mRNA encoding a checkpoint cancer vaccine and uses thereof

Abstract

The present disclosure features lipid nanoparticle (LNP) compositions comprising checkpoint cancer vaccines and uses thereof. The LNP compositions of the present disclosure comprise therapeutic agents of mRNAs encoding checkpoint cancer vaccines comprising IDO antigenic peptides and PD-L1 antigenic peptides. The LNP compositions of the present disclosure can induce immune responses and stimulate T effector cells in vivo, and are therefore useful in the treatment of cancer. The mRNAs encoding checkpoint cancer vaccines and uses thereof are provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 311,716, filed February 18, 2022, and U.S. Provisional Application No. 63 / 381,460, filed October 28, 2022. The contents of the foregoing applications are incorporated herein by reference in their entireties. [Background technology]

[0002] Melanoma is the fifth most common cancer diagnosis in the United States. It accounts for 5.3% of all new cancer diagnoses and 1.5% of all cancer-related deaths. Cutaneous melanoma begins in the melanocytes (pigment-producing cells) of the skin. If diagnosed at a localized stage, the 5-year survival rate is approximately 95%. However, for regional or metastatic disease (stage IIIB+), the 5-year survival rate drops to approximately 30-60%. Approximately 18,000 new patients are diagnosed with stage IIIB+ cutaneous melanoma in the United States. Advanced melanoma (a rare and serious type of skin cancer) accounts for the majority of skin cancer-related deaths, despite representing only 1% of skin cancer cases. The current standard of care is pembrolizumab, nivolumab, or the combination of nivolumab and ipilimumab.

[0003] NSCLC remains asymptomatic and often goes undetected until it progresses to a later stage. Approximately 115,000 people in the United States are diagnosed with or progress to metastatic NSCLC each year. The current approach to treating metastatic NSCLC depends on the presence of PD-L1 expression. If tumor PD-L1 expression is greater than 50%, pembrolizumab or atezolizumab monotherapy is preferred, while for patients with PD-L1 expression less than 50%, the combination of chemotherapy and pembrolizumab is preferred.

[0004] Although there are multiple treatments for cancer (such as melanoma and NSCLC), there is an unmet need to develop new modalities and therapies that can improve treatment outcomes and extend survival for cancer patients. Summary of the Invention

[0005] The present disclosure provides, inter alia, polynucleotide constructs encoding checkpoint cancer vaccines (e.g., comprising one or more IDO antigenic peptides and one or more PD-L1 antigenic peptides), lipid nanoparticle (LNP) compositions comprising such polynucleotides, and uses thereof. The LNP compositions of the present disclosure comprise one or more mRNA molecules encoding (i) one or more IDO antigenic peptides; and (ii) one or more PD-L1 antigenic peptides, and optionally, an adjuvant amino acid sequence. In one embodiment, the LNP compositions of the present disclosure can stimulate an immune response (e.g., stimulate effector T cells to target and kill IDO- or PD-L1-expressing suppressor immune cells and tumor cells), prime T cells to induce tumor-associated antigen recognition, induce helper T cells, enhance T cell influx into tumor sites, and induce cytotoxic T cell-mediated tumor cell killing. Also disclosed herein are methods of using LNP compositions comprising checkpoint cancer vaccines for the treatment of cancer or for stimulating an immune response in a subject.

[0006] Additional aspects of the disclosure are described in further detail below.

[0007] In one aspect, provided herein is a polynucleotide (e.g., mRNA) encoding a checkpoint cancer vaccine comprising (i) one or more indoleamine-pyrrole 2,3-dioxygenase (IDO) antigenic peptides and (ii) one or more programmed death-ligand 1 (PD-L1) antigenic peptides. Optionally, the polynucleotide may also include a sequence encoding an amino acid sequence for an adjuvant. The present invention also relates to lipid nanoparticle (LNP) compositions comprising such polynucleotides.

[0008] In another aspect, the present disclosure provides a lipid nanoparticle (LNP) composition for immunomodulation, e.g., stimulating an immune response by IDO / PDL1-specific T cells or breaking immune tolerance (e.g., stimulating T effector cells by increasing their activation and / or attracting them to tumor cells that may express IDO and PDL1), the composition comprising (i) mRNA for one or more indoleamine-pyrrole 2,3-dioxygenase (IDO) antigenic peptides and (ii) mRNA for one or more programmed death-ligand 1 (PD-L1) antigenic peptides. Optionally, the mRNA may also encode an amino acid sequence for an adjuvant. In one embodiment, the LNP composition enhances tumor cell infiltration and killing of IDO and / or PDL1-expressing tumor cells by CD4+ and / or CD8+ T cells.

[0009] In one aspect, provided herein is a lipid nanoparticle (LNP) composition for stimulating T effector cells in a subject with melanoma or NSCLC, the composition comprising mRNA encoding a checkpoint cancer vaccine comprising (i) one or more indoleamine-pyrrole 2,3-dioxygenase (IDO) antigenic peptides and (ii) one or more programmed death-ligand 1 (PD-L1) antigenic peptides. Optionally, the mRNA may also encode an amino acid sequence for an adjuvant.

[0010] In one embodiment, administration of the LNP compositions disclosed herein results in amelioration or delay of cancer progression in the subject, e.g., as measured by the assays described herein, e.g., as described herein. In one embodiment, a checkpoint inhibitor (e.g., an anti-PD1 antibody, an anti-CTLA4 antibody, or a combination thereof) may also be administered to the subject.

[0011] In one embodiment of any of the LNP compositions disclosed herein, the LNP compositions include an mRNA encoding a checkpoint cancer vaccine, wherein the mRNA comprises at least one chemical modification.

[0012] In one embodiment of any of the LNP compositions disclosed herein, the LNP composition comprises: (i) an ionizable lipid (e.g., an ionizable amino lipid); (ii) a sterol or other structural lipid; (iii) a non-cationic helper lipid or phospholipid; and (iv) a PEG-lipid.

[0013] In one aspect, provided herein is a pharmaceutical composition comprising the LNP compositions disclosed herein.

[0014] In one aspect, provided herein is a method of modulating (e.g., inducing or enhancing) an immune response in a subject, the method comprising administering to a subject in need thereof an effective amount of an LNP composition comprising mRNA encoding a checkpoint cancer vaccine comprising (i) one or more IDO antigenic peptides and (ii) one or more PD-L1 antigenic peptides. Optionally, the mRNA may also encode an amino acid sequence for an adjuvant.

[0015] In another aspect, the present disclosure provides a method of stimulating T effector cells in a subject, the method comprising administering to the subject an effective amount of an LNP composition comprising mRNA encoding a checkpoint cancer vaccine comprising (i) one or more IDO antigenic peptides and (ii) one or more PD-L1 antigenic peptides. Optionally, the mRNA may also encode an amino acid sequence for an adjuvant.

[0016] In yet another aspect, provided herein are methods for treating or preventing the spread of cancer or metastatic lesions thereof (e.g., cutaneous melanoma (e.g., 1L cutaneous melanoma stage IIIB+) or NSCLC (e.g., 1L NSCLC)), or symptoms thereof, comprising administering to a subject in need thereof an effective amount of an LNP composition comprising mRNA encoding a checkpoint cancer vaccine comprising (i) one or more IDO peptides and (ii) one or more PD-L1 peptides. Optionally, the mRNA may also encode an amino acid sequence for an adjuvant.

[0017] In embodiments of any of the methods disclosed herein, the checkpoint cancer vaccine comprises alternating antigenic peptides (e.g., multimers) of IDO and PD-L1.

[0018] In embodiments of any of the methods disclosed herein, the LNP composition comprising mRNA encoding a checkpoint cancer vaccine comprising (i) one or more IDO antigenic peptides and (ii) one or more PD-L1 antigenic peptides is administered in combination with an additional agent, e.g., an agent that further stimulates the immune response, e.g., a checkpoint inhibitor (such as an anti-PD1 antibody or an anti-CTLA4 antibody).

[0019] In one embodiment, the additional agent is administered in the form of a therapeutic protein. In another embodiment, the additional agent is administered in the form of a polynucleotide encapsulated in the LNP. In one embodiment, the LNP composition and the additional agent are in the same composition or in separate compositions.

[0020] In one embodiment, the LNP composition and the additional agent are administered substantially simultaneously or sequentially. In one embodiment, for sequential administration, the LNP composition is administered before the additional agent is administered. In one embodiment, the order of administration is reversed. In embodiments of any of the methods disclosed herein, the cancer is a solid tumor (e.g., a locally advanced or metastatic solid tumor). In some embodiments, the cancer is selected from cutaneous melanoma (e.g., 1L cutaneous melanoma stage IIIB+), NSCLC (e.g., 1L NSCLC), bladder cancer (e.g., non-muscle-invasive bladder cancer), head and neck cancer (e.g., head and neck squamous cell carcinoma), colorectal cancer (e.g., microsatellite-stable colorectal cancer), basal cell carcinoma, or breast cancer (e.g., triple-negative breast cancer).

[0021] In one embodiment, the cancer is cutaneous melanoma. In one embodiment, the cutaneous melanoma is 1L cutaneous melanoma stage IIIB+. In one embodiment, the melanoma is refractory melanoma. In one embodiment, the cancer is NSCLC. In one embodiment, the NSCLC is 1L NSCLC. In one embodiment, the NSCLC is locally advanced or metastatic NSCLC and / or checkpoint inhibitor refractory NSCLC.

[0022] In some embodiments of any of the methods disclosed herein, the LNP composition comprises (i) an ionizable lipid (e.g., an amino lipid); (ii) a sterol or other structural lipid; (iii) a non-cationic helper lipid or phospholipid; and (iv) a PEG-lipid. In some embodiments, the ionizable lipid comprises Compound 25. In some embodiments of any of the methods disclosed herein, the LNP composition comprises an ionizable lipid comprising Compound 25 and a PEG-lipid comprising PEG DMG.

[0023] In yet another embodiment, disclosed herein is a kit comprising a container containing an LNP composition disclosed herein or a pharmaceutical LNP composition disclosed herein.

[0024] In some embodiments, the kit includes a package insert containing instructions for administration of the LNP composition or pharmaceutical LNP composition for the treatment of cancer.

[0025] In some embodiments, the LNP composition comprises a pharmaceutically acceptable carrier.

[0026] Additional features of any of the LNP compositions, pharmaceutical compositions comprising said LNPs, methods, or compositions for use disclosed herein include the following aspects or embodiments.

[0027] In one aspect, the present disclosure provides an LNP composition comprising a polynucleotide encoding, e.g., a checkpoint cancer vaccine, e.g., comprising, e.g., one or more IDO antigenic peptides and one or more PD-L1 antigenic peptides, e.g., as described herein. Optionally, the polynucleotide can also include a sequence encoding an amino acid sequence for an adjuvant.

[0028] In one aspect, the LNP compositions disclosed herein comprise a polynucleotide encoding a checkpoint cancer vaccine comprising one or more IDO antigenic peptides. In one embodiment, the IDO antigenic peptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an IDO antigenic peptide amino acid sequence provided in Table 1A (e.g., SEQ ID NO: 1) or an antigenic fragment thereof. In one embodiment, the IDO antigenic peptide comprises the amino acid sequence of an IDO amino acid sequence provided in Table 1A (e.g., SEQ ID NO: 1) or an antigenic fragment thereof. In one embodiment, the IDO antigenic peptide comprises the amino acid sequence of SEQ ID NO: 1 or an antigenic fragment thereof.

[0029] In one embodiment, the polynucleotide encoding the IDO antigenic peptide comprises a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 2 or an antigenic fragment thereof. In one embodiment, the polynucleotide (e.g., mRNA) encoding the IDO antigenic peptide comprises the nucleotide sequence of SEQ ID NO: 2 or an antigenic fragment thereof. In one embodiment, the polynucleotide encoding the IDO antigenic peptide comprises a codon-optimized nucleotide sequence.

[0030] In one aspect, the LNP compositions disclosed herein comprise a polynucleotide encoding a checkpoint cancer vaccine comprising one or more PD-L1 antigenic peptides. In one embodiment, the PD-L1 antigenic peptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a PD-L1 antigenic peptide amino acid sequence provided in Table 1A (e.g., SEQ ID NO: 3), or an antigenic fragment thereof. In one embodiment, the PD-L1 molecule comprises the amino acid sequence of a PD-L1 amino acid sequence provided in Table 1A (e.g., SEQ ID NO: 3), or an antigenic fragment thereof. In one embodiment, the PD-L1 molecule comprises the amino acid sequence of SEQ ID NO: 3, or an antigenic fragment thereof.

[0031] In one embodiment, the polynucleotide encoding the PD-L1 antigenic peptide comprises a nucleotide sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 4, or an antigenic fragment thereof. In one embodiment, the polynucleotide (e.g., mRNA) encoding the PD-L1 antigenic peptide comprises the nucleotide sequence of SEQ ID NO: 4, or an antigenic fragment thereof. In one embodiment, the polynucleotide encoding the PD-L1 antigenic peptide comprises a codon-optimized nucleotide sequence.

[0032] In one aspect, the LNP compositions disclosed herein comprise a polynucleotide encoding a checkpoint cancer vaccine, e.g., comprising one or more (e.g., 1, 2, 3, 4, or more) IDO antigenic peptides and one or more (e.g., 1, 2, 3, 4, or more) PD-L1 antigenic peptides. In some embodiments, the checkpoint cancer vaccine comprises alternating IDO antigenic peptides and PD-L1 antigenic peptides. In one embodiment, the checkpoint cancer vaccine comprises one IDO antigenic peptide and one PD-L1 antigenic peptide. In one embodiment, the checkpoint cancer vaccine comprises two IDO antigenic peptides and two PD-L1 antigenic peptides. In one embodiment, the checkpoint cancer vaccine comprises three IDO antigenic peptides and three PD-L1 antigenic peptides. In one embodiment, the checkpoint cancer vaccine comprises four IDO antigenic peptides and four PD-L1 antigenic peptides. In some embodiments, the four IDO antigenic peptides and four PD-L1 antigenic peptides are arranged in an alternating manner. Thus, in one embodiment, the checkpoint cancer vaccine comprises (i) an IDO antigenic peptide, (ii) a PD-L1 antigenic peptide, (iii) an IDO antigenic peptide, (iv) a PD-L1 antigenic peptide, (v) an IDO antigenic peptide, (vi) a PD-L1 antigenic peptide, (vii) an IDO antigenic peptide, and (viii) a PD-L1 antigenic peptide.

[0033] In one embodiment, the alternating IDO antigenic peptide and PD-L1 antigenic peptide comprise an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an IDO amino acid sequence provided in Table 1A (e.g., SEQ ID NO: 5), or an antigenic fragment thereof. In one embodiment, the alternating IDO antigenic peptide and PD-L1 antigenic peptide comprise the amino acid sequence of an amino acid sequence provided in Table 1A (e.g., SEQ ID NO: 5), or an antigenic fragment thereof. In one embodiment, the alternating IDO antigenic peptide and PD-L1 antigenic peptide comprise the amino acid sequence of SEQ ID NO: 5, or an antigenic fragment thereof.

[0034] In one embodiment, the polynucleotide encoding the alternating IDO and PD-L1 antigenic peptides comprises a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 6, 300, 301, or 302, or an antigenic fragment thereof. In one embodiment, the polynucleotide (e.g., mRNA) encoding the alternating IDO and PD-L1 antigenic peptides comprises the nucleotide sequence of SEQ ID NO: 6, 300, 301, or 302, or an antigenic fragment thereof. In one embodiment, the polynucleotide encoding the alternating IDO and PD-L1 antigenic peptides comprises a codon-optimized nucleotide sequence.

[0035] In embodiments of any of the LNP compositions, methods, or compositions for use disclosed herein, the polynucleotide comprises at least one chemical modification. In one embodiment, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine. In one embodiment, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof. In one embodiment, the chemical modification is N1-methylpseudouridine. In one embodiment, each mRNA in the lipid nanoparticle comprises a fully modified N1-methylpseudouridine.

[0036] In embodiments of any of the LNP compositions, methods, or compositions for use disclosed herein, the LNP composition comprises: (i) an ionizable lipid (e.g., an amino lipid); (ii) a sterol or other structural lipid; (iii) a non-cationic helper lipid or phospholipid; and (iv) a PEG-lipid.

[0037] In any of the embodiments of the LNP compositions, methods, or compositions for use disclosed herein, the LNP composition comprises an ionizable lipid, including an amino lipid. In one embodiment, the ionizable lipid comprises any compound of Formula (I), (Ia), (Ib), (Ic), (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), or (III). In one embodiment, the ionizable lipid comprises a compound of Formula (I). In one embodiment, the ionizable lipid comprises Compound 18. In one embodiment, the ionizable lipid comprises Compound 25.

[0038] In some embodiments, the lipid nanoparticles comprise an ionizable amino lipid compound of formula (I): [ka] or its N-oxide, or its salt or isomer [In the formula, R' a is R' 分岐鎖 and; R' 分岐鎖 teeth, [ka] and; [ka] represents the attachment point; R aα , R aβ , R aγ , and R aδ are H and C, respectively. 2-12 Alkyl, and C 2-12 alkenyl; R 2 and R 3 are respectively, C 1-14 Alkyl and C 2-14 alkenyl; R 4 is -(CH2) nOH, where n is selected from the group consisting of 1, 2, 3, 4, and 5; and [ka] is selected from the group consisting of [ka] represents the attachment point; R 10 is N(R)2; each R is C 1-6 Alkyl, C 2-3 alkenyl, and H; n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R 5 is C 1-3 Alkyl, C 2-3 independently selected from the group consisting of alkenyl and H; Each R 6 is C 1-3 Alkyl, C 2-3 independently selected from the group consisting of alkenyl and H; M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-; R' is C 1-12 Alkyl or C 2-12 is alkenyl; l is selected from the group consisting of 1, 2, 3, 4, and 5; m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13. Includes.

[0039] In some embodiments, the ionizable amino lipid compound of formula (I) is [ka] is selected from.

[0040] In some embodiments, the lipid nanoparticle further comprises a phospholipid, a structured lipid, and a PEG-lipid.

[0041] In some embodiments, the PEG-lipid is PEG DMG.

[0042] In some embodiments, the lipid nanoparticles comprise: (i) 40-50 mol % of a compound of formula (I), 30-45 mol % of a structural lipid, 5-15 mol % of a phospholipid, and 1-5 mol % of a PEG-lipid; or (ii) 45 to 50 mol % of the compound of formula (I), 35 to 45 mol % of a structural lipid, 8 to 12 mol % of a phospholipid, and 1.5 to 3.5 mol % of a PEG-lipid. Includes.

[0043] In some embodiments, the lipid nanoparticles comprise compound 25, DSPC, cholesterol, and PEG DMG.

[0044] In embodiments of any of the LNP compositions, methods, or compositions for use disclosed herein, the LNP comprises about 20 mol% to about 60 mol% ionizable lipid, about 5 mol% to about 25 mol% non-cationic helper lipid or phospholipid, about 25 mol% to about 55 mol% sterol or other structured lipid, and about 0.5 mol% to about 15 mol% PEG lipid. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 35 mol% to about 55 mol% ionizable lipid, about 5 mol% to about 25 mol% non-cationic helper lipid or phospholipid, about 30 mol% to about 40 mol% sterol or other structured lipid, and about 0 mol% to about 10 mol% PEG lipid. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 50 mol% ionizable lipid, about 10 mol% non-cationic helper lipid or phospholipid, about 38.5 mol% sterol or other structured lipid, and about 1.5 mol% PEG lipid. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 49.83 mol% ionizable lipid, about 9.83 mol% non-cationic helper lipid or phospholipid, about 30.33 mol% sterol or other structured lipid, and about 2.0 mol% PEG lipid. In any embodiment of the LNP composition, method, or composition for use disclosed herein, the LNP comprises about 45 mol% to about 50 mol% ionizable lipid. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 45.5 mol% to about 49.5 mol% ionizable lipid. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 46 mol% to about 49 mol% ionizable lipids. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 46.5 mol% to about 48.5 mol% ionizable lipids. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 47 mol% to about 48 mol% ionizable lipids.

[0045] In embodiments of any of the LNP compositions, methods, or compositions for use disclosed herein, the LNP comprises about 45 mol% to about 49.5 mol% ionizable lipids. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 45 mol% to about 49 mol% ionizable lipids. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 45 mol% to about 48.5 mol% ionizable lipids. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 45 mol% to about 48 mol% ionizable lipids. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 45 mol% to about 47.5 mol% ionizable lipids. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 45 mol% to about 47 mol% ionizable lipids. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 45 mol% to about 46.5 mol% ionizable lipids. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 45 mol% to about 46 mol% ionizable lipid. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 45 mol% to about 45.5 mol% ionizable lipid.

[0046] In embodiments of any of the LNP compositions, methods, or compositions for use disclosed herein, the LNP comprises about 45.5 mol% to about 50 mol% ionizable lipids. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 46 mol% to about 50 mol% ionizable lipids. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 46.5 mol% to about 50 mol% ionizable lipids. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 47 mol% to about 50 mol% ionizable lipids. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 47.5 mol% to about 50 mol% ionizable lipids. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 48 mol% to about 50 mol% ionizable lipids. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 48.5 mol% to about 50 mol% ionizable lipids. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 49 mol% to about 50 mol% ionizable lipids. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 49.5 mol% to about 50 mol% ionizable lipids.

[0047] In embodiments of any of the LNP compositions, methods, or compositions for use disclosed herein, the LNP comprises about 45 mol% to about 46 mol% ionizable lipids. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 45.5 mol% to about 46.5 mol% ionizable lipids. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 46 mol% to about 47 mol% ionizable lipids. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 46.5 mol% to about 47.5 mol% ionizable lipids. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 47 mol% to about 48 mol% ionizable lipids. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 47.5 mol% to about 48.5 mol% ionizable lipids. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 48 mol% to about 49 mol% ionizable lipids. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 48.5 mol% to about 49.5 mol% ionizable lipids. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 49 mol% to about 50 mol% ionizable lipids.

[0048] In embodiments of any of the LNP compositions, methods, or compositions for use disclosed herein, the LNPs comprise about 45 mol% ionizable lipids. In one embodiment of the LNPs or methods of the present disclosure, the LNPs comprise about 45.5 mol% ionizable lipids. In one embodiment of the LNPs or methods of the present disclosure, the LNPs comprise about 46 mol% ionizable lipids. In one embodiment of the LNPs or methods of the present disclosure, the LNPs comprise about 46.5 mol% ionizable lipids. In one embodiment of the LNPs or methods of the present disclosure, the LNPs comprise about 47 mol% ionizable lipids. In one embodiment of the LNPs or methods of the present disclosure, the LNPs comprise about 47.5 mol% ionizable lipids. In one embodiment of the LNPs or methods of the present disclosure, the LNPs comprise about 48 mol% ionizable lipids. In one embodiment of the LNPs or methods of the present disclosure, the LNPs comprise about 48.5 mol% ionizable lipids. In one embodiment of the LNPs or methods of the present disclosure, the LNPs comprise about 49 mol% ionizable lipids. In one embodiment of the LNPs or methods of the present disclosure, the LNPs comprise about 49.5 mol% ionizable lipids. In one embodiment of the LNPs or methods of the present disclosure, the LNPs comprise about 50 mol% ionizable lipids.

[0049] In embodiments of any of the LNP compositions, methods, or compositions for use disclosed herein, the LNPs comprise about 1 mol% to about 5 mol% PEG lipid. In one embodiment of the LNP or method of the present disclosure, the LNPs comprise about 1.5 mol% to about 4.5 mol% PEG lipid. In one embodiment of the LNP or method of the present disclosure, the LNPs comprise about 2 mol% to about 4 mol% PEG lipid. In one embodiment of the LNP or method of the present disclosure, the LNPs comprise about 2.5 mol% to about 3.5 mol% PEG lipid.

[0050] In embodiments of any of the LNP compositions, methods, or compositions for use disclosed herein, the LNP comprises about 1 mol% to about 4.5 mol% PEG lipid. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 1 mol% to about 4 mol% PEG lipid. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 1 mol% to about 3.5 mol% PEG lipid. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 1 mol% to about 3 mol% PEG lipid. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 1 mol% to about 2.5 mol% PEG lipid. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 1 mol% to about 2 mol% PEG lipid. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 1 mol% to about 1.5 mol% PEG lipid.

[0051] In embodiments of any of the LNP compositions, methods, or compositions for use disclosed herein, the LNP comprises about 1.5 mol% to about 5 mol% PEG lipid. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 2 mol% to about 5 mol% PEG lipid. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 2.5 mol% to about 5 mol% PEG lipid. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 3 mol% to about 5 mol% PEG lipid. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 3.5 mol% to about 5 mol% PEG lipid. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 4 mol% to about 5 mol% PEG lipid. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 4.5 mol% to about 5 mol% PEG lipid.

[0052] In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 1 mol% to about 2 mol% PEG lipid. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 1.5 mol% to about 2.5 mol% PEG lipid. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 2 mol% to about 3 mol% PEG lipid. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 3.5 mol% to about 4.5 mol% PEG lipid. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 4 mol% to about 5 mol% PEG lipid.

[0053] In any embodiment of the LNP compositions, methods, or compositions for use disclosed herein, the LNPs comprise about 1 mol% PEG lipid. In one embodiment of the LNPs or methods of the present disclosure, the LNPs comprise about 1.5 mol% PEG lipid. In one embodiment of the LNPs or methods of the present disclosure, the LNPs comprise about 2 mol% PEG lipid. In one embodiment of the LNPs or methods of the present disclosure, the LNPs comprise about 2.5 mol% PEG lipid. In one embodiment of the LNPs or methods of the present disclosure, the LNPs comprise about 3 mol% PEG lipid. In one embodiment of the LNPs or methods of the present disclosure, the LNPs comprise about 3.5 mol% PEG lipid. In one embodiment of the LNPs or methods of the present disclosure, the LNPs comprise about 4 mol% PEG lipid. In one embodiment of the LNPs or methods of the present disclosure, the LNPs comprise about 4.5 mol% PEG lipid. In one embodiment of the LNPs or methods of the present disclosure, the LNPs comprise about 5 mol% PEG lipid.

[0054] In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 50 mol% Compound 25 and about 10 mol% non-cationic helper lipid or phospholipid. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 50 mol% Compound 25 and about 10 mol% non-cationic helper lipid or phospholipid. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 50 mol% Compound 25 and 10 mol% non-cationic helper lipid or phospholipid. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 50 mol% Compound 25 and 10 mol% non-cationic helper lipid or phospholipid. In one embodiment of the LNP or method of the present disclosure, the LNP comprises about 49.83 mol% Compound 25, about 9.83 mol% non-cationic helper lipid or phospholipid, about 30.33 mol% sterol or other structured lipid, and about 2.0 mol% PEG lipid.

[0055] In one embodiment of the LNPs or methods of the disclosure, the LNPs comprise about 48 mol% Compound 25, about 11 mol% non-cationic helper lipid or phospholipid, about 38.5 mol% sterol or other structured lipid, and about 2.5 mol% PEG-lipid. In one embodiment of the LNPs or methods of the disclosure, the LNPs comprise about 48 mol% Compound 25, about 11 mol% DSPC, about 38.5 mol% cholesterol, and about 2.5 mol% PEG-DMG.

[0056] In any of the embodiments of the LNP compositions, methods, or compositions for use disclosed herein, the LNPs are formulated for intravenous, subcutaneous, or intramuscular delivery. In one embodiment, the LNPs are formulated for intramuscular delivery.

[0057] In one embodiment, the LNP compositions disclosed herein are administered to a subject with cancer. In some embodiments, the cancer is a solid tumor, e.g., a locally advanced or metastatic solid tumor.

[0058] In one embodiment of any of the compositions for the methods or uses disclosed herein, the cancer is melanoma. In some embodiments, the melanoma is cutaneous melanoma. In one embodiment, the cutaneous melanoma is 1L cutaneous melanoma stage IIIB+. In one embodiment of any of the compositions for the methods or uses disclosed herein, the cancer is NSCLC. In one embodiment, the NSCLC is 1L NSCLC. In one embodiment of any of the compositions for the methods or uses disclosed herein, the cancer is bladder cancer. In some embodiments, the bladder cancer is non-muscle-invasive bladder cancer. In one embodiment of any of the compositions for the methods or uses disclosed herein, the cancer is head and neck cancer. In some embodiments, the head and neck cancer is head and neck squamous cell carcinoma. In one embodiment of any of the compositions for the methods or uses disclosed herein, the cancer is colorectal cancer. In some embodiments, the colorectal cancer is microsatellite-stable colorectal cancer. In one embodiment of any of the compositions for the methods or uses disclosed herein, the cancer is basal cell carcinoma. In one embodiment of any of the compositions for the methods or uses disclosed herein, the cancer is breast cancer. In some embodiments, the breast cancer is triple-negative breast cancer.

[0059] In one embodiment of any of the compositions for the methods or uses disclosed herein, the LNP composition is administered to a subject according to a dosing interval. In some embodiments, the dosing interval comprises a three-week cycle. In some embodiments, the LNP composition is administered to a subject once every three weeks in one or more cycles. In some embodiments, the dosing regimen comprises two, three, four, five, six, seven, eight, or nine cycles.

[0060] In some embodiments, the LNP composition contains about 50 μg to about 1 mg, e.g., about 100 μg to about 1 mg, about 200 μg to about 900 μg, about 300 μg to about 800 μg, about 400 μg to about 700 μg, about 500 μg to about 600 μg, about 200 μg to about 1 mg, about 300 μg to about 1 mg, about 400 μg to about 1 mg, about 500 μg to about 1 mg, or about 600 μg to about 1 mg. g, about 700 μg to about 1 mg, about 800 μg to about 1 mg, about 900 μg to about 1 mg, about 100 μg to about 900 μg, about 100 μg to about 800 μg, about 100 μg to about 700 μg, about 100 μg to about 600 μg, about 100 μg to about 500 μg, about 100 μg to about 400 μg, about 100 μg to about 300 μg, or about 100 μg to about 200 μg. In some embodiments, the LNP composition is administered at a dose of about 100 μg to about 200 μg, about 200 μg to about 300 μg, about 300 μg to about 400 μg, about 400 μg to about 500 μg, about 500 μg to about 600 μg, about 600 μg to about 700 μg, about 700 μg to about 800 μg, about 800 μg to about 900 μg, or about 900 μg to about 1 mg.

[0061] In some embodiments, the LNP composition is administered at a dose of about 50 μg to about 75 μg (e.g., about 50 μg). In some embodiments, the LNP composition is administered at a dose of about 50 μg to about 150 μg (e.g., about 100 μg). In some embodiments, the LNP composition is administered at a dose of about 150 μg to about 250 μg (e.g., about 200 μg). In some embodiments, the LNP composition is administered at a dose of about 250 μg to about 350 μg (e.g., about 300 μg). In some embodiments, the LNP composition is administered at a dose of about 350 μg to about 450 μg (e.g., about 400 μg). In some embodiments, the LNP composition is administered at a dose of about 450 μg to about 550 μg (e.g., about 500 μg). In some embodiments, the LNP composition is administered at a dose of about 550 μg to about 650 μg (e.g., about 600 μg). In some embodiments, the LNP composition is administered at a dose of about 650 μg to about 750 μg (e.g., about 700 μg). In some embodiments, the LNP composition is administered at a dose of about 750 μg to about 850 μg (e.g., about 800 μg). In some embodiments, the LNP composition is administered at a dose of about 850 μg to about 950 μg (e.g., about 900 μg). In some embodiments, the LNP composition is administered at a dose of about 950 μg to about 1 mg (e.g., about 1 mg).

[0062] In some embodiments, the LNP composition is administered intramuscularly (IM).

[0063] In one embodiment of any of the methods or compositions for use disclosed herein, the subject is a mammal (eg, a human).

[0064] Additional features of any of the foregoing LNP compositions or methods of using said LNP compositions include one or more of the embodiments listed below. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the embodiments listed below.

[0065] Other embodiments of the present disclosure E1. A lipid nanoparticle (LNP) composition comprising a polynucleotide comprising mRNA encoding a checkpoint cancer vaccine comprising (i) one or more (e.g., 1, 2, 3, 4, or more) indoleamine-pyrrole 2,3-dioxygenase (IDO) antigenic peptides and (ii) one or more (e.g., 1, 2, 3, 4, or more) programmed death-ligand 1 (PD-L1) antigenic peptides.

[0066] A lipid nanoparticle composition for stimulating E2.T effector cells, comprising a polynucleotide comprising mRNA encoding a checkpoint cancer vaccine comprising (i) one or more (e.g., 1, 2, 3, 4, or more) IDO antigenic peptides and (ii) one or more (e.g., 1, 2, 3, 4, or more) PD-L1 antigenic peptides.

[0067] E3. A lipid nanoparticle composition for stimulating T effector cells, the composition comprising a polynucleotide comprising mRNA encoding a checkpoint cancer vaccine comprising (i) one or more (e.g., 1, 2, 3, 4, or more) IDO antigenic peptides and (ii) one or more (e.g., 1, 2, 3, 4, or more) PD-L1 antigenic peptides.

[0068] E4. An mRNA construct comprising a polynucleotide encoding a checkpoint cancer vaccine comprising: (i) one or more (e.g., 1, 2, 3, 4, or more) IDO antigenic peptides and (ii) one or more (e.g., 1, 2, 3, 4, or more) PD-L1 antigenic peptides.

