Sugar-derived lipid nanomaterials and their use

Abstract

The present disclosure relates to compositions and methods for treating cancer and other immune disorders, as well as other prophylactic and therapeutic applications.

Description

Detailed description of the invention

[0001] [Technical field] Cross-reference of related applications This application claims the benefits of U.S. Provisional Patent Application No. 63 / 148,755, filed February 12, 2021, the disclosure of which is expressly incorporated herein by reference. Statement on federally funded research This invention was made with the assistance of the United States Government through grant R35GM119679 from the National Institutes of Health. The United States Government reserves certain rights in this invention.

[0002] Technical field This disclosure relates to compositions and methods for treating cancer and other immune disorders. [Background technology] Efficient mRNA delivery is a critical step and challenge for the application of mRNA-based therapeutics. Despite promising data from ongoing clinical trials, the clinical use of mRNA requires the discovery and development of more efficient delivery systems. What is needed are novel compositions and methods for delivering mRNA to treat cancer and other immune disorders.

[0003] [Overview of the prefecture] In some embodiments, compounds having formula I, II, or III are disclosed herein.

[0004] [ka] or a salt thereof, in the formula, R 1 The following are independently selected from alkyl, alkenyl, alkynyl, hydroxyl, ester, ether, carbonate, alkyl alcohol, alkyl ether, alkyl ester, carbamate, urea, guanidine, disulfide, amide, acetal, ketal, thioketal, trisulfide, and oxime ether.

[0005] In some embodiments, the compound has the following formula:

[0006] [ka] or a salt thereof, in the formula, R 1 The following are independently selected from alkyl, alkenyl, alkynyl, hydroxyl, ester, ether, carbonate, alkyl alcohol, alkyl ether, alkyl ester, carbamate, urea, guanidine, disulfide, amide, acetal, ketal, thioketal, trisulfide, and oxime ether.

[0007] In some embodiments, the compound has the following formula:

[0008] [ka] or a salt thereof, in the formula, R 1 The following are independently selected from alkyl, alkenyl, alkynyl, hydroxyl, ester, ether, carbonate, alkyl alcohol, alkyl ether, alkyl ester, carbamate, urea, guanidine, disulfide, amide, acetal, ketal, thioketal, trisulfide, and oxime ether.

[0009] In some embodiments, the compound has the following formula:

[0010] [ka] or a salt thereof, in the formula, R 1The following are independently selected from alkyl, alkenyl, alkynyl, hydroxyl, ester, ether, carbonate, alkyl alcohol, alkyl ether, alkyl ester, carbamate, urea, guanidine, disulfide, amide, acetal, ketal, thioketal, trisulfide, and oxime ether.

[0011] In some embodiments, R 1 The following:

[0012] [ka] Or selected from those salts.

[0013] In some embodiments, the compound is as follows:

[0014] [ka] In the formula, R 1 teeth

[0015] [ka] That is the case.

[0016] In some embodiments, the compound is as follows:

[0017] [ka] In the formula, R 1 teeth

[0018] [ka] That is the case.

[0019] In some embodiments, disclosed herein are lipid-based nanoparticles comprising a compound of any of the preceding embodiments and a recombinant polynucleotide containing a nucleic acid encoding a co-stimulatory molecule.

[0020] In some embodiments, disclosed herein are antigen-presenting cells comprising lipid-based nanoparticles comprising recombinant polynucleotides containing a compound and a costimulatory molecule of any of the preceding embodiments.

[0021] In some embodiments, The aforementioned compound

[0022] [ka] And, In the formula, R 1 teeth

[0023] [ka] That is the case.

[0024] In some embodiments, the co-stimulatory molecule is selected from ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, SLAM, CD2, CD226, galectin 9, TIM1, LFA1, B7-H2, B7-1, B7-2, CD70, LIGHT, HVEM, CD40, 4-1BBL, OX40L, TL1A, GITRL, CD30L, SLAM, CD48, CD58, CD155, CD112, CD80, CD86, ICOSL, TIM3, TIM4, ICAM1, and LFA3. In some embodiments, the co-stimulatory molecule is CD40.

[0025] In some embodiments, the mRNA encoding a co-stimulatory molecule comprises a heterologous 5' untranslated region (5'UTR). In some embodiments, the mRNA encoding a co-stimulatory molecule comprises a heterologous 3' untranslated region (3'UTR). In some embodiments, the mRNA comprises chemically modified nucleobases. In some embodiments, the chemically modified nucleobase is pseudouridine. In some embodiments, the antigen-presenting cell is a bone marrow-derived dendritic cell.

[0026] In some aspects, described herein is a method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the antigen-presenting cell according to any of the preceding aspects and an antibody.

[0027] In some embodiments, the antibody is selected from an anti-CD40 antibody, an anti-PDL1 antibody, an anti-PD1 antibody, an anti-CTLA4 antibody, or a combination thereof. In some embodiments, the antigen-presenting cell and the antibody are administered intratumorally.

[0028] In some aspects, disclosed herein is a method of treating cancer, comprising administering the following to a subject in need thereof: a therapeutically effective amount of the lipid-based nanoparticle according to any of the preceding aspects, and a therapeutically effective amount of the antigen-presenting cell according to any of the preceding aspects.

[0029] In some embodiments, the nanoparticle comprises the following

[0030]

Chemical Formula

[0031]

Chemical Formula

[0032] In some embodiments, the lipid-based nanoparticles contain recombinant polynucleotides, including the nucleic acid encoding CD40L. The attached drawings are incorporated herein by reference and constitute part of this specification, illustrating several embodiments described below. [Brief explanation of the drawing]

