Acalabrutinib in Patients with Relapsed/Refractory (R/R) and High-Risk, Treatment-Naive (TN) Chronic Lymphocytic Leukemia (CLL)

Abstract Background: Bruton tyrosine kinase (BTK) is a critical component of B-cell receptor signaling and a validated target for CLL. Acalabrutinib is a highly selective, potent, covalent BTK inhibitor, which has shown promising efficacy and safety in patients with CLL, including high-risk patients. We present preliminary efficacy, safety, and pharmacodynamic results from an ongoing single-center, open-label, phase 2 study of acalabrutinib monotherapy in patients with R/R and high-risk, TN CLL. Methods: Patients with R/R or high-risk (chromosome 17p deletion [del17p] or mutation in TP53 or NOTCH1) TN CLL/small lymphocytic lymphoma (SLL) who met International Workshop on Chronic Lymphocytic Leukemia (IWCLL) 2008 criteria for treatment and had an Eastern Cooperative Oncology Group performance status ≤2 were eligible. Patients who had prior BTK inhibitor therapy were excluded. Patients were randomized to receive oral acalabrutinib 100 mg twice daily (BID) or 200 mg daily (QD) until progressive disease or unacceptable toxicity. The primary endpoint was investigator-assessed overall response rate (ORR) by IWCLL 2008 criteria with modification for lymphocytosis. Secondary endpoints included safety and BTK occupancy. BTK occupancy was measured with a biotin-tagged analogue probe in peripheral blood cells at drug trough time points after 3 days of dosing and after 1, 6, and 12 mo of treatment. BTK occupancy in lymph node samples was measured at drug trough time points after 3 days of dosing. Results: Forty-six patients were enrolled and treated (100 mg BID, n=22; 200 mg QD, n=24). The median age was 64 years (range, 45-83), and 35% (16/46) were TN. Approximately 39% of patients (25% of TN) had bulky lymph nodes ≥5 cm, 37% (50% of TN) had Rai stage III-IV disease at baseline, 76% (88% of TN) had unmutatedIGHV, 21% (40% of TN) had del(17p), 21% (23% of TN) had TP53 mutation, and 47% (54% of TN) had NOTCH1 mutation. As of April 13, 2018, the median time on study for all treated patients was 20 mo (range 1-39), with 89% (41/46) remaining on acalabrutinib. Two patients (9%) in the BID group and 3 patients (13%) in the QD group discontinued treatment due to an adverse event (AE; n=1), progressive disease (n=1), and other reasons (n=3). The patient who discontinued due to progressive disease (BID group) achieved partial response at 2 mo and developed Richter transformation at 6 mo. The ORR was 90% (95% CI: 76, 97) for efficacy evaluable patients (N=39), defined per protocol as patients who had ≥ 6 mo of acalabrutinib (Table). ORR was 95% (75, 100) and 84% (60, 97) for the BID and QD group, respectively. For the intent-to-treat population (N=46), ORR was 80% (66, 91). Most AEs were grade 1/2 and did not require dose delays or modifications. The most common AEs (all grades; >25%) were headache (63%), contusion (50%), diarrhea (43%), upper respiratory tract infection (43%), arthralgia (33%), influenza-like illness (28%), maculo-papular rash (28%), myalgia (26%), and nausea (26%). Grade 3/4 AEs occurred in 33% (15/46) of patients (BID, 27% [6/22]; QD, 38% [9/24]), most commonly (>10%) infections (13%; urinary tract infection, lung infection, hepatitis B reactivation, which led to treatment discontinuation and fatal hepatic failure after 10 mo of treatment, and an invasive pulmonary aspergillosis at 2 mo in the setting of prolonged neutropenia and recent systemic corticosteroid use that led to treatment discontinuation) and neutropenia (11%). Approximately 33% (15/46) of patients (BID, 23% [5/22]; QD, 42% [10/24]) reported serious AEs (all grades), most commonly (>5%) lung infection (7%). No atrial fibrillation was reported, and one grade 1 atrial flutter occurred (BID). On day 4 of cycle 1, median trough BTK occupancy was significantly higher for the BID group versus the QD group in the peripheral blood (95% vs 87%; P<0.001) and in the lymph node (98% vs 90%, P<0.001). Median trough BTK occupancy in the peripheral blood was also higher for the BID group at 1, 6, and 12 mo (range, 98%-99% for BID vs 95%-97% for QD; P<0.05 at all time points). Conclusion: Acalabrutinib monotherapy produced high ORR in R/R and high-risk TN CLL, with an acceptable safety profile. The study was not designed to detect a statistically significant difference in clinical outcomes between the dosing groups. Near complete target coverage (>95%) was more rapidly achieved with 100 mg BID than 200 mg QD dosing in the lymph node and peripheral blood. Disclosures Nierman: National Institutes of Health: Employment. Covey:Acerta Pharma: Employment; AstraZeneca: Equity Ownership. Hamdy:Acerta Pharma: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: various patents for ACP-196. Izumi:Acerta Pharma: Employment, Equity Ownership, Patents & Royalties: Acerta Pharma, various patents for ACP-196. Liu:Acerta Pharma: Employment. Patel:Acerta Pharma: Employment, Equity Ownership. Wiestner:Pharmacyclics LLC, an AbbVie Company: Research Funding.

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Next-Generation Sequencing Reveals Clonal Evolution at the Immunoglobulin Loci in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v124.21.3302.3302 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3302-3302 ◽

Cited By ~ 1

Author(s):

Jennifer R. Brown ◽

Stacey M. Fernandes ◽

Siddha Kasar ◽

Kevin Hoang ◽

Martin Moorhead ◽

...

Keyword(s):

