B-cell Receptor Pathway Inhibitors Research Articles

MALT1-dependent cleavage of CYLD promotes NF-κB signaling and growth of aggressive B-cell receptor-dependent lymphomas

The paracaspase mucosa-associated lymphoid tissue 1 (MALT1) is a protease and scaffold protein essential in propagating B-cell receptor (BCR) signaling to NF-κB. The deubiquitinating enzyme cylindromatosis (CYLD) is a recently discovered MALT1 target that can negatively regulate NF-κB activation. Here, we show that low expression of CYLD is associated with inferior prognosis of diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) patients, and that chronic BCR signaling propagates MALT1-mediated cleavage and, consequently, inactivation and rapid proteasomal degradation of CYLD. Ectopic overexpression of WT CYLD or a MALT1-cleavage resistant mutant of CYLD reduced phosphorylation of IκBα, repressed transcription of canonical NF-κB target genes and impaired growth of BCR-dependent lymphoma cell lines. Furthermore, silencing of CYLD expression rendered BCR-dependent lymphoma cell lines less sensitive to inhibition of NF-κΒ signaling and cell proliferation by BCR pathway inhibitors, e.g., the BTK inhibitor ibrutinib, indicating that these effects are partially mediated by CYLD. Taken together, our findings identify an important role for MALT1-mediated CYLD cleavage in BCR signaling, NF-κB activation and cell proliferation, which provides novel insights into the underlying molecular mechanisms and clinical potential of inhibitors of MALT1 and ubiquitination enzymes as promising therapeutics for DLBCL, MCL and potentially other B-cell malignancies.

Safety and Effectiveness of Venetoclax/Rituximab in Bcri-Naïve and Bcri-Exposed Patients with Relapsed/Refractory CLL Under Real-Life Conditions - Data from the Observational Verve Study

BackgroundIn clinical trials, venetoclax has shown high efficacy and good tolerability in patients with chronic lymphocytic leukemia (CLL). Retrospective cohort studies have examined outcomes with venetoclax in B-cell receptor pathway inhibitor (BCRi) naïve and exposed patients with relapsed/refractory (R/R) CLL in the real world setting. However, prospective real-world data on the sequencing of new agents in the R/R setting are limited. The prospective non-interventional observational study VeRVe is assessing effectiveness, safety, and quality of life in CLL patients treated with venetoclax in Austria, Germany, and Switzerland according to local label. Here we report outcomes in patients treated with venetoclax in combination with rituximab (VenR) in BCRi-naïve and BCRi-exposed patients.MethodsAdult patients with CLL requiring therapy treated with VenR according to local label were included in this analysis. Patient visits are scheduled at the physician's discretion and according to clinical practice. Study documentation is possible at baseline, weekly during ramp-up, monthly until the end of 6 months and 3-monthly afterwards up to a maximum of 3 years. Response assessment according to iwCLL criteria can be documented at the end of ramp-up, after 3, 12, and 24 months. Analyses were performed for subgroups of BCRi-naïve and BCRi-exposed patients.ResultsUntil April 26, 2021, 94 patients treated with VenR had been enrolled in the ongoing study and had received at least one dose of venetoclax (safety population). For 72 patients, treatment response had been documented at least once (effectiveness population). Enrolled patients had a median of 1 (range 1-10) previous lines of therapy. 57 patients (61%) had previously been treated with chemoimmunotherapy (CIT) and were BCRi-naïve, 32 patients (34%) had received at least one prior BCRi and 7 (7%) had received other prior treatment options. BCRi-naïve patients had a median of 1 prior therapies (range 1-4), while BCRi-exposed patients had received a median of 3 prior therapies (range 1-10). Risk factors were more common in the BCRi-exposed group (38% of patients had a documented del(17p), 34% a TP53 mutation, and 41% an unmutated IGHV status) in comparison to the BCRi-naïve group (14% del(17p), 16% TP53 mutation and 40% unmutated IGHV). Median age at baseline was 72 and 71 years for BCRi-naïve and BCRi-exposed patients, respectively. 65% vs. 84% of BCRi-naive and BCRi-exposed patients had at least one comorbidity.In the BCRi-exposed group, 29 patients (91%) had a BCRi as last prior therapy before VenR initiation with a median duration of prior BCRi therapy of 18 months (range 1 - 61 month). Most frequent reasons for discontinuation of prior BCRi therapy were AE/SAE (11 patients) and disease progression (12 patients).The reported best overall response at 12 months after VenR initiation was 83% (CR+CRi 55%; PR: 28%) for BCRi-naïve and 85% (CR+CRi 30%; PR: 55%) for BCRi-exposed patients (figure 1). After a median follow-up of 315 days the estimated 12-months OS and PFS rate were both 94.8% for BCRi-naïve patients, whereas the estimated 12 months OS and PFS rate for BCRi-exposed patients were 79.1% and 75.7%, respectively, with a median follow-up of 371 days. 79% / 25% of BCRi-naïve and 90% / 44% of BCRi-exposed patients had AEs / SAEs. No new safety signals were observed.ConclusionIn this analysis of real-world use of venetoclax in R/R CLL, patients receiving venetoclax in combination with rituximab were predominantly BCRi-naïve. BCRi-exposed patients were more heavily pre-treated, exhibited more genetic high-risk factors, and a higher proportion of patients with comorbidities. Yet, these patients achieved comparable overall response rates. However, complete remissions were more common and the 12 months OS and PFS rates were higher in BCRi-naïve patients. In both groups, VenR treatment was well tolerated. VenR represents a suitable treatment option in BCRi-naïve as well as BCRi-exposed patients. [Display omitted] DisclosuresRossi: Gilead: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Verastem: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Cellestia: Honoraria, Research Funding. Hebart: AstraZeneca: Honoraria; Janssen: Honoraria; BMS: Honoraria; AbbVie: Honoraria; Roche: Honoraria. Losem: AbbVie: Honoraria; Amgen: Honoraria. Wolff: Bayer: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Teva: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Roche: Honoraria, Research Funding. Schmidt: AbbVie: Current Employment. Famulla: AbbVie: Current Employment. Schmidt: Takeda: Honoraria; Biotest: Honoraria; Alexion: Honoraria; Sanofi-Aventis: Honoraria; Novartis: Honoraria; Incyte: Honoraria; AbbVie: Honoraria; Janssen: Honoraria. Noesslinger: Jansen: Honoraria; Roche: Honoraria; Celgene: Honoraria; Gilead: Honoraria; AstraZeneca: Honoraria; AbbVie: Honoraria. Schwaner: AbbVie: Honoraria; Amgen: Honoraria; AstraZeneca: Honoraria; Celgene: Honoraria; Janssen: Honoraria; Novartis: Honoraria; Roche: Honoraria; Servier: Honoraria.

