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Christian Klein - One of the best experts on this subject based on the ideXlab platform.

  • CD16 pre-ligation by defucosylated tumor-targeting mAb sensitizes human NK cells to γ_c cytokine stimulation via PI3K/mTOR axis
    Cancer Immunology Immunotherapy, 2020
    Co-Authors: Cristina Capuano, Christian Klein, Chiara Pighi, Roberta Maggio, Simone Battella, Stefania Morrone, Gabriella Palmieri, Angela Santoni, Ricciarda Galandrini
    Abstract:

    Obinutuzumab is a glycoengineered tumor-targeting anti-CD20 mAb with a modified crystallizable fragment (Fc) domain designed to increase the affinity for the FcγRIIIA/CD16 receptor, which was recently approved for clinical use in CLL and follicular lymphoma. Here we extend our previous observation that, in human NK cells, the sustained CD16 ligation by Obinutuzumab-opsonized targets leads to a markedly enhanced IFN-γ production upon a subsequent cytokine re-stimulation. The increased IFN-γ competence in response to IL-2 or IL-15 is attributable to post-transcriptional regulation, as it does not correlate with the upregulation of IFN-γ mRNA levels. Different from the reference molecule rituximab, we observe that the stimulation with Obinutuzumab promotes the upregulation of microRNA (miR)-155 expression. A similar trend was also observed in NK cells from untreated CLL patients stimulated with Obinutuzumab-opsonized autologous leukemia. miR-155 upregulation associates with reduced levels of SHIP-1 inositol phosphatase, which acts in constraining PI3K-dependent signals, by virtue of its ability to mediate phosphatidylinositol 3,4,5-trisphosphate (PIP3) de-phosphorylation. Downstream of PI3K, the phosphorylation status of mammalian target of rapamycin (mTOR) effector molecule, S6, results in amplified response to IL-2 or IL-15 stimulation in Obinutuzumab-experienced cells. Importantly, NK cell treatment with the PI3K or mTOR inhibitors, idelalisib and rapamycin, respectively, prevents the enhanced cytokine responsiveness, thus, highlighting the relevance of the PI3K/mTOR axis in CD16-dependent priming. The enhanced IFN-γ competence may be envisaged to potentiate the immunoregulatory role of NK cells in a therapeutic setting.

  • PKPD Assessment of the Anti-CD20 Antibody Obinutuzumab in Cynomolgus Monkey is Feasible Despite Marked Anti-Drug Antibody Response in This Species.
    Journal of pharmaceutical sciences, 2019
    Co-Authors: Hans Peter Grimm, Christian Klein, Eginhard Schick, Dominik Hainzl, Nicole Justies, Elisabeth Husar, Wolfgang F. Richter
    Abstract:

    Abstract The pharmacokinetics (PK) of the anti-CD20 monoclonal antibody Obinutuzumab was assessed after single intravenous dosing to cynomolgus monkeys. In addition, the pharmacokinetic-pharmacodynamic (PKPD) relationship for B-cell depletion was characterized. The PKPD model was used to estimate the B-cell repopulation during the recovery phase of chronic toxicology studies, thereby supporting the study design, in particular planning the recovery phase duration. Marked immunogenicity against Obinutuzumab was observed approximately 10 days after single dose, leading to an up to ∼30-fold increase in Obinutuzumab clearance in the affected monkeys. Despite this accelerated clearance, the PK could be characterized, either by disregarding the clearance in noncompartmental PK analysis or by capturing it explicitly as an additional time-dependent clearance process in compartmental modeling. This latter step was crucial to model the PKPD of B-cells as an indirect response to Obinutuzumab exposure, showing that—without immune response—the limiting factor is Obinutuzumab elimination with concentrations below 0.02 μg/mL required for initiation of B-cell recovery. Overall, the results demonstrate that despite a marked anti-drug antibody response in the nonclinical animal species, the PK and PKPD of Obinutuzumab could be characterized successfully by appropriately addressing the immune-modulated clearance pathway in data analysis and modeling.

  • Abstract 1788: Enhanced in vitro/in vivo cytotoxicity against Burkitt lymphoma/primary mediastinal large B cell lymphoma by polatuzumab vedotin (hu- anti-CD79b-vc-MMAE, PV) alone or in combination with Obinutuzumab
    Cancer Research, 2018
    Co-Authors: Aradhana Awasthi Tiwari, Christian Klein, Dina Edani, Janet Ayello, Christeen Azmy, Mitchell S. Cairo
    Abstract:

    Background: Mature B-NHL, including Burkitt lymphoma (BL) and primary mediastinal large B cell lymphoma (PMBL) express CD20+/CD79b+ and have an excellent prognosis, however, subset of patients relapse secondary to chemoimmunotherapy resistant disease and have a dismal prognosis (≤ 20% 5 yr. EFS, Cairo et al. Blood. 2007; Gerrard/Cairo et al., Blood, 2013, Goldman/Cairo et al. Leukemia, 2013). PV has been demonstrated to possess significant preclinical activity against indolent CD79b+NHL (Polson et. al.Can. Res.2009). We previously observed that Obinutuzumab (Anti-CD20 mAb) significantly enhanced cell death and increased overall survival against BL (Awasthi/Cairo et al., BJH 2015) in xenografted NSG mice. However, additive/synergistic effects of PV with Obinutuzumab against mature PMBL/BL are unknown. Objective: To determine the efficacy of the PV or Obinutuzumab/RTX alone or in combination against PMBL and rituximab (RTX) sensitive/resistant BL cell lines. Methods: Raji4RH (provided by M. Barth, MD, Roswell Park Cancer Institute) and Raji/ Karpas1106P (ATCC, USA) were cultured in RPMI. Tumor cells were incubated with PV, and/or anti-CD79b, MMAE (generously supplied by Genentech Inc.) with Obinutuzumab /rituximab (100ug/ml) for 4 hr with NK cells at 10:1 E: T ratio and cytotoxicity was determined by DELFIA cytotoxicity assay. Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ), were divided into 5 groups: PBS, isotype control, PV, antiCD79B mAb and MMAE (5mg/kg). Mice were xenografted with intravenous injections of Luc+ BL and PMBL cells and tumor burden was monitored by IVIS spectrum system. Results: OS of mice receiving PV alone was significantly increased compared to antiCD79b or isotype control in Raji (35.5 vs.17 vs.19.5 days, p=0.0001, 0.0003), Raji4RH (50 vs.18 vs.18.5 days, p=0.0001, 0.0001) and Karpas1106P (150 vs 89 vs 64 days, p=0.03, 0.003), respectively. Obinutuzumab+NK, rituximab+NK compared to PV+NK cells significantly enhanced cell lysis in Raji, 65.9±2.4% vs. 38.9±5.4% vs. 44.24±8.1% (p=0.001 & p=0.001), Raji4RH, 52.8±9.4% vs. 16.04±7.2% vs.47.0±8.2% (p=0.03 & p=NS) and Karpas1106P, 66.10±5.3% vs.48.2±3.9% vs. 61.6±10.06% (p=0.004 & NS), respectively. PV+ Obinutuzumab+NK, significantly improved cytotoxicity compared to PV+ rituximab+NK in Raji, 93.6±6.1% vs 79.9±5.3% (p=0.007), Raji4RH, 78.07±2.05% vs 63.5±0.16% (p=0.004) and Karpas1106P, 88.3±6.3 %vs.73.03±3.03% (p=0.003), respectively. Conclusion: Our preliminary data indicates that PV significantly increased survival in BL and PMBL NSG xenografts compared to anti-CD79b Ab alone. Furthermore, PV in combination with Obinutuzumab significantly enhances cytotoxicity in BL and PMBL compared to Obinutuzumab or PV alone. Citation Format: Aradhana Awasthi Tiwari, Dina Edani, Christeen Azmy, Janet Ayello, Christian Klein, Mitchell S. Cairo. Enhanced in vitro/in vivo cytotoxicity against Burkitt lymphoma/primary mediastinal large B cell lymphoma by polatuzumab vedotin (hu- anti-CD79b-vc-MMAE, PV) alone or in combination with Obinutuzumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1788.

  • abstract 1788 enhanced in vitro in vivo cytotoxicity against burkitt lymphoma primary mediastinal large b cell lymphoma by polatuzumab vedotin hu anti cd79b vc mmae pv alone or in combination with Obinutuzumab
    Cancer Research, 2018
    Co-Authors: Aradhana Awasthi Tiwari, Christian Klein, Dina Edani, Janet Ayello, Christeen Azmy, Mitchell S. Cairo
    Abstract:

    Background: Mature B-NHL, including Burkitt lymphoma (BL) and primary mediastinal large B cell lymphoma (PMBL) express CD20+/CD79b+ and have an excellent prognosis, however, subset of patients relapse secondary to chemoimmunotherapy resistant disease and have a dismal prognosis (≤ 20% 5 yr. EFS, Cairo et al. Blood. 2007; Gerrard/Cairo et al., Blood, 2013, Goldman/Cairo et al. Leukemia, 2013). PV has been demonstrated to possess significant preclinical activity against indolent CD79b+NHL (Polson et. al.Can. Res.2009). We previously observed that Obinutuzumab (Anti-CD20 mAb) significantly enhanced cell death and increased overall survival against BL (Awasthi/Cairo et al., BJH 2015) in xenografted NSG mice. However, additive/synergistic effects of PV with Obinutuzumab against mature PMBL/BL are unknown. Objective: To determine the efficacy of the PV or Obinutuzumab/RTX alone or in combination against PMBL and rituximab (RTX) sensitive/resistant BL cell lines. Methods: Raji4RH (provided by M. Barth, MD, Roswell Park Cancer Institute) and Raji/ Karpas1106P (ATCC, USA) were cultured in RPMI. Tumor cells were incubated with PV, and/or anti-CD79b, MMAE (generously supplied by Genentech Inc.) with Obinutuzumab /rituximab (100ug/ml) for 4 hr with NK cells at 10:1 E: T ratio and cytotoxicity was determined by DELFIA cytotoxicity assay. Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ), were divided into 5 groups: PBS, isotype control, PV, antiCD79B mAb and MMAE (5mg/kg). Mice were xenografted with intravenous injections of Luc+ BL and PMBL cells and tumor burden was monitored by IVIS spectrum system. Results: OS of mice receiving PV alone was significantly increased compared to antiCD79b or isotype control in Raji (35.5 vs.17 vs.19.5 days, p=0.0001, 0.0003), Raji4RH (50 vs.18 vs.18.5 days, p=0.0001, 0.0001) and Karpas1106P (150 vs 89 vs 64 days, p=0.03, 0.003), respectively. Obinutuzumab+NK, rituximab+NK compared to PV+NK cells significantly enhanced cell lysis in Raji, 65.9±2.4% vs. 38.9±5.4% vs. 44.24±8.1% (p=0.001 & p=0.001), Raji4RH, 52.8±9.4% vs. 16.04±7.2% vs.47.0±8.2% (p=0.03 & p=NS) and Karpas1106P, 66.10±5.3% vs.48.2±3.9% vs. 61.6±10.06% (p=0.004 & NS), respectively. PV+ Obinutuzumab+NK, significantly improved cytotoxicity compared to PV+ rituximab+NK in Raji, 93.6±6.1% vs 79.9±5.3% (p=0.007), Raji4RH, 78.07±2.05% vs 63.5±0.16% (p=0.004) and Karpas1106P, 88.3±6.3 %vs.73.03±3.03% (p=0.003), respectively. Conclusion: Our preliminary data indicates that PV significantly increased survival in BL and PMBL NSG xenografts compared to anti-CD79b Ab alone. Furthermore, PV in combination with Obinutuzumab significantly enhances cytotoxicity in BL and PMBL compared to Obinutuzumab or PV alone. Citation Format: Aradhana Awasthi Tiwari, Dina Edani, Christeen Azmy, Janet Ayello, Christian Klein, Mitchell S. Cairo. Enhanced in vitro/in vivo cytotoxicity against Burkitt lymphoma/primary mediastinal large B cell lymphoma by polatuzumab vedotin (hu- anti-CD79b-vc-MMAE, PV) alone or in combination with Obinutuzumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1788.