B Cell ◽

Peripheral Blood ◽

Clonal Evolution ◽

Post Treatment ◽

Lymphocytic Leukemia ◽

Clinical Relapse ◽

Employment Equity ◽

Time Points ◽

Equity Ownership ◽

Generation Sequencing

Abstract Background: Immunoglobulin (Ig) gene rearrangement is a hallmark of early B-cell development. Chronic lymphocytic leukemia (CLL) is typically considered a malignancy of mature B-cells and is thought to originate from the oncogenic transformation of a single pre- or post-germinal B-cell. Activation-induced deaminase (AID), an enzyme that induces somatic hypermutation (SHM) at the heavy and light chain Ig loci, has been shown to be active in CLL cells in vitro (Patten et al., Blood 2012). Previous studies suggest that multiple CLL-specific Ig clonotypes related by SHM may be present in patients (pts) with dominant CLL clones possessing somatically mutated or unmutated Ig loci (Logan et al., PNAS 2011; Campbell et al., PNAS 2008). To our knowledge, evolution of the dominant CLL-specific Ig clonotype over the course of treatment has not been demonstrated. Here we utilized the LymphoSIGHT™ method, a next-generation sequencing-based method for lymphocyte characterization and quantification, to quantify clonal evolution at the Ig heavy and kappa chain (IGH and IGK) loci in 63 pts with CLL. Methods: Samples were collected at Stanford University and the Dana-Farber Cancer Institute. Peripheral blood mononuclear cells were isolated, and genomic DNA was extracted. Using unbiased universal primer sets, we amplified IGH and IGK variable, diversity, and joining gene segments. Amplified products were sequenced and analyzed using standardized algorithms for clonotype determination (Faham et al., Blood 2012). CLL-specific clonotypes were identified for each patient based on their high frequency (>5%) within the B-cell repertoire of a diagnostic (dx) sample. The highest frequency CLL clonotype identified in a dx sample is termed the “index clonotype”. Dx and post-treatment peripheral blood samples were assessed for evidence of evolved CLL clonotypes using LymphoSIGHT. A clonotype was considered “evolved” based on CDR3 sequence homology to the dx “index clonotype.” Results: CLL clonotypes were identified in dx samples from 63 pts (51 unmutated IGHV; 12 mutated), and we assessed post-treatment samples for the presence of CLL clonotype-associated oligoclonality. Two of 63 pts exhibited clonal evolution in post-treatment samples. One patient with unmutated CLL was MRD negative for over 7 years following allogeneic hematopoietic cell transplant (HCT), and subsequently became MRD positive with the evolved clonotype (differing by 1 nucleotide from the index clonotype) leading to clinical relapse 9 months after MRD positivity, while the original index clone remained undetectable. The patient was treated with ibrutinib upon clinical relapse and continues to have detectable MRD with the same evolved CLL clonotype (Fig 1A). In a second patient with mutated IGHV, we observed several evolved clonotypes in the dx sample. Multiple evolved clonotypes, including 5 that exhibited a significant increase in their frequency relative to the index clonotype, were present in the follow-up sample after treatment with fludarabine and rituximab (Fig 1B). These evolved clonotypes differed from the index clonotype by 1-4 nucleotides, but otherwise shared CDR3 identity, excluding independently arisen B cell clonotypes. Conclusions: We observed evidence of clonal evolution at Ig loci in a small subset (3.2%) of pts with CLL undergoing treatment. The presence of evolution in pts with CLL indicates that either the SHM mechanism, including the AID enzyme, remains active after neoplastic transformation, or the evolved clonotypes arose through a mechanism distinct from SHM. These evolved CLL clonotypes may have a selective advantage, and may be useful as surrogate markers for other oncogenic mutations providing resistance to therapy. Additional cases are under investigation and updated results will be presented. Figure 1. CLL clonal evolution during therapy. MRD levels of two related Ig clonotypes, expressed as leukemia molecules per million leukocytes in peripheral blood, are shown at multiple time points following allogeneic HCT (A). In another patient undergoing conventional treatment, the level of each individual evolved clonotype as a fraction of the total CLL molecules is plotted at dx and post treatment time points. The index clone, evolved clones with increasing levels post-treatment, and evolved clones with decreasing levels post-treatment are shown in red, blue, and white, respectively (B). Figure 1A. Figure 1A. Figure 1B. Figure 1B. Disclosures Moorhead: Sequenta, Inc.: Employment, Equity Ownership. Carlton:Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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CAL-101, An Isoform-Selective Inhibitor of Phosphatidylinositol 3-Kinase P110δ, Demonstrates Clinical Activity and Pharmacodynamic Effects In Patients with Relapsed or Refractory Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v116.21.55.55 ◽

2010 ◽

Vol 116 (21) ◽

pp. 55-55 ◽

Cited By ~ 34

Author(s):

Richard R. Furman ◽

John C. Byrd ◽

Jennifer R Brown ◽

Steven E. Coutre ◽

Don M Benson ◽

...

Keyword(s):

Lymph Node ◽

Median Number ◽

Selective Inhibitor ◽

Lymphocytic Leukemia ◽

Clinical Activity ◽

Pi3k Signaling ◽

Employment Equity ◽

Equity Ownership ◽

Grade 3 ◽

Dose Levels

Abstract Abstract 55 Introduction: The class I phosphatidylinositol 3-kinases (PI3Ks) regulate cellular functions relevant to oncogenesis. Expression of the PI3K p110δ isoform (PI3Kδ) is restricted to cells of hematopoietic origin where it plays a key role in B cell proliferation and survival. In chronic lymphocytic leukemia (CLL) the PI3K pathway is constitutively activated and dependent on PI3Kδ. CAL-101 is an isoform-selective inhibitor of PI3Kδ (EC50 of 62 nM in a whole-blood assay with >200-fold selectivity relative to other PI3K isoforms) that inhibits PI3K signaling and induces apoptosis of CLL cells in vitro. Methods and Patients: This Phase 1 study evaluated the safety, pharmacokinetics, pharmacodynamics and clinical activity of CAL-101 in patients with relapsed or refractory hematologic malignancies. Sequential cohorts of patients were enrolled at progressively higher dose levels with cohort expansion based on toxicity profile and plasma exposure. CAL-101 was administered orally one or 2 times per day (QD or BID) continuously for 28-day cycles for up to 12 cycles (with the potential for more prolonged therapy on an extension protocol thereafter). Clinical response was evaluated according to standard criteria. Results: At data cutoff, the study had enrolled 37 patients with CLL. Patients included: males/females n=31 (84%)/6 (16%) with median age of 65 [range 37–82] years, refractory/relapsed disease n=24 (65%)/13 (35%), bulky disease n= 29 (81%), and adverse cytogenetics of del(17p), del(11q) or both n=22 (63%). The median number of prior therapies was 5 [range 2–14]. The number (%) of patients with specific prior therapies included: rituximab n=37 (100%), purine analog n=37 (100%), alkylating agent n= 31 (84%), and alemtuzumab n=12 (32%). CAL-101 dose levels were 50 mg BID (n=1), 100 mg BID (n=4), 150 mg BID (n=11), 200 mg BID (n=10), 350 mg BID (n=7) and 300 mg QD (n=4). The median number of treatment cycles was 9 [range 1–13], with 21 (57%) patients continuing on treatment (11 on study and 10 on the extension protocol after 12 cycles). Symptomatic adverse events were infrequent, usually low-grade, and not clearly CAL-101-related. Grade ≥3 pneumonias occurred in 9 (24%) patients. Grade ≥3 hematological laboratory abnormalities included neutropenia n=9 (24%), thrombocytopenia n=4 (11%) and anemia n=3 (8%) that were not usually considered CAL-101-related. A pharmacokinetic analysis of dose-proportionality showed minimal increases in plasma Cmax and AUC at CAL-101 doses >150 mg BID; these data, taken together with the tumor regression results, have proved helpful in supporting Phase 2–3 dose selection. Flow cytometry of CLL cells from patients showed that CAL-101 reduced constitutive expression of phospho-AKT to background levels when measured after 1 week of treatment (p<0.0001), demonstrating pharmacodynamic inhibition of activated PI3K signaling. Plasma concentrations of chemokines CCL3, CCL4, and CXCL13 were elevated at baseline and decreased significantly within 1 cycle of CAL-101 administration (p<0.001 for all comparisons). CAL-101 reduced lymphadenopathy in all 32 (100%) patients with at least 1 post-treatment tumor assessment; 29/32 (91%) achieved a lymph node response (≥50% reduction in target nodal lesions). An initial increase in peripheral absolute lymphocyte counts of >50% from baseline was observed in 21/35 (60%) patients; increases were maximal during the first 2 cycles and decreased thereafter; the pattern suggested drug-mediated lymphocyte redistribution. Considering nodal and peripheral blood changes together, partial responses were observed in 11/33 (33%) of patients. The median duration of response had not been reached; 7 patients had response durations of ≥6 months. Of 20 patients with CLL-related thrombocytopenia (baseline platelet counts <100,000/μ L), 15 (75%) had either an improvement to >100,000/μ L or a >50% increase from baseline. Conclusions: CAL-101, an oral PI3Kδ isoform-selective inhibitor, shows acceptable toxicity, positive pharmacodynamic effects, and favorable clinical activity in heavily pretreated patients with CLL, including patients with refractory disease, bulky lymphadenopathy, and poor-prognosis cytogenetics. The high level of lymph node regression and prolonged duration of symptomatic tumor control strongly support evaluation of CAL-101 alone and in combination with other chemo/immunotherapy approaches to CLL management. Disclosures: Byrd: Calistoga Pharmaceuticals: Consultancy, Equity Ownership. Brown:Calistoga: Consultancy. Kahl:Calistoga Pharmaceuticals: Consultancy, Research Funding. Lannutti:Calistoga Pharmaceutical Inc.: Employment. Giese:Calistoga Pharmaceuticals: Equity Ownership. Webb:Calistoga Pharmaceuticals: Employment. Ulrich:Calistoga Pharmaceuticals: Employment, Equity Ownership. Peterman:Calistoga Pharmaceuticals: Employment. Holes:Calistoga Pharmaceuticals: Employment. Yu:Calistoga Pharmaceuticals: Employment, Equity Ownership.

