Immune-stimulating micellar compositions

Abstract

The present invention relates to immune-stimulating micellar compositions and their use in the treatment of diseases and disorders, such as cancer. In particular, the present invention relates to micellar compositions comprising a TLR7 agonist, such as 1V270.

Description

[Technical field]

[0001] Technical Field The present invention relates to immune stimulating micellar compositions and their use in the treatment of diseases and disorders, such as cancer. In particular, the present invention relates to micellar compositions comprising a TLR7 agonist, such as 1V270. [Background technology]

[0002] background Toll-like receptors (TLRs) are a class of receptors expressed on various cell types and play an important role in the innate immune system. Upon activation, TLRs activate signaling pathways involved in immune activation. Several mammalian TLRs and many of their agonists have been identified. For example, single-stranded RNA rich in guanine and uridine has been identified as a natural ligand for TLR7. In addition, several low molecular weight activators of TLR7, including imidazoquinolines and purine-like molecules, have been identified. TLR stimulation initiates a common signaling cascade (involving the adaptor protein MyD88, the transcription factor NFκB, and inflammatory and effector cytokines), and although different TLRs are expressed by different cell types, TLR7 is primarily expressed in monocytes, plasmacytoid dendritic cells, myeloid dendritic cells, and B cells and is localized to the endosomal membrane.

[0003] TLR7 has been shown to play an important role in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE) and in regulating antiviral immunity. The TLR7 agonist Aldara (imiquimod), an imidazoquinoline, has been approved for topical use in the treatment of warts caused by papillomaviruses, basal cell carcinomas, and photoactivated keratoses. Due to their ability to induce robust production of anticancer cytokines such as interleukin-12, TLR7 agonists have also been investigated for cancer immunotherapy. Recent examples include TMX-202 delivery via liposomal formulations, and resiquimod delivery via nanoparticles formed from β-cyclodextrin. Summary of the Invention [Problem to be solved by the invention]

[0004] However, repeated injections of therapeutic nanoparticle compositions have been shown to induce the phenomenon of accelerated blood clearance by antibodies (ABC), especially for PEG-based liposomes, which alters the usefulness of therapeutic nanoparticle delivery systems. Thus, there is a need in the art for the development of more effective therapeutic nanoparticle compositions. [Means for solving the problem]

[0005] The present inventors have surprisingly discovered that micellar compositions containing TLR7 agonists exhibit highly potent anti-cancer activity and are devoid of previously recognized undesirable pharmacokinetic behavior. Effect of the Invention

[0006] In a first aspect, the present disclosure provides a compound of formula (I), formula (II), formula (III) or formula (IV); [ka] (In the formula, In the formula, X 1 is -O-, -S-, or NR C and; R 1 is hydrogen, (C1-C 10 ) Alkyl, Substituted (C1-C 10 ) Alkyl, C 6~10 Aryl or substituted C 6~10 Aryl, C 5~9 Heterocyclic, substituted C 5~9 is a heterocyclic ring; R C is hydrogen, C 1~10 Alkyl or substituted C 1~10 alkyl or R C and R 1 together with the nitrogen to which they are attached form a heterocyclic ring or a substituted heterocyclic ring; Each R 2are independently -OH, (C1-C6) alkyl, substituted (C1-C6) alkyl, (C1-C6) alkoxy, substituted (C1-C6) alkoxy, -C(O)-(C1-C6) alkyl (alkanoyl), substituted -C(O)-(C1-C6) alkyl, -C(O)-(C6-C 10 )Aryl (aroyl), substituted -C(O)-(C6-C 10 )Aryl, -C(O)OH (carboxyl), -C(O)O(C1-C6)alkyl (alkoxycarbonyl), substituted -C(O)O(C1-C6)alkyl, -NR a R b , -C(O)NR a R b (carbamoyl), halo, nitro, or cyano, or R 2 does not exist; Each R a and R b are independently hydrogen, (C1-C6)alkyl, substituted (C1-C6)alkyl, (C3-C8)cycloalkyl, substituted (C3-C8)cycloalkyl, (C1-C6)alkoxy, substituted (C1-C6)alkoxy, (C1-C6)alkanoyl, substituted (C1-C6)alkanoyl, aryl, aryl(C1-C6)alkyl, Het, Het(C1-C6)alkyl, or (C1-C6)alkoxycarbonyl; Substituents on any alkyl, aryl or heterocyclic group include hydroxy, C 1~6 Alkyl, Hydroxy C 1~6 Alkylene, C 1~6 Alkoxy, C 3~6 Cycloalkyl, C 1~6 Alkoxy, C 1~6 alkylene, amino, cyano, halo, or aryl; n is 0, 1, 2, 3 or 4; X 2 is a bond or a linking group; and R 3 is a lipid; X 3 is -N- or -CH-; R 4 is -CH2- or -CH(R 2 )-and k is 0 or 1; X 4 -O-, -S-, -NH-, -N(R d )-, -CH2-, or -CH(R 2 )- and; Each R d are independently -OH, (C1-C6) alkyl, substituted (C1-C6) alkyl, (C1-C6) alkoxy, substituted (C1-C6) alkoxy, -C(O)-(C1-C6) alkyl (alkanoyl), substituted -C(O)-(C1-C6) alkyl, -C(O)-(C6-C 10 )Aryl (aroyl), substituted -C(O)-(C6-C 10 )Aryl, -C(O)O(C1-C6)alkyl(alkoxycarbonyl), substituted -C(O)O(C1-C6)alkyl, -C(O)NR a R b (carbamoyl) Toll-like receptor 7 (TLR7) agonists or its tautomer; or a pharma- ceutically acceptable salt or solvate thereof, The ring system of formula (II) contains one heteroatom which is a N atom and X 2 is a piperidine ring having a N atom of the piperidine ring adjacent to A purine group in any of formulas (I), (II), (III), or (IV) undergoes tautomeric rearrangement; and an amphiphilic micelle forming agent.

[0007] In a second aspect, a micelle composition or pharmaceutical composition as defined herein is provided for use in the prevention, treatment or amelioration of a disease or disorder.

[0008] In a third aspect, there is provided a method for the in vivo activation of immune cells in a subject, comprising administering to said subject a micelle composition or pharmaceutical composition as defined herein in an amount sufficient to activate said immune cells.

[0009] In a fourth aspect, there is provided a method for enhancing or enhancing a treatment comprising radiation therapy and / or administration of a chemotherapeutic agent or an immune checkpoint inhibitor, comprising administering to said subject a micelle composition or pharmaceutical composition as defined herein in combination with radiation therapy, administration of a chemotherapeutic agent and / or administration of an immune checkpoint inhibitor. [Brief description of the drawings]

