Methods and Compositions for Treating Neuroblastoma

Abstract

Methods and compositions for treating neuroblastoma are disclosed.

Description

[0001]This application is a continuation-in-part of 12 / 853,834, filed on Aug. 10, 2010, which is a continuation-in-part of PCT / US2009 / 034288, filed on Feb. 17, 2009, which claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 61 / 029,212, filed on Feb. 15, 2008 and to U.S. Provisional Patent Application No. 61 / 123,775, filed on Apr. 11, 2008. The foregoing applications are incorporated by reference herein.FIELD OF THE INVENTION[0002]The present invention relates to the fields of neuroblastoma. More specifically, the invention provides compositions and methods for the identification, diagnosis, and treatment of neuroblastoma.BACKGROUND OF THE INVENTION[0003]Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains.[0004]Each of these citations is incorporated herein by reference as though set forth in full.[0005]Neuroblastoma is a cancer of early childhood tha...

AI Technical Summary

Benefits of technology

[0027]FIG. 18A provides a graph showing ALK antibody-induced growth inhibition and ADCC of neuroblastoma cells. To measure growth inhibition upon antibody exposure, cell lines were plated in 96-well plates and treated with anti-ALK (mAb30 plus mAb49) or a negative control murine IgGl. Cell growth was monitored by Real Time Cell Electronic Sensing (RT-CES) impedance measurement. Growth inhibition of SY5Y cells treated with indicated amounts of anti-ALK as compared to control Ig. FIG. 18B shows the indicated cell lines were treated with 10 μg / ml ALK antibody and growth inhibition was measured after 144 hours. FIG. 18C provides a graph showing the effect of anti-ALK antibody on RPE1 cell growth. ALK-negative RPE-1 cells were plated in 96-well plates and treated with either 10 μg / ml anti-ALK antibody or murine immunoglobulin . Shown is cell growth for each condition as measured by RT-CES. In FIG. 18D, ADCC was measured using an in vitro assay in which normal donor peripheral blood lymphocytes (PBL), pre-incubated overnight with IL-2, were co-incubated for four hours with neuroblastoma cells in the presence (black line) or absence (grey line) of 1 μg / ml ALK antibody. Shown are % cytotoxicity at the indicated effector:target ratios when NB1 cells (left panel), SY5Y cells (middle panel), or ALK-negative SKNAS cells (right panel) were used as targets.

[0028]FIG. 19A shows the effect of crizotinib on cell surface ALK expression. SY5Y cells were incubated with crizotinib or vehicle, harvested and stained for cell surface ALK with mAb14. ViaProbe viability stain was used to exclude non-viable cells. A representative flow cytometry result is shown for SY5Y cells incubated for 72 hours with either vehicle (medium gray line) or 1000 nM crizotinib (black line). The single-peaked light grey line represents the isotype control. FIG. 19B shows the concentration dependence of the percent change in cell surface ALK, as measured by Mean Fluorescence Intensity (MFI), for cells incubated for 72 hours with varying concentrations of crizotinib as compared to vehicle. FIG. 19C provides a time course of percentage change in cell surface ALK levels (over that seen for vehicle treatment) when cells were incubated with 1000 nM crizotinib. FIG. 19D shows a comparison of ALK antibodies for flow cytometry. SY5Y cells were treated with 1000 nM crizotinib or vehicle, harvested 72 hours later, and stained for flow cytometry. Shown is the percent increase in ALK MFI for crizotinib versus vehicle treated cells using either the mAbl4 or mAB46 anti-ALK antibodies.

[0029]FIG. 20 shows dual antibody / TKI targeting of ALK. SY5Y cells were treated with either 333 nM crizotinib or 10 μg / ml anti-ALK antibody (mAb30+mAb49), or both. As negative control, cells were treated with equal volumes of DMSO and 10 μg / ml IgG1. FIG. 20A shows cell growth monitored by RT-CES, revealing clear growth inhibition by the crizotinib / mAb combination. FIG. 20B provides an immunoblot analysis of native ALK protein levels (upper panel) and phospho-ALK (middle panel). β-actin levels are shown as a loading control (lower panel). FIG. 20C shows the effect of crizotinib pre-treatment on anti-ALK antibody mediated ADCC. SY5Y cells were pre-incubated in the presence of crizotinib or vehicle for 48 hours, harvested, and then used as target cells in the in vitro ADCC assay. **, p<0.01; *, p<0.05.

Problems solved by technology

However, because MYCN is so aberrantly dysregulated, and no putative tumor suppressor gene at 1p and 11 q has been shown to harbor inactivating mutations in more than a small percentage of cases, no tractable molecular target approaches currently exist for this disease.

Because of the lethality of the condition prior to reproductive age, previous genetic linkage scans have been underpowered and results difficult to replicate (Longo et al.

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