Immunomodulatory polynucleotide conjugates and methods of use thereof
阅读说明：本技术 免疫调节性多核苷酸缀合物及其使用方法 (Immunomodulatory polynucleotide conjugates and methods of use thereof ) 是由 J·庞斯 H·I·万 C·W·布拉德肖 B·J·希姆 T·C-C·郭 于 2019-10-16 设计创作，主要内容包括：本文提供了用于调节天然杀伤细胞或骨髓细胞的缀合物,其包含靶向部分和免疫调节性多核苷酸。本文还提供了用于调节天然杀伤细胞或骨髓细胞的药物组合物,其包含含有靶向部分和免疫调节性多核苷酸的缀合物,以及药学可接受的赋形剂。本文另外提供了其用于调节天然杀伤细胞或骨髓细胞和治疗增生性疾病的方法。(Provided herein are conjugates for modulating natural killer cells or bone marrow cells comprising a targeting moiety and an immunomodulatory polynucleotide. Also provided herein are pharmaceutical compositions for modulating natural killer cells or bone marrow cells comprising a conjugate comprising a targeting moiety and an immunomodulatory polynucleotide, and a pharmaceutically acceptable excipient. Further provided herein are methods for their use in modulating natural killer cells or myeloid cells and treating proliferative diseases.)
1. A conjugate comprising a targeting moiety, an immunomodulatory polynucleotide, and a linker, wherein the targeting moiety binds to an antigen expressed by an NK cell or a bone marrow cell; and the linker covalently links the targeting moiety to the immunomodulatory polynucleotide.
2. The conjugate of claim 1, wherein the immunomodulatory polynucleotide comprises an internucleoside phosphotriester.
3. The conjugate of claim 1 or 2, wherein the immunomodulatory polynucleotide comprises a nucleotide having a modified nucleobase.
4. The conjugate of any one of claims 1-3, wherein the conjugate has the structure of formula (C):
or a stereoisomer, a mixture of two or more diastereomers, a tautomer, or a mixture of two or more tautomers thereof; or a pharmaceutically acceptable salt, solvate or hydrate thereof;
wherein:
ab is a targeting moiety;
each LNIndependently a linker;
each Q is independently an immunomodulatory polynucleotide;
each e is independently an integer of about 1, about 2, about 3, or about 4; and
f is an integer of about 1, about 2, about 3, or about 4.
5. The conjugate of claim 4, wherein f is an integer of about 1.
6. The conjugate of claim 4 or 5, wherein LNIs a linker comprising polyethylene glycol.
7. The conjugate of claim 6, wherein LNIs thatWherein d is an integer from about 0 to about 50.
8. The conjugate of claim 6, wherein LNIs thatWherein d is an integer from about 0 to about 50.
9. The conjugate of claim 7 or 8, wherein d is an integer from about 0 to about 10.
10. The conjugate of claim 7 or 8, wherein d is an integer from about 0 to about 5.
11. The conjugate of claim 7 or 8, wherein d is an integer of about 0, about 1, or about 3.
12. The conjugate of any one of claims 4-11, wherein e is an integer of about 1.
13. The conjugate according to any one of claims 4-12, wherein each Q independently has the structure of formula (D):
wherein:
each XNIndependently a nucleotide;
X3’is a 3' terminal nucleotide;
X5’is a 5' terminal nucleotide;
YPis between nucleosidesResidues of phosphotriesters; and
b and c are each independently integers of from about 0 to about 25; provided that the sum thereof is not less than 5.
14. The conjugate of claim 13, wherein b is an integer from about 1 to about 15.
15. The conjugate of claim 13, wherein b is an integer of about 3, about 4, about 11, or about 14.
16. The conjugate of claim 13, wherein b is an integer of about 3.
17. The conjugate of claim 13, wherein b is an integer of about 4.
18. The conjugate of claim 13, wherein b is an integer of about 11.
19. The conjugate of claim 13, wherein b is an integer of about 14.
20. The conjugate of any one of claims 13-19, wherein c is an integer from about 0 to about 10.
21. The conjugate of claim 20, wherein c is an integer from about 0 to about 8.
22. The conjugate of claim 20, wherein c is an integer of about 0.
23. The conjugate of claim 20, wherein c is an integer of about 8.
24. The conjugate of any one of claims 13-23, wherein the sum of b and c is about 5 to about 20.
25. The conjugate of claim 24, wherein the sum of b and c is about 5 to about 15.
26. The conjugate of claim 24, wherein the sum of b and c is about 8, about 9, about 10, about 11, about 12, about 13, or about 14.
27. The conjugate of any one of claims 13-26, wherein each X isNIndependently a 2' -deoxyribonucleotide.
28. The conjugate of claim 27, wherein each X is NIndependently 2' -deoxyadenosine, 2' -deoxyguanosine, 2' -deoxycytidine, 5-halo-2 ' -deoxycytidine, 2' -deoxythymidine, 2' -deoxyuridine or 5-halo-2 ' -deoxyuridine.
29. The conjugate of claim 27, wherein each X isNIndependently 2 '-deoxyadenosine, 2' -deoxyguanosine, 2 '-deoxycytidine, 2' -deoxythymidine, 5-bromo-2 '-deoxyuridine or 5-iodo-2' -deoxyuridine.
30. The conjugate according to any one of claims 13-29, wherein X3’Is a 2' -deoxyribonucleotide.
31. The conjugate of claim 30, wherein X3’Is 2' -deoxyadenosine, 2' -deoxyguanosine, 2' -deoxycytidine, 5-halo-2 ' -deoxycytidine, 2' -deoxythymidine, 2' -deoxyuridine or 5-halo-2 ' -deoxyuridine.
32. The conjugate of claim 30, wherein X3’Is 2' -deoxythymidine.
33. The conjugate of claim 30, wherein X3’Is a 2' -modified ribonucleotide.
34. The conjugate of claim 30, wherein X3’Is 2' -methoxyA polyribonucleotide or a 2' -ethoxymethoxyribonucleotide.
35. The conjugate according to any one of claims 13-34, wherein X5’Is a 2' -deoxyribonucleotide.
36. The conjugate of claim 35, wherein X 5’Is 2' -deoxyadenosine, 2' -deoxyguanosine, 2' -deoxycytidine, 5-halo-2 ' -deoxycytidine, 2' -deoxythymidine, 2' -deoxyuridine or 5-halo-2 ' -deoxyuridine.
37. The conjugate of claim 35, wherein X5’Is a 2' -deoxyribonucleotide having a substituted pyrimidine base.
38. The conjugate of claim 35, wherein X5’Is a 2' -deoxyribonucleotide having a 5-substituted pyrimidine base.
39. The conjugate of claim 35, wherein X5’Is 2' -deoxythymidine, 5-halo-2 ' -deoxycytidine or 5-halo-2 ' -deoxyuridine.
40. The conjugate of claim 35, wherein X5’Is 2' -deoxythymidine, 5-bromo-2 ' -deoxycytidine, 5-iodo-2 ' -deoxycytidine, 5-bromo-2 ' -deoxyuridine or 5-iodo-2 ' -deoxyuridine.
41. The conjugate of claim 35, wherein X5’Is 2' -deoxythymidine, 5-bromo-2 ' -deoxyuridine or 5-iodo-2 ' -deoxyuridine.
42. The conjugate according to any one of claims 13-41, wherein X5’Is a 3' -phosphorothioate group.
43. The conjugate of claim 42, wherein the 3' -phosphorothioate is chiral.
44. The conjugate of claim 43, wherein the 3' -phosphorothioate has Rp chirality.
45. The conjugate of claim 43, wherein the 3' -phosphorothioate has Sp chirality.
46. The conjugate according to any one of claims 13-43, wherein X5’3' -phosphorothioate group having chirality Rp and X3’Is 2 '-methoxy ribonucleotide or 2' -ethoxy methoxy ribonucleotide.
47. The conjugate according to any one of claims 13-43, wherein X5’3' -phosphorothioate group with Sp chirality and X3’Is 2 '-methoxy ribonucleotide or 2' -ethoxy methoxy ribonucleotide.
48. The conjugate according to any one of claims 13-47, wherein Y isPThe method comprises the following steps:
wherein Z is O or S; and d is an integer from about 0 to about 50.
49. The conjugate of claim 48, wherein Y isPThe method comprises the following steps:
wherein Z is O or S; and d is an integer from about 0 to about 50.
50. The conjugate of claim 48 or 49, wherein Z is O.
51. The conjugate of claim 48 or 49, wherein Z is S.
52. The conjugate according to any one of claims 48-51, wherein d is an integer from about 0 to about 10.
53. The conjugate of claim 52, wherein d is an integer from about 0 to about 5.
54. The conjugate of claim 52, wherein d is an integer of about 0, about 1, or about 3.
55. The conjugate of any one of claims 1-54, wherein the immunomodulatory polynucleotide comprises an additional internucleoside phosphotriester.
56. The conjugate of claim 55, wherein the additional internucleoside phosphotriester is an alkylphosphotriester.
57. The conjugate of claim 55, wherein said additional internucleoside phosphotriester is ethylphosphate triester.
58. The conjugate of any one of claims 1-57, wherein said immunomodulatory polynucleotide comprises a 5-halo-2' -deoxyuridine.
59. The conjugate of claim 58, wherein the 5-halo-2 ' -deoxyuridine is 5-bromo-2 ' -deoxyuridine or 5-iodo-2 ' -deoxyuridine.
60. The conjugate of any one of claims 1-59, wherein the immunomodulatory polynucleotide comprises three or more 2' -deoxycytidines.
61. The conjugate of claim 60, wherein the immunomodulatory polynucleotide comprises three 2' -deoxycytidines.
62. The conjugate of any one of claims 1-61, wherein the immunomodulatory polynucleotide comprises four or more 2' -deoxyguanosines.
63. The conjugate of claim 62, wherein the immunomodulatory polynucleotide comprises four 2' -deoxyguanosines.
64. The conjugate of any one of claims 1-63, wherein the immunomodulatory polynucleotide comprises three 2 '-deoxycytidines and four 2' -deoxycytidines.
65. The conjugate of any one of claims 1-63, wherein the immunomodulatory polynucleotide comprises three or more 2' -deoxythymidine.
66. The conjugate of claim 65, wherein the immunomodulatory polynucleotide comprises three, four, five, six, seven, or eight 2' -deoxythymidine.
67. The conjugate of claim 65, wherein the immunomodulatory polynucleotide comprises three, four, five, or eight 2' -deoxythymidine.
68. The conjugate of any one of claims 1-67, wherein the immunomodulatory polynucleotide comprises 0, one, or two 2' -deoxyadenosines.
69. The conjugate of any one of claims 1-68, wherein the immunomodulatory polynucleotide comprises one or more internucleoside phosphorothioates.
70. The conjugate of claim 69, wherein the immunomodulatory polynucleotide comprises about 12 internucleoside phosphorothioates.
71. The conjugate of any one of claims 1-70, wherein the targeting moiety is an antibody directed against an antigen expressed on NK cells.
72. The conjugate of any one of claims 1-70, wherein the targeting moiety is an antibody directed against an antigen expressed on bone marrow cells.
73. The conjugate of any one of claims 1-72, wherein the targeting moiety is a human antibody.
74. The conjugate of any one of claims 1-73, wherein the targeting moiety is a human anti-CD 56 antibody.
75. The conjugate of claim 74, wherein the antibody is a human anti-CD 56 antibody derived from clone 5.1H 11.
76. The conjugate of any one of claims 1-73, wherein the antibody is an anti-SIRPa antibody.
77. The conjugate of claim 76, wherein the targeting moiety is a blocking anti-SIRPa antibody.
78. The conjugate of claim 77, wherein the blocking anti-SIRPa antibody comprises a heavy chain Variable (VH) domain comprising a heavy chain Variable (VH) sequence comprising a sequence selected from SEQ ID NOs: 498-500, HVR-H1 comprising the sequence SEQ ID NO: 501, and HVR-H2 comprising the sequence SEQ ID NO: HVR-H3 of 502; the light chain Variable (VL) domain comprises a heavy chain Variable (VL) domain comprising the sequence of SEQ ID NO: 503, HVR-L1 comprising the sequence SEQ ID NO: 504 and HVR-L2 comprising the sequence SEQ ID NO: 505 HVR-L3.
79. The conjugate of claim 78, wherein the blocking anti-SIRPa antibody comprises a heavy chain variable region comprising a sequence selected from SEQ ID NO: 490-495 and a VH domain comprising the sequence of SEQ ID NO: 496 or 497.
80. The conjugate of claim 77, wherein the blocking anti-SIRPa antibody comprises a heavy chain Variable (VH) domain comprising a heavy chain Variable (VH) sequence comprising a sequence selected from SEQ ID NOs: 512-514, HVR-H1 comprising the sequence SEQ ID NO: 515 and an HVR-H2 comprising the sequence SEQ ID NO: 516 HVR-H3; the light chain Variable (VL) domain comprises a heavy chain Variable (VL) domain comprising the sequence of SEQ ID NO: 517, HVR-L1 comprising the sequence SEQ ID NO: 518 and a HVR-L2 comprising the sequence of SEQ ID NO: 519 HVR-L3.
81. The conjugate of claim 80, wherein the blocking anti-sirpa antibody comprises a heavy chain variable region comprising a sequence selected from SEQ ID NOs: 506-509 and a VH domain comprising the sequence of SEQ ID NO: 510 or 511, or a VL domain of a sequence of seq id no.
82. The conjugate of claim 77, wherein the blocking anti-SIRPa antibody comprises a heavy chain Variable (VH) domain comprising a heavy chain Variable (VH) sequence comprising a sequence selected from SEQ ID NOs: 533-H1, the HVR-H1 comprising the sequence of SEQ ID NO: 536 and HVR-H2 comprising the sequence SEQ ID NO: 537, HVR-H3; the light chain Variable (VL) domain comprises a heavy chain variable domain comprising a sequence selected from SEQ ID NOs: 538-542, the HVR-L1 comprising the sequence SEQ ID NO: 543 and HVR-L2 comprising a sequence selected from SEQ ID NO: 544-546.
83. The conjugate of claim 82, wherein the blocking anti-SIRPa antibody comprises a heavy chain variable region comprising a sequence selected from SEQ ID NO: 520-523 and a VH domain comprising a sequence selected from SEQ ID NO: 525 and 532.
84. The conjugate of claim 76, wherein the targeting moiety is a non-blocking anti-SIRPa antibody.
85. The conjugate of claim 84, wherein the non-blocking anti-SIRPa antibody comprises a heavy chain Variable (VH) domain comprising a heavy chain Variable (VH) sequence comprising a sequence selected from SEQ ID NOs: 554-H1 of the sequence of 556, the HVR-H1 comprising the sequence SEQ ID NO: 557 and HVR-H2 comprising the sequence SEQ ID NO: 558 of HVR-H3; the light chain Variable (VL) domain comprises the sequence SEQ ID NO: 559 HVR-L1, comprising the sequence SEQ ID NO: 560 and HVR-L2 comprising the sequence SEQ ID NO: 561 HVR-L3.
86. The conjugate of claim 85, wherein the non-blocking anti-SIRPa antibody comprises a heavy chain variable region comprising a sequence selected from SEQ ID NO: 547-550 and a VH domain comprising a sequence selected from the group consisting of SEQ ID NO: the VL domain of the sequence of 551-553.
87. The conjugate of claim 84, wherein the non-blocking anti-SIRPa antibody comprises a heavy chain variable region comprising SEQ ID NO: 584 and a VH domain comprising a sequence selected from SEQ ID NO: 585. 562 and 563.
88. The conjugate of any one of claims 1-73, wherein the antibody is an anti-SIRP β antibody.
89. The conjugate of claim 88, wherein the targeting moiety is a blocking anti-SIRP β antibody.
90. The conjugate of claim 88, wherein the targeting moiety is a non-blocking anti-SIRP β antibody.
91. The conjugate of any one of claims 1-73, wherein the antibody is an anti-SIRP γ antibody.
92. The conjugate of claim 91, wherein the targeting moiety is a blocking anti-SIRP γ antibody.
93. The conjugate of claim 91, wherein the targeting moiety is a non-blocking anti-SIRP γ antibody.
94. The conjugate of any one of claims 71-93, wherein the antibody comprises a human Fc region.
95. The conjugate of claim 94, wherein the Fc region is a human IgG1, IgG2, or IgG4 Fc region.
96. The conjugate of claim 94, wherein the Fc region is:
(i) a human IgG1Fc region comprising mutations of L234A, L235A, and G237A according to EU numbering;
(ii) a human IgG1Fc region comprising mutations of L234A, L235A, G237A, and N297A according to EU numbering;
(iii) a human IgG1Fc region comprising the N297A mutation according to EU numbering;
(iv) A human IgG1 Fc region comprising the D265A mutation according to EU numbering;
(v) a human IgG1 Fc region comprising the D265A and N297A mutations according to EU numbering;
(vi) a human IgG2Fc region comprising the a330S and P331S mutations according to EU numbering;
(vii) a human IgG2Fc region comprising the a330S, P331S, and N297A mutations according to EU numbering;
(viii) a human IgG2Fc region comprising the N297A mutation according to EU numbering;
(ix) a human IgG4 Fc region comprising the S228P mutation according to EU numbering;
(x) A human IgG4 Fc region comprising the S228P and D265A mutations according to EU numbering;
(xi) A human IgG4 Fc region comprising the S228P and L235E mutations according to EU numbering;
(xii) A human IgG4 Fc region comprising the S228P and N297A mutations according to EU numbering; or
(xiii) A human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations according to EU numbering.
97. The conjugate of claim 94, wherein the Fc region comprises a sequence selected from the group consisting of SEQ ID NO 564-578.
98. The conjugate of any one of claims 71-97, wherein the antibody comprises a human kappa light chain constant domain.
99. The conjugate of claim 98, wherein the antibody comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 579 light chain constant domain.
100. The conjugate of any one of claims 71-97, wherein the antibody comprises a human lambda light chain constant domain.
101. The conjugate of claim 100, wherein the antibody comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 580 or 581, light chain constant domain.
102. The conjugate of any one of claims 1-101, wherein the linker has the structure:
wherein each q is independently an integer from about 0 to about 50; each r is independently an integer from about 0 to about 50; the amino group is linked to an amino acid residue on the targeting moiety; and the hydroxyl group is linked to an internucleoside phosphorothioate of the immunomodulatory polynucleotide.
103. The conjugate of claim 102, wherein each q is independently an integer from about 0 to about 25.
104. The conjugate of claim 102, wherein each q is independently an integer from about 20 to about 25.
105. The conjugate according to any one of claims 102-104, wherein each r is independently an integer from about 0 to about 5.
106. The conjugate of any one of claims 1-101, wherein the linker has the structure:
wherein each q is independently an integer from about 0 to about 50; each r is independently an integer from about 0 to about 50; the carbonyl group is attached to an amino acid residue on the targeting moiety; and the hydroxyl group is linked to an internucleoside phosphorothioate of the immunomodulatory polynucleotide.
107. The conjugate of claim 106, wherein each q is independently an integer from about 0 to about 25.
108. The conjugate of claim 106, wherein each q is independently an integer from about 5 to about 10.
109. The conjugate according to any one of claims 106-108, wherein each r is independently an integer from about 0 to about 5.
110. The conjugate of any one of claims 1-101, wherein the linker has the structure:
wherein q is an integer of from about 0 to about 50; r is an integer from about 0 to about 50; the amino group is linked to an amino acid residue on the targeting moiety; and the hydroxyl group is linked to an internucleoside phosphorothioate of the immunomodulatory polynucleotide.
111. The conjugate of any one of claims 1-101, wherein the linker has the structure:
wherein q is an integer of from about 0 to about 50; r is an integer from about 0 to about 50; the carbonyl group is attached to an amino acid residue on the targeting moiety; and the hydroxyl group is linked to an internucleoside phosphorothioate of the immunomodulatory polynucleotide.
112. The conjugate of any one of claims 1-111, wherein the DAR of the antibody-nucleotide conjugate is about 1 to about 8.
113. The conjugate of claim 112, wherein the DAR of the antibody-nucleotide conjugate is about 1.
114. The conjugate of claim 112, wherein the DAR of the antibody-nucleotide conjugate is about 3 to about 4.
115. The conjugate of any one of claims 1-114, having the structure:
wherein c is 2' -deoxycytidine; g is 2' -deoxyguanosine; t is thymidine; x is 5-bromo-2' -deoxyuridine; and Z is
116. The conjugate of any one of claims 1-114, having the structure:
wherein s is an integer of about 3 or about 4; c is a 2' -deoxycellA glycoside; g is 2' -deoxyguanosine; t is thymidine; x is 5-bromo-2' -deoxyuridine; and Z is
117. A pharmaceutical composition comprising the conjugate of any one of claims 1-116 and a pharmaceutically acceptable excipient.
118. The pharmaceutical composition of claim 117, wherein the composition is formulated for parenteral administration.
119. The pharmaceutical composition of claim 117 or 118, wherein the composition is formulated as a single dosage form.
120. The pharmaceutical composition of any one of claims 117-119, wherein the composition is formulated as an intravenous dosage form.
121. The pharmaceutical composition of any one of claims 117-120, further comprising a second therapeutic agent.
122. A method for treating, preventing, or ameliorating one or more symptoms of a proliferative disease in a subject comprising administering to the subject a conjugate according to any of claims 1-116.
123. The method of claim 122, wherein the proliferative disease is cancer.
124. A method of modulating natural killer cells in a subject comprising administering to the subject a conjugate of any one of claims 1-116.
125. A method of modulating bone marrow cells in a subject comprising administering to the subject a conjugate of any one of claims 1-116.
126. The method of claim 125, wherein the bone marrow cells are monocytes.
Technical Field
Provided herein are conjugates for modulating natural killer cells or bone marrow cells comprising a targeting moiety and an immunomodulatory polynucleotide. Also provided herein are pharmaceutical compositions for modulating natural killer cells or bone marrow cells comprising a conjugate comprising a targeting moiety and an immunomodulatory polynucleotide, and a pharmaceutically acceptable excipient. Further provided herein are methods for their use in modulating natural killer cells or myeloid cells and treating proliferative diseases.
Background
Natural killer cells (NK cells) are cytotoxic lymphocytes critical to the innate immune system, where NK cells respond rapidly to virus-infected cells and tumor formation in the absence of antibodies and MHC. NK cells can also serve as an interface for adaptive immune responses and play a major role in cancer immunotherapy involving tumor antigen targeting of antibodies. In adaptive immune responses, NK cells act as effector cells of the immune system and actively lyse target cells with their membrane surface antigens labeled with specific antibodies. This mechanism of cell-mediated immune defense is known as antibody-dependent cell-mediated cytotoxicity (ADCC). Hashimoto et al, J.Infect.Dis.1983,148, 785-794. NK cell-mediated ADCC is a major mechanism for the therapeutic efficacy of many anti-cancer antibodies used to treat a variety of cancers that overexpress distinct antigens, such as neuroblastoma, breast cancer, and B-cell lymphoma. Wang et al, front.immunol.2015,6,368; zahavi et al, Antibody therapeutics 2018, 1, 7-12. Methods of enhancing NK cell activity will increase ADCC and may enhance the efficacy of such anti-cancer therapies. In addition, NK cells carry natural cytotoxic receptors that detect altered expression of ligands on the surface of tumor cells, which ultimately triggers NK cell activation and tumor cell lysis. NK cells have been reported to prolong development and highlight specific memory to various antigens, Paust et al, nat. Immunol.2011,12, 500-508. Studies have shown that NK cells are often deficient and dysfunctional in malignant patients, suggesting that this may be a key factor in cancer immune evasion and progression. In addition, low cancer cell function was found to be predictive of increased risk of developing cancer. Berrien-Elliot et al, curr. Opin. Organ transfer. 2015,20, 671-680; imai et al, Lancet 2000,356, 1795-. In essence, it would be advantageous to develop strategies to activate and expand NK cells in the treatment of malignancies.
NK cells are derived from common lymphoid progenitor cells that produce B and T lymphocytes. They differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils and thymus before entering the circulation. NK cells exist as classical and non-classical subsets that typically express CD16 and CD56 surface markers. CD56 (also known as Neuronal Cell Adhesion Molecule (NCAM)) is a homotypic glycoprotein involved in cell-cell adhesion, neurite outgrowth, synaptic plasticity, and learning and memory. Normal cells that stain positively for CD56 include NK cells, activated T cells, brain and cerebellum, and neuroendocrine tissue. Tumors that are CD56 positive include myeloma, myeloid leukemia, neuroendocrine tumors, Wilm's tumor, adult neuroblastoma, NK/T cell lymphoma, pancreatic acinar cell carcinoma, pheochromocytoma, and small cell lung carcinoma. Van Acker et al, front. immunol.2017,8,892.
Bone marrow cells are derived from continuous bone marrow cell progenitors produced by Hematopoietic Stem Cells (HSCs) in the bone marrow. Bone marrow cells are the most abundant nucleated hematopoietic cells in the body and consist of several cell types, including neutrophils, monocytes, macrophages, Dendritic Cells (DCs), eosinophils, and mast cells. Following pathogen invasion, bone marrow cells are rapidly recruited into local tissues through various chemokine receptors, where they are activated for phagocytosis and secretion of inflammatory cytokines, playing a major role in innate immunity. Macrophages can directly kill tumor cells by antibody-dependent cellular phagocytosis (ADCP). Bone marrow cells also play a key role in connecting innate and adaptive immunity, primarily through antigen presentation by DCs and macrophages and recruitment of adaptive immune cells. The subset of bone marrow cells also includes tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs). TAMs are tissue macrophages with heterogeneous functions and phenotypes that are abundant in the microenvironment of solid tumors. TAMs promote the initiation and metastasis of tumor cells, suppress T cell-mediated anti-tumor immune responses, and stimulate tumor angiogenesis and subsequent tumor progression. Yang and Zhang, j.hematol.oncol.2017, 10, 58. In addition, TAMs help to suppress adaptive immunity in advanced cancers. MDSCs, which contain monocyte and granulocyte subpopulations, contribute to an immunosuppressive network that helps drive cancer escape by suppressing T cell adaptive immunity. MDSCs accumulate throughout the progression of cancer and are associated with poor clinical outcomes and resistance of the murine tumor system to chemotherapy, radiation therapy and immunotherapy. Waight et al, J.Clin.Investig.2013,123, 4464-4478; alizadeh et al, Cancer Res.2014,74, 104-. Modulation of myeloid cell activity, such as increasing ADCP by macrophages, enhancing APC function by dendritic cells, decreasing immunosuppressive activity of TAMs and MDSCs, may promote anti-tumor innate and adaptive immunity and enhance the efficacy of other anti-cancer drugs (e.g., checkpoint inhibitors), vaccines, and T cell directed immunotherapy.
Signal regulatory proteins (SIRPs) consist of several membrane glycoproteins expressed primarily by immune cells, including SIRP α, SIRP β and SIRP γ. SIRP α is expressed primarily by bone marrow cells. Sirpa acts as an inhibitory receptor through its cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) domain and interacts with the widely expressed transmembrane protein CD 47. This interaction negatively controls the effector functions of the innate immune cell. Sirpa diffuses laterally on the macrophage membrane and accumulates at the phagocytic synapses to bind CD47 and signals "self," which inhibits the macrophage phagocytic cytoskeleton packing process. This is similar to the self-signaling provided by MHC class I molecules to NK cells via Ig-like or Ly49 receptors. Sirpa is also expressed in other bone marrow cells, such as neutrophils, dendritic cells, and MDSCs; and can act as inhibitory receptors to regulate the activation and maturation of these cell populations. SIRP β has overlapping expression in bone marrow cells compared to SIRP α, but has a different cytoplasmic domain and may interact with a different ligand other than CD 47. SIRP γ is expressed in lymphoid cells such as T cells and MK cells. SIRP γ also interacts with CD47, but has a short cytoplasmic domain that is unlikely to have similar signaling properties as SIRP α. Barclay and Brown, nat. Rev. Immunol.2006,6,457-64.
Toll-like receptors (TLRs) are key pattern recognition receptors for innate immunity that recognize pathogens by sensing pathogen-associated molecular patterns (PAMPs) derived from bacteria, viruses, fungi, and protozoa. Akira et al, nat. Rev. Immunol.2004,4, 499-511; zhang et al, Science 2004,303, 1522-. Each TLR contains a transmembrane domain, an extracellular PAMP binding domain with a leucine rich repeat motif and an intracellular Toll-IL-1 receptor domain that initiates a signaling cascade. Gay and Gangloff, Annu, Rev, biochem, 2007,76, 141-165. The recognition of microbial invaders by TLRs leads to the activation of downstream signaling cascades to secrete cytokines and chemokines and ultimately to the activation of innate and adaptive immune responses against pure pathogens. Takeda and Akira, semin. immunol.2004,16, 3-9; shi et al, J.biol.chem.2016, 291, 1243-1250. In humans, ten TLRs have been identified, including TLR-1, TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, TLR-7/8, TLR-9 and TLR-10. D' Arpa and Leung, adv.Wound Care 2017,6, 330-.
Toll-like receptor 9(TLR9), also known as CD289, is an important receptor expressed in cells of the immune system including Dendritic Cells (DCs), B lymphocytes, macrophages, natural killer cells and other antigen presenting cells. TLR9 activation triggers an intracellular signaling cascade leading to activation, maturation, proliferation, and cytokine production of these immune cells, bridging innate and adaptive immunity. Martinez-Campos et al, Viral Immunol.2016, 30, 98-105; notley et al, Sci. Rep.2017,7,42204. Natural TLR-9 agonists include oligodeoxynucleotides (CpG ODN) containing unmethylated cytosine-guanine dinucleotides (CpG).
CpG ODN are generally divided into three classes: class A, class B and class C. A class CpG ODN typically comprises a poly-G tail with a phosphorothioate backbone and a central palindromic sequence including a phosphate backbone at the 3 '-and 5' -ends. A class CpG ODN typically contains CpG in its central palindromic sequence. B class CpG ODNs typically contain an intact phosphorothioate backbone, and sequences at their 5' ends are often critical for TLR9 activation. The C class CpG ODN comprises a complete phosphorothioate backbone with a 3' -terminal sequence, thereby enabling duplex formation. However, CpG ODN are generally easily degraded in serum, and thus the pharmacokinetics of CpG ODN may be one of the limiting factors in its development into therapeutic agents. Similarly, CpG ODN often exhibit heterogeneous tissue distribution in vivo, with major sites of accumulation in the liver, kidney and spleen. This distribution can lead to off-target activity and local toxicity associated with PAMPs. Therefore, there is a need for effective methods to stabilize and deliver CpG ODN for therapeutic applications.
Summary of The Invention
Provided herein is a conjugate for modulating natural killer cells or bone marrow cells comprising a targeting moiety and an immunomodulatory polynucleotide.
Also provided herein is a pharmaceutical composition for modulating natural killer cells or bone marrow cells comprising a conjugate comprising a targeting moiety and an immunomodulatory polynucleotide; and a pharmaceutically acceptable carrier.
Further provided herein is a method of modulating a natural killer cell or a bone marrow cell comprising contacting the cell with a conjugate comprising a targeting moiety and an immunomodulatory polynucleotide.
Further provided herein is a method of treating a proliferative disease in a subject comprising administering to the subject a conjugate comprising a targeting moiety and an immunomodulatory polynucleotide.
Provided herein are conjugates of formula (C):
or a stereoisomer, a mixture of two or more diastereomers, a tautomer, or a mixture of two or more tautomers thereof; or a pharmaceutically acceptable salt, solvate or hydrate thereof; wherein:
ab is an anti-CD 56 or anti-SIRP antibody;
each LNIndependently a linker;
each Q is independently an immunomodulatory polynucleotide;
each e is independently an integer of about 1, about 2, about 3, or about 4; and
f is an integer of about 1, about 2, about 3, or about 4.
Drawings
FIG. 1 shows the results obtained by using anti-CD 56-CpG nucleotide (SEQ. ID NO:425) conjugate (anti-CD 56-CPG) compared to controls: activation of NK cells was measured by the increase in CD69 expression following 24 hours treatment of Peripheral Blood Mononuclear Cells (PBMCs) with CpG nucleotide alone (p425), anti-CD 56 antibody alone (anti-CD 56) and medium (horizontal dashed line).
FIG. 2 shows the relative ratios of the total antigen by using anti-CD 56-CpG nucleotide (SEQ. ID NO:425) conjugate (anti-CD 56-CPG) compared to controls: activation of NK cells was measured by the increase in CD69 expression following treatment of PBMCs with CpG nucleotide alone (p425), anti-CD 56 antibody alone (anti-CD 56) and media (horizontal dashed line) for 48 hours.
FIG. 3 shows the results of the comparison of control samples using anti-SIRP α -CpG nucleotide (SEQ. ID NO:425) conjugates (anti-Sirp α 1-CpG and anti-Sirp α 2-CpG) with either a blocked anti-SIRP α antibody (anti-Sirp α 1) or a non-blocked anti-SIRP α antibody (anti-Sirp α 2): CD14 following PBMC treatment with CpG nucleotides alone (p425), anti-SIRP α antibodies alone (anti-Sirp α 1 and anti-Sirp α 2), and media (horizontal dashed line)+Increase of cells.
FIG. 4 shows the comparison of control with a blocking anti-SIRP α antibody (anti-Sirp α 1) or a non-blocking anti-SIRP α antibody (anti-Sirp α 2) using anti-SIRP α -CpG nucleotide (SEQ. ID NO:425) conjugates (anti-Sirp α 1-CpG and anti-Sirp α 2-CpG): purified CD14 was treated with CpG nucleotides alone (p425), anti-SIRP α antibodies alone (anti-Sirp α 1 and anti-Sirp α 2), and media (horizontal dashed line)+Post-cellular CD14+Increase of cells.
FIG. 5 shows a series of structures, indicating abbreviations with corresponding structures. The abbreviations are those used in table 2.
FIG. 6 shows a series of structures, indicating abbreviations with corresponding structures. The abbreviations are those used in table 2.
FIGS. 7A-7D show that anti-SIRP α -CpG nucleotide conjugates inhibit tumor growth in vivo. FIG. 7A: measurement of mean CT26 tumor size over time following treatment with 10mg/kg anti-sirpa 1 conjugate (blocking antibody) administered twice three days apart or unconjugated anti-sirpa antibody administered twice three days apart compared to PBS control. FIG. 7B: measurement of mean CT26 tumor size over time after treatment with 3mg/kg of either anti-sirpa 1 conjugate (blocking antibody) or anti-sirpa 2 conjugate (non-blocking antibody), both administered at 2q3, compared to PBS. FIG. 7C: measurement of mean CT26 tumor size over time following treatment with 1mg/kg, 0.3 mg/kg, or 0.1mg/kg anti-SIRPa 1 conjugate (blocking antibody), all at 2q3 dosing, compared to PBS control. FIG. 7D: measurement of mean MC38 tumor size over time following treatment with 10mg/kg anti-sirpa 1 conjugate (blocking antibody) dosed with 2q3, compared to PBS control. mpk-mg/kg. 2q3 is 2 doses, spaced 3 days apart. Arrows indicate administration of conjugate or control.
FIGS. 8A and 8B show that anti-SIRP α -CpG nucleotide conjugates inhibit tumor growth in vivo. FIG. 8A: measurement of mean CT26 tumor size over time following treatment with 1mg/kg anti-sirpa 1 conjugate (blocking antibody) three days apart, two doses apart, or seven days apart, compared to PBS control. FIG. 8B: survival curves of mice in the CT26 tumor model dosed as described in figure 8A. mpk-mg/kg. 2q3 is 2 doses, spaced 3 days apart. 2q7 as doses, at 7 day intervals. Arrows indicate administration of conjugate or control.
Detailed Description
Definition of
To facilitate an understanding of the disclosure set forth herein, a number of terms are defined below.
Generally, the nomenclature used herein and the laboratory procedures in biology, biochemistry, medicinal chemistry, organic chemistry, and pharmacology described herein are those well known and commonly employed in the art. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
The term "subject" refers to an animal, including but not limited to primates (e.g., humans), cows, pigs, sheep, goats, horses, dogs, cats, rabbits, rats, and mice. The terms "subject" and "patient" are used interchangeably herein, e.g., to refer to a mammalian subject, e.g., a human subject, in one embodiment a human.
As used herein, the term "base-free spacer" refers to a divalent group having the structure:
R1–L1–[–L2–(L1)n1–]n2–R2,
(I)
wherein:
n1 is an integer of about 0 or about 1,
n2 is an integer of from about 1 to about 6,
R1is a bond to a nucleotide in an immunomodulatory polynucleotide,
R2is a bond to a nucleoside or a capping group in an immunomodulatory polynucleotide,
Each L1Independently a phosphodiester or phosphotriester, and
each L2Is a sugar-like substance that is a saccharide analog,
with the proviso that,
if the abasic spacer is an internucleoside abasic spacer, then each n1 is 1, and R2Is a bond with a nucleoside, and
if the abasic spacer is a terminal abasic spacer, each n1 is independently an integer of about 0 or about 1, and R2Is a bond to the end-capping group.
The term "about" or "approximately" refers to an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term "about" or "approximately" means within 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range.
The term "alk-tetrayl" as used herein, unless otherwise specified, denotes a tetravalent, acyclic, straight-chain or branched saturated hydrocarbon group having from 1 to 16 carbons. As described for alkyl, the alkane-tetrayl group may be optionally substituted.
The term "alk-triyl" as used herein, unless otherwise indicated, refers to a trivalent, acyclic, straight-chain or branched, saturated hydrocarbon group having from 1 to 16 carbons. As described for alkyl, the alkane-triyl may be optionally substituted.
The term "alkanoyl" as used herein denotes a linkage through a carbonyl groupHydrogen or alkyl groups attached to the parent molecular group are exemplified by formyl (i.e., carboxyaldehyde), acetyl, propionyl, butyryl, and isobutyryl. The unsubstituted alkanoyl group contains 1-7 carbons. As described herein for alkyl, alkanoyl groups can be unsubstituted or substituted (e.g., optionally substituted C)1-7Alkanoyl) group. The suffix "-acyl" may be added to another group defined herein, such as aryl, cycloalkyl and heterocyclyl, to define "aroyl", "cycloalkoyl" and "(heterocyclic) acyl". These groups each represent a carbonyl group attached to an aryl, cycloalkyl or heterocyclyl group. Each of "aroyl", "cycloalkoyl", and "(heterocyclic) acyl" may be optionally substituted, as defined in "aryl", "cycloalkyl", or "heterocyclyl".
As used herein, the term "alkenyl" denotes an acyclic monovalent straight or branched chain hydrocarbon group containing one, two or three carbon-carbon double bonds. Non-limiting examples of alkenyl groups include vinyl, prop-1-enyl, prop-2-enyl, 1-methylvinyl, 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.
As used herein, the term "alkenylene" refers to a straight or branched chain alkenyl group that has one hydrogen removed to render the group divalent. Non-limiting examples of alkenylene groups include ethylene-1, 1-diyl; ethylene-1, 2-diyl; prop-1-ene-1, 1-diyl; prop-2-en-1, 1-diyl; prop-1-en-1, 2-diyl; prop-1-en-1, 3-diyl; prop-2-en-1, 1-diyl; prop-2-en-1, 2-diyl; but-1-en-1, 1-diyl; but-1-en-1, 2-diyl; but-1-en-1, 3-diyl; but-1-en-1, 4-diyl; but-2-en-1, 1-diyl; but-2-en-1, 2-diyl; but-2-en-1, 3-diyl; but-2-en-1, 4-diyl; but-2-en-2, 3-diyl; but-3-en-1, 1-diyl; but-3-en-1, 2-diyl; but-3-en-1, 3-diyl; but-3-en-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 butadiene-2, 3-diene-1, 2-diyl. As described for alkyl, alkenylene groups may be unsubstituted or substituted (e.g., optionally substituted alkenylene).
As used herein, unless otherwise indicated, the term "alkoxy" refers to a chemical substituent of the formula-OR, wherein R is C 1-6An alkyl group. In some embodiments, alkyl groups may be further substituted as defined herein. The term "alkoxy" may be combined with other terms defined herein, such as aryl, cycloalkyl, or heterocyclyl, to define "arylalkoxy", "cycloalkylalkoxy", and "(heterocyclyl) alkoxy" groups. These groups each represent alkoxy substituted by aryl, cycloalkyl or heterocyclyl. For each individual moiety, each of "arylalkoxy," "cycloalkylalkoxy," and "(heterocyclyl) alkoxy" may be optionally substituted as defined herein.
As used herein, the term "alkyl" refers to an acyclic, straight or branched chain saturated hydrocarbon group, unless otherwise specified, having from 1 to 12 carbons when unsubstituted. In certain preferred embodiments, the unsubstituted alkyl group has 1 to 6 carbons. Examples of alkyl groups are methyl; an ethyl group; n-propyl and isopropyl; n-, sec-, iso-, and tert-butyl; neopentyl and the like, and may be optionally substituted, where valency permits, by one, two, three, or, where alkyl has two or more carbons, by four or more substituents independently selected from the group consisting of: an amino group; an aryl group; an aryloxy group; an azide group; a cycloalkyl group; a cycloalkoxy group; a cycloalkenyl group; a cycloalkynyl group; halogenating; a heterocyclic group; (heterocyclyl) oxy; a hydroxyl group; a nitro group; a thiol; a silane group; a cyano group; o; (ii) S; NR ', where R' is H, alkyl, aryl or heterocyclyl. Each substituent may itself be unsubstituted or, where valency permits, substituted by an unsubstituted substituent as defined herein for each group.
As used herein, the term "alkylamino" refers to a group having the formula-N (R)N1)2or-NHRN1Wherein R isN1Is an alkyl group as defined herein. As defined for alkyl, alkylThe alkyl portion of the amino group may be optionally substituted. Each optional substituent on a substituted alkylamino group may itself be unsubstituted or, where valency permits, substituted with one or more unsubstituted substituents as defined herein for each group.
As used herein, the term "alkylcycloalkylene" refers to a saturated divalent hydrocarbon radical that is an alkylcycloalkane in which two valencies replace two hydrogen atoms. Preferably, at least one of the two valencies is present on the cycloalkane moiety. The alkane and cycloalkane moieties may be optionally substituted as described herein for the individual groups.
As used herein, the term "alkylene" refers to a saturated divalent hydrocarbon group that is a straight or branched chain saturated hydrocarbon, wherein two valencies replace two hydrogen atoms. The valency of an alkylene group as defined herein excludes optional substituents. Non-limiting examples of alkylene groups 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-yAlkylene "means an alkylene having x-y carbons. Exemplary values for x are 1, 2, 3, 4, 5, and 6, and exemplary values for y are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. Alkylene groups may be optionally substituted as described herein for alkyl groups.
As used herein, the term "alkyloxythio" denotes a group of formula-S- (alkyl). As described for alkyl, the alkylenesulfoxy group may be optionally substituted.
The term "alkylsulfinyl", as used herein, denotes a group of formula-s (o) - (alkyl). As described for alkyl, alkylsulfinyl may be optionally substituted.
As used herein, the term "alkylsulfonyl" refers to the group of formula-S (O)2A group of (C) - (alkyl). Alkylsulfonyl groups may be optionally substituted as described for alkyl.
As used herein, the term "alkynyl" denotes a monovalent straight or branched chain hydrocarbon group of 2 to 6 carbon atoms containing at least one carbon-carbon triple bond, and examples are ethynyl, 1-propynyl, and the like. As defined for alkyl, alkynyl groups can be unsubstituted or substituted (e.g., optionally substituted alkynyl).
As used herein, the term "5-alkynyl uridine" denotes a nucleoside wherein the nucleobase is a 5-alkynyl uracil having the structure:
Wherein R is bonded to the anomeric carbon of the pentafuranose of the nucleoside and X is an alkynyl group. In some embodiments, X is ethynyl or propynyl (e.g., X is ethynyl).
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 alkynylene groups include ethynyl-1, 2-diyl; prop-1-yne-1, 3-diyl; prop-2-yne-1, 1-diyl; but-1-yn-1, 3-diyl; but-1-yn-1, 4-diyl; but-2-yn-1, 1-diyl; but-2-yn-1, 4-diyl; but-3-yn-1, 1-diyl; but-3-yn-1, 2-diyl; but-3-yn-2, 2-diyl; and but-1, 3-diyne-1, 4-diyl. As described for alkynyl groups, an alkynylene group can be unsubstituted or substituted (e.g., an optionally substituted alkynylene group).
As used herein, the term "amino" means-N (R)N1)2Wherein if the amino group is unsubstituted, then two R areN1Are all H; or, if the amino group is substituted, each RN1Independently is H, -OH, -NO2、-N(RN2)2、-SO2ORN2、-SO2RN2、-SORN2、-COORN2An N-protecting group, an alkyl, alkenyl, alkynyl, alkoxy, aryl, arylalkyl, aryloxy, cycloalkyl, cycloalkenyl, heteroalkyl, or heterocyclyl, provided that at least one R N1Is not H, and wherein each RN2Independently H, alkyl or aryl. Each substituent may itself be unsubstituted or unsubstituted as defined herein for each respective groupAnd (4) substituent substitution. In some embodiments, amino is unsubstituted amino (i.e., -NH)2) Or substituted amino (e.g., -NHR)N1) Wherein R isN1Independently is-OH, -SO2ORN2、 -SO2RN2、-SORN2、-COORN2Optionally substituted alkyl or optionally substituted aryl, and each RN2May be an optionally substituted alkyl group or an optionally substituted aryl group. In some embodiments, a substituted amino group can be an alkylamino group, wherein alkyl is optionally substituted as described herein for alkyl. In certain embodiments, amino is-NHRN1Wherein R isN1Is an optionally substituted alkyl group. Wherein R isN1is-NHR of optionally substituted alkylN1Non-limiting examples of (a) include: c of optionally substituted alkylamino, proteinogenic, non-proteinogenic, proteinogenic amino acid1-6C of alkyl esters and non-proteinogenic amino acids1-6An alkyl ester.
The term "aminoalkyl" as used herein, denotes an alkyl group substituted by one, two or three amino groups as defined herein. As described for alkyl, aminoalkyl may be further optionally substituted.
As used herein, the term "arene-tetrayl" refers to a tetravalent group that is an aryl group in which three hydrogen atoms are substituted by valences. The arene-tetrayl group may be optionally substituted as described herein for aryl groups.