[0010] [Figure 1A] FIG. 1A: Overview of micelles used, micelle name, composition, molar ratio, surface charge (zeta), size and polydispersity index (PDI). Micelles were composed of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DOPE-PEG2000 or PEG) and TLR7 agonist 1v270 (2-(4-(6-amino-2-(2-methoxyethoxy)-8-oxo-7H-purin-9((8H)-yl)methyl)benzamido)ethyl 2,3-bis(oleoyloxy)propyl phosphate) (1v270). The composition and molar ratio of each component in the micelle are shown. Micelle surface charge is expressed in mV. Micelle size is measured in nanometers (nm). Errors are expressed as SEM. Micelle names are used in the figures to clarify the exact composition. [Figure 1B] Figure 1B: Micelle size by number distribution of the four micelles tested. Micelle size increases upon addition of 1v207 to the formulation. [Figure 1C] Figure 1C: Micelle size and zeta potential by number distribution for the four micelles tested. Micelle size increases from 11.5 nm to 13-14 nm upon addition of 1v207 to the formulation. The zeta potential of the micelles decreases from -4 mV to -8 mV upon addition of 1v207 to the formulation. [Diagram 2]Structure of the formulated TLR7 agonist 1v270 (also called TMX-201) used in a micellar preparation (Mw=1085.4, name: 2-(4-(6-amino-2-(2-methoxyethoxy)-8-oxo-7H-purin-9((8H)-yl)methyl)benzamido)ethyl 2,3-bis(oleoyloxy)propyl phosphate). [Figure 3A] FIG. 3A compares antitumor studies of micelles containing 1V270 and liposomes containing 1V270. [Figure 3B] FIG. 3B compares antitumor studies of micelles containing 1V270 and liposomes containing 1V270. [Figure 3C] FIG. 3C shows the antitumor activity of the micelle formulation MBS8 at three doses of 50, 200 and 400 nmol injected per mouse with the first injection on day 9, followed by four more doses at four day intervals. [Figure 3D] FIG. 3D shows the benefit of combining low dose MBS8 micelles at 50 nmol / mouse with optimal PD-1 therapy. [Figure 3E] FIG. 3E shows the median survival in the group of mice treated with radiation therapy in combination with liposomes and micelles containing 1V270. [Figure 3F] FIG. 3F shows the tumor-to-control ratio (mean tumor size of the group of mice treated with micelles or liposomes bearing 1V270 in combination with radiotherapy (tumor=T) compared to the mean tumor size of the radiotreated control group (control=C). Calculation is done by T / C×100. Micelles showed significantly better antitumor activity than liposomes (p<0.01). [Figure 3G] Figure 3G shows the tumor growth inhibition calculated by subtracting the T / C ratio and multiplying by 100. Micelles showed significantly better tumor growth inhibition than liposomes (p<0.01). [Figure 4A]Figures 4A-G show cytokine secretion in plasma from mice treated with equal doses of micelles or liposomes. In general, liposome MBS1 showed higher cytokine secretion than micelles for all cytokines measured, and MBS2 liposomes showed higher cytokine secretion than micelles for most cytokines tested. This indicates that micelles reduce the risk of cytokine-related syndrome (CRS) compared to 1V270-containing liposomes. [Figure 4B] Figures 4A-G show cytokine secretion in plasma from mice treated with equal doses of micelles or liposomes. In general, liposome MBS1 showed higher cytokine secretion than micelles for all cytokines measured, and MBS2 liposomes showed higher cytokine secretion than micelles for most cytokines tested. This indicates that micelles reduce the risk of cytokine-related syndrome (CRS) compared to 1V270-containing liposomes. [Figure 4C] Figures 4A-G show cytokine secretion in plasma from mice treated with equal doses of micelles or liposomes. In general, liposome MBS1 showed higher cytokine secretion than micelles for all cytokines measured, and MBS2 liposomes showed higher cytokine secretion than micelles for most cytokines tested. This indicates that micelles reduce the risk of cytokine-related syndrome (CRS) compared to 1V270-containing liposomes. [Figure 4D] Figures 4A-G show cytokine secretion in plasma from mice treated with equal doses of micelles or liposomes. In general, liposome MBS1 showed higher cytokine secretion than micelles for all cytokines measured, and MBS2 liposomes showed higher cytokine secretion than micelles for most cytokines tested. This indicates that micelles reduce the risk of cytokine-related syndrome (CRS) compared to 1V270-containing liposomes. [Figure 4E]Figures 4A-G show cytokine secretion in plasma from mice treated with equal doses of micelles or liposomes. In general, liposome MBS1 showed higher cytokine secretion than micelles for all cytokines measured, and MBS2 liposomes showed higher cytokine secretion than micelles for most cytokines tested. This indicates that micelles reduce the risk of cytokine-related syndrome (CRS) compared to 1V270-containing liposomes. [Figure 4F] Figures 4A-G show cytokine secretion in plasma from mice treated with equal doses of micelles or liposomes. In general, liposome MBS1 showed higher cytokine secretion than micelles for all cytokines measured, and MBS2 liposomes showed higher cytokine secretion than micelles for most cytokines tested. This indicates that micelles reduce the risk of cytokine-related syndrome (CRS) compared to 1V270-containing liposomes. [Figure 4G] Figures 4A-G show cytokine secretion in plasma from mice treated with equal doses of micelles or liposomes. In general, liposome MBS1 showed higher cytokine secretion than micelles for all cytokines measured, and MBS2 liposomes showed higher cytokine secretion than micelles for most cytokines tested. This indicates that micelles reduce the risk of cytokine-related syndrome (CRS) compared to 1V270-containing liposomes. [Figure 5A] Figures 5A-B show the induction of anti-PEG IgM and IgG molecules in the plasma of mice treated once with an IV injection of micelles or liposomes containing 1V270. Anti-PEG IgM and IgG molecules are measured by an ELISA designed for this purpose. Figure 5C shows the specificity of anti-PEG IgM measured in a competitive assay. Only PEGylated liposomes are recognized by the generated IgM, whereas free PEG chains, DSPE-PEG micelles and MBS8 micelles are not recognized by anti-PEG IgM molecules. [Figure 5B]Figures 5A-B show the induction of anti-PEG IgM and IgG molecules in the plasma of mice treated once with an IV injection of micelles or liposomes containing 1V270. Anti-PEG IgM and IgG molecules are measured by an ELISA designed for this purpose. Figure 5C shows the specificity of anti-PEG IgM measured in a competitive assay. Only PEGylated liposomes are recognized by the generated IgM, whereas free PEG chains, DSPE-PEG micelles and MBS8 micelles are not recognized by anti-PEG IgM molecules. [Figure 5C] Figures 5A-B show the induction of anti-PEG IgM and IgG molecules in the plasma of mice treated once with an IV injection of micelles or liposomes containing 1V270. Anti-PEG IgM and IgG molecules are measured by an ELISA designed for this purpose. Figure 5C shows the specificity of anti-PEG IgM measured in a competitive assay. Only PEGylated liposomes are recognized by the generated IgM, whereas free PEG chains, DSPE-PEG micelles and MBS8 micelles are not recognized by anti-PEG IgM molecules. [Figure 6A] Tumor growth and survival of mice bearing subcutaneous CT26 tumors and treated with 50, 100 or 200 nmol micelles in combination with radiotherapy (RT). Mice were treated 12 days after inoculation and received 2 Gy RT for 5 consecutive days (days 12, 13, 14, 15, 16) and micelles every 4 days (days 12, 16, 20, 24, 28) for a total of 5 treatments. Groups contain 8-10 mice. Tumor growth curves are expressed as mean ± SEM. Statistical significance in survival was determined using the Mantel-Cox test. Figure 6A: Tumor growth of mice treated with MBS6 in combination with radiotherapy. [Figure 6B]Tumor growth and survival of mice bearing subcutaneous CT26 tumors and treated with 50, 100 or 200 nmol micelles in combination with radiotherapy (RT). Mice were treated 12 days after inoculation and received 2 Gy RT for 5 consecutive days (days 12, 13, 14, 15, 16) and micelles every 4 days (days 12, 16, 20, 24, 28) for a total of 5 treatments. Groups contain 8-10 mice. Tumor growth curves are expressed as mean ± SEM. Statistical significance in survival was determined using the Mantel-Cox test. Figure 6B: Survival of mice treated with MBS6 in combination with radiotherapy. [Figure 6C] Tumor growth and survival of mice bearing subcutaneous CT26 tumors and treated with 50, 100 or 200 nmol micelles in combination with radiotherapy (RT). Mice were treated 12 days after inoculation and received 2 Gy RT for 5 consecutive days (days 12, 13, 14, 15, 16) and micelles every 4 days (days 12, 16, 20, 24, 28) for a total of 5 treatments. Groups contain 8-10 mice. Tumor growth curves are expressed as mean ± SEM. Statistical significance in survival was determined using the Mantel-Cox test. Figure 6C: Tumor growth of mice treated with MBS7 in combination with radiotherapy. [Figure 6D] Tumor growth and survival of mice bearing subcutaneous CT26 tumors and treated with 50, 100 or 200 nmol micelles in combination with radiotherapy (RT). Mice were treated 12 days after inoculation and received 2 Gy RT for 5 consecutive days (days 12, 13, 14, 15, 16) and micelles every 4 days (days 12, 16, 20, 24, 28) for a total of 5 treatments. Groups contain 8-10 mice. Tumor growth curves are expressed as mean ± SEM. Statistical significance in survival was determined using the Mantel-Cox test. Figure 6D: Survival of mice treated with MBS7 in combination with radiotherapy. [Figure 6E]Tumor growth and survival of mice bearing subcutaneous CT26 tumors and treated with 50, 100 or 200 nmol micelles in combination with radiotherapy (RT). Mice were treated 12 days after inoculation and received 2 Gy RT for 5 consecutive days (days 12, 13, 14, 15, 16) and micelles every 4 days (days 12, 16, 20, 24, 28) for a total of 5 treatments. Groups contain 8-10 mice. Tumor growth curves are expressed as mean ± SEM. Statistical significance in survival was determined using the Mantel-Cox test. Figure 6E: Tumor growth of mice treated with MBS8 in combination with radiotherapy. [Figure 6F] Tumor growth and survival of mice bearing subcutaneous CT26 tumors and treated with 50, 100 or 200 nmol micelles in combination with radiotherapy (RT). Mice were treated 12 days after inoculation and received 2 Gy RT for 5 consecutive days (days 12, 13, 14, 15, 16) and micelles every 4 days (days 12, 16, 20, 24, 28) for a total of 5 treatments. Groups contain 8-10 mice. Tumor growth curves are expressed as mean ± SEM. Statistical significance in survival was determined using the Mantel-Cox test. Figure 6F: Survival of mice treated with MBS8 in combination with radiotherapy. [Figure 7A] The antitumor activity is compared between MBS8 micelles containing DOPE-PEG2000 and TLR7 agonist 1v270 at a molar ratio of 90:10 and administered in three stages in the CT26 model. MBS8 was injected IV at three doses: 50 (small black circle), 200 (grey circle) and 400 (large black circle) nmol per mouse. The two higher doses of 200 and 400 nmol were significantly different (p<0.001) from the control treatment group. The individual growth of each tumor is shown in Figure 7B for 1) control treatment (PBS), 2) low dose MBS8 at 50 nmol / injection / mouse, 3) medium dose MBS8 at 200 nmol / injection / mouse and 4) high dose MBS8 at 400 nmol / injection / mouse. [Figure 7B]The antitumor activity is compared between MBS8 micelles containing DOPE-PEG2000 and TLR7 agonist 1v270 at a molar ratio of 90:10 and administered in three stages in the CT26 model. MBS8 was injected IV at three doses: 50 (small black circle), 200 (grey circle) and 400 (large black circle) nmol per mouse. The two higher doses of 200 and 400 nmol were significantly different (p<0.001) from the control treatment group. The individual growth of each tumor is shown in Figure 7B for 1) control treatment (PBS), 2) low dose MBS8 at 50 nmol / injection / mouse, 3) medium dose MBS8 at 200 nmol / injection / mouse and 4) high dose MBS8 at 400 nmol / injection / mouse. [Figure 8A] MBS8 micelles at three different dose levels were combined with αPD-1 treatment (clone RMP1-14 administered IP at 10 mg / kg) in a CT26 colon cancer syngeneic mouse tumor model. MBS8 was injected IV at three doses of 50, 200 and 400 nmol per mouse with the first injection on day 9, followed by four more doses at four-day intervals on days 13, 17, 21 and 25. αPD-1 mAb was injected IP twice weekly for three weeks, starting on day 11. All PD-1-MBS8 combination treatments were significantly more potent than PD-1 monotherapy (p<0.05, p<0.005, Wilcoxon rank sum test) (Figure 8A). Figure 8B shows individual tumor growth for all 10 mice per group with 1) control (PBS treatment), 2) 50 nmol MBS8, 3) 200 nmol MBS8, 4) 400 nmol MBS8, 5) PD1 treatment alone injected IP at 10 mg / kg, 6) PD-1 and 50 nmol MBS8 in combination therapy, 7) PD-1 and 200 nmol MBS8 in combination therapy, and 8) PD-1 and 400 nmol MBS8 in combination therapy. [Figure 8B]MBS8 micelles at three different dose levels were combined with αPD-1 treatment (clone RMP1-14 administered IP at 10 mg / kg) in a CT26 colon cancer syngeneic mouse tumor model. MBS8 was injected IV at three doses of 50, 200 and 400 nmol per mouse with the first injection on day 9, followed by four more doses at four-day intervals on days 13, 17, 21 and 25. αPD-1 mAb was injected IP twice weekly for three weeks, starting on day 11. All PD-1-MBS8 combination treatments were significantly more potent than PD-1 monotherapy (p<0.05, p<0.005, Wilcoxon rank sum test) (Figure 8A). Figure 8B shows individual tumor growth for all 10 mice per group with 1) control (PBS treatment), 2) 50 nmol MBS8, 3) 200 nmol MBS8, 4) 400 nmol MBS8, 5) PD1 treatment alone injected IP at 10 mg / kg, 6) PD-1 and 50 nmol MBS8 in combination therapy, 7) PD-1 and 200 nmol MBS8 in combination therapy, and 8) PD-1 and 400 nmol MBS8 in combination therapy. [Figure 9A] MBS8 enhances the antitumor effects of doxorubicin (A-B) and doxil (C-D). Mean tumor volumes ± SEM are shown. n=10 for (A) and (C). Wilcoxon rank sum test was used for statistical data treatment. Tumor volumes of individual animals are shown for the indicated groups in (B) and (D). [Figure 9B-1] MBS8 enhances the antitumor effects of doxorubicin (A-B) and doxil (C-D). Mean tumor volumes ± SEM are shown. n=10 for (A) and (C). Wilcoxon rank sum test was used for statistical data treatment. Tumor volumes of individual animals are shown for the indicated groups in (B) and (D). [Figure 9B-2] MBS8 enhances the antitumor effects of doxorubicin (A-B) and doxil (C-D). Mean tumor volumes ± SEM are shown. n=10 for (A) and (C). Wilcoxon rank sum test was used for statistical data treatment. Tumor volumes of individual animals are shown for the indicated groups in (B) and (D). [Figure 9C]MBS8 enhances the antitumor effects of doxorubicin (A-B) and doxil (C-D). Mean tumor volumes ± SEM are shown. n=10 for (A) and (C). Wilcoxon rank sum test was used for statistical data treatment. Tumor volumes of individual animals are shown for the indicated groups in (B) and (D). [Figure 9D] MBS8 enhances the antitumor effects of doxorubicin (A-B) and doxil (C-D). Mean tumor volumes ± SEM are shown. n=10 for (A) and (C). Wilcoxon rank sum test was used for statistical data treatment. Tumor volumes of individual animals are shown for the indicated groups in (B) and (D). [Figure 10A] Tumor growth of mice bearing subcutaneous CT26 tumors treated with radiotherapy (RT) in combination with 100 nmol MBS8 intratumorally or 200 nmol MBS8 intravenously. Where indicated, mice were treated with lipid-compatible vehicle instead of MBS8. Mice were treated 12 days after inoculation, with 2 Gy of RT for 5 consecutive days, and with micelles every 4 days for a total of 5 treatments. Groups contain 8 mice. Tumor growth curves are displayed as mean ± SEM. Figure 10A: Tumor growth of mice treated with MBS8 intratumorally or intravenously in combination with radiotherapy. Figure 10B: Tumor growth of mice treated with vehicle intratumorally or intravenously in combination with radiotherapy. Figure 10C: Survival of mice treated with MBS8 by intratumoral route, either with micelles without TLR7 agonist (vehicle) or with MBS8 with TLR7 agonist 1V270. Groups are shown in combination with or without radiotherapy. Figure 10D: Survival of mice treated with MBS8 by intravenous route, either micelles without TLR7 agonist (vehicle) or MBS8 with TLR7 agonist 1V270. Groups are shown in combination with or without radiotherapy. Groups treated with RT+iv MBS8 showed significantly better antitumor activity than RT alone (p=0.015), whereas groups treated with RT+it MBS8 did not show significantly better antitumor activity than RT alone. n=8 / group. [Figure 10B]Tumor growth of mice bearing subcutaneous CT26 tumors treated with radiotherapy (RT) in combination with 100 nmol MBS8 intratumorally or 200 nmol MBS8 intravenously. Where indicated, mice were treated with lipid-compatible vehicle instead of MBS8. Mice were treated 12 days after inoculation, with 2 Gy of RT for 5 consecutive days, and with micelles every 4 days for a total of 5 treatments. Groups contain 8 mice. Tumor growth curves are displayed as mean ± SEM. Figure 10A: Tumor growth of mice treated with MBS8 intratumorally or intravenously in combination with radiotherapy. Figure 10B: Tumor growth of mice treated with vehicle intratumorally or intravenously in combination with radiotherapy. Figure 10C: Survival of mice treated with MBS8 by intratumoral route, either with micelles without TLR7 agonist (vehicle) or with MBS8 with TLR7 agonist 1V270. Groups are shown in combination with or without radiotherapy. Figure 10D: Survival of mice treated with MBS8 by intravenous route, either micelles without TLR7 agonist (vehicle) or MBS8 with TLR7 agonist 1V270. Groups are shown in combination with or without radiotherapy. Groups treated with RT+iv MBS8 showed significantly better antitumor activity than RT alone (p=0.015), whereas groups treated with RT+it MBS8 did not show significantly better antitumor activity than RT alone. n=8 / group. [Figure 10C]Tumor growth of mice bearing subcutaneous CT26 tumors treated with radiotherapy (RT) in combination with 100 nmol MBS8 intratumorally or 200 nmol MBS8 intravenously. Where indicated, mice were treated with lipid-compatible vehicle instead of MBS8. Mice were treated 12 days after inoculation, with 2 Gy of RT for 5 consecutive days, and with micelles every 4 days for a total of 5 treatments. Groups contain 8 mice. Tumor growth curves are displayed as mean ± SEM. Figure 10A: Tumor growth of mice treated with MBS8 intratumorally or intravenously in combination with radiotherapy. Figure 10B: Tumor growth of mice treated with vehicle intratumorally or intravenously in combination with radiotherapy. Figure 10C: Survival of mice treated with MBS8 by intratumoral route, either with micelles without TLR7 agonist (vehicle) or with MBS8 with TLR7 agonist 1V270. Groups are shown in combination with or without radiotherapy. Figure 10D: Survival of mice treated with MBS8 by intravenous route, either micelles without TLR7 agonist (vehicle) or MBS8 with TLR7 agonist 1V270. Groups are shown in combination with or without radiotherapy. Groups treated with RT+iv MBS8 showed significantly better antitumor activity than RT alone (p=0.015), whereas groups treated with RT+it MBS8 did not show significantly better antitumor activity than RT alone. n=8 / group. [Figure 10D]Tumor growth of mice bearing subcutaneous CT26 tumors treated with radiotherapy (RT) in combination with 100 nmol MBS8 intratumorally or 200 nmol MBS8 intravenously. Where indicated, mice were treated with lipid-compatible vehicle instead of MBS8. Mice were treated 12 days after inoculation, with 2 Gy of RT for 5 consecutive days, and with micelles every 4 days for a total of 5 treatments. Groups contain 8 mice. Tumor growth curves are displayed as mean ± SEM. Figure 10A: Tumor growth of mice treated with MBS8 intratumorally or intravenously in combination with radiotherapy. Figure 10B: Tumor growth of mice treated with vehicle intratumorally or intravenously in combination with radiotherapy. Figure 10C: Survival of mice treated with MBS8 by intratumoral route, either with micelles without TLR7 agonist (vehicle) or with MBS8 with TLR7 agonist 1V270. Groups are shown in combination with or without radiotherapy. Figure 10D: Survival of mice treated with MBS8 by intravenous route, either micelles without TLR7 agonist (vehicle) or MBS8 with TLR7 agonist 1V270. Groups are shown in combination with or without radiotherapy. Groups treated with RT+iv MBS8 showed significantly better antitumor activity than RT alone (p=0.015), whereas groups treated with RT+it MBS8 did not show significantly better antitumor activity than RT alone. n=8 / group. [Figure 11A] Tumor growth and survival of subcutaneous EL4 or MC38 tumor-bearing mice treated with radiation therapy (RT) in combination with 200 nmol MBS8 or vehicle. Tumor growth curves are displayed as mean ± SEM. EL4-bearing mice were treated with 2 Gy of RT for three consecutive days, starting treatment 7 days after inoculation, and MBS8 or vehicle every 4 days for a total of 5 treatments. Groups contain 9-10 mice. Figure 11A: Tumor growth of treated EL4-bearing mice. [Figure 11B]Tumor growth and survival of mice bearing subcutaneous EL4 or MC38 tumors treated with radiation therapy (RT) in combination with 200 nmol MBS8 or vehicle. Tumor growth curves are displayed as mean ± SEM. EL4-bearing mice were treated starting on day 7 post-inoculation with 2 Gy RT for 3 consecutive days, followed by MBS8 or vehicle every 4 days for a total of 5 treatments. Groups contain 9-10 mice. Figure 11B: Survival of treated EL4-bearing mice. MC38-bearing mice were treated starting on day 10 post-inoculation with 2 Gy RT for 5 consecutive days, followed by MBS8 or vehicle every 2 days (q2d), 4 days (q4d), and 7 days (q7d) for a total of 5 treatments. Groups contain 9 mice. [Figure 11C] Tumor growth and survival of subcutaneous EL4 or MC38 tumor-bearing mice treated with radiation therapy (RT) in combination with 200 nmol MBS8 or vehicle. Tumor growth curves are displayed as mean ± SEM. EL4-bearing mice were treated with 2 Gy RT for three consecutive days, starting treatment 7 days after inoculation, and MBS8 or vehicle every 4 days for a total of five treatments. Groups contain 9-10 mice. Figure 11C: Tumor growth of treated MC38-bearing mice. [Figure 11D] Tumor growth and survival of subcutaneous EL4 or MC38 tumor-bearing mice treated with radiation therapy (RT) in combination with 200 nmol MBS8 or vehicle. Tumor growth curves are displayed as mean ± SEM. EL4-bearing mice were treated with 2 Gy RT for three consecutive days, starting treatment 7 days after inoculation, and MBS8 or vehicle every 4 days for a total of 5 treatments. Groups contain 9-10 mice. Figure 11D: Survival of treated MC38-bearing mice. [Figure 12A]MBS8 administration in cynomolgus monkeys. (A) C-reactive protein (CRP) was tested immediately before drug administration and at 8, 24 and 72 hours after infusion. Data represent mean values ​​(n=3). Arrows indicate peak CRP at 24 hours after each injection. Respective doses are as indicated in FIG. 12B. (B) Body temperature was monitored daily. Mean+SD values ​​(n=3) are shown. Days of drug administration and respective doses are indicated. Dashed lines indicate normal range based on baseline measurements during the acclimation period (days -22 to 0). [Figure 12B] MBS8 administration in cynomolgus monkeys. (A) C-reactive protein (CRP) was tested immediately before drug administration and at 8, 24 and 72 hours after infusion. Data represent mean values ​​(n=3). Arrows indicate peak CRP at 24 hours after each injection. Respective doses are as indicated in FIG. 12B. (B) Body temperature was monitored daily. Mean+SD values ​​(n=3) are shown. Days of drug administration and respective doses are indicated. Dashed lines indicate normal range based on baseline measurements during the acclimation period (days -22 to 0). [Figure 13A]In vivo efficacy study using 12 syngeneic subcutaneous tumor models. (A) Table summarizing the results for all 12 models. Mice were randomized when the mean tumor size reached approximately 100 mm3 and treated with the indicated therapeutics. aPD-1 (clone RMP1-14) was administered ip at 10 mg / kg 6 times q4d and MBS8 was administered iv at 300 μg / mouse 5 times q4d. The same dosing regimen was used for combination studies. All groups contained 10 mice / group. TGI, tumor growth inhibition; CR, complete responder; NA, not applicable. (B-D) Effect of MBS8 on subcutaneous tumor growth. Top panels: tumor growth curves for the indicated groups of animals. Mean tumor volumes + SEM are shown. Significant two-sided p-values ​​are indicated (Wilcoxon rank sum test). Middle and bottom panels: tumor growth curves for individual animals of the indicated treatment groups. (B) EMT-6, (C) Hepa 1-6 and (D) Pan02 represent three distinct response patterns seen in a panel of 12 tumors. CR, complete responder; RR, rechallenge resistance; *Two animals died at the start of treatment and were excluded from statistical analysis. (E) Rejection of rechallenged EMT-6 tumors. Mice that showed a CR to MBS8 (n=8) or MBS8+anti-PD-1 (n=10) treatment in the EMT-6 model and were tumor-free for at least 3 weeks were rechallenged with EMT-6 cells in the contralateral flank and tumor growth was monitored for 29 days. Naive mice inoculated with EMT-6 cells (n=5) were used as untreated controls. (F) Immune memory responses in mice treated with MBS8 alone or in combination with aPD-1. All showed rejection of rechallenged tumors, rechallenged to CR from the CT26 model. The presence of tumor-specific T cells in these animals was assessed using ELISPOT. Analysis was performed on splenocytes from untreated CT26 tumor-bearing mice (control), mice pre-treated with MBS8 and resistant to rechallenge (mono), mice treated with MBS8+a-PD-1 Combo (combo) and tumor-free naive mice (naive) stimulated in vitro with the CT26 tumor-specific antigen AH-1 (+AH-1) or not. The number of IFN-γ expressing cells per well was quantified. Mean + SEM is shown; n=5 mice / group. Statistical analysis was performed using the Wilcoxon rank sum test.Significant two-sided p values ​​are shown. [Figure 13B]In vivo efficacy study using 12 syngeneic subcutaneous tumor models. (A) Table summarizing the results for all 12 models. Mice were randomized when the mean tumor size reached approximately 100 mm3 and treated with the indicated therapeutics. aPD-1 (clone RMP1-14) was administered ip at 10 mg / kg 6 times q4d and MBS8 was administered iv at 300 μg / mouse 5 times q4d. The same dosing regimen was used for combination studies. All groups contained 10 mice / group. TGI, tumor growth inhibition; CR, complete responder; NA, not applicable. (B-D) Effect of MBS8 on subcutaneous tumor growth. Top panels: tumor growth curves for the indicated groups of animals. Mean tumor volumes + SEM are shown. Significant two-sided p-values ​​are indicated (Wilcoxon rank sum test). Middle and bottom panels: tumor growth curves for individual animals of the indicated treatment groups. (B) EMT-6, (C) Hepa 1-6 and (D) Pan02 represent three distinct response patterns seen in a panel of 12 tumors. CR, complete responder; RR, rechallenge resistance; *Two animals died at the start of treatment and were excluded from statistical analysis. (E) Rejection of rechallenged EMT-6 tumors. Mice that showed a CR to MBS8 (n=8) or MBS8+anti-PD-1 (n=10) treatment in the EMT-6 model and were tumor-free for at least 3 weeks were rechallenged with EMT-6 cells in the contralateral flank and tumor growth was monitored for 29 days. Naive mice inoculated with EMT-6 cells (n=5) were used as untreated controls. (F) Immune memory responses in mice treated with MBS8 alone or in combination with aPD-1. All showed rejection of rechallenged tumors, rechallenged to CR from the CT26 model. The presence of tumor-specific T cells in these animals was assessed using ELISPOT. Analysis was performed on splenocytes from untreated CT26 tumor-bearing mice (control), mice pre-treated with MBS8 and resistant to rechallenge (mono), mice treated with MBS8+a-PD-1 Combo (combo) and tumor-free naive mice (naive) stimulated in vitro with the CT26 tumor-specific antigen AH-1 (+AH-1) or not. The number of IFN-γ expressing cells per well was quantified. Mean + SEM is shown; n=5 mice / group. Statistical analysis was performed using the Wilcoxon rank sum test.Significant two-sided p values ​​are shown. [Figure 13C]In vivo efficacy study using 12 syngeneic subcutaneous tumor models. (A) Table summarizing the results for all 12 models. Mice were randomized when the mean tumor size reached approximately 100 mm3 and treated with the indicated therapeutics. aPD-1 (clone RMP1-14) was administered ip at 10 mg / kg 6 times q4d and MBS8 was administered iv at 300 μg / mouse 5 times q4d. The same dosing regimen was used for combination studies. All groups contained 10 mice / group. TGI, tumor growth inhibition; CR, complete responder; NA, not applicable. (B-D) Effect of MBS8 on subcutaneous tumor growth. Top panels: tumor growth curves for the indicated groups of animals. Mean tumor volumes + SEM are shown. Significant two-sided p-values ​​are indicated (Wilcoxon rank sum test). Middle and bottom panels: tumor growth curves for individual animals of the indicated treatment groups. (B) EMT-6, (C) Hepa 1-6 and (D) Pan02 represent three distinct response patterns seen in a panel of 12 tumors. CR, complete responder; RR, rechallenge resistance; *Two animals died at the start of treatment and were excluded from statistical analysis. (E) Rejection of rechallenged EMT-6 tumors. Mice that showed a CR to MBS8 (n=8) or MBS8+anti-PD-1 (n=10) treatment in the EMT-6 model and were tumor-free for at least 3 weeks were rechallenged with EMT-6 cells in the contralateral flank and tumor growth was monitored for 29 days. Naive mice inoculated with EMT-6 cells (n=5) were used as untreated controls. (F) Immune memory responses in mice treated with MBS8 alone or in combination with aPD-1. All showed rejection of rechallenged tumors, rechallenged to CR from the CT26 model. The presence of tumor-specific T cells in these animals was assessed using ELISPOT. Analysis was performed on splenocytes from untreated CT26 tumor-bearing mice (control), mice pre-treated with MBS8 and resistant to rechallenge (mono), mice treated with MBS8+a-PD-1 Combo (combo) and tumor-free naive mice (naive) stimulated in vitro with the CT26 tumor-specific antigen AH-1 (+AH-1) or not. The number of IFN-γ expressing cells per well was quantified. Mean + SEM is shown; n=5 mice / group. Statistical analysis was performed using the Wilcoxon rank sum test.Significant two-sided p values ​​are shown. [Figure 13D]In vivo efficacy study using 12 syngeneic subcutaneous tumor models. (A) Table summarizing the results for all 12 models. Mice were randomized when the mean tumor size reached approximately 100 mm3 and treated with the indicated therapeutics. aPD-1 (clone RMP1-14) was administered ip at 10 mg / kg 6 times q4d and MBS8 was administered iv at 300 μg / mouse 5 times q4d. The same dosing regimen was used for combination studies. All groups contained 10 mice / group. TGI, tumor growth inhibition; CR, complete responder; NA, not applicable. (B-D) Effect of MBS8 on subcutaneous tumor growth. Top panels: tumor growth curves for the indicated groups of animals. Mean tumor volumes + SEM are shown. Significant two-sided p-values ​​are indicated (Wilcoxon rank sum test). Middle and bottom panels: tumor growth curves for individual animals of the indicated treatment groups. (B) EMT-6, (C) Hepa 1-6 and (D) Pan02 represent three distinct response patterns seen in a panel of 12 tumors. CR, complete responder; RR, rechallenge resistance; *Two animals died at the start of treatment and were excluded from statistical analysis. (E) Rejection of rechallenged EMT-6 tumors. Mice that showed a CR to MBS8 (n=8) or MBS8+anti-PD-1 (n=10) treatment in the EMT-6 model and were tumor-free for at least 3 weeks were rechallenged with EMT-6 cells in the contralateral flank and tumor growth was monitored for 29 days. Naive mice inoculated with EMT-6 cells (n=5) were used as untreated controls. (F) Immune memory responses in mice treated with MBS8 alone or in combination with aPD-1. All showed rejection of rechallenged tumors, rechallenged to CR from the CT26 model. The presence of tumor-specific T cells in these animals was assessed using ELISPOT. Analysis was performed on splenocytes from untreated CT26 tumor-bearing mice (control), mice pre-treated with MBS8 and resistant to rechallenge (mono), mice treated with MBS8+a-PD-1 Combo (combo) and tumor-free naive mice (naive) stimulated in vitro with the CT26 tumor-specific antigen AH-1 (+AH-1) or not. The number of IFN-γ expressing cells per well was quantified. Mean + SEM is shown; n=5 mice / group. Statistical analysis was performed using the Wilcoxon rank sum test.Significant two-sided p values ​​are shown. [Figure 13E]In vivo efficacy study using 12 syngeneic subcutaneous tumor models. (A) Table summarizing the results for all 12 models. Mice were randomized when the mean tumor size reached approximately 100 mm3 and treated with the indicated therapeutics. aPD-1 (clone RMP1-14) was administered ip at 10 mg / kg 6 times q4d and MBS8 was administered iv at 300 μg / mouse 5 times q4d. The same dosing regimen was used for combination studies. All groups contained 10 mice / group. TGI, tumor growth inhibition; CR, complete responder; NA, not applicable. (B-D) Effect of MBS8 on subcutaneous tumor growth. Top panels: tumor growth curves for the indicated groups of animals. Mean tumor volumes + SEM are shown. Significant two-sided p-values ​​are indicated (Wilcoxon rank sum test). Middle and bottom panels: tumor growth curves for individual animals of the indicated treatment groups. (B) EMT-6, (C) Hepa 1-6 and (D) Pan02 represent three distinct response patterns seen in a panel of 12 tumors. CR, complete responder; RR, rechallenge resistance; *Two animals died at the start of treatment and were excluded from statistical analysis. (E) Rejection of rechallenged EMT-6 tumors. Mice that showed a CR to MBS8 (n=8) or MBS8+anti-PD-1 (n=10) treatment in the EMT-6 model and were tumor-free for at least 3 weeks were rechallenged with EMT-6 cells in the contralateral flank and tumor growth was monitored for 29 days. Naive mice inoculated with EMT-6 cells (n=5) were used as untreated controls. (F) Immune memory responses in mice treated with MBS8 alone or in combination with aPD-1. All showed rejection of rechallenged tumors, rechallenged to CR from the CT26 model. The presence of tumor-specific T cells in these animals was assessed using ELISPOT. Analysis was performed on splenocytes from untreated CT26 tumor-bearing mice (control), mice pre-treated with MBS8 and resistant to rechallenge (mono), mice treated with MBS8+a-PD-1 Combo (combo) and tumor-free naive mice (naive) stimulated in vitro with the CT26 tumor-specific antigen AH-1 (+AH-1) or not. The number of IFN-γ expressing cells per well was quantified. Mean + SEM is shown; n=5 mice / group. Statistical analysis was performed using the Wilcoxon rank sum test.Significant two-sided p values ​​are shown. [Figure 13F]In vivo efficacy study using 12 syngeneic subcutaneous tumor models. (A) Table summarizing the results for all 12 models. Mice were randomized when the mean tumor size reached approximately 100 mm3 and treated with the indicated therapeutics. aPD-1 (clone RMP1-14) was administered ip at 10 mg / kg 6 times q4d and MBS8 was administered iv at 300 μg / mouse 5 times q4d. The same dosing regimen was used for combination studies. All groups contained 10 mice / group. TGI, tumor growth inhibition; CR, complete responder; NA, not applicable. (B-D) Effect of MBS8 on subcutaneous tumor growth. Top panels: tumor growth curves for the indicated groups of animals. Mean tumor volumes + SEM are shown. Significant two-sided p-values ​​are indicated (Wilcoxon rank sum test). Middle and bottom panels: tumor growth curves for individual animals of the indicated treatment groups. (B) EMT-6, (C) Hepa 1-6 and (D) Pan02 represent three distinct response patterns seen in a panel of 12 tumors. CR, complete responder; RR, rechallenge resistance; *Two animals died at the start of treatment and were excluded from statistical analysis. (E) Rejection of rechallenged EMT-6 tumors. Mice that showed a CR to MBS8 (n=8) or MBS8+anti-PD-1 (n=10) treatment in the EMT-6 model and were tumor-free for at least 3 weeks were rechallenged with EMT-6 cells in the contralateral flank and tumor growth was monitored for 29 days. Naive mice inoculated with EMT-6 cells (n=5) were used as untreated controls. (F) Immune memory responses in mice treated with MBS8 alone or in combination with aPD-1. All showed rejection of rechallenged tumors, rechallenged to CR from the CT26 model. The presence of tumor-specific T cells in these animals was assessed using ELISPOT. Analysis was performed on splenocytes from untreated CT26 tumor-bearing mice (control), mice pre-treated with MBS8 and resistant to rechallenge (mono), mice treated with MBS8+a-PD-1 Combo (combo) and tumor-free naive mice (naive) stimulated in vitro with the CT26 tumor-specific antigen AH-1 (+AH-1) or not. The number of IFN-γ expressing cells per well was quantified. Mean + SEM is shown; n=5 mice / group. Statistical analysis was performed using the Wilcoxon rank sum test.Significant two-sided p values ​​are shown. [Figure 14A] Comparative efficacy of MBS8 administered at different dose schedules. The CT26 subcutaneous model was used. Mice were randomized when the mean tumor size reached approximately 100 mm3 and treated iv at 200 nmol / mouse at the indicated dose schedule. (A) Mean tumor volumes + SEM are shown. Significant two-sided p-values ​​are indicated (two-sided Wilcoxon rank sum test). (B) Tumor growth curves of individual animals. Animals with tumors <40 mm3 at last measurement and showing continuous volume reduction are considered complete responders (CR). [Figure 14B] Comparative efficacy of MBS8 administered at different dose schedules. The CT26 subcutaneous model was used. Mice were randomized when the mean tumor size reached approximately 100 mm3 and treated iv at 200 nmol / mouse at the indicated dose schedule. (A) Mean tumor volumes + SEM are shown. Significant two-sided p-values ​​are indicated (two-sided Wilcoxon rank sum test). (B) Tumor growth curves of individual animals. Animals with tumors <40 mm3 at last measurement and showing continuous volume reduction are considered complete responders (CR). [Figure 15A] MBS8 and R848 treatment of mice bearing subcutaneous CT26 tumors. CT26 tumor-bearing mice were treated with MBS8 (100, 200 or 300 nmol) or R848 (200 nmol) intravenously on a q4d schedule for a total of 5 treatments, starting treatment 11 days after inoculation. n=9 mice / group. (A) Mean tumor growth curves ± SEM are displayed. (B) Survival of treated CT26-bearing mice. Statistical significance in survival was determined using the Mantel-Cox test, ****p<0.0001, *p<0.05. (C) Weight change relative to baseline, mean weight ± SEM are displayed. [Figure 15B]MBS8 and R848 treatment of mice bearing subcutaneous CT26 tumors. CT26 tumor-bearing mice were treated with MBS8 (100, 200 or 300 nmol) or R848 (200 nmol) intravenously on a q4d schedule for a total of 5 treatments, starting treatment 11 days after inoculation. n=9 mice / group. (A) Mean tumor growth curves ± SEM are displayed. (B) Survival of treated CT26-bearing mice. Statistical significance in survival was determined using the Mantel-Cox test, ****p<0.0001, *p<0.05. (C) Weight change relative to baseline, mean weight ± SEM are displayed. [Figure 15C] MBS8 and R848 treatment of mice bearing subcutaneous CT26 tumors. CT26 tumor-bearing mice were treated with MBS8 (100, 200 or 300 nmol) or R848 (200 nmol) intravenously on a q4d schedule for a total of 5 treatments, starting treatment 11 days after inoculation. n=9 mice / group. (A) Mean tumor growth curves ± SEM are displayed. (B) Survival of treated CT26-bearing mice. Statistical significance in survival was determined using the Mantel-Cox test, ****p<0.0001, *p<0.05. (C) Weight change relative to baseline, mean weight ± SEM are displayed. [Figure 16] Cell types in tumors from mice treated with MBS8 monotherapy based on gene expression analysis. Mice were treated with 200 nmol MBS8 on a q4d schedule for a total of 1 or 3 injections. Tumors were collected on day 0 (untreated), day 1 after 1st injection, day 2 after 1st injection, day 4 after 1st injection, day 14 after 1st injection, day 2 after 3rd injection, and day 4 after 3rd injection. n=3-6 for all groups. Gene expression analysis was performed on extracted RNA and cell type analysis was performed from the data with NanoString Advance software module. Two-way ANOVA with multiple comparison correction was performed to compare different time points vs. untreated. * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001, and **** indicates p<0.0001. [Figure 17A]Impact of MBS8 on the tumor microenvironment immediately after injection based on flow cytometry analysis. CT26-bearing mice were injected with 200 nmol MBS8 and tumors were assessed by flow cytometry. Tumors were harvested 1, 3 and 6 hours after injection. All graphs are presented as mean ± SEM. Statistical significance compared to untreated tumors was determined using the Kruskal-Wallis test with Dunn's multiple comparison test, where * indicates p<0.05; ** indicates p<0.01; and *** indicates p<0.001. n=3-4. MFI=median fluorescence intensity. [Figure 17B] Impact of MBS8 on the tumor microenvironment immediately after injection based on flow cytometry analysis. CT26-bearing mice were injected with 200 nmol MBS8 and tumors were assessed by flow cytometry. Tumors were harvested 1, 3 and 6 hours after injection. All graphs are presented as mean ± SEM. Statistical significance compared to untreated tumors was determined using the Kruskal-Wallis test with Dunn's multiple comparison test, where * indicates p<0.05; ** indicates p<0.01; and *** indicates p<0.001. n=3-4. MFI=median fluorescence intensity. [Figure 17C] Impact of MBS8 on the tumor microenvironment immediately after injection based on flow cytometry analysis. CT26-bearing mice were injected with 200 nmol MBS8 and tumors were assessed by flow cytometry. Tumors were harvested 1, 3 and 6 hours after injection. All graphs are presented as mean ± SEM. Statistical significance compared to untreated tumors was determined using the Kruskal-Wallis test with Dunn's multiple comparison test, where * indicates p<0.05; ** indicates p<0.01; and *** indicates p<0.001. n=3-4. MFI=median fluorescence intensity. [Figure 17D]Impact of MBS8 on the tumor microenvironment immediately after injection based on flow cytometry analysis. CT26-bearing mice were injected with 200 nmol MBS8 and tumors were assessed by flow cytometry. Tumors were harvested 1, 3 and 6 hours after injection. All graphs are presented as mean ± SEM. Statistical significance compared to untreated tumors was determined using the Kruskal-Wallis test with Dunn's multiple comparison test, where * indicates p<0.05; ** indicates p<0.01; and *** indicates p<0.001. n=3-4. MFI=median fluorescence intensity. [Figure 18A] Impact on the tumor microenvironment during treatment based on flow cytometry analysis. Mice bearing CT26 tumors were treated with 200 nmol MBS6 every 4 days starting on day 15 after cancer cell inoculation, and tumors and spleens were analyzed 2 days after the second injection. All graphs are presented as mean ± SEM. Statistical significance compared to untreated tumors was determined using the Mann-Whitney U test, * indicates p<0.05; ** indicates p<0.01; n=5. [Figure 18B] Impact on the tumor microenvironment during treatment based on flow cytometry analysis. Mice bearing CT26 tumors were treated with 200 nmol MBS6 every 4 days starting on day 15 after cancer cell inoculation, and tumors and spleens were analyzed 2 days after the second injection. All graphs are presented as mean ± SEM. Statistical significance compared to untreated tumors was determined using the Mann-Whitney U test, * indicates p<0.05; ** indicates p<0.01; n=5. [Figure 18C] Impact on the tumor microenvironment during treatment based on flow cytometry analysis. Mice bearing CT26 tumors were treated with 200 nmol MBS6 every 4 days starting on day 15 after cancer cell inoculation, and tumors and spleens were analyzed 2 days after the second injection. All graphs are presented as mean ± SEM. Statistical significance compared to untreated tumors was determined using the Mann-Whitney U test, * indicates p<0.05; ** indicates p<0.01; n=5. [Figure 18D]Impact on the tumor microenvironment during treatment based on flow cytometry analysis. Mice bearing CT26 tumors were treated with 200 nmol MBS6 every 4 days starting on day 15 after cancer cell inoculation, and tumors and spleens were analyzed 2 days after the second injection. All graphs are presented as mean ± SEM. Statistical significance compared to untreated tumors was determined using the Mann-Whitney U test, * indicates p<0.05; ** indicates p<0.01; n=5. [Figure 18E] Impact on the tumor microenvironment during treatment based on flow cytometry analysis. Mice bearing CT26 tumors were treated with 200 nmol MBS6 every 4 days starting on day 15 after cancer cell inoculation, and tumors and spleens were analyzed 2 days after the second injection. All graphs are presented as mean ± SEM. Statistical significance compared to untreated tumors was determined using the Mann-Whitney U test, * indicates p<0.05; ** indicates p<0.01; n=5. [Figure 19] Dosing schedule studies in CT26 tumor-bearing mice treated with Doxil for all groups were performed on days 9, 13 and 17 (filled circles), groups treated before and after Doxil + MBS8 were further administered MBS8 on days 9 and 19, 23, 27 and 31, and groups treated after Doxil + MBS8 were further administered MBS8 on days 19, 23, 27 and 31. All graphs are presented as mean + SEM. Statistical significance of comparisons between the indicated groups was determined using the Mann-Whitney test with n=10 at the indicated significance levels. [Figure 20A] Mean (±SD) plasma concentration-time curves of MBS8 in female and male rats on linear scale (A) and semi-logarithmic scale (B) on day 1 (first dose) and day 15 (one week after the last of two doses, one week apart) after intravenous infusion of MBS8 (1V270) at 3 mg / kg / day over 1 hour. This figure demonstrates the insignificant kinetics of MBS8 administered on day 1 and day 15 (third dose), respectively. This observation supports that the micelles of the present invention do not induce accelerated blood clearance (ABC). [Figure 20B]Mean (±SD) plasma concentration-time curves of MBS8 in female and male rats on linear scale (A) and semi-logarithmic scale (B) on day 1 (first dose) and day 15 (one week after the last of two doses, one week apart) after intravenous infusion of MBS8 (1V270) at 3 mg / kg / day over 1 hour. This figure demonstrates the insignificant kinetics of MBS8 administered on day 1 and day 15 (third dose), respectively. This observation supports that the micelles of the present invention do not induce accelerated blood clearance (ABC). [Figure 21A] Overlay of individual plasma concentration-time curves of 1V270 in one female and one male cynomolgus monkey on day 1 (A) (first dose) and day 15 (B) (one week after the last of two doses, one week apart) after intravenous infusion of 0.3, 1 and 3 mg / kg / day MBS8 (1V270) on a linear scale. This figure demonstrates the insignificant kinetics of MBS8 administered on day 1 (first dose) and day 15 (third dose after weekly administration). This observation supports that the micelles of the present invention do not induce accelerated blood clearance (ABC). [Figure 21B] Overlay of individual plasma concentration-time curves of 1V270 in one female and one male cynomolgus monkey on day 1 (A) (first dose) and day 15 (B) (one week after the last of two doses, one week apart) after intravenous infusion of 0.3, 1 and 3 mg / kg / day MBS8 (1V270) on a linear scale. This figure demonstrates the insignificant kinetics of MBS8 administered on day 1 (first dose) and day 15 (third dose after weekly administration). This observation supports that the micelles of the present invention do not induce accelerated blood clearance (ABC). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Detailed Description definition "Lipid" as disclosed herein refers to a group of substances that contain at least one hydrophobic moiety that is itself insoluble in water. Exemplary groups of lipids can be, but are not limited to, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, glycolipids, and polyketides.

