DNA Aptamer-Polymer Conjugates for Selective Targeting of Integrin α4β1+ T-Lineage Cancers

Abstract

Selective therapeutic targeting of T-cell malignancies is difficult due to the shared lineage between healthy and malignant T cells. Current front-line chemotherapy for these cancers is largely nonspecific, resulting in frequent cases of relapsed/refractory disease. The development of targeting approaches for effectively treating T-cell leukemia and lymphoma thus remains a critical goal for the oncology field. Here, we report the discovery of a DNA aptamer, named HR7A1, that displays low nanomolar affinity for the integrin α4β1 (VLA-4), a marker associated with chemoresistance and relapse in leukemia patients. After truncation of HR7A1 to a minimal binding motif, we demonstrate elevated binding of the aptamer to T-lineage cancer cells over healthy immune cells. Using cryo-EM and competition studies, we find that HR7A1 shares an overlapping binding site on α4β1 with fibronectin and VCAM-1, which has implications for sensitizing blood cancers to chemotherapy. We last characterize barriers to in vivo aptamer translation, including serum stability, temperature-sensitive binding, and short circulation half-life, and synthesize an aptamer-polymer conjugate that addresses these challenges. Future work will seek to validate in vivo targeting of α4β1⁺ tumors with the conjugate, establishing an aptamer-based biomaterial that can be readily adapted for targeted treatment of T-cell malignancies.

Keywords: DNA aptamers; cancer targeting; integrins; polymers; very late antigen-4.

Figure 1.

Failed cell-SELEX fortuitously identifies HR7A1, a highly enriched aptamer that broadly binds lymphoma and lymphocytic leukemia cells. (A) Phylogenetic tree of the top 50 aptamers from round 7 of cell-SELEX with H9 cells and corresponding consensus motifs. Aptamers with different consensus motifs are highlighted by color in the phylogenetic tree. The E-values of the consensus motifs represents their statistical significance. (B) The minimum free energy structure of the HR7A1 aptamer, predicted using NUPACK (temperature = 4 °C; Na⁺ = 137 mM; Mg²⁺ = 5.5 mM). (C) Flow cytometry binding of FAM-labeled HR7A1 to H9 parental, H9 4IgB7H3, Jurkat, and Raji cells at different concentrations, as determined by median fluorescence intensity (MFI). The bars graphs are representative of n = 1 independent experiment. FAM, 6-carboxyfluorescein.

Figure 2.

Integrin α4β1 is identified as a potential target of HR7A1. (A) Schematic representation of aptamer pull-down procedure for identifying candidate target proteins. (B) Colloidal blue-stained 8% SDS-PAGE gel of enriched proteins from aptamer pull-down assay with Jurkat and H9 membrane protein extracts. Protein bands that were enriched by HR7A1 relative to the no aptamer and tJBA8.1 controls are marked by numbers, and the dotted boxes represent the bands that were excised for mass spectrometry analysis. (C) Summary of the top extracellular protein hit for each excised band identified by mass spectrometry. A higher SEQUEST HT score represents a better identification.

Figure 3.

HR7A1 interacts with both CD49d (α4) and CD29 (β1) for selective recognition of α4β1. (A) Overlaid flow cytometry plots of 10 nM Cy5-labeled tJBA8.1 and HR7A1 aptamer co-staining with FITC-labeled anti-CD49d and PE-labeled anti-CD29 antibodies on H9, Jurkat, and K562 cells. Plots are representative of n = 1 independent experiment. (B) Flow cytometry analysis of FITC-labeled anti-CD49d antibody, PE-labeled anti-CD29 antibody, and 20 nM Cy5-labeled HR7A1 binding to Jurkat cells, 42 h after nucleofection with a CD29 siRNA duplex. Red dashed horizonal line represents binding to non-specific (NS) siRNA-treated controls to which the CD29 siRNA data points were normalized. Horizonal lines and error bars represent mean ± SD; n = 3 independent experiment. ns > 0.05, *P < 0.05, ****P < 0.0001 (paired one-way ANOVA with Tukey’s test). (C) Association and dissociation kinetics of 100 nM α4β1 and α4β7 binding to biotinylated HR7A1 and CD8.A3 aptamers immobilized on streptavidin biosensors by BLI. The association phase is illustrated from 0-300 s, and the dissociation phase is illustrated from 300-900 s (separated by the vertical dotted line). Data are representative of n = 1 independent experiment with one individual concentration of proteins. Cy5, cyanine 5; FITC, fluorescein isothiocyanate; PE, phycoerythrin.