[0069] E5. The LNP composition or mRNA construct of any one of embodiments E1-E4, wherein the IDO antigenic peptide comprises a fragment of a naturally occurring IDO molecule or a variant thereof.

[0070] E6. The LNP composition or mRNA construct of any one of embodiments E1-E5, wherein the IDO antigenic peptide is derived from IDO1 or IDO2.

[0071] E7. The LNP composition or mRNA construct of embodiment E6, wherein the IDO antigenic peptide is derived from IDO1.

[0072] E8. The LNP composition or mRNA construct of any one of embodiments E1-E7, wherein the IDO antigenic peptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:1.

[0073] E9. The LNP composition or mRNA construct of any one of embodiments E1-E8, wherein the polynucleotide encoding the IDO antigenic fragment comprises a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:2 or an antigenic fragment thereof.

[0074] E10. The LNP composition or mRNA construct of any one of embodiments E1-E9, wherein the PD-L1 antigenic peptide comprises a fragment of a naturally occurring PD-L1 molecule or a variant thereof.

[0075] E11. The LNP composition or mRNA construct of any one of embodiments E1-E10, wherein the PD-L1 antigenic peptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:3.

[0076] E12. The LNP composition or mRNA construct of any one of embodiments E1-E11, wherein the polynucleotide encoding the PD-L1 antigenic fragment comprises a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 4, or an antigenic fragment thereof.

[0077] E13. The LNP composition or mRNA construct of any one of embodiments E1-E12, wherein the checkpoint cancer vaccine comprises two IDO antigenic peptides and two PD-L1 antigenic peptides.

[0078] E14. The LNP composition or mRNA construct of any one of embodiments E1-E12, wherein the checkpoint cancer vaccine comprises three IDO antigenic peptides and three PD-L1 antigenic peptides.

[0079] E15. The LNP composition or mRNA construct of any one of embodiments E1-E12, wherein the checkpoint cancer vaccine comprises four IDO antigenic peptides and four PD-L1 antigenic peptides.

[0080] E16. The LNP composition or mRNA construct of any one of embodiments E1-E15, wherein the checkpoint cancer vaccine comprises alternating IDO and PD-L1 antigenic peptides.

[0081] E17. The LNP composition or mRNA construct of embodiment E15 or E16, wherein the checkpoint cancer vaccine comprises (i) an IDO antigenic peptide, (ii) a PD-L1 antigenic peptide, (iii) an IDO antigenic peptide, (iv) a PD-L1 antigenic peptide, (v) an IDO antigenic peptide, (vi) a PD-L1 antigenic peptide, (vii) an IDO antigenic peptide, and (viii) a PD-L1 antigenic peptide.

[0082] E18. The LNP composition or mRNA construct of any one of embodiments E15-E17, wherein the alternating IDO antigenic peptide and PD-L1 antigenic peptide comprise an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an IDO amino acid sequence provided in Table 1A (e.g., SEQ ID NO: 5), or an antigenic fragment thereof.

[0083] E19. The LNP composition or mRNA construct of any one of embodiments E16-E18, wherein the polynucleotide encoding the alternating IDO and PD-L1 antigenic peptides comprises a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 6, or an antigenic fragment thereof.

[0084] E20. The LNP composition or mRNA construct of any one of embodiments E16-E19, wherein the polynucleotide encoding the alternating IDO and PD-L1 antigenic peptides comprises a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 300, or an antigenic fragment thereof.

[0085] E21. The LNP composition or mRNA construct of any one of embodiments E16-E19, wherein the polynucleotide encoding the alternating IDO and PD-L1 antigenic peptides comprises a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 301, or an antigenic fragment thereof.

[0086] E22. The LNP composition or mRNA construct of any one of embodiments E16-E19, wherein the polynucleotide encoding the alternating IDO and PD-L1 antigenic peptides comprises a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 302, or an antigenic fragment thereof.

[0087] E23. The LNP composition or mRNA construct of any one of embodiments E16-E19, wherein the polynucleotide encoding the alternating IDO and PD-L1 antigenic peptides comprises, from the 5' to the 3' end, the nucleotide sequence of SEQ ID NO: 300, which comprises a 5' UTR of SEQ ID NO: 56, an ORF sequence of SEQ ID NO: 6, and a 3' UTR of SEQ ID NO: 108.

[0088] E24. The LNP composition or mRNA construct of any one of embodiments E16-E19, wherein the polynucleotide encoding the alternating IDO and PD-L1 antigenic peptides comprises, from the 5' to the 3' end, the nucleotide sequence of SEQ ID NO: 301, which comprises a 5' UTR of SEQ ID NO: 272, an ORF sequence of SEQ ID NO: 6, and a 3' UTR of SEQ ID NO: 108.

[0089] E25. The LNP composition or mRNA construct of any one of embodiments E16-E19, wherein the polynucleotide encoding the alternating IDO and PD-L1 antigenic peptides comprises, from the 5' to the 3' end, the nucleotide sequence of SEQ ID NO: 302, which comprises a 5' UTR of SEQ ID NO: 273, an ORF sequence of SEQ ID NO: 6, and a 3' UTR of SEQ ID NO: 108.

[0090] E26. The LNP composition or mRNA construct of any one of the preceding embodiments, further comprising a polynucleotide encoding one or more adjuvant amino acid sequences.

[0091] E27. (i) stimulating T effector cells; (ii) cytotoxic T cell-mediated killing of suppressor immune cells and tumor cells that overexpress PD-L1 or IDO; (iii) inducing an anti-tumor immune response; and / or (iv) infiltration of tumor cells by CD4+ and / or CD8+ T cells and killing of tumor cells expressing IDO and / or PDL1. The LNP composition or mRNA construct of any one of the preceding embodiments,

[0092] E28. The LNP composition or mRNA construct of any one of the preceding embodiments, which results in amelioration or delay of cancer progression in a subject, e.g., as described herein.

[0093] E29. The LNP composition or mRNA construct of any one of the preceding embodiments, wherein the polynucleotide comprising mRNA encoding the checkpoint cancer vaccine comprising one or more (e.g., 1, 2, 3, 4, or more) indoleamine-pyrrole 2,3-dioxygenase (IDO) antigenic peptides and (ii) one or more (e.g., 1, 2, 3, 4, or more) programmed death-ligand 1 (PD-L1) antigenic peptides comprises at least one chemical modification.

[0094] E30. The LNP composition or mRNA construct of embodiment E29, wherein the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0095] E31. The LNP composition or mRNA construct of embodiment E30, wherein said chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

[0096] E32. The LNP composition or mRNA construct of embodiment E31, wherein the chemical modification is N1-methylpseudouridine.

[0097] E33. The LNP composition or mRNA construct of any one of the preceding embodiments, wherein the mRNA in the lipid nanoparticle comprises fully modified N1-methylpseudouridine.

[0098] E34. The LNP composition of any one of the preceding embodiments, wherein the LNP composition comprises: (i) an ionizable lipid (e.g., an amino lipid); (ii) a sterol or other structural lipid; (iii) a non-cationic helper lipid or phospholipid; and (iv) a PEG-lipid.

[0099] E35. The LNP composition of embodiment E34, wherein the ionizable lipid comprises an amino lipid.

[0100] E36. The LNP composition of embodiment E34 or E35, wherein the ionizable lipid comprises any compound of formula (I), (Ia), (Ib), (Ic), (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), or (III).

[0101] E37. The LNP composition of any one of embodiments E34-E36, wherein the ionizable lipid comprises Compound 18, Compound 25, Compound 301, or Compound 357.

[0102] E38. The LNP composition of embodiment E37, wherein the ionizable lipid comprises compound 25.

[0103] E39. The LNP composition of any one of embodiments E34-E38, wherein the sterol or other structured lipid comprises cholesterol.

[0104] E40. The LNP composition of any one of embodiments E34-E39, wherein the non-cationic helper lipid or phospholipid comprises DSPC.

[0105] E41. The LNP composition of any one of embodiments E34-E40, wherein the PEG lipid comprises PEG DMG.

[0106] E42. The LNP composition of any one of embodiments E34-E41, wherein the LNP comprises, in molar ratios, about 20-60% ionizable lipid: 5-25% phospholipid: 25-55% cholesterol; and 0.5-15% PEG lipid.

[0107] E43. The LNP composition of embodiment E42, wherein the LNP comprises, in a molar ratio, about 48 mol % ionizable lipid: about 11 mol % phospholipid: about 38.5 mol % cholesterol; and about 2.5 mol % PEG lipid.

[0108] E44. The LNP composition of embodiment E43, wherein the LNP comprises, in molar ratios, about 48 mol % Compound 25: about 11 mol % DSPC: about 38.5 mol % cholesterol; and about 2.5 mol % PEG DMG.

[0109] E45. The LNP composition or mRNA construct of any one of the preceding embodiments, formulated for intramuscular, subcutaneous, intravenous, intranasal, intraocular, rectal, or oral delivery.

[0110] E46. The LNP composition or mRNA construct of any one of the preceding embodiments, further comprising a pharmaceutically acceptable carrier or excipient.

[0111] E47. A pharmaceutical composition comprising the LNP composition or mRNA construct of any one of the preceding embodiments.

[0112] E48. A method of modulating (e.g., stimulating) an immune response in a subject, comprising administering to said subject in need thereof an effective amount of an LNP composition comprising a polynucleotide comprising mRNA encoding a checkpoint cancer vaccine comprising one or more (e.g., 1, 2, 3, 4, or more) indoleamine-pyrrole 2,3-dioxygenase (IDO) antigenic peptides and (ii) one or more (e.g., 1, 2, 3, 4, or more) programmed death-ligand 1 (PD-L1) antigenic peptides.

[0113] E49. A method of stimulating T effector cells in a subject, comprising administering to the subject an effective amount of an LNP composition comprising a polynucleotide comprising mRNA encoding a checkpoint cancer vaccine comprising one or more (e.g., 1, 2, 3, 4, or more) indoleamine-pyrrole 2,3-dioxygenase (IDO) antigenic peptides and (ii) one or more (e.g., 1, 2, 3, 4, or more) programmed death-ligand 1 (PD-L1) antigenic peptides.

[0114] E50. A method for treating or preventing cancer or a symptom thereof, comprising administering to a subject in need thereof an effective amount of an LNP composition comprising a polynucleotide comprising mRNA encoding a checkpoint cancer vaccine comprising one or more (e.g., 1, 2, 3, 4, or more) indoleamine-pyrrole 2,3-dioxygenase (IDO) antigenic peptides and (ii) one or more (e.g., 1, 2, 3, 4, or more) programmed death-ligand 1 (PD-L1) antigenic peptides.

[0115] E51. The method of embodiment E50, wherein said cancer is a locally advanced or metastatic solid tumor.

[0116] E52. The method of embodiment E50, wherein said cancer is melanoma.

[0117] E53. The method of embodiment E52, wherein said melanoma is cutaneous melanoma.

[0118] E54. The method of embodiment 34, wherein said cutaneous melanoma is 1L cutaneous melanoma stage IIIB+.

[0119] E55. The method of embodiment E54, wherein said cancer is NSCLC.

[0120] E56. The method of embodiment E55, wherein said NSCLC is 1L NSCLC.

[0121] E57. The method of embodiment E50, wherein said cancer is bladder cancer.

[0122] E58. The method of embodiment E57, wherein said bladder cancer is non-muscle-invasive bladder cancer.

[0123] E59. The method of embodiment E50, wherein said cancer is head and neck cancer.

[0124] E60. The method of embodiment E59, wherein said head and neck cancer is head and neck squamous cell carcinoma.

[0125] E61. The method of embodiment E50, wherein said cancer is colorectal cancer.

[0126] E62. The method of embodiment E61, wherein said colorectal cancer is microsatellite-stable colorectal cancer.

[0127] E63. The method of embodiment E50, wherein said cancer is basal cell carcinoma.

[0128] E64. The method of embodiment E50, wherein said cancer is breast cancer.

[0129] E65. The method of embodiment E64, wherein said breast cancer is triple-negative breast cancer.

[0130] E66. The method of any one of embodiments E48-E65, wherein said IDO antigenic peptide comprises a fragment of a naturally occurring IDO molecule or a variant thereof.

[0131] E67. The method of any one of embodiments E48-E66, wherein said IDO antigenic peptide is derived from IDO1 or IDO2.

[0132] E68. The method of embodiment E67, wherein said IDO antigenic peptide is derived from IDO1.

[0133] E69. The method of any one of embodiments E48-E68, wherein said IDO antigenic peptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:1.

[0134] E70. The method of any one of embodiments E48 to E69, wherein the polynucleotide encoding the IDO antigenic fragment comprises a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 2 or an antigenic fragment thereof.

[0135] E71. The method of any one of embodiments E48-E70, wherein the PD-L1 antigenic peptide comprises a fragment of a naturally occurring PD-L1 molecule or a variant thereof.

[0136] E72. The method of any one of embodiments E48-E71, wherein the PD-L1 antigenic peptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:3.

[0137] E73. The method of any one of embodiments E48-E72, wherein the polynucleotide encoding the PD-L1 antigenic fragment comprises a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 4, or an antigenic fragment thereof.

[0138] E74. The method of any one of embodiments E48-E73, wherein said checkpoint cancer vaccine comprises two IDO antigenic peptides and two PD-L1 antigenic peptides.

[0139] E75. The method of any one of embodiments E48-E73, wherein said checkpoint cancer vaccine comprises three IDO antigenic peptides and three PD-L1 antigenic peptides.

[0140] E76. The method of any one of embodiments E48-E73, wherein said checkpoint cancer vaccine comprises four IDO antigenic peptides and four PD-L1 antigenic peptides.

[0141] E77. The method of any one of embodiments E48-E76, wherein said checkpoint cancer vaccine comprises alternating IDO and PD-L1 antigenic peptides.

[0142] E78. The method of embodiment E76 or E77, wherein the checkpoint cancer vaccine comprises (i) an IDO antigenic peptide, (ii) a PD-L1 antigenic peptide, (iii) an IDO antigenic peptide, (iv) a PD-L1 antigenic peptide, (v) an IDO antigenic peptide, (vi) a PD-L1 antigenic peptide, (vii) an IDO antigenic peptide, and (viii) a PD-L1 antigenic peptide.

[0143] E79. The method of any one of embodiments E76-E78, wherein the alternating IDO antigenic peptide and PD-L1 antigenic peptide comprise an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an IDO amino acid sequence provided in Table 1A (e.g., SEQ ID NO: 5), or an antigenic fragment thereof.

[0144] E80. The method of any one of embodiments E77-E79, wherein the polynucleotide encoding the alternating IDO and PD-L1 antigenic peptides comprises a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 6, or an antigenic fragment thereof.

[0145] E81. The method of any one of embodiments E77-E80, wherein the polynucleotide encoding the alternating IDO and PD-L1 antigenic peptides comprises a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 300, or an antigenic fragment thereof.

[0146] E82. The method of any one of embodiments E77-E80, wherein the polynucleotide encoding the alternating IDO and PD-L1 antigenic peptides comprises a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 301, or an antigenic fragment thereof.

[0147] E83. The method of any one of embodiments E77-E80, wherein the polynucleotide encoding the alternating IDO and PD-L1 antigenic peptides comprises a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 302 or an antigenic fragment thereof.

[0148] E84. The method of any one of embodiments E77-E80, wherein the polynucleotide encoding the alternating IDO and PD-L1 antigenic peptides comprises the nucleotide sequence of SEQ ID NO: 300, which comprises, from the 5' to the 3' end, a 5' UTR of SEQ ID NO: 56, an ORF sequence of SEQ ID NO: 6, and a 3' UTR of SEQ ID NO: 108.

[0149] E85. The method of any one of embodiments E77-E80, wherein the polynucleotide encoding the alternating IDO and PD-L1 antigenic peptides comprises, from the 5' to the 3' end, the nucleotide sequence of SEQ ID NO: 301, which comprises the 5' UTR of SEQ ID NO: 272, the ORF sequence of SEQ ID NO: 6, and the 3' UTR of SEQ ID NO: 108.

[0150] E86. The method of any one of embodiments E77-E80, wherein the polynucleotide encoding the alternating IDO and PD-L1 antigenic peptides comprises, from the 5' to the 3' end, the nucleotide sequence of SEQ ID NO: 302, which comprises the 5' UTR of SEQ ID NO: 273, the ORF sequence of SEQ ID NO: 6, and the 3' UTR of SEQ ID NO: 108.

[0151] E87. The method of any one of the preceding embodiments, wherein said composition further comprises a polynucleotide encoding one or more adjuvant amino acid sequences.

[0152] E88. (i) stimulating T effector cells; (ii) cytotoxic T cell-mediated killing of suppressor immune cells and tumor cells that overexpress PD-L1 or IDO; (iii) inducing an anti-tumor immune response; and / or (iv) infiltration of tumor cells by CD4+ and / or CD8+ T cells and killing of tumor cells expressing IDO and / or PDL1. 10. The method of any one of the preceding embodiments, wherein

[0153] E89. The method of any one of the preceding embodiments, resulting in amelioration or delay of cancer progression in the subject, e.g., as described herein.

[0154] E90. The method of any one of the preceding embodiments, wherein said polynucleotide comprising mRNA encoding said checkpoint cancer vaccine comprising one or more (e.g., 1, 2, 3, 4, or more) indoleamine-pyrrole 2,3-dioxygenase (IDO) antigenic peptides and (ii) one or more (e.g., 1, 2, 3, 4, or more) programmed death-ligand 1 (PD-L1) antigenic peptides comprises at least one chemical modification.

[0155] E91. The method of embodiment E90, wherein the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0156] E92. The method of embodiment E91, wherein said chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

[0157] E93. The method of embodiment E92, wherein said chemical modification is N1-methylpseudouridine.

[0158] E94. The method of any one of the preceding embodiments, wherein the mRNA in the lipid nanoparticle comprises fully modified N1-methylpseudouridine.

[0159] E95. The method of any one of the preceding embodiments, wherein the LNP composition comprises: (i) an ionizable lipid (e.g., an amino lipid); (ii) a sterol or other structural lipid; (iii) a non-cationic helper lipid or phospholipid; and (iv) a PEG-lipid.

[0160] E96. The method of embodiment E95, wherein the ionizable lipid comprises an amino lipid.

[0161] E97. The method of embodiment E95 or E96, wherein the ionizable lipid comprises any compound of formula (I), (Ia), (Ib), (Ic), (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), or (III).

[0162] E98. The method of any one of embodiments E95-E97, wherein said ionizable lipid comprises a compound of formula (I).

[0163] E99. The method of any one of embodiments E95-E98, wherein said ionizable lipid comprises Compound 18, Compound 25, Compound 301, or Compound 357.

[0164] E100. The method of embodiment E99, wherein said ionizable lipid comprises compound 25.

[0165] E101. The method of any one of embodiments E95-E100, wherein said sterol or other structured lipid comprises cholesterol.

[0166] E102. The method of any one of embodiments E95-E101, wherein the non-cationic helper lipid or phospholipid comprises DSPC.

[0167] E103. The method of any one of embodiments E95-E102, wherein the PEG lipid comprises PEG DMG.

[0168] E104. The method of any one of embodiments E95-E103, wherein said LNP comprises, in molar ratios, about 20-60% ionizable lipid: 5-25% phospholipid: 25-55% cholesterol; and 0.5-15% PEG lipid.

[0169] E105. The method of embodiment E104, wherein said LNP comprises, in a molar ratio, about 48 mol % ionizable lipid: about 11 mol % phospholipid: about 38.5 mol % cholesterol; and about 2.5 mol % PEG lipid.

[0170] E106. The method of embodiment E105, wherein said LNP comprises, in molar ratios, about 48 mol % Compound 25: about 11 mol % DSPC: about 38.5 mol % cholesterol; and about 2.5 mol % PEG DMG.

[0171] E107. The method of any one of embodiments E48-E106, wherein the LNP composition is administered at a dose of about 100 μg to about 1 mg.

[0172] E108. The method of embodiment E107, wherein the LNP composition is administered in a dose of 50 μg to 150 μg, 150 μg to 250 μg, 250 μg to 350 μg, 350 μg to 450 μg, 450 μg to 550 μg, 550 μg to 650 μg, 650 μg to 750 μg, 750 μg to 850 μg, 850 μg to 950 μg, or 950 μg to 1 mg.

[0173] E109. The method of embodiment E108, wherein the LNP composition is administered at a dose of 50 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, or 1 mg.

[0174] E110. The method of any one of embodiments E48-E109, wherein the LNP composition is administered intramuscularly.

[0175] E111. The method of any one of embodiments E48-E110, wherein the LNP composition is administered to the subject according to a dosing interval, eg, as described herein.

[0176] E112. The method of embodiment E111, wherein said dosing interval is accomplished for at least 1 week, 2 weeks, 3 weeks, or 4 weeks.

[0177] E113. The method of embodiment E112, wherein said dosing interval comprises a cycle (e.g., a 21-day or 3-week cycle).

[0178] E114. The method of embodiment E112, wherein the LNP composition is administered to the subject on day 1 of a three-week cycle.

[0179] E115. The method of embodiment E114, wherein the LNP composition is administered for at least 2, 3, 4, 5, 6, 7, 8, or 9 cycles.

[0180] E116. The method of any one of embodiments E48-E115, wherein said method further comprises administering to the subject an additional therapeutic agent.

[0181] E117. The method of embodiment E116, wherein said additional therapeutic agent is a checkpoint inhibitor.

[0182] E118. The method of embodiment E117, wherein said checkpoint inhibitor is selected from the group consisting of an anti-PD-1 agent and an anti-CTLA4 agent.

[0183] E119. The method of embodiment E118, wherein said checkpoint inhibitor comprises pembrolizumab.

[0184] E120. The method of E119, wherein said pembrolizumab is administered to the subject according to a dosing interval, e.g., as described herein.

[0185] E121. The method of embodiment E121, wherein said dosing interval is accomplished for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks.

[0186] E122. The method of embodiment E121, wherein the dosing interval comprises a cycle (e.g., a 6-week cycle).

[0187] E123. The method of embodiment E122, wherein the LNP composition is administered to the subject on day 1 of a 6-week cycle.

[0188] E124. The method of embodiment E123, wherein the LNP composition is administered for at least 2, 3, 4, or 5 cycles.

[0189] E125. The method of any one of embodiments E119 to E124, wherein said pembrolizumab is administered at a dose of about 400 mg. [Brief explanation of the drawings]

[0190] [Figure 1] Graph depicting a comparison of antigen-specific IFNγ ELISpot responses to mRNA-4359 with PD-L1 restimulation in HLA-A*02:01 transgenic and wild-type mice. [Figure 2]1 is a graph illustrating a comparison of antigen-specific IFNγ ELISpot responses to mRNA-4359 with IDO1 restimulation in HLA-A*02:01 transgenic and wild-type mice. [Figure 3] 1 is a graph depicting flow cytometry analysis of antigen-specific CD8+IFNγ+ T cell responses to mRNA-4359 in HLA-A*02:01 transgenic and wild-type mice. [Figure 4A] Graph showing post-vaccination T cell responses measured by the number of INFγ spot-forming units (SFU), normalized to one million PBMCs over time. [Figure 4B] Heatmap showing IDO1 responses in peripheral blood before and after vaccination. [Figure 4C] Heatmap showing PD-L1 responses in peripheral blood before and after vaccination. [Figure 4D] Graph showing the change in IFNγ SFU / million PBMCs in IDO-specific cells. [Figure 4E] Graph showing the change in IFNγ SFU / million PBMCs in PD-L1 specific cells. [Figure 5] A diagrammatic representation of the IS / ID model. [Figure 6A] 1 is a graph showing IFNγ SFU / million PBMCs in IDO1 complete responders. [Figure 6B] Graph showing IFNγ SFU / million PBMCs in IDO1 partial responders. [Figure 6C] Graph showing IFNγ SFU / million PBMCs in PD-L1 complete responders. [Figure 6D] Graph showing IFNγ SFU / million PBMCs in PD-L1 partial responders. [Figure 7A] Graph showing first and total order Sobol index for IDO1 complete responders. [Figure 7B]Graph showing the primary and overall Sobol index for IDO1 partial responders. [Figure 7C] Graph showing primary and overall Sobol index for PD-L1 complete responders. [Figure 7D] Graph showing primary and overall Sobol index for PD-L1 partial responders. [Figure 8] FIG. 1 is a pair of graphs showing predictive dose-parameter effect curves for a peptide vaccine (A) and an mRNA vaccine (B). [Figure 9A] Graph showing predicted steady-state peak and steady-state trough at different doses for IDO1 complete responders. [Figure 9B] Graph showing predicted steady-state peak and steady-state trough at different doses for IDO1 partial responders. [Figure 9C] Figure 10 is a graph showing the expected steady-state peak and steady-state trough at different doses for PD-L1 complete responders. [Figure 9D] Figure 10 shows the predicted steady-state peak and steady-state trough at different doses for PD-L1 partial responders. [Figure 10A] 1 is a graph showing a dosing regimen simulation. [Figure 10B] 1 is a graph showing a dosing regimen simulation. [Figure 10C] 1 is a graph showing a dosing regimen simulation. DETAILED DESCRIPTION OF THE INVENTION

[0191] Using the compositions and methods described herein, immune cells (e.g., T cells) can be primed to recognize tumor-associated antigens and / or become activated, e.g., to possess cytotoxic properties. For example, T effector cells can be primed to mediate an anti-cancer response in a subject, resulting in the killing of suppressor immune cells and tumor cells that express IDO or PDL1. In one embodiment, the subject methods and compositions can be used to induce or enhance an anti-tumor immune response in a subject with cancer.

[0192] An exemplary method for treating cancer includes administering to a subject a checkpoint cancer vaccine comprising a polynucleotide comprising an mRNA sequence encoding one or more (e.g., 1, 2, 3, 4, or more) IDO antigenic peptides and one or more (e.g., 1, 2, 3, 4, or more) PD-L1 antigenic peptides, e.g., as described herein. Optionally, the vaccine may also include an mRNA sequence encoding an amino acid sequence for an adjuvant. Without wishing to be bound by theory, it is believed that in some embodiments, systemic PD-1 / PD-L1 blockade can further amplify the effect, leading to greater immune activation and superior disease control. Thus, in one embodiment, a checkpoint inhibitor, such as an anti-PD1 antibody, an anti-CTLA4 antibody, or a combination thereof, may also be administered to the subject.

[0193] Thus, disclosed herein are lipid nanoparticle (LNP) compositions comprising mRNA encoding a checkpoint cancer vaccine comprising one or more (e.g., 1, 2, 3, 4, or more) IDO antigenic peptides and one or more (e.g., 1, 2, 3, 4, or more) PD-L1 antigenic peptides, and optionally one or more adjuvant amino acid sequences, and uses thereof. The LNP compositions of the present disclosure comprise an mRNA therapeutic encoding a checkpoint cancer vaccine comprising (i) one or more IDO antigenic peptides and (ii) one or more PD-L1 antigenic peptides, and optionally one or more adjuvant amino acid sequences. In one embodiment, the LNP compositions of the present disclosure can prime T effector cells and / or stimulate anti-tumor immune responses in vivo. Also disclosed herein are methods of using LNP compositions comprising checkpoint cancer vaccines comprising (i) one or more IDO antigenic peptides and (ii) one or more PD-L1 antigenic peptides to stimulate an immune response by IDO / PDL1-specific T cells to promote an immunostimulatory environment and / or promote killing of IDO / PDL1-expressing cancer cells, thereby treating cancer (e.g., melanoma or NSCLC).

[0194] definition Administer: As used herein, "administer" refers to a method of delivering a composition to a subject or patient. The method of administration is selected to target (e.g., specifically deliver) delivery to a particular region or system of the body. For example, administration can be parenteral (e.g., intramuscular).

[0195] Approximately, about: As used herein, the term "approximately" or "about," when applied to one or more values ​​of interest, refers to a value similar to the stated reference value. In certain embodiments, the term "approximately" or "about," unless otherwise stated or otherwise clear from the context, refers to a range of values ​​that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less than) the stated reference value (except when such a value exceeds 100% of the possible values). For example, when used in the context of the amount of a given compound in the lipid component of an LNP, "about" can mean ±5% of the recited value. For example, an LNP containing a lipid component with about 40% of a given compound may contain 30-50% of that compound. As another example, an LNP containing a lipid component with about 50% of a given compound may contain 45-55% of that compound.

[0196] Chimeric molecule: As used herein, the term "chimeric molecule" refers to a molecule having at least two portions (e.g., an IDO antigenic peptide and a PD-L1 antigenic peptide) from different sources or origins. For example, the two portions can be derived from two different polypeptides. Each portion can be a full-length polypeptide or a fragment thereof (e.g., an antigenic fragment). In certain embodiments, the two polypeptides are derived from two different organisms. In other embodiments, the two polypeptides are derived from the same organism. The two different polypeptides can both be naturally occurring or synthetic, or one can be naturally occurring and the other synthetic. In some embodiments, the two portions of a chimeric molecule have different properties. The property can be a biological property (such as an in vitro, ex vivo, or in vivo function or activity). The property can also be a physical property or a chemical property (such as binding affinity or specificity). In some embodiments, the two portions are linked together by a covalent bond. For example, the two moieties can be linked directly (e.g., by a single covalent bond (e.g., a peptide bond)) or indirectly (e.g., via a linker (e.g., a peptide linker)). In some embodiments, a chimeric molecule is produced through the linkage of two or more polynucleotides that originally encode separate polypeptides. In some embodiments, the two or more polynucleotides form a single open reading frame.

[0197] Conjugated: As used herein, the term "conjugated," when used in reference to two or more moieties, means that the moieties are physically associated or connected to one another, either directly or through one or more additional moieties that serve as linking agents, to form a structure that is sufficiently stable so that the moieties remain physically associated under the conditions (e.g., physiological conditions) in which the structure will be used. In some embodiments, two or more moieties may be conjugated by a direct covalent chemical bond. In other embodiments, two or more moieties may be conjugated by an ionic or hydrogen bond.

[0198] Contacting: As used herein, the term "contacting" means establishing a physical connection between two or more entities. For example, contacting a cell with an mRNA or lipid nanoparticle composition means that the cell and the mRNA or lipid nanoparticle share a physical connection. Methods for contacting a cell with an external entity in vivo, in vitro, and ex vivo are well known in the field of biology. In exemplary embodiments of the present disclosure, contacting a mammalian cell with a composition (e.g., nanoparticles or a pharmaceutical composition of the present disclosure) is performed in vivo. For example, contacting a lipid nanoparticle composition with a cell (e.g., a mammalian cell) that may be located within an organism (e.g., a mammal) can be performed by any suitable administration route (e.g., parenteral administration to the organism, including intravenous administration, intramuscular administration, intradermal administration, and subcutaneous administration). For cells present in vitro, the composition (e.g., lipid nanoparticles) and the cell can be contacted, for example, by adding the composition to the cell's culture medium, which can involve or result in transfection. Furthermore, two or more cells can be contacted with the nanoparticle composition.

[0199] Deliver: As used herein, the term "deliver" means providing an entity to a destination. For example, delivering a therapeutic and / or prophylactic agent to a subject can include administering LNPs containing the therapeutic and / or prophylactic agent to the subject (e.g., by intravenous, intramuscular, intradermal, or subcutaneous routes). Administering LNPs to a mammal or mammalian cells can include contacting one or more cells with lipid nanoparticles.

[0200] Encapsulate: As used herein, the term "encapsulate" means to enclose, surround, or encase. In some embodiments, a compound, polynucleotide (e.g., mRNA), or other composition may be fully encapsulated, partially encapsulated, or substantially encapsulated. For example, in some embodiments, the mRNA of the present disclosure may be encapsulated in a lipid nanoparticle (e.g., a liposome).

[0201] Encapsulation efficiency: As used herein, "encapsulation efficiency" refers to the amount of therapeutic and / or prophylactic agent that becomes part of the LNP compared to the initial total amount of therapeutic and / or prophylactic agent used in preparing the LNP. For example, if 97 mg of therapeutic and / or prophylactic agent out of 100 mg of total therapeutic and / or prophylactic agent initially provided in the composition is encapsulated in the LNP, the encapsulation efficiency would be given as 97%. As used herein, "encapsulation" can refer to complete, substantial, or partial entrapment, entrapment, surrounding, or envelopment.

