Aradhana Awasthi Tiwari

Abstract 2902: Obinutuzumab (GA101) significantly increases overall survival against CD20+ rituximab-sensitive and -resistant Burkitt (BL) and acute lymphoblastic leukemia (B-ALL): potential targeted therapy in patients with high risk BL and pre-B-ALL

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Patients who relapse with CD20+ B-NHL and B cell Acute lymphoblastic leukemia (B-LL) have a dismal prognosis, which is often associated with chemotherapy resistance and may require alternative therapeutic strategies (Cairo et al. JCO, 2012, Barth/Cairo et al. BJH, 2013). Rituximab (RTX) in combination with FAB 96 chemotherapy is a safe and well-tolerated and is associated with > 90% 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+ B-LL to an increase risk of relapse and/or disease progression (Barth/Cairo et al. BJH, 2012; Tsai et al. Cl. Can. Res, 2012). Obinutuzumab, a novel type II glycoengineered CD20 antibody, mediates enhanced cell death and ADCC vs. RTX (Bologna L et al. JI, 2012), and was recently approved by FDA for first line treatment of CLL in combination with chlorambucil. Objective: To evaluate anti-tumor activity of obinutuzumab vs RTX against RTX resistant and sensitive BL and pre-B-ALL in xenografted NSG mice. Methods: Raji (CD20+) and Loucy (T-ALL, CD20-), (ATCC, Manhass, VA), U698-M (CD20+, DSMZ, Germany) and Raji-4RH (provided by M. Barth, Roswell Park Cancer Institute) were cultured in RPMI with 10% FBS. The lentiviral construct, pSico PolII-eGFP-Luc2, was transfected into Raji, Raji 4RH (RTX resistant), U698M and Loucy. Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ), mice were bred in house under pathogen free conditions, NSG mice were divided into 5 groups: PBS only (control), isotype control (IgG), obinutuzumab 10 mg/kg (generously supplied by Christian Klein, PhD (Roche)), obinutuzumab (30 mg/kg), and rituximab (30 mg/kg). Mice were xenografted with intravenous injections of Luc+ Raji, Raji4RH, U698M and Loucy cells at 5x106 tumor cells/mouse. 6-8 days after tumor cell injection, mice were then injected every 7 days with the respective therapy for 8 weeks. Mice were closely monitored for tumor burden and survival for up to 12 weeks ( approx. 80 days) via bioluminescent imaging (BLI) using the IVIS Spectrum system. Results: We demonstrated that obinutuzumab was significantly more effective than RTX when administered at the same doses in BL (RTX resistant/sensitive) and pre-B-ALL xenografts. Overall survival in mice receiving 30 mg/kg of obinutuzumab was significantly increased when compared to mice receiving 30 mg/kg of RTX in BL; Raji (p=0.0002), Raji4RH (p=0.01) and U698-M (p=0.001), respectively. Conclusion: These preliminary studies demonstrate that RTX sensitive/resistant BL and pre-B-ALL xenografted mice display significantly increased survival when given 30 mg/kg of obinutuzumab and decreased tumor burden in BL and Pre-B-ALL xenografts compared to equal dose of RTX. Citation Format: Aradhana A. Tiwari, Janet Ayello, Carmella van de Ven, Matthew J. Barth, Mitchell S. Cairo. Obinutuzumab (GA101) significantly increases overall survival against CD20+ rituximab-sensitive and -resistant Burkitt (BL) and acute lymphoblastic leukemia (B-ALL): potential targeted therapy in patients with high risk BL and pre-B-ALL. [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 2902. doi:10.1158/1538-7445.AM2014-2902

Abstract 516: Obinutuzumab (GA101): A novel type II glycoengineered CD20 antibody exhibits enhanced cell death against Rituximab-resistant and -sensitive cell lines in B-cell non-Hodgkin lymphoma

