Immunomodulatory polynucleotides, their antibody conjugates, and methods of using them

Abstract

Immune modulatory polynucleotides are disclosed. The immune modulatory polynucleotides may contain 5-modified uridines, 5-modified cytidines, a total of 6 to 16 nucleotides, and / or one or more abasic spacers and / or internucleoside phosphotriesters. Also disclosed are conjugates containing a targeting moiety and one or more immune modulatory polynucleotides. The immune modulatory polynucleotides and conjugates may further contain one or more auxiliary moieties. Also disclosed are compositions containing immune modulatory polynucleotides or conjugates containing one or more sterically condensed internucleoside phosphorothioates. Also disclosed are pharmaceutical compositions containing the immune modulatory polynucleotides or conjugates and methods of use thereof. [Selected Figure] Figure 1

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit of U.S. Provisional Application Nos. 62 / 485,748 and 62 / 537,925, filed on April 14, 2017 and July 27, 2017, respectively; the disclosures of each are hereby incorporated by reference in their entirety into this specification.

[0002] (Field of the Invention) The present invention relates to compositions and methods for modulating immune system responses. Provided herein are immunomodulatory polynucleotides. Also provided herein are immunomodulatory polynucleotides that contain 5 - modified uridine or 5 - modified cytidine and have a length in the range of about 6 to about 16 nucleotides. Further provided herein are conjugates that include a targeting moiety and one or more immunomodulatory polynucleotides. Provided herein are pharmaceutical compositions that include an immunomodulatory polynucleotide or a conjugate that includes a targeting moiety and one or more immunomodulatory polynucleotides. Provided herein are methods of using them for treating diseases such as cancer.

Background Art

[0003] (Background) Pathogen - associated molecular patterns (PAMPs) are molecules associated with various pathogens and are recognized by toll - like receptors (TLRs) and other pattern - recognition receptors (PRRs) that activate the innate immune response. The ability of PAMPs to mobilize the immune system in the absence of pathogens provides a strategy (e.g., anti - cancer therapy) for treating various diseases involving cell destruction through the use of the innate immune system response. One class of PAMPs that has been investigated for various therapeutic applications is immunostimulatory polynucleotides such as CpG ODNs (e.g., agatrimod). CpG ODNs are thought to up - regulate cytokines (e.g., type I interferons and interleukins) by mediating TLR9 dimerization in immune cells (e.g., B cells, monocytes, and plasmacytoid dendritic cells (pDCs)), thereby activating natural killer cells.

[0004] CpG ODNs are generally classified into three classes: class A, class B, and class C. Class A CpG ODNs typically contain 3'- and 5'-terminal poly-G tails with a phosphorothioate backbone and a central palindromic sequence containing a phosphate backbone. Class A CpG ODNs typically contain CpGs in the central palindromic sequence. Class B CpG ODNs generally contain a fully phosphorothioate backbone, and the sequence at the 5'-end of class B CpG ODNs is often extremely important for TLR9 activation. Class C CpG ODNs contain a fully phosphorothioate backbone with a 3'-terminal sequence that allows for duplex formation. CpG ODNs are often prone to degradation in serum. Thus, the pharmacokinetics of CpG ODNs can be one of the limiting factors in their development as therapeutic agents. Additionally, CpG ODNs often exhibit heterogeneous tissue distribution in vivo, and the major accumulation sites are within the liver, kidney, and spleen. Such distribution can induce off-target activities and local toxicities associated with PAMPs. Thus, the therapeutic applications of CpG ODNs can be facilitated by addressing the pharmacokinetic / pharmacodynamic issues described herein.

[0005] Thus, there is a need for novel immunomodulatory polynucleotides. SUMMARY OF THE INVENTION

[0006] (Summary of the Invention) Generally, the present invention relates to immunomodulatory (e.g., immunostimulatory) polynucleotides, as well as conjugates containing a targeting moiety and one or more immunomodulatory (e.g., immunostimulatory) polynucleotides.

[0007] In one aspect, disclosed is an immunomodulatory polynucleotide. The immunomodulatory polynucleotide can be an immunostimulatory polynucleotide. Alternatively, the immunomodulatory polynucleotide can be an immunosuppressive polynucleotide.

[0008] In some embodiments, the immunomodulatory polynucleotide contains one or more (e.g., one or two) abasic spacers or phosphotriesters. In certain embodiments, the immunomodulatory polynucleotide contains one or more (e.g., 1 to 5) internucleoside phosphotriesters. In a further embodiment, at least one of the internucleoside phosphotriesters contains a conjugate group. In yet a further embodiment, the immunomodulatory polynucleotide further contains a terminal phosphate ester (e.g., a 5'-terminal phosphate ester or a 3'-terminal phosphate ester). In yet a further embodiment, the terminal phosphate ester contains a conjugate group. In other embodiments, the immunomodulatory polynucleotide includes a 5'-cap or a 3'-cap. In yet other embodiments, the immunomodulatory polynucleotide contains a 5'-cap that is a 5'-5' cap. In yet other embodiments, the 5'-5' cap contains a conjugate group covalently linked to an internucleoside phosphate, internucleoside phosphorothioate, or internucleoside phosphorodithioate. In some embodiments, the immunomodulatory polynucleotide includes a 3'-cap that contains a conjugate group covalently linked to an internucleoside phosphate, internucleoside phosphorothioate, or internucleoside phosphorodithioate.

[0009] In a further embodiment, the immunomodulatory polynucleotide contains a 5'-capping group that is a monophosphate, diphosphate, triphosphate, auxiliary moiety, terminal phosphodiester, terminal phosphotriester, 5'-5' cap, or a group -OR' (wherein R' is a bio-reversible group, non-bio-reversible group, or O-protecting group). In yet a further embodiment, the 5'-capping group is an optionally substituted C bonded to a phosphate, phosphorothioate, or phosphorodithioate 1-6It is a monophosphate or a terminal phosphodiester containing alkyl. In yet further embodiments, the immunomodulatory polynucleotide is a 3'-capping group that is a monophosphate, diphosphate, triphosphate, auxiliary moiety, terminal phosphodiester, terminal phosphorotriester, and a group -OR' (wherein R' is a bioreversible group, a non-bioreversible group, or an O-protecting group). In some embodiments, the 3'-capping group is optionally substituted C bonded to a phosphate, phosphorothioate, or phosphorodithioate 1-6 It is a monophosphate or a terminal phosphodiester containing alkyl.

[0010] In certain embodiments, the immunomodulatory polynucleotide contains one or more (e.g., one or two) abasic spacers. In further embodiments, at least one of the abasic spacers is an internucleoside abasic spacer. In yet further embodiments, at least one of the abasic spacers is a 3'-terminal abasic spacer. In yet further embodiments, at least one of the abasic spacers contains a conjugated group.

[0011] In certain embodiments, the immunomodulatory polynucleotide contains 5-modified uridine (e.g., 5-halouridine (e.g., 5-bromouridine or 5-iodouridine) or 5-modified cytidine). In further embodiments, the 5-modified uridine (e.g., 5-halouridine (e.g., 5-bromouridine or 5-iodouridine)) is at least one of the two 5'-terminal nucleosides or is present in an immunostimulatory sequence (ISS) in the immunomodulatory polynucleotide. In still further embodiments, the 5-modified uridine (e.g., 5-halouridine) includes a 3'-position bonded to an internucleoside phosphodiester phosphate. In certain embodiments, the 5-modified uridine (e.g., 5-halouridine) includes a 3'-position bonded to an internucleoside phosphorothioate. In still further embodiments, the 5-modified uridine (e.g., 5-halouridine) is at the 5'-terminus. In some embodiments, the 5-modified uridine is 5-bromouridine. In specific embodiments, the immunomodulatory polynucleotide contains cytidine and guanosine as the second and third nucleosides or as the third and fourth nucleosides.

[0012] In specific embodiments, the immunomodulatory polynucleotide contains a 5'-terminal immunostimulatory sequence. In certain embodiments, at least one of the internucleoside phosphotriesters is bonded to the 3'-carbon atom of a nucleoside having a 5'-carbon atom bonded to the 5'-terminal immunostimulatory sequence.

[0013] In a further embodiment, the immunomodulatory polynucleotide comprises a total of 6 to 16 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) nucleotides. In yet a further embodiment, at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%) of the internucleoside bridging groups in the immunomodulatory polynucleotide contain phosphorothioate. In yet a further embodiment, at least 50% of the internucleoside bridging groups in the immunomodulatory polynucleotide contain phosphorothioate.

[0014] In some embodiments, the immunomodulatory polynucleotide comprises a conjugate group covalently attached to a nucleobase in the immunomodulatory polynucleotide.

[0015] In one embodiment, the immunomodulatory polynucleotide comprises one or more auxiliary moieties. In certain embodiments, the immunomodulatory polynucleotide comprises a conjugate moiety containing at least one of the auxiliary moieties. In some embodiments, at least one of the auxiliary moieties contains poly(ethylene glycol) (PEG) having a molecular weight of 100 Da to 2,500 Da. In a further embodiment, each PEG independently contains a total of at least 3 ethylene glycol repeating units. In yet a further embodiment, each PEG independently contains a total of at least 20 ethylene glycol repeating units. In yet a further embodiment, each PEG independently contains a total of 50 or fewer ethylene glycol repeating units. In other embodiments, the immunomodulatory polynucleotide contains 1 to 8 PEGs.

[0016] In certain embodiments, the immunomodulatory polynucleotide is a polynucleotide disclosed herein (e.g., in Table 2).

[0017] In another aspect, disclosed is a hybridized immunomodulatory polynucleotide comprising an immunomodulatory polynucleotide hybridized to a complementary polynucleotide.

[0018] In yet another aspect, disclosed is a composition comprising an immunomodulatory polynucleotide, wherein the immunomodulatory polynucleotide comprises at least one stereochemically enriched internucleoside phosphorothioate.

[0019] In some embodiments, at least one stereochemically enriched internucleoside phosphorothioate is positioned between the 5'-terminal nucleoside and the CpG cytidine in the immunostimulatory sequence of the immunomodulatory polynucleotide. In a further embodiment, one stereochemically enriched internucleoside phosphorothioate connects the first and second nucleosides in the immunomodulatory polynucleotide. In yet a further embodiment, one stereochemically enriched internucleoside phosphorothioate is attached to the 5'-carbon atom of the CpG cytidine in the immunostimulatory sequence of the immunomodulatory polynucleotide. In yet a further embodiment, one stereochemically enriched internucleoside phosphorothioate connects the fourth and fifth nucleosides in the immunomodulatory polynucleotide. In one embodiment, the stereochemically enriched internucleoside phosphorothioate is S-stereogenic. In a particular embodiment, the stereochemically enriched internucleoside phosphorothioate is R-stereogenic.

[0020] In yet another aspect, disclosed is a conjugate comprising a targeting moiety and one or more immunomodulatory polynucleotides.

[0021] In some embodiments, the targeting moiety is a group comprising an antigen-binding portion, a polypeptide, an aptamer, or one or more small molecules. In certain embodiments, the targeting moiety is an antigen-binding portion (e.g., an antibody or an antigen-binding fragment thereof). In further embodiments, the antibody or the antibody fragment comprises an N-terminal or C-terminal Q-tag, wherein the immunomodulatory polynucleotide is covalently attached to the N-terminal or C-terminal Q-tag independently. In still further embodiments, the Q-tag is disposed in the heavy or light chain of the antibody or the antibody fragment.

[0022] In certain embodiments, the immunomodulatory polynucleotide is as disclosed in other embodiments.

[0023] In some embodiments, at least one of the immunomodulatory polynucleotides contains a 5-modified uridine or a 5-modified cytidine. In further embodiments, at least one of the immunomodulatory polynucleotides comprises a 5-modified uridine which is 5-halouridine, 5-alkynyluridine, or 5-heterocyclyluridine. In still further embodiments, the 5-modified uridine is 5-halouridine (e.g., 5-bromouridine or 5-iodouridine). In some embodiments, the 5-modified uridine is one of the two 5'-terminal nucleotides of at least one of the immunomodulatory polynucleotides. In other embodiments, the 5-modified uridine comprises the 3'-position linked to an internucleoside phosphate ester phosphate. In still other embodiments, the 5-modified uridine comprises the 3'-position linked to an internucleoside phosphorothioate ester. In still other embodiments, at least one of the immunomodulatory polynucleotides contains cytidine and guanosine as the second and third nucleosides. In certain embodiments, at least one of the immunomodulatory polynucleotides contains cytidine and guanosine as the third and fourth nucleosides.

[0024] In some embodiments, at least one of the immunomodulatory polynucleotides contains a total of 6 to 16 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) nucleotides.

[0025] In further embodiments, at least one of the immunomodulatory polynucleotides contains one or more abasic spacers or internucleoside phosphorothioates. In still further embodiments, at least one abasic spacer or at least one phosphorothioate contains a linker.

[0026] In certain embodiments, at least one of the immunomodulatory polynucleotides contains one or more (e.g., 1 or 2) abasic spacers. In other embodiments, at least one of the abasic spacers is an internucleoside abasic spacer. In still other embodiments, at least one of the abasic spacers is a 3'-terminal abasic spacer.

[0027] In one embodiment, at least one of the immunomodulatory polynucleotides contains one or more (e.g., 1 to 5) internucleoside phosphorothioates.

[0028] In some embodiments, the conjugate further contains one or more auxiliary moieties attached to a linker. In further embodiments, at least one of the auxiliary moieties contains poly(ethylene glycol) (PEG) having a molecular weight of 100 Da to 2,500 Da. In still further embodiments, each PEG independently contains a total of at least 3 (e.g., at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, or at least 20) ethylene glycol repeat units. In still further embodiments, each PEG independently contains a total of 50 or less (e.g., 45 or less, 40 or less, 35 or less, or 30 or less) ethylene glycol repeat units. In certain embodiments, the conjugate contains 1 to 8 PEGs.

[0029] In certain embodiments, the 5'-capping group in at least one of the immunomodulatory polynucleotides is a monophosphate, diphosphate, triphosphate, auxiliary moiety, terminal phosphodiester, terminal phosphorotriester, or group -OR' (where R' is a bioreversible group, a non-bioreversible group, or an O-protecting group). In further embodiments, the 5'-capping group is a monophosphate or a phosphate, phosphorothioate, or phosphorodithioate attached to an optionally substituted C 1-6 alkyl-containing terminal phosphodiester. In still further embodiments, the 3'-capping group in at least one of the immunomodulatory polynucleotides is a monophosphate, diphosphate, triphosphate, auxiliary moiety, terminal phosphodiester, terminal phosphorotriester, or group -OR' (where R' is a bioreversible group, a non-bioreversible group, or an O-protecting group). In other embodiments, the 3'-capping group is a monophosphate or a phosphate, phosphorothioate, or phosphorodithioate attached to an optionally substituted C 1-6 alkyl-containing terminal phosphodiester.

[0030] In certain embodiments, at least one of the immunomodulatory polynucleotides contains nucleobases attached to a linker.

[0031] In further embodiments, the conjugate contains from 1 to 6 (e.g., 1 to 4) immunomodulatory polynucleotides. In still further embodiments, the conjugate contains only one immunomodulatory polynucleotide. In still further embodiments, the conjugate contains only two immunomodulatory polynucleotides. In other embodiments, the conjugate contains one targeting moiety.

[0032] In some embodiments, the immunomodulatory polynucleotide contains a human immunostimulatory sequence within 4 nucleotides of the 5'-end. In certain embodiments, the human immunostimulatory sequence within 4 nucleotides of the 5'-end of the immunomodulatory polynucleotide includes cytidine containing a 5'-carbon atom bonded to a phosphate ester substituted with a nucleoside.

[0033] In certain embodiments, at least one of the immunomodulatory polynucleotides contains 5-modified uridine or 5-modified cytidine.

[0034] In certain embodiments, at least one of the immunomodulatory polynucleotides is hybridized to its complement.

[0035] In further embodiments, at least one of the immunomodulatory polynucleotides contains at least one stereochemically enriched internucleoside phosphorothioate.

[0036] In a further aspect, disclosed is a composition comprising a conjugate comprising a targeting moiety and one or more immunomodulatory polynucleotides, each of the immunomodulatory polynucleotides independently comprising a linker, wherein the targeting moiety is covalently attached to the linker, and at least one of the immunomodulatory polynucleotides comprises at least one stereochemically enriched internucleoside phosphorothioate.

[0037] In some embodiments, the at least one stereochemically enriched internucleoside phosphorothioate is positioned between the 5'-terminal nucleoside and the cytidine of CpG in the immunostimulatory sequence in the immunomodulatory polynucleotide. In one embodiment, the at least one stereochemically enriched internucleoside phosphorothioate is attached to the 5'-carbon atom of the cytidine of CpG in the immunostimulatory sequence in the immunomodulatory polynucleotide. In certain embodiments, the at least one stereochemically enriched internucleoside phosphorothioate connects the first nucleoside and the second nucleoside in the immunomodulatory polynucleotide. In a further embodiment, the at least one stereochemically enriched internucleoside phosphorothioate connects the fourth nucleoside and the fifth nucleoside in the immunomodulatory polynucleotide. In yet a further embodiment, the stereochemically enriched internucleoside phosphorothioate is S-stereogenic. In yet a further embodiment, the stereochemically enriched internucleoside phosphorothioate is R-stereogenic.

[0038] In yet a further aspect, disclosed is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an immunomodulatory polynucleotide of the invention, a stereochemically enriched composition of the invention, or a conjugate of the invention.

[0039] In yet a further aspect, disclosed is a method of modulating an endosomal toll-like receptor in a cell comprising an endosomal toll-like receptor, by contacting the cell with an immunomodulatory polynucleotide of the invention, a composition of the invention, a conjugate of the invention, or a pharmaceutical composition of the invention, under conditions that allow the immunomodulatory polynucleotide to be transported into the cell, wherein after the contacting, the activity of the endosomal toll-like receptor is modulated.

[0040] In some embodiments, the immunomodulatory polynucleotide is an immunostimulatory polynucleotide and the method is for stimulating an endosomal toll-like receptor.

[0041] In certain embodiments, the immunomodulatory polynucleotide is an immunosuppressive polynucleotide and the method is for suppressing an endosomal toll-like receptor.

[0042] In another aspect, disclosed is a method of inducing one or more cytokines in an antigen-presenting cell comprising an endosomal toll-like receptor, by contacting the antigen-presenting cell with an immunomodulatory polynucleotide of the invention, a composition of the invention, a conjugate of the invention, or a pharmaceutical composition of the invention, under conditions that allow the one or more immunomodulatory polynucleotides to be transported into the cell, wherein after the contacting, the level of at least one cytokine within the cell is increased, the targeting moiety targets the antigen-presenting cell, and the immunomodulatory polynucleotide is an immunostimulatory polynucleotide.

[0043] In some embodiments, the antigen-presenting cell is a B cell. In one embodiment, at least one of the one or more cytokines is an inflammatory cytokine. In certain embodiments, the antigen-presenting cell is a plasmacytoid dendritic cell, wherein the targeting moiety targets the plasmacytoid dendritic cell. In one embodiment, the antigen-presenting cell is a macrophage. In a further embodiment, at least one of the cytokines is a type I interferon. In yet a further embodiment, the toll-like receptor is TLR9.

[0044] In another aspect, disclosed is a method of treating a humoral tumor in a patient by administering to the patient an effective amount of an immunomodulatory polynucleotide, a composition of the invention, a conjugate of the invention, or a pharmaceutical composition of the invention, wherein the targeting moiety targets B cells and the immunomodulatory polynucleotide is an immunostimulatory polynucleotide that is a TLR9 agonist.

[0045] In one embodiment, the humoral tumor is a hematological tumor (e.g., the hematological tumor is a lymphoma). In certain embodiments, the lymphoma is a non-Hodgkin B cell lymphoma. In a further embodiment, the lymphoma is mantle cell lymphoma, diffuse large B cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, or multiple myeloma.

[0046] In another aspect, disclosed is a method of treating a solid tumor in a patient by administering to the patient an immunomodulatory polynucleotide, a composition of the invention, a conjugate of the invention, or a pharmaceutical composition of the invention, wherein the targeting moiety targets plasmacytoid dendritic cells and the immunomodulatory polynucleotide is an immunostimulatory polynucleotide that is a TLR9 agonist. In some embodiments, the method of treating a solid tumor in a patient comprises administering to the patient an immunomodulatory polynucleotide disclosed herein, wherein the immunomodulatory polynucleotide targets B cells in the patient.

[0047] It should be understood that the present invention provides for the use of the immunomodulatory polynucleotide of the present invention, the conjugate of the present invention, the composition of the present invention, or the pharmaceutical composition of the present invention in the manufacture of a product (e.g., a pharmaceutical) for the purposes described herein (e.g., for treating liquid or solid tumors in a patient). It should also be understood that the present invention provides for the use of the immunomodulatory polynucleotide of the present invention, the conjugate of the present invention, the composition of the present invention, or the pharmaceutical composition of the present invention for the purposes described herein (e.g., for treating liquid or solid tumors in a patient). Furthermore, it should be understood that the present invention provides the immunomodulatory polynucleotide of the present invention, the conjugate of the present invention, the composition of the present invention, or the pharmaceutical composition of the present invention for use according to the purposes described herein (e.g., for treating liquid or solid tumors in a patient).

[0048] In any aspect of the present invention, the linker can be as disclosed herein (e.g., according to any one of Formulas (II), (V), and (VI)-(XV)). In any aspect of the present invention, the conjugate group can be as disclosed herein.

[0049] Provided herein is an oligonucleotide of Formula (A): or a stereoisomer thereof, a mixture of two or more diastereomers, a tautomer, or a mixture of two or more tautomers; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; X 5' -(X N ) b -Y P -(X N ) c -X 3' (A) (wherein: each X N is independently a nucleotide; X 3' is the 3'-terminal nucleotide; X 5'is the 5'-terminal nucleotide; Y P is a internucleoside phosphotriester; and b and c are each an integer in the range of about 0 to about 25; provided that their sum is 5 or more; wherein the oligonucleotide comprises nucleotides having modified nucleobases).

[0050] Also provided herein is N 1 N 2 CGN 3 CG(T) x GN 4 CGN 5 an oligonucleotide having the sequence of T, or a stereoisomer thereof, a mixture of two or more diastereomers, a tautomer, or a mixture of two or more tautomers; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; (wherein: x is an integer in the range of 1 to 4; N 1 is absent or is 2'-deoxythymidine; N 2 is a 2'-deoxyribonucleotide having a modified nucleobase; N 3 is 2'-deoxyadenosine or 2'-deoxythymidine, each optionally containing a 3'-phosphotriester; N 4 is 2'-deoxyadenosine or 2'-deoxythymidine; and N 5 is 2'-deoxythymidine optionally containing a 3'-phosphotriester).

[0051] Further provided herein is a compound of formula (B): or a stereoisomer thereof, a mixture of two or more diastereomers, a tautomer, or a mixture of two or more tautomers; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; R X -L N -(Q) e (B) (wherein: R x is a conjugate group; L N is a linker; each Q is independently an oligonucleotide containing a phosphotriester; and e is an integer of 1, 2, 3, or 4).

[0052] Further provided herein is a compound of formula (C): or a stereoisomer thereof, a mixture of two or more diastereomers, a tautomer, or a mixture of two or more tautomers; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; [Chemical formula] (wherein: Ab is an antibody; each L N is independently a linker; each Q is independently an oligonucleotide containing a phosphotriester; each e is independently an integer of 1, 2, 3, or 4; and f is an integer of 1, 2, 3, or 4).

[0053] In one aspect, provided herein is a method of treating cancer in a subject having cancer, comprising administering to the subject a therapeutically effective amount of a CpG-Ab immunoconjugate, wherein the CpG-Ab immunoconjugate does not bind to a tumor-associated antigen (TAA). In some embodiments, the CpG-Ab immunoconjugate specifically binds to a target antigen associated with normal immune cells that express at least one toll-like receptor. In some embodiments, the normal immune cells express TLR9. In some embodiments, the normal immune cells are antigen-presenting cells (APCs). In some embodiments, the APCs are B cells, dendritic cells, or macrophages. In some embodiments, the target antigen is selected from the group consisting of MHC molecules, T cell costimulatory molecules, immune checkpoint molecules, B cell-specific antigens, dendritic cell-specific antigens, and macrophage-specific antigens. In some embodiments, the MHC molecule is selected from MHC class I and MHC class II molecules. In some embodiments, the T cell costimulatory molecule is selected from the list consisting of OX40, CD2, CD27, CDS, ICAM-1, LFA-1 / CD11a / CD18, ICOS / CD278, 4-1BB / CD137, GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and CD83. In some embodiments, the immune checkpoint molecule is selected from the list consisting of PD-1, PD-L1, PD-L2, TIM-1, TIM-3, LAG-3, CEACAM-1, CEACAM-5, CLTA-4, VISTA, BTLA, TIGIT, LAIR1, CD47, CD160, 2B4, CD172a, and TGFR.In some embodiments, the target antigen is selected from the group consisting of CD1, CD2, CD3, CD5, CD6, CD9, CD11, CD14, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD30, CD32, CD37, CD38, CD39, CD40, CD44, CD45R (B220), CD49, CD52, CD55, CD56, CD64, CD66 (carcinoembryonic antigen, CEA), CD68, CD70, CD74, CD79b, CD80, CD93, CD115, CD123, CD126, CD127, CD137, CD138, CD163, CD196, CD197, CD200R, CD205, CD206, CD207, CD208, CD209, CD267, CD269, CD274, CD300a, CD301, CD303, CD304, CD319, CD336, CLEC5a, CLEC6, CLEC9a, CXCL16, CX3CR1, and DC-STAMP. In some embodiments, the CpG-Ab immunoconjugate comprises an immunostimulatory polynucleotide selected from Table 2. In some embodiments, the CpG-Ab immunoconjugate comprises an immunostimulatory polynucleotide selected from the group consisting of p236, p238, p243, p246, p275, p276, p308, p313, p347, p361, p362, p425, p433, p434, p435, p436, p437, p438, p477, p478, p479, p480, p481, p482, p483, p484, p485, p486, p487, p488, and p489. In some embodiments, the CpG-Ab immunoconjugate is not conjugated to a T cell epitope. In some embodiments, the T cell epitope is an epitope of ovalbumin (OVA). In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a liquid tumor. In some embodiments of the methods provided herein, the cancer is a recurrent cancer. In some embodiments of the methods provided herein, the administration or co-administration is by systemic administration.In some embodiments of the methods provided herein, a therapeutically effective amount of the CpG-Ab immunoconjugate is not effective to activate the complement pathway in a subject. In some embodiments of the methods provided herein, the amount is not effective to activate complement C3 in a subject.

[0054] In some embodiments, provided herein is a method of treating cancer in a subject having cancer, comprising administering to the subject a therapeutically effective amount of a CpG-Ab immunoconjugate, wherein the CpG-Ab immunoconjugate specifically binds to a tumor-associated antigen (TAA), wherein the TAA is not an antigen selected from the group consisting of CD19, CD20, CD22, exportin 7, Her2, Src, EGFR, CD52, CXCR-4, Muc-1, and DNA. In some embodiments, the binding of the CpG-Ab immunoconjugate to the TAA promotes internalization of the CpG-Ab immunoconjugate into cancer cells expressing the TAA. In some embodiments, the binding of the CpG-Ab immunoconjugate to the TAA promotes transport of the CpG-Ab immunoconjugate to the endosome of cancer cells expressing the TAA. In some embodiments, the binding of the CpG-Ab immunoconjugate to the TAA promotes activation of the TLR9 signaling pathway in cancer cells expressing the TAA. In some embodiments, the TAA and TLR9 are located on the same cell membrane of cancer cells expressing the TAA. In some embodiments, both the TAA and TLR9 are located on the cell membrane of cancer cells expressing the TAA. In some embodiments, both the TAA and TLR9 are located on the endosomal membrane of cancer cells expressing the TAA. In some embodiments, the binding of the CpG-Ab immunoconjugate to the TAA induces apoptosis of cancer cells expressing the TAA. In some embodiments, the TAA is not expressed by normal immune cells. In some embodiments, the TAA is expressed by normal immune cells. In some embodiments, the normal immune cells are antigen-presenting cells (APCs). In some embodiments, the TAA is selected from the group consisting of CD8, CD11b, CD11c, CD14, CD33, CD40, CD123, CD157, CD168, CD169, CD172a, CD200, CD204, CD205, CD301, CD302, CD303, CD304, and CD206.In some embodiments, the CpG-Ab immunoconjugate is not conjugated to a TAA or any other TAA expressed by the cancer. In some embodiments, the CpG-Ab immunoconjugate comprises an immunostimulatory polynucleotide selected from Table 2. In some embodiments, the CpG-Ab immunoconjugate comprises an immunostimulatory polynucleotide selected from the group consisting of p236, p238, p243, p246, p275, p276, p308, p313, p347, p361, p362, p425, p433, p434, p435, p436, p437, p438, p477, p478, p479, p480, p481, p482, p483, p484, p485, p486, p487, p488, and p489. In some embodiments, the CpG-Ab immunoconjugate is not conjugated to a T cell epitope. In some embodiments, the T cell epitope is ovalbumin (OVA). In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a liquid tumor. In some embodiments of the methods provided herein, the cancer is a recurrent cancer. In some embodiments of the methods provided herein, the administration or co-administration is by systemic administration. In some embodiments of the methods provided herein, the therapeutically effective amount of the CpG-Ab immunoconjugate is not effective to activate the complement pathway in the subject. In some embodiments of the methods provided herein, the amount is not effective to activate complement C3 in the subject.

[0055] In some embodiments, provided herein is a method of treating an immune therapy-resistant or refractory cancer in a subject having the immune therapy-resistant or refractory cancer, the method comprising administering to the subject a therapeutically effective amount of a CpG-Ab immunoconjugate. In some embodiments, the CpG-Ab immunoconjugate does not bind to a tumor-associated antigen. In some embodiments, the CpG-Ab immunoconjugate specifically binds to a target antigen associated with normal immune cells that express at least one toll-like receptor. In some embodiments, the CpG-Ab immunoconjugate specifically binds to a tumor-associated antigen. In some embodiments, the cancer is resistant to treatment with an immune checkpoint modulator. In some embodiments, the method further comprises co-administering to the subject an immune checkpoint modulator. In some embodiments, the CpG-Ab immunoconjugate comprises an immunostimulatory polynucleotide selected from Table 2. In some embodiments, the CpG-Ab immunoconjugate comprises an immunostimulatory polynucleotide selected from the group consisting of p236, p238, p243, p246, p275, p276, p308, p313, p347, p361, p362, p425, p433, p434, p435, p436, p437, p438, p477, p478, p479, p480, p481, p482, p483, p484, p485, p486, p487, p488, and p489 shown in Table 2. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a liquid tumor. In some embodiments of the methods provided herein, the cancer is a recurrent cancer. In some embodiments of the methods provided herein, the administration or co-administration is by systemic administration. In some embodiments of the methods provided herein, the therapeutically effective amount of the CpG-Ab immunoconjugate is not effective to activate the complement pathway in the subject. In some embodiments of the methods provided herein, the amount is not effective to activate complement C3 in the subject.

