Tumor antigen ROR1 targeted drug delivery mediated selective leukemic but not normal B-cell cytotoxicity in chronic lymphocytic leukemia

doi:10.1038/leu.2014.199

. 2015 Feb;29(2):346-55.

doi: 10.1038/leu.2014.199. Epub 2014 Jun 20.

Tumor antigen ROR1 targeted drug delivery mediated selective leukemic but not normal B-cell cytotoxicity in chronic lymphocytic leukemia

R Mani¹, Y Mao², F W Frissora³, C-L Chiang³, J Wang⁴, Y Zhao⁵, Y Wu⁶, B Yu⁷, R Yan⁸, X Mo⁹, L Yu⁹, J Flynn¹⁰, J Jones¹⁰, L Andritsos¹⁰, S Baskar¹¹, C Rader¹², M A Phelps¹³, C-S Chen¹⁴, R J Lee², J C Byrd¹⁵, L J Lee¹⁶, N Muthusamy¹

Affiliations

¹ 1] Division of Hematology, Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, OH, USA [2] Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA [3] Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA.
² 1] Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA [2] Division of Pharmaceutics, College of Pharmacy, The Ohio State University, Columbus, OH, USA [3] Center for Affordable Nanoengineering of Polymeric Biomedical Devices, The Ohio State University, Columbus, OH, USA.
³ 1] Division of Hematology, Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, OH, USA [2] Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA.
⁴ Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA.
⁵ Division of Pharmaceutics, College of Pharmacy, The Ohio State University, Columbus, OH, USA.
⁶ Center for Affordable Nanoengineering of Polymeric Biomedical Devices, The Ohio State University, Columbus, OH, USA.
⁷ 1] Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA [2] Center for Affordable Nanoengineering of Polymeric Biomedical Devices, The Ohio State University, Columbus, OH, USA.
⁸ Division of Medicinal Chemistry, College of Pharmacy, The Ohio State University, Columbus, OH, USA.
⁹ Center for Biostatistics, The Ohio State University, Columbus, OH, USA.
¹⁰ Division of Hematology, Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, OH, USA.
¹¹ Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA.
¹² Department of Cancer Biology and Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, Jupiter, FL, USA.
¹³ 1] Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA [2] Division of Pharmaceutics, College of Pharmacy, The Ohio State University, Columbus, OH, USA.
¹⁴ 1] Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA [2] Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA [3] Division of Medicinal Chemistry, College of Pharmacy, The Ohio State University, Columbus, OH, USA.
¹⁵ 1] Division of Hematology, Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, OH, USA [2] Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA [3] Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA [4] Division of Medicinal Chemistry, College of Pharmacy, The Ohio State University, Columbus, OH, USA.
¹⁶ 1] Center for Affordable Nanoengineering of Polymeric Biomedical Devices, The Ohio State University, Columbus, OH, USA [2] Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH, USA.

PMID: 24947019
PMCID: PMC4272672
DOI: 10.1038/leu.2014.199

Tumor antigen ROR1 targeted drug delivery mediated selective leukemic but not normal B-cell cytotoxicity in chronic lymphocytic leukemia

R Mani et al. Leukemia. 2015 Feb.

. 2015 Feb;29(2):346-55.

doi: 10.1038/leu.2014.199. Epub 2014 Jun 20.

