Non-covalent Inhibitors Research Articles

Trial in Progress: A Phase 1b Study of AS-1763, an Oral, Potent and Selective Noncovalent BTK Inhibitor, in Patients with Previously Treated Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma or Non-Hodgkin Lymphoma

Background: Covalent Bruton's tyrosine kinase (BTK) inhibitors such as ibrutinib are approved for treating patients with B-cell malignancies, but their long-term efficacy is limited by toxicity related to off-target kinase inhibition and acquired resistance due to BTK C481 mutation. AS-1763 is a potent, highly selective, orally available, and non-covalent BTK inhibitor, equipotent against both wild-type and C481S-mutated BTK with sub-nanomolar IC50 values (Kawahata et al. J Med Chem 2021; 64,14129-14141). In a Phase 1 single-ascending dose study in healthy volunteers, AS-1763 was well tolerated and safe at doses of 5-600 mg and achieved maximum inhibition of B cell activation at doses of 100 mg and above at 1-2 h post-dose and the duration of inhibitory effect increased with dose [Arimura et al. AACR 2022; Cancer Res 2022;82(12_Suppl)]. In preclinical studies, AS-1763 demonstrated a broad spectrum of inhibition against BTK mutations that confer resistance to other non-covalent BTK inhibitors in addition to the C481 mutation. Study Design and Methods: This study is an open-label, multi-center, Phase 1b study of oral AS-1763 in patients with advanced chronic lymphocytic leukemia (CLL)/small lymphocytic leukemia (SLL) and other B-cell non-Hodgkin lymphomas (NHLs) who have failed or are intolerant to at least two prior lines of systemic therapy. Prior therapy with a covalent BTK inhibitor is permitted; prior use of a noncovalent BTK inhibitor is excluded. This study consists of two parts, i.e., dose escalation and expansion parts. The dose escalation part will follow a 3+3 design with a starting dose of 100 mg BID. Each cycle will be 28 days. Three dose levels will be selected for dose expansion part based on a comprehensive review of data from dose escalation. The dose expansion part consists of 2 cohorts, i.e., Cohort 1 for CLL/SLL and Cohort 2 for B-cell NHL. In each cohort, the first 30 patients will be allocated to the 3 dose levels (10 patients for each), then the same provisional recommended Phase 2 Dose (RP2D) will be selected for Cohort 1 and Cohort 2 after 30 patients have enrolled in either cohort. After the provisional RP2D is selected, up to 28 patients in Cohort 1 and up to 15 patients in Cohort 2 may be treated at the provisional RP2D. In this study, up to 110 patients will be enrolled. Treatment will continue for 24 cycles or until disease progression, occurrence of unacceptable toxicity or discontinuation because of other reasons. Eligible patients include CLL/SLL, Waldenstrӧm macroglobulinemia (WM), marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), and follicular lymphoma (FL). Patients with transformed disease (e.g., Richter's transformation) prior to or during screening are not eligible. Key exclusion criteria include CNS involvement by systemic lymphoma, stem cell transplant or CAR-T therapy <30 days, and clinically significant cardiovascular disease. The primary objective of the dose escalation part is to determine the maximum tolerated dose/dose-limiting toxicity. Key secondary endpoints include the evaluation of safety profile and tolerability, pharmacokinetics properties, and preliminary anti-tumor activity based on overall response rate (ORR) by investigator according to iwCLL 2018 for CLL, IWW6 for WM, and Lugano Treatment Response Criteria for MCL, MZL, SLL and FL. The primary objective of the dose expansion part is to assess the preliminary anti-tumor activity based on ORR by the Safety Monitoring Committee (SMC). Key secondary objectives are to investigate safety, tolerability, and pharmacokinetic profiles and to assess the preliminary antitumor activity based on ORR by investigator and best overall response, duration of response, progression-free survival and overall survival by investigator and the SMC. This trial will evaluate several key exploratory objectives including evaluation of minimal residual disease negativity, characterization of patient subsets defined by mutation status of BTK and phospholipase C gamma 2 (PLCG2), and characterize BTK and PLGC2 gene mutation before and after disease progression. Changes in the level of biomarkers such as CCL3 and CCL4 chemokines and B-cell receptor pathway signaling will be evaluated prior to, during therapy, and at time of disease progression. The RP2D will be determined based on all the data generated in the study. The study has been registered on ClinicalTrials.gov (NCT05602363).

Outcomes of Venetoclax Retreatment after MRD-Guided Venetoclax +/- Ibrutinib: Results of the Improve Study

Data from small cohorts of patients (pts) with chronic lymphocytic leukemia (CLL) relapsing after venetoclax-based fixed-duration regimens suggest that venetoclax retreatment may be a feasible option. Information on the efficacy and tolerability of venetoclax retreatment in this setting is still scanty, making it worth exploring venetoclax retreatment in prospective cohorts of well-characterized pts with CLL. We analyzed the efficacy and safety of venetoclax retreatment in pts with relapsed/refractory CLL enrolled in our clinical trial IMPROVE (NCT04754035). BTK- and BCL2-inhibitor naïve pts enrolled into the study were treated with venetoclax single-agent for 12 months; those with undetectable measurable residual disease (uMRD) discontinued therapy while those with detectable MRD received the combination of ibrutinib and venetoclax until uMRD, toxicity or for up to 12 months. Pts with detectable MRD at the end the combination therapy stopped venetoclax and continued with ibrutinib only. In pts who experienced progressive disease (PD) requiring treatment, venetoclax single-agent was restarted according to the standard schedule and continued until PD or unacceptable toxicity. MRD was tested every 3 months in peripheral blood using the 6-color flow cytometry panel validated by the ERIC (European Research Initiative on CLL). After a median follow-up of 58 months (5-67) from C1D1, 20/38 (52.6%) pts experienced PD requiring treatment after discontinuing therapy, including 19 upon reaching confirmed uMRD (11 treated with venetoclax single-agent, 8 with the combination of venetoclax+ibrutinib), 1 who discontinued both venetoclax and ibrutinib in partial response (PR) with detectable MRD (dMRD) due to an unrelated adverse event. Two out of 20 had Richter transformation (RT), 2 developed autoimmune hemolytic anemia, 2 received other treatments (BTK inhibitors) per investigator's decision and all 6 went off study. The remaining 14 pts (1 with detectable MRD, 7 who previously achieved uMRD with venetoclax monotherapy, 6 who were treated with the combination of venetoclax+ibrutinib) received venetoclax retreatment at a median of 32.5 months (12-44) after the end of initial therapy (Table 1); to note, the pt who stopped treatment in PR with dMRD restarted venetoclax after only 13 months. All 13 uMRD subjects experienced MRD relapse at a median of 5 months (0-41) after stopping treatment, 26 (0-37) months before disease progression requiring therapy. At the time of venetoclax initiation tumor lysis syndrome (TLS) risk was high in 6, medium in 3, low in 5. Best overall response rate was 79% (11/14), all partial responses (PR), 2 pts experienced stable disease (on treatment for 2 and 24 months). With a median time on treatment of 10 months, 2 pts achieved uMRD after 3 and 6 months, the second one maintaining uMRD for 6 months and then becoming MRD positive again (Figure 1). Five out of 14 pts progressed after 2, 7, 9 and 11 (n=2) months on venetoclax retreatment, 3/5 received next-line treatment with ibrutinib, 2/5 received non-covalent BTK inhibitor and all achieved a PR. At last follow-up, 57% of pts remain on venetoclax; 12/14 patients undergoing venetoclax retreatment were alive (1 death due to RT on ibrutinib, 1 due to CLL progression on non-covalent BTK inhibitor). Progression-free survival from venetoclax restart to PD on venetoclax retreatment, last follow-up or death was not reached (0-28 months). Venetoclax retreatment was very well tolerated with no patients experiencing TLS, 6/14 having grade 3-4 neutropenia (only in 1 case leading to treatment discontinuation), 8/14 infections (2 grade 1, 4 grade 2, 2 grade 3). No other grade 3/4 hematologic or non-hematologic toxicities were observed. Venetoclax retreatment was effective and well-tolerated in a cohort of pts with relapsed/refractory CLL who have previously received time limited, MRD-guided treatment with venetoclax+/-ibrutinib. MRD relapse occurred at a median of about 2 years before clinical progression requiring treatment. Venetoclax retreatment was started at a median of 32.5 months after the end of initial therapy. Nearly 80% of patients achieved at least a partial response with venetoclax retreatment, 14% of pts achieved uMRD and 57% remain on venetoclax, with a median time on treatment of 10 months.