As used herein, the term "aryl" denotes a mono-, bi-or polycyclic carbocyclic ring system having one or two aromatic rings. The aryl group may contain 6 to 10 carbon atoms. All atoms in 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. Aryl groups may be unsubstituted or substituted with one, two, three, four or five substituents independently selected from: an alkyl group; an alkenyl group; an alkynyl group; an alkoxy group; an alkylsulfinyl group; an alkylenesulfoxy group; an alkylsulfonyl group; an amino group; an aryl group; an aryloxy group; an azide group; a cycloalkyl group; a cycloalkoxy group; a cycloalkenyl group; a cycloalkynyl group; halogenating; a heteroalkyl group; a heterocyclic group; (heterocyclyl) oxy; a hydroxyl group; a nitro group; a thiol; silyl groups and cyano groups. Each substituent may itself be unsubstituted or substituted with one or more unsubstituted substituents as defined herein for each respective group.
As used herein, the term "arylalkyl" refers to an alkyl group substituted with an aryl group. The individual groups, aryl and alkyl moieties, as described herein, may be optionally substituted.
As used herein, the term "arylalkylene" refers to an arylalkyl group in which one hydrogen atom is replaced by a valence. The arylalkylene group can be optionally substituted as described herein for arylalkyl.
As used herein, the term "arylene" refers to an aryl group in which one hydrogen atom is replaced by a valence. The arylene group can be optionally substituted as described herein for aryl.
As used herein, unless otherwise indicated, the term "aryloxy" refers to a chemical substituent of the formula-OR, wherein R is aryl. In the optionally substituted aryloxy group, the aryl group is optionally substituted as described herein for the aryl group.
As used herein, the term "auxiliary moiety" means a monovalent group comprising a hydrophilic polymer, a positively charged polymer, or a sugar alcohol.
As used herein, the term "optionally substituted N" denotes divalent-N (R)N1) -group or trivalent-N ═ group. The heteroaryl group may be unsubstituted (wherein R isN1Is H or absent), or may be substituted (wherein R isN1As defined for "amino"), except that R is N1Is not H. Two heteroaryl groups may be linked to form a "diaza".
The term "optionally substituted N-protected amino" as used herein denotes a substituted amino group as defined herein, wherein at least one substituent is an N-protecting group and the other substituent is H if the N-protected amino group is unsubstituted, or is other than H if the N-protected amino group is substituted.
As used herein, the term "azido" refers to-N3A group.
The term "bulky group" as used herein denotes any substituent or group of substituents as defined herein, wherein the group bonded to the disulfide bond is a carbon atom if the group is sp3-hybridized, then the carbon atom carries one or less hydrogen atoms; or if the group is sp2-a hybridized carbon, then the carbon atom does not carry a hydrogen atom. This group is not sp-hybridized carbon. Bulky groups are bonded to disulfide bonds only through carbon atoms.
As used herein, the term "5 ' -5 ' end-capping" refers to the formula R ' -Nuc1–O–(LP)n-wherein R' is a phosphate, phosphorothioate, phosphorodithioate, phosphotriester, phosphodiester, hydroxyl or hydrogen; nuc (Nuc) 1Is a nucleoside; each LPIndependently is-P (═ X)E1)(–XE2–RE2A) -O-; and n is 1, 2 or 3;
wherein each XE1And each XE2Independently is O or S, each RE2AIndependently hydrogen, a bioreversible group, a non-bioreversible group, an auxiliary moiety, a conjugated group, a linker bonded to a targeting moiety or a linker bonded to a targeting moiety and one or more (e.g. 1-6) auxiliary moieties; and
wherein R ' is bonded to the 3 ' -carbon of the nucleoside and-O-is bonded to the 5 ' -carbon of the nucleoside.
As used herein, the term "end-capping group" refers to a monovalent or divalent group located at the 5 '-or 3' -end of a polynucleotide. The end capping group is a terminal phosphate; a diphosphate ester; a triphosphate ester; an auxiliary portion; a bioreversible group; a non-bioreversible group; 5 ' end capping (e.g., 5 ' -5 ' end capping); a solid support; a linker bonded to the target moiety and optionally to one or more (e.g. 1-6) auxiliary moieties; OR a-OR 'group, wherein R' is selected from the group consisting of hydrogen, bioreversible groups, non-bioreversible groups, solid supports and O protecting groups. the-OR' group, diphosphate, triphosphate, bioreversible, non-bioreversible, solid support and adjunct moieties are examples of monovalent end capping groups. The terminal phosphate is an example of an end-capping group, which may be monovalent if the terminal phosphate does not include a linker to the targeting moiety, or divalent if the terminal phosphate includes a linker to the targeting moiety. Linkers bonded to targeting moieties (with or without auxiliary moieties) are examples of divalent end capping groups.
As used herein, the term "carbocycle" means optionally substituted C3-16Monocyclic, bicyclic or tricyclic structures, in which the rings, which may be aromatic or non-aromatic, are formed by carbon atoms. Carbocyclic ring structures include cycloalkyl, cycloalkenyl, cycloalkynyl, and certain aryl groups.
As used herein, the term "carbonyl" denotes the-c (o) -group.
As used herein, the expression "Cx-y"means that when unsubstituted, the group whose name immediately follows the expression contains a total of x to y carbon atoms. If the group is a complex group (e.g. arylalkyl), Cx-yMeans that when unsubstituted, the part of its name immediately following the expression contains a total of x-y carbon atoms. For example, (C)6-10-aryl) -C1-6-alkyl is the following group: wherein when unsubstituted, the aryl moiety contains a total of 6 to 10 carbon atoms and when unsubstituted, the alkyl moiety contains a total of 1 to 6 carbon atoms.
As used herein, the term "cyano" denotes a-CN group.
As used herein, the term "cycloaddition reaction" refers to a reaction of two components in which a total of [4n +2] pi electrons participate in bond formation when activation is absent, activated by a chemical catalyst, or activated using thermal energy, and n is 1, 2, or 3. The cycloaddition reaction is also a two component reaction in which [4n ] pi electrons are involved in the presence of photochemical activation and n is 1, 2 or 3. Ideally, [4n +2] pi electrons participate in bond formation, and n is 1. Representative cycloaddition reactions include the reaction of alkenes with 1, 3-dienes (Diels-Alder reactions), the reaction of alkenes with α, β -unsaturated carbonyl groups (hetero Diels-Alder reactions), and the reaction of alkynes with azido compounds (e.g., Huisgen cycloaddition reactions).
As used herein, unless otherwise indicated, the term "cycloalkenyl"Refers to a non-aromatic carbocyclic group having at least one double bond in the ring and 3-10 carbons (e.g., C)3-C10Cycloalkenyl groups). Non-limiting examples of cycloalkenyl groups include cyclopropyl-1-enyl, cyclopropyl-2-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. As described for cycloalkyl groups, cycloalkenyl groups can be unsubstituted or substituted (e.g., optionally substituted cycloalkenyl).
As used herein, the term "cycloalkenylalkyl" denotes an alkyl group each substituted by a cycloalkenyl group as defined herein. Each group, cycloalkenyl and alkyl moiety, as defined herein, may be substituted.
As used herein, the term "cycloalkenylene" denotes a divalent group that is a cycloalkenyl group, wherein one hydrogen atom is replaced by a valence. Cycloalkenyl groups may be optionally substituted as described herein for cycloalkyl groups. Non-limiting examples of cycloalkenylene groups are cycloalkene-1, 3-diyl.
As used herein, unless otherwise indicated, the term "cycloalkoxy" refers to a chemical substituent of the formula-OR, wherein R is cycloalkyl. In some embodiments, cycloalkyl groups may be further substituted as defined herein.
The term "cycloalkyl" as used herein, unless otherwise indicated, refers to a cyclic alkyl group having 3-10 carbons (e.g., C)3-C10Cycloalkyl groups). Cycloalkyl groups may be monocyclic or bicyclic. The bicyclic cycloalkyl can be bicyclo [ p.q.0]Alkyl type, wherein p and q are each 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, bicyclic cycloalkyl groups may include bridged cycloalkyl structures, such as bicyclo [ p.q.r]Alkyl, wherein r is 1, 2 or 3, and p and q are each 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. Cycloalkyl groups may be spirocyclic, e.g. spiro [ p.q ]]Alkyl, wherein p and q are each 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 groups include cyclopropylCyclobutyl, 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 decahydronaphthyl. Cycloalkyl groups may be unsubstituted or substituted with 1, 2, 3, 4 or 5 substituents independently selected from (e.g., optionally substituted cycloalkyl): an alkyl group; an alkenyl group; an alkynyl group; an alkoxy group; an alkylsulfinyl group; an alkylenesulfoxy group; an alkylsulfonyl group; an amino group; an aryl group; an aryloxy group; an azide group; a cycloalkyl group; a cycloalkoxy group; a cycloalkenyl group; a cycloalkynyl group; halogenating; a heteroalkyl group; a heterocyclic group; (heterocyclyl) oxy; a hydroxyl group; a nitro group; a thiol; a silyl group; a cyano group; o; (ii) S; NR ', where R' is H, alkyl, aryl or heterocyclyl. Each substituent may itself be unsubstituted or substituted with an unsubstituted substituent as defined herein for each respective group.
As used herein, the term "cycloalkylalkyl" denotes an alkyl group substituted by a cycloalkyl group as defined herein. The various groups, cycloalkyl and alkyl moieties, as described herein, may be optionally substituted.
As used herein, the term "cycloalkylene" denotes a divalent group that is a cycloalkyl group in which one hydrogen atom is replaced by a valence. A non-limiting example of a cycloalkylene group is cycloalkane-1, 3-diyl. Cycloalkylene groups may be optionally substituted as described herein for cycloalkyl groups.
The term "cycloalkynyl" as used herein, unless otherwise indicated, refers to a monovalent carbocyclic group having one or two carbon-carbon triple bonds and having from 8 to 12 carbons. Cycloalkynyl may comprise a ring-spanning bond or bridge. Non-limiting examples of cycloalkynyl include cyclooctynyl, cyclononynyl, cyclodecynyl, and cyclodecynyl. As defined for cycloalkyl, cycloalkynyl can be unsubstituted or substituted (e.g., optionally substituted cycloalkynyl).
As used herein, the term "dihydropyridazine group" denotes a divalent group obtainable by cycloaddition between a 1,2,4, 5-tetrazine group and a strained cycloalkenyl group.
As used herein, the term "halo" denotes a halogen selected from the group consisting of bromo, chloro, iodo and fluoro.
As used herein, the term "5-halopyrimidine nucleoside" means a nucleoside wherein the nucleobase is a 5-halouracil having the structure:
wherein R is bonded to the anomeric carbon of the pentafuranose of the nucleoside and X is fluorine, chlorine, bromine or iodine. In some embodiments, X is bromine or iodine.
As used herein, the term "heteroalkan-tetrayl" refers to a moiety interrupted once by one heteroatom; twice, each time independently interrupted by one heteroatom; three times, each time independently interrupted by one heteroatom; or four times, each time independently interrupted by one heteroatom. Each heteroatom is independently O, N or S. In some embodiments, the heteroatom is O or N. Unsubstituted CX-YThe heteroalkan-tetrayl contains X to Y carbon atoms as well as heteroatoms as defined herein. As described for heteroalkyl, the heteroalkane-tetrayl can be unsubstituted or substituted (e.g., optionally substituted heteroalkane-tetrayl).
As used herein, the term "heteroalkane-triyl" refers to a moiety interrupted once by one heteroatom; twice, each time independently interrupted by one heteroatom; three times, each time independently interrupted by one heteroatom; or four times, each time independently interrupted by one heteroatom. Each heteroatom is independently O, N or S. In some embodiments, the heteroatom is O or N. Unsubstituted C X-YHeteroalkan-triyl contains X-Y carbon atoms as well as heteroatoms as defined herein. As described for heteroalkyl, the heteroalkane-triyl can be unsubstituted or substituted (e.g., optionally substituted heteroalkane-triyl).
As used herein, the term "heteroalkyl" refers to being interrupted once by one or two heteroatoms; twice, each independently interrupted by one or two heteroatoms; three times, each time independently interrupted by one or two heteroatoms; or four times, each time independently an alkyl, alkenyl or alkynyl group interrupted by one or two heteroatoms. Each heteroatom is independently O, N or S. In thatIn some embodiments, the heteroatom is O or N. Neither heteroalkyl group includes two consecutive oxygen or sulfur atoms. Heteroalkyl groups can be unsubstituted or substituted (e.g., optionally substituted heteroalkyl groups). When the heteroalkyl group is substituted and the substituent is bonded to a heteroatom, the substituent is selected based on the nature and valency of the heteroatom. Thus, where valency permits, substituents bonded to heteroatoms are selected from the group consisting of: o, -N (R)N2)2、-SO2ORN3、-SO2RN2、-SORN3、-COORN3An N-protecting group, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a cycloalkyl group, a cycloalkenyl group, a cycloalkynyl group, a heterocyclic group or a cyano group, wherein each R is N2Independently is H, alkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl or heterocyclyl, each RN3Independently is alkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, or heterocyclyl. Each of these substituents may itself be unsubstituted or substituted with one or more unsubstituted substituents as defined herein for each respective group. When the heteroalkyl group is substituted and the substituent is bonded to a carbon, the substituent is selected from those described for alkyl, provided that the substituent on the carbon bonded to the heteroatom is not Cl, Br, or I. It is understood that the carbon atom is present at the end of the heteroalkyl group.
As used herein, the term "heteroaryloxy" refers to the structure-OR, wherein R is heteroaryl. As defined for heterocyclyl, heteroaryloxy may be optionally substituted.
As used herein, the term "heterocyclyl" denotes a monocyclic, bicyclic, tricyclic or tetracyclic ring system having a fused or bridged 5-, 6-, 7-or 8-membered ring, which, unless otherwise specified, contains 1,2,3 or 4 heteroatoms independently selected from nitrogen, oxygen and sulfur. The heterocyclic group may be aromatic or non-aromatic. Non-aromatic 5-membered heterocyclyls have 0 or 1 double bond, non-aromatic 6-and 7-membered heterocyclyls have 0-2 double bond, and non-aromatic 8-membered heterocyclyls have 0-2 double bond and/or 0 or 1 carbon-carbon triple bond. Unless otherwise specified, heterocyclyl groups include 1-16 carbon atoms. Certain heterocyclic groups may contain up to 9 carbon atoms. Non-aromatic heterocyclic groups include pyrrolinyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, homopiperidinyl, piperazinyl, pyridazinyl, oxazolidinyl, isoxazolinyl, morpholinyl, thiomorpholinyl, thiazolinyl, isothiazolinyl, thiazolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, dihydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, pyranyl, dihydropyranyl, dithiazolyl, and the like. If the heterocyclic system has at least one aromatic resonance structure or at least one aromatic tautomer, such structure is an aromatic heterocyclic group (i.e., heteroaryl). Non-limiting examples of heteroaryl groups include benzimidazolyl, benzofuranyl, benzothiazolyl, benzothienyl, benzoxazolyl, furanyl, imidazolyl, indolyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, purinyl, pyrrolyl, pyridyl, pyrazinyl, pyrimidinyl, quinazolyl, quinolinyl, thiadiazolyl (e.g., 1,3, 4-thiadiazole), thiazolyl, thienyl, triazolyl, tetrazolyl, and the like. The term "heterocyclyl" also denotes heterocyclic compounds 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 diazabicyclo [2.2.2] octane. The term "heterocyclyl" includes bicyclic, tricyclic and tetracyclic groups in which any of the above-described heterocycles are 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 heterocycle. Examples of fused heterocyclic groups include 1,2,3,5,8, 8 a-hexahydroindolizine; 2, 3-dihydrobenzofuran; 2, 3-indoline and 2, 3-dihydrobenzothiophene. A heterocyclyl group may be unsubstituted or substituted with 1,2,3, 4 or 5 substituents independently selected from: an alkyl group; an alkenyl group; an alkynyl group; an alkoxy group; an alkylsulfinyl group; an alkylenesulfoxy group; an alkylsulfonyl group; an amino group; an aryl group; an aryloxy group; an azide group; a cycloalkyl group; a cycloalkoxy group; a cycloalkenyl group; a cycloalkynyl group; halogenating; a heteroalkyl group; a heterocyclic group; (heterocyclyl) oxy; a hydroxyl group; a nitro group; a thiol; a silyl group; a cyano group; o; (ii) S; NR ', where R' is H, alkyl, aryl or heterocyclyl. Each substituent may itself be unsubstituted or substituted with one or more unsubstituted substituents as defined herein for each group.
As used herein, the term "heterocyclylalkyl" denotes an alkyl group substituted with a heterocyclyl group each as defined herein. Each group, heterocyclyl and alkyl moiety as described herein may be optionally substituted.
As used herein, the term "(heterocyclyl) azepine" refers to compounds of the formula-N (R)N1)(RN2) Wherein R isN1Is a heterocyclic group, and RN2Is H, -OH, -NO2、-N(RN2)2、 -SO2ORN2、-SO2RN2、SORN2、-COORN2An N-protecting group, an alkyl, alkenyl, alkynyl, alkoxy, aryl, arylalkyl, aryloxy, cycloalkyl, cycloalkenyl, heteroalkyl, or heterocyclyl. Preferably, RN2Is H.
As used herein, the term "heterocyclylene" refers to a heterocyclic group in which one hydrogen atom is replaced by a valence. The heterocyclylene group may be optionally substituted in the manner described for the heterocyclyl group. A non-limiting example of a heterocyclylene group is heterocycle-1, 3-diyl.
As used herein, unless otherwise indicated, the term "(heterocyclyl) oxy" means a chemical substituent of the formula-OR, wherein R is heterocyclyl. (heterocyclyl) oxy may be optionally substituted in the manner described for heterocyclyl.
The terms "hydroxyl" and "hydroxy" as used interchangeably herein represent an — OH group.
As used herein, the term "immunomodulatory polynucleotide" refers to a polynucleotide construct comprising a total of 6-50 contiguous nucleosides covalently bound together by an internucleoside bridging group independently selected from an internucleoside phosphate and optionally an internucleoside base-free spacer. The immunomodulatory polynucleotides are capped at the 5 '-and 3' -ends with 5 '-and 3' -blocking groups, respectively. The immunomodulatory polynucleotide is capable of modulating an innate immune response, as determined, for example, by a change in activation of nfkb or a change in secretion of at least one inflammatory cytokine or at least one type I interferon in an antigen presenting cell to which the immunomodulatory polynucleotide is delivered (e.g., as compared to another antigen presenting cell to which the immunomodulatory polynucleotide is not delivered). The immunomodulatory polynucleotide may comprise a conjugate group or, if the immunomodulatory polynucleotide is part of a conjugate, a linker that is fused to the targeting moiety, optionally bonded to one or more (e.g., 1-6) accessory moieties (e.g., polyethylene glycol). The conjugate group or linker may be part of a phosphotriester or a terminal end capping group.
As used herein, the term "immunostimulatory polynucleotide" means an immunomodulatory polynucleotide capable of activating an innate immune response, as determined, for example, by increased activation of nfkb or increased secretion of at least one inflammatory cytokine or at least one type I interferon in an antigen presenting cell to which the immunomodulatory polynucleotide is delivered (e.g., as compared to another antigen presenting cell to which the immunostimulatory polynucleotide is not delivered). In some embodiments, the immunostimulatory polynucleotide comprises at least one cytidine-p-guanosine (CpG) sequence, wherein p is an internucleoside phosphodiester (e.g., a phosphate or phosphorothioate) or an internucleoside phosphotriester or a phosphorothioate triester. As used herein, CpG-containing immunostimulatory polynucleotides may occur naturally, e.g., of bacterial or viral origin or synthetic CpG ODNs. For example, in some embodiments, the CpG sequence in the immunostimulatory polynucleotide contains a 2' -deoxyribose. In some embodiments, the CpG sequence in the immunostimulatory polynucleotide is unmethylated. In some embodiments, the immunostimulatory polynucleotide is a polynucleotide of formula (a) provided herein. In some embodiments, the immunostimulatory polynucleotide is a compound of formula (B) provided herein.
As used herein, the term "immunosuppressive polynucleotide" refers to an immunomodulatory polynucleotide capable of antagonizing an innate immune response as determined by a reduction in activation of nfkb or a reduction in secretion of at least one inflammatory cytokine or at least one type I interferon in an antigen presenting cell to which the immunosuppressive polynucleotide is delivered (e.g., as compared to another antigen presenting cell to which the immunosuppressive polynucleotide is not delivered).
As used herein, the term "internucleoside bridging group" means an internucleoside phosphate or an internucleoside base-free spacer.
As used herein, the term "5-modified cytidine" represents a nucleoside in which the nucleobase has the following structure:
wherein R is bonded to the anomeric carbon of the pentafuranose of the nucleoside and X is halogen, alkynyl, alkenyl, alkyl, cycloalkyl, heterocyclyl or aryl. In some embodiments, the 5-modified cytidine is a 5-halocytidine (e.g., 5-iodocytidine or 5-bromocytidine). In other embodiments, the 5-modified cytidine is a 5-alkynylcytidine.
As used herein, the term "5-modified uridine" represents a nucleoside wherein the nucleobase has the following structure:
wherein R is bonded to the anomeric carbon of the pentafuranose of the nucleoside, and X is halogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl or aryl, with the proviso that the 5-modified uridine is not thymidine. In some embodiments, the 5-modified uridine is a 5-halogenated uridine (e.g., 5-iodouridine or 5-bromouridine). In other embodiments, the 5-modified uridine is a 5-alkynyl uridine. In some embodiments, the 5-modified uridine is a nucleoside comprising 2-deoxyribose.
As used herein, the term "non-bioreversible" refers to a chemical group that is resistant to degradation under the conditions present inside an endosome. The non-bioreversible group does not comprise thioesters and/or disulfides.
As used herein, the term "nucleobase" refers to a nitrogen-containing heterocycle that is bound to the 1' position of the sugar moiety of a nucleotide or nucleoside. Ribonucleases can be unmodified or modified. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), as well as 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-propynyl uracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, and, 5-halo is in particular 5-iodo, 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 5-alkynyl (e.g. 5-ethynyl) uracils, 5-acetamido-uracils, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Other nucleobases include those disclosed in U.S. Pat. No. 3,687,808; the sense Encyclopedia Of Polymer Science And Engineering, pages 858-; those disclosed by Englisch et al, Angewandte Chemie, International Edition,1991,30, 613; and those disclosed by Sanghvi, Y.S., Chapter15, Antisense Research and Applications, pages 289302, (crook et al, ed., CRC Press, 1993). Certain nucleobases are particularly useful for increasing the binding affinity of the hybrid polynucleotides of the invention, including 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methyl cytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 ℃. (Sanghvi et al, eds., Antisense Research and Applications 1993, CRC Press, Boca Raton, pages 276-. In particular embodiments, these may be combined with 2' -O-methoxyethyl sugar modifications. U.S. patents teaching the preparation of certain of these modified nucleobases, as well as other modified nucleobases, include, but are not limited to, the aforementioned U.S. Pat. nos. 3,687,808; 4,845,205, respectively; 5,130, 302; 5,134,066, respectively; 5,175,273, respectively; 5,367,066, respectively; 5,432,272; 5,457,187, respectively; 5,459,255; 5,484,908, respectively; 5,502,177, respectively; 5,525,711, respectively; 5,552,540, respectively; 5,587,469, respectively; 5,594,121, respectively; 5,596,091, respectively; 5,614,617, respectively; and 5,681,941. For the purposes of this disclosure, "modified nucleobase" as used herein also refers to natural or non-natural nucleobases, which include one or more of the protecting groups described herein.
As used herein, the term "nucleoside" represents a pentafuranose-nucleobase combination. The pentafuranose is 2-deoxyribose OR modified form thereof, wherein the 2-position is OR, R, halogen (e.g. F), SH, SR, NH2、NHR、NR2Or CN, wherein R is optionally substituted C1-6Alkyl (e.g. C)1-6Alkyl or (C)1-6Alkoxy) -C1-6-alkyl) or optionally substituted (C)6-14Aryl) -C1-4-an alkyl group. In certain embodiments, the 2 position is substituted with OR OR F, wherein R is C1-6Alkyl or (C)1-6-alkoxy) -C1-6-an alkyl group. The pentafuranose is bonded to the nucleobase at the anomeric carbon. In some embodiments, the term "nucleoside" refers to a divalent group having the structure:
wherein B is1Is a nucleobase; y is H, halogen (e.g. F), hydroxy, optionally substituted C1-6Alkoxy (e.g., methoxy or methoxyethoxy) or protected hydroxy; y is1Is H or C1-6Alkyl (e.g., methyl); and each of 3 'and 5' represents a position bonded to another group.
As used herein, the term "nucleotide" refers to a nucleoside bonded to a phosphate, phosphorothioate or phosphorodithioate.
As used herein, the term "phosphate ester" means a group comprising a phosphate ester, a phosphorothioate ester, or a phosphorodithioate ester, wherein 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. Wherein one and only one of the valencies of the phosphate ester covalently bonded to the nucleoside-containing group is a terminal phosphate ester. The phosphate ester in which two valencies are covalently bonded to a group comprising a nucleoside is an internucleoside phosphate ester. The phosphate ester may be a group having the following structure:
Wherein:
XE1and XE2Each is independently O or S;
RE1and RE3Each is independently hydrogen or is bonded to a nucleoside; a sugar analog without a base spacer; a bioreversible group; a non-bioreversible group; an auxiliary portion; a conjugate group; a linker bonded to the targeting moiety; a linker bonded to the targeting moiety and one or more (e.g., 1-6) auxiliary moieties; or formula-P (═ X)E1)(–XE2–RE2A) -a phosphorus atom in O-,
wherein R isE2AIs hydrogen, a bioreversible group; a non-bioreversible group; an auxiliary portion; a conjugate group; a linker bonded to the targeting moiety; a linker bonded to the targeting moiety and one or more (e.g., 1-6) auxiliary moieties; and
RE2is hydrogen, a bioreversible group; a non-bioreversible group; an auxiliary portion; a conjugate group; a linker bonded to the targeting moiety; a linker bonded to the targeting moiety and one or more (e.g., 1-6) auxiliary moieties;
provided that R isE1And RE3Is bonded to a group comprising at least one nucleoside.
If R isE1And RE3Each independently bonded to a group comprising at least one nucleoside, the phosphate ester is an internucleoside phosphate ester. If R isE1And RE3One of which is bonded to a group which does not contain a nucleoside, to form a phosphate ester Is a terminal phosphate.
As used herein, the term "phosphodiester" refers to a phosphate ester in which two of the three valencies are replaced with a non-hydrogen substituent, while the remaining valencies are replaced with hydrogen. The phosphodiester consists of: a phosphate, phosphorothioate or phosphorodithioate; one or two bonds to a nucleoside, an abasic spacer and/or a phosphoryl group; if the phosphodiester contains only one spacer or phosphoryl group independently selected from bioreversible groups with a nucleoside, no base or a phosphoryl group; a non-bioreversible group; an auxiliary portion; a conjugate group; a linker bonded to the targeting moiety; a bond to one group of the linker that is bonded to the targeting moiety and one or more (e.g., 1-6) auxiliary moieties. The terminal phosphodiester comprises a bond to a nucleoside-containing group and is selected from the group consisting of bioreversible groups; a non-bioreversible group; an auxiliary portion; a conjugate group; a linker bonded to the targeting moiety; a group of a linker bonded to the targeting moiety and one or more (e.g., 1-6) auxiliary moieties. An internucleoside phosphodiester comprises two bonds to a group comprising a nucleoside. The phosphodiester may be a group having the following structure:
Wherein:
XE1and XE2Each is independently O or S;
RE1and RE3Each is independently hydrogen or is bonded to a nucleoside; a sugar analog without a base spacer; a bioreversible group; a non-bioreversible group; an auxiliary portion; a conjugate group; a linker bonded to the targeting moiety; a linker bonded to the targeting moiety and one or more (e.g., 1-6) auxiliary moieties; or formula-P (═ X)E1)(–XE2–RE2A) -a phosphorus atom in O-,
wherein R isE2AIs hydrogen, a bioreversible group; a non-bioreversible group; an auxiliary portion; a conjugate group; a linker bonded to the targeting moiety; with targeting moieties and one or more(e.g., 1-6) linkers bonded to the auxiliary moiety; and
RE2is hydrogen, a bioreversible group; a non-bioreversible group; an auxiliary portion; a conjugate group; a linker bonded to the targeting moiety; a linker bonded to the targeting moiety and one or more (e.g., 1-6) auxiliary moieties;
provided that R isE1、XE2And RE3One and only one of (a) is hydrogen; and
provided that R isE1And RE3Is bonded to a group comprising at least one nucleoside.
If R isE1And RE3Are each bonded to a group comprising at least one nucleoside, the phosphodiester is an internucleoside phosphodiester. If R is E1And RE3And only one is bonded to the group comprising the nucleoside, the phosphodiester is a terminal phosphodiester.
As used herein, the term "phosphoryl" refers to a substituent of the formula
–P(＝XE1)(–XE2–RE2A)–O–RE3A,
Wherein:
XE1and XE2Each is independently O or S;
RE2Ais hydrogen, a bioreversible group; a non-bioreversible group; an auxiliary portion; a conjugate group; a linker bonded to the targeting moiety; a linker bonded to the targeting moiety and one or more (e.g., 1-6) auxiliary moieties; and
RE3Ais hydrogen or open valency.
When a group is identified as bonded to a phosphoryl group, the group is bonded to the phosphorus atom of the phosphoryl group.
As used herein, the term "phosphotriester" refers to a phosphate ester in which all three valencies are substituted with a non-hydrogen substituent. The phosphotriester consists of: a phosphate, phosphorothioate or phosphorodithioate; one or two bonds to one or more nucleosides, or a abasic spacer and/or phosphoryl group; independently selected from bioreversible groups; a non-bioreversible group; an auxiliary portion; a conjugate group; and one or two groups of linkers bonded to the targeting moiety and optionally to one or more (e.g. 1-6) auxiliary moieties. The terminal phosphotriester comprises a bond to a nucleoside-containing group and is independently selected from the group consisting of bioreversible groups; a non-bioreversible group; an auxiliary portion; a conjugate group; and two groups of a linker bonded to the targeting moiety and optionally to one or more (e.g. 1-6) auxiliary moieties. In some embodiments, the terminal phosphotriester comprises 1 or 0 linkers bonded to the targeting moiety and optionally to one or more (e.g., 1-6) auxiliary moieties. The internucleoside phosphotriester comprises two bonds to a nucleoside-containing group. The phosphotriester may be a group having the following structure:
Wherein:
XE1and XE2Each is independently O or S;
RE1and RE3Each is independently hydrogen or is bonded to a nucleoside; a sugar analog without a base spacer; a bioreversible group; a non-bioreversible group; an auxiliary portion; a conjugate group; a linker bonded to the targeting moiety; a linker bonded to the targeting moiety and one or more (e.g., 1-6) auxiliary moieties; or formula-P (═ X)E1)(–XE2–RE2A) -a phosphorus atom in O-,
wherein R isE2AIs hydrogen, a bioreversible group; a non-bioreversible group; an auxiliary portion; a conjugate group; a linker bonded to the targeting moiety; a linker bonded to the targeting moiety and one or more (e.g., 1-6) auxiliary moieties; and
RE2is a bioreversible group; a non-bioreversible group; an auxiliary portion; a conjugate group; a linker bonded to the targeting moiety; a linker bonded to the targeting moiety and one or more (e.g., 1-6) auxiliary moieties;
provided that R isE1And RE3At leastOne bonded to a group comprising at least one nucleoside.
If R isE1And RE3Are each bonded to a group comprising at least one nucleoside, the phosphotriester is an internucleoside phosphotriester. If R isE1And RE3And only one is bonded to the group comprising the nucleoside, the phosphotriester is a terminal phosphotriester.
As used herein, the term "pyridin-2-ylhydrazone" denotes a group of the structure:
wherein each R' is independently H or optionally substituted C1-6An alkyl group. The pyridin-2-ylhydrazone may be unsubstituted (i.e. each R' is H).
As used herein, the term "stereochemically enriched" refers to a local stereochemical preference for one of the stereoisomeric configurations of the recited groups, relative to the opposite stereoisomeric configuration of the same group. Thus, a polynucleotide comprising a stereochemically enriched phosphorothioate is a chain in which a phosphorothioate of a predetermined stereochemistry is present in preference to a phosphorothioate of the opposite stereochemistry. The diastereomeric ratio of phosphorothioates of a predetermined stereochemistry can be used to numerically express this preference. The predetermined stereochemical phosphorothioate diastereomer ratio is the molar ratio of the identified phosphorothioate diastereomer having the predetermined stereochemistry relative to the identified phosphorothioate diastereomer having the opposite stereochemistry. The ratio of diastereomers of the phosphorothioates of the predetermined stereochemistry may be greater than or equal to 1.1 (e.g., greater than or equal to 4, greater than or equal to 9, greater than or equal to 19, or greater than or equal to 39).
As used herein, the term "Q-tag" refers to a portion of a polypeptide comprising a glutamine residue that is in contact with a peptide containing-NH2Following transglutaminase mediated reaction of the amine compound, a conjugate is provided comprising the portion of the polypeptide wherein the glutamine residue comprises an acyl modified to include a bond to the compoundSide chains of amines. Q-tags are known in the art. Non-limiting examples of Q tags are LLQGG (SEQ ID NO: 582) and GGGGLQGG (SEQ ID NO: 583).
The term "strained cycloalkenyl" as used herein means that the ring strain energy of a cycloalkenyl group is at least 16kcal/mol if the open valency is replaced by H.
As used herein, the term "carbohydrate analog" refers to a C3-6Monosaccharides or C3-6A divalent or trivalent radical of a sugar alcohol (e.g., glycerol) modified to replace two hydroxyl groups bonded to an oxygen atom in a phosphate, phosphorothioate or phosphorodithioate or capping group. The saccharide analog does not contain nucleobases capable of hydrogen bonding with nucleobases in the complementary strand. The carbohydrate analogs are cyclic or acyclic. Further optional modifications comprised in the carbohydrate analogues are: replacing one, two or three of the remaining hydroxyl groups or carbon-bonded hydrogen atoms with H; optionally substituted C 1-6An alkyl group; as defined herein-LinkA (-T)p(ii) a A conjugate group; - (CH)2)t1–ORZWherein t1 is an integer from 1 to 6, and RZIs optionally substituted C1-6Alkyl, optionally substituted C2-6Alkenyl, optionally substituted C2-6Alkynyl, optionally substituted C6-14Aryl, optionally substituted C3-8Cycloalkyl, optionally substituted (C)1-9Heterocyclyl) -C1-6Alkyl, optionally substituted (C)6-10Aryl) -C1-6-alkyl or optionally substituted (C)3-8Cycloalkyl) -C1-6-an alkyl group; introduction of one or two unsaturated bonds (e.g., one or two double bonds); and one, two or three hydrogen or hydroxy groups are substituted with substituents as defined for alkyl, alkenyl, cycloalkyl, cycloalkenyl or heterocyclyl. Non-limiting examples of carbohydrate analogs are optionally substituted C2-6Alkylene, optionally substituted C2-6Alkenylene, optionally substituted C5Cycloalkane-1, 3-diyl, optionally substituted C5Cycloalkene-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 substitutedIs (C)1-4Alkyl group) - (C3-8Cycloalkylene) (e.g. optionally substituted (C)1Alkyl group) - (C3Cycloalkylene)).
As used herein, the term "sulfide" denotes a divalent-S-or ═ S group. The disulfide is-S-.
As used herein, the term "targeting moiety" refers to a moiety (e.g., a small molecule, such as a carbohydrate) that specifically binds or reactively associates or complexes with a receptor or other receptor moiety that binds to 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 provided herein comprise a targeting moiety. The targeting moiety may be an antibody or antigen-binding fragment thereof or an engineered derivative thereof (e.g., Fcab or a fusion protein (e.g., scFv)). The targeting moiety may be a polypeptide. Alternatively, the targeting moiety can be a small molecule (e.g., mannose) or a cluster of small molecules (e.g., a mannose cluster). Conjugates of the invention comprising a targeting moiety can exhibit a K of less than 100nM for targets bound by the targeting moietyd. Using methods known in the art, e.g. using Surface Plasmon Resonance (SPR), e.g. using BIACORETMSystem (GE Healthcare, Little Chalfount, UK) to measure Kd。
As used herein, the term "1, 2,4, 5-tetrazinyl" denotes a group having the 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 a group-R a–Rb-, wherein RaAnd RbIs independently optionally substituted alkylene, optionally substituted heteroalkylene, optionally substituted arylene, optionally substituted cycloalkylene, or optionally substituted heterocyclylene.
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, the term refers to any substance intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease or the enhancement of physical or intellectual development and disorders required in animals or humans. As used herein, the term "therapeutically effective amount" or "therapeutically effective dose" refers to the amount of immunomodulatory polynucleotide or conjugate necessary to ameliorate, treat, or at least partially inhibit the symptoms of the disease to be treated. The amount effective for this use depends on the severity of the disease and the weight and general condition of the subject. In general, dosages used in vitro may provide useful guidance in the amounts used for in vivo administration of the pharmaceutical composition, and animal models may be used to determine effective dosages for the treatment of particular diseases.
As used herein, the term "thiocarbonyl" denotes a C (═ S) group.
As used herein, the term "thiacyclylene" refers to the group-S-R-, where R is a heterocyclylene. The thiacyclylene group may be optionally substituted in the manner described for the heterocyclic group.
As used herein, the term "thiol" represents an-SH group.
The term "treating" as used with reference to a disease or condition in a patient is intended to mean obtaining a beneficial or desired result, e.g., a clinical result, by administering to the patient a polynucleotide or conjugate of the invention. Beneficial or desired results may include alleviation or amelioration of one or more symptoms of the disease or disorder; impairs the extent of the disease or condition; stable disease or condition (i.e., not worsening); preventing the spread of a disease or disorder; delay or slow the progression of the disease or disorder; alleviating the disease or condition; and mitigation (whether partial or total). By "alleviating" a disease or disorder is meant that the extent and/or adverse clinical manifestations of the disease or disorder are reduced, and/or the time course of progression is slowed, as compared to the extent or time course of treatment without the polynucleotides or conjugates of the invention.
As used herein, the term "triazolencycloalkene" is meant to encompass a 1, 2-membered ring fused to an 8-membered ringHeterocycloalkenyl of the 3-triazole ring, all of the internal ring atoms of which are carbon atoms and the bridgehead atom of which is sp 2-a hybridized carbon atom. The heterocycloalkenylene group may be optionally substituted in the manner described for the heterocyclic group.
As used herein, the term "triazolyl" refers to a heterocyclyl group that includes a 1,2, 3-triazolyl ring fused to an 8-membered ring that includes at least one heteroatom. The bridgehead atom in the triazole heterocyclylene group is a carbon atom. The triazole heterocyclylene group may be optionally substituted in the manner described for the heterocyclyl group.
It is understood that the terms "immunomodulatory polynucleotide," "immunostimulatory polynucleotide," "immunosuppressive polynucleotide," and "conjugate" include salts of immunomodulatory polynucleotides, immunostimulatory polynucleotides, immunosuppressive polynucleotides, and conjugates, respectively. For example, the terms "immunomodulatory polynucleotide", "immunostimulatory polynucleotide", "immunosuppressive polynucleotide" and "conjugate" encompass protonated neutral forms of phosphate, phosphorothioate or phosphorodithioate (P-XH moieties, wherein X is O or S) and deprotonated ionic forms of phosphate, phosphorothioate or phosphorodithioate (PX-moieties, wherein X is O or S). Thus, it is understood that the description as having RE1、RE2And RE3The one or more phosphate esters and phosphate diesters as hydrogens include salts in which the phosphate, phosphorothioate or phosphorodithioate is present in the form of a deprotonated ion.
The terms "innate immune response" and "innate immunity" are well-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 different forms of cellular activity, including cytokines produced by various pathways and cell death. As used herein, an innate immune response includes a cellular response to a CpG-containing immunostimulatory polynucleotide mediated by Toll-like receptor 9(TLR9), including but not limited to increasing production of inflammatory cytokines (e.g., type I interferon or IL-10 production), activating the nfkb pathway, increasing proliferation, maturation, differentiation, and/or survival of immune cells, and in some cases inducing apoptosis. Activation of innate immunity can be detected using methods known in the art, e.g., measuring activation of (NF) - κ B.
The terms "adaptive immune response" and "adaptive immunity" are well-recognized in the art and refer to antigen-specific defense mechanisms initiated by the body's immune system upon recognition of a particular antigen, which include both humoral responses and cell-mediated responses. As used herein, an adaptive immune response includes a cellular response elicited and/or enhanced by a CpG-containing immunostimulatory polynucleotide. In some embodiments, the immunostimulatory polynucleotide or portion thereof is an antigen target of an antigen-specific adaptive immune response. In other embodiments, the immunostimulatory polynucleotide is not an antigen target for an antigen-specific adaptive immune response, but still enhances the adaptive immune response. Activation of the adaptive immune response can be detected using methods known in the art, such as measuring the production of antigen-specific antibodies or the level of antigen-specific cell-mediated cytotoxicity.
The term "Toll-like receptor" (or "TLR") is recognized in the art and refers to a family of pattern recognition receptors that are initially recognized as sensors of the innate immune system that recognize microbial pathogens. TLRs recognize different structures in microorganisms, commonly referred to as "PAMPs" (pathogen-associated molecular patterns). Ligand-bound TLRs activate a series of intracellular signaling pathways, thereby inducing an innate and/or adaptive immune response. As used herein, the term "toll-like receptor" or "TLR" also refers to a functional fragment of a toll-like receptor protein expressed by a cell. In humans, ten TLRs have been identified, including TLR-1, -2, -3, -4, -5, -6, -7/8, -9, and-10. D' Arpa and Leung, adv.Wound Care, 2017,6, 330-. Human genes encoding TLRs are known.
Toll-like receptor 9(TLR9), also known as CD289 (cluster of differentiation 289), is a member of the Toll-like receptor (TLR) family. Du et al, eur. cytokine netw., 11:362-371 (2000). TLR9 is an important receptor expressed in cells of the immune system, including Dendritic Cells (DCs), B lymphocytes, macrophages, natural killer cells and other antigen presenting cells. TLR9 activates a triggering signaling cascade that bridges innate and adaptive immunity. Martinez-Campos et al, Viral Immunol.,30:98-105 (2016); notley et al, Sci. Rep.,7:42204 (2017). Natural TLR-9 agonists include oligodeoxynucleotides (CpG ODN) that include unmethylated cytosine-guanine dinucleotides (CpG). TLR-9 ligands 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, e.g., measuring recruitment of myeloid differentiation antigen 88(MyD88), activation of Nuclear Factor (NF) - κ B, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) signaling pathways, activation of interferon regulatory factor-7, activation of one or more cytokines (e.g., type I Interferon (IFN), Interleukin (IL) -6, IL-10, and IL-12), activation of one or more immune cell populations (e.g., NK cells, natural killer T cells, monocytes), and levels of cytotoxic lymphocyte (CTL) and T helper 1(Th1) responses, as well as levels of immunoglobulin secretion.
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 upon binding of the Toll-like receptor to an agonist. Toll-like receptors 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 phagosome. The TLR-expressing cell may further express one or more cell surface antigens other than a Toll-like receptor. Certain immune cells express TLRs, and activation of TLR signaling pathways in immune cells elicits innate and/or adaptive immune responses. 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 virally infected cells) express TLRs, and activation of the TLR signaling pathway in the diseased cells can lead to death of the diseased cells, e.g., by inducing apoptosis. Examples of cells expressing TLR9 include, but are not limited to, Dendritic Cells (DCs), B cells, T cells, langerhans cells, keratinocytes, mast cells, endothelial cells, myofibroblasts, and primary fibroblasts. Methods known in the art can be used to determine whether a cell expresses any Toll-like receptor (e.g., TLR9), e.g., to detect the mRNA of a Toll-like receptor in a cell.
As used herein, the term "immune cell" is art-recognized and refers to any cell involved in host defense mechanisms, such as cells that produce pro-inflammatory cytokines and cells involved in the pathogenesis of tissue damage and/or disease. 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.
The term "antigen presenting cell" or "APC" is well recognized in the art and refers to a heterogeneous group of immune cells that mediate a cellular immune response by processing and presenting antigen for recognition by certain lymphocytes (e.g., T cells). Exemplary types of antigen presenting cells include, but are not limited to, professional antigen presenting cells, including, for example, B cells, monocytes, dendritic cells, and langerhans cells, as well as other antigen presenting cells, including, for example, keratinocytes, endothelial cells, astrocytes, fibroblasts, and oligodendrocytes. As used herein, the term "antigen presenting cell" includes antigen presenting cells found in vivo and antigen presenting cells found in vitro cell culture derived from cells in vivo. As used herein, antigen presenting cells also include artificially modified APCs, e.g., APCs that have been genetically modified to express a Toll-like receptor (e.g., TLR9) or to modulate the level of expression of a Toll-like receptor (e.g., TLR 9).
The term "dendritic cell" or "DC" is recognized in the art and refers to a heterogeneous group of specialized antigen sensing and Antigen Presenting Cells (APCs). Human DCs are divided into three major subgroups: plasma cell-like dc (pdc), bone marrow dc (mdc), and monocyte-derived dc (mddc). Schramm et al, curr, opin, immunol, 32:13-20 (2015). Subsets of DCs can be identified based on different TLR expression patterns. For example, the bone marrow or "normal" subset of dcs (mdcs) express TLRs 1-8 when stimulated and produce a range of activation markers (e.g., CD80, CD86, MHC class I and II, CCR7), proinflammatory cytokines, and chemokines. The result of this stimulation and resulting expression is antigen-specific CD4+ and CD8+ T cell priming. These DCs acquire an enhanced ability to take up antigen and present it in an appropriate form to T cells. The plasma cell-like subset of dcs (pdcs) expresses TLR7 and TLR9 upon activation, resulting in activation of NK cells and T cells.
As used herein, the term "antigen" refers to a molecule or antigenic fragment thereof that is capable of eliciting an immune response, including innate and adaptive immune responses. As used herein, an antigen can be a protein, peptide, polysaccharide, lipid, nucleic acid (especially RNA and DNA), nucleotide, and other biological or biochemical substance. The term "eliciting an immune response" refers to stimulating immune cells in vivo in response to a stimulus such as an antigen. Immune responses include cellular immune responses, such as T cell and macrophage stimulation, as well as humoral immune responses, such as B cell and complement stimulation and antibody production. Immune responses can be measured using techniques well known in the art, including but not limited to antibody immunoassays, proliferation assays, and the like.
