Immunomodulatory combinations and methods for treating cancer

文档序号：1803115 发布日期：2021-11-05 浏览：21次中文

阅读说明：本技术 用于治疗癌症的免疫调节组合和方法 (Immunomodulatory combinations and methods for treating cancer ) 是由李立新刘华涛于 2019-12-17 设计创作，主要内容包括：本公开的实施方式涉及使用联合疗法治疗癌症的免疫调节组合物和方法。(Embodiments of the present disclosure relate to immunomodulatory compositions and methods for treating cancer using combination therapy.)

1. A method for treating a tumor or abnormal cell proliferation in a subject in need of treatment comprising administering to the subject:

(i) a chemotherapeutic agent in an amount capable of reducing a myeloid-derived suppressor cell (MDSC) population in the blood, spleen, and/or tumor microenvironment of the subject; and

(ii) an effective amount of an immunotherapeutic agent.

2. The method of claim 1, wherein the MDSC inhibitor is selected from the group consisting of paclitaxel, gemcitabine, 5-fluorouracil, oxaliplatin, cisplatin, carboplatin, dasatinib, sunitinib, and doxorubicin.

3. The method of claim 1, wherein the amount of the chemotherapeutic agent is capable of reducing the amount of MDCS in the blood, spleen, and/or tumor microenvironment of the subject by 10% to 95%.

4. The method of claim 1, wherein the amount of the chemotherapeutic agent is less than when used as a monotherapy.

5. The method of claim 1 wherein the chemotherapeutic agent is gemcitabine and is present in an amount of 400-²。

6. The method of claim 1, wherein the MDSCs express CD11b, CD15, CD33, and CD66 b.

7. The method of claim 1, wherein the MDSCs express CD11b, CD14, and CD 33.

8. The method of claim 1, wherein the chemotherapeutic agent is administered prior to the administration of the immunotherapeutic agent and within 7 days of the administration of the immunotherapeutic agent.

9. The method of claim 1, wherein the chemotherapeutic agent is administered prior to the administration of the immunotherapeutic agent.

10. The method of claim 1, wherein the chemotherapeutic agent is administered at least 1,2,3,4, 5,6, or7 days prior to the administration of the immunotherapeutic agent.

11. The method of claim 1, wherein the immunotherapeutic agent is administered after 10% to 95% reduction in the amount of MDCS in the blood and/or tumor microenvironment of the subject following administration of the chemotherapeutic agent.

12. The method of claim 1, wherein the immunotherapeutic agent comprises a TLR7 and/or TLR8 agonist.

13. The method of claim 1, wherein the immunotherapeutic agent is an agonist of TLR7 and TLR 8.

14. The method of claim 1, wherein the immunotherapeutic agent has the structure of formula (I):

wherein the dotted line represents a bond or a bond is absent;

x is S or-NR₁，R₁is-W₀-W₁-W₂-W₃-W4，

W₀Is a bond, alkyl, alkenyl, alkynyl, alkoxy or-alkyl-S-alkyl-,

W₁is a bond, - -O- -or- -NR₂- -, wherein R is₂Is hydrogen, alkyl or alkenyl,

W₂is a bond, - - -O- -, - -C (O) - -C (S) - -or- -S (O)₂-，

W₃Is a bond, - -NR₃- -, wherein R is₃Is hydrogen, alkyl or alkenyl,

W₄is hydrogen, alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, aryl, aryloxy, heteroaryl or heterocyclyl, each of which is optionally substituted with one or more substituents selected from the group consisting of hydroxy, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, - -NH₂Nitro, -alkyl-hydroxy, -alkyl-aryl, -alkyl-heteroaryl, -alkyl-heterocyclyl, -O-R₄- -O-alkyl-R₄- -alkyl-O-R₄、--C(O)-R₄- -alkyl-C (O) -R₄- -alkyl-C (O) -O-R₄、--C(O)-O-R₄、--S-R₄、--S(O)₂-R₄、--NH-S(O)₂-R₄- -alkyl-S-R4, - -alkyl-S (O)₂-R₄、--NHR₄、--NR₄R₄- -NH-alkyl-R₄Halogen, - - -CN, - -NO₂and-SH, wherein R₄Independently hydrogen, alkyl, alkenyl, - - - -alkyl-hydroxy, aryl, heteroaryl, heterocyclyl or haloalkyl;

z is hydrogen, alkyl, alkenyl, alkynyl, alkoxy, aryl, haloalkyl, heteroaryl, heterocyclyl, each of which may be optionally substituted with one or more substituents selected from hydroxy, alkoxy, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, halogen, cyano, nitro, - - (R) N (R)₅)₂- -alkoxy-alkyl, - -alkoxy-alkenyl, - -C (O) -alkyl, - -C (O) -O-alkyl, - -O- -C (O) -alkyl, - -C (O) -N (R)₅)₂Aryl, heteroaryl, - -CO-aryl and- -CO-heteroaryl, wherein each R is R₅Independently hydrogen, alkyl, haloalkyl, -alkyl-aryl or-alkyl-heteroaryl;

r is hydrogenAlkyl, alkoxy, haloalkyl, halogen, aryl, heteroaryl, heterocyclyl, each of which is optionally substituted with one or more substituents selected from the group consisting of hydroxy, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, - - -NH₂Nitro, -alkyl-hydroxy, -alkyl-aryl, -alkyl-heteroaryl, -alkyl-heterocyclyl, -O-R₄- -O-alkyl-R₄- -alkyl-O-R₄、--C(O)-R₄、--C(O)-NH-R₄、--C(O)-NR₄R₄- -alkyl-C (O) -R₄- -alkyl-C (O) -O-R₄、--C(O)-O-R₄、--O-C(O)-R₄、--S-R₄、--C(O)-S-R₄、--S-C(O)-R₄、--S(O)₂-R₄、--NH-S(O)₂-R₄- -alkyl-S-R₄- -alkyl-S (O)₂-R₄、--NHR₄、-NR₄R₄- -NH-alkyl-R₄Halogen, -CN and-SH, wherein R₄Independently hydrogen, alkyl, alkenyl, alkoxy, -alkyl-hydroxy, aryl, heteroaryl, heterocyclyl or haloalkyl;

n is 0,1, 2,3 or 4;

y is-NR₆R₇、-CR₆R₇R₈or-alkyl-NH₂Each of which may be optionally substituted with one or more substituents selected from hydroxy, alkoxy, alkyl, alkenyl, alkynyl, - - -NH₂Halogen, - - (R) - - (Y- -O) - -N₅)₂- -alkoxy-alkyl, - -alkoxy-alkenyl, - -C (O) -alkyl, - -C (O) -O-alkyl, - -C (O) -N (R)₅)₂Aryl, heteroaryl, - -CO-aryl and- -CO-heteroaryl,

wherein R is₆、R₇And R₈Independently hydrogen, alkyl, alkenyl, alkoxy, alkylamino, dialkylamino, alkylthio, arylthio, - - (alkyl) -hydroxy, - - (alkyl-C (O) -O- -R₉- -alkyl-C (O) -R₉or-alkyl-OC (O) -R₉Wherein each R is₅Independently hydrogen, alkyl, haloalkyl, -alkyl-aryl or-alkyl-heteroaryl, which areIn R₉Is hydrogen, alkyl, alkenyl, halogen or haloalkyl;

x and Z together may optionally form a (5-9) membered ring.

15. The method of claim 1, wherein the immunotherapeutic agent is selected from the group consisting of: 2-propylthiazolo [4,5-c ] quinolin-4-amine, 1- (2-methylpropyl) -1H-imidazo [4,5-c ] quinolin-4-amine, 4-amino-2- (ethoxymethyl) -a, a-dimethyl-1H-imidazo [4,5-c ] quinolin-1-ethanol, 1- (4-amino-2-ethylaminomethylimidazo) - [4,5-c ] quinolin-1-yl) -2-methylpropan-2-ol, N- [4- (4-amino-2-ethyl-1H-imidazo [4,5-c ] quinolin-1-yl) butyl- ] methanesulfonamide, and, 4-amino-2-ethoxymethyl-aa-dimethyl-6, 7,8, 9-tetrahydro-1H-imidazo [4,5-c ] quinoline-1-ethanol, 4-amino-aa-dimethyl-2-methoxyethyl-1H-imidazo [4,5-c ] quinoline-1-ethanol, 1- {2- [3- (benzyloxy) propoxy ] ethyl } -2- (ethoxymethyl) -1H-imidazo [4,5-c ] quinolin-4-amine, N- [4- (4-amino-2-butyl-1H-imidazo [4,5-c ] [1,5] naphthyridin-1-yl) butyl ] -N' -butylurea, and mixtures thereof, N1- [2- (4-amino-2-butyl-1H-imidazo [4,5-c ] [1,5] naphthyridin-1-yl) ethyl ] -2-amino-4-methylpentanamide, N- (2- {2- [ 4-amino-2- (2-methoxyethyl) -1H-imidazo [4,5-c ] quinolin-1-yl ] ethoxy } ethyl) -N' -phenylurea, 1- (2-amino-2-methylpropyl) -2- (ethoxymethyl) -1H-imidazo [4,5-c ] quinolin-4-amine, 1- {4- [ (3, 5-dichlorophenyl) sulfonyl ] butyl } -2-ethyl-1H- Imidazo [4,5-c ] quinolin-4-amine, N- (2- {2- [ 4-amino-2- (ethoxymethyl) -1H-imidazo [4,5-c ] quinolin-1-yl ] ethoxy } ethyl) -N '-cyclohexylurea, N- {3- [ 4-amino-2- (ethoxymethyl) -1H-imidazo [4,5-c ] quinolin-1-yl ] propyl } -N' - (3-cyanophenyl) thiourea, N- [3- (4-amino-2-butyl-1H-imidazo [4,5-c ] quinolin-1-yl) -2, 2-dimethylpropyl ] benzamide, N-methyl-1H-imidazo [4,5-c ] quinolin-1-yl ] -N- (3-cyano-propyl) thiourea, 2-butyl-1- [3- (methylsulfonyl) propyl ] -1H-imidazo [4,5-c ] quinolin-4-amine, N- {2- [ 4-amino-2- (ethoxymethyl) -1H-imidazo [4,5-c ] quinolin-1-yl ] -1, 1-dimethylethyl } -2-ethoxyacetamide, 1- [ 4-amino-2-ethoxymethyl-7- (pyridin-4-yl) -1H-imidazo [4,5-c ] quinolin-1-yl ] -2-methylpropan-2-ol, 1- [ 4-amino-2- (ethoxymethyl) -7- (pyridin-3-yl) -1H-imidazo [4,5-c ] quinolin-1-yl ] -2-methylpropan-2-ol H-imidazo [4,5-c ] quinolin-1-yl ] -2-methylpropan-2-ol, N- {3- [ 4-amino-1- (2-hydroxy-2-methylpropyl) -2- (methoxyethyl) -1H-imidazo [4,5-c ] quinolin-7-yl ] phenyl } methanesulfonamide, 1- [ 4-amino-7- (5-hydroxymethylpyridin-3-yl) -2- (2-methoxyethyl) -1H-imidazo [4,5-c ] quinolin-1-yl ] -2-methylpropan-2-ol, 3- [ 4-amino-2- (ethoxymethyl) -7- (pyridin-3-yl) -2-methylpropan-2-ol ) -1H-imidazo [4,5-c ] quinolin-1-yl ] propane-1, 2-diol, 1- [2- (4-amino-2-ethoxymethyl-1H-imidazo [4,5-c ] quinolin-1-yl) -1, 1-dimethylethyl ] -3-propylurea, 1- [2- (4-amino-2-ethoxymethyl-1H-imidazo [4,5-c ] quinolin-1-yl) -1, 1-dimethylethyl ] -3-cyclopentylurea, 1- [ (2, 2-dimethyl-1, 3-dioxolan-4-yl) methyl ] -2- (ethoxymethyl) -7- (4-hydroxymethylphenyl) -1H-imidazo [4,5-c ] quinolin-4-amine, 4- [ 4-amino-2-ethoxymethyl-1- (2-hydroxy-2-methylpropyl) -1H-imidazo [4,5-c ] quinolin-7-yl ] -N-methoxy-N-methylbenzamide, 2-ethoxymethyl-N1-isopropyl-6, 7,8, 9-tetrahydro-1H-imidazo [4,5-c ] quinolin-1, 4-diamine, 1- [ 4-amino-2-ethyl-7- (pyridin-4-yl) -1H-imidazo [4,5-c ] quinolin-1-yl ] -2-methylpropan-2- A compound of the group consisting of an alcohol, N- [4- (4-amino-2-ethyl-1H-imidazo [4,5-c ] quinolin-1-yl) butyl ] methanesulfonamide and N- [4- (4-amino-2-butyl-1H-imidazo [4,5-c ] [1,5] naphthyridin-1-yl) butyl ] -N' -cyclohexylurea.

16. The method of claim 1, wherein the immunotherapeutic agent comprises resiquimod.

17. The method of claim 1, wherein the amount of the immunotherapeutic agent is capable of:

(1) inducing IFN- α in enriched human blood DC;

(2) induction of TNF- α in enriched human blood DC: and/or

(3) IL-12-alpha is induced in enriched human blood DCs.

18. The method of claim 1, wherein the amount of the immunotherapeutic agent is capable of activating human plasmacytoid dendritic cells, myeloid dendritic cells, or NK cells, or a combination thereof.

19. The method of claim 1, wherein the immunotherapeutic agent is administered in combination with a targeted therapeutic agent against cancer.

20. The method of claim 19, wherein the targeted therapeutic is capable of specifically or preferentially binding to tumor cells as compared to non-tumor cells.

21. The method of claim 19, wherein the tumor cell is a carcinoma, sarcoma, lymphoma, myeloma, or a central nervous system cancer.

22. The method of claim 19, wherein the targeted therapeutic is capable of specifically or preferentially binding to a tumor antigen as compared to a non-tumor antigen.

23. The method of claim 22, wherein said tumor antigen is selected from the group consisting of CD2, CD19, CD20, CD22, CD27, CD33, CD37, CD38, CD40, CD44, CD47, CD52, CD56, CD70, CD79, and CD 137.

24. The method of claim 22, wherein the tumor antigen is selected from the group consisting of 4-1BB, 5T4, AGS-5, AGS-16, angiopoietin 2, B7.1, B7.2, B7DC, B7H1, B7H2, B7H3, BT-062, BTLA, CAIX, carcinoembryonic antigen, CTLA4, Cripto, ED-B, ErbB1, ErbB2, ErbB3, ErbB4, EGFL7, EpCAM, EphA2, EphA3, EphB2, FAP, fibronectin, folate receptor, ganglioside GM3, GD2, glucocorticoid-induced tumor necrosis factor receptor (GITR), gp100, gpA, gpb, nmos R, IGF1R, integrin α v β, KIR, not-3, Lewis, mulc, muwic, MET 72, SLC 72, npa 1, psmc 1, npa 1, npx 1, phb 72, phb 72, phc 72, phb 72, phc 72, phb, phc 72, phb, phc 72, phc, TACI, TAG-72, tenascin, TIM3, TRAILR1, TRAILR2, VEGFR-1, VEGFR-2, VEGFR-3, and variants thereof.

25. The method of claim 19, wherein the targeted therapeutic comprises an immunoglobulin, a protein, a peptide, a small molecule, a nanoparticle, or a nucleic acid.

26. The method of claim 19, wherein the targeted therapeutic comprises an antibody or functional fragment thereof.

27. The method of claim 26, wherein the antibody is selected from the group consisting of rituximab, herceptin (trastuzumab), erbitux (cetuximab), victirab (panitumumab), Arzerra (aftumumab), Bentysta (beliewutuzumab), yrenvoy (ipilimumab), Perjeta (pertuzumab), tremelimumab, nivolumab, trastuzumab, durumab, ureuzumab, MPDL3280A, palboceprizumab, bornautrumab, CT-011, MK-3475, BMS-936559, MED14736, MSB001071 0010718C, and magetuzumab (MGAH 22).

28. The method of claim 26, wherein the targeted therapeutic comprises Fab, Fab ', F (ab') 2, single domain antibodies, T and Abs dimers, Fv, scFv, dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, diabodies, triabodies, sc-diabodies, kappa (lamda) bodies, BiTE, DVD-Ig, SIP, SMIP, DART, or antibody analogs comprising one or more CDRs.

29. The method of claim 19, wherein the targeted therapeutic comprises an ATWLPPR polypeptide of VEGFR, a thrombospondin-1 mimetic, an CDCRGDCFCG (cyclic) polypeptide, a SCH 221153 fragment, a NCNGRC (cyclic) polypeptide, a CTTHWGFTLC polypeptide, a CGNKRTRGC polypeptide (LyP-1), octreotide, vapreotide, lanreotide, a C-3940 polypeptide, dabigatran, risperidone, or cetrorelix.

30. The method of claim 19, wherein the targeted therapeutic comprises the extracellular domain (ECD) or soluble form of PD-1, PDL-1, CTLA4, BTLA, KIR, TIM3, 4-1BB, LAG3, partial full length of surface ligand amphiregulin, β -cytokine, EGF, ephrin, epigen, epithelial regulatory protein, IGF, neuregulin, TGF, TRAIL, or VEGF.

31. The method of claim 1, wherein the immunotherapeutic agent is delivered systemically.

32. The method of claim 1, wherein the immunotherapeutic agent is administered orally or by parenteral injection.

33. The method of claim 1, wherein the immunotherapeutic agent is administered by intravenous injection or intratumoral injection.

34. The method of claim 1, wherein the abnormal cell proliferation comprises a precancerous lesion.

35. The method of claim 1, wherein the abnormal proliferation is abnormal proliferation of cancer cells.

36. The method of claim 35, wherein the cancer is selected from the group consisting of Acute Myeloid Leukemia (AML), breast cancer, Chronic Lymphocytic Leukemia (CLL), Chronic Myeloid Leukemia (CML), hodgkin's lymphoma, multiple myeloma, mycosis fungoides, neuroblastoma, non-hodgkin's lymphoma (NHL), ovarian cancer, and retinoblastoma.

37. The method of claim 1, comprising administering to the subject an oral formulation comprising the immunotherapeutic agent twice weekly at a dose of about 0.0005mg/kg, 0.0006mg/kg, 0.0007mg/kg, 0.0008mg/kg, 0.0009mg/kg, 0.001mg/kg, 0.002mg/kg, 0.003mg/kg, 0.004mg/kg, 0.005mg/kg, 0.006mg/kg, 0.007mg/kg, 0.008mg/kg, 0.009mg/kg, or 0.01mg/kg to about 0.02mg/kg, including all.

38. The method of claim 1, comprising administering to the subject an oral formulation comprising the immunotherapeutic agent at a dose of 0.0001mg/kg to less than or about 0.0005mg/kg, 0.0006mg/kg, 0.0007mg/kg, 0.0008mg/kg, 0.0009mg/kg, 0.001mg/kg, 0.002mg/kg, 0.003mg/kg, 0.004mg/kg, 0.005mg/kg, 0.006mg/kg, 0.007mg/kg, 0.008mg/kg, 0.009mg/kg, or 0.01mg/kg twice weekly.

39. The method of claim 1, comprising administering to the subject an intravenous formulation comprising the immunotherapeutic agent once per week at a dose of about 0.0005mg/kg, 0.0006mg/kg, 0.0007mg/kg, 0.0008mg/kg, 0.0009mg/kg, 0.001mg/kg, 0.002mg/kg, 0.003mg/kg, 0.004mg/kg, 0.005mg/kg, or 0.006mg/kg to about 0.015mg/kg, including all.

40. The method of claim 1, comprising administering to the subject an intravenous formulation comprising the immunotherapeutic agent once per week at a dose of 0.0001mg/kg to less than or about 0.003mg/kg, 0.004mg/kg, 0.005mg/kg, or 0.006mg/kg to about 0.01 mg/kg.

41. The method of claim 18, wherein the local concentration of the immunotherapeutic agent in the subject is about 0.005 μ g/ml to about 12 μ g/ml.

42. The method of claim 1, wherein the local concentration of the immunotherapeutic agent in the subject is about 0.05 μ g/ml, 0.1 μ g/ml, 0.15 μ g/ml, 0.2 μ g/ml, 0.3 μ g/ml, or 0.4 μ g/ml to about 0.5 μ g/ml.

43. The method of claim 1, comprising administering to the subject an intravenous formulation comprising the chemotherapeutic agent in a divided dose of about 40-50mg/kg over 2-5 days.

44. The method of claim 2, wherein the immunotherapeutic agent is administered repeatedly at 2-4 week intervals.

45. The method of claim 1, comprising administering to the subject an intravenous formulation comprising the chemotherapeutic agent at a dose of about 10 to 15mg/kg, once every 7 to 10 days.

46. The method of claim 1, comprising administering to the subject an intravenous formulation comprising the chemotherapeutic agent twice weekly at a dose of about 3 to 5 mg/kg.

47. The method of claim 1, comprising administering at about 60-120mg/m²The dose per day continues to administer an intravenous formulation comprising the chemotherapeutic agent to the subject daily.

48. The method as claimed in claim 1, which comprises a concentration of about 400-1000mg/m²Is administered to the subject in an oral formulation comprising the chemotherapeutic agent over 4-5 days.

49. The method of claim 1, comprising administering at about 50-100mg/m²Administering to the subject an intravenous formulation comprising the chemotherapeutic agent at a dose per day or 1-5 mg/kg/day.

Technical Field

The presently disclosed embodiments relate to immunomodulatory combinations and methods of treating cancer using combination therapies.

Background

Myeloid-derived suppressor cells (MDSCs) are a diverse, immature population of bone marrow cells that have potent immunosuppressive activity and are increasingly considered as the major disruptors of antitumor immunity due to accumulation in almost all cancer patients. Myeloid-derived suppressor cells comprise a series of immature developing bone marrow cells at different myelogenic stages.

There are two major subtypes of MDSCs in mice, the monocyte MDSC (M-MDSC) and the granulocyte MDSC (G-MDSC). M-MDSC is mononuclear and G-MDSC is polymorphonuclear. Both types express the myeloid marker CD11b and the granulocyte marker Gr 1. Gr1 comprises two different molecules, Ly6C and Ly 6G. M-MDSC has lower level Gr1 expression and expresses Ly6C, while G-MDSC has higher level Gr1 and expresses Ly 6G.

There are also two types of human MDSCs. Both types express CD11 b; however, there was no equivalent marker to the mouse Gr1 marker. In contrast, human M-MDSC is characterized in that it expresses CD14 and G-MDSC by the expression of CD15 and CD66 b. Both types also express the general myeloid marker CD33 and lack lineage markers for lymphocytes and NK cells. Since these markers are also expressed by monocytes, MDSCs are distinguished from monocytes by their absence of HLA-DR.

Studies in mice and humans have shown that MDSCs accumulate in most cancer patients, promote tumor progression, inhibit anti-tumor immunity, and are a barrier to many cancer immunotherapies. For example, MDSCs promote neovascularization by producing VEGF and tumor growth by producing matrix metalloproteinases to promote invasion and metastasis; MDSCs prevent T cell activation and action by up-regulating Arg1 and inducible NO synthase. In addition, MDSCs down-regulate macrophages to produce the type 1 cytokine IL-12, inhibit NK-mediated tumor cell lysis, and induce T regulatory cells.

Toll-like receptors (TLRs) are a key part of innate immunity and are the first line of defense against pathogens. Resiquimod is a ligand for TLRs 7 and 8 that directly activates innate immune cells, including bone marrow dendritic cells, plasmacytoid dendritic cells, and monocytes/macrophages. This activation may lead to the activation of costimulatory molecules, the production of antiviral cytokines, and the stimulation of cell-mediated NK cell and T cell immune responses.

Disclosure of Invention

In general, the presently disclosed embodiments provide therapeutic combinations and methods for treating cancer.

In one aspect, embodiments of the present disclosure provide a method for treating a tumor or abnormal cell proliferation in a subject in need of such treatment, comprising administering to the subject:

a chemotherapeutic agent in an amount capable of reducing myeloid-derived suppressor cell (MDSC) populations in the blood, spleen, and/or tumor microenvironment of the subject; and

an effective amount of an immunotherapeutic agent.

In some embodiments, the chemotherapeutic agent comprises an MDCS inhibitor.

In some embodiments, the MDSC inhibitor is a molecule that causes MDSC cell apoptosis and/or necrosis, or causes cytotoxicity, or causes c-kit, or VEGFR, or ARG1, or iNOS, or S100 or MMPs function, or is ROS ERK activation or inhibition of antioxidant genes. In some embodiments, the inhibitor is selected from the group consisting of paclitaxel, gemcitabine, 5-fluorouracil, oxaliplatin, cisplatin, carboplatin, dasatinib, sunitinib, and doxorubicin.

In some embodiments, the chemotherapeutic agent is provided in an amount capable of reducing the amount of MDSCs in the blood, spleen and/or tumor microenvironment of the subject by 10% to 95%, preferably by at least 30%.

In some embodiments, the chemotherapeutic agent is provided in an amount less than when used as monotherapy, e.g., 50% of the standard monotherapy dose.

In some embodiments, the chemotherapeutic agent is gemcitabine and the amount is 400-²。

In some embodiments, the MDSCs express CD11b, CD15, CD33, and CD66 b. In some embodiments, the MDSCs express CD11b, CD14, and CD 33.

In some embodiments, the chemotherapeutic agent is administered prior to administration of the immunotherapeutic agent and within 7 days of administration of the immunotherapeutic agent.

In some embodiments, the chemotherapeutic agent is administered prior to administration of the immunotherapeutic agent.

In some embodiments, the chemotherapeutic agent is administered at least one, two, three, four, five, six or seven days prior to administration of the immunotherapeutic agent.

In some embodiments, the immunotherapeutic agent is administered after a 10% to 95%, or at least about 50% reduction in the amount of MDCS in the blood and/or tumor microenvironment in the subject following administration of the chemotherapeutic agent.

In some embodiments, the immunotherapeutic agent comprises a TLR7 agonist but not TLR8 agonist, a TLR8 agonist but not TLR7 agonist, or agonists of both TRL7 and TLR 8.

In some embodiments, the immunotherapeutic agent is an agonist of TLR7 and TLR 8.

In some embodiments, the immunotherapeutic agent has the structure of formula (I):

wherein the dotted line represents a bond or a bond is absent;

x is S or-NR₁，R₁is-W₀-W₁-W₂-W₃-W4，

W₀Is a bond, alkyl, alkenyl, alkynyl, alkoxy or-alkyl-S-alkyl-,

W₁is a bond, - -O- -or- -NR₂- -, wherein R is₂Is hydrogen, alkyl or alkenyl,

W₂is a bond, - - -O- -, - -C (O) - -C (S) - -or- -S (O)₂-，

W₃Is a bond, - -NR₃- -, wherein R is₃Is hydrogen, alkyl or alkenyl,

W₄is hydrogen or alkylA group, an alkenyl group, an alkynyl group, an alkoxy group, a cycloalkyl group, an aryl group, an aryloxy group, a heteroaryl group or a heterocyclic group, each of which is optionally substituted with one or more substituents selected from the group consisting of a hydroxyl group, an alkoxy group, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, a heteroaryl group, a heterocyclic group, - - -NH- -₂Nitro, -alkyl-hydroxy, -alkyl-aryl, -alkyl-heteroaryl, -alkyl-heterocyclyl, -O-R₄- -O-alkyl-R₄- -alkyl-O-R₄、--C(O)-R₄- -alkyl-C (O) -R₄- -alkyl-C (O) -O-R₄、--C(O)-O-R₄、--S-R₄、--S(O)₂-R₄、--NH-S(O)₂-R₄- -alkyl-S-R4, - -alkyl-S (O)₂-R₄、--NHR₄、--NR₄R₄- -NH-alkyl-R₄Halogen, - - -CN, - -NO₂and-SH, wherein R₄Independently hydrogen, alkyl, alkenyl, - - - -alkyl-hydroxy, aryl, heteroaryl, heterocyclyl or haloalkyl;

r is hydrogen, alkyl, alkoxy, haloalkyl, halogen, aryl, heteroaryl, heterocyclyl, each of which is optionally substituted with one or more substituents selected from the group consisting of hydroxy, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, - - -NH₂Nitro, -alkyl-hydroxy, -alkyl-aryl, -alkyl-heteroaryl, -alkyl-heterocyclyl, -O-R₄- -O-alkyl-R₄- -alkyl-O-R₄、--C(O)-R₄、--C(O)-NH-R₄、--C(O)-NR₄R₄- -alkyl-C (O) -R₄- -alkyl-C (O) -O-R₄、--C(O)-O-R₄、--OC(O)-R₄、--S-R₄、--C(O)-S-R₄、--S-C(O)-R₄、--S(O)₂-R₄、--NH-S(O)₂-R₄- -alkyl-S-R₄- -alkyl-S (O)₂-R₄、--NHR₄、--NR₄R₄- -NH-alkyl-R₄Halogen, -CN and-SH, wherein R₄Independently hydrogen, alkyl, alkenyl, alkoxy, -alkyl-hydroxy, aryl, heteroaryl, heterocyclyl or haloalkyl;

n is 0,1, 2,3 or 4;

wherein R is₆、R₇And R₈Independently hydrogen, alkyl, alkenyl, alkoxy, alkylamino, dialkylamino, alkylthio, arylthio, - - (alkyl) -hydroxy, - - (alkyl-C (O) -O- -R₉- -alkyl-C (O) -R₉or-alkyl-OC (O) -R₉Wherein each R is₅Independently is hydrogen, alkyl, haloalkyl, - - -alkyl-aryl or-alkyl-heteroaryl, wherein R is₉Is hydrogen, alkyl, alkenyl, halogen or haloalkyl;

x and Z together may optionally form a (5-9) membered ring.