First Report of Phase 1 Studies of DZD8586, a BBB Penetrant LYN/BTK Dual Inhibitor, in Patients with B-Cell Non-Hodgkin Lymphoma (B-NHL)

Introduction Resistance to covalent (ibrutinib, acalabrutinib and zanubrutinib) and non-covalent BTK inhibitors (pirtobrutinib) is an emerging clinical challenge. DZD8586 was designed as a full blood-brain barrier (BBB) penetrable LYN/BTK dual inhibitor. Preclinical study suggested DZD8586 has a good selectivity against other TEC family kinases (TEC, ITK, TXK, BMX). DZD8586 inhibits BTK-dependent and BTK-independent BCR signaling pathways by targeting LYN and BTK, which could potentially overcome resistance to both covalent and non-covalent BTK inhibitors for the treatment of CLL and MCL, as well as DLBCL. Two phase 1 clinical studies are ongoing in patients with relapsed or refractory (r/r) B-NHL to assess safety, pharmacokinetics (PK), pharmacodynamics (PD) and anti-tumor efficacy of DZD8586. Methods TAI-SHAN1 (NCT05844956; CTR20220558) and TAI-SHAN5 (NCT05824585) are phase 1 dose escalation and expansion studies in patients with r/r B-NHL. Both studies have similar dose escalation strategies. Patients who progressed following or intolerant of prior systemic therapy were enrolled. Pooled safety and efficacy analysis was performed. In addition, modulation of PD biomarkers was also evaluated. Tumor responses were investigator assessed according to Lugano 2014, IPCG 2005, or iwCLL 2018, as appropriate. Results As of June 15, 2023, 17 patients with r/r B-NHL have been enrolled and received DZD8586 at doses ranging from 10 to 100 mg once daily (QD) (10 mg， n=1；25 mg， n=4；50 mg， n=7；100 mg， n=5). The median age was 61 years, 71% were male, and 53% had an ECOG PS of 1. Histological subtypes included CLL, SCNSL, DLBCL, MCL, FL and MZL. The median number of prior therapies was 3 (range 2 - 8). All patients received prior chemotherapy and anti-CD20 therapy. Six patients, including 1 CLL, 2 MCL, 1 MZL, 1 SCNSL and 1 DLBCL had received prior treatment with covalent BTK inhibitors. DZD8586 was well tolerated across the doses investigated. Drug-related treatment emergent adverse events (TEAEs) reported in > 2 patients included thrombocytopenia (10/17), neutropenia (5/17), and upper respiratory tract infection (3/17). The most common ≥ grade 3 TEAE was thrombocytopenia (3/17, all at 100 mg). Other ≥ grade 3 TEAEs, including neutropenia, drug eruption and upper respiratory tract infection, were each reported by one patient. Bleeding, atrial fibrillation, and arthralgia were not reported. Grade 1 diarrhoea was reported in one patient. The maximum tolerated dose was defined as 100 mg QD. All these TEAEs were reversible after dose interruption and/or with supportive care. The longest duration on treatment was 10+ months. Among 13 patients treated at doses ≥ 25 mg QD with at least one tumor assessment, the objective response rate (ORR) was 9/13 (69.2%). Responses were observed across different histological subtypes (1/2 in SCNSL; 4/5 in DLBCL; 1/2 in FL; 1/1 in MCL, 1/2 in MZL and 1/1 CLL carrying C481S mutation). Among the 5 evaluable BTKi treated patients, tumor responses were observed in 3 patients. The longest duration of response (DoR) was 6.4+ months, and the patient was still ongoing with DZD8586 treatment and responding. PK was generally dose-proportional across the dose ranges of 10 to 100 mg. DZD8586 was absorbed with median t max 1.5 hours, and eliminated with mean terminal half-life of ~18 hours. Accumulation of about two folds in AUC was noted at steady state following once daily dosing. Dose dependent pBTK modulation in whole blood cells was detected, and at the doses ≥ 25 mg, sustained pBTK inhibition was observed. The unbound CSF-to-plasma concentration ratio (K puu,CSF) was 1.2 from one CNSL patient with available CSF samples, suggesting excellent CNS penetration of DZD8586 in humans. Conclusion In the preliminary pooled analysis of two ongoing phase 1 trials, DZD8586 has encouraging anti-tumor activity and safety in heavily pre-treated B-NHL patients. The combination of BBB penetration, dual inhibition of LYN/BTK and potential to overcome C481S mutant CLL and other covalent BTKi resistant lymphomas suggest this may be a useful agent. Updated data will be presented at the meeting.

Understanding Resistance Mechanisms and Growth Kinetics of CLL Treated with Covalent and Non-Covalent BTK Inhibitors

Resistance mechanisms to covalent BTK inhibitors (cBTKi) are well studied, with mutations in BTK at the 481 target binding site most commonly identified, and, less commonly, activating mutations in PLCϒ 2. Second site non-481 BTK mutations have been identified among patients on non-covalent BTK inhibitors (ncBTKi). The present study was designed to further evaluate these resistance mechanisms using dense serial sampling during therapy with both classes of BTKi and using mathematical modeling to analyze the rates of growth and decline of specific mutated clones. We performed whole exome sequencing on matched tumor and normal samples from 19 CLL patients treated with BTK inhibitors (Ibrutinib, Ibr, 42% (8/19); Acalabrutinib, Acala, 21% (4/19) and Pirtobrutinib, Pirto, 37% (7/19)). The median number of longitudinal samples per patient per cohort was 6 for ibr (N=51 total samples), 11 for acala (N=46) and 15 for pirto (N=105). The median number of prior therapies in both the ibr and acala cohorts was 2 and high-risk genomic features del(17p) and del (11q) were both at 50% in acala cohort and 50% and 38% in the ibr group respectively. The ibr cohort had no prior BTKi therapy whereas 1 patient in the acala cohort had received spebrutinib, another cBTKi. The average time to progression (TTP) in the ibr and acala groups was comparable at 44.1 and 44.4 months respectively. The pirto cohort was more heavily pretreated with a median of 3 prior therapies and del(17p) in 28.6% and del(11q) in 42.8%. All patients in this cohort had prior cBTKi (4 patients had 1 and 3 patients had ≥2 cBTKi) and average TTP was shorter at 15.1 months. At progression (PD), mutations in BTK C481S were found in 5 /8 patients (55%) in the ibr cohort with cancer cell fraction (CCF) > 80% in 3 patients. Two of these patients also have mutations in PLCϒ 2 (CCF > 30%). One patient in the cohort has an acquired PLCϒ 2 mutation (D1140N) with CCF>70%, without BTK C481S. Investigation of this patient's resistant clone growth kinetics (using WBC counts) demonstrates that the PLCϒ2 clone which was not detected at baseline takes over the population at ibr progression. Two patients in this cohort did not have either BTK or PLCϒ 2 mutations; however, one patient had 2 expanding subclones with known driver mutations ( TP53, BIRC3, del 8p). All 4 patients in the acala cohort have resistant clones harboring BTK C481S (CCF 16-75%) mutations, with T474I mutations present in 2 patients (CCF 4% and 26%) at progression. Growth kinetics analysis of the C481S clones for 3 patients shows steady increase during acala therapy, suggesting CLL progression in these two patients is driven by the C481S mutations. All but one patient in the pirto cohort had developed PD on cBTKi and 71% (5/7) had BTK C481S mutations prior to starting pirto (CCF 26 - 99%). In all 5 cases, the C481S clone declines on pirto and at PD is replaced by the BTK gatekeeper mutation T474I (CCF 76 - 99%) in 3 patients, the kinase-inactive mutation BTK L528W (CCF 30%) in 1 patient, and mutations in other driver genes ( XPO1, MED12) in 1 patient. Growth kinetics analysis for 1 patient shows exponential decay of the C481S clone under pirto treatment, followed by slow steady growth of the T474I clone, with a sharp increase right before PD. In another patient, exponential growth of the T474I clone occurs during pirto treatment while the C481S clone steadily declines. Interestingly this patient also had an existing PLCϒ 2 mutation (L845F, CCF 42%) at baseline, acquired during ibr treatment, which also steadily decreased on pirto. In another patient previously treated with ibr, with baseline BTK mutations in C481R (CCF 29%), C481S (CCF 18%), and L528W (CCF 2%), the L528W clone steadily increases, while both C481S and C481R clones decrease during PD. This patient exhibits extreme multiclonal diversity of BTK mutations, with 8 different BTK mutations at the DNA level at the C481 site present both at baseline and at PD, with 56% reduction in total wild type reads at PD. Of the remaining two patients without baseline C481S mutations, one patient acquired T474I (CCF 99%) and the other patient acquired 1q gain (MCL1) and t(8;14) at progression. Growth kinetics of different BTKi can vary based on their specific mechanism of action. The approach of combining WES data with growth pattern modeling can help unravel the complexities of tumor evolution and drug resistance for the different classes of BTKi. Data on comparative clone growth rates will be presented at the meeting.