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Rituximab (RTX), an antibody targeting the B-cell antigen CD20, in combination with chemotherapy, is safe, well tolerated and associated with > 90% EFS in children with advanced mature B-Cell NHL (Cairo M.S. et al, ASCO 2010). However, RTX resistance has evolved as one of the limiting factors of such therapy and the exact cause of this resistance is not well understood. C-terminal deletion mutations of CD20 may be one possible reason such resistance or for relapse (Mishima Y. et al, Blood Cancer Journal 2011). Obinutuzumab (GA101), a novel type II glycoengineered CD20 antibody of the IgG1 isotype, demonstrates superior cell death induction and its glyco-engineered Fc region has shown to cause significant enhanced ADCC (Mossner et al, Blood 2010; Niederfellner G. et al, Blood 2011). The objective of this study was to evaluate the efficacy of GA101 as compared to RTX against RTX resistant cell lines (Raji-2R and Raji-4RH, generously supplied by M. Barth, MD, Roswell Park Cancer Center, Buffalo, NY) in-vitro. Methods: We evaluated the efficacy of GA101 against Raji (Burkitt Lymphoma), a RTX sensitive cell line (RSCL) and RTX resistant cell lines (RRCL), Raji-2R and Raji-4R (Barth M. et al, ASH 2010). All cell lines were cultured in RPMI with 10% FBS and incubated with dose escalation of GA101 (1-100 µg/ml) for 24 hrs. Cell death was evaluated by staining with AnnexinV/7AAD using a standard kit (BD Biosciences) and flow-cytometry. B-Cell Leukemia-Lymphoma (BLL) U-698-M cells (CD20+) (DSMZ, Germany) were used as the positive control; whereas, Loucy cells (CD20−) (T-ALL) (ATCC, Manhass, VA) were used as the negative control. Results: At 100 µg/ml of GA101 when incubated for 24 hrs, Raji and U-698-M demonstrated 26.5±0.42% and 32.4±1.9% cell death, respectively. Cell death of the RRCL, Raji-2R and Raji-4RH, was 16.3±3.4% and 17.5±0.07% respectively, at 100 µg/ml of GA101 at 24 hrs. Significant cell death was demonstrated with RSCL as compared to the RRCL, Raji vs Raji-2R (p<0.05) and Raji vs Raji-4RH (p<0.01). There was no significant change in cell death with any concentration of GA101 with the Loucy cell line. Conclusion: Based on these findings, GA101 is a promising novel CD20 antibody for the treatment of RTX resistant B-Cell Lymphomas and related B-Cell malignancies. Further studies are aimed to identify novel candidates that may increase the efficacy of GA101 against RTX resistant cell lines in-vitro and in-vivo. One such approach is to combine GA101 with activated NK cells or a chemotherapeutic agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 516. doi:1538-7445.AM2012-516

Abstract 2656: Polatuzumab vedotin significantly enhancesin vitrocell death and overall survival against CD79b+ Burkitt lymphoma (BL)/primary mediastinal large B-cell lymphoma (PMBL) NSG xenograft mice

Background: Mature B-NHL, including Burkitt lymphoma and primary mediastinal large B cell lymphoma express CD79b+ and have an excellent prognosis with chemo-immunotherapy (Cairo et al Blood, 2007, Gerrard/Cairo et al. Blood, 2013). However, a subset of patients with relapsed/refractory mature B-NHL has chemoimmunotherapy resistant disease a dismal prognosis (≤ 10% 5 years, EFS) (Cairo et al. JCO, 2012). The antibody drug conjugates (Polatuzumab Vedotin, PV) has demonstrated significant preclinical activity against indolent CD79b+NHL (Polson et al.Can. Res. 2009). More recently PV has been safe and well tolerated in adult with CD79b refractory CLL (Palanca-Wessels et al. Lancet Oncol, 2014) but its preclinical activity against mature B-NHL (BL/PMBL) is unknown. Objective: To determine the efficacy of the PV against CD79b+ PMBL and rituximab (RTX) sensitive/resistant BL tumor cell lines in-vitro and in-vivo. Design/Methods: Raji/Raji4RH (BL, provided by M. Barth, Roswell Park Cancer Institute) and Karpas1106P and MedB-1(PMBL) were cultured in 10-20% RPMI. Tumor cells were incubated with hu anti-CD79b-vc-MMAE, and/or anti-CD79b, MMAE or huIgG1 (generously supplied by Genentech Inc.) for 24 hrs. Cell death was evaluated by staining with annexin V/7AAD and analyzed by flow cytometry, n=3. Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ), were divided into 5 groups: PBS only (control), isotype control (IgG), PV (5mg/kg), anti-CD79b mAb (5 mg/kg) and MMAE (5 mg/kg). Mice were xenografted with intravenous injections of Luc+ Raji/Raji4RH and Karpas1106P cells as we have previously demonstrated (Awasthi/Cairo et al, BJH, 2015). Mice were treated twice a week for 6 weeks. Tumor burden was monitored by IVIS spectrum system. Results: Anti-CD79b-vc-MMAE compared to anti-CD79b Ab or IgG1 Ab alone (10µg/ml, 24hrs), significantly enhanced cell death in Raji, 47.2±1.3% vs 29.1±6.0% vs. 28.2±4.3%, (p=0.0008 and p=0.00006), Raji4RH, 29.8±9.1% vs 25.4±3.9% vs. 18.0±8.2% (p=NS and p=0.03), Karpas1106P, 46.8±5.3% vs 33.8±3.5% vs. 26.2±0.4% (p=0.02 and 0.006) and MedB-1, 47.4±2.2% vs 27.6±2.4% vs. 23.9±1.7% (p=0.002 and 0.0001), respectively. Further, median survival time in mice receiving 5 mg/kg of PV was significantly increased when compared to mice receiving 5 mg/kg of anti-CD79b Ab or isotype control in Raji, Raji4RH and Karpas1106P (35.5 vs.17 vs. 19.5 days, p=0.0001, 0.0003, 50 vs. 18 vs. 18.5 days and 150 vs 89 vs 64 days, p=0.03 ,0.003, respectively) Conclusions: Our preliminary data indicates that PV significantly enhances cell death in RTX sensitive/ resistant BL and PMBL compared to CD79b Ab or isotype control. Furthermore, PV significantly increased survival in BL and PMBL NSG xenografts Citation Format: Aradhana A. Tiwari, Janet Ayello, Christeen Azmy, Carmella van de Ven, Mitchell S. Cairo. Polatuzumab vedotin significantly enhances in vitro cell death and overall survival against CD79b+ Burkitt lymphoma (BL)/primary mediastinal large B-cell lymphoma (PMBL) NSG xenograft mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2656. doi:10.1158/1538-7445.AM2017-2656