[0033] [Figure 1A] This figure illustrates the termination of the cancer immune cycle (CIC) and the chemical synthesis of ionizable lipids derived from sugar alcohols, demonstrating the closure of the CIC through the integration of lipid nanoparticles and cell therapy (CATCH). [Figure 1B] This diagram illustrates the completion of the cancer immune cycle (CIC) and the chemical synthesis of ionizable lipids derived from sugar alcohols, showing a typical synthesis pathway for ionizable lipids derived from sugar alcohols. [Figure 1C] This figure illustrates the completion of the cancer immune cycle (CIC) and the chemical synthesis of ionizable lipids derived from sugar alcohols, showing the structures of ionizable lipids derived from sugar alcohols (DIS, DIM, LIS series). [Figure 2A]This document shows the screening and characterization of lipid nanoparticle (LNP)-mRNA formulations. A shows the mRNA delivery efficiency of DIS, DIM, and LIS LNPs in BMDC; B shows the effect of each lipid component at different molar ratios of DIM7 LNP on mRNA delivery; C shows the relative luminescence intensity of orthogonal and preferred formulations of DIM7 LNP; D shows the effect of each lipid component at different molar ratios of LIS10 LNP on mRNA delivery; E shows the relative luminescence intensity of orthogonal formulations of LIS10 LNP; F shows a table of preferred formulations; G shows the characterization of DIM7SLNP including size, PDI, encapsulation efficiency, and zeta potential; H shows a Cryo-TEM image of DIM7S (scale bar = 50 nm); I shows the characterization of LIS10W LNP including size, PDI, encapsulation efficiency, and zeta potential; and J shows a Cryo-TEM image of LIS10W (scale bar = 50 nm). A, B, C, D, and E are from three biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by two-tailed Student's t-tests: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 2B-1]This document shows the screening and characterization of lipid nanoparticle (LNP)-mRNA formulations. A shows the mRNA delivery efficiency of DIS, DIM, and LIS LNPs in BMDC; B shows the effect of each lipid component at different molar ratios of DIM7 LNP on mRNA delivery; C shows the relative luminescence intensity of orthogonal and preferred formulations of DIM7 LNP; D shows the effect of each lipid component at different molar ratios of LIS10 LNP on mRNA delivery; E shows the relative luminescence intensity of orthogonal formulations of LIS10 LNP; F shows a table of preferred formulations; G shows the characterization of DIM7SLNP including size, PDI, encapsulation efficiency, and zeta potential; H shows a Cryo-TEM image of DIM7S (scale bar = 50 nm); I shows the characterization of LIS10W LNP including size, PDI, encapsulation efficiency, and zeta potential; and J shows a Cryo-TEM image of LIS10W (scale bar = 50 nm). A, B, C, D, and E are from three biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by two-tailed Student's t-tests: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 2B-2]This document shows the screening and characterization of lipid nanoparticle (LNP)-mRNA formulations. A shows the mRNA delivery efficiency of DIS, DIM, and LIS LNPs in BMDC; B shows the effect of each lipid component at different molar ratios of DIM7 LNP on mRNA delivery; C shows the relative luminescence intensity of orthogonal and preferred formulations of DIM7 LNP; D shows the effect of each lipid component at different molar ratios of LIS10 LNP on mRNA delivery; E shows the relative luminescence intensity of orthogonal formulations of LIS10 LNP; F shows a table of preferred formulations; G shows the characterization of DIM7SLNP including size, PDI, encapsulation efficiency, and zeta potential; H shows a Cryo-TEM image of DIM7S (scale bar = 50 nm); I shows the characterization of LIS10W LNP including size, PDI, encapsulation efficiency, and zeta potential; and J shows a Cryo-TEM image of LIS10W (scale bar = 50 nm). A, B, C, D, and E are from three biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by two-tailed Student's t-tests: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 2C]This document shows the screening and characterization of lipid nanoparticle (LNP)-mRNA formulations. A shows the mRNA delivery efficiency of DIS, DIM, and LIS LNPs in BMDC; B shows the effect of each lipid component at different molar ratios of DIM7 LNP on mRNA delivery; C shows the relative luminescence intensity of orthogonal and preferred formulations of DIM7 LNP; D shows the effect of each lipid component at different molar ratios of LIS10 LNP on mRNA delivery; E shows the relative luminescence intensity of orthogonal formulations of LIS10 LNP; F shows a table of preferred formulations; G shows the characterization of DIM7SLNP including size, PDI, encapsulation efficiency, and zeta potential; H shows a Cryo-TEM image of DIM7S (scale bar = 50 nm); I shows the characterization of LIS10W LNP including size, PDI, encapsulation efficiency, and zeta potential; and J shows a Cryo-TEM image of LIS10W (scale bar = 50 nm). A, B, C, D, and E are from three biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by two-tailed Student's t-tests: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 2D-1]This document shows the screening and characterization of lipid nanoparticle (LNP)-mRNA formulations. A shows the mRNA delivery efficiency of DIS, DIM, and LIS LNPs in BMDC; B shows the effect of each lipid component at different molar ratios of DIM7 LNP on mRNA delivery; C shows the relative luminescence intensity of orthogonal and preferred formulations of DIM7 LNP; D shows the effect of each lipid component at different molar ratios of LIS10 LNP on mRNA delivery; E shows the relative luminescence intensity of orthogonal formulations of LIS10 LNP; F shows a table of preferred formulations; G shows the characterization of DIM7SLNP including size, PDI, encapsulation efficiency, and zeta potential; H shows a Cryo-TEM image of DIM7S (scale bar = 50 nm); I shows the characterization of LIS10W LNP including size, PDI, encapsulation efficiency, and zeta potential; and J shows a Cryo-TEM image of LIS10W (scale bar = 50 nm). A, B, C, D, and E are from three biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by two-tailed Student's t-tests: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 2D-2]This document shows the screening and characterization of lipid nanoparticle (LNP)-mRNA formulations. A shows the mRNA delivery efficiency of DIS, DIM, and LIS LNPs in BMDC; B shows the effect of each lipid component at different molar ratios of DIM7 LNP on mRNA delivery; C shows the relative luminescence intensity of orthogonal and preferred formulations of DIM7 LNP; D shows the effect of each lipid component at different molar ratios of LIS10 LNP on mRNA delivery; E shows the relative luminescence intensity of orthogonal formulations of LIS10 LNP; F shows a table of preferred formulations; G shows the characterization of DIM7SLNP including size, PDI, encapsulation efficiency, and zeta potential; H shows a Cryo-TEM image of DIM7S (scale bar = 50 nm); I shows the characterization of LIS10W LNP including size, PDI, encapsulation efficiency, and zeta potential; and J shows a Cryo-TEM image of LIS10W (scale bar = 50 nm). A, B, C, D, and E are from three biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by two-tailed Student's t-tests: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 2E]This document shows the screening and characterization of lipid nanoparticle (LNP)-mRNA formulations. A shows the mRNA delivery efficiency of DIS, DIM, and LIS LNPs in BMDC; B shows the effect of each lipid component at different molar ratios of DIM7 LNP on mRNA delivery; C shows the relative luminescence intensity of orthogonal and preferred formulations of DIM7 LNP; D shows the effect of each lipid component at different molar ratios of LIS10 LNP on mRNA delivery; E shows the relative luminescence intensity of orthogonal formulations of LIS10 LNP; F shows a table of preferred formulations; G shows the characterization of DIM7SLNP including size, PDI, encapsulation efficiency, and zeta potential; H shows a Cryo-TEM image of DIM7S (scale bar = 50 nm); I shows the characterization of LIS10W LNP including size, PDI, encapsulation efficiency, and zeta potential; and J shows a Cryo-TEM image of LIS10W (scale bar = 50 nm). A, B, C, D, and E are from three biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by two-tailed Student's t-tests: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 2F]This document shows the screening and characterization of lipid nanoparticle (LNP)-mRNA formulations. A shows the mRNA delivery efficiency of DIS, DIM, and LIS LNPs in BMDC; B shows the effect of each lipid component at different molar ratios of DIM7 LNP on mRNA delivery; C shows the relative luminescence intensity of orthogonal and preferred formulations of DIM7 LNP; D shows the effect of each lipid component at different molar ratios of LIS10 LNP on mRNA delivery; E shows the relative luminescence intensity of orthogonal formulations of LIS10 LNP; F shows a table of preferred formulations; G shows the characterization of DIM7SLNP including size, PDI, encapsulation efficiency, and zeta potential; H shows a Cryo-TEM image of DIM7S (scale bar = 50 nm); I shows the characterization of LIS10W LNP including size, PDI, encapsulation efficiency, and zeta potential; and J shows a Cryo-TEM image of LIS10W (scale bar = 50 nm). A, B, C, D, and E are from three biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by two-tailed Student's t-tests: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 2G]This document shows the screening and characterization of lipid nanoparticle (LNP)-mRNA formulations. A shows the mRNA delivery efficiency of DIS, DIM, and LIS LNPs in BMDC; B shows the effect of each lipid component at different molar ratios of DIM7 LNP on mRNA delivery; C shows the relative luminescence intensity of orthogonal and preferred formulations of DIM7 LNP; D shows the effect of each lipid component at different molar ratios of LIS10 LNP on mRNA delivery; E shows the relative luminescence intensity of orthogonal formulations of LIS10 LNP; F shows a table of preferred formulations; G shows the characterization of DIM7SLNP including size, PDI, encapsulation efficiency, and zeta potential; H shows a Cryo-TEM image of DIM7S (scale bar = 50 nm); I shows the characterization of LIS10W LNP including size, PDI, encapsulation efficiency, and zeta potential; and J shows a Cryo-TEM image of LIS10W (scale bar = 50 nm). A, B, C, D, and E are from three biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by two-tailed Student's t-tests: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 2H]This document shows the screening and characterization of lipid nanoparticle (LNP)-mRNA formulations. A shows the mRNA delivery efficiency of DIS, DIM, and LIS LNPs in BMDC; B shows the effect of each lipid component at different molar ratios of DIM7 LNP on mRNA delivery; C shows the relative luminescence intensity of orthogonal and preferred formulations of DIM7 LNP; D shows the effect of each lipid component at different molar ratios of LIS10 LNP on mRNA delivery; E shows the relative luminescence intensity of orthogonal formulations of LIS10 LNP; F shows a table of preferred formulations; G shows the characterization of DIM7SLNP including size, PDI, encapsulation efficiency, and zeta potential; H shows a Cryo-TEM image of DIM7S (scale bar = 50 nm); I shows the characterization of LIS10W LNP including size, PDI, encapsulation efficiency, and zeta potential; and J shows a Cryo-TEM image of LIS10W (scale bar = 50 nm). A, B, C, D, and E are from three biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by two-tailed Student's t-tests: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 2I]This document shows the screening and characterization of lipid nanoparticle (LNP)-mRNA formulations. A shows the mRNA delivery efficiency of DIS, DIM, and LIS LNPs in BMDC; B shows the effect of each lipid component at different molar ratios of DIM7 LNP on mRNA delivery; C shows the relative luminescence intensity of orthogonal and preferred formulations of DIM7 LNP; D shows the effect of each lipid component at different molar ratios of LIS10 LNP on mRNA delivery; E shows the relative luminescence intensity of orthogonal formulations of LIS10 LNP; F shows a table of preferred formulations; G shows the characterization of DIM7SLNP including size, PDI, encapsulation efficiency, and zeta potential; H shows a Cryo-TEM image of DIM7S (scale bar = 50 nm); I shows the characterization of LIS10W LNP including size, PDI, encapsulation efficiency, and zeta potential; and J shows a Cryo-TEM image of LIS10W (scale bar = 50 nm). A, B, C, D, and E are from three biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by two-tailed Student's t-tests: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 2J]This document shows the screening and characterization of lipid nanoparticle (LNP)-mRNA formulations. A shows the mRNA delivery efficiency of DIS, DIM, and LIS LNPs in BMDC; B shows the effect of each lipid component at different molar ratios of DIM7 LNP on mRNA delivery; C shows the relative luminescence intensity of orthogonal and preferred formulations of DIM7 LNP; D shows the effect of each lipid component at different molar ratios of LIS10 LNP on mRNA delivery; E shows the relative luminescence intensity of orthogonal formulations of LIS10 LNP; F shows a table of preferred formulations; G shows the characterization of DIM7SLNP including size, PDI, encapsulation efficiency, and zeta potential; H shows a Cryo-TEM image of DIM7S (scale bar = 50 nm); I shows the characterization of LIS10W LNP including size, PDI, encapsulation efficiency, and zeta potential; and J shows a Cryo-TEM image of LIS10W (scale bar = 50 nm). A, B, C, D, and E are from three biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by two-tailed Student's t-tests: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 3A-1]This study demonstrates dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and re-challenging tumors. A shows the expression of dendritic cell activation markers including CD80, CD86, MHC-II, IL1-β (proform), TNF-α, and IL12; B is a schematic diagram of the B16F10 tumor model and treatment plan; C and D show tumor volume (C) and mouse survival rate over time (D) (n=6 or 7); and E and F show in vitro (E) and in vitro (F) results including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. The image shows the LNP-inducible ICD markers in vivo (F), G is a schematic diagram of the B16F10 tumor model and treatment regimen, H shows the time course survival rate of mice (n=6), I shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 6), J is a schematic diagram of the B16F10 tumor model and treatment regimen, and K shows the time course survival rate of mice (n=6). The graphs show the survival rates (n=5 or 6) of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge, M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the time-course survival rates of mice (n=9), and O shows the survival rates (n=5 or 8) of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge. All data are from a biologically independent sample of n=3 and are expressed as mean ± SEM. Statistical significance of A, C, E, and F was analyzed by two-sided Student's t-test. Statistical significance of D, H, I, K, L, N, and O was analyzed by log-rank (Mantel-Cox) test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 3A-2]This study demonstrates dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and re-challenging tumors. A shows the expression of dendritic cell activation markers including CD80, CD86, MHC-II, IL1-β (proform), TNF-α, and IL12; B is a schematic diagram of the B16F10 tumor model and treatment plan; C and D show tumor volume (C) and mouse survival rate over time (D) (n=6 or 7); and E and F show in vitro (E) and in vitro (F) results including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. The image shows the LNP-inducible ICD markers in vivo (F), G is a schematic diagram of the B16F10 tumor model and treatment regimen, H shows the time course survival rate of mice (n=6), I shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 6), J is a schematic diagram of the B16F10 tumor model and treatment regimen, and K shows the time course survival rate of mice (n=6). The graphs show the survival rates (n=5 or 6) of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge, M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the time-course survival rates of mice (n=9), and O shows the survival rates (n=5 or 8) of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge. All data are from a biologically independent sample of n=3 and are expressed as mean ± SEM. Statistical significance of A, C, E, and F was analyzed by two-sided Student's t-test. Statistical significance of D, H, I, K, L, N, and O was analyzed by log-rank (Mantel-Cox) test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 3A-3]This study demonstrates dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and re-challenging tumors. A shows the expression of dendritic cell activation markers including CD80, CD86, MHC-II, IL1-β (proform), TNF-α, and IL12; B is a schematic diagram of the B16F10 tumor model and treatment plan; C and D show tumor volume (C) and mouse survival rate over time (D) (n=6 or 7); and E and F show in vitro (E) and in vitro (F) results including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. The image shows the LNP-inducible ICD markers in vivo (F), G is a schematic diagram of the B16F10 tumor model and treatment regimen, H shows the time course survival rate of mice (n=6), I shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 6), J is a schematic diagram of the B16F10 tumor model and treatment regimen, and K shows the time course survival rate of mice (n=6). The graphs show the survival rates (n=5 or 6) of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge, M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the time-course survival rates of mice (n=9), and O shows the survival rates (n=5 or 8) of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge. All data are from a biologically independent sample of n=3 and are expressed as mean ± SEM. Statistical significance of A, C, E, and F was analyzed by two-sided Student's t-test. Statistical significance of D, H, I, K, L, N, and O was analyzed by log-rank (Mantel-Cox) test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 3B]This study demonstrates dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and re-challenging tumors. A shows the expression of dendritic cell activation markers including CD80, CD86, MHC-II, IL1-β (proform), TNF-α, and IL12; B is a schematic diagram of the B16F10 tumor model and treatment plan; C and D show tumor volume (C) and mouse survival rate over time (D) (n=6 or 7); and E and F show in vitro (E) and in vitro (F) results including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. The image shows the LNP-inducible ICD markers in vivo (F), G is a schematic diagram of the B16F10 tumor model and treatment regimen, H shows the time course survival rate of mice (n=6), I shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 6), J is a schematic diagram of the B16F10 tumor model and treatment regimen, and K shows the time course survival rate of mice (n=6). The graphs show the survival rates (n=5 or 6) of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge, M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the time-course survival rates of mice (n=9), and O shows the survival rates (n=5 or 8) of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge. All data are from a biologically independent sample of n=3 and are expressed as mean ± SEM. Statistical significance of A, C, E, and F was analyzed by two-sided Student's t-test. Statistical significance of D, H, I, K, L, N, and O was analyzed by log-rank (Mantel-Cox) test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 3C]This study demonstrates dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and re-challenging tumors. A shows the expression of dendritic cell activation markers including CD80, CD86, MHC-II, IL1-β (proform), TNF-α, and IL12; B is a schematic diagram of the B16F10 tumor model and treatment plan; C and D show tumor volume (C) and mouse survival rate over time (D) (n=6 or 7); and E and F show in vitro (E) and in vitro (F) results including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. The image shows the LNP-inducible ICD markers in vivo (F), G is a schematic diagram of the B16F10 tumor model and treatment regimen, H shows the time course survival rate of mice (n=6), I shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 6), J is a schematic diagram of the B16F10 tumor model and treatment regimen, and K shows the time course survival rate of mice (n=6). The graphs show the survival rates (n=5 or 6) of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge, M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the time-course survival rates of mice (n=9), and O shows the survival rates (n=5 or 8) of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge. All data are from a biologically independent sample of n=3 and are expressed as mean ± SEM. Statistical significance of A, C, E, and F was analyzed by two-sided Student's t-test. Statistical significance of D, H, I, K, L, N, and O was analyzed by log-rank (Mantel-Cox) test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 3D]This study demonstrates dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and re-challenging tumors. A shows the expression of dendritic cell activation markers including CD80, CD86, MHC-II, IL1-β (proform), TNF-α, and IL12; B is a schematic diagram of the B16F10 tumor model and treatment plan; C and D show tumor volume (C) and mouse survival rate over time (D) (n=6 or 7); and E and F show in vitro (E) and in vitro (F) results including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. The image shows the LNP-inducible ICD markers in vivo (F), G is a schematic diagram of the B16F10 tumor model and treatment regimen, H shows the time course survival rate of mice (n=6), I shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 6), J is a schematic diagram of the B16F10 tumor model and treatment regimen, and K shows the time course survival rate of mice (n=6). The graphs show the survival rates (n=5 or 6) of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge, M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the time-course survival rates of mice (n=9), and O shows the survival rates (n=5 or 8) of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge. All data are from a biologically independent sample of n=3 and are expressed as mean ± SEM. Statistical significance of A, C, E, and F was analyzed by two-sided Student's t-test. Statistical significance of D, H, I, K, L, N, and O was analyzed by log-rank (Mantel-Cox) test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 3E]This study demonstrates dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and re-challenging tumors. A shows the expression of dendritic cell activation markers including CD80, CD86, MHC-II, IL1-β (proform), TNF-α, and IL12; B is a schematic diagram of the B16F10 tumor model and treatment plan; C and D show tumor volume (C) and mouse survival rate over time (D) (n=6 or 7); and E and F show in vitro (E) and in vitro (F) results including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. The image shows the LNP-inducible ICD markers in vivo (F), G is a schematic diagram of the B16F10 tumor model and treatment regimen, H shows the time course survival rate of mice (n=6), I shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 6), J is a schematic diagram of the B16F10 tumor model and treatment regimen, and K shows the time course survival rate of mice (n=6). The graphs show the survival rates (n=5 or 6) of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge, M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the time-course survival rates of mice (n=9), and O shows the survival rates (n=5 or 8) of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge. All data are from a biologically independent sample of n=3 and are expressed as mean ± SEM. Statistical significance of A, C, E, and F was analyzed by two-sided Student's t-test. Statistical significance of D, H, I, K, L, N, and O was analyzed by log-rank (Mantel-Cox) test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 3F]This study demonstrates dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and re-challenging tumors. A shows the expression of dendritic cell activation markers including CD80, CD86, MHC-II, IL1-β (proform), TNF-α, and IL12; B is a schematic diagram of the B16F10 tumor model and treatment plan; C and D show tumor volume (C) and mouse survival rate over time (D) (n=6 or 7); and E and F show in vitro (E) and in vitro (F) results including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. The image shows the LNP-inducible ICD markers in vivo (F), G is a schematic diagram of the B16F10 tumor model and treatment regimen, H shows the time course survival rate of mice (n=6), I shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 6), J is a schematic diagram of the B16F10 tumor model and treatment regimen, and K shows the time course survival rate of mice (n=6). The graphs show the survival rates (n=5 or 6) of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge, M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the time-course survival rates of mice (n=9), and O shows the survival rates (n=5 or 8) of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge. All data are from a biologically independent sample of n=3 and are expressed as mean ± SEM. Statistical significance of A, C, E, and F was analyzed by two-sided Student's t-test. Statistical significance of D, H, I, K, L, N, and O was analyzed by log-rank (Mantel-Cox) test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 3G]This study demonstrates dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and re-challenging tumors. A shows the expression of dendritic cell activation markers including CD80, CD86, MHC-II, IL1-β (proform), TNF-α, and IL12; B is a schematic diagram of the B16F10 tumor model and treatment plan; C and D show tumor volume (C) and mouse survival rate over time (D) (n=6 or 7); and E and F show in vitro (E) and in vitro (F) results including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. The image shows the LNP-inducible ICD markers in vivo (F), G is a schematic diagram of the B16F10 tumor model and treatment regimen, H shows the time course survival rate of mice (n=6), I shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 6), J is a schematic diagram of the B16F10 tumor model and treatment regimen, and K shows the time course survival rate of mice (n=6). The graphs show the survival rates (n=5 or 6) of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge, M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the time-course survival rates of mice (n=9), and O shows the survival rates (n=5 or 8) of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge. All data are from a biologically independent sample of n=3 and are expressed as mean ± SEM. Statistical significance of A, C, E, and F was analyzed by two-sided Student's t-test. Statistical significance of D, H, I, K, L, N, and O was analyzed by log-rank (Mantel-Cox) test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 3H]This study demonstrates dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and re-challenging tumors. A shows the expression of dendritic cell activation markers including CD80, CD86, MHC-II, IL1-β (proform), TNF-α, and IL12; B is a schematic diagram of the B16F10 tumor model and treatment plan; C and D show tumor volume (C) and mouse survival rate over time (D) (n=6 or 7); and E and F show in vitro (E) and in vitro (F) results including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. The image shows the LNP-inducible ICD markers in vivo (F), G is a schematic diagram of the B16F10 tumor model and treatment regimen, H shows the time course survival rate of mice (n=6), I shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 6), J is a schematic diagram of the B16F10 tumor model and treatment regimen, and K shows the time course survival rate of mice (n=6). The graphs show the survival rates (n=5 or 6) of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge, M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the time-course survival rates of mice (n=9), and O shows the survival rates (n=5 or 8) of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge. All data are from a biologically independent sample of n=3 and are expressed as mean ± SEM. Statistical significance of A, C, E, and F was analyzed by two-sided Student's t-test. Statistical significance of D, H, I, K, L, N, and O was analyzed by log-rank (Mantel-Cox) test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 3I]This study demonstrates dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and re-challenging tumors. A shows the expression of dendritic cell activation markers including CD80, CD86, MHC-II, IL1-β (proform), TNF-α, and IL12; B is a schematic diagram of the B16F10 tumor model and treatment plan; C and D show tumor volume (C) and mouse survival rate over time (D) (n=6 or 7); and E and F show in vitro (E) and in vitro (F) results including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. The image shows the LNP-inducible ICD markers in vivo (F), G is a schematic diagram of the B16F10 tumor model and treatment regimen, H shows the time course survival rate of mice (n=6), I shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 6), J is a schematic diagram of the B16F10 tumor model and treatment regimen, and K shows the time course survival rate of mice (n=6). The graphs show the survival rates (n=5 or 6) of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge, M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the time-course survival rates of mice (n=9), and O shows the survival rates (n=5 or 8) of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge. All data are from a biologically independent sample of n=3 and are expressed as mean ± SEM. Statistical significance of A, C, E, and F was analyzed by two-sided Student's t-test. Statistical significance of D, H, I, K, L, N, and O was analyzed by log-rank (Mantel-Cox) test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 3J]This study demonstrates dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and re-challenging tumors. A shows the expression of dendritic cell activation markers including CD80, CD86, MHC-II, IL1-β (proform), TNF-α, and IL12; B is a schematic diagram of the B16F10 tumor model and treatment plan; C and D show tumor volume (C) and mouse survival rate over time (D) (n=6 or 7); and E and F show in vitro (E) and in vitro (F) results including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. The image shows the LNP-inducible ICD markers in vivo (F), G is a schematic diagram of the B16F10 tumor model and treatment regimen, H shows the time course survival rate of mice (n=6), I shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 6), J is a schematic diagram of the B16F10 tumor model and treatment regimen, and K shows the time course survival rate of mice (n=6). The graphs show the survival rates (n=5 or 6) of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge, M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the time-course survival rates of mice (n=9), and O shows the survival rates (n=5 or 8) of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge. All data are from a biologically independent sample of n=3 and are expressed as mean ± SEM. Statistical significance of A, C, E, and F was analyzed by two-sided Student's t-test. Statistical significance of D, H, I, K, L, N, and O was analyzed by log-rank (Mantel-Cox) test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 3K]This study demonstrates dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and re-challenging tumors. A shows the expression of dendritic cell activation markers including CD80, CD86, MHC-II, IL1-β (proform), TNF-α, and IL12; B is a schematic diagram of the B16F10 tumor model and treatment plan; C and D show tumor volume (C) and mouse survival rate over time (D) (n=6 or 7); and E and F show in vitro (E) and in vitro (F) results including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. The image shows the LNP-inducible ICD markers in vivo (F), G is a schematic diagram of the B16F10 tumor model and treatment regimen, H shows the time course survival rate of mice (n=6), I shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 6), J is a schematic diagram of the B16F10 tumor model and treatment regimen, and K shows the time course survival rate of mice (n=6). The graphs show the survival rates (n=5 or 6) of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge, M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the time-course survival rates of mice (n=9), and O shows the survival rates (n=5 or 8) of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge. All data are from a biologically independent sample of n=3 and are expressed as mean ± SEM. Statistical significance of A, C, E, and F was analyzed by two-sided Student's t-test. Statistical significance of D, H, I, K, L, N, and O was analyzed by log-rank (Mantel-Cox) test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 3L]This study demonstrates dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and re-challenging tumors. A shows the expression of dendritic cell activation markers including CD80, CD86, MHC-II, IL1-β (proform), TNF-α, and IL12; B is a schematic diagram of the B16F10 tumor model and treatment plan; C and D show tumor volume (C) and mouse survival rate over time (D) (n=6 or 7); and E and F show in vitro (E) and in vitro (F) results including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. The image shows the LNP-inducible ICD markers in vivo (F), G is a schematic diagram of the B16F10 tumor model and treatment regimen, H shows the time course survival rate of mice (n=6), I shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 6), J is a schematic diagram of the B16F10 tumor model and treatment regimen, and K shows the time course survival rate of mice (n=6). The graphs show the survival rates (n=5 or 6) of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge, M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the time-course survival rates of mice (n=9), and O shows the survival rates (n=5 or 8) of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge. All data are from a biologically independent sample of n=3 and are expressed as mean ± SEM. Statistical significance of A, C, E, and F was analyzed by two-sided Student's t-test. Statistical significance of D, H, I, K, L, N, and O was analyzed by log-rank (Mantel-Cox) test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 3M]This study demonstrates dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and re-challenging tumors. A shows the expression of dendritic cell activation markers including CD80, CD86, MHC-II, IL1-β (proform), TNF-α, and IL12; B is a schematic diagram of the B16F10 tumor model and treatment plan; C and D show tumor volume (C) and mouse survival rate over time (D) (n=6 or 7); and E and F show in vitro (E) and in vitro (F) results including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. The image shows the LNP-inducible ICD markers in vivo (F), G is a schematic diagram of the B16F10 tumor model and treatment regimen, H shows the time course survival rate of mice (n=6), I shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 6), J is a schematic diagram of the B16F10 tumor model and treatment regimen, and K shows the time course survival rate of mice (n=6). The graphs show the survival rates (n=5 or 6) of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge, M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the time-course survival rates of mice (n=9), and O shows the survival rates (n=5 or 8) of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge. All data are from a biologically independent sample of n=3 and are expressed as mean ± SEM. Statistical significance of A, C, E, and F was analyzed by two-sided Student's t-test. Statistical significance of D, H, I, K, L, N, and O was analyzed by log-rank (Mantel-Cox) test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 3N]This study demonstrates dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and re-challenging tumors. A shows the expression of dendritic cell activation markers including CD80, CD86, MHC-II, IL1-β (proform), TNF-α, and IL12; B is a schematic diagram of the B16F10 tumor model and treatment plan; C and D show tumor volume (C) and mouse survival rate over time (D) (n=6 or 7); and E and F show in vitro (E) and in vitro (F) results including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. The image shows the LNP-inducible ICD markers in vivo (F), G is a schematic diagram of the B16F10 tumor model and treatment regimen, H shows the time course survival rate of mice (n=6), I shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 6), J is a schematic diagram of the B16F10 tumor model and treatment regimen, and K shows the time course survival rate of mice (n=6). The graphs show the survival rates (n=5 or 6) of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge, M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the time-course survival rates of mice (n=9), and O shows the survival rates (n=5 or 8) of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge. All data are from a biologically independent sample of n=3 and are expressed as mean ± SEM. Statistical significance of A, C, E, and F was analyzed by two-sided Student's t-test. Statistical significance of D, H, I, K, L, N, and O was analyzed by log-rank (Mantel-Cox) test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 3O]This study demonstrates dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and re-challenging tumors. A shows the expression of dendritic cell activation markers including CD80, CD86, MHC-II, IL1-β (proform), TNF-α, and IL12; B is a schematic diagram of the B16F10 tumor model and treatment plan; C and D show tumor volume (C) and mouse survival rate over time (D) (n=6 or 7); and E and F show in vitro (E) and in vitro (F) results including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. The image shows the LNP-inducible ICD markers in vivo (F), G is a schematic diagram of the B16F10 tumor model and treatment regimen, H shows the time course survival rate of mice (n=6), I shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 6), J is a schematic diagram of the B16F10 tumor model and treatment regimen, and K shows the time course survival rate of mice (n=6). The graphs show the survival rates (n=5 or 6) of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge, M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the time-course survival rates of mice (n=9), and O shows the survival rates (n=5 or 8) of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge. All data are from a biologically independent sample of n=3 and are expressed as mean ± SEM. Statistical significance of A, C, E, and F was analyzed by two-sided Student's t-test. Statistical significance of D, H, I, K, L, N, and O was analyzed by log-rank (Mantel-Cox) test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 4A]The therapeutic effects on two tumor or T-cell depletion models, and the dynamic expression of cytokines and chemokines in tumor tissue and blood are shown. A shows a schematic diagram of the B16F10 tumor model and treatment regimen, B and C show the distal tumor volume (B) and mouse survival rate (C) over time (n=8 or 10) of individual mice, D shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, E and F show the brain tumor volume (E) and mouse survival rate (F) over time (n=10), G shows a schematic diagram of the B16F10 tumor model and treatment regimen, H and I show the tumor volume (H) and mouse survival rate (I) over time (n=6), J shows a schematic diagram of the treatment regimen and sample collection, and K and L show the dynamic expression of cytokines and chemokines in tumor tissue (K) and blood (L) (n=5). The data for B, E, and H are expressed as mean ± standard error. The statistical significance of B, E, and H was analyzed by a two-tailed Student's t-test. The statistical significance of Figures 7B and 7D was analyzed by a log-rank (Mantel-Cox) test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 4B]The therapeutic effects on two tumor or T-cell depletion models, and the dynamic expression of cytokines and chemokines in tumor tissue and blood are shown. A shows a schematic diagram of the B16F10 tumor model and treatment regimen, B and C show the distal tumor volume (B) and mouse survival rate (C) over time (n=8 or 10) of individual mice, D shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, E and F show the brain tumor volume (E) and mouse survival rate (F) over time (n=10), G shows a schematic diagram of the B16F10 tumor model and treatment regimen, H and I show the tumor volume (H) and mouse survival rate (I) over time (n=6), J shows a schematic diagram of the treatment regimen and sample collection, and K and L show the dynamic expression of cytokines and chemokines in tumor tissue (K) and blood (L) (n=5). The data for B, E, and H are expressed as mean ± standard error. The statistical significance of B, E, and H was analyzed by a two-tailed Student's t-test. The statistical significance of Figures 7B and 7D was analyzed by a log-rank (Mantel-Cox) test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 4C]The therapeutic effects on two tumor or T-cell depletion models, and the dynamic expression of cytokines and chemokines in tumor tissue and blood are shown. A shows a schematic diagram of the B16F10 tumor model and treatment regimen, B and C show the distal tumor volume (B) and mouse survival rate (C) over time (n=8 or 10) of individual mice, D shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, E and F show the brain tumor volume (E) and mouse survival rate (F) over time (n=10), G shows a schematic diagram of the B16F10 tumor model and treatment regimen, H and I show the tumor volume (H) and mouse survival rate (I) over time (n=6), J shows a schematic diagram of the treatment regimen and sample collection, and K and L show the dynamic expression of cytokines and chemokines in tumor tissue (K) and blood (L) (n=5). The data for B, E, and H are expressed as mean ± standard error. The statistical significance of B, E, and H was analyzed by a two-tailed Student's t-test. The statistical significance of Figures 7B and 7D was analyzed by a log-rank (Mantel-Cox) test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 4D]The therapeutic effects on two tumor or T-cell depletion models, and the dynamic expression of cytokines and chemokines in tumor tissue and blood are shown. A shows a schematic diagram of the B16F10 tumor model and treatment regimen, B and C show the distal tumor volume (B) and mouse survival rate (C) over time (n=8 or 10) of individual mice, D shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, E and F show the brain tumor volume (E) and mouse survival rate (F) over time (n=10), G shows a schematic diagram of the B16F10 tumor model and treatment regimen, H and I show the tumor volume (H) and mouse survival rate (I) over time (n=6), J shows a schematic diagram of the treatment regimen and sample collection, and K and L show the dynamic expression of cytokines and chemokines in tumor tissue (K) and blood (L) (n=5). The data for B, E, and H are expressed as mean ± standard error. The statistical significance of B, E, and H was analyzed by a two-tailed Student's t-test. The statistical significance of Figures 7B and 7D was analyzed by a log-rank (Mantel-Cox) test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 4E]The therapeutic effects on two tumor or T-cell depletion models, and the dynamic expression of cytokines and chemokines in tumor tissue and blood are shown. A shows a schematic diagram of the B16F10 tumor model and treatment regimen, B and C show the distal tumor volume (B) and mouse survival rate (C) over time (n=8 or 10) of individual mice, D shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, E and F show the brain tumor volume (E) and mouse survival rate (F) over time (n=10), G shows a schematic diagram of the B16F10 tumor model and treatment regimen, H and I show the tumor volume (H) and mouse survival rate (I) over time (n=6), J shows a schematic diagram of the treatment regimen and sample collection, and K and L show the dynamic expression of cytokines and chemokines in tumor tissue (K) and blood (L) (n=5). The data for B, E, and H are expressed as mean ± standard error. The statistical significance of B, E, and H was analyzed by a two-tailed Student's t-test. The statistical significance of Figures 7B and 7D was analyzed by a log-rank (Mantel-Cox) test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 4F]The therapeutic effects on two tumor or T-cell depletion models, and the dynamic expression of cytokines and chemokines in tumor tissue and blood are shown. A shows a schematic diagram of the B16F10 tumor model and treatment regimen, B and C show the distal tumor volume (B) and mouse survival rate (C) over time (n=8 or 10) of individual mice, D shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, E and F show the brain tumor volume (E) and mouse survival rate (F) over time (n=10), G shows a schematic diagram of the B16F10 tumor model and treatment regimen, H and I show the tumor volume (H) and mouse survival rate (I) over time (n=6), J shows a schematic diagram of the treatment regimen and sample collection, and K and L show the dynamic expression of cytokines and chemokines in tumor tissue (K) and blood (L) (n=5). The data for B, E, and H are expressed as mean ± standard error. The statistical significance of B, E, and H was analyzed by a two-tailed Student's t-test. The statistical significance of Figures 7B and 7D was analyzed by a log-rank (Mantel-Cox) test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 4G]The therapeutic effects on two tumor or T-cell depletion models, and the dynamic expression of cytokines and chemokines in tumor tissue and blood are shown. A shows a schematic diagram of the B16F10 tumor model and treatment regimen, B and C show the distal tumor volume (B) and mouse survival rate (C) over time (n=8 or 10) of individual mice, D shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, E and F show the brain tumor volume (E) and mouse survival rate (F) over time (n=10), G shows a schematic diagram of the B16F10 tumor model and treatment regimen, H and I show the tumor volume (H) and mouse survival rate (I) over time (n=6), J shows a schematic diagram of the treatment regimen and sample collection, and K and L show the dynamic expression of cytokines and chemokines in tumor tissue (K) and blood (L) (n=5). The data for B, E, and H are expressed as mean ± standard error. The statistical significance of B, E, and H was analyzed by a two-tailed Student's t-test. The statistical significance of Figures 7B and 7D was analyzed by a log-rank (Mantel-Cox) test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 4H]The therapeutic effects on two tumor or T-cell depletion models, and the dynamic expression of cytokines and chemokines in tumor tissue and blood are shown. A shows a schematic diagram of the B16F10 tumor model and treatment regimen, B and C show the distal tumor volume (B) and mouse survival rate (C) over time (n=8 or 10) of individual mice, D shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, E and F show the brain tumor volume (E) and mouse survival rate (F) over time (n=10), G shows a schematic diagram of the B16F10 tumor model and treatment regimen, H and I show the tumor volume (H) and mouse survival rate (I) over time (n=6), J shows a schematic diagram of the treatment regimen and sample collection, and K and L show the dynamic expression of cytokines and chemokines in tumor tissue (K) and blood (L) (n=5). The data for B, E, and H are expressed as mean ± standard error. The statistical significance of B, E, and H was analyzed by a two-tailed Student's t-test. The statistical significance of Figures 7B and 7D was analyzed by a log-rank (Mantel-Cox) test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 4I]The therapeutic effects on two tumor or T-cell depletion models, and the dynamic expression of cytokines and chemokines in tumor tissue and blood are shown. A shows a schematic diagram of the B16F10 tumor model and treatment regimen, B and C show the distal tumor volume (B) and mouse survival rate (C) over time (n=8 or 10) of individual mice, D shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, E and F show the brain tumor volume (E) and mouse survival rate (F) over time (n=10), G shows a schematic diagram of the B16F10 tumor model and treatment regimen, H and I show the tumor volume (H) and mouse survival rate (I) over time (n=6), J shows a schematic diagram of the treatment regimen and sample collection, and K and L show the dynamic expression of cytokines and chemokines in tumor tissue (K) and blood (L) (n=5). The data for B, E, and H are expressed as mean ± standard error. The statistical significance of B, E, and H was analyzed by a two-tailed Student's t-test. The statistical significance of Figures 7B and 7D was analyzed by a log-rank (Mantel-Cox) test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 4J]The therapeutic effects on two tumor or T-cell depletion models, and the dynamic expression of cytokines and chemokines in tumor tissue and blood are shown. A shows a schematic diagram of the B16F10 tumor model and treatment regimen, B and C show the distal tumor volume (B) and mouse survival rate (C) over time (n=8 or 10) of individual mice, D shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, E and F show the brain tumor volume (E) and mouse survival rate (F) over time (n=10), G shows a schematic diagram of the B16F10 tumor model and treatment regimen, H and I show the tumor volume (H) and mouse survival rate (I) over time (n=6), J shows a schematic diagram of the treatment regimen and sample collection, and K and L show the dynamic expression of cytokines and chemokines in tumor tissue (K) and blood (L) (n=5). The data for B, E, and H are expressed as mean ± standard error. The statistical significance of B, E, and H was analyzed by a two-tailed Student's t-test. The statistical significance of Figures 7B and 7D was analyzed by a log-rank (Mantel-Cox) test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 4K]The therapeutic effects on two tumor or T-cell depletion models, and the dynamic expression of cytokines and chemokines in tumor tissue and blood are shown. A shows a schematic diagram of the B16F10 tumor model and treatment regimen, B and C show the distal tumor volume (B) and mouse survival rate (C) over time (n=8 or 10) of individual mice, D shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, E and F show the brain tumor volume (E) and mouse survival rate (F) over time (n=10), G shows a schematic diagram of the B16F10 tumor model and treatment regimen, H and I show the tumor volume (H) and mouse survival rate (I) over time (n=6), J shows a schematic diagram of the treatment regimen and sample collection, and K and L show the dynamic expression of cytokines and chemokines in tumor tissue (K) and blood (L) (n=5). The data for B, E, and H are expressed as mean ± standard error. The statistical significance of B, E, and H was analyzed by a two-tailed Student's t-test. The statistical significance of Figures 7B and 7D was analyzed by a log-rank (Mantel-Cox) test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 4L]The therapeutic effects on two tumor or T-cell depletion models, and the dynamic expression of cytokines and chemokines in tumor tissue and blood are shown. A shows a schematic diagram of the B16F10 tumor model and treatment regimen, B and C show the distal tumor volume (B) and mouse survival rate (C) over time (n=8 or 10) of individual mice, D shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, E and F show the brain tumor volume (E) and mouse survival rate (F) over time (n=10), G shows a schematic diagram of the B16F10 tumor model and treatment regimen, H and I show the tumor volume (H) and mouse survival rate (I) over time (n=6), J shows a schematic diagram of the treatment regimen and sample collection, and K and L show the dynamic expression of cytokines and chemokines in tumor tissue (K) and blood (L) (n=5). The data for B, E, and H are expressed as mean ± standard error. The statistical significance of B, E, and H was analyzed by a two-tailed Student's t-test. The statistical significance of Figures 7B and 7D was analyzed by a log-rank (Mantel-Cox) test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 5A] The graph shows the infiltration of immune cells in tumor tissue. A represents the immune cell population within the tumor tissue (n=5), B represents the percentage of activated macrophages and dendritic cells within the tumor tissue (n=5), C represents the percentage of primed CD8 T cells within the tumor tissue (n=5), and D and E represent the percentages of effector memory T cells and central memory T cells in the spleen (D) and blood (E) (n=5). [Figure 5B] The graph shows the infiltration of immune cells in tumor tissue. A represents the immune cell population within the tumor tissue (n=5), B represents the percentage of activated macrophages and dendritic cells within the tumor tissue (n=5), C represents the percentage of primed CD8 T cells within the tumor tissue (n=5), and D and E represent the percentages of effector memory T cells and central memory T cells in the spleen (D) and blood (E) (n=5). [Figure 5C]The graph shows the infiltration of immune cells in tumor tissue. A represents the immune cell population within the tumor tissue (n=5), B represents the percentage of activated macrophages and dendritic cells within the tumor tissue (n=5), C represents the percentage of primed CD8 T cells within the tumor tissue (n=5), and D and E represent the percentages of effector memory T cells and central memory T cells in the spleen (D) and blood (E) (n=5). [Figure 5D] The graph shows the infiltration of immune cells in tumor tissue. A represents the immune cell population within the tumor tissue (n=5), B represents the percentage of activated macrophages and dendritic cells within the tumor tissue (n=5), C represents the percentage of primed CD8 T cells within the tumor tissue (n=5), and D and E represent the percentages of effector memory T cells and central memory T cells in the spleen (D) and blood (E) (n=5). [Figure 5E] The graph shows the infiltration of immune cells in tumor tissue. A represents the immune cell population within the tumor tissue (n=5), B represents the percentage of activated macrophages and dendritic cells within the tumor tissue (n=5), C represents the percentage of primed CD8 T cells within the tumor tissue (n=5), and D and E represent the percentages of effector memory T cells and central memory T cells in the spleen (D) and blood (E) (n=5). [Figure 6A] This shows the characterization of lipid nanoparticle (LNP)-mRNA formulations. A represents nanoparticle size and polydispersity index (PDI), and B represents capture efficiency and zeta potential. All data are expressed as mean ± standard deviation. [Figure 6B] This shows the characterization of lipid nanoparticle (LNP)-mRNA formulations. A represents nanoparticle size and polydispersity index (PDI), and B represents capture efficiency and zeta potential. All data are expressed as mean ± standard deviation. [Figure 7A]This document describes the characterization of lipid nanoparticle (LNP)-mRNA formulations. A shows the L16(4)4 orthogonal array, B shows the Cryo-TEM image of DIM7S (scale bar = 50 nm), C shows the Cryo-TEM image of LIS10W (scale bar = 50 nm), D shows the relative luminescence intensity of electroporation (Electro), DIM7, and DIM7S against lipofectamine 3000 (Lipo3K), E shows the expression dynamics of mRNA delivered by DIM7S, and F shows the expression of CD40 in BMDC. All data are from three biologically independent samples and are expressed as mean ± SEM. Statistical significance was analyzed by two-sided Student's t-test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 7B] This document describes the characterization of lipid nanoparticle (LNP)-mRNA formulations. A shows the L16(4)4 orthogonal array, B shows the Cryo-TEM image of DIM7S (scale bar = 50 nm), C shows the Cryo-TEM image of LIS10W (scale bar = 50 nm), D shows the relative luminescence intensity of electroporation (Electro), DIM7, and DIM7S against lipofectamine 3000 (Lipo3K), E shows the expression dynamics of mRNA delivered by DIM7S, and F shows the expression of CD40 in BMDC. All data are from three biologically independent samples and are expressed as mean ± SEM. Statistical significance was analyzed by two-sided Student's t-test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 7C]This document describes the characterization of lipid nanoparticle (LNP)-mRNA formulations. A shows the L16(4)4 orthogonal array, B shows the Cryo-TEM image of DIM7S (scale bar = 50 nm), C shows the Cryo-TEM image of LIS10W (scale bar = 50 nm), D shows the relative luminescence intensity of electroporation (Electro), DIM7, and DIM7S against lipofectamine 3000 (Lipo3K), E shows the expression dynamics of mRNA delivered by DIM7S, and F shows the expression of CD40 in BMDC. All data are from three biologically independent samples and are expressed as mean ± SEM. Statistical significance was analyzed by two-sided Student's t-test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 7D] This document describes the characterization of lipid nanoparticle (LNP)-mRNA formulations. A shows the L16(4)4 orthogonal array, B shows the Cryo-TEM image of DIM7S (scale bar = 50 nm), C shows the Cryo-TEM image of LIS10W (scale bar = 50 nm), D shows the relative luminescence intensity of electroporation (Electro), DIM7, and DIM7S against lipofectamine 3000 (Lipo3K), E shows the expression dynamics of mRNA delivered by DIM7S, and F shows the expression of CD40 in BMDC. All data are from three biologically independent samples and are expressed as mean ± SEM. Statistical significance was analyzed by two-sided Student's t-test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 7E]This document describes the characterization of lipid nanoparticle (LNP)-mRNA formulations. A shows the L16(4)4 orthogonal array, B shows the Cryo-TEM image of DIM7S (scale bar = 50 nm), C shows the Cryo-TEM image of LIS10W (scale bar = 50 nm), D shows the relative luminescence intensity of electroporation (Electro), DIM7, and DIM7S against lipofectamine 3000 (Lipo3K), E shows the expression dynamics of mRNA delivered by DIM7S, and F shows the expression of CD40 in BMDC. All data are from three biologically independent samples and are expressed as mean ± SEM. Statistical significance was analyzed by two-sided Student's t-test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 7F] This document describes the characterization of lipid nanoparticle (LNP)-mRNA formulations. A shows the L16(4)4 orthogonal array, B shows the Cryo-TEM image of DIM7S (scale bar = 50 nm), C shows the Cryo-TEM image of LIS10W (scale bar = 50 nm), D shows the relative luminescence intensity of electroporation (Electro), DIM7, and DIM7S against lipofectamine 3000 (Lipo3K), E shows the expression dynamics of mRNA delivered by DIM7S, and F shows the expression of CD40 in BMDC. All data are from three biologically independent samples and are expressed as mean ± SEM. Statistical significance was analyzed by two-sided Student's t-test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 8A] The tumor volume of individual mice (n=6 or 7) is shown. [Figure 8B] The tumor volume of individual mice (n=6 or 7) is shown. [Figure 8C] The tumor volume of individual mice (n=6 or 7) is shown. [Figure 8D] The tumor volume of individual mice (n=6 or 7) is shown. [Figure 8E] The tumor volume of individual mice (n=6 or 7) is shown. [Figure 8F] The tumor volume of individual mice (n=6 or 7) is shown. [Figure 8G] The tumor volume of individual mice (n=6 or 7) is shown. [Figure 9A] The following shows cytotoxicity and CD40 and CD40L expression induced by lipid nanoparticles (LNPs). A shows CD40 expression mediated by CD40-DIM7S in in vivo dendritic cells (n=5), B shows in vitro cytotoxicity induced by CD40L-DIM7S and CD40L-LIS10W, C shows in vitro CD40L expression in B16F10 melanoma cells mediated by CD40L-DIM7S and CD40L-LIS10W, and D and E show in vivo CD40L expression mediated by CD40L-LIS10W in tumor cells (D) and immune cells including macrophages, dendritic cells, CD8 T cells, and CD4 T cells (E) (n=3 or 5). Data for B and C are from n=3 biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by a two-tailed Student's t-test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 9B]The following shows cytotoxicity and CD40 and CD40L expression induced by lipid nanoparticles (LNPs). A shows CD40 expression mediated by CD40-DIM7S in in vivo dendritic cells (n=5), B shows in vitro cytotoxicity induced by CD40L-DIM7S and CD40L-LIS10W, C shows in vitro CD40L expression in B16F10 melanoma cells mediated by CD40L-DIM7S and CD40L-LIS10W, and D and E show in vivo CD40L expression mediated by CD40L-LIS10W in tumor cells (D) and immune cells including macrophages, dendritic cells, CD8 T cells, and CD4 T cells (E) (n=3 or 5). Data for B and C are from n=3 biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by a two-tailed Student's t-test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 9C] The following shows cytotoxicity and CD40 and CD40L expression induced by lipid nanoparticles (LNPs). A shows CD40 expression mediated by CD40-DIM7S in in vivo dendritic cells (n=5), B shows in vitro cytotoxicity induced by CD40L-DIM7S and CD40L-LIS10W, C shows in vitro CD40L expression in B16F10 melanoma cells mediated by CD40L-DIM7S and CD40L-LIS10W, and D and E show in vivo CD40L expression mediated by CD40L-LIS10W in tumor cells (D) and immune cells including macrophages, dendritic cells, CD8 T cells, and CD4 T cells (E) (n=3 or 5). Data for B and C are from n=3 biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by a two-tailed Student's t-test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 9D]The following shows cytotoxicity and CD40 and CD40L expression induced by lipid nanoparticles (LNPs). A shows CD40 expression mediated by CD40-DIM7S in in vivo dendritic cells (n=5), B shows in vitro cytotoxicity induced by CD40L-DIM7S and CD40L-LIS10W, C shows in vitro CD40L expression in B16F10 melanoma cells mediated by CD40L-DIM7S and CD40L-LIS10W, and D and E show in vivo CD40L expression mediated by CD40L-LIS10W in tumor cells (D) and immune cells including macrophages, dendritic cells, CD8 T cells, and CD4 T cells (E) (n=3 or 5). Data for B and C are from n=3 biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by a two-tailed Student's t-test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 9E] The following shows cytotoxicity and CD40 and CD40L expression induced by lipid nanoparticles (LNPs). A shows CD40 expression mediated by CD40-DIM7S in in vivo dendritic cells (n=5), B shows in vitro cytotoxicity induced by CD40L-DIM7S and CD40L-LIS10W, C shows in vitro CD40L expression in B16F10 melanoma cells mediated by CD40L-DIM7S and CD40L-LIS10W, and D and E show in vivo CD40L expression mediated by CD40L-LIS10W in tumor cells (D) and immune cells including macrophages, dendritic cells, CD8 T cells, and CD4 T cells (E) (n=3 or 5). Data for B and C are from n=3 biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by a two-tailed Student's t-test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 10A]The table shows the tumor volume of individual mice. A shows the primary tumor volume of individual mice (n=6), B shows the rechallenged subcutaneous tumor volume of individual mice (n=5 or 6), C and D show the tumor volume over time (C) and the primary tumor volume of individual mice (D) (n=6), E shows the rechallenged subcutaneous tumor volume of individual mice (n=5 or 6), F shows the primary tumor volume of individual mice (n=9), and G shows the brain tumor volume over time (n=5 or 8). Data for C and G are expressed as mean ± sem. Statistical significance was analyzed by two-sided Student's t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 10B] The table shows the tumor volume of individual mice. A shows the primary tumor volume of individual mice (n=6), B shows the rechallenged subcutaneous tumor volume of individual mice (n=5 or 6), C and D show the tumor volume over time (C) and the primary tumor volume of individual mice (D) (n=6), E shows the rechallenged subcutaneous tumor volume of individual mice (n=5 or 6), F shows the primary tumor volume of individual mice (n=9), and G shows the brain tumor volume over time (n=5 or 8). Data for C and G are expressed as mean ± sem. Statistical significance was analyzed by two-sided Student's t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 10C] The table shows the tumor volume of individual mice. A shows the primary tumor volume of individual mice (n=6), B shows the rechallenged subcutaneous tumor volume of individual mice (n=5 or 6), C and D show the tumor volume over time (C) and the primary tumor volume of individual mice (D) (n=6), E shows the rechallenged subcutaneous tumor volume of individual mice (n=5 or 6), F shows the primary tumor volume of individual mice (n=9), and G shows the brain tumor volume over time (n=5 or 8). Data for C and G are expressed as mean ± sem. Statistical significance was analyzed by two-sided Student's t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 10D]The table shows the tumor volume of individual mice. A shows the primary tumor volume of individual mice (n=6), B shows the rechallenged subcutaneous tumor volume of individual mice (n=5 or 6), C and D show the tumor volume over time (C) and the primary tumor volume of individual mice (D) (n=6), E shows the rechallenged subcutaneous tumor volume of individual mice (n=5 or 6), F shows the primary tumor volume of individual mice (n=9), and G shows the brain tumor volume over time (n=5 or 8). Data for C and G are expressed as mean ± sem. Statistical significance was analyzed by two-sided Student's t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 10E] The table shows the tumor volume of individual mice. A shows the primary tumor volume of individual mice (n=6), B shows the rechallenged subcutaneous tumor volume of individual mice (n=5 or 6), C and D show the tumor volume over time (C) and the primary tumor volume of individual mice (D) (n=6), E shows the rechallenged subcutaneous tumor volume of individual mice (n=5 or 6), F shows the primary tumor volume of individual mice (n=9), and G shows the brain tumor volume over time (n=5 or 8). Data for C and G are expressed as mean ± sem. Statistical significance was analyzed by two-sided Student's t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 10F] The table shows the tumor volume of individual mice. A shows the primary tumor volume of individual mice (n=6), B shows the rechallenged subcutaneous tumor volume of individual mice (n=5 or 6), C and D show the tumor volume over time (C) and the primary tumor volume of individual mice (D) (n=6), E shows the rechallenged subcutaneous tumor volume of individual mice (n=5 or 6), F shows the primary tumor volume of individual mice (n=9), and G shows the brain tumor volume over time (n=5 or 8). Data for C and G are expressed as mean ± sem. Statistical significance was analyzed by two-sided Student's t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 10G]The table shows the tumor volume of individual mice. A shows the primary tumor volume of individual mice (n=6), B shows the rechallenged subcutaneous tumor volume of individual mice (n=5 or 6), C and D show the tumor volume over time (C) and the primary tumor volume of individual mice (D) (n=6), E shows the rechallenged subcutaneous tumor volume of individual mice (n=5 or 6), F shows the primary tumor volume of individual mice (n=9), and G shows the brain tumor volume over time (n=5 or 8). Data for C and G are expressed as mean ± sem. Statistical significance was analyzed by two-sided Student's t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 11A] The tumor volume of each mouse is shown, and the tumor volume on the treated side of each mouse (n=8 or 10) is also shown. [Figure 11B] The tumor volume of each mouse is shown, and the tumor volume on the treated side of each mouse (n=10) is also shown. [Figure 11C] The tumor volume of each individual mouse is shown, and the tumor volume of each individual mouse (n=6) is shown. [Figure 12-1] This shows the dynamic expression of cytokines and chemokines in mouse melanoma tissue (n=5). [Figure 12-2] This shows the dynamic expression of cytokines and chemokines in mouse melanoma tissue (n=5). [Figure 12-3] This shows the dynamic expression of cytokines and chemokines in mouse melanoma tissue (n=5). [Figure 12-4] This shows the dynamic expression of cytokines and chemokines in mouse melanoma tissue (n=5). [Figure 12-5] This shows the dynamic expression of cytokines and chemokines in mouse melanoma tissue (n=5). [Figure 12-6] This shows the dynamic expression of cytokines and chemokines in mouse melanoma tissue (n=5). [Figure 12-7] This shows the dynamic expression of cytokines and chemokines in mouse melanoma tissue (n=5). [Figure 12-8]This shows the dynamic expression of cytokines and chemokines in mouse melanoma tissue (n=5). [Figure 13-1] This shows the dynamic expression of cytokines and chemokines in mouse blood (n=5). [Figure 13-2] This shows the dynamic expression of cytokines and chemokines in mouse blood (n=5). [Figure 13-3] This shows the dynamic expression of cytokines and chemokines in mouse blood (n=5). [Figure 13-4] This shows the dynamic expression of cytokines and chemokines in mouse blood (n=5). [Figure 13-5] This shows the dynamic expression of cytokines and chemokines in mouse blood (n=5). [Figure 13-6] This shows the dynamic expression of cytokines and chemokines in mouse blood (n=5). [Figure 13-7] This shows the dynamic expression of cytokines and chemokines in mouse blood (n=5). [Figure 13-8] This shows the dynamic expression of cytokines and chemokines in mouse blood (n=5). [Modes for carrying out the invention]