Patient-Reported Outcomes (PROs) Among Patients with Previously Treated Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (CLL/SLL) Receiving Pirtobrutinib: Analysis from the BRUIN Phase 1/2 Study

Background PROs including health-related quality of life (HRQoL) and symptom burden can provide helpful information to support benefit-risk assessment of a new treatment. Pirtobrutinib, a highly selective, non-covalent (reversible) Bruton tyrosine kinase inhibitor (BTKi), inhibits both wildtype and C481-mutant BTK with equal low nM potency, and has favorable oral pharmacology that enables continuous BTK inhibition throughout the dosing interval regardless of intrinsic rate of BTK turnover. Pirtobrutinib recently received regulatory approval in the US for the treatment of patients with relapsed or refractory mantle cell lymphoma after two or more lines of systemic therapy, including a covalent BTKi (cBTKi). BRUIN (NCT03740529) is an ongoing, open-label, multi-center phase 1/2 study investigating the safety and efficacy of pirtobrutinib for the treatment of B-cell malignancies, including CLL/SLL. This current analysis reports PROs among patients enrolled in the BRUIN study with relapsed or refractory (R/R) CLL/SLL treated with prior cBTKi-containing regimens. Methods Pretreated patients with R/R CLL/SLL in the BRUIN study completed PRO assessments at each clinic visit. The European Organization for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire Core-30 (QLQ-C30) was used to measure physical function (PF) and HRQoL via PF and Global Health Status/QoL subscales. Patient-reported CLL/SLL symptoms and fatigue were assessed using 13 and 6 EORTC Item Library (IL) items, respectively. All subscales are scored 0-100. Higher scores represent better PF and HRQoL; and higher scores represent worse CLL/SLL symptoms and fatigue. Pre-specified clinically meaningful within patient change (MWPC) thresholds of at least 10-point changes in each PRO score were used to categorize patients based on their individual change from Cycle 1 Day 1 (i.e., baseline). Mean changes from baseline and proportions of patients with improved, stable, and worsened PROs from baseline were summarized by treatment cycle. PRO data were presented from Cycle 1 to 24 (a 28-day cycle) since the study median progression-free survival by investigator assessment was 19.6 months. The data cutoff date was 22 July 2022. Results There were 263 patients with CLL/SLL at the time of analysis; median (Quartile 1 and 3) age was 69 (62, 74) years and the majority were male (68%). All patients had received prior cBTKi; 21% received 2 or more prior cBTKi lines and 20% discontinued from their most recent cBTKi treatment due to toxicity. The mean overall completion rate of PRO instruments was 83% at baseline. 76% of all patients in the analysis completed at least one subsequent PRO assessment. Baseline mean (±SD [standard deviation]) score was 80.8 (±19.5) for PF, 61.6 (±23.2) for QoL, 24.8 (±18.4) for CLL/SLL symptoms, and 33.5 (±24.7) for fatigue. The majority of patients reported stable or clinically improved outcomes from baseline at each of the post-baseline visits (through Cycle 24) for PF (proportion of stable/improved ranged from 72.5% to 95.5%), QoL (range 80.4% to 95.0%), CLL/SLL symptoms (range 77.8% to 96.3%), and fatigue (range 77.3% to 89.9%). Conclusion Overall, consistent with the favorable safety profile of pirtobrutinib, HRQoL and symptoms were stable or improved for over 70% of patients with pretreated R/R CLL/SLL throughout the first 24 cycles of pirtobrutinib treatment. These PRO data should be interpreted with caution due to single arm trial design and small numbers of patients available for assessment in later cycles.

Determining Clinical Practice Gaps for HCPs in Treating CLL Using CME-Based Surveys

Background: Targeted therapies have revolutionized the care of patients with chronic lymphocytic leukemia (CLL). In this context, new evidence continues to accumulate with the goal of optimizing the clinical use of these treatments as additional novel therapeutics continue to emerge. In this study, we sought to analyze current clinical practices of healthcare professionals (HCPs) regarding the care of patients with CLL, along with their awareness of the latest evidence on novel investigational therapies for these patients. Methods: Between March 2022 and June 2023, we conducted multiple live and online continuing medical education activities designed for experts to provide HCPs with evidence-based recommendations for the treatment of CLL. Baseline polling questions and surveys designed to assess key aspects of HCP knowledge, practice patterns, and confidence in this area were assessed across the activities. Results: Among 124 HCPs participating in educational activities between September 2022 and June 2023, 40% reported high confidence in their ability to plan optimal treatment strategies using BTK inhibitors for patients with CLL based on available evidence and guideline and expert recommendations. There was considerable discordance between HCP and expert treatment choices for various case scenarios in multiple educational activities; for example, for patient cases without del(17p)/ TP53-mutated/ IGHV-mutated CLL andbulky disease with diminished creatinine clearance, experts would recommend acalabrutinib with or without obinutuzumab for 77% of scenarios, while HCPs selected this treatment 35% of the time (Table). Among 57 HCPs who participated in educational programs from March 2022 to February 2023, 26% reported high confidence in their ability to recognize and manage AEs with BTK inhibitors in patients with CLL. Challenging AEs associated with the currently available BTK inhibitors also were self-identified by HCPs (Figure). One half of responding HCPs (52%) were able to identify characteristics that would qualify a patient for inpatient administration of the initial doses of venetoclax because of increased risk of tumor lysis syndrome (n = 48). Finally, among 213 HCPs participating in educational activities from September 2022 to June 2023, 43% were aware of recent data regarding the use of pirtobrutinib, an investigational noncovalent BTK inhibitor, for treating patients with CLL. Conclusion: Our data suggest that many HCPs are challenged to optimally incorporate contemporary treatment recommendations into the care of patients with CLL and struggle to maintain knowledge of investigational therapies that may affect the treatment landscape in the near future. Continuing educational activities designed to improve the knowledge, competence, and confidence of HCPs in the care of CLL would benefit patients with this disease. A more detailed data analysis of treatment trends and knowledge by geographic location and role on the care team will be presented.

