Nucleic acids encoding therapeutic polypeptides and lipid nanoparticle compositions comprising same

Abstract

The present disclosure provides lipid nanoparticle compositions comprising a nucleic acid encoding a therapeutic polypeptide. The disclosure also provides novel IL-15 polypeptides, fusion proteins comprising the IL-15 polypeptides, and nucleic acids encoding the IL-15 polypeptides and the fusion proteins.

Description

TECHNICAL FIELD

[0001] The present disclosure provides lipid nanoparticle compositions comprising a nucleic acid encoding a therapeutic polypeptide (e.g., interleukin 15 (IL-15)), or a fusion protein comprising IL-15. The present disclosure also provides novel IL-15 polypeptides, fusion proteins comprising the IL-15 polypeptides, and nucleic acids encoding the IL-15 polypeptides and the fusion proteins. BACKGROUND

[0002] Interleukin 15 (IL-15) is a cytokine that induces proliferation of natural killer (NK) cells and other cells of the immune system, and is involved in killing of virus-infected cells and cancer cells. IL-15 is structurally similar to interleukin-2 (IL-2), and both can bind to the IL-2 receptor β / γ complex. Cytokine specificity of IL-2 or IL-15 is conferred by additional, specific receptors, namely IL-2 receptor alpha (IL-2Ra) and IL-15 receptor alpha (IL-15Ra). IL-15Ra can associate with the IL-2 receptor β / γ complex to form a functional high-affinity receptor aβγ complex that is specific for IL-15.

[0003] IL-15 plays a multifaceted role in development and control of the immune system, and can be used to treat cancer. However, existing IL-15 drug candidates have significant side effects. There are also difficulties in delivering IL-15-based protein or nucleic acid therapeutic drugs. Therefore, there remains a need to develop compositions and methods to facilitate delivery of IL-15-based protein or nucleic acid therapeutic drugs and / or reduce adverse reactions caused by IL-15-based protein or nucleic acid therapeutic drugs. SUMMARY

[0004] Disclosed herein are lipid nanoparticle compositions comprising a nucleic acid encoding an IL-15 polypeptide or a fusion protein comprising an IL-15 polypeptide. Also disclosed herein are novel IL-15 polypeptides, fusion proteins comprising the IL-15 polypeptides, and nucleic acids encoding the IL-15 polypeptides and the fusion proteins. The present disclosure also provides uses of the lipid nanoparticle compositions, therapeutic polypeptides, and nucleic acids encoding the therapeutic polypeptides for treating diseases or disorders, including cancer.

[0005] In one aspect, the present disclosure provides a lipid nanoparticle composition comprising: a target polynucleotide comprising a first nucleic acid encoding an interleukin 15 polypeptide or a variant thereof, and a lipid nanoparticle comprising a compound having the following formula (I):

[0006]

[0007] or a pharmaceutically acceptable salt thereof, wherein,

[0008] R a is selected from the group consisting of hydrogen, R 5 , alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl are optionally substituted with one or more groups independently selected from halogen, hydroxyl, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, and R 6 ;

[0009] R 1 is

[0010] R 2 is

[0011] R 3 is

[0012] R 4 is

[0013] R 5 if present is

[0014] R 6 if present is

[0015] each W is independently selected from O, S, or NR b , and each R b is independently selected from hydrogen, alkyl, alkoxycarbonyl, acyl, or sulfonyl;

[0016] each Y is independently selected from O, S, NR c , N(R c )Z(W), N(R c )N(R c ), or N(R c )N(R c )Z(W), and each R c is independently selected from hydrogen, alkyl, alkoxycarbonyl, acyl, or sulfonyl;

[0017] each Z is independently selected from C, S, or S(O);

[0018] each n is independently 0, 1, 2, 3, 4, or 5;

[0019] each m is independently 0, 1, 2, or 3;

[0020] each p is independently 1, 2, 3, or 4; and

[0021] R 1c , R 2c , R 3c , and R 4c are each independently selected from the group consisting of alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally substituted with one or more groups independently selected from the group consisting of halogen, hydroxyl, oxo, cyano, cycloalkyl, heterocyclyl, aryl, and heteroaryl, and the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally interrupted with one or more groups independently selected from the group consisting of cycloalkyl, heterocyclyl, aryl, and heteroaryl.

[0022] In one aspect, the present disclosure provides an interleukin 15 (IL-15) polypeptide variant comprising an amino acid sequence of the present disclosure.

[0023] In one aspect, the present disclosure provides a fusion protein comprising an interleukin 15 (IL-15) polypeptide and an IL-15 receptor alpha sushi domain (IL-15Ra sushi), wherein the fusion protein comprises an amino acid sequence of the present disclosure. In some embodiments, the fusion protein further comprises an Fc moiety located at the N-terminus of the fusion protein, or located at the C-terminus of the fusion protein. In some other embodiments, the fusion protein further comprises an antibody moiety that binds to PD-L1 or PD-1.

[0024] In one aspect, the present disclosure provides an isolated nucleic acid sequence encoding an interleukin 15 polypeptide or a variant thereof, the isolated nucleic acid sequence comprising a nucleic acid sequence having at least 85% sequence identity to a nucleic acid sequence of the present disclosure.

[0025] In one aspect, the present disclosure provides an isolated nucleic acid sequence encoding a fusion protein of the present disclosure.

[0026] In one aspect, the present disclosure provides a method of enhancing immune cell activation in a subject, comprising administering to the subject an effective amount of a lipid nanoparticle composition of the present disclosure, an effective amount of an IL-15 polypeptide variant of the present disclosure, an effective amount of a fusion protein of the present disclosure, or an effective amount of an isolated nucleic acid sequence of the present disclosure.

[0027] In one aspect, the present disclosure provides a method of treating a disease or a disorder in a subject, comprising administering to the subject an effective amount of a lipid nanoparticle composition of the present disclosure, an effective amount of an IL-15 polypeptide variant of the present disclosure, an effective amount of a fusion protein of the present disclosure, or an effective amount of an isolated nucleic acid sequence of the present disclosure. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figures 1A-1DIn vitro expression of the test nucleic acid is shown.

[0029] Figures 2A-2E Affinity of the protein expressed by the test nucleic acid to IL15Rβ / γ is shown.

[0030] Figures 3A-3C Activation level of the reporter cell line of 001 to 004 is shown.

[0031] Figure 4 Affinity of the protein expressed by PDL1Ab_014, PDL1Ab_014D43, PDL1Ab_014D56, PDL1Ab_014D61, PDL1Ab_014D65, PDL1Ab_014D66 and PDL1Ab_014D106 to PD-L1 is shown.

[0032] Figures 5A-5D Level of in vitro activation of PBMC subpopulation proliferation by the test nucleic acid is shown.

[0033] Figure 6A IL6 level in cell supernatant is shown. Figure 6B iFN-γ level in cell supernatant is shown. Figure 6C Cell proliferation after activation by the test nucleic acid is shown. Figure 6D STAT5 phosphorylation after activation by the test nucleic acid is shown.

[0034] Figure 7 In vitro delivery characteristics of the test lipid particle are shown.

[0035] Figures 8A-8C In vivo immune activation activity level of the lipid nanoparticle composition comprising the test nucleic acid is shown.

[0036] Figure 9A Tumor growth curve after administration of the lipid nanoparticle composition comprising 001 to 004 is shown. Figure 9B Mouse weight curve after administration of the lipid nanoparticle composition comprising 001 to 004 is shown.

[0037] Figure 10A Tumor growth curve after administration of the lipid nanoparticle composition comprising 001 to 004 is shown. Figure 10B Mouse weight curve after administration of the lipid nanoparticle composition comprising 001 to 004 is shown. Figure 10C Leukocyte portion of mouse whole blood cell count at the end of the experiment is shown. Figure 10D Erythrocyte portion of mouse whole blood cell count at the end of the experiment is shown. Figure 10E Thrombocyte portion of mouse whole blood cell count at the end of the experiment is shown. Figure 10FThe results of liver function tests in mouse blood chemistry at the end of the experiment are shown. Figure 10G The results of renal function tests in mice by blood chemistry at the end of the experiment are shown, in which no statistically significant results were found for any of the indicators related to hematologic toxicity, hepatotoxicity, and nephrotoxicity associated with the formulation or therapeutic target.

[0038] Figure 11A Tumor growth curves after application of the lipid nanoparticle composition disclosed herein are shown. Figure 11B The body weight curves of mice after administration of the lipid nanoparticle composition disclosed herein are shown.

[0039] Figure 12A Tumor growth curves are shown after administration of the lipid nanoparticle composition of this disclosure in combination with a PD-L1 antibody. Figure 12B The body weight curves of mice after administration of the lipid nanoparticle composition of this disclosure in combination with PD-L1 antibody are shown.

[0040] Figure 13A Tumor growth curves after application of the lipid nanoparticle composition disclosed herein are shown. Figure 13B The body weight curves of mice after administration of the lipid nanoparticle composition disclosed herein are shown.

[0041] Figures 14A-14C Pharmacokinetic curves of the lipid nanoparticle compositions disclosed herein after intravenous or intramuscular injection are shown. Detailed Implementation

[0042] Reference will now be made in detail to certain embodiments of this disclosure, examples of which are illustrated in the accompanying structures and molecular formulas. While this disclosure will be described in conjunction with the enumerated embodiments, it should be understood that these embodiments are not intended to limit this disclosure to them. Rather, this disclosure is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of this disclosure as defined by the claims. Those skilled in the art will recognize that many methods and materials similar to or equivalent to those described herein can be used to practice this disclosure. This disclosure is by no means limited to the methods and materials described. In the event that one or more of the incorporated references and similar materials (including, but not limited to, defined terms, usage of terms, described techniques, etc.) differ from or contradict this application, this disclosure shall prevail. All references, patents, and patent applications cited in this disclosure are incorporated herein by reference in their entirety.

[0043] Definitions

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. As used herein, the following terms are intended to have the following meanings.

[0045] As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes a single compound as well as a plurality of different compounds.

[0046] As used herein, the term "about" is intended to indicate that the recited value should not be interpreted as an absolute value and should also consider measurement error, batch-to-batch variation, and / or device-to-device variation.

[0047] The words "comprise," "comprising," "include," "including," and "includes" used in the specification and claims are intended to mean that there are, or consist of, the features, integers, components, or steps listed, but they do not exclude the presence or addition of one or more other features, integers, components, steps, or groups thereof.

[0048] It should be understood that the "compounds" of the present disclosure can exist in solvated as well as unsolvated forms, such as, for example, hydrated forms, solid forms, and the present disclosure is intended to encompass all such solvated and unsolvated forms. It should also be understood that the "compounds" of the present invention can exist in the form of pharmaceutically acceptable salts. In some embodiments, the "compounds" of the present disclosure are ionizable lipids. In some embodiments, the "compounds" of the present invention can exist as cationic lipids at physiological pH.

[0049] The definitions of specific functional groups and chemical terms are described in more detail below. For the purposes of this disclosure, chemical elements are identified according to the CAS version of the periodic table in the 75th edition of the Handbook of Chemistry and Physics, and specific functional groups are generally defined as described herein. In addition, the general principles of organic chemistry, as well as specific functional components and reactivity, are described in the following literature: *Organic Chemistry*, Thomas Sorrell, 2nd ed., University Science Books, Sosalito, 2006; Smith and March, *March's Advanced Organic Chemistry*, 6th ed., John Wiley & Sons, Inc., New York, 2007; Larock, *Comprehensive Organic Transformations*, 3rd ed., VCH Publishers, Inc., New York, 2018; Carruthers, *Some Modern Methods of Organic Synthesis*, 4th ed., Cambridge University Press, Cambridge, 2004; all of which are incorporated herein by reference.

[0050] Linking substituents are described throughout this disclosure. Where the structure explicitly requires a linking group, the Markush variable listed for said group is understood to be the linking group. For example, if the structure requires a linking group and the Markush group definition of said variable lists "alkyl", then "alkyl" should be understood to mean a linked alkylene group.

[0051] When a bond showing the substituent crosses the bond between two atoms in the linking ring, then the substituent can bond to any atom in the ring. When a substituent is listed but not specified via which atom it bonds to the remainder of the compound in the given formula, the substituent can bond to any atom in the formula. Combinations of substituents and / or variables are permitted, but only if such combinations produce a stable compound.

[0052] In any variable (e.g., R) i When a compound appears more than once in any component or formula, its definition for each occurrence is independent of its definition for each subsequent occurrence. Therefore, for example, if the display group is represented by 0 to 2 R...i partially substituted, the group can be optionally substituted with up to two R i partially substituted, and R i is independently selected from R i is as defined above. Also, combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0053] As used herein, the term “C i-j ” indicates a range of the number of carbon atoms, wherein i and j are integers, and the range of the number of carbon atoms includes the endpoints (i.e., i and j) and every integer point in between, and wherein j is greater than i. For example, C 1-6 indicates a range of one to six carbon atoms, including one carbon atom, two carbon atoms, three carbon atoms, four carbon atoms, five carbon atoms, and six carbon atoms. In some embodiments, the term “C 1-24 ” indicates 1 to 24 carbon atoms, particularly 2 to 24 carbon atoms, particularly 4 to 24 carbon atoms, particularly 6 to 24 carbon atoms, particularly 8 to 22 carbon atoms, particularly 10 to 20 carbon atoms, particularly 10 to 18 carbon atoms, or particularly 12 to 18 carbon atoms.

[0054] As used herein, the term “alkyl,” whether used alone or as part of another term, refers to a saturated straight or branched chain hydrocarbon group that can be optionally independently substituted with one or more substituents described below. The term “C i-j alkyl” refers to an alkyl group having i to j carbon atoms. In some embodiments, the alkyl group contains 1 to 24 carbon atoms. In some embodiments, the alkyl group contains 1 to 23 carbon atoms. In some embodiments, the alkyl group contains 1 to 22 carbon atoms. In some embodiments, the alkyl group contains 1 to 21 carbon atoms. In some embodiments, the alkyl group contains 1 to 20 carbon atoms, 1 to 19 carbon atoms, 1 to 18 carbon atoms, 1 to 17 carbon atoms, 1 to 16 carbon atoms, 1 to 15 carbon atoms, 1 to 14 carbon atoms, 1 to 13 carbon atoms, or 1 to 12 carbon atoms. In some embodiments, the alkyl group contains 12 to 18 carbon atoms. In some embodiments, the alkyl group contains 12 to 17 carbon atoms, 12 to 16 carbon atoms, 12 to 15 carbon atoms, or 12 to 14 carbon atoms. In some embodiments, the alkyl group contains 12 to 19 carbon atoms, 12 to 20 carbon atoms, 12 to 21 carbon atoms, 12 to 22 carbon atoms, 12 to 23 carbon atoms, or 12 to 24 carbon atoms. “C 1-10 alkyl” includes, but is not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl. “C 1-6Examples of "alkyl" are methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-l-butyl, 2-methyl-l-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, and the like.

[0055] An alkyl group can be further substituted with substituents that independently replace one or more hydrogen atoms on one or more carbons of the alkyl group. Examples of such substituents can include, but are not limited to, acyl, alkyl, alkenyl, alkynyl, oxo, halo, hydroxyl, alkoxy, haloalkyl, haloalkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, phosphate, phosphonate, phosphinite, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfmyl, sulfonates, sulfamoyl, sulfamidoyl, nitro, trifluoromethyl, cyano, nitro, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups described below can also be similarly substituted.

