Wild-type Bruton's Tyrosine Kinase Research Articles

Discovery of a novel BTK inhibitor S-016 and identification of a new strategy for the treatment of lymphomas including BTK inhibitor-resistant lymphomas.

Bruton's tyrosine kinase (BTK) has emerged as a therapeutic target for B-cell malignancies, which is substantiated by the efficacy of various irreversible or reversible BTK inhibitors. However, on-target BTK mutations facilitating evasion from BTK inhibition lead to resistance that limits the therapeutic efficacy of BTK inhibitors. In this study we employed structure-based drug design strategies based on established BTK inhibitors and yielded a series of BTK targeting compounds. Among them, compound S-016 bearing a unique tricyclic structure exhibited potent BTK kinase inhibitory activity with an IC50 value of 0.5 nM, comparable to a commercially available BTK inhibitor ibrutinib (IC50 = 0.4 nM). S-016, as a novel irreversible BTK inhibitor, displayed superior kinase selectivity compared to ibrutinib and significant therapeutic effects against B-cell lymphoma both in vitro and in vivo. Furthermore, we generated BTK inhibitor-resistant lymphoma cells harboring BTK C481F or A428D to explore strategies for overcoming resistance. Co-culture of these DLBCL cells with M0 macrophages led to the polarization of M0 macrophages toward the M2 phenotype, a process known to support tumor progression. Intriguingly, we demonstrated that SYHA1813, a compound targeting both VEGFR and CSF1R, effectively reshaped the tumor microenvironment (TME) and significantly overcame the acquired resistance to BTK inhibitors in both BTK-mutated and wild-type BTK DLBCL models by inhibiting angiogenesis and modulating macrophage polarization. Overall, this study not only promotes the development of new BTK inhibitors but also offers innovative treatment strategies for B-cell lymphomas, including those with BTK mutations.

BELLWAVE-011: Phase 3 randomized trial of nemtabrutinib versus ibrutinib or acalabrutinib in untreated chronic lymphocytic leukemia/small lymphocytic lymphoma.

TPS7088 Background: Bruton tyrosine kinase (BTK) inhibitors have significantly improved survival in patients (pts) with chronic lymphocytic leukemia (CLL). Nemtabrutinib is a reversible noncovalent inhibitor of wild-type and C481-mutated BTK that has shown promising antitumor activity in pts with CLL (Woyach JA et al. Cancer Discovery 2023). In this ongoing trial, we evaluate the efficacy and safety of nemtabrutinib versus investigator’s choice of ibrutinib or acalabrutinib in pts with untreated CLL/small lymphocytic lymphoma (SLL) requiring therapy (NCT06136559). Methods: This randomized, open-label, parallel group, active-controlled phase 3 trial will enroll approximately 1200 pts aged ≥18 years. Eligible pts will have confirmed CLL/SLL with active disease based on the IWCLL criteria, which include one of the following: 1) progressive marrow failure or lymphocytosis, 2) massive, progressive, or symptomatic splenomegaly or lymphadenopathy, 3) autoimmune complications, 4) extranodal involvement, or 5) disease-related symptoms. The study will enroll patients who are treatment naïve, with an ECOG performance status 0-2, adequate organ function, and provide blood, bone marrow, and/or a lymph node sample. Pts are excluded if they have Richter Transformation, central nervous system (CNS) involvement by CLL/SLL, an active second malignancy, or a severe bleeding disorder. Pts will be randomized 1:1 to receive oral nemtabrutinib 65 mg QD, or investigator’s choice of oral ibrutinib 420 mg QD or acalabrutinib 100 mg BID. Randomization will be stratified by TP53aberration, clinical stage, investigator’s choice of comparator, and region. Treatment will continue on both arms until unacceptable toxicity, disease progression, or withdrawal. Disease response assessments by physical examination, constitutional symptoms, imaging, and evaluation of blood and bone marrow will be performed Q12W up to week 97, and Q24W thereafter. Primary efficacy endpoints are objective response (OR) and progression free survival (PFS) by BICR per iwCLL 2018 criteria. Secondary endpoints are overall survival (OS), duration of response (DOR) by BICR per iwCLL 2018, and safety. OR will be tested via a non-inferiority comparison, while PFS and OS will be tested for superiority. Adverse events will be monitored throughout the trial and graded per NCI CTCAE v5.0 and iwCLL scales. Enrollment in this trial is ongoing. Clinical trial information: NCT06136559 .

Abstract 6069: Discovery and preclinical development of orally bioavailable potent BTK degrader, and its early clinical development for the treatment of relapsed and refractory B-cell malignancies