  • a comparative global phosphoproteomics analysis of Obinutuzumab ga101 versus rituximab rtx against rtx sensitive and resistant burkitt lymphoma bl demonstrates differential phosphorylation of signaling pathway proteins after treatment
    Oncotarget, 2017
    Co-Authors: Aradhana Awasthi, Christian Klein, Janet Ayello, Carmella Van De Ven, Matthew J Barth, Delphine Rolland, Venkatesha Basrur, Kevin P Conlon, Damian Fermin, Kojo S J Elenitobajohnson
    Abstract:

    We recently demonstrated that Obinutuzumab (GA101), a novel glycoengineered type II CD20 Ab compared to rituximab (RTX) mediates significantly enhanced antibody-dependent cell cytotoxicity (ADCC) in vitro and increased overall survival in a Burkitt lymphoma (BL) xenograft non-obese diabetic severe combined immunodeficiency gamma (NSG) model. In this study we compared the phosphoproteomic changes by pathway analysis following Obinutuzumab vs RTX against RTX-sensitive (Raji) and -resistant BL (Raji4RH). Phosphoproteomic analyses were performed by mass-spectrometry (MS)-based label-free quantitative phosphoproteomic profiling. We demonstrated that 418 proteins in Raji and 377 proteins in Raji 4RH, were differentially phosphorylated (>1.5-fold) after Obinutuzumab vs. RTX. Proteins that were significantly differentially phosphorylated included the B cell antigen receptor (BCR) (PLCG2, BTK and GSK3B), Fc gamma phagocytosis (FCRG2B, MAPK1, PLCG2 and RAF1), and natural killer cell-mediated cytotoxicity (MAPK1, RAF1, PLCG2 and MAPK3) signaling pathways. Differential phosphorylation of BCR or cytotoxicity pathway proteins revealed significant up-regulation of BTK, PLCY2 and ERK1/RAF1 after Obinutuzumab compared to RTX. Silencing of PLCG2 in the BCR and MAPK1 in the cytotoxicity pathway significantly increased BL proliferation and decreased BL cytotoxicity after Obinutuzumab compared to RTX. These results in combination with our previous results demonstrating a significant improvement in in vitro BL cytotoxicity and in vivo BL survival by Obinutuzumab compared to RTX may in part be due to differential effects on selected BL protein signaling pathways.

Mitchell S. Cairo - One of the best experts on this subject based on the ideXlab platform.

  • abstract 1788 enhanced in vitro in vivo cytotoxicity against burkitt lymphoma primary mediastinal large b cell lymphoma by polatuzumab vedotin hu anti cd79b vc mmae pv alone or in combination with Obinutuzumab
    Cancer Research, 2018
    Co-Authors: Aradhana Awasthi Tiwari, Christian Klein, Dina Edani, Janet Ayello, Christeen Azmy, Mitchell S. Cairo
    Abstract:

    Background: Mature B-NHL, including Burkitt lymphoma (BL) and primary mediastinal large B cell lymphoma (PMBL) express CD20+/CD79b+ and have an excellent prognosis, however, subset of patients relapse secondary to chemoimmunotherapy resistant disease and have a dismal prognosis (≤ 20% 5 yr. EFS, Cairo et al. Blood. 2007; Gerrard/Cairo et al., Blood, 2013, Goldman/Cairo et al. Leukemia, 2013). PV has been demonstrated to possess significant preclinical activity against indolent CD79b+NHL (Polson et. al.Can. Res.2009). We previously observed that Obinutuzumab (Anti-CD20 mAb) significantly enhanced cell death and increased overall survival against BL (Awasthi/Cairo et al., BJH 2015) in xenografted NSG mice. However, additive/synergistic effects of PV with Obinutuzumab against mature PMBL/BL are unknown. Objective: To determine the efficacy of the PV or Obinutuzumab/RTX alone or in combination against PMBL and rituximab (RTX) sensitive/resistant BL cell lines. Methods: Raji4RH (provided by M. Barth, MD, Roswell Park Cancer Institute) and Raji/ Karpas1106P (ATCC, USA) were cultured in RPMI. Tumor cells were incubated with PV, and/or anti-CD79b, MMAE (generously supplied by Genentech Inc.) with Obinutuzumab /rituximab (100ug/ml) for 4 hr with NK cells at 10:1 E: T ratio and cytotoxicity was determined by DELFIA cytotoxicity assay. Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ), were divided into 5 groups: PBS, isotype control, PV, antiCD79B mAb and MMAE (5mg/kg). Mice were xenografted with intravenous injections of Luc+ BL and PMBL cells and tumor burden was monitored by IVIS spectrum system. Results: OS of mice receiving PV alone was significantly increased compared to antiCD79b or isotype control in Raji (35.5 vs.17 vs.19.5 days, p=0.0001, 0.0003), Raji4RH (50 vs.18 vs.18.5 days, p=0.0001, 0.0001) and Karpas1106P (150 vs 89 vs 64 days, p=0.03, 0.003), respectively. Obinutuzumab+NK, rituximab+NK compared to PV+NK cells significantly enhanced cell lysis in Raji, 65.9±2.4% vs. 38.9±5.4% vs. 44.24±8.1% (p=0.001 & p=0.001), Raji4RH, 52.8±9.4% vs. 16.04±7.2% vs.47.0±8.2% (p=0.03 & p=NS) and Karpas1106P, 66.10±5.3% vs.48.2±3.9% vs. 61.6±10.06% (p=0.004 & NS), respectively. PV+ Obinutuzumab+NK, significantly improved cytotoxicity compared to PV+ rituximab+NK in Raji, 93.6±6.1% vs 79.9±5.3% (p=0.007), Raji4RH, 78.07±2.05% vs 63.5±0.16% (p=0.004) and Karpas1106P, 88.3±6.3 %vs.73.03±3.03% (p=0.003), respectively. Conclusion: Our preliminary data indicates that PV significantly increased survival in BL and PMBL NSG xenografts compared to anti-CD79b Ab alone. Furthermore, PV in combination with Obinutuzumab significantly enhances cytotoxicity in BL and PMBL compared to Obinutuzumab or PV alone. Citation Format: Aradhana Awasthi Tiwari, Dina Edani, Christeen Azmy, Janet Ayello, Christian Klein, Mitchell S. Cairo. Enhanced in vitro/in vivo cytotoxicity against Burkitt lymphoma/primary mediastinal large B cell lymphoma by polatuzumab vedotin (hu- anti-CD79b-vc-MMAE, PV) alone or in combination with Obinutuzumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1788.

  • Abstract 1788: Enhanced in vitro/in vivo cytotoxicity against Burkitt lymphoma/primary mediastinal large B cell lymphoma by polatuzumab vedotin (hu- anti-CD79b-vc-MMAE, PV) alone or in combination with Obinutuzumab
    Cancer Research, 2018
    Co-Authors: Aradhana Awasthi Tiwari, Christian Klein, Dina Edani, Janet Ayello, Christeen Azmy, Mitchell S. Cairo
    Abstract:

    Background: Mature B-NHL, including Burkitt lymphoma (BL) and primary mediastinal large B cell lymphoma (PMBL) express CD20+/CD79b+ and have an excellent prognosis, however, subset of patients relapse secondary to chemoimmunotherapy resistant disease and have a dismal prognosis (≤ 20% 5 yr. EFS, Cairo et al. Blood. 2007; Gerrard/Cairo et al., Blood, 2013, Goldman/Cairo et al. Leukemia, 2013). PV has been demonstrated to possess significant preclinical activity against indolent CD79b+NHL (Polson et. al.Can. Res.2009). We previously observed that Obinutuzumab (Anti-CD20 mAb) significantly enhanced cell death and increased overall survival against BL (Awasthi/Cairo et al., BJH 2015) in xenografted NSG mice. However, additive/synergistic effects of PV with Obinutuzumab against mature PMBL/BL are unknown. Objective: To determine the efficacy of the PV or Obinutuzumab/RTX alone or in combination against PMBL and rituximab (RTX) sensitive/resistant BL cell lines. Methods: Raji4RH (provided by M. Barth, MD, Roswell Park Cancer Institute) and Raji/ Karpas1106P (ATCC, USA) were cultured in RPMI. Tumor cells were incubated with PV, and/or anti-CD79b, MMAE (generously supplied by Genentech Inc.) with Obinutuzumab /rituximab (100ug/ml) for 4 hr with NK cells at 10:1 E: T ratio and cytotoxicity was determined by DELFIA cytotoxicity assay. Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ), were divided into 5 groups: PBS, isotype control, PV, antiCD79B mAb and MMAE (5mg/kg). Mice were xenografted with intravenous injections of Luc+ BL and PMBL cells and tumor burden was monitored by IVIS spectrum system. Results: OS of mice receiving PV alone was significantly increased compared to antiCD79b or isotype control in Raji (35.5 vs.17 vs.19.5 days, p=0.0001, 0.0003), Raji4RH (50 vs.18 vs.18.5 days, p=0.0001, 0.0001) and Karpas1106P (150 vs 89 vs 64 days, p=0.03, 0.003), respectively. Obinutuzumab+NK, rituximab+NK compared to PV+NK cells significantly enhanced cell lysis in Raji, 65.9±2.4% vs. 38.9±5.4% vs. 44.24±8.1% (p=0.001 & p=0.001), Raji4RH, 52.8±9.4% vs. 16.04±7.2% vs.47.0±8.2% (p=0.03 & p=NS) and Karpas1106P, 66.10±5.3% vs.48.2±3.9% vs. 61.6±10.06% (p=0.004 & NS), respectively. PV+ Obinutuzumab+NK, significantly improved cytotoxicity compared to PV+ rituximab+NK in Raji, 93.6±6.1% vs 79.9±5.3% (p=0.007), Raji4RH, 78.07±2.05% vs 63.5±0.16% (p=0.004) and Karpas1106P, 88.3±6.3 %vs.73.03±3.03% (p=0.003), respectively. Conclusion: Our preliminary data indicates that PV significantly increased survival in BL and PMBL NSG xenografts compared to anti-CD79b Ab alone. Furthermore, PV in combination with Obinutuzumab significantly enhances cytotoxicity in BL and PMBL compared to Obinutuzumab or PV alone. Citation Format: Aradhana Awasthi Tiwari, Dina Edani, Christeen Azmy, Janet Ayello, Christian Klein, Mitchell S. Cairo. Enhanced in vitro/in vivo cytotoxicity against Burkitt lymphoma/primary mediastinal large B cell lymphoma by polatuzumab vedotin (hu- anti-CD79b-vc-MMAE, PV) alone or in combination with Obinutuzumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1788.

