Abstract

Background: Aggressive non-Hodgkin lymphoma (NHL) represents >90% of all NHL that occur in children and adolescents. Among all NHLs, Burkitt Lymphoma (BL) is the most common NHL in children and adolescents and has an excellent prognosis (≥80% 5 yrs, EFS) following short but intense multi-agent chemotherapy (Cairo et al. Blood, 2007). Patients who relapse with CD20+ B-NHL and B cell Acute lymphoblastic leukemia (B-ALL) have a dismal prognosis, 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-NHL/ALL 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 glycoengineered type II CD20 antibody, has been shown to enhance cell death and ADCC vs. RTX (Herter et al, Clinc Canc 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 and sensitive BL and pre-B-ALL tumor targets in-vitro and in-vivo 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. 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. Cell death was evaluated by staining with AnnexinV/7AAD and analysis by flow-cytometry. Loucy cells (CD20-) were used as the negative control. ADCC were performed with K562-IL-15-41BBL expanded NK cells (Ayello/Cairo et al. ASH, 2010) at 20:1 effector: target ratio (E: T, n=3) using an europium release assay (Perkin-Elmer).The lentiviral construct, pSico PolII-eGFP-Luc2, was transfected into Raji, Raji 4RH (RTX resistant), U698M and Loucy for in vivo evaluation by BLI. Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ), mice, bred in-house under pathogen free conditions, were divided into 5 groups: PBS only (control), isotype control (IgG), obinutuzumab 10 mg/kg, obinutuzumab (30 mg/kg), and RTX (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 monitored for tumor burden and survival for up to 12 weeks ( approx. 80 days) via bioluminescent imaging (BLI) using the IVIS Spectrum system.

Results: Obinutuzumab compared to RTX (100 mg/ml, 24hrs), significantly enhanced cell death in Raji 45.1±3.3% vs 32.7±6.8%, (p=0.005), Raji4RH 15.8±2.2% vs 2.1±1.5% (p=0.001) and U698-M 40.5±2.9 % vs 26.36±2.6% (p=0.001) n=6. Obinutuzumab vs RTX also elicited a significant increase ADCC with K562-IL15-41BBL expanded NK cells, in 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±6 % vs. 45.56± 0.1% (p=0.001) n=3.

Further, we found that, in vivo, 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=0.05), Raji4RH (p=0.024) and U698-M (p=0.03) (Figure1: A, B and C).

Conclusion: Obinutuzumab significantly enhances cell death and NK mediates ADCC in sensitive and RTX resistant CD20+ B-NHL and B-ALL compared to RTX. These preliminary studies also 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 an equal dose of RTX. Obinutuzumab may be a novel agent to investigate as adjuvant therapy in patients with relapsed refractory CD20+ B-NHL and/or B-ALL.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.