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Tumor Antigen ROR1 Targeted Drug Delivery Mediated Selective Leukemic but Not Normal B-Cell Cytotoxicity in Chronic Lymphocytic Leukemia

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Tumor Antigen ROR1 Targeted Drug Delivery Mediated Selective Leukemic but Not Normal B-Cell Cytotoxicity in Chronic Lymphocytic Leukemia Leukemia (2015) 29, 346–355 & 2015 Macmillan Publishers Limited All rights reserved 0887-6924/15 www.nature.com/leu ORIGINAL ARTICLE Tumor antigen ROR1 targeted drug delivery mediated selective leukemic but not normal B-cell cytotoxicity in chronic lymphocytic leukemia R Mani1,2,3,11, Y Mao2,4,5,11, FW Frissora1,2, C-L Chiang1,2, J Wang2, Y Zhao4,YWu5,BYu2,5,RYan6,XMo7,LYu7, J Flynn1, J Jones1, L Andritsos1, S Baskar8, C Rader9, MA Phelps2,4, C-S Chen2,3,6,RJLee2,4,5, JC Byrd1,2,3,6, LJ Lee5,10 and N Muthusamy1,2,3 Selective cytotoxicity to cancer cells without compromising their normal counterparts pose a huge challenge for traditional drug design. Here we developed a tumor antigen-targeted delivery of immunonanoparticle carrying a novel non-immunosuppressive FTY720 derivative OSU-2S with potent cytotoxicity against leukemic B cells. OSU-2S induces activation of protein phosphatase 2A (PP2A), phosphorylation and nuclear translocation of SHP1S591 and deregulation of multiple cellular processes in chronic lymphocytic leukemia (CLL) resulting in potent cytotoxicity. To preclude OSU-2S-mediated effects on these ubiquitous phosphatases in unintended cells and avoid potential adverse effects, we developed an OSU-2S-targeted delivery of immunonanoparticles (2A2-OSU-2S-ILP), that mediated selective cytotoxicity of CLL but not normal B cells through targeting receptor tyrosine kinase ROR1 expressed in leukemic but not normal B cells. Developing a novel spontaneous CLL mouse model expressing human ROR1 (hROR1) in all leukemic B cells, we demonstrate the therapeutic benefit of enhanced survival with 2A2- OSU-2S-ILP in vivo. The newly developed non-immunosuppressive OSU-2S, its delivery using human CLL directed immunonanoparticles and the novel transgenic (Tg) mouse model of CLL that expresses hROR1 exclusively in leukemic B cell surface are highly innovative and can be applied to CLL and other ROR1 þ malignancies including mantle cell lymphoma and acute lymphoblastic leukemia. Leukemia (2015) 29, 346–355; doi:10.1038/leu.2014.199 INTRODUCTION evaluate potential activators of phosphatases in CLL and other Chronic lymphocytic leukemia (CLL) characterized by accumula- B-cell malignancies. We have recently reported the cytotoxic tion of apoptotic resistance CD19 þ CD5 þ B lymphocytes in activity of FTY720, a sphingosine analog against several B-cell 10–12 peripheral blood and lymphoid organs is the most prevalent malignancies including CLL through modulation of PP2A. adulthood leukemia in the western hemisphere.1,2 Current Despite its promising in vitro and in vivo activity against a variety therapies for CLL are not curative rendering drug adverse effects of solid tumors and heme malignancies,10–15 our attempts to and immunosuppression.3 Several hematological malignancies develop FTY720 for oncology indications was hindered by its including CLL display aberrantly phosphorylated proteins that immunosuppressive property involving T-cell sequestration have led to development of inhibitors of kinases such as SYK, BTK, to lymph nodes through its action on sphingosine-1-phosphate PIM, mTOR and PI3K for CLL therapy.4 Although this approach is receptor. This prompted us to develop OSU-2S, {(S)-2-amino- very promising, limitations associated with simultaneously 2-(4-{(6-methylheptyl)-oxy}phenethyl)pentan-1-ol}, a synthetic deri- targeting multiple survival kinases involved in diverse survival vative of FTY720 by structure–activity relationship with more signaling pathways, kinase independent survival signals and potent anti-tumor activity but lacking the sphingosine-1- selective inhibition in leukemic but not normal B cells that are phosphate receptor-mediated immunosuppressive effects.16 The critical for the normal physiology warrant alternate approaches. promising preclinical activity of OSU-2S in prognostically poor CLL The discovery of predominantly inactive phosphatases in a variety patient cells prompted us to evaluate this molecule for further of cancers5–9 and the potential for phosphatase-targeted therapy studies. Prolonged exposure of unintended normal cells to as an alternative to kinase inhibitors, especially in situations where chemotherapeutic drugs contributes to major adverse effects. the efficacy of the kinase inhibitors are compromised due to Novel therapy options that specifically target leukemic B cells resistance mechanisms attributed to mutations and single- sparing normal B cells are lacking and will be highly promising for nucleotide