Target Occupancy Research Articles

135P First-in-human phase I trial of SRF617, a potent inhibitor of CD39 activity, as monotherapy or in combination, in patients (pts) with advanced solid tumors

135P First-in-human phase I trial of SRF617, a potent inhibitor of CD39 activity, as monotherapy or in combination, in patients (pts) with advanced solid tumors

Abstract P124: JDQ443, a covalent irreversible inhibitor of KRAS G12C, exhibits a novel binding mode and demonstrates potent anti-tumor activity and favorable pharmacokinetic properties in preclinical models

Abstract RAS is the most frequently mutated oncogene in cancer. KRAS G12C mutations are most prevalent in lung adenocarcinoma (~13%) and colorectal adenocarcinoma (~4%), and occur less commonly in other solid tumor malignancies. First generation KRASG12C inhibitors show anti-tumor activity in early phase clinical trials. However, the emergence of resistance, mediated at least in part by RAS gene mutations that disrupt inhibitor binding and reactivation of downstream pathways, limit the duration of response. Here we report the identification of JDQ443 (NVP-JDQ443), a novel KRASG12C inhibitor which binds under the switch II loop with a novel binding mode, exploiting unique interactions with the KRASG12C protein compared to sotorasib and adagrasib. JDQ443 potently inhibits KRASG12C cellular signaling and proliferation in a mutant selective manner by irreversibly trapping the GDP-bound state of KRASG12C through formation of a covalent bond with cysteine at position 12. Consistent with its mechanism as an irreversible inhibitor, JDQ443 shows sustained target occupancy (TO) in vivo (KRASG12C TO t1/2 ~ 66 h in the MiaPaCa2 model) despite a blood half-life of ~ 2 hours, and exhibits a linear PK/PD relationship. JDQ443 has dose-dependent anti-tumor activity in mice bearing KRAS G12C mutated tumor xenografts comparable to sotorasib and adagrasib. In mouse, rat, and dog, JDQ443 is orally bioavailable, achieves exposures in a range predicted to confer anti-tumor activity, and is well-tolerated. Continuous delivery of JDQ443 using mini-pump administration demonstrates that area under the curve (AUC), rather than maximal concentration (Cmax), is the driver of efficacy. Combination of JDQ443 with the SHP2 inhibitor TNO155 further increases KRAS G12C target occupancy in vivo, enhanced pre-clinical anti-tumor activity, and delayed the emergence of resistance in xenografts. A genome-wide CRISPR screen in 5 KRAS G12C mutated lung cancer cell lines identifies novel mechanisms of resistance to the KRAS/SHP2 drug combination. Furthermore, the characterization of JDQ443 alone and in combination with TNO155 in BaF/3 pools addicted to KRAS alleles that have previously been shown to mediate resistance to adagrasib in clinical samples will be discussed. Collectively, these data show that JDQ443 is a potent, mutant-selective, covalent irreversible KRASG12C inhibitor with favorable pharmaceutical properties. A phase Ib/II clinical trial of JDQ443 alone and in combination with TNO155 in patients with advanced solid tumors harboring the KRAS G12C mutation is ongoing (NCT04699188). Citation Format: Saskia M. Brachmann, Andreas Weiss, Daniel A. Guthy, Kim Beyer, Johannes Voshol, Michel Maira, Anirudh Prahallad, Diana Graus Porta, Christian Schnell, Nils Ostermann, Andrea Vaupel, Marc Gerspacher, Catherine Leblanc, Dirk Erdmann, Dario Sterker, Grainne Kerr, Giovannoni Jerome, Victoria Head, Rowan Stringer, Ruben De Kanter, Kearns Jeff, Danielle Roman, Toni Widmer, Peter Wessels, Eloisa Jimenez Nunez, Richard Sedrani, Frederic Zecri, Francesco Hofmann, Jeff Engleman, Edwige Lorthiois, Simona Cotesta. JDQ443, a covalent irreversible inhibitor of KRAS G12C, exhibits a novel binding mode and demonstrates potent anti-tumor activity and favorable pharmacokinetic properties in preclinical models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P124.