The terms "antigen fragment" and "antibody-binding fragment" are used interchangeably herein. An antigenic fragment as used herein is capable of complexing with an antigen binding molecule, such as an antibody, in a particular reaction. Specific reaction as referred to herein means that an antigen or antigen fragment will react in a highly selective manner with its corresponding antibody, but not with a variety of other antibodies that may be elicited by other antigens. The specificity of this response is determined by the presence or absence of one or more epitopes (immunogenic determinants) in the antigen. As used herein, an antigen or antigenic fragment thereof may have one epitope, or more than one epitope.
As used herein, the term "T cell epitope" refers to any epitope of an antigen produced by a T cell.
As used herein, the term "tumor-associated antigen" or "TAA" refers to an antigen expressed by a cancer cell or solid tumor stroma in a cancer patient receiving the therapeutic or prophylactic care provided herein (e.g., receiving a therapeutic dose of an immunostimulatory polynucleotide or CpG-Ab immunoconjugate). TAAs may or may not be targeted in the treatment or prophylaxis provided herein. The TAA need not be overexpressed, mutated, or deregulated on cancer cells, but can have the same characteristics that a TAA would have in normal cells. In some embodiments, the TAA may be overexpressed, mutated, or deregulated in the cancer cell. TAA may be a protein, nucleic acid, lipid or other antigen. The TAA may be a cell surface expressed TAA, an intracellular TAA or an intranuclear TAA. In the case of solid tumors, TAAs may be expressed in the stroma of the solid tumor mass. As used herein, the term "matrix" refers to a component in a solid tumor mass other than cancer cells. For example, the matrix may include fibroblasts, epithelial cells, other vascular components, or extracellular matrix components. As used herein, the term "substrate" does not include components of the immune system, such as immune cells (e.g., B cells, T cells, dendritic cells, macrophages, natural killer cells, etc.). Various TAAs are known in the art. Identification of TAAs can be performed using Methods known in the art, such as the Methods disclosed in Zhang et al, Methods mol. biol.,520:1-10 (2009).
The terms "antibody," "immunoglobulin," and "Ig" are used interchangeably herein and are used in the broadest sense and specifically encompass, for example, a single monoclonal antibody (including agonists, antagonists, neutralizing antibodies, full-length or intact monoclonal antibodies), antibody compositions having multi-epitope or single-epitope specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity) formed from at least two intact antibodies, single chain antibodies, and antibody fragments. Antibodies can be human, humanized, chimeric, and/or affinity matured, as well as antibodies from other species, such as mice and rabbits.
The term "antibody" is intended to include the polypeptide product of a B cell among the polypeptides of the immunoglobulin class, which is capable of binding a particular antigen and consists of two pairs of identical polypeptide chains, wherein each pair has one heavy chain (about 50-70kDa) and one light chain (about 25kDa), and each amino-terminal portion of each chain comprises about 100 to about 130 or moreA variable region of more amino acids, and each carboxy-terminal portion of each chain includes a constant region. See, Borebaeck (ed.) (1995) antibody engineering, SecondEd., Oxford University Press; kuby (1997) Immunology, third edition, W.H.Freeman and Company, New York. Antibodies also include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinant antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments thereof, which refer to a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment is derived. Non-limiting examples of functional fragments of antibodies include single chain fv (scFv) (e.g., including monospecific or bispecific), Fab fragments, F (ab') fragments, F (ab) 2Fragment, F (ab')2Fragments, disulfide-linked Fv (sdFv), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies. In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, e.g., antigen binding domains or molecules that comprise an antigen binding site that binds an antigen (e.g., one or more Complementarity Determining Regions (CDRs) of an anti-CD 56 antibody or an anti-sirpa antibody). Such antibody fragments are described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989); myers (ed.), Molec.biology and Biotechnology A Comprehensive Desk Reference, New York: VCH publishers, Inc.; huston et al, Cell Biophysics1993,22, 189-224; pl ü ckthun and Skerra, meth. Enzymol.1989,178, 497-515; and Day, Advanced biochemistry, second edition, Wiley-Liss, Inc., New York, NY (1990). The antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass of immunoglobulin molecules (e.g., IgG2a and IgG2 b).
The term "antigen" refers to a predetermined antigen to which an antibody can selectively bind. The target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten or other naturally occurring or synthetic compound. In one embodiment, the target antigen is a polypeptide.
The terms "antigen-binding fragment," "antigen-binding domain," and "antigen-binding region" refer to a portion of an antibody (e.g., a Complementarity Determining Region (CDR)) that comprises amino acid residues that interact with an antigen and confer to a binding agent its specificity and affinity for the antigen.
The terms "specifically binds," "specifically binds," or "specific for …" to a particular polypeptide or epitope on a particular polypeptide target, for example, can be defined by having the following dissociation constant (K) for the targetd) The molecule (e.g., antibody) exhibits: at least about 10-4M, at least about 10-5M, at least about 10-6M, at least about 10-7M, at least about 10-8M, at least about 10-9M, at least about 10-10M, at least about 10-11M or at least about 10-12And M. In one embodiment, the term "specifically binds" refers to binding wherein the molecule binds to a particular polypeptide or epitope on a particular polypeptide, but does not substantially bind to any other polypeptide or epitope of the polypeptide.
The 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. For IgG, the 4-chain unit is typically about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds, depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bonds. Each H chain has one variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each alpha and gamma chain, and four CH domains for the mu and epsilon isotypes. Each L chain has a variable domain (VL) at the N-terminus and then a constant domain (CL) at its other end. VL is aligned with VH and CL is aligned with the first constant domain of heavy chain (CH 1). It is believed that particular amino acid residues form an interface between the light and heavy chain variable domains. The pairing of VH and VL together forms a single antigen binding site. For the structure and properties of different classes of antibodies, see Basic and Clinical Immunology, eighth edition, Stits et al (eds.), Appleton & Lange, Norwalk, CT,1994, pp 71 and Chapter 6.
The term "variable region" or "variable domain" refers to a portion of a light or heavy chain of an antibody that is generally located at the amino terminus of the light or heavy chain and is about 120-130 amino acids in the heavy chain and about 100-110 amino acids in the light chain, and is used for the binding and specificity of each particular antibody for its particular antigen. The variable region of the heavy chain may be referred to as "VH". The variable region of the light chain may be referred to as "VL". The term "variable" refers to the fact that certain fragments of the variable region differ greatly in sequence between antibodies. The V region mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed over the 110 amino acid span of the variable region. In contrast, the V region consists of an elongation of less variable (e.g., relatively invariant), termed Framework Regions (FR) of about 15-30 amino acids, separated by a shorter region of greater variable (e.g., extreme variability), termed "hypervariable regions", each of about 9-12 amino acids in length. The variable regions of the heavy and light chains each comprise four FRs, predominantly in the beta-sheet configuration, and are linked by three hypervariable regions which form loops which connect and in some cases form part of the beta-sheet structure. The hypervariable regions in each chain are tightly bound together by the FRs and, together with the hypervariable regions from the other chain, promote the formation of the antigen-binding site of the antibody (see, e.g., Kabat et al, Sequences of Proteins of Immunological Interest, fifth edition, Public Health Service, National Institutes of Health, Bethesda, Md, 1991). The constant regions are not directly involved in binding of the antibody to the antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The variable regions vary greatly in sequence between different antibodies. The variability of the sequence is concentrated in the CDRs, and the smaller variable portions of the variable regions are called Framework Regions (FRs). The CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with the antigen. In particular embodiments, the variable region is a human variable region.
The term "variable region residues as numbered in Kabat" or "amino acid positions as numbered in Kabat" and variants thereof refer to the numbering system used in Kabat et al, Sequences of Proteins of Immunological Interest, fifth edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991) for the variable region of a heavy chain or a variable region of a light chain for antibody assembly. Using this numbering system, the actual linear amino acid sequence may comprise fewer or additional amino acids corresponding to a shortening or insertion of the FRs or CDRs of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat residue numbering of a given antibody can be determined by aligning the regions of sequence homology of the antibody with "standard" Kabat numbered sequences. When referring to residues in the variable domain (about residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system is typically used (e.g., Kabat et al, Sequences of Immunological interest, fifth edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). When referring to residues in the constant region of an immunoglobulin heavy chain, the "EU numbering system" or "EU index" is typically used (e.g., see EU index reported by Kabat et al, supra). "EU index as in Kabat" refers to the residue numbering of the human IgG 1 EU antibody. Other numbering systems have been described including, for example, AbM, Chothia, Contact, IMGT, and AHon.
An "intact" antibody is an antibody comprising an antigen binding site and CL and at least the heavy chain constant regions CH1, CH2, and CH 3. The constant region may comprise a human constant region or an amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions.
The term "antibody fragment" refers to a portion of an intact antibody, preferably the antigen binding or variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab ', F (ab') 2, and Fv fragments; diabodies and di-diabodies (see, e.g., Holliger et al, Proc. Natl. Acad. Sci. U.S.A.1993,90,6444-8; Lu et al, J.biol. chem.2005,280, 19665-72; Hudson et al, nat. Med.2003,9,129-134; WO 93/11161; and U.S. Pat. No. 5,837,242 No. 6,492,123); single chain antibody molecules (see, e.g., U.S. Pat. Nos. 4,946,778; 5,260,203; 5,482,858 and 5,476,786); double variable domain antibodies (see, e.g., U.S. patent No. 7,612,181); single variable domain antibodies (SdAb) (see, e.g., Woolven et al, immunology 1999, 50, 98-101 Streltsov et al, proc. natl. acad. sci. u.s.a.2004,101, 12444-12449); and multispecific antibodies formed from antibody fragments.
The terms "functional fragment," "binding fragment," or "antigen-binding fragment" of an antibody refer to a molecule that exhibits at least one biological function attributed to an intact antibody, including at least binding to a target antigen.
When used with respect to antibodies, the term "heavy chain" refers to a polypeptide chain of about 50-70kDa, wherein the amino terminal portion comprises the variable region of about 120-130 or more amino acids and the carboxy terminal portion comprises the constant region. Based on the amino acid sequence of the heavy chain constant region, the constant region can be one of five different types (e.g., isotypes) called alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ). The different heavy chains vary in size: alpha, delta, and gamma comprise about 450 amino acids, while mu and epsilon comprise about 550 amino acids. When combined with light chains, these different types of heavy chains produce five well-known antibody classes (e.g., isotypes) IgA, IgD, IgE, IgG, and IgM, respectively, including four subclasses of IgG, namely IgG1, IgG2, IgG3, and IgG 4. The heavy chain may be a human heavy chain.
When used with respect to antibodies, the term "light chain" refers to an about 25kDa polypeptide chain, wherein the amino terminal portion comprises a variable region of about 100 to about 110 or more amino acids and the carboxy terminal portion comprises a constant region. The approximate length of the light chain is 211-217 amino acids. Based on the amino acid sequence of the constant domain, there are two different types, called kappa (κ) and lambda (λ). Light chain amino acid sequences are well known in the art. The light chain may be a human light chain.
As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts, and each monoclonal antibody will typically recognize a single epitope on an antigen. In particular embodiments, as used herein, a "monoclonal antibody" is an antibody produced by a single hybridoma or other cell, wherein the antibody binds only to a β -klotho epitope, as determined by ELISA or other antigen binding or competitive binding assays known in the art. The term "monoclonal" is not limited to any particular method of making an antibody. For example, monoclonal antibodies useful in the present disclosure can be prepared by the hybridoma method first described by Kohler et al, Nature 1975, 256, 495; or can be produced in bacterial, eukaryotic animal, or plant cells using recombinant DNA methods (see, e.g., U.S. patent No. 4,816,567). "monoclonal antibodies" can also be isolated from phage antibody libraries, for example, using the techniques described in Clackson et al, Nature 1991, 352, 624-. Other methods of preparing clonal cell lines and monoclonal antibodies expressed therefrom are well known in the art (see, e.g., Short Protocols in Molecular Biology, (2002) fifth edition, Chapter 11, Ausubel et al, eds., John Wiley and Sons, New York). Exemplary methods of producing monoclonal antibodies are provided in the embodiments herein.
"humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies comprising human immunoglobulins (e.g., recipient antibody) in which the native CDR residues are replaced by residues from the corresponding CDR of a non-human species (e.g., donor antibody), such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and capacity. In some cases, one or more FR region residues of a human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may comprise residues not found in the recipient antibody or in the donor antibody. These modifications were made to further improve antibody performance. The humanized antibody heavy or light chain can comprise substantially all of at least one or more variable regions in which all or substantially all of the CDRs correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. In certain embodiments, the humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region. For further details, see Jones et al, Nature 1986,321, 522-525; riechmann et al, Nature 1988,332, 323-329; presta, curr, Opin, Biotechnol.1992, 3, 394-398; carter et al, Proc.Natl.Acad.Sci.U.S.A.1992, 89, 4285-; and U.S. patent nos. 6,800,738, 6,719,971, 6,639,055, 6,407,213, and 6,054,297.
A "human antibody" is an antibody having an amino acid sequence corresponding to an antibody produced by a human and/or an amino acid sequence that has been prepared using any of the techniques disclosed herein for making human antibodies. This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, J.mol.biol.1991,227, 381; Marks et al, J.mol.biol.1991, 222,581) and yeast display libraries (Chao et al, Nature Protocols 2006, 1,755-768) as described in detail below. Also useful for the preparation of human Monoclonal Antibodies are those described in Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985); boerner et al, J.Immunol.1991,147, 86-95. See also van Dijk and van de Winkel, curr, opin, pharmacol, 2001,5, 368-. Human antibodies can be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen challenge, but has a deletion in the endogenous locus, e.g., mouse (see, e.g., Jakobovits, curr. Opin. Biotechnol. 1995,6,561- TMA technique). For human antibodies produced via human B-cell hybridoma technology, see also, for example, Li et al, proc.natl.acad.sci.u.s.a.2006, 103, 3557-3562.
"CDR" refers to one of the three hypervariable regions (H1, H2, or H3) within the non-framework region of an immunoglobulin (Ig or antibody) VH β -sheet framework, or one of the three hypervariable regions (L1, L2, or L3) within the non-framework region of an antibody VL β -sheet framework. Thus, a CDR is a variable region sequence interspersed within a framework region sequence. CDR regions are well known to those skilled in the art and have been defined, for example, by Kabat as the most hypervariable regions within the variable (V) domain of an antibody. Kabat et al, j.biol.chem.1977,252, 6609-6616; kabat, adv. protein chem.1978,32, 1-75. CDR region sequences are also structurally defined by Chothia as those residues that are not conserved beta-sheet frameworks and therefore are able to accommodate different conformations. Chothia and Lesk, J.mol.biol.1987,196, 901-917. Both terms are well known in the art. CDR region sequences are also defined by AbM, Contact, and IMGT. The location of the CDRs within the variable region of a canonical antibody has been determined by comparing a number of structures. Al-Lazikani et Al, J.mol.biol.1997,273, 927-948; morea et al, methods.2000,20, 267-279. Because the number of residues in a hypervariable region varies among antibodies, additional residues relative to the canonical positions are typically numbered a, b, c, etc. next to the residue numbering in the standard variable region numbering scheme. Al-Lazikani et Al, supra (1997). Similarly, such nomenclature is well known to those skilled in the art.
As used herein, the term "hypervariable region", "HVR" or "HV" refers to the regions of an antibody variable region that are hypervariable in sequence and/or form structurally defined loops. Typically, an antibody comprises six hypervariable regions; three of VH (H1, H2, H3) and three of VL (L1, L2, L3). Many hypervariable region descriptions can be used and are encompassed herein. Based on sequence variability, Kabat Complementarity Determining Regions (CDRs) are most commonly used (see, e.g., Kabat et al, Sequences of Proteins of Immunological Interest, fifth edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). In contrast, Chothia refers to the position of the structural loop. See, e.g., Chothia and Lesk, J.mol. biol.1987, 196901-917. When using the Kabat numbering convention, the ends of the Chothia CDR-H1 loops vary according to the length of the loop between H32 and H34 (since the Kabat numbering scheme places insertions on H35A and H35B; if neither 35A nor 35B is present, the loop terminates at 32; if only 35A is present, the loop terminates at 33; if both 35A and 35B are present, the loop terminates at 34). The AbM hypervariable region represents a compromise between the Kabat CDRs and the Chothia structural loops and is used by Oxford Molecular's AbM Antibody modeling software (see, e.g., Martin, in Antibody Engineering, volume 2, chapter 3, Springer Verlag). The "Contact" hypervariable region is based on an analysis of the available complex crystal structure. Residues from each of these hypervariable regions or CDRs are shown below.
Recently, a universal numbering system, the ImmunoGeneTiCs (IMGT) information system, has been developed and widely adopted. Lafranc et al, Dev.Comp. Immunol.2003,27, 55-77. IMGT is an integrated information system dedicated to the Immunoglobulins (IG), T cell receptors (TR) and Major Histocompatibility Complex (MHC) of humans and other vertebrates. Herein, a CDR refers to an amino acid sequence and position within a light chain or heavy chain. Since the "position" of CDRs within an immunoglobulin variable domain structure is conserved across species and exists in a structure called a loop, CDR and framework residues are readily identified by using a numbering system that aligns the variable domain sequences according to structural features. This information can be used to graft and replace CDR residues of immunoglobulins of one species with acceptor frameworks that are typically derived from human antibodies. An additional numbering system (AHon) has been developed by Honegger and Pl ü ckthun, J.Mol.biol.2001,309, 657-. The correspondence between numbering systems, including, for example, the Kabat numbering and the unique numbering system of IMGT, is well known to those skilled in the art (see, e.g., Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al, supra). The exemplary system presented herein combines Kabat and Chothia.
Hypervariable regions may include the following "extended hypervariable regions": 24-36 or 24-34(L1), 46-56 or 50-56(L2) and 89-97 or 89-96(L3) in VL and 26-35 or 26-35A (H1), 50-65 or 49-65(H2) and 93-102, 94-102 or 95-102 (H3) in VH. As used herein, the terms "HVR" and "CDR" are used interchangeably.
Exemplary embodiments of the invention
IMGT
Kabat
AbM
Chothia
Contact
VH CDR1
26-35
27-38
31-35
26-35
26-32
30-35
VH CDR2
50-65
56-65
50-65
50-58
53-55
47-58
VH CDR3
95-102
105-117
95-102
95-102
96-101
93-101
VL CDR1
24-34
27-38
24-34
24-34
26-32
30-36
VL CDR2
50-56
56-65
50-56
50-56
50-52
46-55
VL CDR3
89-97
105-117
89-97
89-97
91-96
89-96
The term "constant region" or "constant domain" refers to the carboxy-terminal portion of the light and heavy chains that are not directly involved in binding of the antibody to the antigen, but exhibit multiple effector functions, such as interaction with an Fc receptor. The term refers to a portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to another portion of the immunoglobulin (the variable region comprising the antigen binding site). The constant region may comprise the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.
The term "framework" or "FR" residues are those variable region residues that flank the CDRs. FR residues are present in, for example, chimeric, humanized, human, domain antibody, diabody, linear antibody and bispecific antibody. FR residues are those variable domain residues other than the hypervariable region residues or CDR residues.
An "affinity matured" antibody is an antibody that has one or more alterations (e.g., amino acid sequence changes, including alterations, additions, and/or deletions) in one or more HVRs thereof that result in an improvement in the affinity of the antibody as compared to a parent antibody that does not have such alterations. Preferred affinity matured antibodies will have nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies are produced by methods known in the art. See reviews Hudson and Souriau, nat. Med.2003,9, 129-; hoogenboom, nat. Biotechnol.2005,23, 1105-1116; quiroz and Sinclair, Revista Ingenieria biomedicala 2010,4, 39-51.
A "blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces antigen binding. In certain embodiments, a blocking antibody or antagonist antibody substantially or completely blocks binding of an antigen. In certain embodiments, a "blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. In other embodiments, the blocking or antagonist antibody substantially or completely inhibits the biological activity of the antigen. For example, a blocking anti-SIRP antibody substantially or completely prevents the interaction between sipra and CD 47.
A "non-blocking" antibody is an antibody that does not inhibit or reduce antigen binding. In certain embodiments, a "non-blocking" antibody is an antibody that does not inhibit or reduce the biological activity of the antigen to which it binds. In other embodiments, the non-blocking antibody binds to a unique and non-overlapping epitope bound by the antigen. In some embodiments, the non-blocking antibody is an agonist antibody.
An "agonist antibody" is an antibody that triggers a response, e.g., a response that mimics at least one functional activity of a polypeptide of interest. Agonist antibodies include antibodies that are ligand mimetics, for example, where the ligand binds to a cell surface receptor and the binding induces cell signaling or activity through an intercellular cell signaling pathway, and where the antibody induces similar cell signaling or activation.
Antibody "effector functions" refer to those biological activities that are attributed to the Fc region of an antibody (e.g., a native sequence Fc region or an amino acid sequence variant Fc region), and that vary with the antibody isotype. Examples of antibody effector functions include: c1q binding and complement dependent cytotoxicity; fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulating cell surface receptors (e.g., B cell receptors); and B cell activation.
The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the Fc region of a human IgG heavy chain is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxy terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding the heavy chain of the antibody. Thus, a composition of intact antibodies may comprise a population of antibodies with all K447 residues removed, a population of antibodies without K447 residues removed, and a population of antibodies with a mixture of antibodies with and without K447 residues.
The term "treating" is intended to include reducing or eliminating a condition, disorder or disease, or one or more symptoms associated with the condition, disorder or disease; or to reduce or eliminate the etiology of the condition, disorder or disease itself.
The term "preventing" is intended to include methods of delaying and/or preventing the onset of a condition, disorder or disease and/or its attendant symptoms; preventing the subject from acquiring the condition, disorder or disease; or reducing the risk of a disorder, condition or disease in a subject.
The term "contacting" refers to bringing the therapeutic agent and the cell or tissue together such that a physiological and/or chemical effect occurs as a result of such contact. The contacting can be performed in vitro, ex vivo, or in vivo. In one embodiment, the therapeutic agent is contacted with cells in cell culture (in vitro) to determine the effect of the therapeutic agent on the cells. In another embodiment, contacting a therapeutic agent with a cell or tissue comprises administering the therapeutic agent to a subject having the cell or tissue to be contacted.
The term "therapeutically effective amount" means an amount of a compound that, when administered, includes that amount sufficient to prevent or to some extent reduce the development of one or more symptoms of the condition, disorder or disease being treated. The term "therapeutically effective amount" also refers to an amount of a compound that is sufficient to elicit the biological or medical response of a biomolecule (e.g., protein, enzyme, RNA or DNA), cell, tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or clinician.
The terms "pharmaceutically acceptable carrier", "pharmaceutically acceptable excipient", "physiologically acceptable carrier" or "physiologically acceptable excipient" refer to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, solvent or encapsulating material. In one embodiment, each component is "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the pharmaceutical formulation and suitable for contact with the tissues or organs of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio. See, Remington, The Science and Practice of Pharmacy, 22 nd edition; allen ed.: philiadelphia, PA, 2012; handbook of Pharmaceutical Excipients, 7 th edition; rowe et al, eds.; the Pharmaceutical Press and The American Pharmaceutical Association: 2012; handbook of Pharmaceutical Additives, third edition; ash and Ash eds.; gower Publishing Company 2007; pharmaceutical preparation and Formulation,2nd ed.; gibson ed.; CRC Press LLC: Boca Raton, FL, 2009.
The term "CpG-Ab immunoconjugate" or "CpG-Ab" as used herein refers to the linkage of an antibody (Ab) or antigen-binding fragment thereof to a CpG-containing immunostimulatory polynucleotide described herein.
As used herein, the term "T cell agonist" refers to any agent that selectively stimulates proliferation, differentiation and/or survival of T cells from a mixed starting cell population. Thus, the resulting cell population is enriched for increased numbers 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, antigenic molecules that specifically bind to T Cell Receptors (TCRs), 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 ligands. In particular embodiments, the T cell agonist is an antibody directed against a T cell costimulatory molecule. In a particular embodiment, the T cell agonist is a Tumor Associated Antigen (TAA). In particular embodiments, the T cell agonist is a pathogenic antigen.
As used herein, an "immune checkpoint" or "immune checkpoint molecule" is a molecule that modulates a signal in the immune system. The immune checkpoint molecule may be a stimulatory checkpoint molecule, i.e. up-regulating signal, or an inhibitory checkpoint molecule, i.e. down-regulating signal. In particular embodiments, the immune checkpoint is a protein expressed by a T cell or by an Antigen Presenting Cell (APC). Certain types of cancer cells express immune checkpoint proteins to evade immune clearance. The use of immune checkpoint modulators to inhibit the interaction between an immune checkpoint protein expressed by cancer cells and an immune checkpoint protein expressed by T cells has been shown to be effective in certain cancer treatments.
As used herein, an "immune checkpoint modulator" is an agent capable of altering 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 may 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 Fab fragment, a bivalent antibody, an antibody drug conjugate, an scFv, a fusion protein, a bivalent antibody, or a tetravalent antibody). In other embodiments, the immune checkpoint modulator is a small molecule. In particular embodiments, the immune checkpoint modulator is an anti-PD 1 or anti-PD-L1 antibody.
As used herein, the term "targeted delivery" or the verb form "target" refers to the process of facilitating delivery of an agent (e.g., an immunostimulatory polynucleotide) to a particular organ, tissue, cell, and/or intracellular compartment (referred to as a targeted location) more than any other organ, tissue, cell, or intracellular compartment (referred to as a non-target location). Targeted delivery can be detected using methods known in the art, e.g., by comparing the concentration of the delivery agent of the targeted cell population to the concentration of the delivery agent of a non-targeted cell population following systemic administration. As provided herein, targeted delivery results in a concentration at the targeted location that is at least 2-fold higher than the non-target location. Targeted delivery may be achieved by specific binding of a targeting moiety to a receiving moiety associated with the targeted cell. As used herein, a receiving moiety associated with a targeted cell may be located on the surface of the targeted cell or within the cytosol. In some embodiments, the receiving moiety is an antigen associated with the targeted cell.
The term "DAR" refers to the drug-to-antibody ratio, more specifically, the immunomodulatory polynucleotide-to-antibody ratio, of an immunomodulatory polynucleotide-antibody conjugate.
Immunomodulatory polynucleotides
In one embodiment, provided herein is an immunomodulatory (e.g., immunostimulatory) polynucleotide.
In certain embodiments, the immunomodulatory polynucleotide comprises a 5-modified uridine or a 5-modified cytidine. In certain embodiments, the inclusion of a 5-modified uridine (e.g., 5-ethynyl-uridine) at the 5 '-terminus (e.g., among the two 5' -terminal nucleosides) of an immunomodulatory polynucleotide enhances the immunomodulatory properties of the polynucleotide. In certain embodiments, the immunomodulatory polynucleotide is shorter (e.g., comprises a total of about 6 to about 16 nucleotides or about 12 to about 14 nucleotides) than a typical CpG ODN that is 18 to 28 nucleotides in length. In certain embodiments, shorter immunomodulatory polynucleotides (e.g., those comprising a total of about 6 to about 16 nucleotides or about 12 to about 14 nucleotides) retain immunomodulatory activity of longer typical CpG ODNs; or a higher immunomodulatory activity (e.g., as measured by NF-. kappa.B activation or by a change in the expression level of at least one cytokine (e.g., IL-6 or IL-10)) as compared to longer CpG ODNs. In certain embodiments, the immunomodulatory polynucleotide comprises a base-free spacer. In certain embodiments, the immunomodulatory polynucleotide comprises an internucleoside phosphotriester, and exemplary descriptions of immunomodulatory polypeptides may be found in WO 2018189382.
In certain embodiments, the immunomodulatory polynucleotides provided herein exhibit stability (e.g., stability to nucleases) that is superior to the stability of CpG ODNs comprising predominantly internucleoside phosphates (e.g., greater than 50% of 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%) of an internucleoside phosphorothioate or phosphorodithioate or by including an internucleoside phosphotriester and/or an internucleoside abasic spacer. Phosphotriesters and base-free spacers are also conveniently conjugated to the targeting moiety. Phosphate-based phosphotriesters and abasic spacers may also be used to reduce off-target activity relative to polynucleotides having a complete phosphorothioate backbone. Without wishing to be bound by theory, this effect may be achieved by reducing self-delivery without disrupting targeting moiety-mediated delivery to the target cell. Thus, the polynucleotides provided herein can comprise about 15 or less, about 14 or less, about 13 or less, about 12 or less, about 11 or less, or about 10 or less contiguous internucleoside phosphorothioates. For example, an immunostimulatory polynucleotide comprising a total of about 12 to about 16 nucleosides can comprise about 10 or fewer consecutive internucleoside phosphorothioates.
Immunostimulatory polynucleotides provided herein can comprise a total of about 50 or fewer, about 30 or fewer, about 28 or fewer, or about 16 or fewer nucleosides. The immunostimulatory polynucleotide may comprise a total of at least 6, about 10 or more, or about 12 or more nucleosides. For example, an immunostimulatory polynucleotide may comprise a total of about 6 to about 30, about 6 to about 28, about 6 to about 20, about 6 to about 16, about 10 to about 20, about 10 to about 16, about 12 to about 28, about 12 to about 20, or about 12 to about 16 nucleosides.
In certain embodiments, the immunostimulatory polynucleotide comprises one or more phosphotriesters (e.g., internucleoside phosphotriesters) and/or phosphorothioates (e.g., about 1 to about 6 or about 1 to about 4) at one or both termini (e.g., within the six 5 '-terminal nucleosides or the six 3' -terminal nucleosides). The inclusion of one or more internucleoside phosphotriesters and/or phosphorothioates may enhance the stability of the polynucleotide by reducing the rate of exonuclease-mediated degradation.
In certain embodiments, the immunostimulatory polynucleotide comprises a phosphotriester or a terminal phosphodiester, wherein the phosphotriester or terminal phosphodiester comprises a linker bonded to a targeting moiety or a conjugate group and optionally to one or more (e.g., about 1 to about 6) auxiliary moieties. In certain embodiments, the immunostimulatory polynucleotide comprises only one linker. In certain embodiments, the immunostimulatory polynucleotide comprises only one conjugate group.
The polynucleotides provided herein can be hybridized polynucleotides, including the strands and partial or complete complements thereof. The hybridized polynucleotide may have at least 6 complementary base pairs (e.g., about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, or about 23) up to the total number of nucleotides present in the shorter chain comprised. For example, the hybridizing portion of the hybridizing polynucleotide may comprise about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, or about 23 base pairs.
In one embodiment, provided herein is an immunostimulatory polynucleotide of formula (a):
X5′-(XN)b-YP-(XN)C-X3′ (A)
or a stereoisomer, a mixture of two or more diastereomers, a tautomer, or a mixture of two or more tautomers thereof; or a pharmaceutically acceptable salt, solvate or hydrate thereof; wherein:
each XNIndependently a nucleotide;
X3’is a 3' terminal nucleotide;
X5’is a 5' terminal nucleotide;
YPis an internucleoside phosphotriester; and
b and c are each independently integers of from about 0 to about 25; provided that the sum thereof is not less than 5.
In certain embodiments, the immunostimulatory polynucleotide comprises a nucleotide having a modified nucleobase.
In certain embodiments, b is an integer from 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.
In certain embodiments, c is an integer from 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.
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.
In certain embodiments, b and c together total from about 5 to about 20. In certain embodiments, b and c together total from about 5 to about 15. In certain embodiments, b and c together total 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 and c together total about 8, about 9, about 10, about 11, about 12, about 13, or about 14. In certain embodiments, b and c together total about 11. In certain embodiments, b and c together total about 12. In certain embodiments, b and c together total about 14.
In certain embodiments, each X isNIndependently a 2 '-deoxyribonucleotide or a 2' -modified ribonucleotide. In certain embodiments, each X isNIndependently 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 isNIndependently a 2' -deoxyribonucleotide. In certain embodiments, each X is NIndependently 2 ' -deoxyadenosine, 2 ' -deoxyguanosine, 2 ' -deoxycytidine, 5-halo-2 ' -deoxycytidine, 2 ' -deoxythymidine, 2 ' -deoxyuridine or 5-halo-2 ' -deoxyuridine. In certain embodiments, each X isNIndependently 2 '-deoxyadenosine, 2' -deoxyguanosine, 2 '-deoxycytidine, 2' -deoxythymidine, 5-bromo-2 '-deoxyuridine or 5-iodo-2' -deoxyuridine.
In certain embodiments, X3’Is a 2 '-deoxyribonucleotide or a 2' -modified ribonucleotide. In certain embodiments, X3’Is a 2' -deoxyribonucleotide. In certain embodiments, X3’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, X3’Is 2 '-deoxyadenosine, 2' -deoxyguanosine, 2 '-deoxycytidine, 5-halo-2' -deoxycytidine, 2 '-deoxythymidine, 2' -deoxyuridine or 5-haloAnd (3) substituted-2' -deoxyuridine. In certain embodiments, X3’Is 2' -deoxythymidine. In certain embodiments, X 3’Is a 2' -deoxyribonucleotide having a substituted pyrimidine base. In certain embodiments, X3’Is a 2' -deoxyribonucleotide having a 5-substituted pyrimidine base. In certain embodiments, X3’Is 2 ' -deoxythymidine, 5-halo-2 ' -deoxycytidine or 5-halo-2 ' -deoxyuridine. In certain embodiments, X3’Is 2 ' -deoxythymidine, 5-bromo-2 ' -deoxycytidine, 5-iodo-2 ' -deoxycytidine, 5-bromo-2 ' -deoxyuridine or 5-iodo-2 ' -deoxyuridine. In certain embodiments, X3’Is 2 ' -deoxythymidine, 5-bromo-2 ' -deoxyuridine or 5-iodo-2 ' -deoxyuridine. In certain embodiments, X3’Is a terminal nucleotide comprising a 3' blocking group. In certain embodiments, the 3' end capping group is a terminal phosphate. In certain embodiments, the 3' end capping group is 3-hydroxy-propylphosphoryl (i.e., -P (O)2)-OCH2CH2CH2OH)。
In certain embodiments, X5’Is a 2 '-deoxyribonucleotide or a 2' -modified ribonucleotide. In certain embodiments, X5’Is a 2' -deoxyribonucleotide. In certain embodiments, X5’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, X5’Is a 2' -deoxyribonucleotide having a substituted pyrimidine base. In certain embodiments, X5’Is a 2' -deoxyribonucleotide having a 5-substituted pyrimidine base. In certain embodiments, X5’Is 2 '-deoxythymidine, 5-halo-2' -deoxycytidineOr 5-halo-2' -deoxyuridine. In certain embodiments, X5’Is 5-halo-2' -deoxycytidine. In certain embodiments, X5’Is 5-halo-2' -deoxyuridine. In certain embodiments, X5’Is 2 ' -deoxythymidine, 5-bromo-2 ' -deoxycytidine, 5-iodo-2 ' -deoxycytidine, 5-bromo-2 ' -deoxyuridine or 5-iodo-2 ' -deoxyuridine. In certain embodiments, X5’Is 2 ' -deoxythymidine, 5-bromo-2 ' -deoxyuridine or 5-iodo-2 ' -deoxyuridine. In certain embodiments, X5’Is 5-bromo-2' -deoxyuridine. In certain embodiments, X5’Is 5-iodo-2' -deoxyuridine. In certain embodiments, X5’Having a 3' -phosphorothioate group. In certain embodiments, X 5’A 3' -phosphorothioate group having chirality Rp. In certain embodiments, X5’A 3' -phosphorothioate group having Sp chirality.
In certain embodiments, YPIs an internucleoside phosphorothioate triester.
In certain embodiments, YPThe method comprises the following steps:
wherein Z is O or S; and d is an integer from about 0 to about 50. In certain embodiments, Z is O. In certain embodiments, Z is S. In certain embodiments, d is an integer from about 0 to about 10. In certain embodiments, d is an integer from about 0 to about 5. In certain embodiments, d is an integer of about 0, about 1, about 2, about 3, about 4, or about 5. In certain embodiments, d is an integer of about 0, about 1, or about 3.
In certain embodiments, YPThe method comprises the following steps:
wherein Z is O or S; and d is an integer from about 0 to about 50. In certain embodiments, YPThe method comprises the following steps:
wherein Z is O or S; and d is an integer from about 0 to about 50. In certain embodiments, YPThe method comprises the following steps:
wherein Z is O or S; and d is an integer from about 0 to about 50. In certain embodiments, Z is O. In certain embodiments, Z is S. In certain embodiments, d is an integer from about 0 to about 10. In certain embodiments, d is an integer from about 0 to about 5. In certain embodiments, d is an integer of about 0, about 1, about 2, about 3, about 4, or about 5. In certain embodiments, d is an integer of about 0, about 1, or about 3.
In certain embodiments, the immunostimulatory polynucleotide of formula (a) comprises one additional internucleoside phosphotriester. In one embodiment, the additional internucleoside phosphotriester is C1-6Alkyl phosphotriester. In another embodiment, the additional internucleoside phosphotriester is ethyl phosphotriester.
In certain embodiments, the immunostimulatory polynucleotide of formula (a) comprises 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 another embodiment, the 5-halo-2 '-deoxyuridine is 5-fluoro-2' -deoxyuridine. In another embodiment, the 5-halo-2 'deoxyuridine is 5-bromo-2' -deoxyuridine. In another embodiment, the 5-halo-2 '-deoxyuridine is 5-iodo-2' -deoxyuridine.
In certain embodiments, the immunostimulatory polynucleotide of formula (a) comprises three or more 2' -deoxycytidines. In certain embodiments, the immunostimulatory polynucleotide of formula (a) comprises three 2' -deoxycytidines.
In certain embodiments, the immunostimulatory polynucleotide of formula (a) comprises four or more 2' -deoxyguanosines. In certain embodiments, the immunostimulatory polynucleotide of formula (a) comprises four 2' -deoxyguanosines.
In certain embodiments, the immunostimulatory polynucleotide of formula (a) comprises three 2 '-deoxycytidines and four 2' -deoxyguanosines. In certain embodiments, the immunostimulatory polynucleotide of formula (a) comprises one, two, or three CG dinucleotides. In certain embodiments, the immunostimulatory polynucleotide of formula (a) comprises three CG dinucleotides.
In certain embodiments, the immunostimulatory polynucleotide of formula (a) comprises three or more 2' -deoxythymidine. In certain embodiments, the immunostimulatory polynucleotide of formula (a) comprises three, four, five, six, seven or eight 2' -deoxythymidine. In certain embodiments, the immunostimulatory polynucleotide of formula (a) comprises three, four, five or eight 2' -deoxythymidine.
In certain embodiments, the immunostimulatory polynucleotide of formula (a) does not comprise 2' -deoxyadenosine. In certain embodiments, the immunostimulatory polynucleotide of formula (a) comprises one or two 2' -deoxyadenosines.
In certain embodiments, the immunostimulatory polynucleotide of formula (a) is about 5 to about 20 or about 6 to about 15 in length. In certain embodiments, the immunostimulatory polynucleotide of formula (a) is about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 in length. In certain embodiments, the immunostimulatory polynucleotide of formula (a) is about 10, about 11, about 12, about 13, about 14, or about 15 in length.
In certain embodiments, the immunostimulatory polynucleotide of formula (a) comprises one or more internucleoside phosphorothioates. In certain embodiments, all of the internucleoside phosphates in the immunostimulatory polynucleotide of formula (a) are internucleoside phosphorothioates. In certain embodiments, the immunostimulatory polynucleotide of formula (a) comprises one or more chiral internucleoside phosphorothioates.
In certain embodiments, the immunostimulatory polynucleotide of formula (a) is p275, p276, p313 or p 347. In certain embodiments, the immunostimulatory polynucleotide of formula (a) is p236, p238, p243, p246, p308, p361, p362 or p 425. In certain embodiments, the immunostimulatory polynucleotide 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 p 489.
In one embodiment, provided herein is a polypeptide having the sequence N1N2CGN3CG(T)xGN4CGN5An immunostimulatory polynucleotide of T (SEQ ID NO:586), or a stereoisomer, a mixture of two or more diastereomers, a tautomer, or a mixture of two or more tautomers thereof; or a pharmaceutically acceptable salt, solvate or hydrate thereof; wherein:
x is an integer from about 1 to about 4;
N1absent or 2' -deoxythymidine;
N2is a 2' -deoxyribonucleotide having a modified nucleobase;
N3is 2 ' -deoxyadenosine or 2 ' -deoxythymidine, each optionally comprising a 3 ' -phosphotriester;
N4is 2 '-deoxyadenosine or 2' -deoxythymidine;
N5is 2 '-deoxythymidine optionally comprising a 3' -phosphotriester; and
c is 2 '-deoxycytidine and G is 2' -deoxyguanosine.
In certain embodiments, at N1N2CGN3CG(T)xGN4CGN5In T (SEQ ID NO:586), x is an integer of about 1, about 2, about 3, or about 4. In certain embodiments, at N1N2CGN3CG(T)xGN4CGN5In T (SEQ ID NO:586), x is an integer of about 1. In certain embodimentsIn N at1N2CGN3CG(T)xGN4CGN5In T (SEQ ID NO:586), x is an integer of about 4.
In certain embodiments, at N1N2CGN3CG(T)xGN4CGN5In T (SEQ ID NO:586), N1Is absent. In certain embodiments, at N1N2CGN3CG(T)xGN4CGN5In T (SEQ ID NO:586), N 1Is 2' -deoxythymidine.
In certain embodiments, at N1N2CGN3CG(T)xGN4CGN5In T (SEQ ID NO:586), N2Is a 2' -deoxyribonucleotide having a substituted pyrimidine base. In certain embodiments, at N1N2CGN3CG(T)xGN4CGN5In T (SEQ ID NO:586), N2Is a 2' -deoxyribonucleotide having a 5-substituted pyrimidine base. In certain embodiments, at N1N2CGN3CG(T)xGN4CGN5In T (SEQ ID NO:586), N2Is 5-halo-2 '-deoxycytidine or 5-halo-2' -deoxyuridine. In certain embodiments, at N1N2CGN3CG(T)xGN4CGN5In T (SEQ ID NO:586), N2Is 5-bromo-2 '-deoxyuridine or 5-iodo-2' -deoxyuridine.
In certain embodiments, at N1N2CGN3CG(T)xGN4CGN5In T (SEQ ID NO:586), N3Is 2 '-deoxyadenosine which comprises a 3' -phosphotriester. In certain embodiments, at N1N2CGN3CG(T)xGN4CGN5In T (SEQ ID NO:586), N3Is 2' -deoxythymidine. In certain embodiments, at N1N2CGN3CG(T)xGN4CGN5In T (SEQ ID NO:586), N3Is a 2 '-deoxythymidine comprising a 3' -phosphotriester.
In certain embodiments, at N1N2CGN3CG(T)xGN4CGN5In T (SEQ ID NO:586), N4Is 2' -deoxyadenosine. In certain embodiments, at N1N2CGN3CG(T)xGN4CGN5In T (SEQ ID NO:586), N4Is 2' -deoxythymidine.
In certain embodiments, at N1N2CGN3CG(T)xGN4CGN5In T (SEQ ID NO:586), N5Is 2' -deoxythymidine. In certain embodiments, at N 1N2CGN3CG(T)xGN4CGN5In T (SEQ ID NO:586), N5Is a 2 '-deoxythymidine comprising a 3' -phosphotriester.
In certain embodiments, N1N2CGN3CG(T)xGN4CGN5The immunostimulatory polynucleotide of T (SEQ ID NO:586) comprises one or more internucleoside phosphorothioates. In certain embodiments, N1N2CGN3CG(T)xGN4CGN5The immunostimulatory polynucleotide of T (SEQ ID NO:586) comprises at least one chiral internucleoside phosphorothioate.
In certain embodiments, N1N2CGN3CG(T)xGN4CGN5The immunostimulatory polynucleotide of T is p275, p276 or p 313. In certain embodiments, N1N2CGN3CG(T)xGN4CGN5The immunostimulatory polynucleotide of T (SEQ ID NO:586) is p236, p238, p243, p246, p308, p361, p362, or p 425. In certain embodiments, N1N2CGN3CG(T)xGN4CGN5The immunostimulatory polynucleotide 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 p 489.
In certain embodiments, an immunostimulatory polynucleotide provided herein is an immunostimulatory polynucleotide. In certain embodiments, the immunostimulatory polynucleotides provided herein are used as PAMS. In certain embodiments, the immunostimulatory polynucleotides provided herein activate an innate immune response or stimulate an adaptive immune response by triggering TLR9 signaling. In certain embodiments, the immunostimulatory polynucleotide provided herein is a TLR9 agonist.
In certain embodiments, the immunostimulatory polynucleotides provided herein are B-class CpG polynucleotides or modifications thereof, including 5-halouridine or 5-alkynyluridine or truncated forms thereof (e.g., those comprising a total of about 6 to about 16 nucleosides). In certain embodiments, the truncated immunostimulatory polynucleotides provided herein comprise a truncated B class CpG polynucleotide sequence (e.g., a B class CpG polynucleotide sequence from which one or more 3' -terminal nucleotides are eliminated or one or more nucleotides within the sequence are deleted).
In certain embodiments, an immunostimulatory polynucleotide provided herein comprises at least one immunostimulatory sequence (ISS). In certain embodiments, an immunostimulatory polynucleotide provided herein comprises about 1, about 2, about 3, or about 4 ISS. The ISS in the immunostimulatory polynucleotides depends on the target organism. A common feature of ISS used in the immunostimulatory polynucleotides provided herein is a cytidine-p-guanosine sequence, where p is an internucleoside phosphodiester (e.g., a phosphate or phosphorothioate) or an internucleoside phosphotriester. In certain embodiments, cytidine and guanosine in the ISS each independently comprise 2' -deoxyribose. In certain embodiments, an immunostimulatory polynucleotide provided herein comprises about 1, about 2, or about 3 human ISS. In certain embodiments, the human ISS is CG or NCG, wherein N is uridine, cytidine, or thymidine, or a modified uridine or cytidine; and G is guanosine or a modified guanosine. In certain embodiments, the modified uridine or cytidine is a 5-halogenated uridine (e.g., 5-iodouridine or 5-bromouridine), a 5-alkynyluridine (e.g., 5-ethynyluridine or 5-propynyluridine), a 5-heteroaryl uridine, or a 5-halogenated cytidine. In certain embodiments, the modified guanosine is 7-deazaguanosine. In certain embodiments, the human ISS is NCG, and in one embodiment, N is 5-halouridine. In certain embodiments, the human ISS is UCG, in one embodiment U is 5-alkynyluridine, in another embodiment U is 5-ethynyluridine. In certain embodiments, the immunostimulatory polynucleotides provided herein that target humans comprise ISS within four consecutive nucleotides comprising a 5' terminal nucleotide. In certain embodiments, the immunostimulatory polynucleotides provided herein that are targeted to humans comprise a 5' -terminal ISS. In certain embodiments, the immunostimulatory polynucleotides provided herein comprise a murine ISS. In certain embodiments, the murine ISS is a hexameric nucleotide sequence: Pu-Pu-CG-Py-Py, wherein each Pu is independently a purine nucleotide and each Py is independently a pyrimidine nucleotide.