[0012] As used herein, the term "prevention" refers to the prevention of a disease or the prevention of the spread of a disease.

[0013] The term "treatment" as used herein refers to combating a disease or disorder. As used herein, "treatment" or "treating" includes any desired effect on the symptoms or pathology of the disease or condition described herein, and may include even minimal changes or improvements in one or more measurable markers of the disease or condition being treated. "Treatment" or "treating" does not necessarily indicate a complete eradication or cure of the disease or condition, or its associated symptoms.

[0014] As used herein, the term "PEG" refers to polyethylene glycol.

[0015] As used herein, the term "amphiphilic micelle forming agent" refers to an agent that is "amphiphilic", i.e., has both hydrophilic (water-loving, polar) and lipophilic (fat-loving) properties and is also capable of forming micelles. The amphiphilic micelle forming agents disclosed herein are capable of forming micelles in mixtures with other compounds in solution.

[0016] The term "linking group" as used herein refers to a grouping of linking atoms, such as a functional group, that covalently bonds at least two portions of a molecule. In one example, the linking group is a carbonyl group, i.e., "-C(O)-", which can attach, for example, an amine "H2N-alkyl(1)" at one end and an alkyl(2) at the other end, thereby forming an amide "alkyl(2)-C(O)-NH-alkyl(1)".

[0017] The terms "anti-PD-1," "α-PD-1," and "a-PD-1" are used interchangeably herein to refer to the anti-PD-1 group of immune checkpoint inhibitors.

[0018] The terms "anti-PD-L1," "α-PD-L1," and "a-PD-L1" are used interchangeably herein to refer to the anti-PD-L1 group of immune checkpoint inhibitors.

[0019] As used herein, the term "MBS6" refers to a micellar composition of DSPE-PEG2000:1V270 in a molar ratio of 80:20.

[0020] As used herein, the term "MBS7" refers to a micellar composition of DSPE-PEG2000:1V270 in a molar ratio of 95:5.

[0021] As used herein, the term "MBS8" refers to a micellar composition of DSPE-PEG2000:1V270 in a molar ratio of 90:10.

[0022] Micellar Micelles are aggregated particles of amphiphilic molecules dispersed in a liquid colloid. Most micelles in aqueous solution form aggregated particles with the hydrophilic head groups in contact with the surrounding hydrophilic solvent, isolating the hydrophobic tail region at the center of the micelle.

[0023] Among micelle-forming compounds, micelles made of polyethylene glycol phosphatidylethanolamine (PEG-PE) have particularly attractive properties such as good stability, lifetime, and the ability to accumulate in areas with abnormal vasculature (e.g., areas with leaky vasculature such as tumors) through enhanced permeability and retention effects. Furthermore, these micelles can be "targeted" by attaching specific targeting ligand molecules to the micelle surface, or can be composed of stimuli-responsive amphiphilic block copolymers. The addition of a second component, such as a surfactant or another hydrophobic material, to the main micelle-forming material further improves the solubilization ability of the micelles without compromising their stability. Micelles are more easily prepared compared to other nanoparticles, e.g., liposomes, and can be generated by lipid mixture and sonication.

[0024] The size of micelles can be determined by various techniques known to those skilled in the art. Dynamic light scattering (DLS) experiments can be carried out, for example, using a Malvern Zetasizer Nano ZS instrument, which is suitable for measuring the size and size distribution of micelles formed in aqueous solutions. The diameter measured by DLS is a value that indicates how the particle spreads in the fluid and is called the hydrodynamic diameter.

[0025] The diameters of the micelles disclosed herein are expressed as numerical averages.

[0026] In one embodiment, the diameter of the micelles disclosed herein is from 5 to 50 nm, such as from 6 to 46 nm, such as from 7 to 42 nm, such as from 8 to 38 nm, such as from 9 to 34 nm, such as from 10 to 34 nm, such as from 11 to 30 nm, such as from 12 nm to 26 nm.

[0027] In one embodiment, the diameter of the micelles is from 5 nm to 39 nm, such as from 5 nm to 20 nm, such as from 20 nm to 30 nm, such as from 30 nm to 35 nm, such as from 35 nm to 39 nm.

[0028] In one embodiment, the diameter of the micelles is from 5 nm to 39 nm, such as from 5 nm to 38 nm, such as from 5 nm to 37 nm, such as from 5 nm to 36 nm, such as from 5 nm to 35 nm, such as from 5 nm to 34 nm, such as from 5 nm to 33 nm, such as from 5 nm to 32 nm, such as from 5 nm to 31 nm.