Safety and Activity of the Highly Specific BTK Inhibitor BGB-3111 in Combination with the PD-1 Inhibitor BGB-A317 in Patients with B-Cell Lymphoid Malignancies

IntroductionBGB-3111 is a potent, highly specific, and irreversible Bruton tyrosine kinase (BTK) inhibitor, with greater selectivity for BTK vs other TEC- and EGFR-family kinases, and demonstrates favorable pharmacokinetic and pharmacodynamics properties (Tam et al. ASH 2016). BGB-A317, a humanized IgG4 variant monoclonal antibody with no Fc gamma receptor binding, targets the programmed cell death-1 (PD-1) receptor and is being developed for the treatment of solid and hematologic malignancies (Friedlander, ASCO, 2017). The combination of PD-1/PD-L1 inhibitors with B-cell receptor pathway inhibitors is being evaluated in different B-cell malignancies with the expectation of greater benefit for patients. Here, we present early safety data from a Phase 1b trial exploring the combination of BGB-3111 and BGB-A317.MethodsThis is an open-label, multicenter, phase 1b study to evaluate safety, tolerability, and preliminary efficacy of BGB-3111 in combination with BGB-A317 in subjects with B-cell malignancies, including Waldenström's macroglobulinemia (WM), aggressive and indolent non-Hodgkin's lymphoma, and transformed chronic lymphocytic leukemia/follicular lymphoma (FL) amongst other B-cell malignancies. The study includes a standard 3+3 dose escalation phase followed by dose expansion. The dose levels are: Cohort 1, BGB-3111 320 mg once a day (QD) combined with BGB-A317 2.0 mg/kg every 3 weeks (Q3W); Cohort 2, BGB-3111 320 mg QD combined with BGB-A317 5.0 mg/kg Q3W; and Cohort 3, BGB-3111 160 mg BID combined with BGB-A317 200 mg (flat dose) Q3W.ResultsAs of 01 June 2017, 25 pts were enrolled in the dose escalation portion of the trial: 15 pts in Cohort 1 and 10 pts in Cohort 2. Key patient characteristics, safety, and efficacy are shown in the Table.Safety: Median follow - up was 2.8 (range, 0.4-10.7) months. There were no dose-limiting toxicities (DLTs) in Cohort 1. Cohort 2 saw hemolysis in 2 pts, both with WM; one qualified as a DLT. These events were not associated with a positive direct antiglobulin test and resolved with immunosuppressive therapy, but resulted in the decision to exclude further enrollment of WM pts in the trial. No further DLTs were observed after exclusion of WM pts.The most frequent adverse events (AEs) of any cause in Cohort 1 were diarrhea (n=4, 26.7%), fatigue (n=4, 26.7%), and pyrexia (n=3, 20%) and in Cohort 2 were contusion (n=3, 30%), anemia, hemolysis, headache, peripheral edema, thrombocytopenia, and upper respiratory tract infection (n=2, 20% each). Frequent AEs ≥ Grade 3 from both Cohorts included anemia (n=32, 12%), and thrombocytopenia and hemolytic transfusion reaction (n=2, 8% each).Serious AEs related to study treatment were seen in 8 patients: hemolysis (n=2), pneumonitis, infusion-related reaction, abscess limb, pneumonia, anemia, ulcerative keratitis, catheter site hemorrhage, autoimmune encephalitis, dyspnea, and eczema (n=1 each). The encephalitis could not be attributed to other causes and resolved with immunosuppressive therapy. No serious hemorrhages or atrial fibrillation have been reported to date.Discontinuation due to AEs occurred in six patients for the following AEs: hemolysis (n=2), nausea, lymph gland infection, ALT increased and GGT increased (same patient), auto-immune encephalitis, and progression-related complications (n=1 each).Efficacy: Efficacy data is very early with limited follow-up and most pts treated at the lowest dose. So far, 30% of pts have responded, including responses in both WM pts as well as complete responses seen in FL and non-GCB DLBCL pts.ConclusionsThe combination of BGB-3111 and BGB-A317 has a manageable toxicity profile in a wide variety of B-cell malignancies. Autoimmune adverse events, consistent with anti-PD-1 therapy, were observed and managed with supportive care. Notably, hemolysis in 2 WM patients suggestlimitations of this combination in indolent lymphoid diseases such as WM, especially given the risk/ benefit profile of single-agent BGB-3111 in indolent settings .Enrollment is now open for Cohort 3, which will be followed by expansion cohorts to further characterize safety and efficacy of the combination. [Display omitted] DisclosuresCull:Amgen Australia: Other: travel expenses Lugano lymphoma conference 2017; Takeda Australia: Other: travel expenses Highlights of ASH march 2017, Brisbane. Opat:Janssen: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Beigene: Research Funding; Mundipharma: Consultancy; Amgen: Research Funding. Trotman:Janssen Cilag: Other: Funding facilitating research paid to third party (BioGrid Australia), Research Funding. Hilger:BeiGene: Employment. Zhang:BeiGene: Employment. Feng:BeiGene: Employment. Ro:BeiGene: Employment. Huang:BeiGene: Employment. Tam:Abbvie: Honoraria, Research Funding; Janssen Cilag: Honoraria, Research Funding; Roche: Honoraria, Research Funding.