In certain embodiments, the 5 '-flanking nucleotides of CpG in the immunostimulatory polynucleotides provided herein do not comprise a 2' -alkoxyribose. In certain embodiments, the 5 '-flanking nucleotides relative to CpG in the immunostimulatory polynucleotides provided herein comprise only 2' -deoxyribose as the sugar.
In certain embodiments, the immunostimulatory polynucleotides provided herein have (1) a high content of phosphorothioates (e.g., at least 50%, at least 60%, at least 70%, or at least 80% of the nucleosides can be linked by phosphorothioates); (2) no poly G tail is present; (3) the nucleoside in the immunostimulatory polynucleotide comprises a 2 '-deoxyribose or a 2' -modified ribose (e.g., 2 '-halo (e.g., 2' -fluoro) or optionally substituted 2 '-alkoxy (e.g., 2' -methoxy)); and/or (4) comprises a 5' -terminal ISS that is an NCG, wherein N is uridine, cytidine, or thymidine, or modified uridine or cytidine, and G is guanosine or modified guanosine.
In certain embodiments, the immunomodulatory polynucleotides provided herein inhibit an adaptive immune response by reducing activation of TLR9 signaling (e.g., by TLR9 antagonism). In certain embodiments, the immunosuppressive polynucleotides provided herein comprise at least two 2 '-alkoxynucleotides that flank 5' -relative to CpG, such as formula N 1-N2-CG of, wherein N1And N2Each independently is a nucleotide comprising a 2 '-alkoxyribose (e.g., 2' -methoxyribose).
Structural characterization of immunomodulatory polynucleotides
Abasic spacer
In certain embodiments, the immunomodulatory polynucleotides provided herein comprise one or more, in one embodiment, one or two abasic spacers, each of which is independently an internucleoside abasic spacer or a terminal abasic spacer. When the immunomodulatory polynucleotide comprises two or more abasic spacers, the structures of the abasic spacers may be the same or different.
In one embodiment, the base-free spacer has the formula (I):
R1–L1–[–L2–(L1)n1–]n2–R2,
(I)
wherein:
n1 is an integer of about 0 or about 1,
n2 is an integer of from about 1 to about 6,
R1is a bond to a nucleotide in an immunomodulatory polynucleotide,
R2is a bond to a nucleoside or a capping group in an immunomodulatory polynucleotide,
each L1Independently a phosphodiester or phosphotriester, and
each L2Is a carbohydrate analog.
In certain embodiments, if the abasic spacer is an internucleoside abasic spacer, then n1 is about 1, and R is2Is a bond to a nucleoside; if the abasic spacer is a terminal abasic spacer, then n1 is about 0 or about 1, and R 2Is a bond to the end-capping group.
In certain embodiments, the abasic spacer is an internucleoside abasic spacer. In certain embodiments, the abasic spacer is a 3' -terminal abasic spacer.In certain embodiments, every two consecutive L2Radical is represented by L1The groups being spaced apart (e.g. for arrangement in two successive L' s2L between radicals1And n1 is 1).
In certain embodiments, the immunostimulatory polynucleotides provided herein comprise an ISS located within four consecutive nucleotides, including the 5' -terminal nucleotide of the immunostimulatory polynucleotide, wherein the ISS is NCG, wherein N is uridine, cytidine, or thymidine, or in one embodiment the modified uridine or cytidine is a 5-halouridine (e.g., 5-iodouridine or 5-bromouridine), a 5-alkynyluridine (e.g., 5-ethynyluridine or 5-propynyluridine), a 5-heteroaryl uridine, or a 5-halocytidine; and wherein N and C are linked to each other through a phosphodiester or phosphotriester.
Sugar analogs
In one embodiment, the saccharide analog is as C3-6Monosaccharides or C3-6A divalent or trivalent group of a sugar alcohol (e.g., glycerol) 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 the other phosphate ester or to a capping group. The carbohydrate analogs are cyclic or acyclic. Other optional modifications comprised in the carbohydrate analogues are: replacing one, two or three of the remaining hydroxyl groups or carbon-bonded hydrogen atoms with H; optionally substituted C 1-6An alkyl group; as defined herein-LinkA (-T)p(ii) a A conjugate group; - (CH)2)t1–ORZWherein t1 is an integer from about 1 to 6, and RZIs optionally substituted C1-6Alkyl, optionally substituted C2-6Alkenyl, optionally substituted C2-6Alkynyl, optionally substituted C6-14Aryl, optionally substituted C3-8Cycloalkyl, optionally substituted (C)1-9Heterocyclyl) -C1-6Alkyl, optionally substituted (C)6-10Aryl) -C1-6-alkyl or optionally substituted (C)3-8Cycloalkyl) -C1-6-an alkyl group; introduction of one or two unsaturated bonds (e.g., one or two double bonds); and one is substituted with a substituent as defined for alkyl, alkenyl, cycloalkyl, cycloalkenyl or heterocyclylOne, two or three hydrogen or hydroxyl groups. In some embodiments, RZIs optionally substituted C1-6Aminoalkyl (e.g., optionally substituted-NH containing)2C of (A)1-6Aminoalkyl).
Non-limiting examples of carbohydrate analogs are optionally substituted C2-6Alkylene, optionally substituted C2-6Alkenylene, optionally substituted C5Cycloalkane-1, 3-diyl, optionally substituted C5Cycloalkene-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) 1-4Alkyl group) - (C3-8Cycloalkylene) (e.g. optionally substituted (C)1Alkyl group) - (C3Cycloalkylene)). Non-limiting examples of carbohydrate analogs are:
wherein:
R1and R2Each is independently a bond to an oxygen atom in the phosphate ester;
R3and R4Each is independently H; optionally substituted C1-6An alkyl group; - (CH)2)t1–ORZ(ii) a or-LinkA-RT；
Wherein t1 is an integer from about 1 to about 6;
RZis optionally substituted C1-6Alkyl, optionally substituted C2-6Alkenyl, optionally substituted C2-6Alkynyl, optionally substituted C6-14Aryl, optionally substituted C3-8Cycloalkyl, optionally substituted (C)1-9Heterocyclyl) -C1-6Alkyl, optionally substituted (C)6-10Aryl) -C1-6Alkyl, optionally substituted (C)3-8Cycloalkyl) -C1-6-an alkyl group;
LinkA is a linker; and
RTis associated with a targeting moiety; a conjugate moiety; optionally substitutedC of (A)1-6Alkyl, optionally substituted C2-6Alkenyl, optionally substituted C2-6Alkynyl, optionally substituted C6-14Aryl, optionally substituted C3-8Cycloalkyl, optionally substituted (C)1-9Heterocyclyl) -C1-6Alkyl, optionally substituted (C)6-10Aryl) -C1-6-alkyl or optionally substituted (C)3-8Cycloalkyl) -C1-6-a bond to an alkyl group.
In some embodiments, RZIs optionally substituted C1-6Aminoalkyl (e.g., optionally substituted-NH containing)2C of (A)1-6Aminoalkyl).
Phosphoric acid esters
In certain embodiments, an immunomodulatory polynucleotide provided herein comprises one or more internucleoside phosphotriesters and/or one or two terminal phosphodiesters and/or phosphotriesters. In certain embodiments, the phosphotriester comprises a phosphate ester, phosphorothioate or phosphorodithioate ester in which one or two valencies are substituted with a nucleoside and/or a base-free spacer and the remaining valencies are bonded to a bioreversible group, a non-bioreversible group, a linker bonded to a targeting moiety, or a conjugation group. In certain embodiments, the internucleoside phosphotriester is bonded to two nucleosides and/or to an abasic spacer, and the remaining valencies are bonded to a reversible group, a non-bioreversible group, a linker bonded to a targeting moiety, or a conjugation group. In certain embodiments, the internucleoside phosphodiester is bonded to two nucleosides and/or to an abasic spacer. In certain embodiments, the terminal phosphodiester comprises a phosphate, phosphorothioate, or phosphorodithioate at the 5 '-or 3' -end of the immunomodulatory polynucleotide, wherein one of the two remaining valencies is bonded to a bioreversible group, a non-bioreversible group, a linker bonded to a targeting moiety, or a conjugate group.
Linker and conjugate moieties
In certain embodiments, an immunomodulatory polynucleotide provided herein comprises a linker bonded to a targeting moiety and optionally one or more accessory moieties. In certain embodiments, the linker is a multivalent group, wherein the first valency is bonded to the internucleoside or terminal phosphate, internucleoside or terminal phosphorothioate, internucleoside or terminal phosphorodithioate, abasic spacer, capping group, or nucleobase, and the second valency is bonded to the targeting moiety. In certain embodiments, the linker further comprises one or more valencies, each valency being independently bonded to the auxiliary moiety. In certain embodiments (e.g., when the targeting moiety is a small molecule), an immunomodulatory polynucleotide provided herein comprises a plurality of linkers linked to a plurality of targeting moieties. In certain embodiments (e.g., when the targeting moiety is an antibody or antigen-binding fragment thereof), the immunomodulatory polynucleotides provided herein comprise a linker attached to the targeting moiety.
In certain embodiments, an immunomodulatory polynucleotide provided herein comprises a conjugate group. The conjugation group is a functional group capable of undergoing a conjugation reaction, such as a cycloaddition reaction (e.g., dipolar cycloaddition), amidation reaction, or nucleophilic aromatic substitution. Upon reaction with a complementary reactive group, the conjugate group creates a linker in the immunomodulatory polynucleotides provided herein.
In certain embodiments, the linker bonded to the targeting moiety is part of an internucleoside phosphotriester. In certain embodiments, the linker bonded to the targeting moiety is part of a base-free spacer.
In certain embodiments, the linker or conjugate group has formula (II):
–Z1–QA1–Z2–(–QA2–Z3–)k–RT,
(II)
wherein:
Z1is a divalent radical, a trivalent radical, a tetravalent radical or a pentavalent radical, one of the valencies of which is linked to QA1Bonded, the second valence being open ring, or with R if formula (II) is used for the linkerTAnd each remaining valence (if present) is independently bonded to an auxiliary moiety;
Z2absent, divalent radicals, trivalent radicalsA group, tetravalent group or pentavalent group, one of the valences of which is related to QA1Bonded, of second valence to QA2Or RTAnd each remaining valence (if present) is independently bonded to an auxiliary moiety;
Z3absent, is a divalent, trivalent, tetravalent, or pentavalent group, one of the valencies of which is related to QA2Bonded, of second valence to RTAnd each remaining valence (if present) is independently bonded to an auxiliary moiety;
RTa bond absent or attached to the targeting moiety;
k is an integer of about 0 or about 1.
If the formula (II) is used for the joint,
then Q isA1And QA2Independently absent, is optionally substituted C2-12Heteroalkylene (e.g., comprising-C (O) -N (H) -, -N (H) -C (O) -, -S (O)2-N (H) -or-N (H) -S (O)2Heteroalkylene of (A), optionally substituted C1-12Sulfoacylidene groups (e.g. heterocyclic group) Optionally substituted C1-12Heterocyclyl (e.g. 1, 2, 3-triazole-1, 4-diyl or) Cyclobut-3-ene-1, 2-dione-3, 4-diyl, pyridin-2-ylhydrazone, optionally substituted C6-16Triazole heterocyclic radicals (e.g. triazoles)) Optionally substituted C8-16Tricycloalkenylene (e.g. for) Or dihydropyridazine radicals (e.g. trans-Trans-) (ii) a And
RTis a bond to the targeting moiety;
provided that Q is presentA1And QA2At least one of (a).
If formula (II) is used for the conjugate group, then
Or
(i)QA2Is absent, and QA1Is a conjugate moiety, e.g. optionally substituted C2-12Alkynyl, optionally substituted N-protected amino, azido, N-maleimido, S-protected thiol,or N-protected forms thereof,Containing an optionally substituted C with an internal ring carbon-carbon triple bond6-16Heterocyclic radical (e.g. of the formula) 1, 2, 4, 5-tetrazine radicals (e.g. of the formula) Or optionally substituted C8-16Cycloalkynyl (e.g. cycloalkynyl))、–NHRN1Optionally substituted C4-8Strained cycloalkenyl (e.g., trans cyclooctenyl or norbornenyl) or optionally substituted-COOR-containing 12or-CHO C1-16An alkyl group; and
k is an integer of about 0;
or
(ii)QA1Is defined for use in a joint, and QA2Is a conjugate moiety, e.g. optionally substituted C2-12Alkynyl radicalOptionally substituted N-protected amino, azido, N-maleimido, S-protected thiol,Or N-protected forms thereof,Containing an optionally substituted C with an internal ring carbon-carbon triple bond6-16Heterocyclic radical (e.g. of the formula) 1, 2, 4, 5-tetrazine radicals (e.g. of the formula) Or optionally substituted C8-16Cycloalkynyl (e.g. cycloalkynyl))，–NHRN1Optionally substituted C4-8Strained cycloalkenyl (e.g., trans cyclooctenyl or norbornenyl) or optionally substituted-COOR-containing12or-CHO C1-16An alkyl group; and
k is an integer of about 1;
wherein:
RN1is an H, N-protecting group or optionally substituted C1-6An alkyl group;
each R12Independently is H or optionally substituted C1-6An alkyl group;
R13is halogen or F; and
Z3and RTIs absent.
In certain embodiments, Z1Having a branching group and two divalent segments, wherein the branching group is bonded to each of the two divalent segments,
wherein:
one of the divalent segments is bonded to an internucleoside or terminal phosphate ester, an internucleoside or terminal phosphorothioate, an internucleoside or terminal phosphorodithioate, a abasic spacer or a nucleobase, and the remaining divalent segments are bonded to Q A1And (4) bonding.
The branched radical being optionally substituted C1-12Alkanetriyl or optionally substituted C2-12Heteroalkanetriyl in which two valencies are replaced by a divalent segment and the remaining valencies are replaced by
Wherein:
p1 is an integer of about 1, about 2, or about 3;
each s2 is independently an integer from about 0 to about 10;
each QBAnd QDIndependently absent, -CO-, -NH-, -O-, -S-, -SO2–、–OC(O)–、–COO–、–NHC(O)–、–C(O)NH–、–CH2–、–CH2NH–、–NHCH2–、–CH2O-or-OCH2–；
Each QCIndependently absent, is optionally substituted C1-12Alkylene, optionally substituted C2-12Alkenylene, optionally substituted C2-12Alkynylene, optionally substituted C2-12Heteroalkylene, optionally substituted C1-9(Z) a heterocyclylene group or-P (OH) -, wherein Z is O or S;
each QGIndependently is optionally substituted C1-6Alkanetriyl, optionally substituted C1-6Alkanetetrayl, optionally substituted C2-6Heteroalkanetriyl or optionally substituted C2-6A heteroalkane tetrayl group; and
each QHIndependently is RM1or-QG[(–QB–QC–QD)s2–RM1]p1Wherein each R isM1Independently a key connected to the auxiliary portion.
In certain embodiments, Z2Having a branching group and two divalent segments, wherein the branching group is bonded to each of the two divalent segments,
wherein:
one of the divalent segments and the targeting moiety or QA2Bonded and the remaining divalent segment is bonded to Q A1And (4) bonding.
The branched radical being optionally substituted C1-12Alkanetriyl or optionally substituted C2-12Heteroalkanetriyl in which two valencies are replaced by a divalent segment and the remaining valencies are replaced by
Wherein:
p1 is an integer of about 1, about 2, or about 3;
each s2 is independently an integer from about 0 to about 10;
each QBAnd QDIndependently absent, -CO-, -NH-, -O-, -S-, -SO2–、–OC(O)–、–COO–、–NHC(O)–、–C(O)NH–、–CH2–、–CH2NH–、–NHCH2–、–CH2O-or-OCH2–；
Each QCIndependently absent, is optionally substituted C1-12Alkylene, optionally substituted C2-12Alkenylene, optionally substituted C2-12Alkynylene, optionally substituted C2-12Heteroalkylene, optionally substituted C1-9(Z) a heterocyclylene group or-P (OH) -, wherein Z is O or S;
each QGIndependently is optionally substituted C1-6Alkanetriyl, optionally substituted C1-6Alkanetetrayl, optionally substituted C2-6Heteroalkanetriyl or optionally substituted C2-6A heteroalkane tetrayl group; and
each QHIndependently is RM1or-QG[(–QB–QC–QD)s2–RM1]p1Wherein each R isM1Independently a key connected to the auxiliary portion.
In certain embodiments, Z3Having a branched radical and twoDivalent segments, wherein a branching group is bonded to each of the two divalent segments,
wherein:
one of the divalent segments is bonded to the targeting moiety, and the remaining divalent segments are bonded to Q A2And (4) bonding.
The branched radical being optionally substituted C1-12Alkanetriyl or optionally substituted C2-12Heteroalkanetriyl in which two valencies are replaced by a divalent segment and the remaining valencies are replaced by
Wherein:
p1 is an integer of about 1, about 2, or about 3;
each s2 is independently an integer from about 0 to about 10;
each QBAnd QDIndependently absent, -CO-, -NH-, -O-, -S-, -SO2–、–OC(O)–、–COO–、–NHC(O)–、–C(O)NH–、–CH2–、–CH2NH–、–NHCH2–、–CH2O-or-OCH2–；
Each QCIndependently absent, is optionally substituted C1-12Alkylene, optionally substituted C2-12Alkenylene, optionally substituted C2-12Alkynylene, optionally substituted C2-12Heteroalkylene, optionally substituted C1-9(Z) a heterocyclylene group or-P (OH) -, wherein Z is O or S;
each QGIndependently is optionally substituted C1-6Alkanetriyl, optionally substituted C1-6Alkanetetrayl, optionally substituted C2-6Heteroalkanetriyl or optionally substituted C2-6A heteroalkane tetrayl group; and
each QHIndependently is RM1or-QG[(–QB–QC–QD)s2–RM1]p1Wherein each R isM1Independently of the auxiliary partA key.
In certain embodiments, Z1、Z2Or Z3The divalent segment in (A) is- (-Q)B–QC–QD–)s1–，
Wherein:
each s1 is independently an integer from about 1 to about 50 or from about 1 to about 30;
each QBAnd QDIndependently absent, -CO-, -NH-, -O-, -S-, -SO 2–、 -OC(O)–、–COO–、–NHC(O)–、–C(O)NH–、–CH2–、–CH2NH–、–NHCH2–、–CH2O-or-OCH2-; and
each QCIndependently absent, is optionally substituted C1-12Alkylene, optionally substituted C2-12Alkenylene, optionally substituted C2-12Alkynylene, optionally substituted C2-12Heteroalkylene, optionally substituted C1-9A heterocyclylene group;
provided that Q is presentB、QCAnd QDAt least one of (a).
In certain embodiments, at least one Q is present in the divalent segmentC. In certain embodiments, QCIs present in each monomer unit of the divalent segment. In certain embodiments, Z1By the presence of QCAnd (4) bonding. In certain embodiments, Z1Is present in each monomer unit ofBAnd QDAt least one of (a). In certain embodiments, Z2Is present in each monomer unit ofBAnd QDAt least one of (a). In certain embodiments, when present, Z1、Z2And Z3Only one of which contains a branched group.
In certain embodiments, Z1、Z2And Z3One, two or three of independently are
–(–QB–QC–QD–)s1–QE–(–QB–QC–QD–)s1–,
(III)
Wherein:
each s1 is independently an integer from about 1 to about 50 or from about 1 to about 30;
each QBAnd QDIndependently absent, -CO-, -NH-, -O-, -S-, -SO2–、–OC(O)–、–COO–、–NHC(O)–、–C(O)NH–、–CH2–、–CH2NH–、–NHCH2–、–CH2O-or-OCH2–；
Each QCIndependently absent, is optionally substituted C1-12Alkylene, optionally substituted C 2-12Alkenylene, optionally substituted C2-12Alkynylene, optionally substituted C2-12Heteroalkylene, optionally substituted C1-9(Z) a heterocyclylene group or-P (OH) -, wherein Z is O or S; and
QEabsent or a branched group of formula (IV):
wherein:
p1 is an integer of about 1, about 2, or about 3;
each s2 is independently an integer from about 0 to about 10;
QFis optionally substituted C1-12Alkanetriyl or optionally substituted C2-12A heteroalkane triyl; and
each QGIndependently is optionally substituted C1-6Alkanetriyl, optionally substituted C1-6Alkanetetrayl, optionally substituted C2-6Heteroalkanetriyl or optionally substituted C2-6A heteroalkane tetrayl group; and
each QHIndependently is RM1or-QG[(–QB–QC–QD)s2–RM1]p1Wherein each R isM1Independently a key connected to the auxiliary portion.
In formula (IV), if p1 is about 1, QGIs absent; and if p1 is 2 or 3, then there is at least one QG。
In certain embodiments, Z1By the presence of QCTo an internucleoside or terminal phosphate, an internucleoside or terminal phosphorothioate, an internucleoside or terminal phosphorodithioate, an abasic spacer, a capping group or a nucleobase.
In certain embodiments, Q is present in the divalent segmentB、QC、QDAnd QEIs (e.g., there is at least one Q)C，QEPresence or QEAbsent). In certain embodiments, each Q BAnd QDIndependently absent, -CO-, -NH-, -O-, -S-, -SO2–、–NHC(O)–、–C(O)NH–、–CH2–、–CH2NH–、-NHCH2–、 -CH2O-or-OCH2–；
In certain embodiments, - (-Q)B–QC–QD–)s1-combine to form a group:
–QB–(CH2)g1–(CH2OCH2)g2–(CH2)g3–QD–，
wherein:
g2 is an integer from about 1 to about 50;
g1 is an integer of about 1, and QBis-NHCO-, -CONH-or-O-; or g1 is an integer of about 0, and QDis-NHCO-; and
g3 is an integer of about 1, and QBis-NHCO-, -CONH-or-O-; or g3 is an integer of about 0, and QDis-CONH-.
The conjugate moiety may be protected until the adjunct moiety is conjugated to the polynucleotide. For example, the protected conjugate moiety may comprise-COORPGOor-NHRPGNWherein R isPGOIs an O-protecting group (e.g., a carboxyl protecting group), and RPGNIs N-protectionAnd (4) a base.
In certain embodiments, linker a is:
wherein:
QA1and QA2Independently absent, is optionally substituted C2-12Heteroalkylene (e.g., comprising-C (O) -N (H) -, -N (H) -C (O) -, -S (O)2-N (H) -or-N (H) -S (O)2Heteroalkylene of (A), optionally substituted C1-12Sulfoacylidene groups (e.g. heterocyclic group) ) Optionally substituted C1-12Heterocyclyl (e.g. 1,2, 3-triazole-1, 4-diyl or) Cyclobut-3-ene-1, 2-dione-3, 4-diyl, pyridin-2-ylhydrazone, optionally substituted C 6-16Triazole heterocyclic radicals (e.g. triazoles)) Optionally substituted C8-16Tricycloalkenylene (e.g. for) Or dihydropyridazine radicals (e.g. trans-Trans-) Provided that Q is presentA1And QA2At least one of (a);
RTis a bond to the targeting moiety;
RPis between and nucleosideA bond to a bridging group, nucleobase, capping group, or abasic spacer;
each QTIndependently is-CO-, -NH-CH2-or-CO-CH2–；
Each QSIndependently is optionally substituted C2-12Alkylene, optionally substituted C2-12Alkenylene, optionally substituted C2-12Alkynylene or optionally substituted (C)6-10Aryl) -C1-6An alkylene group;
each RMIndependently of each other H, an auxiliary moiety, - (CH)2)q7–CO–N(RM1)2or-C [ -CH2O–(CH2)q7–CO–N(RM1)2]3Wherein each q7 is independently an integer from about 1 to about 5, and each RM1Independently is H or an auxiliary moiety;
each X1、X3And X5Independently absent, is-O-, -NH-, -CH2–NH–、–C(O)–、–C(O)–NH–、–NH–C(O)–、–NH–C(O)–NH–、–O–C(O)–NH–、–NH–C(O)–O–、–CH2–NH–C(O)–NH–、–CH2-O-C (O) -NH-or-CH2–NH–C(O)-O-；
X7Absent, -O-P (O) (OH) -O-, -O-P (S) (OH) -O-, -NH-, -CH-2-NH-、-C(O)-、-C(O)-NH-、-NH-C(O)–、-NH-C(O)-NH-、–O-C(O)–NH–、–NH–C(O)-O-、–CH2-NH-C(O)-NH-、 -CH2-O-C (O) -NH-or-CH2-NH–C(O)–O-；
Each X2、X4And X6Independently absent, is-O-, -NH-, -C (O) -NH-, -NH-C (O) -NH-, -O-C (O) -NH-, or-NH-C (O) -O-;
x1 and each x5 is independently an integer of about 0 or about 1;
Each x2 is independently an integer from about 0 to about 50, from about 1 to about 40, or from about 1 to about 30;
each x3 is independently an integer from about 1 to about 11;
x4 is an integer of about 0, about 1, or about 2; and
each x6 is independently an integer from about 0 to about 10 or from about 1 to about 6, provided that the sum of the two x6 s is about 12 or less.
In certain embodiments, LinkA is:
wherein:
QA1is optionally substituted C2-12Heteroalkylene (e.g., comprising-C (O) -N (H) -, -N (H) -C (O) -, -S (O)2-N (H) -or-N (H) -S (O)2Heteroalkylene of (A), optionally substituted C1-12Sulfoacylidene groups (e.g. heterocyclic group) ) Optionally substituted C1-12Heterocyclyl (e.g. 1, 2, 3-triazole-1, 4-diyl or) Cyclobut-3-ene-1, 2-dione-3, 4-diyl, pyridin-2-ylhydrazone, optionally substituted C6-16Triazole heterocyclic radicals (e.g. triazoles) ) Optionally substituted C8-16Tricycloalkenylene (e.g. for) Or twoHydropyridazine radicals (e.g. trans-Trans- )；
Each RM1Independently H or an auxiliary moiety;
each RTIndependently is a bond to the targeting moiety;
each RPIndependently is a bond to an internucleoside bridging group, a nucleobase, a capping group, or a abasic spacer;
each QTIndependently is-CO-, -NH-CH2-or-CO-CH2–；
Each Q Pindependently-C (O) -N (H) -, -C (O) -, -S (O)2-N (H) -or-N (H) -S (O)2–；
Each QSIndependently is optionally substituted C2-12Alkylene, optionally substituted C2-12Alkenylene, optionally substituted C2-12Alkynylene or optionally substituted (C)6-10Aryl) -C1-6An alkylene group;
each q1, q3, and q7 is independently an integer of about 0 or about 1;
each q2 and q8 is independently an integer of from about 0 to about 50, from about 1 to about 40, or from about 1 to about 30;
each q4 is independently an integer from about 0 to about 10;
each q5 and q6 is independently an integer of from about 1 to about 10 or from about 1 to about 6; and
each q9 is independently an integer from about 1 to about 10.
In certain embodiments, LinkA is:
wherein:
in each structural formula, oneRepresents a single bond, and the other oneRepresents a double bond;
each RM1Independently H or an auxiliary moiety;
each RTIndependently is a bond to the targeting moiety;
each RPIndependently is a bond to an internucleoside bridging group, a nucleobase, a capping group, or a abasic spacer;
each QTIndependently is-CO-, -CO-CH2-, -NH-or-NH-CH2–；
Each QPindependently-C (O) -N (H) -, -C (O) -, -S (O)2-N (H) -or-N (H) -S (O)2–；
Each Q SIndependently is optionally substituted C2-12Alkylene, optionally substituted C2-12Alkenylene, optionally substituted C2-12Alkynylene or optionally substituted (C)6-10Aryl) -C1-6An alkylene group;
each q1, q3, and q7 is independently an integer of about 0 or about 1;
each q2 and q8 is independently an integer of from about 0 to about 50, from about 1 to about 40, or from about 1 to about 30;
each q4 is independently an integer from about 0 to about 10;
each q5 and q6 is independently an integer of from about 1 to about 10 or from about 1 to about 6; and
each q9 is independently an integer from about 1 to about 10.
In certain embodiments, q5 is 0. In certain embodiments, q5 is an integer from about 2 to about 6.
In certain embodiments, the conjugate group is:
wherein:
QA1is optionally substituted C2-12Heteroalkylene (e.g., comprising-C (O) -N (H) -, -N (H) -C (O) -, -S (O)2-N (H) -or-N (H) -S (O)2Heteroalkylene of (A), optionally substituted C1-12Sulfoacylidene groups (e.g. heterocyclic group) ) Optionally substituted C1-12Heterocyclyl (e.g. 1,2, 3-triazole-1, 4-diyl or) Cyclobut-3-ene-1, 2-dione-3, 4-diyl, pyridin-2-ylhydrazone, optionally substituted C6-16Triazole heterocyclic radicals (e.g. triazoles) ) Optionally substituted C8-16Tricycloalkenylene (e.g. for) Or dihydropyridazine radicals (e.g. trans-Trans- )；
QA2Is optionally substituted C2-12Alkynyl, optionally substituted N-protected amino, azido, N-maleimido, S-protected thiol, or N-protected forms thereof, Containing an optionally substituted C with an internal ring carbon-carbon triple bond6-16Heterocyclic radical (e.g. of the formula) 1, 2, 4, 5-tetrazine radicals (e.g. of the formula ) Or optionally substituted C8-16Cycloalkynyl (e.g. cycloalkynyl))、–NHRN1Optionally substituted C4-8Strained cycloalkenyl (e.g., trans cyclooctenyl or norbornenyl) or optionally substituted-COOR-containing12or-CHO C1-16An alkyl group;
RN1is an H, N-protecting group or optionally substituted C1-6An alkyl group;
each R12Independently is H or optionally substituted C1-6An alkyl group;
R13is halogen or F;
RPis a bridging group with a nucleosideA nucleobase, a capping group, or a base-free spacer;
each QSIndependently is optionally substituted C2-12Alkylene, optionally substituted C2-12Alkenylene, optionally substituted C2-12Alkynylene or optionally substituted (C)6-10Aryl) -C1-6An alkylene group;
each RMIndependently of each other H, an auxiliary moiety, - (CH)2)q7–CO–N(RM1)2or-C [ -CH2O–(CH2)q7–CO–N(RM1)2]3Wherein each q7 is independently an integer from about 1 to about 5, and each RM1Independently is H or an auxiliary moiety;
each X3And X5Independently absent, is-O-, -NH-, -CH2–NH–、–C(O)–、–C(O)–NH–、–NH–C(O)–、–NH–C(O)–NH–、–O–C(O)–NH–、–NH–C(O)–O–、–CH2–NH–C(O)–NH–、–CH2-O-C (O) -NH-or-CH2–NH–C(O)–O–；
X7Absent, -O-P (O) (OH) -O-, -O-P (S) (OH) -O-, -NH-, -CH- 2–NH-、-C(O)-、-C(O)-NH-、-NH-C(O)–、-NH-C(O)-NH-、–O-C(O)–NH–、–NH–C(O)-O-、–CH2-NH-C(O)-NH-、 -CH2-O-C (O) -NH-or-CH2-NH–C(O)–O-；
Each X2、X4And X6Independently absent, is-O-, -NH-, -C (O) -NH-, -NH-C (O) -NH-, -O-C (O) -NH-, or-NH-C (O) -O-;
x1 and each x5 is independently an integer of about 0 or about 1;
each x2 is independently an integer from 0 to 50 (e.g., 1 to 40 or 1 to 30);
each x3 is independently an integer from 1 to 11;
x4 is an integer of 0, 1 or 2; and
each x6 is independently an integer from 0 to 10 (e.g., 1 to 6), provided that the sum of the two x6 s is about 12 or less.
In certain embodiments, the conjugate group is:
wherein:
QA1is optionally substituted C2-12Alkynyl, optionally substituted N-protected amino, azido, N-maleimido, S-protected thiol, or N-protected forms thereof, Containing an optionally substituted C with an internal ring carbon-carbon triple bond6-16Heterocyclic radical (e.g. of the formula) 1,2, 4, 5-tetrazine radicals (e.g. of the formula ) Or optionally substituted C8-16Cycloalkynyl (e.g. cycloalkynyl))、–NHRN1Optionally substituted C4-8Strained cycloalkenyl (e.g., trans cyclooctenyl or norbornenyl) or optionally substituted-COOR-containing12or-CHO C1-16An alkyl group;
RN1is an H, N-protecting group or optionally substituted C1-6An alkyl group;
each R12Independently is H or optionally substituted C1-6An alkyl group;
R13is halogen (e.g., F);
RPIs a bond to an internucleoside bridging group, a nucleobase, a capping group, or a abasic spacer;
each QSIndependently is optionally substituted C2-12Alkylene, optionally substituted C2-12Alkenylene, optionally substituted C2-12Alkynylene or optionally substituted (C)6-10Aryl) -C1-6An alkylene group;
X7absent, -O-, -NH-, -O-P (O) (OH) -O-, -O-P (S) (OH) -O-, -CH2–NH–、–C(O)–、–C(O)–NH–、–NH–C(O)–、–NH–C(O)–NH–、–O–C(O)–NH–、–NH–C(O)–O-、–CH2–NH–C(O)–NH–、–CH2-O-C (O) -NH-or-CH2–NH–C(O)–O–；
X6Absent, is-O-, -NH-, -C (O) -NH-, -NH-C (O) -NH-, -O-C (O) -NH-, or-NH-C (O) -O-;
x1 is 0 or 1;
each x2 is independently an integer from 0-50, 1-10, or 1-30;
each x3 is independently an integer from 1 to 11; and
x4 is 0, 1 or 2.
In certain embodiments, the conjugate group is:
wherein:
QA1is optionally substituted C2-12Alkynyl, optionally substituted N-protected ammoniaA group, an azido group, an N-maleimido group, an S-protected thiol, or N-protected forms thereof, Containing an optionally substituted C with an internal ring carbon-carbon triple bond6-16Heterocyclic radical (e.g. of the formula) 1,2,4, 5-tetrazine radicals (e.g. of the formula ) Or optionally substituted C8-16Cycloalkynyl (e.g. cycloalkynyl))、–NHRN1Optionally substituted C4-8Strained cycloalkenyl (e.g., trans cyclooctenyl or norbornenyl) or optionally substituted-COOR-containing 12or-CHO C1-16An alkyl group;
RN1is an H, N-protecting group or optionally substituted C1-6An alkyl group;
each R12Independently is H or optionally substituted C1-6An alkyl group;
R13is halogen (e.g., F);
RPis a bond to an internucleoside bridging group, a nucleobase, a capping group, or a abasic spacer;
QPis-C (O) -N (H) -, -N (H) -C (O) -, -S (O)2-N (H) -or-N (H) -S (O)2–；
Each QSIndependently is optionally substituted C2-12Alkylene, optionally substituted C2-12Alkenylene, optionally substituted C2-12Alkynylene or optionally substituted (C)6-10Aryl) -C1-6An alkylene group;
each q1 and q3 is independently 0 or 1;
q2 is an integer from 0 to 50, 1 to 10, or 1 to 30;
q4 is an integer from 0 to 10; and
q5 is an integer from 1 to 10 or from 1 to 6.
In still certain embodiments, the conjugate group is:
wherein:
RPis a bond to an internucleoside bridging group, a nucleobase, a capping group, or a abasic spacer;
QPis-C (O) -N (H) -, -N (H) -C (O) -, -S (O)2-N (H) -or-N (H) -S (O)2–；
Each QSIndependently is optionally substituted C2-12Alkylene, optionally substituted C2-12Alkenylene, optionally substituted C2-12Alkynylene or optionally substituted (C)6-10Aryl) -C1-6An alkylene group;
each q1 and q3 is independently 0 or 1;
q2 is an integer from 0 to 50, 1 to 10, or 1 to 30;
q4 is an integer from 0 to 10; and
q5 is an integer from 1 to 10 or from 1 to 6.
In certain exemplary embodiments, the conjugate group is:
wherein: q2 is an integer of about 1 to about 50 (e.g., about 1 to about 24 or about 1 to about 8 (e.g., about 2 or about 3)), q4 is an integer of about 0 to about 10 (e.g., about 0, about 2, about 3, about 4, about 5, about 6, about 7, or about 8), q10 is an integer of about 0 to about 8 (e.g., about 1, about 2, about 3, about 4, about 5, or about 6), q11 is about 0 or about 1, Z is O or S, and each R is RMIndependently of each other H, an auxiliary moiety, - (CH)2)q7–CO–N(RM1)2or-C [ -CH2O–(CH2)q7–CO–N(RM1)2]3Wherein each q7 is independently an integer from about 1 to about 5, and each RM1Independently H or an auxiliary moiety.
In certain embodiments, the conjugation group for conjugation to the targeting moiety by metal-catalyzed cycloaddition is:
wherein q2 is an integer of about 1 to about 50 (e.g., about 1 to about 24 or about 1 to about 8 (e.g., about 2 or about 3)), q4 is an integer of about 0 to about 10 (e.g., about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8), q10 is an integer of about 0 to about 8 (e.g., about 1, about 2, about 3, about 4, about 5, or about 6), q11 is about 0 or about 1, and Z is O or S.
In certain embodiments, the conjugation group for conjugation to the targeting moiety by metal-free cycloaddition is:
Wherein: q2 is an integer of about 1 to about 50 (e.g., about 1 to about 24 or about 1 to about 8 (e.g., about 2 or about 3)), q4 is an integer of about 0 to about 10 (e.g., about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8), q10 is an integer of about 0 to about 8 (e.g., about 1, about 2, about 3, about 4, about 5, or about 6), q11 is about 0 or about 1, Z is O or S, and each R is RMIndependently of each other H, an auxiliary moiety, - (CH)2)q7–CO–N(RM1)2or-C [ -CH2O–(CH2)q7–CO–N(RM1)2]3Wherein each q7 is independently an integer from about 1 to about 5, and each RM1Independently H or an auxiliary moiety.
In certain embodiments, the conjugate group conjugated to the targeting moiety by amide formation is:
wherein q2 is an integer of from about 0 to about 50 (e.g., an integer of from about 1 to about 8 (e.g., about 2 or about 3)), and q12 is an integer of from about 1 to about 11 (e.g., an integer of from about 1 to about 5 (e.g., about 1, about 2, about 3, about 4, or about 5).
Bioreversible groups
In certain embodiments, the bioreversible group comprises a disulfide (-S-S-). In certain embodiments, the bioreversible group is intracellularly cleavable under physiological conditions.
In certain embodiments, the bioreversible group has the formula (XXII):
R5–S–S–(LinkB)–,
(XXII)
wherein:
LinkB is a divalent group comprising a phosphate, thio group Phosphate or dithiophosphate bonded sp3-a hybridized carbon atom and a carbon atom bonded to-S-S-, and R5Is optionally substituted C1-6Alkyl, optionally substituted C6-10Aryl or-LinkC (-R)M)rOr LinkB is a trivalent linker comprising an sp bonded to a phosphate, phosphorothioate or phosphorodithioate3-hybridized carbon atom and carbon atom bonded to-S-S-, wherein the third valence of LinkB is with-S-S-and R5Combined to form optionally substituted C3-9A heterocyclylene group;
LinkC is a polyvalent group;
each RMIndependently is H, an auxiliary moiety or-QG[(–QB–QC–QD)s2–RM1]p1，
Wherein R isM1Independently of the other, is H, an auxiliary moiety,
each QBAnd each QDIndependently absent, -CO-, -NH-, -O-, -S-, -SO2–、–OC(O)–、–COO–、–NHC(O)–、–C(O)NH–、–CH2–、–CH2NH–、–NHCH2–、–CH2O-or-OCH2–，
Each QCIndependently absent, is optionally substituted C1-12Alkylene, optionally substituted C2-12Alkenylene, optionally substituted C2-12Alkynylene, optionally substituted C2-12Heteroalkylene, optionally substituted C1-9A heterocyclylene group;
each QGIndependently is optionally substituted C1-6Alkanetriyl, optionally substituted C1-6Alkanetetrayl, optionally substituted C2-6Heteroalkanetriyl or optionally substituted C2-6A heteroalkane tetrayl group; and
each s2 is independently an integer from 0-10, and
p1 is 2 or 3;
And
r is an integer from 1 to 6 (e.g., 1, 2, or 3).
In certain embodiments, LinkB and/or R5Including bulky groups attached to-S-. The inclusion of bulky groups attached to the-S-can enhance the stability of the sulfur-sulfur bond, for example, during polynucleotide synthesis.
In further embodiments, LinkB consists of 1, 2, or 3 groups, each group independently selected from optionally substituted C1-12Alkylene, optionally substituted C2-12Alkenylene, optionally substituted C2-12Alkynylene, optionally substituted C2-12Heteroalkylene, optionally substituted C1-9A heterocyclylene group.
In particular embodiments, LinkB and-S-combine to form a structure selected from:
wherein:
each R6Independently is C2-7An alkanoyl group; c1-6An alkyl group; c2-6An alkenyl group; c2-6An alkynyl group; c1-6An alkylsulfinyl group; c6-10An aryl group; amino (C)6-10Aryl) -C1-4-an alkyl group; c3-8A cycloalkyl group; (C)3-8Cycloalkyl) -C1-4-an alkyl group; c3-8A cycloalkenyl group; (C)3-8Cycloalkenyl) -C1-4-an alkyl group; halogen; c1-9A heterocyclic group; c1-9A heteroaryl group; (C)1-9Heterocyclyl) oxy; (C)1-9Heterocyclyl) aza; a hydroxyl group; c1-9A thioalkoxy group; - (CH)2)qCO2RAWherein q is an integer of 0 to 4, and RAIs selected from C1-6Alkyl radical, C6-10Aryl and (C)6-10Aryl) -C1-4An alkyl group; - (CH)2)qCONRBRCWherein q is an integer of 0 to 4, and wherein R BAnd RCIndependently selected from hydrogen, C1-6Alkyl radical, C6-10Aryl and (C)6-10Aryl) -C1-4-an alkyl group; - (CH)2)qSO2RDWherein q is an integer of 0 to 4, and RDIs selected from C1-6Alkyl radical, C6-10Aryl and (C)6-10Aryl) -C1-4-an alkyl group; - (CH)2)qSO2NRERFWherein q is an integer of 0 to 4, and REAnd RFEach independently selected from hydrogen, alkyl, aryl and (C)6-10Aryl) -C1-4-an alkyl group; a thiol; an aryloxy group; a cycloalkoxy group; an arylalkoxy group; (C)1-9Heterocyclyl) -C1-4-an alkyl group; (C)1-9Heteroaryl) -C1-4-an alkyl group; c3-12A silyl group; a cyano group; or-S (O) RHWherein R isHSelected from hydrogen, C1-6Alkyl radical, C6-10Aryl and (C)6-10Aryl) -C1-4-an alkyl group; or two adjacent R6Radical with each R6The atoms to which the groups are attached together form a cyclic group selected from C6Aryl radical, C2-5Heterocyclyl or C2-5Heteroaryl, wherein cyclyl is optionally substituted with 1, 2 or 3 substituents selected from: c2-7An alkanoyl group; c1-6An alkyl group; c2-6An alkenyl group; c2-6An alkynyl group; c1-6An alkylsulfinyl group; c6-10An aryl group; amino (C)6-10Aryl) -C1-4-an alkyl group; c3-8A cycloalkyl group; (C)3-8Cycloalkyl) -C1-4-an alkyl group; c3-8A cycloalkenyl group; (C)3-8Cycloalkenyl) -C1-4-an alkyl group; halogen; c1-9A heterocyclic group; c1-9A heteroaryl group; (C)1-9Heterocyclyl) oxy; (C)1-9Heterocyclyl) aza; a hydroxyl group; c1-6A thioalkoxy group; - (CH)2)qCO2RAWherein q is an integer of 0 to 4, and R AIs selected from C1-6Alkyl radical, C6-10Aryl and (C)6-10Aryl) -C1-4An alkyl group; - (CH)2)qCONRBRCWherein q is an integer of 0 to 4, and wherein RBAnd RCIndependently selected from hydrogen, C1-6Alkyl radical, C6-10Aryl and (C)6-10Aryl) -C1-4-an alkyl group; - (CH)2)qSO2RDWherein q is an integer of 0 to 4, and RDIs selected from C1-6Alkyl radical, C6-10Aryl and (C)6-10Aryl) -C1-4-an alkyl group; - (CH)2)qSO2NRERFWherein q is an integer of 0 to 4, and REAnd RFEach independently selected from hydrogen, alkyl, aryl and (C)6-10Aryl) -C1-4-an alkyl group; a thiol; an aryloxy group; a cycloalkoxy group; an arylalkoxy group; (C)1-9Heterocyclyl) -C1-4-an alkyl group; (C)1-9Heteroaryl) -C1-4-an alkyl group; c3-12A silyl group; a cyano group; or-S (O) RHWherein R isHSelected from hydrogen, C1-6Alkyl radical, C6-10Aryl and (C)6-10Aryl) -C1-4-an alkyl group.
m1 is 0, 1 or 2; and
m2 is 0, 1, 2, 3 or 4;
or LinkB, -S-S-and R5Combined to form a composition comprising(xx) A group of (1).
