CA-IX APTAMERS AND DIAGNOSTIC AND THERAPEUTIC USES THEREOF

Abstract

The present invention provides nucleic acid aptamers binding to the Carbonic Anhydrase IX (CA-IX) enzyme, derivatives and conjugates thereof and their use as diagnostic tools, particularly for the imaging of organs and tissues expressing CA-IX, or as therapeutic agents for prevention or treatment of CA-IX related diseases.

Claims

1. A RNA aptamer that specifically binds to Carbonic Anhydrase IX enzyme (CA-IX), containing a sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2.

2. The aptamer according to claim 1, characterized in that it has a length of up to 100 nucleotides.

3. The aptamer according to claim 1, consisting of SEQ ID NO: 1.

4. The aptamer according to claim 1, consisting of SEQ ID NO: 2.

5. The aptamer according to claim 1, wherein all the pyrimidine residues are modified to 2′-fluoropyrimidines.

6. The aptamer according to claim 5 which is further modified to comprise at least one chemical modification, wherein said modification is a chemical substitution at a position selected from a sugar position, a phosphate position and a base position of the nucleic acid.

7. The aptamer according to claim 6, wherein said modification is selected from the group consisting of incorporation of a modified nucleotide, conjugation to a compound and labelling with a reporter moiety.

8. The aptamer according to claim 7, wherein the reporter moiety is selected from the group consisting of a fluorophore moiety, a magnetic or paramagnetic moiety, a radiolabel moiety, an affinity label, an X-ray moiety, an ultrasound imaging moiety, a photoacoustic imaging moiety and a nanoparticle-based moiety.

9. The aptamer according to claim 8, wherein the affinity label is biotin.

10. A method of diagnosis, therapy or visualization of a cancer disease correlated with the expression of CA-IX in a subject in need thereof comprising administering an aptamer as defined in claim 1 to the subject.

11. The method according to claim 10 wherein said cancer disease is selected from renal cancer, cervical cancer, colon cancer, prostate cancer, breast cancer and head and neck tumor.

12. The method according to claim 10 wherein said diagnosis or visualization involves the imaging of a body tissue or organ system expressing CA-IX.

13. A diagnostic, therapeutic or imaging composition comprising an aptamer as defined in claim 1 with pharmaceutically acceptable carriers and excipients.

14. A composition according to claim 13, which is suitable for the imaging of organs and tissues expressing CA-IX.

15. A composition according to claim 14, wherein said imaging is based on magnetic resonance imaging, positron-emission tomography (PET), computed tomography (CT), ultrasound, photoacoustic imaging (PAI), near-infrared fluorescence (NIRF) or single photon emission computed tomography (SPECT).

16. The aptamer according to claim 2, wherein all the pyrimidine residues are modified to 2′-fluoropyrimidines.

17. The aptamer according to claim 3, wherein all the pyrimidine residues are modified to 2′-fluoropyrimidines.

18. The aptamer according to claim 4, wherein all the pyrimidine residues are modified to 2′-fluoropyrimidines.

19. A diagnostic, therapeutic or imaging composition comprising an aptamer as defined in claim 3 with pharmaceutically acceptable carriers and excipients.

20. A diagnostic, therapeutic or imaging composition comprising an aptamer as defined in claim 4 with pharmaceutically acceptable carriers and excipients.

Description

DESCRIPTION OF THE DRAWINGS

[0049] FIG. 1: Binding evaluation on CA-IX purified protein by ELONA assay. Absorbance at 450 nm for SAM-1.T1 and SAM-2.T1 (in the left panel) and polyclonal antibody anti-HSA (in the right panel).

[0050] FIG. 2: SAM-1.T1 and SAM-2.T1 aptamers stability in human serum: a) SAM-1.T1 and b) SAM-2.T1 samples collected at different times were loaded on a denaturing gel (upper panel) and c) bands were quantified by ImageJ program (lower panel). The first line for both gels indicates the sequence not treated with human serum to assess the right size of the samples.

[0051] FIG. 3: K.sub.d evaluation for HSA by ELONA assay. Absorbance at 450 nm for (a) SAM-1.T1, (b) SAM-2.T1 and (c) anti-HSA polyclonal antibody.

