Margaret N. Whitacre

Effects of interleukin-18 on natural killer cells: Costimulation of activation through Fc receptors for immunoglobulin

The antitumor activity of monoclonal antibodies is mediated by effector cells, such as natural killer (NK) cells, that express Fc receptors for immunoglobulin. Efficacy of monoclonal antibodies, including the CD20 antibody rituximab, could be improved by agents that augment the function of NK cells. Interleukin (IL)-18 is an immunostimulatory cytokine that has antitumor activity in preclinical models. The effects of IL-18 on NK cell function mediated through Fcγ receptors were examined. Human NK cells stimulated with immobilized IgG in vitro secreted IFN-γ as expected; such IFN-γ production was partially inhibited by blocking CD16 with monoclonal antibodies. IL-18 augmented IFN-γ production by NK cells stimulated with immobilized IgG or CD16 antibodies. NK cell IFN-γ production in response to immobilized IgG and/or IL-18 was inhibited by chemical inhibitors of Syk and several other kinases involved in CD16 signaling pathways. IL-18 augmented antibody-dependent cellular cytotoxicity (ADCC) of human NK cells against rituximab-coated Raji cells in vitro. IL-18 and rituximab acted synergistically to promote regression of human lymphoma xenografts in SCID mice. Inasmuch as IL-18 costimulates IFN-γ production and ADCC of NK cells activated through Fc receptors in vitro and augments antitumor activity of rituximab in vivo, it is an attractive cytokine to combine with monoclonal antibodies for treatment of human cancer.

Combination therapy with ofatumumab and bendamustine in xenograft model of chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) is the most common leukaemia, accounting for c. 30% of all leukaemia diagnoses. The median age at diagnosis is 70 years. Therefore the treatment strategies must be tailored to the age-related reduction in tolerance to toxic chemotherapies (Lin, 2010). For decades, the initial front-line CLL therapy has been a monotherapy with cytotoxic agents. Novel CD20-targeting monoclonal antibody (mAb) therapies were recently approved for use in CLL in Europe and the USA (Hallek, 2009). Ofatumumab is a fully human CD20 mAb approved for treatment of patients with fludarabineand alemtuzumab-refractory CLL (Reagan & Castillo, 2010). Ofatumumab binds a unique membraneproximal epitope encompassing both the smalland large-loop of the CD20 molecule (Tsimberidou, 2010). Its mechanism of action includes complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) (de Haij et al, 2010). Bendamustine (4-[5-[Bis(2-chloroethyl)amino]-1methylbenzimidazol-2methyl]butanoic acid) is a chemotherapeutic that has been used in cancer management for almost 50 years in Europe, and was approved for CLL indication in USA in 2008. It is a bi-functional mechlorethamine derivative that kills cancer cells by inducing DNA breaks as an alkylating agent and by acting as a purine analog (Aivado et al, 2002). A possible synergy was indicated between bendamustine and the chimeric CD20 mAb rituximab in relapsed/ refractory CLL (Hallek, 2009). Here we used preclinical lymphoma and leukaemia models to explore the activity of ofatumumab monotherapy, and to evaluate the effect of ofatumumab in combination with bendamustine in CLL. CD20 expression on Ramos and BC-1 lymphoma cell lines, and JVM-3 CLL cell lines was evaluated by flow cytometry. Staining was performed using ofatumumab, rituximab, and a mouse anti-human CD20 mAb from BD Bioscience (San Jose, CA, USA). All three antibodies were directly labelled with a Zenon fluorescent tag. The expression was highest on Ramos (lymphoma cell line), followed by JVM-3 (CLL cell line). The BC-1 lymphoma cell line was CD20-negative. To study the in vivo effect of ofatumumab, Ramos, JVM-3 and BC-1 cells were subcutaneously injected into C.B-17 severe combined immunodeficiency (SCID) female mice, and treated with an ascending dose of Ofatumumab alone, or in combination with bendamustine in suboptimal doses. Tumour volumes were calculated using formula (1⁄2 · length) · width, and analysed using Prism GraphPad (GraphPad Software, Inc., La Jolla, CA, USA) and one-way analysis of variance (anova) with Bonferroni post-test. Therapy started when mean volumes reached 80–120 mm. Ofatumumab was administered intraperitoneally 2·/week, and bendamustine was injected in a single intravenous dose on day 15. Efficacy was determined by calculating % tumour growth inhibition (TGI = 100 · (1 ) [Vt/Vc]); Vt, Vc = mean V in treated or control group) and tumour growth delay (TGD = 100 · ([Tt ) T])/Tc); Tt, Tc = time to end point in treated or control group). Experiments presented are representative studies that confirmed reproducibility of data. All studies were conducted in accordance with the GlaxoSmithKline Institutional Animal Care and Use Committee and Policy on the Care, Welfare and Treatment of Laboratory Animals. Ofatumumab monotherapy completely arrested (2 mg/kg) or significantly reduced Ramos (1 and 0Æ2 mg/kg) tumour growth. Reduction of Ramos tumour volumes on day 28 was significantly different from controls in all ofatumumab groups (**P < 0Æ01 (2 and 1 mg/kg), and *P < 0Æ05 (0Æ2 mg/kg). Ofatumumab monotherapy in the JVM-3 leukaemia model resulted in a significant TGD at doses 2 mg/kg (*P < 0Æ05) and 4 mg/kg (**P < 0Æ01 and ***P < 0Æ001) (Fig 1). Reduction of tumour volumes on day 29 was significantly different in the ofatumumab groups as compared to the control group (*P < 0Æ05 (2 mg/kg) and **P < 0Æ01 (4 mg/kg). TGI and TGD showed dose-dependent improvement with ofatumumab therapy (Fig 1). Suboptimal doses of ofatumumab (2 mg/kg) and bendamustine (50 mg/kg) were used for combination studies in the JVM-3 s.c. xenograft model. Data