Brain target occupancy of LY3372689, an inhibitor of the O‐GlcNAcase (OGA) enzyme, following administration of single and multiple doses to healthy volunteers

AbstractBackgroundLY3372689, an O‐GlcNAcase (OGA) enzyme inhibitor, is being developed as a potential treatment of tauopathies, including Alzheimer’s disease. OGA inhibition is proposed to delay the progression of tau‐related diseases by slowing the accumulation of hyper‐phosphorylated, insoluble tau filaments. We report on single dose (SD) and multiple dose (MD) clinical studies testing the effect of LY3372689 on brain OGA enzyme occupancy (EO).MethodA positron emission tomography (PET) radioligand, 18F‐LY3316612, was used to assess brain OGA EO in healthy volunteers (HV). In the SD study [NCT03944031], 0.25, 1 and 5 mg LY3372689 were evaluated across 4 cohorts (N = 4 HV per cohort). Each HV had PET scans at baseline and two post‐dose intervals. Post‐dose scans occurred at 2 and 24 hours at 0.25, 1, and 5 mg, with additional scans for 1 mg at 30 and 54 hours. The MD study [NCT04392271] consisted of 1 cohort (N = 4 HV) given 1 mg LY3372689 once daily for 14 days. Each HV had a baseline PET scan, and PET scans at 24 hours post‐dose after the 1st and 14th administration of LY3372689. Plasma pharmacokinetics was assessed in these studies.ResultIn the SD study, the mean brain OGA EO at 5 mg was 98% at 2 hours and 93% at 24 hours. At 1 mg, the mean EO was 97% at 2 hours, 81% at 24 hours, 68% at 30 hours, and 30% at 54 hours. The EO at 0.25 mg was lower at 2 hours (26%) compared to 24 hours (46%). The Emax and EC50 values were estimated to be 97% and 0.1 ng/mL, respectively. In the MD study, the OGA EO at 24 hours after the 1st and 14th administration of 1 mg LY3372689 was 84%.ConclusionPET studies in healthy volunteers demonstrated that LY3372689 can achieve high brain target occupancy of the OGA enzyme. Brain OGA EO was maintained after multiple dosing, supporting the durability of target engagement for longer clinical trials. The human PET data will be used to support LY3372869 dose selection for efficacy trials in tauopathies.

The development of [18F]ACI‐12589, a high affinity and selective alpha‐synuclein radiotracer, as a biomarker for Parkinson’s disease and other synucleinopathies

AbstractBackgroundParkinson’s disease (PD) and other alpha‐synucleinopathies are characterized by the progressive accumulation of alpha‐synuclein (a‐syn) inclusions in the brain, which start in the pre‐motor stage of the disease and correlates with its severity. An imaging agent for a‐syn could allow differential diagnosis and support interventional trails. AC Immune has developed and is currently testing in a first in human (FiH) clinical trial the recently discovered compound, ACI‐12589, as a PET tracer with high affinity and selectivity for a‐syn.MethodScreening of our conformation‐specific, small‐molecular weight MorphomerTM library was followed by an iterative medicinal chemistry optimization program. The primary screening strategy included the development and use of a proprietary binding assay, that importantly involved aggregates derived from PD brain samples. Autoradiography experiments performed with human tissue samples from different alpha‐synucleinopathies provided target engagement and signal specificity data. Autoradiography techniques were also employed to assess selectivity, using tissue samples from Alzheimer’s disease or Frontotemporal lobar degeneration‐TDP donors. Selected compounds were 18F radiolabeled and evaluated for their pharmacokinetics in non‐human primates (NHPs) before proceeding to clinical studies.ResultRecent optimization efforts have led to the identification of several new, highly promising chemical scaffolds. From these, the compound ACI‐12589, has emerged with highly favorable characteristics including low nanomolar affinity and excellent target occupancy in saturation binding experiments using PD brain homogenates. Using high‐resolution autoradiography, ACI‐12589 illustrates target engagement across multiple alpha‐synucleinopathies including PD, Multiple System Atrophy, and Dementia with Lewy Bodies. In classical autoradiography experiments, ACI‐12589 demonstrates significantly higher specific signal in diseased versus non‐diseased tissue samples. Furthermore, ACI‐12589 has at least 30‐fold selectivity versus Abeta and shows no binding to Tau nor TDP‐43 aggregates. Studying [18F]ACI‐12589 in NHPs reveals rapid brain uptake and quick wash out. ACI‐12589 was considered safe in the IND‐enabling toxicology study allowing advancement into the FiH clinical study.ConclusionBased on experience with other tracers, [18F]ACI‐12589 meets the target product profile deemed necessary of a highly favorable candidate to succeed as a radiotracer for imaging a‐syn inclusions. As such, [18F]ACI‐12589 has advanced into the First in Human clinical trial in patients with Parkinson’s disease to be followed by evaluation in other alpha‐synucleinopathies.