Molecular Pathways Associated with Ibrutinib Resistance in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is an aggressive and incurable subtype of B-cell lymphoma and constitutive activation of the B-cell receptor pathway is a hallmark of B-cell lymphomas. Bruton's tyrosine kinase (BTK) is a critical component of the B-cell receptor pathway, and ibrutinib, a first-in-class, once-daily, and oral covalent inhibitor of BTK, was developed to reduce/silence B-cell receptor pathway activity, leading to clinically remarkable anti-tumor activity. In our prior multiple-center phase II clinical trial, the overall response rate in relapsed/refractory MCL patients was 68%, with a median progression free survival (PFS) of 13.9 months, surpassing the effectiveness of other therapies. The follow-up data demonstrate the durability of responses and confirm the unprecedented single-agent activity of ibrutinib in relapsed/refractory MCL. Although ibrutinib is extremely efficacious in patients with relapsed/refractory MCL, ibrutinib-treated patients who experience disease progression die within 12 months.We used RNASeq to compare gene expression profiles between ibrutinib-sensitive and -resistant patient samples, and a molecular signature strongly associated with ibrutinib-resistant clinical responses was generated. As a new prognostic predictor for ibrutinib treatment, this molecular signature in combination with the MCL International Prognostic Index (MIPI) stratified the therapeutic outcome of MCL patients. In addition, gene set enrichment analysis (GSEA) revealed the marked upregulation of components of the c-Myc and mTOR signaling pathways in the ibrutinib-resistant patient samples, indicating that the activation of the pathways may mediate ibrutinib resistance. Furthermore, the gene expression of numerous members of metabolic pathways was significantly upregulated, including the OXPHOS pathway, the citric acid cycle, and pyruvate metabolism, suggesting that metabolic reprogramming may play a major role in ibrutinib resistance. To determine the effects of targeting these pathways, the PI3K/AKT/mTOR or OXPHOS signaling pathways were inhibited in both MCL cell lines and ibrutinib-resistant MCL patient-derived xenograft (PDX) models, greatly reducing MCL cell viability and tumor burden, respectively. Furthermore, synergistic combinations using agents targeting these two pathways are currently being investigated in both in vitro and in vivo models.In conclusion, we identified a molecular signature associated with ibrutinib resistance that may eventually be used as a prognostic indicator of ibrutinib response. In addition, these studies indicate that metabolic reprogramming mediated by the mTOR pathway may underlie ibrutinib resistance in MCL. DisclosuresWang:Celgene: Honoraria, Research Funding; Acerta Pharma: Consultancy, Research Funding; Juno Therapeutics: Research Funding; BeiGene: Research Funding; Dava Oncology: Honoraria; Pharmacyclics: Research Funding; Onyx: Research Funding; Kite Pharma: Research Funding; Asana Biosciences: Research Funding; Proteolix: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

How I treat chronic lymphocytic leukemia after venetoclax

AbstractVenetoclax-based regimens have expanded the therapeutic options for patients with chronic lymphocytic leukemia (CLL), frequently achieving remissions with undetectable measurable residual disease and facilitating time-limited treatment without chemotherapy. Although response rates are high and durable disease control is common, longer-term follow-up of patients with relapsed and refractory disease, especially in the presence of TP53 aberrations, demonstrates frequent disease resistance and progression. Although the understanding of venetoclax resistance remains incomplete, progressive disease is typified by oligoclonal leukemic populations with distinct resistance mechanisms, including BCL2 mutations, upregulation of alternative BCL2 family proteins, and genomic instability. Although most commonly observed in heavily pretreated patients with disease refractory to fludarabine and harboring complex karyotype, Richter transformation presents a distinct and challenging manifestation of venetoclax resistance. For patients with progressive CLL after venetoclax, treatment options include B-cell receptor pathway inhibitors, allogeneic stem cell transplantation, chimeric antigen receptor T cells, and venetoclax retreatment for those with disease relapsing after time-limited therapy. However, data to inform clinical decisions for these patients are limited. We review the biology of venetoclax resistance and outline an approach to the common clinical scenarios encountered after venetoclax-based therapy that will increasingly confront practicing clinicians.

A Multicenter, Retrospective Study of Accelerated Venetoclax Ramp-up in Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia

A Multicenter, Retrospective Study of Accelerated Venetoclax Ramp-up in Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia

Somatic Mutations Associated with IgVH4-34 FR1 Region Unmutated QW and Avy Motifs in DLBCL Patients

There is increasing evidence for antigen-driven B-cell receptor (BCR) signalling in diffuse large B-cell lymphoma (DLBCL) and other lymphoid malignancies. This includes antigens from infections e.g.Helicobacter pyloriand Hepatitis C virus, but it is theorised that self-antigens may play a major role in some cases of lymphoid malignancy. IgVH4-34 demonstrates intrinsic autoreactivity to self-antigens on red cells, which appears to be largely mediated by two motifs within the first framework region (FR1); Q6W7 and A24V25Y26. These motifs work together to for a hydrophobic patch which determines red cell antigen binding and are frequently mutated away from self-reactivity in normal B cells. IgVH4-34 has been reported to be over-represented in DLBCL compared with expression in normal B cells. We therefore sought to identify IgVH4-34 DLBCL cases from a local cohort and to screen them for Q6W7 and A24V25Y26 motifs expecting them to be less frequently mutated in DLBCL compared with normal B cells.We also aimed to screen V4-34 cases for associated somatic mutations in other genes using high-throughput sequencing. DLBCL patient samples were obtained via the Haematology Research Tissue Bank (HRTB) in Canberra, Australia, and the Victoria Cancer BioBank. Forty-eight Formalin-Fixed, Paraffin-Embedded (FFPE) samples and 26 fresh frozen samples were screened. All samples were collected at the time of diagnosis. Patients were treated with standard chemoimmunotherapy approaches. IgVH 4-34 gene sequences were determined using an IgVH4 family-specific leader primer in combination with a JH consensus reverse primer. The IgVH region was then sequenced using Sanger sequencing. Sequences were analyzed using the IgBLAST database (National Centre for Biotechnology Information). DNA extracted from FFPE samples generally proved to have low concentration and fragmented DNA. Only 1 IgVH4-34 sequence was obtained from FFPE tissue. Five samples sequenced from fresh tissue were identified as using IgVH4-34. Using Hans criteria, it was possible to classify 3 of the 6 cases as germinal center (GC) and 1 as non-GC origin. Using fresh samples, we estimated the frequency of IgVH4-34 cases at 23%. Within FR1, Q6W7 was unmutated in all 6 samples. One sample had mutations in the A24V25Y26 motif resulting in a change to A24V25F26. The other 5 samples (83.3%) had unmutated AVY motifs. We extracted genomic DNA from and performed next generation sequencing on the 5 samples with unmutated Q6W7 and A24V25Y26 motifs using a customized capture library (SureSelectXT Target Enrichment System, Aqilent Technologies) covering genes involved in lymphomagenesis. The purified libraries were sequenced on the Illumina NextSeq500 platform at AGRF (Australian Genome Research Facility, Australia). Several genes (FCGR3A,NOTCH2andNOTCH2NLR) had mutations in all 5 samples.FCGR3Ais an IgG Fc receptor gene, and mutations inFCGR3Ahave previously been linked to systemic lupus erythematosus (SLE).NOTCH2pathway genes are frequently mutated in DLBCL.CREBBPwas mutated in four of the five samples. Mutations inCREBBPhave previously been linked with DLBCL development and regulation of immune responses. We identified high rates of IgVH4-34 (23%) in our cohort of fresh samples as previously reported. Further, we noted preservation of the Q6W7 and A24V25Y26 motifs in IgVH4-34-expressing DLBCL. This over-representation of unmutated FR1 motifs suggests that the ability to recognise self-antigens likely provides important ongoing BCR signalling that promotes survival in DLBCL. This study also highlights the difficulties in conducting DNA-based research on FFPE clinical samples which have not been collected for research purposes and the importance of tissue banking fresh samples. Studies are currently being conducted into the efficacy of BCR pathway inhibitors e.g. ibrutinib in the treatment of DLBCL and testing for unmutated IgVH4-34 FR1 motifs may present a method to predict patients who are more likely to respond. Mutations in genes such as FCGR3A,NOTCH2andCREBBPmay work in conjunction with the preserved QW and AVY motifs to promote lymphomagenesis in IgVH4-34-expressing B cells and may present targets for future research into treatment therapies. Figure Disclosures Talaulikar: Roche:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding;Janssen:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau;Amgen:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding;Takeda:Research Funding.

