BTK C481S

The first-in-class covalent BTK inhibitor, Ibrutinib, binds to the C481 amino acid residue to block the BTK enzyme and prevent the downstream signaling...To bypass the C481S mutation, non-covalent BTKi, such as Pirtobrutinib, were developed and are active against both wild-type and the C481S mutation...In addition, we discuss the new emerging class of BTK degraders, which utilize the evolution of proteolysis-targeting chimeras (PROTACs) to degrade the BTK protein and constitute an important avenue of overcoming resistance. The moving landscape of resistance to BTKi and the development of new therapeutic strategies highlight the ongoing advances being made towards the pursuit of a cure for B-cell lymphoid malignancies.

One acalabrutinib-treated patient and 4 ibrutinib-treated patients had emergent BTK/PLCG2 co-mutations. While common BTK C481 mutations were observed with both treatments, patterns of mutation and co-mutation frequency, mutation VAF, and uncommon BTK variants varied with acalabrutinib (T474I and E41V) and ibrutinib (L528W, A428D) in this patient population.

Collectively our findings suggest that phosphorylation of Y223 serves as a useful proxy for phosphorylation of phospholipase C2 (PLCG2), the endogenous substrate of BTK. However, in contrast to a frequently held conception, this post-translational modification is dispensable for the function of BTK.

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...ONO-7018 and tirabrutinib were orally administered to the mice twice a day... 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.

The majority of IR DP with BTK/PLCG2 mutations (6/8), were 100% homologous to germline IGHV unmutated, suggesting an inherent higher risk disease profile as a risk factor for acquiring BTKmt with prolonged BTKi therapy. BTK/PLCG2 mutations were enriched amongst very late progressors with 6/8 pts progressing shortly after stopping therapy at 72 mo., which is later than previously reported for relapsed/refractory CLL pts.

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 ibr cohort had no prior BTKi therapy whereas 1 patient in the acala cohort had received spebrutinib, another cBTKi...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.

Ibrutinib, Zanubrutinib, Orelabrutinib were administered in 55.3%,18.3% and 17.9% of patients respectively, in addition to 6 patients with Acalabrutinib and one with Loxo-305, among them 47.7% (125/262) received BTKi as first line treatment...Venetoclax-based regimen might be an effective salvage therapy which could showed a benefit trend in PFS as compared to other post BTKi therapy (10.1 months vs 3.1 months, p= 0.1818)...Acquired BTK/PLCG2 mutations remained to be key drivers of BTKis resistance and BTKC481S mutation was the dominant mutation, while BTKT474 mutation was only detected in Orelabrutinib-resistant patients. The prognosis was rather poor for relapsed patients especially for RTs, treatment strategy after disease progression remains to be optimized.

ABBV-MALT1 has also shown activity in a range of preclinical lymphoma models as both monotherapy and in combination with venetoclax, including robust antitumor activity in malignant B-cell models that are resistant to BTK inhibitors. Pts are being enrolled in 25 sites across the USA, Australia, Belgium, France, Germany, Israel, Spain, and UK. As of August 1, 2023, 2 pts had been treated.

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...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.

Our findings demonstrate that the B cell population in ibrutinib-resistant and ibrutinib sensitive CLL patients exhibit distinct transcriptional states. At the single-cell level, our findings demonstrate a systematic picture of diverse malignancy and microenvironmental changes in CLL. Overall, these findings provide an insight into the complex molecular and cellular interactions of CLL that may have a crucial role in BTKI resistance.

The combination of CDK4/6 inhibitor palbociclib and PI3K inhibitor idelalisib synergistically induced anti-tumor activity in B-cell lymphoma through downregulation of PLK1 expression, suggested a new combination direction for the treatment of B-NHL and even BTK inhibitor-resistant patients.

TMD-8-IbruR cells carrying the C481S mutation were generated by stepwise induction of increasing concentrations of Ibrutinib...In cell lines expressing C481X and pirtobrutinib resistance mutations, DZD8586 demonstrated concentration dependent anti-proliferative effects, with similar GI50s among cells carrying different BTK mutations...The Kpuu,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.

Our data demonstrate the efficacy of the PKCβ inhibitor MS-553 in preclinical models of CLL including that with BTK mutations conferring BTKi resistance. These findings support continued preclinical and clinical investigation of MS-553 in CLL, and the phase 1 trial of this agent is currently underway (NCT03492125) (Blachly et al., 2022).