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Dual SYK/JAK Inhibition Overcomes Ibrutinib Resistance in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v128.22.5377.5377 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5377-5377

Author(s):

Yue Lynn Wang ◽

Pin Lu ◽

Greg P Coffey ◽

Anjali Pandey ◽

Ailin Guo

Keyword(s):

Cell Death ◽

Research Funding ◽

Single Agent ◽

Lymphocytic Leukemia ◽

Prognostic Indicators ◽

Employment Equity ◽

Btk Inhibitor ◽

Equity Ownership ◽

Ibrutinib Resistance ◽

Novel Therapeutic Strategies

Abstract Ibrutinib (a BTK inhibitor) has generated remarkable responses in CLL. However, the drug, to a large extent, does not cause cell death directly and does not eradicate CLL malignant clones. Inability to eradicate CLL has fostered resistance generation. Once patients become resistant, they do poorly with a median survival of 3-4 months. Novel therapeutic strategies are needed to prevent resistance, improve treatment outcome and ultimately cure the disease. Herein, we explore dual targeting of the BCR and JAK-STAT pathways with a novel single agent, cerdulatinib, which selectively inhibits both SYK (a BCR component) and JAK kinases. We demonstrated that cerdulatinib delivered potent tumor inhibition in 60 primary CLL patient samples, especially in those with poor prognostic indicators. Importantly, cerdulatinib, but not ibrutinib, is able to overcome the support of microenvironment and induces CLL cell death at clinically achievable concentrations. Further, cerdulatinib blocked proliferation of ibrutinib-sensitive and ibrutinib-resistant primary CLL cells and of BTKC481S-transfected cells. These anti-tumor effects are correlated with the inhibition of BCR and JAK-STAT signaling and downstream inhibition of the functions of AKT, ERK and NFκB. Collectively, our results show that simultaneous targeting of BCR and JAK-STAT pathways is a more effective strategy relative to single BTK inhibition. Disclosures Coffey: Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Pandey:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding.

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Partial Reconstitution of Humoral and Cellular Immunity in Patients with Chronic Lymphocytic Leukemia Treated with Acalabrutinib

Blood ◽

10.1182/blood-2018-99-111919 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1874-1874 ◽

Cited By ~ 3

Author(s):

Christopher Pleyer ◽

Clare C. Sun ◽

Pia Niermann ◽

Xin Tian ◽

Inhye E. Ahn ◽

...

Keyword(s):