Authors

Affiliations

¹ 1] Division of Hematology, Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, OH, USA [2] Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA [3] Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA.
² 1] Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA [2] Division of Pharmaceutics, College of Pharmacy, The Ohio State University, Columbus, OH, USA [3] Center for Affordable Nanoengineering of Polymeric Biomedical Devices, The Ohio State University, Columbus, OH, USA.
³ 1] Division of Hematology, Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, OH, USA [2] Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA.
⁴ Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA.
⁵ Division of Pharmaceutics, College of Pharmacy, The Ohio State University, Columbus, OH, USA.
⁶ Center for Affordable Nanoengineering of Polymeric Biomedical Devices, The Ohio State University, Columbus, OH, USA.
⁷ 1] Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA [2] Center for Affordable Nanoengineering of Polymeric Biomedical Devices, The Ohio State University, Columbus, OH, USA.
⁸ Division of Medicinal Chemistry, College of Pharmacy, The Ohio State University, Columbus, OH, USA.
⁹ Center for Biostatistics, The Ohio State University, Columbus, OH, USA.
¹⁰ Division of Hematology, Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, OH, USA.
¹¹ Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA.
¹² Department of Cancer Biology and Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, Jupiter, FL, USA.
¹³ 1] Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA [2] Division of Pharmaceutics, College of Pharmacy, The Ohio State University, Columbus, OH, USA.
¹⁴ 1] Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA [2] Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA [3] Division of Medicinal Chemistry, College of Pharmacy, The Ohio State University, Columbus, OH, USA.
¹⁵ 1] Division of Hematology, Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, OH, USA [2] Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA [3] Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA [4] Division of Medicinal Chemistry, College of Pharmacy, The Ohio State University, Columbus, OH, USA.
¹⁶ 1] Center for Affordable Nanoengineering of Polymeric Biomedical Devices, The Ohio State University, Columbus, OH, USA [2] Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH, USA.

PMID: 24947019
PMCID: PMC4272672
DOI: 10.1038/leu.2014.199

Abstract

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 SHP1(S591) 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+ malignancies including mantle cell lymphoma and acute lymphoblastic leukemia.

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Conflict of interest statement

CONFLICT OF INTEREST: A provisional patent has been filed for the use of OSU-2S to treat hematologic malignancies. The authors declare no additional competing financial interests.

Figures

**Figure 1**
OSU-2S mediates cytotoxicity in CLL primary cells. (**a-c**) Cytotoxic effects of OSU-2S in CLL. Freshly isolated CD19+ CLL primary cells (1×10⁶/ml) were treated with OSU-2S (2µM) and viability was assessed 24 hours later. (a) Mixed CLL samples (N=25). (B-C) Activity of OSU-2S in prognostically poor CLL patient derived primary cells (b) IGVH unmutated (N=8) and (c) chromosome 17p deleted (N=5) CLL cells. (d) Comparison of OSU-2S and Fludarabine (2FaraA) cytotoxicity in CLL. CLL cells (10×10⁶/ml) were treated with OUS-2S(8µM) or 2FaraA(10µM) and viability was measured at indicated time points (N=9). (e) OSU-2S activates PP2A. CLL cells treated with OSU-2S or FTY720 were used for an *in-vitro* PP2A phosphatase enzyme assay. Values were normalized to protein levels and vehicle treated control; bars represent SD (N=3).

**Figure 2**
OSU-2S mediates PKC dependent phosphorylation of SHP1^S591 in CLL. (a) OSU-2S induces PKC dependent phosphorylation of SHP1^S591 in CLL. CLL cells treated with OSU-2S(5 hours) were immunoblotted for phospho SHP1^S591. Bisindolylmaleimide (BIS) was used to inhibit PKC. (b) Inverse correlation between phospho SHP1^S591 and viability. Spearman rank-order correlation was calculated for phospho SHP1^S591 induced after 5 hour by immunoblotting and percent change in the viability at 24 hour time point in response to OSU-2S treatment (N=20). (c) PKC inhibitor BIS reduces phospho SHP1^S591 and rescues OSU-2S cytotoxicity. Viability was done at 24 hour time point by PI staining on CLL cells treated with Vehicle or OSU-2S in the presence or absence of PKC inhibitor BIS(2µM) (N=8); bars represent SE. PMA is used as control for BIS. (D-E) OSU-2S activates PKC. (d) PKC activity was assessed in CLL cell lysates collected 4hr after treatment with vehicle or 8µM OSU-2S, (N=6). (e) Addition of OSU-2S to purified rat PKC had a direct stimulatory effect. PMA is used as positive control (N=3).