Abstract Background : Bruton tyrosine kinase (BTK) is an important signaling hub that activates the B-cell receptor (BCR) signaling cascade, which is critical for growth and survival of B-cell lymphoma or leukemia. BTK inhibitors have shown anti-tumor activity in patients with B-NHL. However, nearly half of the patients on current BTK inhibitors discontinue the treatment due to adverse effects and/or mutation-induced drug resistance. A degrader is a heterobifunctional chemical compound aimed at eliminating disease-causing proteins, rather than simply inhibiting proteins’ enzymatic function, through the ubiquitin-proteasome system (UPS)-mediated protein degradation. This approach offers a unique solution for addressing relapse or refractory disease by removing overexpressed and mutated proteins, unlike traditional inhibitors. We thus designed UBX-382, a degrader targeting BTK. Results : UBX-382 showed superior degradation activity for both wild-type (WT) and mutant BTK proteins in a single-digit nanomolar range of half-maximal degradation concentration when compared to the competing degrader programs in diffuse large B-cell lymphoma cell line. UBX-382 effectively addressed 7 out of 8 previously reported resistant BTK mutants in in vitro experiments and outperformed ibrutinib, ARQ-531, and MT-802 in inhibiting tumor growth in murine xenograft models harboring WT or C481S mutant BTK-expressing TMD-8 cells. Remarkably, oral dosing of UBX-382 for <2 weeks led to complete tumor regression in 3 and 10 mg/kg groups in murine xenograft models. Following this, further optimization was carried out for potency and PK profile. We nominated the candidate for IND filing and are planning a phase 1 clinical trial. For the first-in-human, open-label, multicenter, phase 1 study, patients in part 1 (dose escalation with BOIN design) had R/R B-cell malignancies and had received at least 2 prior therapies. The candidate will be administered orally at 5 mg once daily followed by 2 days, for a 7-day schedule over 28-day cycles. Part 2 (dose expansion) consisted of disease-specific cohorts, including CLL/SLL, DLBCL, MCL, FL, MZL, and WM. The primary endpoints were safety and tolerability, along with the definition of the maximum tolerated dose. Secondary endpoints included pharmacokinetics/pharmacodynamics and preliminary efficacy. Conclusions : We have discovered potent BTK degrader exhibiting excellent anti-tumor activity in vitro and in vivo. In pre-clinical safety studies, the candidate of UBX demonstrated an acceptable safety profile. The candidate of UBX is currently preparing phase 1 clinical trials for hematological malignancies. Citation Format: Song Hee Lee, Sun-Mi Yoo, Ye Seul Lim, Han Wool Kim, Je Ho Ryu, Jong Hwan Lim. Discovery and preclinical development of orally bioavailable potent BTK degrader, and its early clinical development for the treatment of relapsed and refractory B-cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6069.

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).

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).

Initial Findings from a First-in-Human Phase 1a/b Trial of NX-5948, a Selective Bruton's Tyrosine Kinase (BTK) Degrader, in Patients with Relapsed/Refractory B Cell Malignancies

Introduction: Although BTK inhibitors (BTKi) are effective therapeutics in the treatment of B cell malignancies, emerging BTK resistance mutations in chronic lymphocytic leukemia (CLL), as well as potential growth-promoting kinase-independent scaffolding function of BTK, present a need for improved or new approaches. NX-5948 is a novel, orally administered small molecule that induces specific BTK protein degradation by the cereblon E3 ligase complex without degradation of other cereblon neo-substrates. Importantly, NX-5948 induces degradation of wild-type and mutant forms of BTK in B-cells [Noviski et al. 2023] at sub-nanomolar potencies and exhibits potent tumor growth inhibition in TMD8 xenograft models that contain either wild-type BTK or BTKi-resistant mutations [Robbins 2021]. Here we provide the first disclosure of preliminary safety and efficacy findings from a Phase 1a trial of NX-5948 in patients with relapsed/refractory B cell malignancies. Methods: NX-5948-301 is a Phase 1, first-in-human, dose-escalation and cohort-expansion trial evaluating the safety, tolerability, and clinical activity of NX-5948 in relapsed/refractory CLL and various subtypes of non-Hodgkin's lymphoma (NHL). Key eligibility criteria: ≥2 prior lines of therapy; measurable or other evaluable disease per indication-specific response criteria; Eastern Cooperative Oncology Group Performance Status (ECOG PS) 0-1. Phase 1a (dose escalation) evaluates safety and tolerability of NX-5948 via a standard 3+3 dose escalation in patients with relapsed/refractory B cell malignancies. Approximately 110 patients (30 in Phase 1a, 80 in Phase 1b) may be enrolled and treated until confirmed disease progression or unacceptable toxicity. Endpoints include dose-limiting toxicities (DLTs); treatment-emergent adverse events (TEAEs); deaths; changes in safety parameters; objective response rate per disease-specific response criteria. Phase 1b (dose expansion) will include up to four expansion cohorts. Results: As of June 9, 2023, 14 patients were enrolled in Phase 1a and received NX-5948 at 50 mg (n=7), 100 mg (n=4), or 200 mg (n=3) orally once daily. Median age was 65 (range 46-79) years; female/male ratio 28.6%/71.4%; white 92.9%; ECOG PS 0/1 21.4%/78.6%; primary diagnoses were CLL (n=4), diffuse large B cell lymphoma (DLBCL, n=4), mantle cell lymphoma (MCL, n=3), marginal zone lymphoma (MZL, n=2), and follicular lymphoma (FL, n=1). Median number of prior therapies was 4.5 (range 2-10), which included: for CLL - BTKi (n=4/4) and BCL2 inhibitor (n=3/4); for NHL - BTKi (n=5/10), bispecific antibody (n=3/10), and CAR-T (n=2/10). NX-5948 was well tolerated with no DLTs and no TEAEs resulting in drug discontinuation or dose reduction. In addition, there were no NX-5948-related grade ≥3 TEAEs or related serious adverse events. The most common TEAEs were purpura/contusion (57.1%, all below grade 3), nausea (35.7%), and thrombocytopenia (35.7%). No atrial fibrillation/flutter or hypertension was reported. Median duration of treatment was 2.8 (range 0.5-9.6) months with 9/14 patients remaining on treatment. Current data indicate that NX-5948 exhibits dose-dependent pharmacokinetics (PK) and a half-life of ~24 hours, supporting once daily dosing (Figure a). Rapid, robust and sustained BTK degradation was observed in all patients, regardless of absolute BTK starting level, tumor type, or NX-5948 dose (Figure b). Of three evaluable patients with CLL receiving the lowest dose of 50 mg, early signs of clinical activity were observed including one confirmed partial response (PR; at 8 and 16 weeks) and 2 patients with stable disease (SD; at 8 weeks). Further treatment responses will be reported at the time of presentation. Summary/conclusion: Current findings in this heavily pre-treated population of patients with CLL and NHL are encouraging and indicate that NX-5948 is safe and well tolerated and has clinical activity, supporting continuation of its development in CLL and NHL. NX-5948 also exhibits dose-proportional PK, resulting in rapid, robust and sustained BTK degradation. Additional data with higher dose levels and longer treatment duration will be presented at the meeting.

