WO2013185241A1 - Aptamères spécifiques à cfa et tnf-alpha et applications thérapeutiques associées - Google Patents

Aptamères spécifiques à cfa et tnf-alpha et applications thérapeutiques associées Download PDF

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WO2013185241A1
WO2013185241A1 PCT/CA2013/050459 CA2013050459W WO2013185241A1 WO 2013185241 A1 WO2013185241 A1 WO 2013185241A1 CA 2013050459 W CA2013050459 W CA 2013050459W WO 2013185241 A1 WO2013185241 A1 WO 2013185241A1
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seq
aptamer
cea
oligonucleotide
tnfa
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PCT/CA2013/050459
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Jean Gariepy
Eric Hai-bo HUANG
Erik William ORAVA
Leigh Peter REVERS
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D5 Pharma Inc.
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate

Definitions

  • This invention relates to the discovery and uses of 2 classes of short DNA aptamers that selectively recognize one of either two well-known targets, namely the pro-inflammatory cytokine, tumour necrosis factor alpha (TNF-a) or the clinically relevant serum marker known as the carcinoembryonic antigen (CEA).
  • TNF-a tumour necrosis factor alpha
  • CEA carcinoembryonic antigen
  • Unregulated immune responses are intimately associated with degenerative diseases such as atherosclerosis, arthritis, encephalitis, and tumours.
  • the primary proinflammatory cytokine, tumour necrosis factor alpha (TNFct) plays a critical regulatory role in enhancing these responses.
  • DMARDs disease-modifying antirheumatic drugs
  • Anti-TNFct protein-based therapies (Enbrel, Humira, Infliximab) have emerged as a dominant category of DMARDs.
  • ⁇ 40% of patients still display moderate to high levels of disease even after treatment with protein therapeutics suggesting a substantial need for improved therapies.
  • TNFct is a pro-inflammatory cytokine that is produced by an array of cells types such as macrophages, monocytes, lymphocytes, keratinocytes and fibroblasts, in response to inflammation, infection, injury and other environmental challenges.
  • TNF-a is a type 2 transmembrane protein with an intracellular amino terminus and is synthesized as a 26-kD membrane-bound protein (pro-TNFa) that is cleaved to release a soluble 17- kD TNFa molecule.
  • pro-TNFa membrane-bound protein
  • TNFa has the ability to signal as a membrane bound protein as well as a soluble cytokine.
  • TNFa is only active as a non-covalently associated homotrimer.
  • Sufficient levels of TNFa as well as other mediators are critical for sustaining normal immune responses. TNFa can initiate host defence mechanisms in response to local injury but it can also cause acute and chronic tissue damage.
  • a non-protein composition that binds to TNFa with high specificity and/or affinity without immune responses.
  • Such a composition may act as a TNFa antagonist or inhibitor, by selectively binding, and hindering the function of, or inactivating, the TNFa, and thus be useful as a disease-modifying antirheumatic drug.
  • composition may also be useful conjugated or otherwise associated with a cytotoxic agent for specifically targeting such an agent to a TNFa expressing or overexpressing cell.
  • a cytotoxic agent for specifically targeting such an agent to a TNFa expressing or overexpressing cell.
  • Carcinoembryonic antigen is a 180 kDa GPI-linked cell glycoprotein and a member of ain immunoglobulin cell adhesion molecule superfamily (CEACAMs). It was originally identified as a surface marker on adenocarcinomas of the human
  • CEA is known to be over-expressed on tumor cells of patients with colon, breast, lung, cervix, ovary, stomach, bladder, pancreas and esophageal cancer. Importantly, CEA is shed at higher levels in the serum of cancer patients.
  • CEA family members are over-expressed in over 50% of all human cancers, and are expressed on the surface of the tumor cells in these cancers. In contrast, the expression of CEA in normal cells is very limited. Thus CEA has a wide clinical use as a blood tumor marker, and represents a selective therapeutic target in a large population of cancer patients.
