Oligonucleotide aptamer-drug conjugates for targeted therapy of acute myeloid leukemia

Abstract

Oligonucleotide aptamers can specifically bind biomarkers on cancer cells and can be readily chemically modified with different functional molecules for personalized medicine. To target acute myeloid leukemia (AML) cells, we developed a single-strand DNA aptamer specific for the biomarker CD117, which is highly expressed on AML cells. Sequence alignment revealed that the aptamer contained a G-rich core region with a well-conserved functional G-quadruplex structure. Functional assays demonstrated that this synthetic aptamer was able to specifically precipitate CD117 proteins from cell lysates, selectively bound cultured and patient primary AML cells with high affinity (Kd < 5 nM), and was specifically internalized into CD117-expressing cells. For targeted AML treatment, aptamer-drug conjugates were fabricated by chemical synthesis of aptamer (Apt) with methotrexate (MTX), a central drug used in AML chemotherapy regimens. The formed Apt-MTX conjugates specifically inhibited AML cell growth, triggered cell apoptosis, and induced cell cycle arrest in G1 phase. Importantly, Apt-MTX had little effect on CD117-negative cells under the same treatment conditions. Moreover, exposure of patient marrow specimens to Apt-MTX resulted in selective growth inhibition of primary AML cells and had no toxicity to off-target background normal marrow cells within the same specimens. These findings indicate the potential clinical value of Apt-MTX for targeted AML therapy with minimal to no side effects in patients, and also open an avenue to chemical synthesis of new, targeted biotherapeutics.

Keywords: Acute myeloid leukemia (AML); Aptamer-drug conjugates; CD117 biomarker; Methotrexate (MTX); Oligonucleotide aptamer; Targeted therapy.

Fig. 1

Development of CD117-specific ssDNA aptamers. (A) The process of aptamer selection using a hybrid cell- and protein-based enrichment approach. (B) Sequencing results of selected ssDNA aptamers, including forward primer region (23 nt), 35 nt random core region, and reverse primer region (23 nt). The top five dominant aptamer sequences with percentage of total sequencing reads. (C) Binding ability of aptamers #1 and #2 to cultured CD117-positive HEL cells (left panel) and patient primary AML cells (right panel) assessed by flow cytometry.

Fig. 2

Characterization of aptamer sequences. (A) Aptamer #1-F (full length), and aptamers #1-3′ Del, #1-5′ Del (with 3′ or 5′ primer region deletion, respectively), and aptamer #1-35 Core (with only the central core sequence) were synthesized. The 2-dimentional structures of aptamers predicted by IDT software. (B) Cell-binding affinity of synthetic aptamers to cultured HEL cells at 4°C. Aptamer #1-F had the highest binding affinity with Kd =4.24 nM. (C) Predicted 3-dimentional G-quadruplex structure of aptamer core sequence. (D) ThT staining assay confirmed the presence of functional G-quadruplex in aptamer #1-F.

Fig. 3

Specific binding of synthetic aptamer #1-F to HEL cells and cellular CD117 proteins. (A) Cultured HEL cells and CD117-negative control cells were stained with aptamer #1-F and cell binding was detected by flow cytometry. (B) A cell mixture of HEL and pre-stained U937 cells were treated with aptamer #1-F and examined by fluorescence microscopy. Aptamer selectively highlighted HEL cells (red fluorescence), but did not react to CD117-negative U937 cells that were pre-stained (green fluorescence). (C) Marrow cells of AML patients were simultaneously stained with both aptamer probe and FITC-labeled anti-CD117 antibody. Aptamer #1-F targeted AML cells that detected by antibody, but aptamer #2 did not. (D) HEL cell lysates were precipitated using synthetic aptamers or ssDNA library, separated on SDS-PAGE, and proteins were detected by Western blotting with anti-CD117 antibody. Whole HEL cell lysate was used as a positive control. (E) To detect intracellular delivery, cell membrane and nuclei were pre-stained in green and blue, respectively. Cells were then treated with aptamer #1-F and examined under confocal microscope. Aptamer signals (red fluorescence) were detected within HEL cells, but not seen within U937 cells.

Fig. 4

Synthesis of aptamer-methotrexate (Apt-MTX) conjugate. (A) MTX with a functional NHS ester group was directly conjugated to aptamer #1-F at 5′ through an NH₂ group to produce Apt-MTX. (B) Characterization of synthesized Apt-MTX products by HPLC. B-1: initial products; B-2: final Apt-MTX products after twice purification containing highly purified Apt-MTX with <3% free MTX; B-3: baseline of water alone; Overlapping of B-2 and B-3 confirmed purity of final products. (C) UV spectroscopy analysis confirmed the presentation of MTX in Apt-MTX products.

Fig. 5

High therapeutic potential of Apt-MTX to treat AML cells. (A) A schematic of Apt-MTX effect on target and off-target cells. (B) Apt-MTX treatment specifically induced 80% inhibition of HEL cell growth at 10 nM final concentration, but had little effect on CD117-negative U937 cells. (C) Both HEL and U937 cells showed similar low sensitivity to free MTX. (D) Apt-MTX specifically stimulated HEL cell apoptosis and had minimal effect on U937 cells. (E) Apt-MTX treatment specifically triggered G1 phase arrest of HEL cells, but not U937 cells.

Fig. 6

High specificity of Apt-MTX treatment to AML cells with minimal toxicity on off-target cells. (A) A schematic of the mixed-cell MTX treatment study. (B) HEL and U937 cells had very similar growth rates under normal culture condition. (C) Cell mixtures of HEL and U937 cells (1:1 ratio) were exposed to Apt-MTX, an equal dose of free MTX, aptamers alone, or non-treatment (−). Resultant changes of each cell population (%) post-treatment in the same cell mixtures were quantified by flow cytometry analysis based on CD15-expresion of U937 cells and CD15-negativity of HEL cells. (D) Total dead cells (%) of cell mixtures under individual treatments.

Fig. 7

Apt-MTX targeted therapy of AML patient marrow specimens. (A) Primary marrow specimens were collected from AML patients #1 to #4. Each specimen was divided into two parts and exposed to Apt-MTX or non-treatment (−) for two days, respectively. Residual CD34+ AML cells and CD34- background normal marrow cells in each specimen were quantified by flow cytometry (%) and compared between Apt-MTX vs. non-treatment control (−). (B) and (C) Ratios of AML cells to background normal marrow cells with or without Apt-MTX treatment were calculated and showed in graphic. (D) A schematic summary of Apt-MTX medicated targeted therapy of AML patient marrow specimens.