Aptamer-targeted antigen delivery

Abstract

Effective therapeutic vaccines often require activation of T cell-mediated immunity. Robust T cell activation, including CD8 T cell responses, can be achieved using antibodies or antibody fragments to direct antigens of interest to professional antigen presenting cells. This approach represents an important advance in enhancing vaccine efficacy. Nucleic acid aptamers present a promising alternative to protein-based targeting approaches. We have selected aptamers that specifically bind the murine receptor, DEC205, a C-type lectin expressed predominantly on the surface of CD8α(+) dendritic cells (DCs) that has been shown to be efficient at facilitating antigen crosspresentation and subsequent CD8(+) T cell activation. Using a minimized aptamer conjugated to the model antigen ovalbumin (OVA), DEC205-targeted antigen crosspresentation was verified in vitro and in vivo by proliferation and cytokine production by primary murine CD8(+) T cells expressing a T cell receptor specific for the major histocompatibility complex (MHC) I-restricted OVA257-264 peptide SIINFEKL. Compared with a nonspecific ribonucleic acid (RNA) of similar length, DEC205 aptamer-OVA-mediated antigen delivery stimulated strong proliferation and production of interferon (IFN)-γ and interleukin (IL)-2. The immune responses elicited by aptamer-OVA conjugates were sufficient to inhibit the growth of established OVA-expressing B16 tumor cells. Our results demonstrate a new application of aptamer technology for the development of effective T cell-mediated vaccines.

Figure 1

Selection, cloning, and characterization of anti-mDEC205 aptamers. (a) Selection scheme. Three rounds of selection were performed against recombinant mDEC205-hIgG1F_C fusion protein, with negative selection against hIgG1F_C included in Rounds 2 and 3. Round 4 was performed against the surface of Chinese hamster ovary (CHO)/mDEC205, while Round 5 selected for sequences internalized by mouse bone marrow–derived dendritic cells (BMDCs). See Materials and Methods for full procedure. (b) Binding of selection rounds to surface-expressed mDEC205. Individual selection rounds were hybridized with a biotinylated oligonucleotide complementary to a portion of the aptamer pool's 3′ constant region, incubated with CHO/mDEC205 or CHO cells, counter-stained with SA-PE, and analyzed by flow cytometry. (c) Predicted secondary structure of minimized anti-mDEC205 aptamer, min.2. The conserved seven-base motif appearing in most clones—“UUCAUAA”—is highlighted in red. (d) Apparent binding constant for min.2 against mDEC205+ A20.Kb cells as determined by flow cytometry. K_{d app} = 23 ± 6 nmol/l. (e) Binding of minimized, Dy649-labeled anti-mDEC205 aptamer min.2 to primary mDEC205+ BMDCs. The identity of each histogram is as indicated in the key adjacent to the plot.

Figure 2

Min.2, but not cntrl.36, binds to and is internalized by DEC205+ cells. Confocal microscopy images of Chinese hamster ovary (CHO)-mDEC205 cells (a, c, d) or primary mDEC205⁺ bone marrow–derived dendritic cells (BMDCs) (b) following incubation with fluorescently labeled min.2 or cntrl.36. (a, b) Cells were incubated with 200 nmol/l Dy649-labeled min.2 or cntrl.36 (red) and incubated for 1 hour at 37 °C to allow endocytosis, at which time the cells were stringently washed and the cell surface was stained with (a) DEC205 or (b) CD11c antibodies labeled with AF488 (green). Panel labeled “min.2” (inset) is a magnification of the white boxed region in (b). (c) Colocalization of AF488-labeled min.2 (green) and AF647 labeled anti-DEC205 antibody (clone 205yekta; red)). Colocalization of AF488-labeled min.2 (top) or AF488-labeled anti-DEC205 antibody (bottom) with (d) RFP-labeled Rab5a (early endosome), or (e) RFP-labeled LAMP-1 (lysosome). (f–i) Binding and uptake of large (~300 kDa) molecule cargoes by BMDCs. Flow cytometry was used to assess binding and internalization of aptamer-SA:PE conjugates. Biotinylated min.2 or cntrl.36 were precomplexed with SA:PE and subsequently incubated with BMDCs at the indicated temperatures. Following a 1-hour incubation, cells were washed and then treated with an RNAse cocktail as indicated.

