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CN-122029282-A - Novel aptamers to EphA2 (hepaplatin receptor 2 a)

CN122029282ACN 122029282 ACN122029282 ACN 122029282ACN-122029282-A

Abstract

The present invention relates to novel aptamers directed against EphA2 (type a ephrin receptor 2), chemically modified versions thereof and/or complexes comprising any of these, and compositions comprising any of these. The invention also relates to their use in a EphA2 detection or quantification method, or alternatively, in a method comprising EphA2 detection or quantification, for example for screening, diagnosis, prognosis or monitoring of EphA 2-related diseases. The invention also relates to the use of the aptamer, complex or composition in a prophylactic and/or therapeutic method for the treatment of EphA 2-related diseases (such as EphA 2-expressing cancers, including ewing's sarcoma), and to related kits.

Inventors

  • G. Lorent Marina
  • G. Fernandez Miranda

Assignees

  • 阿普塔德尔治疗公司

Dates

Publication Date
20260512
Application Date
20240812
Priority Date
20230812

Claims (18)

  1. 1. A nucleic acid aptamer that specifically binds EphA2, preferably human EphA2, said aptamer comprising or consisting of a sequence of formula (I): Wherein nucleotides n 1 、n 2 、n 6 、n 7 、n 9 to n 13 、n 16 、n 17 、n 21 to n 25 、n 28 、n 31 、n 38 to n 42 and n 48 to n 51 are defined as follows: n 1 and n 2 are independently selected from G or-; n 6 is G or-; n 7 is A or-; n 9 is G or-; n 10 is A or-; n 11 is U or-; n 12 is G or-; n 13 is C or-; n 16 is U or-; n 17 is C or-; wherein G, A, U and C are ribonucleotides, including unmodified and modified forms thereof, and "-" means that no nucleotide is present; The precondition is that: n 6 and n 17 are complementary nucleotides or alternatively, neither are present; n 7 and n 16 are complementary nucleotides or alternatively, neither are present; When n 1 is-, then n 12 is-, and When n 7 is-, then n 11 is U; Wherein: n 21 is U or-; n 22 is C or-; n 23 is G or-; n 24 is U or-; n 25 is C or-; n 28 is U or-; n 31 is G or-; n 38 is G or-; n 39 is A or-; n 40 is C or-; n 41 is G or-; n 42 is A or-; The precondition is that: n 21 and n 42 are complementary nucleotides or alternatively, neither are present; n 22 and n 41 are complementary nucleotides or alternatively, neither are present; n 23 and n 40 are complementary nucleotides or alternatively, neither are present; n 24 and n 39 are complementary nucleotides or alternatively are absent, and N 25 and n 38 are complementary nucleotides or alternatively, neither are present; Wherein: n 48 is C or-; n 49 is C or-; n 50 is G or-; n 51 is A or-; The precondition is that: n 1 and n 49 are complementary nucleotides or alternatively, neither are present; And N 2 and n 48 are complementary nucleotides or alternatively are absent, and Wherein the aptamer is selected from the sequences identified in table 2.1 and/or table 2.2, or a variant having at least 85% identity to any of them.
  2. 2. The nucleic acid aptamer of claim 1, wherein n 25 is C and n 28 is U, n 11 and/or n 12 are absent, and/or the aptamer has a length of 50 nucleotides or less.
  3. 3. The nucleic acid aptamer according to claim 1 or 2, wherein the aptamer is selected from the sequences identified in table 2.1 and/or table 2.2, or a variant having at least 90% identity, preferably 95% identity, to any of them.
  4. 4. A complex comprising an aptamer as defined in any one of the preceding claims and one or more moieties, wherein the aptamer is directly or indirectly linked to the one or more moieties, preferably the moiety is selected from the group consisting of: (i) Antisense oligonucleotides (ASO), siRNA, microRNA, shRNA or ribozymes; (ii) A moiety selected from the group consisting of polysaccharides, lipids (e.g., cholesterol or other lipid moieties), non-protein polymers (e.g., one or more polyethylene glycol (PEG) chains), polyamines, peptides, proteins, small molecules, and combinations thereof; (iii) Preferably the label is selected from the group consisting of biotin, an enzyme, a prosthetic group, a fluorescent material, a luminescent material, a bioluminescent material, an electron dense label, a label for magnetic resonance imaging, a radioactive material, and combinations thereof; (iv) A drug; (vi) Nanoparticle, and (Vii) Any combination thereof.