  • compositions that binds to CEA with high specificity and/or affinity may act as a CEA antagonist or inhibitor, by selectively binding, and hindering the function of, or inactivating, CEA, and thus be useful as an anti-cancer compound.
  • a composition may also be useful when conjugated or otherwise associated with a cytotoxic agent for specifically targeting such an agent to a CEA expressing or over-expressing cell.
  • Aptamers are short, single-stranded nucleic acid oligomers (ssDNA or RNA) which adopt a specific tertiary structure allowing them to bind to molecular targets with high specificity and affinities comparable to that of monoclonal antibodies, through interactions other than classic Watson-Crick base pairing.
  • aptamers will display functional properties beyond just binding to their target. For instance, an aptamer to the inflammation factor human neutrophil elastase (hNE) was shown to significantly reduce lung inflammation in rats and displayed greater specificity for their target than an antielastase IgG control.
  • hNE human neutrophil elastase
  • aptamers exhibiting functional attributes include a DNA aptamer to anti-HIV reverse transcriptase and RNA aptamers to the basic fibroblast growth factor and vascular endothelial growth factor. Finally, a single-stranded DNA aptamer selected to bind to thrombin has been shown to inhibit thrombin-catalyzed fibrin-clot formation in vitro using either purified fibrinogen or human plasma.
  • Aptamers have been generated for over 100 proteins including growth factors, transcription factors, enzymes, immunoglobulins, and receptors.
  • a typical aptamer is 10-15 kDa in size (30-45 nucleotides), binds its target with sub-nanomolar affinity, and discriminates against closely related targets. They have several advantages over antibodies. As a class, they have demonstrated therapeutically acceptable toxicity, and a lack of immunogenicity. Aptamers can typically be administered by subcutaneous injection due to their low solubility as compared to antibodies. Aptamers are chemically robust, and can be readily manufactured since they can be chemically synthesized.
  • Membrane impermeant aptamers have the potential to be used as antagonists themselves, or to serve as intracellular delivery agents specific to an internalized surface marker on a cancer cell, for example.
  • Therapeutic cargos such as siRNAs, antisense oligonucleotides, ribozymes as well as low MW drugs, can be directly coupled to aptamers or packaged into particles modified with aptamers.
  • Aptamer-containing conjugates can be constructed by chemically coupling a drug, such as a chemotherapeutic drug, to the aptamer via a linker or by intercalating the drug into the aptamer folded structure creating a physical complex.
  • the cargo is then imported into a target cell due to the aptamer specificity while reducing toxicity towards other cells.
  • Cargos can be conjugated to aptamers during solid-phase synthesis or post- synthesis by incorporating an amino or thiol group at one end of the oligonucleotide during its assembly.
  • a therapeutic protein can also be coupled to the aptamer, to reach an intracellular substrate target.
  • Aptamers can also be conjugated to radionuclides or metal chelators to image or kill cells targeted by the aptamer.
  • aptamers have been conjugated to nanostructures, representing a promising class of new agents for targeted imaging and therapy.
  • cargos can also be encapsulated into such nanopaticles decorated on their surface with aptamers.
  • the targeted structures include nanorods, quantum dots as well as soft and hard nanoparticles.
  • An aptamer that binds with high specificity and/or affinity to TNF-ct would be desirable for its potential as a simple (compared, for example, to an antibody), synthetic, potentially non-immunogenic TNF-ct inhibitor.
  • Such a compound would be useful for a variety of research, diagnostic, and therapeutic uses, for example, for imaging, diagnosis, or for the treatment of atherosclerosis, arthritis, encephalitis, autoimmune disease, and a variety of tumors.
  • Such a compound could also be useful conjugated or otherwise associated with a cytotoxic agent for specifically targeting such an agent to a TNFct-expressing or overexpressing cell.
  • an aptamer that binds with high specificity and/or affinity to CEA would be desirable for its potential as a simple (compared, for example, to an antibody), synthetic, potentially non-immunogenic CEA inhibitor.