[0056] In some embodiments, provided herein is a method of preventing cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CpG-Ab immunoconjugate, wherein the CpG-Ab immunoconjugate specifically binds to a target antigen associated with normal immune cells that express at least one toll-like receptor. In some embodiments, such a method further comprises co-administering a tumor-associated antigen with the CpG-Ab immunoconjugate. In some embodiments, the CpG-Ab immunoconjugate is not conjugated to the tumor-associated antigen. In some embodiments, the normal immune cells express TLR9. In some embodiments, the normal immune cells are antigen-presenting cells (APCs). In some embodiments, the CpG-Ab immunoconjugate comprises an immunostimulatory polynucleotide selected from Table 2. In some embodiments, the CpG-Ab immunoconjugate comprises an immunostimulatory polynucleotide selected from the group consisting of p236, p238, p243, p246, p275, p276, p308, p313, p347, p361, p362, p425, p433, p434, p435, p436, p437, p438, p477, p478, p479, p480, p481, p482, p483, p484, p485, p486, p487, p488, and p489 shown in Table 2. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a liquid tumor. In some embodiments of the methods provided herein, the cancer is a recurrent cancer. In some embodiments of the methods provided herein, the administration or co-administration is by systemic administration. In some embodiments of the methods provided herein, the therapeutically effective amount of the CpG-Ab immunoconjugate is not effective to activate the complement pathway in the subject. In some embodiments of the methods provided herein, the amount is not effective to activate complement C3 in the subject.

[0057] In some embodiments, provided herein is a method of preventing cancer in a subject in need thereof, comprising co-administering a therapeutically effective amount of a CpG-Ab immunoconjugate with a cancer vaccine, wherein the CpG-Ab immunoconjugate specifically binds to a target antigen associated with normal immune cells that express at least one toll-like receptor. In some embodiments, the CpG-Ab immunoconjugate is formulated as an adjuvant to the cancer vaccine. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a liquid tumor. In some embodiments of the methods provided herein, the cancer is a recurrent cancer. In some embodiments of the methods provided herein, the administration or co-administration is by systemic administration. In some embodiments of the methods provided herein, the therapeutically effective amount of the CpG-Ab immunoconjugate is not effective to activate the complement pathway in the subject. In some embodiments of the methods provided herein, the amount is not effective to activate complement C3 in the subject.

[0058] In some embodiments, provided herein is a method of inducing an adaptive immune response in a subject, comprising administering to the subject a therapeutically effective amount of a CpG-Ab immunoconjugate, wherein the CpG-Ab immunoconjugate specifically binds to a target antigen associated with normal immune cells that express at least one toll-like receptor. In some embodiments, the subject has cancer. In some embodiments, the target antigen is not a TAA. In some embodiments, the target antigen is a TAA that is not an antigen selected from the group consisting of CD19, CD20, CD22, STAT3, exportin 7, Her2, Src, EGFR, CD52, CXCR-4, Muc-1, and DNA. In some embodiments, the subject has an infectious disease. In some embodiments, the normal immune cells express TLR9. In some embodiments, the normal immune cells are antigen-presenting cells (APCs). In some embodiments, the adaptive immune response is CD8+ T cell-dependent. In some embodiments, the CpG-Ab immunoconjugate comprises an immunostimulatory polynucleotide selected from Table 2. In some embodiments, the CpG-Ab immunoconjugate comprises an immunostimulatory polynucleotide selected from the group consisting of p236, p238, p243, p246, p275, p276, p308, p313, p347, p361, p362, p425, p433, p434, p435, p436, p437, p438, p477, p478, p479, p480, p481, p482, p483, p484, p485, p486, p487, p488, and p489 shown in Table 2. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a liquid tumor. In some embodiments of the methods provided herein, the cancer is a recurrent cancer. In some embodiments of the methods provided herein, the administration or co-administration is by systemic administration. In some embodiments of the methods provided herein, the therapeutically effective amount of the CpG-Ab immunoconjugate is not effective to activate the complement pathway in the subject.In some embodiments of the methods provided herein, the amount is not effective to activate complement C3 in a subject.

[0059] In some embodiments, provided herein is a method of treating cancer in a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of a CpG-Ab immunoconjugate selected from Table 6. In some embodiments, the CpG-Ab immunoconjugate binds to a tumor-associated antigen (TAA). In some embodiments, the CpG-Ab immunoconjugate binds to a target antigen other than a TAA. In some embodiments, the CpG-Ab immunoconjugate binds to a target antigen associated with normal immune cells that express TLR receptors. In some embodiments, the CpG-Ab immunoconjugate is selected from the group consisting of CpG-Ab immunoconjugates comprising p236, p238, p243, p246, p275, p276, p308, p313, p347, p361, p362, p425, p433, p434, p435, p436, p437, p438, p477, p478, p479, p480, p481, p482, p483, p484, p485, p486, p487, p488, and p489 shown in Table 2. In some embodiments, the method further comprises co-administering a therapeutically effective amount of at least one additional cancer therapeutic agent. In some embodiments, the at least one additional cancer therapeutic agent is selected from a second TAA, a T cell co-stimulatory molecule, and an immune checkpoint modulator. In some embodiments, the second TAA is the same as the TAA. In some embodiments, the second TAA is different from the TAA. In some embodiments, the T cell co-stimulatory molecule is selected from the list consisting of OX40, CD2, CD27, CDS, ICAM-1, LFA-1 / CD11a / CD18, ICOS / CD278, 4-1BB / CD137, GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and CD83, or ligands thereof. In some embodiments, the T cell co-stimulatory molecule is an anti-OX40 antibody, an anti-ICOS / CD278 antibody, or an anti-4-1BB / CD137 antibody, or antigen-binding fragments thereof.In some embodiments, where the immune checkpoint modulator is an inhibitor of an immune checkpoint molecule selected from the list consisting of PD-1, PD-L1, PD-L2, TIM-3, LAG-3, CEACAM-1, CEACAM-5, CLTA-4, VISTA, BTLA, TIGIT, LAIR1, CD47, CD160, 2B4, CD172a, and TGFR. In some embodiments, the immune checkpoint modulator is an anti-CD47 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, or an antigen-binding fragment thereof. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a liquid tumor. In some embodiments of the methods provided herein, the cancer is a recurrent cancer. In some embodiments of the methods provided herein, the administration or co-administration is by systemic administration. In some embodiments of the methods provided herein, the therapeutically effective amount of the CpG-Ab immunoconjugate is not effective to activate the complement pathway in the subject. In some embodiments of the methods provided herein, the amount is not effective to activate complement C3 in the subject.

[0060] In some embodiments of any of the methods provided herein, where the CpG-Ab immunoconjugate comprises an oligonucleotide of formula (A) as defined above. In some embodiments of any of the methods provided herein, where the CpG-Ab immunoconjugate comprises a compound of formula (B) as defined above. In some embodiments of any of the methods provided herein, where the CpG-Ab immunoconjugate is a compound of formula (C) as defined above.

Brief Description of the Drawings

[0061] (Brief Description of the Drawings) This application file includes at least one drawing created in color. Copies of patents or patent applications with color drawings are provided by the Patent Office after request and payment of the required fees.

[0062]

Figure 1

[0063] Figure 1B is a series of structures showing abbreviations having corresponding structures. These abbreviations are the abbreviations used in Table 2.

[0064]

Figure 2

[0065]

Figure 3

[0066] Figure 3B is an image of an ethidium bromide-stained reducing gel of Q-tagged anti-CD38 antibody before (lane A) and after microbial transglutaminase-mediated conjugation with polynucleotides p83, p84, p85, p86, p87, and p88 corresponding to lanes B, C, D, E, F, and G, respectively. HC+1 indicates the band of the heavy chain of the Q-tagged anti-CD38 antibody conjugated to the polynucleotide. HC indicates the band of the heavy chain of the Q-tagged anti-CD38 antibody. LC indicates the light chain of the anti-CD38 antibody.

[0067]

Figure 4

[0068] Figure 4B is a graph showing an AEX-HPLC trace of a crude mixture containing a rituximab-p19 conjugate showing signals based on absorbance at 280 nm and 260 nm. There are three peaks corresponding to the rituximab-p19 conjugate.

[0069] Figure 4C is a graph showing the synthesis of the AEX-HPLC traces of the crude mixture, p19, and rituximab-p19 AEX peaks 1, 2, and 3 listed in Figure 4B.

[0070] Figure 4D is a 24 image of a denaturing SDS PAGE 6% Tris-glycine gel comparing rituximab-PEG

[0071]

Figure 5

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Figure 6

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Figure 7

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Figure 8

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Figure 9

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Figure 10

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Figure 11

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Figure 12

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Figure 18

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Figure 19

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Figure 20

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Figure 21

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Figure 22

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Figure 23

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Figure 24

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Figure 25

[0092]

Figure 26

[0093] Figure 26B is a graph showing a comparison of the immunostimulatory activities of conjugates containing one or more phosphorothioate-based internucleoside phosphorotriesters. The immunostimulatory activity was evaluated by measuring NFκB activation in Ramos-Blue cells, which was measured by an alkaline phosphatase readout.

[0094]

Figure 27

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Figure 28

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Figure 29

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Figure 30

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Figure 31

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Figure 32

[0100]

Figure 33

[0101] Figure 33B is a graph showing the induction of IL6 in A20 mouse B cell lymphoma cells by a conjugate containing an anti-mouse CD22 antibody and an immunostimulatory polynucleotide in the presence of various concentrations of free anti-mouse CD22 antibody.

[0102]

Figure 34

[0103] Figure 34B is a graph showing the induction of interferon-α in human PBMC using conjugate SB-340. SB-341 and p246 of anti-BDCA2 antibody were used as controls in this experiment. The Y-axis provides the optical density in arbitrary units at a wavelength of 450 nm.

[0104] Figure 34C is a graph showing the induction of interferon-α in purified plasmacytoid cells using conjugate SB-342. Anti-BDCA2 antibody, anti-BDCA4 antibody, and anti-SB-343 were used as controls in this experiment.

[0105]

Figure 35

[0106]

Figure 36

[0107]

Figure 37

[0108] Figure 37B is a graph showing the progression of tumor volume increase after inoculation of A20 mouse B cell lymphoma cells into mice and subsequent intravenous administration of vehicle (saline), immunostimulatory polynucleotide (p3), anti-CD22 antibody (CD22), or conjugates SB-338, SB-339, or SB-344 three times. The administration times are indicated by the arrows on the X-axis.

[0109]

Figure 38

[0110] Figure 38B is a graph showing the tumor volume values on day 20 after inoculation of A20 mouse B cell lymphoma cells into mice and subsequent intratumoral administration of vehicle (saline) or immunostimulatory polynucleotide three times.

[0111]

Figure 39

[0112] Figure 39B is a graph showing the tumor volume values on day 20 after inoculation of A20 mouse B cell lymphoma cells into mice and (i) subsequent intravenous administration of a single dose of the conjugate of the present invention (SB-337), or (ii) subsequent intratumoral administration of three doses of vehicle (physiological saline) or immunostimulatory polynucleotide.

[0113]

Figure 40

[0114]

Figure 41

[0115] Figure 41B is an image of a denaturing gel of a sample of polynucleotide incubated in mouse serum at 37°C for up to 24 hours.

[0116] Figure 41C is an image of a denaturing gel of a sample of polynucleotide incubated in rat serum at 37°C for up to 24 hours.

[0117] Figure 41D is an image of a denaturing gel of a sample of polynucleotide incubated in monkey serum at 37°C for up to 24 hours.

[0118] Figure 41E is an image of a denaturing gel of a sample of polynucleotide incubated in human serum at 37°C for up to 24 hours.

[0119]

Figure 42

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Figure 43

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Figure 44

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Figure 45

[0123]

Figure 46

[0124] Figure 46B shows the survival rates of mice with disseminated B-cell lymphoma treated on days 1, 3, and 5 with (i) 3 mg / kg of CpG (p313)-mAb (CD22) conjugate (black-filled diamonds); (ii) 10 mg / kg of CpG-mAb (CD22) (black-filled triangles); (iii) naked CpG ODN (open triangles); (iv) 10 mg / kg of CD22 mAb (black-filled squares); (v) 10 mg / kg of GpC-mAb control conjugate (open squares); or (vi) phosphate-buffered saline (black-filled circles).

[0125] Figure 46C shows the survival rates of mice that survived the first tumor transplantation and then received a second tumor transplantation on day 47. No treatment was given for survival after the second tumor transplantation. Survivors treated with 10 mg / kg of CpG-mAb (CD22) on days 1, 3, and 5 (downward triangles); survivors treated with 3 mg / kg of CpG-mAb (CD22) on days 1, 3, and 5 (diamonds); a second control group in which tumor cells were transplanted on day 47 and treated with phosphate-buffered saline (upward triangles).

[0126] Figure 46D shows an experiment in which survivors from the first and second tumor transplants were then given a third tumor transplantation on day 90. No treatment was given for survival after the second or third tumor transplantation. A third control group had tumor cells transplanted on day 90 and was treated with phosphate-buffered saline. The tumor volumes of the survivors (squares) and the control group (circles) were monitored from day 90 to day 120.

[0127]

Figure 47

[0128] Figure 47B shows the tumor volumes of mice with solid B-cell lymphoma treated on days 9, 12, and 14 with (i) 3 mg / kg of CpG-mAb (CD22) (open diamonds); (ii) 10 mg / kg of CpG-mAb (CD22) (large black triangles); (iii) naked CpG ODN (open triangles); (iv) 10 mg / kg of CD22 mAb (black squares); (v) 10 mg / kg of GpC-mAb control conjugate (small black squares), or (vi) phosphate-buffered saline (black circles).

[0129] Figure 47C shows the tumor volumes of mice with solid B-cell lymphoma treated with 10 mg / kg of SB-337 DAR1 or 10 mg / kg of SB-337 DAR2 compared to controls (phosphate-buffered saline and SB-339).

[0130] Figure 47D 24 shows the tumor volumes of mice with solid B-cell lymphoma treated with 10 mg / kg of SB-337 PEG 24 Bis DAR1 or 10 mg / kg of SB-337 PEG

[0131] Figure 47E shows the tumor volumes of mice with solid B-cell lymphoma treated with 10 mg / kg of PD-1, 10 mg / kg of PD-1 + 3 mg / kg of SB-337 DAR1; or 10 mg / kg of PD-1 + 3 mg / kg of SB-337 DAR2 compared to a phosphate-buffered saline control.

[0132] Figure 47F shows the effects of p347, SB-337 DAR1, and SB-337 DAR2 on the body weight of mice compared to controls (phosphate-buffered saline and mCD22).

[0133]

Figure 48

[0134] Figure 48B shows the tumor volumes of mice with a solid B-cell lymphoma model after receiving (i) 3 mg / kg of CpG-mAb(CD22) (upward triangles); (ii) an anti-PD-1 antibody (black squares); (iii) 3 mg / kg of CpG-mAb(CD22) in combination with an anti-PD-1 antibody (downward triangles); and (iv) a physiological saline solution (black circles).

[0135]

Figure 49

[0136] Figure 49B shows the tumor volumes in immunodeficient Nu / Nu mice with solid B-cell lymphoma after receiving (i) 10 mg / kg of CpG-mAb(CD22) (squares), (ii) naked CpG ODN (triangles), or (iii) a physiological saline solution (circles).

[0137] Figure 49C shows the tumor volumes in immunodeficient SCID mice with solid B-cell lymphoma after receiving (i) 10 mg / kg of CpG-mAb(CD22) (squares), (ii) naked CpG ODN (triangles), or (iii) a physiological saline solution (circles).

[0138]

Figure 50

[0139] Figure 50B shows the survival rates of mice with soluble B-cell lymphoma after receiving (i) CpG-mAb(CD22) only (open circles); (ii) CpG-mAb(CD22) and natural killer (NK) cell depletion treatment (open squares); (iii) NK cell depletion treatment (filled squares); or (iv) physiological saline aqueous solution (filled circles).

[0140] Figure 50C shows the survival rates of mice with soluble B-cell lymphoma after receiving (i) CpG-mAb(CD22) only (open circles); (ii) CpG-mAb(CD22) and CD8+ T cell depletion treatment (open squares); (iii) CD8+ T cell depletion treatment (filled squares); or (iv) physiological saline aqueous solution (filled circles).

[0141]

Figure 51

[0142] Figure 51B shows the percentages of CD4+ or CD8+ viable gate cells in tumors collected from mice with solid B-cell lymphoma treated with (i) CpG-Ab (squares) or (ii) physiological saline aqueous solution (circles).

[0143] Figure 51C shows the correlation between the percentage of CD8+ tumor cells and the tumor volume in mice treated with (i) CpG-Ab (squares) or (ii) physiological saline aqueous solution (circles).

[0144]

Figure 52

[0145] Figure 52B shows the tumor volume in mice with solid B-cell lymphoma after receiving (i) CpG-mAb (CD22) only (open circles); (ii) anti-PD-L1 antibody only (filled triangles); (iii) CpG-mAb (CD22) combined with anti-PD-L1 antibody (open triangles); or (iv) physiological saline aqueous solution (filled circles).

[0146] Figure 52C shows the tumor volume in mice with solid B-cell lymphoma after receiving (i) CpG-mAb (CD22) only (open circles); (ii) anti-PD-1 antibody only (filled squares); (iii) CpG-mAb (CD22) combined with anti-PD-1 antibody (open squares); (iv) CpG-mAb (CD22) combined with anti-PD-1 antibody and CD8+ T cell depletion treatment; or (v) physiological saline aqueous solution (filled circles).

[0147]

Figure 53

[0148] Figure 53B shows the tumor volume in individual mice with solid B-cell lymphoma after receiving anti-PD-1 antibody only.

[0149] Figure 53C shows the tumor volume in individual mice with solid B-cell lymphoma after receiving CpG-mAb(CD22) / anti-PD-1 antibody combination treatment.

[0150] Figure 53D shows the tumor volume of survivors (upward triangles) from the first tumor transplantation and untreated control group (downward triangles) after the second tumor transplantation.

[0151]

Figure 54

[0152] Figure 54B shows the tumor volume in mice with solid B-cell lymphoma after receiving (i) anti-ICOS antibody alone (squares); (ii) CpG-mAb(CD22) combined with anti-ICOS antibody (triangles); or (iii) physiological saline solution (circles).

[0153] Figure 54C shows the tumor volume in mice with solid B-cell lymphoma after receiving (i) anti-4-1BB antibody alone (diamonds); (ii) CpG-mAb(CD22) combined with anti-4-1BB antibody (triangles); or (iii) physiological saline solution (circles).

[0154]

Figure 55

[0155] Figure 55B shows the number of IFN-γ secreting cells in 10 6 spleen cells isolated from mice treated with (i) CpG-mAb(CD22) or (i) physiological saline solution before or after stimulation with AH1 antigen.

[0156]

Figure 56

[0157] Figure 56B shows the tumor volume in individual mice with B cell lymphoma after intravenous administration of 10 mg / kg of CpG-Ab(CD205) on days 10, 12, and 14.

[0158] Figure 56C shows the tumor volume in individual mice with B cell lymphoma after intravenous administration of 10 mg / kg of CpG-Ab(PD-L1) on days 10, 12, and 14.

[0159] Figure 56D shows the tumor volume of survivors from the first tumor transplantation treated with CpG-Ab(CD205) (squares) or survivors from the first tumor administration treated with CpG-Ab(PD-L1) (triangles) and the untreated control group (circles) after the second tumor transplantation given on day 38.

[0160]

Figure 57

[0161] Figure 57B shows the tumor volume in individual mice with solid B cell lymphoma after intravenous administration of 10 mg / kg of anti-CD205 antibody on each of days 10, 12, and 14.

[0162] Figure 57C shows the tumor volume in individual mice with solid B cell lymphoma after receiving intravenous administration of 10 mg / kg of CpG-Ab (CD205) on each of days 10, 12, and 14.

[0163] Figure 57D shows the tumor volume in individual mice with solid B cell lymphoma after receiving intravenous administration of 10 mg / kg of rat IgG2a antibody on each of days 10, 12, and 14.

[0164]

Figure 58

[0165]

Figure 59

[0166]

Figure 60

[0167] Figure 60B shows the average tumor volume on day 20 of mice with A20 mouse B-cell lymphoma cell xenografts after intravenous administration of (i) physiological saline aqueous solution (solid line); (ii) 3 mg / kg of CpG-Ab (SB-337) (checked pattern); (iii) 3 mg / kg of CD19-mAb (horizontal); (iv) 3 mg / kg of CpG-Ab (SB-388) (vertical); (v) 1.9 μg / mouse of naked CpG (P347) (downward slashes) on each of days 10, 12, and 14.

[0168] Figure 60C shows the average body weight change on day 20, excluding tumor weight changes, of mice with A20 mouse B-cell lymphoma cell xenografts after intravenous administration of (i) physiological saline aqueous solution (black); (ii) 3 mg / kg of CpG-Ab (SB-337) (checked pattern); (iii) 3 mg / kg of CD19-mAb (horizontal); (iv) 3 mg / kg of CpG-Ab (SB-388) (vertical); (v) 1.9 μg / mouse of naked CpG (P347) (downward slashes); (vi) 19 μg / mouse of naked CpG (P347) (lattice); (vii) 190 μg / mouse of naked CpG (P347) (upward slashes) on each of days 10, 12, and 14.

[0169]

Figure 61

[0170] Figure 61B shows lung metastases from mice after B16F10 melanoma re - transplantation following intratumoral administration of physiological saline aqueous solution (upper panel) or p347 (lower panel) on each of days 7, 9, 11, and 13, and after re - transplantation on day 14.

[0171] Figure 61C shows the progression of the increase in the average tumor volume of mice inoculated with CT26 colorectal xenografts following intratumoral administration of (i) physiological saline aqueous solution (upward triangles); or (ii) p347 (downward triangles) on each of days 7, 10, 12, and 14.

[0172]

Figure 62

[0173] Figure 62B shows the progression of the increase in the average tumor volume of mice in which B cells were depleted with anti - CD20 mAb using the CT26 colorectal model following intravenous administration of (i) physiological saline aqueous solution (circles); or (ii) 10 mg / kg of CpG - mAb (SB - 337) (squares) on each of days 10, 12, and 14.

[0174]

Figure 63

[0175] Figure 63B shows the progression of tumor volume increase in each mouse using the MC38 colorectal syngeneic model after intravenous administration of phosphate-buffered saline on days 10, 12, and 14.

[0176] Figure 63C shows the progression of tumor volume increase in each mouse using the MC38 colorectal syngeneic model after intravenous administration of 10 mg / kg anti-CD22 mAb on days 10, 12, and 14.

[0177] Figure 63D shows the progression of tumor volume increase in each mouse using the MC38 colorectal syngeneic model after intraperitoneal administration of 10 mg / kg anti-PD-L1 on days 10, 13, and 17.

[0178] Figure 63E shows the progression of tumor volume increase in each mouse using the MC38 colorectal syngeneic model after intravenous administration of 10 mg / kg CD22-CpG (SB-337) on days 10, 12, and 14.

[0179] Figure 63F shows the progression of tumor volume increase in each mouse using the MC38 colorectal syngeneic model after intravenous administration of 10 mg / kg CD22-CpG (SB-337) on days 10, 12, and 14, and further intraperitoneal administration of 10 mg / kg anti-PD-L1 on days 10, 13, and 17.

[0180]

Figure 64

[0181] Figure 64B shows the progression of mean tumor volume increase in mice using the LLC1 Lewis lung cancer model after administration of (i) phosphate-buffered saline (circles); (ii) 10 mg / kg CD22-CpG (SB-337) (circles); (iii) 10 mg / kg anti-PD1 (squares); (iv) 10 mg / kg CD22-CpG (SB-337) + 10 mg / kg anti-PD1 (upward triangles) (v) 10 mg / kg anti-PD-L1 (downward triangles); (vi) 10 mg / kg CD22-CpG + 10 mg / kg anti-PD-L1 (diamonds). Anti-CD22 and CD22-CpG were administered intravenously on days 7, 10, and 13; anti-PD-L1 and anti-PD1 were administered intraperitoneally on days 7, 10, and 14. ** p = 0.023.

[0182]

Figure 65

[0183] Figure 65B shows the progression of tumor volume increase in each mouse using the CT26 colorectal model after intravenous administration of phosphate-buffered saline on each of days 12, 17, 20, and 24.

[0184] Figure 65C shows the progression of tumor volume increase in each mouse using the CT26 colorectal model after intravenous administration of 10 mg / kg of CD22-CpG (SB-337) on each of days 12, 17, 20, and 24.

[0185] Figure 65D shows the progression of tumor volume increase in each mouse using the CT26 colorectal model after intravenous administration of 10 mg / kg of DEC205-CpG (SB-3096) on each of days 12, 17, 20, and 24.

[0186]

Figure 66

[0187] Figure 66B shows the progression of mean tumor volume increase in mice using the A20 lymphoma model after administration of (i) physiological saline aqueous solution (circles); (ii) CD4 depletion (upward triangles); (iii) 3 mg / kg of CD22-CpG (SB-337) (squares); or (iv) CD4 depletion + 3 mg / kg of CD22-CpG (SB-337) (downward triangles). CD22-CpG was intravenously administered on days 10, 12; and 14. CD4 depletion was performed using anti-CD4.

[0188]

Figure 67

[0189] Figure 67B shows the mean fluorescence intensity (MFI) of CD40, CD80, CD86, and MHC II surface expression on CD19+ / B220+ B cells after in vivo administration of saline (solid line); 10 mg / kg of CD22 Ab (checked pattern); 10 mg / kg of CpG (SB-4715) (horizontal line); or 10 mg / kg of CpG-Ab (SB-337) (vertical line).

[0190]

Figure 68

[0191] Figure 68B shows the percentage of activated T cells (Ki67+, CD3+) relative to the total T cell (CD3+) population in mice treated with (i) saline (solid line); (ii) Ab (anti-CD22) (checked pattern); (iii) CpG-Ab (SB-337) (horizontal); or (iv) CpG (SB-4715) (vertical).

[0192]

Figure 69

[0193] Figure 69B shows the progression of mean tumor volume increase in mice using the CT26 colorectal model after adoptive transfer of draining lymph node cells from mice treated with (i) phosphate-buffered saline (small circles); (ii) 10 mg / kg of CD22-CpG (SB-337) (small squares); (iii) 10 mg / kg of CD22 (small upward triangles); or (iv) free CpG (P347) (small downward triangles); or (v) phosphate-buffered saline (diamonds); (vi) 10 mg / kg of CD22-CpG (SB-337) (large circles); (vii) 10 mg / kg of CD22 (large squares); or (viii) free CpG (P347) (large upward triangles) derived from non-draining lymph node cells.

[0194] Figure 69C shows the mean tumor volume on day 24 in mice using the CT26 colorectal model after adoptive transfer of draining lymph node cells from mice treated with (i) phosphate-buffered saline (upward narrow diagonal lines); (ii) 10 mg / kg of CD22-CpG (SB-337) (downward narrow diagonal lines); (iii) 10 mg / kg of CD22 (grid); or (iv) free CpG (P347) (downward wide diagonal lines); or (v) phosphate-buffered saline (solid line); (vi) 10 mg / kg of CD22-CpG (SB-337) (checkered pattern); (vii) 10 mg / kg of CD22 (horizontal); or (viii) free CpG (P347) (empty) derived from non-draining lymph node cells.

[0195]

Figure 70

[0196] Figure 70B shows the plasma concentrations of IL-1β in untreated mice intravenously treated with (i) saline (solid line); (ii) 10 mg / kg of Ab(CD22) (checked pattern); (iii) 5.7 μg / dose of free CpG (p347) (horizontal); or (iv) 10 mg / kg of CpG-mAb (SB-337) (vertical).

[0197] Figure 70C shows the plasma concentrations of IL-10 in untreated mice intravenously treated with (i) saline (solid line); (ii) 10 mg / kg of Ab(CD22) (checked pattern); (iii) 5.7 μg / dose of free CpG (p347) (horizontal); or (iv) 10 mg / kg of CpG-mAb (SB-337) (vertical).

[0198] Figure 70D shows the plasma concentrations of IL-12p70 in untreated mice intravenously treated with (i) saline (solid line); (ii) 10 mg / kg of Ab(CD22) (checked pattern); (iii) 5.7 μg / dose of free CpG (Sp347) (horizontal); or (iv) 10 mg / kg of CpG-mAb (SB-337) (vertical).

[0199] Figure 70E shows the plasma concentrations of IFNγ in untreated mice intravenously treated with (i) saline (solid line); (ii) 10 mg / kg of Ab(CD22) (checked pattern); (iii) 5.7 μg / dose of free CpG (p347) (horizontal); or (iv) 10 mg / kg of CpG-mAb (SB-337) (vertical).

[0200] Figure 70F shows the plasma concentrations of TNFα in untreated mice intravenously treated with (i) saline (solid line); (ii) 10 mg / kg of Ab(CD22) (checked pattern); (iii) 5.7 μg / dose of free CpG (p347) (horizontal); or (iv) 10 mg / kg of CpG-mAb (SB-337) (vertical).

[0201]

Figure 71

[0202] Figure 71B shows the percentage of germinal center (GC) cells (B220 + , IgD lo , Fas + ) relative to total cells in the spleens of mice using the CT26 colorectal model after intravenous administration of (i) physiological saline (circles); or (ii) 10 mg / kg of CpG-mAb (SB-337) (squares) on days 10, 13, and 17, respectively. * p < 0.05

[0203] Figure 71C shows the percentage of T follicular helper (T fh ) cells (CD4 + , CXCR5 + , PD-1 + ) relative to total cells in the spleens of mice using the CT26 colorectal model after intravenous administration of (i) physiological saline (circles); or (ii) 10 mg / kg of CpG-mAb (SB-337) (squares) on days 10, 13, and 17, respectively. * p < 0.05

[0204] Figure 71D shows the relative fold change in IL-21 from mice using the CT26 colorectal model after intravenous administration of (i) physiological saline; or (ii) 10 mg / kg of CpG-mAb (SB-337) on days 10, 13, and 17, respectively. * p < 0.05

[0205] Figure 71E shows the relative fold change in Bcl-6 from mice using the CT26 colorectal model after intravenous administration of (i) physiological saline; or (ii) 10 mg / kg of CpG-mAb (SB-337) on days 10, 13, and 17, respectively. * p < 0.05

[0206] Figure 71F shows the relative fold change of IRF-4 derived from mice using the CT26 colorectal model after intravenous administration of (i) physiological saline; or (ii) 10 mg / kg of CpG-mAb (SB-337) on days 10, 13, and 17 respectively. * p<0.05

[0207]

Figure 72

[0208] Figure 72B shows the relative fold change of IL-10 in the spleen of mice using the CT26 colorectal model after intravenous administration of (i) physiological saline; or (ii) 3 mg / kg of CpG-mAb (SB-337) on days 10, 13, and 17 respectively.

