Aptamers comprising CPG motifs
Materials and methods are provided for producing and using aptamers useful as oncology therapeutics capable of binding to PDGF, PDGF isoforms, PDGF receptor, VEGF, and VEGF receptor or any combination thereof with great affinity and specificity. The compositions of the present invention are particularly useful in solid tumor therapy and can be used alone or in combination with known cytotoxic agents for the treatment of solid tumors. Also disclosed are aptamers having one or more CpG motifs embedded therein or appended thereto.
1. An aptamer comprising a first nucleic acid sequence that binds to a first target and a second nucleic acid sequence that binds to a second target, wherein the first sequence does not stimulate an immune response upon binding to the first target; and the second sequence is an immunostimulatory CpG motif that stimulates an immune response, wherein the CpG motif consists of an oligonucleotide sequence selected from the group consisting of (T/U)GAACG(T/U)(T/U)CGAGA(T/U) (SEQ ID NO: 13), AACG(T/U)(T/U)CGAGA(T/U) (SEQ ID NO: 14), AACG(T/U)(T/U)CGAG (SEQ ID NO: 15) and G(T/U)GAACG(T/U)(T/U)CGAG (SEQ ID NO: 16), wherein the first sequence binds to a first target, wherein the first target is Tumor Necrosis Factor (TNF-α, and wherein the aptamer comprises the structural arrangement in a 5′ to 3′ direction: second nucleic acid sequence-first nucleic acid sequence-3′InvdT, where 3′InvdT represents a 3′ inverted deoxythymidine.
2. An aptamer comprising a first nucleic acid sequence that binds to a first target and a second nucleic acid sequence that binds to a second target, wherein the first sequence does not stimulate an immune response upon binding to the first target; and the second sequence is an immunostimulatory CpG motif that stimulates an immune response, wherein the CpG motif consists of an oligonucleotide sequence selected from the group consisting of (T/U)GAACG(T/U)(T/U)CGAGA(T/U) (SEQ ID NO: 13), AACG(T/U)(T/U)CGAGA(T/U) (SEQ ID NO: 14), AACG(T/U)(T/U)CGAG (SEQ ID NO: 15) and G(T/U)GAACG(T/U)(T/U)CGAG (SEQ ID NO: 16), wherein the first sequence binds to a first target, wherein the first target is selected from the group consisting of IgE and IgE Fcε R1, and wherein the aptamer comprises the structural arrangement in a 5′ to 3′ direction: second nucleic acid sequence-first nucleic acid sequence-3′InvdT, where 3′InvdT represents a 3′ inverted deoxythymidine.
3. An aptamer comprising a first nucleic acid sequence that binds to a first target and a second nucleic acid sequence that binds to a second target, wherein the first sequence does not stimulate an immune response upon binding to the first target; and the second sequence is an immunostimulatory CpG motif that stimulates an immune response, wherein the CpG motif consists of an oligonucleotide sequence selected from the group consisting of (T/U)GAACG(T/U)(T/U)CGAGA(T/U) (SEQ ID NO: 13), AACG(T/U)(T/U)CGAGA(T/U) (SEQ ID NO: 14), AACG(T/U)(T/U)CGAG (SEQ ID NO: 15) and G(T/U)GAACG(T/U)(T/U)CGAG (SEQ ID NO: 16), wherein the first sequence binds to a first target, wherein the first target is CD22, and wherein the aptamer comprises the structural arrangement in a 5′ to 3′ direction: second nucleic acid sequence-first nucleic acid sequence-3′InvdT, where 3′InvdT represents a 3′ inverted deoxythymidine.
4. An aptamer comprising a first nucleic acid sequence that binds to a first target and a second nucleic acid sequence that binds to a second target, wherein the first sequence does not stimulate an immune response upon binding to the first target; and the second sequence is an immunostimulatory CpG motif that stimulates an immune response, wherein the CpG motif consists of an oligonucleotide sequence selected from the group consisting of (T/U)GAACG(T/U)(T/U)CGAGA(T/U) (SEQ ID NO: 13), AACG(T/U)(T/U)CGAGA(T/U) (SEQ ID NO: 14), AACG(T/U)(T/U)CGAG (SEQ ID NO: 15) and G(T/U)GAACG(T/U)(T/U)CGAG (SEQ ID NO: 16), wherein the first sequence binds to a first target, wherein the first target is CTLA4, and wherein the aptamer comprises the structural arrangement in a 5′ to 3′ direction: second nucleic acid sequence-first nucleic acid sequence-3′InvdT, where 3′InvdT represents a 3′ inverted deoxythymidine.