In some embodiments, the immunotherapeutic agent is selected from the group consisting of: 2-propylthiazolo [4,5-c ] quinolin-4-amine, 1- (2-methylpropyl) -1H-imidazo [4,5-c ] quinolin-4-amine, 4-amino-2- (ethoxymethyl) -a, a-dimethyl-1H-imidazo [4,5-c ] quinolin-1-ethanol, 1- (4-amino-2-ethylaminomethylimidazo) - [4,5-c ] quinolin-1-yl) -2-methylpropan-2-ol, N- [4- (4-amino-2-ethyl-1H-imidazo [4,5-c ] quinolin-1-yl) butyl- ] methanesulfonamide, and, 4-amino-2-ethoxymethyl-aa-dimethyl-6, 7,8, 9-tetrahydro-1H-imidazo [4,5-c ] quinoline-1-ethanol, 4-amino-aa-dimethyl-2-methoxyethyl-1H-imidazo [4,5-c ] quinoline-1-ethanol, 1- {2- [3- (benzyloxy) propoxy ] ethyl } -2- (ethoxymethyl) -1H-imidazo [4,5-c ] quinolin-4-amine, N- [4- (4-amino-2-butyl-1H-imidazo [4,5-c ] [1,5] naphthyridin-1-yl) butyl ] -N' -butylurea, and mixtures thereof, N1- [2- (4-amino-2-butyl-1H-imidazo [4,5-c ] [1,5] naphthyridin-1-yl) ethyl ] -2-amino-4-methylpentanamide, N- (2- {2- [ 4-amino-2- (2-methoxyethyl) -1H-imidazo [4,5-c ] quinolin-1-yl ] ethoxy } ethyl) -N' -phenylurea, 1- (2-amino-2-methylpropyl) -2- (ethoxymethyl) -1H-imidazo [4,5-c ] quinolin-4-amine, 1- {4- [ (3, 5-dichlorophenyl) sulfonyl ] butyl } -2-ethyl-1H- Imidazo [4,5-c ] quinolin-4-amine, N- (2- {2- [ 4-amino-2- (ethoxymethyl) -1H-imidazo [4,5-c ] quinolin-1-yl ] ethoxy } ethyl) -N '-cyclohexylurea, N- {3- [ 4-amino-2- (ethoxymethyl) -1H-imidazo [4,5-c ] quinolin-1-yl ] propyl } -N' - (3-cyanophenyl) thiourea, N- [3- (4-amino-2-butyl-1H-imidazo [4,5-c ] quinolin-1-yl) -2, 2-dimethylpropyl ] benzamide, N-methyl-1H-imidazo [4,5-c ] quinolin-1-yl ] -N- (3-cyano-propyl) thiourea, 2-butyl-1- [3- (methylsulfonyl) propyl ] -1H-imidazo [4,5-c ] quinolin-4-amine, N- {2- [ 4-amino-2- (ethoxymethyl) -1H-imidazo [4,5-c ] quinolin-1-yl ] -1, 1-dimethylethyl } -2-ethoxyacetamide, 1- [ 4-amino-2-ethoxymethyl-7- (pyridin-4-yl) -1H-imidazo [4,5-c ] quinolin-1-yl ] -2-methylpropan-2-ol, 1- [ 4-amino-2- (ethoxymethyl) -7- (pyridin-3-yl) -1H-imidazo [4,5-c ] quinolin-1-yl ] -2-methylpropan-2-ol H-imidazo [4,5-c ] quinolin-1-yl ] -2-methylpropan-2-ol, N- {3- [ 4-amino-1- (2-hydroxy-2-methylpropyl) -2- (methoxyethyl) -1H-imidazo [4,5-c ] quinolin-7-yl ] phenyl } methanesulfonamide, 1- [ 4-amino-7- (5-hydroxymethylpyridin-3-yl) -2- (2-methoxyethyl) -1H-imidazo [4,5-c ] quinolin-1-yl ] -2-methylpropan-2-ol, 3- [ 4-amino-2- (ethoxymethyl) -7- (pyridin-3-yl) -2-methylpropan-2-ol ) -1H-imidazo [4,5-c ] quinolin-1-yl ] propane-1, 2-diol, 1- [2- (4-amino-2-ethoxymethyl-1H-imidazo [4,5-c ] quinolin-1-yl) -1, 1-dimethylethyl ] -3-propylurea, 1- [2- (4-amino-2-ethoxymethyl-1H-imidazo [4,5-c ] quinolin-1-yl) -1, 1-dimethylethyl ] -3-cyclopentylurea, 1- [ (2, 2-dimethyl-1, 3-dioxolan-4-yl) methyl ] -2- (ethoxymethyl) -7- (4-hydroxymethylphenyl) -1H-imidazo [4,5-c ] quinolin-4-amine, 4- [ 4-amino-2-ethoxymethyl-1- (2-hydroxy-2-methylpropyl) -1H-imidazo [4,5-c ] quinolin-7-yl ] -N-methoxy-N-methylbenzamide, 2-ethoxymethyl-N1-isopropyl-6, 7,8, 9-tetrahydro-1H-imidazo [4,5-c ] quinolin-1, 4-diamine, 1- [ 4-amino-2-ethyl-7- (pyridin-4-yl) -1H-imidazo [4,5-c ] quinolin-1-yl ] -2-methylpropan-2- A compound of the group consisting of an alcohol, N- [4- (4-amino-2-ethyl-1H-imidazo [4,5-c ] quinolin-1-yl) butyl ] methanesulfonamide, and N- [4- (4-amino-2-butyl-1H-imidazo [4,5-c ] [1,5] naphthyridin-1-yl) butyl ] -N' -cyclohexylurea. In further individual embodiments, the immunotherapeutic agent is selected from any subset of the above list.

In some embodiments, the immunotherapeutic agent comprises resiquimod.

In some embodiments, the immunotherapeutic agent is selected to be capable of: (1) inducing IFN- α in enriched human blood DC; (2) inducing TNF- α in enriched human blood DC; and/or (3) an amount that induces IL-12-alpha in enriched human blood DCs.

In some embodiments, the immunotherapeutic agent is provided in an amount capable of activating human plasmacytoid dendritic cells, myeloid dendritic cells, or NK cells, and any combination thereof.

In some embodiments, the immunotherapeutic agent is administered in combination with a targeted therapeutic agent against cancer.

In some embodiments, the targeted therapeutic is capable of specifically or preferentially binding to tumor cells as compared to non-tumor cells.

In some embodiments, the tumor cell is a tumor cell of a carcinoma, sarcoma, lymphoma, myeloma, or central nervous system cancer.

In some embodiments, the targeted therapeutic is capable of specifically or preferentially binding to a tumor antigen as compared to a non-tumor antigen.

In some embodiments, the tumor antigen is selected from the group consisting of CD2, CD19, CD20, CD22, CD27, CD33, CD37, CD38, CD40, CD44, CD47, CD52, CD56, CD70, CD79, and CD 137.

In some embodiments, the tumor antigen is selected from the group consisting of 4-1BB, 5T4, AGS-5, AGS-16, angiopoietin 2, B7.1, B7.2, B7DC, B7H1, B7H2, B7H3, BT-062, BTLA, CAIX, carcinoembryonic antigen, CTLA4, Cripto, ED-B, ErbB1, ErbB2, ErbB3, ErbB4, EGFL7, EpCAM, EphA2, EphA3, EphB2, FAP, fibronectin, folate receptor, ganglioside GM3, GD2, glucocorticoid-induced tumor necrosis factor receptor (GITR), gp100, gpA33, NMGPB, ICOS, IGF1R, integrin alpha v beta, KIR, LewisY-3, LewisY, mesothelin, MUC-MET, MUC 72, NEYC, SLC 11, TNOX, TNFR 1, PSRCA 1, PSROX 1, PSRCA 1, GEN 1, PSRCA 1, PSROX 1, GEN 1, PSRCA 1, CTD, CTL-1, CTD-1, CTL-1, CTD, CTL-1, and PSRCA 1, TIM3, TRAILR1, TRAILR2, VEGFR-1, VEGFR-2, VEGFR-3, and variants thereof. In further individual embodiments, the tumor antigen is selected from any subset of the above list.

In some embodiments, the targeted therapeutic agent comprises an immunoglobulin, a protein, a peptide, a small molecule, a nanoparticle, or a nucleic acid.

In some embodiments, the targeted therapeutic comprises an antibody or a functional fragment thereof.

In some embodiments, the antibody is selected from the group consisting of: rituximab, herceptin, trastuzumab, erbitutuzumab, erbitux, verterby, pertuzumab, tremelimumab, nivolumab, Dacetuzumab, umeluzumab, MPDL3280A, pambrizumab, banolizumab, CT-011, MK-3475, BMS-936559, MED14736, MSB001071 0010718C, and magetiuximab (MGAH 22). In further individual embodiments, the antibody is selected from any subset of the above list.

In some embodiments, the targeted therapeutic comprises a Fab, Fab ', F (ab') 2, single domain antibody, T and Abs dimer, Fv, scFv, dsFv, ds-scFv, Fd, linear antibody, minibody, diabody, bispecific antibody fragment, diabody, triabody, sc-diabody, kappa (lambda) body, BiTE, DVD-Ig, SIP, SMIP, DART, or antibody analog comprising one or more CDRs.

In some embodiments, the targeted therapeutic comprises an ATWLPPR polypeptide of VEGFR, a thrombospondin-1 mimetic, CDCRGDCFCG (cyclic) polypeptide, a SCH 221153 fragment, a NCNGRC (cyclic) polypeptide, a CTTHWGFTLC polypeptide, a CGNKRTRGC polypeptide (LyP-1), octreotide, vapreotide, lanreotide, a C-3940 polypeptide, dabigatran, lippah, norrad, or cetrorelix.

In some embodiments, the targeted therapeutic comprises the extracellular domain (ECD) or soluble form of PD-1, PDL-1, CTLA4, BTLA, KIR, TIM3, 4-1BB, LAG3, partial full length of surface ligand amphiregulin, β -cytokine, EGF, ephrin, epigen, epithelial regulatory protein, IGF, neuregulin, TGF, TRAIL, or VEGF.

In some embodiments, the immunotherapeutic agent is delivered systemically.

In some embodiments, the immunotherapeutic agent is administered by oral administration or parenteral injection.

In some embodiments, the immunotherapeutic agent is administered by intravenous injection or intratumoral injection.

In some embodiments, the abnormal cell proliferation comprises a precancerous lesion.

In some embodiments, the abnormal proliferation is abnormal proliferation of cancer cells.

In some embodiments, the cancer is selected from the group consisting of Acute Myeloid Leukemia (AML), breast cancer, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), hodgkin's lymphoma, multiple myeloma, mycosis fungoides, neuroblastoma, non-hodgkin's lymphoma (NHL), ovarian cancer, and retinoblastoma.

In some embodiments, the method comprises administering to the subject an oral formulation comprising the immunotherapeutic agent at a dose of about 0.0005mg/kg, 0.0006mg/kg, 0.0007mg/kg, 0.0008mg/kg, 0.0009mg/kg, 0.001mg/kg, 0.002mg/kg, 0.003mg/kg, 0.004mg/kg, 0.005mg/kg, 0.006mg/kg, 0.007mg/kg, 0.008mg/kg, 0.009mg/kg, or 0.01mg/kg to about 0.02mg/kg, including all twice weekly.

In some embodiments, the method comprises administering to the subject an oral formulation comprising the immunotherapeutic agent at a dose of 0.0001mg/kg to less than or about 0.0005mg/kg, 0.0006mg/kg, 0.0007mg/kg, 0.0008mg/kg, 0.0009mg/kg, 0.001mg/kg, 0.002mg/kg, 0.003mg/kg, 0.004mg/kg, 0.005mg/kg, 0.006mg/kg, 0.007mg/kg, 0.008mg/kg, 0.009mg/kg, or 0.01mg/kg twice weekly.

In some embodiments, the method comprises administering to the subject an intravenous formulation comprising the immunotherapeutic agent at a dose of about 0.0005mg/kg, 0.0006mg/kg, 0.0007mg/kg, 0.0008mg/kg, 0.0009mg/kg, 0.001mg/kg, 0.002mg/kg, 0.003mg/kg, 0.004mg/kg, 0.005mg/kg, or 0.006mg/kg to about 0.015mg/kg, including all once a week.

In some embodiments, the method comprises administering to the subject an intravenous formulation comprising the immunotherapeutic agent at a dose of 0.001mg/kg to less than or about 0.003mg/kg, 0.004mg/kg, 0.005mg/kg, or 0.006mg/kg to about 0.01mg/kg once per week.

In some embodiments, the local concentration of the immunotherapeutic agent in the subject is about 0.005 μ g/ml to about 12 μ g/ml.

In some embodiments, the local concentration of the immunotherapeutic agent in the subject is about 0.05 μ g/ml, 0.1 μ g/ml, 0.15 μ g/ml, 0.2 μ g/ml, 0.3 μ g/ml, or 0.4 μ g/ml to about 0.5 μ g/ml.

In some embodiments, the method comprises administering to the subject an intravenous formulation comprising a chemotherapeutic agent in a divided dose of about 40-50mg/kg over 2-5 days.

In some embodiments, the immunotherapeutic agent is administered repeatedly at 2,3, or4 week intervals within each cycle.

In some embodiments, the method comprises administering to the subject an intravenous formulation comprising a chemotherapeutic agent at a dose of about 10 to 15mg/kg, once every 7 to 10 days.

In some embodiments, the method comprises administering to the subject an intravenous formulation comprising a chemotherapeutic agent at a dose of about 3 to 5mg/kg twice weekly.

In some embodiments, the method comprises administering the composition at about 60-120mg/m²The dose per day is administered to the subject as an intravenous formulation comprising a chemotherapeutic agent, with continuous infusion.

In some embodiments, the method comprises treating the sample with about 400-1000mg/m²Administering to the subject an oral formulation comprising a chemotherapeutic agent in 4-5 days.

In some embodiments, the method comprises administering the composition at about 50-100mg/m²Administering to the subject an intravenous formulation comprising a chemotherapeutic agent at a dose per day or 1-5 mg/kg/day.

In another aspect, embodiments of the present disclosure provide kits comprising a therapeutic combination provided by the present disclosure and optionally instructions for use.

Drawings

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the presently disclosed embodiments will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

FIGS. 1A-B depict the evaluation of the antitumor effect of TLRL in combination with anti-PD-Ll and gemcitabine in a NR-S1 syngeneic tumor model. FIG. 1A: NR-S1 cells were inoculated into C3H mice when tumor volume reached 30mm³Treatment is initiated. Control agents (PBS or rat IgG), Resiquimod (RQ) (data not shown), anti-PD-L1 mAb (PD-L1) (data not shown), and resiquimod and antibody (RQ + PD) were all intraperitoneally injected once every three days for 4 times. Gemcitabine (30mg/kg) was administered intraperitoneally one day prior to the initiation of treatment with resiquimod and PD-L1 mAb (+ Gem). Tumor volumes were measured daily and the data shown in the figure are the combined results from two independent experiments (n-11). The three smaller plots on the left show the growth curves of the individuals. Tumor growth curves and tumor volumes are shown at 14 days (day 24) after initiation of treatment. FIG. 1B: 24 days after tumor inoculationSpleens were collected and total cell number was calculated (left panel). Splenocytes and red blood depleted peripheral blood leukocytes were stained with anti-CD 11b and anti-Gr-1 mAb and analyzed by flow cytometry (middle and right panels, respectively). Shows Gr-1highCD11b⁺Percentage of cells. Values for splenocytes are the mean ± SD (n-11) of two independent experiments with similar results. Peripheral blood values are mean ± SD from a group of six mice. Statistical difference (═ p)<0.05)。

FIGS. 2A-B depict CD11B from NR-S1 and SCCVII tumors⁺Cell surface marker profile of cells. These two tumor types showed different characteristics in TME. Ly6GhighLy 6C-F4/80-cells predominate in NR-S1 tumors, while Ly6GlowLy6Clow/-F4/80+ cells predominate in SCCVII tumors. Subcutaneously inoculating NR-S1 or SCCVII tumor cells when the tumor volume reaches about 1500mm³The tumor mass and spleen were removed. FIG. 2A: in CD45⁺FSClow-high Large lymphocytes (CD 45)⁺gate)、FSClowCD3⁺CD11 b-lymphocyte (CD3)⁺T-gate)、FSClow-highCD3-CD11b⁺Bone marrow cells (CD11 b)⁺gate), Ly6GhighLy6Clow-nega (Fr-1gate) and Ly6GlowLy6Clow-nega (Fr-2gate) tumor infiltrating lymphocyte-fractions were stained and electronically gated, showing the percentage or expression profile of the indicated population (as a contour plot). For T cell analysis, CD8 is shown⁺CD4^-(CD8⁺T)、CD8^-CD4⁺Foxp3^-(conventional T, CD4⁺Tcon)、CD8^-CD4⁺Foxp3⁺(Treg). FIG. 2B: total number of cells from spleen was counted and stained with fluorochrome-conjugated anti-CD 3, anti-CD 11b, anti Ly6C and Ly6G mabs and analyzed by flow cytometry. The electronic gate is placed on the CD3^-CD11b⁺On cells (CD11 b)⁺gate), the expression profiles of Ly6C and Ly6G are shown as contour plots. All quadrant markers are positioned to include>95-98% of cells stained with control fluorochrome. Values shown are mean ± SD (n ═ 5). P<0.05。

FIG. 3 depicts tumor growth after inoculation of 4T1 cells into Balb/c mice,when the tumor volume reaches 100mm³Treatment is initiated. Control agents (PBS), Resiquimod (RQ), anti-PD-L1 mab (PD), and immunotherapeutic agents and antibodies (RQ + PD) were injected intravenously twice, one week apart. Gemcitabine (30mg/kg) was administered intraperitoneally one day prior to the initiation of treatment with resiquimod and PD-L1 mAb (+ Gem). Tumor volumes were measured on days 14, 17 and 21 and data shown were from an independent experiment (n-6). The lower five smaller graphs show the growth curves of the individuals.

Detailed Description

Several aspects of the invention are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. One skilled in the relevant art will readily recognize, however, that the invention can be practiced without one or more of the specific details, or with other methods. The presently disclosed embodiments are not limited by the illustrated ordering of acts or events, as some acts may occur in different orders and/or concurrently with other acts or events.

Moreover, not all illustrated acts or events are required to implement a methodology in accordance with the presently disclosed embodiments.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, if the terms "including", "having", "with", or variants thereof are used in the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising".

The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 standard deviation or over 1 standard deviation, according to practice in the art. Alternatively, "about" may represent a range of up to 20%, preferably up to 10%, more preferably up to 5%, more preferably up to 1% of a given value. Alternatively, particularly for biological systems or processes, the term may mean within an order of magnitude, preferably within 5-fold, more preferably within 2-fold. Where particular values are described in the application and claims, the term "about" shall be construed to mean within an acceptable error range for the particular value, unless otherwise specified.

I.Definitions and abbreviations

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry, and hybridization are those well known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. These techniques and procedures are generally performed according to conventional methods in the art and various general references provided throughout this document. The nomenclature used herein and the laboratory procedures in analytical chemistry, as well as the organic syntheses described below, are those well known and commonly employed in the art. Standard techniques or modifications thereof are used for chemical synthesis and chemical analysis.

Unless otherwise indicated, the term "alkyl" by itself or as part of another substituent means a straight or branched chain or cyclic hydrocarbon group, or combinations thereof, which may be fully saturated, mono-unsaturated or poly-unsaturated and may include divalent and polyvalent groups, having the indicated number of carbon atoms (i.e., C)₁-C₁₀Representing one to ten carbons). Examples of saturated hydrocarbon groups include, but are not limited to, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl) methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Unsaturated alkyl is alkyl having one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, ethenyl, 2-propenyl, crotyl, 2-isopentenyl, 2- (butadienyl), 2, 4-pentadienyl, 3- (1, 4-pentadienyl), ethenylAlkynyl, 1-and 3-propynyl, 3-butynyl and higher homologs and isomers. Unless otherwise indicated, the term "alkyl" is also intended to include those alkyl derivatives defined in more detail below, such as "heteroalkyl. Alkyl groups limited to hydrocarbyl groups are referred to as "homo alkyl groups".

The term "alkylene" by itself or as part of another substituent denotes a divalent radical derived from an alkane, such as, but not limited to-CH₂CH₂CH₂CH₂And further includes those groups described below as "heteroalkylene". Typically, the alkyl (or alkylene) groups will have 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the presently disclosed embodiments. "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene, typically having eight or fewer carbon atoms.

The terms "alkoxy", "alkylamino" and "alkylthio" (or thioalkoxy) are used in their conventional sense to refer to those alkyl groups attached to the rest of the molecule through an oxygen atom, an amino group, or a sulfur atom, respectively.

Unless otherwise specified, the term "heteroalkyl," by itself or in combination with another term, means a stable straight or branched chain, or cyclic hydrocarbon group, or combinations thereof, consisting of the stated number of carbon atoms and at least one heteroatom selected from the group consisting of O, N, Si and S, wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatoms O, N and S and Si can be located at any internal position of the heteroalkyl group or at the position where the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to-CH₂-CH₂-O-CH₃、-CH₂-CH₂-NH-CH₃、-CH₂-CH₂-N(CH₃)-CH₃、-CH₂-S-CH₂-CH₃、-CH₂-CH₂、-S(O)-CH₃、-CH₂-CH₂-S(O)₂-CH₃、-CH＝CH-O-CH₃、-Si(CH₃)₃、-CH₂-CH＝N-OCH₃and-CH ═ CH-N (CH)₃)-CH₃. At most two hetero ringsThe atoms may be consecutive, e.g. -CH₂-NH-OCH₃and-CH₂-O-Si(CH₃)₃. Similarly, the term "heteroalkylene" by itself or as part of another substituent means a divalent radical derived from a heteroalkyl radical, such as, but not limited to, -CH₂-CH₂-S-CH₂CH₂-and-CH₂-S-CH₂-CH₂-NH-CH₂-. For heteroalkylene groups, heteroatoms can also occupy one or both of the chain ends (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Further, for alkylene and heteroalkylene linking groups, the written direction of the formula for the linking group does not imply a direction for the linking group. For example, of the formula-C (O)₂R' -represents-C (O)₂R '-and-R' C (O)₂-。

Typically, the "acyl substituent" is also selected from the above groups. As used herein, the term "acyl substituent" refers to a group that is attached to and satisfies the valence of the carbonyl carbon that is directly or indirectly attached to the polycyclic core of a compound of embodiments of the disclosure.

Unless otherwise indicated, the terms "cycloalkyl" and "heterocycloalkyl" by themselves or in combination with other terms represent cyclic forms of "alkyl" and "heteroalkyl," respectively. Further, for heterocycloalkyl, a heteroatom may occupy the position at which the heterocycle is attached to the rest of the molecule. Examples of cycloalkyl groups include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1- (1,2,5, 6-tetrahydropyridinyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiophen-2-yl, tetrahydrothiophen-3-yl, 1-piperazinyl, 2-piperazinyl, and the like.

Unless otherwise indicated, the term "halo" or "halogen" by itself or as part of another substituent means a fluorine, chlorine, bromine or iodine atom. Further, terms such as "haloalkyl" are intended to include monohaloalkyl and polyhaloalkyl. For example, the term "halo (C)₁-C₄) Alkyl "denotes to includeBut are not limited to trifluoromethyl, 2,2, 2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

As used herein, the term "haloalkyl" refers to an alkyl group, as defined herein, substituted with one or more halo groups, as defined herein. Preferably, the haloalkyl group can be a monohaloalkyl, dihaloalkyl, or polyhaloalkyl group, including perhaloalkyl groups. The monohaloalkyl group can have one iodine, bromine, chlorine or fluorine within the alkyl group. Dihaloalkyl and polyhaloalkyl groups may have two or more of the same halogen atoms or a combination of different halogen groups within the alkyl group. Preferably, the polyhaloalkyl contains up to 12, 10 or 8, or 6, or4, or 3, or2 halo groups. Non-limiting examples of haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl, and dichloropropyl. Perhaloalkyl refers to alkyl groups in which all hydrogen atoms are substituted with halogen atoms.

As used herein, the term "heteroaryl" refers to a 5-14 membered monocyclic-or bicyclic-or fused polycyclic-ring system having 1 to 8 heteroatoms selected from N, O, S or Se. Preferably, the heteroaryl group is a 5-10 membered ring system. Typical heteroaryl groups include 2-or 3-thienyl, 2-or 3-furyl, 2-or 3-pyrrolyl, 2-, 4-or 5-imidazolyl, 3-, 4-or 5-pyrazolyl, 2-, 4-or 5-thiazolyl, 3-, 4-or 5-isothiazolyl, 2-, 4-or 5-oxazolyl, 3-, 4-or 5-isoxazolyl, 3-or 5-1,2, 4-triazolyl, 4-or 5-1,2, 3-triazolyl, tetrazolyl, 2-, 3-or 4-pyridyl, 3-or 4-pyridazinyl, 3-, 4-or 5-pyrazinyl, 2-pyrazinyl, 2-, 4-or 5-pyrimidinyl.

