Sugar-derived lipid nanomaterials and uses thereof

Abstract

The present disclosure relates to compositions and methods for the treatment of cancer, immune disorders, and other prophylactic and therapeutic applications.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 148,755, filed February 12, 2021, the disclosure of which is expressly incorporated herein by reference.

[0003] Statement as to Federally Sponsored Research

[0004] This invention was developed with government support under license number R35GM119679 granted by the National Institutes of Health (NIH). The government holds certain rights to this invention. Technical Field

[0005] This disclosure relates to compositions and methods for treating cancer and other immune disorders. Background Technology

[0006] Efficient mRNA delivery is a critical step and challenge in the administration of mRNA therapeutics. While data from ongoing clinical trials are promising, the clinical use of mRNA requires the exploration and development of more efficient delivery systems. Novel compositions and methods are needed to deliver mRNA into cells for the treatment of cancer and other immune disorders. Summary of the Invention

[0007] In some respects, this article discloses a compound having formula I, II, or III:

[0008]

[0009] Or its salt, wherein:

[0010] R 1 It 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.

[0011] In some embodiments, the compound has the following formula:

[0012]

[0013] Or its salt, wherein:

[0014] R 1 It 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.

[0015] In some embodiments, the compound is of the formula:

[0016]

[0017]

[0018] or salts thereof, wherein:

[0019] R 1 is independently selected from the group consisting of 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.

[0020] In some embodiments, the compound is of the formula:

[0021]

[0022] or salts thereof, wherein:

[0023] R 1 is independently selected from the group consisting of 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.

[0024] In some embodiments, R 1 is selected from the group consisting of:

[0025]

[0026]

[0027] or salts thereof.

[0028] In some embodiments, the compound is

[0029] wherein R 1 is

[0030] In some embodiments, the compound is

[0031]

[0032] wherein R 1 is

[0033] In some aspects, disclosed herein is a lipid-based nanoparticle comprising: a compound according to any preceding aspect, and a recombinant polynucleotide comprising a nucleic acid encoding a costimulatory molecule.

[0034] In some aspects, disclosed herein is an antigen presenting cell comprising a lipid-based nanoparticle comprising: a compound according to any foregoing aspect, and a recombinant polynucleotide comprising a nucleic acid encoding a costimulatory molecule.

[0035] In some embodiments,

[0036] The compound is

[0037] wherein R 1 is

[0038] In some embodiments, the costimulatory molecule is selected from the group consisting of 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.

[0039] 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 chemically modified nucleobases. In some embodiments, the chemically modified nucleobases are pseudouridines. In some embodiments, the antigen presenting cell is a bone marrow-derived dendritic cell.

[0040] In some aspects, disclosed herein is a method of treating cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of an antigen presenting cell according to any foregoing aspect and an antibody.

[0041] In some embodiments, the antibody is selected from the group consisting of an anti-CD40 antibody, an anti-PDL1 antibody, an anti-PD1 antibody, an anti-CTLA4 antibody, or a combination thereof.

[0042] In some embodiments, the antigen presenting cell and the antibody are administered intratumorally.

[0043] In some aspects, disclosed herein is a method of treating cancer, the method comprising administering to a subject in need thereof:

[0044] a therapeutically effective amount of a lipid-based nanoparticle according to any preceding aspect, and

[0045] a therapeutically effective amount of an antigen presenting cell according to any preceding aspect.

[0046] In some embodiments, the lipid-based nanoparticle comprises

[0047]

[0048] wherein R 1 is

[0049] In some embodiments, the lipid-based nanoparticle comprises a recombinant polynucleotide comprising a nucleic acid encoding CD40L. BRIEF DESCRIPTION OF DRAWINGS

[0050] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

[0051] Figures 1A-1C . Closing the cancer immune cycle (CIC) and chemical synthesis of sugar alcohol- derived ionizable lipids. a. Schematic of closing the CIC through integrated lipid nanoparticle and cell therapy (CATCH). b. Representative synthetic route of sugar alcohol-derived ionizable lipids. c. Structures of sugar alcohol-derived ionizable lipids (DIS, DIM, and LIS series).

[0052] Figures 2A-2J . Screening and characterization of lipid nanoparticle (LNP)-mRNA formulations. a. mRNA delivery efficacy of DIS, DIM, and LIS LNPs in BMDCs. b. Effect of each lipid component on mRNA delivery under different molar ratios of DIM7 LNPs. c. Relative luminescence intensity of DIM7 LNP orthogonal formulations and preferred formulation. d. Effect of each lipid component on mRNA delivery under different molar ratios of LIS10 LNPs. e. Relative luminescence intensity of LIS10 LNP orthogonal formulations. f. Table of preferred formulations. g. Characterization of DIM7S LNPs, including size, PDI, encapsulation efficiency, and zeta potential. h. Cryo-TEM images of DIM7S (scale bar = 50 nm). i. Characterization of LIS10W LNPs, including size, PDI, encapsulation efficiency, and zeta potential. j. Cryo-TEM images of LIS10W (scale bar = 50 nm). Data in a, b, c, d, and e are from n = 3 biologically independent samples. All data are represented 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.

[0053] Figures 3A-3O a. Expression of dendritic cell activation markers including CD80, CD86, MHC-II, IL1-β (pro-form), TNF-α and IL12. b. Schematic of B16F10 tumor model and treatment regimen. c, d. Tumor volume (c) and mouse survival (d) over time; n = 6 or 7. e, f. LNP-induced markers of ICD, including extracellular HMGB1, extracellular ATP and cell surface calreticulin, in vitro (e) and in vivo (f). g. Schematic of B16F10 tumor model and treatment regimen. h. Mouse survival over time; n = 6. i. Survival of responding mice in the CD40L-LIS10W+CD40-BMDC group following subcutaneous (s.c.) tumor re-challenge; n = 5 or 6. j. Schematic of B16F10 tumor model and treatment regimen. k. Mouse survival over time; n = 6. l. Survival of responding mice in the CD40L-LIS10W+CD40-BMDC group following subcutaneous tumor re-challenge; n = 5 or 6. m. Schematic of B16F10-Luc2 tumor model and treatment regimen. n. Mouse survival over time; n = 9. o. Survival of responding mice in the CD40L-LIS10W+CD40-BMDC group following intracranial (i.c.) tumor re-challenge; n = 5 or 8. Data in a, e and f are from n = 3 biological independent samples and are presented as mean ± standard deviation (s.d.). Data in c are presented as mean ± standard error of the mean (s.e.m.). 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.

[0054] Figures 4A-4LTherapeutic effects on two tumor or T cell exhaustion models, and dynamic expression of cytokines and chemokines in tumor tissues and blood.a. Schematic of B16F10 tumor model and treatment schedule.b, c. Distal tumor volume (b) and mouse survival rate (c) of individual mice over time; n = 8 or 10.d. Schematic of B16F10-Luc2 tumor model and treatment schedule.e, f. Brain tumor volume (e) and mouse survival rate (f) over time; n = 10.g. Schematic of B16F10 tumor model and treatment schedule.h, i. Tumor volume (h) and mouse survival rate (i) over time; n = 6.j. Schematic of treatment schedule and sample collection.k, 1. Dynamic expression of cytokines and chemokines in tumor tissues (k) and blood (1); n = 5. Data in b, e, and h are presented as mean ± standard error of the mean. Statistical significance in b, e, and h was analyzed by two-tailed Student’s t test. Statistical significance in c, f, and i was analyzed by log-rank (Mantel-Cox) test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0055] Figures 5A-5E Immune cell infiltration in tumor tissues.a. Immune cell populations in tumor tissues; n = 5.b. Percentages of activated macrophages and dendritic cells in tumor tissues; n = 5.c. Percentage of primed CD8 T cells in tumor tissues; n = 5.d, e. Percentages of effector memory T cells and central memory T cells in spleen (d) and blood (e); n = 5.

[0056] Figures 6A-6B Characterization of lipid nanoparticle (LNP)-mRNA formulations.a. Nanoparticle size and polydispersity index (PDI).b. Entrapment efficiency and zeta potential. All data are presented as mean ± standard deviation.

[0057] Figures 7A-7F Characterization of lipid nanoparticle (LNP)-mRNA formulations.a. L16(4) 4Orthogonal table.b. Cryo-TEM images of DIM7S (scale bar = 50 nm). c. Cryo-TEM images of LIS10W (scale bar = 50 nm). d. Relative luminescence intensity of Lipofectamine 3000 (Lipo 3K) for electro, DIM7, and DIM7S. e. Expression kinetics of mRNA delivered by DIM7S. f. CD40 expression in BMDCs. Data in d, e, and f are from n = 3 biologically independent samples and are presented as mean ± standard deviation. Statistical significance in d, e, and f was analyzed by two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0058] Figure 8. Tumor volume of individual mice; n = 6 or 7.

[0059] Figures 9A-9E . Lipid nanoparticle (LNP)-induced cytotoxicity and expression of CD40 and CD40L. a. In vivo CD40 expression mediated by CD40-DIM7S in dendritic cells; n = 5. b. In vitro cytotoxicity induced by CD40L-DIM7S and CD40L-LIS10W. c. In vitro CD40L expression in B16F10 melanoma cells mediated by CD40L-DIM7S and CD40L-LIS10W. d, e. 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 presented 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.

[0060] Figures 10A-10G . Tumor volume of individual mice. a. Primary tumor volume of individual mice; n = 6. b. Re-challenged subcutaneous tumor volume of individual mice; n = 5 or 6. c, d. Tumor volume over time (c) and primary tumor volume of individual mice (d); n = 6. e. Re-challenged subcutaneous tumor volume of individual mice; n = 5 or 6. f. Primary tumor volume of individual mice; n = 9. g. Brain tumor volume over time; n = 5 or 8. Data in c and g are presented as mean ± mean standard error. Statistical significance was analyzed by two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0061] Figures 11A-11CTumor volume in individual mice. a. Tumor volume on the treated side of individual mice; n = 8 or 10. b. Tumor volume on the treated side of individual mice; n = 10. c. Tumor volume in individual mice; n = 6.

[0062] Figure 12 Dynamic expression of cytokines and chemokines in mouse melanoma tissue; n = 5.

[0063] Figure 13 Dynamic expression of cytokines and chemokines in mouse blood; n = 5. DETAILED DESCRIPTION

[0064] Disclosed herein are compositions and methods of modulating the immune system to treat cancer and other immune disorders.

[0065] Reference will now be made in detail to implementations of the application, examples of which are illustrated in the accompanying drawings and examples. The application may, however, be embodied in many different forms and should not be construed as limited to the implementations set forth herein.

[0066] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. As used herein the term “comprising” and its variants are used synonymously with the term “including” and its variants, and are open, non-limiting terms. Although the terms “comprising” and “including” are used interchangeably in this disclosure, the terms “consisting essentially of’ and “consisting of’ can be used in place of “comprising” and “including” to provide more particular implementations, and are also disclosed. As used in the disclosure and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0067] The following definitions are provided to facilitate understanding of certain terms used frequently herein.

[0068] Terminology

[0069] As used herein, the terms “may,” “optionally,” and “may optionally” are used interchangeably and are meant to include instances where the condition occurs and instances where the condition does not occur. Thus, for example, the statement that a formulation “may include an excipient” is intended to include instances where the formulation includes an excipient and instances where the formulation does not include an excipient.

[0070] The term “initiator” or “regulatory element” refers to a region or sequence determinant located upstream or downstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. An initiator need not be of bacterial origin, for example, an initiator derived from a virus or other organism can be used in the compositions, systems, or methods described herein. The term “regulatory element” is intended to include initiators, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include regulatory elements that direct constitutive expression of nucleotide sequences in many types of host cells and regulatory elements that direct expression of nucleotide sequences only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific initiators can direct expression primarily in a desired tissue of interest, e.g., muscle, neuronal, skeletal, skin, blood, a particular organ (e.g., liver, pancreas), or a particular cell type (e.g., lymphocytes). Regulatory elements can also direct expression in a time-dependent manner, e.g., in a cell cycle-dependent or developmental stage-dependent manner, which can or can not also be tissue- or cell type-specific. In some embodiments, the vector comprises one or more pol III initiators (e.g., 1, 2, 3, 4, 5, or more pol I initiators), one or more pol II initiators (e.g., 1, 2, 3, 4, 5, or more pol II initiators), one or more pol I initiators (e.g., 1, 2, 3, 4, 5, or more pol I initiators), or a combination thereof. Examples of pol III initiators include, but are not limited to, U6 initiators and H1 initiators. Examples of pol II initiators include, but are not limited to, a Rous sarcoma virus (RSV) LTR initiator (optionally with an RSV enhancer), a cytomegalovirus (CMV) initiator (optionally with a CMV enhancer) [see, e.g., Boshart et al., Cell, 41:521-530 (1985)], an SV40 initiator, a dihydrofolate reductase initiator, a beta-actin initiator, a phosphoglycerol kinase (PGK) initiator, and an EF1 alpha initiator.The term "regulatory element" also encompasses 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), pp. 466-472, 1988); the SV40 enhancer; and the intron sequence between exon 2 and exon 3 of rabbit beta-globin (Proc. Natl. Acad. Sci. USA., vol. 78(3), pp. 1527-31, 1981). Those skilled in the art will appreciate that the design of an expression vector can depend on such factors as the choice of the host cell to be transformed, the level of desired expression, and the like.

[0071] The term "recombinant" refers to a human manipulated nucleic acid (e.g., polynucleotide) or a copy or complement of a human manipulated nucleic acid (e.g., polynucleotide), or, if referring to a protein (i.e., "recombinant protein"), a protein encoded by a recombinant nucleic acid (e.g., polynucleotide). In embodiments, a recombinant expression cassette comprising a primer operably linked to a second nucleic acid (e.g., polynucleotide) can include a primer that is heterologous to the second nucleic acid (e.g., 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, N.Y., (1989) or Current Protocols in Molecular Biology Vols. 1-3, John Wiley & Sons, Inc. (1994-1998)). In another example, a recombinant expression cassette can include nucleic acids (e.g., polynucleotides) combined in a manner that is not naturally occurring for the nucleic acids (e.g., polynucleotides). For example, a restriction site or plasmid vector sequence can flank or separate a primer from a second nucleic acid (e.g., polynucleotide) that is human manipulated. Those skilled in the art will recognize that nucleic acids (e.g., polynucleotides) can be manipulated in a variety of ways and are not limited to the above examples.