[0202] Effective amount: As used herein, the term "effective amount" of a drug refers to an amount sufficient to achieve a beneficial effect or desired result (e.g., a clinical result); therefore, "effective amount" depends on the context in which it is applied. For example, in the context of the amount of target cell delivery-enhancing lipid in a lipid composition (e.g., LNP) of the present disclosure, an effective amount of the target cell delivery-enhancing lipid is an amount sufficient to achieve a beneficial or desired result compared to a lipid composition (e.g., LNP) lacking the target cell delivery-enhancing lipid. Non-limiting examples of beneficial or desired results achieved by a lipid composition (e.g., LNP) include increasing the percentage of transfected cells and / or increasing the expression level of a protein encoded by a nucleic acid associated with / encapsulated by the lipid composition (e.g., LNP). In the context of administering target cell delivery-enhancing lipid-containing lipid nanoparticles such that an effective amount of the lipid nanoparticles is taken up by target cells in a subject, an effective amount of the target cell delivery-enhancing lipid-containing LNP is an amount sufficient to achieve a beneficial or desired result compared to an LNP lacking the target cell delivery-enhancing lipid. Non-limiting examples of beneficial or desired results in a subject include increasing the percentage of transfected cells compared to LNPs lacking the target cell delivery-enhancing lipid, increasing the level of expression of a protein encoded by a nucleic acid associated with / encapsulated by the target cell delivery-enhancing lipid-containing LNPs, and / or increasing the in vivo prophylactic or therapeutic effect of a nucleic acid or its encoded protein associated with / encapsulated by the target cell delivery-enhancing lipid-containing LNPs. In some embodiments, a therapeutically effective amount of the target cell delivery-enhancing lipid-containing LNPs is sufficient to treat, ameliorate the symptoms of, diagnose, prevent, and / or delay the onset of an infection, disease, disorder, and / or condition when administered to a subject suffering from or susceptible to the infection, disease, disorder, and / or condition.In another embodiment, an effective amount of lipid nanoparticles is sufficient to result in expression of a desired protein in at least about 5%, 10%, 15%, 20%, 25%, or more of the target cells. For example, an effective amount of target cell delivery-enhancing lipid-containing LNPs can be an amount that results in transfection of at least 5%, 10%, 15%, 20%, 25%, 30%, or 35% of the target cells after a single intravenous injection.

[0203] Expression: As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end processing); (3) translation of the RNA into a polypeptide or protein; and (4) post-translational modification of the polypeptide or protein.

[0204] Ex vivo: As used herein, the term "ex vivo" refers to an event that occurs outside of an organism (e.g., an animal, plant, or microorganism, or cells or tissues thereof). An ex vivo event can occur in an environment that is minimally altered from the natural (e.g., in vivo) environment.

[0205] Fragment: As used herein, "fragment" refers to a portion. For example, a fragment of a protein can include a polypeptide obtained by subdivision of a full-length protein isolated from cultured cells or a polypeptide obtained via recombinant DNA techniques.

[0206] GC-rich: As used herein, the term "GC-rich" refers to the nucleobase composition of a polynucleotide (e.g., mRNA) or any portion thereof (e.g., RNA element), or derivative or analog thereof, comprising guanine (G) and / or cytosine (C) nucleobases, with a GC content of greater than about 50%. The term "GC-rich" refers to all or a portion of a polynucleotide, including, but not limited to, a gene, non-coding region, 5'UTR, 3'UTR, open reading frame, RNA element, sequence motif, or any discontinuous sequence, fragment, or segment thereof, that comprises about 50% GC content. In some embodiments of the present disclosure, the GC-rich polynucleotide, or any portion thereof, is composed exclusively of guanine (G) and / or cytosine (C) nucleobases.

[0207] GC content: As used herein, the term "GC content" refers to the percentage of nucleobases (out of the total number of nucleobases in DNA and RNA, which may include adenine (A) and thymine (T) or uracil (U), and derivatives or analogs thereof) in a polynucleotide (e.g., mRNA) or portion thereof (e.g., RNA element) that are guanine (G) and cytosine (C) nucleobases, or derivatives or analogs thereof. The term "GC content" refers to all or a portion of a polynucleotide, including, but not limited to, a gene, a non-coding region, a 5' UTR or 3' UTR, an open reading frame, an RNA element, a sequence motif, or any discontinuous sequence, fragment, or segment thereof.

[0208] IDO antigenic peptide: As used herein, the term "IDO antigenic peptide" refers to full-length naturally occurring IDO (e.g., mammalian IDO, e.g., human IDO, e.g., associated with UniProt: P14902 and / or NCBI Gene ID: 3620; or associated with UniProt Q6ZQW0 and / or NCBI Gene ID 169355), a fragment (e.g., an antigenic fragment) of IDO, or a variant of IDO having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to naturally occurring wild-type IDO or a fragment (e.g., an antigenic fragment) thereof. In some embodiments, the IDO molecule is an IDO gene product (e.g., an IDO polypeptide).

[0209] PD-L1 antigenic peptide: As used herein, the term "PD-L1 antigenic peptide" refers to full-length naturally occurring PD-L1 (e.g., mammalian PD-L1, e.g., human PD-L1, e.g., associated with UniProt: Q9NZQ7; NCBI Gene ID: 29126), a fragment (e.g., an antigenic fragment) of PD-L1, or a variant of PD-L1 that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to naturally occurring wild-type PD-L1 or a fragment (e.g., an antigenic fragment) thereof. In some embodiments, the PD-L1 molecule is a PD-L1 gene product (e.g., a PD-L1 polypeptide).

[0210] Heterologous: As used herein, "heterologous" indicates that a sequence (e.g., an amino acid sequence, or a polynucleotide encoding an amino acid sequence) is not normally present in a given polypeptide or polynucleotide. For example, an amino acid sequence corresponding to a domain or motif of one protein may be heterologous to a second protein.

[0211] Isolated: As used herein, the term "isolated" refers to a substance or entity that has been separated from at least some of the components with which it is associated (whether in nature or in an experimental setting). Isolated substances may have varying levels of purity with respect to the substances with which they are associated. Isolated substances and / or entities may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated. In some embodiments, an isolated agent is greater than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components.

[0212] Liposome: As used herein, "liposome" refers to a structure comprising a lipid-containing membrane enclosing an aqueous interior. Liposomes may have one or more lipid membranes. Liposomes include monolayer liposomes (also known in the art as unilamellar liposomes) and multilayer liposomes (also known in the art as multilamellar liposomes).

[0213] Modified: As used herein, "modified" or "modification" refers to an altered state or change in composition or structure of a polynucleotide (e.g., mRNA). Polynucleotides can be modified in a variety of ways, including chemically, structurally, and / or functionally. For example, polynucleotides can be structurally modified by the incorporation of one or more RNA elements, which contain sequence and / or RNA secondary structure(s) that provide one or more functions (e.g., translational regulatory activity). Thus, polynucleotides of the present disclosure can be composed of one or more modifications, including, for example, one or more chemical, structural, or functional modifications, and can include any combination thereof.

[0214] Modified: As used herein, "modified" refers to an altered state or structure of a molecule of the present disclosure. Molecules can be modified in numerous ways, including chemically, structurally, and functionally. In one embodiment, an mRNA molecule of the present disclosure is modified by the introduction of non-natural nucleosides and / or nucleotides, e.g., when related to the natural ribonucleotides A, U, G, and C. Non-canonical nucleotides (such as cap structures), although different in chemical structure from the A, C, G, and U ribonucleotides, are not considered "modified."

[0215] mRNA: As used herein, "mRNA" refers to messenger ribonucleic acid. mRNA can be naturally occurring or non-naturally occurring. For example, mRNA can contain modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. mRNA can contain a cap structure, a chain-terminating nucleoside, a stem-loop, a polyA sequence, and / or a polyadenylation signal. mRNA can have a nucleotide sequence that encodes a polypeptide. Translation of mRNA (e.g., in vivo translation of mRNA inside a mammalian cell) can produce a polypeptide. Traditionally, the basic components of an mRNA molecule include at least a coding region, a 5'-untranslated region (5'-UTR), a 3'UTR, a 5' cap, and a polyA sequence.

[0216] Nanoparticle: As used herein, "nanoparticle" refers to a particle having any one structural feature on a scale of less than about 1000 nm that exhibits novel properties compared to a bulk sample of the same material. Conventionally, nanoparticles have any one structural feature on a scale of less than about 500 nm, less than about 200 nm, or about 100 nm. More conventionally, nanoparticles have any one structural feature on a scale of about 50 nm to about 500 nm, about 50 nm to about 200 nm, or about 70 to about 120 nm. In exemplary embodiments, nanoparticles are particles having one or more dimensions on the order of about 1 to 1000 nm. In other exemplary embodiments, nanoparticles are particles having one or more dimensions on the order of about 10 to 500 nm. In other exemplary embodiments, nanoparticles are particles having one or more dimensions on the order of about 50 to 200 nm. Spherical nanoparticles may have diameters of, for example, about 50 to 100 or 70 to 120 nanometers. Nanoparticles most often behave as units with respect to their transport and properties. The novel properties that distinguish nanoparticles from corresponding bulk materials typically develop at size scales below 1000 nm, or at sizes of about 100 nm, although it should be noted that nanoparticles can be of larger sizes, e.g., ellipsoidal, tubular, and the like. While the size of most molecules falls within the above outline, individual molecules are not usually referred to as nanoparticles.

[0217] Nucleic Acid: As used herein, the term "nucleic acid" is used in its broadest sense to encompass any compound and / or substance comprising a polymer of nucleotides. These polymers are often referred to as polynucleotides. Exemplary nucleic acids or polynucleotides of the present disclosure include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), DNA-RNA hybrids, RNAi inducers, RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, RNA that induces triple helix formation, threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA having a β-D-ribo configuration, α-LNA (a diastereoisomer of LNA) having an α-L-ribo configuration, 2'-amino-LNA having a 2'-amino functional group, and 2'-amino-α-LNA having a 2'-amino functional group), or hybrids thereof.

[0218] Nucleic acid structure: As used herein, the term "nucleic acid structure" (used interchangeably with "polynucleotide structure") refers to the arrangement or organization of atoms, chemical constituents, elements, motifs, and / or sequences of linked nucleotides or derivatives or analogs thereof that comprise a nucleic acid (e.g., mRNA). The term also refers to the two-dimensional or three-dimensional state of a nucleic acid. Thus, the term "RNA structure" refers to the arrangement or organization of atoms, chemical constituents, elements, motifs, and / or sequences of linked nucleotides or derivatives or analogs thereof that comprise an RNA molecule (e.g., mRNA), and / or refers to the two-dimensional and / or three-dimensional state of an RNA molecule. Nucleic acid structure can be further divided into four organizational categories, referred to herein as "molecular structure," "primary structure," "secondary structure," and "tertiary structure," based on increasing organizational complexity.

[0219] Nucleobase: As used herein, the term "nucleobase" (alternatively, "nucleotide base" or "nitrogenous base") refers to a heterocyclic compound of purine or pyrimidine found in nucleic acids, and includes any derivative or analog of naturally occurring purine and pyrimidine that confers improved properties (e.g., binding affinity, nuclease resistance, chemical stability) to a nucleic acid or a portion or segment thereof. Adenine, cytosine, guanine, thymine, and uracil are the nucleobases found predominantly in natural nucleic acids. Other natural, unnatural, and / or synthetic nucleobases known in the art and / or described herein can be incorporated into nucleic acids.

[0220] Nucleoside / Nucleotide: As used herein, the term "nucleoside" refers to a compound containing a sugar molecule (e.g., ribose in RNA or deoxyribose in DNA) covalently linked to a nucleobase (e.g., a purine or pyrimidine) or a derivative or analog thereof (also referred to herein as a "nucleobase"), but lacking an internucleoside linking group (e.g., a phosphate group), or a derivative or analog thereof. As used herein, the term "nucleotide" refers to a nucleoside covalently linked to an internucleoside linking group (e.g., a phosphate group), or any derivative, analog, or modification thereof that confers improved chemical and / or functional properties (e.g., binding affinity, nuclease resistance, chemical stability) to a nucleic acid or a portion or segment thereof.

[0221] Open Reading Frame: As used herein, the term "open reading frame," abbreviated as "ORF," refers to a segment or region of an mRNA molecule that encodes a polypeptide. An ORF contains a continuous stretch of non-overlapping, in-frame codons, beginning with a start codon and ending with a stop codon, and is translated by the ribosome.

[0222] Patient: As used herein, "patient" refers to a subject who may seek or need treatment, who requests treatment, who is undergoing treatment, who is scheduled to receive treatment, or who is under the care of a trained professional for a particular disease or condition. In certain embodiments, the patient is a human patient. In some embodiments, the patient is a patient suffering from an autoimmune disease, e.g., as described herein.

[0223] Pharmaceutically acceptable: The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0224] Pharmaceutically acceptable excipient: As used herein, the phrase "pharmaceutically acceptable excipient" refers to any ingredient other than the compounds described herein (e.g., a vehicle capable of suspending or dissolving an active compound) that has substantially non-toxic and non-inflammatory properties in a patient. Excipients may include, for example, anti-adhesives, antioxidants, binding substances, coatings, compression aids, disintegrating substances, dyes (pigments), emollients, emulsifying substances, fillers (diluents), film-forming substances or film coatings, flavoring substances, fragrances, fluidizing substances (glidants), lubricating substances, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water for hydration. Exemplary excipients include, but are not limited to, butylhydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0225] Pharmaceutically acceptable salts: As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds in which the parent compound is modified by converting an existing acidic or basic moiety into its salt form (e.g., by reacting a free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues (such as amines); alkali or organic salts of acidic residues (such as carboxylic acids); and the like. Representative acid addition salts include acetate, acetic acid, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzenesulfonic acid, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethoxybenzoate, and 2-hydroxy-ethoxybenzoate. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. The pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.The pharmaceutically acceptable salts of the present disclosure can be synthesized from parent compounds containing a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or free base form of these compounds with a stoichiometric amount of an appropriate base or acid in water, an organic solvent, or a mixture of the two (generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred). Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418; Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl and C.G. Wermuth (eds.), Wiley-VCH, 2008; and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977) (each of which is incorporated herein by reference in its entirety).

[0226] Polypeptide: As used herein, the term "polypeptide" or "polypeptide of interest" refers to a polymer of amino acid residues, typically joined by peptide bonds, which may be naturally (e.g., isolated or purified) or synthetically produced.

[0227] RNA: As used herein, "RNA" refers to a ribonucleic acid, whether naturally occurring or non-naturally occurring. For example, RNA may contain modified and / or non-naturally occurring components (such as one or more nucleobases, nucleosides, nucleotides, or linkers). RNA may contain a cap structure, a chain-terminating nucleoside, a stem-loop, a polyA sequence, and / or a polyadenylation signal. RNA may have a nucleotide sequence encoding a polypeptide of interest. For example, RNA may be messenger RNA (mRNA). Translation of an mRNA encoding a specific polypeptide (e.g., translation of an mRNA inside a mammalian cell in vivo) can produce the encoded polypeptide. RNA may be selected from the non-limiting group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, long non-coding RNA (lncRNA), and mixtures thereof.

[0228] RNA element: As used herein, the term "RNA element" refers to a portion, fragment, or segment of an RNA molecule that provides a biological function and / or has biological activity (e.g., translational regulatory activity). Modification of a polynucleotide by incorporating one or more RNA elements, such as those described herein, provides one or more desired functional properties to the modified polynucleotide. The RNA elements described herein can be naturally occurring, non-naturally occurring, synthetic, engineered, or any combination thereof. For example, naturally occurring RNA elements that provide regulatory activity include elements found throughout the transcriptomes of viruses, prokaryotes, and eukaryotes (e.g., humans). RNA elements, particularly eukaryotic mRNA and translated viral RNA, have been shown to be involved in mediating numerous functions in cells.Exemplary naturally occurring RNA elements include translation initiation elements (e.g., internal ribosome entry sites (IRES), see Kieft et al., (2001) RNA 7(2):194-206), translation enhancer elements (e.g., APP mRNA translation enhancer elements, see Rogers et al., (1999) J Biol Chem 274(10):6421-6431), mRNA stability elements (e.g., AU-rich elements (AREs), see Garneau et al., (2007) Nat Rev Mol Cell Biol 8(2):113-126), translation repression elements (see, e.g., Blumer et al., (2002) Mech Dev 110(1-2):97-112), protein-binding RNA elements (e.g., iron-responsive elements, see Selezneva et al., (2013) J Mol Biol 425(18):3301-3310), cytoplasmic polyadenylation elements (Villalba et al., (2011) Curr Opin Genet Dev 21(4):452-457), and catalytic RNA elements (e.g., ribozymes, see Scott et al., (2009) Biochim Biophys Acta 1789(9-10):634-641).

[0229] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting all or nearly all extent or degree of a characteristic or property of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed perfectly, or achieve or avoid absolute results. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0230] Suffering from: An individual "suffering from" a disease, disorder, and / or condition has been diagnosed with or exhibits one or more symptoms of the disease, disorder, and / or condition.

[0231] Therapeutic Agent: The term "therapeutic agent" refers to any agent that has a therapeutic, diagnostic, and / or prophylactic effect and / or elicits a desired biological and / or pharmacological effect when administered to a subject.

[0232] Transfection: As used herein, the term "transfection" refers to a method of introducing a species (e.g., a polynucleotide such as mRNA) into a cell.

[0233] Subject: As used herein, the term "subject" refers to any organism to which a composition according to the present disclosure can be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants. In some embodiments, a subject can be a patient.

[0234] Treat: As used herein, the term "treat" refers to partially or completely alleviating, relieving, ameliorating, relieving, delaying the onset of, inhibiting the progression of, reducing the severity of, and / or reducing the incidence of one or more symptoms or features of a particular infection, disease, disorder, and / or condition. For example, "treating" cancer may refer to inhibiting tumor survival, growth, and / or spread. Treatment may be administered to subjects who do not exhibit symptoms of the disease, disorder, and / or condition and / or to subjects who exhibit only early signs of the disease, disorder, and / or condition for the purpose of reducing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0235] Prevent: As used herein, the term "prevent" refers to partially or completely inhibiting the onset of one or more symptoms or features of a particular infection, disease, disorder, and / or condition.

[0236] Unmodified: As used herein, "unmodified" refers to any substance, compound, or molecule before it has been altered in any way. Unmodified can, but does not always, refer to the wild-type or native form of a biomolecule. A molecule can undergo a series of modifications, whereby each modified molecule can serve as the "unmodified" starting molecule for subsequent modifications.

[0237] Variant: As used herein, the term "variant" refers to a molecule that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity of the wild-type molecule, e.g., as measured by art-recognized assays.

[0238] IDO antigenic peptides Indoleamine-pyrrole 2,3-dioxygenase (IDO) is a monomeric intracellular heme-containing enzyme that regulates the degradation of tryptophan in the kynurenine pathway (Cemil B and Sarisozen C (2017) Journal of Oncological Sciences 3:2 pp.52-56). There are two isoforms of IDO (IDO1 and IDO2), both of which convert tryptophan to kynurenine at different enzymatic rates. For example, IDO2 is narrowly expressed, while IDO1 is more widely expressed, in endothelial cells, antigen-presenting cells, fibroblasts, macrophages, and dendritic cells.

[0239] In one aspect, the present disclosure provides LNP compositions comprising, e.g., polynucleotides encoding, checkpoint cancer vaccines comprising one or more (e.g., 1, 2, 3, 4, or more) IDO antigenic peptides, e.g., derived from IDO1 or IDO2, e.g., as described herein.

[0240] In one embodiment, the IDO antigenic peptide is derived from IDO1. In one embodiment, the one or more IDO antigenic peptides comprise a naturally occurring IDO1 molecule, a fragment (e.g., an antigenic fragment) of a naturally occurring IDO1 molecule, or a variant thereof. In one embodiment, the IDO antigenic peptide comprises a naturally occurring IDO1 variant molecule (e.g., an IDO1 variant) or a fragment thereof. In one embodiment, an LNP composition comprising a polynucleotide encoding a checkpoint cancer vaccine comprising one or more (e.g., 1, 2, 3, 4, or more) IDO1 antigenic peptides and one or more (e.g., 1, 2, 3, 4, or more) PD-L1 antigenic peptides may be administered alone or in combination with additional agents.

[0241] In one aspect, the LNP composition disclosed herein comprises a polynucleotide encoding a checkpoint cancer vaccine comprising one or more (e.g., 1, 2, 3, 4, or more) IDO1 antigenic peptides. In one embodiment, the IDO antigenic peptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an IDO amino acid sequence provided in Table 1A (e.g., SEQ ID NO: 1) or an antigenic fragment thereof. In one embodiment, the IDO antigenic peptide comprises the amino acid sequence of an IDO amino acid sequence provided in Table 1A (e.g., SEQ ID NO: 1) or an antigenic fragment thereof. In one embodiment, the IDO antigenic peptide comprises the amino acid sequence of SEQ ID NO: 1 or an antigenic fragment thereof. In one embodiment, the IDO antigenic peptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1 or an antigenic fragment thereof. In one embodiment, the IDO antigenic peptide comprises SEQ ID NO: 1 or an antigenic fragment thereof.

[0242] In one embodiment, a polynucleotide encoding a checkpoint cancer vaccine comprising one or more (e.g., 1, 2, 3, 4, or more) IDO1 antigenic peptides comprises a nucleotide sequence (e.g., a codon-optimized nucleotide sequence) having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 2 or an antigenic fragment thereof. In one embodiment, a polynucleotide encoding a checkpoint cancer vaccine comprising one or more (e.g., 1, 2, 3, 4, or more) IDO1 antigenic peptides comprises the nucleotide sequence (e.g., a codon-optimized nucleotide sequence) of SEQ ID NO: 2 or an antigenic fragment thereof.

[0243] In one embodiment, a polynucleotide (e.g., mRNA) encoding a checkpoint cancer vaccine comprising one or more (e.g., 1, 2, 3, 4, or more) IDO1 antigenic peptides further comprises one or more elements (e.g., a 5'UTR and / or a 3'UTR). In one embodiment, the 5'UTR and / or 3'UTR comprises one or more microRNA (mIR) binding sites, e.g., as disclosed herein. Exemplary 5'UTRs and 3'UTRs are disclosed herein in the section entitled "5'UTRs and 3'UTRs."

[0244] PD-L1 molecule PD-L1 (also known as programmed death-ligand 1, CD274, and B7-H1) is a membrane-anchored protein expressed on hematopoietic cells, including antigen-presenting cells such as dendritic cells and macrophages. PD-L1 is also expressed on activated T cells, B cells, and monocytes, as well as peripheral non-hematopoietic tissues, including the liver, heart, skeletal muscle, placenta, lung, and kidney (Dai S et al. (2014) Cell Immunol 290, 72-79). PD-L1 binds to its cognate receptor, PD-1, a co-inhibitory transmembrane receptor expressed on T cells, B cells, natural killer cells, and thymocytes. PD-1 engagement with PD-L1 inhibits T cell receptor (TCR) signaling through the recruitment of regulatory phosphatases, potentially resulting in decreased IL2 production and glucose metabolism. The continuous interaction of PD-1 and PD-L1 can lead to the induction of T cell anergy or the conversion of naive cells into inducible regulatory T cells (iTregs). The PD-L1 / PD-1 pathway has important functions in immune regulation (e.g., inhibition of T cell proliferation, cytotoxic activity, and cytokine production) and promotes the development and function of Tregs.

[0245] In one aspect, the present disclosure provides LNP compositions comprising, e.g., polynucleotides encoding, a checkpoint cancer vaccine comprising, e.g., one or more (e.g., 1, 2, 3, 4, or more) PD-L1 antigenic peptides, e.g., as described herein.

[0246] In one embodiment, the one or more PD-L1 antigenic peptides comprise a naturally occurring PD-L1 molecule, a fragment (e.g., an antigenic fragment) of a naturally occurring PD-L1 molecule, or a variant thereof. In one embodiment, the PD-L1 antigenic peptide comprises a variant (e.g., a PD-L1 variant) of a naturally occurring PD-L1 molecule, or a fragment thereof. In one embodiment, an LNP composition comprising a polynucleotide encoding a checkpoint cancer vaccine comprising one or more (e.g., 1, 2, 3, 4, or more) IDO antigenic peptides and one or more (e.g., 1, 2, 3, 4, or more) PD-L1 antigenic peptides may be administered alone or in combination with additional agents.

[0247] In one aspect, the LNP compositions disclosed herein comprise a polynucleotide encoding a checkpoint cancer vaccine comprising one or more (e.g., 1, 2, 3, 4, or more) PD-L1 antigenic peptides. In one embodiment, the IDO antigenic peptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a PD-L1 amino acid sequence provided in Table 1A (e.g., SEQ ID NO: 3), or an antigenic fragment thereof. In one embodiment, the PD-L1 antigenic peptide comprises the amino acid sequence of a PD-L1 amino acid sequence provided in Table 1A (e.g., SEQ ID NO: 3), or an antigenic fragment thereof. In one embodiment, the PD-L1 antigenic peptide comprises the amino acid sequence of SEQ ID NO: 3, or an antigenic fragment thereof. In one embodiment, the PD-L1 antigenic peptide comprises an amino acid sequence of SEQ ID NO: 3, or an antigenic fragment thereof. In one embodiment, the PD-L1 antigenic peptide comprises SEQ ID NO: 3, or an antigenic fragment thereof.

[0248] In one embodiment, the PD-L1 antigenic peptide includes the amino acid sequence for a leader sequence and / or affinity tag (e.g., a leader sequence described herein and / or an affinity tag described herein). In one embodiment, the PD-L1 antigenic peptide does not include the amino acid sequence for a leader sequence and / or affinity tag (e.g., a leader sequence described herein and / or an affinity tag described herein).

[0249] In one embodiment, a polynucleotide encoding a checkpoint cancer vaccine comprising one or more (e.g., 1, 2, 3, 4, or more) PD-L1 antigenic peptides comprises a nucleotide sequence (e.g., a codon-optimized nucleotide sequence) that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 4, or an antigenic fragment thereof. In one embodiment, a polynucleotide encoding a checkpoint cancer vaccine comprising one or more (e.g., 1, 2, 3, 4, or more) PD-L1 antigenic peptides comprises the nucleotide sequence (e.g., a codon-optimized nucleotide sequence) of SEQ ID NO: 4, or an antigenic fragment thereof.

[0250] In one embodiment, a polynucleotide (e.g., mRNA) encoding a checkpoint cancer vaccine comprising one or more (e.g., 1, 2, 3, 4, or more) PD-L1 antigenic peptides comprises a nucleotide sequence encoding a leader sequence and / or affinity tag (e.g., a leader sequence described herein and / or an affinity tag described herein). In one embodiment, a polynucleotide (e.g., mRNA) encoding a checkpoint cancer vaccine comprising one or more (e.g., 1, 2, 3, 4, or more) PD-L1 antigenic peptides does not comprise a nucleotide sequence encoding a leader sequence and / or affinity tag (e.g., a leader sequence described herein and / or an affinity tag described herein).

[0251] In one embodiment, a polynucleotide (e.g., mRNA) encoding a checkpoint cancer vaccine comprising one or more (e.g., 1, 2, 3, 4, or more) PD-L11 antigenic peptides further comprises one or more elements (e.g., a 5'UTR and / or a 3'UTR). In one embodiment, the 5'UTR and / or 3'UTR comprises one or more microRNA (mIR) binding sites, e.g., as disclosed herein. Exemplary 5'UTRs and 3'UTRs are disclosed herein in the section entitled "5'UTRs and 3'UTRs."

[0252] Exemplary Checkpoint Cancer Vaccines Exemplary checkpoint cancer vaccines include, but are not limited to, those that contain one or more (e.g., 1, 2, 3, 4, or more) IDO antigenic peptides and one or more (e.g., 1, 2, 3, 4, or more) PD-L1 antigenic peptides. In one embodiment, the checkpoint cancer vaccine comprises alternating IDO and PD-L1 antigenic peptides.

[0253] In one embodiment, the checkpoint cancer vaccine comprises one IDO antigenic peptide and one PD-L1 antigenic peptide. In one embodiment, the checkpoint cancer vaccine comprises two IDO antigenic peptides and two PD-L1 antigenic peptides. In one embodiment, the checkpoint cancer vaccine comprises three IDO antigenic peptides and three PD-L1 antigenic peptides. In one embodiment, the checkpoint cancer vaccine comprises four IDO antigenic peptides and four PD-L1 antigenic peptides. In some embodiments, the four IDO antigenic peptides and four PD-L1 antigenic peptides are arranged in an alternating manner. Thus, in one embodiment, the checkpoint cancer vaccine comprises (i) an IDO antigenic peptide, (ii) a PD-L1 antigenic peptide, (iii) an IDO antigenic peptide, (iv) a PD-L1 antigenic peptide, (v) an IDO antigenic peptide, (vi) a PD-L1 antigenic peptide, (vii) an IDO antigenic peptide, and (viii) a PD-L1 antigenic peptide.

[0254] In one embodiment, the alternating IDO antigenic peptide and PD-L1 antigenic peptide comprise an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an IDO amino acid sequence provided in Table 1A (e.g., SEQ ID NO: 5), or an antigenic fragment thereof. In one embodiment, the alternating IDO antigenic peptide and PD-L1 antigenic peptide comprise the amino acid sequence of an amino acid sequence provided in Table 1A (e.g., SEQ ID NO: 5), or an antigenic fragment thereof. In one embodiment, the alternating IDO antigenic peptide and PD-L1 antigenic peptide comprise the amino acid sequence of SEQ ID NO: 5, or an antigenic fragment thereof.

[0255] In one embodiment, the alternating IDO antigenic peptide and PD-L1 antigenic peptide comprise the amino acid sequence for a leader sequence and / or an affinity tag. In one embodiment, the alternating IDO antigenic peptide and PD-L1 antigenic peptide do not comprise the amino acid sequence for a leader sequence and / or an affinity tag.

[0256] In one embodiment, a polynucleotide encoding a checkpoint cancer vaccine comprising alternating IDO and PD-L1 antigenic peptides comprises a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 6, 300, 301, or 302, or an antigenic fragment thereof. In one embodiment, a polynucleotide (e.g., mRNA) encoding a checkpoint cancer vaccine comprising alternating IDO and PD-L1 antigenic peptides comprises the nucleotide sequence of SEQ ID NO: 6, 300, 301, or 302, or an antigenic fragment thereof. In one embodiment, a polynucleotide encoding a checkpoint cancer vaccine comprising alternating IDO and PD-L1 antigenic peptides comprises a codon-optimized nucleotide sequence.

[0257] In some embodiments, a polynucleotide comprising an mRNA nucleotide sequence encoding a checkpoint cancer vaccine comprising one or more (e.g., 1, 2, 3, 4, or more) IDO antigenic peptides and one or more (e.g., 1, 2, 3, 4, or more) PD-L1 antigenic peptides comprises the nucleotide sequence of SEQ ID NO:300, which consists, from the 5' to the 3' end, of the 5' UTR of SEQ ID NO:56, the ORF sequence of SEQ ID NO:6, and the 3' UTR of SEQ ID NO:108.

[0258] In some embodiments, a polynucleotide encoding a checkpoint cancer vaccine comprising one or more (e.g., 1, 2, 3, 4, or more) IDO antigenic peptides and one or more (e.g., 1, 2, 3, 4, or more) PD-L1 antigenic peptides comprises, from the 5' to the 3' end: (i) a 5' cap (such as cap C1 provided herein); (ii) 5'UTR (a sequence provided herein, such as SEQ ID NO: 56); (iii) an open reading frame encoding an alternating IDO antigenic peptide and a PD-L1 antigenic peptide (e.g., a sequence-optimized nucleic acid sequence encoding an alternating IDO antigenic peptide and a PD-L1 antigenic peptide shown as SEQ ID NO: 6); (iv) at least one stop codon (if not present at the 5′ end of the 3′ UTR); (v) 3'UTR (sequence provided herein, such as SEQ ID NO: 108); and (vi) a polyA tail provided herein (e.g., SEQ ID NO: 502). Includes.

[0259] In some embodiments, a polynucleotide comprising an mRNA nucleotide sequence encoding a checkpoint cancer vaccine comprising one or more (e.g., 1, 2, 3, 4, or more) IDO antigenic peptides and one or more (e.g., 1, 2, 3, 4, or more) PD-L1 antigenic peptides comprises the nucleotide sequence of SEQ ID NO:301, which consists, from the 5' to the 3' end, of the 5' UTR of SEQ ID NO:272, the ORF sequence of SEQ ID NO:6, and the 3' UTR of SEQ ID NO:108.

[0260] In some embodiments, a polynucleotide encoding a checkpoint cancer vaccine comprising one or more (e.g., 1, 2, 3, 4, or more) IDO antigenic peptides and one or more (e.g., 1, 2, 3, 4, or more) PD-L1 antigenic peptides comprises, from the 5' to the 3' end: (i) a 5' cap (such as cap C1 provided herein); (ii) 5'UTR (a sequence provided herein, such as SEQ ID NO: 272); (iii) an open reading frame encoding an alternating IDO antigenic peptide and a PD-L1 antigenic peptide (e.g., a sequence-optimized nucleic acid sequence encoding an alternating IDO antigenic peptide and a PD-L1 antigenic peptide shown as SEQ ID NO: 6); (iv) at least one stop codon (if not present at the 5′ end of the 3′ UTR); (v) 3'UTR (sequence provided herein, such as SEQ ID NO: 108); and (vi) a polyA tail provided herein (e.g., SEQ ID NO: 502). Includes.

[0261] In some embodiments, a polynucleotide comprising an mRNA nucleotide sequence encoding a checkpoint cancer vaccine comprising one or more (e.g., 1, 2, 3, 4, or more) IDO antigenic peptides and one or more (e.g., 1, 2, 3, 4, or more) PD-L1 antigenic peptides comprises the nucleotide sequence of SEQ ID NO:302, which consists, from the 5' to the 3' end, of the 5' UTR of SEQ ID NO:273, the ORF sequence of SEQ ID NO:6, and the 3' UTR of SEQ ID NO:108.