[0209] Figure 72C shows the relative fold change of IL-1β in the spleen of mice using the CT26 colorectal model after intravenous administration of (i) physiological saline; or (ii) 3 mg / kg of CpG-mAb (SB-337) on days 10, 13, and 17 respectively.

[0210] Figure 72D shows the relative fold change of TNFα in the spleen of mice using the CT26 colorectal model after intravenous administration of (i) physiological saline; or (ii) 3 mg / kg of CpG-mAb (SB-337) on days 10, 13, and 17 respectively.

[0211]

Figure 73

[0212] Figure 73B shows the relative fold change of IL-10 in the draining lymph nodes derived from mice using the CT26 colorectal model after intravenous administration of (i) physiological saline; or (ii) 3 mg / kg of CpG-mAb (SB-337) on each of days 10, 13, and 17.

[0213] Figure 73C shows the relative fold change of IL-1β in the draining lymph nodes derived from mice using the CT26 colorectal model after intravenous administration of (i) physiological saline; or (ii) 3 mg / kg of CpG-mAb (SB-337) on each of days 10, 13, and 17.

[0214] Figure 73D shows the relative fold change of TNFα in the draining lymph nodes derived from mice using the CT26 colorectal model after intravenous administration of (i) physiological saline; or (ii) 3 mg / kg of CpG-mAb (SB-337) on each of days 10, 13, and 17.

[0215]

Figure 74

[0216] Figure 74B shows the concentration of IgG2a in mice using the CT26 colorectal model after intravenous administration of (i) physiological saline; or (ii) 3 mg / kg of CpG-mAb (SB-337) on each of days 10, 13, and 16. * p < 0.05

[0217] Figure 74C shows the concentration of IgG in mice using the CT26 colorectal model after intravenous administration of (i) physiological saline; or (ii) 3 mg / kg of CpG-mAb (SB-337) on each of days 10, 13, and 16. * p < 0.05

[0218]

Figure 75

[0219]

Figure 76

[0220] Figure 76B shows the percentage of regulatory B cells (Breg; CD19 + , B220 + , CD1d hi ) relative to total cells in spleens from mice that received intravenous administration of (i) saline (circles); or (ii) 10 mg / kg of CpG-mAb (SB-337) (squares) weekly. * p < 0.001

[0221]

Figure 77

[0222] Figure 77B shows the percentage of pooled lymph node (LN) myeloid dendritic cells (mDC; B220 - , CD11C + ; CD8 + ) in whole cells derived from mice using the CT26 colorectal model that were intravenously treated with (i) phosphate-buffered saline (circles); (ii) 10 mg / kg of CpG-mAb (SB-337) (squares); or (iii) 10 mg / kg of CpG (triangles) on each of days 14, 17, and 30. Samples were taken from draining lymph nodes (dLN) and non-draining lymph nodes (ndLN).

[0223]

Figure 78

[0224] Figure 78B shows the progression of mean tumor volume increase in mice using the A20 lymphoma model after treatment with (i) phosphate-buffered saline solution (small circles); (ii) plasmacytoid dendritic cell (pDC) depletion (diamonds); (iii) 3 mg / kg of CD22-CpG (SB-337) (squares); or (iv) pDC depletion + 3 mg / kg of CD22-CpG (SB-337) (large circles) on each of days 10, 12, and 14.

[0225]

Figure 79

[0226] Figure 79B shows the relative fold change in gene expression of macrophage genes from mice using a CT26 colorectal model after intravenous administration of (i) physiological saline (solid line); or (ii) 3 mg / kg of CpG-mAb (SB-337) (horizontal) on each of days 10, 12, and 14.

[0227] Figure 79C shows the relative fold change in gene expression of cytokine genes from mice using a CT26 colorectal model after intravenous administration of (i) physiological saline (solid line); or (ii) 3 mg / kg of CpG-mAb (SB-337) (vertical) on each of days 10, 12, and 14.

[0228] Figure 79D shows the relative fold change in gene expression of apoptosis enzyme genes from mice using a CT26 colorectal model after intravenous administration of (i) physiological saline (solid line); (ii) 3 mg / kg of CpG-mAb (SB-337) (upward slashes); (iii) anti-PD-L1 (downward slashes); (iv) 3 mg / kg of CpG-mAb + anti-PD-L1 (SB-337) (grid) on each of days 10, 12, and 14.

[0229]

Figure 80

[0230] Figure 80B shows the dose-response curves for the mean fluorescence intensity (MFI) of MHC II expression on human primary B cells in response to in vitro treatment with (i) CpG (p425) (triangles); or (ii) CpG-Ab (SB-430) (circles) for 24 - 72 hours.

[0231] Figure 80C shows the dose-response curves for the mean fluorescence intensity (MFI) of CD86 expression on human primary B cells in response to in vitro treatment with (i) CpG (p425) (triangles); or (ii) CpG-Ab (SB-430) (circles) for 24 - 72 hours.

[0232] Figure 80D shows the dose-response curves for the mean fluorescence intensity (MFI) of CD70 expression on human primary B cells in response to in vitro treatment with (i) CpG (p425) (triangles); or (ii) CpG-Ab (SB-430) (circles) for 24 - 72 hours.

[0233] Figure 80E shows the dose-response curves for the mean fluorescence intensity (MFI) of CD20 expression on human primary B cells in response to in vitro treatment with (i) CpG (p425) (triangles); or (ii) CpG-Ab (SB-430) (circles) for 24 - 72 hours.

[0234]

Figure 81

[0235]

Figure 82

[0236] Figure 82B shows the progression of mean tumor volume increase in a humanized mouse model in which fresh human peripheral blood mononuclear cells were intraperitoneally injected prior to subcutaneous implantation of Daudi Burkitt lymphoma cells and intravenous (IV) treatment with (i) saline (circles); (ii) 5 mg / kg of hCD22 Ab (squares); (iii) 5.7 μg / dose of CpG (p425) (open triangles); or (iv) 5 mg / kg of hCD22-CpG (SB-430) (filled triangles) on days 12, 14, and 16, respectively.

[0237]

Figure 83

[0238]

Figure 84

[0239]

Figure 85

[0240]

Figure 86

[0241]

Figure 87

Mode for Carrying Out the Invention

[0242] (Detailed Description) (Definitions) As used herein, the term "abasic spacer" refers to a divalent group having the following structure: R 1 -L 1 -[-L 2 -(L 1 ) n1 -] n2 -R 2 , (I) (where: n1 is 0 or 1, n2 is an integer from 1 to 6, R 1 is a bond with a nucleoside in the immunomodulatory polynucleotide, R 2 is a bond with a nucleoside in the immunomodulatory polynucleotide or a bond with a capping group, each L 1 is independently a phosphodiester or phosphotriester, and each L 2 is a sugar analog, provided that when the abasic spacer is an internucleoside abasic spacer, each n1 is 1, and R 2 is a bond with a nucleoside, and when the abasic spacer is a terminal abasic spacer, each n1 is independently 0 or 1, and R 2 is a bond with a capping group).

[0243] As used herein, the term "about" refers to a value that is ±10% of the recited value.

[0244] As used herein, the term "alkane-tetrayl" refers to a tetravalent acyclic straight-chain or branched-chain saturated hydrocarbon group having 1 to 16 carbons, unless otherwise specified. The alkane-tetrayl may be optionally substituted as described for alkyl.

[0245] As used herein, the term "alkanetriyl", unless otherwise specified, represents a trivalent acyclic straight-chain or branched-chain saturated hydrocarbon group having 1 to 16 carbons. The alkanetriyl may be optionally substituted as described for alkyl.

[0246] As used herein, the term "alkanoyl" represents a hydrogen or alkyl group bonded to the parent molecular group through a carbonyl group, and is exemplified by formyl (i.e., carboxaldehyde group), acetyl, propionyl, butyryl, and iso-butyryl. The unsubstituted alkanoyl group contains 1 to 7 carbons. The alkanoyl group may be unsubstituted or may be substituted as described herein for alkyl groups (e.g., optionally substituted C 1-7 alkanoyl). The suffix "-oyl" may be added to another group defined herein, such as aryl, cycloalkyl, and heterocyclyl, to define "aroyl", "cycloalkanoyl", and "(heterocyclyl)oyl". These groups each represent a carbonyl group bonded to aryl, cycloalkyl, or heterocyclyl, respectively. Each of "aroyl", "cycloalkanoyl", and "(heterocyclyl)oyl" may be optionally substituted as defined for "aryl", "cycloalkyl", or "heterocyclyl", respectively.

[0247] As used herein, the term "alkenyl" represents an acyclic monovalent straight-chain or branched-chain hydrocarbon group containing 1, 2, or 3 carbon-carbon double bonds. Non-limiting examples of the alkenyl group include ethenyl, prop-1-enyl, prop-2-enyl, 1-methylethenyl, but-1-enyl, but-2-enyl, but-3-enyl, 1-methylprop-1-enyl, 2-methylprop-1-enyl, and 1-methylprop-2-enyl. The alkenyl group may be optionally substituted as defined herein for alkyl.

[0248] As used herein, the term "alkenylene" refers to a linear or branched alkenyl group from which one hydrogen has been removed, thereby rendering the group divalent. Non-limiting examples of such alkenylene groups include ethene-1,1-diyl; ethene-1,2-diyl; prop-1-ene-1,1-diyl, prop-2-ene-1,1-diyl; prop-1-ene-1,2-diyl, prop-1-ene-1,3-diyl; prop-2-ene-1,1-diyl; prop-2-ene-1,2-diyl; but-1-ene-1,1-diyl; but-1-ene-1,2-diyl; but-1-ene-1,3-diyl; but-1-ene-1,4-diyl; but-2-ene-1,1-diyl; but-2-ene-1,2-diyl; but-2-ene-1,3-diyl; but-2-ene-1,4-diyl; but-2-ene-2,3-diyl; but-3-ene-1,1-diyl; but-3-ene-1,2-diyl; but-3-ene-1,3-diyl; but-3-ene-2,3-diyl; but-1,2-diene-1,1-diyl; but-1,2-diene-1,3-diyl; but-1,2-diene-1,4-diyl; but-1,3-diene-1,1-diyl; but-1,3-diene-1,2-diyl; but-1,3-diene-1,3-diyl; but-1,3-diene-1,4-diyl; but-1,3-diene-2,3-diyl; but-2,3-diene-1,1-diyl; and but-2,3-diene-1,2-diyl. The alkenylene group may be unsubstituted or substituted as described for alkyl (e.g., optionally substituted alkenylene).

[0249] As used herein, the term "alkoxy" represents a chemical substituent of the formula -OR, where R is C, unless otherwise specified. 1-6It is an alkyl group. In some embodiments, the alkyl group may be further substituted as defined herein. The term "alkoxy" can be combined with other terms defined herein, such as aryl, cycloalkyl, or heterocyclyl, to define "arylalkoxy", "cycloalkylalkoxy", and "(heterocyclyl)alkoxy" groups. Each of these groups represents an alkoxy substituted by aryl, cycloalkyl, or heterocyclyl, respectively. Each of "arylalkoxy", "cycloalkylalkoxy", and "(heterocyclyl)alkoxy" may be optionally substituted as defined herein for each individual moiety.

[0250] As used herein, the term "alkyl", when unsubstituted and unless otherwise specified, refers to an acyclic straight-chain or branched-chain saturated hydrocarbon group having 1 to 12 carbons. In a preferred embodiment, the unsubstituted alkyl has 1 to 6 carbons. Alkyl groups are exemplified by methyl; ethyl; n-propyl and iso-propyl; n-butyl, sec-butyl, iso-butyl, and tert-butyl; neopentyl, etc., and, if the valence permits, amino; aryl; aryloxy; azide; cycloalkyl; cycloalkoxy; cycloalkenyl; cycloalkynyl; halo; heterocyclyl; (heterocyclyl)oxy; hydroxy; nitro; thiol; silyl; cyano; =O; =S; =NR' (wherein R' is H, alkyl, aryl, or heterocyclyl): and may be optionally substituted with 1, 2, 3, or, in the case of alkyl groups having 2 or more carbons, 4 or more substituents independently selected from the group consisting of. Each of the substituents may itself be unsubstituted or, if the valence permits, may be substituted with an unsubstituted substituent as defined herein for each respective group.

[0251] As used herein, the term "alkylamino" refers to the formula -N(R N1 )2 or -NHR N1 (wherein R N1refers to a group having (which is alkyl as defined herein). The alkyl portion of alkylamino may be optionally substituted as defined for alkyl. Each optional substituent on substituted alkylamino itself may be unsubstituted or, if the valence permits, may be substituted with an unsubstituted substituent defined herein for each respective group.

[0252] As used herein, the term "alkylcycloalkylene" refers to a saturated divalent hydrocarbon group that is an alkylcycloalkane in which two valences replace two hydrogen atoms. Preferably, at least one of the two valences is present in the cycloalkane moiety. The alkane and cycloalkane moieties may be optionally substituted as individual groups as described herein.

[0253] As used herein, the term "alkylene" refers to a saturated divalent hydrocarbon group that is a straight-chain or branched-chain saturated hydrocarbon in which two valences replace two hydrogen atoms. The valences of alkylene as defined herein do not include any substituents. Non-limiting examples of the alkylene group include methylene, ethane-1,2-diyl, ethane-1,1-diyl, propane-1,3-diyl, propane-1,2-diyl, propane-1,1-diyl, propane-2,2-diyl, butane-1,4-diyl, butane-1,3-diyl, butane-1,2-diyl, butane-1,1-diyl, and butane-2,2-diyl, butane-2,3-diyl. The term "C x-y alkylene" represents an alkylene group having from x to y carbon atoms. Exemplary values of x are 1, 2, 3, 4, 5, and 6, and exemplary values of y are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. Alkylene may be optionally substituted as described herein for alkyl.

[0254] As used herein, the term "alkylsulfenyl" represents a group of the formula -S-(alkyl). The alkylsulfenyl may be optionally substituted as defined for alkyl.

[0255] As used herein, the term "alkylsulfinyl" represents a group of the formula -S(O)-(alkyl). The alkylsulfinyl may be optionally substituted as defined for alkyl.

[0256] As used herein, the term "alkylsulfonyl" represents a group of the formula -S(O)2-(alkyl). The alkylsulfonyl may be optionally substituted as defined for alkyl.

[0257] As used herein, the term "alkynyl" represents a monovalent straight-chain or branched-chain hydrocarbon group of 2 to 6 carbon atoms containing at least one carbon-carbon triple bond, exemplified by ethynyl, 1-propynyl, etc. The alkynyl group may be unsubstituted or may be substituted as defined for alkyl (e.g., optionally substituted alkynyl).

[0258] As used herein, the term "5-alkynyluridine" represents a nucleoside in which the nucleobase is 5-alkynyluracil of the following structure: [Chemical formula] (wherein R is the bond to the anomeric carbon of the pentafuranose of the nucleoside and X is alkynyl). In some embodiments, X is ethynyl or propynyl (e.g., X is ethynyl).

[0259] As used herein, the term "alkynylene" refers to a straight or branched chain divalent substituent that contains one or two carbon-carbon triple bonds and, when unsubstituted, contains only C and H. Non-limiting examples of such alkynylene groups include ethine-1,2-diyl; propa-1-yn-1,3-diyl; propa-2-yn-1,1-diyl; buta-1-yn-1,3-diyl; buta-1-yn-1,4-diyl; buta-2-yn-1,1-diyl; buta-2-yn-1,4-diyl; buta-3-yn-1,1-diyl; buta-3-yn-1,2-diyl; buta-3-yn-2,2-diyl; and buta-1,3-diyne-1,4-diyl. The alkynylene group may be unsubstituted or substituted as described for alkynyl groups (e.g., optionally substituted alkynylene).

[0260] As used herein, the term "amino" represents -N(R N1 )2, where when the amino is unsubstituted, both R N1 are H; or when the amino is substituted, each R N1 is independently H, -OH, -NO2, -N(R N2 )2, -SO2OR N2 , -SO2R N2 , -SOR N2 , -COOR N2 , an N-protecting group, alkyl, alkenyl, alkynyl, alkoxy, aryl, arylalkyl, aryloxy, cycloalkyl, cycloalkenyl, heteroalkyl, or heterocyclyl, provided that at least one R N1 is not H and each R N2 is independently H, alkyl, or aryl. Each of the substituents may itself be unsubstituted or substituted with unsubstituted substituents as defined herein for each respective group. In some embodiments, the amino is an unsubstituted amino (i.e., -NH2) or a substituted amino (e.g., -NHR N1 ), where R N1 is independently -OH, -SO2OR N2, -SO2R N2 , -SOR N2 , -COOR N2 , an optionally substituted alkyl, or an optionally substituted aryl, and each R N2 can be an optionally substituted alkyl or an optionally substituted aryl. In some embodiments, the substituted amino may be alkylamino, where the alkyl group is optionally substituted as described herein for alkyl. In one embodiment, the amino group is -NHR N1 where R N1 is an optionally substituted alkyl. Non-limiting examples of -NHR N1 where R N1 is an optionally substituted alkyl include: optionally substituted alkylamino, amino acids that make up proteins, amino acids that do not make up proteins, C 1-6 alkyl esters of amino acids that make up proteins, and C 1-6 alkyl esters of amino acids that do not make up proteins.

[0261] As used herein, the term "aminoalkyl" refers to an alkyl substituted with 1, 2, or 3 amino groups as defined herein. The aminoalkyl may be further optionally substituted as described for the alkyl group.

[0262] As used herein, the term "allen-tetrayl" refers to a tetravalent group that is an aryl group in which 3 hydrogen atoms have been replaced by valences. The allen-tetrayl may be optionally substituted as described herein for aryl.

[0263] As used herein, the term "aryl" refers to a monocyclic, bicyclic, or polycyclic carbocyclic ring system having one or two aromatic rings. The aryl group may contain 6 to 10 carbon atoms. All atoms within an unsubstituted carbocyclic aryl group are carbon atoms. Non-limiting examples of carbocyclic aryl groups include phenyl, naphthyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, indenyl, and the like. The aryl group may be unsubstituted or substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of alkyl; alkenyl; alkynyl; alkoxy; alkylsulfinyl; alkylsulfenyl; alkylsulfonyl; amino; aryl; aryloxy; azide; cycloalkyl; cycloalkoxy; cycloalkenyl; cycloalkynyl; halo; heteroalkyl; heterocyclyl; (heterocyclyl)oxy; hydroxy; nitro; thiol; silyl; and cyano. Each of the substituents may itself be unsubstituted or may be substituted with an unsubstituted substituent as defined herein for each respective group.

[0264] As used herein, the term "arylalkyl" refers to an alkyl group substituted with an aryl group. The aryl and alkyl moieties may each be optionally substituted as individual groups as described herein.

[0265] As used herein, the term "arylalkylene" refers to an arylalkyl group in which one hydrogen atom has been replaced with a valence. The arylalkylene may be optionally substituted as described herein for arylalkyl.

[0266] As used herein, the term "arylene" refers to an aryl group in which one hydrogen atom has been replaced with a valence. The arylene may be optionally substituted as described herein for aryl.

[0267] As used herein, the term "aryloxy" represents a chemical substituent of the formula -OR, where R is an aryl group unless otherwise specified. In optionally substituted aryloxy, the aryl group is optionally substituted as described herein for aryl.

[0268] As used herein, the term "auxiliary moiety" represents a hydrophilic polymer, a positively charged polymer, or a monovalent group containing a sugar alcohol.

[0269] As used herein, the term "optionally substituted N" represents a divalent -N(R N1 )- group or a trivalent -N= group. The aza group may be unsubstituted when R N1 is H or absent, or may be substituted except when R N1 is not H and R N1 is as defined for "amino". Two aza groups may be connected to form a "diazab".

[0270] As used herein, the term "optionally substituted N-protected amino" represents a substituted amino as defined herein, where at least one substituent is an N-protecting group and the other substituents are H when the N-protected amino is unsubstituted, or substituents other than H when the N-protected amino is substituted.

[0271] As used herein, the term "azide" represents an -N3 group.

[0272] As used herein, the term "bulky group" represents any substituent or group of substituents as defined herein, where the radical bonded to the disulfide has 1 or fewer hydrogen atoms when the radical is sp 3 hybridized carbon, or is a carbon atom having no hydrogen atoms when the radical is sp 2 hybridized carbon. The radical is not sp hybridized carbon. The bulky group is bonded to the disulfide only through a carbon atom.

[0273] As used herein, the term "5'-5' cap" refers to the group of the formula R'-Nuc 1 -O-(L P ) n -, where R' is phosphate, phosphorothioate, phosphorodithioate, phosphotriester, phosphodiester, hydroxyl, or hydrogen; Nuc 1 is a nucleoside; each L P is independently -P(=X E1 )(-X E2 -R E2A )-O-; and n is 1, 2, or 3; where each X E1 and each X E2 is independently O or S, and each R E2A is independently hydrogen, a bioreversible group, a non-bioreversible group, an auxiliary moiety, a conjugate moiety, a linker attached to a targeting moiety, or a linker attached to a targeting moiety and one or more (e.g., 1 to 6) auxiliary moieties; and wherein R' is attached to the 3'-carbon of the nucleoside and -O- is attached to the 5'-carbon of the nucleoside.

[0274] As used herein, the term "capping group" refers to a monovalent or divalent group located at the 5'- or 3'-terminus of a polynucleotide. The capping group is a terminal phosphate ester; diphosphate; triphosphate; auxiliary moiety; bioreversible group; non-bioreversible group; 5' cap (e.g., 5'-5' cap); solid support; targeting moiety and an optional linker (e.g., 1 to 6) attached to one or more auxiliary moieties; or the group -OR' (wherein R' is selected from the group consisting of hydrogen, a bioreversible group, a non-bioreversible group, a solid support, and an O-protecting group). The group -OR', diphosphate, triphosphate, bioreversible group, non-bioreversible group, solid support, and auxiliary moiety are examples of monovalent capping groups. The terminal phosphate ester is an example of a capping group that can be either monovalent (when the terminal phosphate ester does not include a linker to a targeting moiety) or divalent (when the terminal phosphate ester includes a linker to a targeting moiety). A linker (with or without an auxiliary moiety) attached to a targeting moiety is an example of a divalent capping group.

[0275] As used herein, the term "carbocyclic" refers to an optionally substituted C 3-16 representing a monocyclic, bicyclic, or tricyclic structure, where the ring, which can be aromatic or non-aromatic, is formed by carbon atoms. Examples of carbocyclic structures include cycloalkyl, cycloalkenyl, cycloalkynyl, and certain aryl groups.

[0276] As used herein, the term "carbonyl" refers to the -C(O)- group.

[0277] As used herein, the expression "C x-y " indicates that the group whose name follows this expression contains a total of x to y carbon atoms when unsubstituted. When the group is a synthetic group (e.g., arylalkyl), C x-y indicates that the portion whose name follows this expression contains a total of x to y carbon atoms when unsubstituted. For example, (C 6-10 -aryl)-C 1-6-Alkyl is a group that contains a total of 6 to 10 carbon atoms when the aryl moiety is unsubstituted and contains a total of 6 to 10 carbon atoms when the alkyl moiety is unsubstituted.

[0278] As used herein, the term "cyano" represents a -CN group.

[0279] As used herein, the term "addition cyclization reaction" represents a reaction of two components in which any of no activation, activation by a chemical catalyst, or activation using thermal energy is present and when n is 1, 2, or 3, a total of [4n + 2] π electrons are involved in bond formation. The addition cyclization reaction is also a reaction of two components in which photochemical activation is present and when n is 1, 2, or 3, [4n] π electrons are involved. Desirably, [4n + 2] π electrons are involved in bond formation and n = 1. Representative addition cyclization reactions include the reaction of an alkene and a 1,3-diene (Diels-Alder reaction), the reaction of an alkene and an α,β-unsubstituted carbonyl (hetero Diels-Alder reaction), and the reaction of an alkyne and an azide compound (e.g., Huisgen addition cyclization).

[0280] As used herein, the term "cycloalkenyl" refers to a non-aromatic carbocyclic group having at least one double bond and 3 to 10 carbons in the ring (e.g., C3-C 10 cycloalkenyl), unless otherwise specified. Non-limiting examples of cycloalkenyl include cycloprop-1-enyl, cycloprop-2-enyl, cyclobut-1-enyl, cyclobut-1-enyl, cyclobut-2-enyl, cyclopent-1-enyl, cyclopent-2-enyl, cyclopent-3-enyl, norbornen-1-yl, norbornen-2-yl, norbornen-5-yl, and norbornen-7-yl. The cycloalkenyl group may be unsubstituted or substituted as described for cycloalkyl (e.g., optionally substituted cycloalkenyl).

[0281] As used herein, the term "cycloalkenylalkyl" represents an alkyl group substituted with a cycloalkenyl group, each as defined herein. The cycloalkenyl and alkyl moieties may be substituted with the individual groups as defined herein.

[0282] As used herein, the term "cycloalkenylene" represents a divalent group that is a cycloalkenyl group in which one hydrogen atom has been replaced with a valence. Cycloalkenylene may be optionally substituted as described herein for cycloalkyl. A non-limiting example of cycloalkenylene is cycloalkene-1,3-diyl.

[0283] As used herein, the term "cycloalkoxy" represents a chemical substituent of the formula -OR, wherein R is a cycloalkyl group unless otherwise specified. In some embodiments, the cycloalkyl group may be further substituted as defined herein.

[0284] As used herein, the term "cycloalkyl" represents a cyclic alkyl group having 3 to 10 carbons (e.g., C3-C 10refers to cycloalkyl. The cycloalkyl group may be monocyclic or bicyclic. The bicyclic cycloalkyl group may be of the bicyclo[p.q.0]alkyl type, where each of p and q is independently 1, 2, 3, 4, 5, 6, or 7, provided that the sum of p and q is 2, 3, 4, 5, 6, 7, or 8. Alternatively, the bicyclic cycloalkyl group may contain a bridged cycloalkyl structure, such as bicyclo[p.q.r]alkyl, where r is 1, 2, or 3, and each of p and q is independently 1, 2, 3, 4, 5, or 6, provided that the sum of p, q, and r is 3, 4, 5, 6, 7, or 8. The cycloalkyl group may be a spirocyclic group, such as spiro[p.q]alkyl, where each of p and q is independently 2, 3, 4, 5, 6, or 7, provided that the sum of p and q is 4, 5, 6, 7, 8, or 9. Non-limiting examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, 1-bicyclo[2.2.1.]heptyl, 2-bicyclo[2.2.1.]heptyl, 5-bicyclo[2.2.1.]heptyl, 7-bicyclo[2.2.1.]heptyl, and decalinyl. The cycloalkyl group may be unsubstituted or substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of alkyl; alkenyl; alkynyl; alkoxy; alkylsulfinyl; alkylsulfenyl; alkylsulfonyl; amino; aryl; aryloxy; azide; cycloalkyl; cycloalkoxy; cycloalkenyl; cycloalkynyl; halo; heteroalkyl; heterocyclyl; (heterocyclyl)oxy; hydroxy; nitro; thiol; silyl; cyano; =O; =S; =NR' (R' is H, alkyl, aryl, or heterocyclyl): (e.g., optionally substituted cycloalkyl). Each of the substituents may itself be unsubstituted or substituted with an unsubstituted substituent as defined herein for each respective group.

[0285] As used herein, the term "cycloalkylalkyl" represents an alkyl group substituted with a cycloalkyl group, each as defined herein. The cycloalkyl and alkyl moieties may be optionally substituted as the individual groups described herein.

[0286] As used herein, the term "cycloalkylene" represents a divalent group that is a cycloalkyl group in which one hydrogen atom has been replaced with a valence. Non-limiting examples of cycloalkylene are cycloalkan-1,3-diyl. Cycloalkylene may be optionally substituted as described herein for cycloalkyl.

[0287] As used herein, the term "cycloalkynyl" refers to a monovalent carbocyclic group having one or two carbon-carbon triple bonds and having 8 to 12 carbons, unless otherwise specified. Cycloalkynyl may include one exocyclic bond or bridge. Non-limiting examples of cycloalkynyl include cyclooctynyl, cyclononynyl, cyclodecynyl, and cyclodecadynyl. The cycloalkynyl group may be unsubstituted or substituted as defined for cycloalkyl (e.g., optionally substituted cycloalkynyl).

[0288] As used herein, the term "dihydropyridazinyl group" represents a divalent group obtained through an additional cyclization between a 1,2,4,5-tetrazine group and a strained cycloalkenyl.

[0289] As used herein, the term "halo" represents a halogen selected from bromine, chlorine, iodine, and fluorine.

[0290] As used herein, the term "5-halouridine" represents a nucleoside in which the nucleobase is 5-halouracil of the following structure:

Chemical formula

[0291] As used herein, the term "heteroalkane-tetrayl" refers to an alkane-tetrayl group interrupted one time by one heteroatom; two times, each time independently, by one heteroatom; three times, each time independently, by one heteroatom; or four times, each time independently, by one heteroatom. Each heteroatom is independently O, N, or S. In some embodiments, the heteroatom is O or N. Unsubstituted C X-Y Heteroalkane-tetrayl contains X to Y carbon atoms and the heteroatoms as defined herein. The heteroalkane-tetrayl group may be unsubstituted or may be substituted as described for heteroalkyl (e.g., optionally substituted heteroalkane-tetrayl).

[0292] As used herein, the term "heteroalkane-triyl" refers to an alkane-triyl group interrupted one time by one heteroatom; two times, each time independently, by one heteroatom; three times, each time independently, by one heteroatom; or four times, each time independently, by one heteroatom. Each heteroatom is independently O, N, or S. In some embodiments, the heteroatom is O or N. Unsubstituted C X-Y Heteroalkane-triyl contains X to Y carbon atoms and the heteroatoms as defined herein. The heteroalkane-triyl group may be unsubstituted or may be substituted as described for heteroalkyl (e.g., optionally substituted heteroalkane-triyl).