Bone Marrow ◽

T Cell ◽

Lymphocytic Leukemia ◽

Light Chains ◽

Free Light Chains ◽

Employment Equity ◽

Cell Numbers ◽

Serum Iga ◽

Btk Inhibitor ◽

Equity Ownership

Abstract Introduction Immune dysregulation in chronic lymphocytic leukemia (CLL) contributes to a high rate of infections and morbidity. We previously reported that treatment of CLL with ibrutinib, a Bruton tyrosine kinase (BTK) inhibitor, leads to partial reconstitution of humoral immunity and fewer infections, especially in patients who achieved a ≥50% increase in serum IgA levels. Acalabrutinib is also an irreversible BTK inhibitor that is more selective than ibrutinib and has demonstrated safety and efficacy in the treatment of relapsed or refractory CLL. It is currently unclear how the increased specificity of acalabrutinib affects immune reconstitution and infection rates. We assessed the immunological impact of acalabrutinib in patients with CLL treated with single-agent acalabrutinib. Methods Samples originated from a phase 2, single-center trial studying acalabrutinib 100 mg twice daily (BID) or acalabrutinib 200 mg once daily (QD) in patients with relapsed/refractory (RR) CLL or high-risk, treatment naïve (TN) CLL (chromosome 17p deletion or mutation in TP53 or NOTCH1) (NCT02337829). Patients who received at least 6 months of acalabrutinib and had paired longitudinal data available were included in the analyses. Patients receiving IV immunoglobulin replacement were excluded from analysis of IgG levels. Additionally, patients with detectable monoclonal IgG, IgA and/or IgM proteins on serum immunofixation were excluded from analysis of the corresponding immunoglobulin isotype. The analysis of free light chains was stratified based on k or λ restriction of CLL cells determined by flow cytometry. Immunohistochemical staining of bone marrow biopsies was performed: T cell numbers were estimated by CD3 staining and the degree of CD68-positive macrophage extensions in contact with CLL cells were semi-quantitatively assessed on a scale from 0 (no extensions) to 4 (maximum number of extensions). The Wilcoxon signed-rank test and the Mann-Whitney U test were used to compare paired and unpaired data, respectively. Differences in the rate of infection between groups were examined using the Cox regression model for recurrent events. Results Serum IgA levels increased as early as 3 months after the initiation of acalabrutinib (median increase 35.7%, P = .0001), with levels sustained up to 24 months (Figure 1), whereas serum IgG and IgM levels were not affected by acalabrutinib. There was no difference (P > .05) in IgA, IgG, IgM trend between TN or RR CLL. Furthermore, there was no difference (P > .05) in IgA, IgG and IgM trends between patients treated with QD compared to BID dosing of acalabrutinib. Among 20 k-restricted and 18 λ-restricted CLL cases, the involved (tumor-derived) free light chain was elevated at baseline and trended toward the normal range after 3 months of acalabrutinib therapy consistent with an anti-tumor effect (k: median decrease 55.4%; P < .0001 and λ: median decrease 49.1%; P = .0003). The uninvolved free light chain did not change (P > .05). Peripheral blood CD3+, CD4+ and CD8+ T cell counts were elevated above the laboratory reference range at baseline and normalized after 6 months (CD4+ median decrease 49.2%; P = .0074 and CD8+ median decrease 54.8%; P = .003). T cell numbers in the bone marrow did not appreciably change. However, treatment-induced changes of the immune microenvironment were apparent in tumor-macrophage interactions. At baseline, macrophages tightly interacted with CLL cells, often with multiple podocytes making contact with CLL cells. On acalabrutinib, we observed a decrease in these macrophage podocyte interactions (P = .0007). At a median follow-up of 20 months, 31 (68.9%) patients developed a total of 68 infections, including 7 (10.3%) grade 3 and 1 (1.5%) grade 4 infections. Patients with superior immune reconstitution, as defined by an increase in serum IgA of ≥ a median of 36% from baseline to 3 months, had a significantly lower rate of infections (risk ratio = 0.52, P = 0.029). Conclusions These data indicate that acalabrutinib allows for partial reconstitution of humoral and cell-mediated immunity and disrupts macrophage-CLL cell interaction in the bone marrow microenvironment in patients with CLL. Furthermore, acalabrutinib did not interfere with uninvolved free light chains, suggesting that acalabrutinib selectively inhibits CLL B-cells and does not impair normal B-cell function. Disclosures Izumi: Acerta Pharma: Employment, Equity Ownership, Patents & Royalties: Acerta Pharma, various patents for ACP-196. Hamdy:Acerta Pharma: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Acerta Pharma, various patents for ACP-196. Wiestner:Pharmacyclics LLC, an AbbVie Company: Research Funding.

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The Bruton Tyrosine Kinase (Btk) Inhibitor ACP-196: Marked Activity in Relapsed/Refractory CLL with a Favorable Safety Profile

Blood ◽

10.1182/blood.v126.23.831.831 ◽

2015 ◽

Vol 126 (23) ◽

pp. 831-831

Author(s):

John C. Byrd ◽

William Wierda ◽

Jeffrey Jones ◽

Susan O'Brien ◽

Jennifer R. Brown ◽

...

Keyword(s):

High Risk ◽

Safety Profile ◽

Research Funding ◽

Response Rate ◽

Employment Equity ◽

Advisory Committees ◽

Btk Inhibitor ◽

Equity Ownership ◽

Grade 3 ◽

Favorable Safety

Abstract Introduction Btk is a kinase involved in B-cell receptor (BCR) signal transduction and a critical target in chronic lymphocytic leukemia (CLL). ACP-196-a potent, second generation Btk inhibitor that is more selective than the first-in-class Btk inhibitor, ibrutinib (Covey AACR2015)-has demonstrated antitumor activity in preclinical CLL models (Niemann AACR2014). Here, we present preliminary data from patients with relapsed/refractory (R/R) CLL/small lymphocytic lymphoma (SLL) enrolled in an ongoing Phase 1/2 study of single-agent ACP-196 (ClinicalTrials.gov NCT02029443). Methods and Patients This first-in-human study was designed to evaluate the safety, maximum tolerated dose, pharmacokinetics, pharmacodynamics and efficacy of orally administered ACP-196 in patients with R/R CLL/SLL. Patients were continuously treated with ACP-196 at dosages ranging from 100 to 400 mg once daily (QD) as part of the dose-escalation portion of the study (4 cohorts of 6-8 patients per cohort), and 100 mg twice daily (BID) and 200 mg QD as part of the expansion portion of the study (2 cohorts). Of note, CLL patients with any degree of pancytopenia and prior exposure to PI3K inhibitors were allowed. CLL responses were investigator assessed per IWCLL criteria (modified Hallek 2008). SLL responses were investigator assessed per IWG criteria (Cheson 2007). Patients had a median age of 62 years (range 44-84), bulky lymph nodes ≥ 5 cm (47%) and median of 3 prior therapies (1-13). High-risk prognostic factors included del(17)(p13.1) 31% (18/58), del(11)(q22.3) 29% (17/58) and unmutated IGVH genes 75% (38/51). Results Results are presented through 01 June 2015 for the first 61 R/R patients, including 60 evaluable for response. The median time on study (N=61) was 10.3 (0.5-15.9) months. ACP-196 has been well tolerated with 93% (57/61) of patients continuing on study drug. Of the 4 patients who discontinued, 1 patient each discontinued due to withdrawal of consent, physician decision, unrelated AE (pre-existing, active autoimmune hemolytic anemia) and related AE (Grade 3 dyspnea). To date, no dose-related effect has been observed in frequency or severity of AEs or serious adverse events. No dose-limiting toxicities have occurred, and most AEs were Grade ≤ 2. The most common Grade 1/2 AEs (≥ 15%) were headache (39%), diarrhea (33%) and URI (16%). Grade 3/4 AEs that occurred in ≥ 3 patients were anemia (7%), pneumonia (7%) and hypertension (5%). No major hemorrhage (including subdural hematomas), atrial fibrillation, tumor lysis syndrome or pneumonitis have occurred suggesting an improved safety profile compared with other BCR and BCL-2-targeted therapies. Clinical activity has been observed in patients with R/R CLL/SLL at all doses evaluated. All patients experienced rapid reductions in lymphadenopathy. Treatment-related lymphocytosis (defined as ≥ 50% increase from baseline and above absolute lymphocyte count [ALC] of 5 K/µL) occurred in 61% (37/61) of patients and resolved in 81% (31/37) of these patients. In general, lymphocytosis peaked at a median of 3 weeks and resolved by a median of 19 weeks (range 1 to 58 weeks). The rapid decrease in lymphadenopathy and treatment-related lymphocytosis along with concurrent improvement in baseline cytopenias has led to a high proportion of partial responses (PRs) early in treatment (Figure 1). Best overall response rate including PR and PR with lymphocytosis (PR+L) was 93% (PR=70%, PR+L=23%, SD=7%, PD=0%). For patients with del(17)(p13.1), the response rate was 100% (PR=72%, PR+L=28%). In the 4 patients with prior idelalisib therapy, the response rate also was 100% (PR=75%, PR+L=25%). To date, no disease progression or Richter's transformation has occurred (Figure 2). Pharmacokinetic results showed exposure of ACP-196 was dose proportional with no drug accumulation. At dosages as low as 100 mg QD, pharmacodynamic results showed low intra-patient variability, high Btk occupancy (> 90% over 24 hr) and high phospho-Btk inhibition (> 90% over 24 hr). Conclusion ACP-196 is a highly potent and selective oral Btk inhibitor with a favorable safety profile. Responses occur early in treatment with no disease progression to date either in heavily pretreated patients or those with high-risk prognosis factors. ACP-196 is currently in Phase 3 trials for TN (ClinicalTrials.gov NCT0247568) and R/R high-risk CLL (ClinicalTrials.gov NCT02477696). Disclosures Byrd: Acerta Pharma BV: Research Funding. Jones:Acerta Pharma BV: Research Funding. O'Brien:Acerta Pharma BV: Research Funding. Schuh:Acerta Pharma BV: Research Funding. Hillmen:Abbvie: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Acerta Pharma BV: Research Funding; Gilead: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Stephens:Immunomedics: Research Funding; Acerta Pharma BV: Research Funding. Ghia:Pharmacyclics: Consultancy; Janssen: Consultancy; Adaptive: Consultancy; Acerta Pharma BV: Research Funding; Gilead: Consultancy, Research Funding, Speakers Bureau; GSK: Research Funding; Roche: Consultancy, Research Funding; AbbVie: Consultancy. Devereux:Acerta Pharma BV: Research Funding. Chaves:Acerta Pharma BV: Research Funding. Barrientos:Acerta Pharma BV: Research Funding. Wang:Acerta Pharma BV: Employment, Equity Ownership. Huang:Acerta Pharma BV: Employment, Equity Ownership. Covey:Acerta Pharma BV: Employment, Equity Ownership, Patents & Royalties. Navarro:Acerta Pharma BV: Employment, Equity Ownership. Rothbaum:Acerta Pharma BV: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Izumi:Acerta Pharma: Employment, Equity Ownership, Patents & Royalties. Hamdy:Acerta Pharma BV: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Furman:Gilead: Consultancy; Acerta Pharma BV: Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Honoraria, Speakers Bureau.