[0262] In some embodiments, a polynucleotide encoding a checkpoint cancer vaccine comprising one or more (e.g., 1, 2, 3, 4, or more) IDO antigenic peptides and one or more (e.g., 1, 2, 3, 4, or more) PD-L1 antigenic peptides comprises, from the 5' to the 3' end: (i) a 5' cap (such as cap C1 provided herein); (ii) 5'UTR (a sequence provided herein, such as SEQ ID NO: 273); (iii) an open reading frame encoding an alternating IDO antigenic peptide and a PD-L1 antigenic peptide (e.g., a sequence-optimized nucleic acid sequence encoding an alternating IDO antigenic peptide and a PD-L1 antigenic peptide shown as SEQ ID NO: 6); (iv) at least one stop codon (if not present at the 5′ end of the 3′ UTR); (v) 3'UTR (sequence provided herein, such as SEQ ID NO: 108); and (vi) a polyA tail provided herein (e.g., SEQ ID NO: 502). Includes.

[0263] In one embodiment, the polynucleotide encoding the checkpoint cancer vaccine comprises the nucleotide sequence of variant 1, variant 2, or variant 3, as described in Table 2A.

[0264] In one aspect, the LNP compositions disclosed herein comprise a polynucleotide encoding a checkpoint cancer vaccine, e.g., comprising one or more IDO antigenic peptides and one or more PD-L1 antigenic peptides described herein. In one embodiment, the checkpoint cancer vaccine comprises a half-life extender (e.g., a protein (or fragment thereof) that binds to a serum protein (such as albumin, IgG, FcRn, or transferrin)). In one embodiment, the half-life extender is an immunoglobulin Fc region or a variant thereof (e.g., IgG1 Fc).

[0265] In one embodiment, the checkpoint cancer vaccine further comprises a targeting moiety, which in one embodiment comprises an antibody molecule (e.g., a Fab or scFv), a receptor molecule (e.g., a receptor, a receptor fragment, or a functional variant thereof), a ligand molecule (e.g., a ligand, a ligand fragment, or a functional variant thereof), or a combination thereof. [Table 1A]

[0266] In some embodiments, a polynucleotide of the disclosure (e.g., a polynucleotide comprising an mRNA nucleotide sequence encoding a checkpoint cancer vaccine comprising one or more IDO antigenic peptides and one or more PD-L1 antigenic peptides) comprises: (1) a 5' cap (e.g., as disclosed herein, e.g., as provided in Table 2A or described herein); (2) a 5' UTR (e.g., as provided in Table 2A); (3) a nucleotide sequence ORF provided in Table 2A (e.g., SEQ ID NO: 2 or 5); (4) a stop codon; (5) a 3' UTR (e.g., as provided in Table 2A); and (6) a tail (e.g., a polyA tail) (e.g., as disclosed herein, e.g., a polyA tail of about 100 residues (e.g., SEQ ID NO: 502)).

[0267] In some embodiments, a polynucleotide comprising an mRNA nucleotide sequence encoding a checkpoint cancer vaccine comprising one or more IDO antigenic peptides and one or more PD-L1 antigenic peptides comprises the nucleotide sequence of SEQ ID NO:300, which comprises, from the 5' to the 3' end, the 5' UTR of SEQ ID NO:56, the ORF sequence of SEQ ID NO:6, and the 3' UTR of SEQ ID NO:108.

[0268] In some embodiments, a polynucleotide comprising an mRNA nucleotide sequence encoding a checkpoint cancer vaccine comprising one or more IDO antigenic peptides and one or more PD-L1 antigenic peptides comprises the nucleotide sequence of SEQ ID NO:301, which comprises, from the 5' to the 3' end, the 5' UTR of SEQ ID NO:272, the ORF sequence of SEQ ID NO:6, and the 3' UTR of SEQ ID NO:108.

[0269] In some embodiments, a polynucleotide comprising an mRNA nucleotide sequence encoding a checkpoint cancer vaccine comprising one or more IDO antigenic peptides and one or more PD-L1 antigenic peptides comprises the nucleotide sequence of SEQ ID NO:302, which comprises, from the 5' to the 3' end, the 5' UTR of SEQ ID NO:273, the ORF sequence of SEQ ID NO:6, and the 3' UTR of SEQ ID NO:108.

[0270] In some embodiments, all of the 5'UTR sequences, ORF sequences, and / or 3'UTR sequences comprise the modification(s) described in Table 2A. In some embodiments, one, two, or all of the 5'UTR sequences, ORF sequences, and / or 3'UTR sequences do not comprise the modification(s) described in Table 2A. In some embodiments, a 5'UTR described in Table 2A additionally comprises a first nucleotide that is "A" or "G." [Table 2A-1] [Table 2A-2] [Table 2A-3]

[0271] Therapeutic LNP Disclosed herein, among other things, are LNP compositions comprising a polynucleotide encoding a checkpoint cancer vaccine comprising one or more IDO antigenic peptides and one or more PD-L1 antigenic peptides, which may be administered alone or in combination with an additional agent (e.g., a standard medical therapy). In one embodiment, the additional agent is a polypeptide, such as a protein, a fusion protein, a soluble protein, or an antibody (e.g., an antibody fragment, Fab, scFv, single-domain antibody, humanized antibody, bispecific antibody, and / or multispecific antibody). In one embodiment, the LNP composition and the additional agent are in the same composition or separate compositions. In one embodiment, the LNP composition and the additional agent are administered substantially simultaneously or sequentially.

[0272] Lipid content of LNPs As described above with respect to lipids, the LNPs disclosed herein include (i) ionizable lipids; (ii) sterol or other structural lipids; (iii) non-cationic helper lipids or phospholipids; and (iv) PEG-lipids. These categories of lipids are described in more detail below.

[0273] Ionizable lipids The lipid nanoparticles of the present disclosure comprise one or more ionizable lipids.In certain embodiments, the ionizable lipids of the present disclosure comprise a central amine moiety and at least one biodegradable group.The ionizable lipids described herein can be advantageously used in the lipid nanoparticles of the present disclosure to deliver nucleic acid molecules to mammalian cells or organs.The structures of the ionizable lipids described below include the prefix I to distinguish them from other lipids of the present invention.

[0274] In some embodiments, the present disclosure provides an ionizable amino lipid compound of formula (I): [ka] or its N-oxide, or its salt or isomer [In the formula, R' ais R' 分岐鎖 and; R' 分岐鎖 teeth, [ka] and; [ka] represents the attachment point; R aα , R aβ , R aγ , and R aδ are H and C, respectively. 2-12 Alkyl, and C 2-12 alkenyl; R 2 and R 3 are respectively, C 1-14 Alkyl and C 2-14 alkenyl; R 4 is -(CH2) n OH, where n is selected from the group consisting of 1, 2, 3, 4, and 5; and [ka] is selected from the group consisting of [ka] represents the attachment point; R 10 is N(R)2; each R is C 1-6 Alkyl, C 2-3 alkenyl, and H; n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R 5 is C 1-3 Alkyl, C 2-3 independently selected from the group consisting of alkenyl and H; Each R 6 is C 1-3 Alkyl, C 2-3 independently selected from the group consisting of alkenyl and H; M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-; R' is C 1-12 Alkyl or C 2-12 is alkenyl; l is selected from the group consisting of 1, 2, 3, 4, and 5; m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13. Regarding.

[0275] In some embodiments of the compounds of Formula (I), R' a is R' 分岐鎖 and R' 分岐鎖 teeth, [ka] and; [ka] represents the attachment point; R aα , R aβ , R aγ , and R aδ are H; R 2 and R 3 are respectively, C 1-14 alkyl; R 4 is -(CH2) n OH; n is 2; R 5 are H; R 6 are each H; M and M' are each -C(O)O-; R' is C 1-12 is alkyl; l is 5; and m is 7.

[0276] In some embodiments of the compounds of Formula (I), R' a is R' 分岐鎖 and R' 分岐鎖 teeth, [ka] and; [ka] represents the attachment point; R aα , R aβ , R aγ , and R aδ are H; R 2 and R 3 are respectively, C 1-14 alkyl; R 4 is -(CH2) n OH; n is 2; R 5 are H; R 6 are each H; M and M' are each -C(O)O-; R' is C 1-12 is alkyl; l is 3; and m is 7.

[0277] In some embodiments of the compounds of Formula (I), R' a is R' 分岐鎖 and R' 分岐鎖 teeth, [ka] and; [ka] represents the attachment point; R aα is C 2-12 alkyl; R aβ , R aγ , and R aδ are H; R 2 and R 3 are respectively, C 1-14 alkyl; R 4 teeth, [ka] and;R 10 is NH(C 1-6 alkyl); n2 is 2; R 5 is H;R 6 are each H; M and M' are each -C(O)O-; R' is C 1-12 is alkyl; l is 5; and m is 7.

[0278] In some embodiments of the compounds of Formula (I), R' a is R' 分岐鎖 and R' 分岐鎖 teeth, [ka] and; [ka] represents the attachment point; R aα , R aβ , and R aδ are H; R aγ is C 2-12 alkyl; R 2 and R 3 are respectively, C 1-14 alkyl; R 4 is -(CH2) n OH; n is 2; R 5 are H; R 6 are each H; M and M' are each -C(O)O-; R' is C 1-12 is alkyl; l is 5; and m is 7.

[0279] In some embodiments, the compound of formula (I) is [ka] is selected from.

[0280] In some embodiments, the compound of formula (I) is [ka] is.

[0281] In some embodiments, the compound of formula (I) is [ka] is.

[0282] In some embodiments, the compound of formula (I) is [ka] is.

[0283] In some embodiments, the compound of formula (I) is [ka] is.

[0284] In some embodiments, the present disclosure provides a compound of formula (Ia): [ka] or its N-oxide, or its salt or isomer [In the formula, R' a is R' 分岐鎖 and; R' 分岐鎖 teeth, [ka] and; [ka] represents the attachment point; R aβ , R aγ , and R aδ are H and C, respectively. 2-12 Alkyl, and C 2-12 alkenyl; R 2 and R 3 are respectively, C 1-14 Alkyl and C 2-14 alkenyl; R 4 is -(CH2) n OH, where n is selected from the group consisting of 1, 2, 3, 4, and 5; and [ka] is selected from the group consisting of [ka] represents the attachment point; R 10 is N(R)2; each R is C 1-6 Alkyl, C 2-3 alkenyl, and H; n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R 5 is C 1-3 Alkyl, C 2-3 independently selected from the group consisting of alkenyl and H; Each R 6 is C 1-3 Alkyl, C 2-3 independently selected from the group consisting of alkenyl and H; M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-; R' is C 1-12 Alkyl or C 2-12 is alkenyl; l is selected from the group consisting of 1, 2, 3, 4, and 5; m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13. Regarding.

[0285] In some embodiments, the present disclosure provides a compound of formula (Ib): [ka] or its N-oxide, or its salt or isomer [In the formula, R' a is R' 分岐鎖 and; R' 分岐鎖 teeth, [ka] and; [ka] represents the attachment point; R aα , R aβ , R aγ , and R aδ are H and C, respectively. 2-12 Alkyl, and C 2-12 alkenyl; R 2 and R 3 are respectively, C 1-14 Alkyl and C 2-14 alkenyl; R 4 is -(CH2) n OH, where n is selected from the group consisting of 1, 2, 3, 4, and 5; Each R 5 is C 1-3 Alkyl, C 2-3 independently selected from the group consisting of alkenyl and H; Each R 6 is C 1-3 Alkyl, C 2-3 independently selected from the group consisting of alkenyl and H; M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-; R' is C 1-12 Alkyl or C 2-12 is alkenyl; l is selected from the group consisting of 1, 2, 3, 4, and 5; m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13. Regarding.

[0286] In some embodiments of Formula (I) or (Ib), R' a is R' 分岐鎖 and R' 分岐鎖 teeth, [ka] and; [ka] represents the attachment point; R aβ , R aγ , and R aδ are H; R 2 and R 3 are respectively, C 1-14 alkyl; R 4 is -(CH2) n OH; n is 2; R 5 are H; R 6 are each H; M and M' are each -C(O)O-; R' is C 1-12 is alkyl; l is 5; and m is 7.

[0287] In some embodiments of Formula (I) or (Ib), R' a is R' 分岐鎖 and R' 分岐鎖 teeth, [ka] and; [ka] represents the attachment point; R aβ , R aγ , and R aδ are H; R 2 and R 3 are respectively, C 1-14 alkyl; R 4 is -(CH2) n OH; n is 2; R 5 are H; R 6 are each H; M and M' are each -C(O)O-; R' is C 1-12 is alkyl; l is 3; and m is 7.

[0288] In some embodiments of Formula (I) or (Ib), R' a is R' 分岐鎖 and R' 分岐鎖 teeth, [ka] and; [ka] represents the attachment point; R aβ and R aδ are H; R aγ is C 2-12 alkyl; R 2 and R 3 are respectively, C 1-14 alkyl; R 4 is -(CH2) n OH; n is 2; R 5 are H; R 6 are each H; M and M' are each -C(O)O-; R' is C 1-12 is alkyl; l is 5; and m is 7.

[0289] In some embodiments, the present disclosure provides a compound of formula (Ic): [ka] or its N-oxide, or its salt or isomer [In the formula, R' a is R' 分岐鎖 and; R' 分岐鎖 teeth, [ka] and; [ka] represents the attachment point; R aα , R aβ , R aγ , and R aδ are H and C, respectively. 2-12 Alkyl, and C 2-12 alkenyl; R 2 and R 3 are respectively, C 1-14 Alkyl and C 2-14alkenyl; R 4 teeth, [ka] and [ka] represents the attachment point; R 10 is N(R)2; each R is C 1-6 Alkyl, C 2-3 alkenyl, and H; n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R 5 is C 1-3 Alkyl, C 2-3 independently selected from the group consisting of alkenyl and H; Each R 6 is C 1-3 Alkyl, C 2-3 independently selected from the group consisting of alkenyl and H; M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-; R' is C 1-12 Alkyl or C 2-12 is alkenyl; l is selected from the group consisting of 1, 2, 3, 4, and 5; m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13. Regarding.

[0290] In some embodiments, R' a is R' 分岐鎖 and R' 分岐鎖 teeth, [ka] and; [ka] represents the attachment point; Raβ , R aγ , and R aδ are H; R aα is C 2-12 alkyl; R 2 and R 3 are respectively, C 1-14 alkyl; R 4 teeth, [ka] and; [ka] represents the attachment point; R 10 is NH(C 1-6 alkyl); n2 is 2; R 5 are H; R 6 are each H; M and M' are each -C(O)O-; R' is C 1-12 is alkyl; l is 5; and m is 7.

[0291] In some embodiments, the compound of Formula (Ic) is [ka] is.

[0292] In some embodiments, the present disclosure provides a compound of formula (II): [ka] or its N-oxide, or its salt or isomer [In the formula, R' a is R' 分岐鎖 or R' 環式 and; R' 分岐鎖 teeth, [ka] and R' 環式 teeth, [ka] and; R' b teeth, [ka] and; [ka] represents the attachment point; R aγ and R aδ are H and C, respectively. 1-12 Alkyl, and C 2-12 alkenyl; R aγ and R aδ At least one of the 1-12 Alkyl and C 2-12 alkenyl; R bγ and R bδ are H and C, respectively. 1-12 Alkyl, and C 2-12 alkenyl; R bγ and R bδ At least one of the 1-12 Alkyl and C 2-12 alkenyl; R 2 and R 3 are respectively, C 1-14 Alkyl and C 2-14 alkenyl; R 4 is -(CH2) n OH, where n is selected from the group consisting of 1, 2, 3, 4, and 5; and [ka] is selected from the group consisting of [ka] represents the attachment point; R 10 is N(R)2; each R is C1-6 Alkyl, C 2-3 alkenyl, and H; n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R' is independently C 1-12 Alkyl or C 2-12 is alkenyl; Y a is C 3-6 It is a carbocyclic ring; R * ” a is C 1-15 Alkyl and C 2-15 alkenyl; s is 2 or 3; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9. Regarding.

[0293] In some embodiments, the present disclosure provides a compound of formula (II-a): [ka] or its N-oxide, or its salt or isomer [In the formula, R' a is R' 分岐鎖 or R' 環式 and; R' 分岐鎖 teeth, [ka] and R' b teeth, [ka] and; [ka] represents the attachment point; R aγ and R aδ are H and C, respectively. 1-12Alkyl, and C 2-12 alkenyl; R aγ and R aδ At least one of the 1-12 Alkyl and C 2-12 alkenyl; R bγ and R bδ are H and C, respectively. 1-12 Alkyl, and C 2-12 alkenyl; R bγ and R bδ At least one of the 1-12 Alkyl and C 2-12 alkenyl; R 2 and R 3 are respectively, C 1-14 Alkyl and C 2-14 alkenyl; R 4 is -(CH2) n OH, where n is selected from the group consisting of 1, 2, 3, 4, and 5; and [ka] is selected from the group consisting of [ka] represents the attachment point; R 10 is N(R)2; each R is C 1-6 Alkyl, C 2-3 alkenyl, and H; n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R' is independently C 1-12 Alkyl or C 2-12 is alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9. Regarding.

[0294] In some embodiments, the present disclosure provides a compound of formula (II-b): [ka] or its N-oxide, or its salt or isomer [In the formula, R' a is R' 分岐鎖 or R' 環式 and; R' 分岐鎖 teeth, [ka] and R' b teeth, [ka] and; [ka] represents the attachment point; R aγ and R bγ are respectively, C 1-12 Alkyl and C 2-12 alkenyl; R 2 and R 3 are respectively, C 1-14 Alkyl and C 2-14 alkenyl; R 4 is -(CH2) n OH, where n is selected from the group consisting of 1, 2, 3, 4, and 5; and [ka] is selected from the group consisting of [ka] represents the attachment point; R 10 is N(R)2; each R is C 1-6 Alkyl, C2-3 alkenyl, and H; n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R' is independently C 1-12 Alkyl or C 2-12 is alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9. Regarding.

[0295] In some embodiments, the present disclosure provides a compound of formula (II-c): [ka] or its N-oxide, or its salt or isomer [In the formula, R' a is R' 分岐鎖 or R' 環式 and; R' 分岐鎖 teeth, [ka] and R' b teeth, [ka] and; [ka] represents the attachment point; R aγ is C 1-12 Alkyl and C 2-12 alkenyl; R 2 and R 3 are respectively, C 1-14 Alkyl and C 2-14 alkenyl; R 4 is -(CH2) nOH, where n is selected from the group consisting of 1, 2, 3, 4, and 5; and [ka] is selected from the group consisting of [ka] represents the attachment point; R 10 is N(R)2; each R is C 1-6 Alkyl, C 2-3 alkenyl, and H; n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; R' is C 1-12 Alkyl or C 2-12 is alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9. Regarding.

[0296] In some embodiments, the present disclosure provides a compound of formula (II-d): [ka] or its N-oxide, or its salt or isomer [In the formula, R' a is R' 分岐鎖 or R' 環式 and; R' 分岐鎖 teeth, [ka] and R' b teeth, [ka] and; [ka] represents the attachment point; R aγ and R bγ are respectively, C 1-12 Alkyl and C 2-12 alkenyl; R 4 is -(CH2) n OH, where n is selected from the group consisting of 1, 2, 3, 4, and 5; and [ka] is selected from the group consisting of [ka] represents the attachment point; R 10 is N(R)2; each R is C 1-6 Alkyl, C 2-3 alkenyl, and H; n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R' is independently C 1-12 Alkyl or C 2-12 is alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9. Regarding.

[0297] In some embodiments, the present disclosure provides a compound of formula (II-e): [ka] or its N-oxide, or its salt or isomer [In the formula, R' a is R' 分岐鎖 or R' 環式 and; R' 分岐鎖 teeth, [ka] and R' bteeth, [ka] and; [ka] represents the attachment point; R aγ is C 1-12 Alkyl and C 2-12 alkenyl; R 2 and R 3 are respectively, C 1-14 Alkyl and C 2-14 alkenyl; R 4 is -(CH2) n OH, where n is selected from the group consisting of 1, 2, 3, 4, and 5; R' is C 1-12 Alkyl or C 2-12 is alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9. Regarding.

[0298] In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), m and l are each independently selected from 4, 5, and 6. In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), m and l are each 5.

[0299] In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), each R' is independently C 1-12 In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), each R' is independently C2-5 It is alkyl.

[0300] In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' b teeth, [ka] and R 2 and R 3 are each independently, C 1-14 In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' is alkyl. b teeth, [ka] and R 2 and R 3 are each independently, C 6-10 In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' is alkyl. b teeth, [ka] and R 2 and R 3 are each C8 alkyl.

[0301] In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' 分岐鎖 teeth, [ka] and R' b teeth, [ka] and R aγ is C 1-12 alkyl, and R2 and R 3 are each independently, C 6-10 In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' is alkyl. 分岐鎖 teeth, [ka] and R' b teeth, [ka] and R aγ is C 2-6 alkyl, and R 2 and R 3 are each independently, C 6-10 In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' is alkyl. 分岐鎖 teeth, [ka] and R' b teeth, [ka] and R aγ is C 2-6 alkyl, and R 2 and R 3 are each C8 alkyl.

[0302] In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' 分岐鎖 teeth, [ka] and R' b teeth, [ka] and Raγ and R bγ are respectively, C 1-12 In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' is alkyl. 分岐鎖 teeth, [ka] and R' b teeth, [ka] and R aγ and R bγ are respectively, C 2-6 It is alkyl.

[0303] In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), m and l are each independently selected from 4, 5, and 6; and each R′ is independently selected from C 1-12 In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), m and l are each 5 and each R' is independently C 2-5 It is alkyl.

[0304] In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' 分岐鎖 teeth, [ka] and R' b teeth, [ka] wherein m and l are each independently selected from 4, 5, and 6; and each R′ is independently selected from C 1-12 alkyl, and R aγ and R bγ are respectively, C 1-12In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' is alkyl. 分岐鎖 teeth, [ka] and R' b teeth, [ka] wherein m and l are each 5, and each R' is independently C 2-5 alkyl, and R aγ and R bγ are respectively, C 2-6 It is alkyl.

[0305] In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' 分岐鎖 teeth, [ka] and R' b teeth, [ka] wherein m and l are each independently selected from 4, 5, and 6; and R′ is C 1-12 alkyl, and R aγ is C 1-12 alkyl, and R 2 and R 3 are each independently, C 6-10 In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' is alkyl. 分岐鎖 teeth, [ka] and R' b teeth, [ka] wherein m and l are each 5; and R' is C 2-5 alkyl, and R aγ is C 2-6 alkyl, and R 2 and R 3 are each C8 alkyl.

[0306] In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R 4 teeth, [ka] and R 10 is NH(C 1-6 In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R 4 teeth, [ka] and R 10 is NH(CH3) and n2 is 2.

[0307] In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' 分岐鎖 teeth, [ka] and R' b teeth, [ka] wherein m and l are each independently selected from 4, 5, and 6; and each R′ is independently selected from C 1-12 alkyl, and R aγ and R bγ are respectively, C 1-12 alkyl, and R 4 teeth, [ka] and R 10 is NH(C 1-6 alkyl) and n2 is 2. In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' 分岐鎖 teeth, [ka] and R' b teeth, [ka] wherein m and l are each 5, and each R' is independently C 2-5 alkyl, and R aγ and R bγ are respectively, C 2-6 alkyl, and R 4 teeth, [ka] and R 10 is NH(CH3) and n2 is 2.

[0308] In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' 分岐鎖 teeth, [ka] and R' b teeth, [ka] wherein m and l are each independently selected from 4, 5, and 6; and R′ is C 1-12 alkyl, and R 2 and R 3 are each independently, C 6-10 alkyl, and R aγ is C 1-12 alkyl, and R4 teeth, [ka] and R 10 is NH(C 1-6 alkyl) and n2 is 2. In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' 分岐鎖 teeth, [ka] and R' b teeth, [ka] wherein m and l are each 5; and R' is C 2-5 alkyl, and R aγ is C 2-6 alkyl, and R 2 and R 3 are each C8 alkyl, and R 4 teeth, [ka] and R 10 is NH(CH3) and n2 is 2.

[0309] In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R 4 is -(CH2) n OH and n is 2, 3, or 4. In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R 4 is -(CH2) n OH and n is 2.

[0310] In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R'分岐鎖 teeth, [ka] and R' b teeth, [ka] wherein m and l are each independently selected from 4, 5, and 6; and each R′ is independently selected from C 1-12 alkyl, and R aγ and R bγ are respectively, C 1-12 alkyl, and R 4 is -(CH2) n OH and n is 2, 3, or 4. In some embodiments of compounds of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R' 分岐鎖 teeth, [ka] and R' b teeth, [ka] wherein m and l are each 5, and each R' is independently C 2-5 alkyl, and R aγ and R bγ are respectively, C 2-6 alkyl, and R 4 is -(CH2) n OH and n is 2.

[0311] In some embodiments, the present disclosure provides a compound of formula (II-f): [ka] or its N-oxide, or its salt or isomer [In the formula, R' a is R' 分岐鎖 or R' 環式 and; R' 分岐鎖 teeth, [ka] and R' b teeth, [ka] and; [ka] represents the attachment point; R aγ is C 1-12 is alkyl; R 2 and R 3 are each independently, C 1-14 is alkyl; R 4 is -(CH2) n OH, where n is selected from the group consisting of 1, 2, 3, 4, and 5; R' is C 1-12 is alkyl; m is selected from 4, 5, and 6; l is selected from 4, 5, and 6. Regarding.

[0312] In some embodiments of compounds of Formula (II-f), m and l are each 5; and n is 2, 3, or 4.

[0313] In some embodiments of compounds of Formula (II-f), R' is C 2-5 alkyl, and R aγ is C 2-6 alkyl, and R 2 and R 3 are respectively, C 6-10 It is alkyl.

[0314] In some embodiments of compounds of Formula (II-f), m and l are each 5; n is 2, 3, or 4; and R′ is C 2-5 alkyl, and R aγ is C 2-6 alkyl, and R2 and R 3 are respectively, C 6-10 It is alkyl.

[0315] In some embodiments, the present disclosure provides a compound of formula (II-g): [ka] [In the formula, R aγ is C 2-6 is alkyl; R' is C 2-5 is alkyl; R 4 is -(CH2) n OH, where n is selected from the group consisting of 3, 4, and 5; and [ka] is selected from the group consisting of [ka] represents the attachment point, and R 10 is NH(C 1-6 alkyl), and n2 is selected from the group consisting of 1, 2, and 3. Regarding.

[0316] In some embodiments, the present disclosure provides a compound of formula (II-h): [ka] [In the formula, R aγ and R bγ are each independently, C 2-6 is alkyl; Each R' is independently C 2-5 is alkyl; R 4 is -(CH2) n OH, where n is selected from the group consisting of 3, 4, and 5; and [ka] is selected from the group consisting of [ka] represents the attachment point, and R 10 is NH(C 1-6 alkyl), and n2 is selected from the group consisting of 1, 2, and 3. Regarding.

[0317] In some embodiments of compounds of Formula (II-g) or (II-h), R 4 teeth, [ka] and R 10 is NH(CH3) and n2 is 2.

[0318] In some embodiments of compounds of Formula (II-g) or (II-h), R 4 is -(CH2)2OH.

[0319] In some embodiments, the present disclosure provides a compound having formula (III): [ka] or a salt or isomer thereof [wherein: R1, R2, R3, R4, and R5 are C 5-20 Alkyl, C 5-20 Alkenyl, -R"MR', -R * YR”, -YR”, and -R * independently selected from the group consisting of "OR"; each M is independently selected from the group consisting of -C(O)O-, -OC(O)-, -OC(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH), -P(O)(OR')O-, -S(O)-, an aryl group, and a heteroaryl group; X 1 , X 2 , and X3 are independently selected from the group consisting of a bond, —CH—, —(CH)—, —CHR—, —CHY—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)—CH—, —CH—C(O)—, —C(O)O—CH—, —OC(O)—CH—, —CH—C(O)O—, —CH—OC(O)—, —CH(OH)—, —C(S)—, and —CH(SH)—; Each Y is independently C 3-6 It is a carbocyclic ring; Each R * is C 1-12 Alkyl and C 2-12 alkenyl; Each R is C 1-3 Alkyl and C 3-6 independently selected from the group consisting of: carbocycle; Each R' is C 1-12 Alkyl, C 2-12 independently selected from the group consisting of alkenyl, and H; Each R” is C 3-12 Alkyl and C 3-12 alkenyl; i)X 1 , X 2 , and X 3 is not —CH—; and / or ii) at least one of R1, R2, R3, R4, and R5 is -R"MR'] Regarding.

[0320] In some embodiments, R1, R2, R3, R4, and R5 are each C 5-20 is alkyl; X 1 is -CH2-; X 2 and X 3 are each -C(O)-.

[0321] In some embodiments, the compound of formula (III) is [ka] is.

[0322] The central amine moiety of a lipid according to any of the formulas herein, such as a compound having any of formulas (I), (Ia), (Ib), (Ic), (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), or (III) (each of which is preceded by the letter I for clarity), can be protonated at physiological pH. Thus, the lipid can have a positive or partial positive charge at physiological pH. Such lipids can be referred to as cationic lipids or ionizable (amino) lipids. Lipids can also be zwitterionic, i.e., neutral molecules having both positive and negative charges.

[0323] In some embodiments, the amount of ionizable amino lipids of the invention, e.g., compounds having any of formulas (I), (Ia), (Ib), (Ic), (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), or (III) (each of which is preceded by the letter I for clarity), ranges from about 1 mol% to 99 mol% in the lipid composition.

[0324] In one embodiment, the amount of ionizable amino lipids of the invention, e.g., compounds having any of formulas (I), (Ia), (Ib), (Ic), (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), or (III) (each of which is preceded by the letter I for clarity), in the lipid composition is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 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, or 99 mol%.

[0325] In one embodiment, the amount of ionizable amino lipids of the invention, e.g., compounds having any of formulas (I), (Ia), (Ib), (Ic), (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), or (III) (each of which is preceded by the letter I for clarity), in the lipid composition ranges from about 30 mol% to about 70 mol%, from about 35 mol% to about 65 mol%, from about 40 mol% to about 60 mol%, and from about 45 mol% to about 55 mol%.

[0326] In one particular embodiment, the amount of ionizable amino lipids of the invention, e.g., compounds having any of formulas (I), (Ia), (Ib), (Ic), (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), or (III) (each of which is preceded by the letter I for clarity), is about 45 mol % in the lipid composition.

[0327] In one particular embodiment, the amount of ionizable amino lipids of the invention, e.g., compounds having any of formulas (I), (Ia), (Ib), (Ic), (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), or (III) (each of which is preceded by the letter I for clarity), is about 40 mol % in the lipid composition.

[0328] In one particular embodiment, the amount of ionizable amino lipids of the invention, e.g., compounds having any of formulas (I), (Ia), (Ib), (Ic), (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), or (III) (each of which is preceded by the letter I for clarity), is about 50 mol % in the lipid composition.

[0329] In addition to the ionizable amino lipids disclosed herein, e.g., compounds having any of formulas (I), (Ia), (Ib), (Ic), (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), or (III) (each of which is preceded by the letter I for clarity), the lipid-based compositions (e.g., lipid nanoparticles) disclosed herein can include additional components, such as cholesterol and / or cholesterol analogs, non-cationic helper lipids, structural lipids, PEG-lipids, and any combination thereof.

[0330] Additional ionizable lipids of the present invention include 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl 15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMAC ... ), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), Heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-Dioleyloxy-N,N-dimethylaminopropane (DODMA), (13Z,1 65Z)-N,N-Dimethyl-3-nonydocosa-13-16-dien-1-amine (L608), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-Dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)- The ionizable amino lipid may be selected from the non-limiting group consisting of N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2R)), and (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2S)). In addition, the ionizable amino lipid may also be a lipid containing a cyclic amine group.

[0331] The ionizable lipids of the present invention can also be compounds disclosed in International Publication No. WO2017 / 075531A1, which is incorporated herein by reference in its entirety.

[0332] The ionizable lipids of the present invention can also be compounds disclosed in International Publication No. WO2015 / 199952A1, which is incorporated herein by reference in its entirety.

[0333] In any of the above or related embodiments, the ionizable lipids of the LNPs of the present disclosure include, for example, compounds contained in any compound having any of formulas (I), (Ia), (Ib), (Ic), (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), or (III) (each of which is preceded by the letter I for clarity).

[0334] In any of the above or related embodiments, the ionizable lipid of the LNP of the present disclosure comprises a compound, including any of Compound Nos. 18, 25, 301, and 357.

[0335] In any of the above or related aspects, the ionizable lipid of the LNPs of the present disclosure comprises at least one compound selected from the group consisting of Compound Nos. 18, 25, 301, and 357. In another embodiment, the ionizable lipid of the LNPs of the present disclosure comprises a compound selected from the group consisting of Compound Nos. 18, 25, 301, and 357. In another embodiment, the ionizable lipid of the LNPs of the present disclosure comprises Compound 18. In another embodiment, the ionizable lipid of the LNPs of the present disclosure comprises Compound 25.

[0336] In any of the above or related embodiments, the synthesis of compounds of the invention (e.g., compounds including any of Compound Nos. 18, 25, 301, and 357) follows the synthesis descriptions in U.S. Provisional Patent Application No. 62 / 733,315, filed September 19, 2018.