[0293] As used herein, the term "heteroalkyl" refers to an alkyl, alkenyl, or alkynyl group interrupted one time by one or two heteroatoms; two times, each time independently, by one or two heteroatoms; three times, each time independently, by one or two heteroatoms; or four times, each time independently, by one or two heteroatoms. Each heteroatom is independently O, N, or S. In some embodiments, the heteroatom is O or N. None of the heteroalkyl groups contain two adjacent oxygen or sulfur atoms. The heteroalkyl group may or may not be substituted (e.g., optionally substituted heteroalkyl). When the heteroalkyl is substituted and the substituent is attached to a heteroatom, the substituent is selected according to the nature and valence of the heteroatom. Thus, a substituent attached to a heteroatom is, if the valence permits, =O, -N(R N2 )2, -SO2OR N3 , -SO2R N2 , -SOR N3 , -COOR N3 , an N-protecting group, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, or cyano, where each R N2 is independently H, alkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, or heterocyclyl, and each R N3 is independently alkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, or heterocyclyl. Each of these substituents may itself be unsubstituted or may be substituted with the unsubstituted substituents defined herein for each of their respective groups. When the heteroalkyl is substituted and the substituent is attached to a carbon, the substituent is selected from those described for alkyl, provided that the substituent on a carbon atom attached to a heteroatom is not Cl, Br, or I. It is understood that the carbon atom is found at the end of the heteroalkyl group.

[0294] As used herein, the term "heteroaryloxy" refers to a structure -OR where R is heteroaryl. The heteroaryloxy may be optionally substituted as defined for heterocyclyl.

[0295] As used herein, the term "heterocyclyl", unless otherwise specified, refers to a monocyclic, bicyclic, tricyclic, or tetracyclic ring system having a fused or bridged 5-, 6-, 7-, or 8-membered ring and containing 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. Heterocyclyl can be aromatic or non-aromatic. Non-aromatic 5-membered heterocyclyl has 0 or 1 double bond, non-aromatic 6- and 7-membered heterocyclyl groups have 0 to 2 double bonds, and non-aromatic 8-membered heterocyclyl groups have 0 to 2 double bonds and / or 0 or 1 carbon-carbon triple bond. Unless otherwise specified, a heterocyclyl group contains 1 to 16 carbon atoms. Certain heterocyclyl groups can contain up to 9 carbon atoms. Examples of non-aromatic heterocyclyl groups include pyrrolinyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, homopiperidinyl, piperazinyl, pyridazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, isothiazolidinyl, thiazolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, dihydroindolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, pyranyl, dihydropyranyl, dithiazolyl, and the like. When a heterocyclic ring system has at least one aromatic resonance structure or at least one aromatic tautomer, such a structure is an aromatic heterocyclyl (i.e., heteroaryl). Non-limiting examples of heteroaryl groups include benzimidazolyl, benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, furyl, imidazolyl, indolyl, isoindazolyl, isoquinolinyl, isothiazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, purinyl, pyrrolyl, pyridinyl, pyrazinyl, pyrimidinyl, qunazolinyl, quinolinyl, thiadiazolyl (e.g., 1,3,4-thiadiazole), thiazolyl, thienyl, triazolyl, tetrazolyl, and the like.The term "heterocyclyl" also represents a heterocyclic compound having a bridged polycyclic structure in which one or more carbon and / or heteroatoms bridge two non-adjacent members of a monocyclic ring, for example, quinuclidine, tropane, or diaza-bicyclo[2.2.2]octane. The term "heterocyclyl" includes bicyclic, tricyclic, and tetracyclic groups in which any of the above heterocyclic rings is fused to one, two, or three carbocyclic rings, for example, an aryl ring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring, or another monocyclic heterocyclic ring. Examples of fused heterocyclyls include 1,2,3,5,8,8a-hexahydroindolizine; 2,3-dihydrobenzofuran; 2,3-dihydroindole; and 2,3-dihydrobenzothiophene. The heterocyclyl group may be unsubstituted or substituted with one, two, three, four, or five substituents independently selected from the group consisting of alkyl; alkenyl; alkynyl; alkoxy; alkylsulfinyl; alkylsulfenyl; alkylsulfonyl; amino; aryl; aryloxy; azide; cycloalkyl; cycloalkoxy; cycloalkenyl; cycloalkynyl; halo; heteroalkyl; heterocyclyl; (heterocyclyl)oxy; hydroxy; nitro; thiol; silyl; cyano; =O; =S; =NR' (wherein R' is H, alkyl, aryl, or heterocyclyl). Each of the substituents may itself be unsubstituted or substituted with an unsubstituted substituent as defined herein for each respective group.

[0296] As used herein, the term "heterocyclylalkyl" represents an alkyl group substituted with a heterocyclyl group, each as defined herein. The heterocyclyl and alkyl moieties may optionally be substituted with the individual groups described herein.

[0297] As used herein, the term "(heterocyclyl)aza" represents a chemical substituent of the formula -N(R N1 )(R N2 ), wherein R N1is a heterocyclyl group and R N2 is H, -OH, -NO2, -N(R N2 )2, -SO2OR N2 , -SO2R N2 , -SOR N2 , -COOR N2 , an N-protecting group, alkyl, alkenyl, alkynyl, alkoxy, aryl, arylalkyl, aryloxy, cycloalkyl, cycloalkenyl, heteroalkyl, or heterocyclyl. Preferably, R N2 is H.

[0298] As used herein, the term "heterocyclylene" represents a heterocyclyl group in which one hydrogen atom has been replaced by a valence. The heterocyclylene may be optionally substituted as described for heterocyclyl. Non-limiting examples of heterocyclylene are heterocyclic-1,3-diyl.

[0299] As used herein, the term "(heterocyclyl)oxy" represents a chemical substituent of the formula -OR, where R is a heterocyclyl group unless otherwise specified. (Heterocyclyl)oxy may be optionally substituted as described for heterocyclyl.

[0300] As used interchangeably herein, the terms "hydroxyl" and "hydroxy" represent the -OH group.

[0301] As used herein, the term "immunomodulatory polynucleotide" refers to a polynucleotide construct containing a total of 6 to 50 contiguous nucleosides covalently linked by internucleoside bridging groups independently selected from the group consisting of internucleoside phosphate esters and optionally internucleoside abasic spacers. The immunomodulatory polynucleotide is capped at the 5'- and 3'-ends by 5'- and 3'-capping groups, respectively. The immunomodulatory polynucleotide is capable of modulating the innate immune response, such as by a change in the activation of NFκB (e.g., compared to another antigen-presenting cell not delivered with the immunomodulatory polynucleotide) or a change in the secretion of at least one inflammatory cytokine or at least one type I interferon in an antigen-presenting cell to which the immunomodulatory polynucleotide has been delivered. The immunomodulatory polynucleotide may contain a conjugate group, or, if the immunomodulatory polynucleotide is part of a conjugate, a targeting moiety and optionally one or more (e.g., 1 to 6) auxiliary moieties (e.g., polyethylene glycol) linked by a linker. The conjugate group or the linker may be part of a phosphotriester or a terminal capping group.

[0302] As used herein, the term "immunostimulatory polynucleotide" refers to an immunomodulatory polynucleotide capable of activating the innate immune response, such as determined by an increase in the activation of NFκB (e.g., compared to another antigen-presenting cell not delivered with the immunostimulatory polynucleotide) or an increase in the secretion of at least one inflammatory cytokine or at least one type I interferon in an antigen-presenting cell to which the immunostimulatory polynucleotide has been delivered. In some embodiments, the immunostimulatory polynucleotide contains at least one cytidine-p-guanosine (CpG) sequence (where p is an internucleoside phosphodiester (e.g., phosphate or phosphorothioate) or internucleoside phosphotriester or phosphorothiotriester). As used herein, a CpG-containing immunostimulatory polynucleotide can be a naturally occurring one, such as a CpG ODN of bacterial or viral origin, or a synthesized one. For example, in some embodiments, the CpG sequence in the immunostimulatory polynucleotide contains 2'-deoxyribose. In some embodiments, the CpG sequence in the immunostimulatory polynucleotide is not methylated. In some embodiments, the immunostimulatory polynucleotide is an oligonucleotide of formula (A) provided herein. In some embodiments, the immunostimulatory polynucleotide is an oligonucleotide of formula (B) provided herein.

[0303] As used herein, the term "immunosuppressive polynucleotide" refers to an immunomodulatory polynucleotide capable of suppressing the innate immune response, such as determined by a decrease in the activation of NFκB (e.g., compared to another antigen-presenting cell not delivered with the immunosuppressive polynucleotide) or a decrease in the secretion of at least one inflammatory cytokine or at least one type I interferon in an antigen-presenting cell to which the immunosuppressive polynucleotide has been delivered.

[0304] As used herein, the term "internucleoside bridging group" refers to an internucleoside phosphate ester or an internucleoside abasic spacer.

[0305] As used herein, the term "5-modified cytidine" refers to a nucleoside in which the nucleobase has the following structure:

Chemical formula

[0306] As used herein, the term "5-modified uridine" refers to a nucleoside in which the nucleobase has the following structure:

Chemical formula

[0307] As used herein, the term "nitro" refers to the -NO2 group.

[0308] As used herein, the term "abiotic reversibility" refers to chemical groups that are resistant to degradation under conditions that exist inside endosomes. Abiotic reversible groups do not contain thioesters and / or disulfides.

[0309] As used herein, the term "nucleobase" refers to a nitrogen-containing heterocyclic ring attached to the 1'-position of the sugar moiety of a nucleotide or nucleoside. The nucleobase may or may not be modified. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C or m5c), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azauracil, cytosine, and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-iodo, 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 5-alkynyl (e.g., 5-ethynyl) uracil, 5-acetamido-uracil, 7-methylguanine, and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.Additional nucleobases include those disclosed in U.S. Patent No. 3,687,808; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858 - 859, edited by Kroschwitz, J. I., John Wiley & Sons, 1990; those disclosed in the literature of Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and those disclosed in the literature of Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289 - 302 (edited by Crooke et al., CRC Press, 1993). Certain nucleobases, including 5 - substituted pyrimidines, 6 - azapyrimidines, and N - 2, N - 6, and O - 6 substituted purines including 2 - aminopropynyladenine, 5 - propynyluracil, and 5 - propynylcytosine, are particularly useful for increasing the binding affinity of the hybridized polynucleotides of the present invention. 5 - Methylcytosine substitution has been shown to increase the stability of nucleic acid duplexes by 0.6 - 1.2 °C (edited by Sanghvi et al., Antisense Research and Applications, 1993, CRC Press, Boca Raton, pages 276 - 278). These can be combined, in certain embodiments, with 2'-O - methoxyethyl sugar modifications.U.S. patents that teach the preparation of some of these modified nucleobases and other modified nucleobases include, but are not limited to, the above-mentioned U.S. Patent Nos. 3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; and 5,681,941. For the purposes of the present disclosure, as used herein, "modified nucleobase" further refers to a natural or unnatural nucleobase that includes one or more protecting groups described herein.

[0310] As used herein, the term "nucleoside" refers to a combination of a pentafuranose-nucleobase. The pentafuranose has the 2-position substituted with OR, R, halo (e.g., F), SH, SR, NH2, NHR, NR2, or CN, where R is optionally substituted C 1-6 alkyl (e.g., C 1-6 alkyl or (C 1-6 (alkoxy)-C 1-6 -alkyl) or optionally substituted (C 6-14 aryl)-C 1-4 -alkyl, and is 2-deoxyribose or a modified form thereof. In certain embodiments, the 2-position is substituted with OR or F, where R is C 1-6 alkyl or (C 1-6 (-alkoxy)-C 1-6 -alkyl. The pentafuranose is attached to the nucleobase at the anomeric carbon. In some embodiments, the term "nucleoside" refers to a divalent group having the following structure:

Chemical formula

[0311] As used herein, the term "nucleotide" refers to a nucleoside linked to phosphate, phosphorothioate, or phosphorodithioate.

[0312] As used herein, the term "oxo" represents a divalent oxygen atom (e.g., the structure of oxo may be shown as =O).

[0313] As used herein, the term "patient" refers to a human or non-human animal (e.g., a mammal). In some embodiments, the subject may have a tumor (e.g., a liquid tumor or a solid tumor) as determined by a qualified professional (e.g., a physician or a nurse practitioner) with or without clinical tests known in the art on a sample derived from the patient.

[0314] As used herein, the term "Ph" represents phenyl.

[0315] As used herein, the term "phosphoester" represents a group containing phosphate, phosphorothioate, or phosphorodithioate in which at least one valence is covalently bonded to a non-hydrogen substituent, provided that at least one non-hydrogen substituent is a group containing at least one nucleoside. A phosphoester in which only one valence is covalently bonded to a group containing a nucleoside is a terminal phosphoester. A phosphoester in which two valences are covalently bonded to nucleoside-containing groups is an internucleoside phosphoester. The phosphoester may be a group having the following structure:

Chemical formula

[0316] As used herein, the term "phosphodiester" refers to a phosphate ester in which two of the three valences are substituted with non-hydrogen substituents while the remaining valence is substituted with hydrogen. The phosphodiester is selected from the group consisting of phosphate, phosphorothioate, or phosphorodithioate; one or two bonds with a nucleoside, abasic spacer, and / or phosphoryl group; and, when the phosphodiester contains only one bond with a nucleoside, abasic spacer, or phosphoryl group, a bioreversible group; a non-bioreversible group; an auxiliary moiety; a conjugate group; a linker attached to a targeting moiety; and a linker attached to a targeting moiety and one or more (e.g., 1 to 6) auxiliary moieties. A terminal phosphodiester contains one bond with a group containing a nucleoside and one group selected from the group consisting of a bioreversible group; a non-bioreversible group; an auxiliary moiety; a conjugate group; a phosphoryl group; and a linker attached to a targeting moiety and optionally one or more (e.g., 1 to 6) auxiliary moieties. An internucleoside phosphodiester contains two bonds with a nucleoside-containing group. The phosphodiester may be a group having the following structure: [Chemical Formula] (wherein X E1 and X E2 each independently is O or S; R E1 and R E3 each independently is hydrogen or a bond with a nucleoside; a sugar analog of an abasic spacer; a bioreversible group; a non-bioreversible group; an auxiliary moiety; a conjugate group; a linker attached to a targeting moiety; a linker attached to a targeting moiety and one or more (e.g., 1 to 6) auxiliary moieties; or a phosphorus atom in a group of the formula -P(=X E1 )(-X E2 -R E2A ), and wherein R E2A is hydrogen, a bioreversible group, a non-bioreversible group, an auxiliary moiety, a conjugate group, a linker attached to a targeting moiety, or a linker attached to a targeting moiety and one or more (e.g., 1 to 6) auxiliary moieties; and R E2 is a hydrogen, a bio-reversible group, a non-bio-reversible group, an auxiliary moiety, a conjugate group, a linker attached to a targeting moiety, or a linker attached to a targeting moiety and one or more (e.g., 1 to 6) auxiliary moieties; provided that E1 R E2 and E3 R provided that E1 R E3 and at least one of

[0317] R E1 and E3 R E1 and E3 R

[0318] The term "phosphoryl" as used herein refers to a substituent of the formula -P(=X E1 )(-X E2 -R E2A )-O-R E3A wherein herein each of E1 X E2 and X E2A is independently O or S; R E3A is hydrogen, a bio-reversible group, a non-bio-reversible group, an auxiliary moiety, a conjugate group, a linker attached to a targeting moiety, or a linker attached to a targeting moiety and one or more (e.g., 1 to 6) auxiliary moieties; and

[0319] When a group is specified as being attached to a phosphoryl, the group is attached to the phosphorus atom of the phosphoryl.

[0320] As used herein, the term "phosphotriester" refers to a phosphate ester in which all three valences are substituted with non-hydrogen substituents. The phosphotriester is selected independently from the group consisting of phosphate, phosphorothioate, or phosphorodithioate; one or two linkages with a nucleoside or abasic spacer, and / or a phosphoryl group; and a bioreversible group; a non-bioreversible group; an auxiliary moiety; a conjugate group; and a targeting moiety and optionally one or more (e.g., 1 to 6) auxiliary moieties attached thereto by a linker. A terminal phosphotriester comprises one linkage with two groups independently selected from the group consisting of a nucleoside-containing group, and a bioreversible group; a non-bioreversible group; an auxiliary moiety; a conjugate group; a phosphoryl group; and a targeting moiety and optionally one or more (e.g., 1 to 6) auxiliary moieties attached thereto by a linker. In some embodiments, the terminal phosphotriester contains 1 or 0 linkers attached to a targeting moiety and optionally one or more (e.g., 1 to 6) auxiliary moieties. An internucleoside phosphotriester comprises two linkages with a nucleoside-containing group. The phosphotriester may be a group having the following structure: [Chemical formula] (wherein X E1 and X E2 each independently is O or S; R E1 and R E3 each independently is a nucleoside; a sugar analog of an abasic spacer; a bioreversible group; a non-bioreversible group; an auxiliary moiety; a conjugate group; a linker attached to a targeting moiety; a linker attached to a targeting moiety and one or more (e.g., 1 to 6) auxiliary moieties; or a bond with a phosphorus atom in a group of the formula -P(=X E1 )(-X E2 -R E2A ), and wherein R E2A is hydrogen; a bioreversible group; a non-bioreversible group; an auxiliary moiety; a conjugate group; a linker attached to a targeting moiety; or a linker attached to a targeting moiety and one or more (e.g., 1 to 6) auxiliary moieties; and R E2 is a bioreversible group; a non-bioreversible group; an auxiliary moiety; a conjugate group; a linker attached to a targeting moiety; or a linker attached to a targeting moiety and one or more (e.g., 1 to 6) auxiliary moieties; provided that E1 R E3 and at least one of R R E1 and R E3 are bonds with a group containing at least one nucleoside, the phosphotriester is an internucleoside phosphotriester. If only one of R E1 and R E3 is a bond with a group containing a nucleoside, the phosphotriester is a terminal phosphotriester.

[0321] As used herein, the term "physiological conditions" refers to conditions that may exist inside professional antigen-presenting cells of a living mammal. Such physiological conditions include a temperature of about 35°C to about 42°C and an aqueous pH of about 6 to about 8.

[0322] As used herein, the term "protecting group" refers to a group that is intended to protect a hydroxy, amino, or carbonyl group from involvement in one or more unwanted reactions during chemical synthesis. As used herein, the term "O - protecting group" refers to a group that is intended to protect a hydroxy or carbonyl group from involvement in one or more unwanted reactions during chemical synthesis. As used herein, the term "N - protecting group" refers to a group that is intended to protect a nitrogen - containing (e.g., amino or hydrazine) group from involvement in one or more unwanted reactions during chemical synthesis. Commonly used O - and N - protecting groups are disclosed in Greene's literature, "Protective Groups in Organic Synthesis", 3rd Edition (John Wiley & Sons, New York, 1999), which is incorporated herein by reference. Exemplary O - and N - protecting groups include alkanoyl, aroyl, or carbamyl groups, such as formyl, acetyl, propionyl, pivaloyl, t - butylacetyl, 2 - chloroacetyl, 2 - bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o - nitrophenoxyacetyl, α - chlorobutyryl, benzoyl, 4 - chlorobenzoyl, 4 - bromobenzoyl, t - butyldimethylsilyl, tri - iso - propylsilyloxymethyl, 4,4'-dimethoxytrityl, isobutyryl, phenoxyacetyl, 4 - isopropylpehenoxyacetyl, dimethylformamidine, and 4 - nitrobenzoyl.

[0323] Exemplary O - protecting groups for protecting carbonyl - containing groups include, but are not limited to: acetals, acylals, 1,3 - dithians, 1,3 - dioxanes, 1,3 - dioxolanes, and 1,3 - dithiolanes.

[0324] Other O-protecting groups include: substituted alkyl, aryl, and aryl-alkyl ethers (e.g., trityl; methylthiomethyl; methoxymethyl; benzyloxymethyl; siloxymethyl; 2,2,2-trichloroethoxymethyl; tetrahydropyranyl; tetrahydrofuranyl; ethoxyethyl; 1-[2-(trimethylsilyl)ethoxy]ethyl; 2-trimethylsilylethyl; t-butyl ether; p-chlorophenyl, p-methoxyphenyl, p-nitrophenyl, benzyl, p-methoxybenzyl, and nitrobenzyl); silyl ethers (e.g., trimethylsilyl; triethylsilyl; triisopropylsilyl; dimethylisopropylsilyl; t-butyldimethylsilyl; t-butyldiphenylsilyl; tribenzylsilyl; triphenylsilyl; and diphenylmethylsilyl); carbonates (e.g., methyl, methoxymethyl, 9-fluorenylmethyl; ethyl; 2,2,2-trichloroethyl; 2-(trimethylsilyl)ethyl; vinyl, allyl, nitrophenyl; benzyl; methoxybenzyl; 3,4-dimethoxybenzyl; and nitrobenzyl), but are not limited thereto

[0325] Other N-protecting groups include chiral auxiliaries such as protected or unprotected D, L, or D, L-amino acids such as alanine, leucine, phenylalanine, etc.; sulfonyl-containing groups such as benzenesulfonyl, p-toluenesulfonyl, etc.; carbamate-forming groups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, etc., aryl-alkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl, etc., and silyl groups such as trimethylsilyl, etc., but are not limited thereto. Useful N-protecting groups are formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc), and benzyloxycarbonyl (Cbz).

[0326] As used herein, the term "pyrid-2-ylhydrazone" represents a group of the following structure:

Chemical formula

[0327] As used herein, the term "stereochemically enriched" refers to the local stereochemical preference of a given stereoisomeric configuration of a listed group over the opposite stereoisomeric configuration of the same group. Thus, a polynucleotide containing a stereochemically enriched phosphorothioate is a chain in which a phosphorothioate of a given stereochemistry preferentially exists over a phosphorothioate of the opposite stereochemistry. This preference can be numerically represented using the diastereomeric ratio for a phosphorothioate of a given stereochemistry. The diastereomeric ratio for a phosphorothioate of a given stereochemistry is the molar ratio of the diastereomer having a specified phosphorothioate of a given stereochemistry to the diastereomer having a specified phosphorothioate of the opposite stereochemistry. The diastereomeric ratio for a phosphorothioate of a given stereochemistry can be 1.1 or greater (e.g., 4 or greater, 9 or greater, 19 or greater, or 39 or greater).

[0328] As used herein, the term "Q-tag" refers to a portion of a polypeptide containing a glutamine residue that provides a conjugate containing a portion of the polypeptide upon a transglutaminase-mediated reaction with a compound containing an -NH2 amine, where the glutamine residue contains a side chain modified to include an amide bonded to the compound. Q-tags are known in the art. Non-limiting examples of Q-tags are LLQGG and GGGLLQGG

[0329] As used herein, the term "strained cycloalkenyl" refers to a cycloalkenyl group having at least 16 kcal / mol of ring strain energy when a vacant valence is substituted with H.

[0330] As used herein, the term "sugar analog" refers to a C that is modified such that two hydroxyl groups are replaced with a bond to an oxygen atom in phosphate, phosphorothioate, or phosphorodithioate, or a capping group 3-6 monosaccharide or C 3-6 represents a divalent or trivalent group that is an alditol (e.g., glycerol). A sugar analog does not contain a nucleobase capable of participating in hydrogen bonding with a nucleobase in a complementary strand. A sugar analog is cyclic or acyclic. Any additional modifications included in the sugar analog are: one, two, or three of the remaining hydroxyl groups or carbon-bonded hydrogen atoms with H; optionally substituted C 1-6 alkyl; -Link A(-T) as defined herein p ; a conjugated group; -(CH2) t1 -OR Z (where t1 is an integer from 1 to 6 and R Z is optionally substituted C 1-6 alkyl, optionally substituted C 2-6 alkenyl, optionally substituted C 2-6 alkynyl, optionally substituted C 6-14 aryl, optionally substituted C 3-8 cycloalkyl, optionally substituted (C 1-9 heterocyclyl)-C 1-6 -alkyl, optionally substituted (C 6-10 aryl)-C 1-6 -alkyl, or optionally substituted (C 3-8 cycloalkyl)-C 1-6 -alkyl); the introduction of one or two unsaturations (e.g., one or two double bonds); and substitution of one, two, or three hydrogens or hydroxyl groups with substituents defined for alkyl, alkenyl, cycloalkyl, cycloalkenyl, or heterocyclyl. Non-limiting examples of sugar analogs are optionally substituted C 2-6 alkylene, optionally substituted C 2-6Alkenylene, optionally substituted C5 cycloalkan-1,3-diyl, optionally substituted C5 cycloalken-1,3-diyl, optionally substituted heterocycle-1,3-diyl (e.g., optionally substituted pyrrolidine-2,5-diyl, optionally substituted tetrahydrofuran-2,5-diyl, or optionally substituted tetrahydrothiophene-2,5-diyl), or optionally substituted (C 1-4 alkyl)-(C 3-8 cycloalkylene) (e.g., optionally substituted (C1 alkyl)-(C3 cycloalkylene)).

[0331] As used herein, the term "sulfide" represents a divalent -S- or =S group. A disulfide is -S-S-.

[0332] As used herein, the term "targeting moiety" refers to a moiety (e.g., a small molecule, e.g., a carbohydrate) that specifically binds to, or reacts associatively with, or forms a complex with a receptor or other receptive moiety associated with a given target cell population (e.g., an antigen-presenting cell (APC); e.g., a professional APC (e.g., a B cell, pDC, or macrophage)). The conjugates of the present invention contain a targeting moiety. The targeting moiety can be an antibody or antigen-binding fragment or a modified derivative thereof (e.g., Fcab or a fusion protein (e.g., scFv)). The targeting moiety can be a polypeptide. Alternatively, the targeting moiety can be a small molecule (e.g., mannose) or a cluster of small molecules (e.g., a cluster of mannose). The conjugates of the present invention containing a targeting moiety can exhibit a K d less than 100 nM for the target to which the targeting moiety binds. K d is measured using methods known in the art, e.g., using surface plasmon resonance (SPR), e.g., using a BIACORE™ system (GE Healthcare, Little Chalfont, the United Kingdom).

[0333] As used herein, the term "1,2,4,5-tetrazine group" refers to a group of the following formula. [Chemical formula] (wherein R' is optionally substituted alkyl, optionally substituted aryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl; and R'' is optionally substituted alkylene, optionally substituted heteroalkylene, optionally substituted arylene, optionally substituted cycloalkylene, optionally substituted heterocyclylene, or the group -R a -R b -(wherein R a and R b each independently is optionally substituted alkylene, optionally substituted heteroalkylene, optionally substituted arylene, optionally substituted cycloalkylene, or optionally substituted heterocyclylene)).

[0334] The term "therapeutic effect" refers to a local or systemic effect in a subject, particularly a mammal, more particularly a human, caused by a pharmacologically active substance. Thus, this term means any substance intended for use in the diagnosis, cure, mitigation, treatment, or prevention of a disease or in the promotion of desirable physical or mental development and conditions in animals or humans. As used herein, the term "therapeutically effective amount" or "therapeutically effective dosage" refers to the amount of an immunomodulatory polynucleotide or conjugate necessary to ameliorate, treat, or at least partially arrest the symptoms of a disease to be treated. The amount effective for this use is determined by the severity of the disease and the weight and general condition of the subject. Usually, the dosage used in vitro can provide useful guidance regarding the amount useful for in vivo administration of the pharmaceutical composition, and animal models can be used to determine the effective dosage for the treatment of a particular disease.

[0335] As used herein, the term "thiocarbonyl" refers to a C(=S) group.

[0336] As used herein, the term "thioheterocyclylene" represents the group -S-R-, where R is a heterocyclylene. Thioheterocyclylene may be optionally substituted as described for heterocyclyl.

[0337] As used herein, the term "thiol" represents the -SH group.

[0338] As used in connection with a disease or condition in a patient, the term "treat" is intended to mean obtaining a beneficial or desirable result, e.g., a clinical result, in a patient by administering a polynucleotide or conjugate of the invention to the patient. Beneficial or desirable results include reduction or amelioration of one or more symptoms of the disease or condition; diminution in the extent of the disease or condition; stabilization of the disease or condition (i.e., non-worsening); prevention of expansion of the disease or condition; delay or deceleration of progression of the disease or condition; palliation of the disease or condition; and remission (whether partial or total). To "palliate" a disease or condition means that the extent and / or undesirable clinical symptoms of the disease or condition are reduced and / or the time course of progression is slowed as compared to the extent or time course in the absence of treatment with a polynucleotide or conjugate of the invention.

[0339] As used herein, the term "triazolocyclalkenylene" refers to a heterocyclylene containing a 1,2,3-triazole ring fused to an 8-membered ring in which all of the ring atoms are carbon atoms and the bridgehead atoms are sp 2 hybridized carbon atoms. Triazolocyclalkenylene may be optionally substituted as described for heterocyclyl.

[0340] As used herein, the term "triazolocycloheterocylene" refers to a heterocyclene containing a 1,2,3-triazole ring fused to an 8-membered ring containing at least one heteroatom. The bridgehead atom in the triazolocycloheterocylene is a carbon atom. The triazolocycloheterocylene may be optionally substituted as described for heterocyclyl.

[0341] The terms "immunomodulatory polynucleotide", "immunostimulatory polynucleotide", "immunosuppressive polynucleotide", and "conjugate" are each to be understood to include salts of the immunomodulatory polynucleotide, immunostimulatory polynucleotide, immunosuppressive polynucleotide, and conjugate, respectively. For example, the terms "immunomodulatory polynucleotide", "immunostimulatory polynucleotide", "immunosuppressive polynucleotide", and "conjugate" include both the protonated neutral form of phosphate, phosphorothioate, or phosphorodithioate (P-XH moiety, where X is O or S) and the deprotonated ionic form of phosphate, phosphorothioate, or phosphorodithioate (P-X - moiety, where X is O or S). Thus, it should be understood that phosphoesters and phosphodiesters described as having one or more of R E1 , R E2 , and R E3 as hydrogen include salts in which the phosphate, phosphorothioate, or phosphorodithioate is present in the deprotonated ionic form.

[0342] The terms "innate immune response" and "innate immunity" are recognized in the art and refer to the non-specific defense mechanisms initiated by the body's immune system upon recognition of pathogen-associated molecular patterns, which involve various forms of cellular activity, including cytokine production and cell death via various pathways. As used herein, the innate immune response includes the cellular response to CpG-containing immunostimulatory polynucleotides mediated by toll-like receptor 9 (TLR9), which includes, but is not limited to, increased production of inflammatory cytokines (e.g., production of type I interferons or IL-10), activation of the NFκB pathway, increased proliferation, maturation, differentiation, and / or survival of immune cells, and in some cases, induction of cell apoptosis. Activation of the innate immunity can be detected by methods known in the art, such as measurement of (NF)-κB activation.