5. An aptamer comprising a first nucleic acid sequence that binds to a first target and a second nucleic acid sequence that binds to a second target, wherein the first sequence does not stimulate an immune response upon binding to the first target; and the second sequence is an immunostimulatory CpG motif that stimulates an immune response, wherein the CpG motif consists of an oligonucleotide sequence selected from the group consisting of (T/U)GAACG(T/U)(T/U)CGAGA(T/U) (SEQ ID NO: 13), AACG(T/U)(T/U)CGAGA(T/U) (SEQ ID NO: 14), AACG(T/U)(T/U)CGAG (SEQ ID NO: 15) and G(T/U)GAACG(T/U)(T/U)CGAG (SEQ ID NO: 16), wherein the first sequence binds to a first target, wherein the first target is selected from the group consisting of PD-1, PD-L1, and PD-L2, and wherein the aptamer comprises the structural arrangement in a 5′ to 3′ direction: second nucleic acid sequence-first nucleic acid sequence-3′InvdT, where 3′InvdT represents a 3′ inverted deoxythymidine.
6. An aptamer comprising a first nucleic acid sequence that binds to a first target and a second nucleic acid sequence that binds to a second target, wherein the first sequence does not stimulate an immune response upon binding to the first target; and the second sequence is an immunostimulatory CpG motif that stimulates an immune response, wherein the CpG motif consists of an oligonucleotide sequence selected from the group consisting of (T/U)GAACG(T/U)(T/U)CGAGA(T/U) (SEQ ID NO: 13), AACG(T/U)(T/U)CGAGA(T/U) (SEQ ID NO: 14), AACG(T/U)(T/U)CGAG (SEQ ID NO: 15) and G(T/U)GAACG(T/U)(T/U)CGAG (SEQ ID NO: 16), wherein the first sequence binds to a first target, wherein the first target is FcRIIB, and wherein the aptamer comprises the structural arrangement in a 5′ to 3′ direction: second nucleic acid sequence-first nucleic acid sequence-3′InvdT, where 3′InvdT represents a 3′ inverted deoxythymidine.
7. An aptamer comprising a first nucleic acid sequence that binds to a first target and a second nucleic acid sequence that binds to a second target, wherein the first sequence does not stimulate an immune response upon binding to the first target; and the second sequence is an immunostimulatory CpG motif that stimulates an immune response, wherein the CpG motif consists of an oligonucleotide sequence selected from the group consisting of (T/U)GAACG(T/U)(T/U)CGAGA(T/U) (SEQ ID NO: 13), AACG(T/U)(T/U)CGAGA(T/U) (SEQ ID NO: 14), AACG(T/U)(T/U)CGAG (SEQ ID NO: 15) and G(T/U)GAACG(T/U)(T/U)CGAG (SEQ ID NO: 16), wherein the first sequence binds to a first target, wherein the first target is BTLA, and wherein the aptamer comprises the structural arrangement in a 5′ to 3′ direction: second nucleic acid sequence-first nucleic acid sequence-3′InvdT, where 3′InvdT represents a 3′ inverted deoxythymidine.
8. An aptamer comprising a first nucleic acid sequence that binds to a first target and a second nucleic acid sequence that binds to a second target, wherein the first sequence does not stimulate an immune response upon binding to the first target; and the second sequence is an immunostimulatory CpG motif that stimulates an immune response, wherein the CpG motif consists of an oligonucleotide sequence selected from the group consisting of (T/U)GAACG(T/U)(T/U)CGAGA(T/U) (SEQ ID NO: 13), AACG(T/U)(T/U)CGAGA(T/U) (SEQ ID NO: 14), AACG(T/U)(T/U)CGAG (SEQ ID NO: 15) and G(T/U)GAACG(T/U)(T/U)CGAG (SEQ ID NO: 16), wherein the first sequence binds to a first target, wherein the first target is transmembrane protein containing immunoglobulin and mucin-like domains (TIM-3), and wherein the aptamer comprises the structural arrangement in a 5′ to 3′ direction: second nucleic acid sequence-first nucleic acid sequence-3′InvdT, where 3′InvdT represents a 3′ inverted deoxythymidine.