  • polatuzumab vedotin alone or in combination with Obinutuzumab synergistically enhances in vitro cytotoxicity and cytokine release against cd20 cd79b burkitt lymphoma bl primary mediastinal large b cell lymphoma pmbl
    Blood, 2017
    Co-Authors: Aradhana Awasthi Tiwari, Christian Klein, Dina Edani, Janet Ayello, Mitchell S. Cairo
    Abstract:

    Background: Pediatric Burkitt Lymphoma (PBL) represents the most common malignancy in childhood and adolescent non-Hodgkin Lymphoma (NHL) but occurs in less than 5% of adult NHL cases (Hochberg/Cairo et al, BJH 2009; Miles/Cairo, BJH. 2012). Mature B-cell NHL, including Burkitt lymphoma (BL) and primary mediastinal large B cell lymphoma (PMBL) express CD20+/CD79b+ and have an excellent prognosis with rituximab based immunochemotherapy (Cairo et al Blood. 2007,Goldman/Cairo et al. Leukemia, 2013, Gerrard/Cairo et al. Blood. 2013). The prognosis of PBL has significantly improved over the last 40 years through the use of short and intense multi-agent immunochemotherapy, however, a subset of patients with relapsed/refractory disease has immunochemotherapy resistant disease and a dismal prognosis (≤ 20% 5 yr. EFS),(Cairo et al.Blood. 2007; Cairo et al. JCO.2012). It is therefore critical to investigate and to develop targeted translational strategies in BL/PMBL in order to reduce acute morbidities, decrease late effects, and provide new options for those with recurrent disease. The hu-anti-CD79b-vc-MMAE (Polatuzumab Vedotin, PV) an antibody drug conjugate (ADC) has demonstrated significant preclinical activity against indolent CD79b+NHL (Polson et. al.Can. Res.2009). More recently PV has well tolerated in adults with CD79b refractory CLL (Palanca-Wessels et al. Lancet Oncol, 2014). Obinutuzumab, a glycoengineered type II CD20 antibody, has been shown to enhance cell death and antibody dependent cytotoxicity assay (ADCC) vs. rituximab (RTX) (Herter et al, Clinc Can Res, 2013). We previously observed that PV or Obinutuzumab alone (Awasthi/Cairo et al., AACR 2017, Awasthi/Cairo et al., BJH 2015) significantly enhanced tumor cell death and increased overall survival against BL and PMBL xenografted mice. However, additive/synergistic effects of PV with Obinutuzumab against mature PMBL/BL are unknown. Objective: To determine the efficacy of the PV, Obinutuzumab or RTX alone or in combination with PV+ Obinutuzumab vs. PV+RTX against PMBL and RTX sensitive/resistant BL tumor cell lines. Methods: Raji/Raji4RH (provided by M. Barth, MD, Roswell Park Cancer Institute) and PMBL: Karpas1106P (ATCC, USA) tumor cells were cultured in RPMI with 10 or 20% FBS.Tumor cells were incubated with PV (generously supplied by Genentech Inc.) with Obinutuzumab (generously provided by Roche) or rituximab (100ug/ml) for 4 hrs with NK cells.PBMCs were expanded with lethally irradiated K562-mbIL21-41BBL cells (Lee et al, PLoS One, 2012).CD56+/CD3- isolated and expanded NK cells were used for cytotoxicity assay. Cytotoxicity was determined by DELFIA cytotoxicity assay at 10:1 E: T ratio and cytokine secretion was measured by multiplex ELISA assay kit. Results: Obinutuzumab+NK, RTX+NK (100µg/ml) compared to PV+NK cells (20ug/ml, 10:1E:T ratio) significantly enhanced cell lysis in Raji, 65.9±2.4% vs. 38.9±5.4% vs. 44.24±8.1%, (p=0.001 and p=0.001), Raji4RH, 52.8±9.4% vs. 16.04±7.2% vs.47.0±8.2% (p=0.03 and p=NS), respectively and Karpas1106P, 66.10±5.3% vs.48.2±3.9% vs. 61.6±10.06% (p=0.004 and NS), respectively. PV+ Obinutuzumab+NK, further significantly improved in-vitro cytotoxicity compared to PV+ rituximab+NK, Raji, 93.6±6.1% vs 79.9±5.3% (p=0.007, Figure 1A), Raji4RH, 78.07±2.05% vs 63.5±0.16% (p=0.004, Figure1B) and Karpas1106P, 88.3±6.3 %vs.73.03±3.03% (p=0.003, Figure1C), respectively. Furthermore, IFN-g level from supernatant of PV+Obinutuzumab+NK was significantly increased compared to Obinutuzumab+NK alone, Raji, 22.14±2.1 vs. 9.41±6.5 ug/ml (p=0.01), Raji4RH, 20.30±9.8vs.17.02±2.5 ug/ml (p=0.05) and Karpas1106P, 18.10±1.5vs.9.56±5.5 ug/ml (p=0.03), respectively. Conclusion: Our preliminary data indicates thatPV in combination with Obinutuzumab significantly enhances cell death in RTX sensitive/resistant CD79b+/CD20+ BL and PMBL compared to Obinutuzumab or PV alone. Furthermore, PV+ Obinutuzumab significantly enhanced cytokine secretion in BL (RTX sensitive/resistant) and PMBL compared to Obinutuzumab alone. Disclosures Klein: Roche: Employment, Equity Ownership, Other: patents. Lee: Miltenyi: Speakers Bureau; Cyto-Sen Therapeutics, Inc: Other: Founder, Vice President, Medical Director; Intellia Therapeutics, Inc: Consultancy; Courier Therapeutics, Inc: Membership on an entity9s Board of Directors or advisory committees. Cairo: Jazz Pharmaceuticals: Speakers Bureau.

  • Obinutuzumab ga101 compared to rituximab significantly enhances cell death and antibody dependent cytotoxicity and improves overall survival against cd20 rituximab sensitive resistant burkitt lymphoma bl and precursor b acute lymphoblastic leukaemia
    British Journal of Haematology, 2015
    Co-Authors: Aradhana Awasthi, Christian Klein, Janet Ayello, Carmella Van De Ven, Mona Elmacken, Anthony Sabulski, Matthew J Barth, Myron S Czuczman, Humayun Islam, Mitchell S. Cairo
    Abstract:

    Obinutuzumab is a novel glycoengineered Type-II CD20 monoclonal antibody. CD20 is expressed in approximately 100% of children and adolescents with Burkitt lymphoma (BL) and 40% with precursor B-cell acute lymphoblastic leukaemia (pre-B-ALL). We evaluated the anti-tumour activity of Obinutuzumab versus rituximab against rituximab-resistant (Raji 4RH) and -sensitive (Raji) BL and pre-B-ALL (U698-M) cells in vitro and in human BL or Pre-B-ALL xenografted mice. We demonstrated that Obinutuzumab compared to rituximab significantly enhanced cell death against Raji 35·6 ± 3·1% vs. 25·1 ± 2·0%, (P = 0·001), Raji4RH 19·7 ± 2·2% vs. 7·9 ± 1·5% (P = 0·001) and U-698-M 47·3 ± 4·9% vs. 23·2 ± 0·5% (P = 0·001), respectively. Obinutuzumab versus rituximab also induced a significant increase in antibody-dependent cellular cytotoxicity (ADCC) with K562-IL15-41BBL expanded NK cells against Raji 73·8 ± 8·1% vs. 56·81 ± 4·6% (P = 0·001), Raji-4RH 40·0 ± 1·6% vs. 0·5 ± 1·1% (P = 0·001) and U-698-M 70·0 ± 1·6% vs. 45·5 ± 0·1% (P = 0·001), respectively. Overall survival in tumour xenografted mice receiving 30 mg/kg of Obinutuzumab was significantly increased when compared to those receiving 30 mg/kg of rituximab in BL; Raji (P = 0·05), Raji4RH (P = 0·02) and U698-M (P = 0·03), respectively. These preclinical data suggest Obinutuzumab is significantly superior to rituximab in inducing cell death, ADCC and against rituximab-sensitive/-resistant BL and pre-B-ALL xenografted mice. Taken together, these preclinical results provide evidence to suggest that future investigation of Obinutuzumab is warranted in patients with relapsed/refractory CD20(+) BL and/or pre-B-ALL.

  • Abstract 3096: Obinutuzumab (GA101) significantly induces antiproliferative effects and programmed cell death, and significantly downregulates cell signaling pathways in primary mediastinal B-cell lymphoma (PMBL): Obinutuzumab may be a future potenti
    Tumor Biology, 2014
    Co-Authors: Changhong Yin, Sanghoon Lee, Janet Ayello, Carmella Van De Ven, Timmy O'connell, Mitchell S. Cairo
    Abstract:

    BACKGROUND: Primary Mediastinal large B-cell lymphoma (PMBL) is a rare form of non-Hodgkin Lymphoma (NHL) representing 2% of mature B-cell NHL in patients ≤ 18 years of age (Lones/Cairo et al., JCO, 2000). PMBL is classified as a distinct mature B-Cell lymphoma and is considered midway in the biologic spectrum between Diffuse Large B-Cell Lymphoma (DLBCL) and classical HL (Abramson et al., Blood, 2005). We have recently reported a significant decrease in EFS among children and adolescent PMBL patients compared with other stage III non-PMBL pediatric DLBCL patients following FAB/LMB 96 therapy, suggesting that children and adolescent PMBL may be an inherently different B-NHL (Gerrard/Cairo et al., Blood, 2013). Since 98% of PMBL express CD20, targeting the CD20 receptor with a CD20 antibody is of high clinical interest. Obinutuzumab (GA101) is a novel, anti-CD20 targeted monoclonal antibody recognizing a unique CD20 type II epitope and has been demonstrated to have efficacy in reducing tumor size and improving survival in other B-NHL xenograft models (Mossner et al., Blood, 2010). OBJECTIVES: We hypothesize that Obinutuzumab may be a future potential targeted agent for the treatment of PMBL, and therefore, we investigated the effect of Obinutuzumab on cell proliferation, programmed cell death, and cell signaling pathways in PMBL. METHODS: CD20+ PMBL, Karpas-1106P cells (Karpas) were obtained from the DSMZ, Germany and Obinutuzumab was generously provided by C. Klein, Roche. Cell proliferation and apoptotic rate were assessed using MTS and FACS analysis, respectively. Statistical significance was determined by one-tailed Student9s t-test. RESULTS: There was a significant decrease of cell proliferation in Obinutuzumab-treated Karpas vs PBS-treated Karpas (1.000 ± 0.000) at 48 hours with 1ug/ml (0.723 ± 0.005, p=0.0007), 10ug/ml (0.688 ± 0.025, p=0.0033), and 20ug/ml (0.627 ± 0.042, p=0.0092) Obinutuzumab treatment. There was a significant increase in cell death in Karpas following 10ug/ml Obinutuzumab treatment (37.790 ± 10.096, p=0.018) as compared to IgG isotope (1.040 ± 0.834, p=0.340) and PBS control (1.190 ± 0.762) at 48 hours by FACS analysis. We also observed significant decreases in the phosphorylation of Stat6 (0.730 ± 0.005, p=0.0001), Akt (0.691 ± 0.002, p CONCLUSIONS: Obinutuzumab significantly induced antiproliferative effects and cell death in PMBL and may be a future potential targeted agent for the treatment of PMBL. The effect(s) of Obinutuzumab will be evaluated in vivo in a NOD/SCID PMBL xenograft mouse model. Citation Format: Changhong Yin, Timmy O9Connell, Janet Ayello, Carmella van de Ven, Sanghoon Lee, Mitchell Cairo. Obinutuzumab (GA101) significantly induces antiproliferative effects and programmed cell death, and significantly downregulates cell signaling pathways in primary mediastinal B-cell lymphoma (PMBL): Obinutuzumab may be a future potential targeted agent for treatment of PMBL. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3096. doi:10.1158/1538-7445.AM2014-3096

Janet Ayello - One of the best experts on this subject based on the ideXlab platform.

  • Obinutuzumab ga101 vs rituximab significantly enhances cell death antibody dependent cytotoxicity and improves overall survival against cd20 primary mediastinal b cell lymphoma pmbl in a xenograft nod scid il2rgnull nsg mouse model a potential target
    Oncotarget, 2020
    Co-Authors: Yaya Chu, Aradhana Awasthi, Sanghoon Lee, Dina Edani, Changhong Yin, Jessica Hochberg, Tishi Shah, Taehoon Chung, Janet Ayello, Carmella Van De Ven
    Abstract:

    Primary mediastinal large B-cell lymphoma (PMBL), a distinct mature B-cell lymphoma, expresses CD20 and has recently been successfully treated with the combination of a type I anti-CD20 monoclonal antibody, rituximab, with multiple combination chemotherapy regimens. Obinutuzumab is a glycoengineered type II anti-CD20 monoclonal antibody (mAb), recognizing a unique CD20 extracellular membrane epitope with enhanced antibody dependent cellular cytotoxicity (ADCC) vs rituximab. We hypothesize that Obinutuzumab vs rituximab will significantly enhance in-vitro and in-vivo cytotoxicity against PMBL. PMBL cells were treated with equal dose of Obinutuzumab and rituximab for 24 hours (1-100 μg/ml). ADCC were performed with ex-vivo expanded natural killer cells at 10:1 E: T ratio. Mice were xenografted with intravenous injections of luciferase expressing Karpas1106P cells and treated every 7 days for 8 weeks. Tumor burden was monitored by IVIS spectrum system. Compared with rituximab, Obinutuzumab significantly inhibited PMBL cell proliferation (p = 0.01), promoted apoptosis (p = 0.05) and enhanced ADCC (p = 0.0002) against PMBL. Similarly, in PMBL xenografted NOD scid gamma mice, Obinutuzumab significantly enhanced survival than rituximab when treated with equal doses (p = 0.05). Taken together our results suggest that Obinutuzumab significantly enhanced natural killer cytotoxicity, reduced PMBL proliferation and prolonged the overall survival in humanized PMBL xenografted NOD scid gamma mice.

  • abstract 1788 enhanced in vitro in vivo cytotoxicity against burkitt lymphoma primary mediastinal large b cell lymphoma by polatuzumab vedotin hu anti cd79b vc mmae pv alone or in combination with Obinutuzumab
    Cancer Research, 2018
    Co-Authors: Aradhana Awasthi Tiwari, Christian Klein, Dina Edani, Janet Ayello, Christeen Azmy, Mitchell S. Cairo
    Abstract:

    Background: Mature B-NHL, including Burkitt lymphoma (BL) and primary mediastinal large B cell lymphoma (PMBL) express CD20+/CD79b+ and have an excellent prognosis, however, subset of patients relapse secondary to chemoimmunotherapy resistant disease and have a dismal prognosis (≤ 20% 5 yr. EFS, Cairo et al. Blood. 2007; Gerrard/Cairo et al., Blood, 2013, Goldman/Cairo et al. Leukemia, 2013). PV has been demonstrated to possess significant preclinical activity against indolent CD79b+NHL (Polson et. al.Can. Res.2009). We previously observed that Obinutuzumab (Anti-CD20 mAb) significantly enhanced cell death and increased overall survival against BL (Awasthi/Cairo et al., BJH 2015) in xenografted NSG mice. However, additive/synergistic effects of PV with Obinutuzumab against mature PMBL/BL are unknown. Objective: To determine the efficacy of the PV or Obinutuzumab/RTX alone or in combination against PMBL and rituximab (RTX) sensitive/resistant BL cell lines. Methods: Raji4RH (provided by M. Barth, MD, Roswell Park Cancer Institute) and Raji/ Karpas1106P (ATCC, USA) were cultured in RPMI. Tumor cells were incubated with PV, and/or anti-CD79b, MMAE (generously supplied by Genentech Inc.) with Obinutuzumab /rituximab (100ug/ml) for 4 hr with NK cells at 10:1 E: T ratio and cytotoxicity was determined by DELFIA cytotoxicity assay. Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ), were divided into 5 groups: PBS, isotype control, PV, antiCD79B mAb and MMAE (5mg/kg). Mice were xenografted with intravenous injections of Luc+ BL and PMBL cells and tumor burden was monitored by IVIS spectrum system. Results: OS of mice receiving PV alone was significantly increased compared to antiCD79b or isotype control in Raji (35.5 vs.17 vs.19.5 days, p=0.0001, 0.0003), Raji4RH (50 vs.18 vs.18.5 days, p=0.0001, 0.0001) and Karpas1106P (150 vs 89 vs 64 days, p=0.03, 0.003), respectively. Obinutuzumab+NK, rituximab+NK compared to PV+NK cells significantly enhanced cell lysis in Raji, 65.9±2.4% vs. 38.9±5.4% vs. 44.24±8.1% (p=0.001 & p=0.001), Raji4RH, 52.8±9.4% vs. 16.04±7.2% vs.47.0±8.2% (p=0.03 & p=NS) and Karpas1106P, 66.10±5.3% vs.48.2±3.9% vs. 61.6±10.06% (p=0.004 & NS), respectively. PV+ Obinutuzumab+NK, significantly improved cytotoxicity compared to PV+ rituximab+NK in Raji, 93.6±6.1% vs 79.9±5.3% (p=0.007), Raji4RH, 78.07±2.05% vs 63.5±0.16% (p=0.004) and Karpas1106P, 88.3±6.3 %vs.73.03±3.03% (p=0.003), respectively. Conclusion: Our preliminary data indicates that PV significantly increased survival in BL and PMBL NSG xenografts compared to anti-CD79b Ab alone. Furthermore, PV in combination with Obinutuzumab significantly enhances cytotoxicity in BL and PMBL compared to Obinutuzumab or PV alone. Citation Format: Aradhana Awasthi Tiwari, Dina Edani, Christeen Azmy, Janet Ayello, Christian Klein, Mitchell S. Cairo. Enhanced in vitro/in vivo cytotoxicity against Burkitt lymphoma/primary mediastinal large B cell lymphoma by polatuzumab vedotin (hu- anti-CD79b-vc-MMAE, PV) alone or in combination with Obinutuzumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1788.

  • Abstract 1788: Enhanced in vitro/in vivo cytotoxicity against Burkitt lymphoma/primary mediastinal large B cell lymphoma by polatuzumab vedotin (hu- anti-CD79b-vc-MMAE, PV) alone or in combination with Obinutuzumab
    Cancer Research, 2018
    Co-Authors: Aradhana Awasthi Tiwari, Christian Klein, Dina Edani, Janet Ayello, Christeen Azmy, Mitchell S. Cairo
    Abstract:

    Background: Mature B-NHL, including Burkitt lymphoma (BL) and primary mediastinal large B cell lymphoma (PMBL) express CD20+/CD79b+ and have an excellent prognosis, however, subset of patients relapse secondary to chemoimmunotherapy resistant disease and have a dismal prognosis (≤ 20% 5 yr. EFS, Cairo et al. Blood. 2007; Gerrard/Cairo et al., Blood, 2013, Goldman/Cairo et al. Leukemia, 2013). PV has been demonstrated to possess significant preclinical activity against indolent CD79b+NHL (Polson et. al.Can. Res.2009). We previously observed that Obinutuzumab (Anti-CD20 mAb) significantly enhanced cell death and increased overall survival against BL (Awasthi/Cairo et al., BJH 2015) in xenografted NSG mice. However, additive/synergistic effects of PV with Obinutuzumab against mature PMBL/BL are unknown. Objective: To determine the efficacy of the PV or Obinutuzumab/RTX alone or in combination against PMBL and rituximab (RTX) sensitive/resistant BL cell lines. Methods: Raji4RH (provided by M. Barth, MD, Roswell Park Cancer Institute) and Raji/ Karpas1106P (ATCC, USA) were cultured in RPMI. Tumor cells were incubated with PV, and/or anti-CD79b, MMAE (generously supplied by Genentech Inc.) with Obinutuzumab /rituximab (100ug/ml) for 4 hr with NK cells at 10:1 E: T ratio and cytotoxicity was determined by DELFIA cytotoxicity assay. Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ), were divided into 5 groups: PBS, isotype control, PV, antiCD79B mAb and MMAE (5mg/kg). Mice were xenografted with intravenous injections of Luc+ BL and PMBL cells and tumor burden was monitored by IVIS spectrum system. Results: OS of mice receiving PV alone was significantly increased compared to antiCD79b or isotype control in Raji (35.5 vs.17 vs.19.5 days, p=0.0001, 0.0003), Raji4RH (50 vs.18 vs.18.5 days, p=0.0001, 0.0001) and Karpas1106P (150 vs 89 vs 64 days, p=0.03, 0.003), respectively. Obinutuzumab+NK, rituximab+NK compared to PV+NK cells significantly enhanced cell lysis in Raji, 65.9±2.4% vs. 38.9±5.4% vs. 44.24±8.1% (p=0.001 & p=0.001), Raji4RH, 52.8±9.4% vs. 16.04±7.2% vs.47.0±8.2% (p=0.03 & p=NS) and Karpas1106P, 66.10±5.3% vs.48.2±3.9% vs. 61.6±10.06% (p=0.004 & NS), respectively. PV+ Obinutuzumab+NK, significantly improved cytotoxicity compared to PV+ rituximab+NK in Raji, 93.6±6.1% vs 79.9±5.3% (p=0.007), Raji4RH, 78.07±2.05% vs 63.5±0.16% (p=0.004) and Karpas1106P, 88.3±6.3 %vs.73.03±3.03% (p=0.003), respectively. Conclusion: Our preliminary data indicates that PV significantly increased survival in BL and PMBL NSG xenografts compared to anti-CD79b Ab alone. Furthermore, PV in combination with Obinutuzumab significantly enhances cytotoxicity in BL and PMBL compared to Obinutuzumab or PV alone. Citation Format: Aradhana Awasthi Tiwari, Dina Edani, Christeen Azmy, Janet Ayello, Christian Klein, Mitchell S. Cairo. Enhanced in vitro/in vivo cytotoxicity against Burkitt lymphoma/primary mediastinal large B cell lymphoma by polatuzumab vedotin (hu- anti-CD79b-vc-MMAE, PV) alone or in combination with Obinutuzumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1788.