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Variable Bruton Tyrosine Kinase (BTK) Resynthesis across Patients with Chronic Lymphocytic Leukemia (CLL) on Acalabrutinib Therapy Affect Target Occupancy and Reactivation of B-Cell Receptor (BCR) Signaling

Blood ◽

10.1182/blood-2018-99-110391 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4401-4401 ◽

Cited By ~ 2

Author(s):

Anfal A. Alsadhan ◽

Jean Cheung ◽

Michael Gulrajani ◽

Erika M. Cook ◽

Stefania Pittaluga ◽

...

Keyword(s):

Peripheral Blood ◽

De Novo ◽

Cell Receptor ◽

Lymphocytic Leukemia ◽

Synthesis Rate ◽

De Novo Synthesis ◽

Employment Equity ◽

Blood Samples ◽

Bcr Signaling ◽

Equity Ownership

Abstract Inhibitors of B-cell receptor (BCR) signaling, in particular of Bruton tyrosine kinase (BTK), are effective in patients with chronic lymphocytic leukemia (CLL). Acalabrutinib is a highly selective, covalent BTK inhibitor with rapid absorption and fast elimination (Barf et al., J Pharmacol Exp Ther 2017; 363:240-252). In a previous clinical trial of patients with relapsed/refractory CLL, acalabrutinib therapy was well tolerated and efficacious and had high target occupancy (Byrd et al., N Engl J Med 2016; 374:323-21). Here, we report on BTK occupancy, BTK resynthesis rates, and the relationship between BTK occupancy and BCR signaling in patients with CLL enrolled in a single center, phase 2 study of acalabrutinib monotherapy (NCT02337829). Patients were randomized to receive either 200 mg of acalabrutinib once daily (QD) or 100 mg twice daily (BID). The percentage of BTK bound by acalabrutinib (target occupancy) was measured with the aid of a biotin-tagged analogue probe. After 3 days of dosing, the median BTK occupancy in peripheral blood at peak (4 hours postdose) was 99% in both dosing groups (n=21 per group). However, at the drug trough time points (12 hours for BID; 24 hours for QD), median occupancy was greater in the BID than QD group (95% vs 87%, respectively; P<0.0001), with less interpatient variance. As acalabrutinib binds covalently to BTK, reactivation of the pathway requires de novo synthesis of BTK. We estimated the rate of BTK synthesis in vivo by measuring the accumulation of free BTK (% unbound by acalabrutinib) in serial blood samples taken 4, 12, 24, 36, and 48 hours postdose during a preplanned dose interruption on days 4 and 5. BTK was synthesized at a median rate of 13.4% per day, which was highly variable (fast vs slow) across patients (Figure). Interestingly, we found the median BTK de novo synthesis rate to be roughly twice as fast as observed in B cells from healthy volunteers (P<0.0001). BTK occupancy levels between matched peripheral blood and lymph node samples in individual patients were highly correlated (R=0.83; P<0.0001), with less than a 3% difference overall, suggesting a similar BTK synthesis rate in the lymph nodes and peripheral blood of patients with CLL. Similar results were observed for matched bone marrow and peripheral blood samples. We next correlated BTK occupancy levels and activity of BCR signaling in 10 representative patients. On Day 3, four hours after receiving the drug, significant reductions were observed in phospho-BTK (P=0.05), pNF-κB (P=0.03), and CD69 expression (P=0.001) compared with pretreatment. Next, we evaluated the change in BCR signaling from trough (for each dose group) to 36 (BID) or 48 (QD) hours after dosing (i.e., during the short window of drug withholding). A trend for increased signaling was driven by individual patients with low occupancy (≲70%). To mimic microenvironmental activation, we stimulated overnight with anti-IgM, all the peripheral blood samples collected during drug withholding (4 to 48 hours postdose). We found that upon BCR activation, B-CLL cells showed the ability to reactivate BCR signaling as measured by an increase in CD69 expression. Moreover, the extent of reactivation in BCR signaling positively correlated with the percentage of free BTK (R=0.73; P=0.0001). In conclusion, higher target coverage was achieved when acalabrutinib was administered 100 mg BID compared with 200 mg QD. Given that BTK de novo synthesis rates do vary across patients with CLL, and that reactivation of BCR signaling correlates with lower occupancy (higher free BTK), BID dosing provides the highest target coverage for a greater number of patients. This work was supported by the Intramural Research Program of National Heart, Lung, and Blood Institute, the National Institutes of Health, and Acerta Pharma. We thank our patients for donating blood and tissue samples to make this research possible. Figure. Figure. Disclosures Cheung: Acerta Pharma: Employment, Equity Ownership; AstraZeneca: Equity Ownership. Gulrajani:Acerta Pharma: Employment, Equity Ownership. Davies-Hill:NIH/NCI: Employment. Izumi:Acerta Pharma: Employment, Equity Ownership, Patents & Royalties: Acerta Pharma, various patents for ACP-196. Covey:AstraZeneca: Equity Ownership; Acerta Pharma: Employment. Wiestner:Pharmacyclics LLC, an AbbVie Company: Research Funding.