9. An aptamer comprising a first nucleic acid sequence that binds to a first target and a second nucleic acid sequence that binds to a second target, wherein the first sequence does not stimulate an immune response upon binding to the first target; and the second sequence is an immunostimulatory CpG motif that stimulates an immune response, wherein the CpG motif consists of an oligonucleotide sequence selected from the group consisting of (T/U)GAACG(T/U)(T/U)CGAGA(T/U) (SEQ ID NO: 13), AACG(T/U)(T/U)CGAGA(T/U) (SEQ ID NO: 14), AACG(T/U)(T/U)CGAG (SEQ ID NO: 15) and G(T/U)GAACG(T/U)(T/U)CGAG (SEQ ID NO: 16), wherein the first sequence binds to a first target, wherein the first target is B lymphocyte activating factor (BAFF), and wherein the aptamer comprises the structural arrangement in a 5′ to 3′ direction: second nucleic acid sequence-first nucleic acid sequence-3′InvdT, where 3′InvdT represents a 3′ inverted deoxythymidine.
10. An aptamer comprising a first nucleic acid sequence that binds to a first target and a second nucleic acid sequence that binds to a second target, wherein the first sequence does not stimulate an immune response upon binding to the first target; and the second sequence is an immunostimulatory CpG motif that stimulates an immune response, wherein the CpG motif consists of an oligonucleotide sequence selected from the group consisting of (T/U)GAACG(T/U)(T/U)CGAGA(T/U) (SEQ ID NO: 13), AACG(T/U)(T/U)CGAGA(T/U) (SEQ ID NO: 14), AACG(T/U)(T/U)CGAG (SEQ ID NO: 15) and G(T/U)GAACG(T/U)(T/U)CGAG (SEQ ID NO: 16), wherein the first sequence binds to a first target, wherein the first target is B7-X, and wherein the aptamer comprises the structural arrangement in a 5′ to 3′ direction: second nucleic acid sequence-first nucleic acid sequence-3′InvdT, where 3′InvdT represents a 3′ inverted deoxythymidine.
11. An aptamer comprising a first nucleic acid sequence that binds to a first target and a second nucleic acid sequence that binds to a second target, wherein the first sequence does not stimulate an immune response upon binding to the first target; and the second sequence is an immunostimulatory CpG motif that stimulates an immune response, wherein the CpG motif consists of an oligonucleotide sequence selected from the group consisting of (T/U)GAACG(T/U)(T/U)CGAGA(T/U) (SEQ ID NO: 13), AACG(T/U)(T/U)CGAGA(T/U) (SEQ ID NO: 14), AACG(T/U)(T/U)CGAG (SEQ ID NO: 15) and G(T/U)GAACG(T/U)(T/U)CGAG (SEQ ID NO: 16), wherein the first sequence binds to a first target, wherein the first target is CD25, and wherein the aptamer comprises the structural arrangement in a 5′ to 3′ direction: second nucleic acid sequence-first nucleic acid sequence-3′InvdT, where 3′InvdT represents a 3′ inverted deoxythymidine.
12. A pharmaceutical composition comprising the aptamer of any one of claim 1 or 2 - 11 , a cytotoxic agent and a pharmaceutically acceptable carrier.
13. The composition of claim 12 , wherein the cytotoxic agent belongs to a class of cytotoxic agents selected from the group consisting of tubulin stabilizers, tubulin destabilizers, anti-metabolites, purine synthesis inhibitors, nucleoside analogs, DNA alkylating agents, DNA modifying agents and vascular disrupting agents.
14. The composition of claim 12 , wherein the cytotoxic agent is selected from the group consisting of calicheamycin, doxorubicin, taxol, methotrexate, gemcitabine, cytarabine, vinblastin, daunorubicin, docetaxel, irinotecan, epothilone B, epothilone D, cisplatin, carboplatin and 5-fluoro-U.