  • Such a compound would be useful for a variety of research, diagnostic, and therapeutic uses, for example, for imaging, diagnosis, or for the treatment of a wide variety of tumors.
  • Such a compound could also be useful conjugated or otherwise associated with a cytotoxic agent for specifically targeting such an agent to a CEA-expressing or overexpressing cell.
  • Figure 1 is a graph showing aptamer binding to TNFct.
  • Figure 2 is a figure showing aptamer inhibition of rCEA N domain homotypic binding and binding to rCEA A3B3 domain by ELISA.
  • Figure 3 shows aptamer inhibition of MC38.CEA cells binding to rCEA N.
  • Figure 4A shows a list of the aptamer sequences used to determine minimum binding regions for inhibition of CEA-dependent binding.
  • FIG. 4B illustrated is the inhibition of CEA-dependent binding of
  • Figure 5 shows aptamer binding to CEA+ and CEA- cells using Cy5 labelled aptamers by flow cytometry.
  • Figure 6 shows a summary of implantation studies of MC38.CEA cells in C57/BL6 mice.
  • Figure 7a-c shows, in schematic form, chemical structures of various PEG2o-aptamer conjugates of the present invention.
  • Figure 7d shows the general formula for making PEG2o-aptamer conjugates.
  • Figure 8 shows the change in elution profiles of PEGylated and free TNFa aptamer (TSR1 1 ).
  • FIG. 9 shows the change in molecular weight of TNFa aptamer (TSR1 1 ) following PEGylation.
  • Figure 10 shows the change in elution profiles of PEGylated and free CEA aptamer (CEA54).
  • Figure 1 1 shows the change in molecular weight of CEA aptamer (CEA54) following PEGylation.
  • TNFa aptamers with high specificity and binding affinity to TNFa.
  • the 10 TNFa aptamers disclosed herein at Table 1 (FL1 , FL2, FL4, FL5, FL6,
  • FL8, FL1 1 , FL12, FL16, and FL20 having as their nucleotide sequences the SEQ ID NOs.: 1 -10, respectively, all show high specificity and binding affinity to TNFa, and in one case (FL1 1 ) have been shown capable of inhibiting TNFa functions in vitro.
  • TNFa-specific regions of these aptamers are shown herein at Table 2, (TSR1 , TSR2,
  • TSR2 ATCACAGCGGGTACGAATGGCAGTG (SEQ ID NO. : 12)
  • CEA aptamers with high specificity and binding affinity to CEA.
  • the 10 CEA aptamers disclosed herein at Table 3 (CEA 8, CEA59, CEA56-1 , CEA54, CEA65, CEA57-1 , CEA57-2, CEA42-2, CEA47, and CEA 49), having as their nucleotide sequences the SEQ ID NOs.: 21 -30, respectively, all show high specificity and binding affinity to CEA, and have been shown in the case of CEA54 and CEA56-1 , capable of inhibiting CEA functions in vitro.
  • CEA-specific regions of these aptamers are shown herein at Table 4 (CSR8, CSR59, CSR56-1 , CSR54, CSR65, CSR57-1 , CSR57-2, CSR42-2, CSR47, and CSR49), having as their nucleotide sequences the SEQ ID NOs.: 31 -40, respectively.
  • GTA TGC CGC TTC CGT CCG TCG CTC SEQ ID NO. : 27
  • CEA42-2 GAC GAT AGC GGT GAC GGC ACA GAC GAA TTG GGA GTT AGT ATA CAT CTT ACC GTA TGC CGC TTC CGT CCG TCG CTC (SEQ ID NO. : 28)
  • Binding specificity to TNFa was determined using an aptamer-based enzyme linked binding assay.
  • a 6-well ELISA microtiter-plate (BD Falcon) was coated overnight at 4°C with 10 ⁇ of TNFa (10 g/ml) prepared in coating buffer (0.2 M carbonate/bicarbonate, pH 9.4). The plate was then washed 3 times with PBS-T (0.1 M phosphate, 0.15M sodium chloride, pH 7.0 containing 0.05% Tween-20) and blocked overnight at 4°C in 150 ⁇ of blocking solution (PBS-T + 1 % (w/v) BSA).
