of therapeutic antibodies to their targets on the cell surface. This method can be used to in early drug discovery to determine the antibody binding capacity (ABC) of drug candidates to cells in culture to hone the desired drug characteristics during candidate screening or lead optimization. This method can also be used in xenografts or patient tissues to create pharmacodynamic (PD) data that directs the efficacy hypothesis and biomarker selection for clinical trials. The current approaches use flow cytometry on fresh specimens, which introduces many technical and logistical challenges and does not retain spatial information about the tissue. Here we introduce a new, simple method which can be performed on Formalin Fixed, Paraffin Embedded (FFPE) cells or tissues. This approach eliminates the challenges of flow cytometry methods, provides the type of quantitative data needed, and maintains the tissue contexture to study the effects of immune-directing antibodies in patient tissue samples. We describe a method for developing a standard curve to determine antibody binding capacity (ABC) using our Quanticell ™ fluorescent nanoparticle technology. We demonstrate this in a typical bispecific antibody use case, where both the immune-directing (CD3) and targeting (Her2) arms of the bispecific are modulated to create the desired efficacy characteristics. We demonstrate the establishment of a standard calibration curve for Her2 (BT474/Her2+) and CD3 (Jurkat/CD3+) in cells with titrated amounts of anti-Her2 and anti-CD3 antibodies which target the extracellular domain necessary for these studies. We also show how the same Quanticell assay can be used in clinical tissue, to evaluate the localization of the drug targets and immune cells within the tissue context of human patient samples necessary to understand how pharmacodynamic properties of the drug can modulate immune contexture changes. The Quanticell™ method is a simple immunohistochemistry (IHC) based method which allows extremely sensitive detection of therapeutic antibodies bound on FFPE fixed cells or tissue, which thus does not require the use of flow cytometry in fresh tissues, allowing simple use from early discovery through clinical research studies. Importantly, the Quanticell™ method is capable of detecting a very large and linear dynamic range of analyte concentration to allow full quantification of ABC for RO studies. Critically, the Quanticell™ method retains the tissue context, allowing for simultaneous and spatial evaluation of multiple analytes such as the target of the antibody and the immune contexture. This method allows for a consistent method for RO studies that can be applied to early discovery, translational, and clinical research to optimize drug efficacy. Citation Format: Atsuro Tatsumi, Keisuke Morichika, Caroline Morel, Omid Ghasemi, Hiroyuki Yokota, Joseph S. Krueger. A simple and quantitative immunoassay for evaluation of receptor occupancy and antibody binding capacity in fixed cells or tissue samples for immune-directing antibody development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1870.

Abstract 4041: Use of Receptor Occupancy Assays on cells from Peripheral Blood as a Surrogate Model of the Tumor Microenvironment

Abstract In the development of monoclonal antibodies for the treatment of solid tumors, monitoring receptor occupancy (RO) on peripheral blood lymphocytes can help illustrate potential therapeutic activity occurring in the tumor environment as an assessment of pharmacodynamics (PD). There is increased interest in using these PD assessments on cells from the periphery to assess the effectiveness and efficacy of investigational therapies. In this area of therapeutic development, much focus has been placed on checkpoint inhibitors proteins/receptors such as PD-1, PD-L1, TIM3, CTLA-4, TIGIT, and multiple others. These immune checkpoint inhibitors are suitable targets since they can be upregulated and/or modulated on exhausted T cells in cancer patients. Assessing the expression of these markers in the tumor itself would be an invasive process and provide little information to correlate with pharmacokinetic results throughout the course of a clinical trial to determine optimal dose selection. However, assessment of the receptor expression and occupancy in combination with pharmacokinetics can lead to a better understanding of drug levels required to achieve optimal therapeutic performance. Monitoring the expression and binding of these molecules by drug can determine specific treatment based on the ability of the patient’s own immune system to act, while providing information to identify therapeutic responses against cancer. This poster will present an example of one such RO assessment in its development and implementation. Citation Format: Nicholas Jones, Brian Ngo, Benjamin Fancke, Jessica Limson, Floyd Davis. Use of Receptor Occupancy Assays on cells from Peripheral Blood as a Surrogate Model of the Tumor Microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4041.