[0343] The terms "adaptive immune response" and "adaptive immunity" are recognized in the art and refer to the antigen-specific defense mechanisms initiated by the body's immune system upon recognition of specific antigens, which include both humoral and cellular responses. As used herein, the adaptive immune response includes the cellular response induced and / or enhanced by CpG-containing immunostimulatory polynucleotides. In some embodiments, the immunostimulatory polynucleotide or a portion thereof is the antigen target of an antigen-specific adaptive immune response. In other embodiments, the immunostimulatory polynucleotide is not the antigen target of an antigen-specific adaptive immune response, but nevertheless enhances the adaptive immune response. Activation of the adaptive immune response can be detected by methods known in the art, such as measurement of antigen-specific antibody production or the level of cytotoxicity mediated by antigen-specific cells.

[0344] The term "Toll-like receptor" (or "TLR") is recognized in the art and originally refers to a family of pattern recognition receptors that were identified as sensors of the innate immune system for recognizing microbial pathogens. TLRs recognize unique structures within microorganisms, often called "PAMPs" (pathogen-associated molecular patterns). Binding of a ligand to a TLR activates a cascade of intracellular signaling pathways that induce an innate immune response and / or an adaptive immune response. As used herein, the term "toll-like receptor" or "TLR" also refers to functional fragments of toll-like receptor proteins expressed by cells. In humans, ten TLRs have been identified, including TLR-1, -2, -3, -4, -5, -6, -7 / 8, and -9. The content of D'Arpa and Leung, Adv. Wound Care, 6:330-343 (2017), which is hereby incorporated by reference in its entirety. Human genes encoding TLRs are known.

[0345] Toll-like receptor 9 (TLR9), also designated as CD289 (cluster of differentiation 289), is a member of the toll-like receptor (TLR) family. The content of Du et al., Eur. Cytokine Netw., 11:362-371 (2000), which is hereby incorporated by reference in its entirety. TLR9 is an important receptor expressed in immune system cells including dendritic cells (DCs), B lymphocytes, macrophages, natural killer cells, and other antigen-presenting cells. TLR9 activation induces a signaling cascade that bridges innate and adaptive immunity. The content of Martinez-Campos et al., Viral Immunol., 30:98-105 (2016); Notley et al., Sci. Rep., 7:42204 (2017); each of which is hereby incorporated by reference in its entirety. Natural TLR-9 agonists include unmethylated cytosine-guanine dinucleotide (CpG)-containing oligodeoxynucleotides (CpG ODNs). TLR-9 ligands found to be useful in the present disclosure include, but are not limited to, naturally occurring or synthetic CpG ODNs, as well as other CpG-containing immunostimulatory polynucleotides and / or immunoconjugates provided herein. Activation of the TLR9 signaling pathway can be detected using methods known in the art, such as mobilization of myeloid differentiation antigen 88 (MyD88), activation of the nuclear factor (NF)-κB, c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) signaling pathways, activation of interferon regulatory factor-7, the expression level of one or more cytokines, such as type I interferon (IFN), interleukin (IL)-6, IL-10, and IL-12, activation of one or more immune cell populations, such as NK cells, natural killer T cells, monocytes, and the levels of cytotoxic lymphocyte (CTL) and T helper-1 (Th1) responses, and the level of immunoglobulin secretion.

[0346] As used herein, the term "TLR-expressing cell" refers to a cell that expresses a toll-like receptor and is capable of activating a toll-like receptor signaling pathway when the toll-like receptor binds to an agonist. The toll-like receptor may be expressed on the cell surface and / or on the membrane of one or more intracellular compartments of the cell, such as an endosome or a phagosome. TLR-expressing cells may further express one or more cell surface antigens other than the toll-like receptor. Certain immune cells express TLRs, and activation of the TLR signaling pathway in these immune cells induces an innate immune response and / or an adaptive immune response. Immune cells activated by the TLR signaling pathway can help eliminate other diseased cells from the body. Certain diseased cells (e.g., cancer cells or virus-infected cells) express TLRs, and activation of the TLR signaling pathway in these diseased cells can result in the death of the diseased cells, for example, through induction of apoptosis. Examples of TLR9-expressing cells include, but are not limited to, dendritic cells (DCs), B cells, T cells, Langerhans cells, keratinocytes, mast cells, endothelial cells, myofibroblasts, and primary fibroblasts. Determination of whether a cell expresses any toll-like receptor (e.g., TLR9) can be carried out using methods known in the art, such as detection of the mRNA of the toll-like receptor in the cell.

[0347] The term "immune cell" is recognized in the art and, as used herein, refers to any cell involved in the host defense mechanism, such as a cell that produces pro-inflammatory cytokines, and a cell involved in tissue damage and / or disease pathogenesis. Examples of immune cells include, but are not limited to, T cells, B cells, natural killer cells, neutrophils, mast cells, macrophages, antigen-presenting cells (APCs), basophils, and eosinophils.

[0348] The term "antigen-presenting cell" or "APC" is recognized in the art and refers to a heterogeneous population of immune cells that mediate a cellular immune response by processing and presenting an antigen for recognition by specific lymphocytes, such as T cells. Exemplary types of antigen-presenting cells include professional antigen-presenting cells, such as B cells, monocytes, dendritic cells, and Langerhans cells, and other antigen-presenting cells, such as keratinocytes, endothelial cells, astrocytes, fibroblasts, and oligodendrocytes, but are not limited thereto. As used herein, the term "antigen-presenting cell" includes antigen-presenting cells found in vivo and antigen-presenting cells found in in vitro cell cultures derived from in vivo cells. As used herein, antigen-presenting cells also include APCs that have been artificially modified, for example, genetically modified to express a toll-like receptor (e.g., TLR9) or to regulate the expression level of a toll-like receptor (e.g., TLR9).

[0349] The term "dendritic cell" or "DC" is recognized in the art and refers to a heterogeneous population of specialized antigen-sensing and antigen-presenting cells (APCs). Human DCs are classified into three major subsets: plasmacytoid DCs (pDCs), myeloid DCs (mDCs), and monocyte-derived DCs (MDDCs). Schraml et al., Curr. Opin. Immunol., 32:13-20 (2015); the contents of which are hereby incorporated by reference in their entirety. DC subsets can be identified based on their unique TLR expression patterns. As an example, the myeloid or "conventional" subset of DCs (mDCs) expresses TLRs 1-8 when stimulated and produces a set of activation markers (e.g., CD80, CD86, MHC class I and II, CCR7), pro-inflammatory cytokines, and chemokines. The result of this stimulation and the resulting expression is the priming of antigen-specific CD4+ and CD8+ T cells. These DCs acquire an enhanced ability to take up antigen and present it to T cells in an appropriate form. The plasmacytoid subset of DCs (pDCs) expresses TLR7 and TLR9 when activated, resulting in the activation of NK cells and T cells.

[0350] As used herein, the term "antigen" refers to a molecule or an antigenic fragment thereof that can induce an immune response, including both innate and adaptive immune responses. As used herein, an antigen can be a protein, peptide, polysaccharide, lipid, nucleic acid, particularly RNA and DNA, nucleotide, and other biological or biochemical substances. The term "induce an immune response" refers to the stimulation of immune cells in vivo in response to a stimulus such as an antigen. The immune response consists of both a cellular immune response, e.g., T cell and macrophage stimulation, and a humoral immune response, e.g., B cell and complement stimulation and antibody production. The immune response can be measured using techniques well known in the art, including, but not limited to, antibody immunoassays, proliferation assays, and others.

[0351] The terms "antigenic fragment" and "antigen-binding fragment" are used interchangeably herein. An antigenic fragment as used herein can form a complex in a specific reaction with an antigen-binding molecule, such as an antibody. The specific reaction referred to herein indicates that the antigen or antigenic fragment reacts with its corresponding antibody in a highly selective manner and does not react with a number of other antibodies that can be induced by other antigens. The specificity of such a reaction is determined by the presence of one or more epitopes (immunogenic determinants) in the antigen. As used herein, an antigen or its antigenic fragment may have one epitope or may have a plurality of epitopes.

[0352] The term "T cell epitope" as used herein refers to any epitope of an antigen produced by T cells.

[0353] As used herein, the term "tumor-associated antigen" or "TAA" refers to an antigen that is expressed in the stroma of solid tumors of cancer cells or of cancer patients receiving the treatment or prophylactic care provided herein (e.g., receiving a therapeutic dose of an immunostimulatory polynucleotide or a CpG-Ab immunoconjugate). TAAs may or may not be targeted in the treatment or prophylactic care provided herein. A TAA need not be overexpressed, mutated, or misregulated on cancer cells and can have the same characteristics as would be found in normal cells if it were a TAA. In some embodiments, a TAA may be overexpressed, mutated, or misregulated in cancer cells. A TAA can be a protein, nucleic acid, lipid, or other antigen. A TAA can be a cell surface-expressed TAA, an intracellular TAA, or a nuclear TAA. In the context of solid tumors, a TAA can be expressed in the stroma of the solid tumor mass. As used herein, the term "stroma" refers to the components in a solid tumor mass other than cancer cells. For example, stroma can include fibroblasts, epithelial cells, other vascular components, or extracellular matrix components. As used herein, the term "stroma" does not include components of the immune system, e.g., immune cells (e.g., B cells, T cells, dendritic cells, macrophages, natural killer cells, etc.). A variety of TAAs are known in the art. Identification of TAAs can be carried out using methods known in the art, e.g., as disclosed in Zhang et al., Methods Mol. Biol., 520:1-10 (2009); the content of this reference is incorporated herein by reference.

[0354] As used herein, the term "antibody" refers to a polypeptide of the immunoglobulin family that can bind to a corresponding antigen in a non-covalent, reversible, and specific manner. For example, a native IgG antibody is a tetramer containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated as VL herein) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH region and the VL region can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) that are interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate binding to host tissues or factors, including immunoglobulins and various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

[0355] As used herein, antibodies include, but are not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, and anti-idiotype (anti-Id) antibodies (e.g., including anti-Id antibodies against the antibodies of the present invention). The antibody can be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0356] Both the light and heavy chains are classified into structurally and functionally homologous regions. The terms "constant" and "variable" are used in a functional sense. In this regard, it will be understood that the variable domains of both the light chain (VL) portion and the heavy chain (VH) portion determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the heavy chain (CH1, CH2, or CH3) confer important biological properties such as, for example, secretion, transplacental transfer, Fc receptor binding, complement binding, etc. By convention, the numbering of the constant region domains increases as the domain is further from the antigen-binding site or amino terminus of the antibody. The N-terminus is the variable region and the C-terminus has the constant region; the CH3 domain and the CL domain actually contain the carboxy-terminal domains of the heavy and light chains, respectively.

[0357] As used herein, depending on the context, the term "antibody" can also refer to an antigen-binding fragment of an antibody molecule. As used herein, the term "antigen-binding fragment" refers to one or more portions of an antibody that retain the ability to specifically interact with an epitope of an antigen (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution). Examples of binding fragments include single-chain Fv (scFv), disulfide-linked Fv (sdFv), Fab fragments, F(ab') fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; F(ab)2 fragments, divalent fragments containing two Fab fragments linked by a disulfide bridge in the hinge region; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single arm of an antibody; dAb fragments consisting of VH or VL domains (Ward et al., Nature, 341:544-546 (1989)); single-domain antibodies (VHH), as well as isolated complementarity-determining regions (CDRs), or other epitope-binding fragments of an antibody, but are not limited thereto.

[0358] The terms "specifically bind" or "selectively bind", or similar terms, refer to a chemical interaction between two molecules, compounds, cells, and / or particles, where the first entity binds to a second target entity with greater specificity and affinity than it binds to a third non-target entity. In some embodiments, "specific binding" can refer to an affinity of a first entity for a second target entity that is at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, or more than the affinity of the first entity for a third non-target entity. In some embodiments, "specific binding" is used in the context of describing an interaction between an antigen (or an antigenic fragment thereof) and an antibody (or an antigen-binding fragment thereof). In certain embodiments, "specific binding" has a dissociation constant (KD) of 10 -5 M or less, 10 -6 M or less, or 10 -7Refers to the binding of an antibody with an M value below a certain level to a predetermined antigen, or the binding of an antibody with a KD at least two-fold smaller than its KD for binding to a non-specific antigen other than the predetermined antigen to the predetermined antigen. In some embodiments, specific binding can be used to determine the presence of a predetermined antigen in a heterogeneous protein population and other biologic agents, such as in a biological sample, e.g., blood, serum, plasma, or tissue sample. Thus, under certain specified immunoassay conditions, an antibody or binding agent having a specific binding specificity binds at least two-fold to a specific antigen relative to background and does not bind appreciably to other antigens present in the sample. In one embodiment, under certain specified immunoassay conditions, an antibody or binding agent having a specific binding specificity binds at least 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold to a specific antigen relative to background and does not bind appreciably to other antigens present in the sample. Specific binding to an antibody or binding agent under such conditions may require that the antibody or agent be selected for its specificity for a particular protein. Desirable or appropriate, this selection can be achieved by removing antibodies or other subtypes that cross-react with molecules from other species (e.g., mouse or rat). Alternatively, in some embodiments, an antibody or antibody fragment that cross-reacts with a particular desired molecule is selected.

[0359] The terms "cancer" or "tumor" refer to the presence of cells that possess characteristics typical of cancer-causing cells, such as uncontrolled growth, immortality, metastatic ability, rapid growth and proliferation rates, and certain characteristic morphological features. In some embodiments, such cells exhibit some or all of such characteristics due to the expression and activity of immune checkpoint inhibitors such as PD-1, PD-L1, and / or CTLA-4. Cancer cells can be detected based on tumor masses, for example, by means such as CAT scans, MR imaging, X-rays, ultrasounds, or palpation, and / or can be detected due to the expression of one or more cancer-specific antigens in samples obtained from a patient, and are often in the form of solid tumors. In some embodiments, the solid tumor need not have measurable dimensions. Cancer cells can also be in the form of liquid tumors, and the cancer cells can be present alone or disseminated within an animal. As used herein, the terms "disseminated tumor" and "liquid tumor" are used interchangeably and include, but are not limited to, leukemia and lymphoma and other blood cell cancers.

[0360] The term "leukemia" refers to a type of cancer of the blood or bone marrow characterized by an abnormal increase in immature white blood cells called "blasts". Leukemia is a broad term encompassing a wide variety of diseases. Further, it is part of a broader group of diseases that affect the blood, bone marrow, and lymphatic system, all known as hematological neoplasms. Leukemia can be divided into four major classifications: acute lymphocytic (or lymphoblastic) leukemia (ALL), acute myeloid (or myeloid or non-lymphocytic) leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML). Additional types of leukemia include hairy cell leukemia (HCL), T-cell prolymphocytic leukemia (T-PLL), large granular lymphocytic leukemia, and adult T-cell leukemia.

[0361] The term "lymphoma" refers to a group of blood cell tumors that originate from lymphoid cells. The two main categories of lymphoma are Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL). Lymphomas include any neoplasm of lymphoid tissue. The main class is a cancer of lymphocytes, a type of white blood cell that belongs to both lymph and blood and spreads to both.

[0362] As used herein, the term "cancer" includes pre-malignant and malignant cancers, primary tumors (e.g., tumors whose cells have not metastasized to a site in the subject's body other than the site of the original tumor) and secondary tumors (e.g., tumors resulting from metastasis, which is the migration of tumor cells to a secondary site different from the site of the original tumor), recurrent cancers, and refractory cancers.

[0363] The terms "cancer recurrence" and "cancer relapse" are used interchangeably and refer to the return of signs, symptoms, or disease after remission. Recurrent cancer cells can reappear at the same site as the primary tumor or at another location, e.g., as a secondary cancer. Cancer cells can recur in the same disease form as the primary cancer or in a different disease form. For example, in some embodiments, the primary cancer is a solid tumor and the recurrent cancer is a liquid tumor. In other embodiments, the primary cancer is a liquid tumor and the recurrent cancer is a solid tumor. In still other embodiments, both the primary cancer and the recurrent cancer are solid tumors or both are liquid tumors. In some embodiments, the recurrent tumor expresses at least one tumor-associated antigen that is also expressed by the primary tumor.

[0364] As used herein, the term "refractory cancer" refers to cancer that does not respond to treatment, e.g., cancer that is resistant at the start of treatment (e.g., treatment by immunotherapy) or that can become resistant during treatment. The terms "respond", "response", or "responsive" refer to an anti-cancer response, e.g., in the sense of a decrease in tumor size or inhibition of tumor growth. These terms can also refer to an improvement in prognosis reflected, e.g., in an increase in the time to recurrence or death without recurrence, which is the period until the first recurrence at which discontinuation occurs due to a second primary cancer as the first event, or an increase in overall survival, which is the period from treatment until death from any cause. To respond or have a response means to have a beneficial endpoint achieved when exposed to a stimulus. Alternatively, negative or adverse symptoms are minimized, alleviated, or attenuated when exposed to the stimulus. It will be understood that assessing the likelihood that a tumor or subject will exhibit a desired response is equivalent to assessing the likelihood that the tumor or subject will not exhibit a desired response (i.e., will exhibit a lack of response or be non-responsive).

[0365] As used herein, cancers include, but are not limited to, B-cell cancers such as multiple myeloma, Waldenström macroglobulinemia, heavy chain diseases such as alpha-chain disease, gamma-chain disease, and mu-chain disease, benign monoclonal gammopathy, and immunocytic amyloidosis, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, cancers of the blood tissue, etc. Other non-limiting examples of cancer types suitable for the methods encompassed by the present invention include human sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, hepatocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias such as acute lymphocytic leukemia and acute myelogenous leukemia (myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia); chronic leukemias (chronic myelogenous (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphomas (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström macroglobulinemia, and heavy chain disease. In some embodiments, the cancer is epithelial and includes, but is not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancer, kidney cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In still other embodiments, the epithelial cancer is non-small cell lung cancer, non-papillary renal cell carcinoma, cervical cancer, ovarian cancer (e.g., serous ovarian cancer), or breast cancer.Epithelial cancer can be characterized in a variety of other ways, including but not limited to serous, endometrial, mucinous, clear cell, Brenner type, or undifferentiated types.

[0366] As used herein, the terms "cancer therapy" or "cancer therapeutic agent" refer to a therapy or agent that can exert an anti-tumor effect or have anti-tumor activity. Such anti-tumor effect or anti-tumor activity can be manifested as a decrease in the rate of tumor cell proliferation, viability, or metastatic activity. Conventional methods of demonstrating anti-tumor activity include showing a decrease in the rate of abnormal cell proliferation or a stabilization or decrease in tumor size during treatment. Such activity can be evaluated using generally recognized in vitro or in vivo tumor models, including but not limited to xenograft models, allograft models, MMTV models, and other known models in the art for examining anti-tumor activity.

[0367] As used herein, the terms "prevent," "preventing," or "prevention" of any disease or disorder mean the total or partial prevention of the onset, recurrence, or spread of the disease or disorder described herein, or its symptoms.

[0368] As used herein, a subject "is in need of" such treatment if the subject would benefit biologically, medically, or in terms of quality of life from such treatment.

[0369] The term "therapeutic agent" is recognized in the art and refers to any substance that, when administered to a subject in need thereof, is biologically, physiologically, or pharmacologically active and acts locally or systemically to exert a beneficial therapeutic effect on the subject.

[0370] As used herein, the terms "immunoconjugate" or "antibody-drug-conjugate (ADC)" refer to the linkage of an antigen-binding portion (e.g., an antibody or an antigen-binding fragment thereof) with an immunomodulatory polynucleotide described herein. The linkage can be a covalent or non-covalent interaction and can include chelation. Various linkers known in the art or provided herein can be utilized to form the immunoconjugate. In some embodiments, the immunoconjugate is a conjugate of formula (C) provided herein.

[0371] As used herein, the term "antigen-binding portion" refers to a portion capable of specifically binding to an antigen and includes, but is not limited to, antibodies and antigen-binding fragments.

[0372] As used herein, the terms "CpG-Ab immunoconjugate" or "CpG-Ab" refer to the linkage of an antibody (Ab) or an antigen-binding fragment thereof with a CpG-containing immunostimulatory polynucleotide described herein.

[0373] As used herein, the term "T cell agonist" refers to any agent that selectively stimulates the proliferation, differentiation, and / or survival of T cells derived from a mixed starting cell population. Thus, the resulting cell population is enriched with an increase in the number of T cells compared to the starting cell population. T cell agonists found to be useful in the present disclosure include, but are not limited to, antigen molecules that specifically bind to the T cell receptor (TCR) and T cell costimulatory molecules. Examples of T cell costimulatory molecules include, but are not limited to, OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and CD83 ligand. In certain embodiments, the T cell agonist is an antibody against a T cell costimulatory molecule. In certain embodiments, the T cell agonist is a tumor-associated antigen (TAA). In certain embodiments, the T cell agonist is a pathogenic antigen.

[0374] As used herein, "immune checkpoint" or "immune checkpoint molecule" is a molecule of the immune system that modulates a signal. An immune checkpoint molecule can be a stimulatory checkpoint molecule, i.e., one that enhances a signal, or an inhibitory checkpoint molecule, i.e., one that weakens a signal. In a specific embodiment, an immune checkpoint is a protein expressed by either a T cell or an antigen-presenting cell (APC). Certain types of cancer cells express immune checkpoint proteins to avoid immune clearance. The use of immune checkpoint modulators to inhibit the interaction between immune checkpoint proteins expressed by cancer cells and immune checkpoint proteins expressed by T cells has proven effective in certain cancer treatments.

[0375] As used herein, an "immune checkpoint modulator" is an agent that can alter the activity of an immune checkpoint in a subject. In certain embodiments, the immune checkpoint modulator alters the function of one or more immune checkpoint molecules, including but not limited to PD-1, PD-L1, PD-L2, TIM-3, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD160, CD47, 2B4, and TGFR. The immune checkpoint modulator can be an agonist or antagonist of an immune checkpoint. In some embodiments, the immune checkpoint modulator is an immune checkpoint binding protein (e.g., an antibody, an antibody Fab fragment, a bispecific antibody, an antibody-drug conjugate, an scFv, a fusion protein, a bispecific antibody, or a tetravalent antibody). In other embodiments, the immune checkpoint modulator is a small molecule. In certain embodiments, the immune checkpoint modulator is an anti-PDl or anti-PD-Ll antibody.

[0376] As used herein, the term "targeted delivery" or the verb form "targeting" refers to the process of facilitating the delivery of an agent (e.g., an immunostimulatory polynucleotide) to a particular organ, tissue, cell, and / or intracellular compartment (referred to as the target location) that is greater than any other organ, tissue, cell, or intracellular compartment (referred to as the non-target location). Targeted delivery can be detected using methods known in the art, for example, by comparing the concentration of the delivered agent in the target cell population to the concentration of the delivered agent in the non-target cell population after systemic administration. As provided herein, targeted delivery results in at least a two-fold higher concentration at the target location compared to the non-target location. Targeted delivery can be achieved by specific binding of a targeting moiety to a receptor moiety associated with the target cell. As used herein, the receptor moiety associated with the target cell can be located on the surface or within the cytosol of the target cell. In some embodiments, the receptor moiety is an antigen associated with the target cell.

[0377] As used in connection with a biological entity, tissue, cell, or component thereof, the term "abnormal" refers to a biological entity, tissue, cell, or component thereof that has at least one observable or detectable characteristic (e.g., age, treatment, time, etc.) that is different from a "normal" (expected) respective characteristic of the biological entity, tissue, cell, or component thereof. A characteristic that is normal for or expected of a particular cell or tissue type may be abnormal for a different cell or tissue type. In some embodiments, an abnormal cell is a cancer cell.

[0378] The term "combination therapy" refers to the administration of two or more therapeutic agents for treating a condition or disorder (e.g., cancer) described in the present disclosure. Such administration includes co-administration of these therapeutic agents in a substantially simultaneous manner, e.g., administering a single formulation having a fixed ratio of therapeutic agents, or administering separate formulations (e.g., capsules and / or intravenous formulations) for each therapeutic agent. Further, such administration includes the use of each type of therapeutic agent in a sequential or separate manner, either almost simultaneously or at different times. Such administration also includes each component being formulated as separate formulations that can be administered at different times and / or via different routes of administration. In any event, the treatment regimen of the combination therapy provides a beneficial therapeutic effect in treating the condition or disorder described herein.

[0379] As used herein, the terms "co-administering" or "co-administration" and like terms refer to the act of administering two or more therapeutic agents (e.g., immunoconjugates and immune checkpoint modulators), compounds, therapies, etc. simultaneously or nearly simultaneously. Co-administering can refer to co-administration where various therapeutic agents of the present disclosure, such as immunoconjugates, T cell agonists, immune checkpoint modulators, or other chemotherapeutic agents, are combined into the same formulation or formulated separately for co-administration to a subject. Co-administering can also refer to sequential administration. The order or sequence in which the various therapeutic agents of the present invention, such as immunoconjugates, T cell agonists, immune checkpoint modulators, or other chemotherapeutic agents, are administered may vary and is not limited to any particular order. Co-administering can refer to situations where two or more agents are administered to different body regions or by different delivery schemes, e.g., a first agent is administered systemically and a second agent is administered intratumorally, or a first agent is administered intratumorally and a second agent is administered systemically into the blood or peritumorally. Co-administering can also refer to two or more agents that are administered by the same delivery scheme, e.g., a first agent is administered intratumorally and a second agent is administered intratumorally.

[0380] "Intratumoral injection" refers to the direct administration of the agents provided herein into tumor cell masses and / or the tumor microenvironment. As used herein, the tumor microenvironment includes the neoplastic stroma that creates the structural and / or functional environment for the neoplastic process to survive, expand, or proliferate. The tumor microenvironment is composed of cells, molecules, fibroblasts, extracellular matrix, and blood vessels that surround and nourish one or more neoplastic cells forming the tumor. Examples of cells or tissues within the tumor microenvironment include, but are not limited to, tumor vasculature, tumor infiltrating lymphocytes, fibroblastic reticular cells, endothelial progenitor cells (EPCs), cancer-associated fibroblasts, pericytes, other stromal cells, components of the extracellular matrix (ECM), dendritic cells, antigen-presenting cells, T cells, regulatory T cells, macrophages, neutrophils, and other immune cells located near the tumor. Examples of cellular functions that affect the tumor microenvironment include, but are not limited to, the production of cytokines and / or chemokines, responses to cytokines, antigen processing and presentation of peptide antigens, regulation of leukocyte chemotaxis and migration, regulation of gene expression, complement activation, regulation of signaling pathways, cellular cytotoxicity, cellular immunity, humoral immune responses, and other innate or adaptive immune responses. Measuring the effects of modulating these cellular functions.

[0381] The terms "subject", "patient", "individual" and similar terms are used interchangeably herein and refer to any animal or its cells suitable for the methods provided herein, whether in vitro or in vivo. In one non-limiting embodiment, the patient, subject, or individual is a mammal, such as a human, or another animal, such as a wild animal (e.g., goat, pigeon, crane, etc.), a domestic animal (e.g., duck, goose, etc.), or a laboratory animal (e.g., orangutan, monkey, rat, mouse, rabbit, guinea pig, marmot, gerbil, etc.).

[0382] The term "survival" as used in the context of cancer includes any of the following: survival until death, also known as overall survival (where the death may be unrelated to the relevant cause or tumor); "recurrence-free survival" (where the term recurrence is taken to include both local and distant recurrence); metastasis-free survival; disease-free survival (where the term disease is taken to include cancer and associated diseases). The length of such survival can be calculated with reference to a defined starting point (e.g., at diagnosis or at the start of treatment) and end point (e.g., death, recurrence, or metastasis). Further, the criteria for the efficacy of treatment can be broadened to include response to chemotherapy, probability of survival, probability of metastasis within a given period, and probability of tumor recurrence.

[0383] The present invention provides immunomodulatory (e.g., immunostimulatory) polynucleotides and conjugates containing a targeting moiety and one or more immunomodulatory (e.g., immunostimulatory) polynucleotides. The immunomodulatory polynucleotide may contain 5-modified uridine or 5-modified cytidine. Inclusion of 5-modified uridine (e.g., 5-ethynyl-uridine) at the 5'-end of the immunomodulatory polynucleotide (e.g., between two 5'-terminal nucleosides) may enhance the immunomodulatory properties of the polynucleotide. The immunomodulatory polynucleotide may be shorter than a typical CpG ODN that is 18 - 28 nucleotides in length (e.g., contains a total of 6 - 16 nucleotides or 12 - 14 nucleotides). The shorter immunomodulatory polynucleotides of the present invention (e.g., those containing a total of 6 - 16 nucleotides or 12 - 14 nucleotides) may retain the immunomodulatory activity of longer typical CpG ODNs and may exhibit higher immunomodulatory activity (e.g., as measured by NFκB activation or by a change in the expression level of at least one cytokine (e.g., IL-6 or IL-10)) compared to longer CpG ODNs. Advantageously, the shorter immunomodulatory polynucleotides are simple and economical to prepare as their synthesis involves fewer polynucleotide synthesis steps than that of full-length typical CpG ODNs. The immunomodulatory polynucleotide may contain one or more abasic spacers and / or internucleoside phosphotriesters.

[0384] The immunomodulatory polynucleotides of the present invention can exhibit stability (e.g., stability against nucleases) that is superior to that of CpG ODNs containing mostly internucleoside phosphates (e.g., more than 50% internucleoside phosphates) without substantially sacrificing their immunostimulatory activity. This effect can be achieved, for example, by incorporating at least 50% (e.g., at least 70%) internucleoside phosphorothioates or phosphorodithioates, or by the inclusion of internucleoside phosphotriesters and / or internucleoside abasic spacers. Phosphotriesters and abasic spacers are also convenient for conjugation with targeting moieties. Phosphate-based phosphotriesters and abasic spacers can also be used for reducing off-target activity as compared to polynucleotides having a complete phosphorothioate backbone. Without wishing to be bound by theory, this effect can be achieved by reducing self-delivery without interfering with delivery to target cells mediated by the targeting moiety. Thus, the polynucleotides of the present invention can include 15 or fewer consecutive internucleoside phosphorothioates (e.g., 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, or 10 or fewer consecutive internucleoside phosphorothioates). For example, an immunostimulatory polynucleotide containing a total of 12 to 16 nucleosides can contain 10 or fewer consecutive internucleoside phosphorothioates.