Safety of venetoclax rapid dose escalation in CLL patients previously treated with B-cell receptor signaling antagonists

AbstractVenetoclax has efficacy in patients relapsing after B-cell receptor pathway inhibitors (BCRis); however, because of the risk of tumor lysis syndrome (TLS), a 5-week dose ramp-up is required to attain the target dose. Patients relapsing after BCRis frequently have proliferative disease, requiring a faster time to target dose than this scheme allows. This limitation can potentially be overcome with rapid dose escalation (RDE). We analyzed 33 chronic lymphocytic leukemia patients who underwent venetoclax RDE after prior BTKi treatment. Median time to target dose was 9 days. Seventeen patients (52%) developed laboratory TLS, and 5 (15%) developed clinical TLS, all as a result of renal injury. TLS was seen in more patients with a higher initial tumor burden. TLS occurred at all dose levels, with most episodes occurring at the 50- and 100-mg doses. Most interestingly, a decrease in absolute lymphocyte count (ALC) from pre–venetoclax dose to 24 hours post–venetoclax dose of 10 × 103/μL was associated with an increased risk of TLS (hazard ratio, 1.32; P = .02), after controlling for venetoclax dose level. Venetoclax RDE with close in-hospital monitoring at experienced centers and in select patients is feasible. The rapidity with which ALC drops helps predict TLS and could help guide dose-escalation decisions.

Refractory and 17p-deleted chronic lymphocytic leukemia: improving survival with pathway inhibitors and allogeneic stem cell transplantation.

Refractory/early relapsed and 17p deletion/p53 mutation (del(17p)/TP53mut)-positive chronic lymphocytic leukemia (CLL) has been conventionally considered a high-risk disease, potentially eligible for treatment with allogeneic stem cell transplantation (alloSCT). In this multicenter retrospective analysis of 157 patients, we compared the outcomes of patients with high-risk CLL treated with alloSCT, a B-cell receptor pathway inhibitor (BCRi), and both. Seventy-one patients were treated with BCRis, 67 patients underwent reduced-intensity conditioning alloSCT, and 19 received alloSCT with a BCRi before and/or after transplantation. Inverse probability of treatment weighting analyses were performed to compare the alloSCT and no-alloSCT groups; in the 2 groups, 5-year OS, PFS, and cumulative incidence of nonrelapse mortality (NRM) and relapse were 40% versus 60% (P = .096), 34% versus 17% (P = .638), 28% versus 5% (P = .016), and 38% versus 83% (P = .005), respectively. Patients treated with alloSCT plus BCRi had a 3-year OS of 83%. The 3-year OS and NRM by year of alloSCT, including patients treated with BCRi, were 53% and 17% in 2000 to 2007, 55% and 30% in 2008 to 2012, and 72% and 18% in 2013 to 2018. In conclusion, the combination of pathway inhibitors and alloSCT is feasible and may further improve the outcome of high-risk CLL patients.

Rapid and Durable Responses with the SYK/JAK Inhibitor Cerdulatinib in a Phase 2 Study in Relapsed/Refractory Follicular Lymphoma-Alone or in Combination with Rituximab

Rapid and Durable Responses with the SYK/JAK Inhibitor Cerdulatinib in a Phase 2 Study in Relapsed/Refractory Follicular Lymphoma-Alone or in Combination with Rituximab

An Update on Safety and Preliminary Efficacy of Highly Specific Bruton Tyrosine Kinase (BTK) Inhibitor Zanubrutinib in Combination with PD-1 Inhibitor Tislelizumab in Patients with Previously Treated B-Cell Lymphoid Malignancies

An Update on Safety and Preliminary Efficacy of Highly Specific Bruton Tyrosine Kinase (BTK) Inhibitor Zanubrutinib in Combination with PD-1 Inhibitor Tislelizumab in Patients with Previously Treated B-Cell Lymphoid Malignancies

CXCR4 upregulation is an indicator of sensitivity to B-cell receptor/PI3K blockade and a potential resistance mechanism in B-cell receptor-dependent diffuse large B-cell lymphomas.