  • a comparative global phosphoproteomics analysis of Obinutuzumab ga101 versus rituximab rtx against rtx sensitive and resistant burkitt lymphoma bl demonstrates differential phosphorylation of signaling pathway proteins after treatment
    Oncotarget, 2017
    Co-Authors: Aradhana Awasthi, Christian Klein, Janet Ayello, Carmella Van De Ven, Matthew J Barth, Delphine Rolland, Venkatesha Basrur, Kevin P Conlon, Damian Fermin, Kojo S J Elenitobajohnson
    Abstract:

    We recently demonstrated that Obinutuzumab (GA101), a novel glycoengineered type II CD20 Ab compared to rituximab (RTX) mediates significantly enhanced antibody-dependent cell cytotoxicity (ADCC) in vitro and increased overall survival in a Burkitt lymphoma (BL) xenograft non-obese diabetic severe combined immunodeficiency gamma (NSG) model. In this study we compared the phosphoproteomic changes by pathway analysis following Obinutuzumab vs RTX against RTX-sensitive (Raji) and -resistant BL (Raji4RH). Phosphoproteomic analyses were performed by mass-spectrometry (MS)-based label-free quantitative phosphoproteomic profiling. We demonstrated that 418 proteins in Raji and 377 proteins in Raji 4RH, were differentially phosphorylated (>1.5-fold) after Obinutuzumab vs. RTX. Proteins that were significantly differentially phosphorylated included the B cell antigen receptor (BCR) (PLCG2, BTK and GSK3B), Fc gamma phagocytosis (FCRG2B, MAPK1, PLCG2 and RAF1), and natural killer cell-mediated cytotoxicity (MAPK1, RAF1, PLCG2 and MAPK3) signaling pathways. Differential phosphorylation of BCR or cytotoxicity pathway proteins revealed significant up-regulation of BTK, PLCY2 and ERK1/RAF1 after Obinutuzumab compared to RTX. Silencing of PLCG2 in the BCR and MAPK1 in the cytotoxicity pathway significantly increased BL proliferation and decreased BL cytotoxicity after Obinutuzumab compared to RTX. These results in combination with our previous results demonstrating a significant improvement in in vitro BL cytotoxicity and in vivo BL survival by Obinutuzumab compared to RTX may in part be due to differential effects on selected BL protein signaling pathways.

  • polatuzumab vedotin alone or in combination with Obinutuzumab synergistically enhances in vitro cytotoxicity and cytokine release against cd20 cd79b burkitt lymphoma bl primary mediastinal large b cell lymphoma pmbl
    Blood, 2017
    Co-Authors: Aradhana Awasthi Tiwari, Christian Klein, Dina Edani, Janet Ayello, Mitchell S. Cairo
    Abstract:

    Background: Pediatric Burkitt Lymphoma (PBL) represents the most common malignancy in childhood and adolescent non-Hodgkin Lymphoma (NHL) but occurs in less than 5% of adult NHL cases (Hochberg/Cairo et al, BJH 2009; Miles/Cairo, BJH. 2012). Mature B-cell NHL, including Burkitt lymphoma (BL) and primary mediastinal large B cell lymphoma (PMBL) express CD20+/CD79b+ and have an excellent prognosis with rituximab based immunochemotherapy (Cairo et al Blood. 2007,Goldman/Cairo et al. Leukemia, 2013, Gerrard/Cairo et al. Blood. 2013). The prognosis of PBL has significantly improved over the last 40 years through the use of short and intense multi-agent immunochemotherapy, however, a subset of patients with relapsed/refractory disease has immunochemotherapy resistant disease and a dismal prognosis (≤ 20% 5 yr. EFS),(Cairo et al.Blood. 2007; Cairo et al. JCO.2012). It is therefore critical to investigate and to develop targeted translational strategies in BL/PMBL in order to reduce acute morbidities, decrease late effects, and provide new options for those with recurrent disease. The hu-anti-CD79b-vc-MMAE (Polatuzumab Vedotin, PV) an antibody drug conjugate (ADC) has demonstrated significant preclinical activity against indolent CD79b+NHL (Polson et. al.Can. Res.2009). More recently PV has well tolerated in adults with CD79b refractory CLL (Palanca-Wessels et al. Lancet Oncol, 2014). Obinutuzumab, a glycoengineered type II CD20 antibody, has been shown to enhance cell death and antibody dependent cytotoxicity assay (ADCC) vs. rituximab (RTX) (Herter et al, Clinc Can Res, 2013). We previously observed that PV or Obinutuzumab alone (Awasthi/Cairo et al., AACR 2017, Awasthi/Cairo et al., BJH 2015) significantly enhanced tumor cell death and increased overall survival against BL and PMBL xenografted mice. However, additive/synergistic effects of PV with Obinutuzumab against mature PMBL/BL are unknown. Objective: To determine the efficacy of the PV, Obinutuzumab or RTX alone or in combination with PV+ Obinutuzumab vs. PV+RTX against PMBL and RTX sensitive/resistant BL tumor cell lines. Methods: Raji/Raji4RH (provided by M. Barth, MD, Roswell Park Cancer Institute) and PMBL: Karpas1106P (ATCC, USA) tumor cells were cultured in RPMI with 10 or 20% FBS.Tumor cells were incubated with PV (generously supplied by Genentech Inc.) with Obinutuzumab (generously provided by Roche) or rituximab (100ug/ml) for 4 hrs with NK cells.PBMCs were expanded with lethally irradiated K562-mbIL21-41BBL cells (Lee et al, PLoS One, 2012).CD56+/CD3- isolated and expanded NK cells were used for cytotoxicity assay. Cytotoxicity was determined by DELFIA cytotoxicity assay at 10:1 E: T ratio and cytokine secretion was measured by multiplex ELISA assay kit. Results: Obinutuzumab+NK, RTX+NK (100µg/ml) compared to PV+NK cells (20ug/ml, 10:1E:T ratio) significantly enhanced cell lysis in Raji, 65.9±2.4% vs. 38.9±5.4% vs. 44.24±8.1%, (p=0.001 and p=0.001), Raji4RH, 52.8±9.4% vs. 16.04±7.2% vs.47.0±8.2% (p=0.03 and p=NS), respectively and Karpas1106P, 66.10±5.3% vs.48.2±3.9% vs. 61.6±10.06% (p=0.004 and NS), respectively. PV+ Obinutuzumab+NK, further significantly improved in-vitro cytotoxicity compared to PV+ rituximab+NK, Raji, 93.6±6.1% vs 79.9±5.3% (p=0.007, Figure 1A), Raji4RH, 78.07±2.05% vs 63.5±0.16% (p=0.004, Figure1B) and Karpas1106P, 88.3±6.3 %vs.73.03±3.03% (p=0.003, Figure1C), respectively. Furthermore, IFN-g level from supernatant of PV+Obinutuzumab+NK was significantly increased compared to Obinutuzumab+NK alone, Raji, 22.14±2.1 vs. 9.41±6.5 ug/ml (p=0.01), Raji4RH, 20.30±9.8vs.17.02±2.5 ug/ml (p=0.05) and Karpas1106P, 18.10±1.5vs.9.56±5.5 ug/ml (p=0.03), respectively. Conclusion: Our preliminary data indicates thatPV in combination with Obinutuzumab significantly enhances cell death in RTX sensitive/resistant CD79b+/CD20+ BL and PMBL compared to Obinutuzumab or PV alone. Furthermore, PV+ Obinutuzumab significantly enhanced cytokine secretion in BL (RTX sensitive/resistant) and PMBL compared to Obinutuzumab alone. Disclosures Klein: Roche: Employment, Equity Ownership, Other: patents. Lee: Miltenyi: Speakers Bureau; Cyto-Sen Therapeutics, Inc: Other: Founder, Vice President, Medical Director; Intellia Therapeutics, Inc: Consultancy; Courier Therapeutics, Inc: Membership on an entity9s Board of Directors or advisory committees. Cairo: Jazz Pharmaceuticals: Speakers Bureau.

Aradhana Awasthi Tiwari - One of the best experts on this subject based on the ideXlab platform.

  • abstract 1788 enhanced in vitro in vivo cytotoxicity against burkitt lymphoma primary mediastinal large b cell lymphoma by polatuzumab vedotin hu anti cd79b vc mmae pv alone or in combination with Obinutuzumab
    Cancer Research, 2018
    Co-Authors: Aradhana Awasthi Tiwari, Christian Klein, Dina Edani, Janet Ayello, Christeen Azmy, Mitchell S. Cairo
    Abstract:

    Background: Mature B-NHL, including Burkitt lymphoma (BL) and primary mediastinal large B cell lymphoma (PMBL) express CD20+/CD79b+ and have an excellent prognosis, however, subset of patients relapse secondary to chemoimmunotherapy resistant disease and have a dismal prognosis (≤ 20% 5 yr. EFS, Cairo et al. Blood. 2007; Gerrard/Cairo et al., Blood, 2013, Goldman/Cairo et al. Leukemia, 2013). PV has been demonstrated to possess significant preclinical activity against indolent CD79b+NHL (Polson et. al.Can. Res.2009). We previously observed that Obinutuzumab (Anti-CD20 mAb) significantly enhanced cell death and increased overall survival against BL (Awasthi/Cairo et al., BJH 2015) in xenografted NSG mice. However, additive/synergistic effects of PV with Obinutuzumab against mature PMBL/BL are unknown. Objective: To determine the efficacy of the PV or Obinutuzumab/RTX alone or in combination against PMBL and rituximab (RTX) sensitive/resistant BL cell lines. Methods: Raji4RH (provided by M. Barth, MD, Roswell Park Cancer Institute) and Raji/ Karpas1106P (ATCC, USA) were cultured in RPMI. Tumor cells were incubated with PV, and/or anti-CD79b, MMAE (generously supplied by Genentech Inc.) with Obinutuzumab /rituximab (100ug/ml) for 4 hr with NK cells at 10:1 E: T ratio and cytotoxicity was determined by DELFIA cytotoxicity assay. Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ), were divided into 5 groups: PBS, isotype control, PV, antiCD79B mAb and MMAE (5mg/kg). Mice were xenografted with intravenous injections of Luc+ BL and PMBL cells and tumor burden was monitored by IVIS spectrum system. Results: OS of mice receiving PV alone was significantly increased compared to antiCD79b or isotype control in Raji (35.5 vs.17 vs.19.5 days, p=0.0001, 0.0003), Raji4RH (50 vs.18 vs.18.5 days, p=0.0001, 0.0001) and Karpas1106P (150 vs 89 vs 64 days, p=0.03, 0.003), respectively. Obinutuzumab+NK, rituximab+NK compared to PV+NK cells significantly enhanced cell lysis in Raji, 65.9±2.4% vs. 38.9±5.4% vs. 44.24±8.1% (p=0.001 & p=0.001), Raji4RH, 52.8±9.4% vs. 16.04±7.2% vs.47.0±8.2% (p=0.03 & p=NS) and Karpas1106P, 66.10±5.3% vs.48.2±3.9% vs. 61.6±10.06% (p=0.004 & NS), respectively. PV+ Obinutuzumab+NK, significantly improved cytotoxicity compared to PV+ rituximab+NK in Raji, 93.6±6.1% vs 79.9±5.3% (p=0.007), Raji4RH, 78.07±2.05% vs 63.5±0.16% (p=0.004) and Karpas1106P, 88.3±6.3 %vs.73.03±3.03% (p=0.003), respectively. Conclusion: Our preliminary data indicates that PV significantly increased survival in BL and PMBL NSG xenografts compared to anti-CD79b Ab alone. Furthermore, PV in combination with Obinutuzumab significantly enhances cytotoxicity in BL and PMBL compared to Obinutuzumab or PV alone. Citation Format: Aradhana Awasthi Tiwari, Dina Edani, Christeen Azmy, Janet Ayello, Christian Klein, Mitchell S. Cairo. Enhanced in vitro/in vivo cytotoxicity against Burkitt lymphoma/primary mediastinal large B cell lymphoma by polatuzumab vedotin (hu- anti-CD79b-vc-MMAE, PV) alone or in combination with Obinutuzumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1788.

  • Abstract 1788: Enhanced in vitro/in vivo cytotoxicity against Burkitt lymphoma/primary mediastinal large B cell lymphoma by polatuzumab vedotin (hu- anti-CD79b-vc-MMAE, PV) alone or in combination with Obinutuzumab
    Cancer Research, 2018
    Co-Authors: Aradhana Awasthi Tiwari, Christian Klein, Dina Edani, Janet Ayello, Christeen Azmy, Mitchell S. Cairo
    Abstract:

    Background: Mature B-NHL, including Burkitt lymphoma (BL) and primary mediastinal large B cell lymphoma (PMBL) express CD20+/CD79b+ and have an excellent prognosis, however, subset of patients relapse secondary to chemoimmunotherapy resistant disease and have a dismal prognosis (≤ 20% 5 yr. EFS, Cairo et al. Blood. 2007; Gerrard/Cairo et al., Blood, 2013, Goldman/Cairo et al. Leukemia, 2013). PV has been demonstrated to possess significant preclinical activity against indolent CD79b+NHL (Polson et. al.Can. Res.2009). We previously observed that Obinutuzumab (Anti-CD20 mAb) significantly enhanced cell death and increased overall survival against BL (Awasthi/Cairo et al., BJH 2015) in xenografted NSG mice. However, additive/synergistic effects of PV with Obinutuzumab against mature PMBL/BL are unknown. Objective: To determine the efficacy of the PV or Obinutuzumab/RTX alone or in combination against PMBL and rituximab (RTX) sensitive/resistant BL cell lines. Methods: Raji4RH (provided by M. Barth, MD, Roswell Park Cancer Institute) and Raji/ Karpas1106P (ATCC, USA) were cultured in RPMI. Tumor cells were incubated with PV, and/or anti-CD79b, MMAE (generously supplied by Genentech Inc.) with Obinutuzumab /rituximab (100ug/ml) for 4 hr with NK cells at 10:1 E: T ratio and cytotoxicity was determined by DELFIA cytotoxicity assay. Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ), were divided into 5 groups: PBS, isotype control, PV, antiCD79B mAb and MMAE (5mg/kg). Mice were xenografted with intravenous injections of Luc+ BL and PMBL cells and tumor burden was monitored by IVIS spectrum system. Results: OS of mice receiving PV alone was significantly increased compared to antiCD79b or isotype control in Raji (35.5 vs.17 vs.19.5 days, p=0.0001, 0.0003), Raji4RH (50 vs.18 vs.18.5 days, p=0.0001, 0.0001) and Karpas1106P (150 vs 89 vs 64 days, p=0.03, 0.003), respectively. Obinutuzumab+NK, rituximab+NK compared to PV+NK cells significantly enhanced cell lysis in Raji, 65.9±2.4% vs. 38.9±5.4% vs. 44.24±8.1% (p=0.001 & p=0.001), Raji4RH, 52.8±9.4% vs. 16.04±7.2% vs.47.0±8.2% (p=0.03 & p=NS) and Karpas1106P, 66.10±5.3% vs.48.2±3.9% vs. 61.6±10.06% (p=0.004 & NS), respectively. PV+ Obinutuzumab+NK, significantly improved cytotoxicity compared to PV+ rituximab+NK in Raji, 93.6±6.1% vs 79.9±5.3% (p=0.007), Raji4RH, 78.07±2.05% vs 63.5±0.16% (p=0.004) and Karpas1106P, 88.3±6.3 %vs.73.03±3.03% (p=0.003), respectively. Conclusion: Our preliminary data indicates that PV significantly increased survival in BL and PMBL NSG xenografts compared to anti-CD79b Ab alone. Furthermore, PV in combination with Obinutuzumab significantly enhances cytotoxicity in BL and PMBL compared to Obinutuzumab or PV alone. Citation Format: Aradhana Awasthi Tiwari, Dina Edani, Christeen Azmy, Janet Ayello, Christian Klein, Mitchell S. Cairo. Enhanced in vitro/in vivo cytotoxicity against Burkitt lymphoma/primary mediastinal large B cell lymphoma by polatuzumab vedotin (hu- anti-CD79b-vc-MMAE, PV) alone or in combination with Obinutuzumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1788.

  • polatuzumab vedotin alone or in combination with Obinutuzumab synergistically enhances in vitro cytotoxicity and cytokine release against cd20 cd79b burkitt lymphoma bl primary mediastinal large b cell lymphoma pmbl
    Blood, 2017
    Co-Authors: Aradhana Awasthi Tiwari, Christian Klein, Dina Edani, Janet Ayello, Mitchell S. Cairo
    Abstract:

    Background: Pediatric Burkitt Lymphoma (PBL) represents the most common malignancy in childhood and adolescent non-Hodgkin Lymphoma (NHL) but occurs in less than 5% of adult NHL cases (Hochberg/Cairo et al, BJH 2009; Miles/Cairo, BJH. 2012). Mature B-cell NHL, including Burkitt lymphoma (BL) and primary mediastinal large B cell lymphoma (PMBL) express CD20+/CD79b+ and have an excellent prognosis with rituximab based immunochemotherapy (Cairo et al Blood. 2007,Goldman/Cairo et al. Leukemia, 2013, Gerrard/Cairo et al. Blood. 2013). The prognosis of PBL has significantly improved over the last 40 years through the use of short and intense multi-agent immunochemotherapy, however, a subset of patients with relapsed/refractory disease has immunochemotherapy resistant disease and a dismal prognosis (≤ 20% 5 yr. EFS),(Cairo et al.Blood. 2007; Cairo et al. JCO.2012). It is therefore critical to investigate and to develop targeted translational strategies in BL/PMBL in order to reduce acute morbidities, decrease late effects, and provide new options for those with recurrent disease. The hu-anti-CD79b-vc-MMAE (Polatuzumab Vedotin, PV) an antibody drug conjugate (ADC) has demonstrated significant preclinical activity against indolent CD79b+NHL (Polson et. al.Can. Res.2009). More recently PV has well tolerated in adults with CD79b refractory CLL (Palanca-Wessels et al. Lancet Oncol, 2014). Obinutuzumab, a glycoengineered type II CD20 antibody, has been shown to enhance cell death and antibody dependent cytotoxicity assay (ADCC) vs. rituximab (RTX) (Herter et al, Clinc Can Res, 2013). We previously observed that PV or Obinutuzumab alone (Awasthi/Cairo et al., AACR 2017, Awasthi/Cairo et al., BJH 2015) significantly enhanced tumor cell death and increased overall survival against BL and PMBL xenografted mice. However, additive/synergistic effects of PV with Obinutuzumab against mature PMBL/BL are unknown. Objective: To determine the efficacy of the PV, Obinutuzumab or RTX alone or in combination with PV+ Obinutuzumab vs. PV+RTX against PMBL and RTX sensitive/resistant BL tumor cell lines. Methods: Raji/Raji4RH (provided by M. Barth, MD, Roswell Park Cancer Institute) and PMBL: Karpas1106P (ATCC, USA) tumor cells were cultured in RPMI with 10 or 20% FBS.Tumor cells were incubated with PV (generously supplied by Genentech Inc.) with Obinutuzumab (generously provided by Roche) or rituximab (100ug/ml) for 4 hrs with NK cells.PBMCs were expanded with lethally irradiated K562-mbIL21-41BBL cells (Lee et al, PLoS One, 2012).CD56+/CD3- isolated and expanded NK cells were used for cytotoxicity assay. Cytotoxicity was determined by DELFIA cytotoxicity assay at 10:1 E: T ratio and cytokine secretion was measured by multiplex ELISA assay kit. Results: Obinutuzumab+NK, RTX+NK (100µg/ml) compared to PV+NK cells (20ug/ml, 10:1E:T ratio) significantly enhanced cell lysis in Raji, 65.9±2.4% vs. 38.9±5.4% vs. 44.24±8.1%, (p=0.001 and p=0.001), Raji4RH, 52.8±9.4% vs. 16.04±7.2% vs.47.0±8.2% (p=0.03 and p=NS), respectively and Karpas1106P, 66.10±5.3% vs.48.2±3.9% vs. 61.6±10.06% (p=0.004 and NS), respectively. PV+ Obinutuzumab+NK, further significantly improved in-vitro cytotoxicity compared to PV+ rituximab+NK, Raji, 93.6±6.1% vs 79.9±5.3% (p=0.007, Figure 1A), Raji4RH, 78.07±2.05% vs 63.5±0.16% (p=0.004, Figure1B) and Karpas1106P, 88.3±6.3 %vs.73.03±3.03% (p=0.003, Figure1C), respectively. Furthermore, IFN-g level from supernatant of PV+Obinutuzumab+NK was significantly increased compared to Obinutuzumab+NK alone, Raji, 22.14±2.1 vs. 9.41±6.5 ug/ml (p=0.01), Raji4RH, 20.30±9.8vs.17.02±2.5 ug/ml (p=0.05) and Karpas1106P, 18.10±1.5vs.9.56±5.5 ug/ml (p=0.03), respectively. Conclusion: Our preliminary data indicates thatPV in combination with Obinutuzumab significantly enhances cell death in RTX sensitive/resistant CD79b+/CD20+ BL and PMBL compared to Obinutuzumab or PV alone. Furthermore, PV+ Obinutuzumab significantly enhanced cytokine secretion in BL (RTX sensitive/resistant) and PMBL compared to Obinutuzumab alone. Disclosures Klein: Roche: Employment, Equity Ownership, Other: patents. Lee: Miltenyi: Speakers Bureau; Cyto-Sen Therapeutics, Inc: Other: Founder, Vice President, Medical Director; Intellia Therapeutics, Inc: Consultancy; Courier Therapeutics, Inc: Membership on an entity9s Board of Directors or advisory committees. Cairo: Jazz Pharmaceuticals: Speakers Bureau.