In other embodiments, the LinkC may comprise 0-3 multivalent monomers (e.g., optionally substituted C)1-6Alkanetriyl, optionally substituted C1-6Alkane tetra-or trivalent nitrogen atoms) and one or more divalent monomers (e.g., 1 to 40), wherein each divalent monomer is independently optionally substituted C1-6An alkylene group; optionally substituted C2-6An alkenylene group; optionally substituted C2-6An alkynylene group; optionally substituted C 3-8A cycloalkylene group; optionally substituted C3-8Cycloalkenylene; optionally substituted C6-14An arylene group; optionally substituted C with 1-4 heteroatoms selected from N, O and S1-9A heteroarylene group; optionally substituted C with 1-4 heteroatoms selected from N, O and S1-9A heterocyclylene group; an imino group; optionally substituted N; o; or S (O)mWherein m is 0, 1 or 2. In some embodiments, each monomer is independently optionally substituted C1-6An alkylene group; optionally substituted C3-8A cycloalkylene group; optionally, theSubstituted C3-8Cycloalkenylene; optionally substituted C6-14An arylene group; optionally substituted C with 1-4 heteroatoms selected from N, O and S1-9A heteroarylene group; optionally substituted C with 1-4 heteroatoms selected from N, O and S1-9A heterocyclylene group; an imino group; optionally substituted N; o; or S (O)mWherein m is 0, 1 or 2 (e.g., m is 2). In certain embodiments, each monomer is independently optionally substituted C1-6An alkylene group; optionally substituted C3-8A cycloalkylene group; optionally substituted C3-8Cycloalkenylene; optionally substituted C6-14An arylene group; optionally substituted C with 1-4 heteroatoms selected from N, O and S1-9A heteroarylene group; optionally substituted C with 1-4 heteroatoms selected from N, O and S1-9A heterocyclylene group; optionally substituted N; o; or S (O) mWherein m is 0, 1 or 2 (e.g., m is 2). The non-bioreversible linker attaching the auxiliary moiety to the conjugate moiety or reaction product thereof may comprise 2 to 500 (e.g. 2 to 300, 2 to 200, 2 to 100 or 2 to 50) such monomers. The LinkC may comprise one or more polyethylene glycols (e.g., the polyethylene glycol may have a molecular weight of 88Da to 1kDa (e.g., 88Da to 500 Da).
A group-LinkC (-R) which can be used for the preparation of the compounds of the formula (IIa) is described here and in WO 2015/188197M)rThe compound of (1). -LinkC (-R)M)rNon-limiting examples of (a) include:
wherein:
R14is a bond to-S-S-,
RMis an auxiliary moiety or-QG[(–QB–QC–QD)s2–RM1]p1，
Each one of which isRM1Independently of the other, is H or an auxiliary moiety,
each QBAnd QDIndependently absent, -CO-, -NH-, -O-, -S-, -SO2–、–OC(O)–、–COO–、–NHC(O)–、–C(O)NH–、–CH2–、–CH2NH–、–NHCH2–、–CH2O-or-OCH2–；
Each QCIndependently absent, is optionally substituted C1-12Alkylene, optionally substituted C2-12Alkenylene, optionally substituted C2-12Alkynylene, optionally substituted C2-12Heteroalkylene, optionally substituted C1-9(Z) a heterocyclylene group or-P (OH) -, wherein Z is O or S;
each QGIndependently is optionally substituted C1-6Alkanetriyl, optionally substituted C1-6Alkanetetrayl, optionally substituted C2-6Heteroalkanetriyl or optionally substituted C2-6A heteroalkane tetrayl group;
Each s2 is independently an integer from 0-10, and
p1 is 2 or 3;
each r4 is independently an integer from 1-6; and
each r5 is independently an integer from 0-10.
In certain embodiments, RMIs the auxiliary part. In some embodiments, at least one R isM1Is the auxiliary part.
In certain embodiments, the bioreversible linker group isWherein the group is linked at one end to a polynucleotide and at the other end to a target moiety (in one embodiment, an antibody).
Non-bioreversible groups
A non-bioreversible group is a monovalent substituent that does not contain a bond (e.g., an ester, thioester, or disulfide) that is cleavable in serum or endosomes under physiological conditions. The non-bioreversible group may be optionally substitutedSubstituted C2-16An alkyl group; optionally substituted C3-16An alkenyl group; optionally substituted C3-16An alkynyl group; optionally substituted C3-8A cycloalkyl group; optionally substituted C3-8A cycloalkenyl group; optionally substituted (C)3-8Cycloalkyl) -C1-4-an alkyl group; optionally substituted (C)3-8Cycloalkenyl) -C1-4-an alkyl group; optionally substituted C6-14An aryl group; optionally substituted (C)6-14Aryl) -C1-4-an alkyl group; optionally substituted C with 1-4 heteroatoms selected from N, O and S1-9A heteroaryl group; optionally substituted (C) having 1-4 heteroatoms selected from N, O and S 1-9Heteroaryl) -C1-4-an alkyl group; optionally substituted C with 1-4 heteroatoms selected from N, O and S2-9Heterocyclyl, wherein the heterocyclyl does not contain an S-S bond; optionally substituted (C) having 1-4 heteroatoms selected from N, O and S2-9Heterocyclyl) -C1-4-alkyl, wherein the heterocyclyl does not comprise an S-S bond; or a group of formula (XXIII):
wherein:
L3is C2-6An alkylene group;
R7is optionally substituted C2-6An alkyl group; optionally substituted C6-14An aryl group; optionally substituted (C)6-14Aryl) -C1-4-an alkyl group; optionally substituted C3-8A cycloalkyl group; optionally substituted (C)3-8Cycloalkyl) -C1-4-an alkyl group; optionally substituted C with 1-4 heteroatoms selected from N, O and S1-9A heteroaryl group; optionally substituted (C) having 1-4 heteroatoms selected from N, O and S1-9Heteroaryl) -C1-4-an alkyl group; optionally substituted C with 1-4 heteroatoms selected from N, O and S2-9Heterocyclyl, wherein the heterocyclyl does not contain an S-S bond; optionally substituted (C) having 1-4 heteroatoms selected from N, O and S2-9Heterocyclyl) -C1-4-alkyl, wherein the heterocyclyl does not comprise an S-S bond; and with-OH, C1-6Alkoxy or-COOH-terminated poly (ethylene glycol); and
R8is H or C1-6An alkyl group.
The non-bioreversible phosphotriester may be a phosphate or phosphorothioate substituted with a substituent being a conjugation group, C 2-16Alkyl, aryl, heteroaryl, and heteroaryl, A group formed by a cycloaddition reaction with an azido-containing substrate,
wherein:
n is an integer of 1 to 6;
R9is optionally substituted C6An aryl group; optionally substituted C4-5Heteroaryl, which is a six-membered ring containing 1 or 2 nitrogen atoms; or optionally substituted C4-5A heterocyclic group which is a six-membered ring containing 1 or 2 nitrogen atoms;
R10is H or C1-6An alkyl group;
R11is halogen, -COOR11Aor-CON (R)11B)2Wherein R is11AAnd R11BEach independently of the other being H, optionally substituted C1-6Alkyl, optionally substituted C6-14Aryl, optionally substituted1-9Heteroaryl or optionally substituted C2-9A heterocyclic group; and
the substrate containing an azido group is
In some embodiments, the non-bioreversible group is-LinkD (-R)M1)r1Wherein LinkD is a multivalent linker, each RM1Independently H or an auxiliary moiety, and r1 is an integer from 1 to 6.
In some cases, -LinkD (-R)M1)r1Having the formula (XXIV):
–QR–Q3([–Q4–Q5–Q6]r2–Q7–RM1)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 repeat units are the same or different;
QRis [ -Q ]4–Q5–Q6]r2–QL-, wherein QLIs optionally substituted C2-12Heteroalkylene (e.g., comprising-C (O) -N (H) -, -N (H) -C (O) -, -S (O)2-N (H) -or-N (H) -S (O)2Heteroalkylene of (A), optionally substituted C 1-12Sulfoacylidene groups (e.g. heterocyclic group)) Optionally substituted C1-12Heterocyclyl (e.g. 1,2, 3-triazole-1, 4-diyl or) Cyclobut-3-ene-1, 2-dione-3, 4-diyl, pyridin-2-ylhydrazone, optionally substituted C6-16Triazole heterocyclic radicals (e.g. triazoles)) Optionally substituted C8-16Tricycloalkenylene (e.g. for) Or dihydropyridazine radicals (e.g. trans-Trans- )；
If r1 is 1, then Q3Is a straight-chain group (e.g., [ -Q ]4–Q5–Q6]r2-, or Q if r1 is an integer from 2 to 63Is a branched group (e.g., [ -Q ]4–Q5–Q6]s–Q8([–Q4–Q5–Q6]r2–(Q8)r3)r4Wherein r3 is 0 or 1, r4 is 0, 1,2 or 3); each r2 is independently an integer from 0 to 50 (e.g., 0 to 30), where the repeat units are the same or different;
each Q4And each Q6Independently absent, -CO-, -NH-, -O-, -S-, -SO2–、–OC(O)–、–COO–、–NHC(O)–、–C(O)NH–、–CH2–、–CH2NH–、–NHCH2–、–CH2O-or-OCH2–；
Each Q5Independently absent, is optionally substituted C1-12Alkylene, optionally substituted C2-12Alkenylene, optionally substituted C2-12Alkynylene, optionally substituted C2-12Heteroalkylene, optionally substituted C1-9A heterocyclylene group;
each Q7Independently absent, -CO-, -NH-, -O-, -S-, -SO2–、–CH2–、–C(O)O-、–OC(O)-、-C(O)NH-、-NH-C(O)-、-NH-CH(Ra) -C (O) -or-C (O) -CH (R)a)–NH–；
Each Q8Independently is optionally substituted C1-6Alkanetriyl, optionally substituted C1-6Alkanetetrayl, optionally substituted C 2-6Heteroalkanetriyl or optionally substituted C2-6A heteroalkane tetrayl group; and
each RaIndependently is H or an amino acid side chain; and
each RM1Independently H or an auxiliary moiety.
In formula (XXIV), Q is present4、Q5And Q6At least one of (a). In formula (XXIV), LinkD may contain a single branch point (if each r3 is0) Or a plurality of branch points (if at least one r3 is 1). In formula (XXIV), QRCan be-Q5–Q4–QL-, wherein Q5Is optionally substituted C2-12Heteroalkylene or optionally substituted C1-12Alkylene, and Q4is-CO-, -NH-or-O-. In formula (XXIV), QLCan be as follows:trans-Trans-
In formula (XXIV), Q3May be of the formula [ -Q4–Q5-Q6]r2A linear group of (a), wherein Q4、Q5And Q6The same as defined for formula (XXIV). Or, Q3May be a branched group [ -Q ]4-Q5-Q6]r2-Q8([-Q4-Q5–Q6]r2-(Q8)r3)r4Wherein each Q8Independently is optionally substituted C1-6Alkanetriyl, optionally substituted C1-6Alkanetetrayl, optionally substituted C2-6Heteroalkanetriyl or optionally substituted C2-6A heteroalkane tetrayl group;
wherein:
each r2 is independently an integer from 0 to 50 (e.g., 0 to 30), where the repeat units are the same or different;
r3 is 0 or 1;
r4 is 0, 1, 2 or 3;
wherein the content of the first and second substances,
LinkD is a trivalent or tetravalent group when r3 is 0, and
when r3 is 1, LinkD is a tetravalent, pentavalent, or hexavalent group.
In certain embodiments, r3 is 0.
In some instancesIn the embodiment, Q8The method comprises the following steps:
groups useful in the preparation of formula (I) -LinkD (-R) are described herein and in WO 2015/188197M1)pThe compound of (1).
In certain embodiments, the non-bioreversible linker group is
Wherein the group is linked at one end to a polynucleotide and at the other end to a target moiety (in one embodiment, an antibody).
Auxiliary part
The auxiliary moiety is a monovalent group comprising 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 (ethylene imine)), or a sugar alcohol (e.g., glucitol)). The theoretical molecular weight of the auxiliary moiety may be between 100Da and 2.5kDa (e.g. between 350Da and 2.5kDa, between 100Da and 1200Da, or between 1kDa and 2.5 kDa).
To visualize uptake or to monitor movement of the conjugates of the invention within the cell (e.g., using Fluorescence Recovery After Photobleaching (FRAP)), a dye may be included in the phosphate group. Dyes known in the art may be included as an adjunct moiety to the attachment of a polynucleotide via a phosphate or phosphorothioate at the 5 '-or 3' -terminus or via a phosphate or phosphorothioate which binds two consecutive nucleosides together. Non-limiting examples of useful structures that can be used as dyes include FITC, RD1, Allophycocyanin (APC), aCFTM dye (Biotium, Hayward, CA), BODIPY (Invitrogen 10, Life Technologies, Carlsbad, CA), (InvitrogenTM,Life Technologies,Carlsbad,CA)、DyLight Fluor(Thermo Scientific Pierce Protein Biology Products, Rockford, IL), ATTO (ATTO-TEC GmbH, Siegen, Germany), FluoProbe (Interchim SA,france) and abbero Probes (abbero GmbH,germany).
Hydrophilic polymers and positively charged polymers that can be used as an adjunct moiety in the immunomodulatory polynucleotides of the invention and the conjugates of the invention are known in the art. A non-limiting example of a hydrophilic polymer is poly (ethylene glycol). A non-limiting example of a positively charged polymer is poly (ethyleneimine).
The sugar alcohol based auxiliary moiety may be, for example, an amino-terminal glucitol or a glucitol cluster. The amino-terminal glucitol co-moiety is:
non-limiting examples of glucitol clusters are:
in one embodiment, provided herein is a compound of formula (B):
Rx-LN-(Q)e (B)
or a stereoisomer, a mixture of two or more diastereomers, a tautomer, or a mixture of two or more tautomers thereof; or a pharmaceutically acceptable salt, solvate or hydrate thereof;
wherein:
Rxis a conjugate group;
LNis a joint;
each Q is independently a polynucleotide comprising a phosphotriester; and
e is an integer of 1, 2, 3 or 4.
In certain embodiments, in formula (B), R xIs that
In certain embodiments, in formula (B), LNIs a linker comprising polyethylene glycol.
In certain embodiments, in formula (B), LNIs thatWherein d is an integer from about 0 to about 50. In certain embodiments, d is an integer from about 0 to about 10. In certain embodiments, d is an integer from about 0 to about 5. In certain embodiments, d is an integer of about 0, about 1, or about 3.
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):
wherein each XN、X3’、X5’、YPB and c are as defined herein.
Targeting moieties
The targeting moieties used in the conjugates provided herein are directed to target-specific cells and tissues in the body for targeted delivery of the conjugated payload polynucleotides. In certain embodiments, the cells targeted by the conjugates provided herein are natural killer cells. In certain embodiments, the cells targeted by the conjugates provided herein are bone marrow cells. In certain embodiments, the cells targeted by the conjugates provided herein are neutrophils. In certain embodiments, the cells targeted by the conjugates provided herein are monocytes. In certain embodiments, the cell targeted by the conjugates provided herein is a macrophage. In certain embodiments, the cells targeted by the conjugates provided herein are Dendritic Cells (DCs). In certain embodiments, the cell targeted by the conjugates provided herein is a mast cell. In certain embodiments, the cell targeted by the conjugates provided herein is a Tumor Associated Macrophage (TAM). In certain embodiments, the cells targeted by the conjugates provided herein are myeloid-derived suppressor cells (MDSCs).
In certain embodiments, the targeting moiety is an antigen binding moiety. In certain embodiments, the targeting moiety is an antibody or antigen binding fragment thereof.
In certain embodiments, the antigen-binding portion in the conjugates provided herein is an antibody or antigen-binding fragment thereof (e.g., f (ab)2Or Fab) or an engineered derivative thereof (e.g., Fcab or a fusion protein (e.g., scFv)). In certain embodiments, the antigen binding portion in the conjugates provided herein is a human or chimeric (e.g., humanized) antibody.
The antigen binding portion targets cells having a surface antigen that is recognized by the antigen binding portion.
In certain embodiments, the targeting moiety is an antibody that binds to an antigen expressed by NK cells. Exemplary antigens expressed BY NK cells and that may be targeted BY the conjugates provided herein include, but are not limited to, CD11B, CD11c, CD16/32, CD49B, CD56(NCAM), CD57, CD69, CD94, CD122, CD158(Kir), CD161(NK-1.1), CD244(2B 94), CD314(NKG2 94), CD319(CRACC), CD328(Siglec-7), CD335 (NKp 94), Ly 94, Ly108, V α 24-J α 18TCR iNKT), granulysin, granzyme, perforin, SIRP- α, LAIR 94, SIEC-3 (CD 94), SIGLEC-7, SIGLEC-9, LIR 94 (ILT 94, LILILILIB 94), NKR-P1 RB (KL3672), CD94-NKG 2DL 72, NKR-94, CD KIDL 2, NKR 94, NKR 2DS (NKR 94, CD94, NKR 94 DL 72, CD94, NKR 2DL 72, CD94, NKR 94, CD94, NKR 2DS (NCR 94, NKR 94, CD94, NKR 2DL 72, CD94, NKR 2DL 2DS (NKR 94, CD94, NKR 2DL 72, CD94, NKR 2DS 94, CD94, NKR 94, CD 36DL 2DS (NKR 94, CD 36DL 2DS 94, CD94, NKR 2D 94, CD94, CRTAM, CD2, CD7, CD11a, CD18, CD25, CD27, CD28, NTB-A (SLAMF6), PSGL1, CD96(Tactile), CD100(SEMA4D), NKp80(KLRF1, CLEC5C), SLAMF7(CRACC, CS1, CD319) and CD244(2B4, SLAMF 4).
In certain embodiments, the targeting moiety is an antibody that binds to an antigen expressed by bone marrow cells. Exemplary antigens expressed by bone marrow cells and that can be targeted by the conjugates provided herein include, but are not limited to, siglec-3, siglec 7, siglec 9, siglec 15, CD200R, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, M-CSF, CSF-1R, GM-CSF R, IL 4R, arginase, IDO, TDO, MPO, EP2, COX-2, CCR2, CCR-7, CXCR1, CX3CR1, CXCR2, CXCR3, CXCR4, CXCR7, c-Kit, CD244, L-selectedin/CD 62L, CD11b, CD11c, CD68, CD163, CD204, DEC205, IL-1R, CD31, sirpa, SIRP β, PD-L1, acam-8/bdcm 66, CD 11/BDCA 103, CD11, CD66b, CD 3-3 a-3, CD 36123.
In certain embodiments, the targeting moiety is an antibody that binds to an antigen expressed by the MDSCs. Exemplary antigens expressed by MDSCs and that can be targeted by the conjugates provided herein include, but are not limited to, Siglec-3, Siglec 7, Siglec 9, Siglec 15, CD200R, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, M-CSF, CSF-1R, GM-CSF R, IL 4R, arginase, IDO, TDO, MPO, EP2, COX-2, CCR2, CCR-7, CXCR1, CX3CR1, CXCR2, CXCR3, CXCR4, CXCR7, c-Kit, CD244, L-selectin/CD62L, CD11b, CD11c, CD68, CD163, CD204, DEC205, IL-1R, CD31, sirpa, sirpp β, PD-L1, cem-468/CD 103, BDCA 66b, BDCA2, BDCA 123-3, BDCA 123.
In certain embodiments, the targeting moiety is an antibody that binds to an antigen expressed by the TAM. Exemplary antigens expressed by TAMs and that can be targeted by the conjugates provided herein include, but are not limited to, Siglec-3, Siglec 7, Siglec 9, Siglec 15, CD200R, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, M-CSF, CSF-1R, GM-CSF R, IL 4R, arginase, IDO, TDO, MPO, EP2, COX-2, CCR2, CCR-7, CXCR1, CX3CR1, CXCR2, CXCR3, CXCR4, CXCR7, c-Kit, CD244, L-selectin/CD 62L, CD11b, CD11c, CD68, CD163, CD204, DEC205, IL-1R, CD31, sirpa, sirpp β, PD-L1, cem-468/CD 103, BDCA 66b, BDCA2, BDCA 123-3, BDCA 123.
In certain embodiments, the targeting moiety is an antibody that binds to an NK cell-specific antigen. In certain embodiments, the anti-CD 56 antibody targets NK cells. In certain embodiments, the targeting moiety is an anti-CD 56 antibody. In certain embodiments, the antibody is a monoclonal anti-CD 56 antibody. In certain embodiments, the antibody is a murine anti-CD 56 antibody. In certain embodiments, the murine anti-CD 56 antibody is clone 5.1H11(BioLegend, Cat No: 362502). In certain embodiments, the murine anti-CD 56 antibody is clone MEM-188(BioLegend, 304601). In certain embodiments, the murine anti-CD 56 antibody is clone QA17A16(BioLegend, Cat No: 392402). In certain embodiments, the antibody is a humanized anti-CD 56 antibody. In certain embodiments, the antibody is a human anti-CD 56 antibody. In certain embodiments, the antibody is a humanized anti-CD 56 antibody.
In certain embodiments, the targeting moiety is an antibody that binds a myeloid cell-specific antigen. In certain embodiments, the anti-sirpa antibody targets bone marrow cells. In certain embodiments, the targeting moiety is an anti-sirpa antibody. In certain embodiments, the antibody is a monoclonal anti-sirpa antibody. In certain embodiments, the antibody is a murine anti-sirpa antibody. In certain embodiments, the antibody is a humanized anti-sirpa antibody. In certain embodiments, the antibody is a human anti-sirpa antibody.
In certain embodiments, an anti-sirpa antibody (119 or 119 germline mutation) is a human antibody comprising a VH and a VL, wherein the VH is independently selected from the group consisting of the following sequences:
and VL is independently selected from the following sequences:
in certain embodiments, an anti-SIRPa antibody (119 or 119 germline mutation) is a human antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, each of which is independently selected from the following table.
The 119 human antibody is a CD 47-blocker, which is described in table P of WO 2018/057669 a1, the disclosure of which is incorporated herein by reference in its entirety.
In certain embodiments, an anti-sirpa antibody (135 or 135 germline mutation) is a human antibody comprising a VH and a VL, wherein the VH is independently selected from the group consisting of the following sequences:
And VL is independently selected from the following sequences:
in certain embodiments, an anti-SIRPa antibody (135 or 135 germline mutation) is a human antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, each of which is independently selected from the following table.
The 135 human antibody is a CD 47-blocker, which is described in table P of WO 2018/057669 a1, the disclosure of which is incorporated herein by reference in its entirety.
In certain embodiments, an anti-sirpa antibody (AB21, AB21 germline mutation, or a humanized version of AB 21) is an antibody comprising a VH and a VL, wherein the VH is independently selected from the following sequences:
and VL is independently selected from the following sequences:
in certain embodiments, an anti-SIRPa antibody (AB21, AB21 germline mutation, or a humanized form of AB 21) is a humanized antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, each of which is independently selected from the following table.
The AB21 humanized antibody is a CD 47-blocker, which is described in table P of WO 2018/057669 a1, the disclosure of which is incorporated herein by reference in its entirety.
In certain embodiments, an anti-sirpa antibody (136 or 136 germline mutation) is a human antibody comprising a VH and a VL, wherein the VH is independently selected from the group consisting of the following sequences:
and VL is independently selected from the following sequences:
in certain embodiments, an anti-SIRPa antibody (136 or 136 germline mutation) is a human antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, each of which is independently selected from the following table.
136 human antibodies are non-blocking agents, which are described in table P of WO 2018/057669 a1, the disclosure of which is incorporated herein by reference in its entirety.
In certain embodiments, an anti-SIRPa antibody (218 or humanized 218) is an antibody comprising a VH and a VL, wherein the VH has the sequence DVQLVESGGGVVRPGESLTLSCTASGFTFTSSTMNWVRQAPGEGL DWVSSISTSGVITYYADSVKGRATISRDNSKNTLYLRLFSLRADDT AIYYCATDTFDHWGPGTLVTVSS (SEQ ID NO: 584); and VL is independently selected from the following sequences:
218 human antibodies are non-blocking agents, which are described in table P of WO 2018/057669 a1, the disclosure of which is incorporated herein by reference in its entirety.
In some embodiments, an anti-sirpa antibody comprises a heavy chain Variable (VH) domain comprising a heavy chain Variable (VH) sequence comprising a sequence selected from SEQ ID NOs: 498-500, HVR-H1 comprising the sequence SEQ ID NO: 501, and HVR-H2 comprising the sequence SEQ ID NO: HVR-H3 of 502; the light chain Variable (VL) domain comprises a heavy chain Variable (VL) domain comprising the sequence of SEQ ID NO: 503, HVR-L1 comprising the sequence SEQ ID NO: 504 and HVR-L2 comprising the sequence SEQ ID NO: 505 HVR-L3. In some embodiments, an anti-sirpa antibody comprises a heavy chain Variable (VH) domain comprising a heavy chain Variable (VH) comprising the sequence of SEQ ID NO: 498 HVR-H1, comprising the sequence SEQ ID NO: 501 and HVR-H2 comprising the sequence SEQ ID NO: HVR-H3 of 502; the light chain Variable (VL) domain comprises a heavy chain Variable (VL) domain comprising the sequence of SEQ ID NO: 503, HVR-L1 comprising the sequence SEQ ID NO: 504 and HVR-L2 comprising the sequence SEQ ID NO: 505 HVR-L3. In some embodiments, an anti-sirpa antibody comprises a heavy chain Variable (VH) domain comprising a heavy chain Variable (VH) comprising the sequence of SEQ ID NO: 499 HVR-H1, comprising the sequence SEQ ID NO: 501 and HVR-H2 comprising the sequence SEQ ID NO: HVR-H3 of 502; the light chain Variable (VL) domain comprises a heavy chain Variable (VL) domain comprising the sequence of SEQ ID NO: 503, HVR-L1 comprising the sequence SEQ ID NO: 504 and HVR-L2 comprising the sequence SEQ ID NO: 505 HVR-L3. In some embodiments, an anti-sirpa antibody comprises a heavy chain Variable (VH) domain comprising a heavy chain Variable (VH) comprising the sequence of SEQ ID NO: 500, HVR-H1 comprising the sequence SEQ ID NO: 501 and HVR-H2 comprising the sequence SEQ ID NO: HVR-H3 of 502; the light chain Variable (VL) domain comprises a heavy chain Variable (VL) domain comprising the sequence of SEQ ID NO: 503, HVR-L1 comprising the sequence SEQ ID NO: 504 and HVR-L2 comprising the sequence SEQ ID NO: 505 HVR-L3.
In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising a sequence selected from SEQ ID NOs: 490-495 and/or a VH domain comprising the sequence of SEQ ID NO: 496 or 497. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 490 and/or a VH domain comprising the sequence of SEQ ID NO: 496. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 491 and/or a VH domain comprising the sequence of SEQ ID NO: 496. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 492 and/or a VH domain comprising the sequence of SEQ ID NO: 496. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 493 and/or a VH domain comprising the sequence of SEQ ID NO: 496. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 494 and/or a VH domain comprising the sequence of SEQ ID NO: 496. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 495 and/or a VH domain comprising the sequence of SEQ ID NO: 496. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 490 and/or a VH domain comprising the sequence of SEQ ID NO: 497 sequence of VL domain. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 491 and/or a VH domain comprising the sequence of SEQ ID NO: 497 sequence of VL domain. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 492 and/or a VH domain comprising the sequence of SEQ ID NO: 497 sequence of VL domain. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 493 and/or a VH domain comprising the sequence of SEQ ID NO: 497 sequence of VL domain. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 494 and/or a VH domain comprising the sequence of SEQ ID NO: 497 sequence of VL domain. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 495 and/or a VH domain comprising the sequence of SEQ ID NO: 497 sequence of VL domain.
In some embodiments, an anti-sirpa antibody comprises a heavy chain Variable (VH) domain comprising a heavy chain Variable (VH) sequence comprising a sequence selected from SEQ ID NOs: 512-514, HVR-H1 comprising the sequence SEQ ID NO: 515 and an HVR-H2 comprising the sequence SEQ ID NO: 516 HVR-H3; the light chain Variable (VL) domain comprises a heavy chain Variable (VL) domain comprising the sequence of SEQ ID NO: 517, HVR-L1 comprising the sequence SEQ ID NO: 518 and a HVR-L2 comprising the sequence of SEQ ID NO: 519 HVR-L3. In some embodiments, an anti-sirpa antibody comprises a heavy chain Variable (VH) domain comprising a heavy chain Variable (VH) comprising the sequence of SEQ ID NO: 512, HVR-H1 comprising the sequence SEQ ID NO: 515 and HVR-H2 comprising the sequence SEQ ID NO: 516 HVR-H3; the light chain Variable (VL) domain comprises a heavy chain Variable (VL) domain comprising the sequence of SEQ ID NO: 517, HVR-L1 comprising the sequence SEQ ID NO: 518 and a HVR-L2 comprising the sequence of SEQ ID NO: 519 HVR-L3. In some embodiments, an anti-sirpa antibody comprises a heavy chain Variable (VH) domain comprising a heavy chain Variable (VH) comprising the sequence of SEQ ID NO: 513, HVR-H1 comprising the sequence SEQ ID NO: 515 and HVR-H2 comprising the sequence SEQ ID NO: 516 HVR-H3; the light chain Variable (VL) domain comprises a heavy chain Variable (VL) domain comprising the sequence of SEQ ID NO: 517, HVR-L1 comprising the sequence SEQ ID NO: 518 and a HVR-L2 comprising the sequence of SEQ ID NO: 519 HVR-L3. In some embodiments, an anti-sirpa antibody comprises a heavy chain Variable (VH) domain comprising a heavy chain Variable (VH) comprising the sequence of SEQ ID NO: 514, HVR-H1 comprising the sequence SEQ ID NO: 515 and HVR-H2 comprising the sequence SEQ ID NO: 516 HVR-H3; the light chain Variable (VL) domain comprises a heavy chain Variable (VL) domain comprising the sequence of SEQ ID NO: 517, HVR-L1 comprising the sequence SEQ ID NO: 518 and a HVR-L2 comprising the sequence of SEQ ID NO: 519 HVR-L3.
In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising a sequence selected from SEQ ID NOs: 506-509 and/or a VH domain comprising the sequence of SEQ ID NO: 510 or 511, or a VL domain of a sequence of seq id no. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 506 and/or a VH domain comprising the sequence of SEQ ID NO: 510, VL domain of the sequence of seq id no. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 507 and/or a VH domain comprising the sequence of SEQ ID NO: 510, VL domain of the sequence of seq id no. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 508 and/or a VH domain comprising the sequence of SEQ ID NO: 510, VL domain of the sequence of seq id no. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 509 and/or a VH domain comprising the sequence of SEQ ID NO: 510, VL domain of the sequence of seq id no. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 506 and/or a VH domain comprising the sequence of SEQ ID NO: 511 of seq id no. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 507 and/or a VH domain comprising the sequence of SEQ ID NO: 511 of seq id no. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 508 and/or a VH domain comprising the sequence of SEQ ID NO: 511 of seq id no. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 509 and/or a VH domain comprising the sequence of SEQ ID NO: 511 of seq id no.
In some embodiments, an anti-sirpa antibody comprises a heavy chain Variable (VH) domain comprising a heavy chain Variable (VH) sequence comprising a sequence selected from SEQ ID NOs: 533-H1, the HVR-H1 comprising the sequence of SEQ ID NO: 536 and HVR-H2 comprising the sequence SEQ ID NO: 537, HVR-H3; the light chain Variable (VL) domain comprises a heavy chain variable domain comprising a sequence selected from SEQ ID NOs: 538-542, the HVR-L1 comprising the sequence SEQ ID NO: 543 and HVR-L2 comprising a sequence selected from SEQ ID NO: 544-546. In some embodiments, an anti-sirpa antibody comprises a heavy chain Variable (VH) domain comprising a heavy chain Variable (VH) comprising the sequence of SEQ ID NO: 534, HVR-H1 comprising the sequence SEQ ID NO: 536 and HVR-H2 comprising the sequence SEQ ID NO: 537, HVR-H3; the light chain Variable (VL) domain comprises a heavy chain Variable (VL) domain comprising the sequence of SEQ ID NO: 539, HVR-L1 comprising the sequence SEQ ID NO: 543 and HVR-L2 comprising the sequence SEQ ID NO: 545 HVR-L3. In some embodiments, an anti-sirpa antibody comprises a heavy chain Variable (VH) domain comprising a heavy chain Variable (VH) comprising the sequence of SEQ ID NO: 533, HVR-H1 comprising the sequence SEQ ID NO: 536 and HVR-H2 comprising the sequence SEQ ID NO: 537, HVR-H3; the light chain Variable (VL) domain comprises a heavy chain Variable (VL) domain comprising the sequence of SEQ ID NO: 542, HVR-L1 comprising the sequence SEQ ID NO: 543 and HVR-L2 comprising the sequence SEQ ID NO: 546, HVR-L3. In some embodiments, an anti-sirpa antibody comprises a heavy chain Variable (VH) domain comprising a heavy chain Variable (VH) comprising the sequence of SEQ ID NO: 498 HVR-H1, comprising the sequence SEQ ID NO: 501 and HVR-H2 comprising the sequence SEQ ID NO: HVR-H3 of 502; the light chain Variable (VL) domain comprises a heavy chain Variable (VL) domain comprising the sequence of SEQ ID NO: 503, HVR-L1 comprising the sequence SEQ ID NO: 504 and HVR-L2 comprising the sequence SEQ ID NO: 505 of HVR-L. In some embodiments, an anti-sirpa antibody comprises a heavy chain Variable (VH) domain comprising a heavy chain Variable (VH) comprising the sequence of SEQ ID NO: 554, HVR-H1 comprising the sequence of SEQ ID NO: 557 and HVR-H2 comprising the sequence SEQ ID NO: 558 of HVR-H3; the light chain Variable (VL) domain comprises a heavy chain Variable (VL) domain comprising the sequence of SEQ ID NO: 559 HVR-L1, comprising the sequence SEQ ID NO: 560 and HVR-L2 comprising the sequence SEQ ID NO: 561 HVR-L3.
In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising a sequence selected from SEQ ID NOs: 520-523 and/or a VH domain comprising a sequence selected from SEQ ID NO: 525 and 532.
In some embodiments, an anti-sirpa antibody comprises a heavy chain Variable (VH) domain comprising a heavy chain Variable (VH) sequence comprising a sequence selected from SEQ ID NOs: 554-H1 of the sequence of 556, the HVR-H1 comprising the sequence SEQ ID NO: 557 and HVR-H2 comprising the sequence SEQ ID NO: 558 of HVR-H3; the light chain Variable (VL) domain comprises the sequence SEQ ID NO: 559 HVR-L1, comprising the sequence SEQ ID NO: 560 and HVR-L2 comprising the sequence SEQ ID NO: 561 HVR-L3. In some embodiments, an anti-sirpa antibody comprises a heavy chain Variable (VH) domain comprising a heavy chain Variable (VH) comprising the sequence of SEQ ID NO: 554, HVR-H1 comprising the sequence of SEQ ID NO: 557 and HVR-H2 comprising the sequence SEQ ID NO: 558 of HVR-H3; the light chain Variable (VL) domain comprises a heavy chain Variable (VL) domain comprising the sequence of SEQ ID NO: 559 HVR-L1, comprising the sequence SEQ ID NO: 560 and HVR-L2 comprising the sequence SEQ ID NO: 561 HVR-L3. In some embodiments, an anti-sirpa antibody comprises a heavy chain Variable (VH) domain comprising a heavy chain Variable (VH) comprising the sequence of SEQ ID NO: 555 HVR-H1, comprising the sequence SEQ ID NO: 557 and HVR-H2 comprising the sequence SEQ ID NO: 558 of HVR-H3; the light chain Variable (VL) domain comprises a heavy chain Variable (VL) domain comprising the sequence of SEQ ID NO: 559 HVR-L1, comprising the sequence SEQ ID NO: 560 and HVR-L2 comprising the sequence SEQ ID NO: 561 HVR-L3. In some embodiments, an anti-sirpa antibody comprises a heavy chain Variable (VH) domain comprising a heavy chain Variable (VH) comprising the sequence of SEQ ID NO: 556, HVR-H1 comprising the sequence of SEQ ID NO: 557 and HVR-H2 comprising the sequence SEQ ID NO: 558 of HVR-H3; the light chain Variable (VL) domain comprises a heavy chain Variable (VL) domain comprising the sequence of SEQ ID NO: 559 HVR-L1, comprising the sequence SEQ ID NO: 560 and HVR-L2 comprising the sequence SEQ ID NO: 561 HVR-L3.
In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising a sequence selected from SEQ ID NOs: 547-550 and/or a VH domain comprising a sequence selected from SEQ ID NO: the VL domain of the sequence of 551-553. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 547 and/or a VH domain comprising the sequence of SEQ ID NO: 551, and VL domain of the sequence of seq id no. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 548 and/or a VH domain comprising the sequence of SEQ ID NO: 551, and VL domain of the sequence of seq id no. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: a VH domain of the sequence of SEQ ID NO: 551, and VL domain of the sequence of seq id no. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 550 and/or a VH domain comprising the sequence of SEQ ID NO: 551, and VL domain of the sequence of seq id no. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 547 and/or a VH domain comprising the sequence of SEQ ID NO: 552, VL domain. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 548 and/or a VH domain comprising the sequence of SEQ ID NO: 552, VL domain. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: a VH domain of the sequence of SEQ ID NO: 552, VL domain. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 550 and/or a VH domain comprising the sequence of SEQ ID NO: 552, VL domain. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 547 and/or a VH domain comprising the sequence of SEQ ID NO: 553, or a VL domain of the sequence of seq id no. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 548 and/or a VH domain comprising the sequence of SEQ ID NO: 553, or a VL domain of the sequence of seq id no. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: a VH domain of the sequence of SEQ ID NO: 553, or a VL domain of the sequence of seq id no. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 550 and/or a VH domain comprising the sequence of SEQ ID NO: 553, or a VL domain of the sequence of seq id no.
In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 584 and/or a VH domain comprising a sequence selected from SEQ ID NO: 585. 562 and 563. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 584 and/or a VH domain comprising the sequence of SEQ ID NO: 585 of the sequence VL domain. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 584 and/or a VH domain comprising the sequence of SEQ ID NO: 562. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 584 and/or a VH domain comprising the sequence of SEQ ID NO: 563, or a VL domain of a sequence of seq id no. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 584 and/or a VH domain of three HVRs comprising the sequence of SEQ ID NO: VL domain of three HVRs of sequence 585. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 584 and/or a VH domain of three HVRs comprising the sequence of SEQ ID NO: 562, VL domain of three HVRs of sequence. In some embodiments, an anti-sirpa antibody comprises a heavy chain variable region comprising SEQ ID NO: 584 and/or a VH domain of three HVRs comprising the sequence of SEQ ID NO: 563, VL domain of three HVRs of sequence.
Other anti-SIRP antibodies are disclosed in US 2018/0037652 a 1; WO 2016/205042 a 1; WO 2017/178653 a 2; WO 2018/107058A 1 and WO 2018/057669A 1; the disclosure of each of which is incorporated herein by reference in its entirety.
In some embodiments, the antibodies provided herein comprise a human Fc region, for example a human IgG1, IgG2, or IgG4 Fc region.
In some embodiments, the Fc region of an antibody provided herein comprises one or more mutations that affect one or more antibody properties, such as stability, glycosylation or other modification patterns, effector cell function, pharmacokinetics, and the like. In some embodiments, the antibodies provided herein have reduced or minimized glycosylation. In some embodiments, the antibodies provided herein have eliminated or reduced effector function. Exemplary Fc mutations include, but are not limited to, (i) human IgG1 Fc region mutations L234A, L235A, G237A, and N297A; (ii) human IgG2 Fc region mutations a330S, P331S, and N297A; (iii) human IgG4 Fc region mutations S228P, E233P, F234V, L235A, delG236, and N297A (EU numbering). In some embodiments, the human IgG2 Fc region comprises the a330S and P331S mutations. In some embodiments, the human IgG4 Fc region comprises the S288P mutation. In some embodiments, the human IgG4 Fc region comprises the S288P and L235E mutations.
In some embodiments, the antibodies provided herein comprise a human IgG1Fc region comprising L234A, L235A, and G237A mutations, according to EU numbering. In some embodiments, the antibodies provided herein comprise a human IgG1Fc region comprising L234A, L235A, G237A, and N297A mutations according to EU numbering. In some embodiments, the antibodies provided herein comprise a human IgG1Fc region comprising the N297A mutation, according to EU numbering. In some embodiments, the antibodies provided herein comprise a human IgG1Fc region comprising a D265A mutation, according to EU numbering. In some embodiments, the antibodies provided herein comprise a human IgG1Fc region comprising the D265A and N297A mutations according to EU numbering. In some embodiments, the antibodies provided herein comprise a human IgG2 Fc region comprising a330S and P331S mutations, according to EU numbering. In some embodiments, the antibodies provided herein comprise a human IgG2 Fc region comprising a330S, P331S, and N297A mutations, according to EU numbering. In some embodiments, the antibodies provided herein comprise a human IgG2 Fc region comprising the N297A mutation, according to EU numbering. In some embodiments, the antibodies provided herein comprise a human IgG4 Fc region comprising the S228P mutation, according to EU numbering. In some embodiments, the antibodies provided herein comprise a human IgG4 Fc region comprising S228P and D265A mutations according to EU numbering. In some embodiments, the antibodies provided herein comprise a human IgG4 Fc region comprising the S228P and L235E mutations according to EU numbering. In some embodiments, the antibodies provided herein comprise a human IgG4 Fc region comprising the S228P and N297A mutations according to EU numbering. In some embodiments, the antibodies provided herein comprise a human IgG4 Fc region comprising the S228P, E233P, F234V, L235A, delG236, and N297A mutations, according to EU numbering. In some embodiments, the antibodies provided herein comprise a polypeptide comprising a sequence selected from SEQ ID NOs: 564-578.
In some embodiments, the antibodies provided herein comprise a human kappa light chain constant domain, e.g., comprising SEQ ID NO: 579. In some embodiments, the antibodies provided herein comprise a human lambda light chain constant domain, such as IGLC1 or IGLC2 (as shown in the exemplary Fc region sequences of SEQ ID nos: 580 and 581, respectively).
Antibodies that target cell surface antigens can trigger immune stimulation and effector functions associated with Fc receptor (FcR) binding on immune cells. There are many Fc receptors that are specific for a particular class of antibodies, including IgG (gamma receptor), IgE (eta receptor), IgA (alpha receptor), and IgM (mu receptor). Binding of the Fc region to cell surface Fc receptors can trigger a number of biological responses, including phagocytosis of antibody-coated particles (antibody-dependent cell-mediated phagocytosis or ADCP), clearance of immune complexes, lysis of antibody-coated cells by killer cells (antibody-dependent cell-mediated cytotoxicity or ADCC), and release of inflammatory mediators, placental transfer, and control of immunoglobulin production. In addition, binding of the C1 component of complement to the antibody activates the complement system. Activation of complement may be important for lysis of cellular pathogens. However, complement activation can also stimulate inflammatory responses, and can also be involved in autoimmune hypersensitivity or other immune system diseases. Variant Fc regions with reduced or ablated ability to bind certain Fc receptors can be used to develop therapeutic antibodies and Fc fusion polypeptide constructs that function by targeting, activating, or neutralizing ligand function without damaging or destroying local cells or tissues.
In some embodiments, an Fc domain monomer refers to a polypeptide chain that includes second and third antibody constant domains (e.g., CH2 and CH 3). In some embodiments, the Fc domain monomer further comprises a hinge domain. In some embodiments, the Fc domain monomer has any immunoglobulin antibody isotype including IgG, IgE, IgM, IgA, and IgD. Additionally, in some embodiments, the Fc domain monomer has any IgG subtype (e.g., IgG1, IgG2, IgG2a, IgG2b, IgG2c, IgG3, and IgG 4). In some embodiments, the Fc domain monomer includes up to ten changes (e.g., 1-10, 1-8, 1-6, 1-4 amino acid substitutions, additions or insertions, deletions, or combinations thereof) to the wild-type Fc domain monomer sequence that alter the interaction between the Fc domain and the Fc receptor.
In some embodiments, an Fc domain monomer or fragment of an Fc domain monomer of an immunoglobulin is capable of forming an Fc domain with another Fc domain monomer. In some embodiments, an Fc domain monomer or fragment of an Fc domain monomer of an immunoglobulin is incapable of forming an Fc domain with another Fc domain monomer. In some embodiments, an Fc domain monomer or fragment of an Fc domain is fused to a polypeptide of the present disclosure to increase the serum half-life of the polypeptide. In some embodiments, an Fc domain monomer or fragment of an Fc domain monomer fused to a polypeptide of the present disclosure dimerizes with a second Fc domain monomer to form an Fc domain that binds an Fc receptor, or alternatively, the Fc domain monomer binds an Fc receptor. In some embodiments, an Fc domain or fragment of an Fc domain fused to a polypeptide to increase the serum half-life of the polypeptide does not induce any immune system-related response. The Fc domain comprises two Fc domain monomers that dimerize via interaction between the constant domains of the CH3 antibody.
The wild-type Fc domain forms the smallest structure that binds to an Fc receptor (e.g., Fc γ RI, Fc γ RIIa, Fc γ RIIb, Fc γ RIIIa, Fc γ RIIIb, and Fc γ RIV). In some embodiments, the Fc domain in the antibodies of the present disclosure comprises one or more amino acid substitutions, additions or insertions, deletions, or any combination thereof, which results in reduced effector function, e.g., reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced complement-dependent cytolysis (CDC), reduced antibody-dependent cell-mediated phagocytosis (ADCP), or any combination thereof. For example, antibodies of the present disclosure can exhibit reduced binding (e.g., minimal or no binding) to human Fc receptors and reduced binding (e.g., minimal or no binding) to complement protein C1 q; reduced binding (e.g., minimal or no binding) to human Fc γ RI, Fc γ RIIA, Fc γ RIIB, Fc γ RIIIB, or any combination thereof; an alteration or reduction of antibody-dependent effector function, such as ADCC, CDC, ADCP or any combination thereof; and the like. Exemplary mutations include, but are not limited to, one or more amino acid substitutions at E233, L234, L235, G236, G237, D265, D270, N297, E318, K320, K322, a327, a330, P331, or P329 (numbering according to the EU index of Kabat ((Sequences of Proteins of Immunological Interest, fifth edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).
In some embodiments, binding of the antibodies of the present disclosure to CD16a, CD32a, CD32b, CD32c, and CD64 fey receptors is reduced or eliminated. In some embodiments, the C1q binding of an antibody having a non-native Fc region described herein is reduced by at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more as compared to an antibody comprising a wild-type Fc region. In some embodiments, the CDC of an antibody having a non-native Fc region described herein is reduced by at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more as compared to an antibody comprising a wild-type Fc region.
In some embodiments, the Fc variants herein are minimally glycosylated or have reduced glycosylation relative to the wild-type sequence. In some embodiments, deglycosylation is accomplished by mutation of N297A or by mutation of N297 to any amino acid other than N.
