Sugar-derived lipid nanomaterials and their uses

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 TO RELATED APPLICATIONS This application claims the benefit 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 Sponsored Research This invention was made with United States Government support under Grant R35GM119679 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.

[0002] Technical Field The present disclosure relates to compositions and methods for treating cancer and other immune disorders. [Background technology] Efficient delivery of mRNA is a key step and challenge for the application of mRNA therapeutics. Despite encouraging 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] [Summary of the Invention] In some embodiments, disclosed herein are compounds having formula I, II, or III.

[0004] [ka] or a salt thereof, wherein R 1 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 formula:

[0006] [ka] or a salt thereof, wherein R 1 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 formula:

[0008] [ka] or a salt thereof, wherein R 1 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 formula:

[0010] [ka] or a salt thereof, wherein R 1are 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 are the following:

[0012] [ka] or a salt thereof.

[0013] In some embodiments, the compound is:

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

[0015] [ka] It is.

[0016] In some embodiments, the compound is:

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

[0018] [ka] It is.

[0019] In some embodiments, disclosed herein is a lipid-based nanoparticle comprising a compound of any of the preceding embodiments and a recombinant polynucleotide comprising a nucleic acid encoding a costimulatory molecule.

[0020] In some aspects, disclosed herein is an antigen presenting cell comprising a lipid-based nanoparticle comprising a compound of any of the preceding aspects and a recombinant polynucleotide comprising a nucleic acid encoding a costimulatory molecule.

[0021] In some embodiments, The compound

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

[0023] [ka] It is.

[0024] In some embodiments, the costimulatory 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 costimulatory molecule is CD40.

[0025] In some embodiments, the mRNA encoding the costimulatory molecule comprises a heterologous 5' untranslated region (5'UTR). In some embodiments, the mRNA encoding the costimulatory molecule comprises a heterologous 3' untranslated region (3'UTR). In some embodiments, the mRNA comprises a chemically modified nucleobase. 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 are methods of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of the antigen presenting cells and antibodies of any of the preceding aspects.

[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 cells and antibodies are administered intratumorally.

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

[0029] In some embodiments, the nanoparticles comprise:

[0030] [ka] In the formula, R 1 teeth

[0031] [ka] It is.

[0032] In some embodiments, the lipid-based nanoparticles comprise a recombinant polynucleotide comprising a nucleic acid encoding CD40L. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects described below. [Brief description of the drawings]

