Mona Elmacken

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 3155: Neuroblastoma (NB), Medulloblastoma (MB), and Ewing's sarcoma (ES) express ROR1 and can be effectively targeted with NK cells modified to express an anti ROR1 chimeric antigen receptor (CAR)

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: Metastatic pediatric neuroectodermal solid tumors especially NB, MB, and ES have a dismal prognosis (Perkins et al, PLoS One, 2014; Smoll, Cancer, 2012). Targeted cellular therapy with T or NK cells modified with CARs is a novel approach to chemo-resistant childhood solid tumors (Grupp SA, Clin Cancer Res, 2012; Mackall C,Front Oncol, 2012). NK cells can be significantly expanded by co-culture with genetically engineered K562 cells overexpressing mb-IL21 (Lee D, PLOS, 2012). ROR1 has been identified as a novel target on B cell tumors in which CARs can be developed and utilized for targeted cellular therapy (Hudecek et al., Blood, 2010). Objective: To evaluate the in-vitro cytotoxic activity and function of PBNK expanded ex-vivo with K562 mb-IL21 and nucleofected with mRNA encoding an anti ROR1-CAR against NB, MB, and ES. Material and Methods: PBNK were expanded with irradiated K562 Clone 9.mb-IL21 (generously provided by Lee D, MD, PhD, MD Anderson, TX). Ex-vivo expanded PBNK (ExPBNK) cells were electroporated with anti ROR1-CD28-41BBl-CD3ζ-tEGFR-mRNA. The cell surface expression of the ROR1-CAR on NK cells was detected using anti-mouse IgG, F(ab’)2. Cytotoxicity of ROR1 CAR-NK cells was investigated against NB (SKNBE2, SKNFI & SHSY5Y), MB (DAOY) and ES (TC71, EWS 502 & A673) cell lines by DELFIA cytotoxicity assay at an E:T ratio of 10:1. Intracellular staining of CD107a, interferon gamma, perforin and granzyme B was performed using a 10:1 E:T ratio of ROR1-CAR-NK cells against tumor targets and analyzed on the MACSQuant flow cytometer. Results: NB, MB, and ES cell lines expressed ROR1 (50.2±15.6%), (55.5±5.1%), and (31.5±12%), respectively. Expansion of NK cells was significantly increased 3988 ± 435 fold (p = 0.00001) at day 14 vs day 0. CAR expression after nucleofection was measured by F(ab’)2 staining and showed a significant increase in anti-ROR1-CAR- (88.3±1.7%) vs mock-electroporated NK cell populations (8.1± 6.9%) p = 0.0001 at 36-48 hours. Anti-ROR1-CAR-NK cells exhibited significantly increased lysis of ROR1 expressing tumor cell lines compared to mock NK cells (93±4.6% vs 63.6±7.4%) p = 0.00001 at an E:T of 10:1. Similarly, expression of CD107a (46.1±9.1 vs 27.6±2.4%) p = 0.001, interferon gamma (34.1±11.6 vs 16.7±6.7%) p = 0.003, granzyme B (68.5±8.9 vs 46±7.2%) p = 0.002, and perforin (51.3±7.7 vs 30.3±11.9%) p = 0.002 were significantly increased in anti-ROR1-CAR-NK cells vs Mock-NK cells at 10:1 E:T ratio against the ROR1 expressing targets. Conclusion: Anti-ROR1-CAR-ex-PBNK cells had significant enhanced cytotoxicity and significantly increased CD107a, interferon gamma, perforin, and granzyme B activity against ROR1 expressing tumors. Future directions include investigating the ex-PBNK anti ROR1-CAR cells in-vivo against ROR1 expressing pediatric solid tumors. Citation Format: Mona Elmacken, Aradhana Awasthi, Janet Ayello, Carmella VanDeVen,, Wen Luo, Yanling Liao, Stanley Riddell, Mitchell S. Cairo. Neuroblastoma (NB), Medulloblastoma (MB), and Ewings sarcoma (ES) express ROR1 and can be effectively targeted with NK cells modified to express an anti ROR1 chimeric antigen receptor (CAR). [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 3155. doi:10.1158/1538-7445.AM2015-3155

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