  • PBS-T 0.1 M phosphate, 0.15M sodium chloride, pH 7.0 containing 0.05% Tween-20
  • the plate was washed 3 times with PBS-T and incubated overnight at 4°C with 100 ⁇ (10 g/ml) of 5' biotinylated aptamers dissolved in PBS, 0.005% (v/v) Tween-20.
  • the plates were washed 3 times with PBS-T and incubated for 1 hour at 4°C with 100 ⁇ of streptavidin- HRP (1 :2000), washed 5 times with PBS-T. Plates were read on a plate reader at 450 nm following the dispensing of 100 ⁇ of TMB substrate [1 TMB tablet (Sigma-Aldrich
  • Electrophoretic mobility shift assays were performed with TNFa aptamers FL1 , FL2, FL4, FL5, FL6, FL8, FL1 1 , FL12, FL16, and FL20, as described in Table 1 , above, to confirm their specific binding to TNFa. Briefly, the aptamers were 5' end labeled with ⁇ - 32 ⁇ using PNK according to manufacturers protofol (New England
  • 32 P - labeled aptamers [ ⁇ 100 ng] were then incubated for 1 hour at 37 °C with 2[ig of TNFa or a control protein [CEA] in PBS with the addition of 50 g/mL BSA and 10 g/ml poly(dldC). The complexes were resolved on a 6% acrylamide DNA retardation gel (Invitrogen) and the radiolabeled species detected using a phosphorous screen.
  • TNFa were not due to the non-specific DNA binding properties of the protein.
  • the TNFa aptamers also did not bind to the control N-domain of the carcinoembryonic antigen (rCEA).
  • rCEA carcinoembryonic antigen
  • aptamers were diluted in PBS-T and injected for 30 sec at 30ul/min. Any unbound aptamer was removed by regenerating the surface with a 30 sec injection of NaOH followed by 30 sec injection of 1 M NaCI. Aptamer loading on the chip yielded approximately 10-12 response units which represented ⁇ 1 ng of aptamer.
  • TNFa was diluted in two-fold dilutions from 2 ⁇ to 3.9nM in PBS-T (0.05%
  • Example 3 Inhi bition of TN Fa induced cytotoxicity
  • L929 cells are seeded 24 hours before experiments in 96-well flat- bottom microtiter plates at a density of 1 .0x10 4 cells/well in DMEM medium containing
  • TNFa samples are added to the cells for 2 hours. Cells are then washed with warm PBS and incubated in complete medium for another 48 hours. The viability of adherent cells are subsequently determined using a sulforhodamine B assay. The absorbance of the sulforhodamine B signal in each well is read at 570 nm on a plate reader.
  • Example 4 Inhibition of TNFa - dependent NO?- production in macrophages
  • RAW 264.7 cells ATCC are seeded at a density of 1 .0 ⁇ 10 5 cells/well in a 12-well plate in RPMI 1640 + 10% FBS.
  • Cells are pre-treated for 1 hour with 2U/ml IFN- ⁇ (Peprotech, Rocky Hill, NJ) then treated with 100ng/ml TNFa in 1 ml of medium with TNFa aptamers or a control aptamer at a final concentration of 2 ⁇ or with 10 g/ml anti-TNF mAb. Aliquots of the medium [1 ⁇ ] were removed at each time point and the
  • NO2 level determined using the Griess reagent kit for nitrite determination (Invitrogen, Carlsbad, CA). All experiments are done in triplicate and repeated three times.
  • TNFa alone does not elicit the release of nitrates from macrophages.
  • binding specificity to CEA was determined using an aptamer-based enzyme linked binding assay, for CEA aptamers (CEA8, CEA59, CEA56-1 , CEA54, CEA65, CEA57-1 , CEA57-2, CEA42-2, CEA47, and CEA49).