[0033] [Figure 1A] Figure 1. Closure of the Cancer-Immunity Cycle (CIC) and Chemical Synthesis of Sugar Alcohol-Derived Ionizable Lipids. Closure of the CIC by Integration of Lipid Nanoparticles and Cellular Therapy (CATCH). [Figure 1B] FIG. 1 shows the end of the cancer immunity cycle (CIC) and the chemical synthesis of sugar alcohol-derived ionizable lipids. FIG. 2 shows a representative synthetic pathway for sugar alcohol-derived ionizable lipids. [Figure 1C] FIG. 1 shows the end of the cancer-immunity cycle (CIC) and the chemical synthesis of ionizable lipids derived from sugar alcohols. The structures of ionizable lipids derived from sugar alcohols (DIS, DIM, and LIS series) are shown. [Figure 2A]Screening and characterization of lipid nanoparticle (LNP)-mRNA formulations are shown. A shows the mRNA delivery efficiency of DIS, DIM, and LIS LNPs in BMDCs. B shows the effect of each lipid component at different molar ratios of DIM7 LNPs on mRNA delivery. C shows the relative emission intensity of orthogonal formulations and preferred formulations of DIM7 LNPs. D shows the effect of each lipid component at different molar ratios of LIS10 LNPs on mRNA delivery. E shows the relative emission intensity of orthogonal formulations of LIS10 LNPs. F shows a table of preferred formulations. G shows the characterization of DIM7SLNPs including size, PDI, encapsulation efficiency, and zeta potential. H shows the Cryo-TEM image of DIM7S (scale bar = 50 nm). I shows the characterization of LIS10W LNPs including size, PDI, encapsulation efficiency, and zeta potential. J shows the Cryo-TEM image of LIS10W (scale bar = 50 nm). A, B, C, D, and E are from n=3 biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by two-tailed Student's t-test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 2B-1]Screening and characterization of lipid nanoparticle (LNP)-mRNA formulations are shown. A shows the mRNA delivery efficiency of DIS, DIM, and LIS LNPs in BMDCs. B shows the effect of each lipid component at different molar ratios of DIM7 LNPs on mRNA delivery. C shows the relative emission intensity of orthogonal formulations and preferred formulations of DIM7 LNPs. D shows the effect of each lipid component at different molar ratios of LIS10 LNPs on mRNA delivery. E shows the relative emission intensity of orthogonal formulations of LIS10 LNPs. F shows a table of preferred formulations. G shows the characterization of DIM7SLNPs including size, PDI, encapsulation efficiency, and zeta potential. H shows the Cryo-TEM image of DIM7S (scale bar = 50 nm). I shows the characterization of LIS10W LNPs including size, PDI, encapsulation efficiency, and zeta potential. J shows the Cryo-TEM image of LIS10W (scale bar = 50 nm). A, B, C, D, and E are from n=3 biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by two-tailed Student's t-test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 2B-2]Screening and characterization of lipid nanoparticle (LNP)-mRNA formulations are shown. A shows the mRNA delivery efficiency of DIS, DIM, and LIS LNPs in BMDCs. B shows the effect of each lipid component at different molar ratios of DIM7 LNPs on mRNA delivery. C shows the relative emission intensity of orthogonal formulations and preferred formulations of DIM7 LNPs. D shows the effect of each lipid component at different molar ratios of LIS10 LNPs on mRNA delivery. E shows the relative emission intensity of orthogonal formulations of LIS10 LNPs. F shows a table of preferred formulations. G shows the characterization of DIM7SLNPs including size, PDI, encapsulation efficiency, and zeta potential. H shows the Cryo-TEM image of DIM7S (scale bar = 50 nm). I shows the characterization of LIS10W LNPs including size, PDI, encapsulation efficiency, and zeta potential. J shows the Cryo-TEM image of LIS10W (scale bar = 50 nm). A, B, C, D, and E are from n=3 biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by two-tailed Student's t-test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 2C]Screening and characterization of lipid nanoparticle (LNP)-mRNA formulations are shown. A shows the mRNA delivery efficiency of DIS, DIM, and LIS LNPs in BMDCs. B shows the effect of each lipid component at different molar ratios of DIM7 LNPs on mRNA delivery. C shows the relative emission intensity of orthogonal formulations and preferred formulations of DIM7 LNPs. D shows the effect of each lipid component at different molar ratios of LIS10 LNPs on mRNA delivery. E shows the relative emission intensity of orthogonal formulations of LIS10 LNPs. F shows a table of preferred formulations. G shows the characterization of DIM7SLNPs including size, PDI, encapsulation efficiency, and zeta potential. H shows the Cryo-TEM image of DIM7S (scale bar = 50 nm). I shows the characterization of LIS10W LNPs including size, PDI, encapsulation efficiency, and zeta potential. J shows the Cryo-TEM image of LIS10W (scale bar = 50 nm). A, B, C, D, and E are from n=3 biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by two-tailed Student's t-test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 2D-1]Screening and characterization of lipid nanoparticle (LNP)-mRNA formulations are shown. A shows the mRNA delivery efficiency of DIS, DIM, and LIS LNPs in BMDCs. B shows the effect of each lipid component at different molar ratios of DIM7 LNPs on mRNA delivery. C shows the relative emission intensity of orthogonal formulations and preferred formulations of DIM7 LNPs. D shows the effect of each lipid component at different molar ratios of LIS10 LNPs on mRNA delivery. E shows the relative emission intensity of orthogonal formulations of LIS10 LNPs. F shows a table of preferred formulations. G shows the characterization of DIM7SLNPs including size, PDI, encapsulation efficiency, and zeta potential. H shows the Cryo-TEM image of DIM7S (scale bar = 50 nm). I shows the characterization of LIS10W LNPs including size, PDI, encapsulation efficiency, and zeta potential. J shows the Cryo-TEM image of LIS10W (scale bar = 50 nm). A, B, C, D, and E are from n=3 biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by two-tailed Student's t-test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 2D-2]Screening and characterization of lipid nanoparticle (LNP)-mRNA formulations are shown. A shows the mRNA delivery efficiency of DIS, DIM, and LIS LNPs in BMDCs. B shows the effect of each lipid component at different molar ratios of DIM7 LNPs on mRNA delivery. C shows the relative emission intensity of orthogonal formulations and preferred formulations of DIM7 LNPs. D shows the effect of each lipid component at different molar ratios of LIS10 LNPs on mRNA delivery. E shows the relative emission intensity of orthogonal formulations of LIS10 LNPs. F shows a table of preferred formulations. G shows the characterization of DIM7SLNPs including size, PDI, encapsulation efficiency, and zeta potential. H shows the Cryo-TEM image of DIM7S (scale bar = 50 nm). I shows the characterization of LIS10W LNPs including size, PDI, encapsulation efficiency, and zeta potential. J shows the Cryo-TEM image of LIS10W (scale bar = 50 nm). A, B, C, D, and E are from n=3 biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by two-tailed Student's t-test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 2E]Screening and characterization of lipid nanoparticle (LNP)-mRNA formulations are shown. A shows the mRNA delivery efficiency of DIS, DIM, and LIS LNPs in BMDCs. B shows the effect of each lipid component at different molar ratios of DIM7 LNPs on mRNA delivery. C shows the relative emission intensity of orthogonal formulations and preferred formulations of DIM7 LNPs. D shows the effect of each lipid component at different molar ratios of LIS10 LNPs on mRNA delivery. E shows the relative emission intensity of orthogonal formulations of LIS10 LNPs. F shows a table of preferred formulations. G shows the characterization of DIM7SLNPs including size, PDI, encapsulation efficiency, and zeta potential. H shows the Cryo-TEM image of DIM7S (scale bar = 50 nm). I shows the characterization of LIS10W LNPs including size, PDI, encapsulation efficiency, and zeta potential. J shows the Cryo-TEM image of LIS10W (scale bar = 50 nm). A, B, C, D, and E are from n=3 biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by two-tailed Student's t-test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 2F]Screening and characterization of lipid nanoparticle (LNP)-mRNA formulations are shown. A shows the mRNA delivery efficiency of DIS, DIM, and LIS LNPs in BMDCs. B shows the effect of each lipid component at different molar ratios of DIM7 LNPs on mRNA delivery. C shows the relative emission intensity of orthogonal formulations and preferred formulations of DIM7 LNPs. D shows the effect of each lipid component at different molar ratios of LIS10 LNPs on mRNA delivery. E shows the relative emission intensity of orthogonal formulations of LIS10 LNPs. F shows a table of preferred formulations. G shows the characterization of DIM7SLNPs including size, PDI, encapsulation efficiency, and zeta potential. H shows the Cryo-TEM image of DIM7S (scale bar = 50 nm). I shows the characterization of LIS10W LNPs including size, PDI, encapsulation efficiency, and zeta potential. J shows the Cryo-TEM image of LIS10W (scale bar = 50 nm). A, B, C, D, and E are from n=3 biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by two-tailed Student's t-test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 2G]Screening and characterization of lipid nanoparticle (LNP)-mRNA formulations are shown. A shows the mRNA delivery efficiency of DIS, DIM, and LIS LNPs in BMDCs. B shows the effect of each lipid component at different molar ratios of DIM7 LNPs on mRNA delivery. C shows the relative emission intensity of orthogonal formulations and preferred formulations of DIM7 LNPs. D shows the effect of each lipid component at different molar ratios of LIS10 LNPs on mRNA delivery. E shows the relative emission intensity of orthogonal formulations of LIS10 LNPs. F shows a table of preferred formulations. G shows the characterization of DIM7SLNPs including size, PDI, encapsulation efficiency, and zeta potential. H shows the Cryo-TEM image of DIM7S (scale bar = 50 nm). I shows the characterization of LIS10W LNPs including size, PDI, encapsulation efficiency, and zeta potential. J shows the Cryo-TEM image of LIS10W (scale bar = 50 nm). A, B, C, D, and E are from n=3 biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by two-tailed Student's t-test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 2H]Screening and characterization of lipid nanoparticle (LNP)-mRNA formulations are shown. A shows the mRNA delivery efficiency of DIS, DIM, and LIS LNPs in BMDCs. B shows the effect of each lipid component at different molar ratios of DIM7 LNPs on mRNA delivery. C shows the relative emission intensity of orthogonal formulations and preferred formulations of DIM7 LNPs. D shows the effect of each lipid component at different molar ratios of LIS10 LNPs on mRNA delivery. E shows the relative emission intensity of orthogonal formulations of LIS10 LNPs. F shows a table of preferred formulations. G shows the characterization of DIM7SLNPs including size, PDI, encapsulation efficiency, and zeta potential. H shows the Cryo-TEM image of DIM7S (scale bar = 50 nm). I shows the characterization of LIS10W LNPs including size, PDI, encapsulation efficiency, and zeta potential. J shows the Cryo-TEM image of LIS10W (scale bar = 50 nm). A, B, C, D, and E are from n=3 biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by two-tailed Student's t-test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 2I]Screening and characterization of lipid nanoparticle (LNP)-mRNA formulations are shown. A shows the mRNA delivery efficiency of DIS, DIM, and LIS LNPs in BMDCs. B shows the effect of each lipid component at different molar ratios of DIM7 LNPs on mRNA delivery. C shows the relative emission intensity of orthogonal formulations and preferred formulations of DIM7 LNPs. D shows the effect of each lipid component at different molar ratios of LIS10 LNPs on mRNA delivery. E shows the relative emission intensity of orthogonal formulations of LIS10 LNPs. F shows a table of preferred formulations. G shows the characterization of DIM7SLNPs including size, PDI, encapsulation efficiency, and zeta potential. H shows the Cryo-TEM image of DIM7S (scale bar = 50 nm). I shows the characterization of LIS10W LNPs including size, PDI, encapsulation efficiency, and zeta potential. J shows the Cryo-TEM image of LIS10W (scale bar = 50 nm). A, B, C, D, and E are from n=3 biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by two-tailed Student's t-test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 2J]Screening and characterization of lipid nanoparticle (LNP)-mRNA formulations are shown. A shows the mRNA delivery efficiency of DIS, DIM, and LIS LNPs in BMDCs. B shows the effect of each lipid component at different molar ratios of DIM7 LNPs on mRNA delivery. C shows the relative emission intensity of orthogonal formulations and preferred formulations of DIM7 LNPs. D shows the effect of each lipid component at different molar ratios of LIS10 LNPs on mRNA delivery. E shows the relative emission intensity of orthogonal formulations of LIS10 LNPs. F shows a table of preferred formulations. G shows the characterization of DIM7SLNPs including size, PDI, encapsulation efficiency, and zeta potential. H shows the Cryo-TEM image of DIM7S (scale bar = 50 nm). I shows the characterization of LIS10W LNPs including size, PDI, encapsulation efficiency, and zeta potential. J shows the Cryo-TEM image of LIS10W (scale bar = 50 nm). A, B, C, D, and E are from n=3 biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by two-tailed Student's t-test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 3A-1]Dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and rechallenge 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 regimen. C and D show the tumor volume (C) and survival rate of mice over time (D) (n=6 or 7). E and F show the expression of in vitro (E) and in vivo (F) markers, including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. Figure 1 shows LNP-induced ICD markers in vivo (F); G is a schematic diagram of the B16F10 tumor model and treatment regimen; H shows the survival rate of mice over time (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; K shows the survival rate of mice over time (n = 6). L shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge (n=5 or 6), M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the survival rate of mice over time (n=9), and O shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 8). All data are from n=3 biologically independent samples and are expressed as mean ± SEM. Statistical significance in A, C, E, and F was analyzed by two-tailed Student's t-test. Statistical significance in 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]Dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and rechallenge 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 regimen. C and D show the tumor volume (C) and survival rate of mice over time (D) (n=6 or 7). E and F show the expression of in vitro (E) and in vivo (F) markers, including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. Figure 1 shows LNP-induced ICD markers in vivo (F); G is a schematic diagram of the B16F10 tumor model and treatment regimen; H shows the survival rate of mice over time (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; K shows the survival rate of mice over time (n = 6). L shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge (n=5 or 6), M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the survival rate of mice over time (n=9), and O shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 8). All data are from n=3 biologically independent samples and are expressed as mean ± SEM. Statistical significance in A, C, E, and F was analyzed by two-tailed Student's t-test. Statistical significance in 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]Dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and rechallenge 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 regimen. C and D show the tumor volume (C) and survival rate of mice over time (D) (n=6 or 7). E and F show the expression of in vitro (E) and in vivo (F) markers, including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. Figure 1 shows LNP-induced ICD markers in vivo (F); G is a schematic diagram of the B16F10 tumor model and treatment regimen; H shows the survival rate of mice over time (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; K shows the survival rate of mice over time (n = 6). L shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge (n=5 or 6), M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the survival rate of mice over time (n=9), and O shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 8). All data are from n=3 biologically independent samples and are expressed as mean ± SEM. Statistical significance in A, C, E, and F was analyzed by two-tailed Student's t-test. Statistical significance in 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]Dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and rechallenge 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 regimen. C and D show the tumor volume (C) and survival rate of mice over time (D) (n=6 or 7). E and F show the expression of in vitro (E) and in vivo (F) markers, including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. Figure 1 shows LNP-induced ICD markers in vivo (F); G is a schematic diagram of the B16F10 tumor model and treatment regimen; H shows the survival rate of mice over time (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; K shows the survival rate of mice over time (n = 6). L shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge (n=5 or 6), M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the survival rate of mice over time (n=9), and O shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 8). All data are from n=3 biologically independent samples and are expressed as mean ± SEM. Statistical significance in A, C, E, and F was analyzed by two-tailed Student's t-test. Statistical significance in 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]Dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and rechallenge 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 regimen. C and D show the tumor volume (C) and survival rate of mice over time (D) (n=6 or 7). E and F show the expression of in vitro (E) and in vivo (F) markers, including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. Figure 1 shows LNP-induced ICD markers in vivo (F); G is a schematic diagram of the B16F10 tumor model and treatment regimen; H shows the survival rate of mice over time (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; K shows the survival rate of mice over time (n = 6). L shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge (n=5 or 6), M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the survival rate of mice over time (n=9), and O shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 8). All data are from n=3 biologically independent samples and are expressed as mean ± SEM. Statistical significance in A, C, E, and F was analyzed by two-tailed Student's t-test. Statistical significance in 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]Dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and rechallenge 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 regimen. C and D show the tumor volume (C) and survival rate of mice over time (D) (n=6 or 7). E and F show the expression of in vitro (E) and in vivo (F) markers, including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. Figure 1 shows LNP-induced ICD markers in vivo (F); G is a schematic diagram of the B16F10 tumor model and treatment regimen; H shows the survival rate of mice over time (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; K shows the survival rate of mice over time (n = 6). L shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge (n=5 or 6), M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the survival rate of mice over time (n=9), and O shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 8). All data are from n=3 biologically independent samples and are expressed as mean ± SEM. Statistical significance in A, C, E, and F was analyzed by two-tailed Student's t-test. Statistical significance in 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]Dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and rechallenge 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 regimen. C and D show the tumor volume (C) and survival rate of mice over time (D) (n=6 or 7). E and F show the expression of in vitro (E) and in vivo (F) markers, including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. Figure 1 shows LNP-induced ICD markers in vivo (F); G is a schematic diagram of the B16F10 tumor model and treatment regimen; H shows the survival rate of mice over time (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; K shows the survival rate of mice over time (n = 6). L shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge (n=5 or 6), M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the survival rate of mice over time (n=9), and O shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 8). All data are from n=3 biologically independent samples and are expressed as mean ± SEM. Statistical significance in A, C, E, and F was analyzed by two-tailed Student's t-test. Statistical significance in 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]Dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and rechallenge 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 regimen. C and D show the tumor volume (C) and survival rate of mice over time (D) (n=6 or 7). E and F show the expression of in vitro (E) and in vivo (F) markers, including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. Figure 1 shows LNP-induced ICD markers in vivo (F); G is a schematic diagram of the B16F10 tumor model and treatment regimen; H shows the survival rate of mice over time (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; K shows the survival rate of mice over time (n = 6). L shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge (n=5 or 6), M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the survival rate of mice over time (n=9), and O shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 8). All data are from n=3 biologically independent samples and are expressed as mean ± SEM. Statistical significance in A, C, E, and F was analyzed by two-tailed Student's t-test. Statistical significance in 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]Dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and rechallenge 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 regimen. C and D show the tumor volume (C) and survival rate of mice over time (D) (n=6 or 7). E and F show the expression of in vitro (E) and in vivo (F) markers, including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. Figure 1 shows LNP-induced ICD markers in vivo (F); G is a schematic diagram of the B16F10 tumor model and treatment regimen; H shows the survival rate of mice over time (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; K shows the survival rate of mice over time (n = 6). L shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge (n=5 or 6), M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the survival rate of mice over time (n=9), and O shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 8). All data are from n=3 biologically independent samples and are expressed as mean ± SEM. Statistical significance in A, C, E, and F was analyzed by two-tailed Student's t-test. Statistical significance in 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]Dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and rechallenge 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 regimen. C and D show the tumor volume (C) and survival rate of mice over time (D) (n=6 or 7). E and F show the expression of in vitro (E) and in vivo (F) markers, including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. Figure 1 shows LNP-induced ICD markers in vivo (F); G is a schematic diagram of the B16F10 tumor model and treatment regimen; H shows the survival rate of mice over time (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; K shows the survival rate of mice over time (n = 6). L shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge (n=5 or 6), M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the survival rate of mice over time (n=9), and O shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 8). All data are from n=3 biologically independent samples and are expressed as mean ± SEM. Statistical significance in A, C, E, and F was analyzed by two-tailed Student's t-test. Statistical significance in 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]Dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and rechallenge 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 regimen. C and D show the tumor volume (C) and survival rate of mice over time (D) (n=6 or 7). E and F show the expression of in vitro (E) and in vivo (F) markers, including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. Figure 1 shows LNP-induced ICD markers in vivo (F); G is a schematic diagram of the B16F10 tumor model and treatment regimen; H shows the survival rate of mice over time (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; K shows the survival rate of mice over time (n = 6). L shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge (n=5 or 6), M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the survival rate of mice over time (n=9), and O shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 8). All data are from n=3 biologically independent samples and are expressed as mean ± SEM. Statistical significance in A, C, E, and F was analyzed by two-tailed Student's t-test. Statistical significance in 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]Dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and rechallenge 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 regimen. C and D show the tumor volume (C) and survival rate of mice over time (D) (n=6 or 7). E and F show the expression of in vitro (E) and in vivo (F) markers, including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. Figure 1 shows LNP-induced ICD markers in vivo (F); G is a schematic diagram of the B16F10 tumor model and treatment regimen; H shows the survival rate of mice over time (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; K shows the survival rate of mice over time (n = 6). L shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge (n=5 or 6), M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the survival rate of mice over time (n=9), and O shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 8). All data are from n=3 biologically independent samples and are expressed as mean ± SEM. Statistical significance in A, C, E, and F was analyzed by two-tailed Student's t-test. Statistical significance in 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]Dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and rechallenge 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 regimen. C and D show the tumor volume (C) and survival rate of mice over time (D) (n=6 or 7). E and F show the expression of in vitro (E) and in vivo (F) markers, including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. Figure 1 shows LNP-induced ICD markers in vivo (F); G is a schematic diagram of the B16F10 tumor model and treatment regimen; H shows the survival rate of mice over time (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; K shows the survival rate of mice over time (n = 6). L shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge (n=5 or 6), M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the survival rate of mice over time (n=9), and O shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 8). All data are from n=3 biologically independent samples and are expressed as mean ± SEM. Statistical significance in A, C, E, and F was analyzed by two-tailed Student's t-test. Statistical significance in 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]Dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and rechallenge 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 regimen. C and D show the tumor volume (C) and survival rate of mice over time (D) (n=6 or 7). E and F show the expression of in vitro (E) and in vivo (F) markers, including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. Figure 1 shows LNP-induced ICD markers in vivo (F); G is a schematic diagram of the B16F10 tumor model and treatment regimen; H shows the survival rate of mice over time (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; K shows the survival rate of mice over time (n = 6). L shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge (n=5 or 6), M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the survival rate of mice over time (n=9), and O shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 8). All data are from n=3 biologically independent samples and are expressed as mean ± SEM. Statistical significance in A, C, E, and F was analyzed by two-tailed Student's t-test. Statistical significance in 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]Dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and rechallenge 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 regimen. C and D show the tumor volume (C) and survival rate of mice over time (D) (n=6 or 7). E and F show the expression of in vitro (E) and in vivo (F) markers, including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. Figure 1 shows LNP-induced ICD markers in vivo (F); G is a schematic diagram of the B16F10 tumor model and treatment regimen; H shows the survival rate of mice over time (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; K shows the survival rate of mice over time (n = 6). L shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge (n=5 or 6), M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the survival rate of mice over time (n=9), and O shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 8). All data are from n=3 biologically independent samples and are expressed as mean ± SEM. Statistical significance in A, C, E, and F was analyzed by two-tailed Student's t-test. Statistical significance