[0072] The term "expression cassette" or "vector" refers to a nucleic acid construct that, when introduced into a host cell, results in the transcription and / or translation of RNA or polypeptide, respectively. In embodiments, an expression cassette comprising a primer operably linked to a second nucleic acid (e.g., polynucleotide) can include a primer that is heterologous to the second nucleic acid (e.g., polynucleotide) as a result of human manipulation (e.g., by performing human manipulation as described in Sambrook et al., Molecular Cloning— A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989) or Current Protocols in Molecular Biology Vols. 1-3, John Wiley & Sons, Inc. (1994-1998)). In some embodiments, an expression cassette comprising a terminator (or termination sequence) operably linked to a second nucleic acid (e.g., polynucleotide) can include a terminator that is heterologous to the second nucleic acid (e.g., polynucleotide) as a result of human manipulation. In some embodiments, an expression cassette comprises a primer operably linked to a second nucleic acid (e.g., polynucleotide) and a terminator operably linked to the second nucleic acid (e.g., polynucleotide) as a result of human manipulation. In some embodiments, an expression cassette comprises an endogenous primer. In some embodiments, an expression cassette comprises an endogenous terminator. In some embodiments, an expression cassette comprises a synthetic (or non-native) primer. In some embodiments, an expression cassette comprises a synthetic (or non-native) terminator.

[0073] In the context of two or more nucleic acid or polypeptide sequences, the terms "identical" or percent "identity," mean that two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (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 higher identity over a specified region when compared and aligned for maximum correspondence over the comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters or by manual alignment and visual inspection (see, e.g., NCBI website, etc.). Such sequences are referred to as "substantially identical." This definition also refers to or applies to the complement of a test sequence. The definition also includes sequences with deletions and / or additions, as well as those that have substitutions. As described below, preferred algorithms can take into account gaps and the like. Preferably, identity is present over a region that is at least about 10 amino acids or 20 nucleotides in length, or more preferably over a region that is 10 to 50 amino acids or 20 to 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 with the amino acids in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art will appreciate that optimal alignment of sequences will take into account the identity of amino acids and that optimal alignment with consideration of amino acid identity will provide the best estimate of sequence identity.

[0074] For sequence comparison, typically one sequence is compared to a reference sequence, and the test sequence is compared to the reference sequence. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters are used unless otherwise specified. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence against the reference sequence, based on the program parameters.

[0075] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST and BLAST 2.0 algorithms, which are 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 / ). The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words in the query sequence that either match or exceed the specified positive- valued threshold or cut-off scores, T. 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 for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or 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 word length (W) of 11, an expectation value (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, an expectation value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation value (E) of 10, M=5, N=-4, and a comparison of both strands.

[0076] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01.

[0077] The phrase "codon optimization" refers to a gene or coding region of a nucleic acid molecule for transformation of various hosts, and refers to altering the codons in a gene or coding region of a polynucleic acid molecule to reflect the typical codon usage of the selected organism without changing the DNA encoded polypeptide. Such optimization includes replacing at least one, more than one, or a large number of codons with one or more codons that are more frequently used in genes of the selected organism.

[0078] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a prologue or enhancer is operably linked to a sequence if it influences the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are close enough to each other to be under the control of the same promoter. However, operably linked nucleic acids (e.g., an enhancer and a coding sequence) need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. In embodiments, a prologue is operably linked to a coding sequence (i.e., the coding sequence is under the transcriptional control of the prologue) when the prologue is capable of influencing (e.g., modulating relative to the absence of the prologue) expression of a protein from the coding sequence.

[0079] The term "nucleobase" refers to a nucleotide moiety having Watson / Crick base pairing functionality. The most common naturally occurring nucleobases, adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T), have hydrogen bonding functionality that can bind one nucleic acid strand to another in a sequence-specific manner.

[0080] As used throughout, a "subject" (or "host") refers to an individual. Thus, a "subject" can include, for example, domesticated animals (e.g., cats, dogs, etc.), 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, avians, reptiles, amphibians, fish, and any other animals. A subject can be a mammal, such as a primate or a human. Administration of a therapeutic agent can be at a dose and for a period of time effective to treat the subject.

[0081] The term "about" as used herein when referring to a measurable value such as an amount, a percentage, and the like, is meant to encompass variations of ± 20%, ± 10%, ± 5%, or ± 1% of the measurable value.

[0082] A nucleic acid sequence is "heterologous" to a second nucleic acid sequence if the nucleic acid sequence originates from a foreign species, or if the nucleic acid sequence is modified from its original form due to human action, e.g., the heterologous initiator (or heterologous 5' untranslated region (5' UTR)) operably linked to a coding sequence refers to the coding sequence being from a different species than the initiator is derived from, or if from the same species, the coding sequence is different from a naturally occurring allelic variant (e.g., a 5' UTR or 3' UTR from a different gene operably linked to a nucleic acid encoding a costimulatory molecule).

[0083] The term "nanoparticle" as used herein refers to a particle or structure that is biocompatible with 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 application or treatment and the size of the nanoparticles is in the nanometer range. In some embodiments, the range of nanoparticles is typically from about 1 nm to about 1000 nm, from about 50 nm to about 500 nm, from about 50 nm to about 350 nm, from about 100 nm to about 250 nm, or from about 110 nm to about 150 nm.

[0084] A“therapeutically effective amount” or“therapeutically effective dose” of a composition refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of a given therapeutic agent will typically vary with factors such as the type and severity of the condition or disease being treated, and the age, gender, and weight of the subject. This term can also refer to the amount or rate of delivery of a therapeutic agent (e.g., amount over time) effective to promote a desired therapeutic effect. The precise desired therapeutic effect will vary depending on the condition to be treated, the subject’s tolerance, the agent to be administered and / or the agent formulation (e.g., potency of the therapeutic agent, concentration of the agent in the formulation, etc.), and various other factors as understood by one of ordinary skill in the art. In some cases, the desired biological or medical response is achieved after administration of multiple doses of the composition to the subject over a period of days, weeks, or years.

[0085] As used herein, the terms“treat” or“treatment” of a subject include administering a drug to a subject to cure, heal, alleviate, relieve, alter, remedy, improve, stabilize, or influence the disease or condition, or symptoms of the disease or condition. The terms“treat” and“treatment” can also refer to reducing the severity and / or frequency of symptoms, eliminating symptoms and / or underlying cause, and improving or remedying damage.

[0086] As used herein, the term“prevent” or“prevention” of a disease, condition, or unwanted physiological event in a subject refers to preventing the disease, condition, or unwanted physiological event, or preventing symptoms of the disease, condition, or unwanted physiological event.

[0087] An“effective amount” of an agent refers to the amount of the agent that is sufficient to provide a desired effect. The amount of an agent that is“effective” will vary from subject to subject, depending on factors such as the age and general condition of the subject, the one or more particular agents, and the like. Thus, it is not always possible to specify an“effective amount” quantitatively. However, one of ordinary skill in the art could determine an appropriate“effective amount” in any particular case using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an“effective amount” of an agent can also refer to an amount that encompasses both a therapeutically effective amount and a prophylactically effective amount. The“effective amount” of an agent needed to achieve a therapeutic effect can vary according to factors such as the age, gender, and weight of the subject. Dosage regimens can be adjusted to provide the optimum 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.

[0088] A "pharmaceutically acceptable" component can refer to a component that is not biologically or otherwise undesirable, i.e., the component can be incorporated into a pharmaceutical preparation of the application and administered to a subject without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the preparation in which it is contained. The term is generally intended to refer to components that are approved or approved by the U.S. Food and Drug Administration, or listed in the Inactive Ingredient Guide compiled by the U.S. Food and Drug Administration, for use in humans and animals.

[0089] A "pharmaceutically acceptable carrier" (sometimes referred to as a "carrier") refers to a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable, and includes a carrier or vehicle that is employed in the veterinary and / or human pharmaceutical or therapeutic art. The term "carrier" or "pharmaceutically acceptable carrier" can include but is not limited to a phosphate buffered saline solution, water, emulsions (such as an oil / water or water / oil emulsion) and / or various types of wetting agents. The term "carrier" as used herein encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material known in the art to be useful in pharmaceutical formulations, and as further described herein.

[0090] A "therapeutic agent" refers to any composition having a beneficial biological effect. Beneficial biological effects include both therapeutic effects (e.g., treating a disorder or other undesirable physiological condition) and prophylactic effects (e.g., preventing a disorder or other undesirable physiological condition). The term also includes pharmaceutically acceptable, pharmacologically active derivatives of the beneficial agents specifically mentioned herein, including but not limited to salts, esters, amides, prodrugs, active metabolites, isomers, fragments, analogs, and the like. When the term "therapeutic agent" is used, or when a particular agent is specifically identified, it is understood that the term includes the agent itself as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, conjugates, active metabolites, isomers, fragments, analogs, and the like.

[0091] The term "controlled release" or "controlled release drug delivery" or "sustained release" as used herein refers to the release or administration of a drug from a given dosage form in a controlled manner to achieve desired in vivo pharmacokinetic characteristics. One aspect of "controlled" drug delivery is the ability to manipulate the formulation and / or dosage form to establish the desired drug release kinetics.

[0092] The phrase "concurrent administration," "co-administration," "simultaneous administration," or "simultaneously administering" as used herein means that the compounds are administered at the same point in time or immediately after one another.

[0093] The term "polypeptide" refers to a compound composed of single chains of D-amino acids or L-amino acids or a mixture of D-amino acids and L-amino acids joined by peptide bonds.

[0094] The term "antibody" is used herein in the broadest sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, the term "antibody" includes fragments or aggregates of immunoglobulin molecules, as well as human forms or humanized forms of immunoglobulin molecules or fragments thereof. Antibodies can be tested for desired activity using in vitro assays described herein or by analogous methods, after which they are tested for their in vivo therapeutic and / or prophylactic activity according to known clinical testing methods. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into "subclasses" (isotypes), e.g., IgG-l, IgG-2, IgG-3, and IgG-4; IgA-l and IgA-2. Those of skill in the art will recognize comparable classes for mouse. The heavy chain constant regions that correspond to the different immunoglobulin classes are called a, d, e, g, and m, respectively.

[0095] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies in the population are identical except for possible naturally occurring mutations that can be present in minor amounts. Monoclonal antibodies herein specifically include "chimeric" antibodies and fragments of such antibodies in which a portion of the heavy and / or light chain is identical with or homologous to a corresponding sequence in an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to a corresponding sequence in an antibody from another species or belonging to another antibody class or subclass, so long as they exhibit the desired antagonistic activity.

[0096] The disclosed monoclonal antibodies can be produced using any procedure which produces monoclonal antibodies. For example, the disclosed monoclonal antibodies can be prepared using the hybridoma method, such as described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0097] Monoclonal antibodies can also be made by recombinant DNA methods. DNA encoding the disclosed monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are specific for the murine heavy and light chain genes). Libraries can also be used to produce and screen libraries of antibodies or active antibody fragments using phage display techniques, such as described in U.S. Patent Nos. 5,804,440 to Burton et al. and 6,096,441 to Barbas et al.

[0098] In vitro methods are also suitable for making monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly Fab fragments, can be accomplished using conventional techniques known in the art. For example, digestion can be performed using papain. Examples of papain digestion are described in WO 94 / 29348, published December 22, 1994, and U.S. Patent No. 4,342,566. Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment produces fragments with two antigen binding sites and which are still capable of cross-linking antigen.

[0099] As used herein, the term "antibody or antigen binding fragment thereof" or "antibody or fragment thereof" includes chimeric antibodies and hybrid antibodies having dual or multiple antigen or epitope specificity, as well as fragments, such as F(ab')2, Fab', Fab, Fv, sFv, scFv, and the like, including hybrid fragments. Thus, antibody fragments that retain the ability to bind to their specific antigen are provided. For example, antibody fragments that maintain binding activity are included within the meaning of the term "antibody or antigen binding fragment thereof." Such antibodies and fragments can be prepared by techniques known in the art, and can be screened for specificity and activity according to the methods set forth in the Examples and general methods for producing antibodies and screening antibodies for specificity and activity (see Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988)).

[0100] Also included within the meaning of "antibody or antigen binding fragment thereof" are conjugates of antibody fragments and antigen binding proteins (single chain antibodies). Also included within the meaning of "antibody or antigen binding fragment thereof" are immunoglobulin single variable domains, such as nanobodies.

[0101] A fragment, whether attached to other sequences or not, can also include insertions, deletions, substitutions or other selected modifications of a particular region or particular amino acid residues, provided that the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the unmodified antibody or antibody fragment. These modifications can provide for additional properties, such as removal / addition of amino acids capable of disulfide bonding, increasing the biological lifetime of the amino acids, altering the secretion properties of the amino acids, 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 an antibody or antibody fragment can be identified by performing mutagenesis of specific regions of the protein, followed by expression and testing of the expressed polypeptides. Such methods are readily apparent to one skilled in the art, and can include site-specific mutagenesis of nucleic acids encoding the antibody or antibody fragment. (Zoller, M. J. Curr. Opin. Biotechnol. 3:348-354, 1992).

[0102] As used herein, the term "antibody" can also refer to human antibodies and / or humanized antibodies. Many non-human antibodies (e.g., antibodies derived from mice, rats, or rabbits) are naturally antigenic in humans, and thus can elicit an undesirable immune response when administered to humans. Thus, the use of human antibodies or humanized antibodies in the methods can reduce the chance that the antibody administered to a human elicits an undesirable immune response.

[0103] The term "nucleic acid" as used herein refers to a polymer composed of nucleotides (e.g., deoxyribonucleotides or ribonucleotides).

[0104] The terms "ribonucleic acid" and "RNA" as used herein mean a polymer composed of ribonucleotides.

[0105] The terms "deoxyribonucleic acid" and "DNA" as used herein mean a polymer composed of deoxyribonucleotides.

[0106] The term "polynucleotide" refers to a single-stranded polymer or a double-stranded polymer composed of nucleotide monomers.