  • CEA aptamers CEA8, CEA59, CEA56-1 , CEA54, CEA65, CEA57-1 , CEA57-2, CEA42-2, CEA47, and CEA49.
  • a 6-well ELISA microtiter- plate (BD Falcon) was coated overnight at 4°C with 10 ⁇ of CEA (10 g/ml) prepared in coating buffer (0.2 M carbonate/bicarbonate, pH 9.4). The plate was then washed 3 times with PBS-T (0.1 M phosphate, 0.15M sodium chloride, pH 7.0 containing 0.05%
  • Electrophoretic mobility shift assays were performed with CEA aptamers CEA8, CEA59, CEA56-1 , CEA54, CEA65, CEA57-1 , CEA57-2, CEA42-2, CEA47, and CEA49, as described in Table 1 , above, to confirm their specific binding to
  • CEA CEA-labeled aptamers
  • PNK PNK according to manufacturers protocol (New England Biolabs, Inc.) and free 32 P was removed using a nucleotide cleanup kit (Qiagen).
  • 32 P -labeled aptamers [ ⁇ 100 ng] were then incubated for 1 hour at 37 °C with 2[ig of CEA or a control protein [TNFct] in PBS with the addition of 50 g/ml_ BSA and 10 g/ml poly(dldC).
  • the complexes were resolved on a 6% acrylamide DNA retardation gel (Invitrogen) and the radiolabeled species detected using a phosphorous screen.
  • aptamers CEA8, CEA59, CEA56-1 , CEA54, CEA65, CEA57-1 , CEA57-2, CEA42-2, CEA47, and CEA49 possess reversible binding properties that are specific to CEA with a range of measurable affinities and thus have potential value as agents capable of blocking CEA-mediated interactions.
  • the aptamers were synthesized with a 5' biotin and a standard C spacer.
  • a ProteOn NLC sensor chip, coated with NeutrAvidin for coupling of biotinylated molecules (Bio-Rad) was preconditioned with three injections of 50mM NaOH in 1 M NaCI with a contact time of 30 sec and a flow rate of
  • aptamers were diluted in PBS-T and injected for 30 sec at 30ul/min. Any unbound aptamer was removed by regenerating the surface with a 30 sec injection of NaOH followed by 30 sec injection of 1 M NaCI. Aptamer loading on the chip yielded approximately 10-12 response units which represented ⁇ 1 ng of aptamer.
  • CEA was diluted in two-fold dilutions from 2 ⁇ to 3.9nM in PBS-T (0.05% Tween-20) pH 7.4.
  • Protein concentrations and a buffer control were injected in the analyte channel with a contact time of 120 sec, dissociation time of 800 sec, and a flow rate of 100 ⁇ /min.
  • the ligand channels were regenerated with a 30 sec injection of 1 M H 3 PO 4 followed by a 30 sec injection of 1 M NaCI. All experiments were run at 25°C and repeated in triplicate.
  • the sensorgrams from the CEA aptamers were x and y transformed, and nonspecific binding was referenced using the interspot reference. Sensorgrams were then double- referenced by subtracting the buffer response. The sensorgrams were fitted globally to a 1 : 1 Langmuir binding model, and the refractive index value was kept constant. The kinetic parameters for the association (k a ), dissociation (k d ), and binding constant (K D ) were derived from the fitted curves. Calculated K d were between 5 and 15 nM for the aptamers, indicating strong binding affinity.
  • Example 8 Aptamer inhibition of rCEA homotypic binding events by ELISA
  • Example 9 Aptamer inhibition of MC38.CEA cells binding to rCEA N and rCEA A3B3 domains
  • addition of aptamers N54 and N64 also referred to as CEA54 and CEA64, respectively, in this document
  • CEA54 and CEA64 significantly inhibited CEA- dependent binding of CEA-expressing MC38.CEA cells to wells coated either with rCEA N domain (red bars) or with rCEA A3B3 domain (blue bars), whereas no inhibitory effect was observed for cells binding to BSA coated wells (green bars).