[0056] As used herein, the term "alkenyl," whether used alone or as part of another term, refers to a straight or branched chain hydrocarbon having at least one carbon-carbon double bond that can be optionally independently substituted with one or more substituents described herein, and includes groups having "cis" orientation and "trans" orientation or alternatively "E" orientation and "Z" orientation. In some embodiments, the alkenyl group contains 2 to 24 carbon atoms. In some embodiments, the alkenyl group contains 2 to 23 carbon atoms. In some embodiments, the alkenyl group contains 2 to 22 carbon atoms, 2 to 21 carbon atoms, 2 to 20 carbon atoms, 2 to 19 carbon atoms, 2 to 18 carbon atoms, 2 to 17 carbon atoms, 2 to 16 carbon atoms, 2 to 15 carbon atoms, 2 to 14 carbon atoms, 2 to 13 carbon atoms, 2 to 12 carbon atoms, 2 to 11 carbon atoms, and in some embodiments, the alkenyl group contains 2 carbon atoms. In some embodiments, the alkenyl group contains 12 to 18 carbon atoms. In some embodiments, the alkenyl group contains 12 to 17 carbon atoms, 12 to 16 carbon atoms, 12 to 15 carbon atoms, or 12 to 14 carbon atoms. In some embodiments, the alkenyl group contains 12 to 19 carbon atoms, 12 to 20 carbon atoms, 12 to 21 carbon atoms, 12 to 22 carbon atoms, 12 to 23 carbon atoms, or 12 to 24 carbon atoms. In some embodiments, the alkenyl group contains one or more "Z" carbon-carbon double bonds. Examples of alkenyl groups include, but are not limited to, ethylenyl (or vinyl), propenyl, butenyl, pentenyl, 1 -methyl-2-buten- 1 -yl, 5-hexenyl, and the like. In some embodiments, the alkenyl group has at least one carbon-carbon double bond. In some embodiments, the alkenyl group has at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten carbon-carbon double bonds. In some embodiments, two or more of the carbon-carbon double bonds in the alkenyl group are conjugated. In some embodiments, two or more of the carbon-carbon double bonds in the alkenyl group are unconjugated. In some embodiments, two or more of the carbon-carbon double bonds in the alkenyl group are isolated, cumulative, or conjugated. The term "alkenyl," whether used alone or as part of another term, also means to include straight chain or branched chain hydrocarbons having at least one carbon-carbon triple bond.

[0057] As used herein, the term "alkynyl," whether used alone or as part of another term, refers to a straight or branched chain hydrocarbon having at least one carbon-carbon triple bond that can be optionally independently substituted with one or more substituents described herein. In some embodiments, the alkynyl group contains 2 to 24 carbon atoms. In some embodiments, the alkynyl group contains 2 to 23 carbon atoms. In some embodiments, the alkynyl group contains 2 to 22 carbon atoms, 2 to 21 carbon atoms, 2 to 20 carbon atoms, 2 to 19 carbon atoms, 2 to 18 carbon atoms, 2 to 17 carbon atoms, 2 to 16 carbon atoms, 2 to 15 carbon atoms, 2 to 14 carbon atoms, 2 to 13 carbon atoms, 2 to 12 carbon atoms, 2 to 10 carbon atoms, and in some embodiments, the alkynyl group contains 2 carbon atoms. In some embodiments, the alkynyl group contains 12 to 18 carbon atoms. In some embodiments, the alkynyl group contains 12 to 17 carbon atoms, 12 to 16 carbon atoms, 12 to 15 carbon atoms, or 12 to 14 carbon atoms. In some embodiments, the alkynyl group contains 12 to 19 carbon atoms, 12 to 20 carbon atoms, 12 to 21 carbon atoms, 12 to 22 carbon atoms, 12 to 23 carbon atoms, or 12 to 24 carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl, 1 -propynyl, 2-propynyl, and the like. In some embodiments, the alkynyl group has at least one carbon-carbon triple bond. In some embodiments, the alkynyl group has at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten carbon-carbon triple bonds. In some embodiments, two or more of the carbon-carbon triple bonds in the alkynyl group are conjugated. In some embodiments, two or more of the carbon-carbon triple bonds in the alkynyl group are unconjugated. The term "alkynyl," whether used alone or as part of another term, also means to include straight chain or branched chain hydrocarbons having at least one carbon-carbon triple bond and at least one carbon-carbon double bond.

[0058] As used herein, the term "alkoxy," whether used alone or as part of another term, refers to an alkyl group as previously defined attached to the parent molecule through an oxygen atom. The term "C i-j alkoxy" means that the alkyl portion of the alkoxy group has i to j carbon atoms. In some embodiments, the alkoxy group contains 1 to 10 carbon atoms. In some embodiments, the alkoxy group contains 1 to 9 carbon atoms. In some embodiments, the alkoxy group contains 1 to 8 carbon atoms, 1 to 7 carbon atoms, 1 to 6 carbon atoms, 1 to 5 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms. The term "C 1-6 Examples of "C

[0059] As used herein, the term“amino” means -NH2. In some embodiments, an amino group can be substituted with any possible substituent on nitrogen.

[0060] As used herein, the term“aryl,” whether used alone or as part of another term, means a monocyclic and polycyclic ring system having from 5 to 20 ring members in total, wherein at least one ring in the system is aromatic, and wherein each ring in the system contains from 3 to 12 ring members. Examples of“aryl” include, but are not limited to, phenyl, biphenyl, naphthyl, anthryl, and the like, which can bear one or more substituents. As used herein, the scope of the term“aryl” also includes groups in which an aromatic ring is fused to one or more non-aromatic rings, such as fused to one or more cycloalkyl rings. In the case of polycyclic ring systems, only one ring need be aromatic (e.g., 2,3-dihydroindole), but all rings can be aromatic (e.g., quinoline). The second ring can also be fused, bridged, or spiro. Examples of polycyclic aryl groups include, but are not limited to, benzofuranyl, indanyl, phthalimidyl, naphthalimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like. An aryl group can be substituted at one or more ring positions with substituents as described above.

[0061] As used herein, the term“cycloalkyl,” whether used alone or as part of another term, means a monovalent non-aromatic saturated or partially unsaturated monocyclic and polycyclic ring system, wherein all ring atoms are carbon, and the system contains at least three ring-forming carbon atoms. In some embodiments, a cycloalkyl group can contain from 3 to 12 ring-forming carbon atoms, from 3 to 11 ring-forming carbon atoms, from 3 to 10 ring-forming carbon atoms, from 3 to 9 ring-forming carbon atoms, from 3 to 8 ring-forming carbon atoms, from 3 to 7 ring-forming carbon atoms, from 3 to 6 ring-forming carbon atoms, from 3 to 5 ring-forming carbon atoms, from 3 to 4 ring-forming carbon atoms, from 4 to 12 ring-forming carbon atoms, from 4 to 11 ring-forming carbon atoms, from 4 to 10 ring-forming carbon atoms, from 4 to 9 ring-forming carbon atoms, from 4 to 8 ring-forming carbon atoms, from 4 to 7 ring-forming carbon atoms, from 4 to 6 ring-forming carbon atoms, from 4 to 5 ring-forming carbon atoms. A cycloalkyl group can be saturated or partially unsaturated. A cycloalkyl group can be substituted. In some embodiments, a cycloalkyl group can be a saturated cyclic alkyl group. In some embodiments, a cycloalkyl group can be a partially unsaturated cyclic alkyl group containing at least one double or triple bond in its ring system.

[0062] In some embodiments, a cycloalkyl group can be monocyclic or polycyclic. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, and cyclododecyl.

[0063] In some embodiments, cycloalkyl groups can be saturated or partially unsaturated polycyclic (e.g., bicyclic and tricyclic) carbocyclic ring systems, which can be arranged as fused, spiro, or bridged ring systems. As used herein, the term “fused” refers to ring systems in which two rings share two adjacent atoms, the term “spiro” refers to ring systems in which two rings are connected by a single common atom, and “bridged” refers to ring systems in which two rings share three or more atoms. Examples of fused carbocyclic groups include, but are not limited to, naphthyl, benzopyrenyl, anthryl, acenaphthyl, fluorenyl, and the like. Examples of spiro carbocyclic groups include, but are not limited to, spiro[5.5]undecyl, spiro- pentadienyl, spiro[3.6]-decyl, and the like. Examples of bridged carbocyclic groups include, but are not limited to, bicyclo[1,1,1]pentenyl, bicyclo[2,2,1]heptenyl, bicyclo[2,2,1]heptanyl, bicyclo[2,2,2]octanyl, bicyclo[3,3,1]nonanyl, bicyclo[3,3,3]undecanyl, and the like.

[0064] As used herein, the term “cyano” means -CN.

[0065] As used herein, the term “oxo” means =O, which replaces two hydrogen atoms attached to one atom. For example, an oxo-substituted ethyl group is CH3C(=O)- or -C(=O)CH2-.

[0066] As used herein, the term “halogen” means an atom selected from fluorine (fluoro), chlorine (chloro), bromine (bromo), and iodine (iodo).

[0067] As used herein, the term “haloalkyl,” whether used as a part of another term or standing alone, means an alkyl group having one or more halogen substituents. Examples of haloalkyl include, but are not limited to, trifluoromethyl (-CF3), pentafluoroethyl (-C2F5), difluoromethyl (-CHF2), trichloromethyl (-CCl3), dichloromethyl (-CHCl2), pentachloroethyl (-C2Cl5), and the like.

[0068] As used herein, the term “haloalkoxy,” whether used as a part of another term or standing alone, means an alkoxy group having one or more halogen substituents. Thus, the term “halo-C i-j alkoxy,” whether used as a part of another term or standing alone, means a C i-j alkoxy group having one or more halogen substituents. Examples of haloalkoxy include, but are not limited to, -O-CF3, -O-C2F5, -O-CHF2, -O-CCl3, -O-CHCl2, -O-C2Cl5, and the like.

[0069] As used herein, the term "heteroatom" refers to nitrogen (N), oxygen (O), sulfur (S), and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen including ammonium ions. In some embodiments, a heteroatom is a nitrogen atom.

[0070] As used herein, the term "heteroalkyl," "heteroalkenyl," or "heteroalkynyl," whether used alone or as part of another term, refers to an alkyl, alkenyl, or alkynyl group that contains one or more heteroatoms. Thus, the term "hetero-C i-j alkyl," "hetero-C i-j alkenyl," or "hetero-C i-j alkynyl," whether used alone or as part of another term, refers to a C i-j alkyl, C i-j alkenyl, or C i-j alkynyl group that contains one or more heteroatoms. For example, the term "hetero-C 1-6 alkyl," whether used alone or as part of another term, refers to a C 1-6 alkyl group that contains one or more heteroatoms. In some embodiments, a heteroalkyl, heteroalkenyl, or heteroalkynyl contains at least one heteroatom. In some embodiments, a heteroalkyl, heteroalkenyl, or heteroalkynyl contains at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten heteroatoms. In some embodiments, two or more of the heteroatoms in a heteroalkyl, heteroalkenyl, or heteroalkynyl are the same. In some embodiments, two or more of the heteroatoms in a heteroalkyl, heteroalkenyl, or heteroalkynyl are different. In some embodiments, two or more of the heteroatoms in a heteroalkyl, heteroalkenyl, or heteroalkynyl are directly bound. In some embodiments, two or more of the heteroatoms in a heteroalkyl, heteroalkenyl, or heteroalkynyl are not directly bound.

[0071] As used herein, the term "heteroaryl," whether used alone or as part of another term, refers to an aryl group containing one or more heteroatoms in addition to carbon atoms. The heteroaryl group can be monocyclic. Examples of monocyclic heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, naphthyridinyl, benzofuranyl, and pteridinyl. Heteroaryl groups also include polycyclic groups in which a heteroaromatic ring is fused with one or more aryl, heteroaryl, alicyclic, or heterocyclic rings, where the attachment is through a ring atom of the heteroaromatic ring or another ring. Examples of polycyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, benzothienyl, benzofuranyl, benzo[l,3]dioxolyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, dihydroquinolyl, dihydroisoquinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenoxazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.

[0072] As used herein, the term "heterocyclyl" refers to a saturated or partially unsaturated carbocyclyl group in which one or more ring atoms are heteroatoms independently selected from oxygen, sulfur, nitrogen, phosphorus, and the like, with the remaining ring atoms being carbon, wherein one or more ring atoms can be optionally independently substituted with one or more substituents. In some embodiments, the heterocyclyl group is a saturated heterocyclyl group. In some embodiments, the heterocyclyl group is a partially unsaturated heterocyclyl group having one or more double bonds in its ring system. In some embodiments, the heterocyclyl group can contain any oxidized form of carbon, nitrogen or sulfur, and any quaternized form of a basic nitrogen. Where possible, the heterocyclyl group can be carbon-linked or nitrogen-linked. In some embodiments, the heterocycle is carbon-linked. In some embodiments, the heterocycle is nitrogen-linked. For example, a group derived from pyrrole can be a pyrrol-l-yl group (nitrogen-linked) or a pyrrol-3-yl group (carbon-linked). Further, a group derived from imidazole can be an imidazol-l-yl group (nitrogen-linked) or an imidazol-3-yl group (carbon-linked).

[0073] The heterocyclyl group can be monocyclic. Examples of monocyclic heterocyclyl groups include, but are not limited to, oxetanyl, 1,1-dioxothietanyl pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothienyl, azetidinyl, pyrrolyl, furanyl, thienyl, pyrazolyl, imidazolyl, triazolyl, oxazolyl, thiazolyl, piperidinyl, piperazinyl, morpholinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, pyridonyl, pyrimidonyl, pyrazinonyl, pyrrolidonyl, triazinonyl, and the like.

[0074] Heterocyclyl groups can be polycyclic, including fused, spiro, and bridged ring systems. Fused heterocyclyl groups include groups in which a heterocyclyl group is fused to a saturated, partially unsaturated, or fully unsaturated (i.e., aromatic) carbocyclic or heterocyclic ring. Examples of fused heterocyclyl groups include, but are not limited to, phenyl fused or pyridyl fused rings such as quinolinyl, isoquinolinyl, quinoxalinyl, quinolizinyl, quinazolinyl, azaindolizinyl, pteridinyl, chromenyl, isochromenyl, indolyl, isoindolyl, indolizinyl, indazolyl, purinyl, benzofuranyl, isobenzofuranyl, benzimidazolyl, benzothiophenyl, benzothiazolyl, carbazolyl, phenoxazinyl, phenothiazinyl, phenanthridinyl, imidazo[l,2-a]pyridinyl, [l,2,4]triazolo[4,3-a]pyridinyl, [l,2,3]triazolo[4,3-a]pyridinyl, and the like. Examples of spiro heterocyclyl groups include, but are not limited to, spirofuran, spirooxazine, 5-aza-spiro[2.4]heptanyl, 6-aza-spiro[2.5]octanyl, 6-aza-spiro[3.4]octanyl, 2-oxa-6-aza-spiro[3.3]heptanyl, 2-oxa-6-aza-spiro[3.4]octanyl, 6-aza-spiro[3.5]nonanyl, 7-aza-spiro[3.5]nonanyl, l-oxa-7-aza-spiro[3.5]nonanyl, and the like. Examples of bridged heterocyclyl groups include, but are not limited to, 3-aza-bicyclo[3.1.0]hexanyl, 8-aza-bicyclo[3.2.1]octanyl, l-aza-bicyclo[2.2.2]octanyl, 2-aza-bicyclo[2.2.1]heptanyl, l,4-diaza-bicyclo[2.2.2]octanyl, and the like.