B-cell receptor (BCR) signaling pathway components represent promising treatment targets in multiple B-cell malignancies including diffuse large B-cell lymphoma (DLBCL). In in vitro and in vivo model systems, a subset of DLBCLs depend upon BCR survival signals and respond to proximal BCR/phosphoinositide 3 kinase (PI3K) blockade. However, single-agent BCR pathway inhibitors have had more limited activity in patients with DLBCL, underscoring the need for indicators of sensitivity to BCR blockade and insights into potential resistance mechanisms. Here, we report highly significant transcriptional upregulation of C-X-C chemokine receptor 4 (CXCR4) in BCR-dependent DLBCL cell lines and primary tumors following chemical spleen tyrosine kinase (SYK) inhibition, molecular SYK depletion or chemical PI3K blockade. SYK or PI3K inhibition also selectively upregulated cell surface CXCR4 protein expression in BCR-dependent DLBCLs. CXCR4 expression was directly modulated by fork-head box O1 via the PI3K/protein kinase B/forkhead box O1 signaling axis. Following chemical SYK inhibition, all BCR-dependent DLBCLs exhibited significantly increased stromal cell-derived factor-1α (SDF-1α) induced chemotaxis, consistent with the role of CXCR4 signaling in B-cell migration. Select PI3K isoform inhibitors also augmented SDF-1α induced chemotaxis. These data define CXCR4 upregulation as an indicator of sensitivity to BCR/PI3K blockade and identify CXCR4 signaling as a potential resistance mechanism in BCR-dependent DLBCLs.

PS1165 VENETOCLAX FOR CHRONIC LYMPHOCYTIC LEUKEMIA (CLL): A RETROSPECTIVE CHART REVIEW OF SAFETY AND EFFICACY FROM PRE‐APPROVAL ACCESS (PAA) PROGRAMS IN THE EUROPEAN UNION

Background:Venetoclax (Ven) is an orally bioavailable potent, selective BCL‐2 inhibitor that achieves an overall response rate (ORR) of 77%, with 20% complete response (CR), as a monotherapy in patients (pts) with CLL (Stilgenbauer et al. JCO 2018). In 2016, the European Medicines Agency approved Ven monotherapy for the treatment of pts with CLL with or without del17p or TP53 mutation who either are unsuitable for or have failed a prior B‐cell receptor (BCR) pathway inhibitor, or who have failed chemoimmunotherapy and a BCR pathway inhibitor, respectively. Before approval, Ven was available to pts through PAA programs.Aims:A retrospective chart review was conducted to determine the real‐world effectiveness, safety, and tolerability of Ven in pts enrolled in PAA programs in France, Germany, and the United Kingdom.Methods:Pts with CLL with or without del17p or TP53 mutation who failed or were ineligible for approved therapies or reasonable alternatives were treated on PAA programs in 2016. Pts were included if they received ≥1 dose of Ven. The data cutoff was 1 October 2018. Dosing started at 20 mg/d and ramped up weekly to 50, 100, 200, and 400 mg/d over 5 weeks. The objective was to determine safety, ORR, overall survival (OS), and pts remaining on treatment at 52 weeks.Results:Forty‐seven pts were enrolled with a median age of 68 y (range, 48–90) and a median of 4 prior therapies (range, 0–11) of which 92% received at least 1 prior line of BCR inhibitor; 36% had del17p, 30% had TP53 mutation, and 21% had a high risk for tumor lysis syndrome (TLS) before Ven treatment. Forty‐six pts (98%) received TLS prophylaxis with anti‐uric acid drugs including 16 pts with rasburicase. During Ven ramp‐up, 2 pts (4%) required dose reductions and 6 (13%) required dose interruptions, primarily due to adverse events (AEs). Thirty‐six pts (77%) received doses of ≥400 mg of Ven. The most common AEs were neutropenia in 7 (15%), diarrhea in 5 (11%), and asthenia in 3 (6%) pts; 2 pts (4%) had febrile neutropenia, and 1 pt had a fatal AE of progressive multifocal leukoencephalopathy. Laboratory TLS occurred in 4 pts (9%; only 1 was clinical TLS) with a median time to onset of 2.5 wks (range, 1–4). With a median time on drug of 41 wks (range, 1–90), the ORR was 57%, with 12 pts (26%) achieving a CR or CR with incomplete marrow recovery (Table). Eighteen MRD tests (flow cytometry or PCR) for minimal residual disease (MRD) were done in 11 pts, with 7 tests done at <24 wks, 4 between 24 and 36 wks and 4 beyond 36 wks. Seven tests were MRD negative (6 by peripheral blood and 1 by bone marrow assessment) at 52 wks. Eighteen pts (38%) remained on treatment and 29 discontinued therapy at 52 weeks: 8 from progressive disease, 4 from AEs, and 17 from other reasons including death (7), pt decision (5), other treatment options (2), progression post‐allogenic stem cell transplant (1), stable disease (1) and unknown (1). Median OS was 73 wks (95% CI: 42, not reached).Summary/Conclusion:Outside of clinical studies, the safety profile of Ven, including the rate of TLS, was consistent with that observed in clinical trials. TLS prophylaxis was used in 98% of pts. ORR and OS were lower than results seen in other real‐world evidence trials, such as that reported by Mato, et al. (57% vs 72%; Mato, et al. Haematologica 2018), which could be the result of unknown responses in 21% of pts and the heavily pre‐treated population studied here. CR/CRi rates were consistent with previously published experiences with Ven in CLL (26% vs 20%; Stilgenbauer, et al. J Clin Oncol 2018).image

