1. Introduction
CD19 is a B cell-specific type I transmembrane glycoprotein, belonging to the immunoglobulin (Ig) superfamily, which functions as a major co-stimulatory molecule for the amplification of the B-cell receptor (BCR)-dependent responses. CD19 is expressed exclusively on B cells, from the stage of pro-B cells to that of early plasma cells, while it is not expressed on haematopoietic stem cells or on B cells before the pro-B cell differentiation stage [
1,
2,
3]. Importantly, the expression of CD19 is maintained during malignant transformation of B cells, and it is found highly expressed in the majority of B cell-derived malignancies [
4,
5,
6], but not in other normal body tissues or cells. Given the restricted lineage expression, CD19 has emerged as an attractive targetable marker for B-cell malignancy diagnosis and treatment [
7,
8]. CD19 is, for instance, the target antigen of blinatumomab (Blincyto), a CD19-CD3 bispecific antibody, and of Tisagenlecleucel (CTL019), a CAR-T product, both approved by the FDA for the treatment of relapsed and refractory B cell acute lymphocytic leukaemia (B-ALL) by immunotherapy [
9,
10,
11]. Moreover, given the capacity to induce ligand internalisation, CD19 is also a suitable target for the development of innovative molecules for the targeted delivery of secondary reagents for diagnostic or therapeutic purposes.
Small structured nucleic acid aptamers have emerged as effective tools for targeting relevant markers in cancer pathogenesis, with a great potential for both diagnostic and therapeutic uses [
12]. Indeed, aptamers against receptors overexpressed in cancer may specifically recognise their proper target and are generally endowed with an inhibitory activity, competing with the endogenous ligand for the binding to the target receptor. Moreover, aptamers possess many important advantages over monoclonal antibodies, including low immunogenicity, small size, high batch fidelity, easy production, increased chemical stability, and high versatility [
13]. All these features render aptamers suitable to receive various chemical modifications for their development and improvement as drugs or molecular imaging probes as well. In addition, in some cases, aptamers against cell surface receptors, upon binding to their proper target, demonstrate to undergo target receptor-mediated intracellular uptake, thus representing effective carrier molecules for the targeted delivery of secondary reagents of both diagnostic and therapeutic interest [
14,
15].
In this work, we address the generation and the characterisation of a nuclease resistant 2′fluoro-pyrimidine (2′F-Py) RNA aptamer, selectively binding to the human CD19 glycoprotein, as a potential tool for experimental studies, diagnosis, and treatment of B-cell malignancies.
2. Materials and Methods
2.1. Cell Cultures and Transfection
Used cell lines were from ATCC (LG Standards, Milan, Italy). COS-7, T98G, and U87MG cells were grown in Dulbecco’s modified Eagle’s medium (DMEM), while MEC-1, Jurkat, A549, CLL178, and CLL185 cells were grown in Roswell Park Memorial Institute (RPMI) 1640 medium, both supplemented with 10% of foetal bovine serum (FBS). We added 100 U/mL penicillin/streptomycin for continuous non-primary cell lines. All the transfections were performed by using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. The CD19-expressing plasmid vector (CAT#: SC328905) was purchased from OriGene Technologies (9620 Medical Center Drive, Suite 200, Rockville, MD 20850, USA).
2.2. Cell-SELEX
The starting aptamer pool (G0) (TriLink BioTechnologies, San Diego, CA, USA) contains 2′F-Py RNAs showing a central degenerated region (45 mer) and two fixed regions at the extremities necessary for the RT-PCR amplification and the in vitro transcription procedure. The forward selection primer sequence was 5′-TAATACGACTCACTATAGGGAGACAAGAATAAACGCTCAA-3′; the reverse selection primer sequence was 5′- GCCTGTTGTGAGCCTCCTGTCGAA-3′. Parental COS-7 cells (CD19
−) were used in the counter-selection step, while COS-7 transiently transfected with the human CD19 glycoprotein were used in the positive selection step (COS/CD19
+). The aptamer library was first incubated with COS-7 cells, and then unbound sequences were incubated with COS/CD19
+ cells. Bound aptamers were recovered, and RT-PCR and in vitro transcription were performed. After eight rounds of conventional cell-SELEX, two additional rounds of ‘cell-internalising SELEX’ were performed. In these two rounds, the library was incubated only with COS-7/CD19
+, without any prior incubation with non-target cells (counter-selection step), and following five washes with DMEM serum-free medium to remove unbound aptamers, cells were treated with proteinase K (Roche Diagnostics, IN, USA), for 30 min at 0.5 µg/µL final concentration, in order to deplete for aptamers bound to CD19 expressed on the cell surface. Cells were then washed with serum-free DMEM, and internalised RNA aptamers were recovered by RNA extraction. HTS was performed at rounds III, IV, V, VI, VII, VIII, IX, and X with MySeq Illumina sequencer, following the Illumina MySeq sequence preparation. Raw count reads were generated using python in-house script, then imported in R environment to produce the correlation plot [
16] (The R Project for Statistical Computing. URL
https://www.R-project.org/, (accessed on 21 May 2020).