[0107] Chemical Definitions

[0108] As used herein, the term “substituted” is intended to include all permissible substituents of an organic compound. In a broad sense, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and non-aromatic substituents of an organic compound. Illustrative substituents include those described below, for example. For a suitable organic compound, permissible substituents may be one or more and may be the same or different. For the purposes of this disclosure, heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible substituents of the organic compound described herein that satisfy the valence of the heteroatom. This disclosure is not intended to be limited in any way to permissible substituents of an organic compound. Furthermore, the terms “substituted” or “substituted” include the implicit condition that such substitution is consistent with the permissible valence of the substituted atom and substituent, and that said substitution produces a stable compound, such as a compound that does not spontaneously undergo transformations (e.g., by rearrangement, cyclization, elimination, etc.).

[0109] “Z 1 “Z” 2 “Z” 3 "and "Z 4 "These symbols are used in this document as general symbols to denote various specific substituents. These symbols can be any substituents, not limited to those disclosed herein, and while they are defined as certain substituents in one case, they can be defined as some other substituents in another case."

[0110] As used in this article, the term "aliphatic" refers to a non-aromatic hydrocarbon group, including branched and unbranched alkyl, alkenyl, or alkynyl groups.

[0111] As used herein, the term "alkyl" refers to a branched or unbranched saturated hydrocarbon group having 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetradecyl, etc. Alkyl groups can also be substituted or unsubstituted. An alkyl group may be substituted with one or more groups, including but not limited to alkyl, haloalkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxyl, ketone, nitro, silyl, sulfoxy, sulfonyl, sulfone, sulfoxide, or thiol, as described below.

[0112] Throughout the specification, "alkyl" is used to refer to both unsubstituted alkyl groups and substituted alkyl groups; however, substituted alkyl groups are also specifically referred to herein by specifying the particular substituents on the alkyl group. For example, the term "haloalkyl" specifically refers to alkyl groups substituted with one or more halogens (e.g., fluorine, chlorine, bromine, or iodine). The term "alkoxyalkyl" specifically refers to alkyl groups substituted with one or more alkoxy groups, as described below. The term "alkylamino" specifically refers to alkyl groups substituted with one or more amino groups, as described below, and the like. When "alkyl" is used in one instance and a specific term such as "alkyl alcohol" is used in another instance, it is not meant to imply that the term "alkyl" does not also refer to the specific term such as "alkyl alcohol" and the like.

[0113] This practice is also used for other groups described herein. That is, although a term such as "cycloalkyl" refers to both unsubstituted and substituted cycloalkyl moieties, the substituted moieties can additionally be specifically referred to herein; for example, a particular substituted cycloalkyl can be referred to as, for example, "alkylcycloalkyl." Similarly, a substituted alkoxy group can be specifically referred to as, for example, "haloalkoxy," a particular substituted alkenyl group can be, for example, "alkenyl alcohol," and the like. Furthermore, the use of a general term such as "cycloalkyl" and a specific term such as "alkylcycloalkyl" does not imply that the general term does not also include the specific term.

[0114] The term "alkoxy" as used herein is an alkyl group bonded through a single terminal ether linkage; that is, "alkoxy" can be defined as -OZ 1 where Z 1 is an alkyl group as defined above.

[0115] The term "alkenyl" as used herein is a hydrocarbon group of 2 to 24 carbon atoms having a formula containing at least one carbon-carbon double bond. Asymmetric structures such as (Z 1 Z 2 )C=C(Z 3 Z 4 ) are intended to include both E and Z isomers. This can be assumed in the formulae herein where there is an asymmetric alkene, or it can be explicitly indicated by the bond symbol C=C. The alkenyl group can be substituted with one or more groups including, but not limited to, alkyl, haloalkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxyl, ketone, nitro, silyl, sulfoxy, sulfonyl, sulfone, or thiol, as described below.

[0116] The term "alkynyl" as used herein is a hydrocarbon group of 2 to 24 carbon atoms having a structural formula containing at least one carbon-carbon triple bond. The alkynyl group can be substituted with one or more groups including, but not limited to, alkyl, haloalkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxyl, ketone, nitro, silyl, sulfoxy, sulfonyl, sulfone, sulfoxide, or thiol, as described below.

[0117] The term "aryl" as used herein is a group containing 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 containing an aromatic group having at least one heteroatom bound 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" included in the term "aryl" defines a group containing an aromatic group that does not contain a heteroatom. The aryl or heteroaryl group can be substituted or unsubstituted. The aryl or heteroaryl group can be substituted with one or more groups including, but not limited to, alkyl, haloalkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxyl, ketone, nitro, silyl, sulfoxy, sulfonyl, sulfone, sulfoxide, or thiol, as described herein. The term "biaryl" is a specific type of aryl and is included in the definition of aryl. Biaryl refers to two aryl groups that are bound together via a fused ring structure (as in naphthalene) or linked via one or more carbon-carbon bonds (as in biphenyl).

[0118] The term "cycloalkyl" as used herein is a non-aromatic carbon-based ring consisting 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" is a cycloalkyl group as defined above, wherein at least one carbon atom of the ring is substituted with a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl and heterocycloalkyl groups can be substituted or unsubstituted. The 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, hydroxyl, ketone, nitro, silyl, sulfoxy, sulfonyl, sulfone, sulfoxide, or thiol, as described herein.

[0119] The term "cycloalkenyl" as used herein is 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 as defined above and is included within the meaning of the term "cycloalkenyl" wherein at least one carbon atom of the ring 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. Cycloalkenyl and heterocycloalkenyl 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, hydroxyl, ketone, nitro, silyl, sulfoxy, sulfonyl, sulfone, sulfoxide, or thiol, as described herein.

[0120] The term "cyclic group" is used herein to refer to either aryl, non-aryl (i.e., cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl), 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.

[0121] The term "aldehyde" as used herein is represented by the formula -C(O)H. Throughout the specification, "C(O)" or "CO" is the shorthand notation for C=O.

[0122] The term "amine" or "amino" as used herein is represented by the formula -NZ 1 Z 2 wherein Z 1 and Z 2 may each be a substituent as described herein, such as hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl as described above.

[0123] The term "carboxylic acid" as used herein is represented by the formula -C(O)OH. The "carboxylate" or "carboxyl" group as used herein is represented by the formula -C(O)O - .

[0124] The term "ester" as used herein is represented by the formula -OC(O)Z 1 or -C(O)OZ 1 wherein Z 1 may be alkyl, haloalkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl as described above.

[0125] The term "ether" as used herein is represented by the formula Z 1 OZ 2 wherein Z 1 and Z2 may be independently alkyl, haloalkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl as described above.

[0126] The term "ketone" as used herein is represented by the formula Z 1 C(O)Z 2 wherein Z 1 and Z 2 may be independently alkyl, haloalkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl as described above.

[0127] The term "halide" or "halogen" as used herein refers to fluorine, chlorine, bromine, and iodine.

[0128] The term "hydroxyl" as used herein is represented by the formula -OH.

[0129] The term "nitro" as used herein is represented by the formula -NO2.

[0130] The term "silyl" as used herein is represented by the formula -SiZ 1 Z 2 Z 3 wherein Z 1 , Z 2 and Z 3 may be independently hydrogen, alkyl, haloalkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl as described above.

[0131] The term "sulfonyl" as used herein refers to a sulfoxy group represented by the formula -S(O)2Z 1 wherein Z 1 may be hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl as described above.

[0132] The term "sulfonamido" or "sulfonamide" as used herein is represented by the formula -S(O)2NH-.

[0133] The term "phosphono" is used herein to refer to a phosphoroxy group represented by the formula -P(O)(OZ 1 )2wherein Z 1 may be hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl as described above.

[0134] The term "thiol" as used herein is represented by the formula -SH.

[0135] The term "thio" as used herein is represented by the formula -S-.

[0136] As used herein, "R 1"R 2 "R 3 "R n ", etc. (where n is some integer) can independently have one or more of the above groups. For example, if R 1 is a straight chain alkyl group, one hydrogen atom of the alkyl group can optionally be replaced with a hydroxyl, alkoxyl, amine, alkyl, halide, etc. Depending on the group chosen, the first group can be incorporated within the second group, or alternatively, the first group can be pendant (i.e., attached) to the second group. For example, for the phrase "alkyl group comprising an amino group", the amino group can be incorporated within the backbone of the alkyl group. Alternatively, the amino group can be attached to the backbone of the alkyl group. The nature of the group chosen will determine whether the first group is embedded or attached to the second group.

[0137] Unless otherwise indicated, formulas having bonds shown only as lines and not as wedgeshaped or phantom lines consider each possible isomer, e.g., each enantiomeric, diastereomeric, and meso compound, as well as mixtures of isomers, such as racemic or scalemic mixtures.

[0138] Reference will now be made in detail to specific aspects of the disclosed material, compounds, compositions, articles, and methods, examples of which are illustrated in the accompanying Examples and Figures.

[0139] Compounds

[0140] In some aspects, disclosed herein is a compound of Formula I, II, or III:

[0141]

[0142] or a salt thereof, wherein:

[0143] R 1 is independently selected from the group consisting of 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.

[0144] In some embodiments, the compound is of the following formula:

[0145]

[0146] or a salt thereof, wherein:

[0147] R 1 is independently selected from the group consisting of 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.

[0148] In some embodiments, the compound is of the following formula:

[0149]

[0150]

[0151] or salts thereof, wherein:

[0152] R 1 is independently selected from the group consisting of 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.

[0153] In some embodiments, the compound is of the formula:

[0154]

[0155] or salts thereof, wherein:

[0156] R 1 is independently selected from the group consisting of 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.

[0157] In some embodiments, R 1 is selected from the group consisting of:

[0158]

[0159]

[0160] or salts thereof.

[0161] In some embodiments, R 1 is In some embodiments, R 1 is In some embodiments, R 1 is In some embodiments, R 1 is In some embodiments, R 1 is In some embodiments, R 1 is In some embodiments, R 1 is In some embodiments, R 1 is In some embodiments, R 1 is In some embodiments, R 1 is

[0162] In some embodiments, the compound is

[0163]

[0164] wherein R 1 is

[0165] In some embodiments, the compound is

[0166]

[0167] wherein R 1 is

[0168] In some embodiments, the compound is

[0169]

[0170] wherein each R 1 is

[0171] In some embodiments, the compound is

[0172]

[0173] wherein each R 1 is

[0174] 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 1 is hydroxyl. In some embodiments, R 1 is ester. In some embodiments, R 1 is ether. In some embodiments, R 1 is carbonate. In some embodiments, R 1 is alkyl alcohol. In some embodiments, R 1 is alkyl ether. In some embodiments, R 1 is alkyl ester. In some embodiments, R 1 is carbamate. In some embodiments, R 1 is urea. In some embodiments, R 1 is guanidine. In some embodiments, R 1 is disulfide. In some embodiments, R 1is 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 thio ketal. In some embodiments, R 1 is a trisulfide. In some embodiments, R 1 is an oxime ether.

[0175] In some embodiments, each R 1 is an alkyl. In some embodiments, each R 1 is an alkenyl. In some embodiments, each R 1 is an alkynyl. In some embodiments, each R 1 is a 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 R 1 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 a urea. In some embodiments, each R 1 is a 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 thio ketal. In some embodiments, each R 1 is a trisulfide. In some embodiments, each R 1 is an oxime ether.

[0176] In some embodiments, the alkyl is a branched alkyl. In some embodiments, the alkyl is an unbranched alkyl.

[0177] In some embodiments, the alkyl is a C5alkyl. In some embodiments, the alkyl is a C6alkyl. In some embodiments, the alkyl is a C7alkyl. In some embodiments, the alkyl is a C8alkyl. In some embodiments, the alkyl is a C9alkyl. In some embodiments, the alkyl is a C 10 alkyl. In some embodiments, the alkyl is a C 11 alkyl. In some embodiments, the alkyl is a C12 Alkyl group. In some embodiments, the alkyl group is C10. 13 Alkyl group. In some embodiments, the alkyl group is C10. 14 Alkyl group. In some embodiments, the alkyl group is C10. 15 Alkyl group. In some embodiments, the alkyl group is C10. 16 Alkyl group. In some embodiments, the alkyl group is C10. 17 Alkyl group. In some embodiments, the alkyl group is C10. 18 Alkyl group. In some embodiments, the alkyl group is C10. 19 alkyl.

[0178] Nanoparticles

[0179] In one aspect, this disclosure provides a nanoparticle comprising a compound of formula I, II, or III as described herein.

[0180] Various compounds of formula I, II, or III are described in the above Compounds section. In some embodiments, the nanoparticles comprise a molar ratio of about 1% to about 99% of a compound of formula I, II, or III. In some embodiments, the nanoparticles comprise a molar ratio of about 10% to about 80% of a compound of formula I, II, or III. In some embodiments, the nanoparticles comprise a molar ratio of about 10% to about 40% of a compound of formula I, II, or III. In some embodiments, the nanoparticles comprise a molar ratio of about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40% of a compound of formula I, II, or III. In one embodiment, the nanoparticles comprise a molar ratio of about 20% of a compound of formula I, II, or III.

[0181] In some embodiments, the nanoparticle comprises a non-cationic lipid. In some embodiments, the non-cationic lipid interacts with the lipid as a helper lipid. In some embodiments, the non-cationic lipid can include, but is not limited to, 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l-stearoyl-2-oleoyl-sn- glycero-3-phosphoethanolamine (SOPE), DPPC (1,2-dipalmitoyl-sn-glycero-3- phosphocholine), 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE), 1,2-dioleoyl-5 / 7-glycero-3-phospho-(l'-rac-glycerol) (DOPG), or combinations 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 l-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 l-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.

[0182] In some embodiments, the nanoparticle comprises a non-cationic lipid in a molar ratio of about 10% to about 80%. In some embodiments, the nanoparticle comprises a non-cationic lipid in a molar ratio of about 10% to about 40%. In some embodiments, the nanoparticle comprises a non-cationic lipid in a molar ratio of about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40%. In one embodiment, the nanoparticle comprises a non-cationic lipid in a molar ratio of about 30%.

[0183] In some embodiments, the nanoparticle comprises a polyethylene glycol-lipid (PEG-lipid). PEG-lipids are incorporated to form a hydrophilic outer layer and to stabilize the particle. 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-glycero, methoxypolyethylene glycol (DMG-PEG). In one embodiment, the polyethylene glycol-lipid is 1,2-dimyristoyl-sn-glycero, methoxypolyethylene glycol-2000 (DMG-PEG2000). DMG-PEGXXXX means 1,2-dimyristoyl-sn-glycero, methoxypolyethylene glycol-XXXX, where XXXX represents the molecular weight of the polyethylene glycol moiety, for example DMG-PEG2000 or DMG-PEG5000.