The term "heteroaryl" also refers to a group in which a heteroaryl ring is fused to one or more aryl, alicyclic, or heterocycloalkyl rings, where the group or point of attachment is on the heteroaryl ring. Non-limiting examples include, but are not limited to, 1-, 2-, 3-, 5-, 6-, 7-or 8-indolinyl, 1-, 3-, 4-, 5-, 6-or 7-isoindolyl, 2-, 3-, 4-, 5-, 6-or 7-indolyl, 2-, 3-, 4-, 5-, 6-or 7-indazolyl, 2-, 4-, 5-, 6-, 7-or 8-purinyl, 1-, 2-, 3-, 4-, 6-, 7-, 8-or 9-quinolinyl, 2-, 3-, 4-, 5-, 6-, 7-or 8-quinoline, 1-, 3-, 4-, 5-, 6-, 7-or 8-isoquinoline, 1-, 4-, 5-, 6-, 7-or 8-phthalazinyl, 2-, 3-, 4-, 5-or 6-naphthyridinyl, 2-, 3-, 5-, 6-, 7-or 8-quinazolinyl, 3-, 4-, 5-, 6-, 7-or 8-cinnamyl, 2-, 4-, 6-or 7-pteridinyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-or 8-4 aH-carbazolyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, or 8-carbazolyl, 1-, 3-, 4-, 5-, 6-, 7-, 8-, or 9-carbolinyl, 1-, 2-, 3-, 4-, 6-, 7-, 8-, 9-or 10-phenanthridinyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-or 9-acridinyl, 1-, 2-, 4-, 5-, 6-, 7-, 8-or 9- -pyridinyl, 2-, 3-, 4-, 5-, 6-, 8-, 9-or 10-phenanthrolinyl, 1-, 2-, or, 3-, 4-, 6-, 7-, 8-or 9-phenazinyl, 1-, 2-, 3-, 4-, 6-, 7-, 8-, 9-or 10-phenothiazinyl, 1-, 2-, 3-, 4-, 6-, 7-, 8-, 9-or 10-phenoxazinyl, 2-, 3-, 4-, 5-, 6-or 1-, 3-, 4-, 5-, 6-, 7-, 8-, 9-or 10-benzisoquinolinyl, 2-, 3-, 4-or 5-thieno [2,3-b ] furyl, 2-, 3-, 5-, (a) phenothiazinyl, a salt thereof, or a salt thereof, 6-, 7-, 8-, 9-, 10-or 11-7H-pyrazino [2,3-c ] carbazolyl, 2-, 3-, 5-, 6-or 7-2H-furo [3,2-b ] -pyranyl, 2-, 3-, 4-, 5-, 7-or 8-5H-pyrido [2,3-d ] -o-oxazinyl, 1-, 3-or 5-1H-pyrazolo [4,3-d ] -oxazolyl, 2-, 4-or 54H-imidazo [4,5-d ] thiazolyl, 3-, 5-or 8-pyrazino [2,3-d ] pyridazinyl, 2-, (meth) acrylic acid, or mixtures thereof, 3-, 5-or 6-imidazo [2,1-b ] thiazolyl, 1-, 3-, 6-, 7-, 8-or 9-furo [3,4-c ] cinnamyl, 1-, 2-, 3-, 4-, 5-, 6-, 8-, 9-, 10-or 11-4H-pyrido [2,3-c ] carbazolyl, 2-, 3-, 6-or 7-imidazo [1,2-b ] [1,2,4] triazinyl, 7-benzo [ b ] thienyl, 2-, 4-, 5-, 6-or 7-benzoxazolyl, 2-, 4-, 5-, 6-or 7-benzimidazolyl, 2-, 4-, 5-, 6-or 7-benzothiazolyl, 1-, 2-, 4-, 5-, 6-, 7-, 8-or 9-benzoxazinyl, 2-, 4-, 5-, 6-, 7-or 8-benzoxazinyl, 1-, 2-, 3-, 5-, 6-, 7-, 8-, 9-, 10-or 11-1H-pyrrolo [1,2-b ] [2] benzazepinyl. Typical fused heteroaryl groups include, but are not limited to, 2-, 3-, 4-, 5-, 6-, 7-or 8-quinolinyl, 1-, 3-, 4-, 5-, 6-, 7-or 8-isoquinolinyl, 2-, 3-, 4-, 5-, 6-or 7-indolyl, 2-, 3-, 4-, 5-, 6-or 7-benzo [ b ] thienyl, 2-, 4-, 5-, 6-or 7-benzoxazolyl, 2-, 4-, 5-, 6-or 7-benzimidazolyl, 2-, 4-, 5-, 6-or 7-benzothiazolyl.

As used herein, the term "heterocyclyl" or "heterocycle" refers to an optionally substituted, fully saturated or unsaturated, aromatic or non-aromatic cyclic group, for example, that is a 4-to 7-membered monocyclic, 7-to 12-membered bicyclic, or 10-to 15-membered tricyclic ring system, having at least one heteroatom in at least one carbon atom-containing ring. Each ring of the heterocyclic group containing a heteroatom may have 1,2 or 3 heteroatoms selected from nitrogen atoms, oxygen atoms and sulfur atoms, where the nitrogen and sulfur heteroatoms may also be optionally oxidized. The heterocyclic group may be attached at a heteroatom or carbon atom.

Exemplary monocyclic heterocyclic groups include pyrrolidinyl, pyrrolyl, pyrazolyl, oxetanyl, pyrazolinyl, imidazolyl, imidazolinyl, imidazolidinyl, triazolyl, oxazolyl, oxazolidinyl, isoxazolinyl, isoxazolyl, thiazolyl, thiadiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, furyl, tetrahydrofuryl, thienyl, oxadiazolyl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, 2-oxazayl, azepinyl, 4-piperidinyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone, 1, 3-dioxolane and tetrahydro-1, 1-dioxothienyl, 1,4 trioxo-1, 2, 5-thiadiazol-2-yl, and the like.

Exemplary bicyclic heterocyclic groups include indolyl, indolinyl, benzothiazolyl, benzoxazinyl, benzoxazolyl, benzothienyl, benzothiazinyl, quinuclidinyl, quinolinyl, tetrahydroquinolinyl, decahydroquinolinyl, isoquinolinyl, tetrahydroisoquinolinyl, decahydroisoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuranyl, chromonyl, coumarinyl, benzopyranyl, cinnamyl, quinoxalinyl, indazolyl, pyrrolopyridyl, furopyridinyl (e.g., furo [2,3-c ] pyridinyl, furo [3,2-b ] -pyridinyl ] or furo [2,3-b ] pyridinyl), isoindolyl, 1, 3-dioxo-1, 3-dihydroisoindol-2-yl, dihydroquinazolinyl (e.g., 3, 4-dihydro-4-oxo-quinazolinyl), phthalazinyl, and the like.

Exemplary tricyclic heterocyclic groups include carbazolyl, dibenzoazanyl, dithienoaazanyl, benzindolyl, phenanthrolinyl, acridinyl, phenanthridinyl, phenoxazinyl, phenothiazinyl, xanthenyl, carbolinyl, and the like.

The term "heterocyclyl" also refers to heterocyclic groups as defined herein which are substituted with 1,2 or 3 substituents selected from:

(a) an alkyl group;

(b) hydroxyl (or protected hydroxyl);

(d) oxo, i.e., ═ O;

(e) amino, alkylamino or dialkylamino;

(f) an alkoxy group;

(g) a cycloalkyl group;

(h) a carboxyl group;

(i) heterocyclyloxy, wherein heterocyclyloxy represents a heterocyclic group bonded through an oxygen bridge;

(j) alkyl-O-C (O) -;

(k) a mercapto group;

(l) A nitro group;

(m) cyano;

(n) a sulfonamide or sulfonamido group;

(o) an aryl group;

(p) alkyl-C (O) -O- -;

(q) aryl-C (O) -O- -;

(r) aryl-S-;

(s) aryloxy;

(t) alkyl-S- -;

(u) formyl, i.e., HC (O) -;

(v) a carbamoyl group;

(w) aryl-alkyl-; and

(x) Aryl substituted with alkyl, cycloalkyl, alkoxy, hydroxy, amino, alkyl-C (O) -NH-, alkylamino, dialkylamino or halogen.

As used herein, the term "alkenyl" refers to a straight or branched chain hydrocarbon group having 2 to 20 carbon atoms and comprising at least one double bond. The alkenyl group preferably has about 2 to 8 carbon atoms.

Unless otherwise indicated, the term "aryl" refers to a polyunsaturated aromatic hydrocarbon substituent which may be monocyclic or polycyclic (preferably 1 to 3 rings) which are fused together or linked covalently. The term "heteroaryl" refers to an aryl (or ring) containing one to four heteroatoms selected from N, O and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atoms are optionally quaternized. The heteroaryl group may be attached to the rest of the molecule through a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-benzothiazolyl, Purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalyl, 5-quinoxalyl, 3-quinolyl and 6-quinolyl. The substituents for each of the above aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below.

For brevity, the term "aryl" when used in combination with other terms (e.g., aryloxy, arylthioxy, aralkyl) includes aryl and heteroaryl rings as defined above. Thus, the term "arylalkyl" is intended to include those groups in which the aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, picolyl, and the like), including those alkyl groups in which a carbon atom (e.g., methylene) has been substituted with, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3- (1-naphthyloxy) propyl, and the like).

Each of the above terms (e.g., "alkyl," "heteroalkyl," "aryl," and "heteroaryl") includes both substituted and unsubstituted forms of the indicated group. Preferred substituents for each type of group are provided below.

Substituents for alkyl and heteroalkyl (including those groups commonly referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) are commonly referred to as "alkyl substituents" and "heteroalkyl substituents," respectively, and they may be one or more of a variety of groups selected from, but not limited to: OR ', -O, ═ NR ', -N-OR ', -NR ' R ", -SR ', -halogen, -SiR ' R" R ' ", -oc (O) R ', -c (O) R ', -CO₂R’、-CONR’R”、-OC(O)NR’R”、-NR”C(O)R’、-NR’-C(O)NR”R”’、-NR”C(O)₂R’、-NR-C(NR’R”R’”)＝NR””、-NR-C(NR’R”)＝NR’”、-S(O)R’、-S(O)₂R’、-S(O)₂NR’R”、-NRSO₂R', -CN and-NO₂The number of which ranges from zero to (2m '+ 1), where m' is the total number of carbon atoms in such a group. R ', R ", R'" and R "" each preferably independently mean hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, for example aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy, or aralkyl. For example, when a compound of the invention includes more than one R group, each R group is independently selected, and when more than one R ', R ", R'" and R "" groups are present, these groups are also independently selected. When R' and R "are attached to the same nitrogen atom, they may combine with the nitrogen atom to form a 5,6 or7 membered ring. For example, -NR' R "is intended to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, those skilled in the art will understand that the term "alkyl" is intended to include carbon atom-containing groups bonded to groups other than hydrogen groups, such as haloalkyl (e.g., -CF)₃and-CH₂CF₃) And acyl (e.g., -C (O) CH)₃、-C(O)CF₃、-C(O)CH₂OCH₃Etc.).

Similar to the substituent descriptions for alkyl, aryl and heteroaryl substituents are commonly referred to as "aryl substituents" and "heteroaryl substituents," respectively, and may varyAnd is selected from, for example: halogen, -OR ', - (O), - (NR '), - (N-OR ', - (NR ') R ", -SR ', -halogen, -SiR ' R" R ' ", -oc (O) R ', -c (O) R ', -CO₂R’、-CONR’R”、-OC(O)NR’R”、-NR”C(O)R’、-NR’-C(O)NR”R”’、-NR”C(O)₂R’、-NR-C(NR’R”)＝NR’”、-S(O)R’、-S(O)₂R’、-S(O)₂NR’R”、-NRSO₂R ', -CN, -NO, -R', -N₃、-CH(Ph)₂Fluorine (C)₁-C₄) Alkoxy, and fluorine (C)₁-C₄) Alkyl groups in an amount from zero to the total number of ring-opening valences on the aromatic ring system; wherein R ', R ", R'" and R "" are preferably independently selected from hydrogen, (C)₁-C₈) Alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl) - (C)₁-C₄) Alkyl and (unsubstituted aryl) oxy- (C)₁-C₄) An alkyl group. For example, when a compound of the invention includes more than one R group, each R group is independently selected, and when more than one R ', R ", R'" and R "" groups are present, these groups are also independently selected.

Two aryl substituents on adjacent atoms of an aryl or heteroaryl ring may optionally be substituted by a group of formula-TC (O) - (CRR')_q-U-, wherein T and U are independently-NR-, -O-, -CRR' -or a single bond, and q is an integer from 0 to 3. Alternatively, two substituents on adjacent atoms of an aryl or heteroaryl ring may be optionally substituted by a group of formula-A- (CH)₂)_r-substituent substitution of-B, wherein A and B are independently-CRR' -, -O-, -NR-, -S (O) -, S (O)₂-、-S(O)₂NR' -or a single bond, r is an integer of 1 to 4. One of the single bonds of the new ring so formed may optionally be replaced by a double bond. Alternatively, two substituents on adjacent atoms of the aryl or heteroaryl ring may be optionally substituted by a group of formula- (CRR')_s-X-(CR”R”’)_d-wherein S and d are independently integers from 0 to 3, X is-O-, -NR' -, -S (O)₂-or-S (O)₂NR' -. The substituents R ', R ", R'" and R "" are preferably independently selected from hydrogen or substituted or unsubstituted (C)₁-C₆) An alkyl group.

As used herein, the term "heteroatom" includes oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).

The term "aryloxy" as used herein refers to-O-aryl and-O-heteroaryl, wherein aryl and heteroaryl are defined herein.

As used herein, the term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness and properties of the compounds of the present invention and is not biologically or otherwise undesirable. In many cases, compounds of the disclosed embodiments are capable of forming acid and/or base salts due to the presence of amino and/or carboxyl groups or groups similar thereto (e.g., phenol or hydroxyamidic acid). Can form pharmaceutically acceptable acid addition salts with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Can form pharmaceutically acceptable base addition salts with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like; particularly preferred are ammonium, potassium, sodium, calcium and magnesium salts. Organic bases from which salts can be derived include, for example, primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine, among others. Pharmaceutically acceptable salts in the disclosed embodiments of the invention can be synthesized from the parent compound, basic or acidic moiety, by conventional chemical methods. In general, such salts can be prepared by reacting the free acid forms of these compounds with a stoichiometric amount of the appropriate base (e.g., Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, etc.), or by reacting the free base forms of these compounds with a stoichiometric amount of the appropriate acid. Such reactions are generally carried out in water or an organic solvent or in a mixture of the two. Generally, nonaqueous media such as diethyl ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred where feasible. Lists of other suitable salts can be found, for example, in Remington's Pharmaceutical Sciences,20th ed., Mack Publishing Company, Easton, Pa., (1985), which is incorporated herein by reference.

As used herein, the term "pharmaceutically acceptable carrier/excipient" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal agents), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, materials such as are known to those of ordinary skill in the art, and combinations thereof (see, e.g., Remington's Pharmaceutical Sciences,18th ed. mack Printing Company,1990, pp.1289-1329, incorporated herein by reference). Unless any conventional carrier is incompatible with the active ingredient, it is contemplated that it may be used in therapeutic or pharmaceutical compositions.

As used herein, the term "subject" refers to an animal. Preferably, the animal is a mammal. Subjects also refer to, for example, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, and the like. In a preferred embodiment, the subject is a human.

As used herein, the term "therapeutic combination" or "combination" refers to a combination of one or more active drug substances, i.e., compounds having therapeutic utility, particularly a combination of chemotherapeutic and immunotherapeutic agents, or chemotherapeutic, immunotherapeutic and targeted therapeutic agents as described herein. Typically, each such compound in the therapeutic combination of the presently disclosed embodiments will be present in a pharmaceutical composition comprising the compound and a pharmaceutically acceptable carrier. The compounds of the therapeutic combinations of the disclosed embodiments may be administered simultaneously or separately as part of a regimen. In particular embodiments, the various components of the combination may be formulated separately or together, administered by the same or different routes of administration, or administered according to the same or different schedules. However, even if administered by different routes or on different schedules, co-administration may be possible, resulting in greater benefit in terms of greater therapeutic effect and/or reduced adverse side effects for the subject than if the components were not administered fully or were not co-administered.

II.Composition comprising a metal oxide and a metal oxide

In general, the presently disclosed embodiments provide therapeutic combinations, pharmaceutical compositions, and methods of treating cancer using combination therapy; more specifically, a combination of chemotherapeutic agents and immunotherapy (e.g., using Toll-like receptor ligand "TLRL") (e.g., TLR7 and 8 dual agonists to activate innate DC immunity) that can reduce myeloid-derived suppressor cells (MDSCs). In further embodiments, the combination will also include a targeted therapeutic agent.

In one aspect, the presently disclosed embodiments provide a method for treating a tumor or abnormal cell proliferation in a subject in need of such treatment, comprising administering to the subject: a chemotherapeutic agent provided in an amount capable of reducing the population of MDSCs in the blood, spleen and/or tumor microenvironment of the subject; and an effective amount of an immunotherapeutic agent. In some embodiments, when used as part of a combination, the effective amount of the immunotherapeutic agent is less than when used alone or in combination with the use of a chemotherapeutic agent.

More than one pharmaceutical composition may be used to provide a therapeutic combination. In such embodiments, the chemotherapeutic agent may be provided in one pharmaceutical composition and the immunotherapeutic agent may be provided in a second pharmaceutical composition, such that the two compounds may be administered separately, e.g., at different times, by different routes of administration, etc. Thus, chemotherapeutic agents and immunotherapeutic agents may also be provided in different dosing regimens. Similarly, when a targeted therapy is additionally included in the combination, it may be provided in a third pharmaceutical composition, which, while still in a coordinated manner, may be administered independently of one or both of the other components of the combination.

Unless otherwise indicated, the compounds may include the compounds in any pharmaceutically acceptable form, including any isomer (e.g., diastereoisomer or enantiomer), salt, solvate, polymorph, and the like. In particular, if a compound is optically active, the compound may include each enantiomer of the compound as well as a racemic mixture of the enantiomers.

Typically, the chemotherapeutic agent and the immunotherapeutic agent are not linked to each other, e.g., by a covalent linker. In some embodiments, the chemotherapeutic agent and/or immunotherapeutic agent is not co-formulated with tumor cells or another immunogenic composition containing a tumor-associated antigen. In still further embodiments, administration of the chemotherapeutic agent and/or immunotherapeutic agent is not coordinated with administration of tumor cells or another immunogenic composition containing a tumor-associated antigen.

A. Chemotherapeutic agents

In general, the methods provided by the present disclosure include first administering to a subject a chemotherapeutic agent provided in an amount capable of reducing MDSC populations in the subject's blood, spleen, and/or tumor microenvironment, prior to administration of an immunotherapeutic agent and optionally a targeted therapeutic agent.

In some embodiments, the chemotherapeutic agent comprises a myeloid-derived suppressor cell (MDSC) inhibitor.

By "myeloid-derived suppressor cell (MDSC) inhibitor" herein is meant a therapeutic agent capable of modulating immune suppressor cells, such as MDSCs. In general, MDSC inhibitors cause induction of MDSC apoptosis and/or necrosis or cytotoxicity by inhibiting the function of MDSCs c-kit, or VEGFR, or ARG1, or iNOS, or S100 or MMP, and ROS ERK activation or antioxidant genes. In some embodiments, the MDSC inhibitor is selected from: paclitaxel, gemcitabine, 5-fluorouracil, oxaliplatin, cisplatin, carboplatin, dasatinib, sunitinib, and doxorubicin.

B. Immunotherapeutic agent

In general, the combination or composition of the presently disclosed embodiments comprises an immunotherapeutic agent.

As used herein, an "immunotherapeutic agent" refers to a compound, molecule or agent that is capable of stimulating or enhancing the immune system of the body. Immunotherapeutics are used to treat diseases by inducing, enhancing or suppressing an immune response. The immunotherapeutic agents of the presently disclosed embodiments are generally designed to elicit or amplify an immune response, rather than to suppress an immune response. However, in some embodiments, the primary role of the immunotherapeutic agent may be to inhibit differentiation or proliferation of immunosuppressive cells, rather than to more directly elicit or amplify an anti-tumor immune response.

In general, the immunotherapeutic agents of the presently disclosed embodiments act directly or indirectly on a toll-like receptor, a nucleotide oligomerization domain-like receptor, a RIG-I-like receptor, a c-type lectin receptor, or a cytoplasmic DNA sensor, or a combination thereof. In particular, the immunotherapeutic agent of the presently disclosed embodiments is capable of activating human plasmacytoid dendritic cells, myeloid dendritic cells, NK cells, or tumor cells, or a combination thereof.

In some embodiments, the immunotherapeutic agent of the presently disclosed embodiments activates human immune cells, including but not limited to dendritic cells, macrophages, monocytes, myeloid derived suppressor cells, NK cells, B cells, T cells, or tumor cells, or a combination thereof.

Dendritic cells are the most potent antigen presenting cells. Dendritic cells play an important role in the initiation of innate and adaptive immune responses. Dendritic cells also play a key role in the induction and maintenance of immune tolerance.

The term "dendritic cell" (DC) as used herein is meant to include two major subtypes: i.e., heterogeneous cell populations of bone marrow dc (mdc) and plasma cell dc (pdc) (Steinman et al, 1979, j.exp.med.,149, 1-16). These two blood DC subsets were initially distinguished by their expression of CD11c (integrin complement receptor) and CD123(IL-3R α). The pDC and mDC populations each constitute from about 0.2% to about 0.6% of the population of human PBMCs.

"pDC" herein refers to plasmacytoid dendritic cells and they represent a subset of dendritic cells found in blood and peripheral lymphoid organs. These cells express the surface markers CD123, BDCA-2(CD303), BDCA-4(CD304) and HLA-DR, but do not express CD11c, CD14, CD3, CD20 or CD56, which distinguishes them from conventional dendritic cells, monocytes, T cells, B cells and NK cells. As part of the innate immune system, these cells express intracellular Toll-like receptors 7 and 9, which are capable of detecting viral and bacterial nucleic acids, such as ssRNA or CpG DNA motifs. Upon stimulation and subsequent activation, these cells produce large amounts of type I interferons (primarily IFN- α and IFN- β) and type III interferons (e.g., IFN- λ), which are key pleiotropic antiviral compounds that mediate a wide range of effects. Plasmacytoid dendritic cells are widely involved in innate and adaptive immune responses in the body by producing large amounts of type I interferons, cytokines and chemokines. They can regulate NK cells, T cells, B cells and other cells involved in the intensity, duration and response pattern of the immune response, thereby playing a very important role in tumors, infections and autoimmune diseases. (Liu YJ. IPC: developmental type 1 interaction-producing cells and plasmid dentritic cell safeners. Annual Rev Immunol.2005; 23:275-306.Gilliet M, Cao W, Liu YJ. plasmid dentritic cells: sensing nucleic acids in viral infection and autoimmunity diseases. Nat Rev Immunol.2008 Aug; 8(8): 594).

"mdcs" herein refer to myeloid dendritic cells and they represent a subset of circulating dendritic cells found in blood and peripheral lymphoid organs. These cells expressed the surface markers CD11c, CD1a, HLA-DR and BDCA-1(CD1c) or BDCA-3(CD 141). They do not express BDCA-2 or CD123, which distinguishes their pdcs. mdcs also do not express CD3, CD20, or CD 56. As part of the innate immune system, mdcs express Toll-like receptors (TLRs), including TLRs 2,3,4, 5,6, and 8, which can detect bacterial and viral components. Upon stimulation and subsequent activation, these cells are the most potent antigen presenting cells, activating antigen-specific CD4 and CD 8T cells. In addition, mdcs have the ability to produce large amounts of IL-12 and IL23, which is critical for the induction of Th 1-mediated or Th17 cell-mediated immunity.

It has been found that many solid tumors, such as breast, head and neck and ovarian cancers, have resident pDC (Trelleux I, Blay JY, Bendriss-Vermark N et al. Dendritic cell priming and diagnosis of early stage rupture Cancer, Clin Cancer Res 2004,10:7466-, clin Cancer Res 1997,3: 483-490; bell D, Chomuraat P, Broyles D et al, In break cardio tissue, anatomical resistive cells stress with the term, where the areas of the areas are located In the qualitative objects, J Exp Med 1999,190: 1417-; Meniere-Caux C, Montmain G, Dieu MC et al, inhibition of the differentiation of dendritic cells from CD34(+) precursors by cells, role of interfacial-6 and macrotopographic binding-stimulating factor, Blood 1998,92: 4778-4791). These immature DCs did not function to promote anti-tumor immunity. In contrast, DCs in the tumor microenvironment promote tumor growth by inhibiting anti-tumor immunity and promoting angiogenesis. There is evidence that Toll-like receptor 7 agonist Imiquimod and Toll-like receptor 9 agonist CpG drugs can stimulate pDC within the tumor microenvironment to inhibit tumor development (Dummer R, Urosevic M, Kempf W et al, Imiquode in basal cell carcinosoma: how bit work, Br J Dermatol 2003,149: 57-58; Miller RL, Gerster JF, Owens ML et al, Imiquode amplified morphology: a novel immune response modifier and new class of drug, Int J immunopharmacological, 21: 1-14; Hofmann MA, Kors C, Australine H et al, Imode 1 evaluation of intraviral infection 9-agonist CpG drugs, Imidamole II, Imiquid in basal cell carcinosis P. P5, P.D. pDC, Br J Dermatory, Br J Dermacological model, P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.No.P.P.P.P.P.P.P.P.P.No.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.No.No.P.P.P.P.P.No.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.No.No.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P..

Natural Killer (NK) cells are a type of cytotoxic lymphocytes that constitute a major component of the immune system. NK cells are a subset of peripheral blood lymphocytes, defined by expression of CD56 or CD16 and deletion of the T cell receptor (CD 3). They recognize and kill transformed cell lines without being initiated in an MHC-unrestricted manner. NK cells play a major role in rejecting tumors and virus-infected cells. The process by which NK cells recognize target cells and transmit sufficient signals to trigger target cell lysis is determined by a series of inhibitory and activating receptors on the cell surface. NK differentiates self from altered self-involvement in inhibitory receptor recognition of MHC-I molecules and non-MHC ligands such as CD48 and Clr-1 b. NK recognition of infected or damaged cells (self-alterations) is coordinated by stress-induced ligands (e.g. MICA, MICB, Rae1, H60, Mult1) or virus-encoded ligands recognized by various activating receptors (e.g. m157, hemagglutinin), including NKG2D, Ly49H and NKp46/Ncr 1.

Within months after allogeneic or autologous stem cell transplantation, NK cells represent the major lymphocytes in the peripheral Blood, during which they play a major role in the immunity to pathogens (Reititie et al, (1989) Blood 73: 1351-. The role of NK cells in transplantation, graft versus host disease, anti-leukemic activity and post-transplantation infection is reviewed in Lowdell (2003) transfer Medicine 13: 399-404.

Human NK cells mediate the lysis of tumor cells and virus-infected cells by natural cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC).

Human NK cells are controlled by positive and negative cytolytic signals. Negative (inhibitory) signals are transduced by the C-lectin domain containing receptor CD94/NKG2A and some killer immunoglobulin-like receptors (KIR). The regulation of NK lysis by inhibitory signals is called the "self-deletion" hypothesis, in which a specific HLA class I allele expressed on the surface of a target cell is linked to an inhibitory receptor on NK cells. Down-regulation of HLA molecules on tumor cells and some virally infected cells (e.g., CMV) reduces this inhibition below the target threshold, which may be susceptible to NK cell-mediated lysis if the target cells also carry NK triggering and activating molecules. TLR7, TLR8, or TLR9 agonists can activate mdcs and pdcs to produce type I IFNs and express co-stimulatory molecules, such as GITR ligands, followed by activation of NK cells to produce IFN- γ and effectively promote NK cell killing functions.

Inhibitory receptors are divided into two groups, those of the Ig superfamily, known as killer immunoglobulin-like receptors (KIRs), and those of the lectin family NKG2, which forms dimers with CD94 on the cell surface. KIR has an extracellular structure of 2 or 3 domains and binds to HLA-A, -B or-C. HLA-E is a ligand for the NKG2/CD94 complex.

Inhibitory KIRs have up to 4 ITIM-containing intracellular domains, and the best characterized are KIR2DL1, KIR2DL2, and KIR2DL3, which are known to bind HLA-C molecules. KIR2DL2 and KIR2DL3 bind to the group 1HLA-C allele, while KIR2DL1 binds to the group 2 allele. Certain leukemia/lymphoma cells express both group 1 and group 2HLA-C alleles and are known to be resistant to NK-mediated cell lysis.

With respect to positive activation signals, ADCC is thought to be mediated by CD16, and a number of trigger receptors responsible for natural cytotoxicity have been identified, including CD2, CD38, CD69, NKRP-1, CD40, B7-2, NK-TR, NKp46, NKp30, and NKp 44. Several KIR molecules with short intracytoplasmic tails are also stimulatory. These KIRs (KIR2DS1, KIR2DS2 and KIR2DS4) are known to bind HLA-C; their fine exodomains are identical to their associated inhibitory KIRs. Activating KIRs lack ITIM, but are associated with DAP12, leading to NK cell activation. The control mechanisms for inhibitory and activating KIR expression remain unknown.