Abstract 579: Antibody drug conjugates (anti-CD79b-vc-MMAE, Polatuzumab Vedotin) exhibit enhanced cell death targeted to CD79b+ Burkitt lymphoma (BL) and primary mediastinal large B-cell lymphoma (PMBL)

Background: Mature B-NHL, including Burkitt lymphoma (BL) and primary mediastinal large B cell lymphoma (PMBL) express CD79b+ and have an excellent prognosis with chemo-immunotherapy (Cairo et al Blood, 2007,Goldman/Cairo et al. Leukemia, 2013, Gerrard/Cairo et al.Blood, 2013). However, a subset of patients with relapsed/refractory mature B-NHL has chemoimmunotherapy resistant disease a dismal prognosis (≤ 10% 5 years, EFS) (Cairo et al. JCO, 2012, Miles/Cairo et al. BJH, 2012). The anti-CD79b-vc-MMAE antibody drug conjugates (Polatuzumab Vedotin, PV) has demonstrated significant preclinical activity against indolent CD79b+NHL (Polson et. al.Can. Res. 2009, Dornan et al. Blood, 2009). More recently PV has been safe and well tolerated in adult with CD79b refractory NHL (Palanca-Wessels et al. Lancet oncol, 2015) but its preclinical activity against mature BL/PMBL is unknown. Objective: To determine the efficacy of ADC (anti-CD79b-vc-MMAE) against RTX sensitive/resistant CD79+ BL and PMBL tumor cell lines in-vitro. Methods: BL: Raji/Raji4RH (provided by M. Barth, Roswell Park Cancer Institute) and PMBL: Karpas1106p and MedB-1 were cultured in RPMI with 10 or 20% FBS. Tumor cells were incubated with hu anti- CD79b-vc-MMAE, and/or anti-CD79b, MMAE or huIgG1 (generously supplied by Genentech Inc.) for 24 hrs. Cell death was evaluated by staining with annexin V/7AAD and cell proliferation was analyzed by alamar blue by flow-cytometry, n = 3. Results: Hu- anti -CD79b-vc-MMAE compared to hu anti-CD79b Ab or control hu IgG1 Ab alone (10μg/ml, 24hrs), significantly enhanced cell death (apoptosis) in Raji, 47.2±1.3% vs 29.1±6.0% vs. 28.2±4.3%, (p = 0.0008 and p = 0.00006), Raji4RH, 29.8±9.1% vs 25.4±3.9% vs. 18.0±8.2% (p = NS and p = 0.03), Karpas1106p, 46.8±5.3% vs 33.8±3.5% vs. 26.2±0.4% (p = 0.02 and 0.006) and MedB-1, 47.4±2.2% vs 27.6±2.4% vs. 23.9±1.7% (p = 0.002 and 0.0001), respectively. Hu anti- CD79b-vc-MMAE also significantly reduced cell proliferation compared to hu anti- CD79b Ab or control hu anti -IgG1 Ab alone (20μg/ml, 24hrs). Raji, 56.1±5.1% vs 38.1±0.7% vs. 14.8±0.4% (p = 0.001 and p = 0.0008), Raji4RH, 53.4±5.4% vs 23.4±5.51% vs. 11.3±0.8% (p = 0.004 and 0.006), Karpas1106p, 46.4±0.3%vs 29.0±1.5% vs. 15.9±0.6% (p = 0.005 and 0.00007) and MedB-1, 47.2±7.5% vs 27.7±8.5% vs. 12.3±0.5% (p = 0.01 and p = 0.0005), respectively. Conclusion: Our preliminary data indicates that hu anti- CD79b-vc-MMAE significantly enhances cell death and reduced cell proliferation in sensitive/ RTX resistant CD79b+ BL and PMBL compared to CD79b Ab or isotype control IgG1 Ab alone. Hu anti- CD79b-vc-MMAE may be a novel agent to investigate as immunotherapeutic agent in patients with relapsed refractory CD79b+ BL and/or PMBL. Citation Format: Aradhana Awasthi Tiwari, Janet Ayello, Carmella van de Ven, Lisa Kurien, Matthew J. Barth, Mitchell S. Cairo. Antibody drug conjugates (anti-CD79b-vc-MMAE, Polatuzumab Vedotin) exhibit enhanced cell death targeted to CD79b+ Burkitt lymphoma (BL) and primary mediastinal large B-cell lymphoma (PMBL). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 579.