Identification and Characterization of ACP-5862, the Major Circulating Active Metabolite of Acalabrutinib: Both Are Potent and Selective Covalent Bruton Tyrosine Kinase Inhibitors .

Acalabrutinib is a covalent Bruton tyrosine kinase (BTK) inhibitor approved for relapsed/refractory mantle cell lymphoma and chronic lymphocytic leukemia/small lymphocytic lymphoma. A major metabolite of acalabrutinib (M27, ACP-5862) was observed in human plasma circulation. Subsequently, the metabolite was purified from an in vitro biosynthetic reaction and shown by nuclear magnetic resonance spectroscopy to be a pyrrolidine ring-opened ketone/amide. Synthesis confirmed its structure, and covalent inhibition of wild-type BTK was observed in a biochemical kinase assay. A twofold lower potency than acalabrutinib was observed but with similar high kinase selectivity. Like acalabrutinib, ACP-5862 was the most selective toward BTK relative to ibrutinib and zanubrutinib. Because of the potency, ACP-5862 covalent binding properties, and potential contribution to clinical efficacy of acalabrutinib, factors influencing acalabrutinib clearance and ACP-5862 formation and clearance were assessed. rCYP (recombinant cytochrome P450) reaction phenotyping indicated that CYP3A4 was responsible for ACP-5862 formation and metabolism. ACP-5862 formation Km (Michaelis constant) and Vmax were 2.78 μM and 4.13 pmol/pmol CYP3A/min, respectively. ACP-5862 intrinsic clearance was 23.6 μL/min per mg. Acalabrutinib weakly inhibited CYP2C8, CYP2C9, and CYP3A4, and ACP-5862 weakly inhibited CYP2C9 and CYP2C19; other cytochrome P450s, UGTs (uridine 5'-diphospho-glucuronosyltransferases), and aldehyde oxidase were not inhibited. Neither parent nor ACP-5862 strongly induced CYP1A2, CYP2B6, or CYP3A4 mRNA. Acalabrutinib and ACP-5862 were substrates of multidrug resistance protein 1 and breast cancer resistance protein but not OATP1B1 or OATP1B3. Our work indicates that ACP-5862 may contribute to clinical efficacy in acalabrutinib-treated patients and illustrates how proactive metabolite characterization allows timely assessment of drug-drug interactions and potential contributions of metabolites to pharmacological activity. SIGNIFICANCE STATEMENT: This work characterized the major metabolite of acalabrutinib, ACP-5862. Its contribution to the pharmacological activity of acalabrutinib was assessed based on covalent Bruton tyrosine kinase binding kinetics, kinase selectivity, and potency in cellular assays. The metabolic clearance and in vitro drug-drug interaction potential were also evaluated for both acalabrutinib and ACP-5862. The current data suggest that ACP-5862 may contribute to the clinical efficacy observed in acalabrutinib-treated patients and demonstrates the value of proactive metabolite identification and pharmacological characterization.

CLL-114 BRUIN CLL-321: A Phase 3 Open-Label, Randomized Study of Pirtobrutinib Versus Investigator's Choice of Idelalisib Plus Rituximab or Bendamustine Plus Rituximab in BTK Inhibitor–Pretreated Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (Trial in Progress)

Covalent Bruton tyrosine kinase (BTK) inhibitors (BTKi) have transformed the management of patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), but these treatments are not curative. Covalent BTKi share pharmacologic liabilities (e.g., low oral bioavailability, short half-life) that collectively may lead to suboptimal BTK target coverage, ultimately manifesting as acquired resistance in some patients. To address these limitations, pirtobrutinib, a highly selective, non-covalent BTKi that inhibits both wild-type (WT) and C481-mutated BTK with equally low nM potency, was developed. Demonstrate the superiority of continued BTK pathway inhibition with pirtobrutinib compared with other available therapies in patients with BTKi-treated CLL/SLL. BRUIN CLL-321 is a randomized, open-label, global phase 3 study comparing pirtobrutinib monotherapy versus the investigator's choice of idelalisib plus rituximab or bendamustine plus rituximab in patients with CLL/SLL who have been treated with a prior covalent BTKi. Approximately 250 patients will be randomized 1:1. Randomization will be stratified by 17p deletion (yes/no) and prior venetoclax (yes/no). Patients receiving the investigator's choice are eligible to cross over to pirtobrutinib monotherapy if they experience progressive disease per iwCLL 2018 (determined by an independent review committee [IRC]). Global; community hospitals, academic medical centers. Eligible patients are adults aged ≥18 years with a diagnosis of CLL/SLL who require therapy per iwCLL 2018 criteria and who have received prior covalent BTKi. An unlimited number of lines of prior therapy is allowed. Key exclusion criteria include CNS involvement by CLL/SLL, Richter transformation at any time pre-enrollment, a major bleeding event on prior covalent BTKi, and a history of allogeneic or autologous stem cell transplant or chimeric antigen receptor (CAR) T-cell therapy within 60 days of randomization. Pirtobrutinib monotherapy versus investigator's choice of idelalisib plus rituximab or bendamustine plus rituximab. The primary endpoint is progression-free survival (PFS) per iwCLL assessed by an IRC. Secondary endpoints include overall survival, overall response rate, duration of response, safety and tolerability, and patient-reported outcomes. The global study is currently enrolling patients (NCT04666038). This study is a trial in progress. This study is a trial in progress.