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Development of Molecular Mutations during the Evolution of Therapy-Related MDS and Therapy-Related AML

Blood ◽

10.1182/blood.v124.21.4624.4624 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4624-4624

Author(s):

Wolfgang Kern ◽

Manja Meggendorfer ◽

Tamara Alpermann ◽

Andreia de Albuquerque ◽

Claudia Haferlach ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Gene Panel ◽

Employment Equity ◽

Time Points ◽

Number Of Patients ◽

Patients At Risk ◽

Previously Treated ◽

Equity Ownership ◽

Time Point

Abstract Introduction: Therapy-related myelodysplastic syndrome (t-MDS) and acute myeloid leukemia (t-AML) develop after the application of chemotherapy for malignancies in a significant number of patients (pts). Mutations in TP53 have been described recently to be present even before chemotherapy for the prior malignancy and thus also before any sign of t-MDS or t-AML. Data suggested that chemotherapy selected the TP53mutated clone which evolved to t-MDS/t-AML. More comprehensive genetic analyses, however, have been lacking so far. Aim: To identify molecular mutations by a comprehensive gene panel in pts at t-MDS/t-AML diagnosis and to backtrack them to prior time points. Patients and Methods: We searched our database for pts diagnosed with t-MDS or t-AML for whom in addition ≥1 prior peripheral blood or bone marrow sample from assessment of a previously treated malignancy was stored. Diagnosis of t-MDS and t-AML was performed by cytomorphology, cytochemistry and cytogenetics according to WHO classification 2008 in all cases. A total of 11 pts were identified (3/8 females/males; median age at t-MDS/t-AML diagnosis 72 years, range 50-81 years). 8 pts had t-MDS and 3 had t-AML. All pts had received chemotherapy for CLL before. All pts underwent mutation analysis at t-MDS/t-AML diagnosis by a 26 gene panel targeting ASXL1, BCOR, BRAF, CBL, DNMT3A, ETV6, EZH2, FLT3-TKD, GATA1, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NPM1, NRAS, PHF6, RUNX1, SF3B1, SRSF2, TET2, TP53, U2AF1, and WT1. The library was generated with the ThunderStorm (RainDance Technologies, Billerica, MA) and sequenced on MiSeq instruments (Illumina, San Diego, CA). Specific mutations identified at t-MDS/t-AML diagnosis were selectively analyzed in prior samples of the respective patients. Mutations were considered for this analysis only if they were present at t-MDS/t-AML diagnosis at mutation loads clearly higher than residual CLL infiltration. Accordingly, mutations were excluded from this analysis if their load was in the range of residual CLL infiltration or lower. One not yet described genetic variant was also excluded. Results: 13 mutations were identified at t-MDS/t-AML diagnosis in 8/11 pts. While in 3 pts no mutations were found, 5 pts had 1 mutation, 2 had 2, and 1 had 4 mutations. Mean number of mutations per pt was 1.6. TP53 was mutated most frequently (n=5), RUNX1 was mutated in 2 pts, and FLT3-TKD, IDH2, KRAS, NPM1, NRAS, and U2AF1 in 1 pt each. Mean mutation load was 27% (range 4-48%) while mean CLL infiltration at the same time point was 2% (range 0-4%). Thus, the attribution of the described mutations to t-MDS/t-AML is highly likely. We then analyzed a total of 13 samples (8 bone marrow, 5 peripheral blood) drawn prior to t-MDS/t-AML diagnosis from the 8 pts for the respective mutations identified at t-MDS/t-AML diagnosis. In 5/8 patients the respective specific mutations identified at t-MDS/t-AML diagnosis were found in at least one prior sample. Genes found mutated in the prior samples were TP53 in 2 cases and IDH2, KRAS, NPM1, RUNX1, and U2AF1 in 1 case each. Mutation loads in general were lower in prior samples as compared to samples at t-MDS/t-AML diagnosis (median 54-fold lower, range 1.5 to 205-fold), except for one sample with a similar load at both time points which both times was clearly higher than the residual CLL infiltration (50% and 42% vs. 9% and 4%). Specifically, in 3/4 patients with samples available from the time point of CLL diagnosis all of these mutations (n=4) were not detectable at a sensitivity level of 1% while in 1 patient 2 mutations were not detectable and a U2AF1mutation was identified with a 1.9% load. This further supports the concept of these mutations being related to a pre-malignant clone which in the majority of cases might have been present at undetectable levels at the time point of CLL diagnosis or which even developed only during chemotherapy and later evolved into t-MDS/t-AML. The mean interval from first detection of the respective mutations to t-MDS/t-AML diagnosis was 10 months (range 4-25 months). Conclusions: Mutational screening applying a 26 gene panel identified molecular mutations in the majority of pts. These mutations were present up to 2 years before t-MDS/t-AML diagnosis. Further studies focusing on patients at risk of t-MDS/t-AML should clarify the role of early molecular screening helping to potentially improve diagnosis and management of t-MDS/t-AML. Disclosures Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. de Albuquerque:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Mechanisms of Adaptation to Ibrutinib in High Risk Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood-2018-99-116739 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 585-585 ◽

Cited By ~ 4

Author(s):

Valeria Spina ◽

Gabriela Forestieri ◽

Antonella Zucchetto ◽

Alessio Bruscaggin ◽

Tamara Bittolo ◽

...