[0184] In some embodiments, the nanoparticle comprises a polyethylene glycol-lipid in a molar ratio of about 0% to about 5%. In some embodiments, the nanoparticle comprises a polyethylene glycol-lipid in 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 nanoparticle comprises a polyethylene glycol-lipid in a molar ratio of about 0.75%.

[0185] In some embodiments, the nanoparticle comprises a sterol. Sterols are well known to those of skill in the art and generally refer to those compounds having a cyclopentanoperhydrophenanthrene ring system and having one or more OH substituents. Examples of sterols include, but are not limited to, cholesterol, campesterol, ergosterol, sitosterol, and the like.

[0186] In some embodiments, the sterol is selected from cholesterol-based lipids. In some embodiments, the one or more cholesterol-based lipids are selected from cholesterol, pegylated cholesterol, DC-Choi (N,N-dimethyl-N-ethylformamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine, or a combination thereof.

[0187] Sterols can be used to modulate particle permeability and fluidity according to their function in cell membranes. In one embodiment, the sterol is cholesterol.

[0188] In some embodiments, the nanoparticle comprises sterol in a molar ratio of about 25% to about 50%. In some embodiments, the nanoparticle comprises sterol in a molar ratio of about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%. In one embodiment, the nanoparticle comprises sterol in a molar ratio of about 40%.

[0189] In one embodiment, the nanoparticle further comprises an agent. In one embodiment, the nanoparticle further comprises a therapeutic agent. In one embodiment, the nanoparticle further comprises a diagnostic agent.

[0190] In some embodiments, the nanoparticle comprises one of the formulations as described in Figure 2F In some embodiments, the nanoparticle comprises one of the formulations as described in Figure 2F In some embodiments, the nanoparticle comprises components (lipid, DOPE, cholesterol, and PEG) in molar ratios as disclosed in Figure 7A In some embodiments, the nanoparticle comprises one of the formulations as described in Figure 7A In some embodiments, the nanoparticle comprises components (lipid, DOPE, cholesterol, and PEG) in molar ratios as disclosed in

[0191] The agent delivered into the cell can be a polynucleotide. Polynucleotides or oligonucleotides that can be introduced according to the methods herein include all types of DNA, cDNA, and RNA sequences. For example, the polynucleotide can 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 can also be a DNA construct, such as an expression vector, an expression vector encoding a desired gene product (e.g., a gene product that is homologous or heterologous to the subject into which it is to be introduced), and the like. In one embodiment, the agent is mRNA. In some embodiments, the polynucleotide is encapsulated by the nanoparticle.

[0192] In some embodiments, the polynucleotide comprises a nucleotide encoding a costimulatory molecule. In some embodiments, the costimulatory molecule is selected from the group consisting of 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.

[0193] Sequences of co-stimulatory molecules include, for example (for human sequences): 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_003327.4), 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_001328609.1), CD226 (NCBI Reference Sequence: NM_006566.3), Galectin-9 (GenBank: AB040130.2), TIM1 (GenBank: U02082.1), B7-H2 (NCBI Reference Sequence: NM_015259.5), 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), 4-1BBL (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_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), ICAM1 (NCBI Reference Sequence: NM_000201.3).

[0194] In some embodiments, the mRNA encoding the co-stimulatory molecule comprises a heterologous 5' untranslated region (5' UTR). In some embodiments, the mRNA encoding the co-stimulatory molecule comprises a heterologous 3' untranslated region (3' UTR).

[0195] In some embodiments, the nucleic acid disclosed herein (e.g., the mRNA encoding the co-stimulatory molecule) comprises at least one chemically modified nucleotide. In some embodiments, the at least one chemically modified nucleotide comprises a chemically modified nucleobase, a chemically modified ribose, a chemically modified phosphodiester bond, or a combination thereof.

[0196] In one embodiment, the at least one chemically modified nucleotide is a chemically modified nucleobase.

[0197] In one embodiment, the chemically modified nucleobase is selected from 5-formylcytidine (5fC), 5-methylcytidine (5meC), 5-methoxy cytidine (5moC), 5-hydroxyl cytidine (5hoC), 5-hydroxymethyl cytidine (5hmC), 5-formyluridine (5fU), 5-methyluridine (5-meU), 5-methoxy uridine (5moU), 5-carboxymethyl ester uridine (5camU), pseudouridine (Ψ), N 1 -methylpseudouridine (m 1 Ψ), N 6 -methyladenosine (m 6 A), or thienoguanosine (G). th

[0198] In some embodiments, the chemically modified nucleobase is 5-methoxy uridine (5moU). In some embodiments, the chemically modified nucleobase is pseudouridine (Ψ). In some embodiments, the chemically modified nucleobase is N 1 -methylpseudouridine (m 1 Ψ).

[0199] The structures of these modified nucleobases are shown below:

[0200]

[0201] In one embodiment, the at least one chemically modified nucleotide is a chemically modified ribose.

[0202] ​In one embodiment, the chemically modified ribose is selected from 2'-0-methyl (2'-0-Me), 2'-fluoro (2'-F), 2'-deoxy-2'-fluoro-beta-D-arabino-nucleic acid (2'F-ANA), 4'-S, 4'-SFANA, 2'-azido, UNA, 2'-0-methoxy-ethyl (2'-0-ME), 2'-0-allyl, 2'-0-ethylamine, 2'-0-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'-0-methyl (2'-0-Me). In one embodiment, the chemically modified ribose is 2'-fluoro (2'-F).

[0203] The structures of these modified riboses are shown below:

[0204]

[0205] In one embodiment, the at least one chemically modified nucleotide is a chemically modified phosphodiester linkage.

[0206] In one embodiment, the chemically modified phosphodiester linkage is selected from phosphorothioate (PS), boronophosphate, 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.

[0207] The structures of these modified phosphodiester linkages are shown below:

[0208]

[0209] Antigen presenting cell

[0210] In some aspects, disclosed herein is an antigen presenting cell comprising a lipid-based nanoparticle disclosed herein, the lipid-based nanoparticle comprising: a compound according to any foregoing aspect, and a recombinant polynucleotide comprising a nucleic acid encoding a costimulatory molecule.

[0211] In some embodiments,

[0212] The compound is

[0213] wherein R 1 is

[0214] In some embodiments, the costimulatory molecule is selected from the group consisting of 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.

[0215] In some embodiments, the antigen presenting cell comprises a macrophage or a dendritic cell.

[0216] It will be appreciated that the term "antigen presenting cell" or "APC" as used herein refers to a heterogeneous population of immune cells that can process and present antigens to stimulate the response of certain lymphocytes (e.g., T cells and B cells). Classical APCs include, for example, dendritic cells, macrophages, B cells, and neutrophils.

[0217] It is understood herein that macrophages are often referred to as phagocytic immune cells (Meszaros et al., 1999). They also secrete factors such as chemotactic factors or cytokines. In addition to phagocytosis and antigen presentation, these cells can also exert supportive roles through various plasma membrane and secreted molecules (Gordon 1995, BioEssays, Vol. 17, No. 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 properties typically described for macrophages, including phagocytosis, expression of specific cell surface markers, such as CD64, CD14, and HLA-DR antigen expression. Macrophages according to the present application can be isolated from tissue, or preferably differentiated from blood monocytes (also referred to herein as “monocyte-derived macrophages”), bone marrow precursor cells (also referred to herein as “bone marrow-derived macrophages”), or from any other possible precursor, as well as by using any differentiation method, precursor, and methods known to one of skill in the art. Thus, in some embodiments, macrophages include bone marrow-derived macrophages. In some embodiments, macrophages include monocyte-derived macrophages. In some embodiments, macrophages include iPSC-derived macrophages. In some embodiments, macrophages include macrophage cell lines, including, for example, RAW264.7, THP-1, U937, IC-21, J774A.1, MV-4-11, or KG1.

[0218] It is also understood herein that “dendritic cells” or “DCs” as used herein refer to a type of antigen presenting cell that is typically identified by 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 dendritic cells are mature DCs. In some embodiments, the dendritic cells are immature DCs. The DCs according to the present application can be isolated from tissue, or preferably differentiated from blood monocytes (also referred to herein as “monocyte-derived dendritic cells”), bone marrow precursor cells (also referred to herein as “bone marrow-derived dendritic cells”), or from any other possible precursor, and isolated by using any differentiation method, precursor and methods known to the person skilled in the art. Thus, in some embodiments, the dendritic cells include bone marrow-derived dendritic cells. In some embodiments, the dendritic cells include monocyte-derived dendritic cells. In some embodiments, the dendritic cells include iPSC-derived dendritic cells. In some embodiments, the dendritic cells are conventional dendritic cell 1 (or cDC1, lymphoid DC), which is 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 cells are conventional dendritic cell 2 (or cDC2, myeloid DC), which is typically identified by expression of one or more of the following markers on its cell surface: CD1c and CD172a. In some embodiments, the dendritic cells are plasmacytoid DCs (or pDCs), which are typically identified by expression of one or more of the following markers on its cell surface: CD123, CD303, and CD304.

[0219] In some aspects, disclosed herein are methods of expressing a polypeptide in antigen presentation, the method 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.

[0220] Compositions and methods

[0221] In some aspects, disclosed herein is a method of treating cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of an antigen presenting cell and an antibody disclosed herein.

[0222] 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.

[0223] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds a co-stimulatory molecule is BMS986178. In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds a co-stimulatory molecule is GSK3174998. In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds a co-stimulatory molecule is PF-04518600. In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds a co-stimulatory molecule is MOXR0916. In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds a co-stimulatory molecule is PF-04518600. In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds a co-stimulatory molecule is MEDI6383. In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds a co-stimulatory molecule is MEDI0562. In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds a co-stimulatory molecule is INCAGN01949. In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds a co-stimulatory molecule is InVivoPlus Anti-Mouse OX40 (Clone OX-86) (Company: BioXcell, Catalog: BP0031).

[0224] Other antibodies or antigen-binding fragments thereof that specifically bind a co-stimulatory molecule can include, for example: for mouse, 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); for human, Anti-Human OX40, BMS 986178, GSK3174998, PF-04518600, MOXR0916, PF-04518600, MEDI6383, MEDI0562, INCAGN01949; Anti-Human 4-1BB, Utomilumab, Urelumab; Anti-Human CD40, CP-870893, APX005M, ADC-1013, JNJ-64457107, SEA-CD40, RO7009789.

[0225] In some embodiments, the antigen presenting cell and the antibody are administered intratumorally.

[0226] In some aspects, disclosed herein is a method of treating a cancer, the method comprising administering to a subject in need thereof:

[0227] a therapeutically effective amount of a lipid-based nanoparticle disclosed herein, and

[0228] a therapeutically effective amount of an antigen presenting cell disclosed herein.

[0229] In some embodiments, the lipid-based nanoparticle comprises

[0230]

[0231] wherein R 1 is

[0232] In some embodiments, the lipid-based nanoparticle comprises a recombinant polynucleotide comprising a nucleic acid encoding CD40L.

[0233] In one aspect, the methods described herein are used to treat cancer, such as melanoma, lung cancer (including lung adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, large cell carcinoma, bronchioloalveolar carcinoma, bronchogenic carcinoma, non-small cell carcinoma, small cell carcinoma, mesothelioma); breast cancer (including ductal carcinoma, lobular carcinoma, inflammatory breast cancer, clear cell carcinoma, mucinous carcinoma, serosal cavity breast cancer); colorectal cancer (colon cancer, rectal cancer, colorectal adenocarcinoma); anal cancer; pancreatic cancer (including pancreatic adenocarcinoma, islet cell carcinoma, neuroendocrine tumor); prostate cancer; prostate adenocarcinoma; ovarian cancer (ovarian epithelial cancer or surface epithelial-stromal tumor, including serous tumor, endometrioid tumor, and mucinous cystadenocarcinoma, sex cord-stromal tumor); liver and bile duct cancer (including hepatocellular carcinoma, cholangiocarcinoma, hemangioma); esophageal cancer (including esophageal adenocarcinoma and squamous cell carcinoma); oral and oropharyngeal squamous cell carcinoma; salivary gland adenoid cystic carcinoma; bladder cancer; bladder tumor; uterine tumor (including endometrial adenocarcinoma, ocular cancer, uterine papillary serous carcinoma, uterine clear cell carcinoma, uterine sarcoma, leiomyosarcoma, mixed Mullerian tumor); glioma, glioblastoma, medulloblastoma, and other brain tumors; kidney cancer (including renal cell carcinoma, clear cell carcinoma, Wilms' tumor); head and neck cancer (including squamous cell carcinoma); stomach cancer (gastric cancer, gastric adenocarcinoma, gastrointestinal stromal tumor); testicular cancer; germ cell tumor; neuroendocrine tumor; cervical cancer; carcinoid tumors of the gastrointestinal tract, breast, and other organs; plasmacytoid carcinoma; mesenchymal tumors, including sarcoma, fibrosarcoma, hemangioma, angiomatosis, hemangiopericytoma, pseudangiomatous stromal hyperplasia, myofibroblastoma, fibromatosis, inflammatory myofibroblastic tumor, lipoma, angiolipoma, granular cell tumor, neurofibroma, schwannoma, angiosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma, leiomyoma, leiomyosarcoma, skin cancer (including melanoma), cervical cancer, retinoblastoma, head and neck cancer, pancreatic cancer, brain cancer, thyroid cancer, testicular cancer, kidney cancer, bladder cancer, soft tissue cancer, adrenal cancer, urethral cancer, penile cancer, myxosarcoma, chondrosarcoma, osteosarcoma, chordoma, malignant fibrous histiocytoma, lymphangiosarcoma, mesothelioma, squamous cell carcinoma; epidermoid carcinoma, malignant cutaneous adnexal neoplasms, adenocarcinoma, hepatocarcinoma, hepatocellular carcinoma, renal cell carcinoma, adrenal gland tumor, cholangiocarcinoma, transitional cell carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, anaplastic glioma; glioblastoma multiforme, neuroblastoma, medulloblastoma, malignant meningioma, malignant schwannoma, neurofibrosarcoma, parathyroid cancer, medullary thyroid carcinoma, bronchial carcinoid, pheochromocytoma, islet cell carcinoma, malignant carcinoid tumor, malignant paraganglioma, melanoma, Merkel cell carcinoma, phyllodes tumor of the breast, salivary gland carcinoma, thymic carcinoma, and vaginal cancer, etc.