[0337] Representative synthesis routes: Compound I-182: heptadecan-9-yl 8-((3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

[0338] 3-Methoxy-4-(methylamino)cyclobut-3-ene-1,2-dione [ka] To a solution of 3,4-dimethoxy-3-cyclobutene-1,2-dione (1 g, 7 mmol) in 100 mL of diethyl ether was added 2 M methylamine solution in THF (3.8 mL, 7.6 mmol), resulting in the formation of a precipitate. The mixture was stirred at room temperature for 24 hours and then filtered to collect the solid. The solid was washed with diethyl ether, air-dried, and then dissolved in hot EtOAc and filtered. The filtrate was cooled to room temperature and then to 0° C. to give a precipitate, which was isolated via filtration, washed with cold EtOAc, air-dried, and then dried under vacuum to give 3-methoxy-4-(methylamino)cyclobut-3-ene-1,2-dione (0.70 g, 5 mmol, 73%) as a solid. 1 H NMR (300 MHz, DMSO-d6) δ: ppm 8.50 (br. d, 1H, J = 69 Hz);4.27 (s, 3H);3.02 (sdd, 3H, J = 42 Hz, 4.5 Hz).

[0339] Heptadecan-9-yl 8-((3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate [ka] To a solution of heptadecan-9-yl 8-((3-aminopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (200 mg, 0.28 mmol) in 10 mL of ethanol was added 3-methoxy-4-(methylamino)cyclobut-3-ene-1,2-dione (39 mg, 0.28 mmol). The reaction mixture was stirred at room temperature for 20 hours and then concentrated in vacuo to give a residue. The residue was purified by silica gel chromatography (0-100% in dichloromethane (a mixture of 1% NH4OH, 20% MeOH in dichloromethane)) to give heptadecan-9-yl 8-((3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (138 mg, 0.17 mmol, 60%) as a solid. UPLC / ELSD: RT = 3 min. MS (ES): m / z (MH + ) C 51 H 95N3 For O6, 833.4. 1 H NMR (300 MHz, CDCl3) δ: ppm 7.86 (br. s., 1H);4.86 (quintet, 1H, J = 6 Hz);4.05 (t, 2H, J = 6 Hz);3.92 (d, 2H, J = 3 Hz);3.20 (s, 6H);2.63 (br. s, 2H);2.42 (br. s, 3H);2.28 (m, 4H);1.74 (br. s, 2H);1.61 (m, 8H);1.50 (m, 5H);1.41 (m, 3H);1.25 (br. m, 47H);0.88 (t, 9H, J = 7.5 Hz).

[0340] Compound I-301: heptadecan-9-yl 8-((3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(8-oxo-8-(undecan-3-yloxy)octyl)amino)octanoate [ka] Compound I-301 was prepared similarly to compound 182, except that heptadecan-9-yl 8-((3-aminopropyl)(8-oxo-8-(undecan-3-yloxy)octyl)amino)octanoate (500 mg, 0.66 mmol) was used instead of heptadecan-9-yl 8-((3-aminopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate. After aqueous workup, the residue was purified by silica gel chromatography (0-50% in dichloromethane (a mixture of 1% NH4OH, 20% MeOH in dichloromethane)) to give heptadecan-9-yl 8-((3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(8-oxo-8-(undecan-3-yloxy)octyl)amino)octanoate (180 mg, 32%) as a solid. HPLC / UV (254 nm): RT = 6.77 min. MS (CI): m / z (MH + ) 860.7 for C 52 H 97N3 O6. 1 H NMR (300 MHz, CDCl3): δ ppm 4.86-4.79 (m, 2H);3.66 (bs, 2H);3.25 (d, 3H, J = 4.9 Hz);2.56-2.52 (m, 2H);2.42-2.37 (m, 4H);2.28 (dd, 4H, J = 2.7 Hz, 7.4 Hz);1.78-1.68 (m, 3H);1.64-1.50 (m, 16H);1.48-1.38 (m, 6H);1.32-1.18 (m, 43H);0.88-0.84 (m, 12H).

[0341] Cholesterol / Structural Lipids The LNPs described herein comprise one or more structured lipids.

[0342] As used herein, the term "structured lipid" refers to sterol, and also refers to lipids containing a sterol moiety. The incorporation of structured lipids into lipid nanoparticles can help alleviate the aggregation of other lipids in the particles. Structured lipids may include, but are not limited to, cholesterol, fecosterol, ergosterol, basicasterol, tomatidine, tomatine, ursolic acid, α-tocopherol, and mixtures thereof. In certain embodiments, the structured lipid is cholesterol. In certain embodiments, the structured lipid includes cholesterol and corticosteroids (e.g., prednisolone, dexamethasone, prednisone, hydrocortisone, etc.), or a combination thereof.

[0343] In some embodiments, the structured lipid is a sterol. As defined herein, "sterol" is a subgroup of steroids consisting of steroid alcohols. In certain embodiments, the structured lipid is a steroid. In certain embodiments, the structured lipid is cholesterol. In certain embodiments, the structured lipid is a cholesterol analog. In certain embodiments, the structured lipid is α-tocopherol. Examples of structured lipids include the following: [ka] These include, but are not limited to:

[0344] The target cell delivery LNPs described herein comprise one or more structural lipids.

[0345] As used herein, the term "structured lipid" refers to sterols and also to lipids containing sterol moieties. The incorporation of structured lipids into lipid nanoparticles can help alleviate the aggregation of other lipids in the particles. In certain embodiments, the structured lipids include cholesterol and corticosteroids (e.g., prednisolone, dexamethasone, prednisone, and hydrocortisone), or combinations thereof.

[0346] In some embodiments, the structured lipid is a sterol. As defined herein, "sterol" is a subgroup of steroids consisting of steroid alcohols. Structured lipids can include, but are not limited to, sterols (e.g., plant sterols or animal sterols).

[0347] In certain embodiments, the structural lipid is a steroid. For example, the sterol may include, but is not limited to, cholesterol, β-sitosterol, fecosterol, ergosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid, α-tocopherol, or the like.

[0348] In certain embodiments, the structured lipid is cholesterol. In certain embodiments, the structured lipid is a cholesterol analog.

[0349] Compound ratio The lipid nanoparticles of the present invention may comprise structural components as described herein. The structural components of the lipid nanoparticles may be individual compounds or mixtures of one or more structural compounds of the present invention.

[0350] Non-cationic helper lipids / phospholipids In some embodiments, the lipid-based compositions (e.g., LNPs) described herein comprise one or more non-cationic helper lipids. In some embodiments, the non-cationic helper lipid is a phospholipid. In some embodiments, the non-cationic helper lipid is a phospholipid substitute or phospholipid replacement.

[0351] As used herein, the term "non-cationic helper lipid" refers to a lipid comprising at least one fatty acid chain of at least 8 carbons in length and at least one polar head group moiety. In one embodiment, the helper lipid is not phosphatidylcholine (PC). In one embodiment, the non-cationic helper lipid is a phospholipid or phospholipid substitute. In some embodiments, the phospholipid or phospholipid substitute may be, for example, one or more saturated phospholipids or (poly)unsaturated phospholipids, or phospholipid substitutes, or a combination thereof. Generally, a phospholipid comprises a phospholipid moiety and one or more fatty acid moieties.

[0352] The phospholipid moiety can be selected from the non-limiting group consisting of, for example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin.

[0353] The fatty acid moiety may be selected from the non-limiting group consisting of, for example, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0354] Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. Phospholipids also include phosphosphingolipids such as sphingomyelin.

[0355] In some embodiments, the non-cationic helper lipid is a DSPC analog, a DSPC substitute, oleic acid, or an oleic acid analog.

[0356] In some embodiments, the non-cationic helper lipid is a non-phosphatidylcholine (PC) zwitterionic lipid, a DSPC analog, oleic acid or an oleic acid analog, or a 1,2-distearoyl-i77-glycero-3-phosphocholine (DSPC) substitute.

[0357] phospholipids phospholipids The lipid composition of the lipid nanoparticle compositions disclosed herein can include one or more phospholipids (e.g., one or more saturated or (poly)unsaturated phospholipids, or a combination thereof). Generally, a phospholipid comprises a phospholipid moiety and one or more fatty acid moieties.

[0358] The phospholipid moiety can be selected from the non-limiting group consisting of, for example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin.

[0359] The fatty acid moiety may be selected from the non-limiting group consisting of, for example, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0360] Certain phospholipids can promote fusion with membranes.For example, cationic phospholipids can interact with one or more negatively charged phospholipids of membranes (for example, cell membranes or intracellular membranes).Fusion of phospholipids with membranes can allow one or more components (for example, therapeutic agents) of lipid-containing compositions (for example, LNPs) to pass through the membrane, for example, allowing one or more components to be delivered to target tissues.

[0361] Non-natural phospholipid species are also contemplated, including natural species with modifications and substitutions (including branching, oxidation, cyclization, and alkynes). For example, phospholipids can be functionalized with one or more alkynes (e.g., alkenyl groups in which one or more double bonds are replaced by triple bonds) or crosslinked to one or more alkynes. Under appropriate reaction conditions, alkyne groups can undergo copper-catalyzed cycloaddition upon exposure to azide. Such reactions can be useful in functionalizing the lipid bilayer of nanoparticle compositions to promote membrane permeation or cell recognition, or in conjugating nanoparticle compositions to useful components such as targeting or imaging moieties (e.g., dyes).

[0362] Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidyglycerol, and phosphatidic acid. Phospholipids also include phosphosphingolipids such as sphingomyelin.

[0363] In some embodiments, the phospholipids of the present invention are 1,2-distearoyl-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glysialo-phosphocholine (DMPC), 1,2-dioleoyl-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilinoleoyl-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glysialo-phosphocholine (DMPC), 1,2-dioleoyl-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-glycero-3-phosphocholine (DMPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DL ... Glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2 cholesterylhemisuccinoyl-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-glycero-3-phosphocholine (C16 LysoPC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-arachidonoyl-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3- Phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-arachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof.

[0364] In certain embodiments, phospholipids useful or potentially useful in the present invention are analogs or variants of DSPC. In certain embodiments, phospholipids useful or potentially useful in the present invention are compounds of formula (IV): [ka] or a salt thereof [wherein: Each R 1 are independently optionally substituted alkyl; or optionally, two R 1 are joined together with the intervening atoms to form an optionally substituted monocyclic carbocyclyl or an optionally substituted monocyclic heterocyclyl; or optionally, three R 1 are joined together with the intervening atoms to form an optionally substituted bicyclic carbocyclyl or an optionally substituted bicyclic heterocyclyl; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is a group having the formula: [ka] and; L 2 Each instance of is independently a bond or an optionally substituted C 1-6 alkylene and optionally substituted C 1-6 One methylene unit of alkylene is O, N(R N ), S, C(O), C(O)N(R N ), NR N C(O), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), NR N C(O)O, or NR N C(O)N(R N ) optionally replaced by; R 2 Each instance of may independently be optionally substituted C 1-30 Alkyl, optionally substituted C 1-30 alkenyl, or optionally substituted C 1-30 alkynyl; optionally, R 2one or more methylene units of optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R N ), O, S, C(O), C(O)N(R N ), -NR N C(O), NR N C(O)N(R N ), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), NR N C(O)O, C(O)S, SC(O), -C(=NR N ), C(=NR N )N(R N ), NR N C(=NR N ), NR N C(=NR N )N(R N ), C(S), C(S)N(R N ), NR N C(S), -NR N C(S)N(R N ), S(O), OS(O), S(O)O, OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(R N )S(O), S(O)N(R N ), -N(R N )S(O)N(R N ), OS(O)N(R N ), N(R N )S(O)O, S(O)2, N(R N )S(O)2, S(O)2N(R N ), N(R N )S(O)2N(R N ), -OS(O)2N(R N ), or N(R N ) independently replaced by S(O)2O; R N each instance of is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group; Ring B is an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl; p is 1 or 2] and; provided that the compound has the formula: [ka] [In the formula, R 2 Each instance of is independently an unsubstituted alkyl, an unsubstituted alkenyl, or an unsubstituted alkynyl. isn't it.

[0365] In some embodiments, the phospholipid can be one or more of the phospholipids described in US Application No. 62 / 520,530.

[0366] Modification of phospholipid head groups In certain embodiments, phospholipids useful or potentially useful in the present invention contain a modified phospholipid head group (e.g., a modified choline group). In certain embodiments, the phospholipid with the modified head group is a DSPC with a modified quaternary amine, or an analog thereof. For example, in some embodiments of formula (IV), R 1 At least one of R 1 In certain embodiments, the compound of formula (IV) has the following formula: [ka] or a salt thereof [wherein: each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each u is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each v is independently 1, 2, or 3] It is one of them.

[0367] In certain embodiments, the compound of formula (IV) has the formula (IV-a): [ka] or a salt thereof.

[0368] In certain embodiments, the phospholipids useful or potentially useful in the present invention comprise cyclic moieties instead of glyceride moieties.In certain embodiments, the phospholipids useful in the present invention are DSPC or its analogs with cyclic moieties instead of glyceride moieties.In certain embodiments, the compound of formula (IV) is represented by formula (IV-b): [ka] or a salt thereof.

[0369] Modification of phospholipid tails In certain embodiments, the phospholipids useful or potentially useful in the present invention comprise modified tails.In certain embodiments, the phospholipids useful or potentially useful in the present invention are DSPC or its analogs with modified tails.As described herein, "modified tails" can be tails with shorter or longer aliphatic chains, branched aliphatic chains, substituted aliphatic chains, aliphatic chains in which one or more methylenes are replaced by cyclic or heteroatom groups, or any combination thereof.For example, in certain embodiments, the compound of (IV) is of formula (IV-a) or a salt thereof, wherein R 2 At least one instance of R 2 Optionally substituted C in each instance of 1-30 alkyl, and R 2 one or more methylene units of optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R N), O, S, C(O), C(O)N(R N ), -NR N C(O), NR N C(O)N(R N ), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), NR N C(O)O, C(O)S, SC(O), -C(=NR N ), C(=NR N )N(R N ), NR N C(=NR N ), NR N C(=NR N )N(R N ), C(S), C(S)N(R N ), NR N C(S), -NR N C(S)N(R N ), S(O), OS(O), S(O)O, OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(R N )S(O), S(O)N(R N ), -N(R N )S(O)N(R N ), OS(O)N(R N ), N(R N )S(O)O, S(O)2, N(R N )S(O)2, S(O)2N(R N ), N(R N )S(O)2N(R N ), -OS(O)2N(R N ), or N(R N )S(O)2O.

[0370] In certain embodiments, the compound of formula (IV) has formula (IV-c): [ka] or a salt thereof [wherein: each x is independently an integer between 0 and 30, inclusive; Illustrative examples of each of G include optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R N ), O, S, C(O), C(O)N(R N ), NR N C(O), NR N C(O)N(R N ), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), NR N C(O)O, C(O)S, SC(O), C(=NR N ), C(=NR N )N(R N ), NR N C(=NR N ), -NR N C(=NR N )N(R N ), C(S), C(S)N(R N ), NR N C(S), NR N C(S)N(R N ), S(O), OS(O), S(O)O, OS(O)O, -OS(O)2, S(O)2O, OS(O)2O, N(R N )S(O), S(O)N(R N ), N(R N )S(O)N(R N ), OS(O)N(R N ), N(R N )S(O)O, S(O)2, N(R N )S(O)2, S(O)2N(R N ), N(R N )S(O)2N(R N ), OS(O)2N(R N ), or N(R N )S(O)2O. Each possibility represents a separate embodiment of the present invention.

[0371] In certain embodiments, phospholipids useful or potentially useful in the present invention comprise a modified phosphocholine moiety, wherein the alkyl chain linking the quaternary amine to the phosphoryl group is not ethylene (e.g., n is not 2). Thus, in certain embodiments, phospholipids useful or potentially useful in the present invention are compounds of formula (IV), where n is 1, 3, 4, 5, 6, 7, 8, 9, or 10. For example, in certain embodiments, compounds of formula (IV) have the following formula: [ka] or one of their salts.

[0372] Alternative lipids In certain embodiments, phospholipids useful or potentially useful in the present invention contain a modified phosphocholine moiety in which the alkyl chain linking the quaternary amine to the phosphoryl group is not ethylene (e.g., n is not 2). Thus, in certain embodiments, phospholipids are useful.

[0373] In certain embodiments, alternative lipids are used in place of the phospholipids of the present disclosure.

[0374] In certain embodiments, an alternative lipid of the present invention is oleic acid.

[0375] In certain embodiments, the alternative lipid is one of the following: [ka] It is one of them.

[0376] PEG lipids The lipid composition of the pharmaceutical compositions disclosed herein may include one or more polyethylene glycol (PEG) lipids.

[0377] As used herein, the term "PEG-lipid" refers to polyethylene glycol (PEG)-modified lipid. Non-limiting examples of PEG-lipid include PEG-modified phosphatidylethanolamine and PEG-modified phosphatidic acid, PEG-ceramide conjugate (for example, PEG-CerC14 or PEG-CerC20), PEG-modified alkylamine, and PEG-modified 1,2-diacyloxypropan-3-amine. Such lipids are also referred to as PEGylated lipids. For example, PEG lipids can be PEG-c-DOMG lipids, PEG-DMG lipids, PEG-DLPE lipids, PEG-DMPE lipids, PEG-DPPC lipids, or PEG-DSPE lipids.

[0378] In some embodiments, PEG-lipids include, but are not limited to, 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), or PEG-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA).

[0379] In one embodiment, the PEG-lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof.

[0380] In some embodiments, the lipid portion of the PEG-lipid is about C 14 ~About C 22 , preferably about C14 ~About C 16 In some embodiments, the PEG moiety (e.g., mPEG-NH2) has a size of about 1000, 2000, 5000, 10,000, 15,000, or 20,000 daltons. In one embodiment, the PEG-lipid is 2k -DMG.

[0381] In one embodiment, the lipid nanoparticles described herein can include a PEG lipid that is a non-diffusible PEG. Non-limiting examples of non-diffusible PEGs include PEG-DSG and PEG-DSPE.

[0382] PEG-lipids are known in the art, such as those described in U.S. Pat. No. 8,158,601 and International Publication No. WO2015 / 130584A2, which are incorporated by reference in their entireties.

[0383] Generally, some of the other lipid components (e.g., PEG lipids) of the various formulas described herein can be synthesized as described in International Patent Application No. PCT / US2016 / 000129, filed December 10, 2016, entitled "Compositions and Methods for Delivery of Therapeutic Agents," which is incorporated by reference in its entirety.

[0384] The lipid component of the lipid nanoparticle composition may include one or more molecules containing polyethylene glycol (such as PEG or PEG-modified lipids). Such species may alternatively be referred to as PEGylated lipids. PEG lipids are lipids modified with polyethylene glycol. The PEG lipid may be selected from a non-limiting group including PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. For example, the PEG lipid may be PEG-c-DOMG lipid, PEG-DMG lipid, PEG-DLPE lipid, PEG-DMPE lipid, PEG-DPPC lipid, or PEG-DSPE lipid.

[0385] In some embodiments, the PEG-modified lipid is a modified form of PEG DMG. PEG-DMG has the following structure: [ka] It has.

[0386] In one embodiment, the PEG lipid useful in the present invention may be a PEGylated lipid described in International Publication No. WO2012099755 (the contents of which are incorporated herein by reference in their entirety). Any of these exemplary PEG lipids described herein may be modified to include hydroxyl groups on the PEG chain. In certain embodiments, the PEG lipid is a PEG-OH lipid. As generally defined herein, a "PEG-OH lipid" (also referred to herein as a "hydroxyPEGylated lipid") is a PEGylated lipid having one or more hydroxyl (-OH) groups on the lipid. In certain embodiments, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain. In certain embodiments, the PEG-OH lipid or hydroxyPEGylated lipid includes an -OH group at the end of the PEG chain. Each possibility represents a separate embodiment of the present invention.

[0387] In certain embodiments, the PEG lipid useful in the present invention is a compound of formula (V): [ka] or a salt thereof [wherein: R 3 -OR O and; R O is hydrogen, an optionally substituted alkyl, or an oxygen protecting group; r is an integer between 1 and 100, inclusive; L 1 is an optionally substituted C 1-10 alkylene and optionally substituted C 1-10 At least one methylene of the alkylene is optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, O, N(R N ), S, C(O), C(O)N(R N ), NR N C(O), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), NR N C(O)O, or NR N C(O)N(R N ) are independently replaced by; D is a moiety obtained by click chemistry or a moiety cleavable under physiological conditions; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is a group having the formula: [ka] and; L 2 Each instance of is independently a bond or an optionally substituted C 1-6 alkylene and optionally substituted C 1-6 One methylene unit of alkylene is O, N(R N ), S, C(O), C(O)N(R N ), NRN C(O), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), NR N C(O)O, or NR N C(O)N(R N ) optionally replaced by; R 2 Each instance of may independently be optionally substituted C 1-30 Alkyl, optionally substituted C 1-30 alkenyl, or optionally substituted C 1-30 alkynyl; optionally, R 2 one or more methylene units of optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R N ), O, S, C(O), C(O)N(R N ), -NR N C(O), NR N C(O)N(R N ), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), NR N C(O)O, C(O)S, SC(O), -C(=NR N ), C(=NR N )N(R N ), NR N C(=NR N ), NR N C(=NR N )N(R N ), C(S), C(S)N(R N ), NR N C(S), -NR N C(S)N(R N ), S(O), OS(O), S(O)O, OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(R N )S(O), S(O)N(R N ), -N(R N )S(O)N(R N ), OS(O)N(R N ), N(R N )S(O)O, S(O)2, N(R N)S(O)2, S(O)2N(R N ), N(R N )S(O)2N(R N ), -OS(O)2N(R N ), or N(R N ) independently replaced by S(O)2O; R N each instance of is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group; Ring B is an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl; p is 1 or 2] is provided herein.

[0388] In certain embodiments, the compound of formula (V) is a PEG-OH lipid (i.e., R 3 HA-OR O and R O is hydrogen). In certain embodiments, the compound of formula (V) has the formula (V-OH): [ka] or a salt thereof.

[0389] In certain embodiments, the PEG lipid useful in the present invention is a PEGylated fatty acid. In certain embodiments, the PEG lipid useful in the present invention is a compound of formula (VI). Compound of formula (VI): [ka] or a salt thereof [wherein: R 3 -OR O and; R O is hydrogen, an optionally substituted alkyl, or an oxygen protecting group; r is an integer between 1 and 100, inclusive; R 5 is an optionally substituted C10-40 Alkyl, optionally substituted C 10-40 alkenyl, or optionally substituted C 10-40 alkynyl; R 5 Optionally, one or more methylene groups in the formula (I) are optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R N ), O, S, C(O), C(O)N(R N ), NR N C(O), NR N C(O)N(R N ), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), NR N C(O)O, C(O)S, SC(O), C(=NR N ), C(=NR N )N(R N ), NR N C(=NR N ), NR N C(=NR N )N(R N ), C(S), C(S)N(R N ), NR N C(S), NR N C(S)N(R N ), S(O), OS(O), S(O)O, OS(O)O, -OS(O)2, S(O)2O, OS(O)2O, N(R N )S(O), S(O)N(R N ), N(R N )S(O)N(R N ), OS(O)N(R N ), N(R N )S(O)O, S(O)2, N(R N )S(O)2, S(O)2N(R N ), N(R N )S(O)2N(R N ), OS(O)2N(R N ), or N(R N ) replaced by S(O)2O; R N Each instance of is independently hydrogen, an optionally substituted alkyl, or a nitrogen protecting group. is provided herein.

[0390] In certain embodiments, the compound of formula (VI) has the formula (VI-OH): [ka] or a salt thereof. In some embodiments, r is 45. In another of the above or related aspects, the PEG lipids of the invention are featured, wherein r is 40-50.

[0391] In yet another embodiment, the compound of formula (VI) is [ka] or a salt thereof.

[0392] In one embodiment, the compound of formula (VI) is [ka] is.

[0393] In some embodiments, the lipid composition of the pharmaceutical compositions disclosed herein does not comprise a PEG-lipid.

[0394] In some embodiments, the PEG-lipid can be one or more of the PEG lipids described in US Application No. 62 / 520,530.

[0395] In some embodiments, the PEG-modified lipids of the present invention include PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. In some embodiments, the PEG-modified lipid is PEG-DMG, PEG-c-DOMG (also referred to as PEG-DOMG), PEG-DSG, and / or PEG-DPG.

[0396] In certain embodiments, the LNPs provided herein exhibit increased PEG shedding compared to existing LNP formulations containing PEG-lipids. "PEG shedding" as used herein refers to the cleavage of PEG groups from PEG-lipids. In many instances, the cleavage of PEG groups from PEG-lipids occurs via serum-driven esterase cleavage or hydrolysis. In certain embodiments, the PEG-lipids provided herein are designed to control the rate of PEG shedding. In certain embodiments, the LNPs provided herein exhibit greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% PEG shedding after about 6 hours in human serum. In certain embodiments, the LNPs provided herein exhibit greater than 50% PEG shedding after about 6 hours in human serum. In certain embodiments, the LNPs provided herein exhibit greater than 60% PEG shedding after about 6 hours in human serum.In certain embodiments, the LNPs provided herein exhibit greater than 70% PEG shedding after about 6 hours in human serum.In certain embodiments, the LNPs provided herein exhibit greater than 80% shedding after about 6 hours in human serum.In certain embodiments, the LNPs provided herein exhibit greater than 90% PEG shedding after about 6 hours in human serum.In certain embodiments, the LNPs provided herein exhibit greater than 90% PEG shedding after about 6 hours in human serum.

[0397] In other embodiments, the LNPs provided herein exhibit less than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% PEG shedding after about 6 hours in human serum. In certain embodiments, the LNPs provided herein exhibit less than 60% PEG shedding after about 6 hours in human serum. In certain embodiments, the LNPs provided herein exhibit less than 70% PEG shedding after about 6 hours in human serum. In certain embodiments, the LNPs provided herein exhibit less than 80% PEG shedding after about 6 hours in human serum.

[0398] In addition to the PEG-lipids provided herein, LNPs may contain one or more additional lipid components. In certain embodiments, the PEG-lipids are present in the LNPs at a molar ratio of 0.15-15% relative to other lipids. In certain embodiments, the PEG-lipids are present at a molar ratio of 0.15-5% relative to other lipids. In certain embodiments, the PEG-lipids are present at a molar ratio of 1-5% relative to other lipids. In certain embodiments, the PEG-lipids are present at a molar ratio of 0.15-2% relative to other lipids. In certain embodiments, the PEG-lipids are present at a molar ratio of 1-2% relative to other lipids. In certain embodiments, the PEG-lipids are present at approximately 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2% relative to other lipids. In certain embodiments, the PEG-lipids are present at a molar ratio of approximately 1.5% relative to other lipids.

[0399] In one embodiment, the amount of PEG-lipid in the lipid composition of the pharmaceutical composition disclosed herein is about 0.1 mol% to about 5 mol%, about 0.5 mol% to about 5 mol%, about 1 mol% to about 5 mol%, about 1.5 mol% to about 5 mol%, about 2 mol% to about 5 mol%, about 0.1 mol% to about 4 mol%, about 0.5 mol% to about 4 mol%, about 1 mol% to about 4 mol%, about 1.5 mol% to about 4 mol%, or about 2 mol% to about 4 mol%. , about 0.1mol% to about 3mol%, about 0.5mol% to about 3mol%, about 1mol% to about 3mol%, about 1.5mol% to about 3mol%, about 2mol% to about 3mol%, about 0.1mol% to about 2mol%, about 0.5m ol% to about 2 mol%, about 1 mol% to about 2 mol%, about 1.5 mol% to about 2 mol%, about 0.1 mol% to about 1.5 mol%, about 0.5 mol% to about 1.5 mol%, and about 1 mol% to about 1.5 mol%.

[0400] In one embodiment, the amount of PEG-lipid in the lipid composition disclosed herein is about 2 mol%. In one embodiment, the amount of PEG-lipid in the lipid composition disclosed herein is about 1.5 mol%.

[0401] In one embodiment, the amount of PEG-lipid in the lipid compositions disclosed herein is at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5 mol%.

[0402] Exemplary Synthesis: Compound:HO-PEG 2000 -Ester-C18 [ka]

[0403] To a nitrogen-filled flask containing palladium on carbon (10 wt%, 74 mg, 0.070 mmol) was added benzyl-PEG 2000 To the resulting mixture was added 1-ester-C18 (822 mg, 0.35 mmol) and MeOH (20 mL). The flask was evacuated and backfilled with H2 three times and stirred at room temperature and 1 atmosphere of H2 for 12 hours. The mixture was filtered through Celite, rinsed with DCM, and the filtrate was concentrated in vacuo to give the desired product (692 mg, 88%). Using this methodology, n = 40-50. In one embodiment, n for the resulting polydispersed mixture is represented by an average of 45.

[0404] For example, the value of r can be determined based on the molecular weight of the PEG moiety in the PEG-lipid. For example, a molecular weight of 2,000 (e.g., PEG2000) corresponds to an n value of approximately 45. For a given composition, polymers are often found as a distribution of different polymer chain lengths, so the value of n can encompass a distribution of values ​​within a range accepted in the art. For example, those skilled in the art who understand the polydispersity of such polymeric compounds will recognize that an n value of 45 (e.g., in a structural formula) can represent a distribution of values ​​of 40 to 50 in an actual PEG-containing composition (e.g., a DMG-PEG200 PEG-lipid composition).

[0405] In some embodiments, the target cell delivery lipid of the pharmaceutical compositions disclosed herein does not comprise a PEG-lipid.

[0406] In one embodiment, the target cell delivery LNP of the present disclosure comprises a PEG-lipid. In one embodiment, the PEG-lipid is not PEG-DMG. In some embodiments, the PEG-lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. In some embodiments, the PEG-lipid is selected from the group consisting of PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, and PEG-DSPE lipids. In other embodiments, the PEG-lipid is PEG-DMG.

[0407] In one embodiment, a target cell delivery LNP of the present disclosure comprises a PEG-lipid with a chain length greater than about 14, or greater than about 10 if branched.

[0408] As used herein, the terms "alkyl," "alkyl group," or "alkylene" mean a straight- or branched-chain saturated hydrocarbon containing one or more carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more carbon atoms), optionally substituted. 1-14 The term "alkyl" means an optionally substituted straight or branched chain saturated hydrocarbon containing 1 to 14 carbon atoms. Unless otherwise specified, alkyl groups described herein refer to both unsubstituted and substituted alkyl groups.

[0409] As used herein, the terms "alkenyl," "alkenyl group," or "alkenylene" mean a straight or branched chain hydrocarbon containing two or more carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more carbon atoms) and at least one double bond, and are optionally substituted. 2-14The term "alkenyl" refers to an optionally substituted straight or branched chain hydrocarbon containing 2 to 14 carbon atoms and at least one carbon-carbon double bond. Alkenyl groups can contain 1, 2, 3, 4, or more carbon-carbon double bonds. For example, C 18 Alkenyl may contain one or more double bonds. C containing two double bonds 18 The alkenyl group may be a linoleyl group. Unless otherwise specified, alkenyl groups described herein refer to both unsubstituted and substituted alkenyl groups.

[0410] As used herein, the terms "alkynyl," "alkynyl group," or "alkynylene" mean a straight or branched chain hydrocarbon containing two or more carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more carbon atoms) and at least one carbon-carbon triple bond, and optionally substituted. 2-14 The term "alkynyl" refers to an optionally substituted straight or branched chain hydrocarbon containing 2 to 14 carbon atoms and at least one carbon-carbon triple bond. Alkynyl groups can contain 1, 2, 3, 4, or more carbon-carbon triple bonds. For example, C 18 An alkynyl may contain one or more carbon-carbon triple bonds. Unless otherwise specified, alkynyl groups described herein refer to both unsubstituted and substituted alkynyl groups.

[0411] As used herein, the term "carbocycle" or "carbocyclic group" means an optionally substituted monocyclic or polycyclic ring system containing one or more rings of carbon atoms. The ring can be a 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, or 20-membered ring. 3-6The term "carbocycle" refers to a carbocycle containing a single ring having 3 to 6 carbon atoms. A carbocycle may contain one or more carbon-carbon double or triple bonds and may be non-aromatic or aromatic (e.g., a cycloalkyl group or an aryl group). Examples of carbocycles include cyclopropyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, and 1,2-dihydronaphthyl groups. The term "cycloalkyl," as used herein, refers to a non-aromatic carbocycle, which may or may not contain any double or triple bonds. Unless otherwise specified, carbocycles described herein refer to unsubstituted carbocycle groups and substituted carbocycle groups (i.e., optionally substituted carbocycles).

[0412] As used herein, the term "heterocycle" or "heterocyclic group" refers to an optionally substituted monocyclic or polycyclic ring system containing one or more rings, at least one of which contains at least one heteroatom. The heteroatom may be, for example, a nitrogen, oxygen, or sulfur atom. The ring may be a 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14-membered ring. The heterocycle may contain one or more double or triple bonds and may be non-aromatic or aromatic (e.g., a heterocycloalkyl group or a heteroaryl group). Examples of heterocycles include imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isothiazolidinyl, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furyl, tetrahydrofuranyl, thiophenyl, pyridinyl, piperidinyl, quinolyl, and isoquinolyl. The term "heterocycloalkyl" as used herein refers to a non-aromatic heterocycle, and may or may not contain any double or triple bond. Unless otherwise specified, the heterocycles described herein refer to unsubstituted and substituted heterocycles (i.e., optionally substituted heterocycles).