  • aptamers N54 and N64 had no effect on CEA-negative MC38 cells adhering to wells coated with rCEA N domain (red bars), rCEA A3B3 domain, or BSA (green bars).
  • Example 10 Inhibition of CEA-dependent binding of MC38.CEA cells to rCEA N with truncated aptamer sequences.
  • Figure 4(A) shows a list of the aptamer sequences used to determine minimum binding regions for inhibition of CEA-dependent binding. Bold letters represent the variable region of the sequences.
  • aptamer CEA56 but not aptamer CEA54, retains its inhibitory ability when truncated down to 32 bases with no significant decrease in the adherence of cells as compared to the full length sequence.
  • Example 11 Binding to CEA+ and CEA- cells using Cy5 labelled aptamers by flow cytometry.
  • Cy5-labelled aptamers N54 (shown in Figure 5 as Z/green), N56 (shown in Figure 5 as X/black) and cApt (control, shown in Figure 5 as Y/blue) were incubated with CEA- cells, MC38 (panel A) and HeLa (panel C), or with CEA+ cells, MC38.CEA
  • MC38.CEA cells were injected i.p. into C57/BL6 mice with or without aptamers and sacrificed after 21 days for analysis. Results were summarized in Figure 6, as follows.
  • B Photographs illustrating the implantation of tumours and presence of tumour nodules in peritoneal cavity after 21 days from implantation.
  • E Cytotoxicity assay of aptamers and rCEA N on MC38.CEA cells at the concentration used for in vivo studies (white bars) and 10-fold higher (black bars).
  • aptamers CEA54 and CEA56 are capable of inhibiting the establishment of tumours in a rodent model when used to pretreat malignant CEA- expressing MC38.CEA cells prior to implantation.
  • aptamers CEA54 and CEA56 have potential value as exogenous inhibitory factors in the treatment of malignancies.
  • Example 13 Synthesis and characterization of monovalent mPEG20-TSR.l l aptamer conjugate rVRl l 1 )
  • Amino or thiol-reactive polyethylene glycol polymers can be used to derivatize an amino- or thiol-modified, 3' and/or 5' end of aptamer TSR1 1 , the TNF-ct- specific region of VR1 1 .
  • PEG polymers can present one or more reactive groups within their structures, yielding linear (7a, 7b) and branched (7c) forms of aptamer-PEG conjugates.
  • the general concept is to react PEG-X with Y- Aptamer as illustrated in Figure 7d, where X on the PEG polymer represents either a thiol-reactive maleimide group (Mai), or an amino reactive ester such as N- Hydroxysuccinimide (NHS), succinimidyl glutarate ester (SG), succinimidyl succinate ester (SS), glutaramide succinimidyl ester (GAS), succinimidyl valerate ester (SVA), succinamide succinimidyl ester (SAS), succinimidyl carboxy methyl ester (SCM), or pentafluorophenyl ester (PfP) and where Y depicts an amino group (NH 2 ) or thiol group (SH) located at the 3' or 5' end of a synthetic aptamer.
  • the PEG polymer will range in mass from 1 kDa up to 60kDa.
  • TRS1 1 aptamer was derivated with the 20 kD, amino reactive compound, mPEG-SG (mPEG-succinimidyl glutarate ester, MW 20kDa, (Creative PEGWorks, Winston Salem, NC, USA)) to yield PEG 20 -TSR1 1 conjugate.
  • TRS1 1 was dissolved in 100mM
  • the PEG20-TSR1 1 conjugate was purified by FPLC using a size exclusion SuperdexTM 75 10/300 GL column equilibrated with 150mM NH 4 HC0 3 (eluent). The flow rate was 0.8mL/min, and oligonucleotide-containing fractions (PEGylated and non- PEGylated forms of TSR1 1 ) were detected at 260nm. The yield of this coupling reaction was established to be ⁇ 90% as determined by integrating the peak areas from the chromatogram ( Figure 8).