[0034] Disclosed herein are compositions and methods for modulating the immune system for treating cancer and other immune disorders. Embodiments of the present invention will be described in detail, examples of which are illustrated in the drawings and embodiments. However, the present invention may be embodied in many different forms and should not be construed as being limited to the embodiments described herein.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. The terms “comprising” and its variations used herein are synonymous with the terms “including” and its variations, and are open, non-restrictive terms. While the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “essentially consisting” and “consisting” may be used in place of “comprising” and “including” to provide more specific embodiments, and are disclosed herein. As used in this disclosure and the appended claims, the singular “a,” “an,” and “the” include plural referents unless the context clearly indicates otherwise.

[0036] The following definitions are provided to ensure a complete understanding of the terms used herein.

[0037] term As used herein, the terms “may,” “optionally,” and “may optionally” are used synonymously and include both cases in which this condition occurs and cases in which this condition does not occur. Therefore, for example, the statement that a formulation “may contain excipients” includes both cases in which the formulation contains excipients and cases in which the formulation does not contain excipients.

[0038] The terms “promoter” or “regulatory element” refer to a region or sequencer located upstream or downstream of transcription initiation that is involved in the recognition and binding of RNA polymerase and other proteins to initiate transcription. Promoters do not need to be of bacterial origin; for example, promoters derived from viruses or other organisms may be used in the compositions, systems, or methods described herein. The term “regulatory element” is intended to include promoters, enhancers, intra-sequence ribosome entry sites (IRESs), and other expression regulatory elements (e.g., transcription termination signals such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. 1990). Regulatory elements include those that direct the constitutive expression of nucleotide sequences in many types of host cells, and those that direct the expression of nucleotide sequences only in certain host cells (e.g., tissue-specific regulatory elements). Tissue-specific promoters can primarily direct expression in desired target tissues such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas), or specific cell types (e.g., lymphocytes). Regulatory elements can also direct expression in a time-dependent manner, such as in a cell cycle-dependent or developmental stage-dependent manner, which may or may not be tissue- or cell-type specific. In some embodiments, the vector includes one or more pol III promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or a combination thereof. Examples of pol III promoters include, but are not limited to, the U6 and H1 promoters.Examples of pol II promoters include, but are not limited to, the retroviral rust sarcoma virus (RSV) LTR promoter (optionally including an RSV enhancer), the cytomegalovirus (CMV) promoter (optionally including a CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter. The term “regulatory element” also includes enhancer elements such as WPRE, the CMV enhancer; the R-U5' segment within the LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); the SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981). It will be understood by those skilled in the art that the design of expression vectors may depend on factors such as the selection of host cells to be transformed and the desired expression level.

[0039] The term “recombinant” refers to an artificially engineered nucleic acid (e.g., polynucleotide) or a replica or complement of an artificially engineered nucleic acid (e.g., polynucleotide), or, in the case of a protein (i.e., a “recombinant protein”), to a protein (e.g., polynucleotide) encoded by a recombinant nucleic acid. In embodiments, a recombinant expression cassette comprising a promoter operably linked to a second nucleic acid (e.g., polynucleotide) may include a promoter that is heterologous to the second nucleic acid (e.g., polynucleotide) as a result of human engineering (e.g., by the methods described in Sambrook et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)). In another example, a recombinant expression cassette may include a nucleic acid (e.g., polynucleotide) that is combined in such a way that the nucleic acid (e.g., polynucleotide) is very unlikely to be found in nature. For example, the restriction enzyme site or plasmid vector sequence manipulated by a human may be positioned adjacent to or separated from the promoter of a second nucleic acid (e.g., a polynucleotide). Those skilled in the art will recognize that nucleic acids (e.g., polynucleotides) can be manipulated in many ways, and are not limited to the examples given above.

[0040] The terms “expression cassette” or “vector” refer to a nucleic acid construct that, when introduced into a host cell, results in the transcription and / or translation of RNA or polypeptide, respectively. In embodiments, an expression cassette comprising a promoter operably ligated to a second nucleic acid (e.g., polynucleotide) may include a promoter that is heterogeneous to the second nucleic acid (e.g., polynucleotide) as a result of human manipulation (e.g., by the methods described in Sambrook et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)). In some embodiments, an expression cassette comprising a terminator (or termination sequence) operably ligated to a second nucleic acid (e.g., polynucleotide) may include a terminator that is heterogeneous to the second nucleic acid (e.g., polynucleotide) as a result of human manipulation. In some embodiments, the expression cassette includes a promoter operably ligated to a second nucleic acid (e.g., a polynucleotide) and a terminator operably ligated to the second nucleic acid (e.g., a polynucleotide) as a result of human manipulation. In some embodiments, the expression cassette includes an endogenous promoter. In some embodiments, the expression cassette includes an endogenous terminator. In some embodiments, the expression cassette includes a synthetic (or unnatural) promoter. In some embodiments, the expression cassette includes a synthetic (or unnatural) terminator.

[0041] The terms "identity" or "percent "identity" for two or more nucleic acid or polypeptide sequences are measured using the BLAST or BLAST2.0 sequence comparison algorithm with the default parameters listed below, or by manual alignment and visual inspection (e.g., NCBI). Refers to two or more sequences or subsequences having identical amino acid residues or nucleotides (i.e., when compared and aligned to obtain the maximum correspondence in a comparison window or specified region, in a specified region, approximately 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity). Such sequences are said to be "substantially identical". This definition refers to or may apply to the complement of the test sequence. This definition also includes sequences with deletions and / or additions, as well as sequences with substitutions. Preferred algorithms, as described below, can account for gaps, etc. Preferably, identity resides in a region of at least about 10 amino acids or 20 nucleotides in length, or more preferably, in a region of 10 to 50 amino acids or 20 to 50 nucleotides in length. As used herein, amino acid sequence identity percentage (%) is defined as the percentage of amino acids in a candidate sequence that are identical to amino acids in a reference sequence after aligning the sequences to obtain the maximum sequence identity percentage and introducing gaps as necessary. Alignment for determining sequence identity percentage can be achieved in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software.Appropriate parameters for measuring alignment, including any algorithm required to achieve the maximum alignment across the entire length of the sequences being compared, can be determined by known methods.

[0042] For sequence comparison, typically one sequence is used as a reference sequence, and the sequence under comparison is performed against it. When using a sequence comparison algorithm, the sequence under comparison and the reference sequence are input into a computer, subsequence coordinates are specified as needed, and sequence algorithm program parameters are specified. Preferably, default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the sequence identity percentage of the sequence under comparison relative to the reference sequence based on the program parameters.

[0043] Preferred examples of algorithms suitable for determining sequence identity percentage and sequence similarity are the BLAST and BLAST 2.0 algorithms (described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402 and Altschul et al., (1990) J. Mol. Biol. 215:403-410, respectively). Software for performing BLAST analysis is generally available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm first identifies high-scoring sequence pairs (HSPs) by identifying shorter words W in the query sequence that match or satisfy some positive threshold score T when aligned with words of the same length in the database sequence. T is referred to as the neighbor word score threshold (Altschul et al. (1990) J.Mol.Biol.215:403-410). These first neighbor word hits serve as seed values ​​to initiate the search for longer HSPs containing them. Word hits are extended in both directions along each sequence as long as the cumulative alignment score can increase. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for matching residue pairs, always greater than 0) and N (penalty score for mismatched residues, always less than 0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. The extension of word hits in each direction stops when the cumulative alignment score decreases by a factor X from its maximum actual value, when the cumulative score becomes zero or less due to the accumulation of one or more negative-scoring residue alignments, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a default word length (W), expected value (E), or 10, M=5, N=-4, and comparison of both strands.For amino acid sequences, the BLASTP program defaults to using a word length of 3 and an expected value of 10 (E), as well as a BLOSUM62 scoring matrix (Henikoff and Henikoff (1989), Proc Natl Acad Sci USA, 89:10915) alignment (B), an expected value of 10 (E), M=5, N=-4, and comparison of both strands.

[0044] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul (1993) Proc. Natl. Acad. Sci.USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indicator of the probability that a match between two nucleotide or amino acid sequences could occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the minimum sum probability when comparing the test nucleic acid with the reference nucleic acid is less than about 0.2, more preferably less than about 0.01.

[0045] The term "codon optimization," when referring to the genes or coding regions of nucleic acid molecules for transformation into various hosts, refers to modifying the codons in the genes or coding regions of a polynucleic acid molecule to reflect the typical codon usage frequencies of a selected organism, without altering the polypeptide encoded by DNA. Such optimization may involve replacing at least one, two or more, or a significant number of codons with one or more codons that are more frequently used in the genes of the selected organism.

[0046] Nucleic acids are "operably linked" if they are placed in a functional relationship with another nucleic acid sequence. For example, a pre-sequence or secretion leader DNA is operably linked to the polypeptide DNA if it is expressed as a preprotein involved in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of that sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to facilitate translation. Generally, "operably linked" means that the linked DNA sequences are in close proximity to each other, and in the case of a secretion leader, they are contiguous and located in the reading phase. However, operably linked nucleic acids (e.g., enhancers and coding sequences) do not need to be contiguous. Linking is achieved by ligation at a convenient restriction enzyme site. If such a site is not present, a synthetic oligonucleotide adapter or linker is used according to conventional practice. In embodiments, a promoter is operably linked to a coding sequence if it can affect the expression of a protein from that coding sequence (e.g., modulate in the absence of the promoter) (i.e., the coding sequence is under the transcriptional control of the promoter).

[0047] The term "nucleic acid base" refers to the portion of a nucleotide that possesses the function of Watson / Crick base pairing. The most common natural nucleic acid bases, adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T), possess the function of hydrogen bonding, which sequence-specifically links one nucleic acid chain to another.

[0048] As used throughout, “subject” (or “host”) means an individual. Therefore, “subject” can include, for example, domesticated animals such as cats and dogs, livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mice, rabbits, rats, guinea pigs, etc.), mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animals. The subject may be a primate or a mammal such as a human. Administration of the therapeutic agent may be carried out in a dose and duration effective for the treatment of the subject.

[0049] As used herein, the term "approximately" when referring to measurable values ​​such as quantities or percentages means that the measurable value is subject to variation of ±20%, ±10%, ±5%, or ±1%.

[0050] A nucleic acid sequence is "heterogeneous" to a second nucleic acid sequence if it originates from an alien species, or if it originates from the same species, if it is modified by human action from its original form. For example, a heterogeneous promoter (or heterogeneous 5' untranslated region (5'UTR)) manipulably ligated to a coding sequence points to a coding sequence from a different species from which the promoter originated, or, if from the same species, to a coding sequence different from a naturally occurring allele variant (e.g., a 5'UTR or 3'UTR from a different gene manipulably ligated to a nucleic acid encoding a co-stimulatory molecule).

[0051] As used herein, the term “nanoparticles” refers to particles or structures that are biocompatible and sufficiently resistant to chemical and / or physical destruction by the environment of use, and whose size is in the nanometer range, such that a sufficient number of nanoparticles remain substantially intact after delivery to the application or treatment site. In some embodiments, nanoparticles are typically in the range of about 1 nm to about 1000 nm, about 50 nm to about 500 nm, about 50 nm to about 350 nm, about 100 nm to about 250 nm, or about 110 nm to about 150 nm.

[0052] The “therapeutically effective amount” or “therapeutic dose” of a composition refers to the amount effective in achieving the desired therapeutic outcome. The therapeutically effective amount of a given therapeutic agent will typically vary depending on factors such as the type and severity of the disorder or disease being treated, the age, sex, and weight of the subject. This term may also refer to the amount of therapeutic agent effective in promoting the desired therapeutic effect, or the rate of delivery of the therapeutic agent (e.g., amount over time). The exact desired therapeutic effect will vary depending on the condition being treated, the subject's tolerance, the administered drug and / or drug formulation (e.g., the potency of the therapeutic agent, the concentration of the drug in the formulation), and various other factors understood by those skilled in the art. In some cases, the desired biological or medical response may be achieved after multiple administrations of the composition, over a period of days, weeks, or years.

[0053] As used herein, the terms “to treat” or “to cure” include administering a drug to a subject for the purpose of curing, restoring, reducing, alleviating, altering, treating, relieving, improving, stabilizing, or influencing a disease or disorder, or the symptoms of a disease or disorder. The terms “to treat” and “to cure” may also mean reducing the severity and / or frequency of symptoms, eliminating symptoms and / or underlying causes, and improving or repairing damage.