in 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]Dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and rechallenge 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 regimen. C and D show the tumor volume (C) and survival rate of mice over time (D) (n=6 or 7). E and F show the expression of in vitro (E) and in vivo (F) markers, including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. Figure 1 shows LNP-induced ICD markers in vivo (F); G is a schematic diagram of the B16F10 tumor model and treatment regimen; H shows the survival rate of mice over time (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; K shows the survival rate of mice over time (n = 6). L shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge (n=5 or 6), M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the survival rate of mice over time (n=9), and O shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 8). All data are from n=3 biologically independent samples and are expressed as mean ± SEM. Statistical significance in A, C, E, and F was analyzed by two-tailed Student's t-test. Statistical significance in 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]Dendritic cell activation, lipid nanoparticle (LNP)-induced immunogenic cell death (ICD), and therapeutic effects on primary and rechallenge 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 regimen. C and D show the tumor volume (C) and survival rate of mice over time (D) (n=6 or 7). E and F show the expression of in vitro (E) and in vivo (F) markers, including extracellular HMGB1, extracellular ATP, and cell surface calreticulin. Figure 1 shows LNP-induced ICD markers in vivo (F); G is a schematic diagram of the B16F10 tumor model and treatment regimen; H shows the survival rate of mice over time (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; K shows the survival rate of mice over time (n = 6). L shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after subcutaneous tumor rechallenge (n=5 or 6), M shows a schematic diagram of the B16F10-Luc2 tumor model and treatment regimen, N shows the survival rate of mice over time (n=9), and O shows the survival rate of responder mice in the CD40L-LIS10W+CD40-BMDC group after tumor rechallenge (n=5 or 8). All data are from n=3 biologically independent samples and are expressed as mean ± SEM. Statistical significance in A, C, E, and F was analyzed by two-tailed Student's t-test. Statistical significance in 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 tissues and blood are shown. A shows a schematic diagram of the B16F10 tumor model and therapeutic regimen. B, C show the distal tumor volume of individual mice (B) and the survival rate of mice over time (C) (n=8 or 10). D shows a schematic diagram of the B16F10-Luc2 tumor model and therapeutic regimen. E, F show the brain tumor volume (E) and the survival rate of mice over time (F) (n=10). G shows a schematic diagram of the B16F10 tumor model and therapeutic regimen. H and I show the tumor volume (H) and the survival rate of mice (I) over time (n=6). J shows a schematic diagram of the therapeutic regimen and sample collection. K and L show the dynamic expression of cytokines and chemokines in tumor tissues (K) and blood (L) (n=5). Data in B, E, and H are expressed as mean ± SEM. Statistical significance in B, E, and H was analyzed by two-tailed Student's t test. Statistical significance in Figure 7B and Figure 7D was analyzed by 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 tissues and blood are shown. A shows a schematic diagram of the B16F10 tumor model and therapeutic regimen. B, C show the distal tumor volume of individual mice (B) and the survival rate of mice over time (C) (n=8 or 10). D shows a schematic diagram of the B16F10-Luc2 tumor model and therapeutic regimen. E, F show the brain tumor volume (E) and the survival rate of mice over time (F) (n=10). G shows a schematic diagram of the B16F10 tumor model and therapeutic regimen. H and I show the tumor volume (H) and the survival rate of mice (I) over time (n=6). J shows a schematic diagram of the therapeutic regimen and sample collection. K and L show the dynamic expression of cytokines and chemokines in tumor tissues (K) and blood (L) (n=5). Data in B, E, and H are expressed as mean ± SEM. Statistical significance in B, E, and H was analyzed by two-tailed Student's t test. Statistical significance in Figure 7B and Figure 7D was analyzed by 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 tissues and blood are shown. A shows a schematic diagram of the B16F10 tumor model and therapeutic regimen. B, C show the distal tumor volume of individual mice (B) and the survival rate of mice over time (C) (n=8 or 10). D shows a schematic diagram of the B16F10-Luc2 tumor model and therapeutic regimen. E, F show the brain tumor volume (E) and the survival rate of mice over time (F) (n=10). G shows a schematic diagram of the B16F10 tumor model and therapeutic regimen. H and I show the tumor volume (H) and the survival rate of mice (I) over time (n=6). J shows a schematic diagram of the therapeutic regimen and sample collection. K and L show the dynamic expression of cytokines and chemokines in tumor tissues (K) and blood (L) (n=5). Data in B, E, and H are expressed as mean ± SEM. Statistical significance in B, E, and H was analyzed by two-tailed Student's t test. Statistical significance in Figure 7B and Figure 7D was analyzed by 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 tissues and blood are shown. A shows a schematic diagram of the B16F10 tumor model and therapeutic regimen. B, C show the distal tumor volume of individual mice (B) and the survival rate of mice over time (C) (n=8 or 10). D shows a schematic diagram of the B16F10-Luc2 tumor model and therapeutic regimen. E, F show the brain tumor volume (E) and the survival rate of mice over time (F) (n=10). G shows a schematic diagram of the B16F10 tumor model and therapeutic regimen. H and I show the tumor volume (H) and the survival rate of mice (I) over time (n=6). J shows a schematic diagram of the therapeutic regimen and sample collection. K and L show the dynamic expression of cytokines and chemokines in tumor tissues (K) and blood (L) (n=5). Data in B, E, and H are expressed as mean ± SEM. Statistical significance in B, E, and H was analyzed by two-tailed Student's t test. Statistical significance in Figure 7B and Figure 7D was analyzed by 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 tissues and blood are shown. A shows a schematic diagram of the B16F10 tumor model and therapeutic regimen. B, C show the distal tumor volume of individual mice (B) and the survival rate of mice over time (C) (n=8 or 10). D shows a schematic diagram of the B16F10-Luc2 tumor model and therapeutic regimen. E, F show the brain tumor volume (E) and the survival rate of mice over time (F) (n=10). G shows a schematic diagram of the B16F10 tumor model and therapeutic regimen. H and I show the tumor volume (H) and the survival rate of mice (I) over time (n=6). J shows a schematic diagram of the therapeutic regimen and sample collection. K and L show the dynamic expression of cytokines and chemokines in tumor tissues (K) and blood (L) (n=5). Data in B, E, and H are expressed as mean ± SEM. Statistical significance in B, E, and H was analyzed by two-tailed Student's t test. Statistical significance in Figure 7B and Figure 7D was analyzed by 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 tissues and blood are shown. A shows a schematic diagram of the B16F10 tumor model and therapeutic regimen. B, C show the distal tumor volume of individual mice (B) and the survival rate of mice over time (C) (n=8 or 10). D shows a schematic diagram of the B16F10-Luc2 tumor model and therapeutic regimen. E, F show the brain tumor volume (E) and the survival rate of mice over time (F) (n=10). G shows a schematic diagram of the B16F10 tumor model and therapeutic regimen. H and I show the tumor volume (H) and the survival rate of mice (I) over time (n=6). J shows a schematic diagram of the therapeutic regimen and sample collection. K and L show the dynamic expression of cytokines and chemokines in tumor tissues (K) and blood (L) (n=5). Data in B, E, and H are expressed as mean ± SEM. Statistical significance in B, E, and H was analyzed by two-tailed Student's t test. Statistical significance in Figure 7B and Figure 7D was analyzed by 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 tissues and blood are shown. A shows a schematic diagram of the B16F10 tumor model and therapeutic regimen. B, C show the distal tumor volume of individual mice (B) and the survival rate of mice over time (C) (n=8 or 10). D shows a schematic diagram of the B16F10-Luc2 tumor model and therapeutic regimen. E, F show the brain tumor volume (E) and the survival rate of mice over time (F) (n=10). G shows a schematic diagram of the B16F10 tumor model and therapeutic regimen. H and I show the tumor volume (H) and the survival rate of mice (I) over time (n=6). J shows a schematic diagram of the therapeutic regimen and sample collection. K and L show the dynamic expression of cytokines and chemokines in tumor tissues (K) and blood (L) (n=5). Data in B, E, and H are expressed as mean ± SEM. Statistical significance in B, E, and H was analyzed by two-tailed Student's t test. Statistical significance in Figure 7B and Figure 7D was analyzed by 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 tissues and blood are shown. A shows a schematic diagram of the B16F10 tumor model and therapeutic regimen. B, C show the distal tumor volume of individual mice (B) and the survival rate of mice over time (C) (n=8 or 10). D shows a schematic diagram of the B16F10-Luc2 tumor model and therapeutic regimen. E, F show the brain tumor volume (E) and the survival rate of mice over time (F) (n=10). G shows a schematic diagram of the B16F10 tumor model and therapeutic regimen. H and I show the tumor volume (H) and the survival rate of mice (I) over time (n=6). J shows a schematic diagram of the therapeutic regimen and sample collection. K and L show the dynamic expression of cytokines and chemokines in tumor tissues (K) and blood (L) (n=5). Data in B, E, and H are expressed as mean ± SEM. Statistical significance in B, E, and H was analyzed by two-tailed Student's t test. Statistical significance in Figure 7B and Figure 7D was analyzed by 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 tissues and blood are shown. A shows a schematic diagram of the B16F10 tumor model and therapeutic regimen. B, C show the distal tumor volume of individual mice (B) and the survival rate of mice over time (C) (n=8 or 10). D shows a schematic diagram of the B16F10-Luc2 tumor model and therapeutic regimen. E, F show the brain tumor volume (E) and the survival rate of mice over time (F) (n=10). G shows a schematic diagram of the B16F10 tumor model and therapeutic regimen. H and I show the tumor volume (H) and the survival rate of mice (I) over time (n=6). J shows a schematic diagram of the therapeutic regimen and sample collection. K and L show the dynamic expression of cytokines and chemokines in tumor tissues (K) and blood (L) (n=5). Data in B, E, and H are expressed as mean ± SEM. Statistical significance in B, E, and H was analyzed by two-tailed Student's t test. Statistical significance in Figure 7B and Figure 7D was analyzed by 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 tissues and blood are shown. A shows a schematic diagram of the B16F10 tumor model and therapeutic regimen. B, C show the distal tumor volume of individual mice (B) and the survival rate of mice over time (C) (n=8 or 10). D shows a schematic diagram of the B16F10-Luc2 tumor model and therapeutic regimen. E, F show the brain tumor volume (E) and the survival rate of mice over time (F) (n=10). G shows a schematic diagram of the B16F10 tumor model and therapeutic regimen. H and I show the tumor volume (H) and the survival rate of mice (I) over time (n=6). J shows a schematic diagram of the therapeutic regimen and sample collection. K and L show the dynamic expression of cytokines and chemokines in tumor tissues (K) and blood (L) (n=5). Data in B, E, and H are expressed as mean ± SEM. Statistical significance in B, E, and H was analyzed by two-tailed Student's t test. Statistical significance in Figure 7B and Figure 7D was analyzed by 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 tissues and blood are shown. A shows a schematic diagram of the B16F10 tumor model and therapeutic regimen. B, C show the distal tumor volume of individual mice (B) and the survival rate of mice over time (C) (n=8 or 10). D shows a schematic diagram of the B16F10-Luc2 tumor model and therapeutic regimen. E, F show the brain tumor volume (E) and the survival rate of mice over time (F) (n=10). G shows a schematic diagram of the B16F10 tumor model and therapeutic regimen. H and I show the tumor volume (H) and the survival rate of mice (I) over time (n=6). J shows a schematic diagram of the therapeutic regimen and sample collection. K and L show the dynamic expression of cytokines and chemokines in tumor tissues (K) and blood (L) (n=5). Data in B, E, and H are expressed as mean ± SEM. Statistical significance in B, E, and H was analyzed by two-tailed Student's t test. Statistical significance in Figure 7B and Figure 7D was analyzed by 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 tissues and blood are shown. A shows a schematic diagram of the B16F10 tumor model and therapeutic regimen. B, C show the distal tumor volume of individual mice (B) and the survival rate of mice over time (C) (n=8 or 10). D shows a schematic diagram of the B16F10-Luc2 tumor model and therapeutic regimen. E, F show the brain tumor volume (E) and the survival rate of mice over time (F) (n=10). G shows a schematic diagram of the B16F10 tumor model and therapeutic regimen. H and I show the tumor volume (H) and the survival rate of mice (I) over time (n=6). J shows a schematic diagram of the therapeutic regimen and sample collection. K and L show the dynamic expression of cytokines and chemokines in tumor tissues (K) and blood (L) (n=5). Data in B, E, and H are expressed as mean ± SEM. Statistical significance in B, E, and H was analyzed by two-tailed Student's t test. Statistical significance in Figure 7B and Figure 7D was analyzed by log-rank (Mantel-Cox) test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 5A] Figure 1 shows immune cell infiltration in tumor tissue. A shows immune cell populations in tumor tissue (n=5), B shows the percentage of activated macrophages and dendritic cells in tumor tissue (n=5), C shows the percentage of primed CD8 T cells in tumor tissue (n=5), and D and E show the percentage of effector memory T cells and central memory T cells in the spleen (D) and blood (E) (n=5). [Figure 5B] Figure 1 shows immune cell infiltration in tumor tissue. A shows immune cell populations in tumor tissue (n=5), B shows the percentage of activated macrophages and dendritic cells in tumor tissue (n=5), C shows the percentage of primed CD8 T cells in tumor tissue (n=5), and D and E show the percentage of effector memory T cells and central memory T cells in the spleen (D) and blood (E) (n=5). [Figure 5C]Figure 1 shows immune cell infiltration in tumor tissue. A shows immune cell populations in tumor tissue (n=5), B shows the percentage of activated macrophages and dendritic cells in tumor tissue (n=5), C shows the percentage of primed CD8 T cells in tumor tissue (n=5), and D and E show the percentage of effector memory T cells and central memory T cells in the spleen (D) and blood (E) (n=5). [Figure 5D] Figure 1 shows immune cell infiltration in tumor tissue. A shows immune cell populations in tumor tissue (n=5), B shows the percentage of activated macrophages and dendritic cells in tumor tissue (n=5), C shows the percentage of primed CD8 T cells in tumor tissue (n=5), and D and E show the percentage of effector memory T cells and central memory T cells in the spleen (D) and blood (E) (n=5). [Figure 5E] Figure 1 shows immune cell infiltration in tumor tissue. A shows immune cell populations in tumor tissue (n=5), B shows the percentage of activated macrophages and dendritic cells in tumor tissue (n=5), C shows the percentage of primed CD8 T cells in tumor tissue (n=5), and D and E show the percentage of effector memory T cells and central memory T cells in the spleen (D) and blood (E) (n=5). [Figure 6A] Figure 1 shows the characterization of lipid nanoparticle (LNP)-mRNA formulations: (A) nanoparticle size and polydispersity index (PDI), (B) entrapment efficiency and zeta potential. All data are expressed as mean ± SD. [Figure 6B] Figure 1 shows the characterization of lipid nanoparticle (LNP)-mRNA formulations: (A) nanoparticle size and polydispersity index (PDI), (B) entrapment efficiency and zeta potential. All data are expressed as mean ± SD. [Figure 7A]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 relative to Lipofectamine 3000 (Lipo3K). E shows the expression kinetics of mRNA delivered by DIM7S. F shows the expression of CD40 in BMDCs. All data are from n = 3 biologically independent samples and are expressed as mean ± SEM. Statistical significance was analyzed by two-tailed Student's t-test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 7B] 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 relative to Lipofectamine 3000 (Lipo3K). E shows the expression kinetics of mRNA delivered by DIM7S. F shows the expression of CD40 in BMDCs. All data are from n = 3 biologically independent samples and are expressed as mean ± SEM. Statistical significance was analyzed by two-tailed Student's t-test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 7C]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 relative to Lipofectamine 3000 (Lipo3K). E shows the expression kinetics of mRNA delivered by DIM7S. F shows the expression of CD40 in BMDCs. All data are from n = 3 biologically independent samples and are expressed as mean ± SEM. Statistical significance was analyzed by two-tailed Student's t-test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 7D] 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 relative to Lipofectamine 3000 (Lipo3K). E shows the expression kinetics of mRNA delivered by DIM7S. F shows the expression of CD40 in BMDCs. All data are from n = 3 biologically independent samples and are expressed as mean ± SEM. Statistical significance was analyzed by two-tailed Student's t-test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 7E]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 relative to Lipofectamine 3000 (Lipo3K). E shows the expression kinetics of mRNA delivered by DIM7S. F shows the expression of CD40 in BMDCs. All data are from n = 3 biologically independent samples and are expressed as mean ± SEM. Statistical significance was analyzed by two-tailed Student's t-test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 7F] 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 relative to Lipofectamine 3000 (Lipo3K). E shows the expression kinetics of mRNA delivered by DIM7S. F shows the expression of CD40 in BMDCs. All data are from n = 3 biologically independent samples and are expressed as mean ± SEM. Statistical significance was analyzed by two-tailed Student's t-test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 8A] Tumor volumes from individual mice (n=6 or 7) are shown. [Figure 8B] Tumor volumes from individual mice (n=6 or 7) are shown. [Figure 8C] Tumor volumes from individual mice (n=6 or 7) are shown. [Figure 8D] Tumor volumes from individual mice (n=6 or 7) are shown. [Figure 8E] Tumor volumes from individual mice (n=6 or 7) are shown. [Figure 8F] Tumor volumes from individual mice (n=6 or 7) are shown. [Figure 8G] Tumor volumes from individual mice (n=6 or 7) are shown. [Figure 9A] Figure 1 shows lipid nanoparticle (LNP)-induced cytotoxicity and CD40 and CD40L expression. A shows CD40 expression mediated by CD40-DIM7S in dendritic cells in vivo (n=5). B shows in vitro cytotoxicity induced by CD40L-DIM7S and CD40L-LIS10W. C shows in vitro CD40L expression mediated by CD40L-DIM7S and CD40L-LIS10W in B16F10 melanoma cells. D and E show in vivo CD40L expression mediated by CD40L-LIS10W in tumor cells (D) and immune cells (E), including macrophages, dendritic cells, CD8 T cells, and CD4 T cells (n=3 or 5). Data in B and C are from n=3 biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by two-tailed Student's t-test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 9B]Figure 1 shows lipid nanoparticle (LNP)-induced cytotoxicity and CD40 and CD40L expression. A shows CD40 expression mediated by CD40-DIM7S in dendritic cells in vivo (n=5). B shows in vitro cytotoxicity induced by CD40L-DIM7S and CD40L-LIS10W. C shows in vitro CD40L expression mediated by CD40L-DIM7S and CD40L-LIS10W in B16F10 melanoma cells. D and E show in vivo CD40L expression mediated by CD40L-LIS10W in tumor cells (D) and immune cells (E), including macrophages, dendritic cells, CD8 T cells, and CD4 T cells (n=3 or 5). Data in B and C are from n=3 biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by two-tailed Student's t-test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 9C] Figure 1 shows lipid nanoparticle (LNP)-induced cytotoxicity and CD40 and CD40L expression. A shows CD40 expression mediated by CD40-DIM7S in dendritic cells in vivo (n=5). B shows in vitro cytotoxicity induced by CD40L-DIM7S and CD40L-LIS10W. C shows in vitro CD40L expression mediated by CD40L-DIM7S and CD40L-LIS10W in B16F10 melanoma cells. D and E show in vivo CD40L expression mediated by CD40L-LIS10W in tumor cells (D) and immune cells (E), including macrophages, dendritic cells, CD8 T cells, and CD4 T cells (n=3 or 5). Data in B and C are from n=3 biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by two-tailed Student's t-test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 9D]Figure 1 shows lipid nanoparticle (LNP)-induced cytotoxicity and CD40 and CD40L expression. A shows CD40 expression mediated by CD40-DIM7S in dendritic cells in vivo (n=5). B shows in vitro cytotoxicity induced by CD40L-DIM7S and CD40L-LIS10W. C shows in vitro CD40L expression mediated by CD40L-DIM7S and CD40L-LIS10W in B16F10 melanoma cells. D and E show in vivo CD40L expression mediated by CD40L-LIS10W in tumor cells (D) and immune cells (E), including macrophages, dendritic cells, CD8 T cells, and CD4 T cells (n=3 or 5). Data in B and C are from n=3 biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by two-tailed Student's t-test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 9E] Figure 1 shows lipid nanoparticle (LNP)-induced cytotoxicity and CD40 and CD40L expression. A shows CD40 expression mediated by CD40-DIM7S in dendritic cells in vivo (n=5). B shows in vitro cytotoxicity induced by CD40L-DIM7S and CD40L-LIS10W. C shows in vitro CD40L expression mediated by CD40L-DIM7S and CD40L-LIS10W in B16F10 melanoma cells. D and E show in vivo CD40L expression mediated by CD40L-LIS10W in tumor cells (D) and immune cells (E), including macrophages, dendritic cells, CD8 T cells, and CD4 T cells (n=3 or 5). Data in B and C are from n=3 biologically independent samples. All data are expressed as mean ± standard deviation. Statistical significance was analyzed by two-tailed Student's t-test. *P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 10A]A shows the tumor volumes of individual mice, A shows the primary tumor volumes of individual mice (n=6), B shows the rechallenged subcutaneous tumor volumes of individual mice (n=5 or 6), C and D show the tumor volumes (C) and primary tumor volumes (D) of individual mice (n=6) over time, E shows the rechallenged subcutaneous tumor volumes of individual mice (n=5 or 6), F shows the primary tumor volumes of individual mice (n=9), and G shows the brain tumor volumes (n=5 or 8) over time. Data in C and G are expressed as mean ± sem. Statistical significance was analyzed by two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 10B] A shows the tumor volumes of individual mice, A shows the primary tumor volumes of individual mice (n=6), B shows the rechallenged subcutaneous tumor volumes of individual mice (n=5 or 6), C and D show the tumor volumes (C) and primary tumor volumes (D) of individual mice (n=6) over time, E shows the rechallenged subcutaneous tumor volumes of individual mice (n=5 or 6), F shows the primary tumor volumes of individual mice (n=9), and G shows the brain tumor volumes (n=5 or 8) over time. Data in C and G are expressed as mean ± sem. Statistical significance was analyzed by two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 10C] A shows the tumor volumes of individual mice, A shows the primary tumor volumes of individual mice (n=6), B shows the rechallenged subcutaneous tumor volumes of individual mice (n=5 or 6), C and D show the tumor volumes (C) and primary tumor volumes (D) of individual mice (n=6) over time, E shows the rechallenged subcutaneous tumor volumes of individual mice (n=5 or 6), F shows the primary tumor volumes of individual mice (n=9), and G shows the brain tumor volumes (n=5 or 8) over time. Data in C and G are expressed as mean ± sem. Statistical significance was analyzed by two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 10D]A shows the tumor volumes of individual mice, A shows the primary tumor volumes of individual mice (n=6), B shows the rechallenged subcutaneous tumor volumes of individual mice (n=5 or 6), C and D show the tumor volumes (C) and primary tumor volumes (D) of individual mice (n=6) over time, E shows the rechallenged subcutaneous tumor volumes of individual mice (n=5 or 6), F shows the primary tumor volumes of individual mice (n=9), and G shows the brain tumor volumes (n=5 or 8) over time. Data in C and G are expressed as mean ± sem. Statistical significance was analyzed by two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 10E] A shows the tumor volumes of individual mice, A shows the primary tumor volumes of individual mice (n=6), B shows the rechallenged subcutaneous tumor volumes of individual mice (n=5 or 6), C and D show the tumor volumes (C) and primary tumor volumes (D) of individual mice (n=6) over time, E shows the rechallenged subcutaneous tumor volumes of individual mice (n=5 or 6), F shows the primary tumor volumes of individual mice (n=9), and G shows the brain tumor volumes (n=5 or 8) over time. Data in C and G are expressed as mean ± sem. Statistical significance was analyzed by two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 10F] A shows the tumor volumes of individual mice, A shows the primary tumor volumes of individual mice (n=6), B shows the rechallenged subcutaneous tumor volumes of individual mice (n=5 or 6), C and D show the tumor volumes (C) and primary tumor volumes (D) of individual mice (n=6) over time, E shows the rechallenged subcutaneous tumor volumes of individual mice (n=5 or 6), F shows the primary tumor volumes of individual mice (n=9), and G shows the brain tumor volumes (n=5 or 8) over time. Data in C and G are expressed as mean ± sem. Statistical significance was analyzed by two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 10G]A shows the tumor volumes of individual mice, A shows the primary tumor volumes of individual mice (n=6), B shows the rechallenged subcutaneous tumor volumes of individual mice (n=5 or 6), C and D show the tumor volumes (C) and primary tumor volumes (D) of individual mice (n=6) over time, E shows the rechallenged subcutaneous tumor volumes of individual mice (n=5 or 6), F shows the primary tumor volumes of individual mice (n=9), and G shows the brain tumor volumes (n=5 or 8) over time. Data in C and G are expressed as mean ± sem. Statistical significance was analyzed by two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 11A] Tumor volumes for individual mice are shown, and tumor volumes for the treated side of individual mice (n=8 or 10) are shown. [Figure 11B] Tumor volumes of individual mice are shown, and tumor volumes of the treated side of individual mice (n=10) are shown. [Figure 11C] Tumor volumes for individual mice are shown. Tumor volumes for individual mice (n=6) are shown. [Figure 12-1] Dynamic expression of cytokines and chemokines in mouse melanoma tissues (n=5). [Figure 12-2] Dynamic expression of cytokines and chemokines in mouse melanoma tissues (n=5). [Figure 12-3] Dynamic expression of cytokines and chemokines in mouse melanoma tissues (n=5). [Figure 12-4] Dynamic expression of cytokines and chemokines in mouse melanoma tissues (n=5). [Figure 12-5] Dynamic expression of cytokines and chemokines in mouse melanoma tissues (n=5). [Figure 12-6] Dynamic expression of cytokines and chemokines in mouse melanoma tissues (n=5). [Figure 12-7] Dynamic expression of cytokines and chemokines in mouse melanoma tissues (n=5). [Figure 12-8]Dynamic expression of cytokines and chemokines in mouse melanoma tissues (n=5). [Figure 13-1] Dynamic expression of cytokines and chemokines in mouse blood (n=5). [Figure 13-2] Dynamic expression of cytokines and chemokines in mouse blood (n=5). [Figure 13-3] Dynamic expression of cytokines and chemokines in mouse blood (n=5). [Figure 13-4] Dynamic expression of cytokines and chemokines in mouse blood (n=5). [Figure 13-5] Dynamic expression of cytokines and chemokines in mouse blood (n=5). [Figure 13-6] Dynamic expression of cytokines and chemokines in mouse blood (n=5). [Figure 13-7] Dynamic expression of cytokines and chemokines in mouse blood (n=5). [Figure 13-8] Dynamic expression of cytokines and chemokines in mouse blood (n=5). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] Disclosed herein are compositions and methods for modulating the immune system to treat cancer and other immune disorders. Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the drawings and examples, but the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. As used herein, the term "comprising" and variations thereof are used synonymously with the term "including" and variations thereof and are open, non-limiting terms. Although the terms "comprising" and "including" have been used herein to describe various embodiments, the terms "consisting essentially of" and "consisting of" may be used in place of "comprising" and "including" to provide more specific embodiments and are disclosed. As used in this disclosure and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0036] The following definitions are provided for a full understanding of terms used herein.