[0413] As used herein, the terms "heteroalkyl," "heteroalkenyl," or "heteroalkynyl" refer to an alkyl group, an alkenyl group, or an alkynyl group, respectively, as defined herein, which further contain one or more (e.g., 1, 2, 3, or 4) heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus) that are inserted between adjacent carbon atoms in a parent carbon chain and / or that are inserted between a carbon atom and the parent molecule (i.e., between the points of attachment). Unless otherwise specified, heteroalkyl, heteroalkenyl, or heteroalkynyl described herein refers to unsubstituted and substituted heteroalkyl, heteroalkenyl, or heteroalkynyl (i.e., optionally substituted heteroalkyl, heteroalkenyl, or heteroalkynyl).

[0414] As used herein, a "biodegradable group" is a group that can promote more rapid metabolism of lipids in mammalian organisms. The biodegradable group may be selected from the group consisting of, but not limited to, -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)-, an aryl group, and a heteroaryl group. As used herein, an "aryl group" is an optionally substituted carbocyclic group containing one or more aromatic rings. Examples of aryl groups include phenyl and naphthyl groups. As used herein, a "heteroaryl group" is an optionally substituted heterocyclic group containing one or more aromatic rings. Examples of heteroaryl groups include pyrrolyl, furyl, thiophenyl, imidazolyl, oxazolyl, and thiazolyl. Both aryl and heteroaryl groups can be optionally substituted. For example, M and M' can be selected from the non-limiting group consisting of optionally substituted phenyl, oxazole, and thiazole. In the formulas herein, M and M' can be independently selected from the list of biodegradable groups above. Unless otherwise specified, the aryl or heteroaryl groups described herein refer to both unsubstituted and substituted groups (i.e., optionally substituted aryl or heteroaryl groups).

[0415] Alkyl, alkenyl, and cyclyl (eg, carbocyclyl and heterocyclyl) groups may be optionally substituted, unless otherwise specified. Optional substituents include halogen atoms (e.g., chloride, bromide, fluoride, or iodide groups), carboxylic acids (e.g., C(O)OH), alcohols (e.g., hydroxyl, OH), esters (e.g., C(O)OR OC(O)R), aldehydes (e.g., C(O)H), carbonyls (C(O)R, alternatively represented by, e.g., C=O), acyl halides (e.g., C(O)X, where X is a halide selected from bromide, fluoride, chloride, and iodide), carbonates (e.g., OC(O)OR), alkoxy (e.g., OR), acetals (e.g., C(OR)R"", where each OR is an alkoxy group, which may be the same or different, and R"" is an alkyl or alkenyl group), phosphates (e.g., P(O) 3- ), thiol (e.g., SH), sulfoxide (e.g., S(O)R), sulfinic acid (e.g., S(O)OH), sulfonic acid (e.g., S(O)2OH), thial (e.g., C(S)H), sulfate (e.g., S(O)4 2- ), sulfonyl (e.g., S(O)), amido (e.g., C(O)NR or N(R)C(O)R), azido (e.g., N), nitro (e.g., NO), cyano (e.g., CN), isocyano (e.g., NC), acyloxy (e.g., OC(O)R), amino (e.g., NR, NRH, or NH), carbamoyl (e.g., OC(O)NR, OC(O)NRH, or OC(O)NH), sulfonamide (e.g., S(O)NR, S(O)NRH, S(O)NH, N(R)S(O)R, N(H)S(O)R, N(R)S(O)H, or N(H)S(O)H), alkyl group, alkenyl group, cyclyl (e.g., carbocyclyl or heterocyclyl) group. In any of the preceding, R is an alkyl or alkenyl group as defined herein. In some embodiments, the substituent itself may be further substituted, for example, with 1, 2, 3, 4, 5, or 6 substituents as defined herein. For example, C 1-6Alkyl groups can be further substituted with 1, 2, 3, 4, 5, or 6 substituents described herein.

[0416] Nitrogen-containing compounds of the present disclosure can be converted to N-oxides by treatment with an oxidizing agent (e.g., 3-chloroperoxybenzoic acid (mCPBA) and / or hydrogen peroxide) to provide other compounds of the present disclosure. Accordingly, all nitrogen-containing compounds shown and claimed are deemed to include both the compound as shown and its N-oxide derivative (which may be depicted as N→O or N+-O-), where permitted by valence and structure. Furthermore, in other instances, nitrogen in compounds of the present disclosure can be converted to N-hydroxy or N-alkoxy compounds. For example, N-hydroxy compounds can be prepared by oxidation of the parent amine with an oxidizing agent (such as mCPBA). All nitrogen-containing compounds shown and claimed are deemed to cover both the compounds as shown and their N-hydroxy derivatives (i.e., N-OH) and N-alkoxy derivatives (i.e., N-OR, where R is a substituted or unsubstituted C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, 3- to 14-membered carbocycle, or 3- to 14-membered heterocycle), where permitted by valence and structure.

[0417] Exemplary Additional LNP Components The lipid composition of the pharmaceutical composition disclosed herein may contain one or more components in addition to those described above. For example, the lipid composition may contain one or more permeability enhancer molecules, carbohydrates, polymers, surface modifiers (e.g., surfactants), or other components. For example, the permeability enhancer molecules may be molecules described by U.S. Patent Application Publication No. 2005 / 0222064. Carbohydrates may include monosaccharides (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogs). Polymers may be included in the pharmaceutical compositions disclosed herein (e.g., pharmaceutical compositions in the form of lipid nanoparticles) and / or may be used to encapsulate or partially encapsulate.

[0418] The polymer may be biodegradable and / or biocompatible and may be selected from, but not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyurethanes, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates.

[0419] LNPs containing checkpoint cancer vaccines Specifically disclosed herein are LNP compositions comprising a polynucleotide encoding a checkpoint cancer vaccine comprising one or more IDO antigenic peptides and one or more PD-L1 antigenic peptides for use in stimulating T cells (e.g., T effector cells) for the treatment of cancer in a subject. In another embodiment, the invention relates to an LNP comprising a polynucleotide comprising mRNA encoding a checkpoint cancer vaccine comprising (i) one or more IDO antigenic peptides and (ii) one or more PD-L1 antigenic peptides. The LNP compositions of the present disclosure can be used to prime T cells, stimulate and activate T effector cells, and / or induce killing of immunosuppressive (regulatory) immune cells and cancer cells that overexpress IDO and PD-L1 in vivo or ex vivo.

[0420] In one embodiment, the LNP composition comprising a polynucleotide encoding a checkpoint cancer vaccine comprises: (i) an ionizable lipid (e.g., an amino lipid); (ii) a sterol or other structural lipid; (iii) a non-cationic helper lipid or phospholipid; and (iv) a PEG-lipid.

[0421] In one embodiment, an LNP composition comprising a polynucleotide encoding IDO (e.g., IDO1 or IDO2) comprises (i) an ionizable lipid (e.g., an amino lipid); (ii) a sterol or other structural lipid; (iii) a non-cationic helper lipid or phospholipid; and (iv) a PEG-lipid.

[0422] In one embodiment, an LNP composition comprising a checkpoint cancer vaccine polynucleotide comprising (i) one or more IDO antigenic peptides and (ii) one or more PD-L1 antigenic peptides comprises (i) an ionizable lipid (e.g., an amino lipid); (ii) a sterol or other structural lipid; (iii) a non-cationic helper lipid or phospholipid; and (iv) a PEG-lipid.

[0423] In another embodiment, the LNP compositions of the present disclosure are used in a method of treating cancer in a subject or a method of stimulating an immune response in a subject.

[0424] In one embodiment, an LNP composition comprising a polynucleotide encoding a checkpoint cancer vaccine comprising (i) one or more IDO antigenic peptides and (ii) one or more PD-L1 antigenic peptides can be administered with an additional agent, e.g., as described herein.

[0425] Additional features of LNP compositions for use in combination therapy are provided in the section entitled "Therapeutic LNPs."

[0426] In one embodiment, the ratio between the lipid composition and the polynucleotide range can be from about 10:1 to about 60:1 (wt / wt).

[0427] Nanoparticle Composition In some embodiments, the pharmaceutical compositions disclosed herein are formulated as lipid nanoparticles (LNPs).Accordingly, the present disclosure also provides (i) a lipid composition comprising a delivery agent (such as a compound described herein), and (ii) a nanoparticle composition comprising a polynucleotide encoding a polypeptide of the present invention.In such nanoparticle compositions, the lipid composition disclosed herein can encapsulate the polynucleotide encoding the polypeptide of the present invention.

[0428] Nanoparticle compositions are typically on the order of micrometers or less in size and may contain a lipid bilayer. Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, nanoparticle compositions may be liposomes having a lipid bilayer with a diameter of 500 nm or less.

[0429] Nanoparticle compositions include, for example, lipid nanoparticles (LNPs), liposomes, and lipoplexes. In some embodiments, the nanoparticle composition is a vesicle comprising one or more lipid bilayers. In certain embodiments, the nanoparticle composition comprises two or more concentric bilayers separated by aqueous compartments. The lipid bilayers may be functionalized and / or crosslinked to each other. The lipid bilayers may comprise one or more ligands, proteins, or channels.

[0430] In one embodiment, the lipid nanoparticles comprise an ionizable amino lipid, a structural lipid, a phospholipid, and mRNA. In some embodiments, the LNPs comprise an ionizable amino lipid, a PEG-modified lipid, a sterol, and a structural lipid. In some embodiments, the LNPs have a molar ratio of about 40-50% ionizable amino lipid; about 5-15% structural lipid; about 30-45% sterol; and about 1-5% PEG-modified lipid.

[0431] In some embodiments, the LNPs have a polydispersity value of less than 0.4. In some embodiments, the LNPs have a net neutral charge at neutral pH. In some embodiments, the LNPs have an average diameter of 50-150 nm. In some embodiments, the LNPs have an average diameter of 80-100 nm.

[0432] As generally defined herein, the term "lipid" refers to a small molecule having hydrophobic or amphipathic properties. Lipids can be naturally occurring or synthetic. Examples of lipid classes include, but are not limited to, fats, waxes, sterol-containing metabolites, vitamins, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, and polyketides, and prenol lipids. In some instances, the amphipathic properties of some lipids lead to the formation of liposomes, vesicles, or membranes in aqueous media.

[0433] In some embodiments, lipid nanoparticles (LNPs) may contain ionizable amino lipids. As used herein, the term "ionizable amino lipid" has its ordinary meaning in the art and may refer to a lipid containing one or more charged moieties. In some embodiments, the ionizable amino lipid may be positively or negatively charged. The ionizable amino lipid may be positively charged, in which case it may be referred to as a "cationic lipid." In certain embodiments, the ionizable amino lipid molecule contains an amine group and may be referred to as an ionizable amino lipid. As used herein, a "charged moiety" is a chemical moiety that carries a formal electronic charge (e.g., monovalent (+1 or -1), divalent (+2 or -2), trivalent (+3 or -3), etc.). The charged moiety may be anionic (i.e., negatively charged) or cationic (i.e., positively charged). Examples of positively charged moieties include amine groups (e.g., primary amines, secondary amines, and / or tertiary amines), ammonium groups, pyridinium groups, guanidine groups, and imidizolium groups. In certain embodiments, the charged moiety comprises an amine group. Examples of negatively charged groups or precursors thereof include carboxylate groups, sulfonate groups, sulfate groups, phosphonate groups, phosphate groups, hydroxyl groups, and the like. The charge of a charged moiety may, in some cases, vary depending on environmental conditions (e.g., changes in pH may alter the charge of the moiety and / or render the moiety charged or uncharged). In general, the charge density of a molecule may be selected as desired.

[0434] It should be understood that the terms "charged" or "charged moiety" do not refer to a "partial negative charge" or a "partial positive charge" on a molecule. The terms "partial negative charge" and "partial positive charge" are given their ordinary meaning in the art. A "partial negative charge" can result when a functional group contains a polar bond and electron density is attracted toward one atom of the bond, creating a partial negative charge on the atom. Those skilled in the art will generally recognize bonds that can be polar in this way.

[0435] Ionizable amino lipids are sometimes referred to in the art as "ionizable cationic lipids." In one embodiment, the ionizable amino lipids may have a positively charged hydrophilic head and a hydrophobic tail connected via a linker structure.

[0436] In addition, the ionizable amino lipid can also be a lipid containing a cyclic amine group.

[0437] In one embodiment, the ionizable amino lipid may be selected from, but is not limited to, the ionizable amino lipids described in International Publication Nos. WO2013086354 and WO2013116126 (the contents of each of which are incorporated herein by reference in their entirety).

[0438] In yet another embodiment, the ionizable amino lipid may be selected from, but is not limited to, formulas CLI-CLXXXXII of U.S. Pat. No. 7,404,969, each of which is incorporated herein by reference in its entirety.

[0439] In one embodiment, the lipid may be a cleavable lipid such as those described in International Publication No. WO2012170889 (incorporated herein by reference in its entirety). In one embodiment, the lipid may be synthesized by methods known in the art and / or as described in International Publication No. WO2013086354 (the contents of each of which are incorporated herein by reference in their entirety).

[0440] Nanoparticle compositions can be characterized by various methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of nanoparticle compositions. Dynamic light scattering or potentiometry (e.g., potentiometric titration) can be used to measure zeta potential. Dynamic light scattering can also be used to determine particle size. Devices such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure multiple characteristics of nanoparticle compositions, such as particle size, polydispersity index, and zeta potential.

[0441] The size of the nanoparticles may help to neutralize biological responses (such as, but not limited to, inflammation) or may increase the biological effect of the polynucleotide.

[0442] As used herein, "size" or "average size," in the context of a nanoparticle composition, refers to the average diameter of the nanoparticle composition.

[0443] In one embodiment, the polynucleotide encoding the polypeptide is about 10 to about 100 nm (about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm, about 30 to about 90 nm, about 3 The lipid nanoparticles may be formulated with lipid nanoparticles having a diameter of, but not limited to, 0 to about 100 nm, about 40 to about 50 nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about 80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 to about 60 nm, about 50 to about 70 nm, about 50 to about 80 nm, about 50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100 nm, about 80 to about 90 nm, about 80 to about 100 nm, and / or about 90 to about 100 nm.

[0444] In one embodiment, the nanoparticles have a diameter of about 10 to 500 nm, hi one embodiment, the nanoparticles have a diameter of greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm, or greater than 1000 nm.

[0445] In some embodiments, the largest dimension of the nanoparticle composition is 1 μm or less (e.g., 1 μm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, or less).

[0446] The nanoparticle composition can be relatively homogeneous. The polydispersity index can be used to indicate the homogeneity of the nanoparticle composition (e.g., the particle size distribution of the nanoparticle composition). A small polydispersity index (e.g., less than 0.3) generally indicates a narrow particle size distribution. The nanoparticle composition can have a polydispersity index of about 0 to about 0.25 (e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25). In some embodiments, the polydispersity index of the nanoparticle compositions disclosed herein can be about 0.10 to about 0.20.

[0447] The zeta potential of a nanoparticle composition can be used to indicate the electrokinetic potential of the composition. For example, the zeta potential can describe the surface charge of a nanoparticle composition. Nanoparticle compositions with a relatively low charge (positive or negative) are generally desirable because more highly charged species can interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of the nanoparticle compositions disclosed herein can be about -10 mV to about +20 mV, about -10 mV to about +15 mV, about 10 mV to about +10 mV, about -10 mV to about +5 mV, about -10 mV to about 0 mV, about -10 mV to about -5 mV, about -5 mV to about +20 mV, about -5 mV to about +15 mV, about -5 mV to about +10 mV, about -5 mV to about +5 mV, about -5 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV to about +15 mV, or about +5 mV to about +10 mV.

[0448] In some embodiments, the zeta potential of the lipid nanoparticles is about 0 mV to about 100 mV, about 0 mV to about 90 mV, about 0 mV to about 80 mV, about 0 mV to about 70 mV, about 0 mV to about 60 mV, about 0 mV to about 50 mV, about 0 mV to about 40 mV, about 0 mV to about 30 mV, about 0 mV to about 20 mV, about 0 mV to about 10 mV, about 10 mV to about 100 mV, about 10 mV to about 90 mV, about 10 mV to about 80 mV, about 10 mV to about 70 mV, about 10 mV to about 60 mV, about 10 mV to about 50 mV, about 10 mV to about 40 mV, about 10 mV to about 30 mV, about 10 mV to about 20 mV, or about 20 mV to about 100mV, about 20mV to about 90mV, about 20mV to about 80mV, about 20mV to about 70mV, about 20mV to about 60mV, about 20mV to about 50 mV, about 20mV to about 40mV, about 20mV to about 30mV, about 30mV to about 100mV, about 30mV to about 90mV, about 30mV to about 80mV, The zeta potential of the lipid nanoparticles can be about 30 mV to about 70 mV, about 30 mV to about 60 mV, about 30 mV to about 50 mV, about 30 mV to about 40 mV, about 40 mV to about 100 mV, about 40 mV to about 90 mV, about 40 mV to about 80 mV, about 40 mV to about 70 mV, about 40 mV to about 60 mV, and about 40 mV to about 50 mV. In some embodiments, the zeta potential of the lipid nanoparticles can be about 10 mV to about 50 mV, about 15 mV to about 45 mV, about 20 mV to about 40 mV, and about 25 mV to about 35 mV. In some embodiments, the zeta potential of the lipid nanoparticles can be about 10 mV, about 20 mV, about 30 mV, about 40 mV, about 50 mV, about 60 mV, about 70 mV, about 80 mV, about 90 mV, and about 100 mV.

[0449] The term "encapsulation efficiency" of a polynucleotide describes the amount of polynucleotide encapsulated by or otherwise associated with a nanoparticle composition after preparation, relative to the initial amount provided. As used herein, "encapsulation" can refer to complete, substantial, or partial enclosing, confinement, surrounding, or enveloping.

[0450] Encapsulation efficiency is desirably high (e.g., close to 100%) and can be measured, for example, by comparing the amount of polynucleotide in a solution containing the nanoparticle composition before and after disruption of the nanoparticle composition with one or more organic solvents or detergents.

[0451] Fluorescence can be used to measure the amount of free polynucleotide in solution.For the nanoparticle compositions described herein, the encapsulation efficiency of polynucleotide is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, 97%, 98%, 99%, or 100%).In some embodiments, the encapsulation efficiency is at least 80%.In certain embodiments, the encapsulation efficiency is at least 90%.

[0452] The amount of polynucleotide present in the pharmaceutical compositions disclosed herein can depend on several factors, such as the size of the polynucleotide, the desired target and / or application, or other characteristics of the nanoparticle composition, as well as the characteristics of the polynucleotide.

[0453] For example, the amount of mRNA useful in a nanoparticle composition can depend on the size (expressed as length or molecular weight), sequence, and other characteristics of the mRNA. The relative amounts of polynucleotides in a nanoparticle composition can also vary.

[0454] The relative amounts of lipid composition and polynucleotide present in the lipid nanoparticle compositions of the present disclosure can be optimized for efficacy and tolerability. For compositions containing mRNA as the polynucleotide, the N:P ratio can serve as a useful metric.

[0455] The N:P ratio of a nanoparticle composition controls both expression and tolerability, so nanoparticle compositions with low N:P ratios and high expression are desirable. The N:P ratio varies according to the ratio of lipid to RNA in the nanoparticle composition.

[0456] Generally, lower N:P ratios are preferred. The RNA(s), lipid(s), and amounts thereof can be selected to provide an N:P ratio of about 2:1 to about 30:1 (e.g., 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1). In certain embodiments, the N:P ratio can be about 2:1 to about 8:1. In other embodiments, the N:P ratio is about 5:1 to about 8:1. In certain embodiments, the N:P ratio is 5:1 to 6:1. In one specific aspect, the N:P ratio is about 5.67:1.

[0457] In addition to providing nanoparticle compositions, the present disclosure also provides methods of producing lipid nanoparticles comprising encapsulating a polynucleotide, such methods comprising using any of the pharmaceutical compositions disclosed herein and producing lipid nanoparticles according to methods of producing lipid nanoparticles known in the art. See, e.g., Wang et al. (2015) "Delivery of oligonucleotides with lipid nanoparticles" Adv. Drug Deliv. Rev. 87:68-80; Silva et al. (2015) "Delivery Systems for Biopharmaceuticals. Part I: Nanoparticles and Microparticles" Curr. Pharm. Technol. 16:940-954; Naseri et al. (2015) "Solid Lipid Nanoparticles and Nanostructured Lipid Carriers: Structure, Preparation and Application" Adv. Pham. Bull. 5:305-13; Silva et al. (2015) "Lipid nanoparticles for the delivery of biopharmaceuticals" Curr. Pharm. Biotechnol. 16:291-302, and references cited therein.

[0458] In some embodiments, the ratio between the lipid composition and the polynucleotide is about 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1, 66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1, 78:1, 79:1, 80:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1, 89:1, 90:1, 91:1, 92 The wt / wt ratio of the lipid composition to the polynucleotide encoding the therapeutic agent can be 3:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, or 60:1 (wt / wt). In some embodiments, the wt / wt ratio of the lipid composition to the polynucleotide encoding the therapeutic agent is about 20:1 or about 15:1.

[0459] In some embodiments, the pharmaceutical compositions disclosed herein may contain two or more polypeptides. For example, the pharmaceutical compositions disclosed herein may contain two or more polynucleotides (e.g., RNA, e.g., mRNA).

[0460] In one embodiment, the lipid nanoparticles described herein incorporate a polynucleotide (e.g., mRNA) in a weight ratio of 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, or 70:1 lipid:polynucleotide, or any range or ratio thereof (5:1 to about 10:1, about 5:1 to about 15:1, about 5:1 to about 20:1, about 5:1 to about 25:1, about 5:1 to about 30:1, about 5:1 to about 35:1, about 5:1 to about 40:1, about 5:1 to about 45:1, about 5:1 to about 50:1, about 5:1 to about 55:1, about 5:1 to about 60:1, about 5:1 to about 60:1, about 5:1 to about 70:1, or any range or ratio thereof). about 70:1, about 10:1 to about 15:1, about 10:1 to about 20:1, about 10:1 to about 25:1, about 10:1 to about 30:1, about 10:1 to about 35:1, about 10:1 to about 40:1, about 10:1 to about 45:1, about 10:1 to about 50:1, about 10:1 to about 55:1, about 10:1 to about 60:1, about 10:1 to about 70:1, about 1 The ratio may be, but is not limited to, 5:1 to about 20:1, about 15:1 to about 25:1, about 15:1 to about 30:1, about 15:1 to about 35:1, about 15:1 to about 40:1, about 15:1 to about 45:1, about 15:1 to about 50:1, about 15:1 to about 55:1, about 15:1 to about 60:1, or about 15:1 to about 70:1.

[0461] In one embodiment, the lipid nanoparticles described herein may contain polynucleotides at a concentration of approximately 0.1 mg / ml to 2 mg / ml (such as, but not limited to, 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml (0.5 mg / ml), 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1.0 mg / ml, 1.1 mg / ml, 1.2 mg / ml, 1.3 mg / ml, 1.4 mg / ml, 1.5 mg / ml, 1.6 mg / ml, 1.7 mg / ml, 1.8 mg / ml, 1.9 mg / ml, 2.0 mg / ml, or greater than 2.0 mg / ml).

[0462] Methods of Using LNP Compositions Comprising Checkpoint Cancer Vaccines In one aspect, the present disclosure provides a composition (e.g., an LNP composition described herein) comprising a lipid nanoparticle (LNP) comprising a polynucleotide comprising mRNA encoding a checkpoint cancer vaccine comprising (i) one or more IDO antigenic peptides and (ii) one or more PD-L1 antigenic peptides, and optionally one or more adjuvant amino acid sequences, in the treatment of cancer in a subject, e.g., according to the methods described herein.

[0463] In another aspect, the present disclosure provides a composition (e.g., an LNP composition described herein) comprising a lipid nanoparticle (LNP) comprising a polynucleotide comprising mRNA encoding a checkpoint cancer vaccine comprising (i) one or more IDO antigenic peptides and (ii) one or more PD-L1 antigenic peptides, and optionally one or more adjuvant amino acid sequences, to stimulate an immune response in a subject, e.g., according to the methods described herein.

[0464] In another aspect, the present disclosure provides a composition (e.g., an LNP composition described herein) comprising a lipid nanoparticle (LNP) comprising a polynucleotide comprising mRNA encoding a checkpoint cancer vaccine comprising (i) one or more IDO antigenic peptides and (ii) one or more PD-L1 antigenic peptides, e.g., to stimulate effector T cells in a subject to target and kill tumor cells that express IDO or PD-L1, e.g., according to the methods described herein.

[0465] In another aspect, the present disclosure provides a composition (e.g., an LNP composition described herein) comprising a lipid nanoparticle (LNP) comprising a polynucleotide comprising mRNA encoding a checkpoint cancer vaccine comprising (i) one or more IDO antigenic peptides and (ii) one or more PD-L1 antigenic peptides, and optionally one or more adjuvant amino acid sequences, or a combination thereof, to stimulate T cells (e.g., T effector cells), e.g., according to the methods described herein.

[0466] In another aspect, the present disclosure provides a composition (e.g., an LNP composition described herein) comprising a lipid nanoparticle (LNP) comprising a polynucleotide comprising mRNA encoding a checkpoint cancer vaccine comprising (i) one or more IDO antigenic peptides and (ii) one or more PD-L1 antigenic peptides, and optionally one or more adjuvant amino acid sequences, to induce T cell-mediated killing of tumor cells by vaccine-activated T cells, e.g., according to the methods described herein.

[0467] In another aspect, the present disclosure provides a composition (e.g., an LNP composition described herein) comprising a lipid nanoparticle (LNP) comprising a polynucleotide comprising mRNA encoding a checkpoint cancer vaccine comprising (i) one or more IDO antigenic peptides and (ii) one or more PD-L1 antigenic peptides, and optionally one or more adjuvant amino acid sequences, for use in treating cancer in a subject.

[0468] In a related aspect, provided herein is a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of lipid nanoparticles (LNPs) (e.g., LNP compositions described herein) comprising a polynucleotide comprising mRNA encoding a checkpoint cancer vaccine comprising (i) one or more IDO antigenic peptides and (ii) one or more PD-L1 antigenic peptides, and optionally one or more adjuvant amino acid sequences.

[0469] In any of the embodiments of the methods disclosed herein, administration of the LNPs results in an amelioration or delay in the progression of cancer in the subject, e.g., as measured by an assay described herein, e.g., as described herein. In embodiments, the amelioration or delay in disease progression is compared to disease progression in an otherwise similar subject (e.g., a subject not contacted with an LNP composition comprising a checkpoint cancer vaccine comprising (i) one or more IDO antigenic peptides and (ii) one or more PD-L1 antigenic peptides, and optionally one or more adjuvant amino acid sequences).

[0470] In embodiments, the delay in cancer progression is at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1.5 years, 2 years, 3 years, 4 years, or 5 years or more.

[0471] In some embodiments, checkpoint cancer vaccines stimulate effector T cells that target and kill suppressive immune cells and tumor cells that express the target antigen. Thus, in some embodiments, IDO- and PD-L1-specific T cells kill immunosuppressive (regulatory) immune cells and cancer cells that overexpress IDO and PD-L1. In some embodiments, treatment results in additional tumor killing by vaccine-activated T cells. Furthermore, in some embodiments, administration of a checkpoint cancer vaccine results in T cell priming, leading to recognition of additional tumor-associated antigens and increased tumor killing by tumor-specific cytotoxic T cells. Without wishing to be bound by theory, systemic PD-1 / PD-L1 blockade may further amplify the effect, leading to greater immune activation and superior disease control.

[0472] In one embodiment, the cancer is a solid tumor, for example a locally advanced or metastatic solid tumor. In one embodiment, the cancer is melanoma. In one embodiment, the melanoma is cutaneous melanoma. In one embodiment, the cutaneous melanoma is 1L cutaneous melanoma stage IIIB+. In one embodiment, the cancer is NSCLC. In one embodiment, the NSCLC is 1L NSCLC. In one embodiment, the cancer is bladder cancer. In some embodiments, the bladder cancer is non-muscle invasive bladder cancer. In one embodiment, the cancer is head and neck cancer. In some embodiments, the head and neck cancer is head and neck squamous cell carcinoma. In one embodiment, the cancer is colorectal cancer. In some embodiments, the colorectal cancer is microsatellite stable colorectal cancer. In one embodiment, the cancer is basal cell carcinoma. In one embodiment, the cancer is breast cancer. In some embodiments, the breast cancer is triple-negative breast cancer.

[0473] LNP Dosage and Dosage Regimen In some embodiments, any of the LNPs disclosed herein can be administered according to dosing intervals, such as those described herein. In some embodiments, the dosing interval includes an initial dose of an LNP composition and one or more subsequent doses of the same LNP composition (e.g., 1-50 doses, 5-50 doses, 10-50 doses, 15-50 doses, 20-50 doses, 25-50 doses, 30-50 doses, 35-50 doses, 40-50 doses, 45-50 doses, 1-45 doses, 1-40 doses, 1-35 doses, 1-30 doses, 1-25 doses, 1-20 doses, 1-15 doses, 1-10 doses, 1-5 doses).

[0474] In some embodiments, the dosing interval comprises one or more doses of the LNP composition and one or more doses of the additional agent.

[0475] In some embodiments, the dosing interval is accomplished over at least 1 week, 2 weeks, 3 weeks, or 4 weeks.

[0476] In some embodiments, the dosing interval comprises a cycle (e.g., a 7-day cycle). In some embodiments, the cycle comprises 3 weeks (e.g., 21 days).

[0477] In some embodiments, the LNP composition is administered once every three weeks for one or more cycles, hi some embodiments, the dosing regimen includes two, three, four, five, six, seven, eight, or nine cycles.

[0478] In some embodiments, the LNP composition contains about 50 μg to about 1 mg, e.g., about 100 μg to about 1 mg, about 200 μg to about 900 μg, about 300 μg to about 800 μg, about 400 μg to about 700 μg, about 500 μg to about 600 μg, about 200 μg to about 1 mg, about 300 μg to about 1 mg, about 400 μg to about 1 mg, about 500 μg to about 1 mg, about 600 μg to about 1 mg, about 700 μg to about 1 mg, about 800 μg to about 1 mg, about 900 μg to about 1 mg, or about 100 μg. The subject is administered a dose of about 100μg to about 900μg, about 100μg to about 800μg, about 100μg to about 700μg, about 100μg to about 600μg, about 100μg to about 500μg, about 100μg to about 400μg, about 100μg to about 300μg, about 100μg to about 200μg, about 200μg to about 400μg, about 300μg to about 500μg, about 400μg to about 600μg, about 500μg to about 700μg, about 600μg to about 800μg, or about 700μg to about 900μg.

[0479] In some embodiments, the LNP composition is administered at a dose of about 100 μg to about 200 μg, about 200 μg to about 300 μg, about 300 μg to about 400 μg, about 400 μg to about 500 μg, about 500 μg to about 600 μg, about 600 μg to about 700 μg, about 700 μg to about 800 μg, about 800 μg to about 900 μg, or about 900 μg to about 1 mg. In some embodiments, the LNP composition is administered at a dose of about 50 μg to about 150 μg, about 150 μg to about 250 μg, about 250 μg to about 350 μg, about 350 μg to about 450 μg, about 450 μg to about 550 μg, about 550 μg to about 650 μg, about 650 μg to about 750 μg, about 750 μg to about 850 μg, about 850 μg to about 950 μg, or about 950 μg to about 1 mg. In some embodiments, the LNP composition is administered at a dose of about 100 μg, about 200 μg, about 300 μg, about 400 μg, about 500 μg, about 600 μg, about 700 μg, about 800 μg, about 900 μg, or about 1 mg.

[0480] In some embodiments of any of the LNP compositions disclosed herein, the LNP composition comprises about 0.1-10 mg per kg, about 0.1-9.5 mg per kg, about 0.1-9 mg per kg, about 0.1-8.5 mg per kg, about 0.1-8 mg per kg, about 0.1-7.5 mg per kg, about 0.1-7 mg per kg, about 0.1-6.5 mg per kg, about 0.1-6 mg per kg, about 0.1-5.5 mg per kg, about 0.1-5 mg per kg, about 0.1-5 mg per kg, about 0.1-6 mg per kg, about 0.1-7.5 mg per kg, about 0.1-7 mg per kg, about 0.1-6.5 mg per kg, about 0.1-6 mg per kg, about 0.1-5.5 mg per kg, about 0.1- ... The dose is administered at a dose (e.g., total dose) of about 0.1 to 4.5 mg per kg, about 0.1 to 4 mg per kg, about 0.1 to 3.5 mg per kg, about 0.1 to 3 mg per kg, about 0.1 to 2.5 mg per kg, about 0.1 to 2 mg per kg, about 0.1 to 1.5 mg per kg, about 0.1 to 1 mg per kg, about 0.1 to 0.9 mg per kg, about 0.1 to 0.8 mg per kg, about 0.1 to 0.7 mg per kg, about 0.1 to 0.6 mg per kg, or about 0.1 to 0.5 mg per kg.