Abstract 3893: Obinutuzumab (GA101) versus rituximab against rituximab-sensitive and -resistant Burkitt lymphoma (BL) differentially phosphorylate BCR, Fc-gamma receptor, and natural killer cell-mediated cytotoxicity signaling pathways

Background: Burkitt Lymphoma (BL) is the most common NHL in children and adolescents and has an excellent prognosis (≥80% 5years, EFS, Cairo et al. Blood, 2007) and further improved with the addition of rituximab (Goldman/Cairo et al, Leukemia, 2013, Cairo et al. JCO, 2012). However, a subset of patients with chemoimmunotherapy resistant disease has a dismal prognosis (≤ 10% 5 years, EFS) (Miles/Cairo et al. BJH, 2012, Barth et al. BJH, 2013). Obinutuzumab, a novel glycoengineered type II CD20 Ab, mediates enhanced cell death & ADCC against B-cell lymphoma vs. RTX (Awasthi/Cairo et al. BJH, 2015), and was recently FDA and EMA approved for first line treatment of CLL in combination with chlorambucil. Objective: To evaluate phosphorylation of signaling pathway altered differentially following obinutuzumab vs RTX against RTX-sensitive/resistant BL Methods: Raji (CD20+) and Raji-4RH) cells were cultured in RPMI with 10% FBS. Tumor cells were incubated with 100 μg/ml obinutuzumab, 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 or isotype control. Silencing of PLCG2 (Dharmacon, USA) and MAPK1 (Sigma Aldrich, USA) in Raji/Raji4RH cell lines was carried out according to the manufacturer9s instructions Results: In all 978 unique phosphorylated proteins were identified. Out of these 661 phosphoproteins were identified after obinutuzumab vs. 615 in RTX treatment, respectively. For the Raji4RH, 534 phosphoproteins were identified after obinutuzumab and 534 in RTX treatment, respectively (Fig.1). Functional annotation of proteins differentially phosphorylated in response to obinutuzumab vs. RTX (>1.5-fold) reveals the involvement of the BCR (PLCG2, BTK & GSK3B), FC gamma phagocytosis (FCRG2B, MAPK1 & RAF1), and Natural killer cell-mediated cytotoxicity (MAPK1, RAF1& PLCG2) signaling pathways (Fig. 2). Differential phosphorylations of proteins involved in BCR or cytotoxicity pathways were validated by western blot after incubation with obinutuzumab vs. RTX in Raji/ Raji4RH cell lines, revealed up regulation of BTK, PLCY2 and ERK1/RAF1 after obinutuzumab vs. RTX treatment in Raji. Silencing, of PLCG2 and MAPK1 pathway, significantly increased cell proliferation and decreased cytotoxicity after obinutuzumab treatment in Raji (P = 0.0002 & 0.000002) but no change in Raji4RH. Conclusions: Obinutuzumab and RTX differentially phosphorylate BCR, and cytotoxicity signaling pathways. Obinutuzumab function differentially in RTX resistant and sensitive BL cell lines which may provide insights into alternate therapeutic strategy in RTX resistant BL. Citation Format: Aradhana Awasthi Tiwari, Delphine C.m. Rolland, Mona Elmacken, Janet Ayello, Lisa Kurien, Carmella van de Ven, Venkatesha Basrur, Kevin Conlon, Damine Fermin, Matthew J. Barth, Christian Klein, Kojo S.j. Elenitoba-Johnson, Megan Lim, Mitchell S. Cairo. Obinutuzumab (GA101) versus rituximab against rituximab-sensitive and -resistant Burkitt lymphoma (BL) differentially phosphorylate BCR, Fc-gamma receptor, and natural killer cell-mediated cytotoxicity signaling pathways. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3893.