[0075] As used herein, the term "hydroxyl" or "hydroxy" means -OH.

[0076] As used herein, the term "alkoxycarbonyl" means alkyl-O-C(=O)-. In some embodiments, the alkoxycarbonyl group can be further substituted on the alkyl group with any of the possible substituents described above. Examples of alkoxycarbonyl groups include, but are not limited to, tert-butoxycarbonyl, benzyloxycarbonyl, allyloxycarbonyl, and 9-fluorenylmethoxycarbonyl.

[0077] As used herein, the term "sulfonyl" means R-SO2-, where R is hydrogen, or any of the possible substituents on sulfur. Examples of sulfonyl groups include, but are not limited to, p-toluenesulfonyl, p-bromobenzenesulfonyl, 2- or 4-nitrobenzenesulfonyl, trifluoromethanesulfonyl, methanesulfonyl, and 5-(dimethylamino)naphthalene-l-sulfonyl.

[0078] As used herein, the term "acyl," whether used by itself, as part of another

[0079] As used herein, the term "partially unsaturated" refers to a group that includes at least one double or triple bond. The term "partially unsaturated" is intended to encompass rings that have multiple sites of unsaturation, but is not intended to encompass aromatic (i.e., completely unsaturated) moieties.

[0080] As used herein, the term "substituted," whether preceded by "optionally" or not, means one or more hydrogens of the designated moiety is replaced with a suitable substituent. It should be understood that "substituted" or "substituted with" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable or chemically feasible compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. An "optionally substituted" group can have an appropriate substituent at each substitutable position of the group, and when more than one position in any given structure can be substituted with more than one substituent selected from a specified group, the substituent may, at each position, be same or different, as appropriate. It is understood by those skilled in the art that substituents themselves can be substituted if appropriate. Unless specifically stated otherwise, reference to a chemical moiety herein is understood to include both substituted and unsubstituted variants. For example, reference to an "aryl" group or moiety implicitly includes both substituted and unsubstituted variants.

[0081] As used herein, the term "interrupted," whether preceded by the term "optionally" or not, means that one or more covalent bonds of the designated moiety are replaced by a suitable linking group, but not at the terminus. In some embodiments, the bond that is replaced is a carbon-carbon bond. In some embodiments, the bond that is replaced is a carbon-heteroatom bond. Unless otherwise noted, a "optionally interrupted" group can have a suitable linking group at every replaceable position of the group, and when more than one position in any given structure can be interrupted by more than one linking group selected from the designated groups, the linking group at each position can be the same or different. In some embodiments, an alkyl group "interrupted" by a cycloalkyl group means alkyl-cycloalkyl-alkyl. In some embodiments, an alkenyl group "interrupted" by a cycloalkyl group means alkenyl-cycloalkyl-alkyl, alkenyl-cycloalkyl-alkenyl, alkenyl-cycloalkyl-alkynyl, alkyl-cycloalkyl-alkenyl, or alkynyl-cycloalkyl-alkenyl. In some embodiments, an alkynyl group "interrupted" by a cycloalkyl group means alkynyl-cycloalkyl-alkyl, alkynyl-cycloalkyl-alkenyl, alkynyl-cycloalkyl-alkynyl, alkyl-cycloalkyl-alkynyl, or alkenyl-cycloalkyl-alkynyl. In some embodiments, an alkyl group "interrupted" by a heterocyclyl group means alkyl-heterocyclyl-alkyl. In some embodiments, an alkenyl group "interrupted" by a heterocyclyl group means alkenyl-heterocyclyl-alkyl, alkenyl-heterocyclyl-alkenyl, alkenyl-heterocyclyl-alkynyl, alkyl-heterocyclyl-alkenyl, or alkynyl-heterocyclyl-alkenyl. In some embodiments, an alkynyl group "interrupted" by a heterocyclyl group means alkynyl-heterocyclyl-alkyl, alkynyl-heterocyclyl-alkenyl, alkynyl-heterocyclyl-alkynyl, alkyl-heterocyclyl-alkynyl, or alkenyl-heterocyclyl-alkynyl. In some embodiments, an alkyl group "interrupted" by an aryl group means alkyl-aryl-alkyl. In some embodiments, an alkenyl group "interrupted" by an aryl group means alkenyl-aryl-alkyl, alkenyl-aryl-alkenyl, alkenyl-aryl-alkynyl, alkyl-aryl-alkenyl, or alkynyl-aryl-alkenyl. In some embodiments, an alkynyl group "interrupted" by an aryl group means alkynyl-aryl-alkyl, alkynyl-aryl-alkenyl, alkynyl-aryl-alkynyl, alkyl-aryl-alkynyl, or alkenyl-aryl-alkynyl. In some embodiments, an alkyl group "interrupted" by a heteroaryl group means alkyl-heteroaryl-alkyl. In some embodiments, an alkenyl group "interrupted" by a heteroaryl group means alkenyl-heteroaryl-alkyl, alkenyl-heteroaryl-alkenyl, alkenyl-heteroaryl-alkynyl, alkyl-heteroaryl-alkenyl, or alkynyl-heteroaryl-alkenyl. In some embodiments, an alkynyl group "interrupted" by a heteroaryl group means alkynyl-heteroaryl-alkyl, alkynyl-heteroaryl-alkenyl, alkynyl-heteroaryl-alkynyl, alkyl-heteroaryl-alkynyl, or alkenyl-heteroaryl-alkynyl.

[0082] As used herein, the term "pharmaceutically acceptable" means, within the scope of sound medical judgment, a compound, lipid nanoparticle, lipid nanoparticle composition, material, composition, and / or dosage form that is suitable for use in contact with the tissues of human and other animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio. In some embodiments, a pharmaceutically acceptable compound, lipid nanoparticle, lipid nanoparticle composition, material, composition, and / or dosage form refers to those that are approved by a regulatory agency such as the U.S. Food and Drug Administration, the National Drug Authority, or the European Medicines Agency or listed in a generally recognized pharmacopeia such as the U.S. Pharmacopeia, the Chinese Pharmacopeia, or the European Pharmacopeia for use in animals, more specifically in humans.

[0083] As used herein, "pharmaceutically acceptable salt" or "pharmaceutically acceptable salts" refers to a derivative of a compound in which the parent compound is modified by converting an existing acid moiety (e.g., carboxyl, etc.) or base moiety (e.g., amine, base, etc.) into its salt form. In many cases, the compounds of the present application are capable of forming acid addition salts and / or base salts with amino, base or similar groups present. And "pharmaceutically acceptable salts" include acid addition salts or base salts that retain the biological effectiveness and properties of the parent compound and that are, in general, non-toxic in the amounts needed to deliver a therapeutically effective dose of the subject compounds. Pharmaceutically acceptable salts are well known in the art. For instance, Berge et al. describe pharmaceutically acceptable salts in detail, J. Pharmaceutical Sciences (1977) 66: 1-19. Pharmaceutically acceptable salts of the compounds provided herein include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, lactic acid, trifluoroacetic acid, benzoic acid, cinnamic acid, mandelic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, malonic acid, fumaric acid, citric acid, malic acid, maleic acid, tartaric acid, succinic acid or methanesulfonic acid, or with salts of bases that are used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, besylate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. In some embodiments, inorganic acids that can be used to derive salts include, for example, hydrochloride, sulfate, phosphate, and the like. In some embodiments, organic acids that can be used to derive salts include, for example, maleate, fumarate, oxalate, p-toluenesulfonate, succinate, L-(+)-tartrate, monohydrochloride, hemisulfate, and the like.

[0084] The term“pharmaceutical composition” refers to a mixture of one or more compounds of the disclosure or one or more lipid nanoparticle or lipid nanoparticle composition of the disclosure with other chemical components such as pharmaceutically acceptable diluents, excipients, or carriers. The purpose of a pharmaceutical composition is to facilitate administration of the compound, lipid nanoparticle, or lipid nanoparticle composition to a subject.

[0085] As used herein, the term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the compounds provided herein from one location, body fluid, tissue, organ (internal or external) or portion thereof to another location, body fluid, tissue, organ or portion thereof. The pharmaceutically acceptable excipient or carrier can be a solvent, diluent, excipient or other material that can be used to contact animal tissue without producing an excessive toxic or adverse effect on the animal. Non-limiting examples of pharmaceutically acceptable excipients or carriers include sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as polyethylene glycol and propylene glycol; esters, such as ethyl oleate and ethyl

[0086] As used herein, "administration" of a disclosed compound, lipid nanoparticle, or lipid nanoparticle composition encompasses delivery of a compound, lipid nanoparticle, or lipid nanoparticle composition described herein, or a prodrug or other pharmaceutically acceptable derivative thereof, to a subject using any suitable formulation or route of administration as discussed herein.

[0087] As used herein, the term "delivering," "deliver," or "delivery" means providing an entity to a destination. For example, delivering a therapeutic agent and / or prophylactic agent to a subject can involve administering a lipid nanoparticle composition comprising the therapeutic agent and / or prophylactic agent to the subject (e.g., by intravenous, intramuscular, intradermal, or subcutaneous routes). Administering a lipid particle or a composition comprising a lipid particle to a mammal or a mammalian cell can involve contacting one or more cells with the lipid particle or composition.

[0088] As used herein, the term "enhanced delivery" means that a lipid particle delivers more (e.g., at least 1.5-fold more, at least 2-fold more, at least 3-fold more, at least 4-fold more, at least 5-fold more, at least 6-fold more, at least 7-fold more, at least 8-fold more, at least 9-fold more, at least 10-fold more, or at least 100-fold more) of a therapeutic agent and / or prophylactic agent to a target tissue of interest (e.g., liver, lung, spleen, or muscle) or a target cell of interest (e.g., a liver cell, a lung cell, a spleen cell, or a muscle cell) compared to the level of delivery of the therapeutic agent and / or prophylactic agent to the target tissue of interest (e.g., liver, lung, spleen, or muscle) or the target cell of interest (e.g., a liver cell, a lung cell, a spleen cell, or a muscle cell) by a control lipid particle (e.g., a lipid particle comprising DLin-MC3-DMA). The level of delivery of a therapeutic agent and / or prophylactic agent to a particular tissue or cell can be measured by comparing the amount of therapeutic agent and / or prophylactic agent in the tissue or cell to the total amount of therapeutic agent and / or prophylactic agent in the tissue or cell, comparing the amount of therapeutic agent and / or prophylactic agent in the tissue or cell to the weight of the tissue, comparing the amount of protein produced in the tissue or cell to the total amount of protein in the tissue or cell, comparing the amount of protein produced in the tissue to the weight of the tissue or cell, or comparing the amount of therapeutic agent and / or prophylactic agent delivered to the tissue or cell to the total amount of therapeutic agent and / or prophylactic agent administered. It will be appreciated that enhanced delivery of a therapeutic agent and / or prophylactic agent to a target tissue or cell need not be determined in a subject receiving treatment, but can be determined in a surrogate, such as an animal model (e.g., a mouse or rat model). In certain embodiments, lipid particle compositions comprising a compound of Formula (I) have substantially the same level of enhanced delivery regardless of the route of administration. For example, certain compounds disclosed herein exhibit similar enhanced delivery when used for intravenous or intramuscular delivery of a therapeutic agent and / or prophylactic agent.

[0089] As used herein, the term "selective delivery" or "selectively delivering" or "selectively delivering" means delivering more (e.g., at least 1.5-fold more, at least 2-fold more, at least 3-fold more, at least 4-fold more, at least 5-fold more, at least 6-fold more, at least 7-fold more, at least 8-fold more, at least 9-fold more, or at least 10-fold more) of a therapeutic and / or prophylactic agent to a target tissue (e.g., liver, lung, spleen, or muscle) or a target cell (e.g., a liver cell, a lung cell, a spleen cell, or a muscle cell) by a lipid particle as compared to a non-target tissue (e.g., liver, lung, spleen, or muscle) or a non-target cell of interest (e.g., a liver cell, a lung cell, a spleen cell, or a muscle cell). The level of therapeutic and / or prophylactic agent delivered to a particular tissue or cell can be measured by comparing the amount of therapeutic and / or prophylactic agent in the tissue or cell to the total amount of therapeutic and / or prophylactic agent in the tissue or cell, comparing the amount of therapeutic and / or prophylactic agent in the tissue or cell to the weight of the tissue or cell, comparing the amount of protein produced in the tissue or cell to the total amount of protein in the tissue or cell, or comparing the amount of protein produced in the tissue or cell to the weight of the tissue or cell. It will be appreciated that the ability of a lipid particle to specifically deliver a therapeutic and / or prophylactic agent to a target tissue or cell need not be determined in a subject being treated, but can be determined in a surrogate, such as an animal model (e.g., a mouse or rat model).

[0090] The terms“effective amount,”“pharmaceutically effective amount,” or“therapeutically effective amount” refer to the amount of a therapeutic agent and / or prophylactic agent or a compound or pharmaceutical composition described herein delivered to a tissue or cell that is sufficient to prevent, treat, alleviate and / or ameliorate symptoms and / or underlying cause of any disorder or disease in a subject, or an amount of an agent that is sufficient to have an intended effect on a target cell, e.g., to reduce cell migration, to increase or inhibit expression of a target nucleic acid in a cell, as compared to the level of normal expression of the nucleic acid detected in the absence of delivery of the therapeutic agent and / or prophylactic agent or a compound or pharmaceutical composition described herein to the tissue or cell. In one embodiment, a“pharmaceutically effective amount” or“therapeutically effective amount” refers to an amount that is sufficient to reduce or eliminate symptoms of a disease. In another embodiment, a“pharmaceutically effective amount” or“therapeutically effective amount” refers to an amount that is sufficient to overcome the disease itself. In some particular embodiments, a“pharmaceutically effective amount” or“therapeutically effective amount” refers to an amount effective for detectably killing or inhibiting the growth or spread of cancer cells, reducing the size or number of tumors; or achieving other indicators of level, stage, progression, or severity of cancer. The pharmaceutically effective amount or therapeutically effective amount will vary depending on the subject and the condition being treated, the subject’s body weight and age, the severity of the condition, the particular composition or excipient chosen, the dosing regimen to be followed, the time of administration, the mode of administration, and the like, all of which can be readily determined by one of ordinary skill in the art. The full therapeutic effect can not be realized until after administration of one or more doses has been completed, and can be realized before administration of one or more doses has been completed. The specific dose will vary depending, for example, on the specific compound chosen, the species and age / existing health condition or risk of the subject, the dosing regimen to be followed, the severity of the disease, whether it is used in combination with other agents, the time of administration, the tissue to which it is administered, and the physical delivery system carrying the drug. Thus, a pharmaceutically effective amount or therapeutically effective amount can be administered in one or more administrations. For example, but not by way of limitation, in the context of treating cancer, a pharmaceutically effective amount or therapeutically effective amount of an agent refers to an amount of the agent that reduces, ameliorates, palliates, or eradicates one or more symptoms of cancer in a patient.

[0091] The term“expression” of a nucleic acid sequence refers to one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and / or 3’ end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein.