[0029] In one embodiment, the diameter of the micelles is from 5 nm to 25 nm, such as from 6 nm to 24 nm, such as from 7 nm to 23 nm, such as from 8 nm to 22 nm, such as from 9 nm to 21 nm, such as from 10 nm to 20 nm, such as from 11 nm to 19 nm, such as from 12 nm to 18 nm, such as from 13 nm to 17 nm, such as from 14 nm to 16 nm, such as from 15 nm.

[0030] The "amphiphilic micelle forming agents" of the present disclosure may, in some embodiments, comprise phospholipids. The structure of a phospholipid generally comprises two hydrophobic fatty acid "tails" and a hydrophilic "head" group that includes a phosphate group. The two components are linked by a glycerol molecule. The phosphate group may be modified with a simple organic molecule such as choline, ethanolamine, or serine. In some embodiments, the simple organic molecule acts as a linking group to a polymer such as PEG.

[0031] In one embodiment, the amphiphilic micelle forming agent is selected from the group consisting of poloxamers, poloxamines, PEG-polyesters, PEG-polyanhydrides, PEG-polyamino acids, phospholipids, polysorbates, and polyoxyethylene alkyl ethers.

[0032] In one embodiment, the PEG-polyester is selected from the group consisting of PEG-poly(lactic acid) (PEG-PLA), PEG-poly(lactic-co-glycolic acid) (PLGA), and PEG-poly(ε-caprolactone) (PCL).

[0033] In one embodiment, the PEG-polyanhydride is PEG-polysebacic anhydride (PSA).

[0034] In one embodiment, a micellar composition is provided comprising a PEG-polyamino acid, wherein the PEG-polyamino acid is selected from PEG-poly(L-histidine), PEG-poly(L-aspartic acid), PEG-poly(L-asparagine), PEG-poly(L-glutamic acid), PEG-poly(L-glutamine), and PEG-poly(L-lysine).

[0035] In one embodiment, the amphiphilic micelle forming agent is a phospholipid conjugated to polyethylene glycol (PEG). In one embodiment, the PEG-conjugated phospholipid is conjugated via a carbonyl group.

[0036] One example of a preferred amphiphilic micelle forming agent is DSPE-PEG2000, exemplified below as the ammonium salt: [ka] (DSPE-PEG2000)

[0037] DSPE-PEG2000 comprises phosphatidylethanolamine linked to PEG via a carbonyl group. Phosphatidylethanolamine comprises glycerol esterified with two fatty acids and phosphoric acid. The phosphoric acid group is combined with choline in phosphatidylcholine, while it is combined with ethanolamine in phosphatidylethanolamine. The two fatty acids can be the same or different, and are usually present in the 1,2 position, but can also be present in the 1,3 position. The terminus of PEG is aminated, i.e., linked to NH2, in some embodiments.

[0038] Polyethylene glycol (PEG) In one embodiment, the micelles of the present disclosure include PEG. In one embodiment, the PEG is in the form of PEG conjugated to a phospholipid. The size of the PEG is PEG350 to PEG30,000.

[0039] In one embodiment, the size of PEG is PEG350 to PEG5000, for example, PEG550 to PEG4000, for example, PEG750 to PEG3000, PEG1000 to PEG3000, etc., and preferably, the size of PEG is PEG2000.

[0040] Phospholipids of the Present Disclosure The phospholipids disclosed herein may be part of a TLR7 agonist or an amphipathic micelle forming agent. Exemplary phospholipids disclosed in the context of an amphipathic micelle forming agent may be used as part of a TLR7 agonist, and vice versa.

[0041] In one embodiment, a micellar composition according to the present disclosure is provided comprising a phospholipid, the phospholipid comprising one or more alkyl chains, such as C8-C24 alkyl(s), C10-C22, such as C12-C20, preferably C14-C18, most preferably C16-C18 saturated or unsaturated alkyl chains.

[0042] In one embodiment, a micellar composition according to the present disclosure is provided comprising a phospholipid, the phospholipid comprising phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidic acid (PA), bisphosphatidylglycerol (DPG), or phosphatidyl alcohol.

[0043] In one embodiment, the phosphatidylethanolamine is selected from the group consisting of 1,2-dioleoylphosphatidylethanolamine, 1,2-dipalmitoylphosphatidylethanolamine, 1,2-dimyristoylphosphatidylethanolamine, 1,2-distearoylphosphatidylethanolamine, 1-oleoyl-2-palmitoylphosphatidylethanolamine, 1-oleoyl-2-stearoylphosphatidylethanolamine, 1-palmitoyl-2-oleoylphosphatidylethanolamine, and 1-stearoyl-2-oleoylphosphatidylethanolamine.

[0044] In one embodiment, the phospholipid conjugated to PEG is selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE)-PEG, and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE)-PEG.

[0045] In one embodiment, a micellar composition is provided comprising a phospholipid conjugated to PEG, where the phospholipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG.

[0046] In one embodiment, the amphiphilic micelle forming agent is DSPE-PEG2000.

[0047] Pharmaceutical Compositions The micelles of the present disclosure are useful as components of pharmaceutical formulations. Thus, in one embodiment, the micellar composition of the present disclosure is a pharmaceutical composition. In one embodiment, a pharmaceutical composition is provided that comprises a micellar composition as defined herein.

[0048] Such formulations in any form suitable for administration to a mammal are contemplated.

[0049] The pharmaceutical formulations of the present disclosure are preferably in the form of a solution, dispersion, suspension, lyophilisate, or in frozen form.

[0050] In one embodiment, the route of administration may be intravenous, intratumoral, oral, subcutaneous, intradermal, intramuscular, intranasal, intraperitoneal, pulmonary, or renal administration.

[0051] In one embodiment, a micelle composition of the present disclosure comprises an amphipathic micelle forming agent and a TLR7 agonist, wherein the molar ratio of the amphipathic micelle forming agent to the TLR7 agonist is 50:50 to 99.5:0.5, such as 60:40 to 99:1, such as 70:30 to 98:2, such as 80:20 to 95:5, for example, 95:5, 90:10, or 80:20.

[0052] In one embodiment, there is provided a micelle composition as disclosed herein, the composition comprising a molar concentration of 1%-25%, such as 1%, such as 2%, such as 3%, such as 4%, such as 5%, such as 6%, such as 7%, such as 8%, such as 9%, such as 10%, such as 11%, such as 12%, such as 13%, such as 14%, such as 15%, such as 16%, such as 17%, such as 18%, such as 19%, such as 20%, such as 21%, such as 22%, such as 23%, such as 24%, such as 25%, of a TLR7 agonist.

[0053] The micellar compositions or pharmaceutical compositions defined herein are further provided for use in the prevention, treatment or amelioration of a disease or disorder.

[0054] Therapeutic Uses and Methods The micelle compositions of the present disclosure may be used to prevent, treat, or ameliorate cancer, infectious diseases, inflammatory conditions or diseases, autoimmune diseases, or allergies, hi one embodiment, the micelle compositions of the present disclosure are used to treat cancer.

[0055] As such, the micelle composition or pharmaceutical composition may be used to treat cancer, an infectious disease, an inflammatory condition or disease, an autoimmune disease, or an allergy.

[0056] In one embodiment, the disease or disorder is cancer, such as colon cancer, hi one embodiment, the disease or disorder is cancer, such as a solid tumor.

[0057] infectious disease Another aspect of the present disclosure is to provide for the prevention or treatment of infectious diseases by administration of a micelle composition disclosed herein to a subject. In a preferred embodiment, the micelle composition used to prevent or treat infectious diseases is MBS8. Prevention or treatment of infectious diseases in both humans and livestock can be facilitated by the micelle composition disclosed herein. In one embodiment, the infectious disease is a viral infection or a bacterial infection. In a preferred embodiment, the treatment of infectious diseases is prophylactic. Thus, in one embodiment, a method is provided for preventing infectious diseases by administration of a micelle composition such as MBS8 to a subject in need thereof. Subjects suitable for prophylactic treatment can be, but are not limited to, medical professionals and / or other humans who work in close contact with infected subjects. These suitable subjects are at increased risk of infection, and therefore prophylactic treatment with the micelle composition disclosed herein is advantageous.

[0058] Combination therapy In one embodiment, the treatment of cancer is augmented by the combination of an existing treatment such as a monoclonal antibody (trastuzumab, rituximab, cetuximab), radiation therapy, chemotherapy, or immune checkpoint inhibitors such as pembrolizumab, ipilimumab, etc. Thus, in one embodiment, the treatment of cancer is a combination treatment further comprising administering a monoclonal antibody to a subject suffering from cancer.

[0059] As demonstrated in Examples 11, 13, 14, 15, 16, 18, and 24, an enhancing or synergistic effect may be obtained when the micelle compositions or pharmaceutical compositions disclosed herein are administered in combination with radiation therapy, chemotherapeutic agents, or immune checkpoint inhibitors. Thus, in one embodiment, the cancer treatment is a combination treatment that further includes radiation therapy.

[0060] Certain chemotherapies are particularly relevant in combination with the benefits of TLR7, these being chemotherapeutic compounds that induce the so-called "immunogenic cell death" (ICD). As shown in Examples 14 and 24, the micelle composition comprising 1V270 significantly enhances the efficacy of doxorubicin and doxil, resulting in an effective treatment. In one embodiment, the cancer treatment is a combination treatment further comprising administering a chemotherapeutic agent such as doxorubicin or doxil. In one embodiment, the chemotherapeutic agent is selected from the group consisting of doxorubicin, doxil, epirubicin, cyclophosphamide, bortezomib, and oxaliplatin. In one embodiment, the cancer treatment is a combination therapy further comprising administering a monoclonal antibody targeting PD-1, PD-L1 or CTLA-4, such as α-PD-1, α-PD-L1 or α-CTLA-4, for example, an immune checkpoint inhibitor, such as atezolizumab, avelumab, durvalumab, nivolumab, tislelizumab, pembrolizumab or ipilimumab. Preferably, the immune checkpoint inhibitor is α-PD-1, such as nivolumab or pembrolizumab. In a preferred embodiment, the micelle composition is MBS8 and the immune checkpoint inhibitor is nivolumab or pembrolizumab.

[0061] As demonstrated in Example 18, the micelle composition is highly effective in treating a variety of cancers. This effect is also demonstrated in the treatment of cancers that do not respond to α-PD-1 monotherapy, both as monotherapy and in combination with α-PD-1. In one embodiment, the combination treatment comprises administering the micelle composition and α-PD-1 for the treatment of cancer, particularly a cancer selected from the group consisting of liver cancer, pancreatic cancer, lymphoma, breast cancer, and colon cancer. In one embodiment, the combination treatment comprises administering the micelle composition and α-PD-1 for the treatment of a cancer selected from the group consisting of prostate cancer and kidney cancer. In particular, the combination of the micelle composition and α-PD-1 is effective in the treatment of cancers that do not respond to α-PD-1 monotherapy.

[0062] In one embodiment, the micelle composition used in the combination therapy is MBS8. A preferred combination therapy is MBS8 and α-PD-1. In particular, this preferred combination therapy is effective in treating cancers that do not respond to anti-PD-1 monotherapy.

[0063] As shown in Example 13, micellar compositions comprising 1V270 are highly potent in combination with α-PD-1, resulting in at least 90% complete remission in CT26 tumor-bearing mouse models treated. In one embodiment, MBS8 is administered to a cancer patient in need of treatment in combination with a therapeutic antibody targeting the PD-1 / PD-L1 pathway. In one embodiment, the therapeutic antibody targeting the PD-1 / PD-L1 pathway is selected from the group consisting of atezolizumab, avelumab, durvalumab, nivolumab, pembrolizumab, spartalizumab / PDR001, tislelizumab, BCD-100, TSR-042, camrelizumab, IBI308, KNO35, and CS1001.

[0064] Monoclonal antibodies are beneficial in combination with TLR7 agonists through activation of complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). This is relevant, for example, for antibodies against CD20, EGFR, CD38, and HER2. ADCC, ADCP, and CDC-mediated tumor cell killing rely on activated NK cells, macrophages, and neutrophils that are activated by the micelle compositions of the present disclosure (FIG. 16), particularly MBS8.

[0065] In one embodiment, MBS8 is administered to a cancer patient in combination with a monoclonal antibody that targets CD20.

[0066] In one embodiment, MBS8 is administered to a cancer patient in combination with a monoclonal antibody that targets the epidermal growth factor receptor (EGFR).

[0067] In one embodiment, MBS8 is administered to a cancer patient in combination with a monoclonal antibody that targets human epidermal growth factor receptor 2 (HER2).

[0068] In one embodiment, MBS8 is administered to a cancer patient in combination with a monoclonal antibody that targets CD38. In a particular embodiment, the monoclonal antibody that targets CD38 is selected from daratumumab and isatuximab.

[0069] In one embodiment, MBS8 is administered to a cancer patient in combination with a monoclonal antibody selected from ublituximab, obinutuzumab, ofatumumab, ibritumomab tiuxetan, rituximab, tositumomab, depatuxizumab mafodotin, necitumumab, panitumumab, cetuximab, trastuzumab, trastuzumab-dkst, trastuzumab emtansine, BAT8001, pertuzumab, margetuximab, trastuzumab deruxtecan, trastuzumab duocarmazine, daratumumab, and isatuximab.

[0070] In one embodiment, MBS8 is administered in combination with an antibody that targets CD47, such as magrolimab.

[0071] In one embodiment, the treatment of cancer is carried out as a monotherapy comprising administration of a micelle composition or pharmaceutical composition disclosed herein.

[0072] As used herein, the term "prevention" refers to the prevention of a disease or the prevention of the spread of a disease.

[0073] The term "treatment" as used herein refers to combating a disease or disorder. As used herein, "treatment" or "treating" includes any desired effect on the symptoms or pathology of the disease or condition described herein, and may include even minimal changes or improvements in one or more measurable markers of the disease or condition being treated. "Treatment" or "treating" does not necessarily indicate a complete eradication or cure of the disease or condition, or its associated symptoms.

[0074] The term "amelioration" as used herein refers to a reduction in the severity of symptoms of a disease or condition. Actions that improve the patient's condition or that attempt to cure or at least make more tolerable intolerable symptoms associated with the patient's condition are considered "ameliorating" treatments.

[0075] In one embodiment, the micelle compositions are used in the prevention, treatment, or amelioration of cancer.

[0076] In one embodiment, the micelle composition is used to prevent, treat, or ameliorate an infectious disease.

[0077] In one embodiment, a method of in vivo activation of immune cells in a subject is provided, comprising administering to said subject a micelle composition or pharmaceutical composition as defined herein in an amount sufficient to activate said immune cells. A preferred subject is a human, e.g., a human suffering from cancer.

[0078] TLR7 agonists Toll-like receptor 7, also known as TLR7, is a protein encoded by the TLR7 gene in humans. It is a member of the toll-like receptor (TLR) family and plays an important role in pathogen recognition and activation of innate immunity. Due to their ability to induce robust production of anticancer cytokines such as interleukin-12, TLR7 agonists have been explored for cancer immunotherapy.

[0079] The micelle composition according to the present disclosure comprises a compound represented by formula (I), formula (II), formula (III) or formula (IV); [ka] (In the formula, X 1 is -O-, -S-, or NR C and; R 1 is hydrogen, (C1-C 10 ) Alkyl, Substituted (C1-C 10) Alkyl, C 6~10 Aryl or substituted C 6~10 Aryl, C 5~9 Heterocyclic, substituted C 5~9 is a heterocyclic ring; R C is hydrogen, C 1~10 Alkyl or substituted C 1~10 alkyl or R C and R 1 together with the nitrogen to which they are attached form a heterocyclic ring or a substituted heterocyclic ring; Each R 2 are independently -OH, (C1-C6) alkyl, substituted (C1-C6) alkyl, (C1-C6) alkoxy, substituted (C1-C6) alkoxy, -C(O)-(C1-C6) alkyl (alkanoyl), substituted -C(O)-(C1-C6) alkyl, -C(O)-(C6-C 10 )Aryl (aroyl), substituted -C(O)-(C6-C 10 )Aryl, -C(O)OH (carboxyl), -C(O)O(C1-C6)alkyl (alkoxycarbonyl), substituted -C(O)O(C1-C6)alkyl, -NR a R b , -C(O)NR a R b (carbamoyl), halo, nitro, or cyano, or R 2 does not exist; Each R a and R b are independently hydrogen, (C1-C6) alkyl, substituted (C1-C6) alkyl, (C3-C8)cycloalkyl, substituted (C3-C8)cycloalkyl, (C1-C6)alkoxy, substituted (C1-C6)alkoxy, (C1-C6)alkanoyl, substituted (C1-C6)alkanoyl, aryl, aryl(C1-C6)alkyl, Het, Het(C1-C6)alkyl, or (C1-C6)alkoxycarbonyl; Substituents on any alkyl, aryl or heterocyclic group include hydroxy, C 1~6 Alkyl, Hydroxy C 1~6 Alkylene, C 1~6 Alkoxy, C 3~6 Cycloalkyl, C1~6 Alkoxy, C 1~6 alkylene, amino, cyano, halo, or aryl; n is 0, 1, 2, 3 or 4; X 2 is a bond or a linking group; and R 3 is a lipid; X 3 is -N- or -CH-; R 4 is -CH2- or -CH(R 2 )-and k is 0 or 1; X 4 -O-, -S-, -NH-, -N(R d )-, -CH2-, or -CH(R 2 )- and; Each R d are independently -OH, (C1-C6) alkyl, substituted (C1-C6) alkyl, (C1-C6) alkoxy, substituted (C1-C6) alkoxy, -C(O)-(C1-C6) alkyl (alkanoyl), substituted -C(O)-(C1-C6) alkyl, -C(O)-(C6-C 10 )Aryl (aroyl), substituted -C(O)-(C6-C 10 )Aryl, -C(O)O(C1-C6)alkyl(alkoxycarbonyl), substituted -C(O)O(C1-C6)alkyl, -C(O)NR a R b (carbamoyl) Toll-like receptor 7 (TLR7) agonists or its tautomer; or a pharma- ceutical acceptable salt or solvate thereof, The ring system of formula (II) contains one heteroatom which is a N atom and X 2 a piperidine ring having a N atom of the piperidine ring adjacent to A purine group in any of formulas (I), (II), (III) or (IV) undergoes tautomeric rearrangement; and an amphiphilic micelle forming agent.

[0080] It should be understood that the purine groups in any of formulas (I), (II), (III) or (IV) are subject to tautomeric rearrangement.

[0081] In one embodiment, a micelle composition according to the present disclosure is provided, wherein the TLR7 agonist has formula (I): [ka] (In the formula, X 1 is -O-, -S-, or NR C and; R 1 is hydrogen, (C1-C 10 ) Alkyl, Substituted (C1-C 10 ) Alkyl, C 6~10 Aryl or substituted C 6~10 Aryl, C 5~9 Heterocyclic, substituted C 5~9 is a heterocyclic ring; R C is hydrogen, C 1~10 Alkyl or substituted C 1~10 alkyl or R C and R 1 together with the nitrogen to which they are attached form a heterocyclic ring or a substituted heterocyclic ring; Each R 2 are independently -OH, (C1-C6) alkyl, substituted (C1-C6) alkyl, (C1-C6) alkoxy, substituted (C1-C6) alkoxy, -C(O)-(C1-C6) alkyl (alkanoyl), substituted -C(O)-(C1-C6) alkyl, -C(O)-(C6-C 10 )Aryl (aroyl), substituted -C(O)-(C6-C 10 )Aryl, -C(O)OH (carboxyl), -C(O)O(C1-C6)alkyl (alkoxycarbonyl), substituted -C(O)O(C1-C6)alkyl, -NR a R b , -C(O)NR a R b (carbamoyl), halo, nitro, or cyano, or R 2 does not exist; Each Ra and R b are independently hydrogen, (C1-C6) alkyl, substituted (C1-C6) alkyl, (C3-C8)cycloalkyl, substituted (C3-C8)cycloalkyl, (C1-C6)alkoxy, substituted (C1-C6)alkoxy, (C1-C6)alkanoyl, substituted (C1-C6)alkanoyl, aryl, aryl(C1-C6)alkyl, Het, Het(C1-C6)alkyl, or (C1-C6)alkoxycarbonyl; Substituents on any alkyl, aryl or heterocyclic group include hydroxy, C 1~6 Alkyl, Hydroxy C 1~6 Alkylene, C 1~6 Alkoxy, C 3~6 Cycloalkyl, C 1~6 Alkoxy, C 1~6 alkylene, amino, cyano, halo, or aryl; n is 0, 1 or 2; X 2 is a bond or a linking group; and R 3 is lipid) or its tautomers; or a pharma- ceutical acceptable salt or solvate thereof, Purine groups undergo tautomeric rearrangement.