[0037] term As used herein, the terms "may," "optionally," and "may optionally" are used interchangeably and are meant to include cases where the condition occurs and cases where the condition does not occur. Thus, for example, a statement that a formulation "may include an excipient" is meant to include cases where the formulation includes the excipient and cases where the formulation does not include the excipient.

[0038] The term "promoter" or "regulatory element" refers to a region or sequence determinant located upstream or downstream from the start of transcription and involved in the recognition and binding of RNA polymerase and other proteins to initiate transcription. Promoters need not be of bacterial origin, e.g., promoters derived from viruses or other organisms can be used in the compositions, systems, or methods described herein. The term "regulatory element" is intended to include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals such as polyadenylation signals and polyU 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 constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). A tissue-specific promoter may direct expression primarily in a desired tissue of interest, such as muscle, neurons, bone, skin, blood, a particular organ (e.g., liver, pancreas), or a particular cell type (e.g., lymphocytes). Regulatory elements may also direct expression in a time-dependent manner, such as 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 comprises 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, U6 and H1 promoters.Examples of pol II promoters include, but are not limited to, the retroviral Ruth Sarcoma Virus (RSV) LTR promoter (optionally containing the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally containing the 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 the WPRE, the CMV enhancer; the R-U5' segment in 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 appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the desired level of expression, and the like.

[0039] The term "recombinant" refers to a nucleic acid (e.g., polynucleotide) that has been artificially engineered or a copy or complement of a nucleic acid (e.g., polynucleotide) that has been artificially engineered, or, in the context of a protein (i.e., a "recombinant protein"), to the protein (e.g., polynucleotide) encoded by a recombinant nucleic acid. In an embodiment, a recombinant expression cassette that includes a promoter operably linked to a second nucleic acid (e.g., polynucleotide) can include a promoter that is heterologous 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 another example, a recombinant expression cassette can include nucleic acids (e.g., polynucleotides) that have been combined such that the nucleic acids (e.g., polynucleotides) are highly unlikely to be found in nature. For example, a human engineered restriction enzyme site or a plasmid vector sequence may flank or separate the promoter from the second nucleic acid (e.g., polynucleotide).Those of skill in the art will recognize that nucleic acids (e.g., polynucleotides) can be engineered in many ways and are not limited to the above examples.

[0040] The term "expression cassette" or "vector" refers to a nucleic acid construct that, when introduced into a host cell, results in transcription and / or translation of an RNA or a polypeptide, respectively. In embodiments, an expression cassette that includes a promoter operably linked to a second nucleic acid (e.g., a polynucleotide) may include a promoter that is heterologous to the second nucleic acid (e.g., a polynucleotide) as a result of human manipulation (e.g., by 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 that includes a terminator (or termination sequence) operably linked to a second nucleic acid (e.g., a polynucleotide) may include a terminator that is heterologous to the second nucleic acid (e.g., a polynucleotide) as a result of human manipulation. In some embodiments, the expression cassette comprises a promoter operably linked to a second nucleic acid (e.g., polynucleotide) and a terminator operably linked to a second nucleic acid (e.g., polynucleotide) as a result of human manipulation. In some embodiments, the expression cassette comprises an endogenous promoter. In some embodiments, the expression cassette comprises an endogenous terminator. In some embodiments, the expression cassette comprises a synthetic (or non-natural) promoter. In some embodiments, the expression cassette comprises a synthetic (or non-natural) terminator.

[0041] The term "identity" or percent "identity" with respect to two or more nucleic acid or polypeptide sequences is determined using the BLAST or BLAST 2.0 sequence comparison algorithms using the default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI "Substantially identical" refers to two or more sequences or subsequences that are identical or have a specified percentage of amino acid residues or nucleotides that are identical (i.e., about 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 over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region. Such sequences are said to be "substantially identical." This definition may refer to or apply to the complement of a subject sequence. This definition also includes sequences with deletions and / or additions, as well as sequences with substitutions. As described below, preferred algorithms can account for gaps and the like. Preferably, the identity is in a region that is at least about 10 amino acids or 20 nucleotides in length, or more preferably, in a region that is 10-50 amino acids or 20-50 nucleotides in length. As used herein, percent amino acid sequence identity (%) 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 and introducing gaps, if necessary, to obtain the maximum percent sequence identity. Alignment to determine percent sequence identity may be accomplished in a variety of ways within the skill 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 algorithms needed to achieve maximal alignment over the full-length of the sequences being compared, can be determined by known methods.

[0042] For sequence comparison, one sequence usually serves as a reference sequence and compares it with test sequence.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, partial sequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.Preferably, default program parameters can be used, or alternative parameters can be designated.Then, sequence comparison algorithm calculates the percent sequence identity of test sequence to reference sequence based on program parameters.

[0043] A preferred example of an algorithm suitable for determining percent sequence identity and sequence similarity is 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 analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words W in the query sequence that match or meet some positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. (1990) J. Mol. Biol. 215:403-410). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence as far as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative scores are calculated using the parameters M (reward score for a pair of matching residues, always greater than 0) and N (penalty score for mismatching residues, always less than 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative scores. The extension of the word hits in each direction is stopped when the cumulative alignment score falls by an amount X from its maximum performance value, when the cumulative score falls below zero 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 as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4, and a comparison of both strands.For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and an expectation (E) of 10, and the BLOSUM62 scoring matrix (Henikoff and Henikoff (1989), Proc Natl Acad Sci USA, 89:10915) of 50, alignment (B) of 10, expectation (E) of 10, M=5, N=-4, and a 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 indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered to be 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 phrase "codon optimization," when referring to genes or coding regions of a nucleic acid molecule for transformation into various hosts, refers to modifying the codons in the genes or coding regions of the polynucleic acid molecule to reflect the typical codon usage of a selected organism without modifying the polypeptide encoded by the DNA. Such optimization includes 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] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA of a presequence or secretory leader is operably linked to DNA of a polypeptide 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 so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous with each other, and, in the case of a secretory leader, contiguous and in reading phase. However, operably linked nucleic acids (e.g., enhancer and coding sequence) need not be contiguous. Linkage is accomplished by ligation at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. In embodiments, a promoter is operably linked to a coding sequence if it is capable of affecting (e.g., regulating relative to the absence of the promoter) expression of a protein from that coding sequence (i.e., when the coding sequence is under the transcriptional control of the promoter).

[0047] The term "nucleobase" refers to the portion of a nucleotide that possesses Watson / Crick base pairing functionality. The most common naturally occurring nucleobases, adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T), possess hydrogen-bonding functionality that binds one nucleic acid strand to another in a sequence-specific manner.

[0048] As used throughout, "subject" (or "host") means an individual. Thus, a "subject" can include, for example, domestic animals such as cats, dogs, livestock (e.g., cows, 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 can be a mammal, such as a primate or a human. Administration of a therapeutic agent can be at a dosage and for a duration effective to treat the subject.

[0049] As used herein, the term "about" when referring to a measurable value, such as an amount, percentage, etc., is meant to encompass a variation of ±20%, ±10%, ±5%, or ±1% from the measurable value.

[0050] A nucleic acid sequence is "heterologous" to a second nucleic acid sequence if it is derived from a foreign species, or if derived from the same species, is modified by human action from its original form. For example, a heterologous promoter (or a heterologous 5' untranslated region (5'UTR)) operably linked to a coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, and if from the same species, refers to a coding sequence that is different from a naturally occurring allelic variant (e.g., a 5'UTR or 3'UTR from a different gene is operably linked to a nucleic acid encoding a costimulatory molecule).

[0051] The term "nanoparticle" as used herein refers to a particle or structure that is biocompatible and sufficiently resistant to chemical and / or physical destruction by the environment of use such that a sufficient number of the nanoparticles remain substantially intact after delivery to the site of use or treatment, and whose size is in the nanometer range. In some embodiments, the 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] A "therapeutically effective amount" or "therapeutically effective dose" of a composition refers to an amount effective to achieve a desired therapeutic result. 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. The term may also refer to the amount of therapeutic agent effective to promote a desired therapeutic effect, or the delivery rate of the therapeutic agent (e.g., amount over time). The exact desired therapeutic effect will vary depending on the condition being treated, the tolerance of the subject, the drug and / or drug formulation being administered (e.g., potency of the therapeutic agent, concentration of the drug in the formulation, etc.), as well as various other factors understood by those skilled in the art. In some cases, the desired biological or medical response is achieved over a period of days, weeks, or years after multiple administrations of the composition.

[0053] As used herein, the term "treating" or "treatment" of a subject includes administering a drug to a subject with the intent to cure, ameliorate, alleviate, relieve, alter, treat, ameliorate, improve, stabilize, or affect a disease or disorder, or a symptom of a disease or disorder. The terms "treat" and "treatment" can also refer to reducing the severity and / or frequency of symptoms, eliminating symptoms and / or underlying causes, and ameliorating or repairing damage.

[0054] As used herein, the term "prevention" of a disease, disorder, or undesired physiological event in a subject refers to the prevention of the disease, disorder, or undesired physiological event, or the prevention of a symptom of the disease, disorder, or undesired physiological event.

[0055] An "effective amount" of a drug refers to an amount of the drug sufficient to provide a desired effect. The amount of a drug that is "effective" will vary from subject to subject, depending on many factors, such as the age and general condition of the subject, the specific drug(s), and the like. Thus, a quantified "effective amount" cannot always be specified. However, an appropriate "effective amount" in any subject's case can be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, unless otherwise specified, an "effective amount" of a drug can refer to an amount that encompasses both a therapeutically effective amount and a prophylactically effective amount. The "effective amount" of a drug required to obtain a therapeutic effect can vary depending on factors such as the age, sex, and weight of the subject. Dosage regimens can also be adjusted to provide an optimal therapeutic response. For example, several divided doses can be administered daily, or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0056] A "pharmaceutical acceptable" component may refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation of the present invention and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation containing the component. When used in relation to human administration, the term generally means that the component has met the necessary standards of toxicological and manufacturing testing or that the component is included in the Inactive Ingredients Guide prepared by the U.S. Food and Drug Administration.

[0057] A "pharmaceutically acceptable carrier" (sometimes referred to as a "carrier") generally means a carrier or excipient useful in preparing a safe and non-toxic pharmaceutical or therapeutic composition, including carriers that are acceptable for veterinary and / or human pharmaceutical or therapeutic use. The term "carrier" or "pharmaceutically acceptable carrier" can 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 for use in pharmaceutical formulations, and as further described herein.

[0058] "Therapeutic agent" refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, such as, for example, the treatment of a disorder or other undesirable physiological condition, and prophylactic effects, such as, for example, the prevention of a disorder or other undesirable physiological condition. These terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of the beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, pro-agents, active metabolites, isomers, fragments, analogs, etc. When the term "therapeutic agent" is used, or when a particular agent is specifically identified, the term includes the agent itself, as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, pro-drugs, conjugates, active metabolites, isomers, fragments, analogs, etc.

[0059] As used herein, the term "controlled release" or "controlled release drug delivery" or "sustained release" refers to the release or administration of a drug from a given dosage form in a controlled manner to achieve desired pharmacokinetic properties in vivo. An aspect of "controlled" drug delivery is the ability to manipulate the formulation and / or dosage form to establish desired kinetics of drug release.

[0060] As used herein, the phrases "concurrent administration," "co-administration," "simultaneous administration," or "administered at the same time" mean that the compounds are administered at the same time or shortly after each other.

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

[0062] The term "antibody" is used broadly herein 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 human 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 is 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 mice. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0063] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of antibody molecules. Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired antagonistic activity.