Chemoproteomic Profiling by Cysteine Fluoroalkylation Reveals Myrocin G as an Inhibitor of the Nonhomologous End Joining DNA Repair Pathway.

Chemoproteomic profiling of cysteines has emerged as a powerful method for screening the proteome-wide targets of cysteine-reactive fragments, drugs, and natural products. Herein, we report the development and an in-depth evaluation of a tetrafluoroalkyl benziodoxole (TFBX) as a cysteine-selective chemoproteomic probe. We show that this probe features numerous key improvements compared to the traditionally used cysteine-reactive probes, including a superior target occupancy, faster labeling kinetics, and broader proteomic coverage, thus enabling profiling of cysteines directly in live cells. In addition, the fluorine "signature" of probe 7 constitutes an additional advantage resulting in a more confident adduct-amino acid site assignment in mass-spectrometry-based identification workflows. We demonstrate the utility of our new probe for proteome-wide target profiling by identifying the cellular targets of (-)-myrocin G, an antiproliferative fungal natural product with a to-date unknown mechanism of action. We show that this natural product and a simplified analogue target the X-ray repair cross-complementing protein 5 (XRCC5), an ATP-dependent DNA helicase that primes DNA repair machinery for nonhomologous end joining (NHEJ) upon DNA double-strand breaks, making them the first reported inhibitors of this biomedically highly important protein. We further demonstrate that myrocins disrupt the interaction of XRCC5 with DNA leading to sensitization of cancer cells to the chemotherapeutic agent etoposide as well as UV-light-induced DNA damage. Altogether, our next-generation cysteine-reactive probe enables broader and deeper profiling of the cysteinome, rendering it a highly attractive tool for elucidation of targets of electrophilic small molecules.

Discovery of a highly specific 18F-labeled PET ligand for phosphodiesterase 10A enabled by novel spirocyclic iodonium ylide radiofluorination

As a member of cyclic nucleotide phosphodiesterase (PDE) enzyme family, PDE10A is in charge of the degradation of cyclic adenosine (cAMP) and guanosine monophosphates (cGMP). While PDE10A is primarily expressed in the medium spiny neurons of the striatum, it has been implicated in a variety of neurological disorders. Indeed, inhibition of PDE10A has proven to be of potential use for the treatment of central nervous system (CNS) pathologies caused by dysfunction of the basal ganglia–of which the striatum constitutes the largest component. A PDE10A-targeted positron emission tomography (PET) radioligand would enable a better assessment of the pathophysiologic role of PDE10A, as well as confirm the relationship between target occupancy and administrated dose of a given drug candidate, thus accelerating the development of effective PDE10A inhibitors. In this study, we designed and synthesized a novel 18F-aryl PDE10A PET radioligand, codenamed [18F]P10A-1910 ([18F]9), in high radiochemical yield and molar activity via spirocyclic iodonium ylide-mediated radiofluorination. [18F]9 possessed good in vitro binding affinity (IC50 = 2.1 nmol/L) and selectivity towards PDE10A. Further, [18F]9 exhibited reasonable lipophilicity (logD = 3.50) and brain permeability (Papp > 10 × 10−6 cm/s in MDCK-MDR1 cells). PET imaging studies of [18F]9 revealed high striatal uptake and excellent in vivo specificity with reversible tracer kinetics. Preclinical studies in rodents revealed an improved plasma and brain stability of [18F]9 when compared to the current reference standard for PDE10A-targeted PET, [18F]MNI659. Further, dose–response experiments with a series of escalating doses of PDE10A inhibitor 1 in rhesus monkey brains confirmed the utility of [18F]9 for evaluating target occupancy in vivo in higher species. In conclusion, our results indicated that [18F]9 is a promising PDE10A PET radioligand for clinical translation.