[0385] The immunostimulatory polynucleotides of the present invention can contain a total of 50 or fewer nucleosides (e.g., 30 or fewer, 28 or fewer, or 16 or fewer nucleosides). The immunostimulatory polynucleotides of the present invention can contain a total of at least 6 nucleosides (e.g., 10 or more or 12 or more nucleosides). For example, the immunostimulatory polynucleotides of the present invention can contain a total of 6 to 30 nucleosides (e.g., a total of 6 to 28 nucleosides, a total of 6 to 20 nucleosides, a total of 6 to 16 nucleosides, a total of 10 to 20 nucleosides, a total of 10 to 16 nucleosides, a total of 12 to 28 nucleosides, a total of 12 to 20 nucleosides, or a total of 12 to 16 nucleosides).

[0386] The immunostimulatory polynucleotides of the present invention can contain one or more phosphotriesters (e.g., internucleoside phosphotriesters) and / or phosphorothioates (e.g., 1 to 6 or 1 to 4) at, for example, one or both ends (e.g., within 6 nucleosides at the 5'-end or 6 nucleosides at the 3'-end). Inclusion of one or more internucleoside phosphotriesters and / or phosphorothioates can enhance the stability of the polynucleotide by reducing the rate of exonuclease-mediated degradation.

[0387] In certain embodiments, the immunostimulatory polynucleotides of the present invention contain a phosphotriester or a terminal phosphodiester, where the phosphotriester or the terminal phosphodiester includes a linker that is bound to a targeting moiety or a conjugate group and optionally one or more (e.g., 1 to 6) auxiliary moieties. In certain embodiments, the immunostimulatory polynucleotide contains only one linker. In some embodiments, the immunostimulatory polynucleotide contains only one conjugate group.

[0388] The polynucleotides of the present invention (e.g., immunostimulatory polynucleotides) can be hybridized polynucleotides comprising a strand and its partial or whole complement. The hybridized polynucleotides can have at least 6 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23) complementary base pairs up to the total number of nucleotides present in the shorter strand that is encompassed. For example, the hybridized portion of the hybridized polynucleotide can contain 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 base pairs.

[0389] The conjugate of the present invention contains a targeting moiety and one or more immunomodulatory (e.g., immunostimulatory) polynucleotides (e.g., 1 to 6 or 1 to 4 (e.g., 1 or 2) immunomodulatory (e.g., immunostimulatory) polynucleotides). In the conjugate, each of the immunomodulatory polynucleotides independently includes a linker. The targeting moiety is covalently bound to the linker. The linker may be bound to a nucleobase, abasic spacer, phosphate, phosphorothioate, or phosphorodithioate in the immunomodulatory polynucleotide. The cells targeted by the conjugate of the present invention are professional APCs (e.g., B cells, pDCs, or macrophages). The targeting moiety can be a group containing an antigen-binding moiety (e.g., an antibody or an antigen-binding fragment thereof), a polypeptide, an aptamer, or one or more small molecules (e.g., mannose). In the conjugate of the present invention, the targeting moiety can be an antibody or an antibody fragment. The conjugate of the present invention can contain an antibody or an antibody fragment and one or more immunomodulatory polynucleotides covalently bound to a Q-tag in the antibody or the antibody fragment. The Q-tag can be at the N-terminus or C-terminus. The Q-tag can be arranged on the heavy chain or light chain of the antibody or the antibody fragment. The use of the delivery of the immunomodulatory polynucleotide of the present invention based on the targeting moiety to specifically targeted tissues and cells can overcome the drawback of the usually non-uniform distribution of the immunomodulatory polynucleotide in vivo. Furthermore, the delivery of the immunomodulatory polynucleotide of the present invention based on the targeting moiety can be advantageous for systemic administration or administration to the target tissue of the immunomodulatory polynucleotide, because systemic administration and administration to the target tissue can result in an undesirable distribution of the immunomodulatory polynucleotide through blood circulation in vivo, whereas the conjugate of the present invention can undergo intracellular delivery mainly in the target tissue or cells even when systemically administered. The advantages related to distribution can be particularly prominent in conjugates containing short immunomodulatory polynucleotides (e.g., immunomodulatory polynucleotides containing a total of 6 to 16 nucleosides (e.g., a total of 10 to 16 or 12 to 16 nucleosides)).

[0390] The conjugate of the present invention may further contain one or more (e.g., 1 to 6) auxiliary moieties (e.g., polyethylene glycol (PEG)). The auxiliary moiety may be part of a capping group, a bio-reversible group, or a non-bio-reversible group. The auxiliary moiety may be bound to a linker (e.g., a linker bound to a phosphate, phosphorothioate, or phosphorodithioate in an immunomodulatory (e.g., immunostimulatory) polynucleotide). Inclusion of an auxiliary moiety (e.g., PEG) in the conjugate of the present invention can improve the pharmacokinetic and / or biodistribution properties of the conjugate compared to a reference conjugate lacking such an auxiliary moiety.

[0391] One or more of the immunomodulatory polynucleotides of the present invention can be conjugated to a targeting moiety (e.g., an antigen-binding moiety) that targets antigen-presenting cells (APCs; e.g., professional APCs (e.g., B cells, pDCs, or macrophages)). Delivery of the immunomodulatory polynucleotide or conjugate of the present invention to cells containing endosomal toll-like receptors (e.g., TLR9) (e.g., antigen-presenting cells (APCs; e.g., professional APCs (e.g., B cells, pDCs, or macrophages))) can be used to stimulate (in the case of immunostimulatory polynucleotides) or inhibit (in the case of immunosuppressive polynucleotides) the endosomal toll-like receptor intracellularly. Without being bound by theory, activation of the endosomal toll-like receptor can induce pro-inflammatory cytokines (e.g., IL-6, IL-10, and / or type I interferons); this activity is thought to be useful for the treatment of various tumors (e.g., solid and liquid tumors in a patient).

[0392] In one embodiment, provided herein is an oligonucleotide of formula (A): or a stereoisomer thereof, a mixture of two or more diastereomers, a tautomer, or a mixture of two or more tautomers; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; X 5' -(X N ) b-Y P -(X N ) c -X 3' (A) (wherein: each X N is independently a nucleotide; X 3' is the 3'-terminal nucleotide; X 5' is the 5'-terminal nucleotide; Y P is a internucleoside phosphotriester; and b and c are each an integer in the range of about 0 to about 25; provided that their sum is 5 or more; wherein the oligonucleotide comprises nucleotides having modified nucleobases).

[0393] In certain embodiments, b is an integer in the range of about 1 to about 15. In certain embodiments, b is an integer of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15. In certain embodiments, b is an integer of about 3, about 4, about 11, or about 14. In certain embodiments, b is an integer of about 3. In certain embodiments, b is an integer of about 4. In certain embodiments, b is an integer of about 11. In certain embodiments, b is an integer of about 14.

[0394] In certain embodiments, c is an integer in the range of about 0 to about 10. In certain embodiments, c is an integer of about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10. In certain embodiments, c is an integer of about 0 or about 8. In certain embodiments, c is an integer of about 0. In certain embodiments, c is an integer of about 8.

[0395] In certain embodiments, b is an integer of about 3 and c is an integer of about 8. In certain embodiments, b is an integer of about 4 and c is an integer of about 8. In certain embodiments, b is an integer of about 11 and c is an integer of about 0. In certain embodiments, b is an integer of about 14 and c is an integer of about 0.

[0396] In certain embodiments, the sum of b and c is in the range of about 5 to about 20. In certain embodiments, the sum of b and c is in the range of about 5 to about 15. In certain embodiments, the sum of b and c is about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15. In certain embodiments, the sum of b and c is about 8, about 9, about 10, about 11, about 12, about 13, or about 14. In certain embodiments, the sum of b and c is about 11. In certain embodiments, the sum of b and c is about 12. In certain embodiments, the sum of b and c is about 14.

[0397] In certain embodiments, each X N is independently a 2'-deoxyribonucleotide or a 2'-modified ribonucleotide. In certain embodiments, each X N is independently 2'-deoxyadenosine (A), 2'-deoxyguanosine (G), 2'-deoxycytidine (C), 5-halo-2'-deoxycytidine, 2'-deoxythymidine (T), 2'-deoxyuridine (U), 5-halo-2'-deoxyuridine, 2'-fluororibonucleotide, 2'-methoxyribonucleotide, or 2'-(2-methoxyethoxy)ribonucleotide. In certain embodiments, each X N is independently a 2'-deoxyribonucleotide. In certain embodiments, each X N is independently 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxycytidine, 5-halo-2'-deoxycytidine, 2'-deoxythymidine, 2'-deoxyuridine, or 5-halo-2'-deoxyuridine. In certain embodiments, each X Nis, independently, 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxycytidine, 2'-deoxythymidine, 5-bromo-2'-deoxyuridine, or 5-iodo-2'-deoxyuridine.

[0398] In certain embodiments, X 3' is a 2'-deoxyribonucleotide or a 2'-modified ribonucleotide. In certain embodiments, X 3' is a 2'-deoxyribonucleotide. In certain embodiments, X 3' is 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxycytidine, 5-halo-2'-deoxycytidine, 2'-deoxythymidine, 2'-deoxyuridine, 5-halo-2'-deoxyuridine, 2'-fluororibonucleotide, 2'-methoxyribonucleotide, or 2'-(2-methoxyethoxy)ribonucleotide. In certain embodiments, X 3' is 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxycytidine, 5-halo-2'-deoxycytidine, 2'-deoxythymidine, 2'-deoxyuridine, or 5-halo-2'-deoxyuridine. In certain embodiments, X 3' is 2'-deoxythymidine. In certain embodiments, X 3' is a 2'-deoxyribonucleotide having a substituted pyrimidine base. In certain embodiments, X 3' is a 2'-deoxyribonucleotide having a 5-substituted pyrimidine base. In certain embodiments, X 3' is 2'-deoxythymidine, 5-halo-2'-deoxycytidine, or 5-halo-2'-deoxyuridine. In certain embodiments, X 3' is 2'-deoxythymidine, 5-bromo-2'-deoxycytidine, 5-iodo-2'-deoxycytidine, 5-bromo-2'-deoxyuridine, or 5-iodo-2'-deoxyuridine. In certain embodiments, X 3'is 2'-deoxythymidine, 5-bromo-2'-deoxyuridine, or 5-iodo-2'-deoxyuridine. In certain embodiments, X 3' is a terminal nucleotide comprising a 3'-capping group. In certain embodiments, the 3'-capping group is a terminal phosphate ester. In certain embodiments, the 3'-capping group is 3-hydroxyl-propylphosphoryl (i.e., -P(O2)-CH2CH2CH2OH).

[0399] In certain embodiments, X 5' is a 2'-deoxyribonucleotide or a 2'-modified ribonucleotide. In certain embodiments, X 5' is a 2'-deoxyribonucleotide. In certain embodiments, X 5' is 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxycytidine, 5-halo-2'-deoxycytidine, 2'-deoxythymidine, 2'-deoxyuridine, 5-halo-2'-deoxyuridine, 2'-fluororibonucleotide, 2'-methoxyribonucleotide, or 2'-(2-methoxyethoxy)ribonucleotide. In certain embodiments, X 5' is 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxycytidine, 5-halo-2'-deoxycytidine, 2'-deoxythymidine, 2'-deoxyuridine, or 5-halo-2'-deoxyuridine. In certain embodiments, X 5' is a 2'-deoxyribonucleotide having a substituted pyrimidine base. In certain embodiments, X 5' is a 2'-deoxyribonucleotide having a 5-substituted pyrimidine base. In certain embodiments, X 5' is 2'-deoxythymidine, 5-halo-2'-deoxycytidine, or 5-halo-2'-deoxyuridine. In certain embodiments, X 5' is 5-halo-2'-deoxycytidine. In certain embodiments, X 5' is 5-halo-2'-deoxyuridine. In certain embodiments, X 5'is 2'-deoxythymidine, 5-bromo-2'-deoxycytidine, 5-iodo-2'-deoxycytidine, 5-bromo-2'-deoxyuridine, or 5-iodo-2'-deoxyuridine. In certain embodiments, X 5' is 2'-deoxythymidine, 5-bromo-2'-deoxyuridine, or 5-iodo-2'-deoxyuridine. In certain embodiments, X 5' is 5-bromo-2'-deoxyuridine. In certain embodiments, X 5' is 5-iodo-2'-deoxyuridine. In certain embodiments, X 5' has a 3'-phosphorothioate group. In certain embodiments, X 5' has a 3'-phosphorothioate group having the chirality of Rp. In certain embodiments, X 5' has a 3'-phosphorothioate group having the chirality of Sp.

[0400] In certain embodiments, Y P is a nucleoside phosphorothioate triester.

[0401] In certain embodiments, Y P is:

Chemical formula

[0402] In certain embodiments, Y P is:

Chemical formula

[0403] In certain embodiments, the oligonucleotide of formula (A) contains one additional internucleoside phosphotriester. In one embodiment, the additional internucleoside phosphotriester is a 1-6 C alkyl phosphotriester. In another embodiment, the additional internucleoside phosphotriester is an ethyl phosphotriester.

[0404] In certain embodiments, the oligonucleotide of formula (A) contains one 5-halo-2'-deoxyuridine. In one embodiment, the 5-halo-2'-deoxyuridine is 5-fluoro-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, or 5-iodo-2'-deoxyuridine. In another embodiment, the 5-halo-2'-deoxyuridine is 5-bromo-2'-deoxyuridine or 5-iodo-2'-deoxyuridine. In yet another embodiment, the 5-halo-2'-deoxyuridine is 5-fluoro-2'-deoxyuridine. In yet another embodiment, the 5-halo-2'-deoxyuridine is 5-bromo-2'-deoxyuridine. In yet another embodiment, the 5-halo-2'-deoxyuridine is 5-iodo-2'-deoxyuridine.

[0405] In certain embodiments, the oligonucleotide of formula (A) contains 3 or more 2'-deoxycytidines. In certain embodiments, the oligonucleotide of formula (A) contains 3 2'-deoxycytidines.

[0406] In certain embodiments, the oligonucleotide of formula (A) comprises 4 or more 2'-deoxyguanosines. In certain embodiments, the oligonucleotide of formula (A) comprises 4 2'-deoxyguanosines.

[0407] In certain embodiments, the oligonucleotide of formula (A) comprises 3 2'-deoxycytidines and 4 2'-deoxyguanosines. In certain embodiments, the oligonucleotide of formula (A) comprises 1, 2, or 3 CG dinucleotides. In certain embodiments, the oligonucleotide of formula (A) comprises 3 CG dinucleotides.

[0408] In certain embodiments, the oligonucleotide of formula (A) comprises 3 or more 2'-deoxythymidines. In certain embodiments, the oligonucleotide of formula (A) comprises 3, 4, 5, 6, 7, or 8 2'-deoxythymidines. In certain embodiments, the oligonucleotide of formula (A) comprises 3, 4, 5, or 8 2'-deoxythymidines.

[0409] In certain embodiments, the oligonucleotide of formula (A) does not contain 2'-deoxyadenosine. In certain embodiments, the oligonucleotide of formula (A) comprises 1 or 2 2'-deoxyadenosines.

[0410] In certain embodiments, the oligonucleotide of formula (A) has a length in the range of about 5 to about 20 or about 6 to about 15 nucleotides. In certain embodiments, the oligonucleotide of formula (A) has a length of about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15. In certain embodiments, the oligonucleotide of formula (A) has a length of about 10, about 11, about 12, about 13, about 14, or about 15.

[0411] In certain embodiments, the oligonucleotide of formula (A) comprises one or more internucleoside phosphorothioates. In certain embodiments, all of the internucleoside phosphoesters in the oligonucleotide of formula (A) are internucleoside phosphorothioates. In certain embodiments, the oligonucleotide of formula (A) comprises one or more chiral internucleoside phosphorothioates.

[0412] In certain embodiments, the oligonucleotide of formula (A) is p275, p276, p313, or p347. In certain embodiments, the oligonucleotide of formula (A) is p236, p238, p243, p246, p308, p361, p362, or p425. In certain embodiments, the oligonucleotide of formula (A) is p236, p238, p243, p246, p275, p276, p308, p313, p347, p361, p362, p425, p433, p434, p435, p436, p437, p438, p477, p478, p479, p480, p481, p482, p483, p484, p485, p486, p487, p488, or p489.

[0413] In certain embodiments, the oligonucleotide of formula (A) is an immunomodulatory oligonucleotide.

[0414] In one embodiment, provided herein is an N 1 N 2 CGN 3 CG(T) x GN 4 CGN 5 oligonucleotide having the sequence T, or a stereoisomer, mixture of two or more diastereomers, tautomer, or mixture of two or more tautomers thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; wherein: x is an integer in the range of 1 to 4; N 1 is absent or is 2'-deoxythymidine; N 2is a 2'-deoxyribonucleotide having a modified nucleobase; N 3 is 2'-deoxyadenosine or 2'-deoxythymidine each optionally containing a 3'-phosphotriester; N 4 is 2'-deoxyadenosine or 2'-deoxythymidine; N 5 is 2'-deoxythymidine optionally containing a 3'-phosphotriester; and C is 2'-deoxycytidine and G is 2'-deoxyguanosine.

[0415] In certain embodiments, N 1 N 2 CGN 3 CG(T) x GN 4 CGN 5 in T, x is an integer of 1, 2, 3, or 4. In certain embodiments, N 1 N 2 CGN 3 CG(T) x GN 4 CGN 5 in T, x is an integer of 1. In certain embodiments, N 1 N 2 CGN 3 CG(T) x GN 4 CGN 5 in T, x is an integer of 4.

[0416] In certain embodiments, N 1 N 2 CGN 3 CG(T) x GN 4 CGN 5 in T, N 1 is absent. In certain embodiments, N 1 N 2 CGN 3 CG(T) x GN 4 CGN 5 in T, N 1 is 2'-deoxythymidine.

[0417] In certain embodiments, N 1 N 2 CGN 3 CG(T) x GN 4 CGN 5 In T, N 2 is a 2'-deoxyribonucleotide having a substituted pyrimidine base. In certain embodiments, N 1 N 2 CGN 3 CG(T) x GN 4 CGN 5 In T, N 2 is a 2'-deoxyribonucleotide having a 5-substituted pyrimidine base. In certain embodiments, N 1 N 2 CGN 3 CG(T) x GN 4 CGN 5 In T, N 2 is 5-halo-2'-deoxycytidine or 5-halo-2'-deoxyuridine. In certain embodiments, N 1 N 2 CGN 3 CG(T) x GN 4 CGN 5 In T, N 2 is 5-bromo-2'-deoxyuridine or 5-iodo-2'-deoxyuridine.

[0418] In certain embodiments, N 1 N 2 CGN 3 CG(T) x GN 4 CGN 5 In T, N 3 is 2'-deoxyadenosine containing a 3'-phosphotriester. In certain embodiments, N 1 N 2 CGN 3 CG(T) x GN 4 CGN 5 In T, N 3 is 2'-deoxythymidine. In certain embodiments, N1 N 2 CGN 3 CG(T) x GN 4 CGN 5 In T, N 3 is 2'-deoxythymidine containing a 3'-phosphotriester.

[0419] In certain embodiments, N 1 N 2 CGN 3 CG(T) x GN 4 CGN 5 In T, N 4 is 2'-deoxyadenosine. In certain embodiments, N 1 N 2 CGN 3 CG(T) x GN 4 CGN 5 In T, N 4 is 2'-deoxythymidine.

[0420] In certain embodiments, N 1 N 2 CGN 3 CG(T) x GN 4 CGN 5 In T, N 5 is 2'-deoxythymidine. In certain embodiments, N 1 N 2 CGN 3 CG(T) x GN 4 CGN 5 In T, N 5 is 2'-deoxythymidine containing a 3'-phosphotriester.

[0421] In certain embodiments, N 1 N 2 CGN 3 CG(T) x GN 4 CGN 5 The oligonucleotide of T contains one or more internucleoside phosphorothioates. In certain embodiments, N 1 N 2CGN 3 CG(T) x GN 4 CGN 5 The oligonucleotide of T contains at least one chiral nucleoside internucleotide phosphorothioate.

[0422] In certain embodiments, N 1 N 2 CGN 3 CG(T) x GN 4 CGN 5 The oligonucleotide of T is p275, p276, or p313. In certain embodiments, N 1 N 2 CGN 3 CG(T) x GN 4 CGN 5 The oligonucleotide of T is p236, p238, p243, p246, p308, p361, p362, or p425. In certain embodiments, N 1 N 2 CGN 3 CG(T) x GN 4 CGN 5 The oligonucleotide of T is p236, p238, p243, p246, p275, p276, p308, p313, p347, p361, p362, p425, p433, p434, p435, p436, p437, p438, p477, p478, p479, p480, p481, p482, p483, p484, p485, p486, p487, p488, or p489.

[0423] (Immunostimulatory polynucleotide) The immunostimulatory polynucleotides of the present invention can function as PAMPs and can stimulate an adaptive immune response by activating the innate immune response or inducing TLR9 signaling (e.g., as a TLR9 agonist). The sequences that can be used in the immunostimulatory polynucleotides of the present invention are those known in the art for class B CpG polynucleotides, or modifications thereof containing 5-halouridine or 5-alkynyluridine, or truncated versions thereof (e.g., those containing a total of 6 to 16 nucleosides). The truncated immunostimulatory polynucleotides of the present invention (e.g., those containing a total of 6 to 16 nucleosides) can contain a truncated class B CpG polynucleotide sequence (e.g., a class B CpG polynucleotide sequence in which one or more 3'-terminal nucleotides have been removed or one or more nucleotides within the sequence have been excised).

[0424] The immunostimulatory polynucleotides of the present invention contain at least one immunostimulatory sequence (ISS). For example, the immunostimulatory polynucleotides of the present invention can contain 1, 2, 3, or 4 ISSs. The ISS in the immunostimulatory polynucleotide is determined by the target organism. A general characteristic of the ISS used in the immunostimulatory polynucleotides of the present invention is a cytidine-p-guanosine sequence, where p is an internucleoside phosphodiester (e.g., phosphate or phosphorothioate) or an internucleoside phosphotriester. Preferably, the cytidine and guanosine in the ISS contain 2'-deoxyribose. In some embodiments, the immunostimulatory polynucleotides of the present invention contain 1, 2, or 3 human ISSs. For example, the human ISS can be CG or NCG, where N is uridine, cytidine, or thymidine, or a modified version of uridine or cytidine disclosed herein (e.g., 5-halouridine (e.g., 5-iodouridine or 5-bromouridine), 5-alkynyluridine (e.g., 5-ethynyluridine or 5-propynyluridine), 5-heteroaryluridine, or 5-halocytidine); and G is guanosine or a modified version thereof disclosed herein (e.g., 7-deazaguanosine). Preferably, the human ISS is NCG (e.g., where N is 5-halouridine). In some embodiments, the human ISS is UCG (e.g., where U is 5-alkynyluridine (e.g., 5-ethynyluridine)). Preferably, the immunostimulatory polynucleotides of the present invention targeting humans contain an ISS within 4 adjacent nucleotides including the 5'-terminal nucleotide (e.g., the immunostimulatory polynucleotides of the present invention contain a 5'-terminal ISS). The mouse ISS is a hexamer nucleotide sequence: Pu-Pu-CG-Py-Py, where each Pu is independently a purine nucleotide and each Py is independently a pyrimidine nucleotide.

[0425] In some embodiments, the 5'-adjacent nucleotide to CpG in the immunostimulatory polynucleotide of the present invention does not contain 2'-alkoxyribose. Preferably, the 5'-adjacent nucleotide to CpG in the immunostimulatory polynucleotide of the present invention contains only 2'-deoxyribose as the sugar.

[0426] Structural features of the immunostimulatory polynucleotide of the present invention include: (1) a high content of phosphorothioate (e.g., at least 50%, at least 60%, at least 70%, or at least 80% of the nucleosides can be linked by phosphorothioate), (2) the absence of a poly-G tail, (3) the nucleosides in the immunostimulatory polynucleotide can contain 2'-deoxyribose or 2'-modified ribose (e.g., 2'-halo (e.g., 2'-fluoro) or optionally substituted 2'-alkoxy (e.g., 2'-methoxy)), and / or (4) the inclusion of a 5'-terminal ISS that is NCG (where N is uridine, cytidine, or thymidine, or a modified version of uridine or cytidine disclosed herein (e.g., 5-halouridine (e.g., 5-iodouridine or 5-bromouridine), 5-alkynyluridine (e.g., 5-ethynyluridine or 5-propynyluridine), 5-heteroaryluridine, or 5-halocytidine); and G is guanosine or a modified version thereof disclosed herein (e.g., 7-deazaguanosine)).

[0427] In some embodiments, the conjugate contains one targeting moiety (e.g., an antibody or an antigen-binding fragment thereof) and one immunomodulatory polynucleotide covalently attached to the targeting moiety.

[0428] (Immunosuppressive polynucleotide) The polynucleotide of the present invention can suppress the adaptive immune response by reducing the activation of TLR9 signaling (e.g., via TLR9 inhibitory action). In some embodiments, the immunosuppressive polynucleotide of the present invention has the following formula: N 1 -N2 -CG (wherein N 1 and N 2 each of which is independently a nucleotide containing 2'-alkoxyribose (e.g., 2'-methoxyribose)), and contains at least two 2'-alkoxynucleotides adjacent to CpG at the 5'-position.

[0429] (Structural Features of Polynucleotides) (Abasic Spacer) The immunomodulatory polynucleotides disclosed herein may contain one or more (e.g., one or two) abasic spacers (e.g., internucleoside abasic spacers and / or terminal abasic spacers). When the immunomodulatory polynucleotide contains two or more abasic spacers, the structures of the abasic spacers may be the same or different.

[0430] The abasic spacer is of formula (I): R 1 -L 1 -[-L 2 -(L 1 ) n1 -] n2 -R 2 ,(I) (wherein n1 is 0 or 1, n2 is an integer from 1 to 6, R 1 is a bond to a nucleoside in the immunomodulatory polynucleotide, R 2 is a bond to a nucleoside in the immunomodulatory polynucleotide or a bond to a capping group, each L 1 is independently a phosphodiester or a phosphotriester, and each L 2 is a sugar analog).

[0431] In certain embodiments, when the abasic spacer is an internucleoside abasic spacer, n1 is 1, and R 2is a bond with a nucleoside, where when the abasic spacer is the terminal abasic spacer, n1 is 0 or 1, and R 2 is a bond with a capping group.

[0432] In some embodiments, the abasic spacer is an abasic spacer between nucleosides or a 3'-terminal abasic spacer. In one embodiment, each of two adjacent L 2 groups is separated by an L 1 group (e.g., when an L 2 is disposed between two adjacent L 1 groups, n1 is 1).

[0433] In one embodiment, the immunostimulatory polynucleotide contains an ISS disposed within four consecutive nucleotides including the 5'-terminal nucleotide of the immunostimulatory polynucleotide, wherein the ISS is NCG, where N is uridine, cytidine, or thymidine, or a modified version of uridine or cytidine disclosed herein (e.g., 5-halouridine (e.g., 5-iodouridine or 5-bromouridine), 5-alkynyluridine (e.g., 5-ethynyluridine or 5-propynyluridine), 5-heteroaryluridine, or 5-halocytidine), and N and C are linked to each other via a phosphodiester or phosphotriester.

[0434] (sugar analog) The sugar analog is a divalent or trivalent group of a C 3-6 monosaccharide or a C 3-6 alditol (e.g., glycerol) that is modified to replace two hydroxyl groups with (i) a bond to an oxygen atom in one phosphate ester and (ii) a bond to an oxygen atom in another phosphate ester or a capping group. The sugar analog is cyclic or acyclic. Any additional modifications included in the analog are: one, two, or three of the remaining hydroxyl groups or carbon-bonded hydrogen atoms with H; optionally substituted C 1-6Alkyl; -Link A(-T) as defined herein p ; Conjugated group; -(CH2) t1 -OR Z (where t1 is an integer from 1 to 6 and R Z is optionally substituted C 1-6 alkyl, optionally substituted C 2-6 alkenyl, optionally substituted C 2-6 alkynyl, optionally substituted C 6-14 aryl, optionally substituted C 3-8 cycloalkyl, optionally substituted (C 1-9 heterocyclyl)-C 1-6 -alkyl, optionally substituted (C 6-10 aryl)-C 1-6 -alkyl, or optionally substituted (C 3-8 cycloalkyl)-C 1-6 -alkyl). Substitution with; introduction of 1 or 2 unsaturations (e.g., 1 or 2 double bonds); and substitution of 1, 2, or 3 hydrogens or hydroxyl groups with substituents defined for alkyl, alkenyl, cycloalkyl, cycloalkenyl, or heterocyclyl. In some embodiments, R Z is optionally substituted C 1-6 aminoalkyl (e.g., optionally substituted C 1-6 aminoalkyl containing -NH2).

[0435] Non-limiting examples of sugar analogs are optionally substituted C 2-6 alkylene, optionally substituted C 2-6 alkenylene, optionally substituted C5 cycloalkane-1,3-diyl, optionally substituted C5 cycloalkene-1,3-diyl, optionally substituted heterocycle-1,3-diyl (e.g., optionally substituted pyrrolidine-2,5-diyl, optionally substituted tetrahydrofuran-2,5-diyl, or optionally substituted tetrahydrothiophene-2,5-diyl), or optionally substituted (C 1-4 alkyl)-(C 3-8It is a cycloalkylene (for example, an optionally substituted (C1 alkyl)-(C3 cycloalkylene)). Non-limiting examples of the sugar analogs are: [Chemical formula] (where R 1 and R 2 each is, independently, a bond with an oxygen atom in the phosphate ester; R 3 and R 4 each is, independently, H; optionally substituted C 1-6 alkyl; -(CH2) t1 -OR Z ; or -linker A-R T ; where t1 is an integer from 1 to 6; R Z is optionally substituted C 1-6 alkyl, optionally substituted C 2-6 alkenyl, optionally substituted C 2-6 alkynyl, optionally substituted C 6-14 aryl, optionally substituted C 3-8 cycloalkyl, optionally substituted (C 1-9 heterocyclyl)-C 1-6 -alkyl, optionally substituted (C 6-10 aryl)-C 1-6 -alkyl, optionally substituted (C 3-8 cycloalkyl)-C 1-6 -alkyl; linker A is a linker; and R T is a targeting moiety; a conjugation moiety; optionally substituted C 1-6 alkyl, optionally substituted C 2-6 alkenyl, optionally substituted C 2-6 alkynyl, optionally substituted C 6-14 aryl, optionally substituted C 3-8 cycloalkyl, optionally substituted (C 1-9 heterocyclyl)-C 1-6 -alkyl, optionally substituted (C 6-10(Aryl)-C 1-6 -alkyl, or optionally substituted (C 3-8 (Cycloalkyl)-C 1-6 -alkyl bond).

