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. 2013 Aug;34(26):6185-93.
doi: 10.1016/j.biomaterials.2013.04.063. Epub 2013 May 28.

Targeted drug delivery and cross-linking induced apoptosis with anti-CD37 based dual-ligand immunoliposomes in B chronic lymphocytic leukemia cells

Bo Yu  1 , Yicheng Mao, Yuan Yuan, Chaofang Yue, Xinmei Wang, Xiaokui Mo, David Jarjoura, Michael E Paulaitis, Robert J Lee, John C Byrd, L James Lee, Natarajan Muthusamy
Affiliations

Targeted drug delivery and cross-linking induced apoptosis with anti-CD37 based dual-ligand immunoliposomes in B chronic lymphocytic leukemia cells

Bo Yu et al. Biomaterials. 2013 Aug.

Abstract

Despite advances in chemo and immunotherapeutic agents for B chronic lymphocytic leukemia (B-CLL), the undesirable adverse side effects due to non-specific cellular uptake remain to be addressed. We identified anti-CD37 monoclonal antibody immunoliposomes (ILs) as vehicles for targeted delivery to B chronic lymphocytic leukemia cells. To achieve maximal benefits for all patients, a new strategy of dual-ligand immunoliposomes (dILs) was developed. A combinatorial antibody microarray technology was adapted to quickly identify optimal antibody combinations for individual patient cells. For proof-of-concept, a B-cell specific antibody, either anti-CD19 or anti-CD20, was combined with anti-CD37 to construct dILs with enhanced selectivity and efficacy. Consistent with data from the antibody microarray, these dILs provided highly specific targeting to both leukemia cell lines and B-CLL patient cells. Compared with the single antibody ILs, the anti-CD19/CD37 dILs clearly demonstrated superior delivery efficiency and apoptosis induction to B-CLL patient cells, whereas the anti-CD20/anti-CD37 dILs were found to be the most efficient for delivery to leukemia cell lines. In addition, it was observed that anti-CD37 ILs without payload drug mediated effective CD37 cross-linking and induced potent apoptosis induction. The anti-CD19/CD20 dILs showed the improved cell apoptosis induction compared to either anti-CD19 ILs or anti-CD20 ILs. Our findings suggest that the dual-ligand ILs may provide a preferred strategy of personalized nanomedicine for the treatment of B-cell malignancies.