Some reports describe the expression of TLRs in mouse or human cancer or cancer cell lines. For example, TLRs 1 through TLR6 are expressed by colon, lung, prostate and melanoma mouse tumor cell lines (Huang B, et al, Toll-like receptors on tumor cells efficacy of animal surveillance, Cancer Res.2005,65(12): 5009-; TLR3 is expressed in human breast cancer cells (Salaun B, Coste I, Rissoan MC, Lebecqe SJ, Renno T, TLR3 can direct trigger apoptosis in human cancer cells, J Immunol.2006; 176(8): 4894-; liver and stomach Cancer cells express TLR2 and TLR4(Huang B, et al, Listeria monocytogenes proteins tumor growth via tumor cell toll-like receptor 2 signaling, Cancer res.2007,67(9):4346 and 4352); TLR9(Droemann D, et al., Human lung cancer cells expression functional Toll-like receiver 9, Respir Res.2005,6: 1); and TLR4(He W, Liu Q, Wang L, Chen W, Li N, Cao X, TLR4 signaling proteins antigens of human lung cells by inducing immune cells and apoptosis resistance, Mol Immunol.2007,44(11):2850 and 2859) are expressed by human lung cancer cells. TLR7 and TLR8 are present in tumor cells of human lung cancer (Cherfils-Vicini J, Platonova S, Gillard M, Laurans L, Validire P, Caliandro R, Magdeleeinat P, Mami-Chouaib F, Dieu-Nosjean MC, Fridman WH, Damotte D, Saut des-Fridman C, Cremer I, J.Clin invest.2010,120(4): 1285) -1297).

TLRs are a family of proteins that sense microbial products and/or initiate adaptive immune responses. TLRs activate Dendritic Cells (DCs). TLRs are conserved transmembrane molecules comprising an extracellular domain rich in leucine repeats, a transmembrane domain, and an intracellular TIR (Toll/interleukin receptor) domain. TLRs recognize different structures in microorganisms, commonly referred to as "PAMPs" (pathogen-associated molecular patterns). Binding of ligands to TLRs triggers a series of intracellular signaling pathways that induce the production of inflammation and immune related factors.

In some embodiments, the immunotherapeutic agent is a TLR7 and/or TLR8 agonist. TLR7 and TLR8 are phylogenetically and structurally related. TLR7 is selectively expressed by human pDC and B cells. TLR8 expresses predominantly mdcs, monocytes, macrophages and myeloid suppressor cells. TLR 7-specific agonists activate plasma cell-like dc (pdc) to produce large amounts of type 1 IFN and express high levels of co-stimulatory molecules, promoting activation of T cells, NK cells, B cells, and mdcs. TLR 8-specific agonists activate myeloid DCs, monocytes, macrophages or myeloid-derived suppressor cells to produce large amounts of IFN type 1, IL-12 and IL-23 and express high levels of MHC class I, MHC class II and co-stimulatory molecules, promoting antigen-specific CD4⁺And CD8⁺Activation of T cells.

Dual agonists for TLR7 and TLR8 include resiquimod, CL075(3M002), CL097, native ssRNA, polyU, poly (dT), GU-rich oligonucleotides such as ssRNA40, ssRNA-DR, and ORN 06.

TLR 7-specific agonists include CL264, CL307, gardiquimod (gardiquimod), imiquimod, and loxoribine. TLR 8-specific agonists include TL8-506 and ORN 02.

In some embodiments, the immunotherapeutic agent is a TLR7 and/or TLR8 agonist represented by the structure of formula (I):

wherein the dotted line represents a bond or a bond is absent;

x is S or-NR₁，R₁is-W₀-W₁-W₂-W₃-W4，

W₀Is a bond, alkyl, alkenyl, alkynyl, alkoxy or-alkyl-S-alkyl-,

W₁is a bond, - -O- -or- -NR₂- -, wherein R is₂Is hydrogen, alkyl or alkenyl,

W₂is a bond, - - -O- -, - -C (O) - -C (S) - -or- -S (O)₂-，

W₃Is a bond, - -NR₃- -, wherein R is₃Is hydrogen, alkyl or alkenyl,

n is 0,1, 2,3 or 4;

wherein R is₆、R₇And R₈Independently hydrogen, alkyl, alkenyl, alkoxy, alkylamino, dialkylamino, alkylthio, arylthio, - - (alkyl) -hydroxy, - - (alkyl-C (O) -O- -R₉- -alkyl-C (O) -R₉or-alkyl-OC (O) -R₉Wherein each R is₅Independently is hydrogen, alkyl, haloalkyl, - - -alkyl-aryl or-alkyl-heteroaryl, wherein R is₉Is hydrogen, alkyl, alkenyl, halogen or haloalkyl;

x and Z together may optionally form a (5-9) membered ring.

In some embodiments, X of formula (I) is S.

In some embodiments, X of formula (I) is-NR₁，R₁Is alkyl, - - - -alkyl-W₄- -alkyl-O- -W₄- -alkyl-NH- -C (O) - -W₄- -alkoxy- -NH- -C (O) - -W₄- -alkyl-NH- -C (O) - -NH- -W₄- -alkoxy- -NH- -C (O) - -NH- -W₄- -alkyl-S (O)₂-W₄or-alkyl-NH-C (S) -W₄Wherein W4 is as defined above.

In some embodiments, Z of formula (I) is hydrogen, alkyl, alkoxy, aryl, heteroaryl, haloalkyl, each of which is optionally substituted with one to three substituents selected from the group consisting of hydroxy, alkyl, aryl, heteroaryl, heterocyclyl, cyano, -alkoxy-alkyl, nitro, and-N (R)₅)₂Group of (I) wherein each R₅Independently hydrogen, alkyl, haloalkyl, -alkyl-aryl or-alkyl-heteroaryl.

In some embodiments, Y of formula (I) is-NH₂- -alkyl-NH₂Each of which is optionally substituted with one to three substituents selected from the group consisting of alkyl, alkoxy, alkenyl, and alkynyl.

In some embodiments, n of formula (I) is 1 or 2.

In some embodiments, R of formula (I) is aryl or heteroaryl, each of which is optionally substituted with one to three substituents selected from hydroxy, alkoxy, -alkyl-hydroxy, -O-R₄- -O-alkyl-R₄- -alkyl-O-R₄、--C(O)-R₄、--C(O)-NH-R₄、--C(O)-NR₄R₄- -alkyl-C (O) -R₄- -alkyl-C (O) -O-R₄、--C(O)-O-R₄、--OC(O)-R₄、--S-R₄、--C(O)-S-R₄、--SC(O)-R₄、--S(O)₂-R₄、--NH-S(O)₂-R₄- -alkyl-S-R₄- -alkyl-S (O)₂-R₄、--NHR₄、-NR₄R₄- -NH-alkyl-R₄Halogen, -CN and-SH, wherein R₄Independently hydrogen, alkyl, alkenyl, alkoxy, -alkyl-hydroxy, aryl, heteroaryl, heterocyclyl or haloalkyl.

In some embodiments, the immunotherapeutic agent is a TLR7 and/or TLR8 agonist selected from table 2. The compounds of Table 2 are described and characterized in more detail in U.S. Pat. No. 4,689,338, U.S. Pat. No. 5,389,640, U.S. Pat. No. 5,226,575, U.S. Pat. No.6,110,929, U.S. Pat. No.6,194,425, U.S. Pat. No. 5,352,784, U.S. Pat. No.6,331,539, U.S. Pat. No. 5,482,936, U.S. Pat. No.6,451810, WO2002/46192, WO2002/46193, WO2002/46194, U.S. Pat. No. 2004/0014779 and U.S. Pat. No. 2004/0162309.

TABLE 2 exemplary TLR7 and/or TLR8 agonists

Preferably, in some embodiments, the immunotherapeutic agent is resiquimod or imiquimod.

In some embodiments, the immunotherapeutic agent is a TLR modulator (e.g., a TLR7 and/or TLR8 agonist) represented by the structure of formula (II):

wherein V is-NR₆R₇Wherein R is₆And R₇Each independently of the others is hydrogen, alkyl, alkenyl, alkoxy, alkylamino, dialkylamino, alkylthio, arylthio, - - -alkyl-hydroxy, - - -alkyl-C (O) -O-R₉- -alkyl-C (O) -R₉or-alkyl-OC (O) -R₉Wherein R is₉Is hydrogen, alkyl, alkenyl, hydrogen or haloalkyl;

R₁₀and R₁₁Independently hydrogen, alkyl, alkenyl, aryl, haloalkyl, heteroaryl, heterocyclyl or cycloalkyl, each of whichOptionally substituted with one or more substituents selected from hydroxy, alkoxy, alkyl, alkenyl, alkynyl, halo, - -N (R)₅)₂- -alkoxy-alkyl, - -alkoxy-alkenyl, - -C (O) -alkyl, - -C (O) -O-alkyl, - -C (O) -N (R)₅)₂Aryl, heteroaryl, - -CO-aryl and- -CO-heteroaryl, wherein each R is R₅Independently hydrogen, alkyl, haloalkyl, -alkyl-aryl or-alkyl-heteroaryl,

or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, the immunotherapeutic agent is a TLR modulator (e.g., a TLR7 and/or TLR8 agonist) represented by the structure of formula (III):

whereinIs a double or single bond; r₂And R₃Independently selected from H and lower alkyl, or R₂And R₃Linked to form a saturated carbocyclic ring having 3 to 7 ring members; r₇And R₈One of them is The other is hydrogen; r₄is-NR_cR_dOR-OR₁₀；R_cAnd R_dIs lower alkyl, wherein the alkyl is optionally substituted with one or more-OH; r₁₀Is alkyl, wherein the alkyl is optionally substituted with one or more-OH; z is C andis a double bond, or Z is N andis a single bond; r_aAnd R_bIndependently selected from H, alkyl, alkenyl, alkynyl and R_eWherein alkyl is optionally substituted by one OR more-OR₁₀Or R_eSubstituted, Re is selected from-NH₂-NH (alkyl) and-N (alkyl)₂(ii) a When in useWhen it is a double bond, R₁Is absent, or whenWhen it is a single bond, N₁—R₁And R_aOr R_bIs linked to form a saturated, partially unsaturated or unsaturated heterocyclic ring having 5 to 7 ring members, Ra or R_bThe other of (a) may be hydrogen or absent to accommodate ring unsaturation; and at least one of the following A-D applies: A) r₇Is not hydrogen; B) r₈Is not hydrogen and R_aAnd R_bIs not hydrogen; C) z is N; or D) N₁—R₁And one of Ra or Rb to form a saturated, partially unsaturated, or unsaturated heterocyclic ring having 5-7 ring members. The disclosure of US20140088085a1 is incorporated herein by reference in its entirety.

In some embodiments, R of the compound of formula (III)₇Is composed of

Furthermore, in the compounds of the formula (III), R_aAnd R_bIs not hydrogen, or, for example, R_aAnd R_bOne is alkyl and the other is hydrogen. Further, the alkyl group of formula (III) is substituted with Re. In a different embodiment, R_aAnd R_bBoth are alkyl, or R_aAnd R_bOne of which is Re and the other is hydrogen. For example, R of the formula (III)₈Is not hydrogen.

In some alternative implementationsIn the formula (III) N₁And R_aOr R_bIs linked to form a saturated, partially unsaturated or unsaturated heterocyclic ring having 5-7 ring members, and R_aOr R_bThe other of which is hydrogen, or is absent as necessary to accommodate ring unsaturation, wherein the ring is a 5-membered ring, or, for example, the ring is:

in some embodiments, R in the compound of formula (III)₂And R₃Is not hydrogen, or, for example, R₂And R₃Linked to form a saturated carbocycle, wherein the saturated carbocycle is cyclopropyl. Alternatively, in the compound of formula (III), Z is N.

In some embodiments, the TLR agonist or modulator has the structure of formula (IV):

wherein R is₄Selected from-NR_cR_dand-OR₁₀；R_cAnd R_dIs lower alkyl, wherein the alkyl is optionally substituted with one or more — OH; r₁₀Is alkyl, wherein the alkyl is optionally substituted with one or more — OH; r_fAnd R_gIs lower alkyl or R_fAnd R_gTogether with the nitrogen atom to which they are attached form a saturated heterocyclic ring having 4-6 ring members. For example, R in the compound of formula (IV)_fAnd R_gTogether with the nitrogen atom to which they are attached form a saturated heterocyclic ring, wherein the heterocyclic ring is pyrrolidine.

In some alternative embodiments, R of formula (III) or formula (IV)₄is-OR₁₀Wherein R is₁₀Is alkyl or ethyl. In another embodiment, R of formula (III) or formula (IV)₄is-NR_cR_dWherein both are alkyl or both are propyl. Furthermore, in certain embodimentsIn, R_cOr R_dAt least one of which is alkyl substituted by one-OH or R_cAnd R_dAt least one of which is

And the remaining R_cOr R_dIs propyl.

In some alternative embodiments, the TLR agonist is selected from

The compound of (1).

Alternatively, the compound is selected from

In some alternative embodiments, the TLR agonist compound is

In some alternative embodiments, the TLR agonist is selected from

The compound of (1).

In some alternative embodiments, the TLR agonist is

In some alternative embodiments, the TLR agonist is selected from:

the compound of (1).

In some embodiments, the immunotherapeutic agent is represented by formula (V):

the structures of (a) represent TLR modulators (e.g., TLR7 and/or TLR8 agonists) and metabolites, solvates, tautomers and prodrugs thereof, wherein:

y is CF₂CF₃、CF₂CF₂R⁶Or an aryl or heteroaryl ring, wherein the aryl and heteroaryl rings are independently selected from alkenyl, alkynyl, Br, CN, OH, NR⁶R⁷、C(＝O)R⁸、NR⁶SO₂R⁷、(C₁-C₆Alkyl) amino, R⁶OC(＝O)CH＝CH₂-、SR⁶And SO₂R⁶And wherein the aryl and heteroaryl rings are optionally further independently selected from F, Cl, CF₃、CF₃O-、HCF₂O-, alkyl, heteroalkyl, and ArO-are substituted with one or more groups;

R¹、R³and R⁴Independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, and heteroaryl, wherein alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, and heteroaryl are optionally independently selected from the group consisting of alkyl, alkenyl, alkynyl, F, Cl₅、Br、I、CN、OR⁶、NR⁶R⁷、C(＝O)R⁶、C(＝O)OR⁶、OC(＝O)R⁶、C(＝O)NR⁶R⁷、(C₁-C₆Alkyl) amino, CH₃OCH₂O-、R⁶OC(^O)CH＝CH₂-、NR⁶SO₂R⁷、SR⁶And SO₂R⁶Is substituted with one or more groups of (a),

or R³And R⁴Together with the atoms to which they are attached form a saturated OR partially unsaturated carbocyclic ring, wherein the carbocyclic ring is optionally independently selected from alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN, OR⁶、NR⁶R⁷、C(＝O)R⁶、C(＝O)OR⁶、0C(＝0)R⁶、C(＝O)NR⁶R⁷、(C₁-C₆Alkyl) amino, CH₃OCH₂O-、R⁶OC(＝O)CH＝CH₂-、NR⁶SO₂R⁷、SR⁶And SO₂R⁶Substituted with one or more groups of (a);

R²and R⁸Independently selected from H, OR⁶、NR⁶R⁷Alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, and heteroaryl, wherein alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, and heteroaryl are optionally independently selected from alkyl, alkenyl, alkynyl, F, Cl, Br₅I、CN、OR⁶、NR⁶R⁷、C(＝O)R⁶、C(＝O)OR⁶、OC(＝O)R⁶、C(^O)NR⁶R⁷、(C₁-C₆Alkyl) amino, CH₃OCH₂O-、R⁶OC(＝O)CH＝CH₂-、NR⁶SO₂R⁷、SR⁶And SO₂R⁶Substituted with one or more groups of (a);

R^5a、R^5band R^5cIndependently H, F, Cl, Br, I₅ OMe₅CH₃、CH₂F₅CHF₂Or CF₃；

R⁶And R⁷Independently selected from H₅Alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, and heteroaryl, wherein said alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, and heteroaryl are optionally independently selected from alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN, OR⁶、NR⁶R⁷、C(＝O)R⁶、C(＝O)OR⁶、OC(＝O)R⁶、C(＝O)NR⁶R⁷、(C₁-C₆Alkyl) amino, CH₃OCH₂O-、R⁶OC(^O)CH＝CH₂-、NR⁶SO₂R⁷、SR⁶And SO₂R⁶Is substituted with one or more groups of (a),

or R⁶And R⁷Together with the atoms to which they are attached form a saturated OR partially unsaturated heterocyclic ring, wherein the heterocyclic ring is optionally independently selected from alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN, OR⁶、NR⁶R⁷、C(＝O)R⁶、C(＝O)OR⁶、OC(＝O)R⁶、C(＝O)NR⁶R⁷、(C₁-C₆Alkyl) amino, CH₃OCH₂O-、R⁶OC(＝O)CH＝CH₂-、NR⁶SO₂R⁷、SR⁶And SO₂R⁶Substituted with one or more groups. In certain embodiments, R¹、R³And R⁴Each is hydrogen. In certain embodiments, R^5a、R^5bAnd R^5cEach is hydrogen. The disclosure of WO2007024612a2 is incorporated herein by reference in its entirety.

In some embodiments of the compound of formula (V), R²Is OR⁶. In some embodiments, R⁶Is alkyl, e.g. (C)_1-4) An alkyl group. In certain embodiments, R₆Is ethyl.

In some embodiments of the compound of formula (V), R²Is NR⁶R⁷. In some embodiments, R⁶And R⁷Independently H, alkyl, e.g. (1-6C) alkyl or heteroalkyl, e.g. (C)_1-4Alkoxy radical C_2-4) An alkyl group. In certain embodiments, R⁶And R⁷Independently H, ethyl, propyl or CH₂CH₂OCH₃. In some embodiments of the compounds of formula V, Y is aryl, e.g., phenyl. In some embodiments, aryl is substituted with C (═ O) R⁸Substituted, e.g. in p-R⁸C (═ O) phenyl. In some embodiments, R⁸Is OR⁶、NR⁶R⁷Or a heterocycloalkyl group. In some embodiments, R⁶And R⁷Independently H or alkyl, e.g. (C)_1-6) An alkyl group. In some other embodiments, R⁶And R⁷Together with the nitrogen atom to which they are attached form a 4-6 membered azacycloalkyl ring, such as pyrrolidinyl. In some embodiments, Y is

In some embodiments of the compounds of formula (V), Y is CF₂CF₃。

In some embodiments, the immunotherapeutic agent is a TLR modulator (e.g., a TLR8 agonist) represented by the structure of formula (VI) and metabolites, solvates, tautomers and pharmaceutically acceptable prodrugs and salts thereof:

wherein:

z is H, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, OR⁶Or NR⁶R⁷Wherein alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally independently selected from alkyl, alkenyl, alkynyl, F, Cl₃Br、I、CN、OR⁶、NR⁶R⁷、C(＝O)R⁶、C(＝O)OR⁶、OC(＝O)R⁶、C(＝O)NR⁶R⁷、(C₁-C₆Alkyl) amino, CH₃OCH₂O-、R⁶OCC＝O)CH＝CH₂-、NR⁶SO₂R⁷、SR⁶And SO₂R⁶Substituted with one or more groups of (a);

R¹、R²、R³and R⁴Independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, and heteroaryl, wherein said alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, and heteroaryl are optionally independently selected from the group consisting of alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN, OR⁶、NR⁶R⁷、CC＝O)R⁶、C(＝O)OR⁶、OC(＝O)R⁶、CC＝O)NR⁶R⁷、(C₁-C₆Alkyl) amino, CH₃OCH₂O-、R⁶OCC＝O)CH＝CH₂-、NR⁶SO₂R⁷、SR⁶And SO₂R⁶Is substituted with one or more groups of (a),

or R¹And R²Together with the atoms to which they are attached form a saturated OR partially unsaturated carbocyclic ring, wherein the carbocyclic ring is optionally independently selected from alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN, OR⁶、NR⁶R⁷、C(＝O)R⁶、CC＝O)OR⁶、OC(＝O)R⁶、CC＝O)NR⁶R⁷、CCi-C₆Alkyl) amino, CH₃OCH₂O-、R⁶OCC＝O)CH＝CH₂-、NR⁶SO₂R⁷、SR⁶And SO₂R⁶Substituted with one or more groups of (a);

or R³And R⁴Together are oxo (oxo);

each R⁵Independently selected from H, F, Cl, Br, I, OMe, CH₃、CH₂F、CHF₂、CF₃And CF₂CF₃；

R⁶And R⁷Independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, and heteroaryl, wherein said alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, and heteroaryl are optionally independently selected from the group consisting of alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN, OR⁶、NR⁶R⁷、CC＝O)R⁶、C(＝0)0R⁶、0C(＝0)R⁶、CC＝O)NR⁶R⁷、(C₁-C₆Alkyl) amino, CH₃OCH₂O-、R⁶OC(＝O)CH＝CH₂-、NR⁶SO₂R⁷、SR⁶And SO₂R⁶Is substituted with one or more groups of (a),

or R⁶And R⁷Together with the atoms to which they are attached form a saturated OR partially unsaturated heterocyclic ring, wherein the heterocyclic ring is optionally independently selected from alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN, OR⁶、NR⁶R⁷、CC＝O)R⁶、C(＝0)0R⁶、0C(＝0)R⁶、CC＝O)NR⁶R⁷、(C₁-C₆Alkyl) amino, CH₃OCH₂O-、R⁶OC(＝O)CH＝CH₂-、NR⁶SO₂R⁷、SR⁶And SO₂R⁶Substituted with one or more groups of (a); n is O, 1,2,3 or 4. The disclosure of WO2007040840a2 is incorporated herein by reference in its entirety.

In some embodiments, the immunotherapeutic agent is a TLR modulator (e.g., a TLR7 and/or 8 agonist) represented by the structure of formula (VI) and metabolites, solvates, tautomers and pharmaceutically acceptable salts and prodrugs thereof:

wherein: z is H, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, OR⁶Or NR⁶R⁷Wherein said alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally independently selected from alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN, OR⁶、NR⁶R⁷、C(＝O)R⁶、C(＝O)OR⁶、OC(＝O)R⁶、C(＝O)NR⁶R⁷、(C₁-C₆Alkyl) amino, CH₃OCH₂O-、R⁶OCC＝O)CH＝CH₂-、NR⁶SO₂R⁷、SR⁶And SO₂R⁶Substituted with one or more groups of (a);

R¹、R²、R³and R⁴Independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, and heteroaryl, wherein said alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, and heteroaryl are optionally independently selected from the group consisting of alkyl, alkenyl, alkynyl, F, Cl, Br, Y, and Z,I₉CN、OR⁶、NR⁶R⁷、CC＝0)R⁶、C(＝O)OR⁶、OC(＝O)R⁶、C(＝O)NR⁶R⁷、(C₁-C₆Alkyl) amino, CH₃OCH₂O-、R⁶OCC＝O)CH＝CH₂-、NR⁶SO₂R⁷、SR⁶And SO₂R⁶Is substituted with one or more groups of (a),

or R¹And R²Together with the atoms to which they are attached form a saturated OR partially unsaturated carbocyclic ring, wherein the carbocyclic ring is optionally independently selected from alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN, OR⁶、NR⁶R⁷、C(＝0)R⁶、C(＝O)OR⁶、OC(＝O)R⁶、C(＝O)NR⁶R⁷、(C₁-C₆Alkyl) amino, CH₃OCH₂O-、R⁶OC(＝O)CH＝CH₂-、NR⁶SO₂R⁷、SR⁶And SO₂R⁶Substituted with one or more groups of (a);

or R³And R⁴Together are oxo (oxo);

R⁵is H, F, Cl, Br, I, OMe, CH₃、CH₂F、CHF₂、CF₃And CF₂CF₃；

R⁶And R⁷Independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, and heteroaryl, wherein said alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, and heteroaryl are optionally independently selected from the group consisting of alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN, OR⁶、NR⁶R⁷、C(＝O)R⁶、C(＝0)0R⁶、0C(＝0)R⁶、C(＝O)NR⁶R⁷、(C₁-C₆Alkyl) amino, CH₃OCH₂O-、R⁶OC(＝O)CH＝CH₂-、NR⁶SO₂R⁷、SR⁶And SO₂R⁶Is substituted with one or more groups of (a),

or R⁶And R⁷Together with the atoms to which they are attached form a saturated OR partially unsaturated heterocyclic ring, wherein the heterocyclic ring is optionally independently selected from alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN, OR⁶、NR⁶R⁷、C(＝O)R⁶、C(＝O)OR⁶、OC(＝O)R⁶、CC＝O)NR⁶R⁷、(C₁-C₆Alkyl) amino, CH₃OCH₂O-、R⁶OC(＝O)CH＝CH₂-、NR⁶SO₂R⁷、SR⁶And SO₂R⁶Substituted with one or more groups of (a);

n is O, 1,2,3 or 4.

In some embodiments, Z is OR⁶. In some embodiments, R⁶Is an alkyl group, such as a (1-6C) alkyl group. In certain embodiments, R⁶Is ethyl, propyl, isopropyl or isobutyl.

In some embodiments, Z is NR⁶R⁷. In some embodiments, R⁶And R⁷Independently H or an alkyl group, such as a (1-6C) alkyl group. In some embodiments, R⁶And R⁷Is ethyl. In some embodiments, n is O or 1.

In some embodiments, R⁵Is CF₂CF₃. In certain embodiments, R³Is H or alkyl, e.g. (1-4C) alkyl, and R⁴Is H. In certain embodiments, R is alkyl, such as (1-4C) alkyl. In some embodiments, R is methyl. In other particular embodiments, R³Is H. In some embodiments, R is H or alkyl, e.g., (1-4C) alkyl and R is H. In some embodiments, R¹Is an alkyl group. In some embodiments, R¹Is methyl. In some particular embodiments, R¹Is H.