A Real-World Study of Zanubrutinib Treatment in Chronic Lymphocytic Leukemia and Small Lymphocytic Lymphoma

Background: Zanubrutinib, a second-generation non-covalent Bruton's Tyrosine Kinase inhibitor (BTKi), with better BTK specificity, and fewer off-target effects. Zanubrutinib has demonstrated the confirmed effectiveness and safety in clinical trials such as SEQUOIA and ALPINE in chronic lymphocytic leukemia and small lymphocytic lymphoma (CLL/SLL), and earlier studies showed its efficacy and safety profile of zanubrutinib monotherapy in the real world. Meanwhile, more valuable insights hope to be gained on zanubrutinib treatment in Chinese CLL/SLL patients (pts) outside of the clinical trial setting. This report provides some outcomes evaluated in a real-world study. Aims: This study further evaluated the effectiveness and safety of zanubrutinib monotherapy/combination treatment in Chinese CLL/SLL pts in a real-world setting. Methods: CLL/SLL pts who received zanubrutinib monotherapy/combination treatment for at least 3 months from Aug. 2017 to Jul. 2023 were enrolled from Jiangsu Province Hospital. The primary outcome was progression free survival (PFS). Baseline characteristics were also collated. Results: 77 pts were analyzed in all. Zanubrutinib was administered orally at 160 mg, twice daily (BID), mono or combined. Among the monotherapy pts, 20 (35.1%) pts were treatment naïve (TN) and 37 (64.9%) pts were refractory/relapsed (R/R). Among the combination treatment pts, 14 (70.0%) pts were TN and 6 (30.0%) pts were R/R. Baseline characteristics were shown in Table 1. The median follow-up time was 31.8 months, and the median PFS was not reached in the entire cohort. The 12-month PFS rate was 95.8% (95%CI 91.3-100.0%) while the 24-month PFS rate was 90.9% (95%CI 84.2-98.2%) (Figure A). After stratified by line of therapy in monotherapy, the estimated 12-month PFS rates of TN and R/R zanubrutinib monotherapy pts were 100.0% (95%CI 100.0-100.0%) and 91.3% (95%CI 82.3-100.0%) (Figure B). The estimated 24-month PFS rates of TN and R/R zanubrutinib monotherapy pts were 93.8% (95%CI 82.6-100.0%) and 84.9% (95%CI 73.5-98.1%). 4 (5.2%) pts interrupted treatment due to AE. 2 pts were TN and 2 pts were R/R. 2 pts received zanubrutinib monotherapy and 2 pts received combination treatment. The AEs included hemoptysis (1, 1.3%), platelet hemoglobin reduction and atrial fibrillation (1, 1.3%), bleeding points in both lower limbs (1, 1.3%), rash and edema (1, 1.3%). All treatment-interrupted pts still received zanubrutinib after interruption by the latest follow-up. 17 (22.1%) pts experienced treatment discontinuation due to progressive disease (PD) or transformation. No pts discontinued zanubrutinib treatment due to AE. PD was the most common reason for treatment discontinuation. 13 (16.9%) pts experienced PD and 4 (5.2%) pts experienced transformations. S ummary/Conclusion: Zanubrutinib shows good efficacy in Chinese CLL/SLL pts in the real-world study. Incidence of AE was low, and no treatment discontinued due to AE, revealing a good safety of zanubrutinib. PD was the main reason for treatment discontinuation. Therefore, the effectiveness and safety of zanubrutinib have been validated in the real world, consistent with clinical trials.

Targeting BCL2 pathways in CLL: a story of resistance and ingenuity.

Chronic lymphocytic leukemia (CLL) is common amongst leukemic malignancies, prompting dedicated investigation throughout the years. Over the last decade, the treatment for CLL has significantly advanced with agents targeting B-cell lymphoma 2 (BCL2), Bruton's tyrosine kinase, and CD20. Single agents or combinations of these targets have proven efficacy. Unfortunately, resistance to one or multiple of the new treatment targets develops. Our review investigates various mechanisms of resistance to BCL2 inhibitors, including mutations in BCL2, alterations in the Bcl protein pathway, epigenetic modifications, genetic heterogeneity, Richter transformation, and alterations in oxidative phosphorylation. Additionally, the review will discuss potential avenues to overcome this resistance with novel agents such as bispecific antibodies, Bruton's tyrosine kinase (BTK) degraders, non-covalent BTK inhibitors, and chimeric antigen receptor T (CART).

Management of Toxicities of BTK Inhibitors in B-Cell Malignancies

Brüton’s tyrosine kinase (BTK) inhibitors have significantly advanced treatment options for patients with B-cell malignancies; however, side effects with these agents are not uncommon, and a multidisciplinary approach is necessary for monitoring and managing adverse events such as bleeding, hypertension, and atrial fibrillation. In the treatment of patients on BTK inhibitors, maintaining communication between patients and all providers is essential, particularly concerning the use of any concurrent medications or supplements, as is educating patients on identifying early risk factors and reporting any signs or symptoms of toxicities. Ongoing clinical studies with both covalent and noncovalent BTK inhibitors are expected to further increase the understanding of these toxicities while improving the efficacy and safety of this class of medications.

Development of a new class of potent and highly selective G protein-coupled receptor kinase 5 inhibitors and structural insight from crystal structures of inhibitor complexes

G protein-coupled receptor kinase 5 (GRK5) is an important drug development target for heart failure, cardiac hypertrophy, and cancer. We have designed and developed a new class of highly selective, potent, and non-covalent GRK5 inhibitors. One of the inhibitors displayed GRK5 IC50 value of 10 nM and exhibited >100,000-fold selectivity over GRK2. The X-ray structure of a ketoamide-derived inhibitor-bound GRK5 showed the formation of a hemithioketal intermediate with active site Cys474 in the GRK5 active site and provided new insights into the ligand-binding site interactions responsible for high selectivity. The current studies serve as an important guide to therapeutic GRK5 inhibitor drug development.

Preclinical Efficacy and Mechanism for a Novel BTK-Protacs Against Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) is a rare and aggressive form of B-cell non-Hodgkin lymphoma (NHL) that exhibits diverse clinical courses and biological contexts. Given the significance of Bruton tyrosine kinase (BTK) as a therapeutic target in MCL, this study aimed to design a range of BTK-PROTACs. The objective was to develop efficient BTK-PROTACs capable of degrading BTK proteins, inhibiting the proliferation of MCL cells, and overcoming resistance to both covalent and non-covalent BTK inhibitors in both in vivo and in vitro settings. Methods:A series of BTK-PROTACs were gradually screened by Western blot and MTS assays, and the best active drug was finally determined. Cell function experiments in vitro were applied to verify the screening results and therapeutic efficacy was tested in vivo in NSG mice. RNA sequencing identified differentially expressed genes and important signaling pathways that influenced by BTK-PROTACs. BTKi-resistant cell lines were constructed to evaluate the ability of BTK-PROTACs to degrade BTK protein and suppress the proliferation of MCL cells. R esults: BTK-PROTACs C23 demonstrated effective degradation of BTK protein and inhibition of tumor cell growth across various MCL cell lines. Additionally, C23 exhibited the ability to induce cell apoptosis, activate the ubiquitin pathway, and inhibit the B-cell receptor (BCR), NF-κB, and PI3K-AKT-mTOR signaling pathways. Importantly, C23 effectively degraded BTK in both Z138-BTK C481S and Z138-BTK L528W cells, suggesting its potential to overcome resistance to both covalent and non-covalent BTK inhibitors. In our in vivo experiments using MCL xenograft NSG mouse models, BTK-PROTACs C23 significantly impeded tumor proliferation in Z138-BTK WT, Z138-BTK C481S, and Z138-BTK L528W MCL models. Additionally, C23 induced the degradation of BTK in tumor tissues. Notably, HE staining revealed no significant adverse effects of BTK-PROTACs C23 on normal organs and tissues. Moreover, routine blood tests and liver function tests showed no impairment of hematopoietic or hepatic function caused by BTK-PROTACs C23. C onclusion: In this study, we successfully designed and synthesized BTK-PROTACs C23, which demonstrated a remarkable ability to degrade BTK protein and effectively suppress the proliferation of MCL cells both in vitro and in vivo. Importantly, BTK-PROTACs C23 showed promising potential to overcome resistance to both covalent and non-covalent BTK inhibitors.