Keyword(s):

High Risk ◽

Chronic Lymphocytic Leukemia ◽

Nuclear Localization ◽

Peripheral Blood ◽

Research Funding ◽

Clonal Evolution ◽

Lymphocytic Leukemia ◽

Adaptation Process ◽

Over Time ◽

Stimulation Of

Abstract Introduction. Ibrutinib inhibits the BTK molecule downstream the B-cell receptor (BCR). Though highly active in high risk chronic lymphocytic leukemia (CLL), the most typical response achievable in patients is a minimal residual disease (MRD) positive partial remission (PR) which is maintained until the development of genetically driven resistance caused by the acquisition of mutations in the BTK or PLCG2 genes. The study aims at characterizing the adaptation process allowing residual CLL cells to persist despite BTK inhibition. Methods. The IOSI-EMA-001 study (NCT02827617) is an observational study consisting in the prospective and longitudinal collection of peripheral blood samples and clinical data from high risk CLL patients treated with ibrutinib. Peripheral blood CLL cells longitudinally drawn from patients before treatment start and at fixed timepoints under ibrutinib were monitored by: i) next generation flow cytometry approaches for changes in proliferation rate, surfaceome, and pathway activation; and ii) CAPP-seq targeted deep next generation (sensitivity ~10-3) for clonal evolution. Results. The study cohort comprised 31 high risk CLL patients, including 15 treatment naïve, 16 relapsed, 80% IGHV unmutated, 42% 17p deleted and 55% TP53 mutated. Median duration of ibrutinib treatment was 45 weeks (24-72 weeks). All patients obtained a MRD positive PR that was maintained in all but one who progressed with a PLCG2 mutation (VAF 3%). Compared to baseline, under ibrutinib therapy CLL cells slowed down their proliferation, as suggested by the decreased expression of Ki-67, the reduction of the proliferating fraction (CXCR4dimCD5bright), and the increase of the resting fraction (CXCR4brightCD5dim). Compared to baseline, under ibrutinib therapy CLL cells also upregulated BCR and adhesion/homing proteins, and decreased the expression of BCR inhibitor proteins. Upon stimulation of the BCR with anti-IgM, the downstream path through pBTK and pPLCG2 was inhibited by ibrutinib, while conversely the downstream path through pAKT and pERK was still inducible throughout all the assessed timepoints. The proportion of CLL cells harboring nuclear localization of NF-kB progressively increased over time under ibrutinib. NF-kB nuclear localization was inducible throughout all the assessed timepoints by CD40L stimulation of the non-canonical NF-kB pathway, but not by anti-IgM stimulation of the BCR/canonical NF-kB pathway. Overall, 880 individual mutations were longitudinally discovered and monitored across a total of 121 sequential timepoints collected during ibrutinib treatment. Clonal evolution was observed in (67.7%) cases, a proportion rate previously documented in CLL treated with chemoimmunotherapy. Clonal evolution appeared to be heterogeneous involving different genes without a stereotypic targeting. Consistently, none of the main driver gene mutations was homogeneously selected or suppressed by ibrutinib suggesting that the biological adaptation of CLL cells under ibrutinib is not genetically driven. Clonal evolution propensity was not associated with any of the biomarkers of the disease, and it did not decrease over time under ibrutinib. Conclusions. Taken together these results suggest that residual CLL cells persisting under ibrutinib therapy adapt their phenotype by upregulating adhesion molecules, chemokine receptors and BCR molecules, and by maintaining a competence of BCR signaling through the PI3K/AKT/ERK pathway. The progressive selection of CLL cells having NF-kB in the nucleus, likely due to the BTK independent non-canonical NF-kB pathway, might explain their survival despite ibrutinib therapy. Finally, clonal evolution is not suppressed by ibrutinib chemotherapy, and despite does not seem to be directly involved in such adaptation process, may ultimately favor the acquisition of BTK and PLCG2 ibrutinib resistance mutations. Disclosures Zucca: Celltrion: Consultancy; AstraZeneca: Consultancy. Ghia:Sunesis: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; AbbVie, Inc: Honoraria, Research Funding; Acerta: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; BeiGene: Honoraria, Research Funding. Montillo:Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Research Funding. Tedeschi:Janssen: Consultancy, Speakers Bureau; Gilead: Consultancy; AbbVie: Consultancy. Gaidano:AbbVie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Morphosys: Honoraria; Roche: Consultancy, Honoraria.

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Itolizumab As a Potential Therapeutic for the Prevention and Treatment of Graft Vs Host Disease

Blood ◽

10.1182/blood-2019-122787 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5603-5603 ◽

Cited By ~ 1

Author(s):

Cherie Tracy Ng ◽

Jeanette Ampudia ◽

Robert J. Soiffer ◽

Jerome Ritz ◽

Stephen Connelly

Keyword(s):

Weight Loss ◽

T Cells ◽

T Cell ◽

Peripheral Blood ◽

Research Funding ◽

Chronic Gvhd ◽

Vehicle Control ◽

Control Group ◽

Employment Equity ◽

Equity Ownership

Background: CD6 is a co-stimulatory receptor, predominantly expressed on T cells, that binds to activated leukocyte cell adhesion molecule (ALCAM), a ligand expressed on antigen presentation cells and various epithelial and endothelial tissues. The CD6-ALCAM pathway plays an integral role in modulating T cell activation, proliferation, differentiation and trafficking and is central to inflammation. While effector T cell (Teff) are CD6hi and upregulate expression upon activation, regulatory T cells (Treg) remain CD6lo/-, making this an attractive target to modulate Teff activity while preserving Treg activity. Early studies by Soiffer and colleagues demonstrated using T12, an anti-CD6 monoclonal antibody (mAb) that ex-vivo depletion of CD6+ donor cells prior to transplantation decreased the incidence of both acute and chronic GVHD, highlighting the importance of CD6+ cells in GVHD pathogenesis and validating it as a therapeutic target. However, it remains to be shown whether modulating the CD6-ALCAM pathway in vivo can attenuate GVHD. We investigated the use of itolizumab, a humanized anti-CD6 mAb that has demonstrated clinical efficacy in other autoimmune diseases, as both a preventive and therapeutic treatment for GVHD, using a humanized xenograft mouse model. Methods: Humanized xenograft mice were generated by intravenous transfer of 2x10^7 human PBMCs into 6-8 weeks old NOD/SCID IL2rγ-null (NSG). To investigate the ability of itolizumab to prevent GVHD, mice were dosed with either 60μg or 300μg of itolizumab, 150μg of abatacept (CTLA4-Ig), or vehicle, starting one day prior to PBMC transplantation. To investigate the therapeutic effect of itolizumab, mice were dosed with either 150μg of itolizumab or vehicle, starting at Day 5 post-PBMC transfer, when transplanted T cells are already activated. All treatments were administered IP every other day. Weight and disease scores were monitored throughout the study. At Days 18 and 35, peripheral blood was evaluated by flow cytometry to examine T cell prevalence, and tissues were collected for histological examination of pathology and T cell infiltration. Results: When administered as prevention (Day -1), treatment with either 60μg or 300μg of itolizumab significantly decreased mortality compared to the vehicle control (100% vs. 10%); this decrease was similar to the positive control group treated with abatacept (Figure 1). At 60μg, itolizumab-treated mice demonstrated significant reductions in the prevalence of human T cells in peripheral blood vs. vehicle-treated mice at Day 18 (<0.2% vs. 74.5%; p < 0.001). The reduction in peripheral T cells was accompanied by reductions in tissue-infiltrating T cells in lung (85-fold) and gut (9.5-fold), as well as reductions in disease scores and weight loss. When administered therapeutically, treatment with itolizumab was associated with a survival rate of 50% compared to 10% in the control group (Figure 2). Similarly, peripheral T cell prevalence (34.3% vs. 65.1%; p < 0.001), weight loss, and disease scores were inhibited by itolizumab compared to vehicle control mice. Conclusions: These data suggest that systemic treatment with itolizumab can modulate pathogenic Teff cell activity, establishing this antibody as a potential therapeutic for patents with GvHD. A phase I/II study using itolizumab as first line treatment in combination with steroids for patients with aGVHD is currently ongoing (NCT03763318). Disclosures Ng: Equillium: Employment, Equity Ownership. Ampudia:Equillium: Employment. Soiffer:Mana therapeutic: Consultancy; Kiadis: Other: supervisory board; Gilead, Mana therapeutic, Cugene, Jazz: Consultancy; Juno, kiadis: Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Cugene: Consultancy; Jazz: Consultancy. Ritz:Equillium: Research Funding; Merck: Research Funding; Avrobio: Consultancy; TScan Therapeutics: Consultancy; Talaris Therapeutics: Consultancy; Draper Labs: Consultancy; LifeVault Bio: Consultancy; Celgene: Consultancy; Aleta Biotherapeutics: Consultancy; Kite Pharma: Research Funding. Connelly:Equillium: Employment, Equity Ownership.