[0082] In one embodiment, a micelle composition according to the present disclosure is provided, wherein the TLR7 agonist has formula (I): [ka] (In the formula, X 1 is -O-, -S-, or NR C and; R 1 is hydrogen, (C1-C 10 ) Alkyl, Substituted (C1-C 10 ) Alkyl, C 6~10 Aryl or substituted C 6~10 Aryl, C 5~9 Heterocyclic, substituted C 5~9is a heterocyclic ring; R C is hydrogen, C 1~10 Alkyl or substituted C 1~10 alkyl or R C and R 1 together with the nitrogen to which they are attached form a heterocyclic ring or a substituted heterocyclic ring; R 2 does not exist; n is 0; X 2 is a bond or a linking group; and R 3 is lipid) or its tautomers; or a pharma- ceutical acceptable salt or solvate thereof, The purine group is subject to tautomeric rearrangement. 2 It will be understood by those skilled in the art that if is not present, then n must be 0, i.e., not present.

[0083] In one embodiment, a micelle composition according to the present disclosure is provided, wherein the TLR7 agonist has formula (I): [ka] (In the formula, X 1 is -O-; R 1 is hydrogen, (C1-C 10 ) Alkyl, Substituted (C1-C 10 ) Alkyl, C 6~10 Aryl or substituted C 6~10 Aryl, C 5~9 Heterocyclic, substituted C 5~9 is a heterocyclic ring; R 2 does not exist; n is 0; X 2 is a bond or a linking group; and R 3 is lipid) or its tautomers; or a pharma- ceutical acceptable salt or solvate thereof, Purine groups are subject to tautomeric rearrangement.

[0084] In one embodiment, a micelle composition according to the present disclosure is provided, wherein the TLR7 agonist has formula (V): [ka] (In the formula, X 1 is -O-; R 1 is hydrogen, (C1-C6) alkyl, substituted (C1-C6) alkyl, C6 aryl, or substituted C6 aryl, C 5~6 Heterocyclic, substituted C 5~6 is a heterocyclic ring; R 2 does not exist; n is 0; X 2 is a bond or a linking group; and R 3 is lipid) or its tautomers; or a pharma- ceutical acceptable salt or solvate thereof, Purine groups are subject to tautomeric rearrangement.

[0085] In one embodiment, a micelle composition according to the present disclosure is provided, wherein the TLR7 agonist has formula (VI): [ka] (In the formula, X 1 is -O-; R 1 is hydrogen, (C1-C6) alkyl, substituted (C1-C6) alkyl, C6 aryl, or substituted C6 aryl, C 5~6 Heterocyclic, substituted C 5~6 is a heterocyclic ring; X 2 is a bond or a linking group; and R 3 is lipid) or its tautomers; or a pharma- ceutical acceptable salt or solvate thereof, Purine groups are subject to tautomeric rearrangement.

[0086] In one embodiment, a micelle composition according to the present disclosure is provided, wherein the TLR7 agonist has formula (VII): [ka] (In the formula, X 2 is a bond or a linking group; and R 3 is lipid) or its tautomers; or a pharma- ceutical acceptable salt or solvate thereof, Purine groups are subject to tautomeric rearrangement.

[0087] In one embodiment, a micelle composition according to the present disclosure is provided, wherein the TLR7 agonist has formula (VIII): [ka] (In the formula, R 3 is lipid) or its tautomers; or a pharma- ceutical acceptable salt or solvate thereof, Purine groups are subject to tautomeric rearrangement.

[0088] In one embodiment, X 2 is: bond, -O-, -C(O)-(carbonyl), (C1-C6) alkyl, substituted (C1-C6) alkyl, (C1-C6) alkoxy, substituted (C1-C6) alkoxy, -C(O)-(C1-C6) alkyl (alkanoyl), substituted -C(O)-(C1-C6) alkyl, -C(O)-(C6-C 10 )Aryl (aroyl), substituted -C(O)-(C6-C 10 )Aryl, -C(O)OH (carboxyl), -C(O)O(C1-C6)alkyl (alkoxycarbonyl), substituted -C(O)O(C1-C6)alkyl, -NRa R b , -C(O)NR a R b (carbamoyl); Each R a and R b are independently hydrogen, (C1-C6) alkyl, substituted (C1-C6) alkyl, (C3-C8)cycloalkyl, substituted (C3-C8)cycloalkyl, (C1-C6)alkoxy, substituted (C1-C6)alkoxy, (C1-C6)alkanoyl, substituted (C1-C6)alkanoyl, aryl, aryl(C1-C6)alkyl, Het, Het(C1-C6)alkyl, or (C1-C6)alkoxycarbonyl; X 1 is -O-, -S-, or NR C and; R 1 is hydrogen, (C1-C6) alkyl, substituted (C1-C6) alkyl; R C is hydrogen, C 1~6 Alkyl or substituted C 1~6 alkyl or R C and R 1 together with the nitrogen to which they are attached form a heterocyclic ring or substituted heterocyclic ring.

[0089] In a preferred embodiment, X 2 is -C(O)-(carbonyl). In a preferred embodiment, X 2 is -C(O)-(carbonyl) and R 3 is 1,2-dioleoylphosphatidylethanolamine according to the formula disclosed herein.

[0090] In one embodiment, R 3 is a lipid selected from the group consisting of phospholipids containing one or two carboxylic acid esters, gonanes such as cholesterol, glycolipids, and glycerides. 3 is a phospholipid containing one or two carboxylic acid esters.

[0091] "Lipid" as disclosed herein refers to a group of substances that contain at least one hydrophobic moiety that is itself insoluble in water. Exemplary groups of lipids can be, but are not limited to, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, glycolipids, and polyketides.

[0092] In a particularly preferred embodiment, the TLR7 agonist according to formula (I) is represented by the formula (IA): [ka] (wherein formula (I) is defined as follows: X 1 is -O-; R 1 is 2-methoxy-1-ethyl; R 2 does not exist; X 2 is carbonyl; and R 3 is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or a tautomer thereof; or a pharma- ceutical acceptable salt or solvate thereof.

[0093] The compound according to formula (IA) is also known in the literature, for example as 1V270 in US Pat. No. 8,357,374.

[0094] Thus, in a preferred embodiment, a micelle composition is provided, wherein the TLR7 agonist has the formula (IA): [ka] or a tautomer thereof.

[0095] A halogen atom referred to herein refers to a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.

[0096] Aryl refers to a C6-10 monocyclic or fused ring aryl group, such as phenyl, indenyl, or naphthyl.

[0097] Heterocyclic or heterocycle (Het) refers to a monocyclic saturated heterocyclic group or an unsaturated monocyclic heterocyclic group or a fused heterocyclic group containing at least one heteroatom, such as 0-3 nitrogen atoms, 0-1 oxygen atoms (-O-), and 0-1 sulfur atoms (-S-). Non-limiting examples of saturated monocyclic heterocyclic groups include 5- or 6-membered saturated heterocyclic groups such as tetrahydrofuranyl, pyrrolidinyl, morpholinyl, piperidyl, piperazinyl, or pyrazolidinyl. Non-limiting examples of unsaturated monocyclic heterocyclic groups include 5- or 6-membered unsaturated heterocyclic groups such as furyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, thienyl, pyridyl, or pyrimidinyl. Non-limiting examples of unsaturated fused heterocyclic groups include unsaturated bicyclic heterocyclic groups such as indolyl, isoindolyl, quinolyl, benzothizolyl, chromanyl, benzofuryl, etc. The Het group can be an unsaturated heterocyclic group such as a saturated heterocyclic group or a heteroaryl group.

[0098] Non-limiting examples of heterocycles include 5- or 6-membered saturated heterocyclic rings such as 1-pyrrolidinyl, 4-morpholinyl, 1-piperidyl, 1-piperazinyl or 1-pyrazolidinyl, 5- or 6-membered unsaturated heterocyclic rings such as 1-imidazolyl.

[0099] R 1The alkyl, aryl and heterocyclic groups in the formula (I) may be optionally substituted with one or more substituents, which may be the same or different and include lower alkyl; cycloalkyl, hydroxyl; hydroxy C1-6 alkylene, such as hydroxymethyl, 2-hydroxyethyl or 3-hydroxypropyl; lower alkoxy; C1-6 alkoxy C1-6 alkyl, such as 2-methoxyethyl, 2-ethoxyethyl or 3-methoxypropyl; amino; alkylamino; dialkylamino; cyano; nitro; acyl; carboxyl; lower alkoxycarbonyl; halogen; mercapto; methylthio, ethylthio, propylthio or butylthio. C1-6 alkylthio; substituted C1-6 alkylthio such as methoxyethylthio, methylthioethylthio, hydroxyethylthio, or chloroethylthio; aryl; substituted C6-10 monocyclic or fused ring aryl such as 4-hydroxyphenyl, 4-methoxyphenyl, 4-fluorophenyl, 4-chlorophenyl, or 3,4-dichlorophenyl; 5-6 membered unsaturated heterocycles such as furyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, thienyl, pyridyl, or pyrimidinyl; and bicyclic unsaturated heterocycles such as indolyl, isoindolyl, quinolyl, benzothiazolyl, chromanyl, benzofuranyl, or phthalimino. In certain embodiments, one or more of the above groups can be explicitly excluded as a substituent of various other groups of the formula. In some embodiments, the 5 membered ring of the formula is a thiazole ring.

[0100] R 2 The alkyl, aryl, and heterocyclic groups of R may be optionally substituted with one or more substituents, which may be the same or different and include hydroxyl; C1-6 alkoxy, such as methoxy, ethoxy, or propoxy; carboxyl; C2-7 alkoxycarbonyl, such as methoxycarbonyl, ethoxycarbonyl, or propoxycarbonyl; and halogen. CThe alkyl, aryl, and heterocyclic groups may be optionally substituted with one or more substituents, which may be the same or different, and include C3-6 cycloalkyl; hydroxyl; C1-6 alkoxy; amino; cyano; aryl; substituted aryl, such as 4-hydroxyphenyl, 4-methoxyphenyl, 4-chlorophenyl, or 3,4-dichlorophenyl; nitro, and halogen.

[0101] R C and R 1 and the heterocycle formed together with the nitrogen atom to which they are attached may be optionally substituted with one or more substituents, which may be the same or different, and include C1-6 alkyl; hydroxy C1-6 alkylene; C1-6 alkoxy C1-6 alkylene group; hydroxyl; C1-6 alkoxy, and cyano.

[0102] In one embodiment there is provided a micelle composition as defined herein, wherein the TLR7 agonist is of formula (IA): The amphiphilic micelle forming agent is DSPE-PEG2000; and The ratio of TLR7 agonist to amphipathic micelle forming agent is 95:5, 90:10, or 80:20.

[0103] In a preferred embodiment, there is provided a micelle composition as defined herein, wherein the TLR7 agonist is of formula (IA); The amphiphilic micelle forming agent is DSPE-PEG2000; and The ratio of TLR7 agonist to amphipathic micelle forming agent is 95:5, 90:10, or 80:20.

[0104] Further activators In one embodiment, there is provided a micelle composition as defined herein further comprising at least one additional active ingredient. In one embodiment, the micelle composition further comprises at least one antigen.

[0105] item I-1. Formula (I), Formula (II), Formula (III) or Formula (IV); [ka] (In the formula, In the formula, X 1 is -O-, -S-, or NR C and; R 1 is hydrogen, (C1-C 10 ) Alkyl, Substituted (C1-C 10 ) Alkyl, C 6~10 Aryl or substituted C 6~10 Aryl, C 5~9 Heterocyclic, substituted C 5~9 is a heterocyclic ring; R C is hydrogen, C 1~10 Alkyl or substituted C 1~10 alkyl or R C and R 1 together with the nitrogen to which they are attached form a heterocyclic ring or a substituted heterocyclic ring; Each R 2 are independently -OH, (C1-C6) alkyl, substituted (C1-C6) alkyl, (C1-C6) alkoxy, substituted (C1-C6) alkoxy, -C(O)-(C1-C6) alkyl (alkanoyl), substituted -C(O)-(C1-C6) alkyl, -C(O)-(C6-C 10 )Aryl (aroyl), substituted -C(O)-(C6-C 10 )Aryl, -C(O)OH (carboxyl), -C(O)O(C1-C6)alkyl (alkoxycarbonyl), substituted -C(O)O(C1-C6)alkyl, -NR a R b , -C(O)NR a R b (carbamoyl), halo, nitro, or cyano, or R 2 does not exist; Each R a and R b are independently hydrogen, (C1-C6) alkyl, substituted (C1-C6) alkyl, (C3-C8)cycloalkyl, substituted (C3-C8)cycloalkyl, (C1-C6)alkoxy, substituted (C1-C6)alkoxy, (C1-C6)alkanoyl, substituted (C1-C6)alkanoyl, aryl, aryl(C1-C6)alkyl, Het, Het(C1-C6)alkyl, or (C1-C6)alkoxycarbonyl; Substituents on any alkyl, aryl or heterocyclic group include hydroxy, C 1~6 Alkyl, Hydroxy C 1~6 Alkylene, C 1~6 Alkoxy, C 3~6 Cycloalkyl, C 1~6 Alkoxy, C 1~6 alkylene, amino, cyano, halo, or aryl; n is 0, 1, 2, 3 or 4; X 2 is a bond or a linking group; and R 3 is a lipid; X 3 is -N- or -CH-; R 4 is -CH2- or -CH(R 2 )-and k is 0 or 1; X 4 -O-, -S-, -NH-, -N(R d )-, -CH2-, or -CH(R 2 )-and; Each R d are independently -OH, (C1-C6) alkyl, substituted (C1-C6) alkyl, (C1-C6) alkoxy, substituted (C1-C6) alkoxy, -C(O)-(C1-C6) alkyl (alkanoyl), substituted -C(O)-(C1-C6) alkyl, -C(O)-(C6-C 10 )Aryl (aroyl), substituted -C(O)-(C6-C 10 )Aryl, -C(O)O(C1-C6)alkyl(alkoxycarbonyl), substituted -C(O)O(C1-C6)alkyl, -C(O)NR a R b (carbamoyl) Toll-like receptor 7 (TLR7) agonists or its tautomer; or a pharma- ceutical acceptable salt or solvate thereof A micelle composition comprising: The ring system of formula (II) contains one heteroatom which is a N atom and X 2 is a piperidine ring having a N atom of the piperidine ring adjacent to A purine group of any of formulas (I), (II), (III) or (IV) undergoes tautomeric rearrangement; and an amphiphilic micelle forming agent.

[0106] I-2. The TLR7 agonist has the formula (I): [ka] (In the formula, X 1 is -O-, -S-, or NR C and; R 1 is hydrogen, (C1-C 10 ) Alkyl, Substituted (C1-C 10 ) Alkyl, C 6~10 Aryl or substituted C 6~10 Aryl, C 5~9 Heterocyclic, substituted C 5~9 is a heterocyclic ring; R C is hydrogen, C 1~10 Alkyl or substituted C 1~10 alkyl or R C and R 1 together with the nitrogen to which they are attached form a heterocyclic ring or a substituted heterocyclic ring; Each R 2 are independently -OH, (C1-C6) alkyl, substituted (C1-C6) alkyl, (C1-C6) alkoxy, substituted (C1-C6) alkoxy, -C(O)-(C1-C6) alkyl (alkanoyl), substituted -C(O)-(C1-C6) alkyl, -C(O)-(C6-C 10 )Aryl (aroyl), substituted -C(O)-(C6-C 10)Aryl, -C(O)OH (carboxyl), -C(O)O(C1-C6)alkyl (alkoxycarbonyl), substituted -C(O)O(C1-C6)alkyl, -NR a R b , -C(O)NR a R b (carbamoyl), halo, nitro, or cyano, or R 2 does not exist; Each R a and R b are independently hydrogen, (C1-C6) alkyl, substituted (C1-C6) alkyl, (C3-C8)cycloalkyl, substituted (C3-C8)cycloalkyl, (C1-C6)alkoxy, substituted (C1-C6)alkoxy, (C1-C6)alkanoyl, substituted (C1-C6)alkanoyl, aryl, aryl(C1-C6)alkyl, Het, Het(C1-C6)alkyl, or (C1-C6)alkoxycarbonyl; Substituents on any alkyl, aryl or heterocyclic group include hydroxy, C 1~6 Alkyl, Hydroxy C 1~6 Alkylene, C 1~6 Alkoxy, C 3~6 Cycloalkyl, C 1~6 Alkoxy, C 1~6 alkylene, amino, cyano, halo, or aryl; n is 0, 1 or 2; X 2 is a bond or a linking group; and R 3 is lipid) or its tautomers; or a pharma- ceutical acceptable salt or solvate thereof, The micelle composition of any one of the preceding claims, wherein the purine groups are subject to tautomeric rearrangement.

[0107] I-3. A TLR7 agonist having the formula (I): [ka] (In the formula, X 1 is -O-, -S-, or NR C and; R 1 is hydrogen, (C1-C 10 ) Alkyl, Substituted (C1-C 10 ) Alkyl, C 6~10 Aryl or substituted C 6~10 Aryl, C 5~9 Heterocyclic, substituted C 5~9 is a heterocyclic ring; R C is hydrogen, C 1~10 Alkyl or substituted C 1~10 alkyl or R C and R 1 together with the nitrogen to which they are attached form a heterocyclic ring or a substituted heterocyclic ring; R 2 does not exist; n is 0; X 2 is a bond or a linking group; and R 3 is lipid) or its tautomers; or a pharma- ceutical acceptable salt or solvate thereof, The micellar composition according to any one of items I-1 to I-2, wherein the purine group is subject to tautomeric rearrangement.

[0108] I-4.X 2 is a bond, -O-, -C(O)-(carbonyl), (C1-C6) alkyl, substituted (C1-C6) alkyl, (C1-C6) alkoxy, substituted (C1-C6) alkoxy, -C(O)-(C1-C6) alkyl (alkanoyl), substituted -C(O)-(C1-C6) alkyl, -C(O)-(C6-C 10 )Aryl (aroyl), substituted -C(O)-(C6-C 10 )Aryl, -C(O)OH (carboxyl), -C(O)O(C1-C6)alkyl (alkoxycarbonyl), substituted -C(O)O(C1-C6)alkyl, -NR a R b , -C(O)NR a R b(carbamoyl); Each R a and R b are independently hydrogen, (C1-C6)alkyl, substituted (C1-C6)alkyl, (C3-C8)cycloalkyl, substituted (C3-C8)cycloalkyl, (C1-C6)alkoxy, substituted (C1-C6)alkoxy, (C1-C6)alkanoyl, substituted (C1-C6)alkanoyl, aryl, aryl(C1-C6)alkyl, Het, Het(C1-C6)alkyl, or (C1-C6)alkoxycarbonyl; X 1 is -O-, -S-, or NR C and; R 1 is hydrogen, (C1-C6) alkyl, substituted (C1-C6) alkyl; R C is hydrogen, C 1~6 Alkyl or substituted C 1~6 alkyl or R C and R 1 The micelle composition according to any one of items I-1 to I-3, wherein, together with the nitrogen to which they are attached, form a heterocycle or a substituted heterocycle.