Abstract 1400: AS-1763: a highly potent, noncovalent and next generation BTK inhibitor for the treatment of patients with B-cell malignancies having C481S mutation in BTK

Abstract Introduction: Bruton's tyrosine kinase (BTK) has been recently recognized as a validated drug target for the treatment of B-cell malignances. The emergence of clinical resistance to the first-generation covalent BTK inhibitors is, however, becoming a serious concern. Patients are reported to develop resistance during the treatment due to substitution of cysteine residue at position 481 with serine (C481S mutation) in BTK, which prevents the covalent binding of the first-generation irreversible BTK inhibitors, resulting in the loss of inhibitory activities. Therefore, there is a high unmet medical need for new therapeutic approaches to overcome the BTK C481S-mediated resistance. Material and methods: Compounds were tested in biochemical assays using recombinant human wildtype (WT) and C481S mutant BTKs. Cellular activity of AS-1763 was evaluated in human diffuse large B cell lymphoma (DLBCL) cell lines, OCI-Ly10 and its BTK[C481S] knock-in cells. The in vivo antitumor activity of AS-1763 was assessed in two tumor xenograft mouse models bearing WT or BTK [C481S] knock-in OCI-Ly10 cells. Results: We have discovered AS-1763 as an orally available, highly potent, selective and reversible BTK inhibitor. AS-1763 showed potent inhibitory activities for WT BTK (an activated form, BTK[A]) and the C481S mutant with sub-nanomolar IC50s (IC50 = 0.85 and 0.99 nM for BTK[A] and BTK[C481S], respectively). In cellular assays, AS-1763 exhibited strong antiproliferative activities for WT and BTK [C481S] knock-in OCI-Ly10 cells. AS-1763 demonstrated significant tumor growth inhibition and tumor regression in WT as well as BTK[C481S] knock-in OCI-Ly10 xenograft model. Conclusions: The preclinical data support the clinical development of AS-1763 in patients with B-cell malignancies both having WT and C481S mutation in BTK. The first-in-human Phase 1 clinical trial of AS-1763 in healthy volunteers (EudraCT 2020-005599-37) is currently ongoing, and the Phase 1b study in patients is expected to begin in 2022. Citation Format: Wataru Kawahata, Tokiko Asami, Takao Kiyoi, Takayuki Irie, Shigeki Kashimoto, Hatsuo Furuichi, Masaaki Sawa. AS-1763: a highly potent, noncovalent and next generation BTK inhibitor for the treatment of patients with B-cell malignancies having C481S mutation in BTK [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1400.

Evaluation of vecabrutinib as a model for noncovalent BTK/ITK inhibition for treatment of chronic lymphocytic leukemia

AbstractCovalent Bruton tyrosine kinase (BTK) inhibitors, such as ibrutinib, have proven to be highly beneficial in the treatment of chronic lymphocytic leukemia (CLL). Interestingly, the off-target inhibition of IL-2-inducible T-cell kinase (ITK) by ibrutinib may also play a role in modulating the tumor microenvironment, potentially enhancing the treatment benefit. However, resistance to covalently binding BTK inhibitors can develop as the result of a mutation in cysteine 481 of BTK (C481S), which prevents irreversible binding of the drugs. In the present study we performed preclinical characterization of vecabrutinib, a next-generation noncovalent BTK inhibitor that has ITK-inhibitory properties similar to those of ibrutinib. Unlike ibrutinib and other covalent BTK inhibitors, vecabrutinib showed retention of the inhibitory effect on C481S BTK mutants in vitro, similar to that of wild-type BTK. In the murine Eμ-TCL1 adoptive transfer model, vecabrutinib reduced tumor burden and significantly improved survival. Vecabrutinib treatment led to a decrease in CD8+ effector and memory T-cell populations, whereas the naive populations were increased. Of importance, vecabrutinib treatment significantly reduced the frequency of regulatory CD4+ T cells in vivo. Unlike ibrutinib, vecabrutinib treatment showed minimal adverse impact on the activation and proliferation of isolated T cells. Lastly, combination treatment with vecabrutinib and venetoclax augmented treatment efficacy, significantly improved survival, and led to favorable reprogramming of the microenvironment in the murine Eμ-TCL1 model. Thus, noncovalent BTK/ITK inhibitors, such as vecabrutinib, may be efficacious in C481S BTK mutant CLL while preserving the T-cell immunomodulatory function of ibrutinib.

Discovery of AS-1763: A Potent, Selective, Noncovalent, and Orally Available Inhibitor of Bruton's Tyrosine Kinase.

Although Bruton's tyrosine kinase (BTK) has been recognized as a validated drug target for the treatment of B-cell malignances, the emergence of clinical resistance to the first-generation covalent BTK inhibitors is becoming a serious concern. As a part of our effort to develop noncovalent BTK inhibitors, a series of novel pyrrolopyrimidines was identified as noncovalent inhibitors of both the wild-type and C481S mutant BTKs. Subsequent lead optimization led to the identification of an orally available, potent, and selective BTK inhibitor 13f (AS-1763) as a next-generation noncovalent BTK inhibitor. With significant efficacies in vivo tumor xenograft models, AS-1763 has advanced to phase 1 clinical trials.