[0481] In some embodiments of any of the LNP compositions disclosed herein, the LNP composition comprises about 0.2-10 mg per kg, about 0.3-10 mg per kg, about 0.4-10 mg per kg, about 0.5-10 mg per kg, about 0.6-10 mg per kg, about 0.7-10 mg per kg, about 0.8-10 mg per kg, about 0.9-10 mg per kg, about 1-10 mg per kg, about 1.5-10 mg per kg, about 2-10 mg per kg, about 2.5-10 mg per kg, about 3-30 mg per kg, about 4-40 mg per kg, about 5-60 mg per kg, about 6-70 mg per kg, about 7-80 mg per kg, about 8-90 mg per kg, about 9-10 mg per kg, about 10-120 mg per kg, about 11-140 mg per kg, about 12-140 mg per kg, about 14-150 mg per kg, about 15-160 mg per kg, about 16-170 mg per kg, about 17-180 mg per kg, about 18-20 mg per kg, about 19-210 mg per kg, about 22-230 mg per kg, about 23-240 mg per kg, about 24-250 mg per kg, about 25-260 mg per kg, about 26-270 mg per kg, about 27-280 mg per kg, about 28-300 mg per kg, about 29-310 mg per kg, about 29-310 mg per kg, about 30-320 mg per kg, about 31-320 mg per kg, about 32-330 mg per kg, about 33-340 mg per kg, about 34-350 mg per kg, about 35-360 10 mg, about 3-10 mg per kg, about 3.5-10 mg per kg, about 4-10 mg per kg, about 4.5-10 mg per kg, about 5-10 mg per kg, about 5.5-10 mg per kg, about 6-10 mg per kg, about 6.5-10 mg per kg, about 7-10 mg per kg, about 7.5-10 mg per kg, about 8-10 mg per kg, about 8.5-10 mg per kg, about 9-10 mg per kg, or about 9.5-10 mg per kg (e.g., total dose).

[0482] In some embodiments, any of the LNPs disclosed herein are administered intramuscularly (IM).

[0483] Diseases and Disorders In one embodiment of any of the methods of treatment or compositions for use disclosed herein, the subject has or is identified as having cancer. In one embodiment, the LNPs disclosed herein are administered to the subject to treat or alleviate the symptoms of cancer. In one embodiment, the LNPs disclosed herein are administered to the subject to stimulate an immune response in the subject.

[0484] In one embodiment, the cancer is a solid tumor (e.g., a locally advanced or metastatic solid tumor). In one embodiment, the cancer is melanoma. In some embodiments, the melanoma is cutaneous melanoma. In some embodiments, the cutaneous melanoma is 1L cutaneous melanoma stage IIIB+. In one embodiment, the cancer is non-small cell lung cancer (NSCLC). In some embodiments, the NSCLC is 1L NSCLC. In one embodiment, the cancer is bladder cancer. In some embodiments, the bladder cancer is non-muscle invasive bladder cancer. In one embodiment, the cancer is head and neck cancer. In some embodiments, the head and neck cancer is head and neck squamous cell carcinoma. In one embodiment, the cancer is colorectal cancer. In some embodiments, the colorectal cancer is microsatellite-stable colorectal cancer. In one embodiment, the cancer is basal cell carcinoma. In one embodiment, the cancer is breast cancer. In some embodiments, the breast cancer is triple-negative breast cancer.

[0485] In one embodiment, the subject is a mammal (eg, a human).

[0486] Further combination therapy In some embodiments, the methods of treatment or compositions for use disclosed herein comprise administering the LNPs disclosed herein in combination with an additional agent. In one embodiment, the additional agent is a standard medical treatment for a disease or disorder (e.g., an autoimmune disease). In one embodiment, the additional agent is mRNA.

[0487] In some embodiments, subjects for the present methods or compositions have been treated with one or more standard medical therapies, hi other embodiments, subjects for the present methods or compositions have not been responsive to one or more standard medical therapies.

[0488] For example, a checkpoint inhibitor (such as an anti-PD1 antibody or an anti-CTLA4 antibody) is additionally administered to the subject. In some embodiments, the anti-PD-1 antibody is pembrolizumab.

[0489] The dosage of the combination therapy can be determined by one of skill in the art. In some embodiments, the anti-PD-1 antibody (e.g., pembrolizumab) is administered (e.g., intravenously) to the subject at a dose of 200 mg once every three weeks (or 400 mg once every six weeks), for example, for a total of 24 weeks. In some embodiments, the anti-PD-1 antibody (e.g., pembrolizumab) is administered (e.g., intravenously) to the subject at a dose of 400 mg once every six weeks (e.g., on day 1 of every three-week cycle, e.g., day 1 of cycle 1, cycle 3, cycle 5, cycle 7, and cycle 9).

[0490] In some embodiments, the additional therapy is administered to the patient simultaneously (e.g., on the same day) with the LNP composition, hi some embodiments, the additional therapy is administered separately (e.g., on separate days) from the LNP composition.

[0491] Array optimization and method thereof In some embodiments, a polynucleotide of the disclosure comprises a sequence-optimized nucleotide sequence encoding a checkpoint cancer vaccine comprising (i) one or more IDO antigenic peptides and (ii) one or more PD-L1 antigenic peptides. In some embodiments, a polynucleotide of the disclosure comprises an open reading frame (ORF) encoding one or more IDO antigenic peptides and one or more PD-L1 antigenic peptides, wherein the ORF is sequence-optimized.

[0492] The sequence-optimized nucleotide sequences disclosed herein differ from the corresponding wild-type nucleotide acid sequences and from other known sequence-optimized nucleotide sequences, eg, these sequence-optimized nucleic acids have unique compositional characteristics.

[0493] In some embodiments, the percentage of uracil or thymine nucleobases in a sequence-optimized nucleotide sequence (e.g., encoding a checkpoint cancer vaccine) is modified (e.g., reduced) relative to the percentage of uracil or thymine nucleobases in a reference wild-type nucleotide sequence. Such sequences are referred to as uracil-modified or thymine-modified sequences. The percentage of uracil or thymine content in a nucleotide sequence can be determined by dividing the number of uracils or thymines in the sequence by the total number of nucleotides and multiplying by 100. In some embodiments, a sequence-optimized nucleotide sequence has a lower uracil or thymine content than the uracil or thymine content in a reference wild-type sequence. In some embodiments, the uracil or thymine content in a sequence-optimized nucleotide sequence of the present disclosure exceeds the uracil or thymine content in a reference wild-type sequence yet still maintains a beneficial effect (e.g., increased expression and / or signaling response) when compared to the reference wild-type sequence.

[0494] In some embodiments, the optimized sequences of the present disclosure contain unique stretches of uracil or thymine (if DNA) within the sequence. The uracil or thymine content of an optimized sequence can be expressed in various ways, such as the uracil or thymine content of the optimized sequence relative to the theoretical minimum (%UTM or %TTM), relative to the wild type (%UWT or %TWT), and relative to the total nucleotide content (%UTL or %TTL). For DNA, it is recognized that thymine will occur in place of uracil and will substitute for T where U appears. Thus, for example, all disclosures relating to %UTM, %UWT, or %UTL with respect to RNA are equally applicable to %TTM, %TWT, or %TTL with respect to DNA.

[0495] Uracil or thymine content relative to the theoretical minimum of uracil or thymine refers to a parameter determined by dividing the number of uracils or thymines in a sequence-optimized nucleotide sequence by the total number of uracils or thymines in a hypothetical nucleotide sequence (in which all codons in the hypothetical sequence are replaced by synonymous codons with the lowest possible uracil or thymine content), and multiplying by 100. This parameter is abbreviated herein as %UTM or %TTM.

[0496] In some embodiments, the uracil-modified sequence encoding the checkpoint cancer vaccine of the present disclosure has a reduced number of consecutive uracils relative to the corresponding wild-type nucleic acid sequence. For example, two consecutive leucines can be encoded by the sequence CUUUUG, which contains a four-uracil cluster. Such a subsequence can be replaced, for example, by CUGCUC, which removes the uracil cluster. Phenylalanine can be encoded by UUC or UUU. Thus, even if the phenylalanine encoded by UUU is replaced by UUC, the synonymous codon still contains a uracil pair (UU). Thus, the number of phenylalanines in the sequence establishes the minimum number of uracil pairs (UU) that cannot be removed without changing the number of phenylalanines in the encoded polypeptide.

[0497] In some embodiments, uracil modified sequences encoding the checkpoint cancer vaccines of the present disclosure have a reduced number of uracil triplets (UUU) relative to the wild-type nucleic acid sequence. In some embodiments, uracil modified sequences encoding the checkpoint cancer vaccines have a reduced number of uracil pairs (UU) relative to the number of uracil pairs (UU) in the wild-type nucleic acid sequence. In some embodiments, uracil modified sequences encoding the checkpoint cancer vaccines of the present disclosure have a number of uracil pairs (UU) that corresponds to the minimum possible number of uracil pairs (UU) in the wild-type nucleic acid sequence.

[0498] The phrase "uracil pairs (UU) compared to uracil pairs (UU) in a wild-type nucleic acid sequence" refers to a parameter determined by dividing the number of uracil pairs (UU) in a sequence-optimized nucleotide sequence by the total number of uracil pairs (UU) in the corresponding wild-type nucleotide sequence and multiplying by 100. This parameter is abbreviated herein as %UUwt. In some embodiments, a uracil-modified sequence encoding a checkpoint cancer vaccine has a %UUwt of less than 100%.

[0499] In some embodiments, a polynucleotide of the present disclosure comprises a uracil-modified sequence encoding a checkpoint cancer vaccine disclosed herein. In some embodiments, the uracil-modified sequence encoding a checkpoint cancer vaccine comprises at least one chemically modified nucleobase (e.g., 5-methoxyuracil). In some embodiments, at least 95% of the nucleobases (e.g., uracils) in a uracil-modified sequence encoding a checkpoint cancer vaccine of the present disclosure are modified nucleobases. In some embodiments, at least 95% of the uracils in a uracil-modified sequence encoding a checkpoint cancer vaccine are 5-methoxyuracil. In some embodiments, a polynucleotide comprising a uracil-modified sequence further comprises an miRNA-binding site (e.g., an miRNA-binding site that binds to miR-122). In some embodiments, a polynucleotide comprising a uracil-modified sequence is formulated with a delivery agent (e.g., a compound having Formula (I), e.g., any of Compound Nos. 18, 25, 301, or 357).

[0500] In some embodiments, a polynucleotide of the disclosure (e.g., a polynucleotide comprising a nucleotide sequence encoding a checkpoint cancer vaccine (e.g., a wild-type sequence, a functional fragment, or a variant thereof)) is sequence optimized.

[0501] A sequence-optimized nucleotide sequence (a nucleotide sequence is also referred to herein as a "nucleic acid") comprises at least one codon modification relative to a reference sequence (e.g., a wild-type sequence encoding a checkpoint cancer vaccine). Thus, in a sequence-optimized nucleic acid, at least one codon differs from the corresponding codon in the reference sequence (e.g., the wild-type sequence).

[0502] Generally, sequence-optimized nucleic acids are generated by steps that include at least replacing codons in a reference sequence with synonymous codons (i.e., codons that encode the same amino acid). Such replacement can be achieved, for example, by applying a codon substitution map (i.e., a table providing the codons that encode each amino acid in the codon-optimized sequence) or by applying a set of rules (e.g., if glycine is next to a neutral amino acid, it is coded for by a particular codon, but if it is next to a polar amino acid, it is coded for by a different codon). In addition to codon substitution (i.e., "codon optimization"), the sequence optimization techniques disclosed herein include additional optimization steps that are not strictly directed at codon optimization, such as removing deleterious motifs (replacement of destabilizing motifs).

[0503] Additional and exemplary methods of sequence optimization are disclosed in International PCT Application WO2017 / 201325, filed May 18, 2017, the entire contents of which are incorporated herein by reference.

[0504] MicroRNA (miRNA) binding site Polynucleotides of the invention can contain regulatory elements (e.g., microRNA (miRNA) binding sites, transcription factor binding sites, structured mRNA sequences and / or motifs, artificial binding sites engineered to act as pseudo-receptors for endogenous nucleic acid binding molecules, and combinations thereof). In some embodiments, polynucleotides containing such regulatory elements are said to contain "sensor sequences."

[0505] In some embodiments, a polynucleotide of the invention (e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)) comprises an open reading frame (ORF) encoding a polypeptide of interest and further comprises one or more miRNA binding site(s). The incorporation or incorporation of the miRNA binding site(s) provides for regulation of the polynucleotide of the invention, and in turn, the polypeptide encoded therefrom, based on tissue- and / or cell-type-specific expression of naturally occurring miRNAs.

[0506] The present invention also provides pharmaceutical compositions and formulations comprising any of the polynucleotides described above. In some embodiments, the composition or formulation further comprises a delivery drug.

[0507] In some embodiments, a composition or formulation encoding a polypeptide may contain a polynucleotide comprising a sequence-optimized nucleic acid sequence disclosed herein. In some embodiments, a composition or formulation may contain a polynucleotide (e.g., RNA, e.g., mRNA) comprising a polynucleotide (e.g., ORF) having significant sequence identity to a sequence-optimized nucleic acid sequence disclosed herein encoding a polypeptide. In some embodiments, the polynucleotide further comprises an miRNA binding site (e.g., a miRNA binding site that binds).

[0508] miRNAs (e.g., naturally occurring miRNAs) are 19-25 nucleotide-long non-coding RNAs that bind to polynucleotides and down-regulate gene expression by reducing their stability or by inhibiting their translation. The miRNA sequence contains a "seed" region (i.e., the sequence in the region of positions 2-8 of the mature miRNA). The miRNA seed can include positions 2-8 or 2-7 of the mature miRNA.

[0509] MicroRNAs are enzymatically derived from regions of RNA transcripts that fold back on themselves to form short hairpin structures often referred to as pre-miRNAs (precursor miRNAs). Pre-miRNAs typically have a two-nucleotide overhang at their 3' end, a 3' hydroxyl group, and a 5' phosphate group. This precursor mRNA is processed in the nucleus and subsequently transported to the cytoplasm, where it is further processed by DICER (RNase III enzyme) to form mature microRNAs of approximately 22 nucleotides. The mature microRNAs are then incorporated into ribonucleoparticles to form RNA-induced silencing complexes (RISCs), which mediate gene silencing. Art-recognized nomenclature for mature miRNAs typically designates the arm of the pre-miRNA from which the mature miRNA is derived, with "5p" indicating that the microRNA is from the 5' arm of the pre-miRNA hairpin and "3p" indicating that the microRNA is from the 3' end of the pre-miRNA hairpin. miRs referenced by numbers herein can refer to either of the two mature microRNAs (e.g., either the 3p or 5p microRNA) that originate from opposite arms of the same pre-miRNA. All miRs referenced herein are intended to include both the 3p and 5p arms / sequences unless otherwise specified by a 3p or 5p designation.

[0510] As used herein, the term "microRNA (miRNA or miR) binding site" refers to a sequence within a polynucleotide (e.g., within a DNA or within an RNA transcript, including in the 5'UTR and / or 3'UTR) that has sufficient complementarity to all or a region of the miRNA to interact with, associate with, or bind to the miRNA. In some embodiments, a polynucleotide of the invention comprising an ORF encoding a polypeptide of interest further comprises one or more miRNA binding site(s). In exemplary embodiments, the 5'UTR and / or 3'UTR of a polynucleotide (e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)) comprises one or more miRNA binding site(s).

[0511] An miRNA-binding site that is sufficiently complementary to an miRNA exhibits a degree of complementarity sufficient to facilitate miRNA-mediated regulation of a polynucleotide (e.g., miRNA-mediated translational repression or degradation of the polynucleotide). In exemplary embodiments of the invention, an miRNA-binding site that is sufficiently complementary to an miRNA exhibits a degree of complementarity sufficient to facilitate miRNA-mediated degradation of a polynucleotide (e.g., miRNA-guide RNA-induced silencing complex (RISC)-mediated mRNA cleavage). The miRNA-binding site can be complementary to, for example, an miRNA sequence that is 19-25 nucleotides in length, an miRNA sequence that is 19-23 nucleotides in length, or an miRNA sequence that is 22 nucleotides in length. The miRNA-binding site can be complementary to only a portion of the miRNA (e.g., to less than 1, 2, 3, or 4 nucleotides of the entire length of a naturally occurring miRNA sequence, or to a portion that is less than 1, 2, 3, or 4 nucleotides shorter than the naturally occurring miRNA sequence). When the desired modulation is mRNA degradation, full or complete complementarity (e.g., full or complete complementarity over all or a significant portion of the length of the naturally occurring miRNA) is preferred. In some embodiments, the miRNA binding site comprises a sequence having complementarity (e.g., partial or complete complementarity) with the miRNA seed sequence. In some embodiments, the miRNA binding site comprises a sequence having complete complementarity with the miRNA seed sequence. In some embodiments, the miRNA binding site comprises a sequence having complementarity (e.g., partial or complete complementarity) with the miRNA sequence. In some embodiments, the miRNA binding site comprises a sequence having complete complementarity with the miRNA sequence. In some embodiments, the miRNA binding site has complete complementarity with the miRNA sequence except for one, two, or three nucleotide substitutions, terminal additions, and / or truncations.

[0512] In some embodiments, the miRNA binding site is the same length as the corresponding miRNA. In other embodiments, the miRNA binding site is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides shorter than the corresponding miRNA at the 5' end, 3' end, or both. In still other embodiments, the binding site of a microRNA is 2 nucleotides shorter than the corresponding microRNA at the 5' end, 3' end, or both. A miRNA binding site that is shorter than the corresponding miRNA can still degrade or prevent the translation of an mRNA that incorporates one or more miRNA binding sites.

[0513] In some embodiments, the miRNA-binding site binds to the corresponding mature miRNA that is part of an active RISC containing Dicer. In another embodiment, binding of the miRNA-binding site to the corresponding miRNA in the RISC degrades the mRNA containing the miRNA-binding site or prevents the mRNA from being translated. In some embodiments, the miRNA-binding site has sufficient complementarity to the miRNA so that a RISC complex containing the miRNA cleaves the polynucleotide containing the miRNA-binding site. In other embodiments, the miRNA-binding site has imperfect complementarity to the miRNA so that a RISC complex containing the miRNA induces instability in the polynucleotide containing the miRNA-binding site. In another embodiment, the miRNA-binding site has imperfect complementarity to the miRNA so that a RISC complex containing the miRNA represses transcription in the polynucleotide containing the miRNA-binding site.

[0514] In some embodiments, the miRNA binding site has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mismatch(es) from the corresponding miRNA.

[0515] In some embodiments, the miRNA binding site has at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, or at least about 21 contiguous nucleotides that are complementary to at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, or at least about 21 contiguous nucleotides, respectively, of the corresponding miRNA.

[0516] By engineering one or more miRNA binding sites into a polynucleotide of the present invention, the polynucleotide can be targeted for degradation or reduced translation, provided that the miRNA in question is available. This can reduce off-target effects during polynucleotide delivery. For example, if a polynucleotide of the present invention is not intended for delivery to a tissue or cell but ultimately arrives there, if one or more miRNA binding sites are engineered into the 5' UTR and / or 3' UTR of the polynucleotide, then abundant miRNAs in the tissue or cell may inhibit expression of the gene of interest. Thus, in some embodiments, incorporating one or more miRNA binding sites into an mRNA of the present disclosure can reduce the risk of off-target effects during nucleic acid molecule delivery and / or enable tissue-specific regulation of the expression of the polypeptide encoded by the mRNA. In yet other embodiments, incorporating one or more miRNA binding sites into an mRNA of the present disclosure can modulate immune responses in vivo during nucleic acid delivery. In further embodiments, incorporation of one or more miRNA binding sites into the mRNA of the present disclosure may modulate the accelerated blood clearance (ABC) of the lipid-containing compounds and compositions described herein.

[0517] Conversely, miRNA binding sites can be removed from the polynucleotide sequence in which they naturally occur to increase protein expression in a particular tissue. For example, a binding site for a specific miRNA can be removed from a polynucleotide to improve protein expression in tissues or cells that contain the miRNA.

[0518] Modulation of expression in multiple tissues can be achieved through the introduction or removal of one or more miRNA binding sites (e.g., one or more distinct miRNA binding sites). The decision to remove or insert a miRNA binding site is made based on miRNA expression patterns and / or profiling in tissues and / or cells during development and / or disease. The identification of miRNAs, miRNA binding sites, and their expression patterns and roles in biology have been reported (e.g., Bonauer et al., Curr Drug Targets 2010 11:943-949; Anand and Cheresh Curr Opin Hematol 2011 18:171-176; Contreras and Rao Leukemia 2012 26:404-413 (2011 Dec 20. doi:10.1038 / leu.2011.356); Bartel Cell 2009 136:215-233; Landgraf et al., Cell, 2007 129:1401-1414; Gentner and Naldini, Tissue Antigens. 2012 80:393-403, and all references therein; each of which is incorporated herein by reference in its entirety).

[0519] Examples of tissues in which miRNAs are known to regulate mRNA and thereby protein expression include, but are not limited to, liver (miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells (miR-17-92, miR-126), myeloid cells (miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart (miR-ld, miR-149), kidney (miR-192, miR-194, miR-204), and lung epithelial cells (let-7, miR-133, miR-126).

[0520] Specifically, miRNAs are known to be differentially expressed in immune cells (also called hematopoietic cells), such as antigen-presenting cells (APCs) (e.g., dendritic cells and macrophages), macrophages, monocytes, B lymphocytes, T lymphocytes, granulocytes, and natural killer cells. Immune cell-specific miRNAs are involved in immune responses to immunogenicity, autoimmunity, infection, and inflammation, as well as unwanted immune responses after gene therapy and tissue / organ transplantation. Immune cell-specific miRNAs also regulate multiple aspects of hematopoietic cell (immune cell) development, proliferation, differentiation, and apoptosis. For example, miR-142 and miR-146 are expressed exclusively in immune cells and are particularly abundant in myeloid dendritic cells. It has been demonstrated that immune responses to polynucleotides can be blocked by adding miR-142 binding sites to the 3'-UTR of polynucleotides, allowing for more stable gene transfer in tissues and cells. miR-142 efficiently degrades exogenous polynucleotides in antigen-presenting cells and suppresses cytotoxic elimination of transduced cells (e.g., Annoni A et al., Blood, 2009, 114, 5152-5161; Brown BD, et al., Nat med. 2006, 12(5), 585-591; Brown BD, et al., Blood, 2007, 110(13):4144-4152, each of which is incorporated herein by reference in its entirety).

[0521] In some embodiments, a polynucleotide of the invention comprises an miRNA binding site, wherein the miRNA binding site comprises one or more nucleotide sequences selected from Table 3C or Table 4A and comprises one or more copies of any one or more of the miRNA binding site sequences. In some embodiments, a polynucleotide of the invention further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the same or different miRNA binding sites selected from Table 3C or Table 4A (including any combination thereof).

[0522] In some embodiments, the miRNA-binding site binds to or is complementary to miR-142. In some embodiments, miR-142 comprises SEQ ID NO: 200. In some embodiments, the miRNA-binding site binds to miR-142-3p or miR-142-5p. In some embodiments, the miR-142-3p-binding site comprises SEQ ID NO: 202. In some embodiments, the miR-142-5p-binding site comprises SEQ ID NO: 204. In some embodiments, the miRNA-binding site comprises a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 202 or SEQ ID NO: 204.

[0523] In some embodiments, the miRNA-binding site binds to or is complementary to miR-126. In some embodiments, miR-126 comprises SEQ ID NO: 205. In some embodiments, the miRNA-binding site binds to miR-126-3p or miR-126-5p. In some embodiments, the miR-126-3p-binding site comprises SEQ ID NO: 207. In some embodiments, the miR-126-5p-binding site comprises SEQ ID NO: 710. In some embodiments, the miRNA-binding site comprises a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 121 or SEQ ID NO: 123.

[0524] In one embodiment, the 3'UTR comprises two miRNA binding sites, the first miRNA binding site binding to miR-142 and the second miRNA binding site binding to miR-126. In a specific embodiment, the 3'UTR that binds to miR-142 and miR-126 comprises, consists of, or consists essentially of the sequence of SEQ ID NO:249. [Table 3C]

[0525] In some embodiments, an miRNA binding site is inserted into a polynucleotide of the invention at any position of the polynucleotide (e.g., the 5'UTR and / or 3'UTR). In some embodiments, the 5'UTR comprises an miRNA binding site. In some embodiments, the 3'UTR comprises an miRNA binding site. In some embodiments, the 5'UTR and the 3'UTR comprise miRNA binding sites. The insertion site in the polynucleotide can be anywhere in the polynucleotide, so long as the insertion of the miRNA binding site in the polynucleotide does not interfere with translation of a functional polypeptide in the absence of the corresponding miRNA, and so long as the insertion of the miRNA binding site in the polynucleotide and the binding of the miRNA binding site to the corresponding miRNA in the presence of the miRNA is capable of degrading the polynucleotide or preventing translation of the polynucleotide.

[0526] In some embodiments, the miRNA binding site is inserted at least about 30 nucleotides downstream from the stop codon of the ORF in a polynucleotide of the invention comprising an ORF. In some embodiments, the miRNA binding site is inserted at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 55 nucleotides, at least about 60 nucleotides, at least about 65 nucleotides, at least about 70 nucleotides, at least about 75 nucleotides, at least about 80 nucleotides, at least about 85 nucleotides, at least about 90 nucleotides, at least about 95 nucleotides, or at least about 100 nucleotides downstream from the stop codon of the ORF in a polynucleotide of the invention. In some embodiments, the miRNA binding site is inserted about 10 to about 100 nucleotides, about 20 to about 90 nucleotides, about 30 to about 80 nucleotides, about 40 to about 70 nucleotides, about 50 to about 60 nucleotides, or about 45 to about 65 nucleotides downstream from the stop codon of the ORF in the polynucleotide of the present invention.

[0527] In some embodiments, the miRNA binding site is inserted into the 3'UTR immediately after the stop codon of the coding region in a polynucleotide (e.g., mRNA) of the invention. In some embodiments, if there are multiple copies of the stop codon in the construct, the miRNA binding site is inserted immediately after the last stop codon. In some embodiments, the miRNA binding site is inserted further downstream of the stop codon, in this case with 3'UTR bases between the stop codon and the miR binding site(s). In some embodiments, three non-limiting examples of possible insertion sites for miRs in the 3'UTR are shown in SEQ ID NOs: 248, 249, and 250, which show 3'UTR sequences with a miR-142-3p site each inserted into one of three different possible insertion sites in the 3'UTR.

[0528] In some embodiments, one or more miRNA binding sites may be located within the 5'UTR at one or more possible insertion sites. For example, three non-limiting examples of possible insertion sites for miRs in the 5'UTR are shown in SEQ ID NOs: 251, 252, and 253, which represent 5'UTR sequences with a miR-142-3p site each inserted into one of three different possible insertion sites within the 5'UTR.

[0529] In one embodiment, the codon-optimized open reading frame encoding the polypeptide of interest comprises a stop codon and at least one microRNA binding site is located within the 3'UTR 1 to 100 nucleotides after the stop codon. In one embodiment, the codon-optimized open reading frame encoding the polypeptide of interest comprises a stop codon and at least one microRNA binding site for a miR expressed in immune cells is located within the 3'UTR 30 to 50 nucleotides after the stop codon. In another embodiment, the codon-optimized open reading frame encoding the polypeptide of interest comprises a stop codon and at least one microRNA binding site for a miR expressed in immune cells is located within the 3'UTR at least 50 nucleotides after the stop codon. In other embodiments, the codon-optimized open reading frame encoding the polypeptide of interest contains a stop codon, and at least one microRNA binding site for a miR expressed in immune cells is located within the 3' UTR immediately following the stop codon, or within 15-20 nucleotides after the stop codon, or within 70-80 nucleotides after the stop codon. In other embodiments, the 3' UTR contains two or more miRNA binding sites (e.g., two to four miRNA binding sites), with a spacer region (e.g., 10-100 nucleotides, 20-70 nucleotides, or 30-50 nucleotides in length) between each miRNA binding site. In another embodiment, the 3' UTR contains a spacer region between the end of the miRNA binding site(s) and the polyA tail. For example, a spacer region 10-100 nucleotides, 20-70 nucleotides, or 30-0 nucleotides in length can be placed between the end of the miRNA binding site(s) and the beginning of the polyA tail.

[0530] In one embodiment, the codon-optimized open reading frame encoding the polypeptide of interest includes a start codon and at least one microRNA binding site is located within the 5'UTR 1-100 nucleotides before (upstream) the start codon. In one embodiment, the codon-optimized open reading frame encoding the polypeptide of interest includes a start codon and at least one microRNA binding site for a miR expressed in immune cells is located within the 5'UTR 10-50 nucleotides before (upstream) the start codon. In another embodiment, the codon-optimized open reading frame encoding the polypeptide of interest includes a start codon and at least one microRNA binding site for a miR expressed in immune cells is located within the 5'UTR at least 25 nucleotides before (upstream) the start codon. In other embodiments, the codon-optimized open reading frame encoding the polypeptide of interest includes a start codon, and at least one microRNA binding site for a miR expressed in immune cells is located within the 5' UTR immediately preceding the start codon, or within 15-20 nucleotides before the stop codon, or within 70-80 nucleotides before the start codon. In other embodiments, the 5' UTR includes two or more miRNA binding sites (e.g., 2-4 miRNA binding sites), with a spacer region (e.g., 10-100 nucleotides, 20-70 nucleotides, or 30-50 nucleotides in length) between each miRNA binding site.

[0531] In one embodiment, the 3'UTR contains two or more stop codons, and at least one miRNA binding site is located downstream of the stop codon. For example, the 3'UTR can contain one, two, or three stop codons. Non-limiting examples of triple stop codons that can be used include UGAUAAUAG, UGAUAGUAA, UAAUGAUAG, UGAUAAUAA, UGAUAGUAG, UAAUGAUGA, UAAUAGUAG, UGAUGAUGA, UAAUAAUAA, and UAGUAGUAG. For example, within the 3'UTR, one, two, three, or four miRNA binding sites (e.g., miR-142-3p binding sites) can be located immediately adjacent to the stop codon(s) or any number of nucleotides downstream of the final stop codon. If the 3'UTR contains multiple miRNA binding sites, these binding sites may be located directly next to each other (i.e., alternating) in the construct, or alternatively, spacer nucleotides may be located between each binding site.

[0532] In one embodiment, the 3'UTR contains three stop codons and a single miR-142-3p binding site is located downstream of the third stop codon. Non-limiting examples of 3'UTR sequences with three stop codons and a single miR-142-3p binding site located at different positions downstream of the final stop codon are shown in SEQ ID NOs: 237, 248, 249, and 250. [Table 4A-1] [Table 4A-2] [Table 4A-3] [Table 4A-4] [Table 4A-5] [Table 4A-6] [Table 4A-7]

[0533] In one embodiment, a polynucleotide of the invention comprises a 5' UTR, a codon-optimized open reading frame encoding a polypeptide of interest, a 3' UTR comprising at least one miRNA binding site for an miR expressed in immune cells, and a 3' tail region of linked nucleosides. In various embodiments, the 3' UTR comprises one to four, at least two, one, two, three, or four miRNA binding sites for miRs expressed in immune cells (preferably abundantly or preferentially expressed in immune cells).

[0534] In one embodiment, at least one miRNA expressed in an immune cell is a miR-142-3p microRNA binding site. In one embodiment, the miR-142-3p microRNA binding site comprises the sequence set forth in SEQ ID NO: 202. In one embodiment, the 3'UTR of an mRNA comprising the miR-142-3p microRNA binding site comprises the sequence set forth in SEQ ID NO: 220.

[0535] In one embodiment, at least one miRNA expressed in immune cells is a miR-126 microRNA binding site. In one embodiment, the miR-126 binding site is a miR-126-3p binding site. In one embodiment, the miR-126-3p microRNA binding site comprises the sequence shown in SEQ ID NO: 207. In one embodiment, the 3'UTR of the mRNA of the present invention comprising the miR-126-3p microRNA binding site comprises the sequence shown in SEQ ID NO: 235.

[0536] Non-limiting exemplary sequences for miRs that can bind to the microRNA binding site(s) of the present disclosure include the following: miR-142-3p (SEQ ID NO: 201), miR-142-5p (SEQ ID NO: 203), miR-146-3p (SEQ ID NO: 221), miR-146-5p (SEQ ID NO: 222), miR-155-3p (SEQ ID NO: 223), miR-155-5p (SEQ ID NO: 224), miR-126-3p (SEQ ID NO: 206), miR-126-5p (SEQ ID NO: 207), miR-146-3p (SEQ ID NO: 208), miR-146-5p (SEQ ID NO: 209), miR-146-5p (SEQ ID NO: 210), miR-146-3p (SEQ ID NO: 211), miR-146-5p (SEQ ID NO: 212), miR-146-3p (SEQ ID NO: 213), miR-146-5p (SEQ ID NO: 214), miR-146-5p (SEQ ID NO: 215), miR-146-3p (SEQ ID NO: 216), miR-146-5p (SEQ ID NO: 217), miR-146-5p (SEQ ID NO: 218), miR-146-3p (SEQ ID NO: 219), miR-146-5p (SEQ ID NO: 220), miR-146-5p (SEQ ID NO: 221), miR-146-3p (SEQ ID NO: 219), miR-146-5p (SEQ ID NO: 222), mi miR-16-3p (SEQ ID NO: 208), miR-16-3p (SEQ ID NO: 225), miR-16-5p (SEQ ID NO: 226), miR-21-3p (SEQ ID NO: 227), miR-21-5p (SEQ ID NO: 228), miR-223-3p (SEQ ID NO: 143), miR-223-5p (SEQ ID NO: 230), miR-24-3p (SEQ ID NO: 231), miR-24-5p (SEQ ID NO: 232), miR-27-3p (SEQ ID NO: 233), and miR-27-5p (SEQ ID NO: 234). Other suitable miR sequences expressed in immune cells (e.g., abundantly or preferentially expressed in immune cells) are known and available in the art, for example, the University of Manchester microRNA database (miRBase). Binding sites for any of the aforementioned miRs can be designed based on Watson-Crick complementarity to the miR (typically 100% complementarity to the miR) and inserted into the mRNA constructs of the present disclosure as described herein.