Parking and Ride Induction Methods for Drivers in Commuting Scenes

Parking and ride is a very effective method to improve the traffic condition of commuter channels, and it is necessary to develop effective parking guidance strategies. In this study, considering the travel time, walking distance, parking cruise time, parking fee, and personal attributes of drivers, a probability model of parking and ride selection in commuter scenarios was proposed, and a dynamic price adjustment method based on the equilibrium of parking occupancy in the region was constructed. The parking price was adjusted by determining the target occupancy, thus affecting the parking choice behavior to guide the commuter to park. The example analysis showed that this method adjusted the selection probability of the parking lot by using the dynamic price adjustment method from the perspective of regional parking occupancy equilibrium, solved the model by symmetric duality algorithm and formulated a reasonable parking replacement induction scheme to achieve the goal of occupancy equilibrium. Compared with parking guidance under static pricing, it can avoid the crowding of commuter vehicles into the city center effectively to reduce the congestion of commuter channels.

New Acalabrutinib Formulation Enables Co-Administration with Proton Pump Inhibitors and Dosing in Patients Unable to Swallow Capsules (ELEVATE-PLUS)

New Acalabrutinib Formulation Enables Co-Administration with Proton Pump Inhibitors and Dosing in Patients Unable to Swallow Capsules (ELEVATE-PLUS)

Safety and Efficacy of Menin Inhibition in Patients (Pts) with MLL-Rearranged and NPM1 Mutant Acute Leukemia: A Phase (Ph) 1, First-in-Human Study of SNDX-5613 (AUGMENT 101)

Safety and Efficacy of Menin Inhibition in Patients (Pts) with MLL-Rearranged and NPM1 Mutant Acute Leukemia: A Phase (Ph) 1, First-in-Human Study of SNDX-5613 (AUGMENT 101)

Evorpacept (ALX148), a CD47-Blocking Myeloid Checkpoint Inhibitor, in Combination with Azacitidine: A Phase 1 / 2 Study in Patients with Myelodysplastic Syndrome (ASPEN-02)

Evorpacept (ALX148), a CD47-Blocking Myeloid Checkpoint Inhibitor, in Combination with Azacitidine: A Phase 1 / 2 Study in Patients with Myelodysplastic Syndrome (ASPEN-02)

Development of ICT01, a first-in-class, anti-BTN3A antibody for activating Vγ9Vδ2 T cell-mediated antitumor immune response.

Gamma delta T (γδ T) cells are among the most potent cytotoxic lymphocytes. Activating anti–butyrophilin 3A (BTN3A) antibodies prime diverse tumor cell types to be killed by Vγ9Vδ2 T cells, the predominant γδ T cell subset in peripheral circulation, by mechanisms independent of tumor antigen–major histocompatibility complex (MHC) complexes. In this report, we describe the development of a humanized monoclonal antibody, ICT01, with subnanomolar affinity for the three isoforms of BTN3A. We demonstrate that ICT01-activated Vγ9Vδ2 T cells kill multiple tumor cell lines and primary tumor cells, but not normal healthy cells, in an efficient process requiring approximately 20% target occupancy. We show that ICT01 activity is dependent on BTN3A and BTN2A but independent of the phosphoantigen (pAg)–binding B30.2 domain. ICT01 delays the growth of hematologic and solid tumor xenografts and prolongs survival of NOD/SCID/IL2rγnull (NSG) mice adoptively transferred with human Vγ9Vδ2 T cells. In single- and multiple-dose safety studies in cynomolgus macaques that received up to 100 mg/kg once weekly, ICT01 was well tolerated. With respect to pharmacodynamic endpoints, ICT01 selectively activated Vγ9Vδ2 T cells without affecting other BTN3A-expressing lymphocytes such as αβ T or B cells. A first-in-human, phase 1/2a, open-label, clinical study of ICT01 was thus initiated in patients with advanced-stage solid tumors (EVICTION: NCT04243499; EudraCT: 2019-003847-31). Preliminary results show that ICT01 was well tolerated and pharmacodynamically active in the first patients. Digital pathology analysis of tumor biopsies of a patient with melanoma suggests that ICT01 may promote immune cell infiltration within the tumor microenvironment.

