ROR1 Targeted Immunoliposomal Delivery of OSU-2S Show Selective Cytotoxicity in t(1;19) Translocated B-ALL
Authors: Goswami Swagata , Chiang Chi-Ling , Zapolnik Kevan , Xie Zhiliang , Lee James L. , Baskar Sivasubramanian , Rader Christoph , Byrd John C. , Phelps Mitch A. , Bhatnagar Bhavana , Muthusamy Natarajan 
Published: November 2019

The receptor tyrosine kinase ROR1 is uniquely expressed on and required for many hematological malignancies such as t(1;19) positive acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL). The t(1;19) is one of the most frequent translocations in B-ALL, observed in both adult and pediatric patients. The translocation has intermediate prognosis on its own, but is associated with a poor prognosis in the unbalanced der(19)t(1;19) form in pediatric ALL, and in the context of hyperdiploid B-ALL. While leukemic cell dependence on ROR1 is known, ROR1 lacks kinase activity making it difficult to target therapeutically. However, we have previously shown that ROR1 can be targeted to deliver therapeutic payload specifically to leukemic cells in CLL, sparing the normal cells from toxic side effects. This encouraged us to develop ROR1 directed immunoliposomal nanoparticles encapsulating a novel small molecule OSU-2S. OSU-2S is a non-immunosuppressive derivative of the sphingosine analogue FTY720, with potent anti-tumor activity against multiple hematological malignancies including CLL, mantle cell lymphoma (MCL) and canine B-cell lymphoma. OSU-2S demonstrated potent dose dependent cytotoxicity in patient derived B-ALL samples with different cytogenetic backgrounds including translocations t(4;11), t(9;22) and t(1;19) as well as hyperdiploid, hypodiploid and normal cytogenetic background [n=7, p= 0.0032 (0 vs 2.5µM), mean decrease in relative viability= 44.51±12.12%] as assessed by Annexin V/Propidium Iodide staining.
We confirmed ROR1 expression on t(1;19) translocated patient samples by flow cytometry, and synthesized ROR1 targeted OSU-2S immunoliposomal nanoparticles (2A2-OSU-2S-ILP) (mean size= 186.9 +/- 0.8 nm, mean concentration= 1.38*1013 particles/ml). 2A2-OSU-2S-ILP was selectively cytotoxic to t(1;19) translocated ALL, including unbalanced der(19)t(1;19), from relapsed patients aged 29-37, but not ROR1-ve, t(1;19) non translocated ALL, as compared to control IgG-OSU-2S-ILP, or 2A2/IgG immunoliposomes without OSU-2S [n=3, p= 0.04, mean decrease in relative viability (IgG-OSU-2S-ILP vs 2A2-OSU-2S-ILP)= 35.14±7.36%]. Similar results were seen in ROR1+ve 697 cells, a B-ALL cell line carrying t(1;19) translocation, where 2A2-OSU-2S-ILP showed selective cytotoxicity [n=8, p=0.004, mean decrease in relative viability (IgG-OSU-2S-ILP vs 2A2-OSU-2S-ILP)= 61.62±14.63%].
To assess the effect of 2A2-OSU-2S-ILP on t(1;19) positive ALL in-vivo, we used a disseminated cell line derived xenograft model. Immunocompromised NSG mice were engrafted with 697 cells, treated with 2A2-OSU-2S-ILP or IgG-OSU-2S-ILP for 14 days and tumor burden was assessed in the spleen and bone marrow. 2A2-OSU-2S-ILP treatment significantly reduced the number of human CD45+CD19+ cells in the bone marrow as compared to IgG-OSU-2S-ILP cohort (n=6 per cohort, p=0.022, mean decrease in 697 cells in marrow= 1.751 ± 0.6372 million cells/ femur). There was also a trend towards decreased tumor burden in spleen (mean decrease in 697 cells in spleen= 1.883 ± 0.9729 million cells). Together, these data show the ability of ROR1 targeted liposomal nanoparticles to selectively deliver its payload to leukemic cells in t(1;19) translocated B-ALL, sparing toxicity to the normal cells. Ongoing studies are directed towards understanding the mechanistic basis of OSU-2S mediated therapeutic benefit in B-ALL in-vitro and in-vivo.
[This work was supported by NIH-R01-CA197844-01. SG is supported by Pelotonia Graduate Fellowship]

Disclosures
Baskar: NIH: Patents & Royalties: ROR1 mAb 2A2. Rader:NIH: Patents & Royalties: ROR1 mAb 2A2. Byrd:Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; Acerta: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau. Bhatnagar:Novartis and Astellas: Consultancy, Honoraria; Cell Therapeutics, Inc.: Other: Research support; Karyopharm Therapeutics: Other: Research support. Muthusamy:Ohio State University: Patents & Royalties: OSU-2S.