Anti-Tumour Efficacy Study Of The Bruton’s Tyrosine Kinase (Btk) Inhibitor, ONO-4059, In Combination With The Glycoengineered Type II Anti-CD20 Monoclonal Antibody, Obinutuzumab (GA101) Demonstrates Superior In Vivo Efficacy Compared To ONO-4059 In Combination With Rituximab

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3069-3069
Author(s):  
Toshio Yoshizawa ◽  
Tomoko Yasuhiro ◽  
Joseph TP Birkett ◽  
Christian Klein ◽  
Kazuhito Kawabata
Keyword(s):  
B Cell ◽  
Xenograft Model ◽  
The Novel ◽  
Type Ii ◽  
Anti Tumour Activity ◽  
Btk Inhibitor ◽  
Anti Cd20 ◽  
Tumour Activity ◽  
Tumor Remission

Abstract Purpose The activated B-cell-like (ABC) sub-type of diffuse large B-cell lymphoma (DLBCL) correlates with poor prognosis. There is still a high medical need for new therapies, preferably chemo-sparing, to help treat patients with ABC-DLBCL and CLL as well as other B-cell malignancies. The B-cell receptor signaling pathway is known to be dysregulated in NHL/CLL and given Btk is a downstream mediator of BCR signalling, Btk constitutes an interesting and obvious therapeutic target. Given the high potency and selectivity of the novel Btk inhibitor ONO-4059, it was hypothesized that, the anti-tumour activity of ONO-4059 could be further enhanced by combining it with the anti-CD20 Abs, Obinutuzumab (GA101) or Rituximab (RTX), as a novel therapeutic strategy. Methods TMD-8 tumour cells, a human ABC-DLBCL cell line, were implanted sub-cutaneously into female SCID mice. Randomization of mice occurred when mean tumour volume was 400-450 mm3. ONO-4059 was administered orally at a dose of 10 mg/kg bid, and GA101 or RTX were administered intravenously both at a sub-optimal dose of 3 mg/kg once weekly. Tumour volumes were measured twice a week after initiation of treatment, and tumour volumes were determined using the formula volume (= width2 x length)/2. Animals were euthanized when the tumours reached a maximum volume of 3,000 mm3. Results Treatment with ONO-4059 combined with either GA101 or RTX resulted in a significant inhibition of tumour growth in the TMD-8 xenograft model. GA101 monotherapy showed superior anti-tumour activity compared with RTX monotherapy in terms of tumor growth inhibition (TGI 78% vs 54% at day 21). ONO-4059 combined with GA101 or RTX at sub-optimal doses was significantly better than the respective Abs or ONO-4059 given as monotherapy with TGI of 90% for the GA101 combination and 86% for the rituximab combination. ONO-4059 as single agent mediated a TGI of 63%. In addition combination treatment with GA101 resulted in tumour remission in 3/10 animals, whereas combination treatment with rituximab resulted in tumor remission in only1/10 animals. GA101 monotherapy resulted in 1/10 tumor-free animals, whereas no tumor free animals were observed in the rituximab and ONO-4059 monotherapy groups. Conclusion Recent studies indicate that targeting Btk is effective in the treatment of B-cell malignancies. Our results demonstrate that treatment with ONO-4059 in combination with GA101 or rituximab results in a combined effect in vivo and is superior to the respective monotherapies. Furthermore, ONO-4059 in combination with the novel glycoengineered Type II CD20 antibody GA101 is more effective than the combination of ONO-4059 with rituximab in this DLBCL xenograft model. When interpreting these data it should be noted that both, obinutuzumab and rituximab were dosed at sub-optimal doses as they can induce tumor remission when dosed at 10 mg/kg. Taken together these data indicate that the combination of ONO-4059 with rituximab, and particularily obinutuzumab may be an effective treatment for ABC-DLBCL. Disclosures: Yoshizawa: Ono Pharmaceutical CO., Ltd: Employment. Yasuhiro:Ono Pharmaceutical Co., Ltd.: Employment. Birkett:Ono Pharma UK: Employment. Klein:Roche Glycart AG: Employment. Kawabata:Ono Pharmaceutical Co., Ltd.: Employment.