Discovery of novel BTK PROTACs for B-Cell lymphomas

Bruton's tyrosine kinase (BTK) is a key drug target for B-cell related malignancies. Irreversible covalent BTK inhibitors have been approved for the treatment of B-cell malignancies, yet BTK C481S mutation at the covalent binding site has caused drug-resistance of BTK covalent binding inhibitors. The proteolysis targeting chimera (PROTAC) technology increases the sensitivity to drug-resistant targets compared to classic inhibitors, which provides a new strategy for mutant BTK related B-cell malignancies. ARQ531, a reversible non-covalent BTK inhibitor that inhibits wild type (WT) and mutated BTK with high selectivity, could be an ideal warhead for PROTACs targeting the mutant BTK. Herein, we designed a novel series of PROTACs using the selective non-covalent BTK inhibitor ARQ531 as warhead, with the goal of improving the degradation of both wild-type and C481S mutant BTKs, and increasing the selectivity of BTK over other kinases. This effort will provide some basis for further preclinical study of BTK PROTACs as a novel strategy for treatment of B-cell lymphomas.

CG '806, a First-in-Class Non-Covalent Pan-FLT3/BTK Inhibitor, Exerts Superior Potency Against B-Cell Malignant Cells

CG'806 is a non-covalent pan-FMS like tyrosine kinase 3 (FLT3)/Bruton's tyrosine kinase (BTK) multi-kinase inhibitor that potently inhibits wild type (WT) and mutant forms of FLT3 and BTK. As described previously (2017 AACR Hematologic Malignancies abstract #25 and #44), CG'806 inhibits on FLT3-ITD-driven acute myeloid leukemia (AML) and other forms of AML. Because CG'806 targets the WT BTK (IC50 = 8 nM) and the C481S-BTK (IC50 = 2.5 nM), which is a mutant form resistant to ibrutinib and other covalent BTK inhibitors, this small molecule was profiled for its actions on various types of B-cell malignancies in which B cell receptor (BCR) and BTK signaling are reported to play pivotal pathogenic roles. Against 10 malignant B-cell lines, while CG'806 reduced the level of BTK-Y223 phosphorylation, it was less potent than ibrutinib. In contrast, CG'806 was 50-6,000 times more potent (lower IC50) in the ability to kill the malignant B cells across all tested cell lines evaluated by MTS-based assays of cell proliferation and inhibition of colony formation. Hence, the efficacy of killing malignant B cells by CG'806 and by ibrutinib did not correlate with the degree of BTK inhibition. Pathway analyses (KinomeScan and immunoblotting) demonstrated that CG'806, in addition to inhibiting intracellular BTK, potently inhibited the activity of aurora kinases A, B and C, phospho-H3S10, FLT3 and ERK that are differentially expressed and operative in various cell lines representing mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), B-acute lymphocytic leukemia (B-ALL) and Burkitt's lymphoma. Indeed, measuring BrdU incorporation and Annexin V by flow cytometry, CG'806 directly induced polyploidy and triggered apoptosis in malignant B cells at picomolar (pM) to low nanomolar (nM) concentrations, while ibrutinib potently inhibited BTK activity but was unable to induce apoptosis at lower than micromolar (µM) concentrations. These data distinguish CG'806 from ibrutinib and other covalent and non-covalent BTK inhibitors, and illustrate that CG'806 has activity across a wide range of malignant B cell lines including those intolerant, refractory or resistant (due to the presence of a BTK-C481S mutation) to ibrutinib and other BTK inhibitors. [Display omitted] DisclosuresZhang:Aptose Biosciences, Inc.: Employment. Local:Aptose Biosciences, Inc.: Employment. Benbatoul:Aptose Biosciences, Inc.: Employment. Folger:Aptose Biosciences, Inc.: Employment. Sheng:Aptose Biosciences, Inc.: Employment. Howell:Aptose Biosciences, Inc.: Membership on an entity's Board of Directors or advisory committees. Rice:Aptose Biosciences, Inc.: Employment, Equity Ownership, Patents & Royalties.

The HCK/BTK inhibitor KIN-8194 is active in MYD88-driven lymphomas and overcomes mutated BTKCys481 ibrutinib resistance

AbstractActivating mutations in MYD88 promote malignant cell growth and survival through hematopoietic cell kinase (HCK)–mediated activation of Bruton tyrosine kinase (BTK). Ibrutinib binds to BTKCys481 and is active in B-cell malignancies driven by mutated MYD88. Mutations in BTKCys481, particularly BTKCys481Ser, are common in patients with acquired ibrutinib resistance. We therefore performed an extensive medicinal chemistry campaign and identified KIN-8194 as a novel dual inhibitor of HCK and BTK. KIN-8194 showed potent and selective in vitro killing of MYD88-mutated lymphoma cells, including ibrutinib-resistant BTKCys481Ser-expressing cells. KIN-8194 demonstrated excellent bioavailability and pharmacokinetic parameters, with good tolerance in rodent models at pharmacologically achievable and active doses. Pharmacodynamic studies showed sustained inhibition of HCK and BTK for 24 hours after single oral administration of KIN-8194 in an MYD88-mutated TMD-8 activated B-cell diffuse large B-cell lymphoma (ABC DLBCL) and BCWM.1 Waldenström macroglobulinemia (WM) xenografted mice with wild-type BTK (BTKWT)– or BTKCys481Ser-expressing tumors. KIN-8194 showed superior survival benefit over ibrutinib in both BTKWT- and BTKCys481Ser-expressing TMD-8 DLBCL xenografted mice, including sustained complete responses of >12 weeks off treatment in mice with BTKWT-expressing TMD-8 tumors. The BCL_2 inhibitor venetoclax enhanced the antitumor activity of KIN-8194 in BTKWT- and BTKCys481Ser-expressing MYD88-mutated lymphoma cells and markedly reduced tumor growth and prolonged survival in mice with BTKCys481Ser-expressing TMD-8 tumors treated with both drugs. The findings highlight the feasibility of targeting HCK, a key driver of mutated MYD88 pro-survival signaling, and provide a framework for the advancement of KIN-8194 for human studies in B-cell malignancies driven by HCK and BTK.