[0092] A polypeptide or polynucleotide molecule of the present disclosure can have some degree of sequence similarity or identity to a reference molecule (e.g., a reference polypeptide or a reference polynucleotide). As known in the art, the term “identity” refers to the relatedness between two or more polypeptide or polynucleotide sequences as determined by comparing the sequences. In the art, identity also means the degree of sequence correlation between two or more sequences as determined by the number of matches between the sequences of two or more amino acid residues or nucleic acid residues. Identity measures the percentage of identical matches between the smaller of two or more sequences, where gaps in the alignment, if any, are handled by a particular mathematical model or computer program (e.g., “algorithm”). When applied to polypeptide or polynucleotide sequences, “% identity,” “% sequence identity,” or “% identical” is defined as the percentage of residues in the candidate amino acid or nucleic acid sequence that are identical with the amino acid sequence or nucleic acid sequence of the second sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for alignment are well known in the art. It will be appreciated that identity depends on the calculation of the percentage of identity, but its value can vary somewhat due to the gaps and penalties introduced in the calculation. In some embodiments, a variant of a particular polynucleotide or polypeptide has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the particular reference polynucleotide or polypeptide, as determined by a sequence alignment program and parameters described herein and known to one of skill in the art.

[0093] The “subject” to which administration is contemplated includes, but is not limited to, a human (i.e., a male or female of any age, e.g., a pediatric subject (e.g., an infant, a child, an adolescent) or an adult subject (e.g., a young adult, a middle-aged adult, or an elderly adult) and / or another primate (e.g., a rhesus monkey, a macaque); a mammal, including commercially relevant mammals, such as a cow, a pig, a horse, a sheep, a goat, a rabbit, a hamster, a mouse, a cat, and / or a dog; and / or a bird, including commercially relevant birds, such as a chicken, a duck, a goose, a quail, and / or a turkey. In some embodiments, the subject has been diagnosed with or is likely to be diagnosed with a disease or a disorder. In some embodiments, the subject has not been diagnosed with a disease or a disorder.

[0094] Lipid

[0095] In one aspect, the present disclosure provides novel compounds useful as lipids.

[0096] The lipids have at least one of the following characteristics: a hydrophilic head group with a different pKa, cationic amines, monoamines, diamines, triamines, oligo / polyamines, imidazoles, pyridines, guanidinium salts, and hydrophobic tails. In some embodiments, the lipids are ionizable lipids. In some embodiments, the lipids are cationic lipids.

[0097] As used herein, the term "cationic lipid" includes lipids having an amino head group and one or more aliphatic chains that can be protonated to form a cationic lipid at physiological pH. In some embodiments, the cationic lipid is an amino lipid. Also included are lipids having one or more protonatable or deprotonatable groups, or zwitterionic lipids. In some embodiments, the lipids of the present disclosure have at least one protonatable group such that the lipid is positively charged at a first pH (e.g., at or below pH 7.4) that is equal to or below physiological pH, and is neutral at a second pH (e.g., at or above physiological pH). In some embodiments, the lipids of the present disclosure have at least two or at least three protonatable groups. It is understood that the addition or removal of protons as a function of pH is a process of equilibrium, and reference to a charged (e.g., protonated) or neutral lipid refers to the properties of the predominant species (e.g., more than 50%, 60%, 70%, 80%, 90%, 95%, or 99%) and does not require that all lipids exist in the charged or neutral form.

[0098] In some embodiments, the pKa of the protonatable group of the cationic lipids of the present disclosure is in the range of about 4 to about 11. In some embodiments, the pKa of the lipids when incorporated into a lipid particle is about 4 to about 7, about 5 to about 7, or about 5.5 to about 6.8. In some embodiments, lipids having such a pKa will be cationic at lower pH, while the particles will be mostly (but not completely) surface neutralized at physiological pH (e.g., pH 7.4). In some embodiments, at least some of the nucleic acids associated with the outer surface of the particles comprising lipids having such a pKa will lose their electrostatic interactions at physiological pH and be removed by simple dialysis; thereby greatly reducing the susceptibility of the particles to being cleared. For example, the pKa of the lipids within a lipid particle can be measured using the method described by Cullis et al. (1986) Chem Phys Lipids (40, 127-144) using the fluorescent probe 2-(p-toluidinyl)-6-naphthalene sulfonic acid (TNS).

[0099] In some embodiments, the lipids of the present disclosure are advantageously used in lipid nanoparticles. In some embodiments, the lipid nanoparticles are used to deliver a therapeutic agent to cells in vivo. In some embodiments, the lipid nanoparticles are used to deliver a therapeutic agent to a tissue in vivo.

[0100] On the other hand, the lipids disclosed herein are compounds having the following formula (I):

[0101]

[0102] Or its pharmaceutically acceptable salt, wherein,

[0103] R a Selected from the following group: hydrogen, R 5 Alkyl, alkenyl, ynyl, heteroalkyl, heteroalkenyl, heteroynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups, wherein the alkyl, alkenyl, ynyl, heteroalkyl, heteroalkenyl, heteroynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups are optionally substituted by one or more groups independently selected from the group consisting of: halogen, hydroxyl, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl, and R. 6 ;

[0104] R 1 for

[0105] R 2 for

[0106] R 3 for

[0107] R 4 for

[0108] R 5 If it exists, then it is...

[0109] R 6 If it exists, then it is...

[0110] Each W is independently selected from O, S, or NR. b And each R b It is independently selected from hydrogen, alkyl, alkoxycarbonyl, acyl or sulfonyl;

[0111] Each Y is independently selected from O, S, NR. c 、N(R c Z(W), N(R) c )N(R c ) or N(R c )N(R c Z(W), and each R c It is independently selected from hydrogen, alkyl, alkoxycarbonyl, acyl or sulfonyl;

[0112] Each Z is independently selected from C, S, or S(O);

[0113] each n is independently 0, 1, 2, 3, 4, or 5;

[0114] each m is independently 0, 1, 2, or 3;

[0115] each p is independently 1, 2, 3, or 4; and

[0116] R 1c , R 2c , R 3c , and R 4c are each independently selected from the group consisting of alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally substituted with one or more groups independently selected from the group consisting of halogen, hydroxyl, oxo, cyano, cycloalkyl, heterocyclyl, aryl, and heteroaryl, and the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally interrupted with one or more groups independently selected from the group consisting of cycloalkyl, heterocyclyl, aryl, and heteroaryl.

[0117] In some embodiments, the lipids of the present disclosure are compounds having the following Formula (A) or Formula (B):

[0118]

[0119]

[0120] In some embodiments, at least two of R 2 , R 3 , and R 4 are the same. In some embodiments, at least three of R 2 , R 3 , and R 4 are the same.

[0121] In some embodiments, one or more of W is O.

[0122] In some embodiments, one or more of Y is O, NR c , or N(R c )N(R c )Z(W).

[0123] In some embodiments, one or more of Z is C or S(O).

[0124] In some embodiments, one or more of R 2c , R 3c , and R 4c is alkyl or alkenyl.

[0125] In some embodiments, one or more of R 2c , R 3c , and R4c One or more of them are C 8-24 Alkyl or alkenyl groups.

[0126] In some implementation schemes, R 2c R 3c and R 4c One or more of them are C 10-24 Alkyl or alkenyl groups.

[0127] In some implementation schemes, R 2c R 3c and R 4c One or more of them are alkenyl groups containing one, two or three C=C double bonds.

[0128] In some implementations, R 2c R 3c and R 4c One or more of them are alkenyl groups containing one or more Z-olefins.

[0129] In some implementations, R 1c It is an alkyl group.

[0130] In some implementation schemes, R 1c C 1-12 alkyl.

[0131] In some implementation schemes, R 1c C 4-10 alkyl.

[0132] In some implementation schemes, R a C 1-6 Alkyl groups, which are optionally substituted by one or more groups independently selected from the group consisting of hydroxyl, cycloalkyl, and heteroaryl groups.

[0133] In some implementation schemes, R a It is methyl, ethyl, propyl, butyl, or pentyl.

[0134] In some implementation schemes, One or more of them are independently selected from the following groups:

[0135] In some implementation schemes, R 1c R 2c R 3c and R 4c One or more of them are independently selected from the following groups:

[0136]

[0137] In some embodiments, R 1c , R 2c , R 3c , R 4c , R 5c , and R 6c each, if present, does not contain two directly bonded heteroatoms.

[0138] In some embodiments, R 1c , R 2c , R 3c , R 4c , R 5c , and R 6c each, if present, contains -N(R c )-N(R c )-, or -S(O)2-N(R c )-.

[0139] In some embodiments, the compound of Formula (I) is a compound listed in Table 1.

[0140] For illustrative purposes, exemplary compounds of the present disclosure and their structure codes are listed in Table 1 below.

[0141] Table 1. Exemplary Compounds

[0142]

[0143]

[0144]

[0145]

[0146] The compounds provided herein are described with reference to general formulas and specific compounds. Additionally, the compounds of the present disclosure can exist in a variety of different forms or derivatives, including but not limited to stereoisomers, racemic mixtures, positional isomers, tautomers, salts, prodrugs, soft drugs, active metabolic derivatives (active metabolites), solvated forms, different crystalline or polymorphic forms, all of which are within the scope of the present disclosure.

[0147] The compounds of the present disclosure can contain one or more asymmetric centers and thus can exist in various stereoisomeric forms. Thus, the compounds of the present disclosure and their compositions can be in the form of a single stereoisomer, or can be in the form of a mixture of stereoisomers. In certain embodiments, the compounds of the present disclosure are enantiomeric compounds. In certain embodiments, mixtures of enantiomers or diastereomers are provided.

[0148] The term "enantiomeric" refers to two stereoisomers of a compound that are non- superimposable mirror images of each other. The term "diastereomeric" refers to a pair of optical isomers that are not mirror images of each other. Diastereomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivities.

[0149] Further, certain compounds as described herein can have one or more double bonds that can exist as either the Z or E isomer, unless otherwise indicated. The present disclosure additionally encompasses the compounds occurring as single isomers substantially free of other isomers, and alternatively, the compounds can occur as mixtures of various isomers, such as racemic mixtures of enantiomeric isomers. In addition to the compounds themselves, the present disclosure also encompasses compositions comprising one or more compounds.

[0150] As used herein, the term "isomer" includes any and all geometric isomers and stereoisomers. For example, "isomers" include cis- and trans-isomers, E- and Z isomers, R- and S- enantiomeric forms, diastereomers, (D)-isomers, (L)-isomers, racemic mixtures, and other mixtures thereof, all of which fall within the scope of the present disclosure. By way of example, in some embodiments, stereoisomers can be provided substantially free of one or more of the corresponding stereoisomers, and can also be referred to as "stereochemically enriched."

[0151] When a particular enantiomer is preferred, in some embodiments, the enantiomer can be provided substantially free of the opposite enantiomer, and can also be referred to as "optically enriched." As used herein, "optically enriched" means that the compound consists of a substantially higher proportion of one enantiomer. In certain embodiments, the compound consists of at least about 90% by weight of the preferred enantiomer. In other embodiments, the compound consists of at least about 95%, 98%, or 99% by weight of the preferred enantiomer. The preferred enantiomer can be separated from a racemic mixture by any method known to those of skill in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts, or prepared by asymmetric synthesis. See, e.g., Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, S.H., et al., Tetrahedron 33:2725 (1977); Eliel, E.L., Stereochemistry of Carbon Compounds (McGraw-Hill, New York, 1962); Wilen, S.H., Tables of Resolving Agents and Optical Resolutions, p. 268 (E.L. Eliel, ed., Univ. of Notre Dame Press, Notre Dame, Ind., 1972).

[0152] The compounds of the present disclosure can also exist in different tautomeric forms, and all such forms are encompassed by the scope of the present disclosure. The term “tautomer” or “tautomeric form” refers to structural isomers that have different energies and can interconvert via a low-energy barrier. The existence and concentration of the isomeric forms depends on the environment the compound is in, and can differ, for example, whether the compound is a solid or in an organic or aqueous solution. By way of example, prototropic tautomers (also known as proton-shift tautomers) include interconversions that occur via migration of a proton, such as keto-enol, amide-imidic acid, lactam-lactim isomerization, and cyclic forms in which a proton can occupy two or more positions of a heterocyclic system. Valence tautomers include interconversions that occur by reorganization of some of the bonding electrons in a bond. Tautomers can be kept in equilibrium or locked in space in one form by appropriate substitution. Unless otherwise specified, a compound identified by name or structure in the present disclosure as one specific tautomeric form is intended to include the other tautomeric forms.

[0153] The present disclosure is also intended to include all isotopes of atoms occurring in the compounds. Isotopes of atoms include atoms having the same atomic number but different mass numbers. For example, unless otherwise specified, hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine, chlorine, bromine, or iodine in a compound of the present disclosure are also intended to include isotopes thereof, such as, but not limited to 1 H, 2 H, 3 H, 11 C, 12 C, 13 C, 14 C, 14 N, 15 N, 16 O, 17 O, 18 O, 31 P, 32 P, 32 S, 33 S, 34 S, 36 S, 17 F, 18 F, 19 F, 35 Cl, 37 Cl, 79 Br, 81 Br, 124 I, 127 I, and 131 I. In some embodiments, hydrogen includes protium, deuterium, and tritium. In some embodiments, carbon includes 12 C, and 13 C.

[0154] Synthesis of compounds

[0155] The synthesis of the compounds provided herein, including pharmaceutically acceptable salts thereof, is illustrated in the synthetic schemes in the Examples. The compounds provided herein can be prepared using any known organic synthesis techniques and can be synthesized according to any of a number of possible synthetic routes, and thus the schemes are merely illustrative and not intended to limit other possible methods that can be used to prepare the compounds provided herein. Additionally, the steps in the schemes are for better illustration and can be altered as appropriate. The embodiments of the compounds in the Examples are synthesized for research purposes and possible submission to regulatory agencies.

[0156] The reactions to produce the compounds of the disclosure can be carried out in suitable solvents which can be readily selected by one of skill in the art of organic synthesis. Suitable solvents can be substantially non-reactive with the starting materials, intermediates, or products at the temperatures at which the reactions are carried out, e.g., temperatures between the melting and boiling points of the solvent. Given the highly selective nature of the chemistry described herein, the reactions can be carried out in one solvent or a mixture of more than one solvent. The choice of a suitable solvent will depend on the specific reaction step described in the scheme.

[0157] The preparation of the compounds of the disclosure can involve protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd Ed., Wiley & Sons, Inc., New York (1999), which is incorporated herein by reference in its entirety.

[0158] The reactions can be monitored by any suitable method known in the art. For example, the progress of the reaction can be monitored by spectroscopic means, such as, nuclear magnetic resonance spectroscopy (e.g.,1H or13C), infrared spectroscopy, and / or mass spectrometry, or by chromatographic means, such as, high performance liquid chromatography or thin layer chromatography. 1 H or 13C), infrared spectroscopy, spectrophotometry (e.g., ultraviolet-visible), mass spectrometry, or chromatography (such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LCMS), or thin layer chromatography (TLC)). Compounds can be purified by various methods by one of skill in the art, including high performance liquid chromatography (HPLC) ("Purification: Improved Compound Specific Method Optimization," Karl F. Blom, Brian Glass, Richard Sparks, Andrew P. Combs, J. Combi. Chem., 2004, 6(6), 874-883, which is incorporated by reference herein in its entirety) and normal phase silica gel chromatography.

[0159] The structure of the compounds in the examples were characterized by nuclear magnetic resonance (NMR) or / and liquid chromatography-mass spectrometry (LC-MS). The units of NMR chemical shifts (δ) are 10 -6 (ppm). 1 H-NMR spectra were recorded in CDCl3, CD3OD or DMSO-d6solution (reported in ppm) using tetramethylsilane (TMS) as the reference standard (0.0 ppm) on a Bruker instrument (400 MHz or 500 MHz).