[0234] EMBODIMENT

[0235] The following examples are set forth to illustrate the compositions, methods and results in accordance with the disclosed subject matter. These examples are not intended to include all aspects of the subject matter disclosed herein, but rather are meant to be representative of representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention that are apparent to one of skill in the art.

[0236] Example 1. Sugar-derived lipid nanoparticles

[0237] Effective cancer immunotherapy depends on the initiation, process, and amplification of the cancer immune cycle (CIC). However, the CIC is often blocked by immunosuppressive factors in the tumor microenvironment (TME), leading to tumor promotion, metastasis, and recurrence. Here, a lipid nanoparticle-mRNA formulation and dendritic cell therapy are combined to substantially enhance the CIC via multiple steps. First, lipid nanoparticles containing CD40 ligand (CD40L) mRNA induce robust immunogenic cell death in tumor tissue, leading to tumor-associated antigen (TAA) release and CD40L expression. Next, dendritic cells overexpressing CD40 are adoptively transferred and then activated by CD40L molecules in the tumor tissue. This promotes secretion of proinflammatory cytokines and chemokines, as well as upregulation of costimulatory molecules on dendritic cells, which are critical for reprogramming the TME and priming T cell responses. After dendritic cells present TAA to T cells, all of the above stepwise events contribute to potent cancer-specific T cell immunity that eradicates established skin tumors, suppresses distant lesions, and prevents tumor rechallenge. In summary, this work closes the cancer immune cycle (CIC) in a mouse melanoma model by integrating lipid nanoparticle and cell therapy (CATCH) and promotes tumor elimination and the construction of long-term antitumor immunity.

[0238] Brief Introduction

[0239] Development of immunotherapy has made significant breakthroughs in clinical outcomes for cancer treatment. Effective antitumor immunity depends on a series of responses in the cancer immune cycle (CIC) 1 that are properly activated. First, dying cancer cells release tumor-associated antigens (TAA), which are captured and processed by antigen-presenting cells (APCs) such as dendritic cells 1 . Next, APCs present antigens to T cells, leading to priming of effector T cell responses against TAA 1 . Activated effector T cells infiltrate into tumor tissue and mediate killing of cancer cells 1 . Finally, dying cancer cells further release antigens to increase the breadth and depth of these immune responses in the CIC 1However, these gradual events are often suppressed by the immunosuppressive tumor microenvironment (TME), leading to uncontrolled tumor growth, metastasis, and recurrence 1,2 For example, the immunosuppressive TME can suppress the recruitment, infiltration, and maturation of dendritic cells 1,2 This reduces cytokine secretion and expression of costimulatory factors by dendritic cells that are essential for priming T cell immunity 1,2 .

[0240] Here, it is investigated whether synergistically inducing cancer cell death to release TAA and improve the number and maturation of dendritic cells in tumor tissue can appropriately enhance CIC. This can further prime systemic and memory anti-tumor immunity, leading to eradication of the primary tumor, suppression of tumor metastasis, and prevention of tumor recurrence. Immunogenic cell death (ICD) is a mode of cell death that is accompanied by the release of damage-associated molecular patterns (DAMPs) and TAA 3,4 Recent studies have revealed that lipid nanoparticles (LNP) with the ability to induce ICD can re-model the TME, leading to enhanced anti-tumor immunity 5 CD40 and CD40 ligand (CD40L) are a pair of costimulatory molecules belonging to the tumor necrosis factor / tumor necrosis factor receptor family 6 The interaction between CD40 on dendritic cells and CD40L on CD4 T cells is essential for dendritic cell maturation, which promotes proinflammatory cytokine secretion and upregulation of other costimulatory molecules 6 These play a central role in triggering CD8 T cells to develop cytotoxic and memory responses 6 Several CD40 agonist antibodies have been investigated in clinical trials 6 Furthermore, intratumoral (i.t.) administration of immunotherapeutics can improve in situ bioavailability and thereby treatment efficiency 7 Correspondingly, several LNP-mRNA formulations and immune cells delivered via intratumoral injection are in clinical trials 7-10 In this study, LNP containing CD40L mRNA (CD40L-LNP) are utilized to simultaneously induce ICD and expression of CD40L in tumor tissue. Then, intratumoral adoptive transfer of bone marrow-derived dendritic cells overexpressing CD40 (CD40-BMDC) is performed, which capture TAA and become mature by stimulation of CD40L. These activated dendritic cells further present antigens to T cells, leading to priming of effector T cell responses against cancer cells Figure 1A

[0241] ​To test this concept, efficient delivery of mRNA into tumor cells and primary dendritic cells was required. Sugar alcohols are carbohydrates that are widely used as sugar substitutes in the food industry, which provide sweetness, contain fewer calories, and do not cause blood sugar spikes compared to regular sugars such as glucose 11 These compounds are naturally occurring or derived from sugars via reduction of carbonyl groups to hydroxyl groups 12 In particular, hexitol sugars, such as sorbitol and mannitol, are six-carbon sugar alcohols that can be doubly dehydrated to produce dianhydrohexitols, a series of unique cyclic ethers that serve as important chemical scaffolds and starting materials 13 Accordingly, three groups of sugar alcohol-derived ionizable lipids were designed and synthesized to prepare LNPs for mRNA delivery. To examine these LNPs in biological assays, melanoma was chosen as a disease model. Melanoma is one of the most aggressive skin cancers with increasing incidence 14 The primary cause of melanoma patient death is extensive metastasis, such as skin and brain metastasis 14 Accordingly, two melanoma cell lines were chosen in this study to construct mouse tumor models. After systemic screening and characterization of sugar alcohol-derived ionizable lipids, two LNP formulations were identified that are preferred for mRNA delivery. One formulation induced robust ICD in melanoma tumor tissues and simultaneously efficiently delivered CD40L mRNA into cancer cells. The other formulation showed significantly higher CD40 mRNA delivery efficiency in mouse bone marrow-derived dendritic cells (BMDCs) compared to Lipofectamine 3000 (Lipo 3K) and electroporation (Electro). Combination of CD40L-LNP treatment with adoptive CD40-BMDC transfer resulted in greater than 80% complete response rate in a subcutaneous (s.c.) melanoma tumor model. In addition, local treatment enabled suppression or elimination of metastasis of skin and brain tumors. Importantly, these responder mice were resistant to s.c. re-challenge and exhibited delayed tumor growth after intracranial re-challenge. In summary, two types of LNP-mRNA formulations were utilized to enhance CIC via induction of ICD and adoptive dendritic cell transfer. This work presents an important immunotherapy strategy that enables broad and effective treatment of primary tumors, tumor metastasis, and tumor recurrence.

[0242] Results

[0243] Dianhydrohexitols, such as isosorbide and isomannide, are a series of biogenic bicyclic compounds prepared by the double dehydration of six-carbon sugar alcohols 15 They consist of two cis-fused tetrahydrofuran rings with two secondary hydroxyl groups located at the 2- and 5-positions 13The different configurations of the two hydroxyl groups lead to different reactivity and steric hindrance towards functionalization, thus making dianhydrohexitols versatile bio-based building blocks for the synthesis of pharmaceutically important compounds and polymers 16 Based on their unique chirality and rigidity, three groups of sugar alcohol-derivatized ionizable lipids (DIS, DIM, and LIS) were designed and synthesized with sorbitol, mannitol, and L-sorbitol as precursors, respectively Figure 1B , Figure 1C As a representative synthetic pathway for LIS lipids Figure 1B ), L-isosorbide (the enantiomer of isosorbide) was prepared from L-sorbitol according to a previously reported dehydration procedure in the presence of dimethyl carbonate and sodium ethoxide 17 Then, L-isosorbide underwent a double Michael addition reaction with acrylonitrile, followed by reduction of the nitrile group with borane to give the core amine. Finally, hydrophobic tails with different functional groups such as hydroxyl (LIS1), hydrocarbon (LIS2 to LIS6), ester (LIS7), carbonate (LIS8), and acetal (LIS9 and LIS10) were installed via reductive amination or epoxide ring-opening reactions to provide the corresponding ionizable lipids. These different hydrophobic domains in the lipids affect the formulation of LNP and their interaction with cell membranes, leading to different mRNA delivery efficiencies. Following a similar synthetic pathway, DIS and DIM lipids were synthesized, and their structures were confirmed by H nuclear magnetic resonance and mass spectrometry (see Methods). 1

[0244] Next, LNP containing firefly luciferase (FLuc) mRNA (FLuc-LNP) were formulated and characterized for size, surface charge, and mRNA encapsulation efficiency Figure 6A By quantifying the luminescence intensity of bone marrow-derived dendritic cells (BMDCs), the structure-activity relationship of sugar alcohol-derivatized ionizable lipids was generalized. The chirality of the amine core can affect the mRNA delivery efficiency of the formulated LNP. The amine cores of DIS and LIS lipids are a pair of enantiomers, and they exhibit similar mRNA delivery capacity, especially for DIS-1 to 8 and LIS-1 to 8. Lipids with a C10 hydrocarbon tail induce more efficient mRNA delivery than lipids containing other hydrocarbon tails. On the other hand, the DIM series presents a different trend. Among the DIM series, DIM-7 is the most efficient in mRNA delivery. Lipids containing a carbonate linker in the hydrophobic domain (DIS8, DIM8, and LIS8) exhibit weaker Fluc mRNA delivery capacity. Among all these LNP, DIM7 and LIS10 show the highest mRNA delivery efficiency compared to other LNP, which is 3-fold and 10-fold of Lipo 3K and Electro, respectively Figure 2A ​). To further investigate the formulations of DIM7 and LIS10, based on L16(4) 4 Orthogonal table performs orthogonal screening assay Figure 7A ). Based on the luminescence intensity of the 16 orthogonal formulations of DIM7 or LIS10 Figure 2C , Figure 2E ), the effect of each lipid component with different molar ratios on mRNA delivery was profiled Figure 2B , Figure 2D ) and several preferred formulations were predicted Figure 2B ). Then, their mRNA delivery efficacy was validated by comparison with the corresponding top formulation DIM7M and LIS10G from the orthogonal screening assay Figure 2C , Figure 2E ). For DIM7, the preferred formulation DIM7S resulted in 2-fold (P<0.001) and 3.5-fold (P<0.001) higher luminescence signal compared to the top orthogonal formulation DIM7M and the initial formulation DIM7, respectively Figure 2C ). For LIS10, the preferred formulation LIS10W resulted in 1.2-fold (P<0.0001) higher luminescence signal compared to the top orthogonal formulation LIS10G and 2.3-fold (P<0.0001) higher luminescence intensity compared to the initial formulation LIS10 Figure 2E . Formulations DIM7S and LIS10W showed similar properties. Their particle size was about 110 nm and the polydispersity index (PDI) was <0.2 Figure 2G , Figure 2I . Their mRNA encapsulation efficiency was about 90% and they were slightly positively charged Figure 2G , Figure 2I . Moreover, when visualized by Cryo-TEM, they both exhibited a spherical morphology Figure 2H , Figure 2J , Figure 7B , Figure 7C . Therefore, formulation DIM7S was selected to deliver mRNA into BMDCs ex vivo for the following studies. At the same mRNA concentration, formulation DIM7S was 10-fold and 30-fold more efficient in mRNA delivery than Lipo 3K and Electro, respectively Figure 7D . The maximum luminescence intensity of formulation DIM7S was observed at 12h over a time course of 6h to 24h Figure 7E . Strikingly, formulation DIM7S encapsulating CD40 mRNA (CD40-DIM7S) resulted in more than 70% of CD40 expression in BMDCs (P<0.0001, Figure 7F .

[0245] Activation of dendritic cells is an important step in CICs to elicit T cell responses 1 . Therefore, it was next tested whether CD40 overexpression promotes BMDC activation in the presence of anti-CD40 agonist antibody (CD40 Ab). The combination of CD40-DIM7S and CD40 Ab (CD40-DIM7S+CD40 Ab) stimulated higher levels of dendritic cell activation markers ( Figure 3A ), including CD80 (P<0.01), CD86 (P<0.01), MHC-II (P<0.01), IL1-β (P<0.05 in the pro-form), TNF-α (P<0.05), and IL12 (P<0.01), indicating a stronger capacity of dendritic cell activation. To evaluate whether enhanced dendritic cell activation improves anti-tumor effects in vivo, intratumoral (i.t) injections were performed every other day for a total of 4 doses ( Figure 3B ) in a B16F10 melanoma mouse model for multiple treatments. Treatment strategies included various combinations, including CD40 Ab, CD40-DIM7S, CD40-DIM7S+CD40 Ab, BMDC+CD40 Ab, CD40-BMDC, and CD40-BMDC+CD40 Ab. CD40-BMDC+CD40 Ab significantly inhibited tumor growth by 5-fold to 15-fold compared to all other treatments (P<0.05, Figure 3C , Figure 8) and significantly prolonged overall survival ( Figure 3D , P<0.01). In fact, CD40-DIM7S can engineer dendritic cells in situ to increase expression of CD40 receptor ( Figure 9A ). However, CD40-DIM7S+CD40 Ab exhibited limited therapeutic effects compared to CD40-BMDC+CD40 Ab ( Figure 3C 、 Figure 3D , Figure 8). This can be partially attributed to the lack of infiltrating dendritic cells in the immunosuppressive TME 1,2 . Although BMDC+CD40 Ab supplemented dendritic cells in the tumor tissue, insufficient BMDC activation can be the cause of the weaker anti-tumor effects 1,2 . Altogether, these results uncovered the importance of sufficient dendritic cell infiltration, CD40 overexpression on BMDCs, and CD40 agonism for the anti-tumor activity of CD40-BMDC+CD40 Ab treatment.