Follicular Lymphoma-associated BTK Mutations are Inactivating Resulting in Augmented AKT Activation.

On the basis of the recent discovery of mutations in Bruton tyrosine kinase (BTK) in follicular lymphoma, we studied their functional properties. We identified novel somatic BTK mutations in 7% of a combined total of 139 follicular lymphoma and 11 transformed follicular lymphoma cases, none of which had received prior treatment with B-cell receptor (BCR) targeted drugs. We reconstituted wild-type (WT) and mutant BTK into various engineered lymphoma cell lines. We measured BCR-induced signal transduction events in engineered cell lines and primary human follicular lymphoma B cells. We uncovered that all BTK mutants destabilized the BTK protein and some created BTK kinase-dead mutants. The phospholipase C gamma 2 (PLCγ2) is a substrate of BTK but the BTK mutants did not alter PLCγ2 phosphorylation. Instead, we discovered that BTK mutants induced an exaggerated AKT phosphorylation phenotype in anti-Ig-treated recombinant lymphoma cell lines. The short hairpin RNA-mediated knockdown of BTK expression in primary human nonmalignant lymph node-derived B cells resulted in strong anti-Ig-induced AKT activation, as did the degradation of BTK protein in cell lines using ibrutinib-based proteolysis targeting chimera. Finally, through analyses of primary human follicular lymphoma B cells carrying WT or mutant BTK, we detected elevated AKT phosphorylation following surface Ig crosslinking in all follicular lymphoma B cells, including all BTK-mutant follicular lymphoma. The augmented AKT phosphorylation following BCR crosslinking could be abrogated by pretreatment with a PI3Kδ inhibitor. Altogether, our data uncover novel unexpected properties of follicular lymphoma-associated BTK mutations with direct implications for targeted therapy development in follicular lymphoma.See related commentary by Afaghani and Taylor, p. 2123.

In Vivo Pre-Clinical Evaluation of LOXO-305 Alone and in Combination with Venetoclax, Rituximab, R-CHOP or Obinutuzumab on Human Xenograft Lymphoma Tumor Models in Mice

Introduction: Bruton's Tyrosine Kinase (BTK) is an essential component of normal and malignant B-cell receptor signaling. Covalent BTK inhibitors have transformed the treatment of B-cell malignancies. Despite the marked efficacy of covalent BTKi, treatment failure can occur through the development of resistance and discontinuation for adverse events. The activity of these covalent BTK inhibitors are markedly reduced or absent in the presence of BTK cysteine binding site (C481) mutations. Moreover, these agents share pharmacologic liabilities (e.g. low oral bioavailability, short half-life) that may sometimes lead to suboptimal BTK target coverage, for example in rapidly proliferating tumors with high BTK protein turnover, ultimately manifesting as acquired resistance for some patients. To address these limitations, LOXO-305, a highly selective, non-covalent BTKi that inhibits both WT and C481-mutated BTK with equal low nM potency was developed. Proof-of-concept Phase I results demonstrated LOXO-305's anti-tumor activity across patients with heavily pretreated B-cell malignancies (Mato et al. ASH 2019). We previously showed pre-clinical data demonstrating that LOXO-305 potently inhibited wild-type (WT) BTK and different variants of the BTK mutation C481 with nanomolar potency and caused regression in BTK-dependent lymphoma mouse xenograft models (Brandhuber et al. SOHO 2018, and Gomez et al. ASH 2019). Here we describe the activity of LOXO-305 alone or in combination with venetoclax (BCL-2 inhibitor), in TMD8 BTK WT and TMD8 BTK C481S human tumor xenograft models of diffuse large B lymphoma and a REC-1 human tumor xenograft model of mantle cell lymphoma in mice. We also report the activity of LOXO-305 alone and in combination with R-CHOP (rituximab, cyclophosphamide, doxorubicin hydrochloride, vincristine sulfate, and prednisone) or obinutuzumab (anti-CD20 antibody), in the TMD8 xenograft tumor model. Methods: In all studies, tested articles were administered alone and in combination, following different dosing regimens. Table 1 shows the tested compound(s), dosing frequency, cell line used, disease, BTK status (WT or C481S), and mouse strain used, for each study presented in this abstract. Human TMD8 BTK WT, TMD8 BTK C481S or REC-1 cells were injected subcutaneously in the right flank of mice. When tumors reached a mean volume between 150 mm3 and 400 mm3, mice were randomized based on their tumor volumes. Mice were next dosed for 17 to 23 days depending on the study design. The potencies of the compounds stand-alone or in combination on the inhibition of the tumor growth were assessed based on the tumor volume changes and weights after collection at the end of the study. Additionally, in the TMD8 studies, the plasma concentrations of tested articles were measured at multiple time points after the last dose. Results: All treatments were well tolerated without any significant body weight loss or clinical signs being observed on the mice. LOXO-305 potently inhibited the growth of BTK WT and BTK C481S driven xenograft tumors. In all combinations tested, significantly greater tumor growth inhibition was observed in groups where LOXO-305 was co-administered with clinically approved agents. Conclusion: These data suggest that the co-administration of LOXO-305 with venetoclax, R-CHOP or obinutuzumab could have an increased benefit for patients with B-cell malignancies compared to stand-alone treatments and warrants further investigation. Disclosures Gomez: Loxo Oncology, Inc, a wholly owned subsidiary of Eli Lilly and Company: Current Employment, Divested equity in a private or publicly-traded company in the past 24 months. Wu:Eli Lilly and Company: Current Employment, Current equity holder in publicly-traded company. Stephens:Eli Lilly and Company: Current Employment, Current equity holder in publicly-traded company. Rosendahl:Loxo Oncology, Inc, a wholly owned subsidiary of Eli Lilly and Company: Current Employment, Divested equity in a private or publicly-traded company in the past 24 months. Brandhuber:Loxo Oncology, Inc, a wholly owned subsidiary of Eli Lilly and Company: Current Employment, Divested equity in a private or publicly-traded company in the past 24 months.