CD86 occupancy in belatacept-treated kidney transplant patients is not associated with clinical and infectious outcomes

The CD86 occupancy assay has been developed to measure the number of CD86molecules unbound to belatacept, but its association with clinical outcomes has not been assessed yet. All kidney transplant patients switched to belatacept in our center between 2016 and 2018 were included. Blood samples were collected before each infusion for 1year to assess CD86 occupancy by CD86 antibody cytometry staining on the surface of CD14+ monocytes. Results were expressed as the median fluorescence intensity (MFI) value of CD86staining. At each infusion, the MFIDay of infusion /MFIDay 0 ratio was calculated. Forty-one patients were consecutively included. After every 2-week infusion period, CD86MFI ratio dropped from 1.00 to 0.73 [0.57-0.98], p=.07. However, this ratio progressively increased to 0.78 [0.53-1.13] at 1year, which was not statistically different from pre-switch ratio, p=.4. Over the first year, the MFI ratio coefficient of variation was 31.58% [23.75-38.31]. MFI ratio was not different between patients with or without opportunistic infections: 0.73 [0.60-0.88] versus 0.80 [0.71-1.00], p=.2, or between patients with or without EBV DNAemia, p=.2. Despite previous in vitro results, the CD86 occupancy assay suffers from a high intra-individual variability and does not appear to be relevant to clinical outcomes.

P019 GS-1069518 is a potent and selective small molecule α4β7 inhibitor

Abstract Background The interaction between the adhesion molecule MAdCAM-1 and the α4β7 integrin heterodimer enables the trafficking of gut-antigen specific lymphocytes to the small and large intestines and to gut-associated lymphoid tissues. The role of the α4β7 integrin in the pathophysiology of IBD has been clinically demonstrated, and targeted therapeutics have been shown to mitigate inflammation and mucosal damage mediated by α4β7+ lymphocytes. The purpose of this study was to determine the potency and selectivity of the small molecule α4β7 inhibitor GS-1069518. Methods The selectivity of GS-1069518 and relevant reference molecules was determined using in vitro cell capture assays with natural integrin ligands and by measuring receptor occupancy in human and cynomolgus monkey whole blood. The number of RPMI-8866 cells binding to MAdCAM-1-coated surfaces (a natural α4β7 ligand) and Jurkat cells binding to VCAM-1-coated surfaces (a natural α4β1 ligand) were quantified with high content imaging. Receptor occupancy on CD3+CD4+CD45RO+β7+ (human) or CD3+CD4+CD45RA-β7+ (cynomolgus monkey) and CD3+CD4+CD45RO+α4+β1+β7- (human) or CD3+CD4+CD45RA-α4+β1+β7- (cynomolgus monkey) memory T cells was measured by flow cytometry with fluorescent probes that detect occupancy in the ligand-binding sites. Results In a MAdCAM-1 cell capture assay, the EC50 of GS-1069518 was 0.034 nM, with 71-fold and >10,000-fold selectivity over α4β1 and αLβ2, respectively. In α4β7 receptor occupancy assays the EC50 of GS-1069518 was 0.36 nM in human whole blood and 0.4 nM in cynomolgus monkey whole blood. GS-1069518 was 482-fold selective for α4β7 over α4β1 in human whole blood and 119-fold selective for α4β7 in cynomolgus monkey blood. Conclusion GS-1069518 is a highly potent and selective small molecule inhibitor of the α4β7-MAdCAM-1 ligand binding interaction.