A Novel Heat Shock Protein (HSP) 90 Inhibitor KW-2478 shows Activity in B-Cell Malignancies in Vitro and in Vivo.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1625-1625
Author(s):  
Simone Juliger ◽  
Takayuki Nakashima ◽  
Lenushka Maharaj ◽  
Toshihiko Ishii ◽  
Hiroshi Nakagawa ◽  
...  
Keyword(s):  
Cell Growth ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Hsp90 Inhibitor ◽  
Anti Tumour Activity ◽  
Client Proteins ◽  
Tumour Activity

Abstract Background : HSP90 plays an important role in chaperoning key proteins implicated in malignant disease and is a promising therapeutic target. We now report the in vitro and in vivo activity of a novel HSP90 inhibitor of non-ansamycin, non-purine analogue class, KW-2478, (Kyowa Hakko Kirin) in B-cell malignancies including multiple myeloma (MM), B-cell lymphoma (BCL) and mantle cell lymphoma (MCL) cells, and in primary tumour cells from MM and BCL patients. Procedures: The binding affinity of KW-2478 to HSP90 was examined using immobilised human HSP90a and a biotinylated HSP90 binding agent, radicicol (bRD). The effect of KW-2478 on cell viability, cell growth and apoptosis induction were evaluated in cell lines, with KW-2478 induced changes in major HSP90 client proteins studied by Western blotting analysis. The in vivo anti-tumour activity of KW-2478 was evaluated in a human MM xenograft mouse model,. Primary MM cells were studied using a co-culture system with the HS-5 bone marrow stromal cell line (BMSCs), while primary BCL samples were cultured on CHO cells stably transfected to produce CD40L. Results: KW-2478 inhibited the binding of bRD to HSP90α in concentration-dependent manner with an IC50 value of 3.8 nM. KW-2478 clearly inhibited cancer cell growth in all cell lines, with EC50 values from 101–252 nM in BCL, 81.4–91.4 nM in MCL and 120–622 nM in MM. The drug also exhibited potent growth inhibitory activity in primary CLL (n=3) and NHL (n=2) cells with EC50 values of 40–170 nM and 200–400 nM, respectively. In 2 of 4 human primary myeloma cells, KW-2478 at 2 μM inhibited cell growth by at least 50%. The presence of BMSCs did not affect drug activity against primary MM cells and importantly there was little or no effect on cell number or viability of normal BMSCs at up to 20 μM KW-2478. Exposure of MM and BCL cell lines to KW-2478 for 24 hours resulted in the degradation of HSP90 client proteins (IGF-1Rβ and Raf-1) and the induction of HSP70. KW-2478 also induced PARP cleavage and dephosphorylated Erk1/2 in NCI-H929 cells. Further studies in selected cell lines showed that exposure to 1 μM KW-2478 or lower resulted in the depletion of p53 and Akt proteins, a reduction in nuclear NFKB, and the cleavage of caspase-3. In the NCI-H929 xenograft model, KW-2478 (qd×5, i.v.) showed a statistically significant suppression of tumour growth at the doses of 25, 50, 100 and 200 mg/kg. Moreover, tumour regressions were observed at doses of 100 and 200 mg/kg, with a significant decrease in serum M protein concentration at doses of 50, 100 and 200 mg/kg. No severe KW-2478 toxicity was observed as assessed by treatment-related mortality and body weight change. Conclusions: The novel HSP90 inhibitor KW-2478 showed a potent anti-tumour activity both in vitro and in vivo, including activity in primary patient samples. The agent retained its activity in primary myeloma cells in the presence of BMSCs, suggesting that KW-2478 can overcome the protective effect of the bone marrow microenvironment. Additional pharmacokinetic and safety data support the further development of KW-2478 and the drug is currently undergoing clinical evaluation in a phase I trial.

scholarly journals Novel type II anti-CD20 monoclonal antibody (GA101) evokes homotypic adhesion and actin-dependent, lysosome-mediated cell death in B-cell malignancies

Blood ◽  
2011 ◽  
Vol 117 (17) ◽  
pp. 4519-4529 ◽  
Author(s):  
Waleed Alduaij ◽  
Andrei Ivanov ◽  
Jamie Honeychurch ◽  
Eleanor J. Cheadle ◽  
Sandeep Potluri ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cell ◽  
Actin Polymerization ◽  
Type I ◽  
Type Ii ◽  
B Cell Malignancies ◽  
Therapeutic Success ◽  
Wide Range ◽  
Anti Cd20