  5. 5. A composition comprising the aptamer of any one of claims 1 to 3 or the complex of claim 4, preferably wherein the composition is a pharmaceutical composition comprising a pharmaceutically acceptable excipient and/or carrier.
  6. 6. The aptamer of any one of claims 1 to 3, the complex of claim 4 or the composition of claim 5, alone or as a combination therapy for use as a medicament.
  7. 7. The aptamer of any one of claims 1 to 3, the complex of claim 4 or the composition of claim 5 for use in an in vivo detection or quantification method of a target protein, or in an in vivo detection or prognosis method of a disease.
  8. 8. Use of the aptamer of any one of claims 1 to 3, the complex of claim 4 or the composition of claim 5 as a detection or quantification agent in an in vitro or ex vivo detection or quantification method of a target protein, or in an in vitro or ex vivo detection or prognosis method of a disease.
  9. 9. A kit for detecting or quantifying EphA2 comprising the aptamer of any one of claims 1 to 3, the complex of claim 4 or the composition of claim 5, and optionally means for detecting the aptamer or complex.
  10. 10. A nucleic acid aptamer as defined in any one of claims 1 to 3, or a complex as defined in claim 4 or a composition as defined in claim 5 for use in Methods for in vivo detection or quantification of EphA2, or Methods comprising in vivo detection or quantification of EphA2 or EphA 2-expressing cells for screening, diagnosis, prognosis or monitoring of EphA 2-related diseases, or Methods of treating cancer or EphA 2-related diseases in a subject.
  11. 11. The use of the aptamer or complex as defined in claim 10 as EphA2 detection or quantification agent, or as a vehicle for internalization of cells targeted to EphA 2-expressing cells.
  12. 12. The aptamer, complex or composition as defined in claim 10 as a detection or quantification agent In an in vitro or ex vivo assay or quantification method for EphA2, or Use in a method comprising in vitro or ex vivo detection or quantification of EphA2 for screening, diagnosis, prognosis or monitoring of EphA 2-related diseases.
  13. 13. The aptamer, complex or composition for use in a method of treating EphA 2-related disease according to claim 10 or the use according to claim 12, wherein the aptamer, complex or pharmaceutical composition is administered in combination with another drug.
  14. 14. A kit comprising an aptamer, complex or composition as defined in claim 10 and optionally means for detecting said aptamer or complex for detecting or quantifying EphA2, In a method for in vitro or ex vivo detection or quantification of EphA2, or Use in a method comprising in vitro or ex vivo detection or quantification of EphA2 for screening, diagnosis, prognosis or monitoring of EphA 2-related diseases.
  15. 15. The aptamer, complex or composition for use according to claim 10, the aptamer, complex or composition for use according to claim 12 or the kit for use according to claim 14, wherein the EphA 2-related disease is a cancer characterized by expression of EphA2, preferably a sarcoma, such as ewing's sarcoma, rhabdomyosarcoma (e.g. acinar rhabdomyosarcoma), colorectal cancer, prostate cancer, breast cancer, ovarian cancer, bladder cancer, gastric cancer, mesothelioma, thyroid cancer, kidney cancer, lung cancer, cholangiocarcinoma, uterine cancer, liver cancer, testicular cancer, thymoma, pheochromocytoma and paraganglioma (also known as "PCPG"), esophageal cancer, pancreatic cancer, melanoma, brain cancer and head and neck cancer.
  16. 16. The aptamer, complex or composition for use according to claim 15, the aptamer, complex or composition for use according to claim 15 or the kit for use according to claim 15, wherein the EphA 2-expressing cancer is metastatic.
  17. 17. A method of producing a nucleic acid aptamer according to any one of claims 1 to 3, wherein the method comprises synthesis of the aptamer by standard solid phase synthesis using the phosphoramidite method, by in vitro transcription using standard or modified reverse transcriptase or as a direct result of an exponential enrichment ligand system evolution (SELEX) process.
  18. 18. A method of producing the composite of claim 4, wherein the method comprises: (i) Providing a nucleic acid aptamer of the first aspect of the invention, and (Ii) The aptamer is directly or indirectly linked to one or more moieties.

