We report the results of a multicenter phase 1 dose-escalation study of the selective Bruton tyrosine kinase (BTK) inhibitor ONO/GS-4059 in 90 patients with relapsed/refractory B-cell malignancies. ...There were 9 dose-escalation cohorts ranging from 20 mg to 600 mg once daily with twice-daily regimens of 240 mg and 300 mg. Twenty-four of 25 evaluable chronic lymphocytic leukemia (CLL) patients (96%) responded to ONO/GS-4059, with a median treatment duration of 80 weeks; 21 CLL patients remain on treatment. Lymph node responses were rapid and associated with a concurrent lymphocytosis. Eleven of 12 evaluable patients with mantle cell lymphoma (92%) responded (median treatment duration, 40 weeks). Eleven of 31 non–germinal center B-cell diffuse large B-cell lymphoma patients (35%) responded but median treatment duration was 12 weeks due to development of progressive disease. ONO/GS-4059 was very well tolerated with 75% of adverse events (AEs) being Common Toxicity Criteria for Adverse Events version 4.0 grade 1 or grade 2. Grade 3/4 AEs were mainly hematologic and recovered spontaneously during therapy. One CLL patient experienced a grade 3 treatment-related bleeding event (spontaneous muscle hematoma) but no clinically significant diarrhea, cardiac dysrhythmias, or arthralgia were observed. No maximal tolerated dose (MTD) was reached in the CLL cohort. In the non-Hodgkin lymphoma cohort, 4 patients developed a dose-limiting toxicity, yielding an MTD of 480 mg once daily. ONO/GS-4059 has significant activity in relapsed/refractory B-cell malignancies without major drug-related toxicity. The selectivity of ONO/GS-4059 should confer advantages in combination therapies. This trial was registered at www.clinicaltrials.gov as #NCT01659255.
•We report a first-in-human dose-escalation study in relapsed/refractory B-cell malignancies with the potent BTK inhibitor ONO/GS-4059.•ONO/GS-4059 induced clinically durable responses in relapsed/refractory B-cell malignancies without significant toxicities.
We sought to determine whether nucleolin, a bcl-2 mRNA-binding protein, has a role in the regulation of bcl-2 mRNA stability in MCF-7 and MDA-MB-231 breast cancer cells. Furthermore, we examined the ...efficacy of the aptamer AS1411 in targeting nucleolin and inducing bcl-2 mRNA instability and cytotoxicity in these cells. AS1411 at 5 micromol/L inhibited the growth of MCF-7 and MDA-MB-231 cells, whereas 20 micromol/L AS1411 had no effect on the growth rate or viability of normal MCF-10A mammary epithelial cells. This selectivity of AS1411 was related to a greater uptake of AS1411 into the cytoplasm of MCF-7 cells compared with MCF-10A cells and to a 4-fold higher level of cytoplasmic nucleolin in MCF-7 cells. Stable siRNA knockdown of nucleolin in MCF-7 cells reduced nucleolin and bcl-2 protein levels and decreased the half-life of bcl-2 mRNA from 11 to 5 hours. Similarly, AS1411 (10 micromol/L) decreased the half-life of bcl-2 mRNA in MCF-7 and MDA-MB-231 cells to 1.0 and 1.2 hours, respectively. In contrast, AS1411 had no effect on the stability of bcl-2 mRNA in normal MCF-10A cells. AS1411 also inhibited the binding of nucleolin to the instability element AU-rich element 1 of bcl-2 mRNA in a cell-free system and in MCF-7 cells. Together, the results suggest that AS1411 acts as a molecular decoy by competing with bcl-2 mRNA for binding to cytoplasmic nucleolin in these breast cancer cell lines. This interferes with the stabilization of bcl-2 mRNA by nucleolin and may be one mechanism by which AS1411 induces tumor cell death.