EC50 images, a novel endpoint from PET target occupancy studies, reveal spatial variation in apparent drug affinity.

We recently introduced voxel-level images of drug occupancy from PET via our "Lassen plot filter." Occupancy images revealed clear dependence of 11C-flumazenil displacement on dose of GABAa inhibitor, CVL-865, but with different scales in different brain regions. We hypothesized that regions requiring higher drug concentrations to achieve desired occupancy would have higher EC50 values. We introduce an "EC50 image" from human data to evaluate this hypothesis. Five healthy subjects were scanned with the nonselective GABAa tracer, 11C-flumazenil, before and (twice) after administration of CVL-865. We created ten occupancy images and applied an Emax model locally to create one EC50 image. We also performed simulations to confirm our observations of regional variation in EC50 and to identify the main source of variability in EC50. As expected, the EC50 image revealed spatial variation in apparent drug affinity. High EC50 was found in areas of low occupancy for a given drug dose. Simulations demonstrated that sampling from an inadequate range of plasma drug concentrations could impair precision. Our results argue for (a) confidence in the ability of the EC50 images to identify regional differences and (b) a need to tailor the range of drug doses in an occupancy study to regularize the precision of the EC50 throughout the brain. The EC50 image could add value to early-phase drug development by identifying regional variation in affinity that might impact therapy or safety and by guiding dose selection for later-phase trials.

Conditional PD-1/PD-L1 Probody Therapeutics Induce Comparable Antitumor Immunity but Reduced Systemic Toxicity Compared with Traditional Anti-PD-1/PD-L1 Agents.

Immune-checkpoint blockade has revolutionized cancer treatment. However, most patients do not respond to single-agent therapy. Combining checkpoint inhibitors with other immune-stimulating agents increases both efficacy and toxicity due to systemic T-cell activation. Protease-activatable antibody prodrugs, known as Probody therapeutics (Pb-Tx), localize antibody activity by attenuating capacity to bind antigen until protease activation in the tumor microenvironment. Herein, we show that systemic administration of anti-programmed cell death ligand 1 (anti-PD-L1) and anti-programmed cell death protein 1 (anti-PD-1) Pb-Tx to tumor-bearing mice elicited antitumor activity similar to that of traditional PD-1/PD-L1-targeted antibodies. Pb-Tx exhibited reduced systemic activity and an improved nonclinical safety profile, with markedly reduced target occupancy on peripheral T cells and reduced incidence of early-onset autoimmune diabetes in nonobese diabetic mice. Our results confirm that localized PD-1/PD-L1 inhibition by Pb-Tx can elicit robust antitumor immunity and minimize systemic immune-mediated toxicity. These data provide further preclinical rationale to support the ongoing development of the anti-PD-L1 Pb-Tx CX-072, which is currently in clinical trials.

SMaSh: A Streptavidin Mass Shift Assay for Rapidly Quantifying Target Occupancy by Irreversible Inhibitors.