In some embodiments, variants of an antibody IgG constant region (e.g., Fc variants) have a reduced ability to specifically bind fey receptors or have a reduced ability to induce phagocytosis. In some embodiments, variants of an antibody IgG constant region (e.g., Fc variants) have a reduced ability to specifically bind fey receptors and have a reduced ability to induce phagocytosis. For example, in some embodiments, the Fc domain is mutated to lack effector function, typically a "dead" Fc domain. For example, in some embodiments, the Fc domain includes specific amino acid substitutions known to minimize the interaction between the Fc domain and the fey receptor. In some embodiments, the Fc domain monomer is from an IgG1 antibody and includes one or more of the amino acid substitutions L234A, L235A, G237A, and N297A (as specified by the EU numbering system according to Kabat et al, 1991). In some embodiments, the Fc domain monomer is from an IgG1 antibody and includes one or more of the amino acid substitutions L234A, L235A, and G237A (as specified by the EU numbering system according to Kabat et al, 1991). In some embodiments, the Fc domain monomer is from an IgG1 antibody and includes N297A (as specified according to the EU numbering system of Kabat et al, 1991). In some embodiments, the Fc domain monomer is from an IgG1 antibody and includes D265A (as specified according to the EU numbering system of Kabat et al, 1991). In some embodiments, the Fc domain monomer is from an IgG1 antibody and comprises one or more of the amino acid substitutions D265A and N297A (as specified according to the EU numbering system of Kabat et al, 1991). In some embodiments, such IgG1Fc variants include one or more additional mutations. Non-limiting examples of such other mutations of the human IgG1Fc variant include E318A and K322A. In some cases, the human IgG1Fc variant has up to 12, 11, 10, 9, 8, 7, 6, 5, or 4 or fewer mutations in total compared to the wild-type human IgG1 sequence. In some embodiments, such IgG1Fc variants include one or more additional deletions. For example, in some embodiments, when the polypeptide is produced in a bacterial or mammalian cell, the C-terminal lysine of the heavy chain constant region of Fc IgG1 is deleted, e.g., to increase homogeneity of the polypeptide. In some cases, the human IgG1Fc variants have up to 12, 11, 10, 9, 8, 7, 6, 5, or 4 or fewer deletions in total compared to the wild-type human IgG1 sequence.
In some embodiments, the Fc domain monomer is from an IgG2 antibody and includes the amino acid substitutions a330S, P331S, or both a330S and P331S. The aforementioned amino acid positions are defined according to Kabat et al (1991). The Kabat numbering of amino acid residues of a given antibody can be determined by aligning regions of homology in the sequence of the antibody with "standard" Kabat numbered sequences. In some embodiments, the Fc variant comprises a human IgG2Fc sequence comprising one or more of a330S, P331S, and N297A amino acid substitutions (as specified according to the EU numbering system of Kabat et al (1991)). In some embodiments, the Fc variant comprises a human IgG2Fc sequence comprising one or more of the amino acid substitutions D265A and N297A (as specified according to the EU numbering system of Kabat et al (1991)). In some embodiments, the Fc variant comprises a human IgG2Fc sequence comprising an N297A amino acid substitution (as specified according to the EU numbering system of Kabat et al (1991)). In some embodiments, such IgG2Fc variants include one or more additional mutations. Non-limiting examples of such other mutations for human IgG2Fc variants include V234A, G237A, P238S, V309L, and H268A (designated according to EU numbering system of Kabat et al (1991)). In some cases, the human IgG2Fc variant has up to 10, 9, 8, 7, 6, 5, 4, 3, or fewer mutations in total compared to the wild-type human IgG2 sequence. In some embodiments, such IgG2Fc variants include one or more additional deletions.
When the Fc variant is an IgG4 Fc variant, in some embodiments, such Fc variant comprises an S228P, E233P, F234V, L235A, L235E, or delG236 mutation (as specified according to Kabat et al (1991)). In other instances, such Fc variants comprise S228P and L235E mutations (as specified according to Kabat et al (1991)). In some cases, the human IgG4 Fc variant has up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutations in total compared to the wild-type human IgG4 sequence.
In some embodiments, the Fc variant exhibits reduced binding to an Fc receptor of the subject as compared to a wild-type human IgG Fc region. In some embodiments, the Fc variant exhibits ablated binding to an Fc receptor of the subject as compared to a wild-type human IgG Fc region. In some embodiments, the Fc variant exhibits reduced phagocytosis compared to a wild-type human IgG Fc region. In some embodiments, the Fc variant exhibits ablated phagocytosis as compared to a wild-type human IgG Fc region.
Antibody-dependent cell-mediated cytotoxicity, also referred to herein as ADCC, refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (fcrs) present on certain cytotoxic cells, such as Natural Killer (NK) cells and neutrophils, thereby allowing these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells. Antibody-dependent cell-mediated phagocytosis, also referred to herein as ADCP, refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (fcrs) present on certain phagocytic cells (e.g., macrophages), thereby allowing these phagocytic effector cells to specifically bind to antigen-bearing target cells and subsequently phagocytose and digest the target cells. Ligand-specific high affinity IgG antibodies directed to the surface of target cells can stimulate cytotoxicity or phagocytosis and can be used for such killing. In some embodiments, a polypeptide construct comprising an Fc variant described herein exhibits reduced ADCC or ADCP as compared to a polypeptide construct comprising a wild-type Fc region. In some embodiments, a polypeptide construct comprising an Fc variant as described herein has at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater reduction in ADCC or ADCP as compared to a polypeptide construct comprising a wild-type Fc region. In some embodiments, an antibody comprising an Fc variant described herein exhibits ablated ADCC or ADCP as compared to a polypeptide construct comprising a wild-type Fc region.
Complement-directed cytotoxicity, also referred to herein as CDC, refers to a cytotoxic form in which the complement cascade is activated by complement component C1q that binds antibody Fc. In some embodiments, a polypeptide construct comprising an Fc variant described herein has at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater reduction in C1q binding as compared to a polypeptide construct comprising a wild-type Fc region. In some cases, a polypeptide construct comprising an Fc variant described herein exhibits reduced CDC as compared to a polypeptide construct comprising a wild-type Fc region. In some embodiments, the CDC of a polypeptide construct comprising an Fc variant as described herein is reduced by at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater as compared to a polypeptide construct comprising a wild-type Fc region. In some cases, an antibody comprising an Fc variant described herein exhibits negligible CDC as compared to a polypeptide construct comprising a wild-type Fc region.
Fc variants herein include those that exhibit reduced binding to Fc γ receptors compared to wild-type human IgGFc regions. For example, in some embodiments, the Fc variant binds less to an fey receptor than the wild-type human IgGFc region binds to an fey receptor. In certain instances, binding of an Fc variant to an Fc γ receptor is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (complete ablation of effector function). In some embodiments, the reduced binding is to any one or more fey receptors, e.g., CD16a, CD32a, CD32b, CD32c, or CD 64.
In some cases, the Fc variants disclosed herein exhibit a decrease in phagocytosis compared to their wild-type human IgG Fc region. Such Fc variants exhibit a decrease in phagocytosis compared to a wild-type human IgG Fc region, wherein the decrease in phagocytosis activity is, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some cases, the Fc variant exhibits ablated phagocytosis as compared to its wild-type human IgG Fc region.
In some embodiments, the Fc variants disclosed herein are conjugated to one or more fusion partners. In certain instances, the fusion partner is a therapeutic moiety, such as a cytotoxic agent of the invention. In some cases, fusion partners are selected to initiate targeting, purification, screening, display, etc., of the expressed protein. In some embodiments, the fusion partner also affects the extent of binding to Fc receptors or the extent of decreased phagocytosis.
In certain embodiments, the targeting moiety is a bispecific antibody. In certain embodiments, the bispecific antibody comprises a first antigen-binding domain that binds to the extracellular domain of a human CD56 polypeptide and a second antigen-binding domain that binds to an antigen expressed by a cancer cell. In certain embodiments, the bispecific antibody comprises a first antigen-binding domain that binds the extracellular domain of a human SIRP-a polypeptide and a second antigen-binding domain that binds an antigen expressed by a cancer cell. In certain embodiments, the antigen expressed by the cancer cell is selected from the group consisting of: CD19, CD20, CD22, CD30, CD33, CD38, CD52, CD56, CD70, CD74, CD79B, CD123, CD138, CS1/SLAMF7, Trop-2, 5T4, EphA4, BCMA, mucin 1, mucin 16, PD-L1, PTK7, STEAP1, endothelin B receptor, mesothelin, EGFRvIII, ENPP3, SLC44A4, GNMB, connexin 4, NaPi2B, LIV-1A, guanylate cyclase C, DLL3, EGFR, HER2, VEGF, VEGFR integrin alpha V beta 3, integrin alpha 5 beta 1, MET, IGF1R, TRAILR1, TRAILR2, RANKL, FAP, tenascin, LeyEpCAM, CEA, gpA33, PSMA, TAG72, mucin, CAIX, EPHA3, folate receptor alpha, GD2, GD3 and MHC/peptide complexes comprising from NY-ESO-1/LAGE, SSX-2, MAGE family proteins, MAGE-A3, gp100/pmel17, Melan-A/MART-1, gp75/TRP1, tyrosinase, TRP2, CEA, PSA, TAG-72, immature laminin receptor, MOK/RAGE-1, WT-1, SAP-1, BING-4, EpCAM, MUC1, PRAME, survivin, BRCA1, BRCA2, CDK4, CML66, MART-2, p53, Ras, beta-catenin, TGF-beta RII, HPV E6, or HPV 7. In certain embodiments, the antibody comprises a first antigen-binding domain that binds to the extracellular domain of a human CD56 polypeptide and a second antigen-binding domain that binds to an antigen expressed by an immune cell. In certain embodiments, the antibody comprises a first antigen-binding domain that binds the extracellular domain of a human SIRP-a polypeptide and a second antigen-binding domain that binds an antigen expressed by an immune cell. In some embodiments, the antigen expressed by the immune cell is selected from the group consisting of: BDCA2, BDCA4, ILT7, LILRB1, LILRB2, LILRB3, LILRB4, CSF-1R, CD40, CD40L, CD163, CD206, DEC205, CD47, CD123, IDO, TDO, 41BB, CTLA4, PD1, PD-L1, PD-L2, BTLA, VISTA, LAG-3, CD28, OX40, GITR, CD137, CD27, HVEM, CCR4, CD25, CD103, KIrg1, Nrp1, CD278, Gpr83, TIGIT, CD154, CD160, PVRIG, DNAM, and ICOS.
In certain embodiments, the antibody comprises a constant region sequence selected from the following table.
In certain embodiments, the targeting moiety is a polypeptide. In certain embodiments, the targeting moiety is an RGD peptide, Rabies Virus Glycoprotein (RVG), or DC3 peptide. In certain embodiments, the targeting moiety is an aptamer. In certain embodiments, the targeting moiety comprises a small molecule. In certain embodiments, the targeting moiety comprises a folate, mannose, or PSMA ligand.
Conjugates
In one embodiment, the conjugates provided herein comprise a targeting moiety and one or more, in certain embodiments, about 1 to about 6 or about 1 to about 4, about 1, or about 2, immunomodulatory polynucleotides. In certain embodiments, the conjugate comprises a linker covalently linking the targeting moiety to the immunomodulatory polynucleotide. In certain embodiments, the linker is bonded to a nucleobase, a abasic spacer, a phosphate, a phosphorothioate, or a phosphorodithioate in the immunomodulatory polynucleotide.
In one embodiment, provided herein is a conjugate of formula (C):
or a stereoisomer, a mixture of two or more diastereomers, a tautomer, or a mixture of two or more tautomers thereof; or a pharmaceutically acceptable salt, solvate or hydrate thereof; wherein Ab is a targeting moiety; f is an integer of 1, 2, 3 or 4; and L is NQ and e are each as defined herein.
In certain embodiments, in formula (C), Ab is an antibody. In certain embodiments, in formula (C), Ab is a monoclonal antibody.
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.
In certain embodiments, in formula (C), e and f are each an integer of 1.
In one embodiment, the DAR of the CpG antibody conjugate is 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 DAR of the CpG antibody conjugate is about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8.
Preparation of conjugates
Conjugation
Reactions for conjugating targeting moieties to immunomodulatory polynucleotides are known in the art, including, but not limited to, azido and alkyne-based conjugation groups (e.g., optionally substituted C containing a ring carbon-carbon triple bond)6-16Heterocyclic ring radicals or optionally substituted C8-16Cycloalkynyl) to form a triazole moiety; a Diels-Alder reaction between a dienophile and a diene/heterodiene; bond formation by a pericyclic reaction such as an olefin reaction; formation of amide or thioamide bonds; formation of sulfonamide linkage (e.g., with an azido compound); alcohol or phenol alkylation (e.g., Williamson alkylation), condensation reactions to form oxime, hydrazone, or semicarbazide groups; by conjugate addition of nucleophiles (e.g., amines and thiols); formation of disulfide bonds; and S at a carbonyl group (e.g., on an activated carboxylic acid ester such as pentafluorophenyl (PFP) ester or Tetrafluorophenyl (TFP) ester) or on an electrophilic aromatic hydrocarbon (e.g., on an subfluorinated aromatic hydrocarbon, a fluorobenzonitrile group, or a fluoronitrophenyl group) NAr) (e.g., substitution with amine, thiol, or hydroxyl nucleophiles).
In certain embodiments, the conjugation reaction is a dipolar cycloaddition, and the conjugation moiety comprises an azido group, an optionally substituted C containing an endocyclic carbon-carbon triple bond6-16Heterocyclic ring radicals or optionally substituted C8-16Cycloalkynyl. The complementary reactive group and conjugate group are selected for their mutual complementarity. For example, an azide is used in one of the conjugated group and the complementary reactive group, and an alkyne is used in the other of the conjugated group and the complementary reactive group.
Preparation of immunomodulatory polynucleotides
The immunomodulatory polynucleotides provided herein can be prepared according to methods known in the art of chemical synthesis of polynucleotides, e.g., from nucleoside phosphoramidites. The phosphoramidite can include a conjugate group covalently attached to a phosphorus atom of the phosphoramidite.
Preparation of targeting moieties
The targeting moiety may be conjugated to the immunomodulatory polynucleotide by forming a bond between a conjugate group in the immunomodulatory polynucleotide and a complementary reactive group bonded to the targeting moiety. In certain embodiments, the targeting moiety inherently has a complementary reactive group (e.g., a Q tag (e.g., a QQ tag (e.g., LLQGG (SEQ ID NO:582) or ggglqgg (SEQ ID NO:583) in an antibody or antigen-binding fragment or engineered derivative thereof).
In certain embodiments, the complementary reactive group is optionally substituted C2-12Alkynyl, optionally substituted N-protected amino, azido, N-maleimido, S-protected thiol,Or N-protected forms thereof,Containing an optionally substituted C with an internal ring carbon-carbon triple bond6-16Heterocyclic radical (e.g. of the formula) 1,2,4, 5-tetrazine radicals (e.g. of the formula) Or optionally substituted C8-16Cycloalkynyl (e.g. cycloalkynyl))、-NHRN1Optionally substituted C4-8Strained cycloalkenyl (e.g., trans cyclooctenyl or norbornenyl) or optionally substituted-COOR-containing12or-CHO C1-16An alkyl group;
wherein:
RN1is an H, N-protecting group or optionally substituted C1-6An alkyl group;
each R12Independently is H, optionally substituted C1-6Alkyl, O-protecting groups (e.g., carboxyl protecting groups); and
R13is halogen (e.g., F).
In certain embodiments, the complementary reactive groups are protected until the conjugation reaction. For example, the complementary reactive group to be protected may comprise-COORPGOor-NHRPGNWherein R isPGOIs an O-protecting group (e.g., a carboxyl protecting group), and RPGNIs an N-protecting group.
In certain embodiments, the complementary reactive group is-Z3-QA3，
Wherein:
Z3is a divalent, trivalent, tetravalent or pentavalent radical, one of the valencies being represented by QA3(ii) substituted, one valency being open, and each remaining valency (if present) is independently substituted with an ancillary moiety;
QA3Is optionally substituted C2-12Alkynyl, optionally substituted N-protected amino, azido, N-maleimido, S-protected thiol, Or N-protected forms thereof, Containing an optionally substituted C with an internal ring carbon-carbon triple bond6-16Heterocyclic radical (e.g. of the formula) 1,2,4, 5-tetrazine radicals (e.g. of the formula ) Or optionally substituted C8-16Cycloalkynyl (e.g. cycloalkynyl))、–NHRN1Optionally substituted C4-8Strained cycloalkenyl (e.g., trans cyclooctenyl or norbornenyl) or optionally substituted-COOR-containing12or-CHO C1-16An alkyl group;
wherein:
RN1is an H, N-protecting group or optionally substituted C1-6An alkyl group;
each R12Independently is H, optionally substituted C1-6An alkyl, O-protecting or carboxyl protecting group; and
R13is halogen or F.
In certain embodiments, Z3Comprising a branching group and two divalent segments, wherein the branching group is bonded to each of the two divalent segments, wherein one of the divalent segments has an open valence and the remaining divalent segments are bonded to QA3(ii) a And the branched group comprises one or two groups independently selected from: optionally substituted C1-12Alkanetriyl, optionally substituted C1-12Alkanetetrayl, optionally substituted C2-12Heteroalkanetriyl or optionally substituted C2-12A heteroalkane tetrayl, wherein two valencies of the branching group are bonded to two divalent segments, and each remaining valency is independently substituted with an auxiliary moiety.
In certain embodiments, Z3The divalent segment in (A) is- (-Q)B-QC-QD-)s1-，
Wherein:
s1 is an integer of from about 1 to about 50 or from about 1 to about 30;
each QBAnd QDIndependently of one another are-CO-, -NH-,–O–、–S–、–SO2–、–OC(O)–、–COO–、–NHC(O)–、–C(O)NH–、–CH2–、–CH2NH–、–NHCH2–、–CH2O-or-OCH2-; and
each QCIndependently absent, is optionally substituted C1-12Alkylene, optionally substituted C2-12Alkenylene, optionally substituted C2-12Alkynylene, optionally substituted C2-12Heteroalkylene, optionally substituted C1-9A heterocyclylene group.
In certain embodiments, QBAnd QDAt least one of (A) is present in Z3Per monomer unit of (a).
In certain embodiments, -Z3–QA3Is that
–(–QB–QC–QD–)s1–QE–(–QB–QC–QD–)s1–QA3,
(Vb)
Wherein:
each s1 is independently an integer from about 1 to about 50 or from about 1 to about 30;
QA3as described herein;
each QBAnd QDIndependently absent, -CO-, -NH-, -O-, -S-, -SO2–、–OC(O)–、–COO–、–NHC(O)–、–C(O)NH–、–CH2–、–CH2NH–、–NHCH2–、–CH2O-or-OCH2-; and
each QCIndependently absent, is optionally substituted C1-12Alkylene, optionally substituted C2-12Alkenylene, optionally substituted C2-12Alkynylene, optionally substituted C2-12Heteroalkylene, optionally substituted C1-9A heterocyclic ring radical, and
QEa branched group of formula (IV) as described herein is absent or is present.
In certain embodiments, - (-)QB–QC–QD–)s1-is a group:
–QB–(CH2)g1–(CH2OCH2)g2–(CH2)g3–QD–，
wherein:
(i) g2 is an integer of from about 1 to about 50, from about 1 to about 40, or from about 1 to about 30;
(ii) g1 is 1, and QBis-NHCO-, -CONH-or-O-; or g1 is 0, and QDis-NHCO-; and
(iii) g3 is 1, and QBis-NHCO-, -CONH-or-O-; or g3 is 0, and QDis-CONH-.
In certain embodiments, the complementary reactive groups are:
wherein:
QA2absent, being optionally substituted C2-12Heteroalkylene (e.g., comprising-C (O) -N (H) -, -N (H) -C (O) -, -S (O)2-N (H) -or-N (H) -S (O)2Heteroalkylene of (A), optionally substituted C1-12Sulfoacylidene groups (e.g. heterocyclic group) ) Optionally substituted C1-12Heterocyclyl (e.g. 1, 2, 3-triazole-1, 4-diyl or) Cyclobut-3-ene-1, 2-dione-3, 4-diyl, pyridin-2-ylhydrazone, optionally substituted C6-16Triazole heterocyclic radicals (e.g. triazoles)) Optionally, selectingSubstituted C8-16Tricycloalkenylene (e.g. for) Or dihydropyridazine radicals (e.g. trans-Trans-)；
Each QA3Independently is optionally substituted C2-12Alkynyl, optionally substituted N-protected amino, azido, N-maleimido, S-protected thiol, or N-protected forms thereof, Containing an optionally substituted C with an internal ring carbon-carbon triple bond6-16Heterocyclic radical (e.g. of the formula) 1, 2, 4, 5-tetrazine radicals (e.g. of the formula ) Or optionally substituted C8-16Cycloalkynyl (e.g. cycloalkynyl))、–NHRN1Optionally substituted C 4-8Strained cycloalkenyl (e.g., trans cyclooctenyl or norbornenyl) or optionally substituted-COOR-containing12or-CHO C1-16An alkyl group;
RN1is an H, N-protecting group or optionally substituted C1-6An alkyl group;
each R12Independently is H or optionally substituted C1-6An alkyl group;
R13is halogen or F;
each RTIndependently is a bond to the targeting moiety;
each QTIndependently is-CO-, -CO-CH2-, -NH-or-NH-CH2–；
Each X1、X3And X5Independently absent, is-O-, -NH-, -CH2–NH–、–C(O)–、–C(O)–NH–、–NH–C(O)–、–NH–C(O)–NH–、–O–C(O)–NH–、–NH–C(O)–O–、–CH2–NH–C(O)–NH–、–CH2-O-C (O) -NH-or-CH2–NH–C(O)–O–；
Each X2And X4Independently absent, is-O-, -NH-, -C (O) -NH-, -NH-C (O) -NH-, -O-C (O) -NH-, or-NH-C (O) -O-;
each x2 is independently an integer from about 0 to about 50, from about 1 to about 40, or from about 1 to about 30;
each x3 is independently an integer from about 1 to about 11;
each x5 is independently an integer of about 0 or about 1;
each x6 is independently an integer from about 0 to about 10 or from about 1 to about 6, provided that the sum of the two x6 s is about 12 or less.
In certain embodiments, the complementary reactive groups are:
wherein:
each QA3Independently is optionally substituted C2-12Alkynyl, optionally substituted N-protected amino, azido, N-maleimido, S-protected thiol, Or N-protected forms thereof, Containing an optionally substituted C with an internal ring carbon-carbon triple bond6-16Heterocyclic radical (e.g. of the formula) 1, 2, 4, 5-tetrazine radicals (e.g. of the formula ) Or optionally substituted C8-16Cycloalkynyl (e.g. cycloalkynyl))、–NHRN1Optionally substituted C4-8Strained cycloalkenyl (e.g., trans cyclooctenyl or norbornenyl) or optionally substituted-COOR-containing12or-CHO C1-16An alkyl group;
each RM1Independently H or an auxiliary moiety;
each RN1Independently is H, N-protecting group or optionally substituted C1-6An alkyl group;
each R12Independently is H or optionally substituted C1-6An alkyl group;
each R13Independently of halogenA peptide or F;
each QTIndependently is-CO-, -NH-CH2-or-CO-CH2–；
Each RTIndependently is a bond to the targeting moiety;
each q5 and q6 is independently an integer of from about 1 to about 10 or from about 1 to about 6
Each q7 is independently an integer of about 0 or about 1;
each q8 is independently an integer of from about 0 to about 50, from about 1 to about 40, or from about 1 to about 30; and
each q9 is independently an integer from about 1 to about 10.
In certain embodiments, the complementary reactive groups are:
wherein:
each RM1Independently H or an auxiliary moiety;
each QTIndependently is-CO-, -NH-CH2-or-CO-CH2–；
Each RTIndependently is a bond to the targeting moiety;
Each q5 and q6 is independently an integer of from about 1 to about 10 or from about 1 to about 6
Each q7 is independently an integer of about 0 or about 1;
each q8 is independently an integer of from about 0 to about 50, from about 1 to about 40, or from about 1 to about 30; and
each q9 is independently an integer from about 1 to about 10.
Pharmaceutical composition
Delivery of the conjugates provided herein can be achieved by contacting the cells with the conjugate using a variety of methods known to those skilled in the art. In certain embodiments, the conjugates provided herein are formulated as pharmaceutical compositions comprising a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition is a liquid or a solid (e.g., lyophilized).
The conjugates provided herein can be administered alone or in admixture with a pharmaceutically acceptable excipient selected with regard to the intended route of administration and standard pharmaceutical practice. Thus, the pharmaceutical compositions employed may be formulated in conventional manner using one or more physiologically acceptable carriers, excipients and auxiliaries which facilitate processing of the conjugates into preparations which can be used pharmaceutically.
Commonly used carriers or excipients include sugars (e.g. lactose, mannitol), milk proteins, gelatin, starch, vitamins, cellulose and its derivatives, polyethylene glycols and solvents such as sterile water, ethanol, glycerol and polyols. Intravenous vehicles may include liquids and nutritional supplements. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients including salts, preservatives, buffers and The like, such as those described in Remington: The Science and Practice of Pharmacy, 21 st edition, Gennaro, Ed., Lippencott Williams & Wilkins (2005), and The United States Pharmacopeia: The National Formulary (USP 36 NF31), published in 2013. The pH and exact concentration of the various components of the pharmaceutical composition can be adjusted according to routine practice in the art. See Goodman and Gilman's, the pharmaceutical basic for Therapeutics.
In preparing pharmaceutical compositions, the active ingredient is typically mixed with an excipient (e.g., in a lyophilized formulation) or diluted with an excipient. When the excipient serves as a diluent, it may be a solid, semi-solid, or liquid material (e.g., phosphate buffered saline) that acts as a vehicle, carrier, or medium for the active ingredient. Thus, the compositions may be in the form of tablets, powders, elixirs, suspensions, emulsions, solutions and syrups. As is known in the art, the type of diluent may vary depending on the intended route of administration. The resulting composition may contain other agents, such as preservatives. The formulation may additionally include: lubricants, such as talc, magnesium stearate and mineral oil; a wetting agent; emulsifying and suspending agents; preservatives, for example, methyl benzoate and propyl hydroxybenzoate; a sweetener; and a flavoring agent. Other exemplary Excipients are described in Handbook of Pharmaceutical Excipients, 6 th edition, Rowe et al, eds., Pharmaceutical Press (2009). Preservatives can include antimicrobials, antioxidants, chelating agents, and inert gases.
These pharmaceutical compositions may be prepared in conventional manner, for example by means of conventional mixing, dissolving, granulating, dragee-making, leaching, emulsifying, encapsulating, entrapping or lyophilizing processes. Methods for preparing formulations well known in The art can be found, for example, in Remington, The Science and Practice of Pharmacy, 21 st edition, Gennaro, Ed., Lippencott Williams & Wilkins (2005), and Encyclopedia of Pharmaceutical Technology, eds. J.Swarbrick and J.C.Boylan, 1988. Anscha 1999, Marcel Dekker, New York. The correct formulation depends on the chosen route of administration. The formulation and preparation of such compositions is well known to those skilled in the art of pharmaceutical formulation. In preparing the formulation, the conjugate may be milled to provide the appropriate particle size prior to combining with the other ingredients.
Route of administration
The pharmaceutical composition may be administered locally or systemically. The therapeutically effective amount will vary depending on a variety of factors, such as the degree of disease progression in the subject, the age, sex and weight of the individual. Dosage regimens may be adjusted to provide the optimal therapeutic response. For example, as indicated by the exigencies of the therapeutic situation, several divided doses may be administered daily or the dose may be proportionally reduced.
As will be appreciated by those skilled in the art, the pharmaceutical composition may be administered to a patient in a variety of forms depending on the route of administration selected. The conjugates used in the methods described herein can be administered, for example, by parenteral administration. Parenteral administration includes intramuscular, intravenous, intraarterial, intracranial, subcutaneous, intraorbital, intraventricular, intraspinal, intrathecal, intraperitoneal, rectal, and topical routes of administration. Topical routes of administration include transdermal, intradermal, buccal and sublingual routes of administration. The pharmaceutical compositions are formulated according to the chosen route of administration. Parenteral administration may be by continuous infusion over a selected period of time.
Formulations for parenteral administration
The conjugates provided herein can be administered to a patient in need thereof in the form of a pharmaceutically acceptable parenteral (e.g., intravenous, intramuscular, or subcutaneous) formulation as described herein. The pharmaceutical preparations may also be administered parenterally (e.g., intravenously, intramuscularly or subcutaneously) in dosage forms or formulations comprising conventional non-toxic pharmaceutically acceptable carriers and adjuvants. In particular, formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats and solutes that render the formulation isotonic with the blood of the patient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. For example, to prepare such compositions, the conjugates provided herein can be dissolved or suspended in a parenterally acceptable liquid vehicle. Acceptable vehicles and solvents that may be used are water, water adjusted to an appropriate pH by the addition of an appropriate amount of hydrochloric acid, sodium hydroxide or an appropriate buffer (e.g., phosphate buffered saline), 1, 3-butanediol, ringer's solution and isotonic sodium chloride solution. The aqueous formulation may also contain one or more preservatives, such as methyl, ethyl or n-propyl paraben. Additional information on parenteral formulations can be found, for example, in United States Pharmacopeia-National Formulary (USP-NF), which is incorporated herein by reference.
The parenteral formulation of the conjugates provided herein can be any of four general types of formulations identified by USP-NF as suitable for parenteral administration:
(1) "medicament for injection": a drug substance (e.g., a conjugate provided herein) as a dry (e.g., lyophilized) solid that will be combined with a suitable sterile vehicle for parenteral administration, such as drug injection;
(2) "drug injection emulsion": liquid formulations in which the drug substance (e.g., a conjugate provided herein) is dissolved or dispersed in a suitable emulsion medium;
(3) "drug injectable suspension": liquid formulations of a drug substance (e.g., a conjugate provided herein) suspended in a suitable liquid medium; and
(4) "drug for injectable suspension": the drug substance (e.g., the conjugate provided herein) as a dry solid, which will be combined with a suitable sterile vehicle for parenteral administration, such as a drug injection suspension.
Exemplary formulations for parenteral administration include solutions of the conjugates provided herein prepared in water, suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid poly (ethylene glycol), DMSO, and mixtures thereof, as well as in oils, with or without ethanol. Under normal conditions of storage and use, these formulations may contain a preservative to prevent the growth of microorganisms. Conventional procedures and ingredients for selecting and preparing suitable formulations are described, for example, in Remington: The Science and Practice of Pharmacy,21 st Ed.,Gennaro,Ed.,Lippencott Williams&Wilkins (2005) and us pharmacopoeia published in 2013: the National Formulary (USP 36 NF 31).
Biocompatible, biodegradable lactide polymers, lactide/glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers can be used to control the release of the conjugates provided herein. Other potentially useful parenteral delivery systems for conjugates provided herein include ethylene-vinyl acetate copolymer particles, osmotic pumps, or implantable infusion systems. The parenteral formulation can be formulated for rapid release or sustained/extended release of the polynucleotide and/or conjugate. Exemplary formulations for parenteral release of the conjugates provided herein include: aqueous solutions, powders for reconstitution, co-solvent solutions, oil/water emulsions, suspensions, microspheres, and polymer gels.
Application method
In one embodiment, provided herein is a method for treating, preventing, or ameliorating one or more symptoms of a proliferative disease in a subject, comprising administering to the subject a therapeutically effective amount of a conjugate disclosed herein.
In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human. In certain embodiments, the subject is a primate other than a human, an animal such as a cow, a sport animal, or a pet such as a horse, dog, or cat.
In certain embodiments, the proliferative disease is a tumor. In certain embodiments, the proliferative disease is a liquid or hematological tumor. In certain embodiments, the proliferative disease is a solid tumor. In certain embodiments, the proliferative disease is a neoplastic disease.
In certain embodiments, the proliferative disease is cancer. In certain embodiments, the cancer is a relapsed cancer. In certain embodiments, the cancer is a drug-resistant cancer. In certain embodiments, the cancer is a relapsed drug-resistant cancer. In certain embodiments, the cancer is a multi-drug resistant cancer. In certain embodiments, the cancer is a relapsed, multi-drug resistant cancer.
In certain embodiments, cancers that can be treated with the conjugates provided herein include, but are not limited to, (1) leukemias, including, but not limited to, acute leukemias, acute lymphocytic leukemias, acute myelogenous leukemias such as myeloblastic, promyelocytic, monocytic, erythrocytic leukemia, and myelodysplastic syndrome or symptoms thereof (e.g., anemia, thrombocytopenia, neutropenia, binuclear cytopenia, or pancytopenia), Refractory Anemia (RA), RA with sideroblasts (RARS), RA with excess blasts (RAEB), transformed RAEB (RAEB-T), leukemias-prophase, and chronic myelomonocytic leukemias (CMML 2) chronic leukemias, including, but not limited to, chronic myelogenous (granulocytic) leukemia, Chronic lymphocytic leukemia and hairy cell leukemia; (3) polycythemia vera; (4) lymphomas, including but not limited to Hodgkin's disease and non-Hodgkin's disease; (5) multiple myeloma, including but not limited to smoldering multiple myeloma, non-secretory myeloma, sclerosing myeloma, plasma cell leukemia, solitary plasmacytoma, and bone marrow plasmacytoma; (6) Waldenstrom's macroglobulinemia; (7) monoclonal gammopathy of unknown significance; (8) benign monoclonal gammopathy; (9) heavy chain disease; (10) bone and connective tissue sarcomas including, but not limited to, osteosarcoma, chondrosarcoma, ewing's sarcoma, malignant giant cell tumor, bone fibrosarcoma, chordoma, periosteal sarcoma, soft tissue sarcoma, angiosarcoma (angioendothelioma), fibrosarcoma, kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, metastatic cancer, neuroblastoma, rhabdomyosarcoma, and synovial sarcoma; (11) brain tumors, including but not limited to glioma, astrocytoma, brain stem glioma, ventricular septal tumor, oligodendroglioma, non-glioma, acoustic neuroma, craniopharyngeal tumor, medulloblastoma, meningioma, dermatoblastoma, dermoblastoma, and primary brain lymphoma; (12) breast cancer, including but not limited to adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, primary cancer, Paget's disease, and inflammatory breast cancer; (13) adrenal cancer including but not limited to pheochromocytoma and adrenocortical carcinoma; (14) thyroid cancer including, but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer, and anaplastic thyroid cancer; (15) pancreatic cancer including, but not limited to, insulinoma, gastrinoma, glucagonoma, uveoma, somatostatin-secreting tumors, and carcinoid or islet cell tumor; (16) pituitary cancers including but not limited to Cushing's disease, prolactin-secreting tumors, acromegaly, and diabetes insipidus; (17) eye cancers including, but not limited to, ocular melanoma such as iris melanoma, choroidal melanoma, ciliary melanoma, and retinoblastoma; (18) vaginal cancers including but not limited to squamous cell carcinoma, adenocarcinoma, and melanoma; (19) vulvar cancers including, but not limited to, squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; (20) cervical cancer including but not limited to squamous cell carcinoma and adenocarcinoma; (21) uterine cancers including but not limited to endometrial carcinoma and uterine sarcoma; (22) ovarian cancers including, but not limited to, ovarian epithelial cancers, borderline tumors, germ cell tumors, and stromal tumors; (23) esophageal cancers including, but not limited to, squamous carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; (24) gastric cancers including but not limited to adenocarcinoma, fungal (polypoid), ulcerative, superficial spread, diffuse spread, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; (25) colon cancer; (26) rectal cancer; (27) liver cancer, including but not limited to hepatocellular carcinoma and hepatoblastoma; (28) gallbladder cancer, including but not limited to adenocarcinoma; (29) cholangiocarcinoma, including but not limited to papillary, nodular, and diffuse; (30) lung cancer including, but not limited to, non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma, and small cell lung cancer; (31) testicular cancer including, but not limited to, germinal tumor, seminoma, anaplastic classical (typical) seminoma, non-seminoma, embryonic carcinoma, teratoma, and choriocarcinoma (oocyst carcinoma); (32) prostate cancer including but not limited to adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; (33) criminal cancer; (34) oral cancer, including but not limited to squamous cell carcinoma; (35) basal carcinoma; (36) salivary gland cancers including but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoid cystic carcinoma; (37) pharyngeal cancer, including but not limited to squamous cell carcinoma and verrucous carcinoma; (38) skin cancers including but not limited to basal cell carcinoma, squamous cell carcinoma and melanoma, superficial diffuse melanoma, nodular melanoma, malignant melanoma of tonsils and chronic melanoma of hands and feet; (39) renal cancers, including but not limited to renal cell carcinoma, adenocarcinoma, suprarenal adenoid tumor, fibrosarcoma, and transitional cell carcinoma (renal pelvis and/or uterus); (40) wilms tumor; (41) bladder cancer including, but not limited to, transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, and carcinosarcoma; and other cancers including, but not limited to, myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and papillary adenocarcinoma (see Fishman et al, 1985, Medicine, 2 nd edition, J.B.Lippincott Co., Philadelphia and Murphy et al, 1997, information Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Peking, Penguhnks U.S.A., Inc., United States of America).
In certain embodiments, cancers that can be treated with the conjugates provided herein include, but are not limited to, B cell cancers such as multiple myeloma, waldenstrom's macroglobulinemia, heavy chain diseases such as alpha chain diseases, gamma chain diseases, and mu chain diseases, benign monoclonal gammopathy, and immune cell 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, and hematologic tissue cancer.
In certain embodiments, cancers that can be treated with the conjugates provided herein include, but are not limited to, human sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, angioendotheliosarcoma, lymphangiosarcoma, 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 cancer, sebaceous gland cancer, papillary adenocarcinoma, cystadenocarcinoma, medullary cancer, bronchial cancer, renal cell cancer, hepatocellular carcinoma, cholangiocarcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, choriocarcinoma, and the like, Glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, such as acute lymphocytic leukemia and acute myelogenous leukemia (myelocytic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelogenous (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphomas (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease.
In certain embodiments, the cancer is epithelial in nature, including, but not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancer, kidney cancer, larynx cancer, lung cancer, oral cavity cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, and skin cancer. In certain embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In certain 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.
In certain embodiments, the proliferative disease is an inflammatory disease. In certain embodiments, the proliferative disease is an immune disease. In certain embodiments, the proliferative disease is an infectious disease. In certain embodiments, the proliferative disease is a viral infection.
In another embodiment, provided herein is a method of modulating natural killer cells in a subject comprising administering to the subject an effective amount of a conjugate disclosed herein.
In another embodiment, provided herein is a method of modulating bone marrow cells in a subject comprising administering to the subject an effective amount of a conjugate disclosed herein.
Depending on the disorder, disease or condition to be treated and the condition of the subject, the conjugates or pharmaceutical compositions provided herein can be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection or implant), inhalation, nasal, vaginal, rectal, sublingual, or topical (e.g., transdermal or topical) routes of administration, and can be formulated alone or with pharmaceutically acceptable excipients, carriers, adjuvants and vehicles appropriate for each route of administration. Also provided is the administration of a conjugate or pharmaceutical composition provided herein in a depot formulation, wherein the active ingredient is released over a predetermined period of time.
In treating, preventing, or ameliorating one or more symptoms of the disorders, diseases, or conditions described herein, suitable dosage levels are typically about 0.001 to 100mg per kilogram subject body weight per day (mg/kg per day), about 0.01 to about 75mg/kg per day, about 0.1 to about 50mg/kg per day, about 0.5 to about 25mg/kg per day, or about 1 to about 20mg/kg per day, which may be administered in single or multiple doses. Within this range, the dose may be about 0.005 to about 0.05, about 0.05 to about 0.5, about 0.5 to about 5.0, about 1 to about 15, about 1 to about 20, or about 1 to about 50mg/kg per day.
It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of the compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.
The conjugates provided herein can also be used in combination or combination with other agents or therapies that can be used to treat, prevent, or ameliorate the symptoms of one or more conditions, disorders, or diseases for which the conjugates provided herein are useful.
Suitable additional therapeutic agents may also include, but are not limited to: (1) an alpha-adrenergic agent; (2) antiarrhythmic agents; (3) anti-atherosclerotic agents, such as ACAT inhibitors; (4) antibiotics, such as anthracyclines, bleomycin, mitomycin, actinomycin and plicamycin; (5) anti-cancer and cytotoxic agents, for example alkylating agents such as nitrogen mustards, alkyl sulfonates, nitrosoureas, ethylenimines, and triazenes; (6) anticoagulants, such as acetocoumarol, argatroban, bivalirudin, recombinant hirudin, fondaparinux sodium, heparin, phenindione, warfarin, and ximelagatran; (7) antidiabetics such as biguanide drugs (e.g., metformin), glucosidase inhibitors (e.g., acarbose), insulin, meglitinide drugs (e.g., repaglinide), sulfonylureas (e.g., glimepiride, glyburide, and glipizide), oxazolidinediones (e.g., troglitazone, rosiglitazone, and pioglitazone), and PPAR-gamma agonists; (8) antifungal agents, such as amorolfine, amphotericin B, anidulafungin, bifonazole, butenafine, butoconazole, caspofungin, ciclopirox, clotrimazole, econazole, fenticonazole, felpine, fluconazole, isoconazole, itraconazole, ketoconazole, micafungin, miconazole, naftifine, natamycin, nystatin, oxiconazole, lavoconazole, posaconazole, rimycin, sertaconazole, sulconazole, terbinafine, terconazole, tioconazole, and voriconazole; (9) anti-inflammatory agents, such as non-steroidal anti-inflammatory agents, for example, aceclofenac, acerbamide, amoxicillin, aspirin, apazone, benorilate, bromfenac, carprofen, celecoxib, choline magnesium salicylate, diclofenac, diflunisal, etodolac, etoricoxib, faxamine, benbufen, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, lornoxicam, loxoprofen, lumiracoxib, meclofenamic acid, mefenamic acid, meloxicam, analgin, methyl salicylate, magnesium salicylate, nabumetone, naproxen, nimesulide, oxyphenbutazone, parecoxib, phenylbutazone, piroxicam, salicylsalicylic acid, sulindac, suprofen, tenoxicam, tiaprofenic acid, and tolmetin; (10) antimetabolites such as folic acid antagonists, purine analogs, and pyrimidine analogs; (11) antiplatelet agents such as GPIIb/IIIa blockers (e.g., abciximab, eptifibatide, and tirofiban), P2Y (AC) antagonists (e.g., clopidogrel, ticlopidine, and CS-747), cilostazol, dipyridamole, and aspirin; (12) antiproliferative agents, such as methotrexate, FK506 (tacrolimus), and mycophenolate mofetil; (13) anti-TNF antibodies or soluble TNF receptors such as etanercept, rapamycin, and leflunomide; (14) an aP2 inhibitor; (15) beta-adrenergic agents, such as carvedilol and metoprolol; (16) bile acid sequestrants, such as cholestyramine; (17) calcium channel blockers such as amlodipine besylate; (18) a chemotherapeutic agent; (19) cyclooxygenase 2(COX-2) inhibitors such as celecoxib and rofecoxib; (20) (ii) a cyclosporin; (21) cytotoxic drugs such as azathioprine and cyclophosphamide; (22) diuretics, such as chlorothiazide, hydrochlorothiazide, fluorocarboxhydrazide, hydrofluorocarbonylhydrazide, benzofluorocarboxhydrazide, methylchlorothiazide, trichlorohydrazide, polythiazide, benzothiazine, ethacrynic acid, tennic acid, chlorthalidone, furanilic acid, moxazolidine, bumetanide, triamterene, amiloride, spironolactone; (23) endothelin-converting enzyme (ECE) inhibitors such as phosphoramidon; (24) enzymes, such as L-asparaginase; (25) factor VIIa inhibitors and factor Xa inhibitors; (26) farnesyl protein transferase inhibitors; (27) a fibrate; (28) growth factor inhibitors, such as modulators of PDGF activity; (29) a growth hormone secretagogue; (30) HMG CoA reductase inhibitors such as pravastatin, lovastatin, atorvastatin, simvastatin, NK-104 (also known as itavastatin, nivastatin or nivastatin) and ZD-4522 (also known as rosuvastatin, atorvastatin or misastatin); neutral Endopeptidase (NEP) inhibitors; (31) hormonal agents, such as glucocorticoids (e.g., cortisone), estrogens/antiestrogens, androgens/antiandrogens, progestins, and luteinizing hormone-releasing hormone antagonists, and octreotide acetate; (32) an immunosuppressant; (33) mineralocorticoid receptor antagonists such as spironolactone and eplerenone; (34) microtubule disrupting agents, such as ecteinascidins (ecteinascidins); (35) microtubule stabilizing agents, such as paclitaxel, docetaxel, and epothilones a-F; (36) an MTP inhibitor; (37) nicotinic acid; (38) phosphodiesterase inhibitors, such as PDEIII inhibitors (e.g., cilostazol) and PDEV inhibitors (e.g., sildenafil, tadalafil and vardenafil); (39) products of plant origin, such as vinca alkaloids, epipodophyllotoxins and taxanes; (40) platelet Activating Factor (PAF) antagonists; (41) platinum coordination complexes, such as cisplatin, satraplatin, and carboplatin; (42) a potassium channel opener; (43) an isoprene protein transferase inhibitor; (44) protein tyrosine kinase inhibitors; (45) a renin inhibitor; (46) a squalene synthetase inhibitor; (47) steroids, such as aldosterone, beclomethasone, betamethasone, deoxycorticosterone acetate, fludrocortisone, hydrocortisone (cortisol), prednisolone, prednisone, methylprednisolone, dexamethasone, and triamcinolone acetonide; (48) TNF-a inhibitors, such as tenidap; (49) thrombin inhibitors, such as hirudin; (50) thrombolytic agents such as anitricin, reteplase, tenecteplase, tissue plasminogen activator (tPA), recombinant tPA, streptokinase, urokinase, prourokinase, and the catabolized plasminogen streptokinase activator complex (APSAC); (51) thromboxane receptor antagonists such as ifetroban; (52) a topoisomerase inhibitor; (53) vasopeptidase inhibitors (dual NEP-ACE inhibitors), such as olpadra and gemotrila; (54) other miscellaneous agents, such as hydroxyurea, procarbazine, mitotane, hexamethylmelamine and gold compounds.