AS1411 is a DNA aptamer that is in phase II clinical trials for relapsed or refractory acute myeloid leukemia and for renal cell carcinoma. AS1411 binds to nucleolin, a protein that is overexpressed ...in the cytoplasm and on the plasma membrane of some tumor cells compared with normal cells. Studies were performed to determine whether cell surface nucleolin is a receptor for AS1411 in the acute myeloid leukemia cell line MV4-11. Biotinylation of MV4-11 cell surface proteins followed by immunoblotting of the biotinylated proteins showed that full-length (106 kDa) and truncated forms of nucleolin were present on the cell surface. In contrast, K-562 cells, which are 4-fold less sensitive than MV4-11 cells to AS1411, showed no full-length nucleolin and lesser amounts of the truncated forms of nucleolin on the cell surface. Incubation of MV4-11 cells with (32)PAS1411 and immunoprecipitation of the plasma membrane fraction with anti-nucleolin antibody demonstrated the presence of (32)PAS1411-nucleolin complexes. Anti-nucleolin antibody inhibited binding of fluorescein isothiocyanate (FITC)-AS1411 to plasma membrane nucleolin 56 +/- 10% SE (P < 0.01) compared with cells incubated with FITC-AS1411 only. Cellular uptake of (32)PAS1411 into MV4-11 cells was blocked by a 20-fold excess of unlabeled AS1411 but not by a 20-fold excess of the biologically inactive oligonucleotide CRO-26. Uptake was approximately 3-fold faster into MV4-11 cells than into K-562 cells. Partial knockdown of plasma membrane and cytosolic nucleolin in MCF-7 cells resulted in a 3-fold decrease in AS1411 uptake. These results provide evidence that plasma membrane nucleolin is a functional receptor for AS1411 in MV4-11 cells.
MUC1 is a cell-surface glycoprotein that establishes a molecular barrier at the epithelial surface and engages in morphogenetic signal transduction. Alterations in MUC1 glycosylation accompany the ...development of cancer and influence cellular growth, differentiation, transformation, adhesion, invasion, and immune surveillance. A 20-amino-acid tandem repeat that forms the core protein of MUC1 is overexpressed and aberrantly glycosylated in the majority of epithelial tumors. AS1402 (formerly R1550) is a humanized IgG1k monoclonal antibody that binds to PDTR sequences within this tandem repeat that are not exposed in normal cells. AS1402 is a potent inducer of antibody-dependent cellular cytotoxicity (ADCC), specifically against MUC1-expressing tumor cells. The objective of this study was to determine the safety, tolerability, and pharmacokinetic (PK) characteristics of AS1402 monotherapy in patients with locally advanced or metastatic MUC1-positive breast cancer that had progressed after anthracyclines- and taxane-based therapy.
Patients received AS1402 over a 1- to 3-hour intravenous (i.v.) infusion at doses between 1 and 16 mg/kg, with repeated dosing every 1 to 3 weeks (based on patient-individualized PK assessment) until disease progression. Serum AS1402 levels were measured at multiple times after i.v. administration. Human anti-human antibody (HAHA) responses were measured to determine the immunogenicity of AS1402. Noncompartmental pharmacokinetic parameters were determined and were used to assess dose dependency across the dose range studied.
Twenty-six patients were treated. AS1402 was generally well tolerated. Two grade 3/4 drug-related adverse events were reported, both at the 3-mg/kg dose. Neither was observed in expanded or subsequent dosing cohorts. No anti-human antibodies were detected. Plasma concentrations of AS1402 appeared to be proportional to dose within the 1- to 16-mg/kg dose range assessed, with a mean terminal half-life of 115.4 +/- 37.1 hours.
Repeated iv administration of AS1402 was well tolerated, with a maximum tolerated dose (MTD) exceeding 16 mg/kg, the highest dose administered in this study. The half-life and exposure of AS1402 were such that weekly dosing could achieve plasma concentrations corresponding to the maximal ADCC activity observed in vitro. A phase II study is ongoing to evaluate the clinical activity of AS1402 in patients with advanced breast cancer.
ClinicalTrials.gov Identifier: NCT00096057.
Proteolytic enzymes have been implicated in driving tumor progression by means of their cancer cell microenvironment activity where they promote proliferation, differentiation, apoptosis, migration, ...and invasion. Therapeutic strategies have focused on attenuating their activity using small molecule inhibitors, but the association of proteases with the cell surface during cancer progression opens up the possibility of targeting these using antibody dependent cellular cytotoxicity (ADCC). Cathepsin S is a lysosomal cysteine protease that promotes the growth and invasion of tumour and endothelial cells during cancer progression. Our analysis of colorectal cancer patient biopsies shows that cathepsin S associates with the cell membrane indicating a potential for ADCC targeting.
