Stephen H. Trulli

IL-12 Induces Monocyte IL-18 Binding Protein Expression Via IFN-γ

IL-18 is a Th1 cytokine that synergizes with IL-12 and IL-2 in the stimulation of lymphocyte IFN-γ production. IL-18 binding protein (IL-18BP) is a recently discovered inhibitor of IL-18 that is distinct from the IL-1 and IL-18 receptor families. In this report we show that IL-18BPa, the IL-18BP isoform with the highest affinity for IL-18, was strongly induced by IL-12 in human PBMC. Other Th1 cytokines, including IFN-γ, IL-2, IL-15, and IL-18, were also capable of augmenting IL-18BPa expression. In contrast, IL-1α, IL-1β, TNF-α, IFN-γ-inducible protein-10, and Th2 cytokines such as IL-4 and IL-10 did not induce IL-18BPa. Although monocytes were found to be the primary source of IL-18BPa, the induction of IL-18BPa by IL-12 was mediated through IFN-γ derived predominantly from NK cells. IL-18BPa production was observed in cancer patients receiving recombinant human IL-12 and correlated with the magnitude of IFN-γ production. The IFN-γ/IL-18BPa negative feedback loop identified in this study may be capable of broadly controlling immune activation by cytokines that synergize with IL-18 to induce IFN-γ and probably plays a key role in the modulation of both innate and adaptive immunity.

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.

High-dose recombinant interleukin-18 induces an effective Th1 immune response to murine MOPC-315 plasmacytoma.

Interleukin (IL)-18 has profound antitumor activity when administered at high doses as a single agent for prolonged periods in BALB/c mice bearing late, well-established MOPC-315 tumors. Management with a qD x 27 schedule resulted in regression of tumors in all animals receiving 5 mg/kg/d. A protracted daily management regimen appears to be necessary to induce regression in this advanced tumor model. Biologic markers were assessed and appear to be potentially useful in evaluating the immunologic and antitumor activity of IL-18. The biomarkers of IL-18s immunologic activity include, but are not limited to, IL-1&agr;, IL-2, IL-8, IL-10, IL-12, IL-13, interferon-&ggr;, tumor necrosis factor-&agr;, and granulocyte-macrophage colony-stimulating factor. The profile of these circulating cytokines and their expression levels at baseline, and after IL-18 delivery, can be measured in the serum, as well as from splenocytes of mice or human peripheral blood mononuclear cells derived from either normal subjects or patients with cancer. We compared IL-18 and IL-12 alone or in combination for their ability to induce cytokine production and natural killer cytolytic activity. Our data support the notion that IL-18 induces a predominantly Th1 response, and that the mechanism of IL-18 activity differs from that of IL-12. The biologic activity of IL-18 management revealed by increases in serum levels of cytokines and enhancement of natural killer cytolytic activity will be useful as clinical trials initiate in 2002. Expression of interferon-&ggr; and granulocyte-macrophage colony-stimulating factor serum levels correlates directly over a broad dose escalation with the level of IL-18. Therefore, this provides a convenient pharmacodynamic reference to the biologic response to IL-18 that may serve to guide the conduct of clinical trials.

A bicistronic expression system for bacterial production of authentic human interleukin-18

Interleukin-18 (IL-18) is activated and released from immune effector cells to stimulate acquired and innate immune responses involving T and natural killer (NK) cells. The release of IL-18 from mammalian cells is linked to its proteolytic activation by caspases including interleukin 1 converting enzyme (ICE). The absence of a signal peptide sequence and the requirement for coupled activation and cellular release have presented challenges for the large-scale recombinant production of IL-18. In this study, we have explored methods for the direct production of authentic human IL-18 toward the development of a large-scale production system. Expression of mature IL-18 directly in Escherichia coli with a methionine initiating codon leads to the production of MetIL-18 that is dramatically less potent in bioassays than IL-18 produced as a pro-peptide and activated in vitro. To produce an authentic IL-18, we have devised a bicistronic expression system for the coupled transcription and translation of ProIL-18 with caspase-1 (ICE) or caspase-4 (ICE-rel II, TX, ICH-2). Mature IL-18 with an authentic N-terminus was produced and has a biological activity and potency comparable to that of in vitro processed mature IL-18. Optimization of this system for the maximal production yields can be accomplished by modulating the temperature, to affect the rate of caspase activation and to favor the accumulation of ProIL-18, prior to its proteolytic processing by activated caspase. The effect of temperature is particularly profound for the caspase-4 co-expression process, enabling optimized production levels of over 150 mg/L in shake flasks at 25 degrees C. An alternative bicistronic expression design utilizing a precise ubiquitin IL-18 fusion, processed by co-expressed ubiquitinase, was also successfully used to generate fully active IL-18, thereby demonstrating that the pro-sequence of IL-18 is not required for recombinant IL-18 production.

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

Manipulation of human B cells to confer immortality

Electroporation was used to deliver genomic DNA from a lymphoid tumor to activated/stimulated human peripheral blood lymphocytes to create immortalized lymphoid cell lines. Activation of the recipient lymphocytes was essential for efficient immortalization. A panel of human B cell transfectant clones, each phenotypically representing specific stages of differentiation, resulted from the transfection. Monoclonal antibody production was measured, and the level produced depended on the phenotype of the cells, with the more mature B cell transfectants secreting up to 10 micrograms/mL of immunoglobulin. The transfectants were stable with respect to their morphological appearance, growth rate, and antibody production. Chromosome analysis indicated that the transfectants displayed a normal karyotype, devoid of abnormalities. We have shown that electroporation is an effective method of immortalizing human lymphocytes at different stages of differentiation. The transfectants provide a panel of cells that can readily be studied with respect to their phenotypic/karyotypic stability, regulation, and production of immunoglobulin, lymphokines, and growth factors. These data demonstrate the feasibility of generating immortalized human B cells to provide an important resource for the study of B cell differentiation and immortalization.

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.