Description

Novel aptamers to EphA2 (hepaplatin receptor 2 a) Technical Field The invention relates to the field of biological medicine. In particular, the present invention relates to novel aptamers to EphA2 (type a hepcidin receptor 2), chemically modified versions thereof and/or complexes comprising any of these, and compositions comprising any of these. The invention also relates to their use in a EphA2 detection or quantification method, or alternatively, in a method comprising EphA2 detection or quantification, for example for screening, diagnosis, prognosis or monitoring of EphA 2-related diseases. The invention also relates to the use of the aptamer, complex or composition in a prophylactic and/or therapeutic method for the treatment of EphA 2-related diseases, such as EphA 2-expressing cancers, including ewing's sarcoma, and to related kits Background The ephrin (Eph) receptor is the most widespread subfamily of receptor tyrosine kinases and is involved in a variety of processes including angiogenesis, tissue border formation, cell migration and cell plasticity. These receptors are recognized mediators in cell-cell interactions and motility and are expressed in human cancers. Of these receptors, ephA2 (type a ephrin receptor 2) has been implicated in a number of processes critical to malignancy progression, such as migration, invasion, metastasis, proliferation, survival, and angiogenesis. Inhibition of EphA2 has been reported to reduce tumor growth, survival, and tumor-induced angiogenesis in a variety of preclinical models of breast, ovarian, and pancreatic cancers (Tandon et al 2011, KASINSKI AND SLACK 2013; and Quinn et al 2016). A variety of solid tumors have been reported to express high levels of EphA2, such as ovarian cancer (Thaker, p.h., et al 2004, lin, y.g., et al 2007), prostate cancer (Walker-Daniels, j., et al 1999, zhao, y. et al 2021), pancreatic cancer (Duxbury, m.s., et al 2004), glioblastoma (Wykosky, j., et al 2005, wang, l.f., et al 2008), lung cancer (Kinch, m.s., et al 2003), melanoma (MARGARYAN, n.v., et 2009, udayakumar, d., et al 2011), esophageal cancer (Miyazaki, t., et 2003), colorectal cancer (Dunne, p.d., et al 2016, kataoka, h., et al 2004, saito, t., et al 2004), osteoma (Giordano, g 20224, et al, j.24, and zen.j., et al 2008, n.v., et al, 2014, and brain tumors (2014, j.2014). Furthermore, the overexpression of EphA2 receptors has been associated with the invasive and metastatic potential of many of these tumors (Zhao, y., et al 2021, zelinski, d.p., et al 2001, brantley-Sieders, d.m., et al 2005). Ewing's Sarcoma (ES) is a rare type of cancer that occurs in bones or soft tissues surrounding bones. It accounts for about 35% of all sarcoma cases, and is a major cause of cancer morbidity and mortality, especially in children. About 25% of these patients already have metastatic disease at diagnosis and their current survival rate is only 20% due to lack of effective treatment. Conventional chemotherapy, such as vincristine, cyclophosphamide and doxorubicin, is currently the only drug approved for the treatment of ES. These nonspecific drugs have limited efficacy and have adverse side effects due to their toxicity. Against this background, there is a need to develop new targeted, selective and effective solutions for the treatment of such destructive cancers. A variety of strategies have been described to target and modulate EphA2 activation. Monoclonal antibodies have been developed that mimic the action of ephrin A1 (Carles-Kinch, k., et al 2002, yang, y., et al 2021) and target the EphA2 large extracellular domain (Biao-xue, r., et al 2011) that is frequently upregulated by tumor cells. Peptides and antibodies that bind to the ephrin-binding pocket have been used to specifically deliver cytotoxins, but their use has been limited due to moderate binding affinity (Jackson, d., et al 2008, koolpe, m., et al 2002, wang, s., et al 2012). Aptamers are short single stranded nucleic acid oligomers (ssDNA or RNA) that form complex three-dimensional shapes and bind to target molecules with high affinity and specificity. Aptamers have key advantages over antibodies, for example, aptamers can be chemically synthesized and can be chemically modified to fine tune their properties for specific applications, such as increasing serum stability, improving pharmacokinetics, and delivering small molecules or imaging agents (Odeh, f., et al 2019). Furthermore, chemically modified RNA aptamers may have little to no immunogenicity, thereby improving safety in clinical applications. Thus, aptamers have become promising candidates for diagnostic and targeted therapeutic applications, typically in the form of aptamer-drug conjugates. WO2020245076 A1 describes a 2' -fluoro modified pyrimidine RNA aptamer that specifically binds EphA2 for use in the treatment or diagnosis of EphA2 expressing tumors. In particular, it describes that the RNA aptamer of SEQ ID NO 1 is capable of binding to the EphA2 recepto