Figure 4.

HR7A1 binds α4β1 with single-digit nanomolar affinity and can be significantly truncated without loss of function. (A) Rational truncation of the HR7A1 sequence to remove stem-forming constant regions (grey), resulting in HR7A1.Tr1 and HR7A1.Tr2. MFE secondary structures for each sequence are shown, predicted using NUPACK (temperature = 4 °C; Na⁺ = 137 mM; Mg²⁺ = 5.5 mM). The dashed lines indicate the sites of truncation. (B) Association and dissociation kinetics of serially diluted α4β1 binding to biotinylated HR7A1, HR7A1.Tr1, and HR7A1.Tr2 aptamers immobilized on streptavidin biosensors by BLI. The association phase is illustrated from 0-400 s, and the dissociation phase is illustrated from 400-1000 s (separated by the vertical dotted line). K_D values were calculated by performing a global fit of the multi-concentration kinetic data to a 1:1 binding model. K_D values represent mean ± SD; n = 5 individual concentrations of protein. (C) Flow cytometry binding curves of Cy5-labeled HR7A1, HR7A1.Tr1, HR7A1.Tr2, and Sgc4f to K562, H9, and Jurkat cells, as determined by MFI. The curves represent a nonlinear regression assuming one-site specific binding with Hill slope. K_D values were calculated by averaging the individual regression values of the independent experiments. Data points and error bars, and K_D values, represent mean ± SD; n = 3 independent experiments. ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (ordinary one-way ANOVA with Tukey’s test). Cy5, cyanine 5.

Figure 5.

HR7A1.Tr2 selectively binds immortalized T-leukemia and T-lymphoma cancer cells over healthy PBMCs. (A) Flow cytometry binding curves of Cy5-labeled HR7A1.Tr2 to T-leukemia Jurkat cells, T-lymphoma H9 cells, healthy donor PBMCs, and erythroleukemia K562 cells, as determined by MFI fold change over unstained controls. The curves represent a nonlinear regression assuming one-site specific binding with Hill slope. Data points and error bars represent mean ± SD; n = 3 independent experiments with different PBMC donors. ns > 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (orange: significance between aptamer binding to Jurkat cells and PBMCs; purple: significance between aptamer binding to H9 cells and PBMCs; ordinary two-way ANOVA with Dunnett’s test). (B) Relative binding of Cy5-labeled HR7A1.Tr2 to H9 and Jurkat cells over PBMCs at each of the concentrations tested. Values that fall above the dashed blue line represent cancer-selective binding over PBMCs. Graph bars and error bars represent mean ± SD; n = 3 independent experiments with different PBMC donors. Cy5, cyanine 5.

Figure 6.

HR7A1.Tr2, fibronectin, and VCAM-1 share an overlapping binding epitope on α4β1. (A) Cryo-EM density map of HR7A1.Tr2-bound α4β1. A frontal view is shown with HR7A1.Tr2 and α4β1 densities in yellow and grey, respectively. α4 (green) and β1 (blue) atomic models were extracted from PDB 3V4P and PDB 7NXD, respectively, and docked and rigid-body refined against the experimental map. (B) Zoomed-in view of protein loops and helices found at the HR7A1.Tr2-α4β1 binding interface, with α4 loops highlighted in magenta and β1 loops highlighted in red. Identified amino acid residues are numbered according to the full translatable α4 and β1 amino acid sequences (including signal peptide). (C and D) Competitive binding of (C) 10 nM Cy5-labeled HR7A1.Tr2 to Jurkat cells pre-incubated with varying concentrations biotinylated fibronectin CS1 peptide (CS1-b) and (D) 100 nM His-tagged VCAM-1 and varying concentrations of HR7A1.Tr2 to Jurkat cells by flow cytometry. A biotinylated scrambled peptide (SCR-b) and TfR1-binding tJBA8.1 were included in the respective assays as negative competitor controls. Binding was normalized to stained cells without competitor. Data points and error bars represent mean ± SD; n = 3 independent experiments. ns > 0.05, *P < 0.05, ****P < 0.0001 (ordinary two-way ANOVA with Sidak correction). Cy5, cyanine 5; APC, allophycocyanin.