The streptavidin mass shift (SMaSh) assay is a robust and fast approach for quantifying target protein occupancy by a covalent inhibitor or ligand. It exploits the biotin-streptavidin bond using the Simple Western platform. One measurement on a single sample determines both total and occupied target protein simultaneously and is, therefore, self-normalizing. The approach works in diverse and complex biological matrices and, with no need for matched vehicle-treated controls, readily applies to tissues from animal pharmacology models. Assessing occupancy is critical in the development of targeted covalent drugs. We demonstrate its use by characterizing and validating a variety of chemical probes for Bruton's tyrosine kinase (BTK, UniprotKB Q10607) and mitogen-activated protein kinase (ERK1/2/MAPK1/2, UniprotKB P28482 and P27361) and determining target engagement of covalent inhibitors for both targets and off-target engagement for ERK. We demonstrated that it works in cell lysates, tissues, and human peripheral blood mononuclear cells. The SMaSh assay is superior to traditional methods and broadly useful as a tool in assessing covalent biological probes or targeted covalent inhibitors.

Avelumab in relapsed/refractory classical Hodgkin lymphoma: phase 1b results from the JAVELIN Hodgkins trial

AbstractThe 9p24.1 chromosomal alteration in classical Hodgkin lymphoma (cHL) is associated with increased expression of programmed death ligand 1 (PD-L1)/PD-L2 and an immunosuppressive tumor microenvironment. Blockade of PD-L1/PD-1 interactions with avelumab (anti–PD-L1) is hypothesized to restore antitumor immunity. JAVELIN Hodgkins was a phase 1b, multiple-dose, open-label, randomized, parallel-arm trial of avelumab in patients with relapsed/refractory (R/R) cHL. Primary end points included avelumab target occupancy by dose/schedule in peripheral blood immune cells and pharmacokinetic parameters. Secondary end points included safety and antitumor activity. Four dose levels and 2 dosing schedules were investigated: 70, 350, and 500 mg administered every 2 weeks; 500 mg every 3 weeks; and 10 mg/kg every 2 weeks. Thirty-one patients with R/R cHL were randomized; 9 (29.0%) and 20 (64.5%) had received 3 or ≥4 prior anticancer treatments, respectively. Target occupancy of >90% was observed across all treatment arms, throughout the dosing interval. Avelumab pharmacokinetic data were similar to those previously reported. The most common treatment-related adverse events of any grade were infusion-related reaction (30.0%), nausea (20.0%), increased alanine aminotransferase and rash (16.7% each), and fatigue (13.3%). The objective response rate (ORR) in all randomized patients was 41.9%, with a complete response rate of 19.4%; ORR in those with prior allogeneic hematopoietic stem cell transplant (allo-HSCT) was 55.6%. Due to decreased use of allo-HSCT in patients with R/R cHL, the expansion phase enrolling post–allo-HSCT patients was terminated. Avelumab was tolerable and demonstrated antitumor activity in heavily pretreated patients with cHL, suggesting that PD-L1 blockade may be sufficient for therapeutic benefit in cHL. This trial was registered at www.clinicaltrials.gov as #NCT02603419.

Discovery of GNE-502 as an orally bioavailable and potent degrader for estrogen receptor positive breast cancer

Fulvestrant is an FDA-approved drug with a dual mechanism of action (MOA), acting as a full antagonist and degrader of the estrogen receptor protein. A significant limitation of fulvestrant is the dosing regimen required for efficacy. Due to its high lipophilicity and poor pharmacokinetic profile, fulvestrant needs to be administered through intramuscular injections which leads to injection site soreness. This route of administration also limits the dose and target occupancy in patients. We envisioned a best-in-class molecule that would function with the same dual MOA as fulvestrant, but with improved physicochemical properties and would be orally bioavailable. Herein we report our progress toward that goal, resulting in a new lead GNE-502 which addressed some of the liabilities of our previously reported lead molecule GNE-149.