EXPERIMENTAL SECTION

Equipment

[0052] RT-qPCR was carried out by StepOne™ Plus Real-Time PCR System (Applied Biosystems). Gel visualization was performed with Gel Doc EZ System (Bio-Rad). ELONA data were acquired by Multiskan™ FC Microplate Photometer (ThermoFisher Scientific).

List of Abbreviations

[0053] CA-IX Carbonic Anhydrase IX [0054] SELEX Systematic Evolution of Ligands by Exponential enrichment [0055] RNA Ribonucleic acid [0056] DNA Deoxyribonucleic acid [0057] DMF Dimethylformamide [0058] DMSO Dimethyl sulfoxide [0059] WT Wild type [0060] nt nucleotides [0061] 2′-F-Py 2′-Fluoro pyrimidine [0062] PAGE PolyAcrylamide gel Electrophoresis [0063] pHe Extracellular pH [0064] PG Proteoglycan-like domain [0065] HPLC High Performance Liquid Chromatography [0066] HSA Human serum albumin [0067] ELONA Enzyme-linked oligonucleotide assay [0068] HIF-1 Hypoxia Inducible Factor 1 [0069] MES 2-(N-morpholino)ethanesulfonic acid [0070] COS7 CV-1 (simian) in Origin with SV40 genetic material cells [0071] Rt-q PCR Real-time polymerase chain reaction

Example 1: Selection and Preparation of Anti-CA-IX Aptamers

[0072] Selection: RNA sequences against CA-IX were screened from a non-naïve library of 84 nt fragments by following a cell-SELEX approach. The method included cycles of counter-selection/selection steps of a pre-enriched pool of aptamers with affinity for CAs on COS7-WT and transiently transfected COS7-CA-IX cells respectively, in which at each round a selective pressure was generated. The enriched pool was incubated on cells in acid condition (in the presence of 60 mM MES buffer) in order to reach the extracellular pH maintained by CA-IX (around 6.8). Before each round of cell-SELEX, the pool was transcribed using a mutant form of T7 RNA polymerase able to incorporate 2′-fluoro pyrimidines in the RNA sequences. The counter-selection step was performed against COS7-WT cells to avoid the selection of aptamers that recognize proteins normally expressed on COS7 cells. The selection step was performed against transient transfected COS7-CA-IX cells in order to select aptamers specific for the target. For each cycle, the pool of 2′-fluoro pyrimidines RNA sequences was firstly incubated on the COS7-WT cells at 37° C., then unbound 2′-fluoro pyrimidines RNA sequences were incubated on COS7-CA-IX cells. After several washes, the sequences were recovered by total RNA extraction. At the end of cell-SELEX protocol, the last cycle was cloned and the samples were sequenced. The resulting sequences were analyzed for enrichment and binding assays by RT-qPCR were performed in order to select the sequences able to bind COS7-CA-IX cells. Essentially, DNA sequences were amplified and transcribed, then RNA sequences were incubated at 100 nM, as final concentration, for 15 minutes at 37° C., after pre-treatment with yeast tRNA 200 μg/mL, on COS7-WT cells and COS7-CA-IX cells in acid condition. Following incubation, cells were washed 3 times with PBS and recovered in TRIsure reagent. An RNA sequence used as reference control was spotted in each point for the normalization. A fold ratio was calculated comparing binding values of COS7-CA-IX over COS7-WT cells. Six sequences representatives of couples or groups of identical sequences were screened. Those with higher fold ratio were chosen for further analysis, performing experimental triplicates. In order to obtain shorter sequences useful for imaging applications, the 84mer original molecules were truncated, selecting the shorter sequences corresponding to SEQ ID NO: 1 (SAM-2.T1) and SEQ ID NO: 2 (SAM-1.T1), by isolating the more structured region and checking that each short sequence maintained the folding of the corresponding portion in the long aptamer. The retention of the binding capability in the truncated sequences was assessed in the following example 2.

[0073] Preparation: The selected aptamers of the invention were then obtained by artificial synthesis. For instance, they were generated synthetically by solid phase synthesis with a RNA synthesizer, according to methods well known in the art. They were, then, conjugated to Biotin at the 3′-end of the sequence.