Design, synthesis and biological evaluation of Proteolysis Targeting Chimeras (PROTACs) as a BTK degraders with improved pharmacokinetic properties

A new series of Proteolysis Targeting Chimeras (PROTACs) targeting Bruton's Tyrosine Kinase (BTK) was synthesized, with the goal of improving the pharmacokinetic properties of our previously reported PROTAC, MT802. We recently described the ability of MT802 to induce degradation of both wild-type and C481S mutant BTK in immortalized cells and patient-derived B-lymphocytes. However, the pharmacokinetic properties of MT802 were not suitable for further in vivo development. Therefore, we undertook a systematic medicinal chemistry campaign to overcome this issue and made a series of PROTACs with structural modifications to the linker and E3-recruiting ligand; more specifically, the new PROTACs were synthesized with different von Hippel-Lindau (VHL) and cereblon (CRBN) ligands while keeping the BTK ligand and linker length constant. This approach resulted in an equally potent PROTAC, SJF620, with a significantly better pharmacokinetic profile than MT802. This compound may hold promise for further in vivo exploration of BTK degradation.

LOXO-305: Targeting C481S Bruton Tyrosine Kinase in Patients with Ibrutinib-Resistant CLL

Clinical success with the targeted Bruton Tyrosine Kinase (BTK) inhibitors ibrutinib and acalabrutinib has greatly improved the outcome of patients with relapsed chronic lymphocytic leukemia (CLL). However toxic side effects and acquired resistance due to C481S mutations in BTK remain an issue and the prognosis of those developing resistance is poor. Hence better therapeutic options are needed for these patients. Here we characterize a next generation highly selective reversible BTK inhibitor, LOXO-305, and describe its effectiveness in vitro in treatment naïve CLL patients and in those with C481S mutations. Unlike the irreversible BTK inhibitors ibrutinib and acalabrutinib, LOXO-305 does not require the C481 site for binding to the ATP binding domain of BTK. In addition, LOXO-305 is highly selective, with minimal activity against non-BTK kinase and non-kinase off targets, including ITK, TEC and EGFR. We evaluated the efficacy of LOXO-305 in BTK wild-type B cell lymphoma lines most similar to CLL, namely MEC1 and OSU-CLL, and in CLL patient samples, by determining its effect on cell viability, apoptosis and BCR signaling. In both cell lines LOXO-305 reduced cell viability and BCR mediated activation of BTK, PLCg2, ERK and AKT similar to ibrutinib. We measured cellular apoptosis in these cell lines with Annexin V APC and PI staining and showed that after 48 hours LOXO-305 treated cells had a significantly higher percentage of apoptotic cells in comparison with both DMSO (mean increase with LOXO-305: MEC1 21% (p = 0.0005) & OSU-CLL 35% (p = 0.0016)) and ibrutinib (mean increase with LOXO305: MEC1 19% (p=0.0006), OSU-CLL 22% (p=0.0013)). Since LOXO-305's mechanism of BTK inhibition does not involve covalent binding to the C481 site, we tested its efficacy in in vitro ibrutinib resistant models of stably transfected HEK293 cells expressing either wild-type (WT) or C481S BTK. In WT BTK HEK cells, both LOXO-305 and ibrutinib showed comparable inhibitory activity in vitro against wild-type BTK, with IC50 values for phospho-BTK inhibition equal to 5.69 nM and 3.33 nM, respectively. LOXO-305 also inhibited phospho-BTK in BTK-C481S-expressing HEK293 cells at an IC50 equal to 21.2 nM, while ibrutinib had no inhibitory effect at concentrations as high as 300nM. When we extended our analysis to treatment naïve CLL patient cells, we observed that both LOXO-305 and ibrutinib potently inhibited IgM-induced phospho-BTK with IC50 values equal to 1.34 ± 1.23 nM for LOXO-305 (n = 7, p < 0.0001) and 1.04 ± 1.26 nM for ibrutinib (n = 7, p < 0.0001). We also found a significant reduction in phosphorylation of PLCγ2 (Y1217), the immediate downstream effector of BTK (IC50 33 nM for each agent, n = 7, p = 0.02 for LOXO-305, p = 0.0017 for ibrutinib). When we compared the cellular cytotoxicity of LOXO-305 with ibrutinib and acalabrutinib, all three agents demonstrated significant induction of apoptosis (annexin V positive) in BTK wild-type cells compared to DMSO treatment: (LOXO-305: median 63.77% n=3, p = 0.0073, ibrutinib: median 57.76%, n=3, p < 0.0001, acalabrutinib: median 67.11%, n=3, p = 0.0397). In CLL patient cells harboring C481S mutations, we observed a decrease in BCR signaling with 0.6 μM LOXO-305 treatment, with 95% reduction in phospho-BTK (Y223) and 50% reduction in phospho-PLCγ2 vs DMSO (n = 3, phospho-BTK p < 0.0001, phospho-PLCγ2 p = 0.01). In comparison ibrutinib at 0.6 μM demonstrated 50% reduction in phospho-BTK (n = 3, p = 0.01) and no significant change in phospho-PLCγ2. We also observed a 73% reduction in phospho-ERK vs DMSO with 10 μM LOXO-305 treatment (n=3, p = 0.004) while ibrutinib and acalabrutinib showed no significant change. In CLL patient cells with C481S variant allele frequencies (VAF) of 87% and 89%, LOXO-305 was 40-fold more potent at inhibiting phospho-BTK than ibrutinib (IC50 for LOXO305 0.02 μM, for ibrutinib 0.9 μM). Even in patient cells with lower C481S VAF of 9%, LOXO-305 still demonstrated 30-fold higher potency than ibrutinib (IC50 LOXO-305 0.2 μM, ibrutinib 0.6 μM). Our findings show that LOXO-305 potently inhibits cell survival and BCR signaling in both treatment naïve and C481S CLL patient cells, and therefore may prove an effective treatment in both treatment naïve and ibrutinib-resistant CLL patients. A phase 1 clinical trial is ongoing. Disclosures Ebata: LOXO Oncology Inc.: Employment, Equity Ownership. Gomez:LOXO Oncology Inc.: Employment, Equity Ownership. Brandhuber:LOXO Oncology Inc.: Employment, Equity Ownership. Rothenberg:LOXO Oncology Inc.: Employment. Brown:Janssen, Teva Pharmaceuticals: Honoraria; Gilead, Loxo, Sun Pharmaceuticals, Verastem: Research Funding; Morphosys, Invectys (Data Safety Monitoring Board): Membership on an entity's Board of Directors or advisory committees; Abbvie, Acerta, Astra-Zeneca, Beigene, Catapult Therapeutics, Dynamo Therapeutics, Genentech/Roche, Gilead, Juno/Celgene, Kite, Loxo, Octapharma, Novartis, Pfizer, Pharmacyclics, Sunesis, TG Therapeutics, Verastem: Consultancy.