[0160] Unless otherwise noted, reactions of the present disclosure were typically conducted under a positive pressure of nitrogen or argon or using dry tubes in dry solvents, and reaction flasks were typically equipped with a rubber septum to allow for the introduction of substrates and reagents via syringe. Glassware was oven- and / or heat-dried.

[0161] Lipid nanoparticle

[0162] The present disclosure also provides lipid particles comprising one or more of the above described lipids. Lipid particles include, but are not limited to, lipid nanoparticles (LNP), liposomes, lipoplexes, and lipopolyplexes (LPP). In some embodiments, the lipid particle is a lipid nanoparticle. The present disclosure also provides a method for making a lipid particle.

[0163] The lipid nanoparticles of the present disclosure can also include one or more additional lipids and / or other ingredients, such as a solid alcohol. Other lipids can be included in the lipid nanoparticles of the present disclosure for various purposes, such as to prevent oxidation of the lipids or to attach ligands to the surface of the particle. Any lipid can be present in the lipid nanoparticles of the present disclosure, including amphipathic lipids, neutral lipids, cationic lipids, and anionic lipids, which can be used individually or in combination. Examples of other lipid components that can be present are described below.

[0164] In some embodiments, the lipid nanoparticle comprises a lipid of the present disclosure (e.g., a compound of Formula (I) or any of the compounds in Table 1). In some embodiments, the lipid nanoparticle comprises two or more lipids of the present disclosure. In some embodiments, the molar fraction of a lipid of the present disclosure (e.g., a compound of Formula (I) or any of the compounds in Table 1) is about 5% to 75%, about 10% to 75%, about 10% to 70%, about 10% to 65%, about 15% to 65%, about 20% to 65%, about 25% to 65%, about 30% to 65%, about 35% to 65%, about 40% to 65%, about 45% to 65%, about 40% to 60%, about 40% to 55%, or about 40% to 50% of the total lipids present in the lipid nanoparticle. In some embodiments, the molar fraction of a lipid of the present disclosure (e.g., a compound of Formula (I) or any of the compounds in Table 1) is about 40% to 50% of the total lipids present in the lipid nanoparticle. In some embodiments, the molar fraction of a lipid of the present disclosure (e.g., a compound of Formula (I) or any of the compounds in Table 1) is about 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%, or 65% of the total lipids present in the lipid nanoparticle.

[0165] In some embodiments, the lipid nanoparticle comprises a neutral lipid. The term "neutral lipid" refers to any of a variety of lipid species that exist in uncharged or neutral zwitterionic form at a selected pH. In some embodiments, the neutral lipid is a phospholipid. Examples of phospholipids include, but are not limited to: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-didodecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (Cl6 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-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 any mixture thereof. In some embodiments, the lipid nanoparticle comprises one neutral lipid. In some embodiments, the lipid nanoparticle comprises two or more neutral lipids. In some embodiments, the neutral lipid is selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and any mixture thereof.In some embodiments, the molar fraction of the neutral lipid is about 1% to 40%. In some embodiments, the molar fraction of the neutral lipid is about 1% to 35%, about 5% to 30%, about 5% to 25%, about 5% to 20%, about 5% to 15%, or about 5% to 10% of the total lipid present in the lipid nanoparticle. In some embodiments, the molar fraction of the neutral lipid is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 15%, 20%, 25%, or 30% of the total lipid present in the lipid nanoparticle.

[0166] In some embodiments, the lipid nanoparticle comprises a structural lipid. In some embodiments, the structural lipid is a steryl, a steryl derivative, or any mixture thereof. Examples of steryl or steryl derivatives include, but are not limited to, cholesterol, coprostanol, sitosterol, beta-sitosterol, ergosterol, campesterol, soysterol, brassicasterol, tomatidine, tomatin, ursolic acid, alpha-tocopherol, and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid comprises cholesterol and a corticosteroid such as prednisolone, dexamethasone, prednisone, and hydrocortisone, or a combination thereof. In some embodiments, the lipid nanoparticle comprises cholesterol. In some embodiments, the molar fraction of the structural lipid is about 5% to 50% of the total lipid present in the lipid nanoparticle. In some embodiments, the molar fraction of the structural lipid is about 10% to 50%, about 15% to 50%, about 25% to 50%, or about 40% to 50% of the total lipid present in the lipid nanoparticle. In some embodiments, the molar fraction of the structural lipid is about 10% to 45%, about 15% to 45%, about 25% to 45%, about 30% to 45%, or about 35% to 45% of the total lipid present in the lipid nanoparticle.In some embodiments, the molar fraction of the structural lipid is about 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 40.1%, 40.2%, 40.3%, 40.4%, 40.5%, 40.6%, 40.7%, 40.8%, 40.9%, 41%, 41.1%, 41.2%, 41.3%, 41.4%, 41.5%, 41.6%, 41.7%, 41.8%, 41.9%, 42%, 42.1%, 42.2%, 42.3%, 42.4%, 42.5%, 42.6%, 42.7%, 42.8%, 42.9%, 43%, 43.1%, 43.2%, 43.3%, 43.4%, 43.5%, 43.6%, 43.7%, 43.8%, 43.9%, 44%, 44.1%, 44.2%, 44.3%, 44.4%, 44.5%, 44.6%, 44.7%, 44.8%, 44.9%, 45%, 45.1%, 45.2%, 45.3%, 45.4%, 45.5%, 45.6%, 45.7%, 45.8%, 45.9%, 46%, 46.1%, 46.2%, 46.3%, 46.4%, 46.5%, 46.6%, 46.7%, 46.8%, 46.9%, 47%, 47.1%, 47.2%, 47.3%, 47.4%, 47.5%, 47.6%, 47.7%, 47.8%, 47.9%, 48%, 48.1%, 48.2%, 48.3%, 48.4%, 48.5%, 48.6%, 48.7%, 48.8%, 48.9%, 49%, 49.1%, 49.2%, 49.3%, 49.4%, 49.5%, 49.6%, 49.7%, 49.8%, 49.9%, 50%, 50.1%, 50.2%, 50.3%, 50.4%, 50.5%, 50.6%, 50.7%, 50.8%, 50.9%, 51%, 51.1%, 51.2%, 51.3%, 51.4%, 51.5%, 51.6%, 51.7%, 51.8%, 51.9%, 52%, 52.1%, 52.2%, 52.3%, 52.4%, 52.5%, 52.6%, 52.7%, 52.8%, 52.9%, 53%, 53.1%, 53.2%, 53.3%, 53.4%, 53.5%, 53.6%, 53.7%, 53.8%, 53.9%, 54%, 54.1%, 54.2%, 54.3%, 54.4%, 54.5%, 54.6%, 54.7%, 54.8%, 54.9%, or 55% of the total lipid present in the lipid nanoparticle.

[0167] In some embodiments, the lipid nanoparticle comprises a lipid selected to reduce aggregation of the lipid nanoparticle during formation of the lipid nanoparticle, which aggregation can be due to steric stabilization of the particles, that prevents charge-induced aggregation during formation. Examples of lipids that reduce aggregation of the particles during formation include, but are not limited to, polyethylene glycol (PEG)-modified lipids, monosialoganglioside Gm1, and polyamide oligomers (PAOs). Other compounds with uncharged, hydrophilic, steric barrier moieties that prevent aggregation during formulation, such as PEG or Gm1, can also be coupled to the lipids for use in the methods and compositions of the disclosure. In some embodiments, the lipid that reduces aggregation of the particles during formation is a surfactant. In some embodiments, the surfactant is a PEG-modified lipid. Examples of PEG-modified lipids include, but are not limited to, 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 1,2-dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol (DMG-PEG), 1,2-distearoyl-rac-glycero-3-methoxypolyethyleneglycol (DSG-PEG), N-(methoxypolyethyleneglycol)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEG-DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethyleneglycol)] (DOPE-PEG), or any mixture thereof. Typically, the molar fraction of the lipid component selected to reduce aggregation is about 1% to 15% of the total lipids present in the lipid nanoparticle. In some embodiments, the molar fraction of the lipid component selected to reduce aggregation is about 1% to 10%, about 1% to 7%, about 1% to 5%, or about 0.5% to 5% of the total lipids present in the lipid nanoparticle. In some embodiments, the molar fraction of the lipid component selected to reduce aggregation is about 1% to 5%, about 1% to 2.5%, or about 1.5% to 2% of the total lipids present in the lipid nanoparticle.In some embodiments, the molar fraction of the lipid component selected to reduce aggregation is 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%, or 4% of the total lipid present in the lipid nanoparticle.

[0168] As used herein, the term "PEG," whether used as part of another term or independently, refers to polyethylene glycol. The term "PEGm" or "PEG-m," where m is an integer, refers to a polyethylene glycol molecule or moiety having a molecular weight of m. For example, "PEG2000" or "PEG-2000" refers to a polyethylene glycol molecule or moiety having a molecular weight of 2000. In some embodiments, the PEG is PEG700, PEG800, PEG900, PEG1000, PEG1100, PEG1200, PEG1300, PEG1400, PEG1500, PEG1600, PEG1700, PEG1800, PEG1900, PEG2000, PEG2100, PEG2200, PEG2300, PEG2400, PEG2500, PEG2600, PEG2700, PEG2800, PEG2900, or PEG3000. In some embodiments, the PEG is PEG2000.

[0169] In some embodiments, the lipid nanoparticles of the present disclosure comprise: a molar fraction of about 10%-65% of the compound of Formula (I); a molar fraction of about 5%-30% of a neutral lipid of the present disclosure; a molar fraction of about 15%-50% of a structural lipid of the present disclosure; a molar fraction of about 0.5%-5% of a lipid component selected to reduce aggregation of the present disclosure; based on the total lipid present in the lipid nanoparticle.

[0170] In some embodiments, the lipid nanoparticles of the present disclosure comprise: a molar fraction of about 20%-65% of the compound of Formula (I); a molar fraction of about 5%-25% of a neutral lipid of the present disclosure; a molar fraction of about 25%-50% of a structural lipid of the present disclosure; a molar fraction of about 1%-5% of a lipid component selected to reduce aggregation of the present disclosure; based on the total lipid present in the lipid nanoparticle.

[0171] In some embodiments, the lipid nanoparticles of the present disclosure comprise, based on total lipids present in the lipid nanoparticle: a molar fraction of about 40-65% of the compound of Formula (I); a molar fraction of about 5-15% of a neutral lipid of the present disclosure; a molar fraction of about 25-50% of a structural lipid of the present disclosure; a lipid component selected to reduce aggregation of the present disclosure in a molar fraction of about 1-2.5%.

[0172] In some embodiments, the lipid nanoparticles of the present disclosure comprise, based on total lipids present in the lipid nanoparticle: a molar fraction of about 40-50% of the compound of Formula (I); a molar fraction of about 5-15% of a neutral lipid of the present disclosure; a molar fraction of about 40-50% of a structural lipid of the present disclosure; a lipid component selected to reduce aggregation of the present disclosure in a molar fraction of about 1.5-2%.

[0173] In some embodiments, the lipid nanoparticles of the present disclosure comprise, based on total lipids present in the lipid nanoparticle:

[0174] a compound of Formula (I) in a molar fraction of about 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%, or 65%;

[0175] a neutral lipid of the present disclosure in a molar fraction of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 15%, 20%, 25%, or 30%;

[0176] The structural lipids of the present disclosure, in a molar fraction of about 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 40.1%, 40.2%, 40.3%, 40.4%, 40.5%, 40.6%, 40.7%, 40.8%, 40.9%, 41%, 41.1%, 41.2%, 41.3%, 41.4%, 41.5%, 41.6%, 41.7%, 41.8%, 41.9%, 42%, 42.1%, 42.2%, 42.3%, 42.4%, 42.5%, 42.6%, 42.7%, 42.8%, 42.9%, 43%, 43.1%, 43.2%, 43.3%, 43.4%, 43.5%, 43.6%, 43.7%, 43.8%, 43.9%, 44%, 44.1%, 44.2%, 44.3%, 44.4%, 44.5%, 44.6%, 44.7%, 44.8%, 44.9%, 45%, 45.1%, 45.2%, 45.3%, 45.4%, 45.5%, 45.6%, 45.7%, 45.8%, 45.9%, 46%, 46.1%, 46.2%, 46.3%, 46.4%, 46.5%, 46.6%, 46.7%, 46.8%, 46.9%, 47%, 47.1%, 47.2%, 47.3%, 47.4%, 47.5%, 47.6%, 47.7%, 47.8%, 47.9%, 48%, 48.1%, 48.2%, 48.3%, 48.4%, 48.5%, 48.6%, 48.7%, 48.8%, 48.9%, 49%, 49.1%, 49.2%, 49.3%, 49.4%, 49.5%, 49.6%, 49.7%, 49.8%, 49.9%, 50%, 50.1%, 50.2%, 50.3%, 50.4%, 50.5%, 50.6%, 50.7%, 50.8%, 50.9%, 51%, 51.1%, 51.2%, 51.3%, 51.4%, 51.5%, 51.6%, 51.7%, 51.8%, 51.9%, 52%, 52.1%, 52.2%, 52.3%, 52.4%, 52.5%, 52.6%, 52.7%, 52.8%, 52.9%, 53%, 53.1%, 53.2%, 53.3%, 53.4%, 53.5%, 53.6%, 53.7%, 53.8%, 53.9%, 54%, 54.1%, 54.2%, 54.3%, 54.4%, 54.5%, 54.6%, 54.7%, 54.8%, 54.9%, or 55%; and a lipid component selected to reduce aggregation of the present disclosure, in a molar fraction of 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%, or 4%.

[0177] In some embodiments, the lipid nanoparticles of the present disclosure comprise the components listed in the following table at the indicated molar fractions.

[0178]

[0179]

[0180] Lipid nanoparticles can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy (TEM) or scanning electron microscopy (SEM)) can be used to examine the morphology and particle size distribution of the lipid nanoparticle composition. Zeta potential can be measured using dynamic light scattering (DLS) or potentiometry (e.g., potentiometric titration). Particle size can also be determined using DLS. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure a variety of properties of the lipid nanoparticles, such as particle size, polydispersity index, and zeta potential. The error of a DLS measurement can depend on a variety of factors, such as the scattering angle and multiple scattering. The typical error of a DLS measurement is 5%.

[0181] For example, the average particle size of a lipid nanoparticle, as measured by DLS, can be between tens of nanometers and hundreds of nanometers. For example, the average particle size of a lipid nanoparticle can be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average particle size of a lipid nanoparticle can be from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm. In some embodiments, the average particle size of a lipid nanoparticle can be about 70 nm to about 100 nm. In some embodiments, the average particle size of a lipid nanoparticle can be about 80 nm. In some embodiments, the average particle size of a lipid nanoparticle can be about 100 nm.

[0182] The lipid nanoparticles can be relatively uniform. The polydispersity index (PDI) can be used to indicate the uniformity of the lipid nanoparticles, e.g., the particle size distribution of the lipid nanoparticles. A smaller PDI (e.g., less than 0.3) generally indicates a narrower particle size distribution. In some embodiments, the PDI of a lipid nanoparticle of the present disclosure can be from about 0 to about 0.30, such as 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, 0.25, 0.26, 0.27, 0.28, 0.29, or 0.30. In some embodiments, the PDI of a lipid nanoparticle of the present disclosure can be from about 0.05 to about 0.20.