Pirtobrutinib, a Non-Covalent (reversible) BTK Inhibitor, in Mantle Cell Lymphoma Patients Previously Treated with a Covalent BTK Inhibitor: Results from a China Phase 2 Study

Background Pirtobrutinib, a highly selective, non-covalent (reversible) Bruton tyrosine kinase inhibitor (BTKi), inhibits both wildtype and C481-mutant BTK and has favorable oral pharmacology that enables continuous BTK inhibition throughout the dosing interval regardless of protein turnover, and shows promising efficacy and favorable safety profile in patients with pre-treated B-cell malignancies including a covalent BTKi (cBTKi). This study (NCT04849416) was a China only phase 2, open-label, multi-center trial aiming to evaluate the safety and efficacy of oral pirtobrutinib as a monotherapy for patients with B-cell malignancies. This abstract summarizes the findings on the efficacy in a cBTKi pre-treated mantle cell lymphoma (MCL) population and safety outcomes among all enrolled patients. Methods Eligible Chinese patients with relapsed/refractory B-cell malignancies were enrolled and assigned to one of three cohorts based on tumor histology and prior treatment history. All enrolled patients received pirtobrutinib monotherapy at a daily dose of 200 mg, until disease progression (PD), unacceptable side effects, or other reasons for discontinuation. The primary analysis set (PAS) comprised patients with centrally confirmed non-blastoid MCL, who received a prior cBTKi, and with measurable lesion(s) at baseline. The efficacy analysis set (EAS) was extended into all MCL patients treated with a prior cBTKi. The safety population included all patients who received at least one dose of pirtobrutinib. The primary objective was to evaluate the effectiveness by assessing the overall response rate (ORR) based on Lugano criteria and was conducted by an independent review committee (IRC). Secondary objectives included duration of response (DOR), progression-free survival (PFS), overall survival (OS) and safety profile. Results As of April 10, 2023, of 39 enrolled MCL patients, 28 were in the PAS and 35 were in the EAS for efficacy analysis. The safety analysis included a total of 87 enrolled patients and the median duration of therapy was 4.2 months. In the PAS population (N=28) with cBTKi pre-treated non-blastoid MCL, patients had a median of 3.0 prior lines of therapy (range, 1-8). The majority received prior chemotherapy (96.4%) and an anti-CD20 antibody (85.7%). The most common reason for discontinuation from the prior cBTKi was PD (85.7%). The IRC-assessed ORR was 71.4% (95% CI, 51.3-86.8), with 4 patients (14.3%) achieving complete responses (CR) and 16 patients (57.1%) achieving partial responses (PR). The median DOR assessed by IRC was not reached at the cut-off date with a 6-month DOR rate of 66.02% (95% CI, 39.29-83.14). The median IRC-assessed PFS was 9.43 months (95% CI: 5.32-not evaluable [NE]), and the median OS was 15.47 months (95% CI, 8.67-NE). The EAS (N=35), demonstrated similar efficacy results with IRC-assessed ORR 62.9% (95% CI, 44.9-78.5) and the 6-month DOR rate was 64.45%. In the safety population (N=87) of all enrolled B-cell malignancy patients, common treatment-emergent adverse events (TEAEs) of any grade included decreased neutrophil count (40.2%*), anemia (31%), and increased bilirubin (23.0%, with 2.3% grade ≥3 and none led to dose modification). The most frequent grade ≥3 TEAE was decreased neutrophil count (25.3%*). All grade hemorrhage was observed in 19.5% patient (n=17, with 2.3% grade ≥3), while all grade hypertension was 5.7% (n=5, with 2.3% grade ≥3), and no patient developed atrial fibrillation/flutter. Three patients (3.4%) stopped treatment due to treatment-related TEAEs, and 2 patients (2.3%) experienced treatment related fatal TEAEs (one infection and one patient with reported tumor lysis syndrome, tumor necrosis, and hemorrhage). Conclusion Pirtobrutinib demonstrated clinically meaningful and durable antitumor activity in a cBTKi pre-treated MCL Chinese population and showed a well-tolerated safety profile in patients with B-cell malignancies. * Aggregate of neutropenia and neutrophil count decreased

ONO-7018, a First-in-Class MALT1 Inhibitor, Provides Novel Therapeutic Strategies for B Cell Malignancies: Overcoming BTK Inhibitor Acquired Resistance and Enhancing the Antitumor Effect of BTK Inhibitors

Introduction: B cell receptor (BCR) signaling plays a crucial role in promoting survival and growth in B cell malignancies, and several compounds targeting the signaling pathways have been approved. Bruton's tyrosine kinase (BTK) is a key molecule in BCR signaling pathways and BTK inhibitors are one of the essential treatment options for chronic lymphocytic leukemia (CLL) and certain subtypes of B cell Non-Hodgkin Lymphoma. However, there are issues that acquired resistance mutations in the BTK kinase domain and gain of function mutation of its downstream molecules, which lead to BTK inhibitor resistance and limit its efficacy. For example, BTK C481S mutation is well known as a resistant mutation to covalent BTK inhibitors and several mutations of BTK (such as T474I and L528W mutation) have recently been reported in relapsed or refractory CLL patients with acquired resistance to pirtobrutinib, a non-covalent BTK inhibitor. In addition, there are unmet medical needs to improve the duration of response of current standard therapies including BTK inhibitors for B cell malignancies. Therefore, therapeutic strategies to overcome the BTK inhibitor resistance and novel combination therapies to enhance the efficacy of BTK inhibitors are required. Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is known as a key growth and survival regulator in BCR signaling and an emerging therapeutic target. ONO-7018 (formerly known as CTX-177), a potent and selective MALT1 inhibitor, demonstrated preclinical efficacy in several lymphoma models. Aim: Firstly, to explore the potential of ONO-7018 to overcome BTK inhibitor acquired resistance, we tested the antitumor effects of ONO-7018 on cell lines harboring BTK inhibitor-resistant mutations. Secondly, we examined the combination antitumor efficacy of ONO-7018 and tirabrutinib, a second generation BTK inhibitor in vitro and in vivo. Methods: Diffuse large B-cell lymphoma (DLBCL) cell lines harboring BTK inhibitor-resistant mutations (BTK T474I, C481S, or L528: PLCG2 R665W) were generated by continuous passage with BTK inhibitor or CRISPR-Cas9 gene editing followed by single cell cloning. In in vivo study, human DLBCL cell line, TMD8 cells were subcutaneously implanted in immune deficient mice. ONO-7018 and tirabrutinib were orally administered to the mice twice a day. Results: Anti-proliferative activities of ONO-7018 on these BTK inhibitor resistant cell lines were evaluated in vitro. ONO-7018 inhibited the proliferation of these cells with comparable IC 50 values to the parent cell line. Anti-proliferative activities of ONO-7018 alone or in combination with tirabrutinib on DLBCL and mantle cell lymphoma (MCL) cell lines were evaluated in vitro. ONO-7018 exhibited synergistic effects with tirabrutinib in both DLBCL and MCL cell lines. In addition, combination treatment of ONO-7018 with tirabrutinib strongly inhibited the expression of interferon regulatory factor 4, which is a downstream signal molecule in BCR signaling pathways, compared to their single treatment. We then evaluated the antitumor effects of ONO-7018 in combination with tirabrutinib in the xenograft model. Combination treatment of ONO-7018 with tirabrutinib showed dramatic antitumor effect with tumor regression. These results suggest that ONO-7018 could demonstrate antitumor effect on patients with B cell malignancies harboring BTK inhibitor acquired resistant mutations and synergistic antitumor effect with BTK inhibitors in clinic. Conclusion: ONO-7018 would provide novel therapeutic strategies to overcome the BTK inhibitor acquired resistance and enhance the antitumor effect of BTK inhibitors in clinic. Phase 1 study of ONO-7018 (NCT05515406) is currently ongoing.

