Leopoldo Luistro

Role of interferon-gamma in mediating the antitumor efficacy of interleukin-12.

Although interleukin-12 (IL-12) has marked antitumor activity against the murine Renca renal cell carcinoma in vivo, no antiproliferative activity with IL-12 was observed against these tumor cells in vitro; in contrast, interferon-gamma (IFN-gamma) had growth inhibitory activity. Since one of the properties of IL-12 is its ability to stimulate production of IFN-gamma, the role of IFN-gamma in mediating the antitumor activity of IL-12 was evaluated. Substantially diminished antitumor activity was observed in mice injected with IL-12 and neutralizing antibody to murine IFN-gamma compared with mice receiving IL-12 alone, indicating that IFN-gamma was required for the optimal antitumor efficacy of IL-12. However, several lines of investigation suggest that the antitumor effect of IL-12 is not mediated solely through the induction of IFN-gamma. Exogenous administration of IFN-gamma to Renca tumor-bearing euthymic mice resulted in less antitumor efficacy than that which could be obtained with IL-12. In addition, the antitumor effect of IL-12 was reduced in nude mice compared with euthymic mice, but an approximately 10-fold higher level of serum IFN-gamma was induced in nude than in euthymic mice. Thus, these results indicate that induction of high serum levels of IFN-gamma is not sufficient to mediate the antitumor efficacy of IL-12.

Preclinical Profile of a Potent γ-Secretase Inhibitor Targeting Notch Signaling with In vivo Efficacy and Pharmacodynamic Properties

Notch signaling is an area of great interest in oncology. RO4929097 is a potent and selective inhibitor of gamma-secretase, producing inhibitory activity of Notch signaling in tumor cells. The RO4929097 IC50 in cell-free and cellular assays is in the low nanomolar range with >100-fold selectivity with respect to 75 other proteins of various types (receptors, ion channels, and enzymes). RO4929097 inhibits Notch processing in tumor cells as measured by the reduction of intracellular Notch expression by Western blot. This leads to reduced expression of the Notch transcriptional target gene Hes1. RO4929097 does not block tumor cell proliferation or induce apoptosis but instead produces a less transformed, flattened, slower-growing phenotype. RO4929097 is active following oral dosing. Antitumor activity was shown in 7 of 8 xenografts tested on an intermittent or daily schedule in the absence of body weight loss or Notch-related toxicities. Importantly, efficacy is maintained after dosing is terminated. Angiogenesis reverse transcription-PCR array data show reduced expression of several key angiogenic genes. In addition, comparative microarray analysis suggests tumor cell differentiation as an additional mode of action. These preclinical results support evaluation of RO4929097 in clinical studies using an intermittent dosing schedule. A multicenter phase I dose escalation study in oncology is under way.

In vitro and in vivo activity of R547: a potent and selective cyclin-dependent kinase inhibitor currently in phase I clinical trials.

The cyclin-dependent protein kinases are key regulators of cell cycle progression. Aberrant expression or altered activity of distinct cyclin-dependent kinase (CDK) complexes results in escape of cells from cell cycle control, leading to unrestricted cell proliferation. CDK inhibitors have the potential to induce cell cycle arrest and apoptosis in cancer cells, and identifying small-molecule CDK inhibitors has been a major focus in cancer research. Several CDK inhibitors are entering the clinic, the most recent being selective CDK2 and CDK4 inhibitors. We have identified a diaminopyrimidine compound, R547, which is a potent and selective ATP-competitive CDK inhibitor. In cell-free assays, R547 effectively inhibited CDK1/cyclin B, CDK2/cyclin E, and CDK4/cyclin D1 (Ki = 1–3 nmol/L) and was inactive (Ki > 5,000 nmol/L) against a panel of >120 unrelated kinases. In vitro, R547 effectively inhibited the proliferation of tumor cell lines independent of multidrug resistant status, histologic type, retinoblastoma protein, or p53 status, with IC50s ≤ 0.60 μmol/L. The growth-inhibitory activity is characterized by a cell cycle block at G1 and G2 phases and induction of apoptosis. R547 reduced phosphorylation of the cellular retinoblastoma protein at specific CDK phosphorylation sites at the same concentrations that induced cell cycle arrest, suggesting a potential pharmacodynamic marker for clinical use. In vivo, R547 showed antitumor activity in all of the models tested to date, including six human tumor xenografts and an orthotopic syngeneic rat model. R547 was efficacious with daily oral dosing as well as with once weekly i.v. dosing in established human tumor models and at the targeted efficacious exposures inhibited phosphorylation of the retinoblastoma protein in the tumors. The selective kinase inhibition profile and the preclinical antitumor activity of R547 suggest that it may be promising for development for use in the treatment of solid tumors. R547 is currently being evaluated in phase I clinical trials. [Mol Cancer Ther 2006;5(11):2644–58]