[0109] I-5.R 3 is a lipid selected from the group consisting of phospholipids containing one or two carboxylic acid esters, gonanes such as cholesterol, glycolipids, and glycerides.

[0110] I-6.R 3 is a phospholipid containing one or two kinds of carboxylic acid esters.

[0111] I-7. The micelle composition according to any one of items I-1 to I-6, wherein the diameter of the micelles is 5 to 50 nm, such as 6 to 46 nm, such as 7 to 42 nm, such as 8 to 38 nm, such as 9 to 34 nm, such as 10 to 34 nm, such as 11 to 30 nm, such as 12 nm to 26 nm.

[0112] I-8. The micelle composition according to any one of items I-1 to I-7, wherein the diameter of the micelle is 5 nm to 25 nm, such as 6 nm to 24 nm, such as 7 nm to 23 nm, such as 8 nm to 22 nm, such as 9 nm to 21 nm, such as 10 nm to 20 nm, such as 11 nm to 19 nm, such as 12 nm to 18 nm, such as 13 nm to 17 nm, such as 14 nm to 16 nm, such as 15 nm.

[0113] I-9. The micelle composition according to any one of items I-1 to I-8, wherein the amphiphilic micelle forming agent is selected from the group consisting of poloxamers, poloxamines, PEG-polyesters, PEG-polyanhydrides, PEG-polyamino acids, phospholipids, polysorbates, and polyoxyethylene alkyl ethers.

[0114] I-10. The micelle composition according to any one of paragraphs I-1 to I-9, wherein the PEG-polyester is selected from the group consisting of PEG-poly(lactic acid) (PEG-PLA), PEG-poly(lactic-co-glycolic acid) (PLGA), and PEG-poly(ε-caprolactone) (PCL).

[0115] I-11. A micellar composition according to any one of paragraphs I-1 to I-10, wherein the PEG-polyanhydride is PEG-polysebacic anhydride (PSA).

[0116] I-12. The micelle composition according to any one of items I-1 to I-11, wherein the PEG-polyamino acid is selected from PEG-poly(L-histidine), PEG-poly(L-aspartic acid), PEG-poly(L-asparagine), PEG-poly(L-glutamic acid), PEG-poly(L-glutamine), and PEG-poly(L-lysine).

[0117] I-13. The micelle composition according to any one of paragraphs I-1 to I-12, wherein the amphiphilic micelle forming agent is a phospholipid conjugated with polyethylene glycol (PEG).

[0118] I-14. A micelle composition according to any one of paragraphs I-1 to I-13, wherein the phospholipid conjugated to PEG is conjugated via a carbonyl group.

[0119] I-15. The micellar composition according to any one of items I-1 to I-14, wherein the size of PEG is PEG350 to PEG5000, for example, PEG550 to PEG4000, for example, PEG750 to PEG3000, PEG1000 to PEG3000, etc., and preferably the size of PEG is PEG2000.

[0120] I-16. The micellar composition according to any one of paragraphs I-1 to I-15, wherein the phospholipid comprises one or more alkyl chains, which are C8 to C24 alkyl(s), C10 to C22 etc, C12 to C20 etc, preferably C14 to C18, most preferably C16 to C18 saturated or unsaturated alkyl chains.

[0121] I-17. The micellar composition according to any one of paragraphs I-1 to I-16, wherein the phospholipid comprises phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidic acid (PA), bisphosphatidylglycerol (DPG), or phosphatidyl alcohol.

[0122] I-18. The micellar composition according to any one of items I-1 to I-17, wherein the phosphatidylethanolamine is selected from the group consisting of 1,2-dioleoylphosphatidylethanolamine, 1,2-dipalmitoyl-phosphatidylethanolamine, 1,2-dimyristoylphosphatidylethanolamine, 1,2-distearoylphosphatidylethanolamine, 1-oleoyl-2-palmitoylphosphatidylethanolamine, 1-oleoyl-2-stearoylphosphatidylethanolamine, 1-palmitoyl-2-oleoylphosphatidylethanolamine, and 1-stearoyl-2-oleoylphosphatidylethanolamine.

[0123] I-19. The micelle composition according to any one of paragraphs I-1 to I-18, wherein the phospholipid conjugated to PEG is selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE)-PEG, and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE)-PEG.

[0124] I-20. The micelle composition according to any one of paragraphs I-1 to I-19, wherein the phospholipid conjugated to PEG is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG.

[0125] I-21. The micelle composition according to any one of paragraphs I-1 to I-20, wherein the amphiphilic micelle forming agent is DSPE-PEG2000.

[0126] I-22. The micelle composition according to any one of items I-1 to I-21, wherein the molar ratio of the amphiphilic micelle-forming agent to the TLR7 agonist is 50:50 to 99.5:0.5, such as 60:40 to 99:1, such as 70:30 to 98:2, such as 80:20 to 95:5, for example, 95:5, 90:10, or 80:20.

[0127] I-23. The micelle composition according to any one of paragraphs I-1 to I-22, wherein the composition comprises a molar concentration of 1% to 25%, such as 1%, such as 2%, such as 3%, such as 4%, such as 5%, such as 6%, such as 7%, such as 8%, such as 9%, such as 10%, such as 11%, such as 12%, such as 13%, such as 14%, such as 15%, such as 16%, such as 17%, such as 18%, such as 19%, such as 20%, such as 21%, such as 22%, such as 23%, such as 24%, such as 25% of the TLR7 agonist.

[0128] I-24. A TLR7 agonist having the formula (IA): [ka] Structure by, its tautomers; or a pharma- ceutical acceptable salt or solvate thereof The micelle composition according to any one of items I-1 to I-23, having the following structure:

[0129] I-25. The TLR7 agonist is of formula (IA); the amphiphilic micelle forming agent is DSPE-PEG2000; and The micelle composition according to any one of paragraphs I-1 to I-24, wherein the ratio of the TLR7 agonist to the amphipathic micelle forming agent is 95:5, 90:10, or 80:20.

[0130] I-26. A micelle composition according to any one of paragraphs I-1 to I-25, wherein the molar ratio of DSPE-PEG2000 to TLR7 agonist according to formula (IA) is 90:10 (MBS8).

[0131] I-27. A micelle composition according to any one of paragraphs I-1 to I-26, further comprising at least one additional active ingredient.

[0132] I-28. The micelle composition according to any one of paragraphs I-1 to I-27, further comprising at least one antigen.

[0133] I-29. A pharmaceutical composition comprising the micelle composition according to any one of items I-1 to I-28.

[0134] I-30. The micelle composition or pharmaceutical composition according to any one of paragraphs I-1 to I-29 for use in the prevention, treatment or amelioration of a disease or disorder.

[0135] I-31. The micelle composition or pharmaceutical composition for use according to any one of items I-1 to I-30, wherein the disease or disorder is selected from the group consisting of cancer, infectious diseases, inflammatory conditions or diseases, autoimmune diseases, and allergies.

[0136] I-32. The micelle composition or pharmaceutical composition for use according to any one of paragraphs I-1 to I-31, wherein the disease or disorder is cancer, such as colon cancer.

[0137] I-33. A method for in vivo activation of immune cells in a subject, comprising administering to the subject a micelle composition or pharmaceutical composition according to any one of paragraphs I-1 to I-32 in an amount sufficient to activate the immune cells.

[0138] I-34. A method for the treatment of cancer in a patient in need of such treatment, comprising administering to said patient a micelle composition according to any one of paragraphs I-1 to I-33.

[0139] I-35. The method according to any one of paragraphs I-1 to I-34, further comprising administering to the patient a chemotherapeutic agent.

[0140] I-36. The method according to any one of items I-1 to I-135, wherein the chemotherapeutic agent is selected from the group consisting of doxorubicin, doxil, epirubicin, cyclophosphamide, bortezomib, and oxaliplatin.

[0141] I-37. The method of any one of paragraphs I-1 to I-36, further comprising administering to the patient an immune checkpoint inhibitor.

[0142] I-38. The method according to any one of items I-1 to I-37, wherein the immune checkpoint inhibitor is selected from the group consisting of atezolizumab, avelumab, durvalumab, nivolumab, tislelizumab, pembrolizumab, or ipilimumab.

[0143] I-39. The method according to any one of items I-1 to I-38, wherein the micelle composition is MBS8 and the immune checkpoint inhibitor is selected from the group consisting of nivolumab and pembrolizumab.

[0144] I-40. The method of any one of paragraphs I-1 to I-39, further comprising administering to the patient a monoclonal antibody that targets CD20.

[0145] I-41. The method according to any one of items I-1 to I-40, comprising administering a monoclonal antibody targeting MBS8 and CD20.

[0146] I-42. The method of any one of paragraphs I-1 to I-41, further comprising administering to the patient a monoclonal antibody that targets epidermal growth factor receptor (EGFR).

[0147] I-43. The method according to any one of items I-1 to I-42, comprising administering MBS8 and a monoclonal antibody targeting epidermal growth factor receptor (EGFR).

[0148] I-44. The method of any one of paragraphs I-1 to I-43, further comprising administering to the patient a monoclonal antibody that targets human epidermal growth factor receptor 2 (HER2).

[0149] I-45. The method according to any one of items I-1 to I-44, comprising administering MBS8 and a monoclonal antibody targeting human epidermal growth factor receptor 2 (HER2).

[0150] I-46. The method of any one of paragraphs I-1 to I-45, further comprising administering to the patient a monoclonal antibody that targets CD38, such as daratumumab or isatuximab.

[0151] I-47. The method according to any one of paragraphs I-1 to I-46, comprising administering MBS8 and a monoclonal antibody targeting CD38, such as daratumumab or isatuximab.

[0152] I-48. The method of any one of paragraphs I-1 to I-47, further comprising administering to the patient a monoclonal antibody that targets CD47, such as magrolimab.

[0153] I-49. The method according to any one of paragraphs I-1 to I-48, comprising administering a monoclonal antibody targeting CD47, such as MBS8 and magrolimab.

[0154] I-50. The method according to any one of paragraphs I-1 to I-49, further comprising radiation therapy.

[0155] Working Example Example 1: Preparation of micelles and liposomes Micelles were made from 1,2-distearyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) obtained from Lipoid GmbH. Briefly, lipids were dissolved in tert-butanol:water (volume ratio 9:1) to a final concentration of 5-10 mM in glass vials and heated to 50 °C with magnetic stirring until complete dissolution. Solvent was removed by freezing the vial in liquid nitrogen followed by lyophilization overnight. Micelles were prepared by dispersing the dried lipid in a buffer containing 150 mM NaCl, 10 mM phosphate (pH = 7.4), subjecting the vial to gentle vortexing to establish initial contact of lipid with solvent, followed by sonication for 30 min to ensure formation of micellar structures. The dispersion was vortexed again, after which the dispersion was further sonicated for 30 min. Micelles were stored at 4° C. prior to use and / or characterization.

[0156] Unilamellar fully hydrated liposomes were prepared from a mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), cholesterol (Chol), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DOPE-PEG2000) and 1v270 (C57H93N6O12P, Mw=1085.4, (2-(4-(6-amino-2-(2-methoxyethoxy)-8-oxo-7H-purin-9(8H)-yl)methyl)benzamido)ethyl 2,3-bis(oleoyloxy)propyl phosphate). The molar ratio of each lipid in the liposomes was MBS1:POPC:Chol:DOTAP:1V270:DOPE-mPEG2k (44.25:30:20:0.75:5) and MBS2:POPC:Chol:POPG:1V270:DSPE-PEG2k (44.25:30:20:0.75:5). All lipids were obtained from Avanti Polar lipids or Lipoid. Briefly, appropriate weights of POPC, POPG, Chol, DOTAP, 1V270 and DOPE-PEG2000 were dissolved in chloroform. The solvent was removed by a gentle stream of N2 and the lipid film was dried overnight under low pressure to remove traces of solvent. Multilamellar vesicles were prepared by dispersing dry lipids in a buffer containing 150 mM KCL, 10 mM HEPES (pH=7.5), 1 mM NaN3, 30 μM CaCl2, and 10 μM EDTA. Multilamellar vesicles were extruded ten times through two stacked 100 nm pore size polycarbonate filters as described by Mayer et al., Biochim. Biophys. Acta, 858, 161-168.

[0157] Example 2: Micelle preparation with incorporation of toll-like receptor 7 (TLR7) agonists Micelles were made from 1,2-distearyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) obtained from Lipoid GmbH, and the TLR7 agonist, 1v270 (C57H93N6O12P, Mw=1085.4, (2-(4-(6-amino-2-(2-methoxyethoxy)-8-oxo-7H-purin-9(8H)-yl)methyl)benzamido)ethyl 2,3-bis(oleoyloxy)propyl phosphate). The chemical structure of 1V270 is outlined in FIG. 2. Briefly, lipids were dissolved in tert-butanol:water (volume ratio 9:1) to a final concentration of 5-10 mM (DSPE-PEG2000) or 1-3 mM (1v270) in glass vials and heated to 50 °C with magnetic stirring until completely dissolved. The two lipid dispersions were then mixed in the desired ratio (DSPE-PEG2000:1v270 molar ratio of 95:5 to 80:20). The solvent was removed by freezing the vial in liquid nitrogen and then lyophilizing overnight. Micelles were prepared by dispersing the dried lipid in a buffer containing 150 mM NaCl, 10 mM phosphate (pH = 7.4), subjecting the vial to gentle vortexing to establish initial contact of lipid with solvent, and then subjecting it to sonication for 30 min to ensure the formation of micellar structures. The dispersion was vortexed again, after which the dispersion was further sonicated for 30 min. Micelles were stored at 4° C. prior to use and / or characterization. The batch name, molar ratio, lipid composition, zeta potential (mV), size (nm) and polydispersity index (PDI) are summarized in FIG.

[0158] Example 3: Characterization of composition-dependent micelle size and surface charge Micelles, as outlined in Figure 1, were prepared in an attempt to create a stable formulation of the TLR7 agonist 1v207 in an aqueous medium, thus allowing injection in saline buffer. Micelles were prepared as described in Examples 1+2, and their size (diameter) was measured in nanometers (nm) by dynamic light scattering in a buffer consisting of 5% (w / w) glucose, 10 mM HEPES, 1 mM CaCl2, pH 7.4 in MilliQ water. Micelles without 1V2207 had an average size of 11.5 nm (by particle number distribution), while micelles containing 1V270 had an average size ranging from 11.5 nm to 13-14 nm (by particle number distribution) with increasing 1V270 content from 5% to 20% (Figures 1B and 1C). The zeta potential of empty micelles was measured in glucose buffer w.CaCl2 to be approximately -4 mV, but incorporation of the anionic 1V270 compound into the micelles made them more negative, thus decreasing to -8 mV for micelles with 10% 1V207 (Figure 1C). Both size and zeta potential were measured with a Malvern Instruments Zetasizer.

[0159] Example 4: Antitumor activity of 1V270-containing micelles and liposomes in the CT26 model Tumor studies were performed in the CT26 model (Adlard et al., Int J Cancer, 135, 820-829, 2014). Briefly, the CT26 model is a colon cancer model established as a subcutaneous model in Balb / C mice, which is frequently used as an immunocompetent tumor model for testing cancer drugs and immunotherapeutic agents. We used this model with a control group and a group that was radiotreated daily for 5 days with 2 Gy starting on day 10. 1V270-containing liposomes or micelles were injected on the first day of radiotherapy and every 4 days after the 5th dose (Figures 3A and 3B). Liposomes containing 5% 1V270 were injected to reach a total dose of 266 nmol of 1V270 per mouse (Figure 3A). Micelles were injected at 3 doses of 50, 100 and 200 nmol of 1V270 per mouse, which showed significant tumor growth inhibition compared to radiotherapy alone. Furthermore, the lowest dose of 50 nmol showed 2 out of 10 mice in complete remission, the 100 nmol dose showed 4 out of 9 mice in complete remission, and the 200 nmol dose showed 8 out of 8 mice in complete remission. In addition, almost all mice in complete remission were able to resist rechallenge with the same tumor cells without tumor regrowth after 100 days, demonstrating that the mice developed an antitumor immune memory response. In monotherapy, MBS8 micelles also showed very potent antitumor activity (Figure 3C), where MBS8 doses of 50, 200, and 400 nmol were injected intravenously into mice on day 9 and four more times at 4-day intervals. The two highest doses showed tumor growth inhibition that persisted until 2 weeks (day 34) after the last dose for all 10 mice in the group. At day 41, there were 3 mice in complete remission in both high dose groups. MBS8 micelles at 50 nmol / mouse / dose demonstrated synergistic activity when combined with anti-PD-1 treatment. This combination treatment demonstrated significantly greater antitumor activity (p<0.05) than both anti-PD-1 and MBS8 alone (Figure 3D). Anti-PD1 was administered IP six times on days 11, 15, 19, 23, and 27.

[0160] Example 5: Antitumor activity of micelles compared to TLR7 agonist-containing liposomes in combination with radiotherapy in the CT26 model Multiple tumor studies were performed with groups of mice treated with radiation therapy, seven groups of liposome-treated mice injected with 1V270 ranging from 40 to 266 nmol per mouse in combination with radiation therapy, and thirteen groups of micelle-treated mice injected with 1V270 ranging from 50 to 200 nmol per mouse. The number of mice per group ranged from seven to nine, and median tumor sizes were compared between groups treated with radiation therapy alone and groups treated with radiation therapy together with either liposomes or micelles. The median survival time for each group of micelles or liposome-treated mice is shown in Figure 3E. The four micelle-treated groups showing a median survival of more than 100 days indicate that more than half of the mice exhibited complete tumor remission (median survival of observed mice that exhibited complete tumor remission). This was not observed for any of the liposome+RT treatment groups. Tumor growth in the liposome + RT and micelle + RT treatment groups, compared to the RT alone treatment group (referred to as T / C ratio) and calculated when the RT alone treatment group was sacrificed, showed that 10 of 13 groups for micelles showed a T / C ratio of less than 50%, while only 1 of 7 mice in the liposome treatment group showed a T / C ratio of less than 50%. Using the Wilcoxon rank sum test, the difference was significant at the p<0.01 level (Figure 3F). The T / C values ​​could be converted to % tumor growth inhibition by subtracting the T / C value from 100% (Figure 3G), with values ​​close to 100% indicating very strong inhibition of tumor growth. 10 of 13 mice in the micelle treatment group showed more than 60% tumor growth inhibition, while only 1 of 7 mice in the liposome treatment group showed more than 50% tumor growth inhibition.

[0161] conclusion In conclusion, 1V270-containing micelles demonstrated significantly greater tumor growth inhibition than the corresponding 1V270-containing liposomes (p<0.01, Wilcoxon rank-sum test).