Abstract The anti-CD20 mAb rituximab has substantially improved the clinical outcome of patients with a wide range of B-cell malignancies. However, many patients relapse or fail to respond to rituximab, and thus there is intense investigation into the development of novel anti-CD20 mAbs with improved therapeutic efficacy. Although Fc-FcγR interactions appear to underlie much of the therapeutic success with rituximab, certain type II anti-CD20 mAbs efficiently induce programmed cell death (PCD), whereas rituximab-like type I anti-CD20 mAbs do not. Here, we show that the humanized, glycoengineered anti-CD20 mAb GA101 and derivatives harboring non-glycoengineered Fc regions are type II mAb that trigger nonapoptotic PCD in a range of B-lymphoma cell lines and primary B-cell malignancies. We demonstrate that GA101-induced cell death is dependent on actin reorganization, can be abrogated by inhibitors of actin polymerization, and is independent of BCL-2 overexpression and caspase activation. GA101-induced PCD is executed by lysosomes which disperse their contents into the cytoplasm and surrounding environment. Taken together, these findings reveal that GA101 is able to potently elicit actin-dependent, lysosomal cell death, which may potentially lead to improved clearance of B-cell malignancies in vivo.

scholarly journals 5-Aza-2'-Deoxycytidine Promotes Cytotoxic Effect of CART-Cells on Leukaemia Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5812-5812
Author(s):  
Alla Dolnikov ◽  
Swapna Rossi ◽  
Ning Xu ◽  
Guy Klamer ◽  
Sylvie Shen ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Target Cells ◽  
Car T Cells ◽  
Anti Tumour Activity ◽  
Car T ◽  
Pre Treatment ◽  
Tumour Activity

Abstract T cells modified to express CD19-specific chimeric antigen receptors (CAR) have shown anti-tumour efficacy in early phase clinical trials in patients with relapsed and refractory B-cell malignancies. In addition to direct cytotoxicity, chemotherapeutic drugs can have an immunomodulatory effect both through enhancing the tumour-specific immune response and increasing the tumour’s susceptibility to immune mediated destruction. Hence, combining immunomodulatory chemotherapy and CAR T-cells is an attractive approach for eliminating tumours, particularly in advanced stages. 5-aza-2'-deoxycytidine (5-AZA) is a hypomethylating agent that induces terminal differentiation, senescence or apoptosis in haematological malignancies. Here, we have explored a CAR-based immunotherapy combined with 5-AZA to maximise the effect of the CAR T-cells against CD19+ B-cell leukaemia. A second generation CAR including CD3zeta and the CD28 co-stimulatory domain was cloned into the PiggyBac-transposon vector and was used to generate CAR19 -T cells. Cord blood -derived mononuclear cells (MNC) were transfected with CAR19-transposon/transposase plasmids and expanded with CD3/28 beads for 2 weeks in the presence of 20ng/ml IL2 and 10ng/ml IL7. CAR19 T-cells efficiently induced cytolysis of CD19+ leukaemia cells in vitro and exhibited anti-tumour activity in vivo in a xenograft mouse model of leukaemia. Pre-treatment with 5-AZA produced greater leukaemia cell cytolysis in vitro and maximised anti-tumour activity of CAR19 T-cells in vivo against xenograft primary leukaemia compared to 5-AZA or CAR19 T-cells alone. In vitro analysis revealed that pre-treatment with 5-AZA up-regulates CD19 expression in leukaemia cells and improves CAR19 T-cell recognition of target cells increasing the formation of effector/ target cell conjugates and target cell cytolysis. Therefore using 5-AZA pre-treatment can be particularly useful for B-cell leukaemias with reduced expression of CD19. We have also demonstrated that pre-treatment of target cells with 5-AZA potentiates the effect of CAR19 T-cells used at low dose or low effector:target (E:T) suggesting that even small numbers of CAR19 T-cells can mediate a potent antitumor effect when combined with 5-AZA pre-treatment of target cells. This is particularly important for patients receiving limited numbers of CAR T-cells or for patients with large leukaemic burden. In addition, we speculate that the enhanced cellular cytotoxicity produced by 5-AZA-conditioning may allow the infusion of decreased numbers of CAR19 T-cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Type II (tositumomab) anti-CD20 monoclonal antibody out performs type I (rituximab-like) reagents in B-cell depletion regardless of complement activation

Blood ◽  
2008 ◽  
Vol 112 (10) ◽  
pp. 4170-4177 ◽  
Author(s):  
Stephen A. Beers ◽  
Claude H. T. Chan ◽  
Sonya James ◽  
Ruth R. French ◽  
Kathrine E. Attfield ◽  
...  
Keyword(s):  
B Cell ◽  
Type I ◽  
Type Ii ◽  
Effector Functions ◽  
Equivalent Efficacy ◽  
Anti Cd20 ◽  
Secondary Lymphoid Organs ◽  
Mouse Igg2a ◽  
Human B Cell