In some embodiments, the TLR7 and/or TLR8 agonist is represented by the structure of formula (XV):

wherein the ring A represents a 6-10 membered aromatic carbocyclic ring or a 5-10 membered heteroaromatic ring;

r represents a halogen atom, an alkyl group, a hydroxyalkyl group, a haloalkyl group, an alkoxy group, a hydroxyalkoxy group, a haloalkoxy group, an amino group, an alkylamino group, a dialkylamino group or a 4-7 membered cyclic group containing 1-2 hetero atoms selected from 1-2 nitrogen atoms and optionally 0-1 oxygen atom or 0-1 sulfur atom in the ring;

n represents an integer of 0 to 2, and when n is 2, Rs may be the same or different;

Z¹represents a substituted or unsubstituted alkylene group or a substituted or unsubstituted cycloalkylene group;

X²represents oxygen atom, sulfur atom, SO₂、NR⁵、CO、CONR⁵、NR⁵CO、SO₂NR⁵、NR⁵SO₂、NR⁵CONR⁶Or NR⁵CSNR⁶(wherein R is⁵And R⁶Each independently is a hydrogen atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted cycloalkyl group);

Y¹、Y²and Y³Each independently represents a single bond or an alkylene group;

X¹represents oxygen atom, sulfur atom, SO₂、NR⁴(wherein R is⁴Is a hydrogen atom or an alkyl group) or a single bond;

R²represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, or a substituted or unsubstituted cycloalkyl group; and

R¹represents a hydrogen atom, a hydroxyl group, an alkoxy group, an alkoxycarbonyl group, a haloalkyl group, a haloalkoxy group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group or a substituted or unsubstituted cycloalkyl group. The linker is attached to one of the possible attachment sites of the agonist, e.g. to-NH₂。

In some embodiments, R¹Represents hydrogen, hydroxy or C₁-C₆Alkoxy radical, C₂-C₅Alkoxycarbonyl group, C₁-C₆Haloalkyl, C₁-C₆Haloalkoxy, C₆-C₁₀Aryl radical, C₅-C₁₀Heteroaryl or C₃-C₈Cycloalkyl, each group optionally independently selected from halogen, hydroxy, C₁-C₆Alkyl radical, C₁-C₆Haloalkyl, C₁-C₆Alkoxy radical, C₁-C₆Haloalkoxy, C₂-C₅Alkoxycarbonyl, amino (NH)₂) And (mono) -C₁-C₆Alkylamino and (di) -C₁-C₆(ii) one or more substituents of alkylamino;

Y¹represents a single bond or C₁-C₆An alkylene group;

X¹represents a single bond, an oxygen, sulfur atom, a sulfonyl group (SO)₂) Or NR³；

Z¹Represents C₂-C₆Alkylene or C₃-C₈Cycloalkylene, each optionally substituted with at least one hydroxyl group;

X²represents NR⁴；

Y²Represents a single bond or C₁-C₆An alkylene group;

Y³represents a single bond or C₁-C₆An alkylene group;

n is an integer 0,1 or 2;

r represents halogen or C₁-C₆Alkyl radical, C₁-C₆Hydroxyalkyl radical, C₁-C₆Haloalkyl, C₁-C₆Alkoxy radical, C₁-C₆Hydroxyalkoxy, C₁-C₆Haloalkoxy, amino (NH)₂) And (mono) -C₁-C₆Alkylamino, (di) -C₁-C₆Alkylamino or C₃-C₈A saturated heterocyclic ring comprising a ring nitrogen atom and optionally independently selected from nitrogen, and mixtures thereof,Oxygen and one or more other heteroatoms of sulfur, said heterocycle being optionally independently selected from halogen, hydroxy, oxo, C₁-C₆Alkyl radical, C₁-C₆Alkoxy radical, C₂-C₅Alkylcarbonyl and C₂-C₅One or more substituents of alkoxycarbonyl;

R²represents hydrogen or C₁-C₆Alkyl radical, C₂-C₆Alkenyl radical, C₂-C₆Alkynyl or C₃-C₈Cycloalkyl, each group optionally being independently selected from halogen, hydroxy or C₁-C₆Alkoxy radical, C₂-C₁₀Substituted by one or more substituents of acyloxy, C₂-C₁₀Acyloxy is selected from C_2-5Alkyl carbonyl oxy, C₂-C₅Alkenyl carbonyloxy, C₂-C₅Alkynyl carbonyloxy, C₆-C₉Aryl carbonyloxy and C₅-C₉Heteroarylcarbonyloxy, wherein each acyloxy group may optionally be independently selected from halogen, hydroxy, C₁-C₃Alkoxy and phenyl, provided that the total number of carbon atoms in the acyloxy groups does not exceed 10, amino (NH)₂) And (mono) -C₁-C₆Alkylamino, (di) -C₁-C₆Alkylamino and C comprising a ring nitrogen atom and optionally one or more further heteroatoms independently selected from nitrogen, oxygen and sulfur₃-C₈A saturated heterocycle which in turn is optionally independently selected from halogen, hydroxy, oxo, C₁-C₆Alkyl radical, C₁-C₆Alkoxy radical, C₂-C₅Alkylcarbonyl and C₂-C₅One or more substituents of alkoxycarbonyl;

R³represents hydrogen or C₁-C₆An alkyl group;

R⁴represents CO₂R⁵、SO₂R⁵、COR⁵、SO₂NR⁶R⁷And CONR⁶R⁷；

R⁵Independently represent

(i) Containing 1 or2 radicals selected from cyclic radicals NR⁸、S(O)_mOr a heteroatom of oxygen, 3 to 8 membered heterocyclic ring, the 3 to 8 membered heterocyclic ring optionally being independently selected from halogen, hydroxy or C₁-C₆Alkyl and C₁-C₆Substituted by one or more substituents of alkoxy, or

(ii)C₆-C₁₀Aryl or C₅-C₁₀Heteroaryl, each of which may optionally be independently selected from halogen, cyano, C₁-C₆Alkyl radical, C₁-C₃Haloalkyl, carboxyl, S (O)_mR⁹、OR¹⁰、CO₂R¹⁰、SO₂NR¹⁰R¹¹、CONR¹⁰R¹¹、NR¹⁰R¹¹、NR¹⁰SO₂R⁹、NR¹⁰CO₂R⁹、NR¹⁰COR⁹Is substituted by one or more substituents of (a), or

(iii)C₁-C₆Alkyl radical, C₂-C₆Alkenyl radical, C₂-C₆Alkynyl or C₃-C₈Cycloalkyl, each of which may optionally be independently selected from halogen, CN, C₃-C₈Cycloalkyl, S (O)_pR¹²、OR¹³、COR¹³、CO₂R¹³、SO₂NR¹³R¹⁴、CONR¹³R¹⁴、NR¹³R¹⁴、NR¹³SO₂R¹²、NR¹³CO₂R¹²、NR¹³COR¹²、NR¹³SO₂R¹²Or C₆-C₁₀Aryl or C₅-C₁₀Heteroaryl or heterocyclic, the last three groups being optionally substituted by one or more substituents independently selected from C₁-C₆Alkyl (optionally substituted by hydroxy, C₁-C₆Alkoxy radical, C₁-C₆Alkoxycarbonyl, amino, C₁-C₆Alkylamino radical, di-C₁-C₆Alkylamino radical, NH₂C(O)—、C₁-C₆Alkyl NHC (O), di-C₁-C₆Alkyl radicals NC (O) -OCH₂CH₂OH, pyrrolidinyl, pyrrolidinylcarbonyl, furyl, piperidinyl, methylpiperidinyl or phenyl substitution), C₂-C₆Alkenyl (optionally substituted with phenyl), halogen, hydroxy, cyano, carboxy, amino, C₁-C₆Alkylamino radical, di-C₁-C₆Alkylamino radical, NH₂C(O)—、C₁-C₆Alkyl NHC (O) -, di-C₁-C₆Alkyl radicals NC (O), C₁-C₆Alkoxycarbonyl group, C₁-C₆Alkylsulfonyl radical, C₁-C₆Alkylcarbonylamino, C₁-C₆Alkylcarbonylmethylamino, phenyl (optionally substituted with hydroxy, fluoro or methyl), pyrrolidinyl, pyridinyl, piperidinyl, benzothiazolyl or pyrimidinyl;

R⁶represents hydrogen or C₁-C₆Alkyl radical, C₂-C₆Alkenyl radical, C₂-C₆Alkynyl, C₃-C₈Cycloalkyl or heterocycle, each of which may be optionally independently selected from halogen, hydroxy, oxo, cyano, C₁-C₆Alkyl radical, C₂-C₆Alkenyl radical, C₂-C₆Alkynyl, C₃-C₈Cycloalkyl, OR¹⁵、S(O)_qR¹⁵、CO₂R¹⁶、COR¹⁶、NR¹⁶R¹⁷、CONR¹⁶R¹⁷、NR¹⁶COR¹⁷、NR¹⁶CO₂R¹⁵、SO₂NR¹⁶R¹⁷、NR¹⁶SO₂R¹⁵、C₆-C₁₀Aryl or C₅-C₁₀Heteroaryl or heterocyclic, the last three groups optionally being independently selected from C₁-C₆Alkyl radical, C₃-C₈Cycloalkyl, halogen, S (O)_qR¹⁵、CO₂R¹⁶、COR¹⁶Hydroxy or cyano; and

R⁷represents hydrogen, C₁-C₆Alkyl radical, C₂-C₆Alkenyl radical, C₂-C₆Alkynyl or C₃-C₈Cycloalkyl, each group optionally being independently selected from halogen, C₃-C₈Cycloalkyl radical, C₆-C₁₀Aryl or C₅-C₁₀Heteroaryl, carboxy, cyano, OR¹⁵Hydroxy or NR¹⁸R¹⁹Is substituted by one or more substituents of (a), or

R⁶And R⁷Together with the nitrogen atom to which they are attached form a 3-to 8-membered saturated or partially saturated heterocyclic ring, optionally further comprising a nitrogen atom, S (O)_mOR oxygen, said heterocycle being optionally independently selected from halogen, hydroxy, carboxy, cyano, OR²⁰、NR²¹R²²、S(O)_qR²³、COR²⁴、CO₂R²⁴、NR²⁴R²⁵、CONR²⁴R²⁵、NR²⁴COR²⁵、NR²⁴CO₂R²³、SO₂NR²⁴R²⁵、NR²⁴SO₂R²³、C₆-C₁₀Aryl radical, C₅-C₁₀Heteroaryl, heterocycle, C₁-C₆Alkyl radical, C₂-C₆Alkenyl radical, C₂-C₆Alkynyl or C₃-C₈Cycloalkyl optionally substituted with one OR more substituents independently selected from halogen, hydroxy, oxo, cyano, OR²⁰、S(O)_qR²³、COR²⁴、CO₂R²⁴、NR²⁴R²⁵、CONR²⁴R²⁵、NR²⁴CO₂R²³、NR²⁴COR²⁵、SO₂NR²⁴R²⁵、NR²⁴SO₂R²³Hetero ring or C₆-C₁₀Aryl or C₅-C₁₀Heteroaryl, the last three groups being optionally substituted by one or more substituents independently selected from C₁-C₆Alkyl, halogen, hydroxy and cyano;

R⁸represents hydrogen, CO₂R²⁶、COR²⁶、SO₂R²⁶、C₁-C₆Alkyl or C₃-C₆Cycloalkyl, each group optionally being independently selected from halogen, hydroxy and NR²⁷R²⁸Substituted with one or more substituents of (a);

R¹⁰、R¹¹、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²¹、R²²、R²⁶、R²⁷or R²⁸Each independently represents hydrogen, C₁-C₆Alkyl or C₃-C₆A cycloalkyl group;

R²⁴and R²⁵Each independently represents hydrogen or C₁-C₆Alkyl or C₃-C₆A cycloalkyl group; or

R²⁴And R²⁵Together with the nitrogen atom to which they are attached form a 3-to 8-membered saturated or partially saturated heterocyclic ring, optionally further comprising a nitrogen atom, S (O)_mOr other heteroatoms or heterogroups of oxygen;

R⁹、R¹²、R¹⁵and R²³Represents C₁-C₆Alkyl or C₃-C₆A cycloalkyl group;

R¹³and R¹⁴Are each as defined as R⁶And R⁷；

R²⁰Represents optionally independently selected from halogen, hydroxy OR OR²³C substituted by one or more substituents of₁-C₆An alkyl group;

m, p, q and r each independently represent an integer of 0,1 or 2; and

a represents C₆-C₁₀Aryl or C₅-C₁₂A heteroaryl group. See WO2008004948a1, US8,138,172 and US8,575,180, the disclosures of which are incorporated by reference in their entirety.

In some embodiments, the TLR7 and/or TLR8 agonist has the following structure:

wherein R is Me or H.

In some embodiments, the TLR7 and/or TLR8 agonist has the following structure:

in some embodiments, the TLR7 and/or TLR8 agonist has the structure of formula (XVI):

wherein: r¹Independently is H, -C (O) R³Or a racemic, L-or D-amino acid radical

-C(O)CHNH₂R⁴Wherein R is³Is substituted or unsubstituted alkyl, R⁴Is H, or substituted or unsubstituted alkyl;

R²is H, O, OR⁵Or N (R)⁶)₂Wherein R is⁵Independently is H or alkyl, and wherein R⁶Independently is H, substituted or unsubstituted alkyl, cycloalkyl or together with the nitrogen forms a substituted or unsubstituted heterocycloalkyl ring; and wherein if R is-OH, at least one of the R groups is a racemic, L-or D-amino acid group-C (O) CHNH₂R⁴. See US6,924,271, the disclosure of which is incorporated by reference in its entirety.

In some embodiments, R¹In the groupAt least one being a racemic, L-or D-amino acid group-C (O) CHNH₂R⁴Wherein R is⁴Is a substituted or unsubstituted alkyl group, and wherein the remainder of R¹The group is H; r²Is OR⁵Or N (R)⁶)₂Wherein R is⁵Independently selected from H or alkyl, and wherein R is independently H, substituted or unsubstituted alkyl, cycloalkyl or together with the nitrogen forms a substituted or unsubstituted heterocycloalkyl ring.

In some embodiments, at least one R is¹The group being L-amino acid group-C (O) CHNH₂R⁴Wherein R is⁴Is a substituted or unsubstituted alkyl group, and wherein the remainder of R¹The group is H; r²Is OR⁵Or N (R)⁶)₂Wherein R is⁴Is a substituted alkyl group, and wherein R⁶Independently is H or substituted or unsubstituted alkyl.

In some embodiments, at least one R is¹The group being L-amino acid group-C (O) CHNH₂R, wherein R⁴is-CH (CH)₃)₂And wherein the remaining R¹The group is H; r²Is OH.

In some embodiments, the TLR7 and/or agonist is selected from the group consisting of:

group (d) of (a).

In some embodiments, the TLR7 and/or TLR8 agonist has:

in the structure of (a) to (b),

wherein:

each R¹Is H, or a substituted or unsubstituted alkyl, alkenyl or alkynyl group which may be interrupted by one or more O, S or N heteroatoms, or a substituted or unsubstituted aryl or heteroaryl group;

R²is H, OH, SH, halogen or a substituted or unsubstituted alkyl, alkenyl or alkynyl group, which may be interrupted by one or more O, S or N heteroatoms, or substituted or unsubstituted-O- (alkyl), -O- (aryl), -O- (heteroaryl), -S- (alkyl), -S- (aryl), -S- (heteroaryl), aryl or heteroaryl;

R³is H, OH or SH, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, -O- (alkyl), -O- (aryl), -O- (heteroaryl), -S- (alkyl), -S- (aryl), -S- (heteroaryl), -NH (alkyl), -NH (aryl), -NH (heteroaryl), -NH (R) (alkyl)⁴) (alkyl) - (NH) (R)⁴) (aryl) or-NH (R)⁴) (heteroaryl) wherein R is⁴Is a substituted or unsubstituted alkyl group;

x is O or S;

y is H, halogen, OH, OR⁴、SH、SR⁴Or a substituted or unsubstituted alkyl or aryl group;

z is H, halogen, OH, OR⁴SH or SR⁴. See US 7,576,068, the disclosure of which is incorporated by reference in its entirety.

In some embodiments, the TLR7 and/or TLR8 agonist has the structure of formula (XVIII):

wherein:

Y-Z is-CR⁴R⁵—、—CR⁴R⁵—CR⁴R⁵—、—C(O)CR⁴R⁵—、—CR⁴R⁵C(O)—、—NR⁸C(O)—、—C(O)NR⁸—、—CR⁴R⁵S(O)₂-or-CR⁵═CR⁵—；

L¹is-NR⁸—、—O—、—S—、—N(R⁸)C(O)—、—S(O)₂—、—S(O)—C(O)N(R⁸)—、—N(R⁸)S(O)₂—、—S(O)₂N(R⁸) -or a covalent bond;

R¹is alkyl, substituted alkyl, haloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, heteroalkyl, substituted heteroalkyl, carbocyclyl, substituted carbocyclylalkyl, heterocyclic, substituted heterocyclic, heterocyclylalkyl, or substituted heterocyclylalkyl, arylalkyl, substituted arylalkyl, heteroarylalkyl, substituted heteroarylalkyl, carbocyclylheteroalkyl, substituted carbocyclylheteroalkyl, heterocyclylheteroalkyl, substituted heterocyclylheteroalkyl, arylheteroalkyl, substituted arylheteroalkyl, heteroarylheteroalkyl, or substituted heteroarylheteroalkyl;

X¹is alkylene, substituted alkylene, heteroalkylene, substituted heteroalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, carbocycle, substituted carbocycle, heterocyclylene, substituted heterocyclylene, — NR⁸—、—O—、—C(O)—、—S(O)—、S(O)₂-or a bond;

d is carbocyclyl, substituted carbocyclyl, heterocyclyl or substituted heterocyclyl wherein said carbocyclyl, substituted carbocyclyl, heterocyclyl or substituted heterocyclyl is substituted with one or two-L²-NR⁶R⁷Substitution; or

D is a heterocyclyl, substituted heterocyclyl, heteroaryl, or substituted heteroaryl, wherein the heterocyclyl, substituted heterocyclyl, heteroaryl, or substituted heteroaryl contains one to four nitrogen atoms;

each L²Independently is alkylene, substituted alkylene, heteroalkylene, substituted heteroalkylene, or a covalent bond;

each R³Independently halogen, cyano, azido, nitro, alkyl, substituted alkyl, hydroxy, amino, heteroalkyl, substituted heteroalkyl, alkoxy, haloalkyl, haloalkoxy, — CHO, — c (o) OR⁸、—S(O)R⁸、—S(O)₂R⁸；—C(O)NR⁹R¹⁰、—N(R⁹)C(O)R⁸Carbocyclyl, substituted carbocyclyl, carbocyclylalkyl, substituted carbocyclylalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, — s (o)₂NR⁹R¹⁰、—N(R⁹)S(O)₂R⁸、—N(R⁹)S(O)₂OR¹⁰、—OS(O)₂NR⁹R¹⁰；

n is 0,1, 2,3,4 or 5;

R⁴and R⁵Each independently is H, alkyl, substituted alkyl, haloalkyl, heteroalkyl, substituted heteroalkyl, carbocyclyl, substituted carbocyclyl, carbocyclylalkyl, substituted carbocyclylalkyl, heterocyclyl, substituted heterocyclyl, heterocyclylalkyl, substituted heterocyclylalkyl, arylalkyl, substituted arylalkyl, heteroarylalkyl, substituted heteroarylalkyl, carbocyclylheteroalkyl, substituted carbocyclylheteroalkyl, heterocyclylheteroalkyl, substituted heterocyclylheteroalkyl, arylheteroalkyl, substituted arylheteroalkyl, heteroarylheteroalkyl, OR substituted heteroarylheteroalkyl, cyano, azido, OR⁸、—C(O)H、—C(O)R⁸、—S(O)R⁸、—S(O)₂R⁸、—C(O)OR⁸or-C (O) NR⁹R¹⁰(ii) a Or

R⁴And R⁵Together with the carbon to which they are attached form a carbocyclic ring, substituted carbocyclic ring, heterocyclic ring, or substituted heterocyclic ring; or

When on the same carbon atom, R⁴And R⁵Together with the carbon to which they are attached, is-C (O) -or-C (NR)⁸) A; or

Two R on adjacent carbon atoms⁴Or two R⁵Together with the carbon to which they are attached form a 3-to 6-membered carbocyclic ringA substituted carbocyclic ring, heterocyclic ring or substituted heterocyclic ring;

R⁶and R⁷Each independently is H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, haloalkyl, heteroalkyl, substituted heteroalkyl, carbocyclyl, substituted carbocyclyl, carbocyclylalkyl, substituted carbocyclylalkyl, heterocyclyl, substituted heterocyclyl, heterocyclylalkyl, substituted heterocyclylalkyl, aralkyl, substituted aralkyl, heteroaralkyl, substituted heteroaralkyl, carbocyclylheteroalkyl, substituted carbocyclylheteroalkyl, heterocyclylheteroalkyl, arylheteroalkyl, substituted arylheteroalkyl, heteroarylheteroalkyl, or substituted heteroarylheteroalkyl, -C (O) H, -C (O) R⁸、—S(O)R⁸、—S(O)₂R⁸、—C(O)OR⁸or-C (O) NR⁹R¹⁰、S(O)₂NR⁹R¹⁰(ii) a Or

R⁶And R⁷Together with the nitrogen to which they are attached form a substituted or unsubstituted heterocyclic ring which may contain one or more additional heteroatoms selected from N, O, P or S; or

R⁷And L²And the N to which they are attached, together form a substituted or unsubstituted 3 to 8 membered heterocyclic ring which may contain one or more additional heteroatoms selected from N, O, S or P;

R⁸is H, alkyl, substituted alkyl, haloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, heteroalkyl, substituted heteroalkyl, carbocyclyl, substituted carbocyclyl, carbocyclylalkyl, substituted carbocyclylalkyl, heterocyclyl, substituted heterocyclyl, heterocyclylalkyl, substituted heterocyclylalkyl, arylalkyl, substituted arylalkyl, heteroarylalkyl, substituted heteroarylalkyl, carbocyclylheteroalkyl, substituted carbocyclylheteroalkyl, heterocyclylheteroalkyl, substituted heterocyclylheteroalkyl, arylheteroalkyl, substituted arylheteroalkyl, heteroarylheteroalkyl, or substituted heteroarylheteroalkyl;

R⁹and R¹⁰Each independently is H, alkyl, substituted alkyl, alkenyl, substitutedAlkenyl, alkynyl, substituted alkynyl, haloalkyl, heteroalkyl, substituted heteroalkyl, carbocyclyl, substituted carbocyclyl, carbocyclylalkyl, substituted carbocyclylalkyl, heterocyclyl, substituted heterocyclyl, heterocyclylalkyl, substituted heterocyclylalkyl, arylalkyl, substituted arylalkyl, heteroarylalkyl, substituted heteroarylheteroalkyl, carbocyclylheteroalkyl, substituted carbocyclylheteroalkyl, heterocyclylheteroalkyl, substituted heterocyclylheteroalkyl, arylheteroalkyl, substituted arylheteroalkyl, heteroarylheteroalkyl, or substituted heteroarylheteroalkyl of (a); or

R⁹And R¹⁰Together with the nitrogen to which they are bonded form a substituted or unsubstituted heterocyclic ring;

wherein each substituted alkyl, substituted alkenyl, substituted alkynyl, substituted heteroalkyl, substituted carbocyclyl, substituted carbocyclylalkyl, substituted heterocyclyl, substituted heterocyclylalkyl, substituted arylalkyl, substituted heteroarylalkyl, substituted carbocyclylheteroalkyl, substituted heterocyclylheteroalkyl, substituted arylheteroalkyl, substituted heteroarylheteroalkyl, substituted alkylene, substituted heteroalkylene, substituted alkenylene, substituted alkynylene, substituted carbocyclylene, or substituted heterocyclylene is independently selected from-halo, -R, -O, and^-、═O、—OR、—SR、—S^-、—NR₂、—N(+)R₃═ NR, — C (halogen)₃-CR (halogen)₂、—CR₂(halogen), -CN, -OCN, -SCN, -N ═ C ═ O, -NCS, -NO₂、═N₂、—N₃、—NRC(═O)R、—NRC(═O)OR、—NRC(═O)NRR、—C(═O)NRR、—C(═O)OR、—OC(═O)NRR、—OC(═O)OR、—C(═O)R、—S(═O)₂OR、—S(═O)₂R、—OS(═O)₂OR、—S(═O)₂NR、—S(═O)R、—NRS(═O)₂R、—NRS(═O)₂NRR、—NRS(═O)₂OR、—OP(═O)(OR)₂、—P(═O)(OR)₂、—P(O)(OR)(O)R、—C(═O)R、—C(═S)R、—C(═O)OR、—C(═S)OR、—C(═O)SR、—C(═S)SR、—C(═O)NRR、—C(═S)NRR、

-C (═ NR) NRR and-NRC (═ NR) NRR; wherein each R is independently H, alkyl, cycloalkyl, aryl, arylalkyl, or heterocyclyl. See US 20100143301 a1, the disclosure of which is incorporated by reference in its entirety.

In some embodiments, the TLR7 and/or TLR8 agonist has the structure:

wherein:

L¹is-NH-or-O-;

R¹is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, heterocyclylalkyl, substituted heterocyclylalkyl, carbocyclylalkyl, or substituted carbocyclylalkyl;

R⁴and R⁵Each independently is H or C₁-C₆Alkyl or R⁴And R⁵Together with the carbon to which they are attached is-c (o) -;

X¹is C₁-C₆Alkylene radical, C₁-C₆Heteroalkylene or C₁-C₆Substituted heteroalkylene groups;

d is phenyl, biphenyl or pyridyl, wherein said phenyl, biphenyl or pyridyl is substituted by-L²-NR⁶R⁷Substitution; or

D is pyridyl, piperidyl, piperazinyl or1, 2,3, 4-tetrahydroisoquinolinyl;

n is 0 or 1;

R³is halogen, cyano, alkyl, carbocyclyl, carbocyclylalkyl, haloalkyl, -C (O) OR⁶、—C(O)NR⁹R¹⁰Or — CHO;

L²is C₁-C₆An alkylene or covalent bond;

R⁶and R⁷Each independently is H, alkyl or heteroaryl; or

R⁶And R⁷Together with the nitrogen to which they are attached form a substituted or unsubstituted 4-6 membered heterocyclic ring containing 0 to 2 heteroatoms selected from N, O or S.

In some embodiments, the TLR7 and/or TLR8 agonist has the following structure:

C. amount of immunotherapeutic agent in therapeutic combination

In another aspect, embodiments of the present disclosure provide a therapeutic combination comprising a chemotherapeutic agent and an immunotherapeutic agent in amounts suitable for combination therapy treatment of diseases such as tumors and cancers.

In some embodiments, the amount of immunotherapeutic agent is capable of: (1) inducing IFN- α in enriched human blood DC; (2) inducing TNF- α in enriched human blood DC; and/or (3) inducing IL-12-alpha in enriched human blood DCs.

Methods of measuring the activity of an immunotherapeutic agent include: 1) an assay that measures cytokine release from human dendritic cells stimulated by an immunotherapeutic agent; and 2) efficacy studies in immunotherapeutic-treated tumor models.

In some embodiments, an immunotherapeutic agent (e.g., resiquimod or an analog thereof) is administered orally or intravenously using an oral formulation or an intravenous formulation, respectively, in amounts such that the local concentration of the immunotherapeutic agent (e.g., near or at the tumor site of a solid tumor) is from about 0.005 μ g/ml to about 1,2,3,4, 5,6, 7,8,9, 10, 11, or 12 μ g/ml (all inclusive).

Local concentrations of immunotherapeutic agents (e.g., at or near the tumor site of a solid tumor) can be measured using methods known in the art, e.g., measuring tissue or serum concentrations. The locally effective concentration of the therapeutic agent depends on its absorption from various pathways, tissue distribution, and metabolic processes, and the plasma pharmacokinetics of the agent and tissue concentrations can be routinely measured using methods known in the art.

In some embodiments, the immunotherapeutic agent is administered in an amount such that the local concentration of the immunotherapeutic agent (e.g., at or near the tumor site of a solid tumor) is from about 0.05 μ g/ml, 0.1 μ g/ml, 0.15 μ g/ml, 0.2 μ g/ml, 0.3 μ g/ml, or 0.4 μ g/ml, to about 0.5 μ g/ml (all inclusive).

In some embodiments, the subject is administered an oral formulation comprising an immunotherapeutic agent (e.g., resiquimod or an analog thereof) twice weekly at a dose of about 0.001mg/kg to about 0.0005mg/kg, 0.0006mg/kg, 0.0007mg/kg, 0.0008mg/kg, 0.0009mg/kg, 0.001mg/kg, 0.002mg/kg, 0.003mg/kg, 0.004mg/kg, 0.005mg/kg, 0.006mg/kg, 0.007mg/kg, 0.008mg/kg, 0.009mg/kg, 0.01mg/kg, or 0.015mg/kg, to about 0.02mg/kg, all inclusive. In some embodiments, the subject is administered an oral formulation comprising an immunotherapeutic agent (e.g., resiquimod or an analog thereof) twice weekly at a dose of about 0.0001 to about 0.0005, to about 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.015, or 0.02mg/kg, all inclusive.

In some embodiments, the subject administers an intravenous formulation comprising an immunotherapeutic agent (e.g., resiquimod or an analog thereof) at a dose of about 0.0005mg/kg, 0.0006mg/kg, 0.0007mg/kg, 0.0008mg/kg, 0.0009mg/kg, 0.001mg/kg, 0.002mg/kg, 0.003mg/kg, 0.004mg/kg, 0.005mg/kg, 0.006mg/kg, 0.007mg/kg, 0.008mg/kg, 0.009mg/kg, 0.01mg/kg, or about 0.015mg/kg, including to about 0.02mg/kg once a week. In some embodiments, the subject administers an intravenous formulation comprising an immunotherapeutic agent (e.g., resiquimod or an analog thereof) at a dose of about 0.0005mg/kg, to about 0.0006mg/kg, 0.0007mg/kg, 0.0008mg/kg, 0.0009mg/kg, 0.001mg/kg, 0.002mg/kg, 0.003mg/kg, 0.004mg/kg, 0.005mg/kg, 0.006mg/kg, 0.007mg/kg, 0.008mg/kg, 0.009mg/kg, 0.01mg/kg, 0.015mg/kg, or 0.02mg/kg, inclusive, once a week.

In some embodiments, the method comprises administering to the subject an intravenous formulation comprising the immunotherapeutic agent (e.g., resiquimod or an analog thereof) at a dose of about 0.0008mg/kg to about 0.0133mg/kg once a week.