Here we report the cell surface characterization of cathepsin S and the development of a humanized antibody (Fsn0503h) with immune effector function and a stable in vivo half-life of 274 hours. Cathepsin S is expressed on the surface of tumor cells representative of colorectal and pancreatic cancer (23%-79% positive expression). Furthermore the binding of Fsn0503h to surface associated cathepsin S results in natural killer (NK) cell targeted tumor killing. In a colorectal cancer model Fsn0503h elicits a 22% cytotoxic effect.
This data highlights the potential to target cell surface associated enzymes, such as cathepsin S, as therapeutic targets using antibodies capable of elicitingADCC in tumor cells.
Integrins are cell-surface glycoproteins found in different forms on all cells except erythrocytes. Integrins bind to cell adhesion molecules and to proteins found in the extracellular matrix. A ...tripeptidic sequence Arg-Gly-Asp (RGD) is often the primary site of recognition by integrins which are expressed on tumour cells and are responsible for tumour invasion and metastasis. A synthetic decapeptide designated alpha P2 containing two RGD sequences radiolabelled with technetium-99m was used to image malignant melanoma in vivo. Fourteen patients previously diagnosed with metastatic melanoma underwent gamma camera imaging 20-180 min following intravenous administration of the radiolabelled synthetic decapeptide alpha P2. Six out of eight (6/8) of the lymph node metastases (75%) and all other neoplastic sites (11 sites) were successfully imaged, with the exception of three sites in the mediastinal area which were not positively imaged. In two cases there was false positive uptake in the rounded pigmented areolar/nipple area. In three cases (seven sites) the peptide scan confirmed the absence of disease in suspected lesions (true-negative). The synthetic peptide was rapidly removed from the circulation by filtration through the kidneys and excretion in the urine. No toxicity or adverse events were recorded. Radiolabelled alpha P2 peptide, which binds specifically to adhesion molecules on tumours, can be used for the in vivo detection of neoplastic metastases.
ED-B fibronectin (FN) is a FN isoform derived from alternative splicing of the primary transcript of a single gene. Its expression on tumor stroma and neoformed tumor vasculature and its absence, ...with few exceptions, in normal adult tissues imply a prognostic and diagnostic value for ED-B FN. We investigated the location and source of ED-B FN because this will be of importance both in understanding its role in tumor development and in designing strategies to target this molecule. We have confirmed that ED-B FN is expressed in the majority of breast and colorectal carcinoma tissue samples, with strong immunohistochemical staining around the tumor cells and in the tumor stroma. No staining of tumor neovasculature was seen. ED-B FN is produced by a range of tumor and endothelial (both primary and transformed) cell lines, as detected by reverse transcription-PCR, but is not expressed at the plasma membrane. Strong expression of human ED-B FN is seen in tumor xenografts. These data indicate that neoplastic cells can act as the source of ED-B FN in tumors. The lack of cell surface expression on tumor cell lines has clear implications for the design of therapeutic strategies which target this molecule.
► A single-chain Fv can be successful constructed from a humanized BC-1 immunoglobulin. ► The scFv (HuBC-1) is more stable the its murine counterpart. ► The HuBC-1 scFv retains full binding function ...by ELISA, BIACore analyses and flow cytometry. ► The HuBC-1 scFv is stable and localizes to tumours in an animal model.
Tumour-associated splice variants of fibronectin are a major source of tumour-matrix associated targets and are proving very successful in the development of clinical agents to treat cancer. One of the first monoclonal antibodies to be produced to this target, murine BC-1, recognises a cryptic epitope in domain 7 of the B-form splice variant (EDB-FN). Antibody fragments based on this immunoglobulin (IgG) were unstable, but BC-1 humanisation provided an opportunity to produce a more stable single-chain Fv (scFv). The variable domains of the humanized BC-1 IgG were sub-cloned and constructed into a scFv (HuBC-1 scFv) which was successfully expressed in Escherichia coli. The scFv retained its conformationally-sensitive epitope recognition and demonstrated a good affinity to the target of around 50nM as measured by ELISA, Surface Plasmon Resonance and Flow Cytometry. Furthermore, the scFv was thermostable and stable in serum allowing substantial localisation to human tumours grown in mouse xenograft models. This scFv could form the basis of future tumour-specific biopharmaceuticals.