Targeting Covalent and Non-Covalent Btki-Resistant CLL Using the Dual Irreversible/Reversible 4 th Generation BTK Inhibitor LP-168

Background: Treatment of chronic lymphocytic leukemia (CLL) has been transformed with targeted therapies including inhibitors of Bruton's tyrosine kinase (BTKi). Currently three covalent BTK inhibitors (cBTKi) are approved for CLL, but most patients eventually relapse, commonly through acquisition of the C481S BTK mutation (Woyach et al. 2014). Pirtobrutinib and nemtabrutinib are non-covalent BTK inhibitors (ncBTKi) developed to target and inhibit C481S mutant BTK. However novel secondary site mutations in BTK, namely T474I and L528W, have been found to confer resistance to both cBTKi and ncBTKi (Wang et al. 2022). Regardless of these mutations, BCR signaling remains intact, suggesting that inhibition of BTK maintains its therapeutic importance. LP-168 is a novel ultra-selective 4 th generation BTKi with an active warhead capable of covalent interaction with WT BTK or non-covalent binding when a BTK C481 mutation is present. Methods: To determine the target selectivity of LP-168 we screened the compound against 468 kinases in the scanMAX Kinase Assay. For validation of covalent binding, dialysis against WT BTK was performed. Primary CLL B cells were isolated by negative selection and treated with LP-168 for all experiments. BCR signaling alterations were assessed via immunoblot to observe changes in target protein phosphorylation. Changes in CLL migration towards CXCL12 or CXCL13 were interrogated by transwell migration. Cytotoxicity towards primary CLL cells was measured via Annexin V/PI flow cytometry with and without HS-5 stromal cell support. Experiments using TMD8 cells were performed following CRISPR modification to insert WT, C481S, or T474I BTK. In vivo survival studies were performed using both the Eμ-TCL1 and Eμ-MTCP1 mouse engraftment models. Results: LP-168 demonstrated selectivity towards BTK with roughly 700-fold selectivity for BTK vs its next off-target. In an enzymatic assay, LP-168 displayed nanomolar potency towards both WT BTK (IC 50=0.11 nM) and C481S BTK (IC 50=1.0 nM). Following 2-hour drugging, LP-168 inhibited BCR signaling in primary CLL B cells at increasing concentrations, demonstrated by reduced phosphorylation of BTK (92%, p<0.0001, n=8) and its immediate downstream target PLCγ2 (41%, p=0.0013, n=8). Migration capacity of primary CLL cells towards CXCL12 and CXCL14 was also found to decrease after 2-hours of treatment with LP-168 (52%, p<0.001, n=10 and 51%, p<0.001, n=10, respectively). After 48 hours of exposure, LP-168 was able to induce cytotoxicity of primary CLL cells alone and in co-culture with HS-5 stromal cells in a dose-dependent manner, with similar or improved efficacy to other BTKi. LP-168 decreased CLL cell production of CCL3 and CCL4 chemokines (CCL3: 93%, p<0.001, n=8; CCL4: 93%, p<0.001, n=8). LP-168 also demonstrated modest cytotoxicity towards TMD8 cells harboring either C481S BTK (24%, p=0.0008 , n=3) or T474I BTK (13%, p=0.0690, n=3), as well as decreased production of CCL3 (C481S: 95%, p<0.0001, n=3; T474I: 99%, p<0.0001, n=3) and CCL4 (C481S: 97%, p<0.0001, n=3; T474I: 98%, p<0.0001, n=3) (Figure 1A). Further, we observed inhibition of downstream BCR signaling in primary CLL B cells harboring C481S via immunoblotting and cytokine production. Finally, to evaluate the in vivo efficacy of LP-168 we tested this compound in both the Eµ-TCL1 and Eµ-MTCP1 models. We treated mice daily via oral gavage and found that 50 mg/kg of LP-168 significantly improved survival in the Eµ-TCL1 model when compared to vehicle (median 51 vs 44 days; p=0.0018) or ibrutinib at 50 mg/kg (median 51 vs 45 days; p=0.0098) and the Eµ-MTCP1 model when compared with vehicle (median 122 vs 62 days; p<0.0001) or ibrutinib at 50 mg/kg (median 122 vs 96 days; p=0.0162) (Figure 1B). Conclusions: Collectively, our data demonstrate that LP-168 is a potent and selective inhibitor of BTK with activity against CLL, even in the presence of mutations that mediate resistance to cBTKi and ncBTKi. These data support the continued preclinical and clinical investigation of LP-168, which is currently being studied in the phase 1 setting of CLL (NCT04775745) and NHL (NCT04993690).

Preclinical Study of DZD8586, a Non-Covalent LYN/BTK Dual Inhibitor with Excellent BBB Penetration, for the Treatment of B-Cell Non-Hodgkin Lymphoma (B-NHL)

Introduction Bruton's Tyrosine Kinase (BTK) inhibitors have been approved for the treatment of B-NHL, such as CLL and MCL. However, resistance to BTK inhibitors still inevitably occurred, and different types of mutations were identified post covalent and non-covalent BTK inhibitor treatment. Although BTK inhibitors showed some activities in the treatment of relapsed or refractory non-germinal center B-cell (non-GCB) DLBCL, no drugs have been approved due to unsustainable tumor response. It is hypothesized that blockage of BTK pathway alone is not sufficient to achieve optimal anti-tumor efficacy in DLBCL. Due to limited blood-brain barrier (BBB) penetration of the current therapies, treatment of central nervous system lymphoma (CNSL) remains a clinical challenge. DZD8586 is a rationally designed, oral, non-covalent, LYN and BTK dual inhibitor with excellent BBB penetration as a potential treatment option for B-NHL. Here we report the preclinical activity of DZD8586 in lymphoma cell lines and animal models which supports its clinical development to address the above unmet medical needs. Methods The enzymatic activity of DZD8586 against multiple kinases was assessed at Eurofins panel with purified enzymes. TMD-8-IbruR cells carrying the C481S mutation were generated by stepwise induction of increasing concentrations of Ibrutinib. Cells carrying pirtobrutinib resistance mutations were obtained by transfection of different BTK mutations into RI-1 cells using lentivirus. Cell growth inhibition was determined by CellTiter-Glo. A pBTK enzyme-linked immunosorbent assay (ELISA) was used to evaluate the pharmacodynamic profile of DZD8586. Western blot was used to test the modulations of signaling pathways with DZD8586 treatment. Animal models were established by subcutaneous and intracranial inoculation of tumor cells, respectively, and animals were treated with DZD8586 or other BTK inhibitors. The anti-tumor efficacy was assessed by comparing the change in mean tumor volume between the different treatment groups and vehicle controls. Results Kinase panel profiling showed that DZD8586 potently inhibited LYN and BTK, with good selectivity against other kinases. In cell lines expressing C481X and pirtobrutinib resistance mutations, DZD8586 demonstrated concentration dependent anti-proliferative effects, with similar GI 50s among cells carrying different BTK mutations. In a panel of DLBCL cell lines, DZD8586 exhibited potent cell growth inhibition and induced cell death in a variety of cell lines, and its effect correlated with modulations of both LYN and BTK pathways. In both subcutaneous and CNS tumor models, DZD8586 exhibited profound tumor growth inhibition in a dose-dependent manner, and induced tumor regression at the doses of 50 mg/kg and above. There was a good correlation between plasma concentrations of DZD8586 and modulations of pBTK in whole blood and tumor tissue. DZD8586 has good cell permeability and is not significantly transported by P-gp and BCRP expressed at the BBB. The K puu,CSF of DZD8586 in rat and monkey were 1.2 and 1.3, respectively, suggesting its excellent BBB penetration in humans. Conclusion DZD8586 is a novel non-covalent LYN/BTK dual inhibitor, which could overcome resistance mutations to the approved covalent and non-covalent BTK inhibitors, and derive clinical benefit to patients with DLBCL and CNSL by blocking both BTK-dependent and BTK-independent signaling pathways with excellent BBB penetration.

