A comparative analysis of cell surface targeting aptamers

Abstract

Aptamers represent a potentially important class of ligands for the development of diagnostics and therapeutics. However, it is often difficult to compare the function and specificity of many of these molecules as assay formats and conditions vary greatly. Here, with an interest in developing aptamer targeted therapeutics that could effectively deliver cargoes to cells, we chemically synthesize 15 aptamers that have been reported to target cell surface receptors or cells. Using standardized assay conditions, we assess each aptamer's binding properties on a panel of 11 different cancer cell lines, correlate aptamer binding to antibody controls and use siRNA transfection to validate each aptamer's binding to reported target receptors. Using a subset of these molecules known to be expressed on prostate cancers, we use near-infrared in vivo imaging to assess the tumor localization following intravenous injection. Our data demonstrate some surprising differences in the reported specificity and function for many of these molecules and raise concerns regarding their cell targeting capabilities. They also identify an anti-human transferrin aptamer, Waz, as a robust candidate for targeting prostate cancers and for future development of aptamer-based therapeutics.

Fig. 1. Effects of time and blocking on aptamer uptake.

Aptamers labeled with DY650 were incubated at 37 °C with HeLa and HeLa-PSMA cell lines at 500 nM in full growth media with increasing concentrations of ssDNA as a non-specific inhibitor. Cells were allowed to bind/internalize the aptamers for 1, 6, or 24 h. The median fluorescence signal of each aptamer is shown relative to unstained cells. HeLa cells with A C36, B A9.min, C A10-3, D C2.min, E AS1411 and HeLa PSMA cells with F C36, G A9.min, H A10-3, I C2.min, J AS1411. Bar color corresponds to the concentration of added ssDNA. Purple = no competitor, green = 0.01 mg/mL, red = 0.1 mg/mL, and blue = 1.0 mg/mL. Flow cytometry histograms for the corresponding data can be found in Supplementary Figs. 2–6. Data represents the mean of four experimental replicates ±SD. Source data are provided as a Source Data file.

Fig. 2. Variation of non-specific binding/uptake across cell types.

The indicated cell line was incubated at 37 °C with 50 nM, 100 nM, 500 nM, and 1000 nM of a DY650 labeled non-binding control aptamer, C36, with or without 1 mg/mL ssDNA in full growth media for 1 h. The fold change in median fluorescence relative to unstained cells are shown. Samples with ssDNA blocking added (blue) are superimposed on data derived from the same experiment performed in the absence of ssDNA (red). Data represent three independent experimental replicates. Source data are provided as a Source Data file.

Fig. 3. Correlation of aptamer and antibody binding.

The median fluorescence staining of all aptamers at 500 nM are shown relative to the median fluorescence of C36 for each cell type. Each aptamer is compared to a commercially available antibody against the aptamer’s reported target using either the binding (red) incubated at room temperature or internalization and binding with blocking (green) protocol incubated at 37 °C or internalization and binding without blocking (purple) incubated at 37 °C. Antibody signals are shown relative to an isotype control on each cell line (blue). Aptamers A C2.min and B Waz compared to hTfR antibody. Aptamers C A9.min, D A10-3, and E A10-3.2 compared to PSMA antibody. Aptamers F E07 and G CL4 compared to EGFR antibody. Aptamer H GL21.T compared to AXL antibody. Aptamers I SE15-8-mini and J 2-2(t) compared to HER2 antibody. Aptamer K EpDT3 compared to EpCAM antibody. Data represent the mean of three independent experimental replicates for antibodies and four independent experimental replicates for aptamers ±SD. Source data are provided as a Source Data file.

Fig. 4. Validation of specificity using target-specific siRNA.

Cell lines were transfected with siRNA specific for each aptamer’s reported target (red) or a non-specific siRNA control (blue) and subsequently stained with a target-specific antibody or aptamer. Aptamer or antibody binding to untreated cells are shown in green and non-targeted controls (isotype antibody or the non-targeting aptamer C36) are shown in black. Untreated cells are indicated in gray. All assays were performed at 37 °C using our internalization and binding with blocking protocol except for those denoted with an asterisk. The asterisk denotes experiments performed in the absence of blocking agents (see text for details). The cell type and aptamer concentration used for each experiment were: A LNCaP cells: A9.min (100 nM), A10-3 (100 nM), and A10-3.2 (500 nM); B A431 cells: SGC8c (100 nM); C HeLa cells: Waz (100 nM) and C2.min (100 nM); D HeLa cells: E07 (500 nM) and CL4 (1000 nM); E SKBR3 cells: SE15-8-mini (1000 nM) and 2-2(t) (1000 nM); F MCF7 cells: EpDt3 (1000 nM); and G HeLa cells: GL21.T (1000 nM). Data are representative of two independent experimental replicates.

Fig. 5. In vivo imaging and validation of aptamer function.

Nude mice bearing subcutaneous xenograft tumors on their right flank were injected with 2 nmol of AF750 labeled aptamer (A). Dorsal and lateral images of mice bearing 22Rv1 tumors 12 h post injection. The location of the tumor is circled in purple. Injected aptamers are as indicated. Three animals from each cohort (n = 6) are shown. Additional animals in the cohorts can be found in Supplementary Fig. 188. A time course for a subset of animals (n = 3) used in this experiment can be found in Supplementary Fig. 189. B Dorsal and lateral images of mice bearing PC3-PSMA tumors 12 h post injection. The location of the tumor is circled in purple. Injected aptamers are as indicated (n = 3). A time course for a subset of animals used in this experiment can be found in Supplementary Fig. 190. C Predicted secondary structure of Waz and location of base mutations used to generate mutant variants Waz X and Waz GGG. D Relative binding affinity of Waz, Waz.X and Waz.GGG on 22Rv1 cells. E Dorsal and lateral images of mice bearing 22Rv1 tumors 12 h post injection. The location of the tumor is circled in purple. F Quantitation of fluorescence signal within tumors following injection with Waz, Waz.X, Waz.GGG, or C36 (t = 12 h, n = 3). Data represents the mean ± SD. Source data are provided as a Source Data file.