Targeting of Leukemic Stem Cells By Antibody Functionalized Mesoporous Silica Nanoparticles in a Mouse Model of CALM-AF10 Positive Acute Myeloid Leukemia
Authors: Mandal Tamoghna , Beck Michaela , Lindén Mika , Buske Christian 
Published: October 2019

Abstract
Acute myeloid leukemia has still a dismal prognosis with up to 8 of 10 patients relapsing despite dose intense chemotherapies. AML is propagated by so called leukemic stem cells (LSCs), which are considered to be the cell population responsible for relapse. Based on this it is one major goal to develop treatments which are able to eradicate LSCs, but spare normal hematopoietic stem cells (HSCs) in this disease. We now demonstrate that antibody functionalized mesoporous silica nanoparticles (MSN) are able to target LSCs in a mouse model of CALM-AF10 (CA10) positive AML. We previously showed that in this model AML is initiated and maintained by LSCs being negative for stem cell and myeloid markers, but positive for the lymphoid associated antigen B220 (Deshpande et al., Cancer Cell, 2006). To specifically target B220+ LSCs in this model we generated a multifunctional particle system based on differently charged mesoporous silica nanoparticles (MSN) functionalized with the anti- mouse B220 antibody. To achieve this first amine functionalized and pure silica particles were synthesized and functionalized with a carboxysilane and treated with different amounts of succinic anhydride. In a next step the MSN were loaded with the anthracyline drug daunorubicin. Finally, the particles were further modified with the anti-mouse B220 antibody to obtain specific targeting to murine B220 positive LSCs. MSN coated with the anti-human CD9 antibody served as control. To follow cellular uptake by flow cytometry the antibody tagged MSNs were labelled with the fluorescent dye Atto594. To track the intracellular release of the anthracyline drug daunorubicin, the lipophilic dye DiR was used. First, we tested 7 zwitter-ionic MSNs, with varying surface charge tagged with anti-B220 antibody (conc. 10 µg/mL) for cellular uptake at 4 and 24 hrs in CA10 positive cells. The cellular uptake of the B220 antibody tagged MSN by the CA10 cells was 20.7% and 24.7% higher than in B220-negative AML control cells at 4 hrs and 24 hrs, respectively. The ratio of particle uptake in CA10 cells was 1.3 and 1.9-fold at 4 hrs and at 24 hrs, respectively, compared to the control. This correlated to a 4.6-fold higher mean fluorescence intensity (MFI) of the fluorescent dye Atto594 in the CA10 cells at 4 hrs compared to the control. To demonstrate selectivity, cellular uptake of the anti-B220 MSN was compared to the anti-human CD9 antibody tagged MSN control with a difference in uptake up to 30.8% (particle concentration 100 µg/ml) and a MFI difference and DiR difference of 35.7% and a 34%, respectively after 24 hrs. Loading of the anti-B220 MSN particle with 1.6 µg of daunorubicin per 100 µg of particles induced 100% cell death of CA10 cells at a concentration of 100ug/ml. To confirm that induced cell death was mediated by interaction of the B220 antigen with the anti-B220 antibody-labeled MSN, we blocked the B220 antigen on CA10 cells with unlabeled anti-mouse B220 antibody (81.6% blocking efficiency after 24 hrs). Blocked and unblocked CA10 cells were treated with the anti-B220 MSN loaded with 1.6 µg of daunorubicin/100 µg particles (corresponding to a final concentration of 300ng daunorubicin per ml medium) or free daunorubicin (300 ng/ml). At a particle concentration of 100 µg/ml, 96.5% of the unblocked CA10 cells were eradicated by the anti-B220 MSN compared to 46.5% of the B220 antigen blocked CA10 cells. Anti-B220 MSNs were also more effective than free drug which killed 67% of the cells. Importantly, treatment of CA10 cells with anti-B220 MSN in vitro was able to significantly delay leukemia development after transplantation into lethally irradiated mice with a median onset of disease of 160 days (n=6) compared to a median of 22 days after treatment with the anti-CD9 MSN (n=3)(p<0.0001). Collectively, these data demonstrate the potential of antibody functionalized targeted MSN to selectively kill AML stem cells. This system allows rapid exchange of the antibody coated to MSN, thereby opening the door to target a wide range of LSC associated surface antigens.

Disclosures
Buske: Celltrion, Inc.: Consultancy, Honoraria.