Characterization and Preclinical Evaluation of AS-1763, an Oral, Potent and Selective Noncovalent BTK Inhibitor, in Chronic Lymphocytic Leukemia

Background: Covalent Bruton's tyrosine kinase (BTK) inhibitors (cBTKi) have transformed treatment landscape of chronic lymphocytic leukemia (CLL). These inhibitors bind to C481 residue in the kinase domain of BTK. This is also the site for the most common mutations rendering cells resistant to cBTKi. To circumvent this limitation of the cBTKi, non-covalent BTKi (ncBTKi) such as pirtobrutinib have been developed. Recently, non-C481 BTK mutations have been reported in patients with CLL at the time of disease progression during pirtobrutinib treatment. These observations underscore the need for ncBTKi that can target C481 as well as non-C481 mutations of BTK. AS-1763 is a potent, highly selective, orally available, and ncBTKi, equipotent against both wild-type and C481S-mutated BTK when tested in biochemical assays. In vivo, AS-1763 demonstrated significant antitumor effects in OCI-LY10 tumor xenograft models harboring wild-type or C481S mutant BTK (Kawahata et al. J Med Chem 64:14129, 2021). Study Design and Methods: In the present project, first we used cell free assay systems to evaluate selectivity and potency of AS-1763 by a kinome-wide profiling and inhibitory effect of AS-1763 in enzyme assays using recombinant mutant BTK. Second, we examined dose- and time-dependent inhibition of mutant BTK that is expressed in HEK293 cell line. Third, we tested biological, biochemical, and molecular impact of AS-1763 in primary CLL cells. Fourth, we determined sensitivity of CLL cells to AS-1763 when combined with other targeted agents. Results: AS-1763 showed a highly selective profile for BTK in a panel of 291 kinase assays with >260-fold selectivity except 3 Tec family kinases (BMX, ITK, and TEC). We have generated a total of 17 recombinant BTK mutant proteins (C481, T474, L528 variants and other BTK mutants; Table 1) reported in the literature or predicted by single nucleotide change in the codon, and established assay methods to measure inhibitory potency of BTKi for those BTK mutants (Table 1). AS-1763 showed potent inhibitory activities for those BTK mutants while inhibitory potencies of other cBTKi and ncBTKi were diminished against some BTK mutants such as T474 and/or L528 mutations. AS-1763 exhibited dose-dependent and slow-off rate inhibitions of BTK autophosphorylation (pY223) in HEK293 cells transfected with various BTK mutants. Furthermore, the observed inhibitory effects of AS-1763 on the BTK autophosphorylation (pY223) in HEK293 cells were continued up to 24 h after washing out of AS-1763. In primary CLL samples, AS-1763, pirtobrutinib or ibrutinib induced a modest apoptosis. AS-1763 effectively inhibited the BCR signaling pathway in a dose dependent manner as evidenced by downregulation of pY223-BTK expression which was also observed in cBTKi and ncBTKi relapsed/refractory CLL samples. In vitro incubations with AS-1763 inhibited CLL cell spontaneous migration, decreased CCL3/CCL4 levels in culture supernatant and was accompanied with the inhibition of intracellular calcium release and B-cell activation, as measured by surface CD86 expression. Besides, AS-1763 incubation for 24 hours was shown to modulate the expression of BCL-2 family proteins with the downregulation of MCL-1 and BCL-xl. Interestingly, in vitro treatment of CLL patient samples with AS-1763 demonstrated a notable elevation in cellular ROS and mitochondrial superoxide levels starting at 1 µM, concurrently impacting SOD1 expression in CLL patient samples. Evaluation of drug interaction models utilizing Compusyn and Synergy Finder applications predominantly indicated additive effects between AS-1763 with BCL-2 inhibitor, venetoclax as well as p53 activator APR-246. Consistent with this data, AS-1763 and venetoclax combinations showed high-rates of apoptosis in samples that were relapsed/refractory to cBTKi and ncBTKi. Conclusions: AS-1763 is a selective ncBTKi that inhibits both wild-type and mutant BTKs listed in Table 1. In CLL cells, AS-1763 was effective in inhibiting BCR pathway signaling and sensitized cells to other agents such as venetoclax. Based on these encouraging data we have initiated a clinical trial to test AS-1763 in patients with CLL and other B cell malignancies who have failed or are intolerant to at least two prior lines of systemic therapy, including cBTKi (NCT05602363 Clinical Trials.gov).

A Phase 1a/b Trial of Luxeptinib (CG-806) in Patients with Relapsed/Refractory B-Cell Malignancies or Acute Myeloid Leukemia and Evaluation of New G3 Formulation

INTRODUCTION: Luxeptinib (CG-806; LUX) is an orally active noncovalent kinase inhibitor of Bruton's tyrosine kinase (BTK), wild type and mutant forms of Fms-like tyrosine kinase 3 (FLT3) (including the internal tandem duplicates (ITD), tyrosine kinase domain (TKD), and F691L mutant forms), and growth receptors like KIT, CSF1R, PDGFRα, and TRKs. In prior studies, LUX has been shown to suppress aberrant proliferative signaling in B cell malignancies and acute myeloid leukemia (AML) via regulation of BTK, LYN, SYK, AKT, ERK, and MAPK. LUX is cytotoxic to primary AML cells insensitive to other FLT3 inhibitors and to malignant B-cells insensitive to ibrutinib, at pM and nM concentrations, respectively. AIMS: The primary objectives of these studies are to assess the safety, tolerability, and pharmacokinetics (PK) of LUX and determine recommended phase 2 doses for relapsed or refractory (R/R) AML and B cell malignancy patients. METHODS: In two studies (NCT04477291; NCT03893682), LUX (original formulation, G1) was administered continuously as oral capsules BID in 28-day cycles of ascending cohorts (relapsed or refractory de novo, secondary, or therapy-related AML or higher risk myelodysplastic syndrome (MDS), and relapsed and refractory for B cell lymphoma and chronic lymphocytic/small lymphocytic leukemia). A novel generation 3 (G3) formulation of LUX designed to increase bioavailability was tested at a single-dose (sub-study) for relative bioavailability (RBA), followed by continuous dosing in subsequent R/R AML patients. RESULTS: In the B-cell study (as of 15 th May 2023), LUX has been administered to 36 patients at dose levels from 150 mg to 900 mg BID in patients with a median of 3 lines of prior treatment, with 47.2% having received a BTK-inhibitor. Only one (2.8%) patient had a DLT of hypertension and 14 (38.9%) experienced at least one drug related ≥Grade3 treatment emergent adverse event (TEAE). Two patients (900 mg) are currently on study with no DLT and no ≥Grade 3 related TEAEs. A Follicular lymphoma patient is at cycle 28, and achieved a best response as partial response (PR) with a decrease in lesion size of 76.2%; an SLL patient is at cycle 13, and achieved stable disease (SD) showing a decrease in lesion size of 43.1% (Figure A). The overall best response achieved among the 17 patients who have been on treatment for more than 12 weeks are stable disease (SD) (n=11), partial response (PR) (n=3), minor response (n=2) and complete response (CR) (n=1). As of 5 th June 2023, in the AML trial, a total of 40 patients (16 (40%) FLT3-ITD, 21 (52.5%) FLT3-WT, 2 (5.0%) FLT3-TKD, and 1 (2.5%) unknown mutations) with a median of 3 prior treatments (range, 1 - 10) have been treated with LUX G1 formulation at dose levels from 450 mg - 900 mg BID (n=34) or with 50 mg BID of G3 formulation (n=6). Four (10%) experienced drug related SAE and 7 (17.5%) had a drug related grade ≥3 TEAEs. The most common related TEAEs were lymphocyte count decrease, platelet count decrease, and anemia (n=2; 5% patients each). One (2.5%) patient at 450 mg dose, reported complete remission without minimum residual disease (CRmrd) and remained on study for 56 weeks. In the RBA sub-study, G3 (50 mg) produced comparable exposures to G1 (450-900 mg range) (Figure B) and was, therefore, selected as the starting dose for use in the R/R AML study. Six patients were treated with a continuous dosing of 50 mg BID G3; with plasma levels of 274 ng/mL and 195 ng/mL LUX observed by the end of C1D15 and C1D22 respectively. No drug related Grade ≥3 TEAEs or DLTs were observed in 50 mg BID G3 LUX treated patients; with no new safety signals observed for the G3 formulation. Based on these findings and expected exposures for higher dose levels, the cohort safety review committee has approved escalation of G3 dosing to 200 mg BID at which dosing is ongoing. CONCLUSIONS: LUX has a favorable safety profile in patients at all tested dose levels, for multiple cycles, for both studies. Antitumor activity was observed in a heavily pretreated relapsed AML patient and in multiple B-NHL subtypes and CLL/SLL patients including several with prior ibrutinib exposure. Continuous dosing of patients with R/R AML and Higher-Risk MDS with the G3 formulation is ongoing; with updated clinical data (200 mg dose level) to be presented at the meeting.