2.3. Aptamer Sequences
B88:
5′-GGGAGACAAGAAUAAACGCUCAAUGAUAGACAUUCGGUGCUCUCUUUCAUUGACCGUUCACCUGUUGUUCGACAGGAGGCUCACAACAGGC-3′
B85:
5′-GGGAGACAAGAAUAAACGCUCAACGUUGACAACAAAUGACGAUCGUCAACAUGAUGCUUGAGCCCUGUUCGACAGGAGGCUCACAACAGGC-3′
B97:
5′-GGGAGACAAGAAUAAACGCUCAACGUGCAACGCACAAAUUCUUGAUCAUCUCAAUGAUGUGUGCUUUCGACAGGAGGCUCACAACAGGC-3′
B146:
5′-GGGAGACAAGAAUAAACGCUCAACGACACGUUGCCAGCCGGAGCCUUAGUAACGUGCUUUGAUGUCGAUUCGACAGGAGGCUCACAACAGGC-3′
B88.T1:
5′-AACGCUCAAUGAUAGACAUUCGGUGCUCUCUUUCAUUGACCGUU-3′
B88.T2:
5′-CCUGUUGUUCGACAGGAGGCUCACAACAGG-3′
B85.T1:
5′-GCUCAACGUUGACAACAAAUGACGAUCGUCAACAUUGAUGC-3′
B85.T2:
5′-UGAGCCCUGUUCGACAGGAGGCUCA-3′
B85.T2 (FAM):
5′-(FAM)UGAGCCCUGUUCGACAGGAGGCUCA-3′
B85.T2 (Biotin):
5′-UGAGCCCUGUUCGACAGGAGGCUCA(BioBB)-3′
Ctrl Apt:
5′-UUCGUACCGGGUAGGUUGGCUUGCACAUAGAACGUGUCA-3′
Ctrl Apt (Biotin):
5′-UUCGUACCGGGUAGGUUGGCUUGCACAUAGAACGUGUCA(BioBB)-3′
scraApt:
5′-GCUCAACGUUGACAACAAAUGACGA-3′
B85.T2 stick:
5′-UGAGCCCUGUUCGACAGGAGGCUCAXXXXGUACAUUCUAGAUAGCC-3′
SCRA 6 stick (SCRA6):
5′-CUUGUCAGUCAAGGAGGGUGCCACCXXXXGUACAUUCUAGAUAGCC-3′
Scra stick (SCRA):
5′-GCUCAACGUUGACAACAAAUGACGAXXXXGUACAUUCUAGAUAGCC-3′
A9g stick (A9g):
5′-GGGACCGAAAAAGACCUGACUUCUAUACUAAGUCUACGUUCCCXXXXGUACAUUCUAGAUAGCC-3′
miR-16 passenger stick:
5′-ACUCCAGUAUUAACUGUGCUGCUGAGGGGCUAUCUAGAAUGUAC-3′
miR-16 guide:
5′-CCUUAGCAGCACGUAAAUAUUGGCGU-3′
miR-15a passenger stick:
5′-CGCAGGCCAUAUUGUGCUGCCUCAUGGCUAUCUAGAAUGUAC-3′
miR-15a guide:
5′-AAGUAGCAGCACAUAAUGGUUUGUGGG-3′
Scrambled miR passenger stick:
5′-UUAUCGUACUAUCACCUAAGAUGCCGGCUAUCUAGAAUGUAC-3′
Scrambled miR guide:
5′-GGCAUCUUAGGUGAUAGUACGAUAAGG-3′
RNA sequences contain 2′F-Py. Aptamers B88, B85, B97, and B85.T2 (Biotin) were synthesised by ChemGenes Corporation (Wilmington, MA, USA) or produced by in vitro transcription. The other aptamer sequences were produced by TriLink Bio Technologies (San Diego, CA, USA) or Synthetic and Biopolymer Chemistry Core at the Beckman Research Institute of City of Hope (Duarte, CA, USA). The stick portions (underlined) contain 2′F-Py and 2′-oxygen-methyl purines. The X indicate a three-carbon linker spacer ([CH2]3).