Use of Receptor Occupancy Assays on Cells from Peripheral Blood As a Model to Determine Therapeutic Efficacy

In the development of monoclonal antibodies for the treatment of liquid or solid tumors, monitoring receptor occupancy (RO) on peripheral blood lymphocytes can help illustrate potential therapeutic activity occurring systemically, or in the tumor environment. There is increased interest in using these assessments on cells from the periphery to assess the efficacy of investigational therapies. In this area of therapeutic development, much focus has been placed on checkpoint inhibition proteins/receptors such as PD-1, PD-L1, TIM3, CTLA-4, TIGIT, and multiple others. These immune checkpoint inhibitors are suitable targets since they can be highly modulated on exhausted T cells in cancer patients. In the case of solid tumors, assessing the expression of these markers in the tumor itself via biopsy would be an invasive process that could not be performed sequentially and, therefore, would provide little information to correlate with pharmacokinetic results throughout the course of a clinical trial to determine timing and optimal dose selection. However, assessment of the receptor expression and occupancy in combination with pharmacokinetics can lead to a better understanding of drug levels required to achieve optimal therapeutic performance. Monitoring the expression and binding of these molecules by a drug can determine specific treatment based on the ability of the patient's own immune system to act, while providing information to identify therapeutic responses against cancer. This poster will present an example of receptor occupancy (RO) assessment in its development and implementation. DisclosuresNo relevant conflicts of interest to declare.

Pharmacodynamics of SEA-BCMA, a Nonfucosylated Antibody Targeting BCMA, in Patients with Relapsed/Refractory Multiple Myeloma

Patients with relapsed/triple class refractory (refractory to a proteasome inhibitor, immunomodulatory drug or anti-CD38 antibody) multiple myeloma (MM) have limited treatment options. Versatile therapies, such as unconjugated antibodies (Abs), that are well tolerated and can be combined with multiple modalities are critical for the MM treatment landscape. SEA-BCMA is an investigational, humanized, nonfucosylated IgG1 monoclonal Ab targeting B-cell maturation antigen (BCMA) on malignant plasma cells. Preclinical data show that SEA-BCMA blocks BCMA-mediated pro-survival and proliferative cell signaling and mediates antibody-dependent cellular phagocytosis and enhanced antibody-dependent cellular cytotoxicity via increased binding to activating Fc receptor, FcγRIIIa (Van Epps 2018). A phase 1, open-label, multicenter study to evaluate the safety, tolerability, and antitumor activity of SEA-BCMA in adults with relapsed or refractory MM (SGNBCMA-001; NCT03582033) is ongoing. To support maximal ligand blocking and immune effector engagement by SEA-BCMA, we investigated its binding and saturation pharmacodynamics (PD) in patients enrolled in dose escalation and currently recruiting dose expansion cohorts.Novel quantitative assays were developed to assess the BCMA target and the availability of SEA-BCMA in patients enrolled in the study. These assessments include: a liquid chromatography-mass spectrometry (LC-MS) soluble BCMA (sBCMA) assay, a BCMA receptor occupancy assay on malignant plasma cells in bone marrow aspirates, and a receptor binding assay to assess the direct binding and saturation capacity of SEA-BCMA in patients' serum samples and bone marrow aspirates to a BCMA-expressing cell in vitro. Results reported as median (min-max) values unless specified.At baseline, the median sBCMA level from patients enrolled in SGNBCMA-001 was 8.5(0.5-217.0) ng/mL. After the first dose of SEA-BCMA, a rapid and sustained increase in sBCMA was observed [32.0 (3.0-139.0) fold]. Mechanistically, because SEA-BCMA can bind to sBCMA, accumulation of sBCMA may occur due to formation of the sBCMA:SEA-BCMA complex, resulting in reduced clearance. Importantly, for patients at the dose selected for expansion (1600mg), the molar ratio of SEA-BCMA to sBCMA remained in excess (10:1-400:1), thereby overcoming the liabilities of this complex formation and supporting malignant plasma cell drug exposure. Results of a receptor binding assay using patients' serum samples also demonstrated a dose-dependent ability to saturate BCMA-expressing cells. Evaluation of receptor occupancy in patient bone marrow aspirates showed that at baseline, median unoccupied membrane BCMA (unbound by ligand) was 3,500(1,500-30,000) copies per cell with one excluded outlier that exhibited 270,000 copies per cell. On-treatment, unoccupied membrane BCMA reductions were observed ranging from 50% to 100% from baseline in majority of evaluable patients at the 1600mg dose. An on-treatment increase in total BCMA expression was also observed, which is being further investigated.Maximal ligand blocking and immune effector engagement is best achieved through saturation of a target receptor. Overall, these PD results provide insight into the saturation potential of plasma cell membrane BCMA by SEA-BCMA and informed the dose selection and schedule in expansion cohorts. Further evaluation of these relationships to patient response is ongoing as the Phase 1 study continues to enroll. DisclosuresTaft: Seagen, Inc: Current Employment, Current equity holder in publicly-traded company. Henderson: Seagen, Inc: Current Employment, Current equity holder in publicly-traded company. O'Day: Seagen, Inc: Current Employment, Current equity holder in publicly-traded company. Yu: Seagen, Inc: Current Employment, Current equity holder in publicly-traded company. Li: Seagen Inc.: Current Employment, Current equity holder in publicly-traded company. Ho: Seagen Inc.: Current Employment, Current equity holder in publicly-traded company. Van Epps: Seagen, Inc: Current Employment, Current equity holder in publicly-traded company.