[0054] As used herein, the term “prevention” of a disease, disorder, or undesirable physiological event in the subject matter means prevention of a disease, disorder, or undesirable physiological event, or prevention of symptoms of a disease, disorder, or undesirable physiological event.

[0055] The “effective dose” of a drug refers to the amount of drug sufficient to provide the desired effect. The amount of drug that is “effective” will vary from subject to subject, depending on many factors such as the subject’s age and general condition, and the specific drug(s) involved. Therefore, it is not always possible to specify a quantifiable “effective dose.” However, an appropriate “effective dose” for any subject can be determined by those skilled in the art using routine experiments. Also, as used herein, unless otherwise specified, the “effective dose” of a drug may refer to an amount that encompasses both the therapeutic effective dose and the prophylactic effective dose. The “effective dose” of a drug required to obtain a therapeutic effect may vary depending on factors such as the subject’s age, sex, and weight. The dose regimen can also be adjusted to provide the optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be reduced proportionally as indicated by the urgency of the treatment situation.

[0056] A “pharmaceutically acceptable” ingredient can mean an ingredient that is not biologically or otherwise undesirable, i.e., the ingredient is incorporated into the pharmaceutical formulation of the present invention and administered to the subjects described herein without causing any significant undesirable biological effects or without adverse interactions with any of the other ingredients in the formulation containing the ingredient. When used in reference to administration to humans, this term generally means that the ingredient meets the required standards of toxicological and manufacturing testing, or that the ingredient is included in the Inactive Ingredients Guide created by the U.S. Food and Drug Administration.

[0057] A “pharmaceutically acceptable carrier” (sometimes referred to as “carrier”) generally means a carrier or excipient useful in preparing a safe and non-toxic pharmaceutical or therapeutic composition, and includes carriers acceptable for veterinary and / or human pharmaceutical or therapeutic use. The term “carrier” or “pharmaceutically acceptable carrier” may include, but is not limited to, phosphate-buffered saline, water, emulsions (such as oil / water or water / oil emulsions) and / or various types of wetting agents. As used herein, the term “carrier” includes, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material known in the art and further described herein for use in pharmaceutical formulations.

[0058] "Therapeutic agent" refers to any composition having a beneficial biological effect. Beneficial biological effects include both therapeutic effects, such as the treatment of a disorder or other undesirable physiological condition, and preventive effects, such as the prevention of a disorder or other undesirable physiological condition. These terms also encompass, but are not limited to, pharmaceutically acceptable pharmacologically active derivatives of beneficial agents specifically referred to herein, such as salts, esters, amides, proagents, active metabolites, isomers, fragments, and analogs. Where the term "therapeutic agent" is used, or where a particular agent is specifically identified, the term includes the agent itself, as well as pharmaceutically acceptable pharmacologically active salts, esters, amides, precursors, conjugates, active metabolites, isomers, fragments, and analogs.

[0059] As used herein, the terms “controlled release,” “controlled release drug delivery,” or “sustained release” refer to releasing or administering a drug from a given dosage form in a controlled manner to achieve desired pharmacokinetic properties in vivo. A “controlled” aspect of drug delivery is the ability to manipulate the formulation and / or dosage form to establish the desired kinetics of drug release.

[0060] As used herein, the phrases “concurrent administration,” “combined administration,” “simultaneous administration,” or “administer simultaneously” mean that the compounds are administered at the same time or immediately after each other.

[0061] The term "polypeptide" refers to a compound composed of a single chain of D-amino acids or L-amino acids, or a mixture of D-amino acids and L-amino acids linked by peptide bonds.

[0062] The term “antibody” is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, the term “antibody” also includes fragments or polymers of those immunoglobulin molecules, as well as humanized or humanized forms of immunoglobulin molecules or fragments thereof. Antibodies can be tested for their desired activity using the in vitro assays described herein or by similar methods, and then their in vivo therapeutic and / or prophylactic activity can be tested according to known clinical trial methods. There are five major classes of human immunoglobulins, namely IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. Those skilled in the art will recognize the equivalent classes in mouse. The heavy chain constant domains corresponding to the different classes of immunoglobulin are called α, δ, ε, γ, and μ, respectively.

[0063] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, that is, individual antibodies within the population are identical except for possible naturally occurring variations that may exist in a small subset of antibody molecules. Monoclonal antibodies as used herein specifically include “chimeric” antibodies in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody originating from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody originating from another species or belonging to another antibody class or subclass, as well as in a fragment of such an antibody, insofar as they exhibit the desired antagonistic activity.

[0064] The disclosed monoclonal antibodies can be prepared using any procedure for producing monoclonal antibodies. For example, the disclosed monoclonal antibodies can be prepared using the hybridoma method, e.g., the method described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, mice or other suitable host animals are usually immunized with an immunizer to induce lymphocytes that produce antibodies that specifically bind to the immunizer, or lymphocytes capable of producing such antibodies. Alternatively, lymphocytes may be immunized in vitro.

[0065] Monoclonal antibodies may also be produced by recombinant DNA methods. The DNA encoding the disclosed monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of the mouse antibody). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques, for example, as described in U.S. Patent No. 5,804,440 (Burton et al.) and U.S. Patent No. 6,096,441 (Barbas et al.).

[0066] The in vitro method is also suitable for the preparation of monovalent antibodies. Digestion of antibodies to produce fragments, particularly Fab fragments, can be achieved using predetermined methods known in the art. For example, digestion can be carried out using papain. Examples of papain digestion are described in WO94 / 29348, published December 22, 1994, and U.S. Patent No. 4,342,566. Papain digestion of antibodies typically produces two identical antigen-binding fragments, called Fab fragments, each having a single antigen-binding site and a residual Fc fragment. Pepsin treatment yields fragments having two antigen-binding sites and still capable of crosslinking antigens.

[0067] As used herein, the terms “antibody or its antigen-binding fragment” or “antibody or its fragment” encompass fragments such as F(ab')2, Fab', Fab, Fv, sFv, scFv, and hybrid fragments, including chimeric antibodies and hybrid antibodies having dual or multiple antigen or epitope specificity. Thus, we provide antibody fragments that retain the ability to bind to specific antigens. For example, antibody fragments that maintain binding activity are included within the meaning of the term “antibody or its antibody-binding fragment.” Such antibodies and fragments can be produced by techniques well known in the art and can be screened for specificity and activity according to the methods described in the examples and general methods for generating antibodies and screening them for specificity and activity (see Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988)).

[0068] The term "antibody or its antigen-binding fragment" includes both antibody fragments and conjugates of antigen-binding proteins (single-chain antibodies). It also includes, for example, immunoglobulin single variable domains such as nanobodies.

[0069] The fragment may include insertions, deletions, substitutions, or other selected modifications of specific regions or specific amino acid residues, whether or not they are bound to other sequences, as long as the activity of the antibody or fragment is not significantly altered or impaired compared to an unmodified antibody or antibody fragment. These modifications may provide several additional properties, such as removing / adding disulfide-bondable amino acids, extending lifespan, or altering secretory properties. In any case, the antibody or antibody fragment must possess bioactive properties, such as specific binding to its homologous antigens. The functional or active region of the antibody or antibody fragment may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to those skilled in the art and may include site-directed mutagenesis of the nucleic acid encoding the antibody or antibody fragment (Zoller, MJ Curr. Opin. Biotechnol. 3: 348-354, 1992).

[0070] As used herein, the term “antibody” (plural) may also refer to human antibodies and / or humanized antibodies. Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans and, therefore, can cause undesirable immune responses when administered to humans. Thus, the use of human antibodies or humanized antibodies in these methods helps reduce the chances that antibodies administered to humans will cause undesirable immune responses.

[0071] As used herein, the term “nucleic acid” means a polymer composed of nucleotides, such as deoxyribonucleotides or ribonucleotides. As used herein, the terms "ribonucleic acid" and "RNA" refer to polymers composed of ribonucleotides.

[0072] As used herein, the terms "deoxyribonucleic acid" and "DNA" mean a polymer composed of deoxyribonucleotides. The term "polynucleotide" refers to a single-stranded or double-stranded polymer consisting of nucleotide monomers.

[0073] Chemical Definition As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and non-aromatic substituents of organic compounds. Exemplary substituents include, for example, the substituents described below. One or more permissible substituents may be present for a suitable organic compound, and may be the same or different. For the purposes of the present disclosure, a heteroatom such as nitrogen may have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valency of the heteroatom. The present disclosure is not intended to be limited in any way by permissible substituents of organic compounds. The terms "substitution" or "substituted with" also include the implicit condition that such substitution is in accordance with the permitted valency of the substituted atom and the substituent, and that the substitution results in a compound, e.g., a stable compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

[0074] "Z 1 ", "Z 2 ", "Z 3 ", and "Z 4 " are used herein as generic terms to represent various specific substituents. These symbols may be any substituent, not limited to the substituents disclosed herein, and when they are defined as specific substituents in one example, they may be defined as several other substituents in another example.

[0075] As used herein, the term "aliphatic" refers to a non-aromatic hydrocarbon group, and includes branched and unbranched alkyl, alkenyl, or alkynyl groups. As used herein, the term "alkyl" refers to a branched or unbranched saturated hydrocarbon group having 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like. The alkyl group may also be substituted or unsubstituted. The alkyl group may be substituted with one or more groups including, but not limited to, alkyl, alkyl halide, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfooxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below.

[0076] Throughout the present specification, "alkyl" is generally used to refer to both unsubstituted alkyl groups and substituted alkyl groups, provided that in the present specification, substituted alkyl groups are specifically referred to by identifying the particular substituent(s) on the alkyl group. For example, the term "alkyl halide" specifically means an alkyl group substituted with one or more halogens, for example fluorine, chlorine, bromine or iodine. The term "alkoxyalkyl" specifically refers to an alkyl group substituted with one or more alkoxy groups, as described hereinafter. The term "alkylamino" specifically refers to an alkyl group substituted with one or more amino groups, as described hereinafter, and the like. Where "alkyl" is used in one instance and a specific term such as "alkyl alcohol" is used in another instance, it is not intended that the term "alkyl" does not also encompass such specific terms as "alkyl alcohol".

[0077] This practice is also applied to other groups described herein. That is, terms such as “cycloalkyl” refer to both unsubstituted and substituted cycloalkyl moieties, but substituted moieties can be further specifically identified herein; for example, certain substituted cycloalkyls may be referred to as, for example, “alkylcycloalkyl.” Similarly, substituted alkoxys may be specifically referred to as, for example, “halogenated alkoxy,” and certain substituted alkenyls may be referred to as, for example, “alkenyl alcohols.” Again, the practice of using general terms such as “cycloalkyl” and specific terms such as “alkylcycloalkyl” is not intended to mean that the general terms do not include specific terms.

[0078] As used herein, the term "alkoxy" refers to an alkyl group linked via a single terminal ether bond, i.e., the "alkoxy" group is -OZ 1 It can be defined as, in the formula, Z 1 This is the alkyl group defined above.

[0079] As used herein, the term "alkenyl" refers to a hydrocarbon group of 2 to 24 carbon atoms having a structural formula containing at least one carbon-carbon double bond. (Z 1 Z 2 )C=C(Z 3 Z 4 Asymmetric structures such as ) are intended to include both E and Z isomers. This can be inferred from the structural formulas herein where the asymmetric alkene is present, or it may be explicitly indicated by the bond symbol C=C. The alkenyl group may be substituted with one or more groups, including but not limited to alkyl, alkyl halide, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as listed below.

[0080] As used herein, the term "alkynyl" refers to a hydrocarbon group of 2 to 24 carbon atoms having a structural formula containing at least one carbon-carbon triple bond. The alkynyl group may be substituted with one or more groups, including but not limited to alkyl, alkyl halide, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol.

[0081] As used herein, the term “aryl” refers to any carbon-based aromatic group, including but not limited to benzene, naphthalene, phenyl, biphenyl, and phenoxybenzene. The term “heteroaryl” is defined as a group containing an aromatic group having at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus. The term “nonheteroaryl,” included within the term “aryl,” defines a group containing an aromatic group that does not contain a heteroatom. Aryl groups or heteroaryl groups may be substituted or unsubstituted. Aryl groups or heteroaryl groups may be substituted with one or more groups, including but not limited to alkyl, alkyl halide, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described herein. The term “biaryl” is a special type of aryl group and is included in the definition of aryl. A biaryl refers to two aryl groups that are linked via a fused ring structure, as in naphthalene, or linked via one or more carbon-carbon bonds, as in biphenyl.

[0082] As used herein, the term “cycloalkyl” refers to a non-aromatic carbon-based ring consisting of at least three carbon atoms. Examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. The term “heterocycloalkyl” refers to a cycloalkyl group as defined above, wherein at least one carbon atom of the ring is substituted with a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. Cycloalkyl groups and heterocycloalkyl groups may be substituted or unsubstituted. Cycloalkyl groups and heterocycloalkyl groups may be substituted with one or more groups, including but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described herein.

[0083] As used herein, the term "cycloalkenyl" refers to a non-aromatic carbon-carbon ring consisting of at least three carbon atoms and containing at least one double bond, i.e., a C=C bond. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, and cyclohexadienyl. The term "heterocycloalkenyl" is a type of cycloalkenyl group as defined above and is included in the meaning of the term "cycloalkenyl," in which at least one carbon atom of the ring is substituted with a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. Cycloalkenyl and heterocycloalkenyl groups may be substituted or unsubstituted. Cycloalkenyl and heterocycloalkenyl groups may be substituted with one or more groups, including but not limited to alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol groups, as described herein.

[0084] The term “cyclic group” is used herein to refer to aryl groups, nonaryl groups (i.e., cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl groups), or both. A cyclic group has one or more ring systems, which may be substituted or unsubstituted. A cyclic group may consist of one or more aryl groups, one or more nonaryl groups, or one or more aryl groups and one or more nonaryl groups.

[0085] As used herein, the term “aldehyde” is represented by the formula -C(O)H. Throughout this specification, “C(O)” or “CO” is an abbreviation for C=O. As used herein, the terms "amine" or "amino" refer to formula -NZ 1 Z 2 It is expressed as, in the formula, Z 1 and Z 2Each of these substituents may be one of the substituents described herein, for example, the hydrogen, alkyl, alkyl halide, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group.

[0086] As used herein, the term "carboxylic acid" is represented by the formula -C(O)OH. As used herein, the term "carboxylate" or "carboxyl" group is represented by the formula -C(O)OH - It is represented as follows.

[0087] As used herein, the term "ester" refers to the formula -OC(O)Z 1 Or -C(O)OZ 1 It is expressed as, in the formula, Z 1 This can be the alkyl, alkyl halide, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

[0088] As used herein, the term "ether" refers to the formula Z 1 OZ 2 It is expressed as, in the formula, Z 1 and Z 2 These can independently be the alkyl, alkyl halide, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl groups.

[0089] As used herein, the term "ketone" refers to formula Z 1 C(O)Z 2 It is expressed as, in the formula, Z 1 and Z 2 These can independently be the alkyl, alkyl halide, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl groups.

[0090] As used herein, the terms “halides” or “halogens” refer to fluorine, chlorine, bromine, and iodine. As used herein, the term "hydroxyl" is represented by the formula -OH.

[0091] As used herein, the term "nitro" is represented by the formula -NO2. As used herein, the term "silyl" refers to the formula -SiZ 1 Z 2 Z 3 It is expressed as, in the formula, Z 1 , Z 2 , and Z 3 This can independently be hydrogen, the alkyl, alkyl halide, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group.

[0092] The term "sulfonyl" is derived from the formula -S(O)2Z 1 In this specification, Z is used to refer to a sulfoxo group represented by the formula, where Z 1 This can be hydrogen, the alkyl group, alkyl halide, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group.

[0093] As used herein, the terms "sulfonylamino" or "sulfonamide" are represented by the formula -S(O)2NH-. The term "phosphonyl" is derived from the formula -P(O)(OZ 1 In this specification, Z is used to refer to the phosphooxo group represented by 2 in the formula, where Z 1 This can be hydrogen, the alkyl group, alkyl halide, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group.

[0094] As used herein, the term "thiol" is represented by the formula -SH. As used herein, the term “thio” is represented by the formula -S-. As used herein, “R 1 ”, “R 2 ”, “R 3 ”, “R n ”, where n is any integer, may each independently have one or more of the above groups. For example, when R 1 is a linear alkyl group, one of the hydrogen atoms of the alkyl group may optionally be substituted with hydroxyl, alkoxyl, amine, alkyl, halide or the like. Depending on the group selected, a first group may be incorporated within a second group, or alternatively the first group may be pendant (i.e., attached) to the second group. For example, in the phrase “alkyl group comprising an amino group”, the amino group may be incorporated within the backbone of the alkyl group. Alternatively, the amino group may be attached to the backbone of the alkyl group. The nature of the selected group(s) will determine whether the first group is embedded within or attached to the second group.

[0095] Unless stated otherwise, formulas with chemical bonds shown only as solid lines and not as wedges or dashed lines are intended to encompass each possible isomer, such as each enantiomer, diastereomer, and meso compound, as well as mixtures of isomers such as racemic or scalemic mixtures.

[0096] Reference will now be made in detail to certain embodiments of the disclosed materials, compounds, compositions, articles, and methods, examples of which are illustrated in the accompanying examples and drawings. Compounds In some embodiments, disclosed herein are compounds having Formula I, II, or III

[0097] ## STR ## or a salt thereof, wherein R 1The following are independently selected from alkyl, alkenyl, alkynyl, hydroxyl, ester, ether, carbonate, alkyl alcohol, alkyl ether, alkyl ester, carbamate, urea, guanidine, disulfide, amide, acetal, ketal, thioketal, trisulfide, and oxime ether.

[0098] In some embodiments, the compound has the following formula:

[0099] [ka] or a salt thereof, in the formula, R 1 The following are independently selected from alkyl, alkenyl, alkynyl, hydroxyl, ester, ether, carbonate, alkyl alcohol, alkyl ether, alkyl ester, carbamate, urea, guanidine, disulfide, amide, acetal, ketal, thioketal, trisulfide, and oxime ether.

[0100] In some embodiments, the compound has the following formula:

[0101] [ka] or a salt thereof, in the formula, R 1 The following are independently selected from alkyl, alkenyl, alkynyl, hydroxyl, ester, ether, carbonate, alkyl alcohol, alkyl ether, alkyl ester, carbamate, urea, guanidine, disulfide, amide, acetal, ketal, thioketal, trisulfide, and oxime ether.

[0102] In some embodiments, the compound has the following formula:

[0103] [ka] or a salt thereof, in the formula, R 1 The following are independently selected from alkyl, alkenyl, alkynyl, hydroxyl, ester, ether, carbonate, alkyl alcohol, alkyl ether, alkyl ester, carbamate, urea, guanidine, disulfide, amide, acetal, ketal, thioketal, trisulfide, and oxime ether.

[0104] In some embodiments, R 1 The following:

[0105] [ka] or its salt.

[0106] In some embodiments, R 1 teeth

[0107] [ka] In some embodiments, R 1 teeth

[0108] [ka] In some embodiments, R 1 teeth

[0109] [ka] In some embodiments, R 1 teeth

[0110] [ka] In some embodiments, R 1 teeth

[0111] [ka] In some embodiments, R 1 teeth

[0112] [ka] In some embodiments, R 1 teeth

[0113] [ka] In some embodiments, R 1 teeth

[0114] [ka] In some embodiments, R 1 teeth

[0115] [ka] In some embodiments, R 1 teeth

[0116] [ka] That is the case.

[0117] In some embodiments, the compound is as follows:

[0118] [ka] In the formula, R 1 teeth

[0119] [ka] That is the case.

[0120] In some embodiments, the compound is as follows:

[0121] [ka] In the formula, R 1 teeth

[0122] [ka] That is the case.

[0123] In some embodiments, the compound is as follows:

[0124] [ka] In the formula, each R 1 teeth

[0125] [ka] That is the case.

[0126] In some embodiments, the compound is as follows:

[0127] [ka] In the formula, each R 1 teeth

[0128] [ka] That is the case.

[0129] In some embodiments, R 1 is alkyl. In some embodiments, R 1 is an alkenyl. In some embodiments, R 1 is an alkynyl. In some embodiments, R 1is hydroxyl. In some embodiments, R 1 is an ester. In some embodiments, R 1 is an ether. In some embodiments, R 1 is a carbonate. In some embodiments, R 1 is an alkyl alcohol. In some embodiments, R 1 is an alkyl ether. In some embodiments, R 1 is an alkyl ester. In some embodiments, R 1 is a carbamate. In some embodiments, R 1 is urea. In some embodiments, R 1 is guanidine. In some embodiments, R 1 is a disulfide. In some embodiments, R 1 is an amide. In some embodiments, R 1 is an acetal. In some embodiments, R 1 is a ketal. In some embodiments, R 1 is a thioketal. In some embodiments, R 1 is a trisulfide. In some embodiments, R 1 is an oxime ether.

[0130] In some embodiments, each R 1 is alkyl. In some embodiments, each R 1 is alkenyl. In some embodiments, each R 1 is alkynyl. In some embodiments, each R 1 is hydroxyl. In some embodiments, each R 1 is an ester. In some embodiments, each R 1 is an ether. In some embodiments, each R 1 is a carbonate. In some embodiments, each R 1 is an alkyl alcohol. In some embodiments, each R1 is an alkyl ether. In some embodiments, each R 1 is an alkyl ester. In some embodiments, each R 1 is a carbamate. In some embodiments, each R 1 is urea. In some embodiments, each R 1 is guanidine. In some embodiments, each R 1 is a disulfide. In some embodiments, each R 1 is an amide. In some embodiments, each R 1 is an acetal. In some embodiments, each R 1 is a ketal. In some embodiments, each R 1 is a thioketal. In some embodiments, each R 1 is a trisulfide. In some embodiments, each R 1 It is an oxime ether.

[0131] In some embodiments, the alkyl is a branched alkyl. In some embodiments, the alkyl is an unbranched alkyl. In some embodiments, the alkyl is a C5 alkyl. In some embodiments, the alkyl is a C6 alkyl. In some embodiments, the alkyl is a C7 alkyl. In some embodiments, the alkyl is a C8 alkyl. In some embodiments, the alkyl is a C9 alkyl. In some embodiments, the alkyl is a C 10 It is alkyl. In some embodiments, alkyl is C 11 It is alkyl. In some embodiments, alkyl is C 12 It is alkyl. In some embodiments, alkyl is C 13 It is alkyl. In some embodiments, alkyl is C 14 It is alkyl. In some embodiments, alkyl is C 15 It is alkyl. In some embodiments, alkyl is C 16It is alkyl. In some embodiments, alkyl is C 17 It is alkyl. In some embodiments, alkyl is C 18 It is alkyl. In some embodiments, alkyl is C 19 It is alkyl.

[0132] nanoparticles In one embodiment, the present disclosure provides nanoparticles comprising a compound of formula I, II, or III as described herein.

[0133] Various compounds of formula I, II, or III are described in the section on compounds above. In some embodiments, the nanoparticles contain compounds of formula I, II, or III in a molar ratio of about 1% to about 99%. In some embodiments, the nanoparticles contain compounds of formula I, II, or III in a molar ratio of about 10% to about 80%. In some embodiments, the nanoparticles contain compounds of formula I, II, or III in a molar ratio of about 10% to about 40%. In some embodiments, the nanoparticles contain compounds of formula I, II, or III in molar ratios of about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40%. In some embodiments, the nanoparticles contain compounds of formula I, II, or III in a molar ratio of about 20%.

[0134] In some embodiments, the nanoparticles contain non-cationic lipids. In some embodiments, the non-cationic lipids interact with lipids as helper lipids. In some embodiments, the non-cationic lipids include 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), and DPPC (1,2-dipalmitoyl-sn- The noncationic lipid may include, but is not limited to, 1,2-glycero-3-phosphocholine (DOPE), 1,2-dioleyl-sn-glycero-3-phosphotidylcholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dioleoyl-5 / 7-glycero-3-phospho(1'-rac-glycerol) (DOPG), or combinations thereof. In one embodiment, the noncationic lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In one embodiment, the noncationic lipid is 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE). In one embodiment, the noncationic lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In one embodiment, the noncationic lipid is 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE). While several noncationic lipids are described herein, additional noncationic lipids can be used in combination with the compounds disclosed herein.

[0135] In some embodiments, the nanoparticles contain noncationic lipids in a molar ratio of about 10% to about 80%. In some embodiments, the nanoparticles contain noncationic lipids in a molar ratio of about 10% to about 40%. In some embodiments, the nanoparticles contain noncationic lipids in molar ratios of about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40%. In one embodiment, the nanoparticles contain noncationic lipids in a molar ratio of about 30%.

[0136] In some embodiments, the nanoparticles contain polyethylene glycol lipids (PEG lipids). PEG lipids are incorporated to form a hydrophilic outer layer and stabilize the particles. Non-limiting examples of polyethylene glycol lipids include PEG-modified lipids such as PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. Representative polyethylene glycol lipids include DMG-PEG, DLPE-PEG, DMPE-PEG, DPPC-PEG, and DSPE-PEG. In one embodiment, the polyethylene glycol lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG). In another embodiment, the polyethylene glycol lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol-2000 (DMG-PEG2000). DMG-PEGXXXX means 1,2-dimyristoyl-sn-glycerol,methoxypolyethylene glycol-XXXX, where XXXX represents the molecular weight of the polyethylene glycol portion, for example, DMG-PEG2000 or DMG-PEG5000.