In some embodiments, the subject is administered an intravenous formulation comprising an immunotherapeutic agent (e.g., resiquimod or an analog thereof) once per week at a dose of less than or about 0.003mg/kg, 0.004mg/kg, 0.005mg/kg, or 0.006mg/kg to about 0.01 mg/kg. Reference data on safe dosages of immunotherapeutic agents are found in Jurk et al, Nature Immunology, Vol.4, No.6:499(2002) and Pockros et al, J.hepatology,47:174-182(2007), the disclosures of which are incorporated by reference in their entirety.

D. Targeted therapy

In some embodiments, the immunotherapeutic agent is administered in combination with the targeted therapeutic agent, either in the same formulation or separately.

By "targeted therapeutic agent" herein is meant a therapeutic agent that specifically or selectively binds to a target molecule, cell, particle, tissue, or aggregate, which is generally referred to as a "target" or "marker," as further detailed herein.

By "therapeutic agent" herein is meant an agent having a therapeutic effect, for example in the amelioration or treatment of cancer.

In some embodiments, the targeted therapeutic agent comprises an immunoglobulin, a protein, a peptide, a small molecule, a nanoparticle, or a nucleic acid. In other embodiments, one or more of these are explicitly excluded.

In some embodiments, the targeted therapeutic comprises an Antibody Drug Conjugate (ADC) as provided by the present disclosure.

In some embodiments, the targeted therapeutic comprises an immunoglobulin, protein, or peptide, but not a small molecule.

In some embodiments, the targeted therapeutic is not an ADC.

Exemplary targeted therapeutic agents such as antibodies (e.g., chimeric, humanized, and human) are well recognized in the art and are useful and not limiting in practicing embodiments of the present disclosure.

In some embodiments, the targeted therapeutic is an antibody, antibody fragment, bispecific antibody, or other antibody-based molecule or compound.

In some embodiments, the targeted therapeutic is an aptamer, a high affinity multimer, a receptor binding ligand, a nucleic acid, a biotin-avidin binding pair, a binding peptide or protein, or the like, all of which specifically or preferentially bind to the target molecule and have a therapeutic effect, e.g., against a cancer or tumor.

By "target" or "marker" herein is meant any entity capable of specifically binding to a particular targeted therapeutic agent, such as Her 2/Neu. In some embodiments, the target is specifically associated with one or more specific cell or tissue types. In some embodiments, the target is specifically associated with one or more specific disease states. In some embodiments, the target is specifically associated with one or more specific developmental stages. For example, the expression level of a cell type specific marker in that cell type is typically at least 2-fold higher than in a reference cell population. In some embodiments, the cell-type specific marker is present at a level at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, or at least 1000 fold greater than its average expression in the reference population. Detection or measurement of cell type specific markers may make it possible to distinguish the cell type or types of interest from many, most, or all other types of cells. In some embodiments, the target may comprise a protein, a carbohydrate, a lipid, and/or a nucleic acid, as described herein.

"specific binding" or "preferential binding" in this context means two binding partnersBinding between the partners (e.g., between the targeting moiety and its binding partner) is selective for both binding partners and can be distinguished from unwanted or non-specific interactions. For example, the ability of an antigen-binding moiety to bind to a particular epitope can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (analysis on BIAcore instruments) (Liljeblad et al, Glyco J17, 323-]Antibodies "and" with [ antigens ]]A bound antibody "refers to an antibody that is capable of binding a corresponding antigen with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting the antigen. In some embodiments, the anti- [ antigen]The degree of binding of the antibody to the unrelated protein is less than about 10% of the binding of the antibody to the antigen as measured by, for example, a Radioimmunoassay (RIA). In some embodiments, the dissociation constant (KD) of an antibody that binds an antigen<1μM、<100nM、<10nM、<1nM、<0.1nM、<0.01nM or<0.001nM (e.g., 10)^-8M or less, from 10^-8M to 10^-13M, or from 10^-9M to 10^-13M). It will be appreciated that the above definitions also apply to non-antibody antigen-binding portions that bind to an antigen.

In certain specific embodiments, the target is a tumor marker. In some embodiments, a tumor marker is an antigen present in a tumor that is not present in normal organs, tissues, and/or cells. In some embodiments, a tumor marker is an antigen that is more prevalent in a tumor than in a normal organ, tissue, and/or cell. In some embodiments, the tumor marker is an antigen that is more prevalent in malignant cancer cells than in normal cells.

By "tumor antigen" herein is meant an antigenic substance produced in tumor cells, which may also trigger an immune response in a host. Normal proteins in vivo are not immunogenic due to self-tolerance, during which self-reactive Cytotoxic T Lymphocytes (CTL) and autoantibody producing B lymphocytes are "central" in primary lymphoid tissues (BM) and "peripheral" in secondary lymphoid tissues (mainly T cell thymus and B cell spleen/lymph node). Thus, any protein that is not exposed to the immune system during maturation may elicit an immune response. This may include normal proteins that are well-isolated from the immune system, proteins that are usually produced in very small amounts, proteins that are usually produced only at specific developmental stages, or proteins that change structure due to mutations.

In some embodiments, the target is preferentially expressed in tumor tissue and/or cells as compared to normal tissue and/or cells.

In some embodiments, the marker is a tumor marker. The marker may be a polypeptide that is expressed at a higher level by dividing than by non-dividing cells. For example, Her-2/neu (also known as ErbB-2) is a member of the EGF receptor family and is expressed on the surface of tumor cells associated with breast cancer. Another example is the peptide named F3, which is a suitable targeting agent for directing nanoparticles to nucleolin (Porkka et al, 2002, Proc. Natl. Acad. Sci., USA,99: 7444; and Christian et al, 2003, J.cell biol.,163: 871). Targeting particles comprising nanoparticles and a10 aptamer (specifically binding to PSMA) have been shown to be capable of specifically and efficiently delivering docetaxel to prostate cancer tumors.

Antibodies or other drugs specifically targeting these tumor targets can specifically interfere with and modulate the signaling pathway of tumor cell biological behavior, directly modulate or block the signaling pathway, inhibit tumor cell growth or induce apoptosis. To date, there are dozens of targeted drugs approved for the treatment of solid tumors or hematologic malignancies.

In some embodiments, the tumor antigen (or tumor target or tumor marker) is selected from the group consisting of CD2, CD19, CD20, CD22, CD27, CD33, CD37, CD38, CD40, CD44, CD47, CD52, CD56, CD70, CD79, and CD 137.

In some embodiments, the tumor antigen (or tumor target or tumor marker) is selected from the group consisting of 4-1BB, 5T4, AGS-5, AGS-16, angiopoietin 2, B7.1, B7.2, B7DC, B7H1, B7H2, B7H3, BT-062, BTLA, CAIX, carcinoembryonic antigen, CTLA4, Criptto, ED-B, ErbB1, ErbB2, ErbB3, ErbB4, EGFL7, EpCAM, EphA2, EphA3, EphB2, FAP, fibronectin, folate receptor, ganglioside GM3, GM 2, glucocorticoid-induced tumor necrosis factor receptor (GITR), gp100, gpA 7, GPNMB, ICOS 874, IGF1R, integrin α β, KIR, LAG-3, MUVY-1, MUVY, MUVX, EGV-72, PGD, PGX, HB72, HB11, HB8, HBsA 1, HB11, HBsP 1, HBsP-1, HBsP, HBsC-1, HBsP-1, HBsP-1, HBsP-1, HB, TACI, TAG-72, tenascin, TIM3, TRAILR1, TRAILR2, VEGFR-1, VEGFR-2, VEGFR-3, and variants thereof. Variants of these tumor antigens include various mutants or polymorphisms known in the art and/or occurring in nature.

In some embodiments, the targeted therapeutic comprises an antibody or a functional fragment thereof.

An "immunoglobulin" or "antibody" herein refers to a full-length (e.g., naturally occurring or formed by the process of recombination of normal immunoglobulin gene fragments) immunoglobulin molecule (e.g., an IgG antibody) or immunologically active (i.e., specifically binding) portion of an immunoglobulin molecule, such as an antibody fragment. Within the scope of the claimed subject matter, antibodies or antibody fragments may be conjugated or otherwise derivatized. Such antibodies include IgG1, IgG2a, IgG3, IgG4 (and IgG4 subtypes), and IgA subtypes.

The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain comprising an Fc region. Antibodies also include immunoglobulins that have been designed with additional domains, e.g., whole antibodies or antibody fragments fused to additional variable domains, as may be useful in the production of bispecific antibodies.

"native antibody" herein refers to a naturally occurring immunoglobulin molecule having a different structure (i.e., an immunoglobulin molecule having a structure that can be produced from animal immunization). For example, a native IgG antibody is a heterotetrameric glycoprotein of about 150000 daltons, consisting of two identical light chains and two identical heavy chains linked by disulfide bonds. From N-terminus to C-terminus, each heavy chain has a variable region (VH), also known as the variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3), also known as heavy chain constant regions. Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL), also known as a variable light domain or light chain variable domain, followed by a Constant Light (CL) domain, also known as a light chain constant region. The light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

An "antibody fragment" herein refers to a molecule other than an intact antibody, which comprises a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab '-SH, F (ab') 2, diabodies, linear antibodies, single chain antibody molecules (e.g., scFv), single domain antibodies, and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Hudson et al, Nat Med 9, 129-. For reviews on scFv fragments see, for example, Pliickthun, in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds, Springer-Verlag, New York, pp.269-315 (1994); see also WO 93/16185; and U.S. patent nos. 5,571,894 and 5,587,458. See U.S. Pat. No. 5,869,046 for a discussion of Fab and F (ab') 2 fragments comprising salvage receptor binding epitope residues and having increased half-life in vivo. Diabodies are antibody fragments with two antigen-binding sites, which may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; hudson et al, Nat Med 9, 129-; and Hollinger et al, Proc Natl Acad Sci USA 90, 6444-. Trisomy and tetrasomy are also described in Hudson et al, Nat Med 9, 129-. Single domain antibodies are antibody fragments that comprise all or part of the heavy chain variable domain or all or part of the light chain variable domain of the antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No.6,248,516 Bl). Antibody fragments can be prepared by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies and production of recombinant host cells (e.g., E.coli or phage), as described herein.

An "antigen binding domain" herein refers to a portion of an antibody that comprises a region that specifically binds to and is complementary to part or all of an antigen. The antigen binding domain may be provided by, for example, one or more antibody variable domains (also referred to as antibody variable regions). In particular, the antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

The term "variable region" or "variable domain" as used herein refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, each domain comprising four conserved Framework Regions (FR) and three hypervariable regions (HVRs). See, e.g., Kindt et al, Kuby Immunology,6th ed., w.h.freeman and co, page 91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity.

By "hypervariable region" or "HVR" herein is meant that each region of the antibody variable domain is hypervariable in sequence and/or forms a structurally defined loop "hypervariable loop"). Typically, a native four-chain antibody comprises six HVRs; three of VH (HI, H2, H3) and three of VL (LI, L2, L3). HVRs typically contain amino acid residues from hypervariable loops and/or from Complementarity Determining Regions (CDRs) that have the highest sequence variability and/or are involved in antigen recognition. In addition to the CDR 1in VH, the CDR typically comprises amino acid residues that form a hypervariable loop. Hypervariable regions (HVRs) are also referred to as "complementarity determining regions" (CDRs), which terms are used interchangeably herein to refer to the variable region portions that form the antigen-binding regions. This particular region is described by Kabat et al, U.S. Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and Chothia et al, J Mol Biol 196:901-917(1987), wherein these definitions include overlapping or subset sets of amino acid residues when compared to each other. However, applying either definition to refer to the CDRs of an antibody or variant thereof is intended to fall within the scope of the terms defined and used herein. The exact number of residues comprising a particular CDR will vary depending on the sequence and size of the CDR. Given the variable region amino acid sequence of an antibody, one skilled in the art can routinely determine which residues comprise a particular CDR.

The antibodies of the embodiments of the present disclosure may be chimeric antibodies, humanized antibodies, human antibodies, or antibody fusion proteins.

By "chimeric antibody" herein is meant a recombinant protein comprising antibody heavy and light chain variable domains, including Complementarity Determining Regions (CDRs) of an antibody derived from one species, preferably a rodent antibody, more preferably a murine antibody, while the constant domains of the antibody molecule are derived from the constant domains of a human antibody. For veterinary applications, the constant domains of the chimeric antibody may be derived from constant domains of other species, such as a sub-human primate, cat or dog.

"humanized antibody" herein refers to recombinant proteins in which the CDRs are from an antibody of one species; for example, rodent antibodies, transfer from the heavy and light chain variable chains of a rodent antibody into the human heavy and light chain variable domains. The constant domains of the antibody molecule are derived from those of a human antibody. In some embodiments, certain residues of the framework regions of the humanized antibody, particularly those contacting or near the CDR sequences, may be modified, e.g., replaced with the corresponding residues from the original rodent, sub-human primate, or other antibody.

"human antibody" herein refers to an antibody obtained from, for example, a transgenic mouse that has been "engineered" to produce a specific human antibody in response to an antigenic challenge. In this technique, elements of the human heavy and light chain loci are introduced into mouse strains derived from embryonic stem cell lines containing targeted disruptions of endogenous heavy and light chain loci. Transgenic mice can synthesize human antibodies specific for human antigens, and mice can be used to generate human antibody-secreting hybridomas. Green et al, Nature Genet.7:13(1994), Lonberg et al, Nature 368:856(1994) and Taylor et al, int.Immun.6:579(1994) describe methods for obtaining human antibodies from transgenic mice. Fully human antibodies can also be constructed by genetic or chromosomal transfection methods as well as phage display techniques, all of which are known in the art. For the in vitro production of human antibodies and fragments thereof from immunoglobulin variable domain gene libraries from uninmmunized donors, see, e.g., McCafferty et al, Nature348:552-553 (1990). In this technique, antibody variable domain genes are cloned in-frame into the major or minor coat protein genes of filamentous phage and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selection based on functional properties of the antibody will also result in selection of genes encoding antibodies that exhibit these properties. In this way, the phage mimics some of the characteristics of the B cell. Phage display can be performed in a variety of formats, for a review of them, see, e.g., Johnson and Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993). Human antibodies can also be produced by in vitro activated B cells. See U.S. Pat. nos. 5,567,610 and 5,229,275, the entire contents of which are incorporated herein by reference.

An "antibody fusion protein" herein refers to a recombinantly produced antigen-binding molecule in which two or more identical or different natural antibodies, single chain antibodies, or antibody fragment segments with identical or different specificities are linked. The fusion protein comprises at least one specific binding site. The titer of the fusion protein represents the total number of binding arms or sites of the fusion protein to the antigen or epitope; i.e. monovalent, divalent, trivalent or polyvalent. The multivalency of an antibody fusion protein means that it can bind to an antigen using a variety of interactions, thereby increasing avidity of binding to the antigen or to a different antigen. Specificity indicates how many different types of antigens or epitopes the antibody fusion protein is capable of binding; i.e., monospecific, bispecific, trispecific, multispecific. Using these definitions, a natural antibody, such as IgG, is bivalent because it has two binding arms, but it is monospecific because it binds to one type of antigen or epitope. Monospecific, multivalent fusion proteins have more than one binding site for the same antigen or epitope. For example, a monospecific diabody is a fusion protein with two binding sites that react with the same antigen. The fusion protein may comprise multivalent or multispecific combinations of different antibody components or multiple copies of the same antibody component. The fusion protein may further comprise a therapeutic agent.

In some embodiments, the targeting moiety comprises a precursor, such as, for example, U.S. patent nos.: 8,518,404, respectively; 8,513,390, respectively; and U.S. patent application publication nos.: 20120237977a1, 20120149061a1, 20130150558a1, the disclosures of which are incorporated by reference in their entirety.

The precursor is a monoclonal antibody that is selectively activated in the cancer microenvironment, focusing the activity of the therapeutic antibody on the tumor and protecting healthy tissue.

Generally, an antibody comprises at least an antibody or antibody fragment thereof (collectively "AB") capable of specifically binding to a target, wherein the AB is modified by a Masking Moiety (MM). When the AB is modified with MM and a target is present, specific binding of the AB to its target is reduced or inhibited as compared to specific binding of the AB that is not modified with MM or to the target of the parent AB. The dissociation constant (Kd) of MM to AB is typically greater than the Kd of AB to the target. When the AB is modified with MM and a target is present, specific binding of the AB to its target can be reduced or inhibited as compared to specific binding of the AB that is not modified with MM or to the target of the parent AB. When the AB is coupled to or modified by MM, the MM may "mask" or reduce or inhibit specific binding of the AB to its target. When an AB is coupled to or modified by MM, such coupling or modification can affect a structural change, thereby reducing or inhibiting the ability of the AB to specifically bind to its target.

In some embodiments, the precursor is an Activatable Antibody (AA), wherein the AB modified by the MM may further comprise one or more Cleavable Moieties (CMs). Such AA show activatable/switchable binding to the target of AB. An AA generally includes an antibody or antibody fragment (AB) modified or conjugated by a Masking Moiety (MM) and a modifiable or Cleavable Moiety (CM). In some embodiments, the CM contains an amino acid sequence that serves as a substrate for the protease of interest. In other embodiments, CM provides a cysteine-cysteine disulfide bond that is cleavable by reduction. In other embodiments, the CM provides a photolytic substrate that can be activated by photolysis.

The CM and AB of AA can be selected such that AB represents a binding moiety for the target of interest and CM represents a substrate for a protease that is co-localized with the target at the treatment site of the subject. Alternatively or additionally, CM is a cysteine-cysteine disulfide bond, which is cleavable by reduction of this disulfide bond. The AA comprises at least one of a protease cleavable CM or a cysteine-cysteine disulfide bond, and in some embodiments includes both CMs. The AA may alternatively or further comprise a photolabile substrate which can be activated by a light source. The AA disclosed herein has particular utility, for example, where a protease capable of cleaving a site in the CM is present at a relatively higher level in a target-containing tissue at a treatment site (e.g., diseased tissue; e.g., for therapeutic or diagnostic treatment) than in a tissue at a non-treatment site (e.g., healthy tissue). The AA disclosed herein also have particular utility, e.g., reducing agents capable of reducing CM sites are present at relatively higher levels in target-containing tissues at a therapeutic or diagnostic site as compared to tissues at a non-therapeutic, non-diagnostic site. The AA disclosed herein also has particular utility where a light source capable of photolyzing a site in the CM is introduced, for example by means of a laser, into target-containing tissue at a treatment or diagnostic site.

In some embodiments, the AA can provide reduced toxicity and/or adverse side effects that can result from binding of the AB at a non-treatment site if the AB is not masked or otherwise inhibited from binding to its target. When an AA contains CM that can be cleaved by a reducing agent that promotes disulfide bond reduction, the AB of such AA can be selected to take advantage of the activation of the AB, where the target of interest is present at a desired treatment site characterized by elevated reducing agent levels, such that the environment has a higher reduction potential than, for example, the environment of the non-treatment site.

Typically, an AA can be designed by selecting the AB of interest and constructing the remainder of the AA such that, when conformationally constrained, the MM provides masking of the AB or reduces binding of the AB to its target. Structural design criteria are considered to provide this functional feature.

In some embodiments, the targeted therapeutic is an antibody or antibody fragment that is selected based on its specificity for an antigen expressed on a target cell of interest or at a target site of interest. A variety of tumor-specific or other disease-specific antigens have been identified, and antibodies to these antigens have been used or proposed for the treatment of such tumors or other diseases. Antibodies known in the art are useful as targeted therapeutic agents in therapeutic combinations, particularly for treating diseases associated with a target antigen. Examples of target antigens (and their associated diseases) that can be targeted by a target therapeutic include: CD2, CD19, CD20, CD22, CD27, CD33, CD37, CD38, CD40, CD44, CD47, CD52, CD56, CD70, CD79, CD137, 4-1BB, 5T4, AGS-5, AGS-16, angiopoietin 2, B7.1, B7.2, B7 4, B7H 4, BT-062, BTLA, CAIX, carcinoembryonic antigen, CTLA4, Criptto, ED-4, ErbB4, EGFL 4, EphA 4, EphB 4, FAP, fibronectin, receptor, ganglioside 4, GM hormone-induced tumor receptor (TNF receptor, GIgGIgP receptor), TNF receptor, PGHA-4, PGAS-4, PGA-4, PGAS-4, PGA-4, PGAS-4, PGA-4, PGAS-4, PGA-4, PGX, PGA-3, PGAS-4, PGX, PGA-4, PGA-72, PGX, PGA-4, PGA-3, PGA-4, PGA, PGX, PGA-4, PGA-72, PGA-4, PGA, PGS-4, PGX, PGA-4, PGA-72, PGX, PGA-4, PGA-72, PGA-4, PGA-III, PGA-4, PGS-4, PGA-72, PGA-72, PGA-72, PGA-3, PGA, PGS, PGA-72, PGA, PGS, PGA, PGX, PGA, PGS, PGA, PGS, PGA, PGS, PG, TACI, TAG-72, tenascin, TIM3, TRAILR1, TRAILR2, VEGFR-1, VEGFR-2, VEGFR-3.

In some embodiments, the antibody is selected from the group consisting of rituximab, herceptin (trastuzumab), erbitux (cetuximab), victirib (panitumumab), Arzerra (ofatumumab), Bentysta (beliewuzumab), yrervoy (ipilimumab), Perjeta (pertuzumab), Tremelimumab (Tremelimumab), Opdivo (niuliuzumab, ONO-4538, BMS-936558, or MDX1106), daclizumab (Dacetuzumab), revuzumab (Urelumab), MPDL3280A, parbollizumab and bornauzumab, CT-011, curitant (parbollizumab, MK-3475), BMS-936559, MPDL32 3280A, MED 3214736, or 071001 0010718C.

Rituximab (rituximab) is a chimeric antibody used to treat B-cell non-hodgkin's lymphoma. It acts on the surface of B cells expressing the CD20 antigen, which is expressed on 90% of B cell non-hodgkin's lymphomas. Rituximab binds to CD20 to induce B cell lysis through CDC and ADCC and sensitizes human lymphocytes resistant to certain cytotoxic chemotherapeutic drugs.

Herceptin (trastuzumab) is a humanized monoclonal antibody that acts on the extracellular domain of the human epidermal growth factor receptor Her2 and is expressed in 25% to 30% of breast cancers. Trastuzumab is thought to induce apoptosis by (1) down-regulating Her2 receptors, inhibiting Her2 intracellular signaling pathways; (2) immune mechanism-associated antibody-dependent ADCC and CDC killing of tumor cells; (3) can enhance the effect of chemotherapy and has antitumor effect.

Erbitux (cetuximab) is a chimeric antibody that acts on Epidermal Growth Factor Receptor (EGFR). Erbitux binds EGFR, inhibiting its signal transduction pathway, affecting cell proliferation, invasion and metastasis as well as angiogenesis. Inhibition of the EGFR signaling pathway can enhance the efficacy of chemotherapeutic drugs and radiation therapy.

Avastin (bevacizumab) is a humanized monoclonal antibody targeting Vascular Endothelial Growth Factor (VEGF). Its binding to VEGFR inhibits VEGF and signal transduction, thereby inhibiting tumor angiogenesis.

Other antibodies currently under development may also be used as targeted therapies. For example, therapeutic monoclonal antibodies directed against the following targets are being developed for the treatment of tumors: CD2, CD19, CD20, CD22, CD27, CD33, CD37, CD38, CD40, CD44, CD47, CD52, CD56, CD70, CD79 and CD137 and therapeutic targets for the following tumors: 4-1BB, 5T4, AGS-5, AGS-16, angiopoietin 2, B7.1, B7.2, B7DC, B7H1, B7H2, B7H3, BT-062, BTLA, CAIX, carcinoembryonic antigen, CTLA4, Cripto, ED-B, ErbB1, ErbB2, ErbB3, ErbB4, EGFL7, EpCAM, EphA2, EphA3, EphB2, FAP, fibronectin, folate receptor, ganglioside GM3, GD2, glucocorticoid-induced tumor necrosis factor receptor (GITR), gp100, g 33, GPNMB, ICOS, IGF1R, integrin α v β, KIR, LAG-3, Lewis, mesothelin, c-MET, MN carbonic anhydrase IX, MUC1, MUC16, Nectin-4, NKGD2, NOTCH, OX40, OX40L, PD-1, PDL1, PSCA, PSMA, RANKL, ROR1, ROR2, SLC44A4, syndecan-1, TACI, TAG-72, tenascin, TIM3, TRAILR1, TRAILR2, VEGFR-1, VEGFR-2, VEGFR-3, and variants thereof. (Scott AM, Wolchok JD, Old LJ, Antibody Therapy of Cancer., Nat Rev Cancer 2012 Mar 22,12(4): 278-87).

In some embodiments, the targeted therapeutic comprises a Fab, Fab ', F (ab') 2, single domain antibody, T and Abs dimer, Fv, scFv, dsFv, ds-scFv, Fd, linear antibody, minibody, diabody, bispecific antibody fragment, diabody, triabody, sc-diabody, kappa (lamda) body, BiTE, DVD-Ig, SIP, SMIP, DART, or an antibody analog comprising one or more CDRs.

Table 1 shows the various antibody structures and targets under investigation.

TABLE 1

Targeted therapeutic comprising a targeting moiety

In some aspects, according to embodiments of the present disclosure, targeted therapeutic agents of embodiments of the present disclosure comprise a targeting moiety, such as an ADC.

"Targeting Moiety (TM)" or "targeting agent" refers herein to a molecule, complex or aggregate that specifically or selectively binds to a target molecule, cell, particle, tissue or aggregate, which is generally referred to as a "target" or "label," which are discussed in further detail herein.

In some embodiments, the targeting moiety comprises an immunoglobulin, a protein, a peptide, a small molecule, a nanoparticle, or a nucleic acid.

Exemplary targeting agents, such as antibodies (e.g., chimeric, humanized and human), ligands for receptors, lectins and carbohydrates, and substrates for certain enzymes, are art-recognized and are useful and not limiting in practicing embodiments of the present disclosure. Other targeting agents include a class of compounds that do not include specific molecular recognition motifs, including nanoparticles, macromolecules such as poly (ethylene glycol), polysaccharides, and polyamino acids, which add molecular weight to the active moiety. The additional molecular weight affects the pharmacokinetics of the activated moiety, such as serum half-life.

In some embodiments, the targeting moiety is an antibody, antibody fragment, bispecific antibody, or other antibody-based molecule or compound. However, other examples of targeting moieties are known in the art and may be used, such as aptamers, avimers, receptor binding ligands, nucleic acids, biotin-avidin binding pairs, binding peptides or proteins, and the like. The terms "targeting moiety" and "binding moiety" are used synonymously herein.

By "target" or "label" herein is meant any entity capable of specifically binding to a particular targeting moiety. In some embodiments, the target is specifically associated with one or more specific cell or tissue types. In some embodiments, the target is specifically associated with one or more specific disease states. In some embodiments, the target is specifically associated with one or more specific developmental stages. For example, the expression level of a cell type specific marker in that cell type is typically at least 2-fold higher than in a reference cell population. In some embodiments, the expression level of the cell-type specific marker is at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 50-fold, at least 100-fold, or at least 1000-fold greater than it is in the reference population. Detection or measurement of cell type specific markers may make it possible to distinguish the cell type or types of interest from many, most, or all other types of cells. In some embodiments, the target may comprise a protein, a carbohydrate, a lipid, and/or a nucleic acid, as described herein.

A substance is considered "targeted" for the purposes described herein if it specifically binds to a nucleic acid targeting moiety. In some embodiments, the nucleic acid targeting moiety specifically binds to the target under stringent conditions. A complex or compound of the invention comprising a targeting moiety is considered "targeted" if the targeting moiety specifically binds to the target, thereby delivering the entire complex or compound composition to a particular organ, tissue, cell, extracellular matrix component, and/or intracellular compartment.