[0436] In certain embodiments, R Z is optionally substituted C 1-6 aminoalkyl (e.g., optionally substituted C 1-6 aminoalkyl containing -NH2).

[0437] (Phosphoester) The immunomodulatory polynucleotides of the invention may contain one or more internucleoside phosphotriesters and / or one or two terminal phosphodiesters and / or phosphotriesters. The phosphotriester has one or two valences substituted with a nucleoside and / or a abasic spacer, and the remaining valences are bonded to a bio-reversible group, a non-bio-reversible group, a linker bonded to a targeting moiety, or a conjugate group, and may contain phosphate, phosphorothioate, or phosphorodithioate. The internucleoside phosphotriester is bonded to two nucleosides and / or abasic spacers, and the remaining valences are bonded to a bio-reversible group, a non-bio-reversible group, a linker bonded to a targeting moiety, or a conjugate group. The internucleoside phosphodiester is bonded to two nucleosides and / or abasic spacers. The terminal phosphodiester contains phosphate, phosphorothioate, or phosphorodithioate at the 5'- or 3'-terminus of the immunomodulatory polynucleotide, where one of the remaining two valences is bonded to a bio-reversible group, a non-bio-reversible group, a linker bonded to a targeting moiety, or a conjugate group.

[0438] (Linker and conjugation moiety) The immunomodulatory polynucleotide of the present invention may contain a targeting moiety and a linker optionally attached to one or more auxiliary moieties. The linker has a molecular weight of 43 Da to 10 kDa (e.g., 100 Da to 8 kDa, 100 Da to 7 kDa, or 100 Da to 3 kDa). The linker may be designated as Link A herein. The linker may be a polyvalent group in which the first valence is attached to an internucleoside or terminal phosphate, internucleoside or terminal phosphorothioate, internucleoside or terminal phosphorodithioate, abasic spacer, capping group, or nucleobase, and the second valence is attached to the targeting moiety. The linker may further include one or more valences, each of which is independently attached to an auxiliary moiety. In some embodiments (e.g., when the targeting moiety is a small molecule), the immunomodulatory polynucleotide contains multiple linkers for multiple targeting moieties. In other embodiments (e.g., when the targeting moiety is an antibody or an antigen-binding fragment thereof), the immunomodulatory polynucleotide may contain one linker for the targeting moiety.

[0439] The immunomodulatory polynucleotides disclosed herein may contain a conjugation group. The conjugation group is a functional group that can undergo a conjugation reaction (e.g., an addition cyclization reaction (e.g., dipolar addition cyclization), an amidation reaction, or an aromatic nucleophilic substitution), or includes at least one conjugate moiety that can undergo a conjugation reaction when the functional group is deprotected. By reaction with a complementary reactive group, the conjugation group gives rise to a linker in the immunomodulatory polynucleotide of the present invention.

[0440] In certain embodiments, the linker attached to the targeting moiety is a portion of an internucleoside phosphotriester. In one embodiment, the linker attached to the targeting moiety is a portion of an abasic spacer.

[0441] In some embodiments, the linker (e.g., Link A) or the conjugation group is of formula (II): -Z 1-Q A1 -Z 2 -(-Q A2 -Z 3 -) k -R T , (II) (wherein Z 1 is a divalent, trivalent, tetravalent, or pentavalent group, wherein one of the valences is bonded to Q A1 and the second valence is either vacant or, when formula (II) relates to a linker, is bonded to R T and each of the remaining valences, if any, is independently bonded to an auxiliary moiety; Z 2 is absent, a divalent, trivalent, tetravalent, or pentavalent group, wherein one of the valences is bonded to Q A1 and the second valence is bonded to Q A2 or R T and each of the remaining valences, if any, is independently bonded to an auxiliary moiety; Z 3 is absent, a divalent, trivalent, tetravalent, or pentavalent group, wherein one of the valences is bonded to Q A2 and the second valence is bonded to R T and each of the remaining valences, if any, is independently bonded to an auxiliary moiety; R T is absent or is a bond to a targeting moiety; k is 0 or 1).

[0442] When formula (II) relates to a linker, Q A1 and Q A2 are independently absent, optionally substituted C 2-12 heteroalkylene (e.g., heteroalkylene containing -C(O)-N(H)-, -N(H)-C(O)-, -S(O)2-N(H)-, or -N(H)-S(O)2-), optionally substituted C 1-12 thioheterocyclylene (e.g.,

Chemical Structure

[0443] When formula (II) relates to a conjugated group, (i) Q A2 is absent and Q A1 is a conjugation moiety, e.g., optionally substituted C 2-12 alkynyl, optionally substituted N-protected amino, azide, N-maleimide, S-protected thiol, [Chemical formula] or their N-protected versions, [Chemical formula] ), optionally substituted C containing an internal carbon-carbon triple bond 6-16Heterocyclyl (e.g., [Chem.] ), 1,2,4,5 - tetrazinyl (e.g., [Chem.] ), or optionally substituted C 8-16 cycloalkynyl (e.g., [Chem.] ), -NHR N1 , optionally substituted C 4-8 strained cycloalkenyl (e.g., trans - cyclooctenyl or norbornenyl), or -COOR 12 or -CHO - containing optionally substituted C 1-16 alkyl; and k is 0; or (ii) Q A1 is as defined for the linker, and Q A2 is a conjugation moiety, e.g., optionally substituted C 2-12 alkynyl, optionally substituted N - protected amino, azide, N - maleimide, S - protected thiol, [Chem.] or their N - protected versions, [Chem.] , optionally substituted C 6-16 heterocyclyl containing an internal carbon - carbon triple bond (e.g., [Chem.] ), 1,2,4,5 - tetrazinyl (e.g., [Chem.] ), or optionally substituted C 8-16Cycloalkynyl (e.g., [Chemical formula] ), -NHR N1 , optionally substituted C 4-8 strained cycloalkenyl (e.g., trans-cyclooctenyl or norbornenyl), or -COOR 12 or -CHO-containing optionally substituted C 1-16 alkyl; and k is 1; wherein, R N1 is H, an N-protecting group, or optionally substituted C 1-6 alkyl; each R 12 is independently H or optionally substituted C 1-6 alkyl; R 13 is halogen (e.g., F); Z 3 and R T are absent.

[0444] In certain embodiments, Z 1 has a branching group and two divalent segments, where the branching group is attached to each of the two divalent segments, wherein, one of the divalent segments is attached to an internucleoside or terminal phosphate, internucleoside or terminal phosphorothioate, internucleoside or terminal phosphorodithioate, abasic spacer, or nucleobase, and the remaining divalent segment is attached to Q A1 ; the branching group is optionally substituted C 1-12 alkanetriyl or optionally substituted C 2-12 heteroalkanetriyl, where two valences are substituted with the divalent segment and the remaining valences are [Chemical formula] substituted with, Here, p1 is 1, 2, or 3; each s2 is independently an integer from 0 to 10; each Q B and each Q D is independently non - existent, -CO-, -NH-, -O-, -S-, -SO2-, -OC(O)-, -COO-, -NHC(O)-, -C(O)NH-, -CH2-, -CH2NH-, -NHCH2-, -CH2O-, or -OCH2-; and each Q C is independently non - existent, optionally substituted C 1-12 alkylene, optionally substituted C 2-12 alkenylene, optionally substituted C 2-12 alkynylene, optionally substituted C 2-12 heteroalkylene, optionally substituted C 1-9 heterocyclylene, or -P(Z)(OH)-, where Z is O or S; each Q G is independently optionally substituted C 1-6 alkan - triyl, optionally substituted C 1-6 alkan - tetrayl, optionally substituted C 2-6 heteroalkan - triyl, or optionally substituted C 2-6 heteroalkan - tetrayl; and each Q H is independently R M1 or -Q G [(-Q B -Q C -Q D ) s2 -R M1 p1 where each R M1 is independently a bond to an auxiliary moiety.

[0445] In certain embodiments, Z 2 has a branching group and two divalent segments, where the branching group is attached to each of the two divalent segments, Here, ​One of the two divalent segments is attached to the targeting moiety or Q A2 and the remaining divalent segment is attached to Q A1 ; The branching group is optionally substituted C 1-12 alkanetriyl or optionally substituted C 2-12 heteroalkanetriyl, where two valences are substituted with the divalent segment and the remaining valences are

Chemical formula

[0446] In one embodiment, Z 3 has a branching group and two divalent segments, wherein the branching group is attached to each of the two divalent segments, wherein one of the divalent segments is attached to a targeting moiety and the remaining divalent segment is attached to Q A2 ; the branching group is an optionally substituted C 1-12 alkanetriyl or an optionally substituted C 2-12 heteroalkanetriyl, wherein two valences are substituted with the divalent segments and the remaining valences are

Chemical formula

[0447] Z 1 , Z 2 or Z 3 the divalent segment in is -(-Q B -Q C -Q D -) s1 - and may be, wherein each s1 is, independently, an integer from 1 to 50 (e.g., 1 to 30); each Q B and each Q D is, independently, absent, -CO-, -NH-, -O-, -S-, -SO2-, -OC(O)-, -COO-, -NHC(O)-, -C(O)NH-, -CH2-, -CH2NH-, -NHCH2-, -CH2O-, or -OCH2-; and each Q C is, independently, absent, an optionally substituted C 1-12 alkylene, an optionally substituted C 2-12 alkenylene, an optionally substituted C 2-12 alkynylene, an optionally substituted C 2-12 heteroalkylene, or an optionally substituted C 1-9 heterocyclylene; provided that Q B , Q C ​, and Q D At least one of them exists.

[0448] In certain embodiments, at least one Q C is present in the divalent segment. In certain embodiments, Q C is present in each monomer unit of the divalent segment. In some embodiments, Z 1 is bonded through the Q C present. In a further embodiment, Q B and Q D at least one of which is in each monomer unit of Z 1 present. In yet a further embodiment, Q B and Q D at least one of which is in each monomer unit of Z 2 present. In certain embodiments, when present, Z 1 Z 2 and Z 3 only one of which contains a branching group.

[0449] In yet a further embodiment, Z 1 Z 2 and Z 3 one, two, or three of which are independently -(-Q B -Q C -Q D -) s1 -Q E -(-Q B -Q C -Q D -) s1 -, (III) wherein each s1 is independently an integer from 1 to 50 (e.g., 1 to 30); each Q B and each Q D is independently absent, -CO-, -NH-, -O-, -S-, -SO2-, -OC(O)-, -COO-, -NHC(O)-, -C(O)NH-, -CH2-, -CH2NH-, -NHCH2-, -CH2O-, or -OCH2-; and Each Q C is, independently, absent, optionally substituted C 1-12 alkylene, optionally substituted C 2-12 alkenylene, optionally substituted C 2-12 alkynylene, optionally substituted C 2-12 heteroalkylene, optionally substituted C 1-9 heterocyclylene, or -P(Z)(OH)-, where Z is O or S; and Q E is absent or a branching group of formula (IV):

Chemical formula

[0450] In formula (IV), when p1 is 1, Q G is absent; when p1 is 2 or 3, at least one Q G ​exists.

[0451] In certain embodiments, Z 1 exists and Q C is attached to the internucleoside or terminal phosphate, internucleoside or terminal phosphorothioate, internucleoside or terminal phosphorodithioate, abasic spacer, capping group, or nucleobase via.

[0452] In certain embodiments, Q B , Q C , Q D , and Q E at least one of which is present in the divalent segment (e.g., at least one Q C is present, Q E is present, or Q E is absent). In certain embodiments, each Q B and each Q D is independently absent, -CO-, -NH-, -O-, -S-, -SO2-, -NHC(O)-, -C(O)NH-, -CH2-, -CH2NH-, -NHCH2-, -CH2O-, or -OCH2-.

[0453] In some embodiments, -(-Q B -Q C -Q D -) s1 - combines to form the group: -Q B -(CH2) g1 -(CH2OCH2) g2 -(CH2) g3 -Q D - forming where (i) g2 is an integer from 1 to 50; (ii) g1 is 1 and Q B is -NHCO-, -CONH-, or -O-; or g1 is 0 and Q D is -NHCO-; and (iii) g3 is 1 and Q Bis -NHCO-, -CONH-, or -O-; or g3 is 0, Q D is -CONH-.

[0454] The conjugation moiety may be protected until the auxiliary moiety is conjugated to the polynucleotide. For example, protected conjugation moieties include -COOR PGO or -NHR PGN where R PGO is an O-protecting group (e.g., a carboxyl protecting group), and R PGN is an N-protecting group.

[0455] In a further embodiment, Link A is

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chem.

Chem.

[0456] In yet a further embodiment, the linker A is

Chemical formula

Chem.

Chem.

Chem.

Chem.

Chem.

[0457] In yet a further embodiment, Link A is

Chemical formula

Chemical formula

Chemical formula

[0458] In some embodiments, q5 is 0. In other embodiments, q5 is an integer from 2 to 6.

[0459] In certain embodiments, the conjugated group is:

Chemical formula

Chemical formula

Chemical formula

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

[0460] In some embodiments, the conjugate group is:

Chemical formula

Chemical formula

Chemical formula

[0461] In certain embodiments, the conjugate group is:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0462] In yet a further embodiment, the conjugate group is: [Chemical formula] wherein here R P is a bond to a nucleoside backbone group, nucleobase, capping group, or abasic spacer; Q P is -C(O)-N(H)-, -N(H)-C(O)-, -S(O)2-N(H)-, or -N(H)-S(O)2-; each Q S is independently optionally substituted C 2-12 alkylene, optionally substituted C 2-12 alkenylene, optionally substituted C 2-12 alkynylene, or optionally substituted (C 6-10 aryl)-C 1-6 -alkylene; each of q1 and q3 is independently 0 or 1; q2 is an integer from 0 to 50 (e.g., 1 to 40 or 1 to 30); q4 is an integer from 0 to 10; and q5 is an integer from 1 to 10 (e.g., 1 to 6).

[0463] In one exemplary embodiment, the conjugate group is: [Chemical formula] TIFF0007712765000074.tif214170TIFF0007712765000075.tif231170TIFF0007712765000076.tif72170wherein here, q2 is an integer from 1 to 50 (e.g., 1 to 24 or 1 to 8 (e.g., 2 or 3) integer), q4 is an integer from 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, or 8), q10 is an integer from 0 to 8 (e.g., 1, 2, 3, 4, 5, or 6), q11 is 0 or 1, Z is O or S, and each R M is independently H, a moiety, -(CH2)q7 -CO-N(R M1 )2, or -C[-CH2O-(CH2) q7 -CO-N(R M1 )2]3, where each q7 is, independently, an integer from 1 to 5, and each R M1 is, independently, H or an auxiliary moiety.

[0464] The following exemplary conjugated groups can be used for conjugation with a targeting moiety via metal-catalyzed addition cyclization:

Chemical Structure

[0465] The following exemplary conjugated groups can be used for conjugation with a targeting moiety via metal-free addition cyclization:

Chemical Structure

[0466] The following exemplary conjugate groups can be used for conjugation to a targeting moiety via amide formation: [Chemical formula] (where q2 is an integer from 0 to 50 (e.g., an integer from 1 to 8 (e.g., 2 or 3)), and q12 is an integer from 1 to 11 (e.g., an integer from 1 to 5 (e.g., an integer of 1, 2, 3, 4, or 5))).

[0467] (Bioreversible group) The bioreversible group is a monovalent substituent having a molecular weight of 135 Da to 10 kDa (e.g., 135 Da to 5 kDa, 200 Da to 5 kDa, or 200 Da to 2 kDa) and containing a disulfide (-S-S-). In the bioreversible group, the shortest chain of atoms that covalently share a valence with the disulfide of the bioreversible group is 2 to 10 atoms (e.g., 2 to 6 atoms or 4 to 6 atoms (e.g., 4 or 5 atoms)). The bioreversible group is cleavable intracellularly under physiological conditions.

[0468] Including the bioreversible group in the phosphoester can, for example, reduce the overall negative charge of the immunomodulatory polynucleotide of the present invention. The reduction of the overall negative charge of the immunomodulatory polynucleotide can enhance the cellular uptake of the immunomodulatory polynucleotide and / or conjugate of the present invention. The immunomodulatory polynucleotide of the present invention can include one or more bioreversible groups in the phosphoester and / or abasic spacer. In some embodiments, the immunomodulatory polynucleotide of the present invention can include 1 to 6 bioreversible groups (e.g., 1 to 4 bioreversible groups (e.g., 1, 2, or 3 bioreversible groups)).

[0469] The bioreversible group can be of formula (XXII): R 5-S-S-(Link B)-, (XXII) (wherein Link B is a divalent group containing a sp 3 hybrid carbon atom bonded to a phosphate, phosphorothioate, or phosphorodithioate and a carbon atom bonded to -S-S-, and R 5 is optionally substituted C 1-6 alkyl, optionally substituted C 6-10 aryl, or -Link C(-R M ) r or Link B is a trivalent linker containing a sp 3 hybrid carbon atom bonded to a phosphate, phosphorothioate, or phosphorodithioate and a carbon atom bonded to -S-S-, wherein the third valence of Link B combines with -S-S- and R 5 to form an optionally substituted C 3-9 heterocyclylene; Link C is a polyvalent group; each R M is independently H, an auxiliary moiety, or -Q G [(-Q B -Q C -Q D ) s2 -R M1 p1 and wherein each R M1 is independently H or an auxiliary moiety, each Q B and each Q D are independently absent, -CO-, -NH-, -O-, -S-, -SO2-, -OC(O)-, -COO-, -NHC(O)-, -C(O)NH-, -CH2-, -CH2NH-, -NHCH2-, -CH2O-, or -OCH2-, each Q C is independently absent, optionally substituted C 1-12 alkylene, optionally substituted C 2-12 alkenylene, optionally substituted C 2-12 alkynylene, optionally substituted C 2-12 ​A heteroalkylene, or an optionally substituted C 1-9 heterocyclylene, wherein each Q G is independently an optionally substituted C 1-6 alkanetriyl, an optionally substituted C 1-6 alkanetetrayl, an optionally substituted C 2-6 heteroalkanetriyl, or an optionally substituted C 2-6 heteroalkanetetrayl, wherein each s2 is independently an integer from 0 to 10, and p1 is 2 or 3; and r is an integer from 1 to 6 (e.g., 1, 2, or 3).

[0470] In certain embodiments, Link B and / or R 5 comprises a bulky group attached to -S-S-. Inclusion of a bulky group attached to -S-S- can enhance, for example, the stability of sulfur-sulfur bonds during polynucleotide synthesis.

[0471] In further embodiments, Link B consists of 1, 2, or 3 groups, each of which is independently an optionally substituted C 1-12 alkylene, an optionally substituted C 2-12 alkenylene, an optionally substituted C 2-12 alkynylene, an optionally substituted C 6-10 arylene, an optionally substituted C 2-12 heteroalkylene, and an optionally substituted C 1-9 heterocyclylene selected from the group consisting of.

[0472] In certain embodiments, Link B and -S-S- combine to form:

Chemical formula

Chemical formula

[0473] In yet a further embodiment, the link C can include from 0 to 3 polyvalent monomers (e.g., optionally substituted C 1-6 alkane-triyl, optionally substituted C 1-6 alkane-tetrayl, or a trivalent nitrogen atom) and one or more divalent monomers (e.g., from 1 to 40), where each divalent monomer is independently optionally substituted C 1-6 alkylene; optionally substituted C 2-6 alkenylene; optionally substituted C 2-6 alkynylene; optionally substituted C 3-8 cycloalkylene; optionally substituted C 3-8 cycloalkenylene; optionally substituted C 6-14 arylene; optionally substituted C having from 1 to 4 heteroatoms selected from N, O, and S 1-9 heteroarylene; optionally substituted C having from 1 to 4 heteroatoms selected from N, O, and S 1-9 heterocyclylene; imino; optionally substituted N; O; or S(O) m (where m is 0, 1, or 2). In some embodiments, each monomer is independently optionally substituted C 1-6 alkylene; optionally substituted C 3-8 cycloalkylene; optionally substituted C 3-8 cycloalkenylene; optionally substituted C 6-14 arylene; optionally substituted C having from 1 to 4 heteroatoms selected from N, O, and S 1-9Heteroarylene; optionally substituted C having 1 to 4 heteroatoms selected from N, O, and S 1-9 Heterocyclylene; imino; optionally substituted N; O; or S(O) m (wherein m is 0, 1, or 2 (e.g., m is 2)). In certain embodiments, each monomer is independently optionally substituted C 1-6 Alkylene; optionally substituted C 3-8 Cycloalkylene; optionally substituted C 3-8 Cycloalkenylene; optionally substituted C 6-14 Arylene; optionally substituted C having 1 to 4 heteroatoms selected from N, O, and S 1-9 Heteroarylene; optionally substituted C having 1 to 4 heteroatoms selected from N, O, and S 1-9 Heterocyclylene; optionally substituted N; O; or S(O) m (wherein m is 0, 1, or 2). The non-cleavable reversible linker that connects the auxiliary moiety to the conjugation moiety or to its reaction product can comprise 2 to 500 (e.g., 2 to 300, 2 to 200, 2 to 100, or 2 to 50) such monomers. Linker C can comprise one or more polyethylene glycols (e.g., the polyethylene glycol can have a molecular weight of 88 Da to 1 kDa (e.g., 88 Da to 500 Da).

[0474] The group -Linker C(-R M ) r Compounds that can be used in the preparation of can be found described herein and in WO 2015 / 188197. -Linker C(-R M ) r Non-limiting examples of include:

Chemical formula

[0475] In certain embodiments, R M is a moiety. In some embodiments, at least one R M1 is a moiety.

[0476] In certain embodiments, the bio-reversible linker group is​ [Chemical formula] wherein one end of the group is connected to a polynucleotide and the other end is connected to a target moiety (in one embodiment, an antibody).

[0477] (abioreversible group) The abioreversible group is a monovalent substituent that does not contain a bond cleavable under physiological conditions, in serum, or within an endosome (e.g., an ester, thioester, or disulfide). The abioreversible group is an optionally substituted C 2-16 alkyl; an optionally substituted C 3-16 alkenyl; an optionally substituted C 3-16 alkynyl; an optionally substituted C 3-8 cycloalkyl; an optionally substituted C 3-8 cycloalkenyl; an optionally substituted (C 3-8 (cycloalkyl)-C 1-4 -alkyl; an optionally substituted (C 3-8 (cycloalkenyl)-C 1-4 -alkyl; an optionally substituted C 6-14 aryl; an optionally substituted (C 6-14 (aryl)-C 1-4 -alkyl; an optionally substituted C having 1 to 4 heteroatoms selected from N, O, and S 1-9 heteroaryl; an optionally substituted (C having 1 to 4 heteroatoms selected from N, O, and S 1-9 (heteroaryl)-C 1-4 -alkyl; an optionally substituted C having 1 to 4 heteroatoms selected from N, O, and S 2-9 heterocyclyl (wherein the heterocyclyl does not contain an S-S bond); an optionally substituted (C having 1 to 4 heteroatoms selected from N, O, and S 2-9 (heterocyclyl)-C 1-4 -alkyl (wherein the heterocyclyl does not contain an S-S bond); or a group of formula (XXIII): [Chemical formula] may also be; wherein, L 3 is C 2-6 alkylene; R 7 is optionally substituted C 2-6 alkyl; optionally substituted C 6-14 aryl; optionally substituted (C 6-14 aryl)-C 1-4 -alkyl; optionally substituted C 3-8 cycloalkyl; optionally substituted (C 3-8 cycloalkyl)-C 1-4 -alkyl; optionally substituted C 1-9 heteroaryl having 1 to 4 heteroatoms selected from the group consisting of N, O, and S; optionally substituted (C 1-9 heteroaryl)-C 1-4 -alkyl; optionally substituted C 2-9 heterocyclyl (wherein the heterocyclyl does not contain an S-S bond); optionally substituted (C 2-9 heterocyclyl)-C 1-4 -alkyl (wherein the heterocyclyl does not contain an S-S bond); and poly(ethylene glycol) terminated with -OH, C 1-6 alkoxy, or -COOH; and R 8 is H or C 1-6 alkyl.

[0478] The non-biodegradable phosphotriester is a conjugate group, C 2-16 alkyl,

Chemical formula

Chemical formula

Chemical formula

[0479] In some embodiments, the abiotic reversibility group is -Link D(-R M1 ) r1 , wherein Link D is a polyvalent linker, each R M1 is independently H or an auxiliary moiety, and r1 is an integer from 1 to 6.

[0480] Optionally, -Link D(-R M1 ) r1 is of formula (XXIV): -Q R -Q 3 ([-Q 4 -Q 5 -Q6 ) r2 -Q 7 -R M1 ) r1 , (XXIV) (wherein r1 is an integer from 1 to 6; each r2 is independently an integer from 0 to 50 (e.g., 0 to 30), where the repeating units are the same or different; Q R is [-Q 4 -Q 5 -Q 6 ) r2 -Q L - and here, Q L is optionally substituted C 2-12 heteroalkylene (e.g., heteroalkylene containing -C(O)-N(H)-, -N(H)-C(O)-, -S(O)2-N(H)-, or -N(H)-S(O)2-), optionally substituted C 1-12 thioheterocyclylene (e.g., [Chemical formula] ), optionally substituted C 1-12 heterocyclylene (e.g., 1,2,3-triazole-1,4-diyl or [Chemical formula] ), cyclobuta-3-ene-1,2-dione-3,4-diyl, pyrido-2-ylhydrazone, optionally substituted C 6-16 triazolocycloheterocyclylene (e.g., [Chemical formula] ), optionally substituted C 8-16 triazolocycloalkenylene (e.g., [Chemical formula] ), or dihydropyridazine group (e.g., [Chemical formula] ) and; Q 3 is, when r1 is 1, a linear group (e.g., [-Q 4 -Q 5 -Q 6 ) or, when r1 is an integer from 2 to 6, a branched group (e.g., [-Q r2 -Q 4 -Q 5 -Q 6 ) s -Q 8 ([-Q 4 -Q 5 -Q 6 ) r2 -(Q 8 ) r3 ) r4 (where r3 is 0 or 1 and r4 is 0, 1, 2, or 3); each r2 is independently an integer from 0 to 50 (e.g., 0 to 30), where the repeating units are the same or different; each Q 4 and each Q 6 is independently absent, -CO-, -NH-, -O-, -S-, -SO2-, -OC(O)-, -COO-, -NHC(O)-, -C(O)NH-, -CH2-, -CH2NH-, -NHCH2-, -CH2O-, or -OCH2-; each Q 5 is independently absent, optionally substituted C 1-12 alkylene, optionally substituted C 2-12 alkenylene, optionally substituted C 2-12 alkynylene, optionally substituted C 2-12 heteroalkylene, or optionally substituted C 1-9 heterocyclylene; each Q 7 is independently absent, -CO-, -NH-, -O-, -S-, -SO2-, -CH2-, -C(O)O-, -OC(O)-, -C(O)NH-, -NH-C(O)-, -NH-CH(R a )-C(O)-, or -C(O)-CH(R a )-NH-; each Q 8 is independently optionally substituted C1-6 alkan-triyl, optionally substituted C 1-6 alkan-tetrayl, optionally substituted C 2-6 heteroalkan-triyl, or optionally substituted C 2-6 heteroalkan-tetrayl; and each R a is, independently, H or an amino acid side chain; and each R M1 is, independently, H or an auxiliary moiety).

[0481] In formula (XXIV), Q 4 , Q 5 , and Q 6 at least one of which is present. In formula (XXIV), the linker D may contain a single branch point when each r3 is 0, or may contain multiple branch points when at least one r3 is 1. In formula (XXIV), Q R is, -Q 5 -Q 4 -Q L - and may be, where Q 5 is optionally substituted C 2-12 heteroalkylene or optionally substituted C 1-12 alkylene, and Q 4 is, -CO-, -NH-, or -O-. In formula (XXIV), Q L is:

Chemical formula

[0482] In formula (XXIV), Q 3 is a linear group of the formula [-Q 4 -Q 5 -Q 6 r2 - and may be, where Q 4 , Q 5 , and Q 6 are as defined for formula (XXIV). Alternatively, Q 3 is a branched group [-Q 4 -Q 5 -Q 6 ​​r2 -Q 8 ([-Q 4 -Q 5 -Q 6 ) r2 -(Q 8 ) r3 ) r4 may also be, wherein each Q 8 is independently an optionally substituted C 1-6 alkanetriyl, an optionally substituted C 1-6 alkanetetrayl, an optionally substituted C 2-6 heteroalkanetriyl, or an optionally substituted C 2-6 heteroalkanetetrayl; wherein each r2 is independently an integer from 0 to 50 (e.g., 0 to 30), wherein the repeating units are the same or different; r3 is 0 or 1; r4 is 0, 1, 2, or 3; wherein when r3 is 0, the linker D is a trivalent or tetravalent group, and when r3 is 1, the linker D is a tetravalent, pentavalent, or hexavalent group.

[0483] In certain embodiments, r3 is 0.

[0484] In some embodiments, Q 8 is: [Chemical formula] is.

[0485] The compounds that can be used in the preparation of the group - linker D(-R M1 ) p in formula (I) are described herein and in WO 2015 / 188197.

[0486] In certain embodiments, the non-biodegradable linker group is [Chemical formula] wherein one end of the group is connected to the polynucleotide and the other end is connected to the target moiety (in one embodiment, an antibody).

[0487] (Auxiliary moiety) The auxiliary moiety is a monovalent group containing a dye or a hydrophilic group or a combination thereof (e.g., a hydrophilic polymer (e.g., poly(ethylene glycol) (PEG)), a positively charged polymer (e.g., poly(ethyleneimine)), or a sugar alcohol (e.g., glucitol)). The auxiliary moiety can have a theoretical molecular weight of 100 Da to 2.5 kDa (e.g., 350 Da to 2.5 kDa, 100 Da to 1,200 Da, or 1 kDa to 2.5 kDa).

[0488] The pigment can be included in the phosphate ester group for visualizing the uptake of the conjugate of the present invention into cells or monitoring the movement (for example, using fluorescence recovery after photobleaching (FRAP)). Pigments known in the art can be included as auxiliary moieties linked to polynucleotides via 5'- or 3'-terminal phosphates or phosphorothioates or via phosphates or phosphorothioates that link two consecutive nucleosides. Non-limiting examples of useful structures that can be used as pigments include FITC, RD1, allophycocyanin (APC), aCFTM pigments (Biotium, Hayward, CA), BODIPY (Invitrogen™ 10, Life Technologies, Carlsbad, CA), AlexaFluor® (Invitrogen™, Life Technologies, Carlsbad, CA), DyLight Fluor (Thermo Scientific Pierce Protein Biology Products, Rockford, IL), ATTO (ATTO-TEC GmbH, Siegen, Germany), FluoProbe (Interchim SA, Motlucon, France), and Abberior Probes (Abberior GmbH, Gottingen, Germany).