P037 Nonclinical pharmacokinetics and absorption, distribution, metabolism, and excretion properties of MORF-057 support its clinical development as an oral selective α4β7 integrin inhibitor

Abstract Background MORF-057 is a potent and selective small molecule inhibitor of the α 4β 7 integrin. Using a receptor occupancy (RO) assay under physiologically relevant conditions, MORF-057 achieves 90% α 4β 7 RO at approximately 10 nM in human whole blood. The current studies evaluate nonclinical pharmacokinetics (PK) and properties of absorption, distribution, metabolism, and excretion (ADME) to enable dose/exposure projection to humans. Methods PK studies were conducted in mouse, rat, dog, and monkey following intravenous (IV) and oral administration. ADME studies were conducted in vitro and in rats using carbon-14 [14C] labeled MORF-057. MORF-057 levels were quantified using liquid chromatography coupled with tandem mass spectrometry. Human PK was predicted based on body weight allometry, well-stirred and semi-physiological models. Results MORF-057 exhibited low to moderate clearance (CL) in animals with species dependent volume of distribution (Vdss) resulting in half-lives of 1.1 to 2.7 hours (Table 1). Following an oral dose, absorption was high with bioavailability ranging from 15% to 49%. MORF-057 is highly protein bound and distribution of [14C]MORF-057 derived radioactivity in rat was predominantly in the small intestine wall, liver, and stomach wall. Rifampin, an inhibitor of organic anion transporting polypeptides, decreased MORF-057 clearance by 3-fold in monkeys suggesting MORF-057 elimination involves hepatic uptake transport. MORF-057 is further cleared via CYP3A metabolism followed by biliary/fecal elimination of metabolites. MORF-057 is predicted to have moderate bioavailability (40%) and CL (6.5 mL/min/kg) in humans. Wajima transformation shows good agreement of the normalized PK across animal species, with a predicted human concentration-time profile supporting >90% α 4β 7 RO at trough following 200 mg twice daily dose (Figure 1). Conclusion These data demonstrate that MORF-057 is well absorbed and PK properties in animals support the potential for achieving high α4β7 RO following oral administration in humans. These nonclinical results provided a basis for the progression of MORF-057 into a first-in-human Phase 1 clinical study assessing safety, pharmacokinetics, and receptor occupancy (results being reported separately).

Modeling relationship of pharmacokinetics, in vitro potency, and α4β7 receptor occupancy with intestinal cell trafficking in a gut-homing mouse model of IBD with MORF-057

