Efficient Gene Delivery to Chronic Lymphocytic Leukemia (CLL) Cells with Microbubbles Bearing ROR1 Antibody
Authors: Zhang Suping , Cui Wenjin , Mattrey Robert , Kipps Thomas J. 
Published: October 2019

Abstract 2857

Patients of chronic lymphocytic leukemia (CLL) typically develop progressive immune deficiency, which impairs their response to vaccines. Prior studies showed that infusions of autologous CLL cells transduced ex vivo with adenovirus encoding CD154 could elicit anti-leukemia immune responses. However, the need for high amounts of high-titer adenovirus complicates this approach and handicaps clinical development. A recently developed technology, known as microbubbles, could be activated using safe ultrasound (U/S) energy, potentially providing a new tool with which to affect gene delivery. In this study, we examined whether microbubble-technology could effectively transfect CLL B cells ex vivo or in vivo.

To target CLL cells with microbubbles, we used newly generated mAb specific for ROR1, a surface antigen expressed on CLL B cells, but not on normal B cells or most other adult tissues. Anti-ROR1 mAb was incorporated into the lipid shell of microbubble, which was composed with DSPC and DSPE-PEG2000-Maleimide via maleimide chemistry. We examined whether targeting the microbubbles with anti-ROR1 mAb improved our transfection efficiency for ROR1+ CLL cells. For this we generated microbubbles tagged with anti-ROR1 mAb or control Ig and incubated these with fresh whole blood from patients with CLL. We found that targeted bubbles specifically bound to CD19+CD5+ CLL cells, but not to other cells, including RBCs. Next we examined whether anti-ROR1-tagged microbubbles or non-targeted microbubbles could transfect CLL cells with an expression plasmid (pGFP) encoding green-fluorescence protein (GFP). Isolated CLL cells were incubated with pGFP-laden microbubbles for 1 hour at 37° C. Following this the preparations were exposed to ultrasound at 2w/cm2 and 50% duty cycle or left untreated. After 48 hours culture, the cells were collected and stained with propidium iodide (PI) and examined via flow cytometry. Using these conditions, CLL cells in all treatment groups retained high viability (>80%). CLL cells incubated with anti-ROR1 mAb-tagged microbubbles expressed high levels of GFP provided they had also been exposed to ultrasound; CLL cells treated with anti-ROR1-mAb-tagged microbubbles that did not receive ultrasound treatment remained negative for expression of GFP. Similarly, CLL cells treated with non-targeted pGFP-laden microbubbles did not express GFP regardless of whether they also received ultrasound treatment.

We also examined whether anti-ROR1-mAb-tagged microbubbles could transfect CLL cells in vivo. For this 107 human CLL B cells were injected into the peritoneum of each RAG-2−/−/ γc−/− mouse. Five minutes later, anti-ROR1-mAb-tagged microbubbles or non-targeted microbubbles laden with expression vectors encoding GFP and luciferase were injected into the peritoneum of each animal. Ten minutes later groups of animals did or did not receive treatment with ultrasound at 2w/cm2 and 50% duty cycle for 1 min. Forty-eight hours later mice were examined for reporter gene expression via whole body imaging. The group of animals that received anti-ROR1-mAb-tagged microbubbles and ultrasound treatment all had high-level expression of luciferase. Cells were recovered from the mice via peritoneal lavage. Immunofluorescence studies confirmed that only ROR1+CD5+ cells expressed GFP. In contrast mice injected with anti-ROR1-mAb-tagged microbubbles but did not receive treatment with ultrasound had no detectable luciferase activity. Groups of mice that were treated with non-targeted microbubbles also had negligible leuciferase activity regardless of whether they received ultrasound treatment. This study reveals that anti-ROR1-mAb tagged microbubbles that are activated by extracorporeal ultrasound treatment can be effective vehicles for specific delivery of genes into CLL cells ex vivo and in vivo.

Disclosures:

Kipps: Igenica: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Abbott Industries: Research Funding; Genentech: Research Funding; GSK: Research Funding; Gilead Sciences: Consultancy, Research Funding; Amgen: Research Funding.