**Figure 3**
Gene expression analysis of CLL cells treated with OSU-2S. (a) Heat map of 266 differently expressed genes in OSU-2S treated CLL. RNA was isolated from CLL cells (N=3) treated with vehicle or OSU-2S(8µM) at 16 hours and subjected to microarray analyses using GeneChip Human Genome U133 plus 2.0 (Affymetrix, GPL570). After statistical analyses, genes that changed by ≥ 2 fold and p<0.0005 in response to OSU-2S treatment were used for constructing a heat map. (b) Top five molecular and cellular functions revealed through Ingenuity Pathway analysis (IPA) of genes modulated by OSU-2S in CLL. (c) OSU-2S down modulates TCL1A. RNA was extracted from vehicle or OSU-2S treated (16hr) CLL patients cells (N=7) and TCL1A expression relative to 18S was determined by real-time RT-PCR analysis and values are normalized to vehicle control; bars represent SD. Immunoblot of TCL1A in CLL cells treated with vehicle or OSU-2S for 24 hours is also shown.

**Figure 4**
Immunonanoparticle 2A2-ILPs targeting ROR1+ CLL cells for OSU-2S drug delivery. (a) Internalization curve for 2A2-IgG bound ROR1 in CLL cells. Internalization involving time-dependent increase in mean fluorescent intensity (MFI) of labeled antibody on CLL cells treated with 2A2-IgG, αCD19 or αCD37 is shown. Bars represent mean ± SD, P<0.001 (N=7). (b) Internalization of 2A2-ILP. MFI showing the binding and uptake of 2A2-ILP calcein by CLL cells, normal B cells and T cells (N=8, P<0.0001). (**c-d**) 2A2-OSU-2S-ILP mediates specific cytotoxicity in CLL cells. 1×10⁶ normal B cells from healthy donors or CLL cells were incubated with different formulation at 5µM of OSU-2S and 0.1µg/ml mAbs for 24hr before viability was analyzed by flow cytometry (mean ± SEM). (c) No difference in cytotoxicity between 2A2-OSU-2S-ILP and IgG-OSU-2S-ILP formulations was found for normal B cells (N=4). (d) 2A2-OSU-2S-ILP significantly induced improved cytotoxicity in CLL cells compared to non-targeting IgG-OSU-2S-ILP (N=8, P<0.01).

**Figure 5**
Immunonanoparticle 2A2-ILPs selectively bind to hROR1+ B cells from Eµ-ROR1 transgenic mice. Splenocytes (1×10⁶) from Eµ-ROR1 transgenic(Tg) or age matched non-transgenic(Non Tg) mice were treated with 2A2-ILP FAM ODN for 30 minutes at 37°C, washed and stained with mouse B220-PE or CD8-PE. Flowcytometric analysis revealed FAM ODN signal only in B220+ Eµ-ROR1 transgenic mice but not to B cells from non-transgenic mice.

**Figure 6**
ROR1 targeted delivery of OSU-2S prolongs survival in hROR1+ CLL mouse model. (a) Specific depletion of ROR1+ B cells in peripheral blood of Eµ-ROR1 single transgenic animals after three doses of 2A2-OSU-2S-ILP intravenous treatment (10mg/kg every other day). (b) Flowcytometric dot plot of hROR1+ splenic cells from Eµ-ROR1-TCL1 animal with leukemia. (**c-d**) Comparison of specific cytotoxicity of 2A2-OSU-2S-ILP in Eµ-ROR1-TCL1(N=8) (c) and Eµ-TCL1(N=6) (d) mice derived splenocytes cultured ex-vivo with different ILPs(5µg/ml) and viability was assessed after 16hr by flowcytometry. (**e-f**) ROR1 targeted delivery of OSU-2S prolongs survival in hROR1+ CLL mouse model. (e) Survival curve of Eµ-ROR1-TCL1 splenocytes engrafted mice treated with 2A2-OSU-2S-ILP(N=11); IgG-OSU-2S-ILP(N=11), OSU-2S-LP(N=7) or 2A2-Empty-ILP(N=10). (f) Peripheral blood analysis of leukemic burden in the engrafted mice by flowcytometry.