Abstract Objective: MORF-057 is an orally bioavailable, selective, and potent small molecule inhibitor of α4β7 integrin being developed for inflammatory bowel diseases (IBD) that is currently in phase 1 clinical testing. We have previously presented work that characterized its nonclinical pharmacologic profile. The current study integrates data to generate a pharmacokinetic (PK) and pharmacodynamic (PD) model of MORF-057. Methods: To determine in vivo potency, MORF-057 was tested in murine gut homing assays and the PD response was determined relative to the non-protein bound drug in mouse plasma. A cell adhesion assay (CAA) for α4β7 was refined to enable detection of picomolar-level sensitivity. Murine receptor occupancy (RO) assays for α4β7 and α4β1 were established under physiologic conditions, and MORF-057 was evaluated for its potency and selectivity in fresh mouse whole blood. These datasets were used to build and validate predictive models of PD response. Results: MORF-057 strongly inhibited the homing of α4β7hi cells to murine gut lymphoid tissues with an IC90 of 7.9 nM. MORF-057 showed high potency in CAA with an IC90 of 8.8 nM. Similarly, RO assays confirmed MORF-057 to be a highly potent inhibitor of α4β7 in mouse whole blood with an IC90 of 20.5 nM and over 1500-fold selectivity vs. α4β1. The predictive models built upon these datasets revealed a strong PK-PD relationship of α4β7 inhibitors in vivo. Conclusions: We observed consistently high potency of MORF-057 across multiple assay platforms. Integrated modeling based on these assays, particularly the RO assay, successfully predicted the PD response to MORF-057. These data begin to establish the relationship between PK, target engagement, and PD with MORF-057.

Evaluation of monoamine oxidase A and B type enzyme occupancy using non-radiolabelled tracers in rat brain

Monoamine oxidase (MAO) enzymes, type A and B metabolise the amine neurotransmitters of the body. Selective inhibition of either enzyme is an approach for treating neurodegenerative and stress-induced disorders, and inhibition of an enzyme is proportional to the binding of the MAO inhibitor. Conventionally, the binding of test compounds to enzymes is assessed by radiolabelled ligands in ex vivo and in vivo occupancy assays. Regulatory restrictions and turnaround time are the limitations of the methods that use radiolabelled ligands. But the use of non-radiolabelled tracers and sensitive mass spectrometry (LC-MS/MS) based assays accelerated the determination of target occupancy in pre-clinical species. A report on use of non-radiolabelled ligand in in vivo MAO occupancy assay is not available. The objectives of the present study were to optimise non-radiolabelled harmine and deprenyl as selective tracers in MAO-A and MAO-B occupancy assays and evaluate MAO occupancy of test compounds in rat brain. Tracer optimisation resulted in a detectable, stable, and low ratio (<3.0) of tracer concentrations between any two brain tissues. In occupancy assay, tracer was intravenously administered (10 μg/kg, harmine or 60 μg/kg, L-deprenyl) after the treatment with test compound (clorgyline or tranylcypromine or pargyline or phenelzine or thioperamide). Specific brain tissues were isolated at a defined interval and tracer concentrations were quantified using LC-MS/MS method. Pre-treatment with MAO inhibitors resulted in a decrease (maximum, 80–85%) in harmine or an increase (maximum, 85–300%) in L-deprenyl concentrations. But we considered the change in tracer concentration, relative to the vehicle and positive control groups to calculate MAO occupancy. The observed selectivity and ratio of occupancies (ED50) of test compound towards MAO-A and MAO-B are comparable with the results from in vitro radiolabelled ligand-based inhibition assay. The results demonstrated the application of these non-radiolabelled tracers as suitable pre-clinical tools to determine MAO occupancy.

Abstract PO076: Development of a CD137 (4-1BB) receptor occupancy assay using fluorescently labelled Bicycles®