Abstract Anti-CD20 monoclonal antibodies (mAbs) are classified into type I (rituximab-like) or type II (tositumomab-like) based on their ability to redistribute CD20 molecules in the plasma membrane and activate various effector functions. To compare type I and II mAbs directly in vivo and maximize Fc effector function, we selected and engineered mAbs with the same mouse IgG2a isotype and assessed their B-cell depleting activity in human CD20 transgenic mice. Despite being the same isotype, having similar affinity, opsonizing activity for phagocytosis, and in vivo half-life, the type II mAb tositumomab (B1) provided substantially longer depletion of B cells from the peripheral blood compared with the type I mAb rituximab (Rit m2a), and 1F5. This difference was also evident within the secondary lymphoid organs, in particular, the spleen. Failure to engage complement did not explain the efficacy of the type II reagents because type I mAbs mutated in the Fc domain (K322A) to prevent C1q binding still did not display equivalent efficacy. These results give support for the use of type II CD20 mAbs in human B-cell diseases.

Development of a Bruton's Tyrosine Kinase (Btk) Inhibitor, ONO-WG-307: Efficacy in ABC-DLBCL Xenograft Model – Potential Treatment for B-Cell Malignancies,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3731-3731 ◽  
Author(s):  
Ryohei Kozaki ◽  
Toshio Yoshizawa ◽  
Shuji Tohda ◽  
Tomoko Yasuhiro ◽  
Shingo Hotta ◽  
...  
Keyword(s):  
B Cell ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Xenograft Model ◽  
B Cell Lymphoma ◽  
Lymphoid Cells ◽  
B Cell Malignancies ◽  
Pharmacodynamic Marker ◽  
Btk Inhibitor

Abstract Abstract 3731 Purpose: ONO-WG-307 is a small molecule inhibitor that covalently binds to Btk. Signals from B cell receptors (BCR) play a central role in signal transduction pathways regulating survival, activation, proliferation, and differentiation of B-lineage lymphoid cells. BCR signaling is implicated in the survival of malignant B cells and recent studies indicate that targeting Btk, an essential component of the BCR pathway, may be effective in the treatment of B-cell lymphoma. The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) correlates with poor prognosis and new therapies, preferably chemo-sparing therapies, or as add-on to existing treatment regimens are required to help treat patients with ABC-DLBCL. Therefore, Btk constitutes an interesting therapeutic target, thus the activity of ONO-WG-307 was evaluated in an ABC-DLBCL xenograft model. Methods: Tumor cells (TMD-8) were implanted subcutaneously into female SCID mice. Tumors were allowed to grow to a volume of 100–200 mm3 before the mice were randomized into groups based on tumor size. ONO-WG-307 was administered orally at doses up to 10 mg/kg bid. Tumors were measured two or three times weekly after initiation of treatment, and tumor volumes were determined using the formula volume (=width2xlength)/2. Animals were euthanized when the tumors reached a maximum volume of 2,000 mm3 or after a maximum period of 2 months. In parallel, an exploratory pharmacodynamic marker of Btk inhibition (Phosphorylated-Btk [P-Btk]) was also investigated in vivo. Results: Treatment with ONO-WG-307 resulted in a dose-dependent inhibition of tumor growth in a TMD-8 xenograft model. Furthermore, parallel analysis of a pharmacodynamic marker, P-Btk, supported that Btk was inhibited and the level of P-Btk inhibition was correlated with the decreased tumor volumes observed in the TMD-8 model. Conclusion: ONO-WG-307 is a highly potent and selective oral Btk inhibitor with evidence of efficacy in the ABC-DLBCL xenograft model, with Btk inhibition further supported using a PD marker. Given the need to treat and overcome disease resistance especially in ABC-DLBCL, the use of a Btk inhibitor is a novel, mechanistic approach to treating B cell malignancies. Additional work is underway, combining ONO-WG-307 with chemotherapy and other targeted agents. Disclosures: No relevant conflicts of interest to declare.

Obinutuzumab (GA101) Significantly Improves Survival In CD20 Positive Pre-B Cell Lymphoblastic Leukemia (pre-B-ALL) Xenograft Models Compared To Rituximab (RTX): Potential Targeted Therapy In Patients With High Risk Pre-B-ALL

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3068-3068 ◽  
Author(s):  
Aradhana Awasthi ◽  
Janet Ayello ◽  
Carmella van de Ven ◽  
Stefan Hovy ◽  
Mitchell S. Cairo
Keyword(s):  
B Cells ◽  
Mouse Model ◽  
B Cell ◽  
Tumor Volume ◽  
Tumor Burden ◽  
Scid Mice ◽  
Type I ◽  
Type Ii ◽  
Anti Cd20