Abstract 2479: Obinutuzumab compared to Rituximab significantly enhances cell death, antibody dependent cytotoxicity (ADCC) and improves overall survival against CD20+ rituximab-sensitive/-resistant Burkitt Lymphoma (BL) and precursor Lymphoblastic L

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: CD20, expressed on normal and malignant B-cells, has proven to be a useful target for immunotherapeutic approaches in hematological malignancies. (Dalle, S et al. Mol. Can. Ther., 2011) Patients who relapse with CD20+ BL/ B-ALL have a dismal prognosis, often associated with chemotherapy resistance and may require alternative therapeutic strategies (Cairo et al. Blood, 2007, Cairo et al. JCO, 2012, Barth/Cairo et al. BJH, 2013,Goldman/Cairo et al. Leukemia, 2013). Obinutuzumab, a novel glycoengineered type II CD20 antibody, has been shown to enhance cell death and ADCC vs. RTX (Herter et al, Clinc Can Res, 2013), and was recently approved by FDA and EMA for first line treatment of CLL in combination with chlorambucil. Objective: To evaluate anti-tumor activity of obinutuzumab vs RTX against RTX resistant/sensitive BL and pre-B-ALL tumor targets in-vitro/ in-vivo in xenografted NSG mice. Methods: Raji (CD20+), (ATCC, Manhass, VA), U698-M (CD20+, DSMZ, Germany) and Raji-4RH (provided by M. Barth, Roswell Park Cancer Institute) were cultured in RPMI with 10% FBS. Tumor cells were incubated with 100 μg/ml obinutuzumab (Hoffmann La Roche (Switzerland)), and/or RTX for 48 hrs. Cell death was evaluated by staining with Ann.V/7AAD by flow-cytometry. ADCC were performed with K562-IL-15-41BBL expanded NK cells at 20:1E: T ratio. Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ), were divided into 5 groups: PBS only (control), isotype control (IgG), obinutuzumab (10 and 30 mg/kg), and RTX (30 mg/kg). Mice were xenografted with intravenous injections of Luc+ Raji, Raji4RH and U698M cells at 5×106 tumor cells/mouse. Mice were treated every 7 days for 8 weeks. Tumor burden was monitored by IVIS spectrum system. Results: Obinutuzumab vs. RTX (100 μg/ml), significantly enhanced cell death in Raji, 35.6 ± 3.1 vs. 25.1± 2.0% (p = 0.001), Raji2R, 18.2 ± 0.9 vs. 7.8 ± 2.4% (p = 0.001), Raji4RH, 19.7 ± 2.2 vs. 7.97 ± 3.4% (p = 0.001) and U698-M 47.3 ± 4.9 vs 23.2 ± 0.50%, (p = 0.001). Obinutuzumab vs RTX also elicited a significant increase in ADCC in expanded NK cells, Raji 73.8± 8.1% vs 56.81± 4.6% (p = 0.001), Raji-2R, 38.0 ± 2.0 vs 21.6 ± 1.2% (p = 0.0001), Raji-4RH 40.0±1.6% vs 0.5±1.1%, (p = 0.001), and U-698-M 70.0±6% vs. 45.56± 0.1%, (p = 0.001). Further, overall survival in mice receiving 30 mg/kg of obinutuzumab was significantly increased when compared to mice receiving 30 mg/kg of RTX; Raji (p = 0.05), Raji4RH (p = 0.024) and U698-M (p = 0.03). Conclusion: Obinutuzumab significantly enhances cell death and NK mediated ADCC in RTX sensitive/ resistant CD20+ BL and pre-B-ALL vs. RTX. Furthermore, obinutuzumanb significantly increased survival and decreased tumor burden in BL and Pre-B-ALL xenografts compared to an equal dose of RTX. Citation Format: Aradhana Awasthi Tiwari, Janet Ayello, Carmella Vandeven, Mona Elmacken, Matthew J. Barth, Christian Klein, Mitchell S. Cairo. Obinutuzumab compared to Rituximab significantly enhances cell death, antibody dependent cytotoxicity (ADCC) and improves overall survival against CD20+ rituximab-sensitive/-resistant Burkitt Lymphoma (BL) and precursor Lymphoblastic L [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2479. doi:10.1158/1538-7445.AM2015-2479