Abstract LB-317: Combination therapy with ofatumumab and bendamustine in xenograft model of chronic lymphocytic leukemia

Ofatumumab (OFA) is a fully human CD20 monoclonal antibody (mAb) recently approved for treatment of patients with fludarabine- and alemtuzumab-refractory chronic lymphocytic leukemia (CLL). OFA binds a unique membrane-proximal epitope encompassing both the small- and large-loop on the CD20 receptor. Its mechanism of action includes complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). Combining OFA with cytotoxic chemotherapy may lead to elimination of additional tumor cells by alternate mechanisms such as apoptosis via alkylating agents. We conducted preclinical studies to determine: 1) OFA activity in B-cell lymphoma and leukemia murine xenograft models with differential CD20 expression; and 2) activity of combining OFA with bendamustine in a CLL model. CD20 expression on tumor cell lines was determined by flow cytometry, and lymphoma (Ramos, Daudi) and CLL (JVM-3) lines with differential CD20 expression were selected for in vivo animal model development. CD20-negative BC-1 lymphoma model served as a control. Tumor cells were implanted s.c. into immunodeficient 4-6 weeks old C.B-17 SCID female mice (Taconic), and tumor volumes were determined twice a week based on caliper measurements (tumor volume = width 2 × length/2). Mice were randomized and therapy was initiated when tumors reached mean volume (V) = 80 mm 3 at two weeks after tumor implantation. OFA was administered i.p. twice a week, and bendamustine was injected in a single i.v. dose on day 15 of the study. Efficacy of therapy was determined by evaluating % tumor growth inhibition (TGI=100x(1-(Vt/Vc), where Vt, Vc = mean V in the treated, or control group, respectively) and tumor growth delay (TGD=100x((Tt-Tc)/Tc) where Tt, Tv = time to end-point in the treated, or control group). CD20 expression ranged from highest to lowest on Ramos, JVM-3 and BC-1 (CD20 negative) cells. All cell lines were tumorigenic in C.B-17 SCID mice. OFA monotherapy was effective in the Ramos and JVM-3, but not in the control BC-1 s.c. xenograft model. OFA dose-response was performed and suboptimal doses of OFA (2 mg/kg i.p.) and bendamustine (50 mg/kg i.v.) were used for chemotherapy combination studies in the JVM-3 s.c. xenograft model. Combination therapy resulted in a synergistic anti-tumor activity with TGI = 96% and TGD = 42%, compared to bendamustin (TGI = 9%, TGD = 14%) or OFA alone (TGI = 16%, TGD = 14%) in the CLL model. In conclusion, single-agent therapy with OFA demonstrated significant and dose-dependent activity in preclinical animal xenograft models of human B-cell lymphoma and CLL. The combination of suboptimal OFA and bendamustine doses in the CLL model resulted in significant antitumor activity, compared exposure to either agent alone. These preclinical findings provide a rationale for clinical studies of the combination of OFA and bendamustine in patients with CLL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-317.