[0537] In another embodiment, a polynucleotide of the invention (e.g., and mRNA, e.g., its 3'UTR) may comprise at least one miRNA binding site for reducing or inhibiting accelerated blood clearance, e.g., by reducing or inhibiting production of IgM by B cells, e.g., against PEG, and / or reducing or inhibiting proliferation and / or activation of pDCs, and may comprise at least one miRNA binding site for modulating tissue expression of the encoded protein of interest.

[0538] miRNA gene regulation can be influenced by the sequences surrounding the miRNA, including, but not limited to, the species of the surrounding sequence, the type of sequence (e.g., heterologous, homologous, foreign, endogenous, or artificial), regulatory elements in the surrounding sequence, and / or structural elements in the surrounding sequence. miRNAs can be influenced by the 5' UTR and / or 3' UTR. As a non-limiting example, a non-human 3' UTR can increase the regulatory effect of the miRNA sequence on the expression of a polypeptide of interest compared to a human 3' UTR of the same sequence type.

[0539] In one embodiment, other regulatory and / or structural elements in the 5'UTR may affect miRNA-mediated gene regulation. One example of a regulatory and / or structural element is a structured IRES (internal ribosome entry site) in the 5'UTR, which is necessary for the binding of translation elongation factors that initiate protein translation. EIF4A2 binding to this secondary structured element in the 5'UTR is necessary for miRNA-mediated gene expression (Meijer HA et al., Science, 2013, 340, 82-85, incorporated herein by reference in its entirety). The polynucleotide of the present invention may further comprise this structured 5'UTR to promote microRNA-mediated gene regulation.

[0540] At least one miRNA binding site can be engineered into the 3'UTR of a polynucleotide of the present invention. In this context, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more miRNA binding sites can be engineered into the 3'UTR of a polynucleotide of the present invention. For example, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 2, or 1 miRNA binding site can be engineered into the 3'UTR of a polynucleotide of the present invention. In one embodiment, the miRNA binding sites incorporated into a polynucleotide of the present invention can be the same or different miRNA sites. Combinations of different miRNA binding sites incorporated into a polynucleotide of the present invention can include combinations in which any two or more copies of different miRNA sites are incorporated. In another embodiment, the miRNA binding sites incorporated into a polynucleotide of the present invention can target the same or different tissues in the body. As a non-limiting example, the level of expression in specific cell types (e.g., myeloid cells and endothelial cells) can be reduced through the introduction of tissue-, cell type-, or disease-specific miRNA binding sites into the 3'-UTR of the polynucleotides of the invention.

[0541] In one embodiment, an miRNA binding site can be engineered into a polynucleotide of the present invention near the 5' end of the 3'UTR, midway between the 5' and 3' ends of the 3'UTR, and / or near the 3' end of the 3'UTR. As a non-limiting example, an miRNA binding site can be engineered near the 5' end of the 3'UTR and midway between the 5' and 3' ends of the 3'UTR. As another non-limiting example, an miRNA binding site can be engineered near the 3' end of the 3'UTR and midway between the 5' and 3' ends of the 3'UTR. In another non-limiting example, an miRNA binding site can be engineered near the 5' end of the 3'UTR and near the 3' end of the 3'UTR.

[0542] In another embodiment, the 3'UTR can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 miRNA binding sites. The miRNA binding sites can be complementary to the miRNA, the miRNA seed sequence, and / or the miRNA sequence adjacent to the seed sequence.

[0543] In some embodiments, expression of a polynucleotide of the invention can be controlled by incorporating at least one sensor sequence into the polynucleotide and formulating the polynucleotide for administration. As a non-limiting example, a polynucleotide of the invention can be targeted to a tissue or cell by incorporating an miRNA binding site and formulating the polynucleotide in a lipid nanoparticle comprising an ionizable lipid (including any of the lipids described herein).

[0544] The polynucleotides of the present invention can be engineered for more targeted expression in specific tissues, cell types, or biological conditions based on the expression patterns of miRNAs in different tissues, cell types, or biological conditions. The polynucleotides of the present invention can be designed for optimal protein expression in a tissue or cell, or in the context of a biological condition, through the introduction of tissue-specific miRNA binding sites.

[0545] In some embodiments, polynucleotides of the present invention can be designed to incorporate miRNA binding sites that are either 100% identical to a known miRNA seed sequence or less than 100% identical to the miRNA seed sequence. In some embodiments, polynucleotides of the present invention can be designed to incorporate miRNA binding sites that are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a known miRNA seed sequence. The miRNA seed sequence can be partially mutated to reduce miRNA binding affinity and therefore reduce down-regulation of the polynucleotide. In short, the degree of match or mismatch between the miRNA binding site and the miRNA seed can act as a rheostat to finely adjust the ability of the miRNA to regulate protein expression. In addition, mutations in the non-seed region of the miRNA binding site can also affect the ability of the miRNA to regulate protein expression.

[0546] In one embodiment, the miRNA sequence may be incorporated into the loop of the stem-loop.

[0547] In another embodiment, the miRNA seed sequence may be incorporated into the loop of the stem-loop and the miRNA binding site may be incorporated into the 5' or 3' stem of the stem-loop.

[0548] In one embodiment, miRNA sequences in the 5'UTR may be used to stabilize the polynucleotides of the invention described herein.

[0549] In another embodiment, an miRNA sequence in the 5' UTR of a polynucleotide of the invention can be used to reduce the accessibility of a translation initiation site (such as, but not limited to, the start codon). See, for example, Matsuda et al., PLoS One. 2010 11(5):e15057 (incorporated herein by reference in its entirety), which reduced the accessibility of the first start codon (AUG) using an antisense locked nucleic acid (LNA) oligonucleotide and an exon junction complex (EJC) near the start codon (-4 to +37, if the A in the AUG codon is +1). Matsuda showed that altering the sequence near the start codon with an LNA or EJC affected the efficiency, length, and structural stability of the polynucleotide. The polynucleotide of the invention can include an miRNA sequence near the translation initiation site instead of the LNA or EJC sequence described by Matsuda et al. to reduce the accessibility of the translation initiation site. The site of translation initiation can be before, after, or within the miRNA sequence. As a non-limiting example, the site of translation initiation can be within the miRNA sequence (such as the seed sequence or binding site).

[0550] In some embodiments, the polynucleotide of the present invention may contain at least one miRNA to attenuate antigen presentation by antigen-presenting cells. The miRNA may be a complete miRNA sequence, a miRNA seed sequence, a miRNA sequence without a seed, or a combination thereof. As a non-limiting example, the miRNA incorporated into the polynucleotide of the present invention may be specific to the hematopoietic system. As another non-limiting example, the miRNA incorporated into the polynucleotide of the present invention to attenuate antigen presentation is miR-142-3p.

[0551] In some embodiments, a polynucleotide of the present invention can comprise at least one miRNA to attenuate expression of the encoded polypeptide in a tissue or cell of interest. As a non-limiting example, a polynucleotide of the present invention can comprise at least one miR-142-3p binding site, a miR-142-3p seed sequence, a miR-142-3p binding site without a seed, a miR-142-5p binding site, a miR-142-5p seed sequence, a miR-142-5p binding site without a seed, a miR-146 binding site, a miR-146 seed sequence, and / or a miR-146 binding site without a seed sequence.

[0552] In some embodiments, the polynucleotides of the present invention may contain at least one miRNA binding site in the 3'UTR to selectively degrade mRNA therapeutics in immune cells to suppress unwanted immunogenic responses caused by therapeutic delivery. As a non-limiting example, the miRNA binding site may make the polynucleotides of the present invention less stable in antigen-presenting cells. Non-limiting examples of these miRNAs include miR-142-5p, miR-142-3p, miR-146a-5p, and miR-146-3p.

[0553] In one embodiment, the polynucleotide of the invention comprises at least one miRNA sequence in a region of the polynucleotide that is capable of interacting with an RNA binding protein.

[0554] In some embodiments, a polynucleotide (e.g., RNA, e.g., mRNA) of the invention comprises (i) a sequence-optimized nucleotide sequence (e.g., ORF) encoding a checkpoint cancer vaccine comprising (i) one or more IDO antigenic peptides and (ii) one or more PD-L1 antigenic peptides, and (ii) an miRNA-binding site (e.g., an miRNA-binding site that binds to miR-142) and / or an miRNA-binding site that binds to miR-126.

[0555] IVT Polynucleotide Architecture In some embodiments, a polynucleotide of the present disclosure comprising an mRNA encoding a checkpoint cancer vaccine comprising (i) one or more IDO antigenic peptides and (ii) one or more PD-L1 antigenic peptides is an IVT polynucleotide. Traditionally, the basic components of an mRNA molecule include at least a coding region, a 5' UTR, a 3' UTR, a 5' cap, and a poly-A tail. While the IVT polynucleotide of the present disclosure can function as an mRNA, it is distinguished from wild-type mRNA in functional and / or structural design features that help overcome existing problems with effective polypeptide production using, for example, nucleic acid-based therapeutics.

[0556] The primary construct of an IVT polynucleotide comprises a first region of linked nucleotides flanked by a first flanking region and a second flanking region. This first region may include, but is not limited to, an encoded checkpoint cancer vaccine. The first flanking region may comprise a sequence of linked nucleosides that functions as a 5' untranslated region (UTR) (such as the native 5' UTR of a polypeptide or any 5' UTR of a nucleic acid encoding a non-native 5' UTR, such as, but not limited to, a heterologous 5' UTR or a synthetic 5' UTR). An IVT encoding a checkpoint cancer vaccine comprising (i) one or more IDO antigenic peptides and (ii) one or more PD-L1 antigenic peptides may include a signal sequence region encoding one or more signal sequences at its 5' end. The flanking region may comprise a region of linked nucleotides that includes one or more complete or incomplete 5' UTR sequences. The flanking region may also include a 5' cap. The second flanking region can comprise a region of linked nucleotides comprising one or more complete or incomplete 3'UTRs, which can encode the native or non-native 3'UTR of the checkpoint cancer vaccine (such as, but not limited to, a heterologous 3'UTR or a synthetic 3'UTR). The flanking region can also comprise a 3' tail sequence. The 3' tail sequence can be, but is not limited to, a polyA tail sequence, a polyAG quartet sequence, and / or a stem-loop sequence.

[0557] Additional and exemplary features of IVT polynucleotide architecture are disclosed in International PCT Application WO2017 / 201325, filed May 18, 2017, the entire contents of which are incorporated herein by reference.

[0558] 5'UTR and 3'UTR The UTRs can be homologous or heterologous to the coding region in the polynucleotide. In some embodiments, the UTRs are homologous to an ORF encoding a checkpoint cancer vaccine comprising (i) one or more IDO antigenic peptides and (ii) one or more PD-L1 antigenic peptides.

[0559] In some embodiments, the polynucleotide comprises two or more 5' UTRs or functional fragments thereof, each of which has the same or different nucleotide sequences. In some embodiments, the polynucleotide comprises two or more 3' UTRs or functional fragments thereof, each of which has the same or different nucleotide sequences.

[0560] In some embodiments, the 5'UTR or a functional fragment thereof, the 3'UTR or a functional fragment thereof, or any combination thereof is sequence-optimized.

[0561] In some embodiments, the 5'UTR or a functional fragment thereof, the 3'UTR or a functional fragment thereof, or any combination thereof comprises at least one chemically modified nucleobase (e.g., N1 methylpseudouracil or 5-methoxyuracil).

[0562] UTRs can have features that provide a regulatory role (e.g., stability, localization, and / or increased or decreased translation efficiency). A polynucleotide containing a UTR can be administered to a cell, tissue, or organism, and one or more regulatory features can be measured using routine methods. In some embodiments, a functional fragment of a 5' or 3' UTR comprises one or more regulatory features of the full-length 5' or 3' UTR, respectively.

[0563] Naturally occurring 5'UTRs are characterized by their role in translation initiation. These UTRs possess signatures such as the Kozak sequence, which is commonly known to be involved in the process of ribosomal translation initiation of many genes. The Kozak sequence has the consensus CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another "G." 5'UTRs are also known to form secondary structures involved in elongation factor binding.

[0564] Manipulation of features typically found in highly expressed genes of specific target organs can enhance polynucleotide stability and protein production. For example, introduction of the 5' UTR of a liver-expressed mRNA (such as albumin, serum amyloid A, apolipoprotein A / B / E, transferrin, alpha-fetoprotein, erythropoietin, or factor VIII) can enhance polynucleotide expression in hepatocyte cell lines or the liver. Similarly, using 5UTRs from other tissue-specific mRNAs to improve expression in tissues is possible in muscle (e.g., MyoD, myosin, myoglobin, myogenin, herculin), endothelial cells (e.g., Tie-1, CD36), myeloid cells (e.g., C / EBP, AML1, G-CSF, GM-CSF, CD11b, MSR, Fr-1, i-NOS), leukocytes (e.g., CD45, CD18), adipose tissue (e.g., CD36, GLUT4, ACRP30, adiponectin), and lung epithelial cells (e.g., SP-A / B / C / D).

[0565] In some embodiments, UTRs are selected from a family of transcripts in which proteins share common functions, structures, characteristics, or properties. For example, the encoded polypeptide may belong to a family of proteins (i.e., it shares at least one function, structure, characteristic, localization, origin, or expression pattern) that are expressed in a particular cell, tissue, or at a certain time during development. Any UTR from a gene or mRNA can be exchanged with another UTR from the same or different protein family to generate a new polynucleotide.

[0566] In some embodiments, the 5'UTR and 3'UTR may be heterologous. In some embodiments, the 5'UTR may be derived from a different species than the 3'UTR. In some embodiments, the 3'UTR may be derived from a different species than the 5'UTR.

[0567] Co-owned International Patent Application No. PCT / US2014 / 021522 (Publication No. WO / 2014 / 164253, incorporated herein by reference in its entirety) provides a list of exemplary UTRs that can be utilized as flanking regions to an ORF in the polynucleotides of the invention.

[0568] Additional exemplary UTRs of the present application include those derived from globin (such as α-globin or β-globin (e.g., Xenopus, mouse, rabbit, or human globin)); strong Kozak translation initiation signals; CYBA (e.g., human cytochrome b-245 α polypeptide); albumin (e.g., human albumin 7); HSD17B4 (hydroxysteroid (17-β) dehydrogenase); viruses (e.g., tobacco etch virus (TEV), Venezuelan equine encephalitis virus (VEEV), dengue virus, cytomegalovirus (CMV) (e.g., CMV immediate early 1 (IE1)), hepatitis viruses (e.g., hepatitis B virus), Sindbis virus, or PAV barley yellow dwarf virus); heat shock proteins proteins (e.g., hsp70); translation initiation factors (e.g., eIF4G); glucose transporters (e.g., hGLUT1 (human glucose transporter 1)); actin (e.g., human α-actin or β-actin); GAPDH; tubulin; histones; citric acid cycle enzymes; topoisomerases (e.g., the 5'UTR of the TOP gene lacking the 5'TOP motif (oligopyrimidine tract)); ribosomal protein large 32 (L32); ribosomal proteins (e.g., human or mouse ribosomal proteins such as rps9); ATP synthases (e.g., ATP5A1 or mitochondrial H +beta subunit of ATP synthase; growth hormone (e.g., bovine (bGH) or human (hGH)); elongation factors (e.g., elongation factor 1 alpha 1 (EEF1A1)); manganese superoxide dismutase (MnSOD); myocyte enhancer factor 2A (MEF2A); beta-F1-ATPase, creatine kinase, myoglobin, granulocyte colony-stimulating factor (G-CSF); collagen (e.g., type I collagen alpha 2 chain (Col1A2), type I collagen alpha 1 chain (Col1A1), type VI collagen alpha 2 chain (Col6A2), These include, but are not limited to, one or more 5'UTRs and / or 3'UTRs derived from the nucleic acid sequences of type VI collagen alpha 1 chain (Col6A1); ribophorin (e.g., ribophorin I (RPNI)); low-density lipoprotein receptor-related protein (e.g., LRP1); cardiotrophin-like cytokine factor (e.g., Nnt1); calreticulin (Calr); procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (Plod1); and nucleobindin (e.g., Nucb1).

[0569] In some embodiments, the 5'UTR is selected from the group consisting of a β-globin 5'UTR; a 5'UTR containing a strong Kozak translation initiation signal; a cytochrome b-245 alpha polypeptide (CYBA) 5'UTR; a hydroxysteroid (17-beta) dehydrogenase (HSD17B4) 5'UTR; a tobacco etch virus (TEV) 5'UTR; a Venezuelan equine encephalitis virus (TEEV) 5'UTR; a 5' proximal open reading frame of rubella virus (RV) RNA encoding a nonstructural protein; a dengue virus (DEN) 5'UTR; a heat shock protein 70 (Hsp70) 5'UTR; an eIF4G 5'UTR; a GLUT1 5'UTR; a functional fragment thereof, and any combination thereof.

[0570] In some embodiments, the 3'UTR is selected from the group consisting of β-globin 3'UTR; CYBA 3'UTR; albumin 3'UTR; growth hormone (GH) 3'UTR; VEEV 3'UTR; hepatitis B virus (HBV) 3'UTR; α-globin 3'UTR; DEN 3'UTR; PAV barley yellow dwarf virus (BYDV-PAV) 3'UTR; elongation factor 1 alpha 1 (EEF1A1) 3'UTR; manganese superoxide dismutase (MnSOD) 3'UTR; mitochondrial H(+)-ATP synthase beta subunit (β-mRNA) 3'UTR; GLUT1 3'UTR; MEF2A 3'UTR; β-F1-ATPase 3'UTR; functional fragments thereof, and combinations thereof.

[0571] Wild-type UTRs from any gene or mRNA can be incorporated into the polynucleotides of the present invention. In some embodiments, UTRs can be altered compared to wild-type or native UTRs, for example, by changing the orientation or location of the UTR relative to the ORF, or by incorporating additional nucleotides, deleting nucleotides, exchanging nucleotides, or rearranging nucleotides, to produce variant UTRs. In some embodiments, variants of 5'UTR or 3'UTR can be used, such as mutations of wild-type UTRs, or variants in which one or more nucleotides are added to or removed from the end of the UTR.

[0572] Additionally, one or more synthetic UTRs may be used in combination with one or more non-synthetic UTRs, see, e.g., Mandal and Rossi, Nat. Protoc. 2013 8(3):568-82, the contents of which are incorporated herein by reference in their entirety.

[0573] UTRs or portions thereof may be placed in the same orientation as in the selected transcript, or their orientation or location may be altered. Thus, the 5'UTR and / or 3'UTR may be inverted, shortened, extended, or combined with one or more other 5'UTRs or 3'UTRs. In some embodiments, the polynucleotide contains multiple UTRs, such as duplicated, tripled, or quadrupled 5'UTRs or 3'UTRs. For example, a duplicated UTR contains two copies of the same UTR, contiguous or substantially contiguous. For example, a duplicated β-globin 3'UTR may be used (see US2010 / 0129877, the contents of which are incorporated herein by reference in their entirety).

[0574] In certain embodiments, the 5'UTR and / or 3'UTR sequences of the present invention comprise a nucleotide sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a sequence selected from the group consisting of 5'UTR sequences including any of the 5'UTR or 3'UTR sequences disclosed herein (e.g., in Table 3A or Table 3B), and any combination thereof.

[0575] Polynucleotides of the invention can comprise a combination of features. For example, an ORF can be flanked by a 5' UTR containing a strong Kozak translation initiation signal and / or a 3' UTR containing an oligo(dT) sequence for templated addition of a polyA tail. The 5' UTR can comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and / or different UTRs (see, e.g., US2010 / 0293625, incorporated herein by reference in its entirety).

[0576] Other non-UTR sequences can be used as regions or subregions within the polynucleotides of the invention. For example, introns or portions of intron sequences can be incorporated into the polynucleotides of the invention. Incorporation of an intron sequence can increase protein production and polynucleotide expression levels. In some embodiments, the polynucleotides of the invention contain an internal ribosome entry site (IRES) in place of or in addition to a UTR (see, e.g., Yakubov et al., Biochem. Biophys. Res. Commun. 2010 394(1):189-193, the contents of which are incorporated herein by reference in their entireties). In some embodiments, the polynucleotide contains an IRES in place of a 5'UTR sequence. In some embodiments, the polynucleotide contains an ORF and a viral capsid sequence. In some embodiments, the polynucleotide contains a synthetic 5'UTR in combination with a non-synthetic 3'UTR.

[0577] In some embodiments, a UTR may also include at least one translation-enhancing polynucleotide, translation enhancer element, or translational enhancer element (collectively "TEE," which refers to a nucleic acid sequence that increases the amount of a polypeptide or protein produced from a polynucleotide). As a non-limiting example, a TEE may be located between the transcription promoter and the start codon. In some embodiments, the 5'UTR includes a TEE.

[0578] In one embodiment, a TEE is a conserved element in a UTR that can enhance the translation activity of a nucleic acid (such as, but not limited to, cap-dependent or cap-independent translation).

[0579] a. 5'UTR sequence The 5'UTR sequence is reported to be important for ribosome recruitment to mRNA and plays a role in translation (Hinnebusch A, et al., (2016) Science, 352:6292:1413-6).

[0580] Specifically disclosed herein is a polynucleotide (e.g., mRNA) encoding a polypeptide comprising an open reading frame encoding a checkpoint cancer vaccine comprising (i) one or more IDO antigenic peptides and (ii) one or more PD-L1 antigenic peptides (e.g., as described herein), the polynucleotide having a 5' UTR that confers increased half-life, increased expression, and / or increased activity of the polypeptide encoded by the polynucleotide or the polynucleotide itself. In one embodiment, the polynucleotide disclosed herein comprises, and LNP compositions comprise, (a) a 5' UTR (e.g., as provided in Table 3A or a variant or fragment thereof); (b) a coding region comprising a termination element (e.g., as described herein); and (c) a 3' UTR (e.g., as described herein). In one embodiment, the polynucleotide comprises a 5' UTR comprising a sequence provided in Table 3A or a variant or fragment thereof (e.g., a functional variant or fragment thereof).

[0581] In one embodiment, a polynucleotide having a 5' UTR sequence provided in Table 3A, or a variant or fragment thereof, has an increased polynucleotide half-life (e.g., about a 1.5-20 fold increase in the half-life of the polynucleotide). In one embodiment, the increase in half-life ...

Claims

1. A lipid nanoparticle (LNP) composition comprising a polynucleotide comprising an mRNA encoding a checkpoint cancer vaccine comprising (i) one or more (e.g., 1, 2, 3, 4, or more) indoleamine-pyrrole 2,3-dioxygenase (IDO) antigenic peptides and (ii) one or more (e.g., 1, 2, 3, 4, or more) programmed death-ligand 1 (PD-L1) antigenic peptides.

2. A lipid nanoparticle (LNP) composition for immunomodulation, e.g., for inducing an immune response and / or breaking immune tolerance (e.g., stimulating T effector cells), comprising a polynucleotide comprising an mRNA encoding a checkpoint cancer vaccine comprising (i) one or more (e.g., 1, 2, 3, 4, or more) IDO antigenic peptides and (ii) one or more (e.g., 1, 2, 3, 4, or more) PD-L1 antigenic peptides.

3. A lipid nanoparticle composition for stimulating T effector cells, the composition comprising a polynucleotide comprising an mRNA encoding a checkpoint cancer vaccine comprising (i) one or more (e.g., 1, 2, 3, 4, or more) IDO antigenic peptides and (ii) one or more (e.g., 1, 2, 3, 4, or more) PD-L1 antigenic peptides.

4. An mRNA construct comprising a polynucleotide encoding a checkpoint cancer vaccine comprising (i) one or more (e.g., 1, 2, 3, 4, or more) IDO antigenic peptides and (ii) one or more (e.g., 1, 2, 3, 4, or more) PD-L1 antigenic peptides.

5. 2. The LNP composition or mRNA construct of any one of the preceding claims, wherein the IDO antigenic peptide comprises a fragment of a naturally occurring IDO molecule or a variant thereof.

6. The LNP composition or mRNA construct of any one of the preceding claims, wherein the IDO antigenic peptide is derived from IDO1 or IDO2.

7. The LNP composition or mRNA construct of claim 6 , wherein the IDO antigenic peptide is derived from IDO1.

8. The LNP composition or mRNA construct of any one of the preceding claims, wherein the IDO antigenic peptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:

1.

9. 2. The LNP composition or mRNA construct of any one of the preceding claims, wherein the polynucleotide encoding the IDO antigenic fragment comprises a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:2 or an antigenic fragment thereof.

10. The LNP composition or mRNA construct of any one of the preceding claims, wherein the PD-L1 antigenic peptide comprises a fragment of a naturally occurring PD-L1 molecule or a variant thereof.

11. 3. The LNP composition or mRNA construct of any one of the preceding claims, wherein the PD-L1 antigenic peptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:

3.

12. 4. The LNP composition or mRNA construct of any one of the preceding claims, wherein the polynucleotide encoding the PD-L1 antigenic fragment comprises a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:4 or an antigenic fragment thereof.

13. The LNP composition or mRNA construct of any one of the preceding claims, wherein the checkpoint cancer vaccine comprises two IDO antigenic peptides and two PD-L1 antigenic peptides.

14. The LNP composition or mRNA construct of any one of claims 1 to 12, wherein the checkpoint cancer vaccine comprises three IDO antigenic peptides and three PD-L1 antigenic peptides.

15. The LNP composition or mRNA construct of any one of claims 1 to 12, wherein the checkpoint cancer vaccine comprises four IDO antigenic peptides and four PD-L1 antigenic peptides.

16. 2. The LNP composition or mRNA construct of any one of the preceding claims, wherein the checkpoint cancer vaccine comprises alternating IDO and PD-L1 antigenic peptides.

17. The LNP composition or mRNA construct of claim 15 or 16, wherein the checkpoint cancer vaccine comprises (i) an IDO antigenic peptide, (ii) a PD-L1 antigenic peptide, (iii) an IDO antigenic peptide, (iv) a PD-L1 antigenic peptide, (v) an IDO antigenic peptide, (vi) a PD-L1 antigenic peptide, (vii) an IDO antigenic peptide, and (viii) a PD-L1 antigenic peptide.

18. The LNP composition or mRNA construct of any one of claims 15-17, wherein the alternating IDO antigenic peptide and PD-L1 antigenic peptide comprise an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an IDO amino acid sequence provided in Table 1A (e.g., SEQ ID NO:5), or an antigenic fragment thereof.

19. The LNP composition or mRNA construct of any one of claims 16-18, wherein the polynucleotide encoding the alternating IDO and PD-L1 antigenic peptides comprises a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 6, 300, 301, or 302, or an antigenic fragment thereof.

20. (i) stimulating T effector cells; (ii) cytotoxic T cell-mediated killing of suppressor immune cells and tumor cells that overexpress PD-L1 or IDO; and / or (iii) inducing an anti-tumor immune response The LNP composition or mRNA construct of any one of the preceding claims,

21. The LNP composition or mRNA construct of any one of the preceding claims, which results in the amelioration or delay of cancer progression in a subject, for example as described herein.

22. 10. The LNP composition or mRNA construct of any one of the preceding claims, wherein the polynucleotide comprising an mRNA encoding the checkpoint cancer vaccine comprising one or more (e.g., 1, 2, 3, 4, or more) indoleamine-pyrrole 2,3-dioxygenase (IDO) antigenic peptides and (i) one or more (e.g., 1, 2, 3, 4, or more) programmed death-ligand 1 (PD-L1) antigenic peptides comprises at least one chemical modification.

23. The LNP composition or mRNA construct of claim 22, wherein the chemical modification is N1-methylpseudouridine.

24. The LNP composition of any one of the preceding claims, wherein the LNP composition comprises: (i) an ionizable lipid (e.g., an amino lipid); (ii) a sterol or other structured lipid; (iii) a non-cationic helper lipid or phospholipid; and (iv) a PEG-lipid.

25. 25. The LNP composition of claim 24, wherein the ionizable lipid comprises compound 25.

26. 25. The LNP composition of claim 24, wherein the PEG lipid is PEG DMG.

27. 25. The LNP composition of claim 24, wherein the LNP composition comprises (i) Compound 25, (ii) cholesterol, (iii) DSPC, and (iv) PEG DMG.

28. A pharmaceutical composition comprising the LNP composition or mRNA construct of any one of claims 1 to 27.

29. 1. A method of modulating (e.g., stimulating) an immune response in a subject, comprising administering to said subject in need thereof an effective amount of an LNP composition comprising a polynucleotide comprising an mRNA encoding a checkpoint cancer vaccine comprising one or more (e.g., 1, 2, 3, 4, or more) indoleamine-pyrrole 2,3-dioxygenase (IDO) antigenic peptides and (i) one or more (e.g., 1, 2, 3, 4, or more) programmed death-ligand 1 (PD-L1) antigenic peptides.

30. 1. A method of stimulating T effector cells in a subject, the method comprising administering to the subject an effective amount of an LNP composition comprising a polynucleotide comprising an mRNA encoding a checkpoint cancer vaccine comprising one or more (e.g., 1, 2, 3, 4, or more) indoleamine-pyrrole 2,3-dioxygenase (IDO) antigenic peptides and (i) one or more (e.g., 1, 2, 3, 4, or more) programmed death-ligand 1 (PD-L1) antigenic peptides.

31. A method of treating or preventing cancer or a symptom thereof, comprising administering to a subject in need thereof an effective amount of an LNP composition comprising a polynucleotide comprising an mRNA encoding a checkpoint cancer vaccine comprising one or more (e.g., one, two, three, four, or more) indoleamine-pyrrole 2,3-dioxygenase (IDO) antigenic peptides and (i) one or more (e.g., one, two, three, four, or more) programmed death-ligand 1 (PD-L1) antigenic peptides.

32. 32. The method of claim 31 , wherein the cancer is a locally advanced or metastatic solid tumor.

33. 32. The method of claim 31 , wherein the cancer is melanoma.

34. 34. The method of claim 33, wherein the melanoma is cutaneous melanoma.

35. 35. The method of claim 34, wherein the cutaneous melanoma is 1L cutaneous melanoma stage IIIB+.

36. 32. The method of claim 31 , wherein the cancer is NSCLC.

37. 37. The method of claim 36, wherein the NSCLC is 1L NSCLC.

38. 32. The method of claim 31 , wherein the cancer is bladder cancer.

39. 39. The method of claim 38, wherein the bladder cancer is non-muscle invasive bladder cancer.

40. 32. The method of claim 31 , wherein the cancer is head and neck cancer.

41. 41. The method of claim 40, wherein the head and neck cancer is head and neck squamous cell carcinoma.

42. 32. The method of claim 31 , wherein the cancer is colorectal cancer.

43. 43. The method of claim 42, wherein the colorectal cancer is microsatellite-stable colorectal cancer.

44. 32. The method of claim 31 , wherein the cancer is basal cell carcinoma.

45. 32. The method of claim 31 , wherein the cancer is breast cancer.

46. 46. ​​The method of claim 45, wherein the breast cancer is triple-negative breast cancer.

47. 47. The method of any one of claims 29-46, wherein the mRNA encoding the checkpoint cancer vaccine comprises the nucleic acid sequence of SEQ ID NO: 300, 301, or 302.

48. The method of any one of claims 29-47, wherein the LNP composition comprises: (i) an ionizable lipid (e.g., an amino lipid); (ii) a sterol or other structured lipid; (iii) a non-cationic helper lipid or phospholipid; and (iv) a PEG-lipid.

49. 49. The method of claim 48, wherein the ionizable lipid comprises compound 25.

50. 49. The method of claim 48, wherein the LNP composition comprises (i) Compound 25, (ii) cholesterol, (iii) DSPC, and (iv) PEG DMG.

51. 49. The method of claim 48, wherein the method further comprises administering to the subject a checkpoint inhibitor selected from the group consisting of anti-PD-1 and anti-CTLA4.

52. 52. The method of claim 51 , wherein the checkpoint inhibitor comprises pembrolizumab.

53. The method of any one of claims 29 to 52, wherein the LNP composition is administered at a dose of about 100 μg to about 1 mg.

54. 54. The method of claim 53, wherein the LNP composition is administered in a dose of 50 μg to 150 μg, 150 μg to 250 μg, 250 μg to 350 μg, 350 μg to 450 μg, 450 μg to 550 μg, 550 μg to 650 μg, 650 μg to 750 μg, 750 μg to 850 μg, 850 μg to 950 μg, or 950 μg to 1 mg.

55. 55. The method of claim 54, wherein the LNP composition is administered in a dose of 50 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, or 1 mg.

56. The method of any one of claims 29 to 55, wherein the LNP composition is administered intramuscularly.

57. The method of any one of claims 29 to 56, wherein the LNP composition is administered once every three weeks.

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