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Duohexabody-CD37, a Novel Bispecific Antibody with a Hexamerization-Enhancing Mutation Targeting CD37, Demonstrates Superior Complement-Dependent Cytotoxicity in Preclinical B-Cell Malignancy Models

Blood ◽

10.1182/blood-2018-99-114723 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4170-4170

Author(s):

Simone C. Oostindie ◽

Hilma J. Van Der Horst ◽

Marije B. Overdijk ◽

Kristin Strumane ◽

Sandra Verploegen ◽

...

Keyword(s):

B Cell ◽

Research Funding ◽

Bispecific Antibody ◽

Lymphocytic Leukemia ◽

Single Point Mutation ◽

B Cell Malignancies ◽

Employment Equity ◽

Healthy Human ◽

Complement Dependent Cytotoxicity ◽

Equity Ownership

Abstract CD37 is a tetraspanin plasma membrane protein abundantly expressed on B-cells and represents a promising therapeutic target for the treatment of B-cell malignancies. Although complement-dependent cytotoxicity (CDC) has proven to be a powerful Fc-mediated effector function for killing hematological cancer cells, CD37 antibody-based therapeutics currently in clinical development are poor inducers of CDC. Here we present DuoHexaBody-CD37, a novel humanized IgG1 bispecific antibody targeting two different CD37 epitopes, with an E430G hexamerization-enhancing mutation, for the potential treatment of B-cell malignancies. The natural process of antibody hexamer formation through intermolecular Fc-Fc interactions between IgG molecules after cell surface antigen binding can be improved by introducing a single point mutation such as E430G in the IgG Fc domain, thereby facilitating more efficient C1q binding and complement activation (Diebolder et al., Science 2014; de Jong et al., PLoS Biol 2016). The hexamerization-enhancing mutation E430G was introduced into two humanized CD37 monoclonal antibodies (mAbs) that bind non-overlapping CD37 epitopes. Different antibody formats and combinations, including the single antibodies, combinations of the mAbs and bispecific mAbs were tested for their capacity to induce CDC and antibody-dependent cellular cytotoxicity (ADCC). The bispecific hexamerization-enhanced antibody variant DuoHexaBody-CD37, showed superior CDC activity compared to the single hexamerization-enhanced mAbs and the combination thereof, both in vitro over a range of different B-cell lines, and ex vivo in tumor cell samples obtained from patients with chronic lymphocytic leukemia (CLL). In a CDC assay using tumor cells obtained from a relapsed/refractory CLL patient who received prior treatment with rituximab, ibrutinib and idelalisib, DuoHexaBody-CD37 induced almost complete lysis (84% lysis at a concentration 100 µg/mL), thereby outperforming the single HexaBody molecules (15% and 23% lysis) and the combination (57%) (Figure 1). In addition to its potent CDC activity, DuoHexaBody-CD37 was also capable of inducing potent ADCC of Daudi cells (EC50 = 12.3 ± 9.5 ng/mL), as assessed using peripheral blood mononuclear cells from 8 healthy human donors in a standard chromium release assay. In assays using whole blood from 6 healthy human donors, DuoHexaBody-CD37 showed efficient B-cell binding and potent and specific depletion of the B-cell population (98% ± 1.3% depletion at 10 µg/mL, EC50 = 0.85 ± 0.284 µg/mL). Furthermore, DuoHexaBody-CD37 induced significant inhibition of tumor growth in vivo in Daudi-luc Burkitt's lymphoma and JVM-3 CLL mouse xenograft models, at doses as low as 0.1 and 1 mg/kg (p<0.05), respectively. In summary, we present a novel therapeutic antibody that, for the first time, combines proprietary DuoBody® and HexaBody® platforms. DuoHexaBody-CD37 induced highly potent CDC and efficient ADCC in preclinical models, suggesting that DuoHexaBody-CD37 may serve as a potential therapeutic mAb for the treatment of human B-cell malignancies. Disclosures Oostindie: Genmab: Employment, Equity Ownership. Van Der Horst:Genmab: Research Funding. Overdijk:Genmab: Employment, Equity Ownership. Strumane:Genmab: Employment, Equity Ownership. Verploegen:Genmab: Employment, Equity Ownership. Lindorfer:Genmab: Research Funding. Cook:Genmab: Research Funding. Chamuleau:Gilead: Research Funding; BMS: Research Funding; celgene: Research Funding; Genmab: Research Funding. Mutis:Gilead: Research Funding; Celgene: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genmab: Research Funding; Novartis: Research Funding; OnkImmune: Research Funding. Schuurman:Genmab: Employment, Other: Warrants. Sasser:Genmab: Employment, Equity Ownership. Taylor:Genmab: Research Funding. Parren:Genmab: Equity Ownership; Lava Therapeutics: Employment. Beurskens:Genmab: Employment, Equity Ownership. Breij:Genmab: Employment, Equity Ownership.

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