[0183] The zeta potential of a lipid nanoparticle can be used to indicate the zeta potential. For example, the zeta potential can describe the surface charge of the lipid nanoparticle. Lipid nanoparticles having a relatively low charge (positive or negative) are generally desired, as more highly charged substances can have undesirable interactions with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of a lipid nanoparticle of the present disclosure can be from about -10 mV to about +25 mV, from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +25 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +25 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +25 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

[0184] Lipid nanoparticle composition

[0185] In another aspect, the present disclosure also provides a lipid nanoparticle composition, which refers to a composition comprising one or more lipid particles as described above and one or more therapeutic agents.

[0186] As used herein, the term "therapeutic agent" includes any molecule or compound capable of exerting an intended effect on a cell, tissue, organ, or subject. Such effect can be a biological, physiological, or cosmetic effect. The therapeutic agent can be any type of molecule or compound, including but not limited to nucleic acids, peptides, and polypeptides, e.g., antibodies, cytokines, growth factors, differentiation-inducing factors, apoptosis factors, cell surface receptors and their ligands, hormones, and small molecules (including small organic molecules or compounds). In some embodiments, the therapeutic agent is a target polynucleotide. In some embodiments, the therapeutic agent is a target polynucleotide comprising a first nucleic acid encoding an interleukin 15 polypeptide or a variant thereof.

[0187] As used herein, the term "polynucleotide" refers to a polymer of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars, and intemucleosidic (backbone) linkages. The term "polynucleotide" also includes polymers comprising non-natural monomers or functional analogs of such backbones or linkages that are synthetic, naturally occurring, or non-naturally occurring, and which have similar binding properties to reference nucleic acids. Such modified or substituted polynucleotides are often preferred over naturally-occurring forms because of properties such as, for example, enhanced cellular uptake, increased stability in the presence of a nuclease, and the like. As used herein, the term "nucleotide" includes a sugar (deoxyribose (DNA) or ribose (RNA)), a base, and a phosphate group. Nucleotides are linked together by phosphate groups. "Bases" include purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, including but not limited to modifications that introduce new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and halogenated alkanes. In some embodiments, the polynucleotide comprises more than 50 nucleotides or nucleoside monomers.

[0188] The polynucleotides present in the disclosed compositions (e.g., lipid nanoparticle compositions comprising one or more target polynucleotides) include any known form of nucleic acid. Polynucleotides used herein include, but are not limited to, single-stranded DNA or RNA, or double-stranded DNA or RNA, and DNA-RNA hybrids. Examples of double-stranded DNA include, but are not limited to, structural genes, genes comprising control and termination regions, and self-replicating systems such as viral or plasmid DNA. Examples of double-stranded RNA include, but are not limited to, siRNA and other RNA interference agents. Single-stranded nucleic acids include, but are not limited to, messenger RNA (mRNA), antisense oligonucleotides, ribozymes, microRNAs, and triple- stranded forming oligonucleotides. Polynucleotides used herein also include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, or non-naturally occurring, and which have similar binding properties to reference nucleic acids. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unless otherwise indicated, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences, as well as the sequence explicitly indicated. Nucleic acids present in the compositions of the disclosure can comprise one or more modifications.

[0189] Nucleic acids of the present disclosure can have a variety of lengths, generally depending on the particular form of the nucleic acid. In some embodiments, the nucleic acid is 10-5000 nucleotides in length. In some embodiments, the nucleic acid is about 4000 nucleotides in length. In some embodiments, the nucleic acid is about 3000 nucleotides in length. In some embodiments, the nucleic acid is about 2500 nucleotides in length. In some embodiments, the nucleic acid is about 2400, 2300, 2200, 2100, 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 750, 700, 650, 600, 550, 450, 400, 350, 300, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, or 30 nucleotides in length.

[0190] As used herein, the term “variant” of a polypeptide refers to a molecule whose amino acid sequence differs from that of a native or reference sequence. The amino acid sequence variant can have substitutions, mutations, deletions, and / or insertions at certain positions within the amino acid sequence compared to the native sequence or reference sequence. In some embodiments, a variant has at least 50%, or at least 60% or at least 70% sequence identity to the native sequence or reference sequence. In some embodiments, a variant has at least 80% identity, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98% sequence identity to the native sequence or reference sequence.

[0191] In some embodiments, the therapeutic agent (e.g., target polynucleotide) is encapsulated inside the lipid nanoparticle. In some embodiments, the therapeutic agent is present within one or more lipid layers of the lipid nanoparticle. In some embodiments, the therapeutic agent is bound to the external or internal lipid surface of the lipid nanoparticle.

[0192] As used herein, “encapsulation,” “encapsulated,” “encapsulating,” and “loaded” and “associated” can refer to complete, substantial, or partial enclosure, confinement, surrounding, or wrapping. As used herein, “encapsulation” or “association” can refer to the process of confining a single nucleic acid within a lipid nanoparticle and / or establishing a physicochemical relationship between the single nucleic acid and the lipid nanoparticle.

[0193] The“encapsulation efficiency” of a therapeutic agent refers to the amount of the therapeutic agent encapsulated or otherwise associated with the lipid nanoparticles after preparation relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., greater than 80%, greater than 85%, greater than 90%, or greater than 95%). The encapsulation efficiency can be measured, for example, by comparing the amount of therapeutic agent in a solution containing the lipid nanoparticles before and after the lipid nanoparticles are disrupted with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free therapeutic agent (e.g., RNA) in solution. In some embodiments, the encapsulation efficiency of a therapeutic agent can be at least 50%, e.g., at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%.

[0194] The amount of therapeutic agent (e.g., a target polynucleotide) in a lipid nanoparticle can depend on the particle size, composition, desired target and / or application, or other properties of the lipid nanoparticle, as well as the properties of the therapeutic agent. For example, the amount of RNA available in a lipid particle can depend on the size, sequence, and other characteristics of the RNA. The relative amounts of therapeutic agent and other components (e.g., lipids) in a lipid nanoparticle can also vary. In some embodiments, the mass ratio of lipids (e.g., cationic lipids, neutral lipids, structural lipids (e.g., sterols), and lipids selected to reduce aggregation such as surfactants) to therapeutic agent (e.g., RNA) in a lipid nanoparticle composition can be about 5: 1 to about 60: 1, such as about 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 11: 1, 12: 1, 13: 1, 14: 1, 15: 1, 16: 1, 17: 1, 18: 1, 19: 1, 20: 1, 25: 1, 30: 1, 35: 1, 40: 1, 45: 1, 50: 1, and 60: 1. For example, the mass ratio of lipids to therapeutic agent can be about 10: 1 to about 50: 1. In some embodiments, the mass ratio of lipids to therapeutic agent is about 40: 1. In some embodiments, the mass ratio of lipids to therapeutic agent is about 20: 1. In some embodiments, the mass ratio of lipids to therapeutic agent is calculated by dividing the total mass of cationic lipids, neutral lipids, structural lipids such as sterols, and surfactants free of solvent, by the mass of anhydrous therapeutic agent (e.g., RNA). For example, the amount of therapeutic agent in a lipid nanoparticle composition can be measured using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).

[0195] In some embodiments, the target polynucleotide is an RNA. In some embodiments, the target polynucleotide is a messenger RNA (mRNA). The mRNA can be natural, or can comprise modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. “Messenger RNA” (mRNA) refers to any polynucleotide that encodes at least one polypeptide (e.g., a naturally occurring, non-naturally occurring, or modified amino acid polymer), and can be translated in vitro, in vivo, in situ, or ex vivo to produce the encoded polypeptide. Translation of the mRNA (e.g., in vivo translation of mRNA within a mammalian cell) can produce a polypeptide.

[0196] It will be understood by those skilled in the art that, unless otherwise specified, the polynucleotide sequences set forth in this application will be represented in representative DNA sequences with “T,” but when the sequence represents RNA (e.g., mRNA), the “T” will be replaced with “U,” and vice versa. Thus, any RNA polynucleotide identified by a particular sequence identification number can also comprise the corresponding RNA (e.g., mRNA) sequence encoded by that DNA, with each “T” in the DNA sequence replaced with “U.”

[0197] The mRNA of the present disclosure can be transcribed in vitro from a template DNA. In vitro transcription of RNA is known in the art, and one of skill in the art can readily and affirmatively obtain an mRNA sequence based on the provided template DNA sequence. In some embodiments, the RNA transcript is synthesized from a non-amplified linear DNA template of the gene of interest via an in vitro enzymatic transcription reaction that utilizes T7 bacteriophage, RNA polymerase, and nucleotide triphosphates with the desired chemical properties.

[0198] The mRNA molecule can comprise a cap structure, a chain-terminating nucleoside, a stem loop, a poly A sequence, and / or a polyadenylation signal. In some embodiments, the basic components of the mRNA molecule include at least a coding region, a 5'-untranslated region (5'-UTR), a 3' UTR, and a poly A sequence. In some embodiments, the 5' untranslated region (UTR) comprises a nucleic acid selected from the group consisting of SEQ ID NOs: 261-263. In some embodiments, the 3' untranslated region (UTR) comprises a nucleic acid selected from the group consisting of SEQ ID NOs: 264-267. In some embodiments, the poly-A region is 50-120 nucleotides in length. For example, the poly A region can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 adenosine monophosphates. In some embodiments, the mRNA of the present application comprises a 5' terminal cap. In some embodiments, the 5' terminal cap can be cap0GG, cap1GG, and cap1AG. In some embodiments, the mRNA of the present application comprises one or more modified nucleotides selected from the group consisting of pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), 5-methyluridine (m5U), 2-thiouridine (s2U), 5-methylcytidine (m5C), and 5-methoxyuridine (5moU).

[0199] In some embodiments, the target polynucleotide is an mRNA comprising a first nucleic acid encoding an interleukin 15 polypeptide or a variant thereof.

[0200] In some embodiments, the first nucleic acid encodes a human interleukin 15 polypeptide comprising the amino acid sequence of SEQ ID NO: 8 or 299-406, or an amino acid sequence having at least 95% (96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%) identity to SEQ ID NO: 8 or 299-406.

[0201] In some embodiments, the first nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 9 or 407-514, or a nucleic acid sequence having at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%) identity to SEQ ID NO: 9 or 407-514, the first nucleic acid encoding a human interleukin 15 polypeptide.

[0202] In some embodiments, the target polynucleotide further comprises a second nucleic acid encoding an IL-15Ra sushi domain. In some embodiments, the target polynucleotide is an mRNA comprising a first nucleic acid encoding an interleukin 15 polypeptide or a variant thereof, and a second nucleic acid encoding an IL-15Ra sushi domain. The IL-15Ra sushi domain is critical for the functional activity of IL-15Ra. The extracellular region of IL-15Ra comprises a sushi domain, which is a common motif for protein-protein interactions and contains four cysteines that form two disulfide bonds in the 1-3 and 2-4 pattern. An exemplary IL-15Ra sushi domain comprises the amino acid sequence of amino acids 31-95 of SEQ ID NO: 721, the amino acid sequence of amino acids 31-105 of SEQ ID NO: 721, or the amino acid sequence of amino acids 31-107 of SEQ ID NO: 721 (e.g., the amino acid sequence of SEQ ID NO: 6).

[0203] In some embodiments, the first nucleic acid and the second nucleic acid are linked together directly or via a linker.

[0204] In some embodiments, the IL-15Ra sushi domain comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence that is at least 95% (e.g., 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%) identical to SEQ ID NO: 6.

[0205] In some embodiments, the second nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 7, or a nucleic acid sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%) identical to SEQ ID NO: 7, the second nucleic acid encoding an IL-15Ra sushi domain.

[0206] In some embodiments, the target polynucleotide further comprises a third nucleic acid encoding an Fc moiety. In some embodiments, the target polynucleotide is an mRNA comprising a first nucleic acid encoding an interleukin 15 polypeptide or variant thereof, a second nucleic acid encoding an IL-15Ra sushi domain, and a third nucleic acid encoding an Fc moiety. In some embodiments, the third nucleic acid is directly linked to the first nucleic acid and / or the second nucleic acid, optionally, the third nucleic acid is linked to the 5’ end of the first nucleic acid or the second nucleic acid, or linked to the 3’ end of the first nucleic acid or the second nucleic acid. In some embodiments, the 3’ end of the first nucleic acid is linked to the 5’ end of the second nucleic acid, and the 3’ end of the third nucleic acid is linked to the 5’ end of the first nucleic acid. In some embodiments, the 3’ end of the first nucleic acid is linked to the 5’ end of the second nucleic acid, and the 5’ end of the third nucleic acid is linked to the 3’ end of the second nucleic acid. In some embodiments, the 5’ end of the first nucleic acid is linked to the 3’ end of the second nucleic acid, and the 3’ end of the third nucleic acid is linked to the 5’ end of the second nucleic acid. In some embodiments, the 5’ end of the first nucleic acid is linked to the 3’ end of the second nucleic acid, and the 5’ end of the third nucleic acid is linked to the 3’ end of the first nucleic acid. Each of the two nucleic acids can be directly linked or linked via a linker.

[0207] In some embodiments, the Fc moiety is derived from human IgGl, IgG2, or IgG4.

[0208] In some embodiments, the Fc moiety described herein is derived from human IgGl. In some embodiments, the Fc moiety has one or more mutations selected from the group consisting of L234A and L235A (EU numbering). In some embodiments, the Fc moiety comprises L234A and L235A mutations (“LALA”). In some embodiments, the Fc moiety comprises knobs-into-holes mutations.

[0209] In some embodiments, the target polynucleotide further comprises a fourth nucleic acid encoding an antibody or antigen binding fragment thereof, the fourth nucleic acid is directly linked to the first nucleic acid and / or the second nucleic acid and / or the third nucleic acid, or linked via a linker. In some embodiments, the target polynucleotide is an mRNA comprising a first nucleic acid encoding an interleukin 15 polypeptide or variant thereof, a second nucleic acid encoding an IL-15Ra sushi domain, a third nucleic acid encoding an Fc moiety, and a fourth nucleic acid encoding an antibody or antigen binding fragment thereof.

[0210] In some embodiments, the antibody binds to PD-L1 or PD-1. In some embodiments, the antibody can be of any heavy chain isotype (e.g., IgG, IgA, IgM, IgE, or IgD) or subtype (e.g., IgGl, IgG2, IgG3, or IgG4). In some embodiments, the antibody can be of any light chain isotype (e.g., kappa or lambda). In some embodiments, the antibody is a single domain antibody. In some embodiments, the antibody is a single chain antibody. In some embodiments, the antibody can be a monoclonal antibody, a polyclonal antibody, a multispecific antibody, a bispecific antibody, an anti-idiotypic antibody, or a bifunctional hybrid antibody. In some embodiments, the antibody can be a human antibody, a non-human antibody, a chimeric antibody (e.g., having non-human variable regions and human constant regions), or a humanized antibody (e.g., having non-human CDRs and human framework and constant regions). In some embodiments, the antibody is a derivatized antibody. The term "antigen-binding fragment" of an antibody refers to a polypeptide or a set of interacting polypeptides of an antibody that specifically binds an antigen, and includes, but is not limited to, Fab, Fab', F(ab')2, Fv, disulfide linked Fv, scFv, or a single domain antibody.

[0211] In some embodiments, the anti-PD-L1 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 268, 270, 272, 274, 275, 277, 279, 281, 283, or 285. In some embodiments, the anti-PD-L1 antibody further comprises a light chain having the amino acid sequence of SEQ ID NO: 269, 271, 273, 276, 278, 280, 282, 284, or 286.