[0162] Example 6: Micelles exhibit a safer cytokine profile with reduced risk of cytokine release syndrome (CRS) Micelles MBS6, MBS7 and MBS8 containing the TLR7 agonist 1V270 were compared to empty micelles (MBS0 = vehicle), PBS and two different liposomes containing 1V270 by IV injection into Balb / C mice at a dose of 100 nmol of 1V270 for all samples except PBS and vehicle. Plasma samples were taken at 2 and 6 hours post-injection and cytokines related to anti-tumor immune response and toxicity were measured by multiplex or ELISA analysis. Both interferon gamma (Figure 4A) and interferon alpha (Figure 4B) were reduced in all three micelle formulations MBS6-8 compared to the two liposomal formulations at either 2 or 6 hours post-treatment, supporting a reduced risk of cytokine storm with the micelle formulations. IL-12p70, which is important in inducing type 1 cytotoxic T cell responses, showed a slight but significant reduction with MBS6 and MBS8 treatment compared to MBS1 liposomes, but still showed cytokine levels expected to induce an anti-tumor response (Figure 4C). IL-6, a cytokine important in the initiation of CRS, shows reduced IL-6 levels in plasma from mice treated with MBS6-8 compared to liposomes MBS1 and MBS2 at the 2 hour time point. After 6 hours, all mice showed lower IL-6 levels than after 2 hours, with IL-6 levels either the same or slightly lower than liposomes, demonstrating that micelles showed an overall reduction in IL-6 levels and not just a delay. TNF-alpha, which is also associated with the initiation of CRS syndrome, was also measured, showing an overall reduction in cytokines with micelles, especially compared to MBS1, and most notable with MBS6 and MBS8. The cytokine IL-1b and chemokine GRO were measured but did not show significantly different cytokine levels.

[0163] conclusion In conclusion, administration of micelles MBS6-8 in mice resulted in a safer cytokine profile with a reduced risk of cytokine release syndrome (CRS) compared to the corresponding 1V270-containing liposomal MBS1 and MBS2.

[0164] Example 7: Micelles exhibit a better toxicity profile compared to 1V270-containing liposomes Multiple studies were performed with liposomes of different charges and liposomes containing 1V270 at 0.75-5% content, injected at doses ranging from 13 to 266 nmol / mouse / mouse per mouse in groups of 7-9 mice / group. At the first two injections on days 0 and 4, mice tolerated the liposomes well without any adverse events and signs of toxicity. However, at the third injection on day 8, mice showed adverse events and toxicity in 81% of the studies performed. This toxicity was associated with a transient period starting 10-15 minutes after administration, and a prolonged period of 30-40 minutes, lack of movement, piloerection, weight loss, and overall poor well-being. This observation was seen in 30 of 37 studies using liposomes across the entire dose range. In contrast, micelles did not show this toxicity in any of the current 30 of 30 studies, indicating that micellar formulations of 1V270 show a better toxicity profile than liposomal formulations of 1V270.

[0165] conclusion In conclusion, administration of micelles MBS6-8 in mice resulted in a more favorable toxicity profile compared to the corresponding 1V270-containing liposomes.

[0166] Example 8: Micelles induce lower IgM and IgG anti-PEG antibodies when injected into mice To investigate toxicity associated with 1V270-containing liposomes but not with 1V270-containing micelles, 1V270-containing liposomes and 1V270-containing micelles were injected once by IV injection into mice and blood samples were taken for 28 days. Micelles without 1V270 induced low levels of anti-PEG IgM antibodies in mouse plasma as measured by an ELISA kit for anti-PEG IgM antibodies (Figure 5A, empty micelles). MBS8 micelles induced anti-PEG IgM molecules in plasma on day 5, but at a level less than half the amount induced by 1V270-containing liposomes (Figure 5A, MBS8 vs. MBS liposomes). For anti-PEG IgG antibodies, which, if induced, are usually more specific and present for a longer time in mammals, induction was very high with liposomes 1-2 weeks after administration and then decreased, but was still present throughout the study (Figure 5B). MBS8 micelles induced much lower IgG anti-PEG antibodies (FIG. 5B, about 17-fold lower) than 1V270-containing liposomes. This observation indicates that micelles induce lower levels of IgM and IgG anti-PEG responses, thus reducing immune-related toxicity in mice and potentially other mammals, which is important for reducing toxicity in patients treated with micelles and liposomes bearing immune-enhancing reagents.

[0167] FIG. 5C shows the specificity of anti-PEG antibodies generated against MBS8. Healthy mice were treated with a single dose of MBS8 micelles at a total dose of 200 nmol of 1V270. Blood was drawn and plasma obtained 5 days after injection. Plasma was then incubated with either PBS, PEGylated liposomes, free mPEG2000 chains, DSPE-PEG micelles without 1V270, or MBS8 micelles. PEG concentration was 1 μM in all preincubations. Plasma was then added to microscopy wells containing immobilized fluorescently labeled PEGylated liposomes and incubated for 10 min. Microscopy wells were washed and a fluorescent secondary antibody against mouse IgM was added. Liposomes were imaged by confocal microscopy to determine IgM surface density on single liposomes. When plasma was preincubated with PBS (no competition), the surface density was approximately 60 A.U. Similar IgM binding was measured when plasma was preincubated with free mPEG, DSPE-PEG micelles, or MBS8 micelles, showing high IgM surface density (in the 60-70 AU range) and no competition for binding with anti-PEG IgM present in plasma. When plasma was preincubated with PEG-liposomes (positive control for competition), anti-PEG IgM binding to immobilized PEG-liposomes was almost completely lost, showing strong competition for binding. Thus, anti-PEG IgM detected by ELISA in Figure 5A induced by MBS8 micelles can only recognize PEG chains attached to planar or liposomal surfaces, but not PEG micelles (including MBS8) or free PEG chains.

[0168] conclusion Anti-PEG IgM generated against MBS8 micelles after injection into mice was generally unable to recognize and bind to MBS8 micelles or DSPE-PEG micelles, supporting the unlikely generation of anti-PEG IgM antibodies in patients that bind to MBS8 upon multiple injections.

[0169] Example 9: Treatment of mammals with cancer using immunostimulatory 1v270 micelles (non-antigen specific) To obtain immune stimulating micelles suitable for cancer therapy, micelles can be produced by mixing 1v270:DOPE-PEG2k (10:90=MBS8). The compounds are mixed in an organic solvent and dried into a lipid film. The film is hydrated in a buffer suitable for intravenous administration, including, for example, saline. The micelles are administered intravenously, for example at 1-2 week intervals, to cancer patients suffering from, for example, lung cancer, breast cancer, prostate cancer, HNC, leukemia, lymphoma or melanoma. Combination with clinically approved therapies is likely to enhance antitumor efficacy. In particular, in combination with immune checkpoint inhibitors, for example, mAb therapies such as rituximab and trastuzumab, in combination with radiation therapy to enhance the abscopal effect in lung cancer patients, to boost antibody-dependent cellular cytotoxicity (ADCC), to enhance the response to immunogenic cell death induced by certain chemotherapies such as doxorubicin, oxaliplatin, cyclophosphamide and mitoxantrone.

[0170] Example 10: Treatment of mammals with cancer using immunostimulatory 1v270 micelles (antigen-specific) To obtain an antigen-specific immune response, micelles are prepared as in Example 9, for example, by adding an antigen peptide containing all or part of the antigen of interest linked to a lipid anchor such as DOPE. The peptide antigen associated with the lipid anchor ensures sufficient micelle association, as seen for a peptide sequence of 25 amino acids from the MUC1 tumor antigen, and palmitoylated lysine residues ensure sufficient liposome association of the antigen (Sangha and Butts, Clin Cancer Res 2007;13,15 Suppl, 2007,4652-54s). The antigen can be, for example, a MAGE antigen for the treatment of melanoma, PSA for the treatment of prostate cancer, a neoantigen or a third antigen or combination of antigens. The antigen is administered together with 1v270 to a cancer patient expected to express the loaded antigen in his tumor. The micelles are administered to the same patient several times, preferably at intervals of 1-2 weeks, to enhance the antigen-specific response.

[0171] Example 11: Antitumor activity of MBS8 micelles in combination with radiotherapy Mice bearing CT26 subcutaneous tumors were treated with radiotherapy (RT) and 50, 100 or 200 nmol of 1V270-containing micelles. Micelles were administered intravenously every 4 days starting on day 12 after tumor inoculation for a total of 5 treatments. 2 Gy of RT was irradiated to the tumor-bearing flank daily for 5 consecutive days starting on day 12 after tumor inoculation. The number of mice per group was 8-10, and mice were rechallenged with CT26 on the opposite flank on day 101 after primary inoculation. Tumor growth curve data are mean tumor size ± SEM. Combination of 50 or 100 nmol MBS6 with RT provided negligible tumor control, while 200 nmol MBS6 combined with RT provided good tumor control (Figure 6A). With RT alone, 1 of 9 mice were complete responders and 1 of 1 mouse rejected rechallenge. For 50 nmol MBS6 combined with RT, 2 of 9 mice were complete responders and 2 of 2 mice rejected rechallenge. For 200 nmol MBS6 combined with RT, 7 of 9 mice were complete responders and 7 of 9 mice rejected rechallenge (Figure 6A-B).

[0172] The combination of MBS7 with RT showed synergistic effects. For 50 nmol MBS7 combined with RT, 5 out of 9 mice were complete responders and 5 out of 5 mice rejected rechallenge. For 100 nmol MBS7 as monotherapy, 3 out of 9 mice were complete responders and 3 out of 3 mice rejected rechallenge. The combination of 100 nmol MBS7 with RT resulted in 8 out of 10 complete responders and 3 out of 3 mice that rejected rechallenge, providing significantly improved survival compared to monotherapy (p=0.02, Mantel-Cox test; Figure 6C-D). The combination of 50 nmol MBS8 with RT resulted in 2 out of 10 complete responders and 2 out of 2 mice that rejected rechallenge. For 100 nmol MBS8 in combination with RT, 4 out of 9 mice were complete responders and 4 out of 4 mice rejected rechallenge. For 200 nmol MBS8 in combination with RT, 8 out of 8 mice were complete responders and 7 out of 8 mice rejected rechallenge. Furthermore, a dose-dependence could be observed for MBS8 in combination with RT (p<0.3, Mantel-Cox test; Figure 6E-F).

[0173] conclusion Micelles containing 1V270 at molar ratios of 80:20, 90:10 and 95:5 all exhibit strong antitumor activity both as monotherapy and in combination with radiotherapy.

[0174] Example 12: Antitumor activity of MBS8 micelles in monotherapy The efficacy of MBS8 micelles was investigated as monotherapy in a CT26 syngeneic subcutaneous colon cancer model. Mice were randomized and treated at 10-20% CI, 0.01 to 0.25, with a mean tumor volume of 100 mm. 3Treatment was started when the tumor reached a mean tumor volume of 1000 mg / kg / day (day 0). Groups of 10 mice were each treated with PBS as a vehicle control, or with three doses of MBS8 by intravenous bolus at 50, 200, or 400 nmol / mouse / injection (Figure 7A). MBS8 was administered q4d (days 9, 13, 17, 21, and 25) starting on day 9 for a total of five injections. Tumor growth was measured twice weekly. The mean tumor volume of treated mice is shown in Figure 7A, and tumor volumes of individual animals are shown in Figure 7B (1-4). Statistical analysis using Wilcoxon rank sum test revealed a significant enhancement (p<0.01) of efficacy of MBS8 at the medium and high doses compared to controls. Eight of 10 mice showed complete remission in the medium and high dose MBS8 treatment groups.

[0175] conclusion MBS8 micelles containing 1V270 at 10 molar content showed significant antitumor activity at doses of 200 and 400 nmol in the CT26 model and were highly potent in inducing complete responses.

[0176] Example 13: Antitumor activity of MBS8 micelles in combination with PD-1 therapy The efficacy of MBS8 micelles in combination with αPD-1 therapy was investigated with a fixed dose of αPD-1 at 10 mg / kg IP injection and increasing doses of MBS8 at 50, 200 and 400 nmol / mouse / injection. Using the CT26 model, MBS8 treatment was performed as described in Example 12. MBS8 treatment was started on day 9 and αPD-1 on day 11, then twice weekly for 3 weeks (Figure 8A). Low dose MBS8 in combination with αPD-1 showed significant tumor growth delay (p<0.05) versus low dose MBS8 alone and αPD-1 alone (Figures 8A and 8B, graphs 1-4). At medium and high doses of MBS8, the combination therapy did not show any difference initially, as all 10 mice in each group showed remission or very small tumors (Figure 8A). However, between days 30 and 50, some tumors began to grow in the MBS8 monotherapy group, and two mice escaped the tumors (Figures 8A and 8B, graphs 3 and 4). In the αPD-1 combination group, none of these escapees were observed (Figure 8B, graphs 7 and 8), and all tumors eventually disappeared or became very small (<30 mm). 3 ).

[0177] conclusion MBS8 micelles containing 1V270 were highly potent in combination with αPD-1, resulting in at least 90% complete remission in treated CT26 tumor-bearing mouse models.

[0178] Example 14: Antitumor activity of MBS8 micelles in combination with chemotherapy (doxorubicin and doxil) in the CT26 model The efficacy of MBS8 micelles was investigated in combination with standard of care chemotherapy, including doxorubicin and doxil, and compared to the efficacy of monotherapy with these chemotherapeutic agents in the CT26 syngeneic subcutaneous colon cancer model. Mice were randomized and treated at 10-20% CI, 0.01 to 0.25, with tumors of ... 3Treatment was started when the tumor volume reached 100 μg / mL (day 0). Groups of 10 mice each were treated with PBS as a vehicle control, doxorubicin (4 mg / kg) or a combination of MBS8 (400 nmol / mouse) and doxorubicin (4 mg / kg) (Figure 9A), or doxil (4 mg / kg) or a combination of MBS8 (200 nmol / mouse) and doxil (4 mg / kg) (Figure 9C). All drugs were administered by intravenous bolus. Doxorubicin or doxil was injected q4d for a total of three times. MBS8 was injected q4d for a total of five times. Doxorubicin or doxorubicin was administered on days 10, 14, and 18, and MBS8 was administered on days 10, 20, 24, 28, and 32. Tumor growth was measured twice weekly. The mean tumor volumes of doxorubicin and / or MBS8 treated animals are shown in Figure 9A, and the tumor volumes of individual animals are shown in Figure 9B. The mean tumor volumes of doxil and / or MBS8 treated animals are shown in Figure 9C, and the tumor volumes of individual animals are shown in Figure 9D. Statistical analysis using Wilcoxon rank sum test revealed a significant enhancement of the efficacy of chemotherapy drugs when combined with MBS8. All animals treated with MBS8 / doxil combination showed a complete response, with 8 out of 10 complete responders in the MBS8 / doxorubicin treated group. No complete responders were found in the single chemotherapy groups.

[0179] conclusion Micelles containing 1V270 at 10 molar loading significantly enhanced the efficacy of doxorubicin and doxil, resulting in complete responders.

[0180] Example 15: Antitumor activity of MBS8 micelles using intravenous versus intratumoral administration CT26 subcutaneous tumor-bearing mice were treated with radiotherapy (RT) and injected intravenously or intratumorally with MBS8. MBS8 or lipid-compatible micellar vehicle without TLR7 agonist 1V270 was administered every 4 days starting from day 12 after tumor inoculation for a total of 5 doses. 2Gy RT was irradiated to the tumor-bearing flank daily for 5 consecutive days starting from day 12 after tumor inoculation. Intratumoral MBS8 injections were performed as 100 nmol of 1V270 by volume restriction, and intravenous MBS8 injections were performed as 200 nmol of 1V270 (Figure 10A). The number of mice per group was 8, and mice in complete remission were rechallenged with CT26 on the opposite flank on day 103 after the primary challenge. Data for tumor growth curves are mean tumor size ± SEM. MBS8 provided good tumor control as monotherapy and in combination with radiotherapy regardless of the route of administration, although the IV route was slightly more efficacious than IT administration (Figure 10A). Intravenous injection of MBS8 alone resulted in 3 of 8 complete responders, of which 3 of 3 refused rechallenge. Intravenous injection of MBS8 in combination with RT resulted in 6 of 8 complete responders, of which 5 of 6 refused rechallenge. Intratumoral injection of MBS8 alone resulted in 3 of 8 complete responders, of which 3 of 3 refused rechallenge. Intratumoral injection of MBS8 in combination with radiotherapy resulted in 4 of 8 complete responders, of which 4 of 4 refused rechallenge. In contrast, injection of vehicle did not improve tumor control (Figure 10B). Radiotherapy alone or in combination with vehicle did not result in any complete responders. Survival of mice in each group is shown for the intratumoral (FIG. 10C) and intravenous (FIG. 10D) groups. Median survival was 43.5 days for MBS8 treatment as monotherapy administered IT versus 63 days for IV administration. For MBS8 in combination with radiation therapy, median survival was 56 days for IT versus 100+ days for IV administration, with 6 of 8 mice achieving complete remission.

[0181] conclusion 1V270-loaded MBS8 micelles provided good tumor control when injected either intravenously or intratumorally and demonstrated synergistic effects with radiation therapy regardless of the route of administration.

[0182] Example 16: Antitumor activity of MBS8 micelles in combination with radiotherapy Mice bearing MC38, EL4, or B16-F10 tumors were treated with radiotherapy (RT) and intravenous MBS8. 200 nmol of MBS8 or lipid-matched vehicle was administered q4d for a total of five doses. In EL4-bearing mice, treatment began 7 days after inoculation, micelles were given q4d, and radiotherapy was administered to the tumor-bearing flank of 9–10 mice per group for three consecutive days at 2 Gy. In MC38-bearing mice, treatment began 10 days after inoculation, and radiotherapy was administered to the tumor-bearing flank of 9 mice per group for five consecutive days at 2 Gy. MC38-bearing complete responders were rechallenged with MC38 on the opposite flank 80 days after the primary challenge.

[0183] MBS8 did not provide tumor control with EL4 as monotherapy, but in combination with RT, it resulted in 4 of 10 complete responders. RT alone resulted in only 1 of 9 complete responders, and vehicle combined with RT resulted in 2 of 10 complete responders (Figure 11A-B). MBS8 provided moderate tumor control in MC38-bearing mice when combined with radiation therapy. RT combined with MBS8 resulted in 3 of 9 complete responders, of which 2 of 3 refused rechallenge (Figure 11C-D).

[0184] conclusion In conclusion, MBS8 provides good tumor control in EL4 and MC38 when combined with RT.

[0185] Example 17: Tolerance of MBS8 micelles in monkeys The tolerability of MBS8 micelles was evaluated in cynomolgus monkeys (macaca fascicularis). MBS8 was administered to three naive male monkeys weighing approximately 3 kg every 14 days with the following dose escalation scheme: 0.01 mg / kg → 0.03 mg / kg → 0.1 mg / kg → 0.3 mg / kg → 0.9 mg / kg → 2.7 mg / kg. Drug administration was performed by intravenous infusion at a rate of 0.25 ml / min for a total of 5 ml. During the acclimation period, blood samples were taken 14 and 7 days before as baseline controls, and during the treatment period at 2, 4, 8, 24, 72 and 14 days. Blood was analyzed at 8, 24, 72 and 14 days. Blood chemistry was analyzed 14 days after each drug administration, i.e., immediately before the next administration. C-reactive protein (CRP) was measured at 4, 8, 24, 72 and 14 days after dosing. Body temperature was monitored daily. Blood pressure was measured 2, 4, 8 hours after each drug administration and then daily until the next dose. Food consumption was monitored daily and body weight was measured weekly. All doses were well tolerated. A transient dose-dependent increase in CRP levels was detected at 24 hours after infusion, which returned to baseline levels within 3 days (Figure 12A). There were no significant changes in body temperature during the study (Figure 12B). Based on blood chemistry, there was no indication of liver toxicity or changes in electrolyte profile.

[0186] conclusion Intravenous infusion of MBS8 was well tolerated in cynomolgus monkeys within the dose range of 0.01 to 2.7 mg / kg.