In certain embodiments, other therapies that can be used in combination with the conjugates provided herein include anti-cancer agents and cytotoxic agents, for example alkylating agents, such as nitrogen mustards, alkyl sulfonates, nitrosoureas, ethylenimines, and triazenes; for example, alkylating agents such as nitrogen mustards, alkyl sulfonates, nitrosoureas, ethyleneimines, and triazenes.
In certain embodiments, other therapies that can be used in combination with the conjugates provided herein include, but are not limited to, immune checkpoint modulators. In certain embodiments, the immune checkpoint modulator is a PD-1 inhibitor. In certain embodiments, the immune checkpoint modulator is a PD-L1 inhibitor. In certain embodiments, the PD-1 inhibitor is an anti-PD-1 antibody or antigen-binding fragment thereof. In certain embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody or antigen-binding fragment thereof. In certain embodiments, the immune checkpoint modulator blocks the interaction between PD-1 and PD-L1.
In certain embodiments, other therapies that can be used in combination with the conjugates provided herein include, but are not limited to, T cell costimulatory molecules and immune checkpoint modulators. In certain embodiments, the T cell co-stimulatory molecule is OX40, CD2, CD27, CDS, ICAM-1, LFA-1/CD11a/CD18, ICOS/CD278, 4-1BB/CD137, GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKAMG 2C, SLAMF7, NKp80, CD160, B7-H3, or CD83, or a ligand thereof. In certain embodiments, the T cell co-stimulatory molecule is an anti-OX 40 antibody, an anti-ICOS/CD 278 antibody, or an anti-4-1 BB/CD137 antibody or antigen binding fragment thereof. In certain embodiments, the immune checkpoint modulator is an inhibitor of an immune checkpoint molecule selected from 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 certain embodiments, the immune checkpoint modulator is an anti-CD 47 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, or an antigen-binding fragment thereof.
In certain embodiments, other therapies that can be used in combination with the conjugates provided herein include, but are not limited to, surgery, endocrine therapy, biological response modifiers (e.g., interferons, interleukins, and Tumor Necrosis Factor (TNF)), hyperthermia and cryotherapy, and agents that can mitigate any adverse effects (e.g., antiemetics).
Such other agents or drugs can be administered simultaneously or sequentially with the conjugates provided herein by the route and amount in which they are typically used. When the conjugates provided herein are used concurrently with one or more other drugs, pharmaceutical compositions containing such other drugs may be used, but are not required, in addition to the conjugates provided herein. Accordingly, the pharmaceutical compositions provided herein include those that contain one or more other active ingredients or therapeutic agents in addition to the conjugates provided herein.
In certain embodiments, the conjugates provided herein are administered in combination with a second antibody, e.g., an antibody that binds to an antigen expressed by a cancer (e.g., an effective amount of the second antibody). Exemplary antigens expressed by cancer are known in the art and include, but are not limited to: CD19, CD20, CD22, CD30, CD33, CD38, CD52, CD56, CD70, CD74, CD79B, CD123, CD138, CS1/SLAMF7, Trop-2, 5T4, EphA4, BCMA, mucin 1, mucin 16, PD-L1, PTK7, STEAP1, endothelin B receptor, mesothelin, EGFRvIII, ENPP3, SLC44A4, GNMB, connexin 4, NaPi2B, LIV-1A, guanylate cyclase C, DLL3, EGFR, HER2, VEGF, VEGFR, integrins α V β 3, integrins α 5 β 1, MET, IGF1R, TRAILR1, TRAILR2, RANKL, FAP, tenascin, Le yEpCAM, CEA, gpA33, PSMA, TAG72, mucin, CAIX, EPHA3, folate receptor alpha, GD2, GD3 and MHC/peptide complexes comprising from NY-ESO-1/LAGE, SSX-2, MAGE family proteins, MAGE-A3, gp100/pmel17, Melan-A/MART-1, gp75/TRP1, tyrosinase, TRP2, CEA, PSA, TAG-72, immature laminin receptor, MOK/RAGE-1, WT-1, SAP-1, BING-4, EpCAM, MUC1, PRAME, survivin, BRCA1, BRCA2, CDK4, CML66, MART-2, p53, Ras, beta-catenationA cyclic protein, TGF- β RII, HPV E6 or HPV E7. In certain embodiments, the conjugates provided herein are administered in combination with a monoclonal antibody that binds CD123 (also known as IL-3 receptor alpha), such as trastuzumab (also known as CSL362 and JNJ-56022473). In certain embodiments, the conjugates provided herein are administered in combination with a monoclonal antibody that binds EGFR (e.g., cetuximab). In certain embodiments, the second antibody comprises one or more effector functions, such as effector functions associated with Fc receptor (FcR) binding on immune cells, including but not limited to ADCC or ADCP, and/or Complement Dependent Cytotoxicity (CDC). Without wishing to be bound by theory, it is believed that it is particularly advantageous to combine such antibodies with the conjugates provided herein, e.g., to direct FcR expressing leukocytes to target tumor cells bound by the second antibody while modulating NK or bone marrow cell activity.
In certain embodiments, the conjugates provided herein are administered in combination with an immunotherapeutic agent (e.g., an effective amount of an immunotherapeutic agent). An immunotherapeutic agent may refer to any therapeutic agent that targets the immune system and facilitates therapeutic redirection of the immune system, e.g., modulators of co-stimulatory pathways, cancer vaccines, recombinantly modified immune cells, and the like. Exemplary and non-limiting immunotherapeutic agents are described below. Without wishing to be bound by theory, it is believed that the complementary mechanism of action, for example in the activation of macrophages and other immune cells (e.g., T)EffectorCells) to target tumor cells, the conjugates provided herein are suitable for use with immunotherapeutic agents.
In certain embodiments, the immunotherapeutic agent comprises an antibody. Exemplary antigens for immunotherapeutic antibodies are known in the art and include, but are not limited to, BDCA2, BDCA4, ILT7, LILRB1, LILRB2, LILRB3, LILRB4, CSF-1R, CD40, CD40L, CD163, CD206, DEC205, CD47, CD123, IDO, TDO,41BB, CTLA4, PD1, PD-L1, PD-L2, TIM-3, BTLA, VISTA, LAG-3, CD28, OX40, GITR, CD137, CD27, HVEM, CCR4, CD25, CD103, KIrg1, Nrp1, CD278, Gpr83, TIGIT, 154, dnacd 160, PVRIG, and os. Immunotherapeutics approved or in later clinical testing include, but are not limited to, ibritumomab, pembrolizumab, nigulumab, azilizumab, avelizumab, Devolumab, and the like. In certain embodiments, the antibodies of the present disclosure are administered in combination with an inhibitor of the PD-L1/PD-1 pathway, such as an anti-PD-L1 or anti-PD-1 antibody. As demonstrated herein, the combined administration of an anti-SIRP-a antibody of the invention and an inhibitor of the PD-L1/PD-1 pathway can result in synergistic anti-tumor activity.
In certain embodiments, the immunotherapeutic agent comprises a vaccine, an oncolytic virus, an adoptive cell therapy, a cytokine, or a small molecule immunotherapeutic agent. Examples of such immunotherapeutic agents are known in the art. For example, adoptive cell therapies and therapeutic agents can include, but are not limited to, chimeric antigen receptor T cell therapy (CAR-T), Tumor Infiltrating Lymphocytes (TILs), TCR-engineered NK cells, and macrophage products. Vaccines can include, but are not limited to, polynucleotide vaccines, polypeptide vaccines, or cell-based (e.g., tumor or dendritic cell-based) vaccines. Various cytokines useful for treating cancer are known and include, but are not limited to, IL-2, IL-15, IL-7, IL-10, and IFN. Small molecule immunotherapeutic agents may include, but are not limited to, IDO/TDO inhibitors, arginase inhibitors, A2aR inhibitors, TLR agonists, STING agonists, and Rig-1 agonists.
In certain embodiments, the conjugates provided herein are administered in combination with a therapeutic agent, including, but not limited to, methotrexate (m: (m))Methotrexate), cyclophosphamideThalidomideAcridine formamide,Actinomycin, 17-N-allylamino-17-demethoxygeldanamycin, aminopterin, amsacrine, anthracycline, antitumor agent, 5-azacytidine, azathioprine, BL22, bendamustine, bicoda, bleomycin, bortezomib, and phentermine Pravastatin, flutriafol, mexicamine, camptothecin, capecitabine, carboplatin, cetuximab, chlorambucil, cisplatin, cladribine, clofarabine, cytarabine, dacarbazine, dasatinib, daunorubicin, decitabine, dichloroacetic acid, discotide olide, docetaxel, doxorubicin, epirubicin, epothilone, eribulin, estramustine, etoposide, irinotecan, epsiprovalbumin, floxuridine, fludarabine, fluorouracil, fosfestrol, temustine, ganciclovir, gemcitabine, hydroxyurea, IT-101, idarubicin, ifosfamide, imiquimod, irinotecan, iloufofumesaten, ixaromafen, ixaromauron, piliquar, lapatinib, lenalidomide, lomustine, tolmetin, tolyturgic, tolnaftate, melphalan, meclol, mechlorethamine, mecloethamine, meclizine, mitomycin, doxycycline, and a, Mitotane, mitoxantrone, nelarabine, nilotinib, orlistat, oxaliplatin, PAC-1, paclitaxel, pemetrexed, pentostatin, pipobroman, pixelstron, plicamycin, procarbazine, proteasome inhibitors (e.g., bortezomib), Letitrexib, rebeccamycin, and mixtures thereof, rubite-can, SN-38, salinosporamide A, satraplatin, streptozotocin, swainsonine, tariquidar, a taxane, efadine, temozolomide, testolactone, thiotepA, thioguanine, topotecan, trabectedin, tretinoin, triplatin tetranitrate, tris (2-chloroethyl) amine, troxacitabine, uracil mustard, valrubicin, vinblastine, vincristine, vinorelbine, vorinostat or zoquidar.
In certain embodiments, the conjugates provided herein are administered in combination with a therapeutic agent including, but not limited to, 3F8, 8H9, abamectin, abciximab, abituzumab, alemtuzumab, adouximab, adalimumab, aducaumab, aphidikumab, aphidimab, atomab, peratuzumab, pemirolizumab, ALD518, alemtuzumab, aleukumab, aleutizumab, pertuzumab, almitumomab, amoximab, mabamumazumab, ranibizumab-alemtuzumab, aninflutumab, albuzumab (IMA-638), aprepirubizumab, acipimox mab, alemtuzumab, acymumab, asexuzumab, alemtuzumab, adoitumumab, adonituzumab, basiliximab, gemtuzumab, gobitumumab, belimumab, dolab, and belimumab, Benralizumab, patitmab, bevacizumab, beztuzumab, biitumumab, bimagrumab, bimagribulizumab, bivacizumab, rituximab, bruzonuzumab, boceprituzumab, bevacizumab, budralumab, breloxizumab, broncutuzumab, canamumab, macrantuzumab, carpuzumab, cBR 96-doxorubicin immunoconjugate, CC49, cetilizumab, trastuzumab, cetuximab, Ch.14.18, placocizumab, cetuximab, cleuzumab, clexituzumab, clinuzumab, clinicifutuzumab, clexituzumab, curuzumab, ranibixin-Raxin-gac, Finaretuzumab, Conatuzumab, Coxituzumab, CR6261, CR 61, CR, Daratuzumab, pego-dapaglutizumab, dalargimumab, Dectrekumab, demomicuzumab, Denitumumab mafodotin, Denitumumab, Destuzumab biotin, demomicab, dartuximab, Dituzumab, Atomozumab, Attatuozumab, Delotuzumab, Doliguotu mab, Durleut-Lupulumab, Dewauzumab, Duchentuzumab, Eimeliximab, Ekulizumab, Ebaruzumab, Efavizumab, Engumab, Edreuuzumab, Elgemtuzumab, Estrozumab, Eximazumab, Imitzemazumab, Imitamab, Emituzumab, Eimazemazumab, Evzemazumab, Enpekuzumab, Ensuluzumab, Immunotuzumab, Evonizumab, Evonivolumumab, Esprutuzumab, Espertimazumab, Esperituzumab, Esperitukulizumab, EsperitujevE, Esperitukumab, Esperozumab, Esperozi, Avomab, Faxomab, Farmumab, Faradoumab, Fachimumab, FBTA05, Novizumab, Nozazumab, Nozakunzumab, Fisherculella, Finitumumab, Firivumab, Frantozumab, Fuletuzumab, Farluzumab, Floruzumab, Furazumab, Frazimab, Francizumab, Fuliximab, Fulvitamab, Fuvizumab, Riviximab, Galiximab, Ganitafizumab, Ganituzumab, Gituzumab ozolomide, Cawaduzumab, Virtin mAb, golimumab, Goliximab, Guxizumab, Guxivumab, Tinituzumab, ibritumumab, Ekularuzumab, Imatuzumab, Iabvuv mAb, Immunumab, Eviitumumab, Goutuzumab, Gouzumab, Indoutumutumumab, Indoxib, Indoxituzumab, Indovutuzumab, Indovub, Indovutuzumab, Ituzumab, Eszomicin, infliximab, rituximab, Isatuximab, eltromumab, chikulizumab, Kylliximab, labeprizumab, lambrolizumab, lammepartuzumab, lemazumab, Lenzilumab, Lenzilumab, lexulizumab, lexanumab, lexulizumab, Virgizumab, Vittin-Rifalizumab, Riglilizumab, Lilotumab satetraxene, Lintuzumab, riluzumab, Logevimab, Lauralizumab, regolizumab, Pego-lullizumab, Luximab, rituzumab, Marupuzumab, Margeitumumab, Maruguzumab, Melimuzumab, Maruzumab, Metulizumab, Memituzumab, Mirasuzumab, Millimulus, Murituzumab, Murituximab, Murrax, Murmuci, Murraya kolomuci, Mutamuci, Murraya, Mutamimumab, and Murmuci, Moxitemmumab, Moluomamab-CD 3, tanacetumab, Nanoluzumab, tanamazumab, Narituzumab, Nasutuzumab, Neubakumab, Neiximab, Nemilizumab, Nerimuzumab, Centivimab, nimotuzumab, Niveluzumab, Suminomumab, Otositumumab, Okauzumab, Oudelizumab, Olaruzumab, Oudelizumab, Opimuzumab, Oudelizumab, Olympuzumab, Moolouzumab, Ogovolumab, Ontaumab, Oxiximab, Outalizumab, Palizumab, Pakinuzumab, Pacuriumab, Pacurizumab, Pacuriab, Satuzumab, Satsuma, Neurizumab, Ourizumab, Outab, Outalizumab, Outau-kui, Outau-C, Outalizumab, and E, Pertuzumab, panitumumab, pertuzumab, perkatlizumab, pertuzumab, peckulizumab, Petulizumab, Pinnitzezumab, Pinnituzumab, Poratuzumab, Pratuzumab, Perbizumab, Pritumumab, Prtuximab, Prolizumab, Pro 140, Kulizumab, Radermatamab, Ravirumab, Rapantozezumab, Ramurumab, Ramurumumab, Raoxicumab, Rafavizumab, Rigeuzumab, Rakageuzumab, Rakalizumab, Rakulizumab, Rituzumab, Rituximab, Rouzumab, Rovilizumab, Ravilizumab, Samatuzumab, Seruzumab, Securizumab, Rituzumab, Rispertib, Rituzumab, and Rituzumab, Sevimab, Cetuzumab, SGN-CD19A, SGN-CD33A, Cefavulizumab, Cetuzumab, Celizumab, Serratuzumab, Sotuzumab vedotin, Sulangzumab, Sutuzumab, Sonpevizumab, Sontuzumab, Setuzumab, Setatuzumab, Thiozumab, Sovizumab, Tabeuzumab, Talizumab, Tanilizumab, Pertuzumab, Tatuzumab, Te bazumab, Attitumumab, Tituzumab, tenenximab, Cetuzumab, Tilizumab, Tesidolub, TGN1412, Ticimumab (Ticimumab) (Trimetulimumab (tremelimumab)), Tetuzumab pegamulizumab (Tituzumab), Tituzumab (Tituzumab), TNX-650, Totuzumab (Totuzumab, Totuzumab (Totuzumab), Totuzumab (Toutuzumab), Totuzumab (Tituzumab (Titugakib, Titugakib), TNX-650, Toutuzumab), Toutuzumab (Toutuzumab), Toutuzumab, Touttatuzumab (Touttatuzumab), Toutuzumab), Touttatuzumab (Touttatuzumab), Toutuzumab (Tituzumab), Touttatuzumab), Teutuzumab), Teuttatuzumab), Teutuzumab (Titutemozu, Tauttatuzumab), Teuttatuzumab (Titutemozu, Tautuzumab), Teutu, Tautu, Teuttatuzumab), Teutuzumab (Titutemozu, Teutu, Teutuzumab), Teutu, Teutuzumab (Titutemozu, Teutuzumab), Teutu, Teutuzumab (Titutemozu, Teutu, Teutuzu, Teutu, Teutuzu, Teutu, Teut, TRBS07, trastuzumab, tremelimumab, simon interleukin mab, tuvirumab, ubelix mab, Ulocuplumab, umeclizumab, ubuzumab, ustrocumab, utegravizumab, vildagliptin-Wandototuzumab, vatitzeugumab, valnussezumab, valliximab, varliulimumab, vetritlizumab, vedolizumab, vetuzumab, vepamumab, vesikozumab, vesizumab, valloximab, volitumumab, zalutumumab, zatuzumab, zilaramumab, or aziumumab.
Any type of cancer known in the art may be included, such as, but not limited to, carcinomas, sarcomas, lymphomas, leukemias, lymphomas, and blastomas. More specific examples of such cancers include, but are not limited to, lung cancer, squamous cell carcinoma, brain tumor, glioblastoma, head and neck cancer, hepatocellular carcinoma, colorectal cancer (e.g., colon or rectal cancer), liver cancer, bladder cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, urinary tract cancer, breast cancer, peritoneal cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, anal cancer, penile cancer, melanoma, multiple myeloma, and B-cell lymphoma (including non-hodgkin's lymphoma (NHL)); acute Lymphocytic Leukemia (ALL); chronic Lymphocytic Leukemia (CLL); acute Myeloid Leukemia (AML); merkel cell carcinoma; chronic Myelogenous Leukemia (CML); and associated diversion.
The conjugates provided herein can also be provided as articles of manufacture using packaging materials well known to those skilled in the art. See, for example, U.S. patent nos. 5,323,907; 5,052,558, respectively; and 5,033,252. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, and any packaging material suitable for the selected formulation and intended mode of administration and treatment.
Also provided herein are kits that, when used by a practitioner, can simplify administration of appropriate amounts of the active ingredients to a subject. In certain embodiments, a kit provided herein comprises a container and a dosage form of a conjugate provided herein.
In certain embodiments, a kit comprises a container comprising a conjugate dosage form provided herein in a container comprising one or more other therapeutic agents described herein.
The kits provided herein can further comprise a device for administering the active ingredient. Examples of such devices include, but are not limited to, syringes, needleless syringes, drop bags, patches, and inhalers. The kits provided herein can further comprise a rubber boot for administering the active ingredient.
The kits provided herein can further include a pharmaceutically acceptable vehicle that can be used to administer one or more active ingredients. For example, if the active ingredient is provided in a solid form that must be reconstituted for parenteral administration, the kit may include a sealed container of a suitable vehicle in which the active ingredient may be dissolved to form a sterile, particle-free solution suitable for parenteral administration. Examples of pharmaceutically acceptable excipients include, but are not limited to: aqueous vehicles including, but not limited to, water for injection USP, sodium chloride injection, ringer's injection, dextrose and sodium chloride injection, and lactated ringer's injection; water-miscible vehicles including, but not limited to, ethanol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles including, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.
The disclosure will be further understood by the following non-limiting examples.
Detailed Description
Example 1 Synthesis and purification of nucleotides and polynucleotides
Exemplary syntheses of immunomodulatory polynucleotides and precursors thereof are described below.
Precursor body
Precursors for the preparation of polynucleotides of the invention are provided in WO 2015/188197 (e.g., phosphoramidites, targeting moieties and bioreversible groups comprising PEG chains).
Phosphoramidites and other monomers
Nucleoside-containing intermediates useful in the synthesis of polynucleotides of the invention are disclosed in WO 2015/188197 (e.g. compounds U1-U54, A1-A15, C1-9 and G1-G12 in WO 2015/188197).
Commercially available phosphoramidites are available from Glen Research (Sterling, Va.) or ChemGenes (Wilmington, Mass.). If desired, other phosphoramidites can be prepared from appropriately protected nucleosides using standard reaction conditions described herein or elsewhere.
Compound S61B
To a solution of S61(0.48g,2.0mmol) in DCM (5.0mL) were added S61A (0.60g, 2.0mmol) and ETT (0.25M in acetonitrile, 4.8mL, 1.2 mmol). The mixture was stirred for 2 hours. Evaporation of volatiles gave a residue which was subjected to Flash silica gel column purification using ethyl acetate/hexanes (0-30% gradient on Combi Flash Rf instrument) to give compound S61B (0.49g, 55%) as a colorless oil. 31P NMR(202MHz,CDCl3； ppm):δ147.83(s)。
Compound S108
To 2- [2- (2-aminoethoxy) ethoxy]A stirred mixture of ethanol (S108A, 25.0g, 167mmol) and N-methylmorpholine (21.0mL, 191mmol) in dioxane (100mL) was added dropwise to a solution of Fmoc-OSu (62.2g, 184mmol) in dioxane (50 mL). After stirring overnight, the reaction was concentrated in vacuo to give a pale yellow oil. The crude product was redissolved in EtOAc and washed with saturated NaHCO3(aq.) and brine wash. The organic layer was removed in vacuo to give an oil which was passed through SiO2Chromatographic purification to give FmocNH-PEG2-OH (S108,55g, 88% yield). ESI + M/z Called 371.4, found 372.2[ M + H ]]+。
X1 and X2 base spacer free synthesis-general scheme:
compound S110
To a suspension of NaH (13.2g, 60% in mineral oil, 230.0mmol) in THF (40mL) under argon at 0 deg.C was added dropwise a solution of a diol (S109, 4.92g, 22.0mmol) in THF (20 mL); the resulting mixture was warmed to room temperature and stirred for 1 h. The reaction mixture was cooled to 0 ℃, propargyl bromide (18.6g, 158.4mmol) in THF (25mL) was added slowly, and the resulting mixture was warmed to room temperature and stirred at 40 ℃ overnight. After the product was exhausted by TLC, the reaction was quenched by dropwise addition of water at 0 ℃ and the resulting mixture was extracted with dichloromethane (50mL × 2). The combined organic layers were washed with brine, over anhydrous Na 2SO4Dried, filtered and evaporated to give a residue which was purified by flash silica gel column using ISCO partner (hexane/ethyl acetate, 0-30%) to give 5.92g (89.5%) of compound S110 as an oil.1H NMR(500MHz,CDCl3；ppm): δ7.49-7.47(dd,J8.0,1.5Hz,2H),7.38-7.34(m,3H),5.43(s,1H),4.21 (d,J2.5Hz,2H),4.12(t,J2.5Hz,4H),4.10(s,1H),3.91(s,1H),3.89 (s,1H),3.37(s,2H)；C18H20O4ESIMS calculated value of 300.34, observed value [ M + H ]]+301.3。
Compound S111
The dipropargyl compound S110(5.9g, 19.64mmol) was dissolved in an acetic acid/water mixture (60mL, 75: 25) and the reaction was continued for 2 h at 50 ℃. After completion of the reaction, the solution was evaporated and co-evaporated with toluene (2 × 20 mL). The residue was directly purified using a flash silica gel column using an ISCO partner (hexane/ethyl acetate, 20-80%) without any treatment to give 3.02g (72.5%) of compound S111 as an oil.1H NMR(500MHz,CDCl3； ppm):δ4.15(d,J 2.5Hz，4H),3.68(s,4H),3.59(s,4H),2.44(t,J 2.5 Hz,2H),2.30-2.40(br,2H)；C11H16O4ESI MS of calculated value 212.24, observed value [ M + H]+213.2。
Compound S112
A solution of dimethoxytrityl chloride (4.8g, 14.2mmol) in dichloromethane (40mL) was added dropwise to a solution of diol S111(3.0g, 14.2mmol), N-diisopropylethylamine (3.15mL, 17.0mmol) and DMAP (0.36g, 2.83mmol) in dichloromethane (25mL) at 0 deg.C, and the reaction was continued overnight at room temperature. The mixture was diluted with dichloromethane, washed with water and brine, and the organic layer was washed with anhydrous Na2SO4Dried, filtered and evaporated. The resulting residue was purified by flash silica gel column using ISCO partner (hexane/ethyl acetate, 0-40%) to give 5.29g (73%) of mono DMT protected compound S112 as a white solid. 1H NMR(500MHz,CDCl3；ppm):δ7.4-7.42(m,2H), 7.32-7.31(m,4H),7.28-7.25(m,2H),6.84-6.81(m,4H),4.09(d,J2.5 Hz,4H),3.79(s,6H),3.67(d,J6.0Hz,2H),3.64-3.56(m,4H),3.13(s, 2H),2.39(t,J2.5Hz,2H)；C32H34O6ESI MS of (E) calculated 514.6, observed [ M + Na ]]+537.4。
Compound S113
To a solution of DMT protected compound S112(0.5g, 0.98mmol) in dichloromethane (4mL) was added dropwise a solution of 2 ' -cyanoethyl-N, N, N ', N ' -tetraisopropyl phosphoramidite (0.58 g,1.95mmol) in dichloromethane (3mL) at room temperature, followed by the addition of 5-benzylthio-1H-tetrazole (BTT; 0.25M in acetonitrile, 0.78mL, 0.18mmol) under an argon atmosphere. The reaction was continued until the starting material disappeared (2h) and the crude mixture was diluted with 20mL of dichloromethane, followed by saturated NaHCO3The solution (10mL) and brine (10mL) were washed with anhydrous Na2SO4And (5) drying. The solvent was evaporated in vacuo and the crude mixture was purified by silica gel column chromatography using ethyl acetate/hexane with 3% triethylamine as co-solvent (0-30% gradient on Combi Flash Rf instrument) to give 0.53g of compound S113 as an oil (75%). ESI MS: c41H51N2O7P calculated 714.82, observed 715.6[ M + H ]]+；31P NMR (202MHz,CDCl3):δ147.89。
Compound S114
To a solution of DMT protected compound S112(0.98g, 1.9mmol) and N, N-diisopropylethylamine (0.39mL, 2.09mmol) in 8.0mL of anhydrous dichloromethane at-78 deg.C was added dropwise a solution of bis- (N, N-diisopropylamino) -chlorophosphine (0.56g, 2.09mmol) in dichloromethane (4.0mL) under an argon atmosphere. The reaction mixture was allowed to warm to room temperature while maintaining stirring for 1 h. A solution of 3-butyn-1-ol (0.14g, 1.9mmol) in 2.0mL of anhydrous dichloromethane was added at room temperature; the resulting mixture was stirred for 10 minutes at which time a 0.25M solution of ETT in acetonitrile (4.6mL, 1.15mmol) was added and stirring continued for an additional 3 h. After completion of the reaction, the crude mixture was diluted with 20mL dichloromethane and in turn with saturated NaHCO as observed by the disappearance of the starting material by TLC 3The solution (10mL) and brine (10mL) were washed with anhydrous Na2SO4And (5) drying. The volatiles were evaporated in vacuo and the crude mixture was purified by silica gel column chromatography using ethyl acetate/hexane and 3% triethylamine as solvent system (0-40% gradient on Combi Flash Rf instrument) to give 0.33g of compound S114 as an oil (25%). ESI MS: c42H52NO7P calculated 713.83, observed 714.7 [ M + H ]]+；31P NMR(202MHz,CDCl3):δ146.89。
X3 and X4 base spacer free synthesis-general scheme:
compound S116
Use ofCompound S116 was prepared as an oil in 91% yield according to the protocol described for compound S110.1H NMR(500MHz,CDCl3；ppm):δ7.51(d,J 7.5Hz,2H), 7.37-7.32(m,3H),5.56(s,1H),3.37-3.35(m,4H),4.10-4.07(dd,J 13.0 Hz,J 2.5Hz，2H),3.65-3.64(m,1H),2.43-2.42(t,J 6.5Hz，1H)；ESI MS：C13H14O3Calculated value 218.24, observed value [ M + H]+219.2。
Compound S117
Compound S117 was prepared as an oil in 91% yield using the protocol described for compound S111.1H NMR(500MHz,CDCl3；ppm):δ4.33(s,2H),3.83-3.70(m, 5H),2.48(s,1H),2.04(br,2H)；ESIMS：C6H10O3Calculated value 130.14, observed value [ M + Na]+153.0。
Compound S118
Compound S118 was prepared as a white solid in 54% yield using the protocol described for compound S112.1H NMR(500MHz,CDCl3；ppm):δ7.43(d,J7.5Hz,2H), 7.37-7.27(m,5H),7.23-7.16(m,2H),6.83(d,J9.0Hz,3H),6.78-6.76 (ddJ 8.5Hz,1H),4.35-4.22(m,2H),3.77(s,6H)3.76-3.72(m,2H), 3.71-3.64(m,1H),3.27-3.19(m,2H),2.48(t,J4.5Hz，1H),2.03-1.96 (m,1H)；ESIMS：C27H28O5Calculated value 432.50, observed value [ M + Na]+455.4。
Compound S119
Compound S119 was prepared as an oil using the protocol described for compound S113, in 86% yield. ESI MS: c36H45N2O6P calculated 432.72, observed 433.5[ M + H ]]+；31P NMR(202MHz,CDCl3):δ149.05,148.96。
Compound S120
Compound S120 was prepared as an oil using the protocol described for compound S114 in 47% yield. ESI MS: c 37H46NO6P calculated 431.73, observed 432.5[ M + H ]]+；31P NMR(202MHz,CDCl3):δ147.80,147.71。
X5 and X6 base spacer free synthesis-general scheme:
compound S121
To a solution of S116(4.0g, 22.2mmol) in dioxane (25mL) was added a solution of KOH (0.12g, 2.2mmol) dissolved in a minimum amount of water, and the resulting mixture was stirred at room temperature for at least 30 minutes. The mixture was cooled to 0 ℃, a solution of acrylonitrile (2.35g, 44.4mmol) in dioxane (15mL) was added dropwise, and the resulting mixture was reacted at room temperature overnight. The volatiles were evaporated in vacuo, the residue was diluted with water and the pH was adjusted to near neutral. The crude product was extracted with ethyl acetate (2 × 50mL) and the combined organic layers were washed with brine, over anhydrous Na2SO4Dry, filter, evaporate to give a residue, which is purified by flash silica gel column using ISCO partner (dichloromethane/methanol, 0-5%) to give 3.1g (60%) of compound S121 as a white solid.1H NMR(500MHz,CDCl3；ppm): δ7.49(d,J 7.0Hz,2H),7.36-7.34(m,3H),5.56(s,1H),3.36(d,J 13.0 Hz 2H),4.10-4.07(dd,J 13.0Hz,J 2.0Hz,2H),3.84(t,J 6.5Hz,2H), 3.42(m,1H),3.69(t,J 6.5Hz，2H)；ESIMS：C13H15NO3Calculated value 233.2, observed value [ M + Na]+256.3。
Compound S122
To a suspension of lithium aluminium hydride (0.83g, 4.0mmol) in THF (10mL) at 0 deg.C was added dropwise a solution of compound S121(1.28g, 5.5mmol) in THF (15mL), the resulting mixture was warmed to room temperature and stirring continued for 3 h. After completion of the reaction, the reaction mixture was cooled to 0 ℃ and quenched by dropwise addition of water (about 2-3mL) as needed. An additional 8mL of water was added and the crude product was extracted into ethyl acetate (2 × 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na 2SO4Dried, filtered and evaporated to give compound S122, which was used without further purification in the subsequent step.1H NMR(500MHz, CDCl3；ppm):δ7.49(d,J 7.0Hz,2H),7.40-7.32(m,3H),5.55(d,J 5.0 Hz,1H),4.34(d,J 13.0Hz,1H),4.20-4.11(dd,J 12.0Hz4H), 4.05-4.03(d,J 13.0Hz,J 2.0Hz,1H),3.66-3.62(m,2H),3.27(m,1H), 2.86(t,J 6.5Hz,1H),2.16(br,2H)；ESI MS：C13H19NO3Calculated value 237.2, observed value [ M + H]+238.2。
Compound S123
To a solution of compound S122(1.0g, 4.2mmol) and N, N-diisopropylethylamine (2.3mL, 12.6mmol) in dichloromethane (8mL) was added dropwise a solution of Fmoc-OSu (1.7g, 5.0mmol) at 0 deg.C, and the resulting mixture was reacted at room temperature for 3 hours. Upon completion, the reaction mixture was diluted with dichloromethane (10mL) and washed with water and then brine. Separating the organic layer with anhydrous Na2SO4Dried, filtered and evaporated to give a residue. The residue was purified by ISCO companion (hexane/ethyl acetate, 0-50%)Purification on a flash silica gel column gave 0.65g (35%) of compound S123 as a white solid.1H NMR(500MHz, CDCl3；ppm):δ7.75(d,J 7.5Hz,2H),7.58(d,J 7.5Hz,2H),7.51(d,J 7.5Hz,2H),7.37(t,J 7.5Hz,2H),7.31-7.26(m,5H),5.57(s,1H),5.48 (br,1H),4.46-4.32(m,4H),4.15(d,J 7.0Hz,1H),4.06(t,J 12.5Hz 2H),3.67(m,2H),3.54(m,2H),3.41(s,1H),1.88(t,J 6.0Hz,2H)； ESIMS：C28H29NO5Calculated value 459.5, observed value [ M + Na]+482.5。
Compound S124
Compound S124 was prepared as an oil using the protocol described for compound S111, with quantitative yield.1H NMR(500MHz,CDCl3；ppm):δ7.76(d,J 7.5Hz,2H),7.58 (d,J 7.5Hz，2H),7.39(t,J 7.5Hz，2H),7.32(t,J 7.5Hz,2H),5.18(br， 1H),4.44(d,J 6.5Hz,2H),4.21(t,J 6.5Hz,1H),4.76-4.73(dd,J 11.5, 3.5Hz 2H),3.67-60(m,4H),3.42(m,1H),3.37(br,2H),2.07(m,2H), 1.75(br,2H)；ESI MS：C21H25NO5Calculated value 371.4, observed value [ M + Na]+ 394.3。
Compound S125
Compound S125 was prepared as a white solid using the protocol described for compound S112, with a 48% yield of product (S125).1H NMR(500MHz,CDCl3；ppm):δ7.75(t,J 7.5Hz,2H),7.58(t,J 7.5Hz,2H),7.40-7.38(m,3H),7.32-27(m,7H), 7.18-7.16(m,3H),6.83(t,J 7.0Hz,4H),5.16(br,1H),4.44(d,J 6.5 Hz,2H),4.20(m,1H),3.80(s,3H),3.79(m,1H),3.76(s,3H),3.74(m, 2H),3.66-3.62(m,4H),3.43-3.37(m,2H),2.31(br,1H),1.76(br,2H)； C42H43NO7ESI MS of calculated value 673.7, observed value [ M + Na]+696.7。
Compound S126
Compound S126 was prepared as an oil using the protocol described for compound S113, with 78% yield of product (S126). ESI MS: c 51H60N3O8P calculated 874.0, observed 896.9 [ M + Na]+,913.0[M+K]+；31P NMR(202MHz,CDCl3；ppm):δ148.90, 148.76。
Synthesis of base-free spacer S131-general scheme:
compound S127
To a solution of S109(2.56g, 11.4mmol) in dichloromethane (50mL) was added bromoacetonitrile (3.01g, 25.1mmol), silver (I) oxide (5.28g, 22.8mmol) and tetrabutylammonium iodide (0.84g, 2.28mmol) under argon and the resulting mixture was stirred overnight. Passing the mixture throughFiltration and evaporation of the filtrate gave a black residue which was subjected to flash silica gel column purification (hexane/ethyl acetate, 15-90%) on an ISCO partner to give 1.34g (39%) of the desired compound S127 as a viscous oil. ESI MS: c16H18N2O4Calculated value 302.3, observed value [ M + H]+303.3。
Compound S128
To a solution of compound S127(1.34g, 4.43mmol) in THF (30mL) under argon was addedAdding LiAlH4Was added to the reaction solution, and the solution was dissolved in THF (2M, 8.9mL, 17.7mmol), and the mixture was heated to 55 ℃ for 4 hours. Adding another portion of LiAlH4Was added to the solution of THF (2M, 4mL, 8.0mmol) and stirring was continued for 4 hours. After completion of the reaction, the mixture was cooled to room temperature and taken with Na2SO4.10H2And O quenching. The solid was filtered off and washed with ethyl acetate. Passing the filtrate through anhydrous Na2SO4And (5) drying. The mixture was filtered and evaporated to give a residue, which was dissolved in dichloromethane (20 mL). To this solution were added Fmoc-OSu (1.5g, 4.43mmol) and DIEA (0.87mL, 5.0 mmol). The mixture was stirred for 1 hour and then evaporated to give a residue which was subjected to flash silica gel column purification (hexane/ethyl acetate, 20-90%) on an ISCO partner to give 1.04g (31%) of the compound S128 as a white foam. 1H NMR (500MHz,CDCl3；ppm):δ7.75(4H，dd,J 7.5,4.5Hz)，7.58(4H,t,J 7.0 Hz),7.48(2H,d,J7.0Hz),7.41-7.34(7H,m),7.32-7.26(4H,m),5.44 (1H,s),5.15-5.05(2H,m),4.44(2H,d,J5.5Hz),4.38(2H,d,J6.0Hz), 4.25-4.15(2H,m),4.10(2H,d,J11.5Hz),3.82(2H,d,J 11.5Hz),3.78 (2H,s),3.53(2H,s),3.42(2H,s),3.36-3.27(4H，m),3.25(2H,s)；ESI MS：C46H46N2O8Calculated value 754.9, observed value [ M + H]+755.3。
Compound S129
Compound S128(1.1g, 1.51mmol) is dissolved in AcOH/H2O mixture (10mL, 3:1) and reaction was continued at 55 ℃ for 5 h. After completion of the reaction, the volatiles were evaporated and co-evaporated with toluene (2 × 20mL) and the residue was subjected to flash silica gel column purification on an ISCO partner (hexane/ethyl acetate, 30-100%) to give 0.54g (54%) of compound S129 as a white foam.1H NMR(500MHz,CDCl3；ppm):δ7.75(4H,d,J 7.5Hz), 7.58(4H,d,J 7.5Hz),7.39(4H,t,J 7.5Hz),7.30(4H,t,J 7.5Hz), 5.20-5.05(2H,m),4.41(4H,d，J 6.5Hz),4.21(4H,t,J 6.5Hz),3.64 (4H,s),3.48(8H,s),3.36(4H,s)；ESI MS：C39H42N2O8Calculated value 666.7, observed value [ M + H]+667.3。
Compound S130
A solution of DMTrCl (0.34g, 0.99mmol) in dichloromethane (1mL) was added dropwise to a solution of diol S129(0.73g, 1.1mmol), DIPEA (0.19mL, 1.1mmol) and DMAP (0.013g, 0.11mmol) in dichloromethane (6mL) at 0 deg.C. The resulting mixture was warmed to room temperature and stirred overnight. The mixture was evaporated to give a residue which was subjected to flash silica gel column purification on ISCO (hexane/ethyl acetate, 20-100%) to give 0.47g (44%) of the mono-dimethoxytrityl protected compound S130 as a white foam.1H NMR(500MHz，CDCl3；ppm):δ7.75(4H,d,J 7.5Hz),7.58(4H,d,J 7.5Hz),7.39(4H,t,J 7.5Hz),7.32-7.25(8H,m),7.17(4H,d,J 6.5Hz), 6.83(4H,d,J 6.5Hz),5.20-5.05(2H,m),4.41(4H,d,J 6.5Hz),4.21 (4H,t,J 6.5Hz),3.82(6H,s),3.64(4H,s),3.48(8H,s),3.36(4H,s)； ESI MS：C60H60N2O10Calculated value 969.1, observed value [ M + Na]+991.3。
Compound S131
3-Fmoc-amino-propan-1-ol (0.090g,0.30mmol) and N, N-diisopropylethylamine (0.18mL, 1.05mmol) in anhydrous CH at-78 deg.C2Cl2(3.0mL) solution was added dropwise bis- (N, N-diisopropylamino) -chlorophosphine (0.085g, 0.32mmol) in anhydrous CH 2Cl2(1.0mL) of the solution. The reaction mixture was warmed to room temperature and stirred for 1.5 hours. 1.0mL of anhydrous CH as compound S130(0.30g, 0.30mmol) was added2Cl2The solution, and the resulting mixture was stirred for 10 minutes. To the reaction mixture was added a solution of ETT (0.72mL, 0.25M in acetonitrile, 0.18mmol) and the resulting mixture was stirred for 3 h. Subjecting the mixture to CH2Cl2Diluting (20mL) and diluting with waterAnd aqueous sodium bicarbonate (20mL) and brine (20 mL). The organic layer was dried over anhydrous sodium sulfate and the filtrate was evaporated in vacuo to give a residue which was flash silica gel column purified on an ISCO partner using ethyl acetate/hexane and 3% triethylamine as co-solvent (0-30% gradient) to give 0.12g of the product S131 as a white foam (32%). ESIMS: c84H91N4O13P calculated 1395.6, observed 1395.7[ M ]]+；31P NMR(202MHz,CDCl3):δ146.41。
Compound dT4
Synthesis of FmocNH-PEG 2-hydroxy-diisopropylamino-dT (5' -DMT) phosphoramidite (dT 4). 5' -DMT-deoxythymidine (4.30g, 7.89mmol) and DIEA (1.51mL, 8.68mmol) in CH under argon2Cl2The stirred suspension in (40mL) was cooled to-78 ℃. Bis (diisopropylamino) chlorophosphine (2.32g, 8.68mmol) in CH was added dropwise2Cl2(10mL) of the solution. The mixture was removed from the cooling bath and stirred for 1 h. To the reaction mixture is added CH 2Cl2FmocNH-PGE2-OH (S108,2.93g,7.89mmol) in (15mL) and then ETT (0.25M in acetonitrile, 18.9mL) solution was added. After stirring overnight, the mixture was concentrated in vacuo, redissolved in EtOAc and diluted with saturated NaHCO3(aq.) and brine wash. The organic layer was removed in vacuo to give a white foam. Passing the crude material through SiO2Purification by chromatography afforded the title phosphoramidite (dT4, 4.1g, 50% yield).
The above synthesis scheme is used to synthesize other phosphoramidite precursors for different triesters.
Compound dU6
To dU1(3.3g, 5.0mmol), 1-methylimidazole (1.2mL, 15.0mmol) and iodine (1.9g, 15.0 m) under stirring at room temperature under Ar (g)mol) in THF (10 mL) was added dropwise a solution of tert-butyldimethylsilyl chloride (0.8g, 5.5mmol) in THF (5 mL). The reaction was stirred at room temperature for 1 hour. TLC confirmed the reaction was complete. The solvent was removed in vacuo, the crude dissolved in ethyl acetate and concentrated NaHCO3And (4) washing with an aqueous solution. Passing the organic phase over Na2SO4The liquid was dried, filtered and evaporated. The crude product was purified through a flash silica gel column using an ISCO partner (hexane/ethyl acetate, 0-50%) to provide dU2 as a solid in quantitative yield. NMR was consistent with that published. Nucleic Acids Research,2011, Vol.39, No.9, 3962-.
A solution of dU2(3.9g, 5.0mmol) dissolved in 80% aqueous acetic acid (40mL) with triisopropylsilane (1.0mL, 5.0mmol) was stirred at room temperature for 1 hour. TLC confirmed the reaction was complete. The solvent was removed in vacuo. The crude product was purified through a flash silica gel column using an ISCO partner (hexane/ethyl acetate, 0-60%) to give 1g (43%) of the desired compound dU3 as a solid. ESI MS: c15H25IN2O5Calculated Si 468.4, observed [ M + Na ]]+491.0。
To a solution of dU3(1.0g, 2.2mmol) in THF (20mL) cooled to 0 ℃ in an ice-water bath was added sodium hydride (60% dispersion, 0.2g, 4.7mmol) under ar (g). The reaction was stirred at 0 ℃ for 30 minutes. Methyl iodide (0.7mL, 10.8mmol) was added dropwise and the reaction stirred at 0 ℃ for 3 h. RP-HPLC/MS confirmed the reaction was complete. The reaction was quenched with 20mL methanol at 0 ℃ and warmed to room temperature. Addition of saturated NaHCO3Aqueous solution, and the mixture is treated with CH2Cl2And (4) extracting. Passing the organic phase over Na2SO4Dried, filtered and the liquid concentrated in vacuo. Purification by silica gel column chromatography (hexanes/ethyl acetate, 0-50%) gave dU4 as a solid (0.6g, 58% yield). ESI MS: c16H27IN2O5Calculated Si 482.4, observed [ M + H ]]+483.1。
To a cooled solution of dU4(0.6g, 1.3mmol) in THF (20mL) under Ar (g) was added dropwise tert-butylammonium fluoride (1M THF, 3mL, 3.0mmol) with stirring. The cooled solution was stirred for 30 minutes and then warmed to room temperature. After 3.5 hours, RP-HPLC/MS confirmed the reaction was complete. Will be provided with The crude product was purified by silica gel column chromatography (dichloromethane/methanol, 0-10%) to give dU5 as a solid (0.4g, 92% yield). ESIMS: c10H13IN2O5Calculated value 368.1, observed value [ M + H]+369.0。
To a solution of dU5(0.4g, 1.2mmol) in dichloromethane (5mL) at room temperature under Ar (g) was added dropwise a solution of 2 ' -cyanoethyl-N, N, N ', N ' -tetraisopropyl phosphoramidite (0.4mL, 1.3mmol) in dichloromethane (5mL) with stirring. The reaction was stirred at room temperature for 30 minutes. Thioethyltetrazole (0.25M solution in ACN, 2.9mL, 0.7mmol) was then added and the reaction was allowed to continue overnight. TLC confirmed the reaction was complete. The solvent was removed in vacuo and the crude mixture was diluted with 20mL of dichloromethane, followed by saturated NaHCO3The solution (10mL) and brine (10mL) were washed. Passing the organic phase over Na2SO4The liquid was dried, filtered and evaporated. The crude mixture was dissolved in ethyl acetate and purified by silica gel column using an Isco partner (hexane/ethyl acetate, 0-100%) to give 0.3g (49.9%) of the desired compound dU6 as a solid. ESIMS: c19H30N4O6P calculated 568.3, observed 567.3[ M-H ]]-；31P NMR(202MHz,CDCl3,ppm):δ149.25。
Compound dU9
The title compound was prepared by reacting dU3 under standard reaction conditions shown below.31P-NMR(202mHz,CDCl3In ppm) delta 149.42,149.31; measured value of MSESI-M/z 667.1[ M-H ]MSESI + M/z found 669.2[ M + H ]],691.3 [M+Na]。
Preparation of linkers bonded to the auxiliary moiety:
compounds PP2, PP3 and PP4
Preparation of (5-azidovaleryl) -N-Boc lysineAcid (PP 1). Mixing epsilon-N-Boc lysine (9.46g, 38.4mmol) and K2CO3(2.67g,19.3mmol) was dissolved in 1:1 THF: H2O (60 mL). Pentafluorophenyl-5-azidovalerate (10.8g, 34.9mmol) in THF (10mL) was added and the reaction was stirred at room temperature overnight. By RP-HPLC-MS 394.2[ M + Na]The desired product was observed. The reaction was acidified to pH 5 by titration with 1N HCl (aq) and the product extracted with EtOAc (3X 100 mL). The organic layer was successively treated with H2O (50mL) and brine (50 mL). The organic layer was purified over MgSO4Dried and concentrated in vacuo to a thick slurry. The crude product was purified by silica gel column chromatography to give the desired white needle product PP2(8.1g, 62% yield). ESI MS + mass calculated value C16H29N5O5371.4, found 394.2[ M + Na]+。
General protocol for pegylation of PP 1: preparation of (5-azidovaleryl) - ε -N- (NH-Boc PEG24) lysine (PP 4). PP1(0.74g, 2.0mmol) was treated with HCl (2mL, 4N in dioxane) for 4 hours. HPLC-MS showed complete deprotection of 272.2[ M + H]+. With 1: 1H2Dilution of the reaction with acetonitrile (10mL), freezing and lyophilization overnight gave PP2 as a white solid in quantitative yield. NHBoc-PEG24 acid (1.1g, 0.88mmol) in DMF (3mL) was activated with HATU (0.34g, 0.88mmol), HOBt (0.14g, 0.88mmol) and DIEA (0.7mL, 4.0mmol) and treated with PP2(0.24g, 0.8mmol) for 2 h. RP-HPLCMS showed the formation of the desired PP 4. The crude product was purified by RP-HPLC to give PP4 as a white solid (0.55g, 46% yield). ESI MS + mass calculated value C 67H130N6O301499.77, found 1499.9[ M + H ]]+,1400.8[M-Boc]+。
bissegx-NH 2 and trissegx-NH 2 (where X is various PEG lengths) were prepared from commercially available starting materials using the procedure described in WO 2015/188197.