[0212] In some embodiments, the anti-PD-L1 antibody comprises a heavy chain and / or a light chain listed in the table below.

[0213] Table 2. Exemplary anti-PD-L1 antibodies

[0214]

[0215]

[0216]

[0217]

[0218] In some embodiments, the target polynucleotide encodes a polypeptide comprising an amino acid selected from the group consisting of SEQ ID NOs: 8, 10, 13, 15, 17, 23, 26, 28, 30-137, 287-298, 299-406, and 515-617.

[0219] In some embodiments, the target polynucleotide comprises a nucleic acid having SEQ ID NO: 9, 11-12, 14, 16, 18-19, 21-22, 24-25, 27, 29, 138-260, 407-514, 618-720, or 724-728, or a nucleic acid sequence having at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%) identity to SEQ ID NO: 9, 11-12, 14, 16, 18-19, 21-22, 24-25, 27, 29, 138-260, 407-514, 618-720, or 724-728, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 11-12, 14, 16, 18-19, 21-22, 24-25, 27, 29, 138-260, 407-514, 618-720, or 724-728. In some embodiments, the target polynucleotide comprises a nucleic acid having SEQ ID NO: 19, or a nucleic acid sequence having at least 85% identity to SEQ ID NO: 19, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 19. In some embodiments, the target polynucleotide comprises a nucleic acid having SEQ ID NO: 246, or a nucleic acid sequence having at least 85% identity to SEQ ID NO: 246, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 246. In some embodiments, the target polynucleotide comprises a nucleic acid having SEQ ID NO: 247, or a nucleic acid sequence having at least 85% identity to SEQ ID NO: 247, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 247. In some embodiments, the target polynucleotide comprises a nucleic acid having SEQ ID NO: 248, or a nucleic acid sequence having at least 85% identity to SEQ ID NO: 248, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 248. In some embodiments, the target polynucleotide comprises a nucleic acid having SEQ ID NO: 249, or a nucleic acid sequence having at least 85% identity to SEQ ID NO: 249, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 249. In some embodiments, the target polynucleotide comprises a nucleic acid having SEQ ID NO: 250, or a nucleic acid sequence having at least 85% identity to SEQ ID NO: 250, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 250.In some embodiments, the target polynucleotide comprises a nucleic acid having a nucleic acid sequence of SEQ ID NO: 252, or at least 85% identity to SEQ ID NO: 252, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 252. In some embodiments, the target polynucleotide comprises a nucleic acid having a nucleic acid sequence of SEQ ID NO: 253, or at least 85% identity to SEQ ID NO: 253, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 253. In some embodiments, the target polynucleotide comprises a nucleic acid having a nucleic acid sequence of SEQ ID NO: 254, or at least 85% identity to SEQ ID NO: 254, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 254. In some embodiments, the target polynucleotide comprises a nucleic acid having a nucleic acid sequence of SEQ ID NO: 255, or at least 85% identity to SEQ ID NO: 255, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 255. In some embodiments, the target polynucleotide comprises a nucleic acid having a nucleic acid sequence of SEQ ID NO: 256, or at least 85% identity to SEQ ID NO: 256, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 256. In some embodiments, the target polynucleotide comprises a nucleic acid having a nucleic acid sequence of SEQ ID NO: 257, or at least 85% identity to SEQ ID NO: 257, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 257. In some embodiments, the target polynucleotide comprises a nucleic acid having a nucleic acid sequence of SEQ ID NO: 258, or at least 85% identity to SEQ ID NO: 258, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 258.

[0220] In some embodiments, the target polynucleotide comprises:

[0221] (i) a 5’ untranslated region (UTR) comprising a nucleic acid selected from the group consisting of SEQ ID NOs: 261-263;

[0222] (ii) a 3’ untranslated region (UTR) comprising a nucleic acid selected from the group consisting of SEQ ID NOs: 264-267; and / or

[0223] (iii) a poly-A region having a length of 50-120 nucleotides, preferably wherein the poly-A region comprises a nucleic acid selected from the group consisting of SEQ ID NOs: 729-734.

[0224] Table 3. UTR

[0225]

[0226]

[0227] In alternative embodiments, the target polynucleotide further comprises a signal peptide coding sequence, a termination sequence, and a linker coding sequence. In some embodiments, the target polynucleotide further comprises a nucleic acid sequence encoding a signal peptide operably linked, directly or through a linker, to the first nucleic acid, the second nucleic acid, the third nucleic acid, or the fourth nucleic acid.

[0228] The term "signal peptide" as used herein refers to a peptide comprising the N-terminal 15-60 amino acids of a protein that is usually necessary for transmembrane transport in the secretory pathway and thus universally controls the process of most proteins in eukaryotes and prokaryotes into the secretory pathway. Signal peptides typically include three regions: an N-terminal region of varying length, which usually contains positively charged amino acids; a hydrophobic region; and a short carboxy-terminal peptide region.

[0229] In some embodiments, the signal peptide used herein can be the F7 signal peptide (SEQ ID NO: 4, MVSQALRLLCLLLGLQGCLA), which is encoded by the sequence of SEQ ID NO: 5 (AUGGUCUCCCAGGCCCUCAGGCUCCUCUGCCUUCUGCUUGGGCUUCAGGGCUGCCUGGCU) or by the sequence of SEQ ID NO: 722 (AUGGUGAGCCAGGCCCUGAGACUGCUGUGCCUGCUGCUCGGCCUGCAGGGCUGUCUGGCC). The signal peptide can be the IL15Ra signal peptide (SEQ ID NO: 2, MAPRRARGCRTLGLPALLLLLLLRPPATRG), which is encoded by the sequence of SEQ ID NO: 3 (AUGGCCCCGCGGCGGGCGCGCGGCUGCCGGACCCUCGGUCUCCCGGCGCUGCUACUGCUGCUGCUGCUCCGGCCGCCGGCGACGCGGGGC).

[0230] In some embodiments, the target polynucleotide comprises:

[0231] a) a nucleic acid having SEQ ID NO: 9, 11-12, 14, 16, 18-19, 21-22, 24-25, 27, 29, 138-260, 407-514, 618-720, or 724-728, or a nucleic acid sequence having at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%) identity to SEQ ID NO: 9, 11-12, 14, 16, 18-19, 21-22, 24-25, 27, 29, 138-260, 407-514, 618-720, or 724-728, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 11-12, 14, 16, 18-19, 21-22, 24-25, 27, 29, 138-260, 407-514, 618-720, or 724-728; and

[0232] b) a nucleic acid having SEQ ID NO: 5 operably linked to the nucleic acid of (a).

[0233] In some embodiments, the target polynucleotide comprises:

[0234] a) a nucleic acid having SEQ ID NO: 9, 11-12, 14, 16, 18-19, 21-22, 24-25, 27, 29, 138-260, 407-514, 618-720, or 724-728, or a nucleic acid sequence having at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%) identity to SEQ ID NO: 9, 11-12, 14, 16, 18-19, 21-22, 24-25, 27, 29, 138-260, 407-514, 618-720, or 724-728, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 11-12, 14, 16, 18-19, 21-22, 24-25, 27, 29, 138-260, 407-514, 618-720, or 724-728; and

[0235] b) a nucleic acid having SEQ ID NO: 722 operably linked to the nucleic acid of (a).

[0236] In some embodiments, the target polynucleotide comprises:

[0237] a) a nucleic acid having SEQ ID NO: 9, 11-12, 14, 16, 18-19, 21-22, 24-25, 27, 29, 138-260, 407-514, 618-720, or 724-728, or a nucleic acid having at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%) identity to SEQ ID NO: 9, 11-12, 14, 16, 18-19, 21-22, 24-25, 27, 29, 138-260, 407-514, 618-720, or 724-728, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 11-12, 14, 16, 18-19, 21-22, 24-25, 27, 29, 138-260, 407-514, 618-720, or 724-728; and

[0238] b) a nucleic acid having SEQ ID NO: 3 operably linked to the nucleic acid of (a).

[0239] In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 19, or at least 85% identity to SEQ ID NO: 19, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 19; and a nucleic acid having SEQ ID NO: 5 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 19, or at least 85% identity to SEQ ID NO: 19, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 19. In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 19, or at least 85% identity to SEQ ID NO: 19, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 19; and a nucleic acid having SEQ ID NO: 722 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 19, or at least 85% identity to SEQ ID NO: 19, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 19. In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 19, or at least 85% identity to SEQ ID NO: 19, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 19; and a nucleic acid having SEQ ID NO: 3 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 19, or at least 85% identity to SEQ ID NO: 19, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 19.

[0240] In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 246, or at least 85% identity to SEQ ID NO: 246, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 246; and a nucleic acid having SEQ ID NO: 5 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 246, or at least 85% identity to SEQ ID NO: 246, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 246. In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 246, or at least 85% identity to SEQ ID NO: 246, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 246; and a nucleic acid having SEQ ID NO: 722 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 246, or at least 85% identity to SEQ ID NO: 246, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 246. In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 246, or at least 85% identity to SEQ ID NO: 246, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 246; and a nucleic acid having SEQ ID NO: 3 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 246, or at least 85% identity to SEQ ID NO: 246, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 246.

[0241] In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 247, or at least 85% identity to SEQ ID NO: 247, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 247; and a nucleic acid having SEQ ID NO: 5 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 247, or at least 85% identity to SEQ ID NO: 247, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 247. In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 247, or at least 85% identity to SEQ ID NO: 247, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 247; and a nucleic acid having SEQ ID NO: 722 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 247, or at least 85% identity to SEQ ID NO: 247, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 247. In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 247, or at least 85% identity to SEQ ID NO: 247, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 247; and a nucleic acid having SEQ ID NO: 3 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 247, or at least 85% identity to SEQ ID NO: 247, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 247.

[0242] In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 248, or at least 85% identity to SEQ ID NO: 248, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 248; and a nucleic acid having SEQ ID NO: 5 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 248, or at least 85% identity to SEQ ID NO: 248, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 248. In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 248, or at least 85% identity to SEQ ID NO: 248, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 248; and a nucleic acid having SEQ ID NO: 722 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 248, or at least 85% identity to SEQ ID NO: 248, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 248. In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 248, or at least 85% identity to SEQ ID NO: 248, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 248; and a nucleic acid having SEQ ID NO: 3 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 248, or at least 85% identity to SEQ ID NO: 248, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 248.

[0243] In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 249, or at least 85% identity to SEQ ID NO: 249, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 249; and a nucleic acid having SEQ ID NO: 5 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 249, or at least 85% identity to SEQ ID NO: 249, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 249. In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 249, or at least 85% identity to SEQ ID NO: 249, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 249; and a nucleic acid having SEQ ID NO: 722 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 249, or at least 85% identity to SEQ ID NO: 249, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 249. In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 249, or at least 85% identity to SEQ ID NO: 249, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 249; and a nucleic acid having SEQ ID NO: 3 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 249, or at least 85% identity to SEQ ID NO: 249, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 249.

[0244] In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 250, or at least 85% identity to SEQ ID NO: 250, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 250; and a nucleic acid having SEQ ID NO: 5 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 250, or at least 85% identity to SEQ ID NO: 250, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 250. In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 250, or at least 85% identity to SEQ ID NO: 250, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 250; and a nucleic acid having SEQ ID NO: 722 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 250, or at least 85% identity to SEQ ID NO: 250, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 250. In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 250, or at least 85% identity to SEQ ID NO: 250, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 250; and a nucleic acid having SEQ ID NO: 3 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 250, or at least 85% identity to SEQ ID NO: 250, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 250.

[0245] In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 252, or at least 85% identity to SEQ ID NO: 252, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 252; and a nucleic acid having SEQ ID NO: 5 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 252, or at least 85% identity to SEQ ID NO: 252, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 252. In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 252, or at least 85% identity to SEQ ID NO: 252, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 252; and a nucleic acid having SEQ ID NO: 722 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 252, or at least 85% identity to SEQ ID NO: 252, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 252. In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 252, or at least 85% identity to SEQ ID NO: 252, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 252; and a nucleic acid having SEQ ID NO: 3 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 252, or at least 85% identity to SEQ ID NO: 252, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 252.

[0246] In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 253, or at least 85% identity to SEQ ID NO: 253, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 253; and a nucleic acid having SEQ ID NO: 5 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 253, or at least 85% identity to SEQ ID NO: 253, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 253. In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 253, or at least 85% identity to SEQ ID NO: 253, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 253; and a nucleic acid having SEQ ID NO: 722 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 253, or at least 85% identity to SEQ ID NO: 253, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 253. In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 253, or at least 85% identity to SEQ ID NO: 253, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 253; and a nucleic acid having SEQ ID NO: 3 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 253, or at least 85% identity to SEQ ID NO: 253, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 253.

[0247] In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 254, or at least 85% identity to SEQ ID NO: 254, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 254; and a nucleic acid having SEQ ID NO: 5 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 254, or at least 85% identity to SEQ ID NO: 254, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 254. In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 254, or at least 85% identity to SEQ ID NO: 254, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 254; and a nucleic acid having SEQ ID NO: 722 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 254, or at least 85% identity to SEQ ID NO: 254, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 254. In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 254, or at least 85% identity to SEQ ID NO: 254, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 254; and a nucleic acid having SEQ ID NO: 3 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 254, or at least 85% identity to SEQ ID NO: 254, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 254.

[0248] In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 255, or at least 85% identity to SEQ ID NO: 255, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 255; and a nucleic acid having SEQ ID NO: 5 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 255, or at least 85% identity to SEQ ID NO: 255, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 255. In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 255, or at least 85% identity to SEQ ID NO: 255, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 255; and a nucleic acid having SEQ ID NO: 722 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 255, or at least 85% identity to SEQ ID NO: 255, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 255. In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 255, or at least 85% identity to SEQ ID NO: 255, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 255; and a nucleic acid having SEQ ID NO: 3 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 255, or at least 85% identity to SEQ ID NO: 255, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 255.

[0249] In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 256, or at least 85% identity to SEQ ID NO: 256, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 256; and a nucleic acid having SEQ ID NO: 5 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 256, or at least 85% identity to SEQ ID NO: 256, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 256. In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 256, or at least 85% identity to SEQ ID NO: 256, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 256; and a nucleic acid having SEQ ID NO: 722 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 256, or at least 85% identity to SEQ ID NO: 256, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 256. In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 256, or at least 85% identity to SEQ ID NO: 256, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 256; and a nucleic acid having SEQ ID NO: 3 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 256, or at least 85% identity to SEQ ID NO: 256, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 256.

[0250] In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 257, or at least 85% identity to SEQ ID NO: 257, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 257; and a nucleic acid having SEQ ID NO: 5 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 257, or at least 85% identity to SEQ ID NO: 257, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 257. In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 257, or at least 85% identity to SEQ ID NO: 257, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 257; and a nucleic acid having SEQ ID NO: 722 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 257, or at least 85% identity to SEQ ID NO: 257, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 257. In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 257, or at least 85% identity to SEQ ID NO: 257, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 257; and a nucleic acid having SEQ ID NO: 3 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 257, or at least 85% identity to SEQ ID NO: 257, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 257.

[0251] In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 258, or at least 85% identity to SEQ ID NO: 258, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 258; and a nucleic acid having SEQ ID NO: 5 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 258, or at least 85% identity to SEQ ID NO: 258, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 258. In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 258, or at least 85% identity to SEQ ID NO: 258, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 258; and a nucleic acid having SEQ ID NO: 722 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 258, or at least 85% identity to SEQ ID NO: 258, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 258. In some embodiments, the target polynucleotide comprises: a nucleic acid having the nucleic acid sequence of SEQ ID NO: 258, or at least 85% identity to SEQ ID NO: 258, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 258; and a nucleic acid having SEQ ID NO: 3 operably linked to the nucleic acid having the nucleic acid sequence of SEQ ID NO: 258, or at least 85% identity to SEQ ID NO: 258, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 258.