Twenty-three (76.7%) of the patients were on ibrutinib, while 7 (23.3%) were on acalabrutinib...Six patients (20.0%) continued on the same BTKi with addition of venetoclax, 10 (33.3%) patients were switched to venetoclax and immunotherapy (either obinutuzumab or rituximab), and 3 (10.0%) patients were started on single agent venetoclax...Three (10.0%) patients were taken to chimeric T cell receptor (CAR-T) cell therapy of which one patient had no response and was started on salvage treatment with duvelisib... Our results reveal that durable responses can be achieved by switching to venetoclax based regimens in patients with BTKi. Though the early results of the use of noncovalent BTKi in this setting aree encouraging, the durability of response is limited as new BTK mutations are selected and future therapeutic alternatives are needed for these subset of patients.

ABBV-101 is currently in phase 1 clinical trial for a variety of B-cell malignancies. ClinicalTrials.gov identifier: NCT05753501

We uncovered known and novel BTK resistance mutations and demonstrated BTK scaffolding activity independent of HCK, highlighting the need for other strategies to disrupt scaffolding-mediated BCR signaling. Beyond BTK mutations, our CRISPR KO screens illustrate a map of genetic modifiers of BTKi response and point to several potential resistance biomarkers for both acala and pirto. A better understanding of resistance mechanisms in the presence and absence of BTK mutations will help augment the use of BTKi treatments in patients.

In primary CLL samples, AS-1763, pirtobrutinib or ibrutinib induced a modest apoptosis...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... 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).

Covalent BTKi (cBTKi) including ibrutinib, acalabrutinib, and zanubrutinib, share a common resistance mechanism, acquisition of a C481 mutation in BTK. 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.

BTK Cysteine 481 substitution is known to contribute to cBTKi acquired resistance to ibrutinib, acalabrutinib, and zanubrutinib. Despite this cohort representing the first relapsing CLL patients from BRUIN and presenting with frequent baseline BTK mutations, response to pirtobrutinib was high, with an ORR of 83%, and substantial clearance of BTK C481 clones. At progression, the majority of pts (56%) either acquired non-BTK mutations or did not acquire any resistance mutations in this targeted panel, suggesting alternative resistance mechanisms. A smaller group of patients (44%) displayed emergence of non-C481 clones, particularly gatekeeper T474 and kinase-impaired L528W mutations.

We used duplex sequencing, a technology that enables detection of mutations at ultra-low allelic frequencies, to identify mutations in five genes associated with drug resistance in CLL and followed their evolution in two patients who received multiple targeted therapies and ultimately developed disease progression on pirtobrutinib...In patient R001, multiple known resistance mutations in both BTK and PLCG2 appeared following progression on zanubrutinib (BTK p.L528W, p.C481S, PLCG2 S707F, L845F, R665W, and D993H). In contrast, patient R002 developed multiple BTK mutations following acalabrutinib treatment including known resistance mutations p.C481R, p.T474I and p.C481S...For example, BTK p.L528W in patient R001 increased in frequency more than 1000-fold (from CCF 0.02% to 35%), and p.T474I in patient R002 increased in CCF from 0.03% to 4.2% (more than 100-fold). Our data illuminates the evolutionary dynamics of resistant clones over the patients' disease course and under selective pressure from different targeted treatments.

This systematic investigation describes development of mutations over time in patients without PD and informs the potential clinical opportunity to optimize ongoing benefits for such patients.

The C481S and C481R mutations eliminated the anti-proliferative effects of covalent inhibitors ibrutinib, acalabrutinib, and zanubrutinib, whereas the V416L, T474I, and L528W mutations had variable effects on the covalent inhibitors. By contrast, the non-covalent inhibitors pirtobrutinib, vecabrutinib, and fenebruitnib maintained activity against C481S but displayed partially reduced potency against the C481R mutation and dramatically reduced potency against the V416L, T474I, and L528W mutations...These degrader molecules may also have utility in earlier lines of therapy due to their ability to suppress scaffold-mediated BTK signaling. Phase 1a/b trials of NX-5948 and NX-2127 in patients with relapsed or refractory B-cell malignancies are ongoing (NX-5948: NCT05131022; NX-2127: NCT04830137).