Introduction
Bruton’s tyrosine kinase (BTK) is a critical kinase involved in B-cell receptor signal transduction. ONO-4059, a highly potent and selective oral BTK inhibitor has demonstrated ...anti-tumour activity in pre-clinical models (Yasuhiro et al, AACR2013) and in the clinic in both CLL and NHL patients (Morschhauser et al, EHA 2014; Rule et al, EHA 2014). Here, we present data from 25 CLL patients enrolled in the ongoing Phase I study ONO-4059POE001.
Methods
Twenty five CLL patients were administered ONO-4059 as monotherapy, given once daily (QD) to determine safety, pharmacokinetics and pharmacodynamics, and preliminary efficacy. Patients can receive ONO-4059 for up to a maximum of 3 years and upon completion of the first 6 months of treatment, intra-patient dose escalation is permitted. Here, we present data for 25 evaluable patients treated with ONO-4059 at doses ranging from 20-600mg (8 cohorts).
Patients had a median age 67 yrs range 40-79, median baseline tumour burden 3,943 mm2 461-19,750 mm2, median of 4 prior therapies 2-8, including fludarabine (23/25) and rituximab-containing therapy (23/25). Baseline median haematology parameters included lymphocytes 34.6 x 109/L 0.1-391, haemoglobins 11.1 g/dL 8.5-15.2, platelets 97 x 109/L 23-185, neutrophils 2.39 x 109/L 0.5-11.1. 9/24 patients were 17p deleted 38% and 6/24 had the 11q deletion 25%, 20/24 were IgVH unmutated status 83%, with 13/24 54% displaying TP53 mutation).
Results
To date, 19 of 25 patients remain on treatment with a median duration of treatment of 363 days 27-659. ONO-4059 was found to be well tolerated with few adverse reactions over a long duration. A total of sixty two ONO-4059-related Grade 1- 2 adverse events were reported in 19 out of 25 patients. Eight dose independent ONO-4059-related G3 or G4 haematological toxicities were reported in 5 patients; with neutropenia in 3 patients, G3 x 1, G4 x 2), febrile neutropenia in 1 patient G3 and leucopenia in 2 patients G3 x 4. Six Grade 3 ONO-4059-related non-haematological events were reported (pyrexia x 1, Hepatitis E reactivation x 1, Lymphocytic infiltration, Purpura, Urinary tract infection and Haematoma in just one patient). Nine ONO-4059-related SAEs were reported in 6 patients (febrile neutropenia G3 and pyrexia G3 at 20 mg, rash G2 at 80 mg, hepatitis E reactivation G3 at 320mg, neutropenia G4 at 320 mg, purpura x 2 G2, G3 and lymphocytic infiltration G3 at 400 mg, haematoma G2 at 500mg).
All patients experienced rapid reductions in lymphadenopathy observed early in treatment between Cycle1 and Cycle3, accompanied by an increase in absolute lymphocyte count in 72% of patients. Almost all patients have durable response over a long duration. Best overall response rate according to IWCLL criteria (including modified PR with lymphocytosis) was 84% based on CT-scan and P/E for 21/25 evaluable patients with 17 PR, 4 PR with lymphocytosis (for 21 responding patients, median reduction of lymph nodes was 83% 51-100), 1 SD and 2 PD, with 89% response rate on 17p deleted. The early tumour shrinkage was associated with a rise in haemoglobin and platelet count. An increase in exposure (as assessed by Cmax and AUC) that was proportional to an increase in dose was seen for all doses of ONO-4059 ranging from 20-600mg.
Conclusion
ONO-4059 is a highly potent and selective oral Btk inhibitor with a favourable safety profile along with promising efficacy over a long duration in this heavily pre-treated population, with responses observed in relapsed, refractory and currently poor prognostic 17p deleted patients.
Fegan:ONO PHARMA: Honoraria. Salles:celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees; roche: Honoraria, Membership on an entity’s Board of Directors or advisory committees; genetech: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Karlin:Janssen: Honoraria; celgene: Consultancy, Honoraria; Sandoz: Consultancy. Rule:Pharmacyclics, J&J: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Morschhauser:ONO PHARMA: Honoraria; Roche: Honoraria; Celgene: Honoraria; Mundipharma: Honoraria. Dyer:ONO PHARMA: Research Funding. Cartron:ONO PHARMA: Honoraria. Honda:Ono Pharmaceutical Co., Ltd.: Employment. Yasuhiro:Ono Pharmaceutical Co., Ltd.: Employment. Yoshizawa:Ono Pharmaceutical Co., Ltd.: Employment.
PDF