In certain embodiments, compounds according to embodiments of the present disclosure comprise targeting moieties that specifically bind to one or more targets (e.g., antigens) associated with an organ, tissue, cell, extracellular matrix component, and/or intracellular compartment. In some embodiments, the compound comprises a targeting moiety that specifically binds to a target associated with a particular organ or organ system. In some embodiments, compounds according to embodiments of the present disclosure comprise a nuclear targeting moiety that specifically binds to one or more intracellular targets (e.g., organelles, intracellular proteins). In some embodiments, the compound comprises a targeting moiety that specifically binds to a target associated with a diseased organ, tissue, cell, extracellular matrix component, and/or intracellular compartment. In some embodiments, the compounds comprise a targeting moiety that specifically binds to a target associated with a particular cell type (e.g., endothelial cells, cancer cells, malignant cells, prostate cancer cells, etc.).

In some embodiments, according to embodiments of the present disclosure, compounds of embodiments of the present disclosure comprise a targeting moiety that binds to a target specific for one or more specific tissue types (e.g., liver tissue vs. prostate tissue). In some embodiments, according to embodiments of the present disclosure, compounds of embodiments of the present disclosure comprise a targeting moiety that binds to a target specific for one or more specific cell types (e.g., T cell vs. In some embodiments, according to embodiments of the present disclosure, compounds of embodiments of the present disclosure comprise a targeting moiety that binds to a target specific for one or more specific disease states (e.g., tumor cells versus healthy cells). In some embodiments, according to embodiments of the present disclosure, compounds of embodiments of the present disclosure comprise a targeting moiety that binds to a target specific for one or more particular developmental stages (e.g., stem cells versus differentiated cells).

In some embodiments, the target may be a marker that is associated exclusively or primarily with one or several cell types, one or several diseases and/or one or several developmental stages. The expression level of a cell-type specific marker in that cell type is typically at least 2-fold higher than in a reference cell population, which may consist of, for example, a mixture containing roughly equal numbers of a plurality (e.g., 5-10 or more) cells from different tissues or organs. In some embodiments, the cell-type specific marker is present at a level at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, or at least 1000 fold greater than its average expression in the reference population. Detection or measurement of cell type specific markers may make it possible to distinguish the cell type or types of interest from many, most, or all other types of cells.

In some embodiments, the target comprises a protein, a carbohydrate, a lipid, and/or a nucleic acid. In some embodiments, the target comprises a protein and/or characteristic portions thereof, such as tumor markers, integrins, cell surface receptors, transmembrane proteins, intercellular proteins, ion channels, membrane transporters, enzymes, antibodies, chimeric proteins, glycoproteins, and the like. In some embodiments, the target comprises a carbohydrate and/or characteristic portions thereof, such as a glycoprotein, a sugar (e.g., a monosaccharide, a disaccharide, a polysaccharide), a glycocalyx (i.e., a carbohydrate-rich peripheral region on the outer surface of most eukaryotic cells), and the like. In some embodiments, the target comprises a lipid and/or characteristic portion thereof, such as an oil, fatty acid, glyceride, hormone, steroid (e.g., cholesterol, bile acid), vitamin (e.g., vitamin E), phospholipid, sphingolipid, lipoprotein, and the like. In some embodiments, the target comprises a nucleic acid and/or characteristic portion thereof, e.g., a DNA nucleic acid; an RNA nucleic acid; a modified DNA nucleic acid; a modified RNA nucleic acid; nucleic acids including any combination of DNA, RNA, modified DNA and modified RNA.

Many markers are known in the art. Typical markers include cell surface proteins, such as receptors. Exemplary receptors include, but are not limited to, transferrin receptor; (ii) an LDL receptor; growth factor receptors, such as epidermal growth factor receptor family members (e.g., EGFR, Her2, Her3, Her4) or vascular endothelial growth factor receptors, cytokine receptors, cell adhesion molecules, integrins, selectins, and CD molecules. The marker may be a molecule, e.g. a tumor antigen, which is present exclusively or in higher amounts on malignant cells.

In some embodiments, the targeting moiety binds specifically or preferentially to tumor cells as compared to non-tumor cells.

Binding of the target moiety to the tumor cell can be measured using assays known in the art.

In some embodiments, the tumor cell is a malignant epithelial tumor, a sarcoma, a lymphoma, a myeloma, or a central nervous system cancer.

In some embodiments, the targeting moiety is capable of specifically or preferentially binding to a tumor antigen as compared to a non-tumor antigen.

In certain specific embodiments, the target is a tumor marker. In some embodiments, a tumor marker is an antigen that is present in a tumor but not in normal organs, tissues, and/or cells. In some embodiments, a tumor marker is an antigen that is more prevalent in a tumor than in a normal organ, tissue, and/or cell. In some embodiments, the tumor marker is an antigen that is more prevalent in malignant cancer cells than in normal cells.

In some embodiments, the targeting moiety comprises folic acid or a derivative thereof.

In recent years, research on folic acid has been greatly advanced. Folic acid is a small molecule vitamin essential for cell division. Tumor cells divide abnormally and the Folate Receptor (FR) is highly expressed on the surface of tumor cells to capture enough folate to support cell division.

The data indicate that FR expression in tumor cells is 20-200 fold higher than in normal cells. The expression rate of FR in various malignancies is: ovarian cancer 82%, non-small cell lung cancer 66%, renal cancer 64%, colon cancer 34%, breast cancer 29% (Xia W, Low PS. late-targeted therapies for cancer, J Med chem.2010; 53(19): 6811-24). The expression rate of FA is positively correlated with the malignancy degree of epithelial tumor invasion and metastasis. FA enters cells through FR-mediated endocytosis, and FA forms an FA complex with the drug entering the cells through its carboxyl group. Under acidic conditions (pH 5), FR separates from FA, which releases the drug into the cytoplasm.

Clinically, the system can be used to deliver drugs that selectively attack tumor cells. The folic acid has small molecular weight, no immunogenicity, high stability and low synthesis cost. More importantly, the chemical coupling between the drug and the carrier is simple, so that the construction of a drug delivery system by using FA as a targeting molecule has become a hot research point for cancer treatment. EC145(FA chemotherapeutic drug conjugates) currently undergoing clinical trials can effectively attack cancer cells (prism P and Edelman MJ. EC145: a novel targeted agent for adenocardioma of the lung, Expert Opin. investig. drugs,2012,21:755 Ack 761).

In some embodiments, the targeting moiety comprises the extracellular domain (ECD) or soluble forms of PD-1, PDL-1, CTLA4, CD47, BTLA, KIR, TIM3, 4-1BB, and LAG3, full-length partial surface ligand amphiregulin, cytokine, EGF, ephrin, epigenin, epithelial regulatory protein, IGF, neuregulin, TGF, TRAIL, or VEGF. In some embodiments, these extracellular domains or soluble forms may be fused to an antibody Fc domain (sometimes referred to as an immunoadhesin).

In some embodiments, the targeting moiety comprises a Fab, Fab ', F (ab') 2, single domain antibody, T and Abs dimer, Fv, scFv, dsFv, ds-scFv, Fd, linear antibody, minibody, diabody, bispecific antibody fragment, diabody, triabody, sc-diabody, kappa (lamda) body, BiTE, DVD-Ig, SIP, SMIP, DART, or an antibody analog comprising one or more CDRs.

In some embodiments, the targeting moiety is an antibody or antibody fragment that is selected based on the specificity of the antigen expressed on the target cell or target site of interest. A variety of tumor-specific or other disease-specific antigens have been identified, and antibodies to these antigens have been used or proposed for the treatment of such tumors or other diseases. Antibodies known in the art can be used as targeted therapeutic agents in the combinations disclosed herein, particularly for the treatment of diseases associated with a target antigen. Examples of target antigens (and their associated diseases) include: CD2, CD19, CD20, CD22, CD27, CD33, CD37, CD38, CD40, CD44, CD47, CD52, CD56, CD70, CD79, CD1374-1BB, 5T4, AGS-5, AGS-16, angiopoietin 2, B7.1, B7.2, B7 4, B7H 4, BT-062, BTLA, CAIX, carcinoembryonic antigen, CTLA4, Criptto, ED-4, ErbB4, EGFL 4, EphA 4, EphB 4, FAP, fibronectin, glucocorticoid receptor, ganglioside 4, GM hormone-induced tumor receptor (TNF receptor), GIgGIgGIgP receptor (TNF receptor), PGHA-100, PGHA 4, PGHA-4, PGA-4, PGAS-4, PGA-4, PGX-4, PGX, PGA-4, PGA-3, PGA-4, PGX, PGA-4, PGA-3, PGA-4, PGX, PGA-3, PGA, PGX, PGA-4, PGA, PGX, PGA-3, PGA-4, PGX, PGA-3, PGA, PGX, PGA-4, PGA-3, PGA-4, PGA-3, PGA, PGX, PGA, TACI, TAG-72, tenascin, TIM3, TRAILR1, TRAILR2, VEGFR-1, VEGFR-2, VEGFR-3. In some embodiments, the targeted therapeutic may comprise an antibody-linker-drug conjugate. In other embodiments, antibody-linker-drug conjugates are specifically excluded.

In some embodiments, the targeting moiety comprises a nucleic acid targeting moiety.

Generally, a nucleic acid targeting moiety is any polynucleotide that binds to an organ, tissue, cell, extracellular matrix component, and/or a component associated with an intracellular compartment (target).

In some embodiments, the nucleic acid targeting moiety is an aptamer.

Aptamers are generally polynucleotides that bind to a specific target structure associated with a specific organ, tissue, cell, extracellular matrix component, and/or intracellular compartment. In general, the targeting function of aptamers is based on the three-dimensional structure of the aptamers. In some embodiments, binding of an aptamer to a target is generally mediated by an interaction between the two-dimensional and/or three-dimensional structure of the aptamer and the target. In some embodiments, the binding of an aptamer to a target is based not only on the primary sequence of the aptamer, but also on the three-dimensional structure of the aptamer and/or target. In some embodiments, aptamers bind to their target through complementary Watson-Crick (Watson-Crick) base pairing that is interrupted by a structure that disrupts base pairing, such as a hairpin loop.

In some embodiments, the nucleic acid targeting moiety is a spiegelmers (PCT publication Nos. WO98/08856, WO02/100442, and WO 06/117217). In general, spiegelmers are synthetic spiegelmers that bind specifically to a target (i.e., spiegelmers). Spiegelmers are characterized by structural features that make them exonuclease and endonuclease insensitive.

One of ordinary skill in the art will recognize that any nucleic acid targeting moiety capable of specifically binding to a target (e.g., an aptamer or spiegelmer) may be used in accordance with embodiments of the present disclosure. In some embodiments, nucleic acid targeting moieties used in accordance with embodiments of the present disclosure may target markers associated with a disease, disorder and/or condition. In some embodiments, a nucleic acid targeting moiety used in accordance with embodiments of the present disclosure may target a cancer-associated target. In some embodiments, nucleic acid targeting moieties used in accordance with embodiments of the present disclosure can target tumor markers. According to embodiments of the present disclosure, any type of cancer and/or any tumor marker may be targeted using a nucleic acid targeting moiety. To name a few examples, the nucleic acid targeting moiety can target markers associated with prostate cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, pancreatic cancer, endometrial cancer, ovarian cancer, bone cancer, esophageal cancer, liver cancer, stomach cancer, brain tumors, skin melanoma, and/or leukemia.

Nucleic acids of embodiments of the present disclosure (including nucleic acid targeting moieties and/or functional RNAs to be delivered, e.g., RNAi-inducing entities, ribozymes, trnas, etc., described in further detail below) can be synthesized according to any available technique, including but not limited to chemical synthesis, enzymatic or chemical cleavage of longer precursors, and the like. Methods for synthesizing RNA are known in the art (see, e.g., Gait, M.J. (ed.) Oligonuclotide synthesis: a practical approach, Oxford [ Oxfordshire ], Washington, D.C.: IRL Press, 1984; and Herdewin, P. (ed.) Oligonuclotide synthesis: Methods and applications, Methods in molecular biology, v.288(Clifton, N.J.) Totowa, N.J.: Humana Press, 2005).

The nucleic acid forming the nucleic acid targeting moiety can comprise a naturally occurring nucleoside, a modified nucleoside, a naturally occurring nucleoside having a hydrocarbon linker (e.g., alkylene) or a polyether linker (e.g., PEG linker) interposed between one or more nucleosides, a modified nucleoside having a hydrocarbon or PEG linker interposed between one or more nucleosides, or a combination thereof. In some embodiments, the nucleotides or modified nucleotides of the nucleic acid targeting moiety may be replaced with a hydrocarbon linker or a polyether linker, provided that the binding affinity and selectivity of the nucleic acid targeting moiety is not substantially reduced by the substitution (e.g., the dissociation constant of the nucleic acid targeting moiety for the target should not be greater than about 1x10^-3M)。

One of ordinary skill in the art will appreciate that nucleic acids according to embodiments of the present disclosure may comprise nucleotides of the type found entirely in naturally occurring nucleic acids, or may alternatively include one or more nucleotide analogs or have a structure that is otherwise different from a naturally occurring nucleic acid. U.S. patent application No.6,403,779; 6,399,754, respectively; 6,225,460, respectively; 6,127,533, respectively; 6,031,086, respectively; 6,005,087, respectively; 5,977,089, respectively; the references therein disclose a variety of specific nucleotide analogs and modifications that can be used. See C crook, S. (ed.) Antisense Drug Technology: Principles, Strategies, and Applications, Marcel Dekker; 0824705661 parts of ISBN; 1st edition (2001) and references therein. For example, 2-modifications include halogen, alkoxy, and allyloxy. In some embodiments, the 2' -OH group is substituted with a group selected from H, OR, R, halogen, SH, SR, NH2, NHR, NR2, OR CN, wherein R is C1-C6 alkyl, alkenyl, OR alkynyl, and halogen is F, Cl, Br, OR I. Examples of modified linkages include phosphorothioate and 5' -N-phosphoramidite linkages.

Nucleic acids comprising a variety of different nucleotide analogs, modified backbones, or non-naturally occurring internucleoside linkages can be used according to embodiments of the present disclosure. Nucleic acids of embodiments of the present disclosure may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) or modified nucleosides. Examples of the modified nucleotide include base-modified nucleosides (e.g., cytarabine, inosine, isoguanosine, nebularine (nebularine), pseudouridine, 2, 6-diaminopurine, 2-aminopurine, 2-thiothymidine, 3-deaza-5-azacytidine, 2' -deoxyuridine, 3-nitropyrrole, 4-methylindole, 4-thiouridine, 4-thiothymidine, 2-aminoadenosine, 2-thiothymidine, 2-thiouridine, 5-bromocytidine, 5-iodouridine, inosine, 6-azauridine, 6-chloropurine, 7-deazaadenosine, 7-deazaguanosine, 8-azaadenosine, 8-azidoadenosine, benzimidazole, M1-methyladenosine, pyrrolopyrimidine, 2-amino-6-chloropurine, 3-methyladenosine, 5-propynyl cytidine, 5-propynyl uridine, 5-bromouridine, 5-fluorouridine, 5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O (6) -methylguanine, and 2-thiocytidine), chemically or biologically modified bases (e.g., methylated bases), modified sugars (e.g., 2-fluororibose, 2-aminoribose, 2-azidoribose, 2-O-methylribose, L-enantiomeric nucleoside arabinose, and hexoses), modified phosphate groups (e.g., phosphorothioate, and 5' -N-phosphoramidite bonds), and combinations thereof. Natural and modified nucleotide monomers for chemical synthesis of nucleic acids are readily available. In some cases, nucleic acids comprising such modifications exhibit improved properties relative to nucleic acids consisting only of naturally occurring nucleotides. In some embodiments, the nucleic acid modifications described herein are used to reduce and/or prevent digestion by nucleases (e.g., exonucleases, endonucleases, etc.). For example, the structure of a nucleic acid can be stabilized by including nucleotide analogs at the 3' end of one or both strands to reduce digestion.

The modified nucleic acid need not be uniformly modified along the entire length of the molecule. Different nucleotide modifications and/or backbone structures may be present at different positions of the nucleic acid. One of ordinary skill in the art will appreciate that nucleotide analogs or other modifications can be located anywhere in a nucleic acid such that the function of the nucleic acid is not substantially affected. As just one example, the modification may be located anywhere on the nucleic acid targeting moiety such that the ability of the nucleic acid targeting moiety to specifically bind to a target is not substantially affected. The modification region may be located at the 5 'end and/or the 3' end of one or both strands. For example, modified nucleic acid targeting moieties have been used in which about 1-5 residues from the 5 'and/or 3' end of either strand are nucleotide analogs and/or have backbone modifications. The modification may be a 5 'or 3' terminal modification. One or both nucleic acid strands may comprise at least 50% unmodified nucleotides, at least 80% unmodified nucleotides, at least 90% unmodified nucleotides, or 100% unmodified nucleotides.

Nucleic acids according to embodiments of the present disclosure may, for example, comprise modifications to sugars, nucleosides, or internucleoside linkages, such as those described in U.S. patent application publications 2003/0175950, 2004/0192626, 2004/0092470, 2005/0020525, and 2005/0032733. Embodiments of the present disclosure include the use of any nucleic acid having one or more of the modifications described herein. For example, many terminal conjugates, such as lipids like cholesterol, lithocholic acid, aluminate or long alkyl branches have been reported to improve cellular uptake. Analogs and modifications can be tested, e.g., using any suitable assay known in the art, e.g., to select those that result in improved delivery of a therapeutic or diagnostic agent, improved specific binding of a nucleic acid targeting moiety to a target, and the like. In some embodiments, a nucleic acid according to embodiments of the disclosure may comprise one or more non-natural nucleoside linkages. In some embodiments, one or more internal nucleotides at the 3 '-end, the 5' -end, or the 3 '-and 5' -ends of the nucleic acid targeting moiety are inverted to create a linkage, e.g., a3 '-3' linkage or a 5 '-5' linkage.

In some embodiments, nucleic acids according to embodiments of the present disclosure are not synthetic, but rather are naturally occurring entities isolated from their natural environment.

Any method can be used to design novel nucleic acid targeting moieties (see, e.g., U.S. Pat. Nos. 6,716,583; 6,465,189; 6,482,594; 6,458,543; 6,458,539; 6,376,190; 6,344,318; 6,242,246; 6,184,364; 6,001,577; 5,958,691; 5,874,218; 5,853,984; 5,843,732; 5,843,653; 5,817,785; 5,789,163; 5,763,177; 5,696,249; 5,660,985; 5,595,877; 5,567,588; and 5,270,163; and U.S. patent application publications 2005/0069910, 2004/0072234, 2004/0043923, 2003/0087301, 2003/0054360 and 2002/0064780). Embodiments of the present disclosure provide methods for designing novel nucleic acid targeting moieties. Embodiments of the present disclosure further provide methods for isolating or identifying novel nucleic acid targeting moieties from a mixture of candidate nucleic acid targeting moieties.

Nucleic acid targeting moieties that bind to proteins, carbohydrates, lipids, and/or nucleic acids can be designed and/or identified. In some embodiments, nucleic acid targeting moieties can be designed and/or identified for the ability to bind proteins and/or characteristic portions thereof, such as tumor markers, integrins, cell surface receptors, transmembrane proteins, intercellular proteins, ion channels, membrane transporters, enzymes, antibodies, chimeric proteins, and the like, in targeted therapies. In some embodiments, nucleic acid targeting moieties can be designed and/or identified for use in targeted therapies that are capable of binding carbohydrates and/or characteristic portions thereof, such as glycoproteins, sugars (e.g., monosaccharides, disaccharides, and polysaccharides), glycocalyx (i.e., a peripheral region rich in carbohydrates on the outer surface of most eukaryotic cells), and the like. In some embodiments, nucleic acid targeting moieties can be designed and/or identified for use in targeted therapies that are capable of binding lipids and/or characteristic moieties thereof, such as oils, saturated fatty acids, unsaturated fatty acids, glycerides, hormones, steroids (e.g., cholesterol, bile acids), vitamins (e.g., vitamin E), phospholipids, sphingolipids, lipoproteins, and the like. In some embodiments, nucleic acid targeting moieties can be designed and/or identified for use in targeting therapies that are capable of binding nucleic acids and/or characteristic portions thereof, such as DNA nucleic acids; an RNA nucleic acid; a modified DNA nucleic acid; a modified RNA nucleic acid; and nucleic acids comprising any combination of DNA, RNA, modified DNA and modified RNA, and the like.

Any available method can be used to design and/or identify nucleic acid targeting moieties (e.g., aptamers or spiegelmers). In some embodiments, the nucleic acid targeting moiety is designed and/or identified by identifying the nucleic acid targeting moiety from a mixture of candidate nucleic acids. Systematic evolution of ligands by exponential enrichment (SELEX) or variants thereof is a common method for identifying nucleic acid targeting moieties that bind to a target from a mixture of candidate nucleic acids.

A nucleic acid targeting moiety that selectively binds to any target can be isolated by the SELEX process or variants thereof, provided that the target can be used as a target in the SELEX process.

III、Pharmaceutical formulations and administration

Embodiments of the present disclosure further relate to pharmaceutical formulations comprising the compounds of the combinations (i.e., chemotherapeutic, immunotherapeutic, or targeted therapeutic) or pharmaceutically acceptable salts thereof and one or more pharmaceutically acceptable carriers.

Various routes or modes of administration can be used to deliver a pharmaceutical formulation described herein comprising a pharmaceutically acceptable carrier to a patient. Suitable routes of administration include inhalation, transdermal, oral, rectal, transmucosal, intestinal, and parenteral administration, including intramuscular, subcutaneous, and intravenous injections. Preferably, the compound comprising the combination of immunomodulators (i.e. immunotherapeutic) is administered parenterally, more preferably intravenously.

As used herein, the term "administration" or "administering" is intended to encompass all manner of delivering a compound directly and indirectly to its intended site of action.

The compounds described herein, or pharmaceutically acceptable salts and/or hydrates thereof, can be administered alone, in combination with other compounds in combination, and/or in the form of a cocktail in combination with other therapeutic agents. Of course, the choice of therapeutic agent that can be co-administered with the combined compounds will depend in part on the condition being treated. In other embodiments, the use of additional therapeutic agents, in particular excluding those of the combinations disclosed herein, is precluded.

For example, when administered to a patient suffering from a disease state caused by an organism that is dependent on an autoinducer, the combined compounds may be administered in the form of a cocktail containing agents useful for treating pain, infections, and other symptoms and side effects commonly associated with disease. Such agents include, for example, analgesics, antibiotics, and the like.

When administered to a patient receiving cancer treatment, the compounds may be administered in the form of a cocktail containing anti-cancer agents and/or complementary potentiators. The compounds may also be administered in the form of cocktails containing agents that treat the side effects of radiation therapy, such as antiemetics, radioprotectants, and the like.

Supplementary potentiating agents which can be co-administered with the compounds of the present invention include, for example, tricyclic antidepressants (e.g., imipramine, desipramine, amitriptyline, clomipramine, trimipramine, doxepin, nortriptyline, protriptyline, amoxapine, and maprotiline); non-tricyclic antidepressants (e.g., sertraline, trazodone and citalopram); ca +2 antagonists (e.g., verapamil, nifedipine, nitrendipine, and caroviline); amphotericin; tripareol analogs (e.g., tamoxifen); antiarrhythmic drugs (e.g., quinidine); antihypertensive drugs (e.g., reserpine); thiol consuming agents (e.g., buthionine and sulfoximines); and calcium folinate.

The combined active compounds are administered as such or in the form of a pharmaceutical composition, wherein the active compounds are mixed with one or more pharmaceutically acceptable carriers, excipients or diluents. Pharmaceutical compositions for use in accordance with embodiments of the present disclosure are generally formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations which can be used pharmaceutically. The appropriate formulation will depend on the route of administration chosen.

For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the compounds can be readily formulated by combining the active compound with pharmaceutically acceptable carriers well known in the art. Such carriers enable the combined compounds to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions, for oral administration to a patient to be treated. The oral pharmaceutical preparations can be obtained as solid excipients, optionally grinding the resulting mixture, and processing the mixture of granules, if desired to obtain tablets or dragee cores, after adding suitable auxiliaries. Suitable excipients are in particular fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations, for example maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents, such as cross-linked polyvinylpyrrolidone, agar or alginic acid or a salt thereof such as sodium alginate, may be added.

Dragee cores are provided with suitable coatings. To this end, concentrated sugar solutions may be used, which may optionally comprise gum arabic, talc, polyvinyl pyrrolidone, carbomer gel, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the tablets or dragee coatings for the purpose of identifying or characterizing different combinations of active compound doses.

Pharmaceutical preparations for oral use include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Push-fit capsules may contain the active ingredients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, for example fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be suitable for such dosages for administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds used in accordance with embodiments of the present disclosure are conveniently delivered in the form of an aerosol spray from a pressurized pack or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin, containing a powder mix of the compound and a suitable powder base such as lactose or starch, may be formulated for use in an inhaler or insufflator.

One or more of the components of the combinations disclosed herein may be formulated for parenteral administration by injection, for example by bolus injection or continuous infusion. Injection is the preferred method of administration. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The formulations may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as the addition of suspending, stabilizing and/or dispersing agents, for example cross-linked polyvinylpyrrolidone, agar or alginic acid or a salt thereof such as sodium alginate.

Pharmaceutical preparations for parenteral administration comprise aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, for example sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound to allow for the preparation of highly concentrated solutions. For injection, the components of the combinations disclosed herein may be formulated in aqueous solution, preferably in a physiologically compatible buffer, such as hanks 'solution, ringer's solution, or physiological saline buffer.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds may also be formulated as depot preparations. Such long acting formulations may be administered by implantation or transdermal delivery (e.g., subcutaneous or intramuscular), intramuscular injection, or transdermal patch. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g., as a sparingly soluble salt.

The pharmaceutical compositions may also comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include calcium carbonate, calcium phosphate, various sugars, starch, cellulose derivatives, gelatin, and polymers such as polyethylene glycol.

In some embodiments, the pharmaceutical composition of embodiments of the present disclosure further comprises an additional therapeutic agent.

In some embodiments, the additional therapeutic agent is an anti-cancer agent.

In some embodiments, the additional anti-cancer agent is selected from an anti-metabolite, topoisomerase I and II inhibitor, an alkylating agent, a microtubule inhibitor, an anti-androgen agent, a GNRh modulator, or a mixture thereof.

In some embodiments, the additional therapeutic agent is a chemotherapeutic agent.

By "chemotherapeutic agent" herein is meant a compound useful in the treatment of cancer. Examples include, but are not limited to: gemcitabine, irinotecan, doxorubicin, 5-fluorouracil, cytarabine ("Ara-C"), cyclophosphamide, thiotepa, busulfan, Cytoxin, TAXOL, methotrexate, cisplatin, melphalan, vinblastine, and carboplatin.

In some embodiments, the second chemotherapeutic agent is selected from the group consisting of tamoxifen, raloxifene, anastrozole, exemestane, letrozole, imatinib, paclitaxel, cyclophosphamide, lovastatin, minoxidil, gemcitabine, cytarabine, 5-fluorouracil, methotrexate, docetaxel, goserelin, vincristine, vinblastine, nocodazole, teniposide etoposide, gemcitabine, epothilone, vinorelbine, camptothecin, daunorubicin, actinomycin D, mitoxantrone, acridine, doxorubicin, epirubicin, or idarubicin.

IV、Reagent kit

In another aspect, embodiments of the present disclosure provide kits comprising a therapeutic combination provided herein and instructions for using the therapeutic combination. The kit may further comprise a container and optionally one or more vials, tubes, flasks, bottles, or syringes. Other forms of kits will be apparent to those skilled in the art and are within the scope of embodiments of the disclosure.

V、Medical use

In another aspect, embodiments disclosed herein provide a method for treating a disease condition in a subject in need of such treatment, comprising: administering to a subject a therapeutic combination or pharmaceutical composition comprising a therapeutically effective amount of a compound of an embodiment of the disclosure, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

In addition to the compositions and constructs described above, embodiments of the present disclosure provide a variety of uses for therapeutic combinations. The combinations of the present disclosure comprise chemotherapeutic agents having one or more functions of a myeloid-derived suppressor cell inhibitor. These uses include administering to an animal, such as a mammal or human, in need thereof an effective amount of a compound of an embodiment of the disclosure.