Figure 3

Min.2 is functional for crosspresentation in vitro. (a) Proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled OT-I cells following incubation of monovalent aptamer:OVA conjugates or controls with primary dendritic cells (DCs). CD11c⁺ DCs were isolated from the spleens of C57BL/6 mice. After isolation, 4 × 10⁵ DCs were incubated with pIC plus lipopolysaccharide for 4 hours (except Dulbecco's phosphate-buffered saline labeled), followed by overnight incubation with 10 nmol/l conjugate or controls for 16–20 hours at 37 °C. The next day, a single-cell suspension of 1 × 10⁵ CFSE-labeled OT-I cells was added, and the cells were incubated for an additional 3 days at 37 °C, washed with flow cytometry buffer, stained with 4′,6-diamidino-2-phenylindole and antibodies against CD8α and TCR-β, and analyzed by flow cytometry. See Materials and Methods for full procedure. (b–d) Intracellular cytokine staining of OT-I cells in (a). Cells were treated with 10 μg/ml brefeldin A, incubated for 5 hours at 37 °C, stained with antibodies against CD25, interleukin (IL)-2, and interferon (IFN)-γ, and analyzed by flow cytometry. OT-I responses following incubation with min.2:OVA versus cntrl.36:OVA and NLDC145:OVA versus IgG2a:OVA were compared by student's t-test (****P < 0.0001). Data shown are representative of three experiments. Secretion of cytokines (e) IFN-γ and (f) IL-2 into the media by cells following OT-I proliferation assays performed in the presence of 10 nmol/l aptamer:OVA or antibody:OVA conjugates, free OVA (OVA) or treatment with 100 nmol/l SIINFEKL peptide. (g) Dose–response analysis of cells to aptamer or control treatments. Proliferation of CFSE-labeled OT-I cells was monitored following incubation with primary DCs with a graded dose of aptamer (min.2 or cntrl.36) or antibody (rIgG2a or NLDC145; where rIgG2a is an isotype control) OVA conjugates. The concentration of conjugate used is indicated. OVA, ovalbumin.

Figure 4

Injection of multivalent min.2 results in uptake by DEC205⁺ dendritic cells that is functional for antigen crosspresentation. (a) Uptake of multivalent aptamer-SA conjugates by splenic dendritic cells (DCs). C57BL/6 mice were injected with 20 µg fluorescently labeled multimerized cntrl.36, min.2 or NLDC145 antibody. Twenty-four hours later, spleens were harvested, and aptamer uptake by CD11c⁺DEC205⁺ cells was determined by flow cytometry. The percentage of CD11c⁺DEC205⁺ cells that have taken up fluorescent aptamer/antibody is reported in each plot. (b) Activation of adoptively transferred OT-I cells following treatment with multivalent aptamer:OVA conjugates. 10⁶ OT-I cells were labeled with carboxyfluorescein succinimidyl ester and transferred into congenic B6.SJL-ptprc mice. Twenty-four hours later, mice were injected with 20 µg of aptamer:OVA plus 25 µg pIC. Three days later, OT-I cell proliferation was determined by flow cytometry. The percentage of TCR-β⁺CD45.2⁺ cells present in the total splenic population (OT-I cells) is reported on each plot. (c, d) OT-I cells in (b) were analyzed for intracellular expression of interferon (IFN)-γ (c) and interleukin (IL)-2 (d). The percentage of OT-I cells that have undergone three or more divisions is reported in each dot plot. Data shown are representative of three experiments. OVA, ovalbumin.

Figure 5

Injection of min.2 inhibits growth of established M05 flank tumors. Mice were injected s.c. with 10⁶ M05 cells in one flank. Four days later, mice received 10⁶ OT-I cells, and the following day mice were injected with 10 µg aptamer:OVA (min.2 or cntrl.36) or 5 µg NLDC145:OVA plus 25 µg pIC. Tumor growth was monitored and tumor area ± SEM is shown. Statistical significance calculated using two-way analysis of variance. Comparison of cntrl.36:OVA versus min.2:OVA, P < 0.02 (*) and cntrl.36:OVA versus NLDC145:OVA, P < 0.05 (*). Treatment groups were min.2:OVA (green), ctrl.36:OVA (black), NLDC145:OVA (blue), and no treatment (red). Each group contained 5 mice. Data shown are representative of two separate experiments. OVA, ovalbumin.