[0137] In some embodiments, the nanoparticles contain polyethylene glycol lipids in a molar ratio of about 0% to about 5%. In some embodiments, the nanoparticles contain polyethylene glycol lipids in molar ratios of about 0%, about 0.25%, about 0.5%, about 0.75%, about 1%, about 1.5%, about 2%, about 3%, about 4%, or about 5%. In one embodiment, the nanoparticles contain polyethylene glycol lipids in a molar ratio of about 0.75%.

[0138] In some embodiments, the nanoparticles contain sterols. Sterols are well known to those skilled in the art and generally refer to compounds having a perhydrocyclopentanophenanthrene ring system and one or more OH substituents. Examples of sterols include, but are not limited to, cholesterol, campesterol, ergosterol, and sitosterol.

[0139] In some embodiments, the sterol is selected from cholesterol-based lipids. In some embodiments, one or more cholesterol-based lipids are selected from cholesterol, PEGylated cholesterol, DC-Choi(N,N-dimethyl-N-ethylcarboxyamide cholesterol), 1,4-bis(3-N-oleylaminopropyl)piperazine, or a combination thereof.

[0140] Sterols can be used to regulate particle permeability and fluidity based on their function in cell membranes. In one embodiment, the sterol is cholesterol. In some embodiments, the nanoparticles contain sterols in a molar ratio of about 25% to about 50%. In some embodiments, the nanoparticles contain sterols in a molar ratio of about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%. In one embodiment, the nanoparticles contain sterols in a molar ratio of about 40%.

[0141] In one embodiment, the nanoparticles further contain a drug. In one embodiment, the nanoparticles further contain a therapeutic agent. In one embodiment, the nanoparticles further contain a diagnostic agent.

[0142] In some embodiments, the nanoparticles contain one of the formulations shown in Figure 2F. In some embodiments, the nanoparticles contain components (lipids, DOPE, cholesterol, and PEG) in molar ratios as disclosed in Figure 2F. In some embodiments, the nanoparticles contain one of the formulations shown in Figure 7A. In some embodiments, the nanoparticles contain components (lipids, DOPE, cholesterol, and PEG) in molar ratios as disclosed in Figure 7A.

[0143] The drug delivered to the cell may be a polynucleotide. Polynucleotides or oligonucleotides that can be introduced according to the methods herein include all types of DNA, cDNA, and RNA sequences. For example, polynucleotides may be double-stranded DNA, single-stranded DNA, complex DNA, encapsulated DNA, naked RNA, encapsulated RNA, messenger RNA (mRNA), tRNA, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), antisense RNA (asRNA), and combinations thereof. Polynucleotides may also be DNA constructs such as expression vectors, expression vectors encoding the desired gene product (e.g., a gene product of the same or different species as the target into which it is introduced). In one embodiment, the drug is mRNA. In some embodiments, the polynucleotide is encapsulated by nanoparticles.

[0144] In some embodiments, the polynucleotide contains a nucleotide acid encoding a co-stimulatory molecule. In some embodiments, the co-stimulatory molecule is selected from ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, SLAM, CD2, CD226, galectin 9, TIM1, LFA1, B7-H2, B7-1, B7-2, CD70, LIGHT, HVEM, CD40, 4-1BBL, OX40L, TL1A, GITRL, CD30L, SLAM, CD48, CD58, CD155, CD112, CD80, CD86, ICOSL, TIM3, TIM4, ICAM1, and LFA3. In some embodiments, the co-stimulatory molecule is CD40. In some embodiments, the co-stimulatory molecule is CD40L.

[0145] Examples of co-stimulatory molecule sequences (in the case of human sequences) include ICOS (NCBI reference sequence: NM_012092.3), CD28 (NCBI reference sequence: NM_006139.4), CD27 (NCBI reference sequence: NM_001242.4), HVEM (NCBI reference sequence: NM_003820.3), LIGHT (NCBI reference sequence: NM_003807.4), CD40L (NCBI reference sequence: NM_000074.2), 4-1BB (NCBI reference sequence: NM_001561.5), OX40 (NCBI reference sequence: NM_003 327.4), DR3 (NCBI reference sequence: NM_148965.1), GITR (NCBI reference sequence: NM_004195.3), CD30 (GenBank: M83554.1), SLAM (NCBI reference sequence: NM_003037.4), CD2 (NCBI reference sequence: NM_003037.4), Reference sequence: NM_001328609.1), CD226 (NCBI reference sequence: NM_006566.3), Galectin-9 (GenBank: AB040130.2), TIM1 (GenBank: U02082.1), B7-H2 (NCBI reference sequence: NM_01 5259.5), B7-1 (NCBI reference sequence: NM_005191.4), B7-2 (NCBI reference sequence: NM_175862.5), CD70 (NCBI reference sequence: NM_001252.5), CD40 (NCBI reference sequence: NM_001250.5), 4-1 BBL (NCBI reference sequence: NM_003811.4), OX40L (NCBI reference sequence: NM_003326.5), TL1A (NCBI reference sequence: NM_005118.4), GITRL (GenBank:AY358868.1), CD30L (NCBI reference sequence: NM_005118.4), Examples include sequence: NM_001244.3), SLAM (GenBank: U33017.1), CD48 (NCBI reference sequence: NM_001778.4), CD58 (NCBI reference sequence: NM_001779.3), CD155 (NCBI reference sequence: NM_006505.5), CD112 (NCBI reference sequence: NM_001042724.2), TIM3 (GenBank: AF450242.1), TIM4 (NCBI reference sequence: NM_138379.3), and ICAM1 (NCBI reference sequence: NM_000201.3).

[0146] In some embodiments, the mRNA encoding the co-stimulatory molecule includes a heterogeneous 5' untranslated region (5'UTR). In some embodiments, the mRNA encoding the co-stimulatory molecule includes a heterogeneous 3' untranslated region (3'UTR).

[0147] In some embodiments, the nucleic acids disclosed herein (e.g., mRNA encoding a co-stimulatory molecule) include at least one chemically modified nucleotide. In some embodiments, the at least one chemically modified nucleotide includes a chemically modified nucleic acid base, a chemically modified ribose, a chemically modified phosphodiester linkage, or a combination thereof.

[0148] In one embodiment, at least one chemically modified nucleotide is a chemically modified nucleic acid base. In one embodiment, the chemically modified nucleic acid bases are 5-formylcytidine (5fC), 5-methylcytidine (5meC), 5-methoxycytidine (5moC), 5-hydroxycytidine (5hoC), 5-hydroxymethylcytidine (5hmC), 5-formyluridine (5fU), 5-methyluridine (5-meU), 5-methoxyuridine (5moU), 5-carboxymethyl ester uridine (5camU), pseudouridine (Ψ), N 1 -methylpsuduridine (me 1 Ψ), N 6 -Methyladenosine (me 6 A) or thienoguanosine ( th Selected from G)

[0149] In some embodiments, the chemically modified nucleic acid base is 5-methoxyuridine (5moU). In some embodiments, the chemically modified nucleic acid base is pseudouridine (Ψ). In some embodiments, the chemically modified nucleic acid base is N 1 - Pseudouridine (me 1 It is Ψ).

[0150] The structures of these modified nucleic acid bases are shown below:

[0151] [ka]

[0152] In one embodiment, at least one chemically modified nucleotide is a chemically modified ribose. In one embodiment, the chemically modified ribose is selected from 2'-O-methyl (2'-O-Me), 2'-fluoro (2'-F), 2'-deoxy-2'-fluoro-β-D-arabino-nucleic acid (2'F-ANA), 4'-S, 4'-SFANA, 2'-azide, UNA, 2'-O-methoxy-ethyl (2'-O-ME), 2'-O-allyl, 2'-O-ethylamine, 2'-O-cyanoethyl, locked nucleic acid (LAN), methylene-cLAN, N-MeO-aminoBNA, or N-MeO-aminooxyBNA. In one embodiment, the chemically modified ribose is 2'-O-methyl (2'-O-Me). In one embodiment, the chemically modified ribose is 2'-fluoro (2'-F).

[0153] The structures of these modified ribose molecules are shown below:

[0154] [ka]

[0155] In one embodiment, at least one chemically modified nucleotide is a chemically modified phosphodiester linkage. In one embodiment, the chemically modified phosphodiester linkage is selected from phosphorothioate (PS), boranophosphate, phosphodithioate (PS2), 3',5'-amide, N3'-phosphoramide (NP), phosphodiester (PO), or 2',5'-phosphodiester (2',5'-PO). In one embodiment, the chemically modified phosphodiester linkage is a phosphorothioate.

[0156] The structures of these modified phosphodiester links are shown below:

[0157] [ka]

[0158] antigen presenting cells In some embodiments, disclosed herein are antigen-presenting cells comprising lipid-based nanoparticles comprising recombinant polynucleotides containing nucleic acids encoding compounds and co-stimulatory molecules of any prior art.

[0159] In some embodiments, the compound

[0160] [ka] And, In the formula, R 1 teeth

[0161] [ka] That is the case.

[0162] In some embodiments, the co-stimulatory molecule is selected from ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, SLAM, CD2, CD226, galectin 9, TIM1, LFA1, B7-H2, B7-1, B7-2, CD70, LIGHT, HVEM, CD40, 4-1BBL, OX40L, TL1A, GITRL, CD30L, SLAM, CD48, CD58, CD155, CD112, CD80, CD86, ICOSL, TIM3, TIM4, ICAM1, and LFA3. In some embodiments, the co-stimulatory molecule is CD40.

[0163] In some embodiments, the antigen-presenting cells include macrophages or dendritic cells. As used herein, the term “antigen-presenting cell” or “APC” should be understood to refer to a heterogeneous group of immune cells capable of processing and presenting antigens to stimulate a response in specific lymphocytes (e.g., T cells and B cells). Classical APCs include, for example, dendritic cells, macrophages, B cells, and neutrophils.

[0164] As used herein, it is understood that macrophages are generally known as phagocytic immune cells (Meszaros et al., 1999). They also secrete factors such as chemokines or cytokines. In addition to phagocytosis and antigen presentation, these cells may play a supportive role via a diverse repertoire of plasma membrane and secreted molecules as previously shown for erythroblasts, hepatocytes and neurons (Gordon 1995, Bioessays, Volume 17, Issue 11) (Sadahira & Morr, Pathol Int. 1999 Oct; 49(10):841-8.)(Takeishi, Hirano, et al. Arch Histol Cytol. December 1999; 62(5):413-22)(Polazzi, Gianni, et al. Glia. December 2001; 36(3):271-80). The term "macrophage" refers to cells that exhibit the properties typically described for macrophages, including phagocytosis and expression of defined cell surface markers such as CD64, CD14 and HLA-DR antigen expression. Macrophages according to the present invention can be isolated from tissues, or preferentially by in vitro differentiation from blood monocytes (also referred to herein as "monocyte-derived macrophages"), bone marrow progenitor cells (also referred to herein as "bone marrow-derived macrophages") or any other possible precursor, and by using any differentiation method, precursor and approach known to a person skilled in the art. Accordingly, in some embodiments, the macrophages comprise bone marrow-derived macrophages. In some embodiments, the macrophages comprise monocyte-derived macrophages. In some embodiments, the macrophages comprise iPSC-derived macrophages. In some embodiments, the macrophages comprise macrophage cell lines including, for example, RAW264.7, THP-1, U937, IC-21, J774A.1, MV-4-11, or KG1.

[0165] Furthermore, it should be understood that as used herein, “dendritic cells” or “DCs” refer to a type of antigen-presenting cell typically identified by the expression of one or more of the following markers on their cell surface: CD1a, CD1b, and CD1c, CD4, CD11c, CD33, CD40, CD80, CD86, CD83, and HLA-DR. In some embodiments, the cells are dendritic cells. In some embodiments, the dendritic cells are immature dendritic cells. The dendritic cells according to the present invention can be isolated from tissue, or preferentially, by in vitro differentiation from blood monocytes (also referred herein as “monocyte-derived dendritic cells”), bone marrow progenitor cells (also referred herein as “bone marrow-derived dendritic cells”) or any other possible precursors, and by using any differentiation methods, precursors, and methods known to those skilled in the art. Thus, in some embodiments, the dendritic cells include bone marrow-derived dendritic cells. In some embodiments, the dendritic cells include monocyte-derived dendritic cells. In some embodiments, the dendritic cells include iPSC-derived dendritic cells. In some embodiments, the dendritic cell is a conventional dendritic cell 1 (or cDC1, lymphoid DC), typically identified by the expression of one or more of the following markers on its cell surface: CD141, CLEC9A, and XCR1. In some embodiments, the dendritic cell is a conventional dendritic cell 2 (or cDC2, myeloid DC), typically identified by the expression of one or more of the following markers on its cell surface: CD1c and CD172a. In some embodiments, the dendritic cell is a plasmacytoid DC (or pDC), typically identified by the expression of one or more of the following markers on its cell surface: CD123, CD303, and CD304.

[0166] In some embodiments, a method for expressing a polypeptide in antigen presentation is disclosed herein, comprising administering an effective amount of nanoparticles disclosed herein to antigen-presenting cells, wherein the nanoparticles comprise a recombinant polynucleotide comprising a nucleic acid encoding the polypeptide. Compositions and methods In some embodiments, the methods described herein include administering a therapeutically effective amount of antigen-presenting cells and antibodies disclosed herein to a subject in need thereof.

[0167] In some embodiments, the immunotherapy agent is selected from anti-CD40 antibodies, anti-PDL1 antibodies, anti-PD1 antibodies, anti-CTLA4 antibodies, or combinations thereof. In some embodiments, the antibody or its antigen-binding fragment that specifically binds to the costimulatory molecule is BMS986178. In some embodiments, the antibody or its antigen-binding fragment that specifically binds to the costimulatory molecule is GSK3174998. In some embodiments, the antibody or its antigen-binding fragment that specifically binds to the costimulatory molecule is PF-04518600. In some embodiments, the antibody or its antigen-binding fragment that specifically binds to the costimulatory molecule is MOXR0916. In some embodiments, the antibody or its antigen-binding fragment that specifically binds to the costimulatory molecule is PF-04518600. In some embodiments, the antibody or its antigen-binding fragment that specifically binds to the costimulatory molecule is MEDI6383. In some embodiments, the antibody or its antigen-binding fragment that specifically binds to the costimulatory molecule is MEDI0562. In some embodiments, the antibody or its antigen-binding fragment that specifically binds to the costimulatory molecule is INCAGN01949. In some embodiments, the antibody or antigen-binding fragment that specifically binds to the costimulatory molecule is InVivoPlus anti-mouse OX40 (clone OX-86) (Company: BioXcell, Catalog: BP0031).

[0168] Examples of additional antibodies or antigen-binding fragments that specifically bind to the costimulatory molecule include: in mice, InVivoPlus anti-mouse 4-1BB (CD137) (clone LOB12.3) (Company: BioXcell, Catalog: BP0169), InVivoPlus anti-mouse CD40 (clone FGK4.5 / FGK45) (Company: BioXcell, Catalog: BP0016-2); in humans, anti-human O Examples include X40, BMS986178, GSK3174998, PF-04518600, MOXR0916, PF-04518600, MEDI6383, MEDI0562, INCAGN01949; anti-human 4-1BB, utomirumab, urerumab; anti-human CD40, CP-870893, APX005M, ADC-1013, JNJ-64457107, SEA-CD40, and RO7009789.

[0169] In some embodiments, antigen-presenting cells and antibodies are administered into the tumor. In some embodiments, disclosed herein are methods for treating cancer, comprising administering the following to a subject in need: A therapeutically effective amount of the lipid-based nanoparticles disclosed herein, and A therapeutically effective amount of antigen-presenting cells disclosed herein.

[0170] In some embodiments, the nanoparticles include the following:

[0171] [ka] In the formula, R 1 teeth

[0172] [ka] That is the case.

[0173] In some embodiments, the lipid-based nanoparticles contain recombinant polynucleotides, including the nucleic acid encoding CD40L. In one aspect, the methods described herein are for cancers, for example, in particular melanoma, lung cancer (adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, large cell carcinoma, bronchoalveolar carcinoma, bronchogenic carcinoma, non-small cell carcinoma, small cell carcinoma, mesothelioma, etc.); breast cancer (ductal carcinoma, lobular carcinoma, inflammatory breast cancer, clear cell carcinoma, mucinous carcinoma, serosal breast cancer, etc.); colorectal cancer (colon cancer, rectal cancer, colorectal adenocarcinoma); anal cancer; pancreatic cancer (pancreatic adenocarcinoma, islet cell carcinoma, neuroendocrine tumor, etc.); prostate cancer; prostate adenocarcinoma; ovarian cancer (surface epithelial stromal tumors, endometrioid tumors and mucinous cystadenocarcinoma, sex cord adenocarcinoma, etc., including ovarian epithelial carcinoma or serous tumors); Squamous tumors; liver and bile duct cancer (hepatocellular carcinoma, bile duct cancer, hemangioma, etc.); esophageal cancer (esophageal adenocarcinoma and squamous cell carcinoma, etc.); oral and oropharyngeal squamous cell carcinoma; salivary gland adenoid cystic carcinoma; bladder cancer; bladder carcinoma; uterine cancer (including endometrial adenocarcinoma, eye, serous carcinoma of the uterine body, clear cell carcinoma of the uterine body, uterine sarcoma, leiomyosarcoma, mixed Müllerian duct tumor); glioma, glioblastoma, medulloblastoma, and other brain tumors; kidney cancer (renal cell carcinoma, clear cell carcinoma, Wilms' tumor, etc.); head and neck cancer (squamous cell carcinoma, etc.); gastric cancer (gastric cancer, gastric adenocarcinoma, gastrointestinal stromal tumor); testicular tumor; germ cell tumor; neurological tumors Cancers of the uterus; cervical cancer; carcinoid tumors of the gastrointestinal tract, chest, and other organs; signet ring cell carcinoma; mesenchymal tumors such as sarcomas, fibrosarcoma, hemangioma, hemangiomatosis, periangiocarcinoma, pseudohemangiomatous stromal hyperplasia, myofibroblastoma, fibromatosis, inflammatory myofibroblastoma, lipoma, angiolipoma, granuloma, neurofibroma, schwannoma, angiosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma, leiomyoma, leiomyosarcoma, skin, melanoma, etc., cervical cancer, retinoblastoma, head and neck cancer, pancreatic, brain, thyroid, testis, kidney, bladder, soft tissue, adrenal gland, urethra, penile cancer, myxosarcoma, chondrosarcoma, osteosarcoma, chordoma It is used to treat malignant fibrous histiocytoma, lymphangiosarcoma, mesothelioma, squamous cell carcinoma; epidermoid carcinoma, malignant adnexal tumors, adenocarcinoma, hepatoma, hepatocellular carcinoma, renal cell carcinoma, adrenal tumor, cholangiocarcinoma, transitional cell carcinoma, choriocarcinoma, seminoma, embryonal cell carcinoma, anaplastic glioma; glioblastoma multiforme, neuroblastoma, medulloblastoma, malignant meningioma, malignant schwannoma, neurofibrosarcoma, parathyroid carcinoma, medullary thyroid carcinoma, bronchial carcinoid, pheochromocytoma, islet cell carcinoma, malignant carcinoid, malignant paraganglioma, melanoma, Merkel cell neoplasm, phyllodes cystic sarcoma, salivary carcinoma, thymic carcinoma, and vaginal cancer. [Examples]

[0174] The following examples are provided below to illustrate the compositions, methods, and results of the disclosed subject matter. These examples are not intended to include all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the invention that would be apparent to those skilled in the art.

[0175] Example 1. Sugar-derived lipid nanoparticles Effective cancer immunotherapy depends on the initiation, progression, and amplification of the cancer immune cycle (CIC). However, CIC is usually blocked by immunosuppressive factors in the tumor microenvironment (TME), resulting in tumor promotion, metastasis, and recurrence. Here, a combination of lipid nanoparticle-mRNA preparations and dendritic cell therapy was used to sufficiently increase CIC through multiple steps. First, lipid nanoparticles containing CD40 ligand (CD40L) mRNA induce potent immunogenic cell death in tumor tissue, leading to the release of tumor-associated antigens (TAAs) and CD40L expression. Next, CD40-overexpressing dendritic cells are adopted and subsequently activated by CD40L molecules within the tumor tissue. This promotes the secretion of pro-inflammatory cytokines and chemokines and facilitates the upregulation of costimulatory molecules on dendritic cells. These are crucial for TME reprogramming and priming of the T cell response. Following the presentation of TAAs by dendritic cells to T cells, all of the above stepwise events contribute to the enhancement of potent cancer-specific T cell immunity, clearing established skin tumors, suppressing distal lesions, and preventing tumor reattack. Overall, this study demonstrates that integrating lipid nanoparticles and cell therapy (CATCH) provides closure of the cancer immune cycle (CIC) in a mouse melanoma model, promoting tumor clearance and building long-term anti-tumor immunity.

[0176] preface The development of immunotherapy has brought about a major breakthrough in the clinical outcomes of cancer treatment. Effective antitumor immunity depends on the proper activation of a series of responses in the Cancer-Immunity Cycle (CIC). 1First, dying cancer cells release tumor-associated antigens (TAAs), which are captured and processed by antigen-presenting cells (APCs) such as dendritic cells. 1 Next, the APC presents the antigen to the T cell, resulting in priming of the effector T cell response to the TAA. 1 These activated effector T cells invade tumor tissue and attack cancer cells. 1 It mediates the killing of the cancer cells. Finally, the dead cancer cells become CIC 1 Further release of antigens to increase the breadth and depth of these immune responses. 1 However, these gradual events are usually attenuated by the immunosuppressive tumor microenvironment (TME), resulting in uncontrolled tumor growth, metastasis, and recurrence. 1,2 For example, immunosuppressive TMEs can inhibit the recruitment, invasion, and maturation of dendritic cells. 1,2 This reduces the secretion of cytokines and the expression of costimulators by dendritic cells, which are important for priming T cell immunity. 1,2 .

[0177] The inventors investigated whether it is possible to appropriately increase CIC by synergistically inducing cancer cell death to release TAA and improving the number and maturity of dendritic cells in tumor tissue. This may further enhance systemic and memory-based antitumor immunity, potentially leading to the removal of primary tumors, suppression of tumor metastasis, and prevention of tumor recurrence. Immunogenic cell death (ICD), a mode of cell death, involves the efflux of damage-associated molecular patterns (DAMPs) and TAAs. 3,4 Recent studies have revealed that lipid nanoparticles (LNPs) capable of inducing ICDs can remodel TMEs, resulting in enhanced antitumor immunity. 5 CD40 and CD40 ligand (CD40L) are a pair of costimulatory molecules belonging to the tumor necrosis factor / tumor necrosis factor receptor family. 6 The interaction between CD40 on dendritic cells and CD40L on CD4 T cells is crucial for dendritic cell maturation, promoting the secretion of pro-inflammatory cytokines and the upregulation of other costimulatory molecules.6 These play a central role in inducing CD8 T cells to produce cytotoxic and memory responses. 6 Several CD40 agonist antibodies are being studied in clinical trials. 6 Furthermore, intratumoral (IT) administration of immunotherapeutic agents can improve in situ bioavailability and, therefore, improve treatment efficiency. 7 Accordingly, several LNP-mRNA preparations and immune cells delivered via intrathecal injection are currently undergoing clinical trials. 7-10 In this study, we used LNPs containing CD40L mRNA (CD40L-LNP) to simultaneously induce ICD expression and CD40L expression in tumor tissue. Next, we captured TAAs and adopted CD40-overexpressing bone marrow-derived dendritic cells (CD40-BMDCs) that matured upon CD40L stimulation. These activated dendritic cells then presented antigens to T cells, leading to priming of the effector T cell response against cancer cells (Figure 1A).

[0178] To test this concept, effective delivery of mRNA to tumor cells and primary dendritic cells was required. Sugar alcohols are carbohydrates widely used in the food industry as a sugar substitute because they provide sweetness, contain fewer calories, and do not cause blood sugar spikes compared to regular sugars such as glucose. 11 These compounds are naturally occurring or derived from sugars by the reduction of the carbonyl group to the hydroxyl group. 12 In particular, hexitols such as sorbitol and mannitol are hexose alcohols that can be double-dehydrated to obtain dianhydrohexitol, i.e., a series of unique cyclic ethers that function as important chemical skeletons and starting materials. 13 Therefore, we designed and synthesized three sets of ionizable lipids derived from sugar alcohols to prepare LNPs for mRNA delivery. To examine these LNPs in a bioassay, we selected melanoma as a disease model. Melanoma is one of the fastest-growing skin cancers with increasing incidence. 14The leading cause of death in melanoma patients is widespread metastasis, such as metastases to the skin and brain. 14 Therefore, in this study, two melanoma cell lines were selected to construct a mouse tumor model. Following systematic screening and characterization of ionizable lipids derived from sugar alcohols, two preferred LNP formulations for mRNA delivery were identified. One formulation induced potent ICD in melanoma tumor tissue, while effectively delivering CD40L mRNA into cancer cells. The other formulation showed dramatically higher CD40 mRNA delivery efficiency in mouse bone marrow-derived dendritic cells (BMDCs) compared to lipofectamine 3000 (Lipo3K) and electroporation (Electro). The combination of CD40L-LNP therapy and adoptive transfer of CD40-BMDCs resulted in a complete response rate of over 80% in the subcutaneous (sc) melanoma tumor model. Furthermore, local treatment enabled the suppression or elimination of tumor metastases to the skin and brain. Importantly, these responder mice were resistant to subcutaneous rechallenge and showed delayed tumor growth after intracranial rechallenge. In summary, we increased CIC by inducing ICD and adaptive dendritic cell transplantation using two types of LNP-mRNA preparations. This study presents an important immunotherapy strategy that enables the broad and effective treatment of primary tumors, tumor metastases, and tumor recurrence.