The combinations of the present disclosure are useful for treating a disease, e.g., cancer, in a subject, e.g., a human. Combinations and uses for treating tumors by providing a subject with the combined compounds in a pharmaceutically acceptable composition and manner, and a pharmaceutically effective amount of the composition of the disclosed embodiments.

"cancer" as used herein refers to a pathological condition in humans characterized by unregulated cell proliferation. Examples include, but are not limited to: carcinomas, sarcomas, lymphomas, blastomas, and leukemias. More specific examples of cancer include, but are not limited to: acute Myeloid Leukemia (AML), breast cancer, Chronic Lymphocytic Leukemia (CLL), Chronic Myeloid Leukemia (CML), hodgkin's lymphoma, multiple myeloma, mycosis fungoides, neuroblastoma, non-hodgkin's lymphoma (NHL), ovarian cancer, and retinoblastoma.

As used herein, "inhibiting" or "treating" or "treatment" refers to reducing, treating, and prophylactic or preventative treatment, wherein the object is to reduce or prevent the targeted pathological disorder or condition. In one example, a cancer patient may experience a reduction in tumor size after administration of a compound of an embodiment of the disclosure. "treating" or "treatment" includes (1) inhibiting a disease in a subject experiencing or exhibiting a pathology or symptom of the disease, (2) ameliorating the disease in a subject experiencing or exhibiting a pathology or symptom of the disease, (3) affecting any measurable reduction in the disease in a subject or patient experiencing or exhibiting a pathology or symptom of the disease. To the extent that a compound of an embodiment of the present disclosure can prevent growth and/or kill cancer cells, it can be cytostatic and/or cytotoxic.

The terms "treat" or "treatment" broadly include any kind of therapeutic activity, including any activity that alleviates or prevents a disease in a human or other animal, or otherwise affects the structure or any function of the human or other animal's body. The therapeutic activities include administration of the medicaments, dosage forms, and pharmaceutical compositions described herein to a patient, particularly according to the various methods of treatment disclosed herein, whether by a healthcare professional, the patient himself, or any other person. The treatment activities include orders, instructions, and recommendations by healthcare professionals, such as doctors, physician assistants, nurse practitioners, and the like, followed by actions by any other person, including other healthcare professionals or the patient himself. For the combinations of the present disclosure, the different ingredients may be administered by different humans; however, the activity should be considered as being performed on the order of a prescription or supervising healthcare professional, such as but not limited to an oncologist or a resident, or on the request of the patient in order for the patient to obtain a combined benefit. In some embodiments, the treatment activity may also include encouraging, inducing, or managing the selection of a particular drug or combination thereof to treat the condition-and the drug is actually used-either by insurance coverage approving the drug, refusal coverage replacing the drug including the drug or combination, or eliminating the replacement drug or combination from a drug prescription set, or providing economic incentive to use the drug or combination, as may be done by an insurance company or a pharmacy welfare authority, or the like. In some embodiments, the treatment activity may also include encouraging, inducing or managing the selection of a particular combination drug to treat the condition-and the drug or combination is actually used-through policy or practice criteria that may be established by a hospital, clinic, health maintenance organization, medical practice or physician community, or the like. In addition to the therapeutic benefits to the patient, the treatments disclosed herein may participate in benefits related to the cost and cost effectiveness of all parties, as well as benefits related to the effect of overall improvements in the patient population produced by healthcare professionals, institutions, and insurers.

Cancer treatment is usually performed cyclically. As used herein, the most basic treatment cycle refers to the time interval during which the drug is administered and the subsequent time interval during which it is not administered. When the administration is restarted, a new cycle begins. The intervals of non-administration provide the body with an opportunity to recover from adverse side effects of treatment and/or to assess the effectiveness of treatment by medical personnel. Further, when two active agents are administered in combination, as in embodiments of the present disclosure, the two active agents may be administered at different schedules, where the schedule of one agent defines the start of a cycle. In some embodiments, the active agent may be administered a fixed number of times, in a fixed cycle, and/or within a fixed time interval. In other embodiments, any one or more of these parameters may be variable. In certain embodiments as described herein, one cycle begins with administration of the chemotherapeutic agent component, followed by a first administration of the immunotherapeutic agent component, e.g., 1-7 days later. The initiation of administration of the immunotherapeutic agent may also be based on the threshold degree to which MDCS reduction is achieved. In some embodiments, the chemotherapeutic agent is administered once per cycle. In some embodiments, administration of the chemotherapeutic agent continues beyond the point where administration of the immunotherapeutic agent begins. In some embodiments, administration of the chemotherapeutic agent is stopped after a certain time interval. In some embodiments, the chemotherapeutic agent is administered repeatedly at certain time intervals within the cycle or when the MDCS number rises above a threshold proportion of its pre-treatment level (pretreatment level), e.g., >30, >40, >50, >60, >70, or > 80% of its pre-treatment level. In some embodiments, administration of the immunotherapeutic agent continues for a period of time within the cycle after administration of the chemotherapeutic agent ceases. In some embodiments, a cycle is defined as the cessation and resumption of administration of the chemotherapeutic agent component. In an alternative embodiment, the concurrent administration of the chemotherapeutic agent and the immunotherapeutic agent is stopped for a period of time before starting a new cycle.

By "therapeutically effective amount" herein is meant an amount of a compound provided by the present disclosure effective to "treat" a condition in a subject or mammal. In the case of cancer, a therapeutically effective amount of the drug may reduce the number of cancer cells, reduce tumor size, inhibit the penetration of cancer cells into peripheral organs, inhibit tumor metastasis, inhibit tumor growth to some extent, and/or alleviate one or more symptoms associated with the cancer to some extent.

The effectiveness of cancer treatment is often measured in terms of "response". Techniques to monitor the response may be similar to tests used to diagnose cancer, such as, but not limited to:

tumors or tumors involving some lymph nodes can be externally palpated and measured by physical examination.

Some internal cancer tumors may appear in an X-ray or CT scan, which can be measured with a ruler.

Blood tests, including those that measure organ function, can be performed.

Certain cancers can be tested for tumor markers.

Whatever test is used, whether it is a blood test, a cell count or a tumor marker test, is repeated at specific time intervals in order to compare the results with earlier tests of the same type.

There are several definitions of response to cancer treatment:

complete response-disappearance of all cancers or tumors; there were no signs of disease. The expression level of a tumor marker (as applicable) may be within the normal range.

Partial response-cancer shrinks by a certain percentage, but the disease remains. The level of a tumor marker (if applicable) may have decreased (or increased, according to the tumor marker, as an indication of a decreased tumor burden), but evidence of disease still exists.

Stable disease-cancer neither increases nor decreases; the number of diseases did not change. Tumor markers (if applicable) did not change significantly.

Disease progression-cancer has progressed; more diseases are present than before treatment. The tumor marker test (if applicable) shows that the tumor marker has increased.

Other measures of cancer treatment effectiveness include overall survival interval (i.e., time to death for any cause measured from diagnosis or from the start of the treatment being evaluated), cancer-free survival (i.e., length of time after complete response to cancer remains undetectable), and progression-free survival (i.e., length of time after disease stabilization or partial response to restoration of tumor growth is undetectable).

There are two standard methods for assessing the response of solid cancer treatments based on tumor size (tumor burden), the WHO and RECIST criteria. These methods measure solid tumors to compare current tumors to past measurements or changes to future measurements and change treatment regimens. In the WHO method, the major and minor axes of a solid tumor are measured, and then the product of these two measurements is calculated; if there are multiple solid tumors, the sum of all products is calculated. In RECIST method, only the long axis is measured. If there are multiple solid tumors, the sum of all long axis measurements is calculated. However, for lymph nodes, the short axis is measured instead of the long axis.

Administration "in combination with" one or more other therapeutic agents includes simultaneous (concurrent) and sequential administration in any order. In some embodiments, a "pharmaceutical composition" refers to a product obtained by mixing or combining active ingredients, and includes fixed and non-fixed combinations of active ingredients. The term "fixed combination" means that the active ingredients are administered to a patient simultaneously in the form of a single entity or dose. The term "non-fixed combination" means that the active ingredients are administered to a patient as separate entities simultaneously, concurrently or sequentially with no specific time limitation, wherein such administration provides therapeutically effective levels of the active ingredients in the patient. The latter also applies to cocktail therapies, such as the administration of three or more active ingredients.

In some embodiments, the disease condition is a tumor or cancer. In some embodiments, the cancer or tumor is selected from the group consisting of gastric, colon, rectal, liver, pancreatic, lung, breast, cervical, uterine corpus, ovarian, testicular, bladder, kidney, brain/CNS, head and neck, throat, hodgkin's disease, non-hodgkin's lymphoma, multiple myeloma, leukemia, melanoma, non-melanoma skin cancer, acute lymphocytic leukemia, acute myeloid leukemia, ewing's sarcoma, small cell lung cancer, choriocarcinoma, rhabdomyosarcoma, wilms' tumor, neuroblastoma, hairy cell leukemia, oral/pharyngeal cancer, esophageal, laryngeal, renal, or lymphoma.

In some embodiments, the disease condition comprises abnormal cell proliferation, such as a precancerous lesion.

The combinations of the present disclosure are particularly useful for treating cancer and inhibiting the proliferation of tumor cells or cancer cells in an animal. Cancer or precancerous conditions, including tumors, metastases, or any disease or disorder characterized by uncontrolled cell growth, can be treated or prevented by administering a chemotherapeutic agent-immunotherapeutic agent combination. In some embodiments comprising a targeted therapeutic, the targeting moiety specifically binds to or is associated with a cancer cell or tumor cell-associated antigen. In some embodiments comprising an immunotherapeutic agent and a targeted therapeutic agent, the two may be conjugated, i.e., they are provided as antibody-drug conjugates (ADCs). In some embodiments, the ADC is internalized upon binding to a cell, e.g., by receptor-mediated endocytosis (TLR7 and TLR8 are endosomal receptors). The antigen may be attached to a tumor cell or a cancer cell, or may be an extracellular matrix protein associated with a tumor cell or a cancer cell. Once inside the cell, the linker is hydrolyzed or enzymatically cleaved by the tumor cell or cancer cell-associated protease, thereby releasing the immunotherapeutic agent. The released immunotherapeutic agent is then free to diffuse and induce or enhance the immune activity of the immune or tumor cells. In another embodiment, the immunotherapeutic agent is lysed in the tumor microenvironment, and the immunotherapeutic agent subsequently permeabilizes the cells.

Representative examples of precancerous conditions that may be targeted by the compounds of the embodiments of the present disclosure include: metaplasia, hyperplasia, dysplasia, colorectal polyps, actinic keratosis, actinic cheilitis, human papilloma virus, leukoplakia, lichen planus, and bowen's disease.

Representative examples of cancers or tumors that may be targeted by the compounds of the disclosed embodiments include: lung cancer, colon cancer, prostate cancer, lymphoma, melanoma, breast cancer, ovarian cancer, testicular cancer, CNS cancer, kidney cancer (renal cancer), kidney cancer (kidney cancer), pancreatic cancer, stomach cancer, oral cancer, nasal cancer, cervical cancer, and leukemia. It will be apparent to the skilled person that the particular targeting moiety used in the compound may be selected to target the active moiety to the tumour tissue to be treated with the drug.

In some embodiments, the abnormal proliferation is abnormal proliferation of cancer cells.

In some embodiments, the cancer is selected from the group consisting of breast cancer, colorectal cancer, diffuse large B-cell lymphoma, endometrial cancer, follicular lymphoma, gastric cancer, glioblastoma, head and neck cancer, hepatocellular carcinoma, lung cancer, melanoma, multiple myeloma, ovarian cancer, pancreatic cancer, prostate cancer, and renal cell carcinoma.

In some embodiments, the cancer treated is selected from the group consisting of Acute Myeloid Leukemia (AML), breast cancer, Chronic Lymphocytic Leukemia (CLL), Chronic Myeloid Leukemia (CML), hodgkin's lymphoma, multiple myeloma, mycosis fungoides, neuroblastoma, non-hodgkin's lymphoma (NHL), and ovarian cancer.

In some embodiments, embodiments of the present disclosure provide compounds for killing cells. Administering a compound to the cells in an amount sufficient to kill the cells. In exemplary embodiments, the compound is administered to a subject bearing cells. In another exemplary embodiment, the administration is for delaying or stopping the growth of a tumor comprising cells (e.g., the cells may be tumor cells). For administration to delay growth, the growth rate of the cells should be at least 10% less than the growth rate prior to administration. Preferably, the growth rate will be delayed by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or stopped completely.

Furthermore, embodiments of the present disclosure provide a compound or pharmaceutical composition of an embodiment of the present disclosure for use as a medicament. Embodiments of the disclosure also provide compounds or pharmaceutical compositions for killing, inhibiting, or delaying the proliferation of tumors or cancer cells, or for treating diseases in which TLR7 and/or TLR8 are involved.

Dosage and mode of administration

In some embodiments, the chemotherapeutic agent is provided in an amount capable of reducing the amount of MDCS in the blood, spleen, and/or tumor microenvironment of the subject by about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95%. Preferably by 30, 40 or 50%.

In some embodiments, the chemotherapeutic agent is provided in an amount that is lower than when used as monotherapy, for example about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 95%, preferably about 50% of the standard monotherapy dose.

In some embodiments, the chemotherapeutic agent is gemcitabine and is present in an amount of 400-625mg/m²。

In some embodiments, the chemotherapeutic agent is administered prior to administration of the immunotherapy and within 7 days of administration of the immunotherapeutic agent.

In some embodiments, the chemotherapeutic agent is administered prior to administration of the immunotherapeutic agent.

In some embodiments, the chemotherapeutic agent is administered at least one, two, three, four, five, six, or seven days prior to administration of the immunotherapeutic agent.

In some embodiments, the immunotherapeutic agent is administered after a 10% to 95% reduction in the amount of MDCS in the blood and/or tumor microenvironment in the subject following administration of the chemotherapeutic agent.

In some embodiments, the immunotherapeutic agent is administered in combination with a targeted therapeutic agent against cancer, either simultaneously, e.g., in a single formulation, or separately, e.g., in separate formulations.

In some embodiments, "providing in an amount" includes administering the specified amount. In some embodiments, "providing in an amount" includes providing a dosage form, including but not limited to a unit dosage form, continuing with the specified amount.

Effective dose

Pharmaceutical compositions suitable for use in embodiments of the present disclosure include compositions in which the active ingredient is contained in a therapeutically effective amount, i.e., in an amount effective to achieve its intended purpose. The actual amount effective for a particular application will depend, inter alia, on the condition being treated.

For any of the compounds described herein, a therapeutically effective amount can be initially determined by cell culture assays. The target plasma concentrations will be those of the active compound capable of inhibiting cell growth or division. In a preferred embodiment, cellular activity is inhibited by at least 25%. The target plasma concentration of the active compound is presently preferably capable of inducing at least about 30%, 50%, 75% or even 90% or more inhibition of cellular activity. The percent inhibition of cellular activity in the patient can be monitored to assess the appropriateness of the achieved plasma drug concentration, and the dose can be adjusted up or down to achieve the desired percent inhibition.

A therapeutically effective amount for use in humans can also be determined from animal models. For example, dosages for humans can be formulated to achieve circulating concentrations that have been found to be effective in animals. As described above, the human dose can be adjusted by monitoring cytostatic and adjusting the dose up or down.

For compounds known to have similar pharmacological activity, a therapeutically effective dose can also be determined from human data. The dosage administered may be adjusted according to the relative bioavailability and potency of the administered compound compared to known compounds.

Adjusting the dosage to achieve maximal efficacy in humans can be accomplished based on the methods described above and other methods known in the art.

In some embodiments, the compositions of embodiments of the present disclosure are delivered locally or regionally to a tumor located within the body of a subject, systemically, or by intratumoral injection or by direct injection into the tumor vasculature.

In some embodiments, the compositions provided by the present disclosure are formulated for systemic delivery. In some embodiments, the pharmaceutical composition is formulated for oral administration or parenteral injection. In some embodiments, the pharmaceutical composition is formulated for intravenous injection or intratumoral injection.

In another aspect, embodiments of the present disclosure provide methods of treating a tumor or abnormal cell proliferation in a subject in need of such treatment comprising administering to the subject a combination provided by the present disclosure.

In some embodiments, the methods provided by the present disclosure comprise administering to the subject an oral formulation comprising the immunotherapeutic agent twice weekly at a dose of about 0.0005mg/kg, 0.0006mg/kg, 0.0007mg/kg, 0.0008mg/kg, 0.0009mg/kg, 0.001mg/kg, 0.002mg/kg, 0.003mg/kg, 0.004mg/kg, 0.005mg/kg, 0.006mg/kg, 0.007mg/kg, 0.008mg/kg, 0.009mg/kg, or 0.01mg/kg, to about 0.02mg/kg, including all.

In some embodiments, the methods provided by the present disclosure comprise administering to the subject an oral formulation comprising the immunotherapeutic agent at a dose of less than or about 0.0005mg/kg, 0.0006mg/kg, 0.0007mg/kg, 0.0008mg/kg, 0.0009mg/kg, 0.001mg/kg, 0.002mg/kg, 0.003mg/kg, 0.004mg/kg, 0.005mg/kg, 0.006mg/kg, 0.007mg/kg, 0.008mg/kg, 0.009mg/kg, or 0.01mg/kg twice weekly.

In some embodiments, the methods provided by the present disclosure comprise administering to the subject an intravenous formulation comprising the immunotherapeutic agent at a dose, including all, of about 0.0005mg/kg, 0.0006mg/kg, 0.0007mg/kg, 0.0008mg/kg, 0.0009mg/kg, 0.001mg/kg, 0.002mg/kg, 0.003mg/kg, 0.004mg/kg, 0.005mg/kg, or 0.006mg/kg to about 0.015mg/kg, including all, once a week.

In some embodiments, the combination is administered repeatedly at 2,3, or4 week intervals within each cycle.

In some embodiments, the methods provided by the present disclosure comprise administering to the subject an intravenous formulation comprising a chemotherapeutic agent at a dose of about 10 to 15mg/kg, once every 7 to 10 days.

In some embodiments, the methods provided by the present disclosure comprise administering an effective amount of about 60-120mg/m²The dose per day is administered to the subject an intravenous formulation comprising a chemotherapeutic agent for a continuous daily period.

In some embodiments, the methods provided by the present disclosure include treating the mammal with about 400-²Administering to the subject an oral formulation comprising a chemotherapeutic agent in 4-5 days.

In some embodiments, the methods provided by the present disclosure comprise administering an effective amount of about 50-100mg/m²Administering to the subject an intravenous formulation comprising a chemotherapeutic agent at a dose per day or 1-5 mg/kg/day.

In some embodiments, the methods provided by the present disclosure comprise administering to the subject an intravenous formulation comprising a chemotherapeutic agent in the form of an intermittent therapy, wherein 40-50mg/kg is divided into doses administered over 2-5 days. Administration may be repeated at 2-4 week intervals during each cycle. In some embodiments, the dose is 10 to 15mg/kg administered every 7 to 10 days; or 3 to 5mg/kg twice weekly.

In some embodiments, the methods provided by the present disclosure comprise administering to the subject an oral formulation comprising a chemotherapeutic agent in the form of an intermittent therapy, wherein a dose of 40-50mg/kg is divided to be administered within 4-5 days.

In some embodiments, the methods provided by the present disclosure comprise administering to the subject an oral formulation comprising a chemotherapeutic agent at a dose of 1-5 mg/kg/day.

In the case of topical administration, the systemic circulating concentration of the administered compound is not particularly important. In such cases, the compound is administered to achieve a concentration in the local area effective to achieve the desired result.

Therapeutic amounts of the specific antibodies disclosed herein can also be administered as a combined component with an immunotherapeutic agent in a single mixture or separately. In some embodiments, the therapeutic amount is an amount that eliminates or reduces tumor burden, or prevents or reduces metastatic cell proliferation, in the patient. The dosage will depend on a number of parameters, including the nature of the tumor, patient history, patient condition, possible co-use of other oncolytic agents, and the method of administration. Methods of administration include injection (e.g., parenteral, subcutaneous, intravenous, intraperitoneal, etc.), wherein the antibody is provided in a non-toxic pharmaceutically acceptable carrier such as water, saline, ringer's solution, dextrose solution, 5% human serum albumin, fixed oil, ethyl oleate, or liposomes. A typical dosage range may be from about 0.01 to about 20mg/kg, for example from about 0.1 to about 10 mg/kg. Other effective methods and dosages of administration can be determined by routine experimentation and are within the scope of the present invention.

For other modes of administration, the dosage and interval may be adjusted individually to provide plasma levels of the administered compound that are effective for the particular clinical indication being treated. For example, in one embodiment, a compound according to an embodiment of the present disclosure may be administered at relatively high concentrations multiple times per day. Alternatively, it may be more desirable to administer the combined compounds in a minimum effective concentration and less frequent dosing regimen. This will provide a treatment regimen commensurate with the severity of the disease in the individual.

Using the teachings provided by the present disclosure, an effective treatment regimen can be planned that does not cause significant toxicity and is completely effective in treating the clinical symptoms exhibited by a particular patient. The program should include careful selection of the active compound by consideration of such factors as the potency of the compound, relative bioavailability, patient weight, presence and severity of adverse side effects, mode of administration preferred and toxicity characteristics of the drug selected.

Toxicity and adverse events were sometimes graded according to a 5-grade system. Grade 1 or mild toxicity is asymptomatic or causes only mild symptoms; may be characterized only by clinical or diagnostic observations; and no intervention is indicated. Grade 2 or moderate toxicity may impair activities of daily living (e.g., preparing meals, shopping, managing money, using telephone, etc.), but indicates that only minor, local or non-invasive intervention is required. Grade 3 toxicity is medically significant, but not immediately life threatening; indicating a need for hospitalization or extended hospitalization; may affect daily activities associated with self-care (e.g., bathing, dressing and undressing, having eaten, going to the toilet, taking medicine, and being bedridden). Grade 4 toxicity is life threatening, indicating that urgent intervention is required. Grade 5 toxicity produces death associated with adverse events. Thus, in various embodiments, use of the disclosed combinations will reduce the magnitude of toxicity associated with the treatment by at least one magnitude as compared to use of a similarly effective dose of the immunotherapeutic agent alone, if such effectiveness can be achieved. Alternatively, the immunotherapeutic agent has greater efficacy as part of the combination, which can be achieved at the maximum tolerated dose of the immunotherapeutic agent used alone. In other embodiments, the use of the disclosed combinations limits toxicity to grade 2 or less, grade 1 or less, or does not produce observations of particular toxicity.

While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments described herein may be employed in practicing the invention.

Examples

Embodiments of the present disclosure are further illustrated by, but not limited to, the following description of embodiments of preparing a combination of compounds.

Example 1

Killing tumor cells in vivo using a combination therapy of TLRL with PDL1 and gemcitabine in immunocompetent mice

Female 6-7 week old C3H/HeN (C3H) mice were purchased from Japanese SLC (Oriental Bittercress). All mice were kept under specific pathogen free conditions at the university of dentistry, tokyo. All experimental procedures were reviewed and approved by the animal care and use committee of the tokyo medical dental university (0170344a and a 2018-262C). NR-S1 and SCC VII are murine Squamous Cell Carcinoma (SCC) cell lines of C3H origin. Cells were cultured for 5-7 days before being used for tumor inoculation. Mixing NR-S1(1x 10)⁶Individual cell) and SCCVII (2X 10)⁵Individual cells) were injected subcutaneously (s.c.) into the right side of the shaved hair of syngeneic C3H mice, and tumor size was measured as described previously. When the tumor volume of the individual mouse reaches 30mm³(6-8 days after tumor inoculation) treatment was initiated. Resiquimod (1.7 μ g/mouse) and/or anti-PD-L1 mAb (MIH5, rat IgG2a, 200 μ g/mouse) were administered intraperitoneally. Tumor diameter was measured along three orthogonal axes (x, y and z) and tumor volume was calculated by (xyz)/2.

In two independent experiments, mice received 1.7 μ g of TLRL (resiquimod) or combination therapy with 200 μ g of PDL1 antibody (clone: MIH5, rat IgG2Ga), once every three days, 4 times in the NR-S1 tumor model shown in FIG. 1, or once weekly in the 4T1 tumor model shown in FIG. 3. Gemcitabine (30mg/kg) was injected intraperitoneally the day before the initial treatment.

Example 2

Analysis of MSDC in NR-S1 and SCCVII tumors

Tumor infiltrating lymphocytes were isolated by enzymatic digestion with collagenase I, hyaluronidase and DNase and density gradient centrifugation. Monoclonal antibodies (mAbs) directed against CD (17A, rat IgG 2), CD (GK1.5, rat IgG 2), CD (53-6.72, rat IgG), CD (30-F, rat IgG 2), IFN-r (XMG1.2, rat IgG), Foxp (-165, rat IgG 2), CD11 (M/70, rat IgG 2), Gr-1(Ly 6/Ly 6, RB-8C, rat IgGa), Ly6 (HK1.4, rat IgG 2), Ly6 (1A, rat IgG 2), F/80 (BM, rat IgG 2), CD (PO3.1, rat IgG 2), MHC class II (M/114, rat IgG 2) and PD-L (MIH, rat IgG 2) were used. All fluorochrome (FITC, PE-Cyanine7, APC-eFluor780, eFluor450, Brmiant Violet 510) conjugated mAbs were from Thermo Fisher Scientific (Calsbarded, Calif.), BD-Biosciences (san Jose, Calif.) or Biolegend (san Diego, Calif.). All stained cells were analyzed using a FACSVerse flow cytometer (BD Biosciences) and FlowJo software (Tree Star, Ashland, OR). Statistical analysis was performed using the Mann-Whitney U test. A value of p <0.05 was considered to indicate significance.

The results depicted in figure 2 indicate that the immune status of tumor associated macrophages differs for the two SCC tumors, especially with respect to CD11b + bone marrow cells. Ly6GhighLy 6C-F4/80-tumor associated neutrophils predominate in NR-S1 tumors, Ly6GlowLy6Clow/-F4/80+ tumor associated macrophages predominate in SCCVII tumors.

The proportion of CD11b + cells in NR-S1 tumors was significantly lower than that of CD11b + cells in SCCVII tumors, with a higher proportion of CD3+ T cells (FIG. 2A).

CD11B + cells showed different levels of Gr-1 expression (FIG. 2B, top right). Most of the CD11b + cells in the NR-S1 tumor had the Ly6GhighLy 6C-phenotype (Fr-1), while most of the CD11b + cells in the SCCVII tumor had the Ly6GlowLy 6C-phenotype (Fr-2). SCCVII Fr-2 cells were FSChigh, SSChigh and F4/80 +.

Expression of F4/80 and Ly6G, cell size of the FSC and intracellular granule status of SSC indicate that Fr-1 and Fr-2 are tumor-associated neutrophils and tumor-associated macrophages, respectively.

Spleens from NR-S1 tumor-bearing mice showed marked splenomegaly and a two-fold greater expression profile for Ly6C and Ly6G (FIG. 2B).

Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about". As used herein, the terms "about" and "approximately" mean within 10% to 15%, preferably within 5% to 10%. Accordingly, unless indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided in this disclosure, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is contemplated that one or more members of a group may be included in or deleted from the group for convenience and/or patentability reasons. When any such inclusion or deletion occurs, the specification is considered to encompass the modified group, thereby fulfilling the written description of all markush groups used in the appended claims.

Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations of those described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

The specific embodiments disclosed herein may be further limited in the claims by the use of language that consists of or consists essentially of language. As used in the claims, the transitional term "consisting of … …, whether filed or added as a modification, excludes any elements, steps or components not specified in the claims. The transitional term "consisting essentially of … …" limits the scope of the claims to particular materials or steps, as well as those materials or steps, which do not materially affect the basic and novel characteristics. Embodiments of the invention so claimed are described and enabled herein either inherently or explicitly.

Further, throughout the specification, numerous references are made to patents and printed publications. Each of the above-cited references and printed publications is individually incorporated by reference herein in its entirety.

Finally, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, and not limitation, alternative configurations of the present invention may be used in accordance with the teachings herein. Accordingly, the invention is not limited to that shown and described.

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