DESIGNER ORGANOIDS FOR MODELING EPIGENETICS, SIGNALING, AND THERAPIES IN LYMPHOMA

Diffuse Large B cell lymphoma (DLBCL) is the most common lymphoma with significant molecular heterogeneity within morphologically indistinguishable tumors of the lymph node. Two subtypes of DLBCL have remarkably poor clinical outcomes: the recently defined extranodal class of activated B cell-like (ABC)-DLBCL and the epigenetic class of germinal center B cell (GCB)-DLBCL (1, 2). The mechanism through which DLBCLs, and potentially other lymphomas, are resistant to current therapies are unknown but may be linked to the particular spectrum of somatic mutations in these tumors, which are in concert with complex growth signals provided by the malignant lymph node tumor microenvironment (Ly-TME). Many of the hallmark ABC-DLBCL mutations result in constitutive activation of protein components of B cell receptor (BCR) and Toll-like receptor (TLR) pathways in these malignant immune cells. Hence these pathways are emerging as a source of therapeutic targets for the treatment of ABC-DLBCLs. However, to date, existing BCR pathway inhibitors such as those targeting Bruton's tyrosine kinase are active in a limited subset of patients and only for a short duration, the causes of which are unknown. The substantial differences in response rates and response duration between ABC-DLBCL patients reflect the variable dependencies on BCR and TLR signaling and/or differential regulation by the Ly-TME. Unfortunately, we do not understand the impact of Ly-TME on cooperative, feed-back, and bypass signaling pathways in ABC-DLBCLs and, consequently, on the efficacy of inhibitors and/or agonists to adequately suppress these networks. A major limitation in the field has been the lack of an experimental therapeutics platform that recapitulates human cell and extracellular matrix components of Ly-TME, fluid characteristics and tissue stiffness that regulate DLBCL signaling, and also could model BCR- Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) paracaspase and TLR signaling cross-talk. In this talk, we will discuss the discoveries enabled by designer lymphoid organoids and on-chip technologies in understanding the epigenetic switches such as enhancer of zeste homolog 2 (EZH2) and components of the B Cell receptor signaling pathway that have emerged as key therapeutic targets in DLBCLs (3-6). We will also discuss the ongoing development of a more accurate 3D on-chip model and test the cooperative signaling between BCR-MALT1-TLR as well as the therapeutic efficacy of select inhibitors alone or in combination with other drugs. Research funding: The authors acknowledge financial support from the National Institutes of Health (1R33CA212968-01 to A.S) and Department of Defense Career Development Award (W81XWH-17-1-0215to A.S.).

Concurrent treatment with two B-cell receptor pathway inhibitors

The prognosis of B-cell lymphoid malignancies has improved after the implementation of ibrutinib and idelalisib: two orally-available drugs targeting the B-cell receptor signalling pathway. Ibrutinib is a Bruton tyrosine kinase inhibitor (BTKi) approved for chronic lymphocytic leukaemia, mantle cell lymphoma, and Waldenstrom macroglobulinaemia. 1 Byrd JC Brown JR O'Brien S et al. Ibrutinib versus ofatumumab in previously treated chronic lymphoid leukemia. N Engl J Med. 2014; 371: 213-223 Crossref PubMed Scopus (1232) Google Scholar , 2 Robak T Burger JA Tedeschi A et al. Single-agent ibrutinib versus chemoimmunotherapy regimens for treatment-naïve patients with chronic lymphocytic leukemia: A cross-trial comparison of phase 3 studies. Am J Hematol. 2018; 93: 1402-1410 Crossref PubMed Scopus (19) Google Scholar , 3 Wang ML Rule S Martin P et al. Targeting BTK with ibrutinib in relapsed or refractory 507 mantle-cell lymphoma. N Engl J Med. 2013; 369: 507-516 Crossref PubMed Scopus (1215) Google Scholar Idelalisib is also an approved B-cell receptor pathway inhibitor. It is a first-in-class, orally-bioavailable, selective competitive inhibitor of phosphoinositol-3-kinase δ (PI3K-δ), which has been approved for the treatment of chronic lymphocytic leukaemia, in association with rituximab, and for follicular lymphoma as monotherapy. 4 Furman RR Sharman JP Coutre SE et al. Idelalisib and rituximab in relapsed chronic lymphocytic leukemia. N Engl J Med. 2014; 370: 997-1007 Crossref PubMed Scopus (1369) Google Scholar However, idelalisib can induce severe hepatotoxicity, diarrhoea or colitis, pneumonitis, fatal or serious infections, and intestinal perforation. To address these issues, next-generation PI3K inhibitors, including umbralisib, duvelisib, copanlisib, and buparlisib, are also being investigated for B-cell disorders. These drugs seem to be more potent and better tolerated than idelalisib. Umbralisib is a next-generation inhibitor of PI3K-δ and casein kinase-1ε, and it appears to be well tolerated, has a reduced inhibitory activity on T regulatory cells, and has a lower rate of hepatotoxicity than idelalisib. 5 Burris 3rd, HA Flinn IW Patel MR et al. Umbralisib, a novel PI3Kδ and casein kinase-1ɛ inhibitor, in relapsed or refractory chronic lymphocytic leukaemia and lymphoma: an open-label, phase 1, dose-escalation, first-in-human study. Lancet Oncol. 2018; 19: 486-496 Summary Full Text Full Text PDF PubMed Scopus (138) Google Scholar Umbralisib also showed a favourable safety profile and good clinical activity when administered as a single drug to patients with B-cell lymphoma in a phase 1 study. 5 Burris 3rd, HA Flinn IW Patel MR et al. Umbralisib, a novel PI3Kδ and casein kinase-1ɛ inhibitor, in relapsed or refractory chronic lymphocytic leukaemia and lymphoma: an open-label, phase 1, dose-escalation, first-in-human study. Lancet Oncol. 2018; 19: 486-496 Summary Full Text Full Text PDF PubMed Scopus (138) Google Scholar In contrast to idelalisib, patients given umbralisib had no observed cases of grade 3 hepatotoxicity or colitis, and the only grade 3 or higher adverse event was neutropenia. Umbralisib in combination with ibrutinib in patients with relapsed or refractory chronic lymphocytic leukaemia or mantle cell lymphoma: a multicentre phase 1–1b studyUmbralisib plus ibrutinib is well tolerated and active in relapsed or refractory chronic lymphocytic leukaemia and mantle cell lymphoma, with a recommended phase 2 dose of umbralisib 800 mg once daily. To the best of our knowledge, these are the first clinical data on a BTKi and PI3K-δi doublet in B-cell malignancies, and the results suggest that this approach is feasible and worthy of further study. Full-Text PDF

The magnitude of improvement in progression-free survival with targeted therapy in relapsed/refractory chronic lymphocytic leukemia based on prognostic risk category: a systematic review and meta-analysis

Chronic lymphocytic leukemia (CLL) guidelines highlight the relevance of cytogenetic and molecular testing to identify patients with high-risk genetic features. However, at the moment, only 17p del/TP53 mutation are universally recognized parameters influencing choice of therapy. We conducted a systematic review and meta-analysis assessing the magnitude of improvement in progression-free survival (PFS) with B-cell receptor (BCR) (i.e. ibrutinib and idelalisib) or BCL2 (i.e. venetoclax) pathway inhibitors based on the presence or absence of 17p deletion/TP53 mutations, 11q deletion and IGHV mutational status in relapsed/refractory (R/R) CLL patients. Meta-analysis of seven randomized trials comprising 2409 patients with R/R CLL revealed that improvement over traditional treatments observed with BCR or BCL2 pathway inhibitors is common to all patients, including those patients with unfavorable and favorable prognostic parameters. These findings provide quantitative evidence to support the choice of therapy in R/R CLL not solely on the basis of 17p del/TP53 mutations.