[0179] result Dianhydrohexitols, such as isosorbide and isomannide, are a series of bio-derived bicyclic compounds prepared by double dehydration of hexose alcohols. 15 They consist of two cis-condensed tetrahydrofuran rings and have two secondary hydroxyl groups located at positions 2 and 5. 13 The different configurations of the two hydroxyl groups result in different reaction reactivity and steric hindrance to functionalization, thus making dianhydrohexitol a versatile bio-derived building block for the synthesis of pharmaceutically important compounds and polymers. 16Based on their unique chirality and rigidity, three sets of ionizable lipids (DIS, DIM, and LIS) derived from sugar alcohols were designed and synthesized using sorbitol, mannitol, and L-sorbitol as precursors, respectively (Figures 1B and 1C). As a representative synthetic route for LIS lipids (Figure 1B), L-isosorbide, an enantiomer of isosorbide, was prepared from L-sorbitol in the presence of dimethyl carbonate and sodium ethoxide following the previously reported dehydration procedure. 17 Next, a coreamine was obtained by a double Michael addition reaction between L-isosorbide and acrylonitrile, followed by reduction of the nitrile group with borane. Finally, a hydrophobic tail with various functional groups such as hydroxyl (LIS1), hydrocarbons (LIS2-LIS6), esters (LIS7), carbonate esters (LIS8), and acetals (LIS9 and LIS10) was installed by reductive amination or epoxide ring-opening to obtain the corresponding ionizable lipids. These diverse hydrophobic domains in the lipids influence the formulation of LNPs and their interaction with the cell membrane, resulting in different mRNA delivery efficiencies. DIS and DIM lipids were synthesized following a similar synthetic route. 1 Its structure was confirmed by 1H nuclear magnetic resonance and mass spectrometry (see Methods).

[0180] Next, LNPs containing firefly luciferase (FLuc) mRNA (FLuc-LNP) were prepared, and their size, surface charge, and mRNA encapsulation efficiency were identified (Figures 6A-6C). The structure-activity relationship of sugar alcohol-derived ionized lipids was generalized by quantifying the luminescence intensity in bone marrow-derived dendritic cells (BMDCs). The chirality of the amine core may affect the mRNA delivery efficiency of the compounded LNP. The amine cores of the DIS and LIS lipids were paired enantiomers, and they exhibited similar mRNA delivery capabilities, particularly DIS-1 to 8 and LIS-1 to 8. Lipids with a C10 hydrocarbon tail induced more effective mRNA delivery than lipids with other hydrocarbon tails. The DIM series, however, showed a different trend. Among the DIM series, DIM-7 was the most effective for mRNA delivery. Lipids containing carbonate ester linkers (DIS8, DIM8, and LIS8) in the hydrophobic domain showed weak Fluc mRNA delivery capability. Among these LNPs, DIM7 and LIS10 showed the highest mRNA delivery efficiency compared to the other LNPs, demonstrating 3-fold and 10-fold efficacy for mRNA delivery compared to Lipo3K and Electro, respectively (Figure 2A). To further investigate the combination of DIM7 and LIS10, an orthogonal screening assay was performed using L16(4). 4The analysis was performed based on an orthogonal table (Figure 7A). The effect of each lipid component on mRNA delivery was examined based on the luminescence intensity of 16 orthogonal formulations of DIM7 or LIS10 (Figures 2C, 2E), different molar ratios (Figures 2B, 2D), and several predicted preferred formulations (Figure 2B). Next, their mRNA delivery efficiency was examined by comparing them with the corresponding top formulations from the orthogonal screening assays DIM7 M and LIS10 G (Figures 2C, 2E). With respect to DIM7, the preferred formulation DIM7 S showed a luminescence signal that was 2 times (P<0.001) and 3.5 times (P<0.001), respectively, compared to the orthogonal top formulation DIM7 M and the initial formulation DIM7 (Figure 2C). Regarding LIS10, the preferred formulation LIS10W yielded a 1.2-fold (P<0.0001) higher luminescence signal compared to the top orthogonal formulation LIS10G, and a 2.3-fold (P<0.0001) higher luminescence intensity compared to the initial formulation LIS10 (Figure 2E). Formulations DIM7S and LIS10W showed similar characteristics. Their particle size was approximately 110 nm with a polydispersity index (PDI) < 0.2 (Figures 2G, 2I). Their mRNA encapsulation efficiency was approximately 90%, and they were slightly positively charged (Figures 2G, 2I). Furthermore, both exhibited a spherical morphology when visualized by Cryo-TEM (Figures 2H, 2J, 7B, 7C). As a result, formulation DIM7S was selected, and its mRNA was delivered ex vivo to BMDCs for the following studies. At the same mRNA concentration, formulation DIM7S was 10-fold and 30-fold superior to Lipo3K and Electro, respectively, in mRNA delivery (Figure 7D). The maximum luminescence intensity of formulation DIM7S was observed at 12:00 over a time course of 6 to 24 hours (Figure 7E). Significantly, when formulation DIM7S encapsulated CD40 mRNA (CD40-DIM7S), CD40 expression increased by more than 70% in BMDCs compared to approximately 17% in untreated BMDCs (P<0.0001, Figure 7F).

[0181] Dendritic cell activation is a crucial step in priming the T cell response in CIC. 1Therefore, we next tested whether CD40 overexpression promotes BMDC activation in the presence of an anti-CD40 agonist antibody (CD40Ab). Compared to PBS, CD40 Ab, and CD40-DIM7S treatment, the combination of CD40-DIM7S and CD40 Ab (CD40-DIM7S + CD40 Ab) stimulated dendritic cell activation markers including CD80 (P<0.01), CD86 (P<0.01), MHC-II (P<0.01), IL1-β (proform, P<0.01), TNF-α (P<0.05), and IL12 (P<0.01) at high levels (Figure 3A), indicating a stronger ability to activate dendritic cells. To evaluate whether enhanced dendritic cell activation improves in vivo antitumor effects, multiple intratumor (it) injections were administered four times daily in a B16F10 melanoma mouse model (Figure 3B). Treatment strategies included various combinations, including CD40Ab, CD40-DIM7S, CD40-DIM7S+CD40Ab, BMDC+CD40Ab, CD40-BMDC, and CD40-BMDC+CD40Ab. Compared to all other treatments, CD40-BMDC+CD40Ab significantly suppressed tumor growth by 5–15 times (23 days post-inoculation, P<0.05, Figure 3C, Figure 8) and significantly extended overall survival (Figure 3D, P<0.01). Indeed, CD40-DIM7S can be used to manipulate dendritic cells in situ to increase CD40 receptor expression (Figure 9A). However, CD40-DIM7S+CD40 Ab showed limited therapeutic efficacy compared to CD40-BMDC+CD40 Ab (Figures 3C, 3D, and 8). This may be partly due to the lack of infiltrating dendritic cells in immunosuppressive TMEs. 1,2 Although dendritic cells in tumor tissue were replenished by BMDC+CD40 Ab, insufficient BMDC activation may be the reason for the weak antitumor effect. 1,2 These results highlight the overall importance of sufficient dendritic cell infiltration, CD40 overexpression on BMDCs, and CD40 agonists for the antitumor activity of CD40-BMDC + CD40 Ab therapy.

[0182] Although CD40-BMDC+CD40 Ab showed promising antitumor effects, the inability to completely eliminate the tumor indicates insufficient progression of CIC. Cancer cell death, the first step in CIC, is presented by dendritic cells, enabling the release of TAAs that promote tumor-specific T cell responses. 1,3-5 Furthermore, clinical studies have revealed that ICDs in cancer cells are beneficial to the antitumor effects of immunotherapy. 3.4Therefore, we investigated whether LNP-induced cancer cell ICDs could enhance the antitumor effect of CD40-BMDC therapy. We also examined LNP-mediated CD40L expression in tumor tissue and whether this could activate adoptively transferred CD40-BMDCs. As a result, the combination of CD40L-LNP-induced cancer cell ICDs and adoptively transferred CD40-BMDCs effectively increased CICs, thereby eliminating tumor lesions (Figure 1A). The cytotoxicity of DIM7S and LIS10W containing CD40L mRNA (CD40L-DIM7S and CD40L-LIS10W) was examined in B16F10 melanoma cells. LIS10W induced potent cytotoxicity (85%) after 18 hours of treatment (Figure 9B). Since both CD40L-DIM7S and CD40L-LIS10W resulted in nearly 100% CD40L expression in B16F10 melanoma cells after 18 hours of incubation (Figure 9C), LIS10W was selected to evaluate the following capabilities. In B16F10 melanoma cells, ICD markers including extracellular high-mobility group box 1 (HMGB1), extracellular ATP, and cell surface calreticulin were selected to evaluate their inducing ability. Compared to PBS treatment, FLuc-LIS10W or CD40L-LIS10W increased the levels of extracellular HMGB1, extracellular ATP, and cell surface calreticulin in vitro by approximately 1.7-fold (P<0.01), 7-fold (P<0.001), and 45-fold (P<0.001), respectively (Figure 3E). ICD induced by FLuc-LIS10W or CD40L-LIS10W has consistently been observed in vivo mouse B16F10 melanoma tissue (Figure 3F), suggesting that LNPs in LIS10W contribute to the ICD effect in addition to mRNA cargo.

[0183] Because the expression profile of CD40L in tumor tissue may influence its biological activity, its in vivo distribution was studied at the cellular level using flow cytometry. A single injection of CD40L-LIS10W resulted in approximately 15% CD40L-positive B16F10 cells in tumor tissue (Figure 9D). Furthermore, immune cells within tumor tissue, including macrophages, dendritic cells, CD4 T cells, and CD8 T cells, showed approximately 1.5 to 2-fold increased CD40L expression (Figure 9E). CD40L expression in both cancer cells and immune cells may contribute to the activation of CD40-BMDCs. 18 .

[0184] To evaluate the antitumor effect of the combination of cancer cell ICD and adoptive CD40-BMDC, CD40-BMDC was administered intratumorally 18 hours after intratumoral injection of CD40L-LIS10W (Figure 3G). Four doses of this treatment regimen resulted in complete tumor regression in 83% of B16F10 tumor-bearing mice (Figure 3H, Figure 10A). This was significantly higher than CD40-BMDC + CD40 Ab treatment (0%, P<0.05) and CD40L-DIM7S + CD40-BMDCs treatment (17%, P<0.05). Importantly, all responder mice in the CD40L-LIS10W + CD40-BMDC group were resistant to B16F10 tumor re-attack in the contralateral abdomen (Figure 3I, Figure 10B), indicating the development of antitumor memory. These results highlight the crucial role of ICD in priming antitumor immunity in CIC. To further confirm the function of CD40-BMDC and CD40L-mediated activation in this treatment plan, two other treatment groups, including FLuc-LIS10W+CD40-BMDC and CD40L-LIS10W+CD40-DIM7S, were further studied (Figure 3J). FLuc-LIS10W and CD40L-LIS10W showed similar in vivo ICD effects (Figure 3F), but CD40L-LIS10W+CD40-BMDC dramatically suppressed tumor growth by approximately 21 times (P<0.05) and 7 times (19 days post-inoculation, P<0.05), respectively, compared to FLuc-LIS10W+CD40-BMDC and CD40L-LIS10W+CD40-DIM7S (Figures 5C, 5D). Furthermore, 83% of mice survived in the CD40L-LIS10W+CD40-BDMC group, while no mice survived 45 days after tumor inoculation in the other two treatment groups (P<0.01, Figure 3K). Additionally, re-administration of B16F10 cells to the contralateral flank resulted in 100% responder mice showing resistance to secondary tumor development (Figure 3L, Figure 10E). These findings suggest that ICD treatment alone is insufficient to eradicate tumor lesions, highlighting the importance of dendritic cell infiltration and activation. Long-term efficacy of immunotherapy requires anti-tumor memory against tumor recurrence. Therefore, the development of anti-tumor memory was further investigated via an intracranial (ic) rechallenge mouse model (Figure 3M).In this experiment, a different melanoma cell line (B16F10-Luc2) containing a luciferase reporter was used, which facilitated monitoring of brain tumor growth by bioluminescence imaging. CD40L-LIS10W+CD40-BDMC treatment achieved complete regression of approximately 88% of primary subcutaneous tumors 45 days after tumor inoculation (Figure 3N, Figure 10F). In particular, surviving mice showed significantly delayed tumor growth in the brain (11 days post-inoculation, P<0.05, Figure 10G). Furthermore, 38% of responder mice remained tumor-free compared to 0% of naive controls (75 days post-ic rechallenge, P<0.01, Figure 3O). These data indicate that a robust population of anti-tumor memory T cells was generated from this topical therapy.

[0185] The effectiveness of local immunotherapy in the treatment of metastatic cancer depends on the development of systemic anti-tumor immunity. 5,7,10 To evaluate this, B16F10 cells were seeded bilaterally in each mouse, followed by CD40L-LIS10W+CD40-BDMC treatment at one tumor site (Figure 4A). This treatment eliminated 80% of the primary tumor (Figure 11A) and 70% of the distal tumor (Figure 4B), with an overall survival rate of approximately 70% (Figure 4C). In two other tumor models (skin + brain tumors), B16F10-Luc2 cells were inoculated subcutaneously and intracytoplasmically (Figure 4D). CD40L-LIS10W+CD40-BDMC treatment eradicated 80% of the subcutaneous tumors (Figure 11B). The treatment also visibly degraded tumor growth in the brain (11 days post-inoculation, P<0.001, Figure 4E) and significantly extended overall survival (P<0.0001, Figure 4F)). These results, taken together, indicate that local treatment resulted in systemic anti-tumor immunity.

[0186] T-cell-mediated cancer cell death is a crucial step in eradicating neoplastic lesions in CIC. To evaluate the role of CD8 and CD4 T cells in therapeutic efficacy, mice treated with CD40L-LIS10W+CD40-BDMC were administered with anti-CD8 antibody, anti-CD4 antibody, or isotype control antibody (Figure 4G). Compared to the control group, CD8 or CD4 T cell depletion significantly impaired tumor regression and mouse survival in CD40L-LIS10W+CD40-BDMC treatment (Figures 4H, 4I, and 11C). In particular, CD8 T cell depletion more significantly reduced therapeutic efficacy compared to CD4 T cell depletion (Figures 4H, 4I, and 11C), suggesting a crucial role of CD8 T cells in this treatment.

[0187] To elucidate the underlying mechanisms of action of the CD40L-LIS10W + CD40-BDMC therapeutic regimen, dynamic expression of cytokines and chemokines was used in mouse melanoma tissue and blood after the initial injection (Figure 4J). In tumor tissue, CD40L-LIS10W induced 29 cytokines and chemokines, including GM-CSF, IFN-γ, TNF-α, CCL5, and CXCL10, in a 32-plex panel over a 6–24-hour time course (Figure 4K, Figure 12). Furthermore, their concentrations increased further after administration of CD40-BDMC (Figure 4K, Figure 12). In blood, the majority of cytokines and chemokines were upregulated 6 hours after CD40L-LIS10W infusion, and their concentrations gradually decreased over the next 18 hours (Figure 4L, Figure 13). However, administration of CD40-BDMC restimulated the production of many cytokines and chemokines over a 24 to 42-hour time course (Figure 4L, Figure 13). These data indicate that CD40L-LIS10W + CD40-BDMC reprogrammed well with immunosuppressive TME and induced a stronger systemic immune response than CD40L-LIS10W alone. This upregulation of inflammatory cytokines and chemokines may not only enhance the recruitment and activation of immune cells but also promote the development of immunological memory. Next, we profiled changes in immune cell populations in tumor tissue after treatment with CD40L-LIS10W + CD40-BDMCs. In this experiment, CD40-BDMCs were labeled with CellTrace Blue before injection, and administered dendritic cells and endogenous dendritic cells could be distinguished by flow cytometry analysis. The treatment resulted in a massive influx of dendritic cells, CD8 T cells, and CD4 T cells, while the recruitment of macrophages and regulatory T (Treg) cells in tumor tissue decreased (Figure 5A). Furthermore, CD80 / 86 + Macrophages (P<0.01) and CD80 / 86 +The proportion of antitumor phenotypes in APCs, including dendritic cells (P<0.0001, Figure 5B), increased with this treatment. Importantly, this treatment stimulated the expression of Ki-67 (P<0.01), IFN-γ / TNF-α (P<0.0001), and granzyme B (P<0.001, Figure 5C) in CD8 T cells, indicating the generation of cytotoxic T cells. Memory T cells were also examined in the spleen and blood of responder mice. Compared to naive controls, the number of effector memory and central memory T cells was significantly increased in both the spleen and blood, suggesting the development of long-term antitumor immunity.

[0188] Consideration The immunosuppressive tumor microenvironment (TME) blocks immune activation in the cancer-immune cycle (CIC), leading to uncontrolled tumor growth, metastasis, and recurrence. To overcome incomplete activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD) was combined with dendritic cell therapy to adequately enhance the CIC through a comprehensive immune response. A library of ionizable lipids derived from sugar alcohols was synthesized, and two preferred LNP formulations, DIM7S and LIS10W, were identified for effective mRNA delivery. DIM7S showed 10-fold and 30-fold higher mRNA delivery efficiencies in BMDCs compared to Lipo3K and Electro, respectively (Figure 7D). LIS10W induced potent ICD in melanoma tissue (Figure 3F) and effectively delivered mRNA to melanoma cells (Figures 9C, 9D). In the treatment of CD40L-LIS10W + CD40-BMDC, CD40L-LIS10W was administered first to induce cancer cell death. This resulted in the release of TAAs and DAMPs (e.g., HMGB1, extracellular ATP, and cell surface calreticulin), and simultaneously, CD40L expression occurred in tumor tissue. Next, CD40-BMDCs were adoptively transferred to CD40-BMDCs engineered in vitro with CD40-DIM7S. Data from both in vitro and in vivo showed that these activated dendritic cells upregulated costimulatory molecules (Figure 3A, Figure 5B) and inflammatory cytokines (Figure 3A, Figure 4K, 4L), and, together with presented TAAs, stimulated the differentiation of naive T cells into cytotoxic CD8 T cells (Figure 5C). This is beneficial, on the one hand, for T cell-mediated cancer cell killing, which is a crucial step in eliminating tumor lesions in CIC. Meanwhile, T cell priming promotes the production of IFN-γ and TNF-α, which activates APCs and induces the production of chemokines such as CCL3, CCL4, CCL5, and CXCL10 (Figures 4K, 4I). In response to these chemokines, additional APCs and T cells are recruited to the tumor tissue (Figure 5A). All of these events contribute to the reprogramming of immunosuppressive TMEs and the continued amplification and expansion of CICs, ultimately resulting in the complete regression of more than 80% of primary cutaneous melanoma tumors (Figures 3H, 3K, 3N).Importantly, boosted CIC beneficially stimulated the generation of memory CD8 T cells in the spleen and blood (Figures 5D, 5E), resulting in nearly 100% and 38% protection, respectively, from subcutaneous melanoma re-attack and IC melanoma re-challenge in responder mice (Figures 3I, 3L, 3O). Finally, this topical treatment prevented approximately 70% of distal skin tumors, significantly reduced distal brain tumors, and suggested effective suppression of metastatic cancer.

[0189] In summary, it has been shown that synergistic induction of cancer cell immunogenic death (ICD) and dendritic cell activation can reprogram the first two steps of the cancer immune cycle (CIC) to immunosuppressive TMEs and promote tumor antigen presentation. This strategy yields a strong effector T cell response and protective immunological memory against cancer-specific antigens, which translates to the eradication of both local and distal tumors and prevention from tumor rechallenge. Overall, the combination of lipid nanoparticle mRNA formulations and dendritic cell therapy provides an important platform for cancer immunotherapy.

[0190] Materials and methods antibody Antibodies used in flow cytometry: CD40-FITC (eBioscience, 11040285), CD40L-FITC (eBioscience, MA516506), CD80-FITC (eBioscience, 11080181), CD86-PE (eBioscience, 12086281), CD86-FITC (eBioscience, 11086281), MHCII-APC (eBioscience, 17532080), Proform IL-1β-FITC (eBioscience, 11711480), Calreticulin-Alexa Fluor 647 (Novus Biologicals, NBP1-47518AF647), Ki-67-FITC (eBioscience, 11569882), Granzyme B-FITC (eBioscience, 11889882), IFNγ-FITC (Biolegend, 505806), TNFα-FITC (eBioscience, 11732181), CD62L-FITC (eBioscience, 11-0621-81), CD44-Alexa Fluor 700 (eBioscience, 56044182) Antibodies used in ELISA: Rat anti-mouse IL-12 p70 (eBioscience, MM121), rat anti-mouse TNFα (eBioscience, 14732581), goat anti-rat IgG-HRP (Cell Signaling, 7077S), mouse anti-mouse HMGB1 (eBioscience, MA120338), goat anti-mouse IgG-HRP (Abcam, ab7068), Antibodies used in vivo: Anti-mouse CD40 antibody (CD40 Ab, BioXcell, BP00162), anti-mouse CD8α (Bioxcell, BE0004-1), anti-mouse CD4 (Bioxcell, BP0003-1), anti-rat IgG2b isotype (Bioxcell, BP0090)

[0191] cell culture The B16F10 melanoma cell line was obtained from the laboratory of Dr. Jianhua Yu. The B16F10-Luc2 melanoma cell line was purchased from the American Type Culture Collection (ATCC). These two cell lines were cultured in an incubator at 37°C in 5% CO2 using Dulbecco's modified Eagle medium (ATCC, 302002) containing 10% fetal bovine serum (FBS, Gibco, 26140079). Mouse bone marrow-derived dendritic cells (BMDCs) were obtained by adapting the previous procedure. 19 In short, monocytes were isolated from mouse bone marrow and cultured in RPMI 1640 medium containing 10% FBS, 50 ng / mL GM-CSF (Shenandoah Biotechnology, 20015), and 50 ng / mL IL4 (Shenandoah Biotechnology, 20018). After 8 days of culture, BMDCs were purified with CD11c beads (Miltenyi Biotec, 130108338). Flow cytometry gating The gate strategy is a previously reported method. 10,19 It was based on that.

[0192] B16F10 cells: CD45 - Macrophage: CD45 + CD11b + F4 / 80 + Activated macrophages: CD45 + CD11b + F4 / 80 + , CD80 + / CD86 + Dendritic cells: CD45 + CD11b + CD11c + Activated dendritic cells: CD45 + CD11b + CD11c + , CD80 + / CD86 + CD4 T cells:CD45 + CD3e + CD4 + FoxP3 - Regulatory T cells: CD45 + CD3e + CD4 + FoxP3 + CD8 T cells: CD45 + CD3e + CD8a + Effector Memory T Cells: CD45 + CD3e + CD8a + CD44 hi CD62L lo Central memory T cells: CD45 + CD3e + CD8a + CD44 hi CD62L hi Activated CD8 T cells: CD45 + CD3e + CD8a + Ki-67 + / Granzyme B + / IFNγ + / TNFα +

[0193] mRNA synthesis Linear dsDNA of firefly luciferase (FLuc), mouse CD40, and mouse CD40L was obtained from Integrated DNA Technologies. Plasmids were generated using the pUC19 vector. mRNA of FLuc, mouse CD40, and mouse CD40L was synthesized using previously reported methods. 20 .

[0194] Preparation and characterization of lipid nanoparticles (LNPs) mRNA LNPs were prepared by mixing an ethanol solution containing ionizable lipids, DOPE, cholesterol, and DMG-PEG2000 with a citrate solution containing mRNA via NanoAssemblr (Precision NanoSystems, Canada). 19 Size, polydispersity index (PDI), and zeta potential were measured using a NanoZS Zetasizer (Malvern, USA). Encapsulation efficiency was detected by a Ribogreen assay. Morphology was observed using a Glacios Cryo-TEM (Thermo Scientific, USA).

[0195] In the initial screening, newly synthesized ionizable lipids were combined with DOPE, cholesterol, and DMG-PEG2000 (lipid:dope:cholesterol = 20:30:40:0.75, molar ratio) and FLuc mRNA (lipid:mRNA = 10:1, mass ratio). 21 LNP is L16(4) 4 Preparations were made based on orthogonal tables and predicted formulations. The lipofectamine 3000 / mRNA complex was prepared according to the recommended protocol. Electroporation for BMDCs was performed using a mouse dendritic cell nuclear reagent kit (Lonza, VAPA1011). mRNA delivery efficacy was determined by luciferase expression assay.

[0196] Expression of CD40 and D40L Approximately 1×10 6 Each BMDC was seeded into a 6-well plate and treated with 2.5 μg of CD40-LNP for 12 hours. Approximately 1 × 10⁶ cells were grown. 5 Individual B16F10 cells were seeded in each well of a 24-well plate and treated with 0.25 μg of CD40L-LNP for 18 hours. The cells were then stained with CD40-FITC or CD40L-FITC antibody and analyzed using an LSRFortessa flow cytometer (Becton Dickinson, USA).

[0197] Tumor volume approximately 500 mm² 2Mice were injected with 10 μg of FLuc-LNP, CD40-LNP, or CD40L-LNP. After 6 hours, the tumors were dissected using a mouse tumor dissociation kit (Miltenyi Biotec, 130-096-730). Infiltrating immune cells were isolated using Ficoll-Paque density gradient medium (Cytiva, 17544602). After staining with antibody combinations based on the flow cytometry gate method described above, the cells were analyzed by flow cytometry.