Novel, non-C481 BTK mutations have been described, including BTK L528W, which has been reported in patients treated with ibrutinib, zanubrutinib, and pirtobrutinib (Maddocks, JAMA Oncol. This is the largest dataset characterizing the incidence and patterns of BTK and PLCG2 mutations in patients with CLL treated with ibrutinib. BTK C481 mutations are the most frequently occurring mutations. PLCG2 mutations occur across the gene at a low incidence per locus (<5%).

main study Substudy patients treated with VenR (n = 194) patients treated with BR (n = 195) patients retreated with VenR (n = 25) Baseline characteristics mean age, years (SD) 63.9 (10.5) 64.4 (9.6) 65.8 (8.3) Number of prior cancer therapy, n (%) 1 111 (57.2) 117 (60.0) 0 (0.0) 2 58 (29.9) 43 (22.1) 20 (80.0) ≥3 25 (12.9) 35 (17.9) 5 (20.0) del(17p) and/or TP53 mutation (aCGh), n (%) mutated 53 (27.3) 55 (28.2) 14 (56.0) unmutated 104 (53.6) 98 (50.3) 9 (36.0) unknown 37 (19.1) 42 (21.5) 2 (8.0) Genomic complexity, n (%) n = 48 n = 46 n = 20 3–4 34 (70.8) 29 (63.0) 3 (15.0) ≥5 14 (29.2) 17 (37.0) 8 (40.0) iGhV, n (%) n = 180 n = 180 n = 23 mutated 53 (29.4) 51 (28.3) 2 (8.7) unmutated 123 (68.3) 123 (68.3) 21 (91.3) unknown 4 (2.2) 6 (3.3) 0 (0.0) efficacy results median follow-up, months 85.7 85.7 33.4 Best oRR, % 93.3 67.7 72.0 umRD at eoCT of main study, n (%) 121 (62.4) 26 (13.3) 16 (64.0) umRD at eoCT of substudy, n (%) N/a N/a 8 (32.0) umRD at eoT of main study, n (%) 83 (70.3)* N/a 14 (56.0) umRD at eoT of substudy, n (%) N/a N/a 0 (0.0) median pFS, months (95% Ci) 54.7 17.0 23.3 (52.3, 59.9) (15.5, 21.7) (15.6, 24.3) 3-year oS rate, % (95% Ci) 88.4 78.9 53.1 (83.8, 93.0) (72.8, 84.9) (25.1, 81.0) *patients who completed 2 years of Ven without pD (n = 118) aCGh: array comparative genomic hybridization; BR: bendamustine- rituximab; Ci: confidence interval; del(17p): deletion in chromosome 17p; eoCT: end of combination treatment; eoT: end of treatment; iGhV immu- noglobulin heavy chain gene; oRR: overall response rate; oS: overall sur- vival; pD: progressive disease; pFS: progression free survival; SD: standard deviation; TP53: tumor protein p53; umRD: undetectable minimal residual disease; Ven(R): venetoclax-(rituximab) cBTKi pre-treated CLL pts treated with pirtobrutinib monotherapy in the phase 1/2 BRUIN trial (NCT03740529) who subsequently developed disease progression (PD) were included in this analysis...Pts received one or more of the following cBTKi: ibrutinib (n = 44, 90%), acalabrutinib (n = 10, 20%), or zanubrutinib (n = 1, 2%)... Pts who progressed on pirtobrutinib showed clearance of BTK C481 clones and the emergence or outgrowth of non-C481 clones, particularly gatekeeper T474 and kinase-impaired L528W mutations, as well as other VUS. Many acquired BTK mutations were shown to pre-exist at baseline at low VAF, reflecting emergence on prior cBTKi. Importantly, these baseline kinase domain BTK mutations did not preclude pirtobrutinib efficacy.

Our findings demonstrate that the B cell population in ibrutinib-resistant and ibrutinib sensitive CLL patients exhibit distinct transcriptional states. At the single-cell level, our findings demonstrate a systematic picture of diverse malignancy and microenvironmental changes in CLL. Overall, these findings provide an insight into the complex molecular and cellular interactions of CLL that may have a crucial role in BTKI resistance.

73 patients had only one BTKi (ibrutinib (IBR), 64; acalabrutinib (ACA), 9). 12 pts had multiple BTKis, 8 with two drugs with IBR first followed by ACA (Nf3, 37.5%), vecabrutinib (Nf1, 12.5%), and PIR (Nf4, 50.0%); and 4 with three or more drugs... Our retrospective report summarizes mutations detected during BTKi treatment and shows that BTK L528W can occur during both covalent and non-covalent BTK inhibitor therapy. Four of six patients who progressed on PIR had T474 mutations. In addition, our results may suggest that activating mutations in RAS/RAF/MAPK pathway are related to BTKi resistance.