  • comparative phosphoproteomics study between Obinutuzumab ga101 vs rituximab rtx against rtx sensitive resistant burkitt lymphoma bl differentially phosphorylated b cell receptor fc gamma receptor phagocytosis and natural killer cell mediated cytotoxi
    Blood, 2015
    Co-Authors: Aradhana Awasthi Tiwari, Janet Ayello, Carmella Van De Ven, Mona Elmacken, Matthew J Barth, Delphine Rolland, Venkatesha Basrur, Kevin P Conlon, Damine Fermin, Christian Klein
    Abstract:

    Background: Burkitt Lymphoma (BL) is the most common form of NHL in children and adolescents and has an excellent prognosis (≥80% 5years, EFS, Cairo et al. Blood, 2007, Cairo et al. JCO, 2012). The prognosis has improved with the addition of targeted immunotherapy with rituximab (Goldman/Cairo et al, Leukemia, 2013, Goldman/Cairo et al. BJH, 2014). However, a subset of patients with chemoimmunotherapy-resistant disease has a dismal prognosis (≤ 10% 5 years, EFS) (Miles/Cairo et al. BJH, 2012). Deregulation of signaling pathways controlled by protein phosphorylation underlies the pathogenesis of B-cell lymphomas, however, the extent to which they contribute to rituximab resistance is largely unknown (Barth et al. BJH, 2013). Obinutuzumab (GA101), a novel glycoengineered type II CD20 Ab vs. RTX, a Type I CD20 Ab, mediates enhanced cell death & ADCC against diffuse B-cell lymphoma vs. RTX (Mossner et al. Blood, 2010), and was recently approved by FDA and EMA for first line treatment of CLL in combination with chlorambucil. Objective: To evaluate phosphorylation of signaling pathways are differentially altered following Obinutuzumab vs RTX against RTX-sensitive/resistant BL. Methods: Raji (CD20+, ATCC, Manhass, VA) and Raji-4RH (provided by M. Barth, Roswell Park Cancer Institute) were cultured in RPMI with 10% FBS. For in-vitro studies, tumor cells were incubated with 100 µg/ml Obinutuzumab (supplied by Christian Klein, PhD, Roche Research & Early Development, Zurich), and/or RTX for 24 hrs. For Phosphoproteomics analysis, we performed a mass spectrometry-based label-free quantitative phosphoproteomic profiling of the BL cell lines Raji/Raji4RH in the presence/absence of Obinutuzumab or rituximab (100µg/ml for 24h) or isotype control. Six milligrams of protein from each condition were digested by trypsin and peptides and subjected to phosphopeptide enrichment using metal oxide affinity chromatography (MOAC) and immunoprecipitation. An LTQ Orbitrap XL, in-line with a Paradigm MS2 HPLC was employed for acquiring high-resolution MS and MS/MS data that were searched with the Swissprot Human taxonomic protein database (McDonnell and Lim et al, Blood, 2013). Silencing of PLCG2 in Raji and Raji4RH cell lines was carried out according to the manufacturer9s instructions (Dharmacon, PA, USA). Results: Four hundred and eighteen out of total 661 proteins in Raji and 377 out of total 534 proteins in Raji4RH were differentially phosphorylated (>1.5 fold) after Obinutuzumab treatment. Of these proteins, 46 were expressed at significantly higher levels in Obinutuzumab vs. RTX in Raji (Figure 1). However, Raji4RH, the RTX resistant cell line did not show significant increase in phosphorylation of protein following in Obinutuzumab vs. RTX. Proteins differentially phosphorylated in response to Obinutuzumab vs. RTX are involved in the BCR (PLCG2, BTK, GSK3B and RAF-1), FC gamma phagocytosis (FCRG2B, MAPK1, PLCG2 and RAF-1), and Natural Killer cell-mediated cytotoxicity (MAPK1, RAF-1, PLCG2 and MAPK3) signaling pathways in Raji vs. Raji4RH (Figure 2). Differential phosphorylation of BCR signaling pathways proteins (BTK, PLCG2 and GSK3B), validated by western blot studies after incubation with Obinutuzumab vs. RTX in Raji/Raji4RH cell lines, revealed up-regulation of BTK and PLCY2 after Obinutuzumab treatment vs. RTX treatment in Raji BL cell line. Silencing one of the BCR signaling pathway protein, PLCG2 significantly increased cell proliferation and decreased cell death after Obinutuzumab vs. RTX treatment in Raji (P=0.0001 & 0.004) however, there was no change in Raji 4RH RTX resistant cell line. Conclusions: Obinutuzumab and RTX differentially phosphorylate BCR, phagocytosis and cytotoxicity signaling pathways in BL. Knockdown of PLCG2 significantly enhanced BL proliferation and reduced cell death after Obinutuzumab vs. RTX treatment. These results offer insights into alternate therapeutic strategies in the treatment of RTX resistant BL. Disclosures Klein:Roche: Employment.

Dina Edani - One of the best experts on this subject based on the ideXlab platform.

  • Obinutuzumab ga101 vs rituximab significantly enhances cell death antibody dependent cytotoxicity and improves overall survival against cd20 primary mediastinal b cell lymphoma pmbl in a xenograft nod scid il2rgnull nsg mouse model a potential target
    Oncotarget, 2020
    Co-Authors: Yaya Chu, Aradhana Awasthi, Sanghoon Lee, Dina Edani, Changhong Yin, Jessica Hochberg, Tishi Shah, Taehoon Chung, Janet Ayello, Carmella Van De Ven
    Abstract:

    Primary mediastinal large B-cell lymphoma (PMBL), a distinct mature B-cell lymphoma, expresses CD20 and has recently been successfully treated with the combination of a type I anti-CD20 monoclonal antibody, rituximab, with multiple combination chemotherapy regimens. Obinutuzumab is a glycoengineered type II anti-CD20 monoclonal antibody (mAb), recognizing a unique CD20 extracellular membrane epitope with enhanced antibody dependent cellular cytotoxicity (ADCC) vs rituximab. We hypothesize that Obinutuzumab vs rituximab will significantly enhance in-vitro and in-vivo cytotoxicity against PMBL. PMBL cells were treated with equal dose of Obinutuzumab and rituximab for 24 hours (1-100 μg/ml). ADCC were performed with ex-vivo expanded natural killer cells at 10:1 E: T ratio. Mice were xenografted with intravenous injections of luciferase expressing Karpas1106P cells and treated every 7 days for 8 weeks. Tumor burden was monitored by IVIS spectrum system. Compared with rituximab, Obinutuzumab significantly inhibited PMBL cell proliferation (p = 0.01), promoted apoptosis (p = 0.05) and enhanced ADCC (p = 0.0002) against PMBL. Similarly, in PMBL xenografted NOD scid gamma mice, Obinutuzumab significantly enhanced survival than rituximab when treated with equal doses (p = 0.05). Taken together our results suggest that Obinutuzumab significantly enhanced natural killer cytotoxicity, reduced PMBL proliferation and prolonged the overall survival in humanized PMBL xenografted NOD scid gamma mice.

  • abstract 1788 enhanced in vitro in vivo cytotoxicity against burkitt lymphoma primary mediastinal large b cell lymphoma by polatuzumab vedotin hu anti cd79b vc mmae pv alone or in combination with Obinutuzumab
    Cancer Research, 2018
    Co-Authors: Aradhana Awasthi Tiwari, Christian Klein, Dina Edani, Janet Ayello, Christeen Azmy, Mitchell S. Cairo
    Abstract:

    Background: Mature B-NHL, including Burkitt lymphoma (BL) and primary mediastinal large B cell lymphoma (PMBL) express CD20+/CD79b+ and have an excellent prognosis, however, subset of patients relapse secondary to chemoimmunotherapy resistant disease and have a dismal prognosis (≤ 20% 5 yr. EFS, Cairo et al. Blood. 2007; Gerrard/Cairo et al., Blood, 2013, Goldman/Cairo et al. Leukemia, 2013). PV has been demonstrated to possess significant preclinical activity against indolent CD79b+NHL (Polson et. al.Can. Res.2009). We previously observed that Obinutuzumab (Anti-CD20 mAb) significantly enhanced cell death and increased overall survival against BL (Awasthi/Cairo et al., BJH 2015) in xenografted NSG mice. However, additive/synergistic effects of PV with Obinutuzumab against mature PMBL/BL are unknown. Objective: To determine the efficacy of the PV or Obinutuzumab/RTX alone or in combination against PMBL and rituximab (RTX) sensitive/resistant BL cell lines. Methods: Raji4RH (provided by M. Barth, MD, Roswell Park Cancer Institute) and Raji/ Karpas1106P (ATCC, USA) were cultured in RPMI. Tumor cells were incubated with PV, and/or anti-CD79b, MMAE (generously supplied by Genentech Inc.) with Obinutuzumab /rituximab (100ug/ml) for 4 hr with NK cells at 10:1 E: T ratio and cytotoxicity was determined by DELFIA cytotoxicity assay. Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ), were divided into 5 groups: PBS, isotype control, PV, antiCD79B mAb and MMAE (5mg/kg). Mice were xenografted with intravenous injections of Luc+ BL and PMBL cells and tumor burden was monitored by IVIS spectrum system. Results: OS of mice receiving PV alone was significantly increased compared to antiCD79b or isotype control in Raji (35.5 vs.17 vs.19.5 days, p=0.0001, 0.0003), Raji4RH (50 vs.18 vs.18.5 days, p=0.0001, 0.0001) and Karpas1106P (150 vs 89 vs 64 days, p=0.03, 0.003), respectively. Obinutuzumab+NK, rituximab+NK compared to PV+NK cells significantly enhanced cell lysis in Raji, 65.9±2.4% vs. 38.9±5.4% vs. 44.24±8.1% (p=0.001 & p=0.001), Raji4RH, 52.8±9.4% vs. 16.04±7.2% vs.47.0±8.2% (p=0.03 & p=NS) and Karpas1106P, 66.10±5.3% vs.48.2±3.9% vs. 61.6±10.06% (p=0.004 & NS), respectively. PV+ Obinutuzumab+NK, significantly improved cytotoxicity compared to PV+ rituximab+NK in Raji, 93.6±6.1% vs 79.9±5.3% (p=0.007), Raji4RH, 78.07±2.05% vs 63.5±0.16% (p=0.004) and Karpas1106P, 88.3±6.3 %vs.73.03±3.03% (p=0.003), respectively. Conclusion: Our preliminary data indicates that PV significantly increased survival in BL and PMBL NSG xenografts compared to anti-CD79b Ab alone. Furthermore, PV in combination with Obinutuzumab significantly enhances cytotoxicity in BL and PMBL compared to Obinutuzumab or PV alone. Citation Format: Aradhana Awasthi Tiwari, Dina Edani, Christeen Azmy, Janet Ayello, Christian Klein, Mitchell S. Cairo. Enhanced in vitro/in vivo cytotoxicity against Burkitt lymphoma/primary mediastinal large B cell lymphoma by polatuzumab vedotin (hu- anti-CD79b-vc-MMAE, PV) alone or in combination with Obinutuzumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1788.