Before use, aptamers are subjected to the following temperature cycle to ensure their correct folding: 5 min at 85 °C, 5 min on ice, 10 min at 37 °C.
2.4. Binding Analysis
Binding assays by qRT-PCR: At 48 h post-transfection, COS-7/Cd19+ and parental COS-7 cells were treated with 200 nM aptamer for 30 min at 37 °C in the presence of 0.1 µg/µL polyinosinic acid (Poly(I)) in serum-free medium. Following the incubation, three washings with ice-cold PBS were performed to remove unbound aptamers. Bound RNA aptamers were then recovered with TRIzol in the presence of 0.5 pmol/mL aptamer CL4 (CL4: 5′-GCCUUAGUAACGUGCUUUGAUGUCGAUUCGACAGGAGGC-3′), used as a control for normalisation. The quantity of aptamer bound to the cells was determined by a two-step qRT-PCR procedure. In a first step, the RNA was reverse-transcribed in vitro with specific 3′ primers, according to the following protocol: 5 min at 65 °C, annealing for 5 min at 22 °C, extension for 30 min at 42 °C, end extension for 30 min at 48 °C, enzyme inactivation for 5 min at 95 °C. In a second step, the samples were amplified by real-time quantitative (q)PCR using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) with the following protocol: 2 min at 95 °C, 40 cycles of heating at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 60 °C for 30 s. Further, a melting curve step was performed by heating at 60–95 °C. The sequences of primers used for PCR amplification were Fw 5′-TAATACGACTCACTATAGGGAGACAAGAATAAACGCTCAA-3′ and Rv 5′-GCCTGTTGTGAGCCTCCTGTCGAA-3′ used for G0 starting library and for B88, B85, and B97 long aptamers; primer CL4 Fw 5′-GCCTTAGTAACGTGCTTT-3′ and primer CL4 Rv 5′-GCCTCCTGTCGAATCG-3′; B88.T1 Fw 5′-AACGCTCAATGATAGACAT-3′ and B88.T1 Rv 5′- AACGGTCAATGAAAGAGA-3′; B88.T2 Fw 5′-CGATCCCTGTTGTTCGA-3′ and B88.T2 Rv 5′-AGGCAATACGACCTGTT-3′; B85.T1 Fw 5′-GCTCAACGTTGACAACAA-3′ and B85.T1 Rv 5′- GCATCAATGTTGACGATC-3′; B85.T2 Fw 5′-ACGATCTGAGCCCTGTT-3′ and B85.T2 Rv 5′-AGGCAATACGATGAGC-3′; Scra Fw 5′-TAATACGACTCACTATAGGGAGACAAGAATAAACGCTCAA-3′ and Scra Rv 5′-GCCTGTTGTGAGCCT CCTGTCGAA-3′. Data were normalised to the CL4 reference control and to the number of cells, as determined by counting cells cultured in conjunction with each experiment.
Binding assay by FACS: A total of 3 × 105 cells were washed three times with serum-free medium, resuspended in 300 µL of serum free medium containing 0.4 µg/µL tRNA and 100 nM of Ctrl Apt (Biotin) as unspecific competitors, and incubated for 30 min at 37 °C with slow shaking. Following pre-treatment with the competitors, cells were treated with 250 nM of FAM-labeled B85.T2 aptamer for 30 min at 37 °C with slow shaking in the presence of the competitors, washed once with ice-cold PBS, and then the mean fluorescence was measured at FACS, the cell auto-florescence was subtracted, and the specific binding on CD19+ cells was calculated.