  • the PEGylated TSR1 1 exhibits similar binding activity to TNF-ct, and an increased half life, as compared to the unconjugated TSR1 1 .
  • Example 14 Synthesis and characterization of monovalent mPEG20-CEA54 aptamer conjugate
  • the PEGylated CEA54 exhibits similar binding activity to CEA54, and an increased half life, as compared to the unconjugated CEA54.

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Abstract

La présente invention concerne de nouveaux aptamères dirigés contre TNFα et CEA, ainsi que leur utilisation dans une variété de méthodes et applications thérapeutiques et diagnostiques.
PCT/CA2013/050459 2012-06-15 2013-06-14 Aptamères spécifiques à cfa et tnf-alpha et applications thérapeutiques associées WO2013185241A1 (fr)

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Cited By (5)

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WO2015139138A1 (fr) * 2014-03-21 2015-09-24 D5Pharma Inc. Aptamères adn spécifiques du cd200r1 et leurs utilisations thérapeutiques
WO2018079864A1 (fr) * 2016-10-24 2018-05-03 김성천 Aptamère de liaison au tnf-alpha, et utilisation thérapeutique
JP2019041672A (ja) * 2017-09-01 2019-03-22 Ｎｅｃソリューションイノベータ株式会社 核酸分子およびその用途
WO2019203904A1 (fr) * 2018-04-20 2019-10-24 Academia Sinica Aptamères ciblant le facteur tnf et leurs utilisations pour le traitement ou le diagnostic de maladies inflammatoires associées au tnf
US10584342B2 (en) 2014-03-21 2020-03-10 D5Pharma Inc. DNA aptamers specific to CD2000R1 and their therapeutic uses

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LEE ET AL.: "An RNA aptamer that binds carcinoembrronic antigen inhibits hepatic metastasis of colon cancer cells in mice", GASTOETEROLOGY, vol. 143, no. 1, 28 March 2012 (2012-03-28), pages 155 - 165 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015139138A1 (fr) * 2014-03-21 2015-09-24 D5Pharma Inc. Aptamères adn spécifiques du cd200r1 et leurs utilisations thérapeutiques
US9938533B2 (en) 2014-03-21 2018-04-10 D5Pharma Inc. DNA aptamers specific to CD200R1 and their therapeutic uses
US10584342B2 (en) 2014-03-21 2020-03-10 D5Pharma Inc. DNA aptamers specific to CD2000R1 and their therapeutic uses
WO2018079864A1 (fr) * 2016-10-24 2018-05-03 김성천 Aptamère de liaison au tnf-alpha, et utilisation thérapeutique
JP2019535249A (ja) * 2016-10-24 2019-12-12 バイオイズ カンパニー リミテッド ＴＮＦ−α結合アプタマー及びその治療的用途
US11028395B2 (en) 2016-10-24 2021-06-08 Biois Co., Ltd. TNF-alpha-binding aptamer, and therapeutic use for same
JP2019041672A (ja) * 2017-09-01 2019-03-22 Ｎｅｃソリューションイノベータ株式会社 核酸分子およびその用途
WO2019203904A1 (fr) * 2018-04-20 2019-10-24 Academia Sinica Aptamères ciblant le facteur tnf et leurs utilisations pour le traitement ou le diagnostic de maladies inflammatoires associées au tnf
EP3781688A4 (fr) * 2018-04-20 2021-06-23 Academia Sinica Aptamères ciblant le facteur tnf et leurs utilisations pour le traitement ou le diagnostic de maladies inflammatoires associées au tnf
TWI733071B (zh) * 2018-04-20 2021-07-11 中央研究院 治療或診斷ｔｎｆ相關發炎疾病的ｔｎｆ靶向適配體及其用途
US11447778B2 (en) 2018-04-20 2022-09-20 Academia Sinica TNF-targeting aptamers and uses thereof for treatment or diagnosing TNF-related inflammatory diseases

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