Figure 7.

Multivalent aptamer-polymer conjugates constructed with metal-free click chemistry address issues with aptamer serum and thermal stability. (A) Schematic representation of dual DBCO-TEG- and Cy5-labeled HR7A1.Tr2.S2E2 aptamer conjugation to biotinylated HPMA-AzP3MA polymer by strain-promoted azide-alkyne cycloaddition (SPAAC). Biotin, HPMA, and AzP3MA monomers within the polymer structure are shown in green, purple, and blue, respectively, whereas DBCO-TEG and Cy5 modifications on the 5’ and 3’ ends of the aptamer are shown in red and pink, respectively. (B) SYBR Gold-stained 15% urea-PAGE gel of aptamer conjugation to polymer at different molar ratios, with fixed 66.7 μM aptamer in each reaction. The large upward shift in the aptamer band size signifies successful conjugation of aptamer onto the polymer via SPAAC, and further shifts in conjugate band size reflects different aptamer valencies on the polymer. (C) Semi-quantitative analysis of aptamer conjugation efficiency to the polymer at different molar ratios, as measured by reduction in SYBR gold signal intensity of the unconjugated aptamer band in the urea-PAGE gel. Data point and error bars represent mean ± SD; n = 2 independent gels ran from the same reactions. (D and E) Flow cytometry binding curves of free HR7A1.Tr2.S2E2 and 1:1, 3:1, and 5:1 HR7A1.Tr2.S2E2-polymer conjugates to Jurkat cells, as determined by MFI of (D) the Cy5 label on the aptamer and (E) SA-PE labeling of the biotinylated polymer minus the binding MFI of matched SCRM controls. The curves represent a nonlinear regression assuming one-site specific binding with Hill slope. K_D values were calculated by averaging the individual regression values of the independent experiments. Data points and error bars, and K_D values, represent mean ± SD; n = 3 independent experiments. ns > 0.05, *P < 0.05 (ordinary one-way ANOVA with Tukey’s test). (F) Flow cytometry binding of 5 nM Cy5-labeled free HR7A1.Tr2.S2E2 and 1:1 and 3:1 HR7A1.Tr2.S2E2-polymer conjugates to Jurkat cells after 4-h incubation in 50% normal mouse serum at 37 °C, normalized to a 0-h no incubation control. The curves represent a nonlinear regression assuming one-phase exponential decay. Serum half-life values were calculated from a single regression of the averaged experimental data. Data points and error bars represent mean ± SD; n = 3 independent experiments. **P < 0.01, ****P < 0.0001 (pink: significance between HR7A1.Tr2.S2E2 and 1:1 HR7A1.Tr2.S2E2-polymer; red: significance between HR7A1.Tr2.S2E2 and 3:1 HR7A1.Tr2.S2E2-polymer; ordinary two-way ANOVA with Dunnett’s test). (G) Flow cytometry binding of 5 nM Cy5-labeled free HR7A1.Tr2.S2E2 and 1:1 and 3:1 HR7A1.Tr2.S2E2-polymer conjugates to Jurkat cells at 4, 20, and 37 °C, normalized to binding at 4 °C. Non-specific binding of matched SCRM controls at each temperature was subtracted before normalizing data. Data points and error bars represent mean ± SD; n = 3 independent experiments. ****P < 0.0001 (ordinary two-way ANOVA with Dunnett’s test). DBCO, dibenzocyclooctyne; TEG, triethylene glycol; PEG, polyethylene glycol; HPMA, 2-hydroxypropyl methacrylamide; AzP3MA; 11-azido-3,6,9-trioxaundecan-1-methacrylamide; CCP, 4-Cyano-4-(dodecylsulfanylthiocarbonyl)sulfanylpentanoic acid; Cy5, cyanine 5; SA-PE, streptavidin phycoerythrin.