[0064] The disclosed monoclonal antibodies can be produced using any procedure that produces monoclonal antibodies. For example, the disclosed monoclonal antibodies can be prepared using the hybridoma method, such as the method described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse or other suitable host animal is typically immunized with an immunizing agent to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0065] Monoclonal antibodies may also be produced by recombinant DNA methods. 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 genes encoding the heavy and light chains of mouse antibodies). Libraries of antibodies or active antibody fragments can also be produced and screened using phage display techniques, for example, as described in U.S. Pat. No. 5,804,440 (Burton et al.) and U.S. Pat. No. 6,096,441 (Barbas et al.).

[0066] In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to generate fragments thereof, particularly Fab fragments, can be accomplished using routine techniques known in the art. For example, digestion can be performed using papain. Examples of papain digestion are described in WO94 / 29348 published December 22, 1994, and U.S. Pat. No. 4,342,566. Papain digestion of antibodies usually generates two identical antigen-binding fragments, called Fab fragments, each with a single antigen-binding site and a remaining Fc fragment. Pepsin treatment results in a fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

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

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

[0069] The fragments may also include insertions, deletions, substitutions, or other selected modifications of specific regions or specific amino acid residues, whether or not linked to other sequences, so long as the activity of the antibody or fragment is not significantly altered or impaired compared to the unmodified antibody or antibody fragment. These modifications may provide some additional properties, such as removing / adding amino acids capable of disulfide bonding, extending biological lifespan, altering secretion properties, etc. In any case, the antibody or antibody fragment must have a biologically active property, such as specific binding to its cognate antigen. Functional or active regions of the antibody or antibody fragment may be identified by mutagenesis of specific regions of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to one 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" or "antibodies" may also refer to human and / or humanized antibodies. Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans and therefore may provoke an unwanted immune response when administered to humans. Thus, the use of human or humanized antibodies in these methods helps to reduce the chance that an antibody administered to a human will provoke an unwanted immune response.

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

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

[0073] chemical definition As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Exemplary substituents include, for example, the substituents described below. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, heteroatoms such as nitrogen can have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valence of the heteroatom. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. The terms "substituted" or "substituted with" also include the implicit proviso that such substitution is in accordance with the permissible valences of the substituted atom and substituent, as well as that the substitution results in a compound (e.g., a stable compound that does not naturally undergo transformation by rearrangement, cyclization, elimination, etc.).

[0074] "Z 1 ","Z 2 ","Z 3 " and "Z 4 " is used herein as a collective term to represent various specific substituents. These symbols can be any substituent not limited to the substituents disclosed herein, and when in one instance they are defined as specific substituents, they may be defined as some other substituent in another instance.

[0075] The term "aliphatic," as used herein, refers to non-aromatic hydrocarbon groups, and includes branched and unbranched alkyl, alkenyl, or alkynyl groups. The term "alkyl" as used herein refers to a branched or unbranched saturated hydrocarbon group of 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, etc. 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, sulfoxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below.

[0076] Throughout this specification, "alkyl" is used generally to refer to both unsubstituted and substituted alkyl groups, but substituted alkyl groups are specifically referred to herein by identifying the specific substituent(s) on the alkyl group. For example, the term "halogenated alkyl" specifically refers to an alkyl group substituted with one or more halogens, such as fluorine, chlorine, bromine, or iodine. The term "alkoxyalkyl" specifically refers to an alkyl group substituted with one or more alkoxy groups, as described below. The term "alkylamino" specifically refers to an alkyl group substituted with one or more amino groups, as described below. When "alkyl" is used in one instance and a specific term such as "alkylalcohol" is used in another instance, it is not intended that the term "alkyl" does not also refer to the specific term such as "alkylalcohol".

[0077] This practice is also used for other groups described herein. That is, a term such as "cycloalkyl" refers to both unsubstituted and substituted cycloalkyl moieties, but the substituted moieties can be further specifically identified herein, for example, a particular substituted cycloalkyl can be referred to as, for example, an "alkylcycloalkyl". Similarly, a substituted alkoxy can be specifically referred to as, for example, a "halogenated alkoxy", a particular substituted alkenyl can be, for example, an "alkenylalcohol", etc. Again, the practice of using a general term such as "cycloalkyl" and a specific term such as "alkylcycloalkyl" is not intended to mean that the general term does not also include the specific term.

[0078] As used herein, the term "alkoxy" refers to an alkyl group attached through a single, terminal ether linkage; i.e., an "alkoxy" group is -OZ. 1 where Z 1 is alkyl as 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. 1 Z 2 )C=C(Z 3 Z 4 Asymmetric structures such as alkene, alkoxy, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol may be included. This may be assumed in structural formulas herein where an asymmetric alkene is present, or may be explicitly indicated by the bond symbol C=C. Alkenyl 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 below.

[0080] The term "alkynyl" as used herein refers to a hydrocarbon group of 2 to 24 carbon atoms having a structural formula containing at least one carbon-carbon triple bond. Alkynyl groups can 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 below.

[0081] The term "aryl" as used herein is a group that includes any carbon-based aromatic group, including, but not limited to, benzene, naphthalene, phenyl, biphenyl, phenoxybenzene, and the like. The term "heteroaryl" is defined as a group that includes an aromatic group with 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 "non-heteroaryl" within the term "aryl" defines a group that includes an aromatic group that does not contain a heteroatom. The aryl or heteroaryl group may be substituted or unsubstituted. The aryl or heteroaryl 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 described herein. The term "biaryl" is a special type of aryl group and is included in the definition of aryl. Biaryl refers to two aryl groups linked through a fused ring structure, as in naphthalene, or through one or more carbon-carbon bonds, as in biphenyl.

[0082] As used herein, the term "cycloalkyl" refers to a non-aromatic carbon-based ring of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. The term "heterocycloalkyl" refers to a cycloalkyl group as defined above, in which at least one of the carbon atoms of the ring is replaced with a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. Cycloalkyl and heterocycloalkyl groups can be substituted or unsubstituted. Cycloalkyl and heterocycloalkyl groups can 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-based ring consisting of at least three carbon atoms and containing at least one double bond, i.e., C=C. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and the like. The term "heterocycloalkenyl" is a type of cycloalkenyl group defined above and is included in the meaning of the term "cycloalkenyl", in which at least one of the ring carbon atoms is replaced with a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. Cycloalkenyl and heterocycloalkenyl groups can be substituted or unsubstituted. The 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, as described herein.

[0084] The term "cyclic group" is used herein to refer to aryl groups, non-aryl groups (i.e., cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl groups), or both. Cyclic groups have one or more ring systems that can be substituted or unsubstituted. Cyclic groups can contain one or more aryl groups, one or more non-aryl groups, or one or more aryl groups and one or more non-aryl groups.

[0085] The term "aldehyde" as used herein is represented by the formula -C(O)H. Throughout this specification, "C(O)" or "CO" is a shorthand notation for C=O. As used herein, the term "amine" or "amino" refers to a group of the formula -NZ 1 Z 2 In the formula, Z 1 and Z 2may each be a substituent as described herein, for example, hydrogen, alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group as described above.

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

[0087] As used herein, the term "ester" refers to an ester of the formula -OC(O)Z 1 or -C(O)OZ 1 In the formula, Z 1 can be an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group as described above.

[0088] As used herein, the term "ether" refers to a group of the formula Z 1 OZ 2 In the formula, Z 1 and Z 2 can be independently an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group as described above.

[0089] As used herein, the term "ketone" refers to a compound of the formula Z 1 C(O)Z 2 In the formula, Z 1 and Z 2 can be independently an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group as described above.

[0090] The term "halide" or "halogen" as used herein refers to fluorine, chlorine, bromine, and iodine. The term "hydroxyl" as used herein is represented by the formula --OH.

[0091] The term "nitro" as used herein is represented by the formula --NO. As used herein, the term "silyl" refers to a group of the formula -SiZ 1 Z 2 Z 3 In the formula, Z 1 , Z 2 , and Z 3 may be independently hydrogen, an alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group as described above.

[0092] The term "sulfonyl" refers to a group of the formula -S(O)Z 1 is used herein to refer to a sulfooxo group represented by the formula 1 can be hydrogen, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group as described above.

[0093] The term "sulfonylamino" or "sulfonamide" as used herein is represented by the formula -S(O)2NH-. The term "phosphonyl" refers to a group of the formula -P(O)(OZ 1 )2, wherein Z 1 can be hydrogen, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group as described above.

[0094] The term "thiol" as used herein is represented by the formula --SH. The term "thio" as used herein is represented by the formula -S-. As used herein, "R 1 ", "R 2 ", "R 3 ", "R n " etc., where n is any integer, may independently carry one or more of the above groups. For example, R 1 When is a straight chain alkyl group, one of the hydrogen atoms of the alkyl group can be optionally replaced with a hydroxyl group, an alkoxyl group, an amine group, an alkyl group, a halide, etc. Depending on the group selected, the first group may be embedded within the second group, or alternatively, the first group may be pendant (i.e., attached) to the second group. For example, in the phrase "an alkyl group that includes an amino group," the amino group may be embedded 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 group(s) selected will determine whether the first group is embedded or attached to the second group.

[0095] Unless stated to the contrary, formulas having chemical bonds shown with solid lines only, and no wedges or dashed lines, contemplate each of the possible isomers, e.g., 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 aspects of the disclosed materials, compounds, compositions, articles and methods, examples of which are illustrated in the accompanying examples and drawings. compound In some embodiments, disclosed herein are compounds having formula I, II, or III:

[0097] [ka] or a salt thereof, wherein R 1are 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 formula:

[0099] [ka] or a salt thereof, wherein R 1 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 formula:

[0101] [ka] or a salt thereof, wherein R 1 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 formula:

[0103] [ka] or a salt thereof, wherein R 1 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 are the following:

[0105] [ka] or a salt thereof.

[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] It is.

[0117] In some embodiments, the compound is

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

[0119] [ka] It is.

[0120] In some embodiments, the compound is

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

[0122] [ka] It is.

[0123] In some embodiments, the compound is

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

[0125] [ka] It is.

[0126] In some embodiments, the compound is

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

[0128] [ka] It is.

[0129] In some embodiments, R 1 is alkyl. In some embodiments, R 1 is alkenyl. In some embodiments, R 1 is 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 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 In some embodiments, alkyl is C 11 In some embodiments, alkyl is C 12 In some embodiments, alkyl is C 13 In some embodiments, alkyl is C 14 In some embodiments, alkyl is C 15 In some embodiments, alkyl is C 16In some embodiments, alkyl is C 17 In some embodiments, alkyl is C 18 In some embodiments, alkyl is C 19 It is an alkyl.

[0132] Nanoparticles In one aspect, the present disclosure provides nanoparticles comprising a compound of Formula I, II, or III described herein.

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

[0134] In some embodiments, the nanoparticles include non-cationic lipids. In some embodiments, the non-cationic lipids interact with the 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), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine ...1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DSPC), 1-stearoyl-sn-glycero-3-phosphoethanolamine (SOPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DSPC), 1-stearoyl-sn-glycero-3-phosphoethanolamine (SOPE), 1,2-dioleoyl-sn-glycero- The non-cationic lipid may include, but is not limited to, 1,2-dioleoyl-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 a combination thereof. In one embodiment, the non-cationic lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In one embodiment, the non-cationic lipid is 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE). In one embodiment, the non-cationic lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In one embodiment, the non-cationic lipid is 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE). Although several non-cationic lipids are described herein, additional non-cationic lipids can be used in combination with the compounds disclosed herein.

[0135] In some embodiments, the nanoparticles comprise non-cationic lipids at a molar ratio of about 10% to about 80%. In some embodiments, the nanoparticles comprise non-cationic lipids at a molar ratio of about 10% to about 40%. In some embodiments, the nanoparticles comprise non-cationic lipids at a molar ratio of about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40%. In one embodiment, the nanoparticles comprise non-cationic lipids at a molar ratio of about 30%.

[0136] In some embodiments, the nanoparticles include polyethylene glycol lipids (PEG lipids). The 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 phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, and PEG-modified dialkylglycerols. 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 one embodiment, the polyethylene glycol lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol-2000 (DMG-PEG2000). DMG-PEGXXXX means 1,2-dimyristoyl-sn-glycerol, methoxypolyethyleneglycol-XXXX, where XXXX represents the molecular weight of the polyethylene glycol moiety, for example, DMG-PEG2000 or DMG-PEG5000.

[0137] In some embodiments, the nanoparticles comprise polyethylene glycol lipids at a molar ratio of about 0% to about 5%. In some embodiments, the nanoparticles comprise polyethylene glycol lipids at a molar ratio 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 comprise polyethylene glycol lipids at a molar ratio of about 0.75%.

[0138] In some embodiments, nanoparticles contain sterol.Sterol is well known to those skilled in the art and generally refers to a compound that has a perhydrocyclopentanophenanthrene ring system and has one or more OH substituents.Examples of sterol include, but are not limited to, cholesterol, campesterol, ergosterol, sitosterol, etc.

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

[0140] Sterols can be used to modulate the permeability and fluidity of particles based on their function in cell membranes, hi 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 comprise a pharmaceutical agent.In one embodiment, the nanoparticles further comprise a therapeutic agent.In one embodiment, the nanoparticles further comprise a diagnostic agent.

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

[0143] The agent delivered to the cell may be a polynucleotide. The polynucleotides or oligonucleotides that can be introduced according to the methods herein include all types of DNA, cDNA, and RNA sequences. For example, the polynucleotide may be double-stranded DNA, single-stranded DNA, complexed 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. The polynucleotide may also be a DNA construct, such as an expression vector, encoding a desired gene product (e.g., a gene product homologous or heterologous to the subject into which it is introduced). In one embodiment, the agent is an mRNA. In some embodiments, the polynucleotide is encapsulated by a nanoparticle.

[0144] In some embodiments, the polynucleotide comprises a nucleotide acid encoding a costimulatory molecule. In some embodiments, the costimulatory 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 costimulatory molecule is CD40. In some embodiments, the costimulatory molecule is CD40L.

[0145] Examples of sequences of costimulatory molecules (for 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), 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 costimulatory molecule comprises a heterologous 5' untranslated region (5'UTR). In some embodiments, the mRNA encoding the costimulatory molecule comprises a heterologous 3' untranslated region (3'UTR).

[0147] In some embodiments, a nucleic acid disclosed herein (e.g., an mRNA encoding a costimulatory molecule) comprises at least one chemically modified nucleotide. In some embodiments, the at least one chemically modified nucleotide comprises 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 nucleobase. In one embodiment, the chemically modified nucleobase is 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-carboxymethylesteruridine (5camU), pseudouridine (Ψ), N 1 -Methylpseudouridine (me 1 Ψ), N 6 -Methyladenosine (me 6 A), or thienoguanosine ( th G).

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

[0150] The structures of these modified nucleobases are shown below:

[0151] [ka]

[0152] In one embodiment, the 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'-azido, 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-amino BNA, or N-MeO-aminooxy BNA. 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 riboses 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'-phosphoramidate (NP), phosphodiester (PO), or 2',5'-phosphodiester (2',5'-PO). In one embodiment, the chemically modified phosphodiester linkage is phosphorothioate.

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

[0157] [ka]

[0158] antigen presenting cells In some aspects, disclosed herein is an antigen presenting cell comprising a lipid-based nanoparticle comprising a compound of any of the preceding aspects and a recombinant polynucleotide comprising a nucleic acid encoding a costimulatory molecule.

[0159] In some embodiments, the compound is

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

[0161] [ka] It is.

[0162] In some embodiments, the costimulatory 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 costimulatory molecule is CD40.