The Magnitude of Improvement in Progression-Free Survival with Targeted Therapy in Relapsed/Refractory Chronic Lymphocytic Leukemia Based on Genetic Risk: A Systematic Review and Meta-Analysis

Background Currently, only genetic testing, del(17p) or TP53, determines choices of therapy in chronic lymphocytic leukemia (CLL) whereas mutational status of IGHV and 11q deletion are not recognized as reliable parameters for treatment selection (with exception for fludarabine, cyclophosphamide and rituximab [FCR] in mutated IGHV).To increase understanding of the predictive value of prognostic parameters in the novel therapy era, we conducted a systematic review and meta-analysis assessing the magnitude of improvement in progression-free survival (PFS) with B-cell receptor (BCR) or BCL2 pathway inhibitors based on the presence or absence of 17p deletion/TP53 mutations, 11q deletion and IGHV mutational status in relapsed refractory (R/R) CLL patients.Methods Randomized trials comparing BCR or BCL2 inhibitors to other treatment regimens were considered eligible for this analysis. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement was utilized as a reporting guideline. The search strategy yielded 1184 records for screening with 97 full-text articles assessed for eligibility. Finally, seven randomized trials were included in the quantitative meta-analysis, all of which compared BCR or BCL2 inhibitors to a different comparator.Results Meta-analysis of seven randomized trials comprising 2415 patients with R/R CLL revealed that treatment with BCR or BCL2 pathway inhibitors improved OS compared with the combined comparator treatments [Hazard ratio (HR) 0.555;95% confidence interval (CI): 0.452-0.680;P=0.000]. Of note, the I2 statistic for heterogeneity (i.e.,8.8% [P=0.36] ) and Q values (i.e., 6.583) indicated a high level of homogeneity of results across studies. Similar results were obtained with PFS (HR, 0.196; 95% CI: 0.144-0.268; P=0.000) although significant statistical heterogeneity existed across the studies (I2 = 78.6%, P<0.00001;Q=28.08).Next, we evaluated the magnitude of improvement in PFS obtained with BCR or BCL2 pathway inhibitors in R/R CLL patients with high-risk genetic features (Fig 1). The risk of progression was lower for 17p deleted patients (n= 450) treated with BCR or BCL2 inhibitors in comparison to combined comparator treatments (HR, 0.206; 95% CI, 0.108-0.392; P = .000; I2 = 76%; P=0.001;Q=20.853). The same applied with the analysis of TP53 which included five studies enrolling 582 R/R CLL patients with mutated TP53 (HR, 0.231 (95% CI:0.1370.390;P=0.000;I2=75.8%,P=0.0002;Q=16.513).Data for PFS by 11q deletion status, restricted to 4 studies (3 of ibrutinib and one of venetoclax) including 433 patients , revealed a significantly higher risk of progression for 11q deleted patients treated with comparator treatments [HR,0.081; 95% CI: 0.054-0.121; I2= 0.0% (P=0.56), Q =2.06]. Finally, data for PFS by IGHV mutational status that relied on 6 studies including 1580 IGHV unmutated patients favoured treatment with BCR or BCL2 inhibitors ( HR, 0.172; 95% CI, 0.109-0.272; P=0.000; I2= 84.8%, P<0.0001; Q= 32.98).An additional pooled meta-analysis restricted to patients with low risk genetic features revealed that the benefit observed with BCR or BCL2 inhibitors in patients with high-risk genetic features may extend to former (data not shown). Of note, the interaction analysis comparing the magnitude of PFS improvement in patients with and without high risk genetic features, respectively, showed that the benefit was independent of mutational status of IGHV (HR,1.39; 95% CI,0.77-2.40;P=0.26), mutational status of TP53 (HR,1.06; 95% CI,0.56-2.00;P=0.43) and 17p deletion (HR,1.10;95% CI,1.10;95%CI, 0.52-2.35;P=0.40). Interestingly, R/R CLL patients who carried 11q deletion treated with BCR or BCL2 inhibitor fared significantly better in terms of the improvement of PFS than patients without 11q deletion (HR,0.39; 95% CI,0.25-0.62;P<0.0001) indicating the particular value of novel therapies in patients with this genetic defect.Conclusions The important information provided by present meta-analysis is that the improvement over traditional treatments observed with BCR or BCL2 pathway inhibitors is common to all patients with R/R CLL, including those patients with unfavorable and favourable prognostic parameters. BCR signal transduction and BCL2 inhibitors have broad utility and overcome many but not all of the traditional poor risk molecular features in CLL. DisclosuresMolica:Gilead: Other: Advisory board; AbbVie: Other: Advisory board; Roche: Other: Advisory board; Jansen: Other: Advisory board. Levato:Novartis: Other: Advisory board. Shanafelt:Genentech: Research Funding; GlaxoSmithKline: Research Funding; Jansen: Research Funding; Pharmacyclics: Research Funding.