[0198] Activation and analysis of dendritic cells CD40-overexpressing BMDCs (CD40-BMDCs) were incubated with 10 μg / mL anti-mouse CD40 antibody for 12 hours. Several activation markers were then stained with CD80, CD86, MHC-II, and proform IL1-β antibodies and analyzed by flow cytometry. Other activation markers, including IL-12 and TNF-α, were detected by ELISA. LNP-induced immunogenic cell death (ICD) ICD markers were detected by adapting previous methods. 5 The cytotoxicity of LNPs in B16F10 cells was investigated by MTT assay. Extracellular ATP was measured using a luminescent ATP detection kit (Abcam, ab113849). Extracellular HMGB1 levels were detected by ELISA. Cell surface calreticulin was detected by flow cytometry.

[0199] Tumor models and treatment regimens C57BL / 6 mice (male and female, 6-8 weeks old) were purchased from Jackson Laboratory and housed in the University Laboratory Animal Resources Biomedical Research Tower at Ohio State University. All mouse studies were approved by the Institutional Animal Care and Use Committee (IACUC) at Ohio State University and complied with local, state, and federal regulations.

[0200] In the case of a unilateral subcutaneous (sc) tumor model, approximately 1 × 10⁻⁶ 5 Individual B16F10 cells or 2 × 10 510 B16F10-Luc2 cells were subcutaneously injected into the right flank of mice. Seven days after tumor inoculation, mice with a maximum tumor size of approximately 0.5 cm in diameter were randomly assigned to different treatment groups. In the bilateral subcutaneous tumor model, approximately 1 × 10⁶ cells were used. 5 A number of B16F10 cells were subcutaneously injected into the right flank of mice. Five days after tumor inoculation into the right flank, approximately 1 × 10⁶ cells were present. 5 B16F10 cells were subcutaneously injected into the left flank of mice. Seven days after tumor inoculation into the right flank, mice with tumors up to approximately 0.5 cm in diameter were randomly assigned to different treatment groups. In the skin + brain 2 tumor model, approximately 2 × 10 5 B16F10-Luc2 cells were subcutaneously injected into the right flank of mice. Five days after tumor inoculation into the right flank, approximately 1 × 10⁶ cells were observed. 4 10 B16F10-Luc2 cells were injected intracranially (ic) at a depth of 3 mm. The injection sites were 2 mm lateral to the sagittal suture and 1 mm prior to the coronal suture. Seven days after tumor implantation in the right flank, mice with tumors up to approximately 0.5 cm in diameter were randomly assigned to different treatment groups. In the subcutaneous tumor re-challenge, approximately 1 × 10⁶ cells were injected 45 days after tumor inoculation. 5 Individual B16F10 cells were subcutaneously injected into the left flank of mice that responded fully. In the brain tumor re-challenge, approximately 1 × 10¹⁶ cells were obtained 45 days after tumor inoculation. 4 Individual B16F10-Luc2 cells were injected into fully responsive mice. In the case of a T cell depletion tumor model, 1 × 10⁶ cells were injected. 5 Individual B16F10 cells were subcutaneously injected into the right flank of mice. Six days after tumor inoculation, the mice were intraperitoneally treated with anti-mouse CD8α, anti-mouse CD4, or anti-rat IgG2b isotype antibodies. Each antibody was administered at a dose of 200 μg per injection, every three days for three doses.

[0201] The treatment regimens in this study were as follows: In a single IT injection, the doses of CD40 Ab and LNP were 50 μg and 5 μg, respectively, and the dose of dendritic cells was 2 million. In the case of CD40-LNP + CD40 Ab treatment, CD40 Ab was administered 6 hours after the injection of CD40-LNP. In the case of dendritic cell + CD40 Ab treatment, CD40 Ab was administered 1 hour after the injection of dendritic cells. In the case of CD40-LNP + CD40L-LNP treatment, CD40-LNP was administered 18 hours after the injection of CD40L-LNP. In the case of CD40L- or FLuc-LNP + CD40-BMDC treatment, CD40-BMDC was administered 18 hours after the injection of CD40L- or FLuc-LNP. All of these treatment regimens included four doses.

[0202] The elimination criteria in this study were as follows: Subcutaneous tumor size was measured every 2-4 days and calculated as volume (length × width × width / 2). Mice were sacrificed when the maximum tumor diameter reached 1.6 cm or when body weight decreased by more than 20%. Brain tumor size was monitored using IVIS Lumina II (Caliper Life Sciences, USA). Mice were euthanized if they exhibited a hunched posture, had difficulty walking or eating, or lost more than 20% of their body weight.

[0203] Analysis of cytokines and chemokines using Luminex Tumor volume approximately 500 mm² 2Mice were injected with a single dose of CD40L-LNP+CD40-BMDC. Tumor tissue and serum from the mice were collected 0 to 42 hours after injection. Tumor tissue was frozen in liquid nitrogen, and the pulverized tissue was extracted at 150 mg / ml in RIPA lysis buffer (Thermo Scientific, 89900) containing a protease inhibitor (Thermo Scientific, 87785). Whole blood was collected in a tube containing sodium citrate, and serum was collected by centrifugation (10000 rpm) at 4°C for 5 minutes. Tumor lysates and serum were stored at -80°C. Cytokines and chemokines in the mice were detected by the mouse cytokine / chemokine discovery assay (Eve Technologies, Canada).

[0204] Analysis of immune cell populations and activation in vivo Tumor volume approximately 250 mm² 2 Mice were treated twice with CD40L-LNP+CD40-BMDC. Tumor tissue was then collected and dissociated using a mouse tumor dissociation kit (Miltenyi Biotec, 130-096-730). For profiling of the immune cell population, all cells were stained with antibody combinations based on the flow cytometry gating method described above, and the number of immune cells was counted by flow cytometry. In this experiment, CD40-BMDC was labeled with CellTrace Blue (Invitrogen, C34568) before injection, allowing for differentiation of administered dendritic cells from endogenous dendritic cells by flow cytometry. To evaluate immune cell activation, infiltrating immune cells were isolated using Ficoll-Paque density gradient medium (Cytiva, 17544602). After staining the antibody combinations based on the flow cytometry gating method described above, T cell activation markers were detected by flow cytometry.

[0205] References 1 Chen, D. S. & Mellman, I. Oncology meets immunology: the cancer-immunitance cycle. Immunity 39, 1-10, doi:10.1016 / j.immuni.2013.07.012 (2013). 2 Wculek, S. K. et al. Dendritic cells in cancer immunology and immunotherapy. Nature Reviews Immunology 20, 7-24 (2020). 3 Galluzzi, L., Buque, A., Kepp, O., Zitvogel, L. & Kroemer, G. Immunogenic cell death in cancer and infectious disease. Nature Reviews Immunology 17, 97-111 (2017). 4 Kroemer, G., Galluzzi, L., Kepp, O. & Zitvogel, L. Immunogenic cell death in cancer therapy. Annual review of immunology 31, 51-72 (2013). 5 Li, Y. et al. Multifunctional oncolytic nanoparticles deliver self-replicating IL-12 RNA to eliminate established tumors and prime systemic immunity. Nature Cancer 1, 882-893 (2020). 6 Vonderheide, R. H. CD40 agonist antibodies in cancer immunotherapy. Annual review of medicine 71, 47-58 (2020). 7 Melero, I., Castanon, E., Alvarez, M., Champiat, S. & Marabelle, A. Intratumoural administration and tumour tissue targeting of cancer immunotherapies. Nature Reviews Clinical Oncology 18、558-576(2021)。 8 Hou, X.、Zaks, T.、Langer, R. & Dong, Y. Lipid nanoparticles for mRNA delivery. Nature Reviews Materials 6、1078-1094(2021)。 9 Zhang, Y.、Sun, C.、Wang, C.、Jankovic, KE & Dong, Y. Lipids and lipid derivatives for RNA delivery. Chemical Reviews 121、12181-12277(2021)。 10 Hewitt, S. L. et al. Durable anticancer immunity from intratumoral administration of IL-23, IL-36γ, and OX40L mRNAs. Science translational medicine 11, eaat9143 (2019). 11 Ghosh, S. & Sudha, M. A review on polyols: new frontiers for health-based bakery products. International journal of food sciences and nutrition 63, 372-379 (2012). 12 Zada, B. et al. Recent advances in catalytic production of sugar alcohols and their applications. Science China Chemistry 60, 853-869 (2017). 13 Stoss, P. & Hemmer, R. 1, 4: 3, 6-Dianhydrohexitols. Advances in carbohydrate chemistry and biochemistry 49, 93-173 (1991). 14 Spagnolo, F. et al. Survival of patients with metastatic melanoma and brain metastases in the era of MAP-kinase inhibitors and immunologic checkpoint blockade antibodies: a systematic review. Cancer treatment reviews 45, 38-45 (2016). 15 Wiggins, L. in Advances in carbohydrate chemistry Vol. 5 191-228 (Elsevier, 1950). 16 Fenouillot、F.、Rousseau、A.、Colomines、G.、Saint-Loup、R.、Pascault、J.-P. Polymers from renewable 1, 4: 3, 6-dianhydrohexitols (isosorbide, isomannide and isoidide): A review. Progress in Polymer Science 35, 578-622(2010). 17 Arico, F., Tundo, P., Maranzana, A. & Tonachini, G. Synthesis of five‐membered cyclic ethers by reaction of 1, 4‐Diols with dimethyl carbonate ChemSusChem 5, 1578-1586(2012)。 18 Kikuchi, T., Moore, M. A. & Crystal, R. G. Dendritic cells modified to express CD40 ligand elicit therapeutic immunity against preexisting murine tumors. Blood、The Journal of the American Society of Hematology 96, 91-99(2000). 19 Lee, W. et al. Biomimetic nanoparticles deliver mRNAs encoding costimulatory receptors and enhance T cell mediated cancer immunotherapy. Nature communication 12, 1-12 (2021). 20 Zeng, C. et al. Leveraging mRNA Sequences and Nanoparticles to Deliver SARS‐CoV‐2 Antigens In Vivo. ADvanced Materials 32, 2004452 (2020). 21 Hou, X. et al. Vitamin lipid nanoparticles enable adoptive macrophage transfer for the treatment of multidrug-resistant bacterial sepsis. Nature Nanotechnology 15, 41-46 (2020).

[0206] Example 2. Chemical synthesis of dianhydrohexitol-derived lipids

[0207] [ka] Chemical structure of ionized lipids derived from dianhydrohexitol Aldehyde synthesis:

[0208] [ka] 1-Hexanol 1 (5.1 g, 50 mmol) was added dropwise to a suspension of paraformaldehyde (1.5 g, 50 mmol) in TMSCl (25 mL, 197 mmol) at room temperature. The reaction mixture was stirred at room temperature for 2 hours. The clean solution was concentrated under reduced pressure to obtain 1-chloromethoxyhexane 2 as a colorless oil, which was used directly without further purification.

[0209] The above chloromethyl ether was added dropwise to a solution of 1,6-hexanediol 3 (11.8 g, 100 mmol) and i-Pr2NEt (17.45 mL, 100 mmol) in CH2Cl2 (150 mL). The reaction mixture was stirred at room temperature for 24 hours, and then the reaction was stopped by adding a saturated solution of NH4Cl (80 mL). Extraction with CH2Cl2 (60 mL x 2 times) was performed, and the combined organic layers were washed with water (30 mL) and saturated brine (30 mL) and dried on Na2SO4. The organic phase was filtered, concentrated under reduced pressure, and the residue was purified by silica gel flash chromatography (20% siRNA in hexane). 6.74 g of 4 was obtained as a colorless oil (yield 58.0%). 1 H NMR (300 MHz, chloroform-d) δ 4.65 (s, 2H), 3.63 (q, J = 6.3 Hz, 2H), 3.51 (td, J = 6.6, 2.9 Hz, 4H), 1.57 (dq, J = 13.5, 6.9 Hz, 6H), 1.41-1.21 (m, 10H), 0.88 (t, J = 3.6 Hz, 3H). HRMS (ESI, m / z): [M+Na] + Calculated value. C 13 H 28N a For O3, 255.1931; measured value: 255.1941.

[0210] [ka] (Diacetoxyiodo)benzene (0.79 g, 2.45 mmol) was added at room temperature to a dry DCM suspension of 4 (518 mg, 2.23 mmol), TEMPO (34.9 mg, 0.22 mmol), and NaHCO3 (412 mg, 4.9 mmol) in 20 mL. The reaction mixture was stirred for 3 hours, and TLC showed that 4 was completely consumed. The mixture was then quenched with a saturated aqueous solution of Na2S2O3 (30 mL) and extracted with DCM (3 × 20 mL). The combined DCM layers were washed with aqueous NaHCO3 solution (20 mL) and saturated saline (20 mL), dried on anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel flash chromatography (10% siRNA in hexane). 510 mg of 5 was obtained as a colorless oil (quantitative). 1 ¹H NMR (300 MHz, chloroform-d) δ 9.76 (s, 1H), 4.64 (s, 2H), 3.51 (q, J = 6.09 Hz, 4H), 2.43 (t, J = 7.2 Hz, 1H), 1.7-1.52 (m, 6H), 1.49-1.19 (m, 8H), 0.88 (t, J = 6.3 Hz, 3H).

[0211] [ka] To a 30 mL solution of 6 (5.0 g, 30.4 mmol) in DCM, 1,5-hexanediol 3 and TEA (16.9 mL, 121 mmol) in a 100 mL solution of DCM were added dropwise at 0°C. After stirring for 30 minutes to 3 hours, the reaction was quenched with 50 mL of water, extracted with 3 x 50 mL DCM solutions, then the organic phases were washed with water, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (20% siRNA in hexane). 3.2 g of 7 was obtained as a colorless oil (yield 45.3%). 1H NMR (300 MHz, chloroform-d) δ 4.11 (td, J = 6.6, 2.7 Hz, 4H), 3.62 (td, J = 6.6, 3.3 Hz, 3H), 1.72-1.62 (m, 4H), 1.60-1.53 ​​(m, 2H), 1.43-1.33 (m, 6H), 1.33-1.26 (m, 4H), 0.91-0.84 (t, J =6.6 Hz, 3H). HRMS (ESI, m / z): [M+Na] + Calculated value. C 13 H 26 N a For O4, 269.1723; measured value: 269.1735.

[0212] [ka] BAIB (1.41 g, 4.38 mmol) was added to a 20 mL DCM suspension of 7 (0.98 g, 3.98 mmol), TEMPO (62.2 mg, 0.4 mmol), and NaHCO3 (736 mg, 8.6 mmol). The reaction mixture was stirred for 3 hours until TLC indicated that A was completely consumed. The mixture was stopped with saturated Na2S2O3 (30 mL) and extracted with DCM (3 × 20 mL). The combined DCM layers were washed with aqueous NaHCO3 (20 mL) and brine (20 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel flash chromatography (10% siRNA in hexane). 0.78 g of 8 was obtained as a pale yellow oily substance (yield 80.2%). 1 H NMR (300 MHz, chloroform-d) δ 9.77 (t, J = 1.5 Hz, 1H), 4.13 (td, J = 6.6, 2.7 Hz, 4H), 2.45 (td, J = 7.2, 1.8 Hz, 2H) ), 1.76-1.58 (m, 6H), 1.48-1.25 (m, 8H), 0.89 (t, J = 6.6 Hz, 3H).

[0213] [ka] 1-Hexanol 1 (5.1 g, 50 mmol) was added dropwise to a solution of acetaldehyde (1.76 g, 40 mmol) in TMSCl (20 mL, 157 mmol) at room temperature. The reaction mixture was stirred at room temperature for 2 hours. The clean solution was concentrated under reduced pressure to obtain 1-chloromethoxyhexane 9 as a colorless oil, which was used directly without further purification.

[0214] The above chloromethyl ether was added dropwise to a solution of 1,6-hexanediol 3 (9.44 g, 80 mmol) and i-Pr2NEt (13.96 mL, 80 mmol) in CH2Cl2 (150 mL). The reaction mixture was stirred at room temperature for 24 hours, and then the reaction was stopped by adding a saturated solution of NH4Cl (80 mL). Extraction with CH2Cl2 (60 mL x 2 times) was performed, and the combined organic layers were washed with water (30 mL) and saturated brine (30 mL) and dried on Na2SO4. The organic phase was filtered, concentrated under reduced pressure, and the residue was purified by silica gel flash chromatography (20% siRNA in hexane). 3.45 g of 10 was obtained as a colorless oil (yield 35.0%). 1 H NMR (300 MHz, chloroform-d) δ 4.64 (q, J = 5.4 Hz, 1H), 3.68-3.48 (m, 4H), 3.38 (dtd, J = 9.3, 6.6, 2.4 Hz, 2H), 1.65-1.47 (m, 7H), 1.43-1.23 (m, 13H), 0.87 (t, J = 6.6 Hz, 3H). HRMS (ESI, m / z): [M+Na] + Calculated value. C 14 H 30 N a For O3, 269.2087; measured value: 269.2075.

[0215] [ka] BAIB (1.77 g, 5.5 mmol) was added to a 20 mL DCM suspension of 10 (1.23 g, 5.0 mmol), TEMPO (78.2 mg, 0.5 mmol), and NaHCO3 (924 mg, 11.0 mmol). The reaction mixture was stirred for 3 hours until TLC indicated that A was completely consumed. The mixture was stopped with saturated Na2S2O3 (30 mL) and extracted with DCM (3 × 20 mL). The combined DCM layers were washed with aqueous NaHCO3 (20 mL) and saturated saline (20 mL), dried on anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel flash chromatography (10% siRNA in hexane). 1.0 g of 11 was obtained as a pale yellow oily substance (yield 81.8%). 1 H NMR (300 MHz, chloroform-d) δ 9.78 (s, 1H), 4.66 (q, J = 5.4 Hz, 1H), 3.57 (dq, J = 9.2, 6.8 Hz, 2H), 3.41 (dtd, J) = 9.8, 6.6, 3.3 Hz, 2H), 2.45 (td, J = 7.2, 1.8 Hz, 2H), 1.73-1.51 (m, 6H), 1.48-1.25 (m, 11H), 0.990 (t, J =6.6 Hz, 3H). HRMS (ESI, m / z): [M+Na] + Calculated value. C 13 H 28 N a For O3, 255.1931; measured value: 255.1941.

[0216] Synthesis of sugar alcohol-derived diamines

[0217] [ka] EtONa (75 mg, 1.1 mmol) was added to a solution of L-isosorbide (2.0 g, 10.98 mmol) and diethyl carbonate (DMC) (3.96 g, 43.9 mmol) in 12 mL of MeOH, and the resulting mixture was refluxed for 48 hours. The reaction was then stopped, cooled to room temperature, and diethyl ether (15 mL) was added to the mixture. The reaction mixture was filtered through a Celite pad, and the solvent was removed by distillation. Finally, the product was purified by silica gel chromatography using dichloromethane / methanol (9:1) as the eluent to obtain 1.2 g of the desired product as a white solid. Yield 75%.

[0218] 1 H NMR (400 MHz, heavy water) δ 4.68 (t, J = 4.7 Hz, 1H), 4.52 (d, J = 4.3 Hz, 1H), 4.43 (td, J = 6.9, 5.0 Hz, 1H), 4.36 (d, J = 3.2 Hz, 1H), 4.02-3.94 (m, 2H), 3.90 (dd, J = 10.5, 3.2 Hz, 1H), 3.52 (dd, J = 9.1, 7.4 Hz, 1H). 13 C NMR (75MHz, heavy water) δ 87.33, 81.40, 75.46, 75.07, 71.76, 71.09. MS (ESI, m / z): [M+H] + Calculated value. C6H 11 For O4, 147.1; measured value: 147.1.

[0219] [ka] The dicyanoethylation of the diol was carried out according to a previously reported method. 1Acrylonitrile 16 (7.96 g, 150 mmol) was added dropwise to a tert-butanol solution of diol (7.3 g, 50 mmol) and a 50% NaOH aqueous solution (2.0 g, 0.5 mol%) at 60°C for 30 minutes. The reaction continued for a total of 6 hours. Tert-butanol and excess acrylonitrile were removed under reduced pressure using a rotary evaporator. The residue was dissolved in 100 mL of DCM, and unreacted isosorbide and monocyanoethylated isosorbide were removed by washing three times with water. The organic phase was dried over anhydrous Na2SO4, filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (0% to 100% hexane in dichloromethane) to obtain the corresponding product as a yellow oil.

[0220] Compound 17: Yield 70.5%. 1 H NMR (400 MHz, DMSO-d6) δ 4.60 (t, J = 4.8 Hz, 1H), 4.42 (dt, J = 4.4, 1.2 Hz, 1H), 4.06 (td, J = 6.4, 4.8 Hz, 1H), 4.02-3.98 (m, 1H), 3.89-3.72 (m, 4H), 3.67-3.60 (m, 3H), 3.47 (dd, J = 8.8, 6.8 Hz, 1H), 2.78-2.72 (m, 4H). MS (ESI, m / z): [M+Na] + Calculated value. C 12 H 17 For N2O4, the measured value was 275.1002; the measured value was 275.1007.

[0221] Compound 18: Yield 68%. 1H NMR (300MHz, DMSO-d6)δ 4.52 (dt, J =4.7, 2.4Hz, 2H), 4.07 (tdd, J =8.0, 3.6, 1.6Hz, 2H), 3.89 (dd, J =8.5), 6.7 Hz, 2H), 3.79-3.73 (m, 1H), 3.71 (d, J = 6.1 Hz, 1H), 3.62 (ddd, J = 9.7, 6.5, 5.7 Hz, 2H), 3.50 (t, J = 8.2 Hz, 2H), 2.75 (ddd, J = 6.6, 5.7, 1.1 Hz, 4H). MS (ESI, m / z): [M+Na] + Calculated value. C 12 H 17 For N2O4, 275.1; measured value: 275.1.

[0222] Compound 19: Yield 93%. 1 H NMR (300MHz, DMSO-d6) δ 4.61 (t, J =4.8Hz, 1H), 4.43 (dt, J =4.8, 0.9Hz, 1H), 4.07 (td, J =6.6, 4.8Hz, 1H), 4.03-3.97 (m, 1H), 3.90-3.72 (m, 4H), 3.70-3.59 (m, 3H), 3.48 (dd, J = 8.7, 6.6 Hz, 1H), 2.82-2.71 (m, 4H). 13 C NMR (75MHz, DMSO-d6) δ 120.07, 120.06, 86.42, 84.49, 80.89, 80.46, 73.44, 70.70, 65.47, 64.54, 19.19, 19.14. MS (ESI, m / z): [M+Na] + Calculated value C 12 H 17 For N2O4, 275.1; measured value: 275.1.

[0223] [ka] The diamine was synthesized according to a previously reported method. 2A THF solution (2.0 M, 29.7 mL, 59.4 mmol) of the BH3·THF complex was to be added dropwise over 1 hour at room temperature to a THF solution (25 mL) of a dinitrile compound (3.0 g, 11.89 mmol). After the addition was complete, the reaction mixture was stirred for 48 hours. Then, methanol (30 mL) was carefully added to stop the reaction, during which hydrogen gas was vigorously generated. After stirring for 3 hours, the solvent was removed under reduced pressure, then 40 mL of THF was added to dissolve the residue, followed by the dropwise addition of a diethyl ether solution of hydrochloric acid (2.0 M, 29.7 mL), which formed a white precipitate. The suspension was then filtered to obtain the crude diamine·HCl salt as a white powder. The diamine·2HCl salt was then dissolved in deionized water (30 mL) to obtain a pale yellow solution. To this solution, freshly washed Amberlyst A26-OH (17 g) was added. The resulting suspension was sonicated in an ultrasonic bath at 30°C for 1.5 hours. The suspension was filtered through a Celite pad, and the resin was thoroughly washed with water (3 × 4 mL). The combined colorless, transparent solution was evaporated to dryness (or freeze-dried) using a rotary evaporator to obtain the extended diamine as a pale yellow oily liquid.

[0224] Isosorbide-derived diamine 20: Yield 70%. 1 H NMR (300 MHz, methanol-d4) δ 4.64 (t, J = 4.5 Hz, 1H), 4.54-4.45 (m, 1H), 4.11-4.00 (m, 1H), 3.98-3.84 (m, 4H) ), 3.80-3.69 (m, 1H), 3.66-3.43 (m, 4H), 2.86-2.61 (m, 4H), 1.83-1.67 (m, 4H). HRMS (ESI, m / z): [M+H] + Calculated value. C 12 H 25 For N2O4, the measured value is 261.1809; the measured value is 261.1819.

[0225] Isomannide-derived diamine 21: Yield 68%. HRMS (ESI, m / z): [M+H] + Calculated value. C 12 H 25For N2O4, the measured value was 261.1809; the measured value was 261.1817. L-isosorbide-derived diamine 22: Yield 71%. 1 H NMR (400 MHz, methanol-d4) δ 4.67 (t, J = 4.4 Hz, 1H), 4.56-4.54 (m, 1H), 4.13-4.10 (m, 1H), 3.98-3.92 (m, 2H) ), 3.92-3.86 (m, 2H), 3.78-3.68 (m, 1H), 3.68-3.50 (m, 4H), 2.94-2.71 (m, 4H), 1.85-1.73 (m, 4H). MS (ESI, m / z): [M+H] + Calculated value. C 12 H 25 For N2O4, 261.2; measured value: 261.2.