[0074] The RNA sequences were conjugated at their 3′-end to the commercial Biotin after insertion of a C.sub.6-amino linker (3′-C.sub.6—NH.sub.2). The linker was inserted at the 3′-terminal phosphate by condensation with a C.sub.6 aliphatic diamine in basic catalysis. The resulting free NH.sub.2 moiety was coupled with Biotin-NHS ester, to form a covalent amide bond. The Biotin-NHS ester was dissolved in high-quality anhydrous DMF or DMSO, and the reaction was carried out in 0.1-0.2 M sodium bicarbonate buffer, pH 8.3, at room temperature. Purification was performed by PAGE followed by HPLC.

Example 2: Binding and Affinity of Aptamers SAM-1.T1 and SAM-2.T1 to CA-IX Positive Cells

[0075] To the aim of confirming that the short aptamers SAM-1.T1 (SEQ ID NO: 2) and SAM-2.T1 (SEQ ID NO: 1) contained the active site of the original molecules and preserved high binding and affinity to COS7-CA-IX cells, binding assays were performed in duplicate comparing the sequences binding capability on COS7-CA-IX over COS7-WT cells. The COS7 cells were seeded and transfected with human CA-IX cDNA. After 24 h, the RNA sequences were incubated at 100 nM, as final concentration, for 15 minutes at 37° C. on COS7-WT and COS7-CA-IX in acid conditions. Samples were analysed by RT-qPCR to quantify the amount of bound aptamers and the fold change over COS7-WT cells was calculated.

[0076] The fold change value of 1.3 and 2.4 was obtained for aptamers SAM-1.T1 and SAM-2.T1 respectively. This result confirmed their ability to bind the target CA-IX in its physiological conformation on the membrane of the cell surface.

Example 3: Binding Assay of Aptamers SAM-1.T1 and SAM-2.T1 to Human CA-IX Purified Protein

[0077] The binding of aptamers SAM-1.T1 (SEQ ID NO: 2) and SAM-2.T1 (SEQ ID NO: 1) was further investigated in a different experiment. Biotinylated aptamers SAM-1.T1 and SAM-2.T1 were tested on human CA-IX purified protein to confirm their ability to recognize the target.

[0078] Sequences SAM-1.T1 and SAM-2.T1 200 nM, biotinylated at the 3′-terminus, were incubated on 96 well microtiter high binding plates previously coated or non-coated (blank) with 50 nM human CA-IX purified protein. For each experiment an anti-CA-IX antibody was used as positive control. Samples were then analyzed by ELONA assay. Results, shown in FIG. 1, indicated that both the aptamers bind to CA-IX human protein as the anti-CA-IX antibody.

Example 4: Aptamers SAM-1.T1 and SAM-2.T1 Stability in Human Serum

[0079] Aptamers SAM-1.T1 and SAM-2.T1 were tested for stability in human serum in order to evaluate their resistance to enzymatic degradation. They were incubated in 87% human serum at 37° C. The experiment was performed in triplicate. The samples were collected at different times (T0, 1, 2, 4, 8, 12, 24, 48, 72 h), incubated with proteinase K for 1 h at 37° C. in order to degrade serum proteins and loaded on a denaturing gel.

[0080] Results, reported in FIG. 2, showed that SAM-1.T1 and SAM-2.T1 aptamers are extremely stable in human serum; in particular, SAM-1.T1 aptamer was stable until 24 hours and SAM-2.T1 aptamer was stable for more than 72 hours.

Example 5: Binding Affinity of Aptamers SAM-1.T1 and SAM-2.T1 to HSA

[0081] The ELONA assay was performed in order to evaluate the binding of SAM-1.T1 and SAM-2.T1 aptamers to human serum albumin (HSA). Biotinylated SAM-1.T1 and SAM-2.T1 aptamers were incubated at increasing concentrations (10-100-1000 nM) on 96 well microtiter high binding plates previously coated or not-coated (blank) with 25 nM HSA. No aptamer binding was detected in any of the conditions used, indicating that SAM-1.T1 and SAM-2.T1 aptamers do not react with HSA up to 1000 nM. In each experiment an anti-HSA biotinylated polyclonal antibody was used as positive control. The results are shown in FIG. 3.