High tumor levels of IL6 and IL8 abrogate preclinical efficacy of the γ-secretase inhibitor, RO4929097

Interest continues to build around the early application of patient selection markers to prospectively identify patients likely to show clinical benefit from cancer therapies. Hypothesis generation and clinical strategies often begin at the preclinical stage where responder and nonresponder tumor cell lines are first identified and characterized. In the present study, we investigate the drivers of in vivo resistance to the γ‐secretase inhibitor RO4929097. Beginning at the tissue culture level, we identified apparent IL6 and IL8 expression differences that characterized tumor cell line response to RO4929097. We validated this molecular signature at the preclinical efficacy level identifying additional xenograft models resistant to the in vivo effects of RO4929097. Our data suggest that for IL6 and IL8 overexpressing tumors, RO4929097 no longer impacts angiogenesis or the infiltration of tumor associated fibroblasts. These preclinical data provide a rationale for preselecting patients possessing low levels of IL6 and IL8 prior to RO4929097 dosing. Extending this hypothesis into the clinic, we monitored patient IL6 and IL8 serum levels prior to dosing with RO4929097 during Phase I. Interestingly, the small group of patients deriving some type of clinical benefit from RO4929097 presented with low baseline levels of IL6 and IL8. Our data support the continued investigation of this patient selection marker for RO4929097 and other types of Notch inhibitors undergoing early clinical evaluation.

Discovery of siRNA Lipid Nanoparticles to Transfect Suspension Leukemia Cells and Provide In Vivo Delivery Capability

As a powerful research tool, siRNAs therapeutic and target validation utility with leukemia cells and long-term gene knockdown is severely restricted by the lack of omnipotent, safe, stable, and convenient delivery. Here, we detail our discovery of siRNA-containing lipid nanoparticles (LNPs) able to effectively transfect several leukemia and difficult-to-transfect adherent cell lines also providing in vivo delivery to mouse spleen and bone marrow tissues through tail-vein administration. We disclose a series of novel structurally related lipids accounting for the superior transfection ability, and reveal a correlation between expression of Caveolins and successful transfection. These LNPs, bearing low toxicity and long stability of >6 months, are ideal for continuous long-term dosing. Our discovery represents the first effective siRNA-containing LNPs for leukemia cells, which not only enables high-throughput siRNA screening with leukemia cells and difficult-to-transfect adherent cells but also paves the way for the development of therapeutic siRNA for leukemia treatment.

RG7212 anti-TWEAK mAb inhibits tumor growth through inhibition of tumor cell proliferation and survival signaling and by enhancing the host antitumor immune response.

Purpose: To explore the role of TWEAK in tumor growth and antitumor immune response and the activity and mechanism of RG7212, an antagonistic anti-TWEAK antibody, in tumor models. Experimental Design: TWEAK-induced signaling and gene expression were explored in tumor cell lines and inhibition of these effects and antitumor efficacy with RG7212 treatment was assessed in human tumor xenograft-, patient-derived xenograft, and syngeneic tumor models and phase I patients. Genetic features correlated with antitumor activity were characterized. Results: In tumor cell lines, TWEAK induces proliferation, survival, and NF-κB signaling and gene expression that promote tumor growth and suppress antitumor immune responses. TWEAK-inducible CD274, CCL2, CXCL-10 and -11 modulate T-cell and monocyte recruitment, T-cell activation, and macrophage differentiation. These factors and TWEAK-induced signaling were decreased, and tumor, blood, and spleen immune cell composition was altered with RG7212 treatment in mice. RG7212 inhibits tumor growth in vivo in models with TWEAK receptor, Fn14, expression, and markers of pathway activation. In phase I testing, signs of tumor shrinkage and stable disease were observed without dose-limiting toxicity. In a patient with advanced, Fn14-positive, malignant melanoma with evidence of tumor regression, proliferation markers were dramatically reduced, tumor T-cell infiltration increased, and tumor macrophage content decreased. Antitumor activity, a lack of toxicity in humans and animals and no evidence of antagonism with standard of care or targeted agents in mice, suggests that RG7212 is a promising agent for use in combination therapies in patients with Fn14-positive tumors. Clin Cancer Res; 19(20); 5686–98. ©2013 AACR.