2.5. Aptamer In Vitro Serum Stability
B88.T2 and B85.T2 aptamers were incubated at 4 µM final concentration in 80% human serum for different times. Type AB Human Serum provided by Euroclone (category ECS0219D) was used. At each time point, 16 pmol of aptamer was recovered and incubated with proteinase K (20 mg/mL) for 1 h at 37 °C to remove serum proteins impairing the electrophoretic migration. Then, the samples were resuspended in a denaturing RNA dye (Invitrogen, Waltham, MA, USA) and loaded on 15% denaturing polyacrylamide gel. Gels were stained with ethidium bromide and displayed under UVs.
2.6. Dose-Response Binding by FACS
A total of 3 × 105 MEC-1 cells were washed two times with 5 mL of RPMI serum-free medium, resuspended in 300 µL of serum-free medium containing 0.4 µg/µL tRNA and 100 nM of biotinylated Ctrl Apt as unspecific competitors, and incubated for 30 min at 37 °C with slow shaking. Following the pre-treatment with the unspecific competitors, cells were treated with FAM-labelled B85.T2 aptamer at increasing concentrations (25 nM, 50 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM) for 30 min at 37 °C with slow shaking. Then, cells were washed once with ice-cold PBS, and the mean fluorescence was measured at FACS; cell auto-florescence was subtracted.
2.7. Binding and KD Determination by Bio-Layer Interferometry (BLI)
BLI measurements were performed according to the manufacturer’s instructions using a BLItz system and related commercial biosensors (ForteBio, Fremont, CA, USA). Assays were performed through immobilising the human recombinant CD19 (rhCD19) glycoprotein (Sino Biological, Wayne, PA, USA) or the human recombinant B Cell Maturation Antigen (rhBCMA) protein (Sino Biological, Wayne, PA, USA) used as a negative control on the surface of ARG2 sensor chips following the amino coupling procedure. After pre-hydration for 10 min in PBS buffer, the AR2G tips were activated with an EDC (0.2 M)/NHS (0.05 M) coupling mixture for 300 s; then, rhCD19 and rhBCMA were exposed for 180 s to distinct biosensors at the concentration of 10 µg/mL in 10 mM Na acetate at pH 5 and pH 4.5, respectively. Unused activated carboxylated groups on the tip’s surface were reacted with 1.0 M ethanolamine hydrochloride, pH 8.5, for 120 s. After activation, a regeneration step with 10 mM NaOH was performed to minimise non-specific binding. Dose–response binding experiments to rhCD19 were performed with both B85.T2 and with an unrelated control aptamer (Ctrl Apt). Experiments with B85.T2 were performed in the concentration range between 2.5 and 100 nM, while Ctrl Apt was used at concentrations between 10 and 1 µM. As a further control experiment, the binding of B85.T2 to the unrelated protein rhBCMA was performed using the aptamer at concentrations between 10 and 1 µM. Each individual assay was completed through performing the following steps: (i) exposure to running buffer to acquire the initial baseline (baseline, exposure time 30 s); (ii) exposure to protein solutions (association, volume 4.0 µL, exposure time 180 s); (iii) exposure to running buffer (dissociation, exposure time 120 s); (iv) exposure to 5 mM NaOH (three times; regeneration, exposure time 20 s). A reduced volume sample cuvette (4 µL) was used for all the experiments with the shaker speed set to 2000 rpm according to the manufacturer’s instructions. Reference interferograms were subtracted from experimental values before data processing to reduce the background. Data were exported from the BLItz Pro 1.2 software (ForteBio, Fremont, CA, USA) and re-plotted with GraphPad Prism, vers. 5.00, GraphPad Software (San Diego, CA, USA). Plateau values of binding as reflected by changes in optical thickness (nm) at 202 s were used to calculate the affinity constant (K
D) by applying a non-linear curve fitting algorithm and the one binding site hyperbola as binding model (GraphPad Prism). Data fitting was carried out using the ‘one site binding hyperbola’ algorithm corresponding to the following equation:
where X is the concentration of the ligand, Y is the specific binding, and Bmax is the maximum binding expressed in the same units as the
y-axis.
2.8. Aptamer-Mediated Pull-Down Assay
A total of 10 × 106 MEC-1 cells were pre-treated with 0.4 µg/µL tRNA for 30 min at 37 °C in serum-free medium and then incubated with 400 nM of B85.T2 biotinylated aptamer or with a biotinylated unrelated aptamer used as a control (Ctrl Apt). Following three PBS washings, cells were lysed with 10 mmol/L Tris-HCl (pH 7.5) containing 200 mmol/L NaCl, 5 mmol/L EDTA, 0.1% Triton X-100, and protease inhibitors.