Ibrutinib-Resistant Mantle Cell Lymphoma Undergoes Metabolic Reprogramming Towards Oxphos

IntroductionMantle cell lymphoma (MCL) is an incurable B-cell lymphoma subtype and constitutive activation of the B-cell receptor pathway is a hallmark of B-cell lymphomas. Bruton's tyrosine kinase (BTK) is a critical component of the B-cell receptor pathway, and ibrutinib, a first-in-class, once-daily, and oral covalent inhibitor of BTK, was developed to reduce/silence B-cell receptor pathway activity, leading to clinically remarkable anti-tumor activity. In our prior multiple-center Phase II clinical trial, the overall response rate in relapsed/refractory MCL patients was 68% (Wang et al., NEJM, 2013), surpassing the effectiveness of other therapies. Although ibrutinib is extremely efficacious in patients with relapsed/refractory MCL, the one-year overall survival rate of ibrutinib-exposed patients who relapse is only 22%.MethodsPatient primary cells were isolated from MCL patients treated with ibrutinib either prior to treatment or at treatment discontinuation. Whole exome sequencing (WES) was performed to determine the mutational landscape of ibrutinib resistance. RNA-seq was employed to compare the gene expression profiles between ibrutinib-sensitive and -resistant patient samples. Gene set enrichment analysis was utilized to identify dysregulated molecular pathways associated with the resistant phenotype. The RNA-seq data were then validated with reverse phase protein array (RPPA) analysis of ibrutinib-sensitive and -resistant MCL cell lines. Metabolic assays including the measurement of mitochondria respiration rates with the Seahorse analyzer and reactive oxygen species (ROS) levels, targeted metabolomics, and ATP analysis. Functional studies targeting this molecular pathway were conducted, including in vitro cell viability and apoptosis assays, as well as in vivo efficacy studies in an ibrutinib-resistant MCL patient-derived xenograft mouse model.ResultsWES data analysis identified frequent inactivating somatic alterations in ATM, KMT2D, and TP53 in both the ibrutinib-sensitive and -resistant tumors. CDKN2A (5/7, 71%) was frequently deleted, and the deletion was only observed in the ibrutinib-resistant tumors (p = 0.010). The RNA-seq analysis identified a total of 63 protein-coding genes as the most differentially expressed genes (DEGs) between the ibrutinib-resistant and -sensitive groups, with a fold change of ≥ 2 or ≤ -2 and the false discovery rate (FDR q-value) ≤ 0.01. Among the DEGs, 26 genes were upregulated in ibrutinib-resistant tumors. In addition, gene set enrichment analysis (GSEA) revealed the marked upregulation of oncogenic pathways including c-MYC, mTOR (mTORC1), Wnt, and NF-ĸb signaling, followed by cell cycle, apoptosis, BCR signaling and DNA repair in the ibrutinib-resistant tumors. Notably, in addition to these oncogenic pathways, the metabolic pathways, including oxidative phosphorylation (OXPHOS), were significantly enriched in the ibrutinib-resistant tumors (normalized enrichment score > 3 and FDR q-value < 1e-5). In further support of this finding, metabolomics analysis and the measurement of ATP production and mitochondrial respiration indicated that the OXPHOS pathway is the predominant metabolic pathway employed by ibrutinib-resistant MCL cells. To determine the effects of targeting these pathways, OXPHOS was inhibited with a novel electron transport complex I inhibitor (IACS-010759, developed by MD Anderson Cancer Center) in both MCL cell lines and ibrutinib-resistant MCL patient-derived xenograft (PDX) models. Single agent IACS-010759 treatment at 10 mg/kg oral gavage for 5 consecutive days/week completely prevented tumor growth compared with the vehicle control as shown by measuring tumor volume (n = 5, p < 0.0001) and human β2M levels (n = 5; p < 0.0001) throughout treatment. No apparent toxicities were observed in the IACS-010759-treated MCL PDX mice.ConclusionThis current study warrants the exploitation of active cancer metabolic pathways, especially OXPHOS, to improve the clinical outcomes of MCL and additional lymphoma, which is actively being investigated in a Phase I lymphoma clinical trial (NCT03291938). DisclosuresWang:AstraZeneca: Consultancy, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Research Funding; Novartis: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; MoreHealth: Consultancy; Acerta Pharma: Honoraria, Research Funding; Kite Pharma: Research Funding; Pharmacyclics: Honoraria, Research Funding; Dava Oncology: Honoraria.

Characterizing the kinetics of lymphocytosis in patients with chronic lymphocytic leukemia treated with single-agent ibrutinib

Increased absolute lymphocyte count (ALC) is a key feature of chronic lymphocytic leukemia (CLL) but is also observed during treatment with B-cell receptor pathway inhibitors including ibrutinib, a first-in-class inhibitor of Bruton’s tyrosine kinase. In patients with CLL treated with single-agent ibrutinib in two multicenter, open-label, randomized, phase 3 studies (RESONATE-2, NCT01722487; RESONATE, NCT01578707), lymphocytosis was observed in 77 of 136 (57%) patients treated in first-line and 133 of 195 (69%) relapsed/refractory patients. On treatment, lymphocytosis resolved in 95% of patients in the first-line and 94% in the relapsed/refractory setting. The median duration of lymphocytosis was 12 and 14 weeks in the first-line and relapsed/refractory settings, respectively. Lymphocytosis is a common and predictable pharmacodynamic effect of ibrutinib treatment, and in the absence of other signs of progression, does not represent disease progression. Lymphocytosis resolves in the majority of patients and does not require interruption or discontinuation of ibrutinib therapy.

Sensitive Detection of the Natural Killer Cell-Mediated Cytotoxicity of Anti-CD20 Antibodies and Its Impairment by B-Cell Receptor Pathway Inhibitors.

The antibody-dependent cell-mediated cytotoxicity (ADCC) of the anti-CD20 monoclonal antibodies (mAbs) rituximab and obinutuzumab against the cell line Raji and isolated CLL cells and its potential impairment by kinase inhibitors (KI) was determined via lactate dehydrogenase release or calcein retention, respectively, using genetically modified NK92 cells expressing CD16-176V as effector cells. Compared to peripheral blood mononuclear cells, recombinant effector cell lines showed substantial alloreactivity-related cytotoxicity without addition of mAbs but afforded determination of ADCC with reduced interassay variability. The cytotoxicity owing to alloreactivity was less susceptible to interference by KI than the ADCC of anti-CD20 mAbs, which was markedly diminished by ibrutinib, but not by idelalisib. Compared to rituximab, the ADCC of obinutuzumab against primary CLL cells showed approximately 30% higher efficacy and less interference with KI. Irreversible BTK inhibitors at a clinically relevant concentration of 1 μM only weakly impaired the ADCC of anti-CD20 mAbs, with less influence in combinations with obinutuzumab than with rituximab and by acalabrutinib than by ibrutinib or tirabrutinib. In summary, NK cell line-based assays permitted the sensitive detection of ADCC of therapeutic anti-CD20 mAbs against CLL cells and of the interference of KI with this important killing mechanism.