Gene expression analysis in biomarker research and early drug development using function tested reverse transcription quantitative real-time PCR assays.

The identification of new biomarkers is essential in the implementation of personalized health care strategies that offer new therapeutic approaches with optimized and individualized treatment. In support of hypothesis generation and testing in the course of our biomarker research an online portal and respective function-tested reverse transcription quantitative real-time PCR assays (RT-qPCR) facilitated the selection of relevant biomarker genes. We have established workflows applicable for convenient high throughput gene expression analysis in biomarker research with cell lines (in vitro studies) and xenograft mouse models (in vivo studies) as well as formalin-fixed paraffin-embedded tissue (FFPET) sections from various human research and clinical tumor samples. Out of 92 putative biomarker candidate genes selected in silico, 35 were shown to exhibit differential expression in various tumor cell lines. These were further analysed by in vivo xenograft mouse models, which identified 13 candidate genes including potential response prediction biomarkers and a potential pharmacodynamic biomarker. Six of these candidate genes were selected for further evaluation in FFPET samples, where optimized RNA isolation, reverse transcription and qPCR assays provided reliable determination of relative expression levels as precondition for differential gene expression analysis of FFPET samples derived from projected clinical studies. Thus, we successfully applied function tested RT-qPCR assays in our biomarker research for hypothesis generation with in vitro and in vivo models as well as for hypothesis testing with human FFPET samples. Hence, appropriate function-tested RT-qPCR assays are available in biomarker research accompanying the different stages of drug development, starting from target identification up to early clinical development. The workflow presented here supports the identification and validation of new biomarkers and may lead to advances in efforts to achieve the goal of personalized health care.

Combination GITR targeting/PD-1 blockade with vaccination drives robust antigen-specific antitumor immunity

Tumor progression is facilitated immunologically by mechanisms that include low antigen expression, an absence of coimmunostimulatory signals, and the presence of regulatory T cells (Tregs), all of which act to suppress and restrict effector T cells in the tumor. It may be possible to overcome these conditions by a combination of modulatory immunotherapy agents and tumor-antigen targeting to activate and drive effective antitumor T cell responses. Here, we demonstrated that co-administration of aGITR and aPD-1 monoclonal antibodies (mAb) in combination with a peptide vaccine (Vax) in mice bearing established tumors significantly delayed tumor growth and induced complete regression in 50% of the mice. This response was associated with increased expansion and functionality of potent Ag-specific polyfunctional CD8+ T cells, reduced Tregs, and the generation of memory T cells. Tumor regression correlated with the expansion of tumor-infiltrating antigen-specific CD8+ effector memory T cells, as depletion of this cell population significantly reduced the effectiveness of the triple combination Vax/aGITR/aPD-1 therapy. These findings support the concept that dual aGITR/aPD-1 combination with cancer vaccines may be a novel strategy against poorly immunogenic tumors.

Interleukin 12: Biology and Preclinical Studies of a New Anti-Tumor Cytokine

Interleukin-12 (IL-12) is a recently cloned cytokine (1,2) that has some unique properties. Two groups working independently simultaneously discovered this cytokine utilizing different assay systems. One group was searching for a cytokine, initially called cytotoxic lymphocyte maturation factor (CLMF) that synergized with IL-2 in the induction of several cytotoxic lymphocyte populations (3–5). The second group was characterizing a cytokine, natural killer cell stimulatory factor (NKSF), that enhanced natural killer (NK) cell activity and induced the secretion of interferon gamma (IFN-γ) from these cells (6). It is now clear that both of these properties, as well as others, are associated with one cytokine, IL-12.

Targeting of CD122 enhances antitumor immunity by altering the tumor immune environment

Mounting evidence demonstrates that CD8+CD122+ T cells have suppressive properties with the capacity to inhibit T cell responses. Therefore, these cells are rational targets for cancer immunotherapy. Here, we demonstrate that CD122 monoclonal antibody (mAb; aCD122) therapy significantly suppressed tumor growth and improved long-term survival in tumor-bearing mice. This therapeutic effect correlated with enhanced polyfunctional, cytolytic intratumoral CD8+ T cells and a decrease in granulocytic myeloid-derived suppressor cells (G-MDSCs). In addition, aCD122 treatment synergized with a vaccine to augment vaccine-induced antigen (Ag)-specific CD8+ T cell responses, reject established tumors and generate memory T cells. Furthermore, aCD122 mAb synergized with an anti-GITR (aGITR) mAb to confer significant control of tumor growth. These results suggest CD122 might be a promising target for cancer immunotherapy, either as a single agent or in combination with other forms of immunotherapy.