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Figures

Fig. 1
Fig. 1. Schematic of dual-antibody immunoliposomes
Simultaneous use of two different antibodies allow dual-Ab immunoliposomes to efficiently bind to and target B cells expressing variable levels of each of the target antigens.
Fig. 2
Fig. 2. CD37 is an optimal candidate for targeted delivery to B-CLL cells
(a) Selective binding of anti-CD37 to CD19+ B cells but not CD3+ T cells in human PBMC. For surface staining, the PBMC cells were incubated with or without anti-CD37-FITC on ice for 30 min and washed twice with cold PBS. Then, the treated cells were further stained with PE labeled anti-CD19 (B-Cell marker) or PE labeled anti-CD3 (T-Cell marker) on ice for another 30 min and rinsed twice with cold PBS. (b) Mean fluorescent intensity of antigen expression levels on cell lines and (c) mean fluorescent intensity of antigen expression levels on CLL patients (n=6). (d) Determination on internalization rates of various mAbs. Cells were incubated with fluorochrome labeled antibodies (anti-CD20-PE, anti-CD19-PE and anti-CD37-PE) at 37°C for 15, 30, 60, 120 and 240 min and extracellular bound antibodies were removed with stripping buffer thus allowing detection of only internalized fluorochrome labeled antibody by flow cytometry. Appropriate IgG isotypes were used as negative controls. Internalization is defined as time-dependent increase in the Mean Fluorescent Intensity (MFI) after acidic washing by stripping buffer, which removed any surface bound antibody (n=3, mean ± SD).
Fig. 3
Fig. 3. Binding efficiency of B-CLL cells on combinatorial antibody microarrays
A library of antibody mixtures containing three antibodies (anti-CD19, anti-CD20 and anti-CD37) at equal total concentrations with all possible combinations was used for microarray assay. Total antibody concentration was maintained at 0.5 mg/ml. The CFSE (carboxy-fluorescein diacetate, succinimidyl ester; Invitrogen, Carlsbad CA) fluorescence labeled B-CLL patient cells (1.5×106/ml) were incubated with the Ab microarray. After removal of unbound cells, the quantified mean spot intensities were acquired and data (n=3, mean ± SD) was presented as relative binding efficiency. (A) Representative of Ab microarrays constructed by printing the anti-CD19, anti-CD20, anti-CD37 antibodies and their dual combinations at different concentrations. The highest binding fluorescence intensity in the Ab microarray was set as 1. (B and C) Quantitative binding profiles of cells from nine B-CLL patients on the combinatorial antibody microarrays (n=9). Linear mixed effect models were used to estimate unrestricted covariance structures and produce robust hypothesis tests. Holm’s method was used to adjust for multiplicity.
Fig. 4
Fig. 4. Optimization of Ab ratio in dILs for efficient delivery to leukemia cell lines and B-CLL cells
(a) Effect of mAb ratio in dILs on Daudi and B-CLL cells. Indicated ratios of anti-CD20 and anti-CD37 antibodies were immobilized onto liposomes with the post insertion method previously described. The binding efficiency was determined by MFI via flow cytometry detection (n=3, P < 0.0001 for all 100/0 vs. 50/50 or 50/50 vs. 0/100 in Daudi or B-CLL cells). Linear mixed effect models were used to estimate unrestricted covariance structures and produce robust hypothesis tests. Holm’s method was used to adjust for multiplicity. (b) Comparison of delivery efficiency of ILs on B cell lines. Fluorescent labeled ILs were incubated with different cell lines for 30 min followed by twice wash, and MFI were determined by flow cytometry. (c) Histogram comparison of anti-CD20 ILs, anti-CD37 ILs and anti-CD20/anti-CD37 (50/50) dILs. MFI for anti-CD20-ILs, anti-CD37-ILs and anti-CD20/anti-CD37-ILs were 12.3, 13.5 and 18.9, respectively. (d) Confocal microscopy analysis on the enhanced cellular uptake by anti-CD20/anti-CD37 (50/50) dILs compared with the two single-Ab ILs. Cells were incubated with anti-CD20 ILs, anti-CD37 ILs and anti-CD20/anti-CD37 dILs for 4 hrs at 37°C and washed twice with PBS, followed by fixation with 2% paraformaldehyde for 30 min. Anti-CD20/anti-CD37 dILs demonstrated the highest cellular uptake.
Fig. 5
Fig. 5. Delivery efficiency evaluation of various ILs on B-CLL cells
(a and b) 1×106 B-CLL cells were incubated with indicated calcein encapsulated ILs and dILs for 1.0 hr at 37°C. Following washing aw ay free ILs, the fluorescence intensity was determined by flow cytometry (n=11). Linear mixed effect models were used to estimate unrestricted covariance structures and produce robust hypothesis tests. Holm’s method was used to adjust for multiplicity.
Fig. 6
Fig. 6. Cytotoxic effect on B-CLL cells with single anti-human CD37 ILs in comparison with cross-linking effect
(a and b) B-CLL cells (1×106) were co-cultured with indicated reagents for 24 hr and cytotoxicity was determined via annexin V FITC/PI analysis by flow cytometry (mean ± SD). Data was analyzed by mixed effect model and p-values were adjusted by Holm’s method.
Fig. 7
Fig. 7. Improved efficiency in delivering of liposomal FTY720 with dILs and enhanced cytotoxicity on B-CLL cells
B-CLL cells were incubated with indicated formulations of IL or dIL FTY720 at 4 μM of FTY720 and 0.1 μg/ml mAbs in total for 24 hr. The viability of the cells were then assessed by annexin V/PI analysis by flow cytometry (mean ± SD). Encapsulating FTY720 into liposomes and targeting it with mAbs can induce potent target cell apoptosis compared to the corresponding IL or dIL groups without FTY720 is shown (n= 6, p-value<0.0001).
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