polymorphisms of the drug targets prompted us to CLL patients. To address this, we entrapped OSU-2S in lipid 1Division of Hematology, Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, OH, USA; 2Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA; 3Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA; 4Division of Pharmaceutics, College of Pharmacy, The Ohio State University, Columbus, OH, USA; 5Center for Affordable Nanoengineering of Polymeric Biomedical Devices, The Ohio State University, Columbus, OH, USA; 6Division of Medicinal Chemistry, College of Pharmacy, The Ohio State University, Columbus, OH, USA; 7Center for Biostatistics, The Ohio State University, Columbus, OH, USA; 8Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA; 9Department of Cancer Biology and Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, Jupiter, FL, USA and 10Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH, USA. Correspondence: Profesor Dr N Muthusamy, Division of Hematology, Department of Internal Medicine, Molecular Virology Immunology and Medical Genetics and Veterinary Biosciences, The Ohio State University Comprehensive Cancer Center, 455E, OSUCCC, 410, West 12th Avenue, Columbus 43210, OH, USA. E-mail: [email protected] 11These authors contributed equally to this work. Received 4 March 2014; revised 8 May 2014; accepted 4 June 2014; accepted article preview online 20 June 2014; advance online publication, 15 July 2014 ROR1-targeted delivery of OSU-2S in B-CLL R Mani et al 347 nanoparticles targeting the tumor antigen ROR1. ROR1 is a were purchased from Sigma. DSPE-PEG-maleimide was obtained from receptor tyrosine kinase expressed in over 95% of CLL but not Avanti Polar Lipids (Alabaster, AL, USA). Protein kinase C (PKC) activity was normal B cells. Its unaltered surface expression related to the measured using PepTag assay kit, Promega. severity of CLL disease,17,18 and its relatively low cell surface Antibodies against phospho S591 SHP1 (ECM Biosciences, Versailles, KY, density and internalization property makes it an attractive target USA); GAPDH, SHP1, PP2Ac (Millipore); Brg1, (Santa Cruz Biotechnology, Dallas, TX, USA); TCL1A (MBL International, Woburn, MA, USA). Goat F(ab0) for armed rather than naked monoclonal antibodies (mAbs). 2 against human IgA þ IgG þ IgM (H þ L) (Jackson Immunoresearch Labora- Liposomal immunonanoparticles (ILPs) have provided a modality tories, West Grove, PA, USA) were commercially obtained. Anti-human for high interactive affinities with target antigens on cell surface CD19-FITC/PE, CD20-PE, CD3-FITC/PE, CD37-FITC, CD86-FITC and mouse and are used to selectively enhance drug payload to target cells, B220-FITC/PE/APC-Cy7, CD19-PE, CD3-FITC/PE, CD3-PE-Cy7, CD5-FITC/PE, favorably alter the drug kinetics, overcome drug off-target effects CD45-APC, Streptavidin-PE (BD Bioscience); hROR1 Biotinylated/Alexa Fluor and potentially the drug efflux pumps.19 To evaluate in vivo the 488 (R&D Systems, Minneapolis, MN, USA) were used for flow cytometric proof-of-principle, we have generated a custom mouse model analyses. 2A2-IgG against hROR1 was from Dr Christoph Rader, CCR, NCI. expressing human ROR1 (hROR1) surface protein on all leukemic B cells, which further closely resembled CLL. Utilizing this in vivo Quantitative RT–PCR model, we show the therapeutic benefit of delivering OSU-2S- Cells suspended in TRizol (Ambion, Life Technologies) were kept at À 70 1C loaded nanoparticles directed against hROR1 expressing leukemic until RNA was extracted following manufacturer’s instruction. cDNA was cells for CLL therapy. made using random primers (Invitrogen, Life Technologies). Real-time PCR was performed using TaqMan (Life Technologies) gene expression assay probe primer sets for Tcl1A (ID:Hs00951350_m1) and 18S MATERIALS AND METHODS (ID:HS03003631_g1) and ViiA 7 Real-Time PCR System (Applied Biosystems, Cells Grand Island, NY, USA). The expression of target genes relative to internal Peripheral blood was obtained from CLL patients after informed consent control gene was calculated using the threshold cycle number (Ct). The under protocol approved by the institution’s internal review board. All relative target gene expression for each condition was normalized to vehicle control and fold change determined using the comparative primary CLL cells used are immunophenotypically defined as outlined by DDCt the modified 1996 National Cancer Institute criteria.20 CLL cells were method (2 ). isolated from fresh patient blood using ‘Rosette-Sep’ kit (Stem Cell Technologies; Vancouver, BC, Canada) and Ficoll density gradient Gene expression microarray analysis centrifugation (Ficoll-Paque Plus; Amersham Biosciences, Piscataway, NJ, USA) according to the manufacturer’s instructions. Normal B cells were RNA extracted from primary CLL cells that responded
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