Initial Results of a Phase 1 Dose Escalation Study of LP-168, a Novel Covalent and Non-Covalent Next-Generation Inhibitor of Bruton's Tyrosine Kinase

Introduction: Inhibitors of Bruton's Tyrosine Kinase (BTKi) have been transformative therapies in chronic lymphocytic leukemia (CLL) and other lymphoid malignancies. Despite extended remissions, most patients (pts) will relapse. Covalent BTKi (cBTKi) including ibrutinib, acalabrutinib, and zanubrutinib, share a common resistance mechanism, acquisition of a C481 mutation in BTK. Non-covalent BTKi (ncBTKi) can overcome this, however, second-site mutations, notably T474 gatekeeper and kinase-dead mutations, have been observed. LP-168 is a selective next generation inhibitor of BTK that can bind wild-type and C481-mutated BTK covalently and non-covalently, respectively, with preclinical activity in resistant CLL models (submitted to ASH 2023). This dual activity is hypothesized to allow efficacy against cells with wild-type BTK through covalent activity while preventing expansion of the most common resistance mechanisms through non-covalent activity. Here we describe initial results of a phase 1 study of LP-168, with safety evaluation focusing on DLT evaluable pts, and efficacy evaluation focusing on the cohort of pts with CLL. Methods: NCT04775745 is a multicenter phase 1 dose escalation study of LP-168 monotherapy in pts with relapsed/refractory B-cell malignancies. CLL pts must have received ≥2 prior therapies. Dose escalation was performed using 3+3 design with expansion at multiple doses. Treatment emergent adverse events (TEAEs) were assessed per NCI CTCAE v5.0. Responses were evaluated using iwCLL 2018 criteria. Intra-patient dose escalation was permitted when dose levels cleared safety evaluation. Results: From 16 Sep 2021 to 10 Jul 2023, 43 DLT-evaluable pts were enrolled, including 35 with CLL, at doses from 100-300 mg daily as well as 150 mg twice daily. Data for this abstract were locked 10 Jul 2023. CLL pts received a median of 3 prior therapies, with 94% receiving a prior cBTKi and 11% a ncBTKi. 20 pts had alterations in TP53, 21 pts had C481S BTK mutations, 7 pts had gatekeeper mutations at T474 BTK, and 5 pts had mutation in PLC γ2 (Table). 33 pts have response data with median follow-up of 12.6 months (Figure). Overall response rate (ORR) including all dose levels was 54.5% (18/33)-all were partial response (PR) or partial response with lymphocytosis (PR-L). When evaluating only pts at doses of 200 mg or higher, ORR was 66.7% (10/15). In the subgroup of pts who had received only one prior BTKi including all dose levels, ORR was 77.8% (14/18). In the subgroup of pts with both BTK C481 and T474 mutations at doses of 150mg BID or 300mg QD, ORR was 75% (3/4). At the time of this report, only 5 pts with CLL have discontinued the study for progressive disease. At data lock, 43 DLT evaluable pts (with all histologies) were evaluable for safety. No DLT have been observed, and most AE have been low grade. TEAE seen in >20% of pts included diarrhea, fatigue, arthralgia, headache, constipation, cough, and dizziness; of those, grade 3 or higher AE included fatigue and constipation in 1 pt each. Serious AE were seen in 11 pts, of which 1 led to discontinuation (intracranial hemorrhage in a mantle cell lymphoma pt in complete remission). No DLT was identified at doses up to 300 mg daily and 150 mg twice daily. No atrial fibrillation/flutter has been reported. Hypertension grade 3 or higher was seen in 1 pt, and 1 pt experienced high-grade bleeding (as described). AE frequency did not increase with increasing doses. Pharmacokinetics showed plasma exposure increases dose-dependently. Steady state AUC 24h was between 47.1-94.6 ug/mL*h with limited accumulation. Steady state half-life was between 15.3-21.9 hours at all doses and C max was between 3500 and 5600 ng/mL. BTK occupancy is nearly at 100% at dose levels that were tested, with little variability among pts. Collective review demonstrates optimal Cmax to be achieved at either 200 or 300 mg dosed daily. Conclusions: LP-168 has been well tolerated in this study, with no DLTs identified at doses up to 300 mg daily. Preliminary efficacy was observed in high risk CLL pts, including those treated with one or more BTKi. Considering safety, efficacy, and PK/PD, dose expansion will occur at 200 mg and 300 mg daily following the FDA Project Optimus guidelines to determine the recommended phase 2 dose. A separate cohort will enroll pts with gatekeeper mutations to further explore efficacy. Updated biomarker, safety, and efficacy data will be presented at the meeting.

A combination of structure-based virtual screening and experimental strategies to identify the potency of caffeic acid ester derivatives as SARS-CoV-2 3CLpro inhibitor from an in-house database

Drug development requires significant time and resources, and computer-aided drug discovery techniques that integrate chemical and biological spaces offer valuable tools for the process. This study focused on the field of COVID-19 therapeutics and aimed to identify new active non-covalent inhibitors for 3CLpro, a key protein target. By combining in silico and in vitro approaches, an in-house database was utilized to identify potential inhibitors. The drug-likeness criteria were considered to pre-filter 553 compounds from 12 groups of natural products. Using structure-based virtual screening, 296 compounds were identified that matched the chemical features of SARS-CoV-2 3CLpro peptidomimetic inhibitor pharmacophore models. Subsequent molecular docking resulted in 43 hits with high binding affinities. Among the hits, caffeic acid analogs showed significant interactions with the 3CLpro active site, indicating their potential as promising candidates. To further evaluate their efficacy, enzyme-based assays were conducted, revealing that two ester derivatives of caffeic acid (4k and 4l) exhibited more than a 30% reduction in 3CLpro activity. Overall, these findings suggest that the screening approach employed in this study holds promise for the discovery of novel anti-SARS-CoV-2 therapeutics. Furthermore, the methodology could be extended for optimization or retrospective evaluation to enhance molecular targeting and antiviral efficacy of potential drug candidates.

A Small Molecule with Big Impact: MRTX1133 Targets the KRASG12D Mutation in Pancreatic Cancer.

KRAS mutations drive oncogenic alterations in numerous cancers, particularly in human pancreatic ductal adenocarcinoma (PDAC). About 93% of PDACs have KRAS mutations, with G12D (∼42% of cases) and G12V (∼32% of cases) being the most common. The recent approval of sotorasib (AMG510), a small-molecule, covalent, and selective KRASG12C inhibitor, for treating patients with non-small cell lung cancer represents a breakthrough in KRAS targeted therapy. However, there is a need to develop other much-needed KRAS-mutant inhibitors for PDAC therapy. Notably, Mirati Therapeutics recently developed MRTX1133, a small-molecule, noncovalent, and selective KRASG12D inhibitor through extensive structure-based drug design. MRTX1133 has demonstrated potent in vitro and in vivo antitumor efficacy against KRASG12D-mutant cancer cells, especially in PDAC, leading to its recent initiation of a phase I/II clinical trial. Here, we provide a summary of the recent advancements related to the use of MRTX1133 for treating KRASG12D-mutant PDAC, focusing on its efficacy and underlying mechanistic actions. In addition, we discuss potential challenges and future directions for MRTX1133 therapy for PDAC, including overcoming intrinsic and acquired drug resistance, developing effective combination therapies, and improving MRTX1133's oral bioavailability and target spectrum. The promising results obtained from preclinical studies suggest that MRTX1133 could revolutionize the treatment of PDAC, bringing about a paradigm shift in its management.