[0187] Example 18: Antitumor activity of MBS8 micelles and αPD-1 in monotherapy and combination therapy in multiple syngeneic tumor models The efficacy of MBS8 as monotherapy or in combination with an anti-PD-1 monoclonal antibody (clone RMP1-14) was examined in a panel of 12 syngeneic mouse models (Figure 13). Mice were inoculated sc with cancer cells and had a mean tumor size of approximately 100 mm. 3When the tumor reached 100 mg / kg, the animals were randomized into four treatment groups of 10 animals each: Group 1, vehicle control (PBS); Group 2, anti-PD-1, 10 mg / kg; Group 3, MBS8, 300 micrograms / mouse; Group 4, MBS8 combined with anti-PD-1. Vehicle or MBS8 was administered by low iv bolus on a q4d schedule for a total of 5 injections starting on the day of randomization. Anti-PD-1 was administered ip on a q4d schedule for a total of 6 injections starting 2 days after randomization. A summary of the results is shown in Figure 13A. TGI(%) tumor growth inhibition was calculated as TGI(%)=100×(1−T / C). T and C are the mean tumor volumes of the treatment and control groups, respectively, on the day the control group was terminated.

[0188] Three distinct response patterns were observed: (1) MBS8 is highly potent in monotherapy, and therefore no benefit of combination with anti-PD-1 is observed due to the excessive activity of MBS8 (Figure 13B). Tumors in this category were CT26 (colon cancer), EMT-6 (breast cancer), A20 (B-cell lymphoma), and H22 (liver cancer). (2) Both anti-PD-1 and MBS8 showed therapeutic activity as monotherapy. Combination therapy showed additional benefit (Figure 13C). This was observed for Hepa1-6 (liver cancer) and M38 (colon cancer). (3) Anti-PD-1 was inactive in monotherapy, but MBS8 monotherapy showed efficacy. The combination of the two drugs resulted in synergistic activity by converting PD-1 non-responsive mice into responsive mice (Figure 13D). This category included RM-1 (prostate cancer), Pan02 (pancreatic cancer), and Renca (renal cancer).

[0189] For the EMT-6 model, complete responders from groups 3 and 4 that had been tumor-free for at least 3 weeks were rechallenged with sc injections of EMT-6 cells in the opposite flank. The mice were followed up for 29 days. All rechallenged animals showed complete rejection of the rechallenged tumors (Figure 13E). For the CT26 model, mice that showed complete responses to MBS8 or the combination of MBS8 with anti-PD-1 therapy and rejected the rechallenged tumors were analyzed for the presence of tumor-specific T cells (Figure 13F, ELISPOT graphs). Spleen cells from untreated mice bearing CT26 tumors, mice treated with either MBS8 or MBS8 with anti-PD-1, or untreated mice without tumors were stimulated in vitro with AH-1 tumor-specific antigen peptide. IFNγ-producing cells were quantified using ELISPOT. All mice that rejected the rechallenged tumors showed increased numbers of IFNγ-positive cells in response to antigenic stimulation, thus confirming the establishment of an immune memory response.

[0190] Conclusion: MBS8 showed therapeutic efficacy in monotherapy. In some tumors that responded weakly to anti-PD-1, combination treatment with MBS8 had an additive effect. In models that did not respond to anti-PD-1 treatment but did respond to MBS8, anti-PD-1 and the latter sensitized tumors to the drug showed a strong synergistic effect. MBS8, either as monotherapy or in combination with anti-PD-1, led to the establishment of an immune memory response.

[0191] Example 19: Dose schedule optimization of MBS8 in the CT26 model and correlation to antitumor activity The effect of dose schedule on the efficacy of MBS8 monotherapy was analyzed using a CT26 colon cancer syngeneic mouse model. Mice with established tumors were treated with 200 nmol / mouse MBS8 (1) single injection; (2) two injections q7d; (3) three injections q7d, (4) four injections q7d, (5) five injections q7d (Figure 14). As controls, vehicle (PBS) treatment was used, as well as MBS8 treatment with five injections q4d, schedules that showed good efficacy in previous studies. Already a single injection showed significant inhibition of tumor growth, while multiple weekly administrations led to complete eradication of tumors.

[0192] Conclusions: A single injection of MBS8 resulted in significant antitumor activity, while booster injections on either a 4- or 7-day schedule resulted in complete remissions in multiple mice.

[0193] Example 20: Antitumor activity of MBS8 micelles compared to R848 monotherapy The efficacy of MBS8 micelles and R848 was investigated as monotherapy in the CT26 syngeneic subcutaneous colon cancer model. 3Randomization and treatment of mice was initiated when the tumor size reached 100 nmol / mouse / injection (day 11). Groups of 9 mice each were treated with vehicle control (MBS0); MBS8 by intravenous bolus at 100, 200 or 300 nmol / mouse / injection or in combination with R848 at 200 nmol / mouse / injection. Treatment was performed q4d (days 11, 15, 19, 23, 27) for a total of 5 injections starting on day 11. Tumor growth was measured twice weekly. The mean tumor volume of treated mice is shown in Figure 15A and survival curves are shown in Figure 15B. Statistical analysis using Wilcoxon rank sum test revealed significant differences between all doses of MBS8 compared to vehicle (p<0.0001). Furthermore, MBS8 at 200 nmol was significantly more effective than R848 at 200 nmol (p<0.05). Weight changes relative to baseline are shown in Figure 15C. For MBS8 micelles, weight loss changes became less severe with increasing number of treatments, while R848 induced the most severe weight loss changes from 25 to 40 days.

[0194] conclusion MBS8 micelles exhibit superior antitumor efficacy to R848 when administered at equimolar doses. Due to enhanced therapeutic activity and less severe weight loss, MBS8 micelles have increased therapeutic index compared to the TLR7 agonist R848.

[0195] Example 21: Nanostring tumor gene expression analysis of CT26 tumor-bearing mice treated with MBS8 Gene expression profiles of CT26 tumors from mice treated with MBS8 as monotherapy were generated to determine gene expression associated with MBS8 treatment and the presence of specific tumor cell types. Gene expression was assessed using a Pan Cancer Immuno Panel (Nanostring) performed on bulk RNA extracted from tumors. Mice were randomized into groups of 10 on day 12 and treated with MBS8 at 200 nmol / mouse / injection by intravenous bolus administered on a q4d schedule for a total of 1 or 3 injections. Mice were sacrificed and tumors were flash frozen at day 0 (untreated), 1 day post-injection on day 1, 2 days post-injection on day 1, 4 days post-injection on day 1, 14 days post-injection on day 1, 2 days post-injection on day 3, and 4 days post-injection on day 3. RNA was extracted and 750 genes were analyzed with the Pan Cancer Immuno Panel. Cell type analysis was performed using the Advanced Analysis module of Nanostring's software. A two-way ANOVA with correction for multiple comparisons was performed to compare the different time points to untreated. At early time points (1, 2, and 4 days after the first injection), a significant increase was observed in both neutrophils and dendritic cells, demonstrating activation of the innate immune system. At later time points (from day 4 after the first injection onwards), T cells, especially CD8 T cells, were increased in the tumor, demonstrating activation of the adaptive immune system. Furthermore, macrophages are also upregulated at later time points. The time point 14 days after the first injection has a cell type profile similar to UT, indicating that a single injection in this setting is not sufficient to induce a sustained immune response.

[0196] conclusion Gene expression profiling of tumors from mice treated with MBS8 monotherapy shows the induction of an innate immune response at early time points, with the presence of large numbers of neutrophils and dendritic cells. An adaptive immune response is seen at later time points dominated by T cells, both of which mediate the antitumor effects of MBS8 treatment.

[0197] Example 22: Acute effects on the tumor microenvironment after MBS8 treatment The acute effect of intravenous injection of 200 nmol of MBS8 on tumors was evaluated by multicolor flow cytometry. CT26 tumor-bearing mice were treated with MBS8 and tumors were evaluated by flow cytometry at 1, 3, and 6 hours after injection. Erythrocytes were determined based on size and Ter-119 expression and excluded from further analysis. All further analysis was performed based on the absence of Ter-119 expression and based on size and staining as live. CD8+ T cells were defined as CD45+CD3+CD8+. Erythrocytes made up the majority of cells in the tumor 6 hours after injection of MBS8 (Figure 17A). Furthermore, the tumor microenvironment was enriched for immune cells (CD45 + ) and a significant and surprising reduction in cancer cells (Figure 17B, graphs 1 and 2). Although immune cells were reduced in the tumor, there was a significant increase in neutrophils at 6 hours post-treatment (Figure 17C, graph 1), whereas T cells, NKT cells, and NK cells did not change significantly (Figure 17C, graphs 2-5). However, when immune activity was monitored in these cell subsets based on CD69 expression, T cells, NKT cells, and NK cells showed significant activation at 3 hours post-treatment (Figure 17D, graphs 1-5).

[0198] conclusion Intravenous injection of MBS8 induced enrichment of neutrophils and erythrocytes in the tumor 6 hours after treatment, accompanied by a decrease in viable tumor and immune cells, while a significant activation was seen for immune cells; CD8 T cells, NKT cells and NK cells.

[0199] Example 23: Effects on the tumor microenvironment and spleen after TLR7 micelles The impact of intravenous injection of MBS6 on the tumor microenvironment was assessed by multicolor flow cytometry. CT26 tumor-bearing mice were treated with 200 nmol MBS6 every 4 days starting on day 15 after cancer cell inoculation, and tumors and spleens were analyzed 2 days after the second injection and 6 days after the first injection (day 21). Viable cells were identified based on cell size and lack of staining with a viability dye. All further analyses were based on viable cells. Neutrophils were stained with CD45+ CD11b + Ly6g + Tumor-associated macrophages (TAMs) were identified as CD45 + Ly6g - CD11C + CD11b + CD64 高 Patrolling monocytes (PMos) were identified as CD45 + Ly6g - CD11b + CD11c - Ly6c - CD64 + Monocytic myeloid-derived suppressor cells (Mo-MDSCs) were identified as CD45 + Ly6g - CD11b + CD11c - Ly6c 高 Classical DC (cDC)1s were identified as CD45 + Ly6g - CD11c + CD64 低 CD11b 低 XCR1 + cDC2s were identified as CD45 + Ly6g - CD11c + CD64 低 XCR1 - CD11b 高 Plasmacytoid DCs (pDCs) were identified as CD45 + Ly6g - CD11c + CD64 低 XCR1 - CD11b - Ly6c 高 Siglec-H + CD8 + T cells were identified as CD45 + Side Scatter 低 CD3 + CD4 - CD8 +Cells per 100 mg were calculated based on the population of interest, tumor weight and total cells. Tumor weight was lower compared to untreated tumors (Figure 18A, p=0.056). Furthermore, cell viability in tumors was significantly decreased after treatment with MBS6, which was hardly reflected in the spleen (Figure 18B, graphs 1 and 2). Among the surviving cells, MBS6 significantly increased the number of immune cells (CD45 + ) (Figure 18C). This is in contrast to what was seen in Figure 17C, but can be explained by the fact that this time point is 6 days after the first treatment. Neutrophils were highly enriched in the tumor microenvironment (versus the absence of other cell types), as seen already 6 hours after MBS6 treatment in Figure 17, which was not reflected in the spleen (Figure 18D, graphs 1 and 2). All other immune populations and cancer cells examined were strongly reduced in the tumor microenvironment (Figure 18E, graphs 1-8).

[0200] conclusion Intravenous injection of MBS6 strongly reduced cell viability in the tumor microenvironment but did not affect survival in the spleen. Moreover, MBS6 induced a strong increase in neutrophils in the tumor microenvironment, which was not observed in the spleen.

[0201] Example 24: Optimal dosing schedule for MBS8 and Doxil The dosing regimen when using combination treatment of MBS8 and chemotherapy is important to achieve optimal tumor control. We wanted to test whether doxil treatment would be improved in combination with MBS8, and if so, how MBS8 should be dosed to achieve optimal response (Figure 19). Doxil treatment alone showed a delay in tumor growth, but the mice did not go into complete remission, with a tumor growth inhibition (TGI) of 80% compared to vehicle-treated mice (Figure 19). When MBS8 was first administered 2 days after the last doxil treatment (post-MBS8 administration), there was a significant tumor growth inhibition of 90% versus vehicle-treated mice, and significant antitumor activity compared to doxil alone (p=0.0007). However, when MBS8 was first administered on the same day as doxil (before and after MBS8 administration), all mice went into complete remission with a TGI of 98%, demonstrating significantly better antitumor activity than doxil alone or than MBS8 alone administered after doxil treatment.

[0202] conclusion These data indicate that MBS8 immunotherapy is significantly more efficient when pre-administration is applied on the same day as chemotherapy, followed by follow-up treatment after chemotherapy has ended, compared to situations in which immunotherapy is initiated before chemotherapy.

[0203] Example 25: Micelles show no signs of accelerated blood clearance (ABC) In mice: The mean (±SD) plasma concentration-time curves of MBS8 in female and male rats on day 1 (first dose) and day 15 (one week after the last of two doses, one week apart) after intravenous infusion of 3 mg / kg / day MBS8 (1V270) over one hour are shown in Figure 20 in linear scale (A) and semi-logarithmic scale (B). This example demonstrates the insignificant kinetics of MBS8 administered on day 1 and day 15 (third dose), respectively. This observation supports that the micelles of the present invention do not induce accelerated blood clearance (ABC).

[0204] In cynomolgus monkeys: The individual plasma concentration-time curves of 1V270 in one female and one male cynomolgus monkey on day 1 (A, first dose) and day 15 (B, third dose after weekly administration of two doses) after intravenous infusion of 0.3, 1 and 3 mg / kg / day MBS8 (1V270) over one hour are shown in Figure 21 on a linear scale. This example demonstrates the insignificant kinetics of MBS8 administered after one day and the third dose after 15 days, respectively. This observation supports that the micelles of the present invention do not induce accelerated blood clearance (ABC).

[0205] conclusion In both mice and cynomolgus monkeys, the micelles of the present disclosure did not induce accelerated blood clearance (ABC) when infused on day 1 (1st dose) and day 15 (3rd dose), supporting the significantly improved therapeutic utility of micelles, at least compared to liposomes that are known to induce ABC events.

Claims

1. Formula (I); 【Chemistry 1】 (In the formula, In the formula, X １ is -O-, -S-, or NR Ｃ and R １ is hydrogen, (C １ -C6) alkyl, or substituted (C １ -C6) alkyl; R Ｃ is hydrogen, C1-6 alkyl, or substituted C1-6 alkyl, or R Ｃ and R １ together with the nitrogen to which they are attached form a heterocyclic or substituted heterocyclic ring; R ２ does not exist; n is 0; X ２ is selected from the group consisting of a bond, —O—, —C(O)-(carbonyl), (C 1 -C 6 )alkyl, substituted (C 1 -C 6 )alkyl, (C 1 -C 6 )alkoxy, substituted (C 1 -C 6 )alkoxy, —C(O)-(C 1 -C 6 )alkyl (alkanoyl), substituted —C(O)-(C 1 -C 6 )alkyl, —C(O)-(C 6 -C 10 )aryl (aroyl), substituted —C(O)-(C 6 -C 10 )aryl, —C(O)OH (carboxyl), —C(O)O(C 1 -C 6 )alkyl (alkoxycarbonyl), substituted —C(O)O(C 1 -C 6 )alkyl, —NR a R b , —C(O)NR a R b (carbamoyl); each R a and R b is independently hydrogen, (C 1 -C 6 )alkyl, substituted (C 1 -C 6 )alkyl, (C 3 -C 8 )cycloalkyl, substituted (C 3 -C 8 )cycloalkyl, (C 1 -C 6 )alkoxy, substituted (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkanoyl, substituted (C 1 -C 6 )alkanoyl, aryl, aryl(C 1 -C 6 )alkyl, Het, Het(C 1 -C 6 )alkyl, or (C 1 -C 6 )alkoxycarbonyl; R 3 is a lipid selected from the group consisting of phospholipids, gonanes, glycolipids, and glycerides, each of which contains one or two carboxylic acid esters. or a tautomer thereof; or a pharma- ceutical acceptable salt or solvate thereof A micelle composition comprising: the purine group undergoes tautomeric rearrangement; Contains an amphiphilic micelle forming agent The micelles of the micellar composition have an average diameter of 5 nm to 25 nm. Micellar compositions.

2. 2. The micelle composition of claim 1, wherein the amphiphilic micelle forming agent is selected from the group consisting of poloxamers, poloxamines, PEG-polyesters, PEG-polyanhydrides, PEG-polyamino acids, phospholipids, polysorbates, and polyoxyethylene alkyl ethers.

3. the PEG-polyester is selected from the group consisting of PEG-poly(lactic acid) (PEG-PLA), PEG-poly(lactic-co-glycolic acid) (PLGA), and PEG-poly(ε-caprolactone) (PCL); the PEG-polyanhydride is PEG-polysebacic anhydride (PSA); and / or The PEG-polyamino acid is selected from PEG-poly(L-histidine), PEG-poly(L-aspartic acid), PEG-poly(L-asparagine), PEG-poly(L-glutamic acid), PEG-poly(L-glutamine), and PEG-poly(L-lysine). The micelle composition of claim 2.

4. The micelle composition of claim 1 , wherein the amphiphilic micelle forming agent is a phospholipid conjugated to polyethylene glycol (PEG).

5. The micelle composition of claim 4 , wherein the PEG-conjugated phospholipid is conjugated via a carbonyl group.

6. The micelle composition according to any one of claims 2 to 5, wherein the size of the PEG is from PEG 350 to PEG 5000.

7. The phospholipid comprising: C8 to C24 alkyl(s); and / or Phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidic acid (PA), bisphosphatidylglycerol (DPG), or phosphatidyl alcohol The micelle composition of any one of claims 2 to 4, comprising:

8. 8. The micellar composition of claim 7, wherein the phosphatidylethanolamine is selected from the group consisting of 1,2-dioleoylphosphatidylethanolamine, 1,2-dipalmitoylphosphatidylethanolamine, 1,2-dimyristoylphosphatidylethanolamine, 1,2-distearoylphosphatidylethanolamine, 1-oleoyl-2-palmitoylphosphatidylethanolamine, 1-oleoyl-2-stearoylphosphatidylethanolamine, 1-palmitoyl-2-oleoylphosphatidylethanolamine, and 1-stearoyl-2-oleoylphosphatidylethanolamine.

9. 6. The micelle composition of claim 5, wherein the PEG-conjugated phospholipid is selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE)-PEG, and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE)-PEG.

10. The micelle composition of claim 1, wherein the amphiphilic micelle forming agent is DSPE-PEG2000.

11. The micelle composition of claim 1, wherein the molar ratio of the amphipathic micelle forming agent to the TLR7 agonist is from 50:50 to 99.5:0.

5.

12. The micelle composition of claim 11 , wherein the molar ratio of the amphipathic micelle forming agent to the TLR7 agonist is 90:

10.

13. The micelle composition of claim 1 , wherein the micelle composition comprises a molar concentration of the TLR7 agonist of 1% to 25%.

14. The TLR7 agonist has formula (IA): 【Chemistry 2】 Structure by or a tautomer thereof; or a pharma- ceutically acceptable salt or solvate thereof.

15. Formula (IA) 【Chemistry 3】 a TLR7 agonist; or a tautomer thereof; or a pharma- ceutically acceptable salt or solvate thereof; and DSPE-PEG2000 and A micelle composition, wherein the molar ratio of DSPE-PEG2000 to said TLR7 agonist is 95:5, 90:10, or 80:

20.

16. 2. The micelle composition of claim 1, wherein the molar ratio of DSPE-PEG2000 to the TLR7 agonist of formula (IA) is 90:10 (MBS8).

17. The micelle composition of claim 1 further comprising at least one additional active ingredient.

18. The micelle composition of claim 1 further comprising at least one antigen.

19. A pharmaceutical composition comprising the micelle composition of claim 1.

20. 20. A micelle composition according to claim 1 or a pharmaceutical composition according to claim 19 for use in the prevention, treatment or amelioration of a disease or disorder, comprising: The disease or disorder is selected from the group consisting of cancer, an infectious disease, an inflammatory condition or disease, an autoimmune disease, and an allergy. A micelle composition or a pharmaceutical composition.

21. 21. The micellar composition or pharmaceutical composition of claim 20, wherein the disease or disorder is cancer.

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