[0226] [ka]

[0227] Synthesis of β-amino alcohols To a solution of diamine (52 mg, 0.2 mmol) in 2 mL of dry EtOH, epoxide 23 (184 mg, 1.0 mmol) was added. The mixture was then heated to 90°C and stirred for 12 hours. TLC showed that A was completely consumed, and EtOH was removed under reduced pressure. The residue was purified by silica gel chromatography (0% to 20% in dichloromethane [a mixture of 3% NH4OH and 22% MeOH in dichloromethane]) to obtain the desired product.

[0228] DIS-1: Yield 61.2%. 1 H NMR (300 MHz, chloroform-d) δ 4.66-4.60 (m, 1H), 4.51 (d, J = 3.9 Hz, 1H), 3.95 (qd, J = 7.5, 3.9 Hz, 5H), 3.88-3.05 (m, 11H), 2.97-2.05 (m, 12H), 1.82-1.60 (m, 4H), 1.50-1.19 (m, 72H), 0.99-0.78 (m, 12H). HRMS (ESI, m / z): [M+H]+ Calculated value. C 60 H 121 For N2O8, the measured value is 997.9118.

[0229] DIM-1: Yield 69.2%. 1 H NMR (300 MHz, chloroform-d) δ 4.56 (dd, J = 4.5, 2.7 Hz, 2H), 4.11-3.89 (m, 4H), 3.89-3.49 (m, 9H), 3.49-3.23 (m, 5H), 2.86-2.14 (m, 12H), 1.85-1.65 (m, 4H), 1.50-1.17 (m, 72H), 0.91-0.80 (m, 12H). MS (ESI, m / z): [M+H] + Calculated value. C 60 H 121 For N2O8, the value is 997.9. Measured value: 998.0.

[0230] LIS-1: Yield 38.1%. 1 H NMR (300 MHz, chloroform-d) δ 4.67−4.61 (m, 1H), 4.55−4.39 (m, 1H), 4.03−3.80 (m, 5H), 3.55 (tdd, J = 34.4, 30.2, 12.9), 6.2 Hz, 12H), 2.90−2.08 (m, 12H), 1.86-1.61 (m, 4H), 1.47-1.19 (m, 72H), 0.87 (t, J = 6.5 Hz, 12H). MS (ESI, m / z): [M+H] + Calculated value. C 60 H 121 For N2O8, the value is 997.9. Measured value: 998.0.

[0231] [ka] General synthetic procedure for reductive amination of diamines: Aldehyde (1.0 mmol) was added to a solution of diamine (52 mg, 0.2 mmol) in THF (4 mL), and the mixture was stirred at room temperature for 30 minutes. Then, NaBH(OAc)3 (254 mg, 1.2 mmol) was added to the above solution, and the resulting mixture was stirred for 12 hours. The reaction was stopped by adding aq. NaHCO3 solution (15 mL). Next, the aqueous solution was extracted with DCM (15 mL x 3), the organic layers were combined, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (0% to 100% [a mixture of 3% NH4OH and 22% MeOH in dichloromethane]) to obtain the desired product.

[0232] Compound DIS-2: Yield 37.0%. 1 H NMR (300 MHz,chloroform-d) δ 4.60 (t,J = 4.2 Hz,1H),4.48 (d,J = 4.2 Hz,1H),4.02-3.84 (m,5H),3.68 (dt,J = 9.3,6.6 Hz,1H),3.62-3.33 (m,4H),2.63-2.18 (m,12H),1.79-1.60 (m,4H),1.49-1.15 (m,48H),0.88 (t,J = 6.6 Hz,12H).MS (ESI,m / z): [M+H] + Calculated value.C 44 H 89 For N2O4, 709.7; measured value: 709.9. Compound DIS-3: Yield 37.0%. 1 H NMR (300 MHz,chloroform-d) δ 4.60 (t,J = 4.2 Hz,1H),4.48 (d,J = 4.2 Hz,1H),4.03-3.87 (m,5H),3.75-3.62 (m ,1H),3.62-3.38 (m,4H),2.61-2.40 (m,4H),2.40-2.25 (m,8H),1.76-1.65 (m,4H),1.48-1.15 (m,64H),0.88 ((t,J = 6.6 Hz,12H).MS (ESI,m / z): [M+H] + Calculated value.C52 H 105 Regarding N2O4, 821.8. Measured value: 821.9. Compound DIS-4: Yield 49.3%. 1 H NMR (300 MHz,chloroform-d) δ 4.59 (t,J = 4.5 Hz,1H),4.48 (d,J = 4.2 Hz,1H),4.01-3.85 (m,5H),3.72-3.65 (m ,1H),3.62-3.37 (m,4H),2.60-2.28 (m,12H),1.83-1.55 (m,4H),1.48-1.32 (m,8H),1.30-1.17 (s,72H),0.97-0.74 (m,12H).. MS (ESI,m / z): [M+H] + Calculated value.C 60 H 121 For N2O4, 933.9321; measured value: 933.9320. Compound DIS-5: Yield 33.0%. 1 H NMR (300 MHz,chloroform-d) δ 4.60 (t,J = 4.2 Hz,1H),4.48 (d,J = 4.2 Hz,1H),4.02-3.85 (m,5H),3.72-3.64 (m ,1H),3.62-3.38 (m,4H),2.54-2.25 (m,12H),1.79-1.61 (m,4H),1.50-1.15 (m,96H),0.98-0.81 (m,12H).MS (ESI,m / z): [M+H] + Calculated value.C 68 H 137 Regarding N2O4, measurement value: 1046.0. Compound DIS-6: Yield 19.1%. 1H NMR (300 MHz, クロロホルム-d) δ 4.59 (t,J = 4.2 Hz,1H),4.48 (d,J = 4.2 Hz,1H),4.02-3.85 (m,5H),3.72-3.65 (m ,1H),3.63-3.41 (m,4H),2.73-2.30 (m,12H),1.84-1.65 (m,4H),1.50-1.35 (m,8H),1.35-1.17 (m,104H),0.92-0.80 (m,12H).MS (ESI,m / z): [M+H] + Calculation value.C 76 H 153 N2O4について,1158.2; measured value: 1158.2. Compound DIS-7: Yield 45.7%. 1 H NMR (400 MHz,クロロホルム-d) δ 4.81 (ddd,J = 12.4,6.8,5.6 Hz,4H),4.59 (t,J = 4.0 Hz,1H),4.48 (d,J = 4.2 Hz,1H) ),4.02―​​3.80 (m,5H),3.68 (dt,J = 9.1,6.5 Hz,1H),3.70―3.63 (m,1H),3.52―3.40 (m,3H),2.54―2.39 (m,4H) ),2.35 (tt,J = 7.2,3.2 Hz,8H),2.28 (t,J = 7.6 Hz,8H),1.86―1.41 (m,28H),1.41-1.05 (m,64H),0.94-0.79 (m,24H).HRMS (ESI,m / z): [M+H] + Calculation value.C 80 H 153 N2O 12 について,1334.1418; measured value: 1334.1404. Compound DIS-8: Yield 35.0%. 1H NMR (300 MHz, クロロホルム-d) δ 4.59 (t,J = 4.2 Hz,1H),4.47 (d,J = 4.2 Hz,1H),4.11 (t,J = 6.9 Hz,16H),4.01-3.86 (m,5H),3.74-3.62 (m,1H),3.60-3.46 (m,4H),2.47-2.30 (m,12H),1.77-1.61 (m,20H),1.43-1.24 (m,48H),1.01-0.79 (m,12H).MS (ESI,m / z): [M+H] + Calculation value.C 64 H 121 N2O 16 について,1173.9. Measured value: 1174.0. Compound DIS-9: Yield 31.0%. 1 H NMR (300 MHz, クロロホルムd) δ 4.66 (s,8H),4.60 (t,J = 4.2 Hz,1H),4.48 (d,J = 4.2 Hz,1H),4.01-3.85 (m,4H) ),3.72-3.64 (m,1H),3.60-3.38 (m,20H),2.60-2.30 (m,12H),1.79-1.52 (m,20H),1.49-1.22 (m,48H),0.95-0.80 (m,12H).MS (ESI,m / z): [M+H] + Calculation value.C 64 H 129 N2O 12 について,1118.0; measured value: 1118.1. Compound DIS-10: Yield 54.5%. 1 H NMR (300 MHz, クロロホルムd) δ 4.65 (d,J = 5.2 Hz,4H),4.58 (t,J = 4.2 Hz,1H),4.47 (d,J = 4.2 Hz,1H),4.02-3.83 (m,5H),3.67-3.46 (m,12H),3.47-3.30 (m,9H),2.53-2.26 (m,12H),1.84-1.47 (m,30H),1.46-1.14 (m,50H) ),0.86 (d,J = 6.9 Hz,12H).MS (ESI,m / z): [M+H] + Calculation value.C68 H 137 N2O 12 について,1174.0; measured value: 1174.2. Compound DIM-2: Yield 54.5%. 1 H NMR (300 MHz, クロロホルム-d) δ 4.59-4.49 (m,2H),4.11-3.92 (m,4H),3.75-3.61 (m,4H),3.51 (dt,J = 9.0,6.6 Hz,2H),2.48 (td,J = MS (ESI,m / z): [M+H] + Calculation value.C 44 H 89 N2O4について,709.7. Measured value: 709.8. Compound DIM-3: Yield 31.7%. 1 H NMR (300 MHz, クロロホルムd) δ 4.52 (dd,J = 3.0,1.2 Hz,2H),4.07-3.93 (m,4H),3.75-3.58 (m,4H),3.49 (dt,J = 9.0,6.6 Hz,2H),2.46 (td,J = 6.9,1.8 Hz,4H),2.41 - 2.27 (m,8H),1.74 (p,J = 6.9 Hz,4H),1.39 (p,J = 6.8) ,6.2 Hz,8H),1.32-1.15 (m,56H),0.87 (t,J = 6.6 Hz,12H).MS (ESI,m / z): [M+H] + Calculation value.C 52 H 105 N2O4について,821.8. Measured value: 821.9. Compound DIM-4: yield 49.3%. 1H NMR (400 MHz, クロロホルム-d) δ 4.53 (dt,J = 4.4,2.0 Hz,2H),4.08-3.94 (m,4H),3.72-3.60 (m,4H),3.49 (dt,J = 9.2,6.8Hz,2H),2.60-2.46(m,4H),2.47-2.30(m,8H),1.76(p,J =7.0Hz,4H),1.49-1.35(m,8H),1.31-1.20 (m,72H),0.88 (t,J = 6.8 Hz,12H).HRMS (ESI,m / z): [M+H] + Calculation value.C 60 H 121 N2O4について,933.9321; measured value: 933.9321. Compound DIM-5: Yield 22.0%. 1 H NMR (300 MHz, クロロホルムd) δ 4.52 (dd,J = 3.0,1.2 Hz,2H),4.09-3.93 (m,4H),3.75-3.59 (m,4H),3.49 (dt,J = 9.0,6.6 Hz,2H),2.46 (td,J = 6.9,1.8 Hz,4H),2.41 - 2.27 (m,8H),1.74 (p,J = 6.9 Hz,4H),1.50 - 1.15(m,96H),0.88 (t,J = 6.6 Hz,12H).MS (ESI,m / z): [M+H] + Calculation value.C 68 H 137 N2O4について,1046.1. Measured value: 1046.0. Compound DIM-6: Yield 37.3%. 1 H NMR (300 MHz, クロロホルム-d) δ 4.55 (dd,J = 3.3,1.5 Hz,2H),4.10-3.93 (m,4H),3.74-3.58 (m,4H),3.49 (dt,J = 9.0,6.6 Hz,2H),2.46 (td,J = 6.9,1.8 Hz,4H),2.35 (dd,J = 8.5,6.3 Hz,8H),1.74 (p,J = 6.9 Hz,4H),1.45-1.15 (m,112H),0.88 (t,J = 6.6 Hz,12H).MS (ESI,m / z): [M+H]+ Calculation value.C 76 H 153 N2O4について,1158.2; measured value: 1158.0. Compound DIM-7: Yield 36.0%. 1 H NMR (400 MHz, クロロホルム-d) δ 4.81 (p,J = 6.4 Hz,4H),4.53 (d,J = 2.8 Hz,2H),4.08-3.94 (m,4H),3.75-3.60 (m ,4H),3.48 (dt,J = 9.0,6.6 Hz,2H),2.51 (t,J = 7.2 Hz,4H),2.39 (t,J = 7.6 Hz,8H),2.27 (t,J = 7.6 Hz) ,8H),1.76 (p,J = 6.8 Hz,4H),1.66 - 1.46 (m,24H),1.45 - 1.35 (m,8H),1.33 - 1.19 (m,56H),0.91 - 0.82 (m,24H). Compound DIM-8: Yield 32.5%. 1 H NMR (300 MHz, クロロホルム-d) δ 4.52 (d,J = 3.0 Hz,2H),4.11 (t,J = 6.6 Hz,15H),4.04-3.91 (m,4H),3.74-3.57 (m ,4H),3.54 - 3.40 (m,2H),2.45 (t,J = 7.2 Hz,4H),2.35 (t,J = 7.2 Hz,8H),1.80 - 1.60 (m,20H),1.46 - 1.19 (m,48H),0.88 (t,J = 6.6 Hz,12H).MS (ESI,m / z): [M+H] + Calculation value.C 64 H 121 N2O 16 について,1173.9. Measured value: 1174.0. Compound DIM-9: Yield 26.2%. 1H NMR (400 MHz,クロロホルム-d)δ 4.65 (s,8H),4.52 (d,J = 3.6 Hz,2H),4.10-3.90 (m,4H),3.69-3.61 (m,4H),3.53-3.45 (m,18H),2.55-2.30 (m,12H),1.80-1.65 (m,4H),1.56 (p,J = 6.8 Hz,16H),1.49-1.14 (m,48H),0.88 (t) ,J = 6.8 Hz,12H).MS (ESI,m / z): [M+H] + Calculation value.C 64 H 129 N2O 12 について,1118.0; measured value: 1118.2. Compound DIM-10: Yield 29.4%. 1 H NMR (300 MHz, クロロホルム-d) δ 4.64 (q,J = 5.4 Hz,4H),4.51 (d,J = 3.0 Hz,2H),4.10-3.90 (m,4H),3.73-3.25 (m ,22H),2.44 (t,J = 7.2 Hz,4H),2.35 (t,J = 7.2 Hz,8H),1.80-1.65 (m,4H),1.54 (p,J = 6.9 Hz,16H),1.45-1.20 (m,60H),0.87 (t,J = 6.6 Hz,12H).MS (ESI,m / z): [M+H] + Calculation value.C 68 H 137 N2O 12 について,1174.0; measured value: 1174.3. Compound LIS-2: Yield 31.0%. 1H NMR (400 MHz, クロロホルム-d) δ 4.60 (t,J = 4.4 Hz,1H),4.48 (d,J = 4.4 Hz,1H),4.01-3.88 (m,5H),3.68 (dt,J = 9.2,6.8 Hz,1H),3.57 (t,J = 8.0 Hz,1H),3.53-3.45 (m,3H),2.55-2.32 (m,12H),1.81-1.65 (m,4H),1.45-1.35 (m,8H),1.33 - 1.20 (s,40H),0.87 (t,J = 6.8 Hz,12H).MS (ESI,m / z): [M+H] + Calculation value.C 44 H 89 N2O4について,709.7. Measured value: 709.8. Compound LIS-3: yield 29.2%. 1 H NMR (400 MHz, クロロホルム-d) δ 4.60 (t,J = 4.4 Hz,1H),4.48 (d,J = 4.0 Hz,1H),4.03-3.86 (m,5H),3.69 (dt,J = 9.4,6.4 Hz,1H),3.62-3.45 (m,4H),2.77-2.20 (m,12H),1.85-1.69 (m,4H),1.52-1.36 (m,8H),1.35-1.15 (s) ,56H),0.88 (t,J = 6.4 Hz,12H).MS (ESI,m / z): [M+H] + Calculation value.C 52 H 105 N2O4について,821.8. Measured value: 822.0. Compound LIS-4: yield 30.3%. 1 H NMR (300 MHz,クロロホルム-d) δ 4.60 (t,J = 4.2 Hz,1H),4.48 (d,J = 4.2 Hz,1H),4.00-3.85 (m,5H),3.68 (dt,J = 9.0,6.3 Hz,1H),3.63-3.43 (m,4H),2.55-2.30 (m,12H),1.80-1.64 (m,4H),1.50-1.15 (m,80H),0.87 (t,J) = 6.6 Hz,12H).MS (ESI,m / z): [M+H] +Calculation value.C 60 H 121 N2O4について,933.9; measured value: 934.2. Compound LIS-5: yield 30.6%. 1 H NMR (300 MHz, クロロホルム-d) δ 4.63 (t,J = 4.2 Hz,1H),4.51 (d,J = 4.2 Hz,1H),4.14-3.85 (m,5H),3.80-3.65 (m ,1H),3.64-3.45 (m,4H),2.70-2.30 (m,12H),1.89-1.62 (m,4H),1.55-1.40 (m,8H),1.40-1.15 (m,88H),0.90 (d,J = 6.3 Hz,12H).MS (ESI,m / z): [M+H] + Calculation value.C 68 H 137 N2O4について,1046.1. Measured value: 1046.3. Compound LIS-6: yield 29.4%. 1 H NMR (400 MHz, クロロホルム-d) δ 4.60 (t,J = 4.4 Hz,1H),4.48 (d,J = 4.4 Hz,1H),4.07-3.83 (m,5H),3.75-3.65 (m ,1H),3.62 - 3.43 (m,4H),2.88 - 2.12 (m,12H),1.85 - 1.65 (m,4H),1.55 - 1.15 (m,112H),0.88 (t,J = 6.8 Hz,12H). Compound LIS-7: Yield 20.2%. 1 H NMR (400 MHz, クロロホルムd) δ 4.80 (p,J = 6.4 Hz,4H),4.59 (t,J = 4.4 Hz,1H),4.47 (d,J = 4.4 Hz,1H),4.08-3.82 (m,5H),3.75-3.65 (m,1H),3.61-3.42 (m,4H),2.58-2.30 (m,12H),2.27 (t,J = 7.6 Hz,8H) 1.77―1.48 (m) ,28H),1.42-1.18(m,64H),0.92-0.82(m,24H).MS (ESI,m / z): [M+2H] 2+ Calculation value.C80 H 154 N2O 12 について,667.6; measured value: 668.0. Compound LIS-8: yield 25.6%. 1 H NMR (400 MHz, クロロホルム-d) δ 4.61 (d,J = 4.4 Hz,1H),4.50 (d,J = 4.4 Hz,1H),4.13 (t,J = 6.8 Hz,16H),4.02-3.90 (m,5H),3.75-3.65 (m,1H),3.60-3.45 (m,4H),2.65-2.28 (m,12H),1.80-1.60 (m,20H),1.55-1.20 (m,48H) ),0.91 (d,J = 6.4 Hz,12H).MS (ESI,m / z): [M+H] + Calculation value.C 64 H 121 N2O 16 In this case, 1173.9. Measured value: 1174.0. Compound LIS-9: Yield 26.4%. 1 H NMR (400 MHz, クロロホルムd) δ 4.66 (s,8H),4.59 (t,J = 4.0 Hz,1H),4.46 (d,J = 4.0 Hz,1H),4.03-3.83 (m,4H) ),3.76-3.63 (m,1H),3.60-3.45 (m,20H),2.60-2.25 (m,12H),1.79-1.22 (m,68H),0.89 (t,J = 6.8 Hz,12H).MS (ESI,m / z): [M+H] + Calculation value.C 64 H 129 N2O 12 について,1118.0; measured value: 1118.0. Compound LIS-10: yield 25.9%. 1H NMR (400 MHz,chloroformd) δ 4.65 (q,J = 5.2 Hz,4H),4.59 (t,J = 4.4 Hz,1H),4.47 (d,J = 4.4 Hz,1H),4.03-3.84 (m,5H),3.67 (dt,J = 9.2,6.4 MS (ESI,m / z): [M+H] + Calculated value.C 68 H 137 N2O 12 Regarding this, 1174.0; measured value: 1174.1.

[0233] References 1 Hong., Radojcic, D., Ionescu, M., Petrovic, ZS, Eastwood, E. Advanced Materials from Corn: Isosorbide-Based Epoxy Resins. Polym. Chem. 5, 5360-5368 (2014). 2 Wroblewska, A., Zych, A., Thiyagarajan, S., Dudenko, D., van Es, D., Hansen, MR, Koning, C., Duchateau, R., Jasinska-Walk, L. Towards Sugar-Derived Polyamides as Environmentally Friendly Materials. Polym. Chem. 6, 4133-4143 (2015). Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the disclosed inventions belong. Publications and materials cited herein are specifically incorporated by reference.

[0234] Those skilled in the art will understand that many changes and modifications can be made to preferred embodiments of the present invention, and that such changes and modifications can be made without departing from the spirit of the invention. Accordingly, the appended claims are intended to cover all such equivalent modifications that fall within the true spirit and scope of the invention.

Claims

1. Compounds having formula I, II, or III: 【Chemistry 1】 or a salt thereof, in the formula: R １ teeth, 【Chemistry 2】 A compound or a salt thereof, independently selected from the above.

2. The aforementioned compound has the following formula: 【Transformation 3】 or a salt thereof, in the formula, R １ teeth, 【Chemistry 4】 A compound according to claim 1, independently selected from the above.

3. The aforementioned compound has the following formula: 【Transformation 5】 or a salt thereof, in the formula, R １ teeth, 【Transformation 6】 A compound according to claim 1, independently selected from the above.

4. The aforementioned compound has the following formula: 【Transformation 7】 or a salt thereof, in the formula, R １ teeth, 【Transformation 8】 【change】 A compound according to claim 1, independently selected from the above.

5. R １ but 【Chemistry 9】 The compound according to claim 1.

6. R １ but 【Chemistry 10】 The compound according to claim 1.

7. The aforementioned compound, 【Chemistry 11】 And, In the formula, R １ teeth 【Chemistry 12】 The compound according to claim 1.

8. The aforementioned compound, 【Chemistry 13】 And, In the formula, R １ teeth 【Chemistry 14】 The compound according to claim 1.

9. Lipid-based nanoparticles, The compound according to any one of claims 1 to 8, and Lipid-based nanoparticles comprising recombinant polynucleotides containing nucleic acids encoding co-stimulatory molecules.

10. Antigen-presenting cells, Lipid-based nanoparticles including the following: The compound according to any one of claims 1 to 8, and The antigen-presenting cell comprising recombinant polynucleotides containing nucleic acids encoding a co-stimulatory molecule.

11. The aforementioned compound 【Chemistry 15】 And, In the formula, R １ is 【Chemistry 16】 The antigen-presenting cell according to claim 10.

12. The aforementioned compound 【Chemistry 17】 And, In the formula, R １ teeth [Chemistry 18] The antigen-presenting cell according to claim 10.

13. The antigen-presenting cell according to any one of claims 10 to 12, wherein the co-stimulatory molecule is selected from ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, SLAM, CD2, CD226, galectin 9, TIM1, LFA1, B7-H2, B7-1, B7-2, CD70, LIGHT, HVEM, CD40, 4-1BBL, OX40L, TL1A, GITRL, CD30L, SLAM, CD48, CD58, CD155, CD112, CD80, CD86, ICOSL, TIM3, TIM4, ICAM1, and LFA3.

14. The antigen-presenting cell according to claim 13, wherein the aforementioned costimulatory molecule is CD40.

15. The antigen-presenting cell according to any one of claims 10 to 14, wherein the nucleic acid encoding the co-stimulatory molecule includes a heterogeneous 5' untranslated region (5'UTR).

16. The antigen-presenting cell according to any one of claims 10 to 15, wherein the nucleic acid encoding the costimulatory molecule includes a heterogeneous 3' untranslated region (3'UTR).

17. The antigen-presenting cell according to any one of claims 10 to 16, wherein the nucleic acid comprises a chemically modified nucleic acid base.

18. The antigen-presenting cell according to claim 17, wherein the chemically modified nucleic acid base is pseudouridine.

19. The antigen-presenting cell according to any one of claims 10 to 18, wherein the antigen-presenting cell is a bone marrow-derived dendritic cell.

20. A pharmaceutical composition comprising an antigen-presenting cell and an antibody according to any one of claims 10 to 19 in a therapeutically effective amount for treating cancer, wherein the treatment comprises administering the antigen-presenting cell and the antibody to a subject in need thereof.

21. The pharmaceutical composition according to claim 20, wherein the antibody is selected from an anti-CD40 antibody, an anti-PD-L1 antibody, an anti-PD1 antibody, an anti-CTLA4 antibody, or a combination thereof.

22. The pharmaceutical composition according to claim 20 or 21, wherein the antigen-presenting cells and the antibody are administered into the tumor.

23. The pharmaceutical composition according to any one of claims 20 to 22, wherein the antigen-presenting cells and the antibody are administered simultaneously.

24. To treat cancer, A pharmaceutical composition comprising a therapeutically effective amount of lipid-based nanoparticles according to claim 9 and a therapeutically effective amount of antigen-presenting cells according to any one of claims 10 to 19, wherein the treatment comprises administering the lipid-based nanoparticles and the antigen-presenting cells to a subject in need thereof.

25. The aforementioned lipid-based nanoparticles 【Chemistry 19】 Includes, In the formula, R １ teeth 【Chemistry 20】 The pharmaceutical composition according to claim 24.

26. The aforementioned lipid-based nanoparticles 【Chemistry 21】 Includes, In the formula, R １ teeth 【Chemistry 22】 The pharmaceutical composition according to claim 24.

27. The pharmaceutical composition according to any one of claims 24 to 26, wherein the lipid-based nanoparticles comprise a recombinant polynucleotide containing a nucleic acid encoding CD40L.

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