Our data demonstrate the efficacy of the PKCβ inhibitor MS-553 in preclinical models of CLL, including BTK inhibitor resistant disease. These findings support continued preclinical and clinical investigation of MS-553 in CLL, and the phase 1 trial of this agent is currently underway (NCT03492125) [7].

Multiple mechanisms are involved in resistance to the BCL2 inhibitor venetoclax, including point mutations that impair drug binding, the upregulation of BCL2-related anti-apoptotic family members, and microenvironmental alterations. Recently, immune checkpoint inhibitors and CAR-T cells have been tested for CLL treatment, obtaining conflicting results. Potential refractoriness biomarkers to immunotherapy were identified, including abnormal levels of circulating IL-10 and IL-6 and the reduced presence of CD27CD45RO CD8 T cells.

Moreover, compound 3e suppressed the cell growth more potently than the small molecule inhibitors ibrutinib and ARQ-531 in several cells. Furthermore, compound 3e with the rigid linker displayed a significantly improved metabolic stability profile with the T increased to more than 145 min. Overall, we discovered a highly potent and selective BTK PROTAC lead compound 3e, which could be further optimized as potential BTK degradation therapy for BTK-associated human cancers and diseases.

There were 29 men, 16 women, median age 65.5 years (range 47-86), 43 patients received ibrutinib and 2 acalabrutinib. Our study shows that BTK and/or PLCG2 mutations were found in 64.4% of patients with progression of CLL during BTKi therapy, and in 35.6% of patients the cause of resistance has not yet been identified. Most mutations in our sample were detected in the C481 codon of BTK gene after 2 years of treatment, suggesting that regular screening with simple PCR tests starting from the second year of treatment is a reasonable approach. NGS may expand data in cases with undetectable mutations.

Nemtabrutinib 65 mg continued to show promising and durable antitumor activity in pts with R/R CLL/SLL and a manageable safety profile in pts with hematologic malignancies. Clinical trial, Chronic lymphocytic leukemia

Inhibition of BTK by covalent BTK inhibitors (BTKi), such as ibrutinib, acalabrutinib, and zanubrutinib, have revolutionized the management of CLL and other B cell malignancies. BGB-16673 is a potent inhibitor against tumors expressing wildtype and clinical-relevant BTK mutations. In addition, it is superior to ibrutinib and LOXO-305 equivalent for more durable anti-tumor activities and less metastasis. Absolute lymphocyte count, Tumor model, Lymphoma, Mouse model

Pts received one or more of the following cBTKi: ibrutinib (n=44, 90%), acalabrutinib (n=10, 20%), or zanubrutinib (n=1, 2%). Pts who progressed on pirtobrutinib showed clearance of BTK C481 clones and the emergence or outgrowth of non-C481 clones, particularly gatekeeper T474 and kinase-impaired L528W mutations, as well as other VUS. Many acquired BTK mutations were shown to pre-exist at baseline at low VAF, reflecting emergence on prior cBTKi. Importantly, these baseline kinase domain BTK mutations did not preclude pirtobrutinib efficacy.

Approximately 300 pts will be enrolled and randomly assigned 1:1 to receive nemtabrutinib (65 mg orally once daily until progressive disease, unacceptable toxicity, or discontinuation) or intravenous infusion of the investigator’s choice of chemoimmunotherapy for 6 cycles lasting 28 days each (FCR: fludarabine 25 mg/m2 and cyclophosphamide 250 mg/m2 on days 1, 2, and 3 of each cycle, plus rituximab 375 mg/m2 initially, followed by 500 mg/m2 on day 1 of each cycle; or BR: bendamustine 70-90 mg/m2 on days 1 and 2 of each cycle plus rituximab 375 mg/m2 initially, followed by 500 mg/m2 on day 1 of each cycle). Enrollment is ongoing. Clinical trial information: NCT05624554.

In pre-clinical safety studies, NX-2127 demonstrates an acceptable safety profile. NX-2127 is currently in phase 1 clinical trials (NCT04830137) for hematological malignancies.

In summary, HZ-Q1070 is a potent and highly selective BTK degrader against both BTK-WT and multiple BTK inhibitors-resistant mutations. In vivo, HZ-Q1070 exhibits excellent pharmacokinetic properties, BTK degradation activity and robust tumor growth inhibition in TMD8 and Mino mouse xenograft tumor model. These findings support further clinical development of HZ-Q1070 for the treatment of B cell malignancies.