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References

1. Rai KR, Sawitsky A, Cronkite EP, Chanana AD, Levy RN, Pasternack BS. Clinical staging of chronic lymphocytic leukemia. Blood. 1975;46:219–234. - PubMed
1. Binet JL, Lepoprier M, Dighiero G, Charron D, D’Athis P, Vaugier G, et al. A clinical staging system for chronic lymphocytic leukemia: prognostic significance. Cancer. 1977;40:855–864. - PubMed
1. Danilov AV. Targeted therapy in chronic lymphocytic leukemia: past, present, and future. Clinical therapeutics. 2013;35:1258–1270. - PMC - PubMed
1. Davids MS, Brown JR. Targeting the B Cell Receptor Pathway in Chronic Lymphocytic Leukemia. Leuk Lymphoma. 2012 - PMC - PubMed
1. Neviani P, Santhanam R, Trotta R, Notari M, Blaser BW, Liu S, et al. The tumor suppressor PP2A is functionally inactivated in blast crisis CML through the inhibitory activity of the BCR/ABL-regulated SET protein. Cancer Cell. 2005;8:355–368. - PubMed

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[1] Rai KR, Sawitsky A, Cronkite EP, Chanana AD, Levy RN, Pasternack BS. Clinical staging of chronic lymphocytic leukemia. Blood. 1975;46:219–234. - PubMed

[2] Rai KR, Sawitsky A, Cronkite EP, Chanana AD, Levy RN, Pasternack BS. Clinical staging of chronic lymphocytic leukemia. Blood. 1975;46:219–234. - PubMed

[3] Binet JL, Lepoprier M, Dighiero G, Charron D, D’Athis P, Vaugier G, et al. A clinical staging system for chronic lymphocytic leukemia: prognostic significance. Cancer. 1977;40:855–864. - PubMed

[4] Binet JL, Lepoprier M, Dighiero G, Charron D, D’Athis P, Vaugier G, et al. A clinical staging system for chronic lymphocytic leukemia: prognostic significance. Cancer. 1977;40:855–864. - PubMed

[5] Danilov AV. Targeted therapy in chronic lymphocytic leukemia: past, present, and future. Clinical therapeutics. 2013;35:1258–1270. - PMC - PubMed

[6] Danilov AV. Targeted therapy in chronic lymphocytic leukemia: past, present, and future. Clinical therapeutics. 2013;35:1258–1270. - PMC - PubMed

[7] Davids MS, Brown JR. Targeting the B Cell Receptor Pathway in Chronic Lymphocytic Leukemia. Leuk Lymphoma. 2012 - PMC - PubMed

[8] Davids MS, Brown JR. Targeting the B Cell Receptor Pathway in Chronic Lymphocytic Leukemia. Leuk Lymphoma. 2012 - PMC - PubMed

[9] Neviani P, Santhanam R, Trotta R, Notari M, Blaser BW, Liu S, et al. The tumor suppressor PP2A is functionally inactivated in blast crisis CML through the inhibitory activity of the BCR/ABL-regulated SET protein. Cancer Cell. 2005;8:355–368. - PubMed

[10] Neviani P, Santhanam R, Trotta R, Notari M, Blaser BW, Liu S, et al. The tumor suppressor PP2A is functionally inactivated in blast crisis CML through the inhibitory activity of the BCR/ABL-regulated SET protein. Cancer Cell. 2005;8:355–368. - PubMed

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Tumor antigen ROR1 targeted drug delivery mediated selective leukemic but not normal B-cell cytotoxicity in chronic lymphocytic leukemia

Affiliations

Tumor antigen ROR1 targeted drug delivery mediated selective leukemic but not normal B-cell cytotoxicity in chronic lymphocytic leukemia

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Affiliations

Abstract

Conflict of interest statement

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Abstract

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