[0246] Although CD40-BMDC+CD40 Ab showed promising anti-tumor effects, the lack of complete tumor burden eradication indicates insufficient progression of CIC. Cancer cell death as an initial step of CIC enables the release of TAA, which is presented by dendritic cells and elicits tumor-specific T cell responses 1,3-5 Furthermore, clinical studies have revealed that cancer cell ICD benefits the anti-tumor effects of immunotherapeutic agents 3,4 Therefore, it was investigated whether LNP-induced cancer cell ICD can promote the anti-tumor effects of CD40-BMDC therapy. It was also investigated whether LNP-mediated CD40L expression in tumor tissue can activate adoptively transferred CD40-BMDC. Thus, the combination of LNP-induced cancer cell ICD with CD40L and adoptive CD40-BMDC transfer can effectively enhance CIC and thereby eradicate tumor lesions Figure 1A The cytotoxicity of DIM7S and LIS10W containing CD40L mRNA (CD40L-DIM7S and CD40L-LIS10W) was investigated in B16F10 melanoma cells. LIS10W induced potent cytotoxicity (85%) Figure 9B Since both CD40L-DIM7S and CD40L-LIS10W resulted in almost 100% CD40L expression in B16F10 melanoma cells after 18 h incubation Figure 9C , LIS10W was chosen to evaluate the ability to induce ICD markers including extracellular high mobility group box 1 (HMGB1), extracellular ATP and cell surface calreticulin in B16F10 melanoma cells. FLuc-LIS10W or CD40L-LIS10W induced about 1.7-fold (P < 0.01), 7-fold (P < 0.001) and 45-fold (P < 0.001) higher levels of extracellular HMGB1, extracellular ATP and cell surface calreticulin, respectively, in vitro compared to PBS treatment Figure 3E Consistently, ICD induced by FLuc-LIS10W or CD40L-LIS10W was observed in vivo in mouse B16F10 melanoma tissue Figure 3F , indicating that LIS10W LNP in addition to the mRNA cargo contributes to the ICD effect.

[0247] Since the expression profile of CD40L in tumor tissue can affect its biological activity, its biodistribution was investigated at the cellular level by flow cytometry. A single injection of CD40L-LIS10W resulted in about 15% of CD40L positive B16F10 cells in tumor tissue Figure 9D). Moreover, immune cells in tumor tissues, including macrophages, dendritic cells, CD4 T cells, and CD8 T cells, showed about 1.5-fold to 2-fold increase in CD40L expression Figure 9E ). Both the expression of CD40L in cancer cells and immune cells can contribute to CD40-BMDC activation 18 .

[0248] To evaluate the anti-tumor effect of the combination of cancer cell ICD and adoptive CD40-BMDC transfer, CD40-BMDCs were intratumorally administered 18 h 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 ), which was significantly higher than that of CD40-BMDC + CD40 Ab treatment (0%, P < 0.05) and CD40L-DIM7S + CD40-BMDC treatment (17%, P < 0.05). Importantly, all the responding mice in the CD40L-LIS10W + CD40-BMDC group were resistant to re-challenge with B16F10 tumor on the opposite flank Figure 3I 、 Figure 10B ), indicating the emergence of anti-tumor memory. These results highlight the critical role of ICD in eliciting anti-tumor immunity in CIC. To further confirm the function of CD40-BMDC and CD40L-mediated activation in this treatment regimen, two additional treatment groups, including FLuc-LIS10W + CD40-BMDC and CD40L-LIS10W + CD40-DIM7S Figure 3J ) were further investigated. Although FLuc-LIS10W and CD40L-LIS10W showed similar ICD effects in vivo Figure 3F ), CD40L-LIS10W + CD40-BDMC significantly inhibited tumor growth by about 21-fold (P < 0.05) and 7-fold (19 days after inoculation, P < 0.05) compared to FLuc-LIS10W + CD40-BDMC and CD40L-LIS10W + CD40-DIM7S, respectively Figure 5C 、 Figure 5D ). Moreover, 83% of mice in the CD40L-LIS10W + CD40-BDMC group survived 45 days after tumor inoculation, while no mice survived in the other two treatment groups (P < 0.01, Figure 3K ). In addition, 100% of the responding mice were resistant to the development of secondary tumors after re-challenge with B16F10 cells on the opposite flank Figure 3L 、 Figure 10E). These findings suggest that ICD alone is insufficient to eradicate tumor lesions, highlighting the importance of dendritic cell infiltration and activation. Long-term benefits of immunotherapy require anti-tumor memory against tumor recurrence. Therefore, a mouse model of re-challenge via intracranial (i.c.) was further validated the development of anti-tumor memory Figure 3M ) In this experiment, another melanoma cell line containing a luciferase reporter gene (B16F10-Luc2) was used, which facilitated monitoring of brain tumor growth via bioluminescence imaging. CD40L-LIS10W+CD40-BDMC treatment achieved approximately 88% complete regression of primary subcutaneous tumors 45 days after tumor inoculation Figure 3N 、 Figure 10F ) Notably, surviving mice showed significantly delayed brain tumor growth (11 days after inoculation, P < 0.05, Figure 10G ). Moreover, 38% of responding mice remained tumor-free compared to 0% of controls in the first experiment (75 days after i.c. re-challenge, P < 0.01, Figure 3O ). These data indicate that a robust anti-tumor memory T cell population was generated from this local therapy.

[0249] Efficacy of local immunotherapy treatment of metastatic cancer depends on development of systemic anti-tumor immunity 5,7,10 . To evaluate this, B16F10 cells were inoculated into both flanks of each mouse, followed by CD40L-LIS10W+CD40-BDMC treatment on one tumor site Figure 4A ). The treatment eliminated 80% of primary tumors Figure 11A ) and 70% of distant tumors Figure 4B ), and overall survival was approximately 70% Figure 4C ). In another dual tumor model (skin + brain tumors), B16F10-Luc2 cells were inoculated subcutaneously and intracranially Figure 4D ). CD40L-LIS10W+CD40-BDMC treatment eradicated 80% of subcutaneous tumors Figure 11B ). At the same time, the treatment significantly inhibited brain tumor growth (11 days after inoculation, P < 0.001, Figure 4E ) and significantly prolonged overall survival time (P < 0.0001, Figure 4F ). In summary, these results indicate that local treatment generates systemic anti-tumor immunity.

[0250] T cell-mediated cancer cell killing is an important step in eradicating tumor lesions in CIC. To evaluate the role of CD8 and CD4 T cells in treatment efficacy, mice receiving anti-CD8, anti-CD4, or isotype control Ab were subjected to CD40L-LIS10W+CD40-BDMC treatmentFigure 4G Compared to the control group, the depletion of CD8 or CD4 T cells significantly impaired tumor regression and mouse survival in patients treated with CD40L-LIS10W+CD40-BDMC. Figure 4H , Figure 4I , Figure 11C In particular, compared to CD4 T cell depletion, CD8 T cell depletion led to a more significantly weakened therapeutic effect. Figure 4H , Figure 4I , Figure 11C This indicates that CD8T cells play a crucial role in this treatment.

[0251] To uncover the potential mechanism of action of the CD40L-LIS10W+CD40-BDMC treatment regimen, the dynamic expression of cytokines and chemokines was analyzed 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 the 32-plex group over a time span of 6 to 24 hours. Figure 4K , Figure 12 Furthermore, their concentrations increased further after intratumoral administration of CD40-BDMC. Figure 4K , Figure 12 In the blood, most cytokines and chemokines are upregulated 6 hours after CD40L-LIS10W injection, while their concentrations gradually decrease over the following 18 hours. Figure 4L , Figure 13 However, CD40-BDMC administration over a timeframe of 24 to 42 hours restimulates the production of many cytokines and chemokines. Figure 4L , Figure 13 These data indicate that CD40L-LIS10W+CD40-BDMC better reprograms the immunosuppressive TME and induces a stronger systemic immune response compared to CD40L-LIS10W alone. The upregulation of these inflammatory cytokines and chemokines not only enhances the recruitment and activation of immune cells but also promotes the development of immune memory. Next, changes in the immune cell population in tumor tissue after CD40L-LIS10W+CD40-BDMC treatment were analyzed. In this experiment, CD40-BDMCs were labeled with CellTraceBlue before intratumoral injection, which allowed for the differentiation of applied dendritic cells from endogenous dendritic cells via flow cytometry analysis. Treatment resulted in a significant influx of dendritic cells, CD8 T cells, and CD4 T cells, while reducing the recruitment of macrophages and regulatory T (Treg) cells in tumor tissue. Figure 5A). In addition, the treatment enhanced the percentage of APCs that included CD80 / 86 + macrophages (P < 0.01) and CD80 / 86 + 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 from the responder mice. The number of effector memory and central memory T cells in both the spleen and blood was significantly increased compared to the control of the first experiment, indicating the development of long-term anti-tumor immunity.

[0252] Discussion

[0253] 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) and dendritic cell therapy were combined to appropriately enhance the CIC via a comprehensive immune response. A library of sugar alcohol-derivatized ionizable lipids was synthesized and two preferred LNP formulations, DIM7S and LIS10W, for efficient mRNA delivery were identified. DIM7S exhibited 10-fold and 30-fold mRNA delivery efficacy in BMDCs compared to Lipo 3K and Electro, respectively Figure 7D LIS10W induced robust ICD in melanoma tissue Figure 3F and efficiently delivered mRNA into melanoma cells Figure 9C , Figure 9D For CD40L-LIS10W + CD40-BMDC treatment, CD40L-LIS10W was first administered intratumorally to induce cancer cell death. This led to the release of TAA and DAMPs (e.g., HMGB1, extracellular ATP, and cell surface calreticulin) and, at the same time, CD40L expression in the tumor tissue. Next, CD40-BMDCs engineered with CD40-DIM7S ex vivo were adoptively transferred intratumorally. 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 , Figure 4L , which, together with the presented TAA, stimulated the differentiation of na'ive T cells into cytotoxic CD8 T cells Figure 5C). On one hand, this facilitates T cell-mediated cancer cell killing, an important step in eradicating tumor lesions in CIC. On the other hand, T cell priming promotes their production of IFN-γ and TNF-α, which in turn activate APCs and induce production of chemokines ( Figure 4K , Figure 4I ), such as CCL3, CCL4, CCL5, and CXCL10. In response to these chemokines, additional APCs and T cells are recruited into the tumor tissue ( Figure 5A ). All these events contribute to reprogramming the immunosuppressive TME and sustain amplification and expansion of CIC, ultimately leading to complete regression of over 80% of primary cutaneous melanoma tumors ( Figure 3H , Figure 3K , Figure 3N ). Importantly, enhanced CIC benefits the generation of memory CD8 T cells in the spleen and blood ( Figure 5D , Figure 5E ), resulting in nearly 100% and 38% protection against subcutaneous and intracranial melanoma re-challenge, respectively, in responding mice ( Figure 3I , Figure 3L , Figure 3O ). Finally, this local treatment prevents ~70% of distant cutaneous tumors and significantly regresses distant brain tumors, indicating effective suppression of metastatic cancer.

[0254] In summary, showing synergistic induction of cancer immunogenic cell death (ICD) and dendritic cell activation (the first two steps of cancer immuno-cycle (CIC)) can reprogram the immunosuppressive TME and promote tumor antigen presentation. This strategy leads to potent effector T cell responses and protective immune memory against cancer-specific antigens, reflected in eradication of both local and distant tumors and prevention of tumor re-challenge. In summary, the combination of lipid-nanoparticle-mRNA formulation and dendritic cell therapy provides an important platform for cancer immunotherapy.

[0255] Materials and Methods

[0256] Antibodies

[0257] Antibodies 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), pro-form IL-1 b-FITC (eBioscience, 11711480), Calreticulin-Alexa Fluor 647 (Novus Biologicals, NBP1-47518AF647), Ki-67-FITC (eBioscience, 11569882), Granzyme B-FITC (eBioscience, 11889882), IFNy-FITC (Biolegend, 505806), TNFa-FITC (eBioscience, 11732181), CD62L-FITC (eBioscience, 11-0621-81), CD44-Alexa Fluor 700 (eBioscience, 56044182).

[0258] Antibodies for ELISA: Rat anti-mouse IL-12p70 (eBioscience, MM121), Rat anti-mouse TNFa (eBioscience, 14732581), Goat anti-rat IgG-HRP (Cell Signaling, 7077S), Mouse anti-mouse HMGB1 (eBioscience, MA120338), Goat anti-mouse IgG-HRP (Abeam, ab7068).

[0259] Antibodies for in vivo: Anti-mouse CD40 antibody (CD40 Ab, BioXcell, BP00162), Anti-mouse CD8a (Bioxcell, BE0004-1), Anti-mouse CD4 (Bioxcell, BP0003-1), Anti-rat IgG2b isotype (Bioxcell, BP0090).

[0260] Cell culture

[0261] The B16F10 melanoma cell line was obtained from Dr. Jianhua Yu's lab. The B16F10-Luc2 melanoma cell line was purchased from the American Type Culture Collection (ATCC). Both cell lines were cultured in Dulbecco's Modified Eagle Medium (ATCC, 302002) containing 10% fetal bovine serum (FBS, Gibco, 26140079) in an incubator at 37°C in 5% CO2. Mouse bone marrow-derived dendritic cells (BMDCs) were obtained by adapting a previous protocol 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).

[0262] Flow cytometry gating

[0263] Gating strategy was based on previously reported methods 10,19 .

[0264] B16F10 cells: CD45 -

[0265] Macrophages: CD45 + , CD11b + , F4 / 80 +

[0266] Activated macrophages: CD45 + , CD11b + , F4 / 80 + , CD80 + / CD86 +

[0267] Dendritic cells: CD45 + , CD11b + , CD11c +

[0268] Activated dendritic cells: CD45 + , CD11b + , CD11c + , CD80 + / CD86 +

[0269] CD4 T cells: CD45 + , CD3e + , CD4 + , FoxP3 -

[0270] Regulatory T cells: CD45 + , CD3e + , CD4 + , FoxP3 +

[0271] CD8 T cells: CD45 + , CD3e + , CD8a +

[0272] Effector memory T cells: CD45 + , CD3e + , CD8a + , CD44 hi , CD62L lo

[0273] Central memory T cells: CD45 + , CD3e + , CD8a + , CD44 hi , CD62L hi

[0274] Activated CD8 T cells: CD45 + , CD3e + , CD8a + , Ki-67 + / Granzyme B + / IFNy + / TNFa +

[0275] mRNA synthesis

[0276] Linear dsDNA for Firefly luciferase (FLuc), mouse CD40, and mouse CD40L were obtained from Integrated DNA Technologies. The pUC19 vector was used for Golden Gate assembly to generate the plasmids. mRNAs for FLuc, mouse CD40, and mouse CD40L were synthesized by previously reported methods 20 .