[0163] In some embodiments, the antigen presenting cells comprise macrophages or dendritic cells. The term "antigen-presenting cells" or "APCs" as used herein should be understood to refer to a heterogeneous group of immune cells that can process and present 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] It is understood herein that macrophages are commonly 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 supporting role through a diverse repertoire of plasma membrane and secreted molecules (Gordon 1995, Bioessays, Volume 17, Issue 11), as previously shown for erythroblasts, hepatocytes and neurons (Sadahira & Morr, Pathol Int. 1999 Oct; 49(10): 841-8.) (Takeishi, Hirano, et al. Arch Histol Cytol. 1999 Dec; 62(5): 413-22) (Polazzi, Gianni, et al. Glia. 2001 Dec; 36(3): 271-80). By "macrophage" is meant a cell that exhibits the characteristics typically described for macrophages, including phagocytosis, expression of defined cell surface markers such as CD64, CD14 and HLA-DR antigen expression. Macrophages according to the present invention may be isolated from tissues or, preferentially, by ex vivo 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 precursors, and by using any differentiation method, precursors and methods known to those skilled in the art. Thus, 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] It should also be understood that "dendritic cells" or "DCs" as used herein refer to a type of antigen-presenting cell typically identified by the expression of one or more of the following markers on its 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. Dendritic cells according to the present invention may be isolated from tissues or preferentially by ex vivo differentiation from blood monocytes (also referred to herein as "monocyte-derived dendritic cells"), bone marrow progenitor cells (also referred to herein as "bone marrow-derived dendritic cells") or any other possible precursors, and by using any differentiation method, precursors, and methods known to those skilled in the art. Thus, in some embodiments, the dendritic cells comprise bone marrow-derived dendritic cells. In some embodiments, the dendritic cells comprise monocyte-derived dendritic cells. In some embodiments, the dendritic cells comprise iPSC-derived dendritic cells. In some embodiments, the dendritic cell is a conventional dendritic cell 1 (or cDC1, lymphoid DC), typically identified by 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 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 expression of one or more of the following markers on its cell surface: CD123, CD303, and CD304.

[0166] In some aspects, disclosed herein is a method for expressing a polypeptide in antigen presentation, comprising administering to an antigen presenting cell an effective amount of a nanoparticle disclosed herein, wherein the nanoparticle comprises a recombinant polynucleotide comprising a nucleic acid encoding the polypeptide. Compositions and methods In some aspects, described herein are methods of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of the antigen presenting cells and antibodies disclosed herein.

[0167] In some embodiments, the immunotherapeutic agent 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 antibody or antigen-binding fragment thereof that specifically binds to a costimulatory molecule is BMS986178. In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to a costimulatory molecule is GSK3174998. In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to a costimulatory molecule is PF-04518600. In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to a costimulatory molecule is MOXR0916. In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to a costimulatory molecule is PF-04518600. In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to a costimulatory molecule is MEDI6383. In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to a costimulatory molecule is MEDI0562. In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to a costimulatory molecule is INCAGN01949. In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to a costimulatory molecule is InVivoPlus anti-mouse OX40 (clone OX-86) (company: BioXcell, catalog: BP0031).

[0168] Additional antibodies or antigen-binding fragments thereof that specifically bind to costimulatory molecules include, for example: 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 X40, BMS986178, GSK3174998, PF-04518600, MOXR0916, PF-04518600, MEDI6383, MEDI0562, INCAGN01949; anti-human 4-1BB, utomirumab, urelumab; anti-human CD40, CP-870893, APX005M, ADC-1013, JNJ-64457107, SEA-CD40, RO7009789.

[0169] In some embodiments, the antigen presenting cells and antibodies are administered intratumorally. In some embodiments, disclosed herein is a method of treating cancer comprising administering to a subject in need thereof: a therapeutically effective amount of a lipid-based nanoparticle disclosed herein, and A therapeutically effective amount of an antigen presenting cell disclosed herein.

[0170] In some embodiments, the nanoparticles comprise:

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

[0172] [ka] It is.

[0173] In some embodiments, the lipid-based nanoparticles comprise a recombinant polynucleotide comprising a nucleic acid encoding CD40L. In one aspect, the methods described herein are directed to the treatment of cancer, such as, among others, melanoma, lung cancer (such as lung adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, large cell carcinoma, bronchioloalveolar carcinoma, bronchogenic carcinoma, non-small cell carcinoma, small cell carcinoma, mesothelioma, etc.); breast cancer (such as ductal carcinoma, lobular carcinoma, inflammatory breast cancer, clear cell carcinoma, mucinous carcinoma, serous cavity breast carcinoma, etc.); colorectal cancer (such as colon carcinoma, rectal carcinoma, colorectal adenocarcinoma); anal cancer; pancreatic cancer (such as pancreatic adenocarcinoma, pancreatic islet cell carcinoma, neuroendocrine tumors, etc.); prostate cancer; prostate adenocarcinoma; ovarian cancer (such as ovarian epithelial or serous tumors, superficial epithelial stromal tumors, endometrioid tumors and mucinous cystadenocarcinomas, sex cord interstitial tumors, etc. interstitial tumors);liver and bile duct cancer (hepatocellular carcinoma, cholangiocarcinoma, 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, uterine serous carcinoma, uterine clear cell carcinoma, uterine sarcoma, leiomyosarcoma, mixed Mullerian tumor);glioma, glioblastoma, medulloblastoma, and other tumors of the brain;kidney cancer (renal cell carcinoma, clear cell carcinoma, Wilms' tumor, etc.);head and neck cancer (squamous cell carcinoma, etc.);stomach cancer (gastric carcinoma, gastric adenocarcinoma, gastrointestinal stromal tumor);testicular tumor;germ cell tumor;neuronal tumor urinary tumors; cervical cancer; carcinoids of the gastrointestinal tract, breast, and other organs; signet ring cell carcinoma; mesenchymal tumors such as sarcomas, fibrosarcoma, hemangiomas, hemangiomatosis, hemangiopericytoma, pseudoangiomatous stromal hyperplasia, myofibroblastoma, fibromatosis, inflammatory myofibroblastic tumor, lipoma, angiolipoma, granular cell tumor, neurofibroma, neurilemmoma, angiosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma, leiomyoma, leiomyosarcoma, skin, melanoma, neck, retinoblastoma, head and neck cancer, pancreatic, brain, thyroid, testicular, kidney, bladder, soft tissue, adrenal, urethral, ​​penile cancer, myxosarcoma, chondrosarcoma, osteosarcoma, chordoma , malignant fibrous histiocytoma, lymphangiosarcoma, mesothelioma, squamous cell carcinoma; epidermoid carcinoma, malignant skin adnexal tumor, adenocarcinoma, hepatoma, hepatocellular carcinoma, renal cell carcinoma, adrenal gland tumor, cholangiocarcinoma, transitional cell carcinoma, choriocarcinoma, seminoma, embryonal cell carcinoma, anaplastic glioma; glioblastoma multiforme, neuroblastoma, medulloblastoma, malignant meningioma, malignant neurilemmoma, neurofibrosarcoma, parathyroid carcinoma, medullary thyroid carcinoma, bronchial carcinoid, pheochromocytoma, islet cell carcinoma, malignant carcinoid, malignant paraganglioma, melanoma, Merkel cell neoplasm, phyllodes cystosarcoma, salivary carcinoma, thymic carcinoma, vaginal carcinoma. EXAMPLES

[0174] The following examples are provided below to illustrate compositions, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of 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 present invention that are 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, we combined lipid nanoparticle-mRNA preparations with dendritic cell therapy 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 the expression of CD40L. Next, CD40-overexpressed dendritic cells are adoptively transferred, which are then activated by CD40L molecules in tumor tissue. This promotes the secretion of proinflammatory cytokines and chemokines and upregulation of costimulatory molecules on dendritic cells, which are important for reprogramming the TME and priming T cell responses. After dendritic cells present TAAs to T cells, all the above stepwise events contribute to the enhancement of potent cancer-specific T cell immunity that clears established skin tumors, suppresses distant lesions, and prevents tumor rechallenge. Overall, this study provides closure of the cancer immune cycle (CIC) in a mouse melanoma model by integrating lipid nanoparticles with cellular therapy (CATCH), promoting tumor elimination and building long-term antitumor immunity.

[0176] preface The development of immunotherapy has brought about major breakthroughs in the clinical outcomes of cancer treatment. Effective antitumor immunity depends on the appropriate 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 APCs then present antigens to T cells, leading to the priming of effector T cell responses against TAAs. 1 These activated effector T cells infiltrate tumor tissue and attack cancer cells. 1 Finally, dead cancer cells are mediated by CICs. 1 release additional antigens to increase the breadth and depth of these immune responses in 1 However, these cascading events are usually attenuated by the immunosuppressive tumor microenvironment (TME), resulting in uncontrolled tumor growth, metastasis, and recurrence. 1,2 For example, an immunosuppressive TME can inhibit dendritic cell recruitment, infiltration, and maturation. 1,2 This reduces the secretion of cytokines and expression of costimulatory agents by dendritic cells that are important for priming T cell immunity. 1,2 .

[0177] We investigated whether CICs could be appropriately elevated by synergistically inducing cancer cell death to release TAAs and improving the number and maturity of dendritic cells in tumor tissues. This could further promote systemic and memory antitumor immunity, leading to the elimination of primary tumors, inhibition of tumor metastasis, and prevention of tumor recurrence. Immunogenic cell death (ICD), a mode of cell death, is accompanied by the shedding of damage-associated molecular patterns (DAMPs) and TAAs. 3,4 Recent studies have demonstrated that lipid nanoparticles (LNPs) with the ability to induce ICD can remodel the TME, thereby enhancing antitumor immunity. 5 CD40 and CD40 ligand (CD40L) are a pair of costimulatory molecules that belong 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 important for dendritic cell maturation, which promotes the secretion of proinflammatory cytokines and upregulation of other costimulatory molecules.6 They play a central role in inducing CD8 T cells to generate 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 therapeutic efficacy. 7 Accordingly, several LNP-mRNA formulations and immune cells delivered via intrathecal injection are underway in clinical trials. 7-10 In this study, we utilized LNPs containing CD40L mRNA (CD40L-LNPs) to simultaneously induce ICD and CD40L expression in tumor tissues. We then captured TAAs and performed adoptive transfer of CD40-overexpressing bone marrow-derived dendritic cells (CD40-BMDCs) that were matured by stimulation of CD40L. These activated dendritic cells further presented antigens to T cells, resulting in the priming of effector T cell responses 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 that are widely used as sugar substitutes in the food industry because they provide a sweet taste, contain fewer calories, and do not cause blood sugar spikes compared to regular sugars such as glucose. 11 These compounds occur naturally or are derived from sugars by reduction of the carbonyl group to a hydroxy group. 12 In particular, hexitols such as sorbitol and mannitol are six-carbon sugar alcohols that can be doubly dehydrated to give dianhydrohexitols, a series of unique cyclic ethers that serve as important chemical scaffolds and starting materials. 13 Therefore, three sets of sugar alcohol-derived ionizable lipids were designed and synthesized to prepare LNPs for mRNA delivery. To examine these LNPs in bioassays, melanoma was selected as a disease model. Melanoma is one of the most aggressive skin cancers with increasing morbidity. 14The main cause of death in melanoma patients is widespread metastasis, e.g., to the skin and brain. 14 Therefore, two melanoma cell lines were selected to construct mouse tumor models in this study. After systematic screening and characterization of sugar alcohol-derived ionizable lipids, two preferred LNP formulations for mRNA delivery were identified. One formulation induced strong ICD in melanoma tumor tissues, during which it effectively delivered CD40L mRNA into cancer cells. The other formulation showed dramatically higher CD40 mRNA delivery efficiency in mouse bone marrow-derived dendritic cells (BMDCs) compared with Lipofectamine 3000 (Lipo3K) and electroporation (Electro). The combination of CD40L-LNP treatment and adoptive transfer of CD40-BMDCs resulted in a complete response rate of over 80% in a subcutaneous (sc) melanoma tumor model. Moreover, local treatment enabled the inhibition or resolution of tumor metastasis in the skin and brain. Importantly, these responder mice were resistant to subcutaneous rechallenge and showed delayed tumor growth after intracranial rechallenge. In summary, two types of LNP-mRNA preparations were utilized to increase CIC by inducing ICD and adaptive dendritic cell engraftment. This study presents an important immunotherapeutic strategy that allows for broad and effective treatment of primary tumors, tumor metastases, and tumor recurrence.

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

[0180] Next, LNPs containing firefly luciferase (FLuc) mRNA (FLuc-LNPs) were prepared, and their size, surface charge and mRNA encapsulation efficiency were identified (Figures 6A-6C). The structure-activity relationship of sugar alcohol-derived ionizable lipids was generalized by quantification of luminescence intensity in bone marrow-derived dendritic cells (BMDCs). The chirality of the amine core may affect the mRNA delivery efficiency of the formulated LNPs. The amine cores of DIS and LIS lipids are a pair of enantiomers, and they showed similar mRNA delivery capabilities, especially DIS-1-8 and LIS-1-8. Lipids with C10 hydrocarbon tails induced more effective mRNA delivery than lipids containing other hydrocarbon tails. Meanwhile, the DIM series 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 capabilities. Among these LNPs, DIM7 and LIS10 showed the highest mRNA delivery efficiency over other LNPs, which were 3-fold and 10-fold more effective for mRNA delivery than Lipo3K and Electro, respectively (Figure 2A). To further explore the formulation of DIM7 and LIS10, an orthogonal screening assay was performed with L16 (4) 4Based on the orthogonal table of (Figure 7A). According to the luminescence intensity of 16 orthogonal formulations of DIM7 or LIS10 (Figure 2C, 2E), the effect of each lipid component on mRNA delivery was examined by different molar ratios (Figure 2B, 2D) and some predicted preferred formulations (Figure 2B). Then, their mRNA delivery efficiency was verified by comparing with the corresponding top formulations from the orthogonal screening assays, DIM7 M and LIS10G (Figure 2C, 2E). With respect to DIM7, the preferred formulation DIM7 S showed 2-fold (P<0.001) and 3.5-fold (P<0.001) higher luminescence signals compared to the orthogonal top formulation DIM7M and initial formulation DIM7, respectively (Figure 2C). As for LIS10, the preferred formulation LIS10W yielded a 1.2-fold (P<0.0001) higher luminescence signal than the top orthogonal formulation LIS10G, and a 2.3-fold (P<0.0001) higher luminescence intensity than the initial formulation LIS10 (Figure 2E). Formulations DIM7S and LIS10W also showed similar properties. Their particle sizes were about 110 nm with a polydispersity index (PDI) <0.2 (Figures 2G, 2I). Their mRNA encapsulation efficiency was about 90%, and they were slightly positively charged (Figures 2G, 2I). Moreover, both of them showed spherical morphology when visualized by Cryo-TEM (Figures 2H, 2J, Figures 7B, 7C). As a result, formulation DIM7S was selected to deliver mRNA ex vivo to BMDCs for the following studies. At the same mRNA concentration, formulation DIM7S was 10- and 30-fold superior in mRNA delivery to Lipo3K and Electro, respectively (FIG. 7D). The maximum luminescence intensity of formulation DIM7S was observed at 12:00 over the 6- to 24-h time course (FIG. 7E). Significantly, formulation DIM7S encapsulation of CD40 mRNA (CD40-DIM7S) resulted in over 70% CD40 expression in BMDCs compared to approximately 17% in untreated BMDCs (P<0.0001, FIG. 7F).

[0181] Dendritic cell activation is a key step for priming T cell responses in CICs 1Therefore, we next tested whether CD40 overexpression promoted BMDC activation in the presence of anti-CD40 agonist antibody (CD40Ab). Compared with PBS, CD40 Ab, and CD40-DIM7S treatment, the combination of CD40-DIM7S and CD40 Ab (CD40-DIM7S+CD40 Ab) stimulated higher levels of 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) (Figure 3A), indicating a stronger capacity for dendritic cell activation. To evaluate whether enhanced activation of dendritic cells improves antitumor efficacy in vivo, intratumoral (it) injections were performed for multiple treatments, four times daily administration, 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 with all other treatments, CD40-BMDC+CD40 Ab significantly inhibited tumor growth by 5-15 fold (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 engineer dendritic cells in situ to increase CD40 receptor expression (Figure 9A). However, CD40-DIM7S+CD40 Ab showed limited therapeutic effect compared with CD40-BMDC+CD40 Ab (Figures 3C, 3D, and 8), which may be partially due to the lack of infiltrating dendritic cells in the immunosuppressive TME. 1,2 Although BMDC+CD40 Ab recruited dendritic cells in tumor tissues, the insufficient activation of BMDCs may be the reason for the weak antitumor effect. 1,2 Collectively, these results highlight the importance of sufficient dendritic cell infiltration, CD40 overexpression on BMDCs, and CD40 agonism for the antitumor activity of CD40-BMDC+CD40 Ab treatment.

