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

Obinutuzumab (GA101) is a type II, glycoengineered, humanized anti-CD20 monoclonal antibody (Ab). Obinutuzumab binds with high affinity and selectivity to a type II binding region of the CD20 protein present on all B cells except stem or plasma cells. Unlike type I anti-CD20 monoclonal Abs, type II anti-CD20 Abs promote direct cell death induction without the need for a cross-linking Ab. Patients who relapse with CD20+ B-NHL and early relapsed pre-B-ALL have a dismal prognosis, often associated with chemotherapy resistance (Cairo et al. JCO, 2012, Barth/Cairo et al. BJH, 2013) and require alternative therapeutic strategies. RTX in combination with FAB/LMB 96 chemotherapy is a safe, well-tolerated treatment that is associated with > 95% EFS in children with newly diagnosed and advanced mature B-Cell NHL (Goldman/Cairo et al. Leukemia, 2013). Resistance to RTX, however, may predispose patients with CD20+ NHL/Pre-B-ALL to an increased risk of relapse and or disease progression (Barth/Cairo et al. BJH, 2013).

To evaluate the anti-tumor activity of obinutuzumab vs RTX against pre-B-LL in-vivo in severe combined immune-deficient IL2 receptor gamma chain knockout (NSG) and NOD/SCID mice.

U698-M (Pre-B cell ALL, CD20⁺, DSMZ, Germany), Loucy cells (T-ALL, CD20- ATCC, Manasass, VA) were maintained in RPMI with 10% FBS at 370C, 5% CO2. The mammalian expression construct ffLUCZeo-pcDNA (kindly provided by L.Cooper, MD,PhD) was transfected into U698M and Loucy (Luc+) tumor targets for later evaluation by bioluminescent imaging (BLI). Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, no functional T, B and NK cells) and NOD/SCID (absence of functional T and B cells) mice were bred in-house and treated in pathogen free conditions at the animal facility. The in vivo cytotoxic activity of obinutuzumab and RTX was determined by dividing NSG and NOD/SCID mice into 5 groups: PBS only (control), isotype control (IgG), obinutuzumab (10 mg/kg) (kindly provided by C. Klein, Roche Glycart AG), obinutuzumab (30 mg/kg), and rituximab (30 mg/kg). Mice received intravenous injections of Luc+ U698M and Loucy cells at 5x106 tumor cells/mouse. 6-8 days after tumor cell injection, mice were injected every 7 days with the respective therapy for 8 weeks. Mice were closely monitored for tumor burden progression/regression and survival for up to 12 weeks (approx. 80 days) via BLI using the IVIS Spectrum System.

We observed that obinutuzumab was significantly more effective than RTX when administered at the same doses in established pre-B-ALL xenografts. In the NSG mouse model, at 30 days tumor volume in mice receiving 30 mg/kg of obinutuzumab was significantly decreased when compared to mice receiving 10 mg/kg obinutuzumab or 30 mg/kg of RTX (p<0.0001, p<0.002 respectively). At 11 weeks, mice receiving 30 mg/kg of obinutuzumab showed no increase in tumor volume compared to week 4. The overall survival following obinutuzumab at 30 mg/kg was 80 days compared to 48 days by obinutuzumab at 10mg/kg (p=0.001) and 30 days following RTX treatment at 30mg/kg)(p=0.002) (Figure-1A). Relative tumor luminescence activity on day 8 was significantly decreased in mice receiving 30 mg/kg of obinutuzumab (8x10⁵ RLU) compared to 10 mg/kg of obinutuzumab (7.7x10⁵ RLU) or 30 mg/kg of RTX (9.9x10⁵ RLU) compared to IgG isotype (2.0x10⁶ RLU, p=0.02,p=0.07,p=0.1, respectively).

Similarly, in the NOD/SCID mouse model, at day 80, we observed a significant decrease in tumor luminescence when mice were treated with 30 mg/kg obinutuzumab compared to 10 mg/kg obinutuzumab or 30 mg/kg rituximab (p=0.001). Furthermore, at day 80 there was 100% survival in animals with 30 mg/kg obinutuzumab compared to 60% following rituximab at 30 mg/kg ( p=0.001) or 40% obinutuzumab (10mg/kg; p=0.001) (Figure1B).

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These preliminary studies in two groups of pre-B-ALL xenograft mice (NSG and NOD/SCID), demonstrated that the antitumor effect of obinutuzumab at 30 mg/kg significantly increases survival and decreases the tumor burden in Pre-B-ALL xenografts compared to equal dose of RTX. Further studies are underway to determine the mechanism(s) responsible for the significantly increased efficacy of obinutuzumab vs. RTX in pre-B-ALL xenografts.

Cairo:Roche/Genentech: advisory board Other.