[0277] Preparation and characterization of lipid nanoparticles (LNP)

[0278] mRNA LNPs were prepared by mixing ethanol solution containing ionizable lipids, DOPE, cholesterol and DMG-PEG2000 with 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). Encapsulation efficiency was detected by Ribogreen assay. Morphology was observed on Glacios Cryo-TEM (Thermo Scientific, USA).

[0279] In the preliminary 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 was based on L16 (4) 4 Orthogonal table and predicted formulation preparation. Lipofectamine 3000 / mRNA complex was prepared based on the recommended protocol. Electroporation was performed on BMDCs using Mouse dendritic cell nucleofection kit (Lonza, VAPA1011). mRNA delivery efficacy was determined by luciferase expression assay.

[0280] CD40 and CD40L expression

[0281] About 1 x 10 6 BMDCs were seeded into each well of a 6-well plate and treated with 2.5 pg of CD40-LNP for 12 h. About 1 x 10 5 B16F10 cells were seeded into each well of a 24-well plate and treated with 0.25 pg of CD40L-LNP for 18 h. Next, cells were stained with CD40-FITC or CD40L-FITC antibody and analyzed by LSR Fortessa flow cytometer (Becton Dickinson, USA).

[0282] Mice with tumor volume of about 500 mm 2 were intratumorally injected with 10 pg of FLuc-LNP, CD40-LNP or CD40L-LNP. After 6 h, tumors were dissociated using Mouse Tumor Dissociation Kit (Miltenyi Biotec, 130-096-730). Infiltrating immune cells were isolated by Ficoll-Paque density gradient medium (Cytiva, 17544602). After staining with antibody panel based on the flow cytometry gating method described above, cells were analyzed by flow cytometry.

[0283] Dendritic cell activation and analysis

[0284] CD40-overexpressing BMDCs (CD40-BMDCs) were incubated with 10 pg / mL of anti-mouse CD40 antibody for 12 h. Next, some activation markers were stained by CD80, CD86, MHC-II, and pro-form IL1 -beta antibodies and analyzed by flow cytometry. Other activation markers, including IL-12 and TNF-a, were detected by ELISA.

[0285] LNP-induced immunogenic cell death (ICD)

[0286] ICD markers were detected by adapting previous methods 5 Cytotoxicity of LNPs in B16F10 cells was examined by MTT assay. Extracellular ATP was measured by luminescent ATP detection kit (Abeam, ab113849). Extracellular HMGB1 levels were detected by ELISA. Cell surface calreticulin was detected by flow cytometry.

[0287] Tumor models and treatment regimens

[0288] C57BL / 6 mice (male and female, 6-8 weeks) were purchased from Jackson Laboratory and housed in the University Laboratory Animal Resources - Biomedical Research Tower of The Ohio State University. All mouse studies were approved by the Institutional Animal Care and Use Committee (IACUC) of The Ohio State University and followed local, state, and federal regulations.

[0289] For a unilateral subcutaneous (s.c.) tumor model, about 1 x 10 5 B16F10 cells or 2 x 10 5 B16F10-Luc2 cells were injected subcutaneously into the right flank of mice. On day 7 after tumor inoculation, mice with tumor size of about 0.5 cm in maximum diameter were randomized into different treatment groups. For a bilateral s.c. tumor model, about 1 x 10 5 B16F10 cells were injected subcutaneously into the right flank of mice. On day 5 after tumor inoculation on the right flank, about 1 x 10 5B16F10 cells were injected subcutaneously into the left flank of mice. On day 7 after tumor inoculation on the right flank, mice with tumor sizes of approximately 0.5 cm in maximum diameter were randomized into different treatment groups. For the skin + brain dual tumor model, approximately 2 x 10 5 B16F10-Luc2 cells were injected subcutaneously into the right flank of mice. On day 5 after tumor inoculation on the right flank, approximately 1 x 10 4 B16F10-Luc2 cells were injected intracranially (i.c.) at a depth of 3 mm. The injection site was 2 mm lateral to the midline suture and 1 mm anterior to the coronal suture. On day 7 after tumor inoculation on the right flank, mice with tumor sizes of approximately 0.5 cm in maximum diameter were randomized into different treatment groups. For the subcutaneous tumor re-challenge, on day 45 after tumor inoculation, approximately 1 x 10 5 B16F10 cells were injected subcutaneously into the left flank of completely responsive mice. For the brain tumor re-challenge, on day 45 after tumor inoculation, approximately 1 x 10 4 B16F10-Luc2 cells were injected intracranially into completely responsive mice. For the T cell-depleted tumor model, 1 x 10 5 B16F10 cells were injected subcutaneously into the right flank of mice. On day 6 after tumor inoculation, mice were treated intraperitoneally with anti-mouse CD8a, anti-mouse CD4, or anti-rat IgG2b isotype antibody. Each antibody was given every 3 days for a total of three doses at 200 pg per injection.

[0290] Treatment regimens in this study are described below. In single intratumoral injection, the doses of CD40 Ab and LNP were 50 pg and 5 pg, respectively; and the dose of dendritic cells was 2 million. For CD40-LNP + CD40 Ab treatment, CD40 Ab was administered intratumorally 6 h after intratumoral injection of CD40-LNP. For dendritic cell + CD40 Ab treatment, CD40 Ab was administered intratumorally 1 h after intratumoral injection of dendritic cells. For CD40-LNP + CD40L-LNP treatment, CD40-LNP was administered intratumorally 18 h after intratumoral injection of CD40L-LNP. For CD40L- or FLuc-LNP + CD40-BMDC treatment, CD40-BMDC was administered intratumorally 18 h after intratumoral injection of CD40L- or FLuc-LNP. All these treatment regimens included four doses.

[0291] The removal criteria in this study are as follows. Subcutaneous tumor size was measured every 2 to 4 days and calculated as volume (length × width × width / 2). Mice were euthanized when the maximum diameter of the tumor reached 1.6 cm or the mouse's body weight decreased by more than 20%. Brain tumor size was monitored using IVIS Lumina II (Caliperlife Sciences, USA). Mice were euthanized if they exhibited kyphosis, difficulty walking and eating, or body weight loss greater than 20%.

[0292] Luminex analysis of cytokines and chemokines

[0293] The tumor volume is approximately 500 mm. 2 Mice were intratumorally injected with a single dose of CD40L-LNP+CD40-BMDC. Tumor tissue and serum were collected from mice between 0 and 42 hours post-injection. Tumor tissue was frozen in liquid nitrogen, and the fragmented tissue was extracted at 150 mg / ml in RIPA lysis buffer (Thermo Scientific, 89900) containing a protease inhibitor (Thermo Scientific, 87785). Whole blood was collected in tubes containing sodium citrate, and serum was collected by centrifugation at 4°C (10000 rpm) for 5 minutes. Tumor lysates and serum were stored at -80°C. Mouse cytokines and chemokines were detected using a mouse cytokine / chemokine exploration assay (Eve Technologies, Canada).

[0294] Immune cell population analysis and in vivo activation

[0295] The tumor volume is approximately 250 mm. 2 Mice were treated intratumorally with two doses of CD40L-LNP + CD40-BMDC. Tumor tissue was then collected and dissected using a mouse tumor dissociation kit (Miltenyi Biotec, 130-096-730). To analyze the immune cell population, total cells were stained with an antibody combination based on the flow cytometry-gated method described above, and the number of immune cells was then counted by flow cytometry. In this experiment, CD40-BMDCs were labeled with CellTraceBlue (Invitrogen, C34568) before intratumoral injection, which allowed flow cytometry to distinguish between administered dendritic cells and endogenous dendritic cells. To evaluate immune cell activation, infiltrating immune cells were isolated using Ficoll-Paque density gradient media (Cytiva, 17544602). After staining with an antibody combination based on the flow cytometry-gated method described above, T cell activation markers were detected by flow cytometry.

[0296] References cited

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[0301] 5 Li, Y. et al. Multifunctional oncolytic nanoparticles deliver self-replicating IL-12 RNA to eliminate established tumots and prime systemic immunity. Nature Cancer 1, 882-893 (2020).

[0302] 6 Vonderheide, R. H. CD40 agonist antibodies in cancer immunotherapy. Annual review of medicine 71, 47-58 (2020).

[0303] 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).

[0304] 8 Hou, X., Zaks, T., Langer, R. & Dong, Y. Lipid nanoparticles for mRNA delivery. Nature Reviews Materials 6, 1078-1094 (2021).

[0305] 9 Zhang, Y., Sun, C., Wang, C., Jankovic, K. E. & Dong, Y. Lipids and lipid derivatives for RNA delivery. Chemical Reviews 121, 12181-12277 (2021).

[0306] 10 Hewitt, S. L. et al. Durable anticancer immunity from intratumoral administration of IL-23, IL-36y, and OX40L mRNAs. Science translational medicine 11, eaat9143 (2019).

[0307] 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).

[0308] 12 Zada, B. et al. Recent advances in catalytic production of sugar alcohols and their applications. Science China Chemistry 60, 853-869 (2017).

[0309] 13 Stoss, P. & Hemmer, R. 1,4:3,6-Dianhydrohexitols. Advances in carbohydrate chemistry and biochemistry 49, 93-173 (1991).

[0310] 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).

[0311] 15 Wiggins, L. in Advances in carbohydrate chemistry Vol. 5 191-228 (Elsevier, 1950).

[0312] 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).

[0313] 17 Aricò, F., Tundo, P., Maranzana, A. & Tonachini, G. Synthesis of five-membered cyclic ethers by reaction of 1,4-Diols with dimethylcarbonate. ChemSusChem 5, 1578-1586 (2012).

[0314] 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).

[0315] 19 Li, W. et al. Biomimetic nanoparticles deliver mRNAs encoding costimulatory receptors and enhance T cell mediated cancer immunotherapy. Nature communications 12, 1-12 (2021).

[0316] 20 Zeng, C. et al. Leveraging mRNA Sequences and Nanoparticles to Deliver SARS-CoV-2 Antigens In Vivo. Advanced Materials 32, 2004452 (2020).

[0317] 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).

[0318] Example 2. Chemical synthesis of dianhydrohexitol derivatized lipids

[0319]

[0320] Chemical structure of dianhydrohexitol derivatized ionizable lipids

[0321] Synthesis of aldehyde

[0322]

[0323] To a suspension of paraformaldehyde (1.5 g, 50 mmol) in TMSCl (25 mL, 197 mmol) was added dropwise 1-hexanol 1 (5.1 g, 50 mmol) at room temperature (RT). The reaction mixture was stirred at RT 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.

[0324] The above chloromethyl ether was added dropwise to a solution of 1,6-hexanediol 3 (11.8 g, 100 mmol) and i-Pr2NEt (17.45 mL, 100 mmol) in CH2Cl2(150 mL). The reaction mixture was stirred at RT for 24 h, then quenched by the addition of a saturated NH4Cl solution (80 mL). Extraction with CH2Cl2(60 mL*2 times) was performed and the combined organic layers were washed with water (30 mL) and brine (30 mL) and dried over Na2SO4. The organic phase was filtered and concentrated under reduced pressure and the residue was purified via flash chromatography on silica gel (20% EtOAc in hexanes). 6.74 g of 4 was obtained as a colorless oil in 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] + C 13 H 28 N a Calculated for O3 255.1931; found 255.1941.

[0325]

[0326] (diacyloxyiodo)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 NaHC03(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 complete consumption of 4. The mixture was then quenched with saturated aqueous Na2S203(30 mL) and extracted with DCM (3 x 20 mL). The DCM phases were combined and washed with aqueous NaHC03(20 mL) and brine (20 mL), dried over Na2S04, filtered, and concentrated under reduced pressure. The residue was purified via silica gel flash chromatography (10% EtOAc in hexanes). 510 mg of 5 was obtained as a colorless oil, quant. 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).

[0327]

[0328] 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 over 30 min. After stirring for 3 h, the reaction was quenched with 50 mL of water and extracted with DCM (50 mL*3), then the organic phases were combined and washed with water, dried over Na2S04and concentrated under reduced pressure. The residue was purified via silica gel chromatography (20% EtOAc in hexanes). 3.2 g of 7 was obtained as a colorless oil, in 45.3% yield. 1 H 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] + C 13 H 26 N a Calculated for C26H42N04: 269.1723; found: 269.1735.

[0329]

[0330] 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 NaHC03(736 mg, 8.6 mmol) in 20 mL of DCM. The reaction mixture was stirred for 3 h until TLC showed A was completely consumed. The mixture was then quenched with saturated aqueous Na2S203(30 mL) and extracted with DCM (3 x 20 mL). The DCM phases were combined and washed with aqueous NaHC03(20 mL) and brine (20 mL), dried over Na2S04, filtered, and concentrated under reduced pressure. The residue was purified via silica gel flash chromatography (10% EtOAc in hexanes). 0.78 g of 8 was obtained as a light yellow oil in 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).

[0331]

[0332] To a solution of acetaldehyde (1.76 g, 40 mmol) in TMSC1 (20 mL, 157 mmol) was added dropwise 1-hexanol 1 (5.1 g, 50 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 9 as a colorless oil, which was used directly without further purification.

[0333] The above chloromethyl ether was added dropwise to a solution of 1,6-hexanediol 3 (9.44 g, 80 mmol) and i-Pr2NEt (13.96 mL, 80 mmol) in CH2Cl2(150 mL). The reaction mixture was stirred at room temperature for 24 h, followed by quenching by the addition of saturated NH4CI solution (80 mL). Extraction was performed with CH2Cl2(60 mL*2 times) and the combined organic layers were washed with water (30 mL) and brine (30 mL) and dried over Na2S04. The organic phase was filtered and concentrated under reduced pressure, and the residue was purified via silica gel flash chromatography (20% EtOAc in hexanes). 3.45 g of 10 was obtained as a colorless oil in 35.0% yield. 1H 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] + C 14 H 30 N a Calculated for C25H45NO3: 255.1931; found: 255.1941.

[0334]

[0335] 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 NaHC03(924 mg, 11.0 mmol) in 20 mL of DCM. The reaction mixture was stirred for 3 h until TLC showed A was completely consumed. The mixture was then quenched with saturated aqueous Na2S203(30 mL) and extracted with DCM (3 x 20 mL). The DCM phases were combined and washed with aqueous NaHC03(20 mL) and brine (20 mL), dried over Na2S04, filtered, and concentrated under reduced pressure. The residue was purified via flash chromatography on silica gel (10% EtOAc in hexanes). 1.0 g of 11 was obtained as a light yellow oil in 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] + C 13 H 28 N a Calculated for C25H45NO3: 255.1931; found: 255.1941.