A total of 800 µg of cell lysates in 800 µL of lysis buffer were incubated with 200 µL of magnetic streptavidin beads (Promega Corporation, Madison, WI, USA) for 2 h with slow shaking. Magnetic beads were washed three times with lysis buffer, and bound proteins were recovered in Laemmli buffer and immunoblotted with anti-CD19 antibody (Cell Signaling Technology, Danvers, MA, USA).
2.9. Internalisation Assays
Internalisation assay by trypsin-EDTA washings and FACS analysis: A total of 5 × 105 MEC-1 cells were washed three times with serum-free medium and then resuspended with 500 µL of serum-free medium containing 250 nM of FAM-labeled B85.T2 aptamer. Incubation with the aptamer was performed at different times (15 min, 1 h, and 2 h). Following incubation, cells were washed three times with ice-cold PBS to recover total aptamers or were incubated with 0.25% trypsin-EDTA for 30 min at 4 °C and then washed two times with ice-cold PBS to remove surface-bound aptamers not internalised. The mean fluorescence was measured at FACS, the cell auto-florescence was subtracted, and the aptamer internalisation rate was calculated.
Internalisation assay by immunofluorescence: A total of 4 × 105 MEC-1 or Jurkat cells were pretreated for 30 min at 37 °C with slow shaking with 0.4 µg/µL tRNA and 100 nM of Ctrl Apt (Biotin), as unspecific competitors, in 400 µL of serum-free RPMI, and then FAM-labeled B85.T2 (5 µM) was added to cells in the presence of the competitors and incubated for two hours at 37 °C with slow shaking. Following incubation, cells were washed three times with ice-cold PBS, fixed with 4% PFA, and transferred in a dish of a 24-well plate containing a cover glass. The plate was incubated for 10 min at 37 °C, centrifuged for 5 min at 2000 r.p.m., and washed once with PBS; then, the cover glasses were mounted with SlowFade Diamond Antifade Mountant with DAPI (Life Technologies, Carlsbad, CA, USA) to mark nuclei
2.10. Aptamer-miRNA Conjugate Production
For aptamer–miRNA conjugation, the miRNA passenger and guide strands, resuspended in a specific buffer (20 mM 2-[4-(2-hydroxyethyl) piperazin-1-yl] ethane sulfonic acid (HEPES; pH 7.5), 150 mM NaCl, 2 mM CaCl2) were annealed by incubation at 95 °C for 10 min, 55 °C for 10 min, and 37 °C for 20 min. Further, the B85.T2 aptamer was correctly refolded by incubation at 85 °C for 5 min, on ice for 5 min, at 37 °C for 10 min, and then incubated with the passenger-guide miRNA strands at 37 °C for 30 min (ratio 1:1) to allow for the annealing of the stick sequences.
2.11. miRNA Delivery Assays
A total of 1 × 106 cells were seeded in 12-well plates and treated with 400 nM of aptamers or conjugates. Following 24 h treatment, the cell culture medium was replaced with fresh medium without aptamers or conjugates. Total cell RNA was recovered 48 h after the starting of the treatment with TRIzol Reagent (Life Technologies, Carlsbad, CA, USA). A total of 50 ng of total RNA was then reverse-transcribed with gene-specific stem-loop reverse transcription primers and the TaqMan microRNA reverse-transcription kit (Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s protocol. Amplification, to evaluate miRNA expression level, was performed with TaqMan miRNA Assays (Life Technologies, Carlsbad, CA, USA) on a Bio-Rad CFX384 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). U6 RNA was used as a reference gene.
2.12. Cell Viability and Propidium Iodide (PI) Staning
MEC-1 cells were seeded in 96-well plates (5 × 103 cells per well) or 6-well plates (2 × 105 cells per well) with or without treatment with 400 nmol/L aptamers or complexes to analyse cell viability or perform PI staining, respectively. After 72 h, treatments were renewed and incubation was prolonged for up to 5 days. Cell viability was monitored by CellTiter 96 Proliferation Assay (Promega, Madison, WI, USA), according to the manufacturer’s instructions. For PI staining, cells were recovered, washed with PBS, and incubated with 40 µg/mL PI (Sigma) for 15 min at room temperature. PI-positive cells were analysed with BD Accuri™ C6 cytometer.