[0182] Although CD40-BMDC+CD40 Ab showed promising antitumor effects, the failure to completely eliminate tumors indicates insufficient progression of CIC. Cancer cell death, the first step in CIC, allows the release of TAAs presented by dendritic cells and promotes tumor-specific T cell responses. 1,3-5 Furthermore, clinical studies have demonstrated that ICD in cancer cells is beneficial for the antitumor effects of immunotherapy. 3.4Therefore, we investigated whether cancer cell ICD induced by LNPs could promote the antitumor effect of CD40-BMDC therapy. The expression of CD40L mediated by LNPs was also examined in tumor tissues to see whether it could activate adoptively transferred CD40-BMDCs. As a result, the combination of cancer cell ICD induced by CD40L-LNPs and adoptive CD40-BMDC transfer can effectively increase 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 strong cytotoxicity (85%) after 18 h of treatment (Figure 9B). Because both CD40L-DIM7S and CD40L-LIS10W resulted in nearly 100% CD40L expression in B16F10 melanoma cells after 18 h of incubation (Figure 9C), LIS10W was chosen to assess its ability to induce ICD markers, including extracellular high-mobility group box 1 (HMGB1), extracellular ATP, and cell surface calreticulin, in B16F10 melanoma cells. Compared with 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). Consistently, ICD induced by FLuc-LIS10W or CD40L-LIS10W was observed in mouse B16F10 melanoma tissues in vivo ( Figure 3F ), ​​suggesting that LIS10W LNPs contributed more than just the mRNA cargo to the ICD effect.

[0183] Because the expression profile of CD40L in tumor tissues may affect its biological activity, its biodistribution was studied at the cellular level by flow cytometry. A single injection of CD40L-LIS10W resulted in approximately 15% CD40L-positive B16F10 cells in tumor tissues (Figure 9D). Furthermore, immune cells in tumor tissues, including macrophages, dendritic cells, CD4 T cells, and CD8 T cells, showed approximately 1.5- to 2-fold increased CD40L expression (Figure 9E). Both the expression of CD40L in 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 adoptively transferred CD40-BMDC, CD40-BMDC were 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 rechallenge in the contralateral flank (Figure 3I, Figure 10B), indicating the development of antitumor memory. These results highlighted the important role of ICD in priming antitumor immunity in CICs. To further confirm the function and CD40L-mediated activation of CD40-BMDCs in this treatment regimen, two other treatment groups including FLuc-LIS10W+CD40-BMDCs and CD40L-LIS10W+CD40-DIM7S were further investigated (Figure 3J). Although FLuc-LIS10W and CD40L-LIS10W showed similar ICD effects in vivo (Figure 3F), CD40L-LIS10W+CD40-BDMC dramatically suppressed tumor growth by approximately 21-fold (P<0.05) and 7-fold (19 days after inoculation, P<0.05) compared with FLuc-LIS10W+CD40-BDMC and CD40L-LIS10W+CD40-DIM7S, respectively (Figures 5C, 5D). Furthermore, 83% of mice from the CD40L-LIS10W+CD40-BDMC group survived, while none from the other two treatments survived 45 days after tumor inoculation (P<0.01, Figure 3K). Furthermore, when re-administered with B16F10 cells in the contralateral flank, 100% responder mice were resistant to the development of secondary tumors (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. Antitumor memory against tumor recurrence is necessary for the long-term efficacy of immunotherapy. Thus, the development of antitumor memory was further validated via an intracranial (ic) rechallenge mouse model (Figure 3M).In this experiment, another melanoma cell line (B16F10-Luc2) was used that contains a luciferase reporter, 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). Notably, surviving mice showed significantly delayed tumor growth in the brain (11 days after inoculation, P<0.05, Figure 10G). Furthermore, 38% of responder mice remained tumor-free compared to 0% of naive controls (75 days after ic rechallenge, P<0.01, Figure 3O). These data indicate that a potent antitumor memory T cell population was generated from this local therapy.

[0185] The efficacy of local immunotherapy in the treatment of metastatic cancer depends on the generation of systemic antitumor immunity 5,7,10 To assess this, B16F10 cells were seeded on both flanks of 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), 70% of the distant tumor (Figure 4B), and the overall survival rate was about 70% (Figure 4C). In another two-tumor model (skin + brain tumor), B16F10-Luc2 cells were inoculated subcutaneously and intravenously (Figure 4D). CD40L-LIS10W+CD40-BDMCs treatment eradicated 80% of the subcutaneous tumor (Figure 11B). Meanwhile, the treatment visibly regressed tumor growth in the brain (11 days after inoculation, P<0.001, Figure 4E) and significantly extended the overall survival time (P<0.0001, Figure 4F)). Collectively, these results indicate that local treatment resulted in systemic antitumor immunity.

[0186] T cell-mediated cancer cell killing is a critical step to eradicate neoplastic lesions in CIC. To evaluate the role of CD8 and CD4 T cells in the therapeutic effect, CD40L-LIS10W+CD40-BDMC treatment was performed on mice administered anti-CD8, anti-CD4, or isotype control antibodies (Figure 4G). Compared with the control group, depletion of CD8 or CD4 T cells significantly impaired tumor regression and mouse survival of CD40L-LIS10W+CD40-BDMCs treatment (Figures 4H, 4I, and 11C). Notably, depletion of CD8 T cells more significantly reduced the therapeutic effect compared with depletion of CD4 T cells (Figures 4H, 4I, and 11C), suggesting the important role of CD8 T cells in this treatment.

[0187] To clarify the underlying mechanism of action of CD40L-LIS10W+CD40-BDMC on the therapeutic regimen, dynamic expression of cytokines and chemokines was used in mouse melanoma tissues and blood after the first injection (Figure 4J). In tumor tissues, CD40L-LIS10W induced 29 cytokines and chemokines, such as GM-CSF, IFN-γ, TNF-α, CCL5, and CXCL10, in a 32-plex panel from a 6- to 24-h time course (Figure 4K, Figure 12). Moreover, their concentrations were further increased after administration of CD40-BDMC (Figure 4K, Figure 12). In blood, most of the cytokines and chemokines were upregulated 6 h after injection of CD40L-LIS10W, and their concentrations gradually decreased over the next 18 h (Figure 4L, Figure 13). However, administration of CD40-BDMCs re-stimulated the production of many cytokines and chemokines over a time course from 24 to 42 hours (Figure 4L, Figure 13). These data indicate that CD40L-LIS10W+CD40-BDMCs successfully reprogrammed the immunosuppressive TME and induced a stronger systemic immune response than CD40L-LIS10W alone. The upregulation of these inflammatory cytokines and chemokines may not only enhance immune cell recruitment and activation but also promote the development of immunological memory. Next, we profiled the changes in immune cell populations in tumor tissues after CD40L-LIS10W+CD40-BDMCs treatment. In this experiment, CD40-BMDCs were labeled with CellTrace Blue before injection, allowing us to distinguish administered dendritic cells from endogenous dendritic cells by flow cytometry analysis. Treatment resulted in a massive influx of dendritic cells, CD8 T cells, and CD4 T cells, whereas recruitment of macrophages and regulatory T (Treg) cells in tumor tissue was reduced (Figure 5A). + Macrophages (P<0.01) and CD80 / 86 +The treatment increased the proportion of antitumor phenotype APCs, including dendritic cells (P<0.0001, Figure 5B). Importantly, the 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 numbers of effector and central memory T cells in both the spleen and blood were significantly increased, 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 CIC via comprehensive immune responses. 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- and 30-fold higher mRNA delivery efficiency in BMDCs than Lipo3K and Electro, respectively (Figure 7D). LIS10W induced potent ICD in melanoma tissues (Figure 3F) and effectively delivered mRNA to melanoma cells (Figures 9C, 9D). For the treatment of CD40L-LIS10W+CD40-BMDCs, 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 concomitant CD40L expression in tumor tissues. CD40-BMDCs were then adoptively transferred into CD40-BMDCs engineered ex vivo with CD40-DIM7S. Both in vitro and in vivo data showed that these activated dendritic cells upregulated costimulatory molecules (Figure 3A, Figure 5B) and inflammatory cytokines (Figure 3A, Figure 4K, 4L), together with the presented TAAs, stimulating the differentiation of naive T cells into cytotoxic CD8 T cells (Figure 5C). On the one hand, this is beneficial for T cell-mediated killing of cancer cells, which is a crucial step for eliminating tumor lesions in CICs. 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 (Figure 4K, 4I). In response to these chemokines, additional APCs and T cells are recruited to the tumor tissue (Figure 5A). All these events contribute to the reprogramming of the immunosuppressive TME and the continuous amplification and expansion of CICs, ultimately resulting in the complete regression of more than 80% of primary cutaneous melanoma tumors (Figure 3H, 3K, 3N).Importantly, boosted CICs provided benefits for the generation of memory CD8 T cells in the spleen and blood (Figure 5D, 5E), resulting in nearly 100% and 38% protection from subcutaneous and IC melanoma rechallenge in responder mice, respectively (Figure 3I, 3L, 3O). Finally, this local treatment prevented distant skin tumors by approximately 70% and significantly regressed distant brain tumors, suggesting effective suppression of metastatic cancer.

[0189] In summary, we show that synergistic induction of cancer cell immunogenic cell death (ICD) and dendritic cell activation, the first two steps in the cancer immune cycle (CIC), can reprogram the immunosuppressive TME and promote tumor antigen presentation. This strategy results in strong effector T cell responses and protective immune memory against cancer-specific antigens, reflected in eradication of both local and distant tumors and protection 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 for 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 for 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 B16F10, a melanoma cell line, was obtained from Dr. Jianhua Yu's laboratory. 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 with Dulbecco's modified Eagle's medium (ATCC, 302002) containing 10% fetal bovine serum (FBS, Gibco, 26140079). Murine bone marrow-derived dendritic cells (BMDCs) were obtained by adapting a previous procedure. 19 Briefly, 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 gating strategy was as previously reported. 10,19 was based on.

[0192] B16F10 cells: CD45 - Macrophages: 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 Firefly luciferase (FLuc), mouse CD40, and mouse CD40L linear dsDNA were obtained from Integrated DNA Technologies. Plasmids were generated using the pUC19 vector. FLuc, mouse CD40, and mouse CD40L mRNA were synthesized by methods previously reported. 20 .

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

[0195] In the initial screening, the newly synthesized ionizable lipids were formulated 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, L16(4) 4 The preparation was based on the orthogonal table and the predicted preparation. Lipofectamine 3000 / mRNA complex was prepared based on the recommended protocol. Electroporation for BMDC was performed using Mouse Dendritic Cell Nucleation Reagent Kit (Lonza, VAPA1011). Delivery efficiency of mRNA was determined by luciferase expression assay.

[0196] Expression of CD40 and D40L Approximately 1×10 6 BMDCs were seeded in each well of a 6-well plate and treated with 2.5 μg of CD40-LNP for 12 h. Approximately 1 × 10 5 B16F10 cells were seeded in each well of a 24-well plate and treated with 0.25 μg of CD40L-LNP for 18 h. Cells were then stained with CD40-FITC or CD40L-FITC antibodies and analyzed on an LSRFortessa flow cytometer (Becton Dickinson, USA).

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

[0198] Activation and analysis of dendritic cells CD40-overexpressing BMDCs (CD40-BMDCs) were incubated with 10 μg / mL of anti-mouse CD40 antibody for 12 h. Then, several activation markers were 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. Immunogenic cell death (ICD) by LNPs ICD markers were detected by adapting a previous method 5 The cytotoxicity of LNPs in B16F10 cells was examined by MTT assay. Extracellular ATP was measured by luminescent ATP detection kit (Abcam, ab113849). Extracellular HMGB1 levels were detected by ELISA. Calreticulin on the cell surface 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 The Ohio State University. All mouse studies were approved by The Ohio State University Institutional Animal Care and Use Committee (IACUC) and complied with local, state, and federal regulations.

[0200] For unilateral subcutaneous (sc) tumor models, approximately 1 × 10 5 1 x 10 B16F10 cells or 2 x 10 5Mice were subcutaneously injected with B16F10-Luc2 in the right flank. Seven days after tumor inoculation, mice with tumor sizes of approximately 0.5 cm in maximum diameter were randomly assigned to different treatment groups. For the bilateral subcutaneous tumor model, approximately 1 × 10 5 Mice were subcutaneously injected with B16F10 cells into the right flank. Five days after tumor inoculation into the right flank, approximately 1 × 10 5 Mice were subcutaneously injected with B16F10 cells into the left flank. On day 7 after tumor inoculation into the right flank, mice with tumors measuring approximately 0.5 cm in diameter were randomly assigned to different treatment groups. For the skin + brain 2 tumor model, approximately 2 × 10 5 Mice were subcutaneously injected with B16F10-Luc2 cells into the right flank. Five days after tumor inoculation into the right flank, approximately 1 × 10 4 B16F10-Luc2 cells were injected intracranially (ic) at a depth of 3 mm. The injection site was 2 mm lateral to the sagittal suture and 1 mm short of the coronal suture. Seven days after tumor inoculation in the right flank, mice with tumor size of approximately 0.5 cm in maximum diameter were randomized into different treatment groups. Subcutaneous tumor rechallenge was performed 45 days after tumor inoculation, with approximately 1 × 10 5 B16F10 cells were injected subcutaneously into the left flank of fully responding mice. For brain tumor rechallenge, approximately 1 × 10 cells were injected 45 days after tumor inoculation. 4 B16F10-Luc2 cells were injected i.c. into fully responding mice. For the T cell-depleted tumor model, 1 × 10 5 B16F10 cells were injected subcutaneously into the right flank of mice. Six days after tumor inoculation, mice were treated intraperitoneally with anti-mouse CD8α, anti-mouse CD4, or anti-rat IgG2b isotype antibodies. Each antibody was administered at 200 μg per injection every 3 days for three doses.

[0201] The treatment regimens in this study were as follows: In one 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. For CD40-LNP+CD40 Ab treatment, CD40 Ab was administered 6 h after the injection of CD40-LNP. For dendritic cell+CD40 Ab treatment, CD40 Ab was administered 1 h after the injection of dendritic cells. For CD40-LNP+CD40L-LNP treatment, CD40-LNP was administered 18 h after the injection of CD40L-LNP. For CD40L- or FLuc-LNP+CD40-BMDC treatment, CD40-BMDC was administered 18 h after the injection of CD40L- or FLuc-LNP. All these treatment regimens included four administrations.

[0202] The removal 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 tumor reached a maximum diameter of 1.6 cm or when body weight was reduced by more than 20%. Brain tumor size was monitored by IVIS LuminaII (Caliper life sciences, USA). Mice were euthanized if they showed a stooped posture, had difficulty walking or eating, or lost more than 20% of their body weight.

[0203] Cytokine and chemokine analysis by Luminex Tumor volume approximately 500mm 2Mice were injected with one dose of CD40L-LNP+CD40-BMDCs. Tumor tissues and serum from mice were collected over a time course from 0 to 42 hours after injection. Tumor tissues were frozen in liquid nitrogen and ground tissues were extracted at 150mg / ml in RIPA lysis buffer (Thermo Scientific, 89900) containing protease inhibitors (Thermo Scientific, 87785). Whole blood was collected in tubes containing sodium citrate and serum was collected by centrifugation (10000rpm) at 4°C for 5 minutes. Tumor lysates and serum were stored at -80°C. Mouse cytokines and chemokines were detected by mouse cytokine / chemokine discovery assay (Eve Technologies, Canada).