Loxo-305, a Highly Selective and Non-Covalent Next Generation BTK Inhibitor, Inhibits Diverse BTK C481 Substitution Mutations

Introduction: Bruton's Tyrosine Kinase (BTK) is an essential component of normal and malignant B-cell receptor signaling. Covalent BTK inhibitors have transformed the treatment of B-cell malignancies but are limited by off-target toxicity and acquired resistance, leading to eventual treatment discontinuation and disease progression. Emerging evidence suggests that acquired resistance is mediated predominantly by BTK C481 substitution mutations at the covalent BTK inhibitors' binding site. There is significant unmet clinical need for new treatment approaches that overcome acquired resistance and minimize toxicity. LOXO-305 is a highly selective, non-covalent, next generation BTK inhibitor. We previously showed that LOXO-305 potently inhibited both wild-type (WT) BTK and BTK C481S -mediated kinase activity in enzyme and cell-based assays with nanomolar potency, caused regression of BTK-dependent lymphoma mouse xenograft models, and was more than 300-fold selective for BTK over 98% of 370 other kinases tested and showed no significant inhibition of non-kinase off targets at 1 mM (Brandhuber et al. SOHO 2018). In addition, ADME and pharmacokinetic experiments in two preclinical species predicted that LOXO-305 will have high human exposure and sustained BTK C481S target coverage in patients at clinically achievable doses. Here we describe the activity of LOXO-305 against additional BTK C481 substitution mutations, including mutations identified in patients with acquired resistance to covalent BTK inhibitors. We further determine equilibrium-binding affinities for LOXO-305 for diverse mutant BTK enzymes in comparison to other clinically available BTK inhibitors. Methods: To assess cellular BTK inhibitor potency, HEK293T cell lines transiently expressing wild-type BTK and BTK C481 substitution mutations were serum starved and incubated with LOXO-305 overnight. Cells were next incubated with serum and orthovanadate for 5 min and the phosphorylated Y223 BTK was analyzed by immunoblot. Bands were quantified and the IC50 values calculated with GraphPad Prism. The equilibrium-binding affinities for targeted BTK inhibitors to BTK enzyme variants were determined by surface plasmon resonance (SPR) using the Biacore T200. Biotinylated BTK variants were immobilized on a docked streptavidin coated sensor chip. Five concentrations of each inhibitor plus blank controls were analyzed. Association/dissociation rate constants were calculated by global fitting of the data to a 1:1 binding interaction model. Results: While BTK C481S possessed similar levels of basal Y223 autophosphorylation as wild-type BTK in cells, BTK C481T autophosphorylation was reduced by ~50%, C481R by ~90%, and mutants C481F, and C481Y were inactive in HEK293T cells. LOXO-305 inhibited Y223 phosphorylation of all active mutants with similar nanomolar potency. In contrast, autophosphorylation of all BTK C481 mutants were resistant to both Ibrutinib and acalabrutinib. Equilibrium-binding affinities of LOXO-305 for select BTK C481 substitution mutations confirmed LOXO-305's superior potency versus commercially available BTK inhibitors (ibrutinib and acalabrutinib). Conclusions: The next generation, non-covalent, highly selective BTK inhibitor LOXO-305 potently inhibited the cellular activity of BTK C481S, T and R mutations and displayed strong equilibrium binding to WT BTK and several BTK C481 substitution mutations. Together with high selectivity and significant BTK target coverage in vivo, these results indicate that LOXO-305 may overcome acquired resistance to covalent BTK inhibitors in patients without significant off-target toxicity. A phase 1 clinical trial of LOXO-305 is currently underway. Disclosures Gomez: LOXO Oncology Inc.: Employment, Equity Ownership. Isabel:Loxo Oncology: Employment. Rosendahal:Loxo Oncology: Employment. Rothenberg:LOXO Oncology Inc.: Employment. Andrews:Loxo Oncology: Employment. Brandhuber:LOXO Oncology Inc.: Employment, Equity Ownership.

Synergistic Targeting of BTK and E-Selectin/CXCR4 in the Microenvironment of Mantle Cell Lymphomas

Synergistic Targeting of BTK and E-Selectin/CXCR4 in the Microenvironment of Mantle Cell Lymphomas