General protocol for pegylation of PP2, PP3 and PP 4: with HATU (37mg, 0.1 mm)ol), N-diisopropylethylamine (49mL,0.3mmol) and mPEG48-NH2(200mg,0.09mmol) lysine PP1(38mg, 0.1mmol) dissolved in DMF (1mL) was treated. RP-HPLC-MS showed complete addition of PEG48 to PP 1. The crude product was purified by RP-HPLC to give NHBoc PP7(97mg, 42% yield) as a white solid. ESI MS + mass calculated value C113H224N6O522499.03, found 833.7 [ M +3H]3+,625.6[M+4H]4+. PP7 was deprotected with HCl (2mL, 4N in dioxane) for 4 h. HPLC-MS showed complete deprotection as observed by the disappearance of the peak with mass of starting material. With 1: 1H2Acetonitrile (10mL) diluted the reaction, frozen, and lyophilized overnight to yield PP8 quantitatively as a white solid. ESI MS + calculated mass value C108H216N6O502398.88, found 1199.8[ M +2H]2+,800.3[M+3H]3+, 600.5[M+4H]4+,480.6[M+5H]5+。
In this scheme, the conditions are:
A) 6-methyltetrazine-OSu, HATU, Hunig's base, DMF; and
B) DBCO-CpG, acetonitrile/H2O；
Wherein the 6-methyltetrazine-OSu has the formula:
and is
DBCO-CpG has the formula:
General protocol for the preparation of linkers loaded with polynucleotides (PP28 and PP 30).
Tetrazine conjugation treatment of PP12 and PP 16: PP12(43mg, 0.12mmol) was dissolved inIn DMF (0.5mL), treated with HATU (4.6mg, 0.12mmol), DIEA (12.7. mu.L, 0.73mmol) and after 5 min with 6-methyl-tetrazine-OSu (19.9mg, 0.61 mmol). The crude reaction was stirred at room temperature for 30 minutes and RP-HPLCMS showed complete coupling of 6-methyl-tetrazine carboxylate to PP 12. The crude product was purified by RP-HPLC and the combined fractions were lyophilized to give PP27 as a purple solid (39mg, 85% yield). ESI MS + mass calculated value: c170H325N11O76:3739.47, found 833.7[ M +3H]3+,625.6 [M+4H]4. Pure PP27 was treated in DBCO-CpG in acetonitrile: water (1:1) and incubated at 37 ℃ for 1-2 hours and then at room temperature for another 1 hour to give PP 28. PP28 was purified by preparative AEX (20mM phosphate and 20mM phosphate-1M sodium bromide).
Another one-pot route to CpG-loaded linkers PP28 and PP 30. PP12 (400nmol) was treated with DBCO-CpG (420nmol) in acetonitrile: water (1:1) and incubated at 37 ℃ for 1-2 hours and then at room temperature for another 1 hour. tetrazine-OSu (4000nmol) in DMSO stock solution was added to the crude PP12-DBCO-CpG solution and the purple solution was reacted at room temperature for 3 hours and 1-2 hours to give PP 28. The crude PP28 was purified by preparative RP-HPLC (water and 10% acetonitrile: 50mM TEAA in water) or preparative AEX (20mM phosphate and 20mM phosphate-1M sodium bromide).
Preparation of tetrazine-PEG 24-OPFP (PP 32). To a solution of amino-PEG 24-carboxylic acid (1.0g, 0.9mmol) and diisopropylethylamine (0.8mL, 4.4mmol) in DMF/water (1:1, 12mL) under Ar (g) with stirring was added methyl tetrazinylphenylacetyl succinimide ester (370mg,1.1mmol) in DMF (3mL) dropwise. The reaction was stirred at room temperature for 2 hours. RP-HPLC/MS indicated the product was formed. The solvent was removed in vacuo and the crude product was purified by RP-HPLC (TFA modifier) to give 1.1g (80%) of PP 31. C62H111N5O27ESI MS of calculated value 1358.56, observed value [ M + H]+1358.8. To a solution of PP31(109mg, 0.08mmol) in dichloromethane (3mL) under Ar (g)Anhydrous pyridine (32mg, 0.4mmol) and pentafluorophenyl trifluoroacetate (67mg, 0.24mmol) were added. The reaction was stirred at room temperature overnight. The solvent was removed in vacuo. The crude product was redissolved in EtOAc and treated with NaHCO3(5% w/v) (3X) aqueous solution and brine (1X). Passing the organic phase over Na2SO4Dried, filtered, and concentrated in vacuo to yield PP32 quantitatively. Used in the next step without further purification. ESIMS: c68H110F5N5O27Calculated value 1524.61, observed value [ M +2H]2+763.0。
PP34 was prepared. To a solution of mPEG 48-amine (2.15g, 1.00mmol) and diisopropylethylamine (0.87mL, 5.00mmol) in DMF/water (1:1, 10mL) under Ar (g) was added dropwise Cbz-Ne-Boc-L-lysine succinimide ester (570mg, 1.2mmol) in DMF (5 mL). The reaction mixture was stirred at room temperature for 2 hours. RP-HPLC/MS indicated the formation of product PP 33. The reaction mixture was concentrated in vacuo and chromatographed on silica gel (CH) 2Cl2MeOH 0-10%) in the sample. The recovered PP33 was used directly in the next reaction. ESI MS: c116H223N3O53Calculated value 2508.0, observed value [ M +3H]3+836.7,[M+4H]4+627.9. A solution of PP33(1.00mmol) in MeOH was sparged with nitrogen (g) and palladium on activated carbon (10% wt, catalyzed) was added. The solution was alternately evacuated and purged with hydrogen (g) (3X). After 2 hours, RP-HPLC/MS showed the formation of PP 34. The heterogeneous mixture was filtered through a celite bed and washed with copious amounts of methanol. The solvent was removed in vacuo to give PP34(2.0g, 84% yield, via 2 steps). ESIMS: c108H217N3O51Calculated value 2373.87, observed value [ M +3H]3+792.0。
PP37 was prepared. To PP32(124mg, 0.08) under stirring under Ar (g)mmol) and diisopropylethylamine (31mg, 0.24mmol) in DMF/water (1:1,10mL) PP34(230mg, 0.1mmol) in DMF/water (1:1,10mL) was added dropwise. The reaction was stirred at room temperature for 2 hours and RP-HPLC/MS indicated the formation of product PP 35. The solvent was removed in vacuo and PP35 was used in the next step without further purification. ESI MS: c170H326N8O77Calculated value 3714.4, observed value [ M +4H]4+929.5,[M+5H]5+743.8. Crude PP35(0.08 mmol) was treated with HCl (4N in dioxane, 5mL) under Ar (g). The reaction was stirred at room temperature for 2 hours and RP-HPLC/MS indicated complete removal of the Boc protecting group. The solvent was removed in vacuo and the amine was acylated with a solution of bis-Peg 3-PFP ester (230mg, 0.4mmol) in DMF (5mL) and diisopropylethylamine (140uL, 0.8 mmol). After 2 hours, RP-HPLC/MS indicated the formation of the product PP 37. The solvent was removed in vacuo and the crude product was purified by RP-HPLC (TFA modifier) to provide PP37 as the tetra TFA salt, 31mg, 8.7% yield. ESI MS: c 181H333F5N8O81Calculated value 4012.56, observed value [ M +3H]3+1338.3,[M+4H]4+1004.0,[M+5H]5+803.4, [M+6H]6+669。
List of linkers containing auxiliary moieties:
in the above table, the groups identified as Y or Z have the following structures:
in the above table, the symbolsIs "p 313+ N3The radical Z of-valeramide "is p313 and has N3Valeramide as a product of a cycloaddition reaction between linkers of Z.
The phosphoramidite monomers shown in table 1 were synthesized using standard synthetic procedures described herein and WO 2015/188197.
Bicyclic oxaazaphosphono monomers for chiral phosphorothioate oligonucleotide synthesis were prepared using the literature protocol reported by Wada, J.am.chem.Soc.130:16031-16037, 2008.
TABLE 1
Chiral abasic spacer-compounds X7, X8, X9 and X10:
x7 and X8 synthesis:
x9 and X10 synthesis:
the following are other hydrophilic nucleoside phosphoramidites that can be prepared using methods known in the art and described herein:
wherein R is OH, optionally substituted amino or-CO2R1(R1Is H or a counterion), and n is an integer from 1 to 4;
wherein R is OH, OAc, OMe, optionally substituted amino or CO2R1(R1Is H or a counter ion), n is an integer from 1 to 51.
The following are further substituted nucleoside phosphoramidites that can be prepared using methods known in the art and described herein:
Wherein R and R1Each independently is H or optionally substituted C1-6Alkyl (e.g., Me, Et, i-Pr or n-Bu))。
The following phosphoramidites were purchased from Glen Research (Sterling, Va.) or Chemgenes (Wilmington, Mass.) or prepared using standard protocols described herein:
these intermediates are useful for preparing polynucleotides of the invention (e.g., polynucleotides containing 5' -terminally modified nucleosides). Non-limiting examples of 5' -terminally modified nucleosides are 5-halogenated uridine, 5-alkynyl uridine, 5-heteroaryl uridine and 5-halogenated cytidine.
5' -end capping
a)5 '-5' -end capping
b) 5' -phosphate or phosphorothioate end-capping:
synthesis of small molecule-based targeting moieties
Exemplary compounds for preparing small molecule-based targeting moieties are described in WO 2015/188197 (e.g., compounds M1-M30 described in WO 2015/188197).
Synthesis of the glucitol Co-moiety
Exemplary compounds for preparing a glucitol based co-moiety are described in WO 2015/188197 (e.g., compounds POH1-POH10 described in WO 2015/188197).
General polynucleotide synthesis:
general scheme
Details of the experiment:
automated polynucleotide synthesis (1 μmol scale) was performed on MerMade 6 or 12 using the following reagents and solvents:
Oxidizing agent-THF/pyridine/H20.02MI in O2(60 s of oxidation per cycle),
sulfurizing reagent II-dithiazole derivative/pyridine/acetonitrile (0.05M in 6:4 pyridine: acetonitrile) (60 s per cycle)
Deblocking-3% trichloroacetic acid (2X 40s deblocking per cycle),
end capping mixture A-THF/2, 6-lutidine/Ac2O (capping 60s per cycle), and
end capping mixture B-16% methylimidazole in THF (60 s end capping per cycle)
The exceptions to standard polynucleotide synthesis conditions are as follows:
using a CPG support with a non-nucleoside linker, called Uny-linker.
Before synthesis, all 2 '-deoxyribose-phosphoramides were resuspended in 100mM 100% anhydrous acetonitrile, except that some modified 2' -deoxyphosphoramides were dissolved in 100mM THF/acetonitrile mixture (1:4), depending on the solubility of the starting material.
Activation of phosphoramidites with a 2.5-fold molar excess of 5-benzylthio-1H-tetrazole (BTT). Each insertion couples the activated 2' -deoxyribose-phosphoramide for 2x1 minutes and each insertion couples the modified phosphoramidite for 2x3 minutes.
Backbone sulfurization with sulfurizing reagent II in 0.05M pyridine/acetonitrile (6:4) for 1 minute.
Polynucleotide deprotection and purification protocol:
after automated polynucleotide synthesis, the solid support and base protecting groups (e.g., A-Bz, C-Ac, G-iBu, etc.) and the methyl ester of the deprotected phosphotriester are cleaved and deprotected (36% ammonia and 40% methylamine in methanol at a ratio of 1: 1) in 1mL of AMA for 2h or more at room temperature, then centrifuged to evaporate.
The crude polynucleotide pellet was resuspended in 100. mu.L of 50% acetonitrile, briefly heated to 65 ℃ and vortexed thoroughly.
To purify the polynucleotide, 100 μ L of the crude polynucleotide was injected onto RP-HPLC with the following buffer/gradient:
buffer a ═ 50mM TEAA in water;
-buffer B ═ 90% acetonitrile; and
-flow rate of 1 mL/min;
-a gradient:
o 0-2 min (100% buffer A/0% buffer B),
o 2-42 min (0% -60% buffer B), and
o 42-55 min (60% -100% buffer B).
DBCO conjugation and purification scheme:
as described herein, DBCONHS esters were conjugated to crude 2' -deoxydmt-polynucleotides. The crude polynucleotide pellet was suspended in 45 μ l LDMSO, briefly heated to 65 ℃, and vortexed extensively. DIPEA was added in 5. mu.L followed by DBCO-NHS ester (30eq) pre-dissolved in DMSO (1M). The reaction was allowed to stand for 10 minutes or until product formation was confirmed by MALDI. A total of 80. mu.L of the crude polynucleotide sample was injected into the RP-HPLC using the following buffer/gradient:
buffer a 50mM TEAA in water
-buffer B90% acetonitrile
-flow rate of 1 mL/min;
-a gradient:
o 0-2 min (90% buffer A/10% buffer B)
o 2-42 min (0% -60% buffer B)
o 42-55 min (60% -100% buffer B).
On the main RP-HPLC peak, a 0.5mL fraction was collected and analyzed by MALDI-TOF mass spectrometry to confirm the presence of the desired mass. The mass selected purified fractions were frozen and lyophilized. Once dried, the fractions were resuspended, combined with the corresponding fractions, frozen and lyophilized.
DMT cleavage: the lyophilized pellet was suspended in 20. mu.L of 50% acetonitrile, and 80. mu.L of acetic acid was added, and the sample was allowed to stand at room temperature for 1h, frozen and lyophilized. The dried sample was redissolved in 20% acetonitrile and passed through NAP 10 (Sephadex)TM-G25DNA Grade) column desalting. The collected pure fractions were frozen and lyophilized to give the final product.
General conjugation scheme using abasic spacers:
chaining reaction-general scheme:
wherein:
each q is 0 or 1;
each m is an integer of 0 to 5;
z is O or S;
ROis a bond in a polynucleotide to a nucleoside;
r is a group that is related to H, a nucleoside in a polynucleotide, a solid support, or a capping group (e.g., - (CH)2)3-OH) linked bonds;
each R' is independently H, -Q1–QA1A bioreversible group or a non-bioreversible group;
each R' is independently H, -Q1–QA–Q2-T, a bioreversible group or a non-bioreversible group;
Each RAIndependently is H OR-ORCWherein R isCis-Q1–QA1A bioreversible group, a non-bioreversible group or a bond to a solid support;
each RBIndependently is H OR-ORDWherein R isDis-Q1–QA–Q2-T, a bioreversible group or a non-bioreversible group;
wherein:
each Q1Independently is a divalent, trivalent, tetravalent, or pentavalent group, one of which valencies is related to QAOr QA1Bonding; the second valency is open, and when present, each remaining valency is independently bonded to the auxiliary moiety;
each Q2Independently a divalent, trivalent, tetravalent or pentavalent radicalOne of the valencies of each group is as defined above for QABonding; the second valency is bonded to T, and when present, each remaining valency is independently bonded to the auxiliary moiety;
QAis 1,2, 3-triazole-1, 4-diyl, optionally substituted C6-16Triazole heterocyclic radicals (e.g. triazoles)) Optionally substituted C8-16Tricycloalkenylene (e.g. for) Or dihydropyridazine radicals (e.g. trans-Or trans-)；
QA1Is optionally substituted C2-12Alkynyl, C containing an internal ring carbon-carbon triple bond optionally substituted6-16Heterocyclic radical (e.g. of the formula) Optionally substituted C8-16Cycloalkynyl (e.g. cycloalkynyl)) Or optionally substituted C4-8Strained cycloalkenyl (e.g., trans cyclooctenyl); and
t is a targeting moiety
With the proviso that the starting material contains at least one-Q1–QA1And the product comprises-Q1–QA–Q2-T; and
provided that the starting material and product comprise 0 or 1 bond to the solid support.
Conjugation process
Cu-catalyzed chain reaction
Preparation of copper-THPTA complex
Adding 5mM of pentahydrateCopper sulfate solution (CuSO)4-5H2O) and 10mM aqueous tris (3-hydroxypropyltriazolylmethyl) amine (THPTA) were mixed at 1:1(v/v) (1:2 molar ratio) and allowed to stand at room temperature for 1 hour. The complexes can be used to catalyze Huisgen cycloaddition reactions, for example, as shown in the general conjugation scheme below.
General protocol (100nM scale)
To a solution of 710. mu.L water and 100. mu.L t-butanol (10% of the final volume) in a 1.7mL Eppendorf tube was added 60. mu.L of the copper-THPTA complex followed by 50. mu.L of 2mM oligonucleotide, 60. mu.L of 20mM aqueous sodium ascorbate solution and 20. mu.L of a 10mM solution of the targeting azide moiety. After thorough mixing, the solution was allowed to stand at room temperature for 1 hour. Completion of the reaction was confirmed by gel analysis. Adding the reaction mixture to a solution containing a 5-to 10-fold molar excessTAAcONa (resin-bound sodium EDTA) screw cap vial. The mixture was stirred for 1 hour. The mixture is then passed through an illustra TMNapTM-10 column SephadexTMAnd (4) eluting. The resulting solution was then frozen and lyophilized overnight.
Conjugation via amide bond:
conjugation via amidation may be performed under amidation reaction conditions known in the art. See, for example, Aaronson et al, Bioconjugate chem.22:1723-1728, 2011.
Wherein:
each q is 0 or 1;
each m is an integer of 0 to 5;
z is O or S;
ROis a bond in a polynucleotide to a nucleoside;
r is a group that is related to H, a nucleoside in a polynucleotide, a solid support, or a capping group (e.g., - (CH)2)3-OH) linked bonds;
each R' is independently H, -Q1–QA1A bioreversible group or a non-bioreversible group;
each R' is independently H, -Q1–QA–Q2-T, a bioreversible group or a non-bioreversible group;
each RAIndependently is H OR-ORCWherein R isCis-Q1–QA1A bioreversible group or a non-bioreversible group;
each RBIndependently is H OR-ORDWherein R isDis-Q1–QA–Q2-T, a bioreversible group or a non-bioreversible group;
wherein:
each Q1Independently is a divalent, trivalent, tetravalent, or pentavalent group, one of which valencies is related to QAOr QA1Bonding; the second valency is open, and when present, each remaining valency is independently bonded to the auxiliary moiety;
Each Q2Independently is a divalent, trivalent, tetravalent, or pentavalent group, one of which valencies is related to QABonding; the second valency is bonded to T, and when present, each remaining valency is independently bonded to the auxiliary moiety;
QAis optionally substituted C comprising-C (O) -N (H) -or-N (H) -C (O) -2-12A heteroalkylene group;
QA1is–NHRN1or-COOR12Wherein R isN1Is H, N-protecting group or optionally substituted C1-6Alkyl, and R12Is H, optionally substituted C1-6An alkyl or O-protecting group; and
t is a targeting moiety and T is a targeting moiety,
with the proviso that the starting material contains at least one-Q1–QA1And the product comprises-Q1–QA–Q2–T。
Solution phase conjugation
Wherein:
m is an integer of 0 to 5;
z is O or S;
ROis a bond in a polynucleotide to a nucleoside;
r is a bond to H, a nucleoside or a capping group in a polynucleotide;
each R' is independently H, -Q1–NH2A bioreversible group or a non-bioreversible group;
each R' is independently H, -Q1–NH–CO–Q2-T, a bioreversible group or a non-bioreversible group;
each RAIndependently is H OR-ORCWherein R isCis-Q1–NH2A bioreversible group or a non-bioreversible group;
each RBIndependently is H OR-ORDWherein R isDis-Q1–NH–CO–Q2-T, a bioreversible group or a non-bioreversible group;
Wherein:
each Q1Independently a divalent, trivalent, tetravalent or pentavalent radical, one of which valencies is in combination with-NH-CO-or-NH2Bonding; the second valency is open, and when present, each remaining valency is independently bonded to the auxiliary moiety;
each Q2Independently a divalent, trivalent, tetravalent, or pentavalent group, wherein one valence is bonded to-NH-CO-; the second valency is bonded to T, and when present, each remaining valency is independently bonded to the auxiliary moiety;
t is a targeting moiety and T is a targeting moiety,
with the proviso that the starting material comprises-Q1–NH2And the product comprises-Q1–NH–CO–Q2–T。
Conjugation on a support:
wherein:
z is O or S;
ROis a bond in a polynucleotide to a nucleoside;
each Q2Independently a divalent, trivalent, tetravalent, or pentavalent group, wherein one valence is bonded to-NH-CO-; the second valency is bonded to T, and when present, each remaining valency is independently bonded to the auxiliary moiety; and
t is a targeting moiety.
Wherein:
n is an integer from 1 to 8;
a is O or-CH2–；
Z is O or S;
ROis a bond in a polynucleotide to a nucleoside;
each Q2Independently a divalent, trivalent, tetravalent, or pentavalent group, wherein one valence is bonded to the azide or triazole; the second valency is bonded to T, and when present, each remaining valency is independently bonded to the auxiliary moiety; and
T is a targeting moiety.
Wherein:
n is an integer from 1 to 8;
a is O or-CH2–；
Z is O or S;
ROis a bond in a polynucleotide to a nucleoside;
each Q2Independently a divalent, trivalent, tetravalent or pentavalent radical, whichOne of the valencies is bonded to an azide or a triazole; the second valency is bonded to T, and when present, each remaining valency is independently bonded to the auxiliary moiety; and
t is a targeting moiety.
Wherein:
n is an integer from 1 to 8;
a is O or-CH2–；
Z is O or S;
ROis a bond in a polynucleotide to a nucleoside;
each Q2Independently a divalent, trivalent, tetravalent, or pentavalent group, wherein one valence is bonded to the azide or triazole; the second valency is bonded to T, and when present, each remaining valency is independently bonded to the auxiliary moiety; and
each T is independently a targeting moiety.
Representative examples of Fmoc deprotection of phosphate triacetate:
subjecting a polynucleotide comprising a phosphotriester with an Fmoc-protected amine to deprotection conditions results in Fmoc deprotection without observing conversion of the phosphotriester to a phosphodiester.
TCCATGACGTTCCTGACGTT (p 68; see Table 2; SEQ ID NO:68)
DBCO-NHS conjugation to p 68-representative examples:
the conjugation of DBCO-NHS to the amino group in the phosphotriester was completed within 10 min at room temperature as demonstrated by mass spectrometry.
RP-HPLC purification of p68 (see table 2) containing DBCO conjugate groups was performed using the following conditions:
buffer a ═ 50mM TEAA in water;
-buffer B ═ 90% acetonitrile; and
-flow rate of 1 mL/min;
-a gradient:
o 0-2 min (100% buffer A/0% buffer B),
o 2-22 min (0% -100% buffer B), and
o 22-25 min (100% buffer B).
Similar procedures can be used to prepare polynucleotides using, for example, 2' -modified nucleoside phosphoramidites, such as those described herein. Such a procedure is provided in International patent application PCT/US 2015/034749. The disclosure of the disulfide phosphotriester oligonucleotide synthesis in PCT/US2015/034749 is incorporated herein by reference.
The general procedure described herein was followed to prepare the immunomodulatory polynucleotides listed in table 2.
In Table 2, column A provides IL-6 Expression (EC) in DB-6 cells50nM); panel B provides IL-10 Expression (EC) in DB cells50nM); column C provides NF-. kappa.B activation (EC) in Ramos blue cells50nM); column D provides NF-. kappa.B activation (EC) of Hela-hTLR 9-NF-. kappa.B-luc cells50nM); e columnProvides NF kB activation (EC) of Hela-mTLR9-NF kB-luc cells50nM); panel F provides IL-6 secretion (EC) in mouse splenocytes50nM); column G provides IL-6 secretion (EC) in mouse splenocytes after 24 hours of preincubation in 95% mouse plasma 50nM); column H provides IL-6 secretion (EC) in mouse bone marrow differentiated DCs50nM); column I provides NF κ B activation in mouse HEK-Blue cells 2h after transfection with RNAiMax; column J provides NF-. kappa.B activation (EC) in human HEK-Blue cells 2h after transfection with RNAiMax50， nM)。
Key descriptors for the sequences provided in all tables included herein are as follows: lowercase ═ nucleoside 3' -phosphorothioate; capitalized nucleoside 3' -phosphate esters; italicized lowercase ═ nucleosides (PS) with 3' tBuDS-Ph (ortho) triesters; italicized upper case nucleoside (PO) with 3' tBuDS-Ph (ortho) triester; dt ═ dt (dbco); bold double underline t ═ DBCO-C6-dT; bolded lower case ═ nucleosides with 3' n-butyl triester (PS); bolded capitalization ═ nucleoside (PO) with 3' n-butyl triester; italicized lower case nucleosides (PS) with 3' homopropargyl triester (hPro);lower case with italic underliningNucleoside (PS) with 3' DBCO-NH-PEG2 triester (N1);italic underlined capitalNucleoside (PO) with 3' DBCO-NH-PEG2 triester (N1); double-underlined t ═ dT PEG2-NH2Triesters (PS); double-underlined T ═ dT PEG2-NH2Triester (PO); italic double-underlined lowercase with 3' PEG2-NH2Nucleosides (PS) of triesters (N1); italic double-underlined capital letter with 3' PEG2-NH 2Nucleosides (PO) of triesters (N1); bold italics underline U ═ 5-iodo-2' -deoxyuridine (PO); bold italics underline lowercase u ═ 5-iodo-2' -deoxyuridine (PS); bold underlined is 2' -fluoro nucleotide (PO); the apostrophe indicates that the nucleotide identified by the left letter of the apostrophe contains a 2' -OMe modified ribose; underlined ng-7-deaza-2' -deoxyguanosine (PS); underlined pT ═ PEG4 dT triester (PO); underlined pt ═ PEG4 dT triester (PS); fT-5-trifluoromethyl thymidine (PO); 5-fluoro-2' -deoxyuridine (PO); 5-bromo-2' -deoxyuridine (PO); ft-5-trifluoromethyl thymidine (PS); fu ═ 5-fluoro-2' -deoxyuridine (PS); bu ═ 5-bromo-2' -deoxyuridine (PS); c3 ═ C3 spacer (- (CH)2)3-OH) (PO); c3 ═ C3 spacer (- (CH)2)3-OH) (PS); c6 ═ hexane-1, 6-diyl; NH (NH)2C6 ═ 6-aminohex-1-yl; te-thymidine (PO) with 3' ethyl triester; ge ═ guanosine (PO) with 3' ethyl triester; cytidine (PO) with 3' ethyl triester; ue ═ 5-iodouridine (PO) with 3' ethyl triester; ue ═ 5-iodouridine (PS) with 3' ethyl triester; 5 ' -5 ' end-capping based on 5-iodo-2 ' -deoxyuridine (PS); 5 ' -5 ' end-capping based on 5-iodo-2 ' -deoxyuridine (PO); x5 ═ X5-dbco (po); x5 ═ x5-dbco (ps); x3 ═ X3 no base spacer (PO); and IR700 is a dye. Here, the descriptor (PO) represents 3' -phosphoric acid; and (PS) represents 3' -phosphorothioate; od ═ 5' -orthodisulfide phosphodiester; o ═ 5' -phosphoric acid (PO); ods ═ 5' -orthothiophosphorodithioate; s ═ 5' -Phosphorothioate (PS); superscript "r" ═ Rp PS; superscript "s" ═ Sp PS; af ═ 2' -fluoroadenosine (PO); csi ═ dC O-silyl triester (PO); tsi ═ dT O-silyl triester (PO); tm-2' -OMe thymidine (PS); t (m) ═ 2' -OMOE thymidine (PS). The structure is shown in figures 5 and 6.
Double-stranded CpG:
annealing and gel analysis:
polynucleotide p88(1mL, 5mM stock) was added to p144(3.3mL, 2mM stock) with DPBS (24.7 mL). Polynucleotide p88 was treated in a similar manner with p 145. The mixture was heated to 65 ℃ for 10 minutes. TBE urea gel analysis indicated that p88 fully annealed (see fig. 2). mu.L of each sample was removed, added to 5. mu.L of formamide loading buffer, and loaded onto 15% TBE-urea gels per well for 40 minutes at 200 volts, followed by ethidium bromide (EtBr) staining. See table 2 for the structures of p88, p144, and p 145.
Double-stranded CpG-representative example (1) using p88/p 144:
TCGTCGTTTTGTCGTTTTGTCGTT(SEQ ID NO:234)
AGCAGCAAAACAGCAAAACAGCAA(SEQ ID NO:468)
double-stranded CpG-representative example (2) using p88/p 145:
TCGTCGTTTTGTCGTTTTGTCGTT(SEQ ID NO:467)
AGCAGCAAAACAGCAAAACAGCAA(SEQ ID NO:468)
example 2: preparation of antibody-CpG conjugates
A. Preparation of anti-SIRP alpha antibody-CpG nucleotide conjugates
Two anti-SIRP α antibodies were selected. An anti-sirpa antibody blocks the binding of CD47 with an epitope that overlaps (blocks) the CD47 binding site. Another anti-sirpa antibody binds to an epitope (non-blocking) different from the binding site of CD 47. See WO 2018/057669, the disclosure of which is incorporated herein by reference in its entirety. anti-SIRP α antibodies were conjugated via a transglutaminase ("mTGase") reaction.
The VH and VL of the blocking antibody selected for conjugation against SIRPa 1 were EVQLVESGGGVVQPGGSLRLSCAASGFTFSSNAMSWVRQAPGKG LEWVAGISAGGSDTYYPASVKGRFTISRDNSKNTLYLQMNSLRAE DTAVYYCARETWNHLFDYWGQGTLVTVSS (SEQ ID NO:521) and SYELTQPPSVSVSPGQTARITC, respectively SGGSYSS YYYAWYQQKPGQAPVT LIYSDDKRPSNIPERFSGSSSGTTVTLTISGVQAEDEADYYCGGYDQSSYTNPFGGGTKLTVL(SEQ ID NO:525)。
The VH and VL of the non-blocking antibody selected for conjugation against SIRPa 2 were EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDVNWVRQAPGKG LEWVSLISGSGEIIYYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCAKENNRYRFFDDWGQGTLVTVSS (SEQ ID NO:548) and ETVLTQSPGTLSLSPGERATLSCRASQSVYTYLAWYQQKPGQAPR LLIYGASSRATGIPDRFSGSGSGTEFTLTISSLQSEDFAVYYCQQYY DRPPLTFGGGTKVEIK (SEQ ID NO:553), respectively.
Anti-sirpa antibody buffer carrying the N297A mutation in human IgG1 was exchanged with 25mM Tris, 150mM NaCl, pH 8 using a desalting column. Adding mTGase and having N to SIRP alpha antibody solution3-CH2CH2(OCH2CH2)23-NH2N of structure3-PEG23-NH2And (4) a joint. In view of the N297A mutation, conjugation may occur through the side chain of glutamine 295 (EU numbering). The resulting mixture was left at room temperature overnight. In the mTGase reaction mixture, the final antibody concentration was 50 μ M; the ratio of antibody to mTGase is about 10, and the ratio of linker to antibody is about 5. mTGase and free PEG linker were removed by protein a purification. Will be repaired by desalting columnThe decorated antibody buffer was exchanged for 1 xPBS. The amino acid sequence of SEQ ID NO: 425H ü isgen cycloaddition of alkynes in CpG nucleotides, provides an anti-SIRP α -CpG nucleotide conjugate having the structure of formula (D) or (E):
Wherein Ab is a blocking or non-blocking anti-sirpa antibody; c is 2' -deoxycytidine; g is 2' -deoxyguanosine; t is thymidine; x is 5-bromo-2' -deoxyuridine; and Z is
B. Preparation of anti-CD 56 antibody-CpG nucleotide conjugates
The murine monoclonal anti-CD 56 antibody (clone 5.1H11) is commercially available. anti-CD 56 antibodies were conjugated via activated pentafluorophenyl (PFP) ester. To a solution of anti-CD 56 antibody in Dulbecco's Phosphate Buffered Saline (DPBS) buffer (. about.2.5. mu.g/. mu.L) was added a solution of N3-CH2CH2(OCH2CH2)8-CH2azido-PEG 8-PFP ester of CO-PFP structure with a linker to antibody ratio of 20. The resulting mixture was left at room temperature overnight to form an azide-containing antibody. Excess azido-PEG 8-PFP was then removed by exchanging the buffer through an Amicon 30kD rotary concentrator using DPBS as the eluent. The amino acid sequence of SEQ ID NO: 425H ü isgen cycloaddition of alkynes in CpG nucleotides, provides anti-CD 56-CpG nucleotide conjugates having the structure of formula (F) or (G):
wherein Ab is an anti-CD 56 antibody; s isAn integer of about 3 or about 4; c is 2' -deoxycytidine; g is 2' -deoxyguanosine; t is thymidine; x is 5-bromo-2' -deoxyuridine; and Z is
Example 3 biological evaluation of antibody-CpG nucleotide conjugates
Trima residues were received from Blood Centers of the Pacific and diluted 1:4 with Phosphate Buffered Saline (PBS). The diluted blood was divided into four tubes and used 15mLPaque Density gradient Medium (GE Healthcare Life Sciences) support. The tube was centrifuged at 400Xg for 30 min. PBMCs were collected from the interface and resuspended in FACS buffer (PBS with 0.5% bovine serum albumin). CD14 was purified by negative selection using the monocyte isolation kit II (Miltenyi Biotec) and an LS column (Miltenyi Biotec) according to the manufacturer's protocol+A monocyte.
Mixing PBMC or CD14+Cells were immediately plated in 96 well format (500K/well) in complete Roswell Park medical Institute medium (RPMI). At 5% CO2Downward from the antibody and antibody-CpG nucleotide conjugates from 100nM-6.4pM and 1 μ M-64pM of SEQ ID NO: 425 for 24 or 48 hours in a five-fold serial dilution. Cells were pelleted by centrifugation at 400Xg for five minutes and stained in 100. mu.L of Fixable visual Dye eFluor 780(Thermo Fisher) diluted 1:2000 in PBS at 4 ℃. Cells were centrifuged and stained for 30 min at 4 ℃ in 100 μ L FACS buffer containing 2 μ LFc γ R blocking reagent, 1.25 μ L anti-CD 14, anti-CD 3, anti-CD 19 (for anti-sirpa assays) and anti-CD 56, anti-CD 16, anti-CD 69, anti-CD 14, anti-CD 3 and anti-CD 19 (for anti-CD 56 assays). Cells were centrifuged and washed twice in 200 μ L FACS buffer and fixed in 100 μ L0.5% paraformaldehyde. CountBright absolute counting beads were added to each well to count the number of cells. Cells were analyzed on an Attune NxT flow cytometer, followed by data analysis by Flowjo 10.7. By passing The eFluor 780 negative population was gated to exclude dead cells. In door control CD14+Lineage specific cells (CD19, CD3) were excluded before cells and gated on CD56+CD16+Cells were previously excluded (CD19, CD3, CD 14).
As shown in fig. 1 and 2, the anti-CD 56-CpG nucleotide conjugate enhanced NK cell activation compared to CpG nucleotide and anti-CD 56 alone at 24 hours and 48 hours, respectively. The anti-CD 56-CpG nucleotide conjugate was able to induce NK cell activation as determined by CD69 expression.
As shown in FIGS. 3 and 4, conjugates of anti-SIRP α -CpG nucleotides with blocking (anti-SIRP α 1) or non-blocking (anti-SIRP α 2) antibodies induced whole PBMC and purified CD14+CD14 in a population+Proliferation of monocytes. CD14 determination using counting beads+Absolute number of monocytes, conjugates of anti-sirpa-CpG nucleotides with blocking antibodies (anti-sirpa 1) or non-blocking antibodies (anti-sirpa 2) induced proliferation compared to CpG nucleotides and anti-sirpa antibodies alone. Using purified CD14+Monocyte experiments have shown that an increase in cell numbers is a result of delivery of CpG nucleotides into cells by anti-SIRP antibodies. Together, these data indicate that anti-sirpa antibodies (blocking and non-blocking) that bind to different epitopes have been shown to deliver CpG nucleotides to CD14 +Monocytes and lead to an expansion of their cell number.
Example 4 determination of KD
The interaction of anti-sirpa antibodies with sipra, SIRP β and SIRP γ from various species (human v1, human v2, cynomolgus monkey, mouse 129, BL6, BALBc, NOD) was analyzed using two methods to directly immobilize the antibodies (via a GLC chip) according to the following protocol. All experiments were performed at 25 ℃ using SPR-based ProteOn XPR36 biosensors (BioRad, inc., Hercules, CA) equipped with GLC or NLC sensor chips. Use FREESTYLETM293-FS cells (Thermo Fisher) express antibodies. Purification was performed by standard protein a affinity column chromatography and the eluted antibody was stored in PBS buffer.
The running buffer was PBS pH7.4(PBST +) with 0.01% TWEEN-20. All analytes were used at nominal concentrations determined by a280 absorbance and calculated extinction coefficients using their molarity. Analytes were injected in a "one-shot" kinetic mode as described (see, e.g., Bravman et al, anal. biochem.2006,358, 281-288).
For the method using the GLC chip, the analyte was injected and the anti-SIRPa antibody immobilized on the GLC chip (. about.1000 RUs) was flowed through using a protein amine conjugate kit. For the immobilization step, the GLC chip was activated with EDAC/Sulpho-NHS 1:1(Biorad) diluted to 1/100 for 300s at 25. mu.L/min. The anti-SIRP α antibody was diluted to 80nM concentration in 10mM sodium acetate buffer pH 4.5 and fixed on the chip at 30 μ L/min for 50 seconds. The chip was inactivated with ethanolamine at 25. mu.L/min for 300 seconds. Analytes (e.g., SIRP- α, SIRP- β, SIRP- γ from different species) were injected at nominal concentrations of 100, 33, 11, 3.7, 1.2, and 0nM in a "one-shot" kinetic mode. The association time was monitored at 100uL/min for 90s and the dissociation time for 1200 s. The surface was regenerated with a 2:1v/v blend of Pierce IgG elution buffer/4M NaCl.
The biosensor data was double referenced by subtracting the inter-spot data (containing no immobilized protein) from the reaction spot data (immobilized protein) and then subtracting the response of the buffer "blank" analyte injection from the response of the analyte injection. The double reference data was globally fitted to a simple Langmuir model and K was calculated from the ratio of the apparent kinetic rate constantsDValue (K)D＝kd/ka)。
The binding kinetics of blocker 119, 135 and non-blocker 136 human antibodies to various SIRP-alpha, SIRP-beta, SIRP-gamma from different species were determined. These antibodies bind SIRP- α from human v1, human v2, and cynomolgus monkeys with high affinity. They do not bind various mouse SIRP-alpha. However, they exhibit high affinity binding to human SIRP-beta and human SIRP-gamma. Thus, these antibodies would be useful as pan-resistant SIRPs for conjugating and delivering CpG immunomodulatory polynucleotides to modulate the activity of various myeloid cell populations. The results are summarized in table 3.
The binding kinetics of humanized AB21 blocking antibodies to various SIRP-alpha, SIRP-beta, SIRP-gamma from different species were determined. The AB21 antibody binds with high affinity to SIRP-alpha from human v1, human v2, cynomolgus monkeys, various mouse SIRP-alpha (NOD, BL6, and BALBC), human SIRP-beta, and SIRP-gamma. Therefore, AB21 blocking antibodies would be useful pan-resistant SIRPs for conjugating and delivering CpG immunomodulatory polynucleotides to modulate the activity of various bone marrow cell populations. The results are summarized in table 4.
Example 5 in vivo evaluation of antibody-CpG nucleotide conjugates
CT26 and MC38 cells were plated in RPMI 1640 (for CT26) or DMEM (for MC38) at 2X 106Concentrations of individual cells/mouse were injected into the right side of BALB/C and C57BL/6 female mice. Monitoring the tumor until the average size of the tumor reaches 75-300 mm3(depending on the study). Mice were randomly divided into PBS control group, anti-SIRP α -CpG nucleotide conjugate with blocking antibody (anti-SIRP α 1) group and anti-SIRP α -CpG nucleotide conjugate with non-blocking antibody (anti-SIRP α 2) group, with 5-7 mice per group depending on the study. The sequence of an anti-sirpa antibody is described in example 2; CpG corresponds to p 313. anti-SIRP alpha-CpG nucleotide conjugate treated mice were dosed with 0.1-10mg/kg twice three days apart. Both drugs were administered intraperitoneally. Tumors were measured with calipers in two dimensions and tumor volume was calculated as: length x width x 0.5, where length is the greater of the two measurements.
Mice bearing CT26 tumors were measured and randomized by tumor volume. On day 4, the mean tumor size of 5 mice per group was 75mm3. Two administrations with 10mg/kg anti-SIRP alpha 1 conjugate at 3 days intervalsTreated mice showed tumor elimination (4/5 mice), while mice treated with 10mg/kg unconjugated control anti-SIRPa antibody twice, 3 days apart, showed suboptimal tumor inhibition compared to PBS (figure 7A). Mice bearing CT26 tumors were measured and randomized by tumor volume. On day 8, the mean tumor size of the tumors of 7 mice per group was 100mm 3Two treatments with 3mg/kg anti-sirpa 1 conjugate and anti-sirpa 2 conjugate separated by 3 days showed complete tumor elimination (figure 7B). On day 24, 7 of 7 mice treated with anti-sirpa 1 conjugate (anti-sirpa blocking antibody conjugate) and 6 of 7 mice treated with anti-sirpa 2 conjugate (anti-sirpa non-blocking antibody conjugate) had intact tumor elimination. As shown in FIG. 7C, the mean tumor size was 300mm treated with 0.1, 0.3, and 1mg/kg anti-SIRPa 1 conjugates3Two mice bearing the CT26 tumor (and 5 mice per group) were isolated at three days. A tumor-suppressing dose response was observed, with 1mg/kg being the most effective. In the group treated with 1mg/kg of the anti-sirpa 1 conjugate, four out of five mice showed tumor elimination on day 21. As shown in FIG. 7D, the average 155mm treated with two doses of 10mg/kg anti-SIRPa 1 conjugate three days apart3Tumor volume MC38 tumor-bearing mice showed complete tumor elimination at day 21. Taken together, these data indicate tumor elimination in various tumor models, specific activity of SIRPa-CpG compared to unconjugated SIRPa antibody, tumor elimination using SIRPa blocking and non-blocking antibody CpG conjugates and tumor elimination when mice were treated with anti-SIRPa 1 conjugates as low as 1 mg/kg.
Tumors were monitored until the average size of the tumors reached 300mm3. Mice were randomly divided into PBS control and anti-SIRP α -CpG nucleotide conjugate with blocking antibody (anti-SIRP α 1), with 5 mice per group. The sequence of an anti-sirpa antibody is described in example 2. CpG corresponds to p 313. For the CT26 model, mice treated with 1mg/kg of anti-SIRP α -CpG nucleotide conjugate were dosed 2 times, 3 or 7 days apart. An anti-SIRP α -CpG nucleotide conjugate is administered intraperitoneally. Tumors were measured with calipers in two dimensions and tumor volume was calculated as: length x width x 0.5, where length is the greater of the two measurements.
Mice bearing CT26 tumors were measured and randomized by tumor volume. On day 10, the mean tumor size of the tumors was 300mm3And treatment with two doses of 1mg/kg anti-SIRP α 1 conjugate three or seven days apart showed tumor elimination (figure 8A). Four of the five mice in both groups were tumor-free on day 25. On day 63, of the four surviving mice treated three days apart, all mice were tumor free, while the mice treated seven days apart also had four surviving mice, but only two were still tumor free (fig. 8B).
The examples set forth above are provided to enable those skilled in the art to obtain a complete disclosure and description of how to make and use the claimed embodiments, and are not intended to limit the scope of what is disclosed herein. Modifications apparent to those skilled in the art are intended to fall within the scope of the appended claims. All publications, patents, and patent applications cited in this specification are herein incorporated by reference as if each such publication, patent, or patent application were specifically and individually indicated to be incorporated by reference.
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