[0489] Hydrophilic polymers and positively charged polymers that can be used as auxiliary moieties in the immunomodulatory polynucleotides of the present invention and in the conjugates of the present invention are known in the art. Non-limiting examples of hydrophilic polymers are poly(ethylene glycol). Non-limiting examples of positively charged polymers are poly(ethylene imine).

[0490] The sugar alcohol-based auxiliary moiety can be, for example, an amino-terminal glucitol or a glucitol cluster. The amino-terminal glucitol auxiliary moiety is:

Chemical formula

Chemical formula

[0491] In one embodiment, provided herein is a compound of formula (B): or a stereoisomer, a mixture of two or more diastereomers, a tautomer, or a mixture of two or more tautomers; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; R X -L N -(Q) e (B) (wherein: R x is a conjugated group; L N is a linker; each Q is independently an oligonucleotide containing a phosphotriester; and e is an integer of 1, 2, 3, or 4).

[0492] In certain embodiments, in formula (B), R x is

Chemical formula

[0493] In certain embodiments, in formula (B), L N is a linker containing polyethylene glycol.

[0494] In certain embodiments, in formula (B), L N is

Chemical formula

[0495] In certain embodiments, in formula (B), e is an integer of 1. In certain embodiments, in formula (B), each Q independently has the structure of formula (D):

Chemical formula

[0496] X N 、X 3' 、X 5' 、Y P 、b, and c are each as defined herein.

[0497] (Targeting moiety) Using the targeting moiety used in the conjugate of the present invention, specific cells and tissues in the body can be targeted for the targeted delivery of the conjugated payload polynucleotide. The cells targeted by the conjugate of the present invention are professional APCs (e.g., B cells, pDCs, or macrophages). The targeting moiety can be a moiety comprising an antigen-binding portion (e.g., an antibody or an antigen-binding fragment thereof), a polypeptide, an aptamer, or one or more small molecules (e.g., mannose). The targeting moiety in the conjugate of the present invention can be effective in addressing the problem of non-uniform distribution of immunomodulatory polynucleotides in vivo.

[0498] (Antigen-binding portion) The antigen-binding portion in the conjugate of the present invention can be an antibody or an antigen-binding fragment thereof (e.g., F(ab)2 or Fab) or a modified derivative thereof (e.g., Fcab or a fusion protein (e.g., scFv)). Human or chimeric (e.g., humanized) antibodies can be used as the antibody in the conjugate of the present invention.

[0499] The antigen-binding portion targets cells having a surface antigen recognized by the antigen-binding portion. In particular, APCs can be targeted by the antigen-binding portion in the conjugates of the present invention. B cells can be targeted by anti-CD38, anti-CD79b, anti-CD30, anti-CD22, or anti-CD20, anti-CD19 antibodies, or antigen-binding fragments thereof, or modified derivatives thereof. Plasmacytoid dendritic cells (pDCs) can be targeted by anti-DEC205, anti-CD304, anti-CD303, anti-CD40, anti-CD74, anti-BDCA2, or anti-CD123 antibodies, or antigen-binding fragments thereof, or modified derivatives thereof. Macrophages can be targeted by anti-CD163, anti-CD40, anti-CD74, anti-CD206, or anti-CD123 antibodies, or antigen-binding fragments thereof, or modified derivatives thereof.

[0500] Non-limiting examples of anti-CD38 antibodies are daratumumab, SAR650984, MOR202, or any one of the antibodies Ab79, Ab19, Ab43, Ab72, and Ab110 disclosed in WO 2012 / 092616, the disclosures of which are incorporated by reference. A non-limiting example of an anti-CD79b antibody is huMA79b v28 disclosed in WO 2014 / 011521. A non-limiting example of an anti-CD22 antibody is 10F4 disclosed in US 2014 / 0127197. A non-limiting example of an anti-CD20 antibody is rituximab. A non-limiting example of an anti-DEC205 antibody is provided in US 2010 / 0098704, the antibody of which is incorporated herein by reference. Non-limiting examples of anti-CD40 antibodies are lucatumumab and dacetuzumab. A non-limiting example of an anti-CD304 antibody is besencumab.

[0501] (polypeptide) The targeting moiety can be a polypeptide having an affinity for cells (e.g., having an affinity for a cell type, e.g., a plasmacytoid cell). Non-limiting examples of polypeptides are RGD peptides, rabies virus glycoprotein (RVG), and DC3 peptides.

[0502] (Small molecule) The targeting moiety can be a small molecule capable of complexing with a receptor expressed on the surface of a target cell. Non-limiting examples of small molecules that can be used as the targeting moiety in the conjugates of the present invention are folic acid, mannose, PSMA ligand, and mannose clusters.

[0503] Folic acid can be used as the targeting moiety. In the conjugates of the present invention, folic acid can have the following structure: [Chemical Structure] .

[0504] The conjugates of the present invention can be targeted to plasmacytoid dendritic cells and macrophages using mannose or mannose clusters, because these cells express mannose receptors on their surface.

[0505] Mannose clusters are known in the art. The mannose auxiliary moiety (e.g., mannose cluster) can have the formula (XXV): -(-Q M1 -Q M2 -Q M3 -) s3 -Q M4 [(-Q M1 -Q M2 -Q M3 -) s3 -Q M5 p3 , (XXV) (wherein, p3 is 1, 2, or 3; each s3 is independently an integer from 0 to 50 (e.g., 0 to 30); each Q M1 and each Q M3 is independently absent, -CO-, -NH-, -O-, -S-, -SO2-, -OC(O)-, -COO-, -NHC(O)-, -C(O)NH-, -CH2-, -CH2NH-, -NHCH2-, -CH2O-, or -OCH2-; and​ each Q M2 is, independently, absent, optionally substituted C 1-12 alkylene, optionally substituted C 2-12 alkenylene, optionally substituted C 2-12 alkynylene, optionally substituted C 2-12 heteroalkylene, or optionally substituted C 1-9 heterocyclylene; Q M4 is, when absent (where p3 is 1), optionally substituted C 1-6 alkanetriyl (where p3 is 2), optionally substituted C 1-6 alkanetetrayl (where p3 is 3), optionally substituted C 2-6 heteroalkanetriyl (where p3 is 2), or optionally substituted C 2-6 heteroalkanetetrayl (where p3 is 3); each Q M5 is, independently, mannose or -Q M6 [(-Q M1 -Q M2 -Q M3 ) s2 -R M2 p1 and wherein each R M2 is, independently, mannose; and each Q M6 when present, is, independently, optionally substituted C 1-6 alkanetriyl, optionally substituted C 1-6 alkanetetrayl, optionally substituted C 2-6 heteroalkanetriyl, or optionally substituted C 2-6 heteroalkanetetrayl).

[0506] Non-limiting examples of mannose clusters are:

Chemical Structure

[0507] (Conjugate) In one embodiment, provided herein is a conjugate of formula (C): or a stereoisomer thereof, a mixture of two or more diastereomers, a tautomer, or a mixture of two or more tautomers; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; [Chemical formula] (wherein Ab is a targeting moiety; f is an integer of 1, 2, 3, or 4; and L N , Q, and e are each as defined herein).

[0508] In certain embodiments, in formula (C), Ab is an antibody. In certain embodiments, in formula (C), Ab is a monoclonal antibody.

[0509] In certain embodiments, in formula (C), f is an integer of 1 or 2. In certain embodiments, in formula (C), f is an integer of 1.

[0510] In certain embodiments, in formula (C), both e and f are each an integer of 1. As used herein, the term "DAR" refers to the drug - antibody ratio of a CpG antibody conjugate, more specifically, the polynucleotide - antibody ratio. In one embodiment, the CpG antibody conjugate has a DAR in the range of about 1 to about 20, about 1 to about 10, about 1 to about 8, about 1 to about 4, or about 1 to about 2. In another embodiment, the CpG antibody conjugate has a DAR of about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8.

[0511] (Preparation of the Conjugate) (Conjugation) Reactions useful for conjugating a targeting moiety to one or more immunomodulatory polynucleotides are described herein and are known in the art (e.g., bioorthogonal reactions). Exemplary reactions that can be used to form this linkage include azide and alkyne-based conjugate groups for forming a triazole moiety (e.g., an optionally substituted C containing an internal carbon-carbon triple bond 6-16 heterocyclylene or an optionally substituted C 8-16 cycloalkynyl); Huisgen cycloaddition (metal-catalyzed or metal-free) between an azide and an alkyne-based conjugate group; Diels-Alder reaction between a dienophile and a diene / hetero-diene; other pericyclic reactions, e.g., bond formation by an ene reaction; amide or thioamide bond formation; sulfonamide bond formation (e.g., by an azide compound); alcohol or phenol alkylation (e.g., Williamson alkylation), condensation reactions for forming an oxime, hydrazone, or semicarbazide group; conjugate addition reactions by nucleophiles (e.g., amines and thiols); disulfide bond formation; and nucleophilic substitution in a carbonyl (e.g., an activated carboxylic acid and ester, e.g., a pentafluorophenyl (PFP) ester or a tetrafluorophenyl (TFP) ester) or an electrophilic arene (e.g., an oligo-fluorinated arene, a fluorobenzonitrile group, or a fluoronitrobenzene group S N Ar) (e.g., by an amine, thiol, or hydroxyl nucleophile). In some embodiments, the conjugation reaction is a dipolar cycloaddition and the conjugation moiety comprises an azide, an optionally substituted C containing an internal carbon-carbon triple bond 6-16 heterocyclylene, or an optionally substituted C 8-16 cycloalkynyl. Complementary reactive groups and conjugate groups are selected for their mutual complementarity. For example, an azide can be used as one of the conjugate group and the complementary reactive group, and simultaneously, an alkyne can be used as the other of the conjugate group and the complementary reactive group.

[0512] (Nucleophilic / electrophilic reaction) Nucleophilic and electrophilic reagents can be involved in bond-forming reactions selected from, but not limited to, insertion of an electrophilic reagent into a C-H bond, insertion of an electrophilic reagent into an O-H bond, insertion of an electrophilic reagent into an N-H bond, addition of an electrophilic reagent across an alkene, addition of an electrophilic reagent across an alkyne, addition to an electrophilic carbonyl center, substitution at an electrophilic carbonyl center, addition to a ketene, nucleophilic addition to an isocyanate, nucleophilic addition to an isothiocyanate, nucleophilic substitution at an electrophilic silyl group, nucleophilic substitution of a leaving group (e.g., a halide or pseudohalide) in an alkyl halide or pseudohalide; nucleophilic addition / elimination at the carbonyl of an activated carboxylic acid ester (e.g., a PFP ester or TFP ester), thioester, anhydride, or acyl halide; 1,4-conjugate addition of a nucleophilic reagent to an α,β-unsaturated carbonyl group, nucleophilic ring-opening of an epoxide, aromatic nucleophilic substitution of an electron-deficient aromatic compound, nucleophilic addition to an activated phosphorus center, nucleophilic substitution at an activated phosphorus center, nucleophilic addition to an activated sulfur center, and nucleophilic substitution at an activated sulfur center.

[0513] The nucleophilic conjugate group can be an optionally substituted alkene, optionally substituted alkyne, optionally substituted aryl, optionally substituted heterocyclyl, hydroxyl, amino, alkylamino, anilide, or thio.

[0514] The electrophilic conjugate group can be an azide, activated carbonyl (e.g., an activated carboxylic acid ester (e.g., a succinimidyl ester or sulfosuccinimidyl ester), thioester, anhydride, or acyl halide), isocyanate, thioisocyanate, Michael acceptor (e.g., maleimide), alkyl halide or pseudohalide, epoxide, episulfide, aziridine, or electron-deficient aryl.

[0515] For example, conjugation can occur via a condensation reaction to form a linkage that is a hydrazone bond.

[0516] Conjugation can involve, for example, activation of a carboxyl-based conjugate group (e.g., carboxylic acid, ester, or -CONH2) and subsequent formation of an amide bond by reaction with a primary amine in the conjugate group. The activator can be various carbodiimide-like ones: EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), EDAC (1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride), DCC (dicyclohexylcarbodiimide), CMC (1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide), DIC (diisopropylcarbodiimide), or Woodward's reagent K (N-ethyl-3-phenylisoxazolium-3'-sulfonate). Activation of a carboxyl-based conjugate group that is -CONH2 can be achieved using transglutaminase. The reaction of an activated NHS-ester-based conjugate group with a primary amine-based conjugate group also results in the formation of an amide bond.

[0517] Polynucleotides can contain a carbonyl-based conjugate group. Conjugation with concomitant formation of a secondary amine can be achieved by reductive amination (i.e., reacting an amine-based conjugate group with an aldehyde-based conjugate group and then reducing with a hydride donor such as sodium cyanoborohydride or sodium triacetoxyborohydride).

[0518] Using ether formation, a targeting moiety can be conjugated to one or more polynucleotides to form the conjugate of the present invention. Ether linkage formation can involve the reaction of an epoxide-based conjugate group with a hydroxy-based conjugate group.

[0519] Thiols can also be used as the conjugate group. For example, conjugation by the formation of a disulfide bond can be achieved by pyridyldisulfide-mediated thiol-disulfide exchange. The introduction of a sulfhydryl-based conjugate group can be mediated by, for example, Traut's reagent (2-iminothiolane), SATA (N-succinimidyl S-acetylthioacetate), SATP (succinimidyl acetylthiopropionate), SPDP (N-succinimidyl 3-(2-pyridyldithio)propionate), SMPT (succinimidyl oxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene), N-acetylhomocysteine thiolactone, SAMSA (S-acetylmercaptosuccinic anhydride), AMBH (2-acetamido-4-mercaptobutyric acid hydrazide), and cystamine (2,2'-dithiobis(ethylamine).

[0520] Thioether linkage formation can be carried out by reacting a sulfhydryl-based conjugate group with a maleimide or iodoacetyl-based conjugate group or with an epoxide-based conjugate group.

[0521] Maleimide-based conjugate groups can be introduced by SMCC (succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate), MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester), sulfo-MBS (m-maleimidobenzoyl-N-sulfohydroxysuccinimide ester), SMPB (succinimidyl-4-(p-maleimidophenyl)butyrate), sulfo-SMPB (sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate), GMBS (N-α-maleimidobutyryl-oxysuccinimide ester), sulfoGMBS (N-α-maleimidobutyryl-oxysulfosuccinimide ester).

[0522] Conjugation by the formation of a carbamate linkage can be carried out by reacting a hydroxy-based conjugate group with CDI (N,N'-carbonyldiimidazole) or DSC (N,N'-disuccinimidyl carbonate) or N-hydroxysuccinimidyl chloroformate and then reacting with an amine-based conjugate group.

[0523] (Addition cyclization reaction) The desired covalent bond can be formed using an addition cyclization reaction. Representative addition cyclization reactions include the reaction of an alkene-based conjugate group with a 1,3-diene-based conjugate group (Diels–Alder reaction), the reaction of an alkene-based conjugate group with an α,β-unsaturated carbonyl-based conjugate group (hetero-Diels–Alder reaction), and the reaction of an alkyne-based conjugate group with an azide-based conjugate group to obtain a triazole moiety (Huisgen addition cyclization, including its metal-catalyzed and metal-free modifications), but are not limited thereto. Non-limiting examples of conjugate groups containing reactants for the addition cyclization reaction are: alkenes, alkynes, 1,3-dienes, α,β-unsaturated carbonyls, and azides. For example, the Huisgen addition cyclization (click reaction) between an azide and an alkyne has been used for the functionalization of various biological entities.

[0524] A strained alkyne-based conjugate group is a carbocyclic or heterocyclic ring system containing one internal carbon-carbon triple bond (e.g., an optionally substituted C 6-16 heterocyclylene or an optionally substituted C 8-16 cycloalkynyl). Strained alkyne-based conjugate groups can be useful for conjugating a targeting moiety to a polynucleotide via a metal-free dipolar addition cyclization with an azide conjugate group.

[0525] (Coupling reaction) Conjugate groups can include, but are not limited to, reactants for hydrosilylation, olefin cross-metathesis, conjugate addition, Stille coupling, Suzuki coupling, Sonogashira coupling, Hiyama coupling, and Heck reaction. Conjugation moieties for these reactions include hydridosilanes, alkenes (e.g., activated alkenes, such as enones or enoates), alkynes, aryl halides, aryl pseudohalides (e.g., triflates or nonaflates), alkyl halides, and alkyl pseudohalides (e.g., triflates, nonaflates, and phosphates). Catalysts for cross-coupling reactions are well known in the art. Such catalysts can be organometallic complexes or metal salts (e.g., Pd(0), Pd(II), Pt(0), Pt(II), Pt(IV), Cu(I), or Ru(II)). Additives such as ligands (e.g., PPh3, PCy3, BINAP, dppe, dppf, SIMes, or SIPr) and metal salts (e.g., LiCl) may be added to facilitate the cross-coupling reaction.

[0526] (Preparation of immunomodulatory polynucleotides) The immunomodulatory polynucleotide can be prepared, for example, according to a method known in the art for chemically synthesizing polynucleotides from nucleoside phosphoramidites.Non-limiting examples of the synthesis of nucleoside phosphoramidites and immunomodulatory polynucleotides are provided in the Examples.The phosphoramidites can include a conjugated group covalently bonded to the P atom of the phosphoramidite.

[0527] Preparation of the Targeting Moiety The targeting moiety can be conjuga...

Claims

Claim 1 An oligonucleotide of formula (A): or a stereoisomer thereof, a mixture of two or more diastereomers, a tautomer, or a mixture of two or more tautomers; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; X 5' -(X N ) b -Y P -(X N ) c -X 3' (A) (wherein: Each X N is, independently, a nucleotide; X 3' is the 3'-terminal nucleotide; X 5' is the 5'-terminal nucleotide; Y P is a phosphate, phosphorothioate, or phosphorodithioate, all three valences of which are substituted with non-hydrogen substituents and two of which are covalently bonded to a nucleoside-containing group; and b and c are each an integer in the range of 0 to 25; provided that the sum thereof is 5 or more; Here, X 5’ contains 5-halo-2'-deoxyuridine; and wherein the oligonucleotide contains 1, 2, or 3 CG dinucleotides). Claim 2 The oligonucleotide according to claim 1, comprising 2 or 3 CG dinucleotides. Claim 3 (i) b is an integer in the range of 1 to 15, or b is an integer of 2, 3, 4, 11, or 14, (ii) c is an integer in the range of 0 to 10, or c is an integer of 0 or 8, and / or (iii) the sum of b and c is in the range of 5 to 20, or the sum of b and c is 8, 9, 10, 11, 12, 13, or 14, The oligonucleotide according to claim 1. Claim 4 (i) each X N is (a) Independently, 2'-deoxyribonucleotide, or (b) Independently, comprising 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxycytidine, 5-halo-2'-deoxycytidine, 2'-deoxythymidine, 2'-deoxyuridine, or 5-halo-2'-deoxyuridine; (ii) X 3' is (a) 2'-deoxyribonucleotide, (b) 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxycytidine, 5-halo-2'-deoxycytidine, 2'-deoxythymidine, 2'-deoxyuridine, or 5-halo-2'-deoxyuridine, (c) 2'-modified ribonucleotide, or (d) Comprising 2'-methoxyribonucleotide or 2'-ethoxymethoxyribonucleotide; and / or (iii) X 5' The oligonucleotide according to claim 1 or 3, wherein X comprises 5-bromo-2'-deoxyuridine or 5-iodo-2'-deoxyuridine. Claim 5 X 5' The oligonucleotide according to any one of claims 1 to 4, wherein X is a 3'-phosphorothioate group. Claim 6 The oligonucleotide according to claim 5, wherein the 3'-phosphorothioate group is chiral. Claim 7 X 5' comprises a 3'-phosphorothioate group having the chirality of Rp and X 3' is a 2'-methoxyribonucleotide or a 2'-ethoxymethoxyribonucleotide, or X 5' comprises a 3'-phosphorothioate group having the chirality of Sp, and X 3' is a 2'-methoxyribonucleotide or a 2'-ethoxymethoxyribonucleotide, the oligonucleotide according to claim 5 or 6. Claim 8 Y P is: (i) 【Chemical Formula 1】 wherein Z is O or S; and d is an integer in the range of 0 to 50); or (ii) 【Chemical 2】 wherein Z is O or S; and d is an integer in the range of 0 to 50), The oligonucleotide according to any one of claims 1 to 7. Claim 9 The oligonucleotide according to any one of claims 1 to 8, further comprising an additional internucleoside phosphate, phosphorothioate, or phosphorodithioate. Claim 10 The oligonucleotide according to claim 9, wherein the additional internucleoside phosphate, phosphorothioate, or phosphorodithioate is an alkyl phosphate, alkyl phosphorothioate, alkyl phosphorodithioate, ethyl phosphate, ethyl phosphorothioate, or ethyl phosphorodithioate.

11. The oligonucleotide according to any one of claims 1 to 10, comprising one or more internucleoside phosphorothioates.

12. A conjugate comprising the oligonucleotide according to any one of claims 1 to 11 and an antibody, wherein the antibody specifically binds to a tumor-associated antigen (TAA).

13. The conjugate according to claim 12, wherein the TAA is Her2 or CD22.

14. A compound of formula (B): or a stereoisomer thereof, a mixture of two or more diastereomers, a tautomer, or a mixture of two or more tautomers; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; R X -L N -(Q) e (B) (wherein: R X is a conjugate base; L N is a linker; each Q is independently an oligonucleotide according to any one of claims 1 to 11; and e is an integer of 1, 2, 3, or 4).

15. Said L N The compound according to claim 14, wherein the linker of N contains polyethylene glycol (PEG) and e is 1.

16. Y P is: 【Chemical Formula 3】 is (wherein: Z is O or S; and d is an integer in the range of 0 to 50), the compound according to claim 15.

17. The compound according to claim 16, wherein Z is S.

18. The compound according to claim 16, wherein d is 1.

19. The compound according to claim 15, wherein the PEG contains a total of at least 20 and a total of 30 or less ethylene glycol repeating units.

20. The compound according to claim 19, wherein the PEG contains a total of 25 ethylene glycol repeating units.

21. The compound according to claim 19, wherein the PEG contains a total of 24 ethylene glycol repeating units.

22. The compound according to claim 19, wherein the PEG contains a total of 23 ethylene glycol repeating units.

23. The compound according to claim 15, wherein b is an integer in the range of 1 to 15, and the sum of b and c is an integer in the range of 5 to 15.

24. The compound according to claim 23, wherein the sum of b and c is 11.

25. The compound according to claim 23, wherein the sum of b and c is 12.

26. R X A compound according to any one of claims 14 to 25, wherein R is conjugated to an antibody.

27. Compound of formula (C); or a stereoisomer thereof, a mixture of two or more diastereomers, a tautomer, or a mixture of two or more tautomers; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; 【Chemical Formula 4】 (Wherein: Ab is an antibody; Each L N is, independently, a linker; Each Q is independently an oligonucleotide according to any one of claims 1 to 11; Each e is independently an integer of 1, 2, 3, or 4; and f is an integer of 1, 2, 3, or 4).

28. N 1 N 2 CGN 3 CG(T) x GN 4 CGN 5 The oligonucleotide according to claim 1, comprising the sequence T (Here: x is an integer in the range of 1 to 4; N 1 is absent or is 2'-deoxythymidine; N 2 is a 2'-deoxyribonucleotide having a modified nucleobase; N 3 is 2'-deoxyadenosine or 2'-deoxythymidine; N 4 is 2'-deoxyadenosine or 2'-deoxythymidine; and N 5 is 2'-deoxythymidine).

29. N 5 The oligonucleotide according to claim 28, wherein N is 2'-deoxythymidine and comprises a 3'-phosphate, 3'-phosphorothioate, or 3'-phosphorodithioate.

30. N 3 The oligonucleotide according to claim 28, wherein the 2'-deoxyadenosine or 2'-deoxythymidine comprises a 3'-phosphate, 3'-phosphorothioate, or 3'-phosphorodithioate.

31. An immunomodulatory polynucleotide comprising an oligonucleotide according to any one of claims 1 to 11.

32. A conjugate comprising an antibody or an antigen-binding fragment thereof and one or more immunomodulatory polynucleotides, wherein the antibody or an antigen-binding fragment thereof comprises a Q-tag containing a glutamine residue, the Q-tag is disposed at the C-terminus of the heavy or light chain of the antibody or an antigen-binding fragment thereof, and each of the immunomodulatory polynucleotides is independently covalently bound to a linker covalently bound to the glutamine residue of the Q-tag, comprising an oligonucleotide according to any one of claims 1 to 11, said conjugate.

33. A conjugate comprising a targeting moiety and one or more immunomodulatory polynucleotides, wherein each of the immunomodulatory polynucleotides comprises an oligonucleotide according to any one of claims 1 to 11 and independently a linker, wherein the targeting moiety is covalently bound to the linker, said conjugate.

34. A conjugate comprising a targeting moiety, one or more polyethylene glycol moieties, and one or more immunomodulatory polynucleotides, wherein each of the immunomodulatory polynucleotides independently comprises an oligonucleotide according to any one of claims 1 to 11 and a linker bound to the targeting moiety, said conjugate.

35. Use of a conjugate in the manufacture of a medicament for treating cancer in a subject in need thereof, wherein the conjugate comprises an oligonucleotide according to any one of claims 1 to 11 and an antibody or an antigen-binding fragment thereof, and (i) the conjugate does not bind to a tumor-associated antigen (TAA); (ii) the conjugate specifically binds to a tumor-associated antigen (TAA), where the TAA is not an antigen selected from the group consisting of CD19, CD20, CD22, STAT3, exportin 7, Her2, Src, EGFR, CD52, CXCR-4, Muc-1, and DNA; or (iii) the cancer is an immune therapy-resistant or refractory cancer, such use. **Claim 36** Use of an oligonucleotide according to any one of claims 1 to 11, or 28 to 30, a compound according to any one of claims 14 to 27, an immunomodulatory polynucleotide according to claim 31, or a conjugate according to any one of claims 12, 13 or 32 to 34 in the manufacture of a medicament for treating cancer in a subject in need thereof. **Claim 37** Use of a conjugate in the manufacture of a medicament for preventing cancer in a subject in need thereof, where the conjugate comprises an oligonucleotide according to any one of claims 1 to 11 and an antibody or an antigen-binding fragment thereof, and (i) the conjugate specifically binds to immune cells expressing at least one toll-like receptor; and / or (ii) the medicament is for co-administration with a cancer vaccine, and the conjugate specifically binds to immune cells expressing at least one toll-like receptor, such use. **Claim 38** Use of a conjugate in the manufacture of a medicament for inducing an adaptive immune response in a subject, where the conjugate comprises an oligonucleotide according to any one of claims 1 to 11 and an antibody or an antigen-binding fragment thereof, and where the conjugate specifically binds to immune cells expressing at least one toll-like receptor. **Claim 39** A pharmaceutical composition for use in the regulation of an endosomal toll-like receptor in a cell comprising the endosomal toll-like receptor, wherein the use comprises contacting the cell with an oligonucleotide according to any one of claims 1 to 11 or 28 to 30, a compound according to any one of claims 14 to 27, an immunomodulatory polynucleotide according to claim 31, or a conjugate according to any one of claims 12, 13 or 32 to 34, under conditions that allow the oligonucleotide, compound, immunomodulatory polynucleotide, or conjugate to be transported into the cell, wherein after the contacting, the activity of the endosomal toll-like receptor is regulated, said pharmaceutical composition.

40. A pharmaceutical composition for use in the induction of one or more cytokines in an antigen-presenting cell comprising the endosomal toll-like receptor, wherein the use comprises contacting the antigen-presenting cell with an oligonucleotide according to any one of claims 1 to 11 or 28 to 30, a compound according to any one of claims 14 to 27, an immunomodulatory polynucleotide according to claim 31, or a conjugate according to any one of claims 12, 13 or 32, under conditions that allow the oligonucleotide, compound, one or more immunomodulatory polynucleotides, or conjugate to be transported into the cell, wherein after the contacting, the level of at least one cytokine in the cell is increased, said pharmaceutical composition.

41. A pharmaceutical composition for the induction of one or more cytokines in an antigen-presenting cell comprising the endosomal toll-like receptor, wherein the induction comprises contacting the antigen-presenting cell with a conjugate according to claim 33 or 34, under conditions that allow the conjugate to be transported into the cell, wherein after the contacting, the level of at least one cytokine in the cell is increased; wherein the targeting moiety targets the antigen-presenting cell; and wherein the immunomodulatory polynucleotide is an immunostimulatory polynucleotide, said pharmaceutical composition.

42. A pharmaceutical composition comprising a conjugate for use in the treatment of cancer in a subject in need thereof, wherein the conjugate comprises an oligonucleotide according to any one of claims 1 to 11 and an antibody or antigen-binding fragment thereof, and (i) The conjugate does not bind to a tumor-associated antigen (TAA); (ii) The conjugate specifically binds to a tumor-associated antigen (TAA), where the TAA is not an antigen selected from the group consisting of CD19, CD20, CD22, STAT3, exportin 7, Her2, Src, EGFR, CD52, CXCR-4, Muc-1, and DNA; or (iii) The cancer is an immune therapy-resistant or refractory cancer, said pharmaceutical composition.

43. For use in the treatment of cancer in a subject in need thereof, an oligonucleotide according to any one of claims 1 to 11, or 28 to 30, a compound according to any one of claims 14 to 27, an immunomodulatory polynucleotide according to claim 31, or a conjugate according to any one of claims 12, 13 or 32 to 34, a pharmaceutical composition.

44. A pharmaceutical composition comprising a conjugate for use in the prevention of cancer in a subject in need thereof, wherein the conjugate comprises an oligonucleotide according to any one of claims 1 to 11 and an antibody or antigen-binding fragment thereof, and (i) The conjugate specifically binds to immune cells expressing at least one toll-like receptor; and / or (ii) The pharmaceutical composition is for co-administration with a cancer vaccine, and the conjugate specifically binds to immune cells expressing at least one toll-like receptor, said pharmaceutical composition.

45. A pharmaceutical composition comprising a conjugate for use in inducing an adaptive immune response in a subject, wherein the conjugate comprises an oligonucleotide according to any one of claims 1 to 11 and an antibody or antigen-binding fragment thereof, and wherein the conjugate specifically binds to immune cells expressing at least one toll-like receptor, said pharmaceutical composition.

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