Adaptation to the market? Status differences between target occupations in the application process and realized training occupation of German adolescents

ABSTRACT Adolescents’ occupational expectations are relevant for occupational status attainment. In strong vocational education and training (VET) systems, such as in Germany, school leavers face the challenge of forming occupational expectations that correspond to the competitive VET market. This study investigates students’ target occupations in the application process and its relation to their first training occupation. Do applicants for VET positions apply for occupations of different socioeconomic status over time? Does the status of the target occupations increasingly fit to the finally achieved training occupation? Are there differences by familial socioeconomic background? Analysis are based on longitudinal data on the application process collected from German students in lower secondary and intermediate secondary schools in one urban area. Overall, the status level of the target occupations at the beginning of the application process differs significantly according to school track, but additionally to school grades or family background. At the end of the application process, the application behaviour becomes diversified: applicants with poor school grades and of low status continue to apply for target occupations at a similar status level but at the same time apply for occupations of relative lower status. This lower level does not, however, translate into training occupations.

Potent Phenylpyridine and Oxodihydrofuran Inhibitors of Cyclooxygenase-2: Optimization toward a Long Residence Time with Balanced Internal Energetics

Long residence time enzyme inhibitors with a two-step binding mechanism are characterized by a high internal energy barrier for target association. This raises the question of whether optimizing residence time via further increasing this internal energy barrier would inevitably lead to insufficient target occupancy in vivo due to slow, time-dependent binding. We attempted to address this question during optimization of cyclooxygenase-2 (COX-2) inhibitors. Defining long residence time drugs with acceptable association and dissociation rate constants required for sufficient target occupancy and sustained efficacy, which we termed "balanced internal energetics", provides an important criterion for successful progression during lead optimization. Despite the advancement of several COX-2 inhibitors to marketed drugs, their detailed inhibition kinetics have been surprisingly limiting especially during the structure-activity relationship process mainly due to the lack of robust kinetic assays. Herein, we describe a reoptimized COX enzymatic assay and a novel MS-based assay enabling detailed mechanistic studies for identifying long residence time COX-2 inhibitors with balanced internal energetics. These efforts led to the discovery of promising leads possessing dissociation half-lives of ≤40 h, much greater than the values of 6 and 0.71 h for two marketed drugs, etoricoxib and celecoxib, respectively. Importantly, the inhibition rate constants remain comparable to those of the marketed drugs and above the lower limits set by the criteria of balanced internal energetics, predicting sufficient target occupancy required for efficacy. Taken together, this study demonstrates the feasibility of increasing the internal energy barrier as a viable approach for lead optimization toward discovering long residence time drug candidates.

Abstract CT034: Activation of the antitumor immune response of gamma9delta2 T cells in patients with solid or hematologic malignancies with ICT01, a first-in-class, monoclonal antibody targeting butyrophilin 3A: The EVICTION study