Abstract Receptor occupancy (RO) assays are designed to quantify the binding of therapeutics to their cell surface targets and are frequently used to generate both pre-clinical and clinical pharmacodynamic biomarker data. Flow cytometry is a commonly used technique to measure receptor occupancy on immune cell populations within fresh blood specimens. Yet, receptor occupancy assays are subject to numerous technical and logistical challenges. To ensure that reliable and high-quality results are generated from receptor occupancy assays, careful assay design and key reagent selection, characterization, and utilization are of critical importance. Antibodies are commonly conjugated with fluorophores and used in receptor occupancy assays for related therapeutics. Bicycles® are a new therapeutic modality - fully synthetic, constrained bicyclic peptides with high affinity and excellent target selectivity. Here, we describe a novel utility for fluorescently-labeled Bicycles in the development of a CD137 (4-1BB) receptor occupancy assay. CD137 is a member of the TNFR superfamily involved in stimulation of several immune cell types, including T cells and NK cells. CD137 is well validated pre-clinically, as agonism with anti-CD137 antibodies is effective in vivo, however, clinical utility to date has been limited by dose dependent hepatotoxicity. We have demonstrated that tumor-targeted immune cell agonists (TICAsTM) comprised of CD137 binding Bicycles coupled to tumor antigen binding Bicycles exert anti tumoral properties with a favorable safety profile. Here, we demonstrate another functionality for the Bicycle platform, as a measurement of CD137 TICA target engagement and receptor occupancy. Using fluorescently-labeled CD137 Bicycles, we measured RO on immune populations in isolated human peripheral blood mononuclear cells (PBMCs), as well as in whole blood. The assay shows high specificity as no Bicycle binding was observed in CD137 negative cell populations. Additionally, RO was shown to be dependent on the presence of CD137-binding Bicycles. Together, this novel approach of measuring receptor occupancy may be used to inform on both the pre-clinical and clinical therapeutic properties of Bicycle TICAs. Citation Format: Drasti N. Kanakia, Punit Upadhyaya, Johanna Lahdenranta, Elizabeth Repash, Eric Haines, Kevin McDonnell, Kristen Hurov, Philip Brandish, Nicholas Keen. Development of a CD137 (4-1BB) receptor occupancy assay using fluorescently labelled Bicycles® [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO076.

Best practices for optimization and validation of flow cytometry-based receptor occupancy assays.

In the development of therapeutic compounds that bind cell surface molecules, it is critical to demonstrate the extent to which the drug engages its target. For cell-associated targets, flow cytometry is well-suited to monitor drug-to-target engagement through receptor occupancy assays (ROA). The technology allows for the identification of specific cell subsets within heterogeneous populations and the detection of nonabundant cellular antigens. There are numerous challenges in the design, development, and implementation of robust ROA. Among the most difficult challenges are situations where there is receptor modulation or when the target-antigen is expressed at low levels. When the therapeutic molecules are bi-specific and bind multiple targets, these challenges are increased. This manuscript discusses the challenges and proposes best practices for designing, optimizing, and validating ROA.

Abstract 4024: NAMPT inhibitor decreases NAMPT capture by an antibody directed against the 5-phosphoribosyl-1-pyrophosphate-binding loop: Rationale for an NAMPT occupancy assay

Abstract Nicotinamide phosphoribosyl transferase (NAMPT) catalyzes the rate-limiting step of nicotinamide adenine dinucleotide (NAD) biosynthesis. As NAMPT inhibitors (NAMPTi) have been shown to be substrates of NAMPT, binding of NAMPTi to NAMPT results in the NAMPTi-phosphoribose adduct (RP) that binds tightly and strongly inhibits the NAMPT enzyme. Structural analyses performed in this study unveiled the proximity of the NAMPTi-RP complex to the phosphoribosyl diphosphate (PRPP)-binding loop within the NAMPT catalytic site. The PRPP-binding loop of NAMPT is subject to conformational flexibility. The PRPP-binding loop domain is oriented to the outer side of the NAMPT dimer and can potentially serve as an antigen-binding site for antibodies. Interestingly, we show that the NAMPTi-RP adduct bound to NAMPT decreases NAMPT capture by an antibody that is directed against the c-terminal 5-phosphoribosyl-1-pyrophosphate-binding loop in NAMPT. This finding was then used to explore cellular NAMPT occupancy. The NAMPTi-RP complex displays a sustained, cellular NAMPT occupancy that correlates with the inhibition of NAMPT activity. Moreover, a good correlation between NAMPT occupancy, NAD decrease, and NAMPTi efficacy was observed in-vivo in a NCI-H82 small cell lung cancer (SCLC) xenograft model. NAMPT-occupancy assay can be used in clinical settings to better define the optimal dose levels and dose regimen for effective NAMPT inhibition. Citation Format: Maysoun Shomali, Sandrine Hilairet, Marianne Duhamel, Frederico Nardi, Thomas Bertrand, Geraldine Penarier, Laurent Debussche, Monsif Bouaboula. NAMPT inhibitor decreases NAMPT capture by an antibody directed against the 5-phosphoribosyl-1-pyrophosphate-binding loop: Rationale for an NAMPT occupancy assay [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 4024.