[0204] Analysis of immune cell populations and activation in vivo Tumor volume approximately 250mm 2 Mice were treated with CD40L-LNP+CD40-BMDCs twice. Then, tumor tissues were collected and dissociated using a mouse tumor dissociation kit (Miltenyi Biotec, 130-096-730). For profiling of immune cell populations, total cells were stained with antibody combinations based on the flow cytometry gating method described above, and immune cell numbers were counted by flow cytometry. In this experiment, CD40-BMDCs were labeled with CellTrace Blue (Invitrogen, C34568) before injection, which allows us to distinguish administered and endogenous dendritic cells by flow cytometry. To evaluate immune cell activation, infiltrated immune cells were isolated by Ficoll-Paque density gradient medium (Cytiva, 17544602). After staining with 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 structures of ionizable lipids derived from dianhydrohexitols. Synthesis of aldehydes:

[0208] [ka] To a suspension of paraformaldehyde (1.5 g, 50 mmol) in TMSCl (25 mL, 197 mmol) was added 1-hexanol 1 (5.1 g, 50 mmol) dropwise at room temperature. The reaction mixture was stirred at room temperature for 2 h. The clear solution was concentrated under reduced pressure to give 1-chloromethoxy-hexane 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 CHCl2 (150 mL). The reaction mixture was stirred at room temperature for 24 h and then quenched by the addition of a saturated solution of NH4Cl (80 mL). Extraction with CHCl2 (2 x 60 mL) was performed and the combined organic layers were washed with water (30 mL) and saturated brine (30 mL) and dried over Na2SO4. The organic phase was filtered and concentrated under reduced pressure, and the residue was purified by silica gel flash chromatography (20% EtOAc in hexane). 6.74 g of 4 was obtained as a colorless oil (58.0% yield). 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; Measurement: 255.1941.

[0210] [ka] (Diacetoxyiodo)benzene (0.79 g, 2.45 mmol) was added to a 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 of dry DCM at room temperature. The reaction mixture was stirred for 3 h 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 x 20 mL). The combined DCM layers were washed with aqueous NaHCO3 (20 ml) and saturated brine (20 ml), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel flash chromatography (10% EtOAc in hexane). 510 mg of 5 was obtained as a colorless oil (quantitative). 1 H 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] A solution of 6 (5.0 g, 30.4 mmol) in DCM (30 mL) was added dropwise to a solution of 1,5-hexanediol 3 and TEA (16.9 mL, 121 mmol) in DCM (100 mL) at 0° C. After stirring for 30 min over 3 h, the reaction was quenched with 50 mL of water and extracted with DCM (50 mL×3), then the combined organic phase was washed with water, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (20% EtOAc in hexane). 3.2 g of 7 was obtained as a colorless oil (45.3% yield). 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; Measurement: 269.1735.

[0212] [ka] BAIB (1.41 g, 4.38 mmol) was added to a suspension of 7 (0.98 g, 3.98 mmol), TEMPO (62.2 mg, 0.4 mmol) and NaHCO3 (736 mg, 8.6 mmol) in 20 mL of DCM. The reaction mixture was stirred for 3 h until TLC showed that A was completely consumed. The mixture was quenched with saturated Na2S2O3 (30 mL) and extracted with DCM (3 x 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% EtOAc in hexanes). 0.78 g of 8 was obtained as a pale yellow oil (80.2% yield). 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 h. The clear solution was concentrated under reduced pressure to give 1-chloromethoxy-hexane 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 CHCl2 (150 mL). The reaction mixture was stirred at room temperature for 24 h, then quenched by the addition of a saturated solution of NH4Cl (80 mL). Extraction with CHCl2 (2 x 60 mL) was performed, and the combined organic layers were washed with water (30 mL) and saturated brine (30 mL) and dried over Na2SO4. The organic phase was filtered and concentrated under reduced pressure, and the residue was purified by silica gel flash chromatography (20% EtOAc in hexane). 3.45 g of 10 was obtained as a colorless oil (35.0% yield). 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; Measurement: 269.2075.

[0215] [ka] BAIB (1.77 g, 5.5 mmol) was added to a suspension of 10 (1.23 g, 5.0 mmol), TEMPO (78.2 mg, 0.5 mmol) and NaHCO3 (924 mg, 11.0 mmol) in 20 mL of DCM. The reaction mixture was stirred for 3 h until TLC showed that A was completely consumed. The mixture was quenched with saturated Na2S2O3 (30 mL) and extracted with DCM (3 x 20 mL). The combined DCM layers were washed with aqueous NaHCO3 (20 ml) and saturated brine (20 ml), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel flash chromatography (10% EtOAc in hexanes). 1.0 g of 11 was obtained as a pale yellow oil (81.8% yield). 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; Measurement: 255.1941.

[0216] Synthesis of diamines derived from sugar alcohols

[0217] [ka] 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 was added EtONa (75 mg, 1.1 mmol) and the resulting mixture was refluxed for 48 h. Then the reaction was stopped and cooled at 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 evaporated. Finally, the product was purified by silica gel chromatography using dichloromethane / methanol (9:1) as eluent to give 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; reading: 147.1.

[0219] [ka] Dicyanoethylation of diols was carried out according to a previously reported method. 1To a solution of the diol (7.3 g, 50 mmol) in tert-butanol and 50% aqueous NaOH (2.0 g, 0.5 mol%) was added acrylonitrile 16 (7.96 g, 150 mmol) dropwise over 30 min at 60 °C. The reaction was continued for a total of 6 h. tert-butanol and excess acrylonitrile were removed by rotary evaporation under reduced pressure. 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 give 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, 275.1002; measured value: 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: 93% yield. 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. 2To a solution of BH3·THF complex in THF (2.0 M, 29.7 mL, 59.4 mmol), a solution of the dinitrile compound (3.0 g, 11.89 mmol) in THF (25 mL) was added dropwise over 1 h at room temperature. After the addition was complete, the reaction was stirred for 48 h. The reaction was then quenched by careful addition of methanol (30 mL), during which time hydrogen gas evolved vigorously. After stirring for 3 h, the solvent was removed under reduced pressure, and then 40 mL of THF was added to dissolve the residue, followed by the dropwise addition of a solution of hydrochloric acid in diethyl ether (2.0 M, 29.7 mL), which formed a white precipitate. The suspension was then filtered to give the crude product of the diamine·HCl salt as a white powder. The diamine·2HCl salt was then dissolved in deionized water (30 mL) to give a pale yellow solution. To this solution was added freshly washed Amberlyst A26-OH (17 g). The resulting suspension was sonicated in an ultrasonic bath at 30° C. for 1.5 h. The suspension was filtered through a Celite pad and the resin was washed thoroughly with water (3×4 mL). The combined clear, colorless solution was evaporated to dryness using a rotary evaporator (or lyophilized) to give the extended diamine as a pale yellow oily liquid.

[0224] Isosorbide derived diamine 20: 70% yield. 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, 261.1809; measured value: 261.1819.

[0225] Isomannide derived diamine 21: 68% yield. HRMS (ESI, m / z): [M+H] + Calculated value. C 12 H 25For N2O4, 261.1809; measured value: 261.1817. L-Isosorbide derived diamine 22: 71% yield. 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 the diamine (52 mg, 0.2 mmol) in 2 mL of dry EtOH was added epoxide 23 (184 mg, 1.0 mmol). The mixture was then warmed to 90 °C and stirred for 12 h. TLC showed that A was completely consumed, and EtOH was removed under reduced pressure. The residue was purified by silica gel chromatography (0%-20% [mixture of 3% NH4OH, 22% MeOH in dichloromethane] in dichloromethane) to give 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, 997.9118; Measured: 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, 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, 997.9. Measured value: 998.0.

[0231] [ka] General synthetic procedure for reductive amination of diamines: To a solution of diamine (52 mg, 0.2 mmol) in THF (4 mL) was added aldehyde (1.0 mmol) and the mixture was kept stirring at room temperature for 30 min. Then NaBH(OAc)3 (254 mg, 1.2 mmol) was added to the above solution and the resulting mixture was stirred for 12 h. The reaction was quenched by adding aq. NaHCO3 solution (15 mL). The aqueous solution was then extracted with DCM (15 mL × 3) and the organic layers were combined, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (0%-100% [mixture of 3% NH4OH, 22% MeOH in dichloromethane] in dichloromethane) to give 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 For 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 For N2O4, 1046.1. Measured value: 1046.0. Compound DIS-6: Yield 19.1%. 11H NMR (300 MHz, chloroform-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] + Calculated value. C 76 H 153 For N2O4, 1158.2; Measured value: 1158.2. Compound DIS-7: Yield 45.7%. 1 1H NMR (400 MHz, chloroform-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] + Calculated value. C 80 H 153 N2O 12 For, 1334.1418; Measured value: 1334.1404. Compound DIS-8: Yield 35.0%. 11H NMR (300 MHz, chloroform-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] + Calculated value for C 64 H 121 N2O 16 is 1173.9. Measured value: 1174.0. Compound DIS-9: Yield 31.0%. 1 1H NMR (300 MHz, chloroform-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] + Calculated value for C 64 H 129 N2O 12 is 1118.0; Measured value: 1118.1. Compound DIS-10: Yield 54.5%. 1 1H NMR (300 MHz, chloroform-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] + Calculated value for C68 H 137 N2O 12 For N2O, 1174.0; measured value: 1174.2. Compound DIM-2: Yield 54.5%. 1 H NMR (300 MHz, chloroform-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 = 6.9, 2.1 Hz, 4H), 2.43 - 2.33 (m, 8H), 1.76 (p, J = 6.9 Hz, 4H), 1.50 - 1.36 (m, 8H), 1.35 - 1.15 (d, J = 2.6 Hz, 40H), 0.89 (t, J = 6.9 Hz, 12H). MS (ESI, m / z): [M+H] + Calculated value. C 44 H 89 For N2O4, 709.7. Measured value: 709.8. Compound DIM-3: Yield 31.7%. 1 H NMR (300 MHz, chloroform-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] + Calculated value. C 52 H 105 For N2O4, 821.8. Measured value: 821.9. Compound DIM-4: Yield 49.3%. 11H NMR (400 MHz, chloroform-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, 6.8 Hz, 2H), 2.60 - 2.46 (m, 4H), 2.47 - 2.30 (m, 8H), 1.76 (p, J = 7.0 Hz, 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] + Calculated for C 60 H 121 N2O4, 933.9321; Found: 933.9321. Compound DIM-5: Yield 22.0%. 1 1H NMR (300 MHz, chloroform-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] + Calculated for C 68 H 137 N2O4, 1046.1. Found: 1046.0. Compound DIM-6: Yield 37.3%. 1 1H NMR (300 MHz, chloroform-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]+ Calculated value.C 76 H 153 For N2O4, 1158.2; Measured value: 1158.0. Compound DIM-7: Yield 36.0%. 1 H NMR (400 MHz, chloroform-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, chloroform-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] + Calculated value.C 64 H 121 N2O 16 For, 1173.9. Measured value: 1174.0. Compound DIM-9: Yield 26.2%. 11H NMR (400 MHz, chloroform-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] + Calculated for C 64 H 129 N2O 12 is 1118.0; found: 1118.2. Compound DIM-10: Yield 29.4%. 1 1H NMR (300 MHz, chloroform-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] + Calculated for C 68 H 137 N2O 12 is 1174.0; found: 1174.3. Compound LIS-2: Yield 31.0%. 11H NMR (400 MHz, chloroform-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] + Calculated value. C 44 H 89 For N2O4, 709.7. Measured value: 709.8. Compound LIS-3: Yield 29.2%. 1 1H NMR (400 MHz, chloroform-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] + Calculated value. C 52 H 105 For N2O4, 821.8. Measured value: 822.0. Compound LIS-4: Yield 30.3%. 1 1H NMR (300 MHz, chloroform-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] +Calculated value.C 60 H 121 For N2O4, 933.9; Measured value: 934.2. Compound LIS-5: Yield 30.6%. 1 H NMR (300 MHz, chloroform-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] + Calculated value.C 68 H 137 For N2O4, 1046.1. Measured value: 1046.3. Compound LIS-6: Yield 29.4%. 1 H NMR (400 MHz, chloroform-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, chloroform 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+ Calculated value.C80 H 154 N2O 12 For, 667.6; Measured value: 668.0. Compound LIS-8: Yield 25.6%. 1 H NMR (400 MHz, chloroform-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] + Calculated value. C 64 H 121 N2O 16 In the case of, 1173.9. Measured value: 1174.0. Compound LIS-9: Yield 26.4%. 1 H NMR (400 MHz, chloroform-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] + Calculated value. C 64 H 129 N2O 12 For, 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 About,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 meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention belongs. Publications and materials cited herein are specifically incorporated by reference.

[0234] Those skilled in the art will understand that numerous changes and modifications may be made to the preferred embodiments of the present invention, and that such changes and modifications may be made without departing from the spirit of the present invention. It is therefore intended that the appended claims cover all such equivalent variations that fall within the true spirit and scope of the present invention.

Claims

1. A compound having formula I, II, or III: 【Chemistry 1】 or a salt thereof, wherein: R １ is 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, or a salt thereof.

2. The compound has the formula: 【Chemistry 2】 or a salt thereof, wherein R １ is 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.

3. The compound has the formula: 【Chemistry 3】 or a salt thereof, wherein R １ is 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.

4. The compound has the formula: 【Chemistry 4】 or a salt thereof, wherein R １ is 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.

5. R １ 5. The compound according to claim 1 , wherein: 【Chemistry 5】

6. R １ but 【Chemistry 6】 6. The compound of claim 5,

7. R １ but 【Chemistry 7】 6. The compound of claim 5,

8. The compound is 【Chemistry 8】 and In the formula, R １ teeth 【Chemistry 9】 2. The compound of claim 1,

9. The compound is 【Chemistry 10】 and In the formula, R １ teeth 【Chemistry 11】 2. The compound of claim 1,

10. 1. A lipid-based nanoparticle, comprising: A compound according to any one of claims 1 to 9, and The lipid-based nanoparticles comprising a recombinant polynucleotide comprising a nucleic acid encoding a costimulatory molecule.

11. An antigen-presenting cell, Lipid-based nanoparticles comprising: A compound according to any one of claims 1 to 9, and The antigen-presenting cell comprising a recombinant polynucleotide comprising a nucleic acid encoding a costimulatory molecule.

12. The compound 【Chemistry 12】 and In the formula, R １ teeth 【Chemistry 13】 The antigen-presenting cell of claim 11 .

13. The compound 【Chemistry 14】 and In the formula, R １ teeth 【Chemistry 15】 The antigen-presenting cell of claim 11 .

14. The antigen-presenting cell of any one of claims 11 to 13, wherein the costimulatory 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.

15. The antigen-presenting cell of claim 14 , wherein the costimulatory molecule is CD40.

16. The antigen-presenting cell of claim 11 , wherein the nucleic acid encoding the costimulatory molecule comprises a heterologous 5′ untranslated region (5′UTR).

17. 17. The antigen-presenting cell of claim 11, wherein the nucleic acid encoding the costimulatory molecule comprises a heterologous 3' untranslated region (3'UTR).

18. The antigen-presenting cell of claim 11 , wherein the nucleic acid comprises a chemically modified nucleic acid base.

19. The antigen-presenting cell of claim 18, wherein the chemically modified nucleic acid base is pseudouridine.

20. The antigen-presenting cell according to any one of claims 11 to 19, wherein the antigen-presenting cell is a bone marrow-derived dendritic cell.

21. A pharmaceutical composition comprising a therapeutically effective amount of an antigen-presenting cell and an antibody described in any one of claims 11 to 20 for treating cancer, wherein the treatment comprises administering the antigen-presenting cell and the antibody to a subject in need thereof.

22. 22. The pharmaceutical composition of claim 21, 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.

23. The pharmaceutical composition of claim 21 or 22, wherein the antigen-presenting cells and the antibody are administered intratumorally.

24. The pharmaceutical composition of any one of claims 21 to 23, wherein the antigen-presenting cells and the antibody are administered simultaneously.

25. To treat cancer, 21. A pharmaceutical composition comprising a therapeutically effective amount of the lipid-based nanoparticles of claim 8 and a therapeutically effective amount of the antigen-presenting cells of any one of claims 11 to 20, wherein the treatment comprises administering the lipid-based nanoparticles and the antigen-presenting cells to a subject in need thereof.

26. The lipid-based nanoparticles are 【Chemistry 16】 Including, In the formula, R １ teeth 【Chemistry 17】 26. The pharmaceutical composition of claim 25,

27. The lipid-based nanoparticles are 【Chemistry 18】 Including, In the formula, R １ teeth 【Chemistry 19】 26. The pharmaceutical composition of claim 25,

28. 28. The pharmaceutical composition of any one of claims 25 to 27, wherein the lipid-based nanoparticle comprises a recombinant polynucleotide comprising a nucleic acid encoding CD40L.

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