Abstract Background: Gamma9 Delta2 (γ9δ2) T cells function at the crossroad of innate and adaptive immunity with important roles in immune responses against pathogens and carcinogenesis, making them an attractive target for cancer immunotherapy. Butyrophilin (BTN) 3A (CD277) is an immune checkpoint molecule expressed on tumors and cells of the immune system that is required for γ9δ2 T cell activation. These observations led to the design and development of ICT01, a humanized, monoclonal antibody that binds the extracellular domain of all 3 isoforms of BTN3A1/A2/A3 and induces pAg-independent γ9δ2 T cell activation and killing of multiple tumors in nonclinical settings. Methods: EVICTION (www.clinicaltrials.gov NCT04243499; EudraCT Number: 2019-003847-31) is a first-in-human, open-label clinical study to assess the safety, tolerability, and activity of a range of IV doses of ICT01 as monotherapy and in combination with pembrolizumab, in patients with advanced-stage, relapsed/refractory cancer. Following Regulatory and Ethics Committee approvals, the study is being conducted at cancer centers in France, Belgium, Spain, Germany, the UK and USA. Following signed informed consent, patients received ICT01 every 3 weeks with blood samples collected at multiple timepoints for immunophenotyping by flow cytometry and cytokine analysis (IFNγ, TNFα, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-13). Tumor biopsies are collected at baseline and on Day 28 for immunohistochemistry (e.g., BTN3A, γ9δ2 T cells) and expression profiling of cancer- and immunity-related genes. Results: Solid tumor (ST) dose cohorts 1-4 (total n=20) were enrolled and treated with ICT01 doses ranging from 20 μg to 20 mg, and dose cohort 1 of hematologic cancer patients (n=3, 200 μg) has been enrolled. ICT01 was well-tolerated, with no dose-limiting toxicities or related SAEs reported. Target occupancy on T cells at 30 min or 4 hours post first dose ranged from 34% (700 μg, n=2), 79% (2 mg, n=5), 93% (7 mg, n=4) and 100% (20 mg, n=1), with activated γ9δ2 T cells (CD69+) migrating from the blood within 30 min of dosing that led to a > 95% decrease from baseline in the number of circulating γ9δ2 T cells for all 4 doses by 24hrs. The 2nd dose of ICT01 induced a similar activation & migration of γ9δ2 T cells from the circulation. There were no effects on CD4/CD8 T cells, NK, or B cells. Available cytokine data from ST cohorts 1 & 2 showed no CRS although transient 2-3x increases in IFNγ, TNFα, & IL-2 were observed 4 hours post dose in patients with higher baseline γ9δ2 T cell counts. A 5-10x increase of IFNγ was observed on Days 7 & 28 in ST cohort 2, consistent with γ9δ2 T cell activation. Digital pathology analysis of tumor biopsies of a cohort 1 melanoma pt demonstrated a 25x and 8x increase of CD3-TCRgamma delta++ and CD3+ cell densities, respectively, as compared to baseline. Analyses of additional cohorts are ongoing and will be presented. Conclusion: The preliminary results demonstrate that ICT01 safely and potently activates the anti-tumor immune response of γ9δ2 T cells mediated by BTN3A. Citation Format: Aurelien Marabelle, Christiane Jungels, Johann De Bono, Norbert Vey, Martin Wermke, Elena Garralda, Steven Le Gouill, Patricia LoRusso, Aude De Gassart, Emmanuel Valentin, Patrick Brune, Marina Iche, Daniel Olive, Paul A. Frohna. Activation of the antitumor immune response of gamma9delta2 T cells in patients with solid or hematologic malignancies with ICT01, a first-in-class, monoclonal antibody targeting butyrophilin 3A: The EVICTION study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT034.

Abstract 2437: Potency and selectivity evaluation of TEAD covalent inhibitors in living cells by two complementary mass spectrometry methods

Abstract The HIPPO pathway plays an important role in cancer progression and among its essential components are the transcription factor proteins from the TEAD family. The TEAD family consists of 4 different members (TEAD1-TEAD4) who modulate gene expression through the interaction with co-activator proteins such as YAP1 and TAZ. Recent studies have shown that the central pocket of TEAD could be an alternative druggable approach for inhibiting YAP1. However, until recently, the irreversible binding mechanism of such a compound in living cells was not clearly established. Here, we report a mass spectrometry-based approach to demonstrate and quantitatively assess the target occupancy of TEADs covalent inhibitors both in vitro cellular and ex vivo tumor models. Applying our method to compounds from different chemical series confirmed that these inhibitors modulate the TEAD central pocket protein in cell cultures and led us to quantify their target occupancy for all TEADs expressed in our cellular models. We extended and strengthened the characterization of our inhibitors by implementing a proteomic LC-MS/MS cysteine profiling strategy - assessing the selectivity of inhibitors across free cysteine residues in the cellular proteome. As a result, we showed a high selectivity of the first inhibitors towards TEADs cysteine binding. To our knowledge, this is the first study proving by direct means and not by indirect biomarker and cell proliferation phenotypes that the central pocket of TEAD is actually available to ligand binding in cells, allowing direct measurement of target engagement for different TEAD proteins and finally demonstrating selectivity of inhibitors. These two-mass spectrometry-based methods developed here are very robust, powerful and can be currently used to help for the development of TEADs inhibitors Citation Format: Armelle Buzy, Pascal Paul, Stephanie Hamon, Olivier Venier, Jean-Claude Guillemot, Vincent Mikol, Jurgen Moll, Laurent Debussche, Iris Valtingojer. Potency and selectivity evaluation of TEAD covalent inhibitors in living cells by two complementary mass spectrometry methods [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2437.