  • Abstract 1788: Enhanced in vitro/in vivo cytotoxicity against Burkitt lymphoma/primary mediastinal large B cell lymphoma by polatuzumab vedotin (hu- anti-CD79b-vc-MMAE, PV) alone or in combination with Obinutuzumab
    Cancer Research, 2018
    Co-Authors: Aradhana Awasthi Tiwari, Christian Klein, Dina Edani, Janet Ayello, Christeen Azmy, Mitchell S. Cairo
    Abstract:

    Background: Mature B-NHL, including Burkitt lymphoma (BL) and primary mediastinal large B cell lymphoma (PMBL) express CD20+/CD79b+ and have an excellent prognosis, however, subset of patients relapse secondary to chemoimmunotherapy resistant disease and have a dismal prognosis (≤ 20% 5 yr. EFS, Cairo et al. Blood. 2007; Gerrard/Cairo et al., Blood, 2013, Goldman/Cairo et al. Leukemia, 2013). PV has been demonstrated to possess significant preclinical activity against indolent CD79b+NHL (Polson et. al.Can. Res.2009). We previously observed that Obinutuzumab (Anti-CD20 mAb) significantly enhanced cell death and increased overall survival against BL (Awasthi/Cairo et al., BJH 2015) in xenografted NSG mice. However, additive/synergistic effects of PV with Obinutuzumab against mature PMBL/BL are unknown. Objective: To determine the efficacy of the PV or Obinutuzumab/RTX alone or in combination against PMBL and rituximab (RTX) sensitive/resistant BL cell lines. Methods: Raji4RH (provided by M. Barth, MD, Roswell Park Cancer Institute) and Raji/ Karpas1106P (ATCC, USA) were cultured in RPMI. Tumor cells were incubated with PV, and/or anti-CD79b, MMAE (generously supplied by Genentech Inc.) with Obinutuzumab /rituximab (100ug/ml) for 4 hr with NK cells at 10:1 E: T ratio and cytotoxicity was determined by DELFIA cytotoxicity assay. Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ), were divided into 5 groups: PBS, isotype control, PV, antiCD79B mAb and MMAE (5mg/kg). Mice were xenografted with intravenous injections of Luc+ BL and PMBL cells and tumor burden was monitored by IVIS spectrum system. Results: OS of mice receiving PV alone was significantly increased compared to antiCD79b or isotype control in Raji (35.5 vs.17 vs.19.5 days, p=0.0001, 0.0003), Raji4RH (50 vs.18 vs.18.5 days, p=0.0001, 0.0001) and Karpas1106P (150 vs 89 vs 64 days, p=0.03, 0.003), respectively. Obinutuzumab+NK, rituximab+NK compared to PV+NK cells significantly enhanced cell lysis in Raji, 65.9±2.4% vs. 38.9±5.4% vs. 44.24±8.1% (p=0.001 & p=0.001), Raji4RH, 52.8±9.4% vs. 16.04±7.2% vs.47.0±8.2% (p=0.03 & p=NS) and Karpas1106P, 66.10±5.3% vs.48.2±3.9% vs. 61.6±10.06% (p=0.004 & NS), respectively. PV+ Obinutuzumab+NK, significantly improved cytotoxicity compared to PV+ rituximab+NK in Raji, 93.6±6.1% vs 79.9±5.3% (p=0.007), Raji4RH, 78.07±2.05% vs 63.5±0.16% (p=0.004) and Karpas1106P, 88.3±6.3 %vs.73.03±3.03% (p=0.003), respectively. Conclusion: Our preliminary data indicates that PV significantly increased survival in BL and PMBL NSG xenografts compared to anti-CD79b Ab alone. Furthermore, PV in combination with Obinutuzumab significantly enhances cytotoxicity in BL and PMBL compared to Obinutuzumab or PV alone. Citation Format: Aradhana Awasthi Tiwari, Dina Edani, Christeen Azmy, Janet Ayello, Christian Klein, Mitchell S. Cairo. Enhanced in vitro/in vivo cytotoxicity against Burkitt lymphoma/primary mediastinal large B cell lymphoma by polatuzumab vedotin (hu- anti-CD79b-vc-MMAE, PV) alone or in combination with Obinutuzumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1788.

  • polatuzumab vedotin alone or in combination with Obinutuzumab synergistically enhances in vitro cytotoxicity and cytokine release against cd20 cd79b burkitt lymphoma bl primary mediastinal large b cell lymphoma pmbl
    Blood, 2017
    Co-Authors: Aradhana Awasthi Tiwari, Christian Klein, Dina Edani, Janet Ayello, Mitchell S. Cairo
    Abstract:

    Background: Pediatric Burkitt Lymphoma (PBL) represents the most common malignancy in childhood and adolescent non-Hodgkin Lymphoma (NHL) but occurs in less than 5% of adult NHL cases (Hochberg/Cairo et al, BJH 2009; Miles/Cairo, BJH. 2012). Mature B-cell NHL, including Burkitt lymphoma (BL) and primary mediastinal large B cell lymphoma (PMBL) express CD20+/CD79b+ and have an excellent prognosis with rituximab based immunochemotherapy (Cairo et al Blood. 2007,Goldman/Cairo et al. Leukemia, 2013, Gerrard/Cairo et al. Blood. 2013). The prognosis of PBL has significantly improved over the last 40 years through the use of short and intense multi-agent immunochemotherapy, however, a subset of patients with relapsed/refractory disease has immunochemotherapy resistant disease and a dismal prognosis (≤ 20% 5 yr. EFS),(Cairo et al.Blood. 2007; Cairo et al. JCO.2012). It is therefore critical to investigate and to develop targeted translational strategies in BL/PMBL in order to reduce acute morbidities, decrease late effects, and provide new options for those with recurrent disease. The hu-anti-CD79b-vc-MMAE (Polatuzumab Vedotin, PV) an antibody drug conjugate (ADC) has demonstrated significant preclinical activity against indolent CD79b+NHL (Polson et. al.Can. Res.2009). More recently PV has well tolerated in adults with CD79b refractory CLL (Palanca-Wessels et al. Lancet Oncol, 2014). Obinutuzumab, a glycoengineered type II CD20 antibody, has been shown to enhance cell death and antibody dependent cytotoxicity assay (ADCC) vs. rituximab (RTX) (Herter et al, Clinc Can Res, 2013). We previously observed that PV or Obinutuzumab alone (Awasthi/Cairo et al., AACR 2017, Awasthi/Cairo et al., BJH 2015) significantly enhanced tumor cell death and increased overall survival against BL and PMBL xenografted mice. However, additive/synergistic effects of PV with Obinutuzumab against mature PMBL/BL are unknown. Objective: To determine the efficacy of the PV, Obinutuzumab or RTX alone or in combination with PV+ Obinutuzumab vs. PV+RTX against PMBL and RTX sensitive/resistant BL tumor cell lines. Methods: Raji/Raji4RH (provided by M. Barth, MD, Roswell Park Cancer Institute) and PMBL: Karpas1106P (ATCC, USA) tumor cells were cultured in RPMI with 10 or 20% FBS.Tumor cells were incubated with PV (generously supplied by Genentech Inc.) with Obinutuzumab (generously provided by Roche) or rituximab (100ug/ml) for 4 hrs with NK cells.PBMCs were expanded with lethally irradiated K562-mbIL21-41BBL cells (Lee et al, PLoS One, 2012).CD56+/CD3- isolated and expanded NK cells were used for cytotoxicity assay. Cytotoxicity was determined by DELFIA cytotoxicity assay at 10:1 E: T ratio and cytokine secretion was measured by multiplex ELISA assay kit. Results: Obinutuzumab+NK, RTX+NK (100µg/ml) compared to PV+NK cells (20ug/ml, 10:1E:T ratio) significantly enhanced cell lysis in Raji, 65.9±2.4% vs. 38.9±5.4% vs. 44.24±8.1%, (p=0.001 and p=0.001), Raji4RH, 52.8±9.4% vs. 16.04±7.2% vs.47.0±8.2% (p=0.03 and p=NS), respectively and Karpas1106P, 66.10±5.3% vs.48.2±3.9% vs. 61.6±10.06% (p=0.004 and NS), respectively. PV+ Obinutuzumab+NK, further significantly improved in-vitro cytotoxicity compared to PV+ rituximab+NK, Raji, 93.6±6.1% vs 79.9±5.3% (p=0.007, Figure 1A), Raji4RH, 78.07±2.05% vs 63.5±0.16% (p=0.004, Figure1B) and Karpas1106P, 88.3±6.3 %vs.73.03±3.03% (p=0.003, Figure1C), respectively. Furthermore, IFN-g level from supernatant of PV+Obinutuzumab+NK was significantly increased compared to Obinutuzumab+NK alone, Raji, 22.14±2.1 vs. 9.41±6.5 ug/ml (p=0.01), Raji4RH, 20.30±9.8vs.17.02±2.5 ug/ml (p=0.05) and Karpas1106P, 18.10±1.5vs.9.56±5.5 ug/ml (p=0.03), respectively. Conclusion: Our preliminary data indicates thatPV in combination with Obinutuzumab significantly enhances cell death in RTX sensitive/resistant CD79b+/CD20+ BL and PMBL compared to Obinutuzumab or PV alone. Furthermore, PV+ Obinutuzumab significantly enhanced cytokine secretion in BL (RTX sensitive/resistant) and PMBL compared to Obinutuzumab alone. Disclosures Klein: Roche: Employment, Equity Ownership, Other: patents. Lee: Miltenyi: Speakers Bureau; Cyto-Sen Therapeutics, Inc: Other: Founder, Vice President, Medical Director; Intellia Therapeutics, Inc: Consultancy; Courier Therapeutics, Inc: Membership on an entity9s Board of Directors or advisory committees. Cairo: Jazz Pharmaceuticals: Speakers Bureau.