[0336] Synthesis of sugar alcohol derived diamines

[0337]

[0338] 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, cooled at room temperature and to the mixture was added diethyl ether (15 mL). The reaction mixture was filtered through a pad of celite and the solvent was evaporated. Finally, the product was purified by silica gel chromatography using dichloromethane / methanol (9:1) as eluent to obtain 1.2 g of the desired product as a white solid with a yield of 75%.

[0339] 1 H NMR (400 MHz, Deuterium oxide) 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 (75 MHz, Deuterium oxide) δ 87.33, 81.40, 75.46, 75.07, 71.76, 71.09. MS (ESI, m / z): [M+H] + C6H 11 C6H4of 147.1 ; found 147.1.

[0340]

[0341] According to the previously reported method 1 Dicyanoethylation of the diol was performed. To a solution of the diol (7.3 g, 50 mmol) in t-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 period of 6 h. The t-butanol and excess acrylonitrile were removed via rotary evaporation under reduced pressure. The residue was dissolved in 100 mL of DCM and the unreacted isosorbide and monocyanoethylated isosorbide were removed by three water washes. The organic phase was dried over anhydrous Na2S04, the solution was filtered and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (0% - 100% hexanes in dichloromethane) to the corresponding product as a yellow oil.

[0342] Compound 17: yield 70.5%. 1H 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] + C 12 H 17 Calculated for N2O4: 275.1; found: 275.1.

[0343] Compound 18: yield 68%. 1 H NMR (300 MHz, DMSO-d6) δ 4.52 (dt, J = 4.7, 2.4 Hz, 2H), 4.07 (tdd, J = 8.0, 3.6, 1.6 Hz, 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] + C 12 H 17 Calculated for N2O4: 275.1; found: 275.1.

[0344] Compound 19: yield 93%. 1 H NMR (300 MHz, DMSO-d6) δ 4.61 (t, J = 4.8 Hz, 1H), 4.43 (dt, J = 4.8, 0.9 Hz, 1H), 4.07 (td, J = 6.6, 4.8 Hz, 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 (75 MHz, 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] + C 12 H17 Calculated for N2O4: 275.1 ; Found: 275.0.

[0345]

[0346] Diamines were synthesized according to previously reported methods 2 To a solution of BH3-THF complex in THF (2.0 M, 29.7 mL, 59.4 mmol) was added dropwise a solution of the dinitrile compound (3.0 g, 11.89 mmol) in THF (25 mL) at room temperature over 1 h. After the addition was complete, the reaction was stirred for 48 h. Then, methanol (30 mL) was carefully added to quench the reaction, during which time hydrogen gas was vigorously evolved. After stirring for 3 h, the solvent was removed under reduced pressure, then 40 mL of THF was added to dissolve the residue, after which a solution of hydrochloric acid in diethyl ether (2.0 M, 29.7 mL) was added dropwise, after which a white precipitate formed. The suspension was then filtered to give the crude diamine HCI salt as a white powder. The diamine 2 HCI salt was then dissolved in deionized water (30 mL) to give a light yellow solution. To this solution was added freshly washed Amberlyst A 26-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 pad of celite, and the resin was thoroughly washed with water (3 x 4 mL). The combined clear colorless solution was evaporated to dryness using a rotary evaporator (or lyophilized) to give the elongated diamine as a light yellow oily liquid.

[0347] Isosorbide-derived diamine 20: yield 70%. 1 H NMR (300 MHz, Methanol-d4) δ 4.64 (t, J = 4.5 Hz, 1H), 4.54 - 4.45 (m, 1H), 4.11 - 4.00 (m, 1H), 3.98 - 3.84 (m, 4H), 3.80 - 3.69 (m, 1H), 3.66 - 3.43 (m, 4H), 2.86 - 2.61 (m, 4H), 1.83 - 1.67 (m, 4H). HRMS (ESI, m / z): [M+H] + C 12 H 25 Calculated for N2O4: 261.1809; Found: 261.1819.

[0348] Isomannide-derived diamine 21 : yield 68%. HRMS (ESI, m / z): [M+H] + C 12 H 25 Calculated for N2O4: 261.1809; Found: 261.1817.

[0349] L-isosorbide-derived diamine 22: yield 71%. 1 H NMR (400 MHz, Methanol-d4) δ 4.67 (t, J = 4.4 Hz, 1H), 4.56-4.54 (m, 1H), 4.13-4.10 (m, 1H), 3.98-3.92 (m, 2H), 3.92-3.86 (m, 2H), 3.78-3.68 (m, 1H), 3.68-3.50 (m, 4H), 2.94-2.71 (m, 4H), 1.85-1.73 (m, 4H). MS (ESI, m / z): [M+H] + C 12 H 25 Calculated for N2O4 261.2; found 261.2.

[0350]

[0351] Synthesis of β-amino alcohols

[0352] To a solution of diamine (52 mg, 0.2 mmol) in 2 mL of anhydrous 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 complete consumption of A, and the EtOH was removed under reduced pressure. The residue was purified via silica gel chromatography (0-100% of [3% NH4OH, 22% MeOH in dichloromethane] in dichloromethane) to give the desired product.

[0353] 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] + C 60 H 121 Calculated for N2O8 997.9118; found 997.9118.

[0354] DIM-1: yield 69.2%. 1H 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] + C 60 H 121 Calculated for N2O8 997.9; found 998.0.

[0355] 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] + C 60 H 121 Calculated for N2O8 997.9; found 998.0.

[0356]

[0357] 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. Aqueous NaHC03(15 mL) was added to quench the reaction. The aqueous solution was extracted with DCM (15 mL*3 times), the organic phases were combined, dried over anhydrous Na2S04, filtered and concentrated under reduced pressure. The residue was purified via silica gel chromatography (0% - 100% of [3% NH4OH, 22% MeOH in dichloromethane] in dichloromethane) to give the desired product.

[0358] Compound DIS-2: yield 37.0%. 1H 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] + C 44 H 89 Calculated for N2O4: 709.7; found: 709.9.

[0359] 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] + C 52 H 105 Calculated for N2O4: 821.8; found: 821.9.

[0360] 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] + C 60 H 121 Calculated for N2O4: 933.9321; found: 933.9320.

[0361] Compound DIS-5: yield 33.0%. 1H 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] + C 68 H 137 Calculated for N2O4: 1046.1; found: 1046.0.

[0362] Compound DIS-6: yield 19.1%. 1 H 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] + C 76 H 153 Calculated for N2O4: 1158.2; found: 1158.2.

[0363] Compound DIS-7: yield 45.7%. 1 H 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] + C 80 H 153 N2O 12Calculated 1334.1418; found 1334.1404.

[0364] Compound DIS-8: yield 35.0%. 1 H 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] + C 64 H 121 N2O 16 Calculated 1173.9; found 1174.0.

[0365] Compound DIS-9: yield 31.0%. 1 H 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] + C 64 H 129 N2O 12 Calculated 1118.0; found 1118.1.

[0366] Compound DIS-10: yield 54.5%. 1 H 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] + C68 H 137 N2O 12 Calculated for N2O4: 709.7; found: 709.8.

[0367] 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] + C 44 H 89 Calculated for N2O4: 709.7; found: 709.8.

[0368] 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] + C 52 H 105 Calculated for N2O4: 821.8; found: 821.9.

[0369] Compound DIM-4: yield 49.3%. 1H 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] + C 60 H 121 Calculated for N2O4 933.9321; found 933.9321.

[0370] Compound DIM-5: yield 22.0%. 1 H NMR (300 MHz, Chloroform-d) δ 4.52 (d, J = 3.0 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] + C 68 H 137 Calculated for N2O4 1046.1; found 1046.0.

[0371] Compound DIM-6: yield 37.3%. 1 H 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] + C 76 H 153 Calculated for N2O4 1158.2; found 1158.0.

[0372] 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).

[0373] 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] + C 64 H 121 N2O 16 Calculated 1173.9; found 1174.0.

[0374] Compound DIM-9: yield 26.2%. 1 H 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] + C 64 H 129 N2O 12 Calculated 1118.0; found 1118.2.

[0375] Compound DIM-10: yield 29.4%. 1 H 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] + C 68 H 137 N2O 12 Calculated for N2O4 709.7; found 709.8.

[0376] Compound LIS-2: yield 31.0%. 1 H NMR (400 MHz, Chloroform-d) δ 4.60 (t, J = 4.4 Hz, 1H), 4.48 (d, J = 4.4 Hz, 1H), 4.01 - 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] + C 44 H 89 Calculated for N2O4 709.7; found 709.8.

[0377] Compound LIS-3: yield 29.2%. 1 H 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] + C 52 H105 Calculated for N2O4: 821.8; found: 822.0.

[0378] Compound LIS-4: yield 30.3%. 1 H NMR (300 MHz, Chloroform-d) δ 4.60 (t, J = 4.2 Hz, 1H), 4.48 (d, J = 4.2 Hz, 1H), 4.00 - 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] + C 60 H 121 Calculated for N2O4: 933.9; found: 934.2.

[0379] 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] + C 68 H 137 Calculated for N2O4: 1046.1; found: 1046.3.

[0380] 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).

[0381] Compound LIS-7: yield 20.2%. 1H 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+ C 80 H 154 N2O 12 Calculated 667.6; found 668.0.

[0382] 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] + C 64 H 121 N2O 16 Calculated 1173.9; found 1174.0.

[0383] 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] + C 64 H 129 N2O 12 Calculated 1118.0; found 1118.0.

[0384] Compound LIS-10: yield 25.9%. 1 H NMR (400 MHz, Chloroform-d) 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 Hz, 1H), 3.61 - 3.30 (m, 20H), 2.62 - 2.28 (m, 12H), 1.80 - 1.63 (m, 6H), 1.60 - 1.50 (m, 16H), 1.49 - 1.18 (m, 58H), 0.88 (t, J = 6.8 Hz, 12H). MS (ESI, m / z): [M+H] + C 68 H 137 N2O 12 Calculated for 1174.0; found 1174.1.

[0385] References

[0386] 1 Hong, J., Radojcic, D., lonescu, M., Petrovic, Z. S., Eastwood, E. Advanced Materials from Corn: lsosorbide-Based Epoxy Resins. Polym. Chem. 5, 5360-5368 (2014).

[0387] 2 Wroblewska, A., Zych, A., Thiyagarajan, S., Dudenko, D., van Es, D., Hansen, M. R., Koning, C., Duchateau, R., Jasinska-Walc, L. Towards Sugar-Derived Polyamides as Environmentally Friendly Materials. Polym. Chem. 6, 4133-4143 (2015).

[0388] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed application belongs. Publications cited herein and the material for which they are cited are specifically incorporated by reference.

[0389] Those skilled in the art will appreciate that many changes and modifications can be made to the preferred embodiments of the application, and such changes and modifications can be made without departing from the spirit of the application. Therefore, the appended claims are intended to cover all such changes and modifications that fall within the true spirit and scope of the application.

Claims

1. A compound of Formula I, Formula II, or Formula III: Formula I; Formula II; Formula III; or a salt thereof, wherein:

2. The compound of claim 1, wherein the compound is of the formula: Formula I; or a salt thereof, wherein:

3. The compound of claim 1, wherein the compound is of the formula: Formula II; or a salt thereof, wherein: R 1 is independently selected from , , , , , , , , , or .

4. The compound of claim 1, wherein the compound is of the formula: Formula III; or a salt thereof, wherein:

7. The compound of claim 1, wherein the compound is R 1 independently selected from , , , , , , , , , or .

8. The compound of claim 1, wherein the compound is 9. A lipid-based nanoparticle comprising: a compound of any one of claims 1-8, and R 1 independently selected from , , , , , , , , , or . a recombinant polynucleotide comprising a nucleic acid encoding a costimulatory molecule.

10. An antigen presenting cell comprising: a lipid-based nanoparticle comprising: R 1 independently selected from 、 、 、 、 、 、 、 、 、 or .

5. The compound of claim 1, 2, or 4, wherein R 1 is .

6. The compound of any one of claims 1 to 3, wherein R 1 is . a compound of any one of claims 1-8, and ， wherein R 1 is . a recombinant polynucleotide comprising a nucleic acid encoding a costimulatory molecule. ， wherein R 1 is .

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

14. The antigen presenting cell of claim 13, wherein the costimulatory molecule is CD40.

15. The antigen presenting cell of claim 10, wherein the nucleic acid encoding the costimulatory molecule comprises a heterologous 5' untranslated region (5' UTR).

16. The antigen presenting cell of claim 10, wherein the nucleic acid encoding the costimulatory molecule comprises a heterologous 3' untranslated region (3' UTR).

17. The antigen presenting cell of claim 10, wherein the nucleic acid comprises chemically modified nucleobases.

18. The antigen presenting cell of claim 17, wherein the chemically modified nucleobases are pseudouridines. ， wherein R 1 is .

19. The antigen presenting cell of claim 10, wherein the antigen presenting cell is a bone marrow-derived dendritic cell. ， wherein R 1 is .

20. Use of an antigen presenting cell and an antibody of any one of claims 10-19 in the manufacture of a preparation for treating cancer. ​ ​ ​ ​ ​ ​ ​ 21. The use of claim 20, wherein the antibody is selected from an anti-CD40 antibody, an anti-PDLl antibody, an anti-PDl antibody, an anti-CTLA4 antibody, or a combination thereof.

22. The use of claim 20 or 21, wherein the antigen presenting cell and the antibody are administered intratumorally.

23. The use of claim 20, wherein the antigen presenting cell and the antibody are administered simultaneously.

24. Use of the lipid-based nanoparticle of claim 9 and the antigen presenting cell of any one of claims 10 to 19 in the manufacture of a medicament for the treatment of cancer.

25. The use of claim 24, wherein the lipid-based nanoparticle comprises , wherein R 1 is .

26. The use of claim 24, wherein the lipid-based nanoparticle comprises , wherein R 1 is .

27. The use of any one of claims 24 to 26, wherein the lipid-based nanoparticle comprises a recombinant polynucleotide comprising a nucleic acid encoding CD40L.

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