Abstract Background Obinutuzumab (GA101) is a type II, glycoengineered, humanized anti-CD20 monoclonal antibody (Ab). Obinutuzumab binds with high affinity and selectivity to a type II binding region of the CD20 protein present on all B cells except stem or plasma cells. Unlike type I anti-CD20 monoclonal Abs, type II anti-CD20 Abs promote direct cell death induction without the need for a cross-linking Ab. Patients who relapse with CD20+ B-NHL and early relapsed pre-B-ALL have a dismal prognosis, often associated with chemotherapy resistance (Cairo et al. JCO, 2012, Barth/Cairo et al. BJH, 2013) and require alternative therapeutic strategies. RTX in combination with FAB/LMB 96 chemotherapy is a safe, well-tolerated treatment that is associated with > 95% EFS in children with newly diagnosed and advanced mature B-Cell NHL (Goldman/Cairo et al. Leukemia, 2013). Resistance to RTX, however, may predispose patients with CD20+ NHL/Pre-B-ALL to an increased risk of relapse and or disease progression (Barth/Cairo et al. BJH, 2013). Objective To evaluate the anti-tumor activity of obinutuzumab vs RTX against pre-B-LL in-vivo in severe combined immune-deficient IL2 receptor gamma chain knockout (NSG) and NOD/SCID mice. Methods U698-M (Pre-B cell ALL, CD20+, DSMZ, Germany), Loucy cells (T-ALL, CD20- ATCC, Manasass, VA) were maintained in RPMI with 10% FBS at 370C, 5% CO2. The mammalian expression construct ffLUCZeo-pcDNA (kindly provided by L.Cooper, MD,PhD) was transfected into U698M and Loucy (Luc+) tumor targets for later evaluation by bioluminescent imaging (BLI). Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, no functional T, B and NK cells) and NOD/SCID (absence of functional T and B cells) mice were bred in-house and treated in pathogen free conditions at the animal facility. The in vivo cytotoxic activity of obinutuzumab and RTX was determined by dividing NSG and NOD/SCID mice into 5 groups: PBS only (control), isotype control (IgG), obinutuzumab (10 mg/kg) (kindly provided by C. Klein, Roche Glycart AG), obinutuzumab (30 mg/kg), and rituximab (30 mg/kg). Mice received intravenous injections of Luc+ U698M and Loucy cells at 5x106 tumor cells/mouse. 6-8 days after tumor cell injection, mice were injected every 7 days with the respective therapy for 8 weeks. Mice were closely monitored for tumor burden progression/regression and survival for up to 12 weeks (approx. 80 days) via BLI using the IVIS Spectrum System. Results We observed that obinutuzumab was significantly more effective than RTX when administered at the same doses in established pre-B-ALL xenografts. In the NSG mouse model, at 30 days tumor volume in mice receiving 30 mg/kg of obinutuzumab was significantly decreased when compared to mice receiving 10 mg/kg obinutuzumab or 30 mg/kg of RTX (p<0.0001, p<0.002 respectively). At 11 weeks, mice receiving 30 mg/kg of obinutuzumab showed no increase in tumor volume compared to week 4. The overall survival following obinutuzumab at 30 mg/kg was 80 days compared to 48 days by obinutuzumab at 10mg/kg (p=0.001) and 30 days following RTX treatment at 30mg/kg)(p=0.002) (Figure-1A). Relative tumor luminescence activity on day 8 was significantly decreased in mice receiving 30 mg/kg of obinutuzumab (8x105 RLU) compared to 10 mg/kg of obinutuzumab (7.7x105 RLU) or 30 mg/kg of RTX (9.9x105 RLU) compared to IgG isotype (2.0x106 RLU, p=0.02,p=0.07,p=0.1, respectively). Similarly, in the NOD/SCID mouse model, at day 80, we observed a significant decrease in tumor luminescence when mice were treated with 30 mg/kg obinutuzumab compared to 10 mg/kg obinutuzumab or 30 mg/kg rituximab (p=0.001). Furthermore, at day 80 there was 100% survival in animals with 30 mg/kg obinutuzumab compared to 60% following rituximab at 30 mg/kg ( p=0.001) or 40% obinutuzumab (10mg/kg; p=0.001) (Figure1B). Conclusion These preliminary studies in two groups of pre-B-ALL xenograft mice (NSG and NOD/SCID), demonstrated that the antitumor effect of obinutuzumab at 30 mg/kg significantly increases survival and decreases the tumor burden in Pre-B-ALL xenografts compared to equal dose of RTX. Further studies are underway to determine the mechanism(s) responsible for the significantly increased efficacy of obinutuzumab vs. RTX in pre-B-ALL xenografts. Disclosures: Cairo: Roche/Genentech: advisory board Other.

Sign in / Sign up

Export Citation Format

Share Document