Digital Receptor Occupancy Assay in Quantifying On- and Off-Target Binding Affinities of Therapeutic Antibodies.

While monoclonal antibodies are the fastest-growing class of therapeutic agents, we lack a method that can directly quantify the on- and off-target binding affinities of newly developed therapeutic antibodies in crude cell lysates. As a result, some therapeutic antibody candidates could have a moderate on-target binding affinity but a high off-target binding affinity, which not only gives a reduced efficacy but triggers unwanted side effects. Here, we report a single-molecule counting method that precisely quantifies antibody-bound receptors, free receptors, and unbound antibodies in crude cell lysates, termed digital receptor occupancy assay (DRO). Compared to the traditional flow cytometry-based binding assay, DRO assay enables direct and digital quantification of the three molecular species in solution without the additional antibodies for competitive binding. When characterizing the therapeutic antibody, cetuximab, using DRO assay, we found the on-target binding ratio to be 65% and the binding constant (Kd) to be 2.4 nM, while the off-target binding causes the binding constant to decrease by 0.3 nM. Other than cultured cells, the DRO assay can be performed on tumor mouse xenograft models. Thus, DRO is a simple and highly quantitative method for cell-based antibody binding analysis which can be broadly applied to screen and validate new therapeutic antibodies.

Relative Selectivity of Covalent Inhibitors Requires Assessment of Inactivation Kinetics and Cellular Occupancy: A Case Study of Ibrutinib and Acalabrutinib.

Kinases form an attractive class of targets for small molecule inhibitors, but similarity among their adenosine triphosphate binding sites presents difficulties for developing selective drugs. Standard methods of evaluating selectivity of most reversibly bound drugs account for binding affinity but not the two-step process, affinity and inactivation, occurring during covalent inhibition. To illustrate this concept, we assessed the selectivity of Bruton's tyrosine kinase (BTK) over TEC kinases by two novel therapeutics: ibrutinib and acalabrutinib. The two-step process and time-dependent inhibition unique to covalent inhibitors were evaluated with two biochemical assays measuring enzymatic function and inhibition kinetics. The selectivity for BTK over TEC found in these biochemical analyses was 1-1.5 for ibrutinib and 3.0-4.2 for acalabrutinib. To further assess drug selectivity in a more physiologically relevant context, we developed cell-based occupancy assays that quantify the percentage of drug-inactivated kinases. Cellular selectivity of BTK over TEC was determined after MWCL-1 cells, and samples from patients with chronic lymphocytic leukemia (CLL) were treated for durations and concentrations based on human pharmacokinetics of each drug. In MWCL-1 cells, BTK/TEC selectivities measured at 0.5, 1, and 3 hours were 2.53, 1.05, and 1.51 for ibrutinib and 0.97, 1.13, and 2.56 for acalabrutinib, respectively. The equivalent selectivity measured in samples from patients with CLL were 1.31 ± 0.27 and 1.09 ± 0.11 for ibrutinib and acalabrutinib, respectively. Collectively, our data show that when properly accounting for time-dependent factors and relevant cellular context, ibrutinib and acalabrutinib demonstrate similar selectivity for BTK over TEC. SIGNIFICANCE STATEMENT: This study shows relative selectivity of covalent inhibitors toward different kinase targets should be assessed with both affinity and inactivation kinetics to accurately account for time-dependent effects of covalent binding and assessed in a cellular matrix to reproduce the physiologic context of target inhibition. This is illustrated with a case study of ibrutinib and acalabrutinib for which selectivity assessment with appropriate assays, as opposed to measuring binding affinity with KINOMEscan alone, corroborate emerging clinical data demonstrating similar safety profiles between the therapies.