[0252] IL-15 polypeptide variants

[0253] In another aspect, the present disclosure provides IL-15 polypeptide variants for use in treating a disease or disorder, including cancer. In some embodiments, the IL-15 polypeptide variants can be used to enhance immune cell activation.

[0254] In some embodiments, at least one amino acid residue of the IL-15 polypeptide variant is substituted with an aspartic acid residue according to SEQ ID NO: 8 (NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLE LQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEK NIKEFLQSFVHIVQMFINTS). In some embodiments, the IL-15 polypeptide variant comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 299-406.

[0255] Table 4. Target IL-15 polypeptides

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272] Also provided herein are polypeptides comprising an IL-15 polypeptide variant and a suitable signal peptide. The signal peptide can be fused to the N-terminus or C-terminus of the IL-15 polypeptide variant. In some embodiments, the signal peptide can be fused to the N-terminus of the IL-15 polypeptide variant. In some embodiments, the signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4.

[0273] Fusion proteins

[0274] In another aspect, the present disclosure provides a fusion protein for treating a disease or disorder, including cancer. In some embodiments, the fusion protein can be used to enhance immune cell activation.

[0275] In another aspect, the present disclosure provides a fusion protein comprising an IL-15 polypeptide and an IL-15Ra sushi, wherein the IL-15 polypeptide is fused to the IL-15Ra sushi directly or through a peptide linker. In some embodiments, the IL-15 polypeptide is fused to the N-terminus of the IL-15Ra sushi. In some embodiments, the IL-15 polypeptide is fused to the C-terminus of the IL-15Ra sushi. In some embodiments, the peptide linker is GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, or GGGGSGGGGSGGGGSGGGGS. In a preferred embodiment, the peptide linker is GGGGSGGGGSGGGGS.

[0276] As used herein, "IL-15 polypeptide" refers to a wild-type of IL-15 (e.g., SEQ ID NO: 8) or an IL-15 polypeptide variant. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and 299-406.

[0277] In some embodiments, the IL-15Ra sushi domain comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 721

[0278] the amino acid sequence of amino acid residues 31-95 of (MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT). In some embodiments, the IL-15Ra sushi domain comprises the amino acid sequence of amino acid residues 31-105 of SEQ ID NO: 721. In some embodiments, the IL-15Ra sushi domain comprises the amino acid sequence of amino acid residues 31-107 of SEQ ID NO: 721 (e.g., the amino acid sequence of SEQ ID NO: 6). SEQ ID NO: 6: ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP.

[0279] In some embodiments, the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 13, and 30-137.

[0280] In some other embodiments, the fusion protein further comprises an Fc moiety located at the N-terminus of the fusion protein, or at the C-terminus of the fusion protein. In some embodiments, the Fc moiety is fused directly or via a peptide linker to the IL-15 polypeptide and / or the IL-15Ra sushi. Optionally, the Fc moiety is fused to the N-terminus of the IL-15 polypeptide or the IL-15Ra sushi, or the Fc moiety is fused to the C-terminus of the IL-15 polypeptide or the IL-15Ra sushi. In some embodiments, the IL-15 polypeptide is fused to the N-terminus of the IL-15Ra sushi, and the Fc moiety is fused to the N-terminus of the IL-15 polypeptide. In some embodiments, the IL-15 polypeptide is fused to the N-terminus of the IL-15Ra sushi, and the Fc moiety is fused to the C-terminus of the IL-15Ra sushi. In some embodiments, the IL-15 polypeptide is fused to the C-terminus of the IL-15Ra sushi, and the Fc moiety is fused to the C-terminus of the IL-15 polypeptide. In some embodiments, the IL-15 polypeptide is fused to the C-terminus of the IL-15Ra sushi, and the Fc moiety is fused to the N-terminus of the IL-15Ra sushi. Each of the two peptide fragments can be fused directly or via a peptide linker. In some embodiments, the peptide linker is GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, or GGGGSGGGGSGGGGSGGGGS. In a preferred embodiment, the peptide linker is GGGGSGGGGSGGGGS.

[0281] In some embodiments, the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 17, 23, 26, 28, 287-291, and 515-617.

[0282] In some other embodiments, the fusion protein further comprises an antibody moiety that binds to PD-L1 or PD-1. In some embodiments, the antibody moiety comprises or consists of a heavy chain variable region. In some embodiments, the antibody moiety comprises or consists of a heavy chain variable region and a light chain variable region. In some embodiments, the heavy chain variable region and the light chain variable region are linked directly or via a peptide linker. In some embodiments, the antibody moiety is a ScFv. In some embodiments, the antibody is a single domain antibody. In some embodiments, the antibody moiety comprises an amino acid sequence of the present disclosure.

[0283] In some embodiments, the fusion protein comprises an amino acid selected from the group consisting of SEQ ID NOs: 292-298.

[0284] Table 5. Target fusion proteins

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345] In another aspect, the present disclosure provides a fusion protein further comprising a suitable signal peptide fused at the N-terminus or C-terminus of any of the above fusion proteins. In some embodiments, the suitable signal peptide is fused at the N-terminus of any of the fusion proteins. In some embodiments, the signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4.

[0346] Nucleic acids

[0347] In another aspect, the present disclosure provides a nucleic acid for use in treating a disease or disorder, including cancer. In some embodiments, the nucleic acid can be used to enhance immune cell activation. In some embodiments, the nucleic acid of the present disclosure is an isolated nucleic acid sequence.

[0348] In another aspect, the present disclosure provides an isolated nucleic acid sequence encoding an interleukin 15 polypeptide or a variant thereof, the isolated nucleic acid sequence comprising a nucleic acid sequence having at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9 and 407-514.

[0349] In another aspect, the present disclosure provides an isolated nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises an IL-15 polypeptide and an IL-15Ra sushi, and wherein the isolated nucleic acid sequence comprises:

[0350] (i) a nucleic acid sequence encoding an IL-15 having at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 9 and 407-514; and

[0351] (ii) a nucleic acid sequence encoding an IL-15Ra sushi having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 7 (AUCACGUGCCCUCCCCCCAUGUCCGUGGAACACGCAGACAUCUGGGUCAAGAGCUACAGCUUGUACUCCAGGGAGCGGUACAUUUGUAACUCUGGUUUCAAGCGUAAAGCCGGCACGUCCAGCCUGACGGAGUGCGUGUUGAACAAGGCCACGAAUGUCGCCCACUGGACAACCCCCAGUCUCAAAUGCAUUAGAGACCCUGCCCUGGUUCACCAAAGGCCAGCGCCACCC).

[0352] In some embodiments, the isolated nucleic acid sequence has at least 85% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11-12, 14, 21-22, and 138-245.

[0353] In some embodiments, the fusion protein further comprises an Fc moiety located at the N-terminus of the fusion protein, or located at the C-terminus of the fusion protein. In some embodiments, the isolated nucleic acid sequence has at least 85% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16, 18-19, 24-25, 27, 29, 246-250, 618-720, and 724-728.

[0354] In some embodiments, the fusion protein further comprises an antibody moiety that binds to PD-L1 or PD-1. In some embodiments, the isolated nucleic acid sequence has at least 85% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 252-258.

[0355] In some embodiments, the isolated nucleic acid sequence further comprises a nucleic acid encoding an isolated antibody or antigen-binding fragment thereof that binds to PD-L1 or PD-1. In some embodiments, the anti-PD-L1 antibody comprises a heavy chain and / or a light chain listed in Table 2.

[0356] In some embodiments, the isolated nucleic acid sequence further comprises a nucleic acid encoding a 2A peptide. In some embodiments, the 2A peptide is P2A, T2A, E2A, or F2A. In some embodiments, the P2A comprises the amino acid sequence GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 735).

[0357] In some embodiments, the isolated nucleic acid sequence has at least 85% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 259-260.

[0358] Table 6. Target nucleic acids

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

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[0610] In another aspect, the present disclosure provides a nucleic acid sequence further comprising a suitable signal peptide coding sequence operably linked at the 5' end or 3' end of any of the above nucleic acids, directly or through a linker. In some embodiments, the signal peptide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 722.

[0611] In some embodiments, the nucleic acid sequence comprises:

[0612] a) a nucleic acid having SEQ ID NO: 9, 11-12, 14, 16, 18-19, 21-22, 24-25, 27, 29, 138-260, 407-514, 618-720, or 724-728, or a nucleic acid sequence having at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%) identity to SEQ ID NO: 9, 11-12, 14, 16, 18-19, 21-22, 24-25, 27, 29, 138-260, 407-514, 618-720, or 724-728, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 11-12, 14, 16, 18-19, 21-22, 24-25, 27, 29, 138-260, 407-514, 618-720, or 724-728; and

[0613] b) a nucleic acid having SEQ ID NO: 5 operably linked to the nucleic acid of (a).

[0614] In some embodiments, the nucleic acid sequence comprises:

[0615] a) a nucleic acid having SEQ ID NO: 9, 11-12, 14, 16, 18-19, 21-22, 24-25, 27, 29, 138-260, 407-514, 618-720, or 724-728, or a nucleic acid sequence having at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%) identity to SEQ ID NO: 9, 11-12, 14, 16, 18-19, 21-22, 24-25, 27, 29, 138-260, 407-514, 618-720, or 724-728, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 11-12, 14, 16, 18-19, 21-22, 24-25, 27, 29, 138-260, 407-514, 618-720, or 724-728; and

[0616] b) a nucleic acid having SEQ ID NO: 3 operably linked to the nucleic acid of (a).

[0617] In some embodiments, the nucleic acid sequence comprises:

[0618] a) a nucleic acid having SEQ ID NO: 9, 11-12, 14, 16, 18-19, 21-22, 24-25, 27, 29, 138-260, 407-514, 618-720, or 724-728, or a nucleic acid sequence having at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%) identity to SEQ ID NO: 9, 11-12, 14, 16, 18-19, 21-22, 24-25, 27, 29, 138-260, 407-514, 618-720, or 724-728, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 11-12, 14, 16, 18-19, 21-22, 24-25, 27, 29, 138-260, 407-514, 618-720, or 724-728; and

[0619] b) a nucleic acid having SEQ ID NO: 3 operably linked to the nucleic acid of (a).

[0620] In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 19, or at least 85% identity to SEQ ID NO: 19, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 19; and a nucleic acid having SEQ ID NO: 5 operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 19, or at least 85% identity to SEQ ID NO: 19, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 19. In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 19, or at least 85% identity to SEQ ID NO: 19, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 19; and a nucleic acid having SEQ ID NO: 722 operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 19, or at least 85% identity to SEQ ID NO: 19, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 19. In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 19, or at least 85% identity to SEQ ID NO: 19, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 19; and a nucleic acid having SEQ ID NO: 3 operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 19, or at least 85% identity to SEQ ID NO: 19, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 19.

[0621] In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 246, or having at least 85% identity to SEQ ID NO: 246, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 246; and a nucleic acid having SEQ ID NO: 5 operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 246, or having at least 85% identity to SEQ ID NO: 246, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 246. In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 246, or having at least 85% identity to SEQ ID NO: 246, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 246; and a nucleic acid having SEQ ID NO: 722 operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 246, or having at least 85% identity to SEQ ID NO: 246, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 246. In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 246, or having at least 85% identity to SEQ ID NO: 246, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 246; and a nucleic acid having SEQ ID NO: 3 operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 246, or having at least 85% identity to SEQ ID NO: 246, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 246.

[0622] In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 247, or having at least 85% identity to SEQ ID NO: 247, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 247; and a nucleic acid having SEQ ID NO: 5, operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 247, or having at least 85% identity to SEQ ID NO: 247, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 247. In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 247, or having at least 85% identity to SEQ ID NO: 247, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 247; and a nucleic acid having SEQ ID NO: 722, operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 247, or having at least 85% identity to SEQ ID NO: 247, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 247. In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 247, or having at least 85% identity to SEQ ID NO: 247, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 247; and a nucleic acid having SEQ ID NO: 3, operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 247, or having at least 85% identity to SEQ ID NO: 247, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 247.

[0623] In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 248, or having at least 85% identity to SEQ ID NO: 248, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 248; and a nucleic acid having SEQ ID NO: 5 operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 248, or having at least 85% identity to SEQ ID NO: 248, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 248. In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 248, or having at least 85% identity to SEQ ID NO: 248, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 248; and a nucleic acid having SEQ ID NO: 722 operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 248, or having at least 85% identity to SEQ ID NO: 248, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 248. In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 248, or having at least 85% identity to SEQ ID NO: 248, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 248; and a nucleic acid having SEQ ID NO: 3 operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 248, or having at least 85% identity to SEQ ID NO: 248, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 248.

[0624] In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 249, or having at least 85% identity to SEQ ID NO: 249, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 249; and a nucleic acid having SEQ ID NO: 5 operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 249, or having at least 85% identity to SEQ ID NO: 249, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 249. In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 249, or having at least 85% identity to SEQ ID NO: 249, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 249; and a nucleic acid having SEQ ID NO: 722 operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 249, or having at least 85% identity to SEQ ID NO: 249, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 249. In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 249, or having at least 85% identity to SEQ ID NO: 249, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 249; and a nucleic acid having SEQ ID NO: 3 operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 249, or having at least 85% identity to SEQ ID NO: 249, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 249.

[0625] In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 250, or having at least 85% identity to SEQ ID NO: 250, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 250; and a nucleic acid having SEQ ID NO: 5 operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 250, or having at least 85% identity to SEQ ID NO: 250, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 250. In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 250, or having at least 85% identity to SEQ ID NO: 250, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 250; and a nucleic acid having SEQ ID NO: 722 operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 250, or having at least 85% identity to SEQ ID NO: 250, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 250. In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 250, or having at least 85% identity to SEQ ID NO: 250, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 250; and a nucleic acid having SEQ ID NO: 3 operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 250, or having at least 85% identity to SEQ ID NO: 250, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 250.

[0626] In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 252, or having at least 85% identity to SEQ ID NO: 252, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 252; and a nucleic acid having SEQ ID NO: 5 operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 252, or having at least 85% identity to SEQ ID NO: 252, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 252. In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 252, or having at least 85% identity to SEQ ID NO: 252, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 252; and a nucleic acid having SEQ ID NO: 722 operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 252, or having at least 85% identity to SEQ ID NO: 252, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 252. In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 252, or having at least 85% identity to SEQ ID NO: 252, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 252; and a nucleic acid having SEQ ID NO: 3 operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 252, or having at least 85% identity to SEQ ID NO: 252, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 252.

[0627] In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 253, or having at least 85% identity to SEQ ID NO: 253, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 253; and a nucleic acid having SEQ ID NO: 5, operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 253, or having at least 85% identity to SEQ ID NO: 253, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 253. In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 253, or having at least 85% identity to SEQ ID NO: 253, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 253; and a nucleic acid having SEQ ID NO: 722, operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 253, or having at least 85% identity to SEQ ID NO: 253, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 253. In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 253, or having at least 85% identity to SEQ ID NO: 253, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 253; and a nucleic acid having SEQ ID NO: 3, operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 253, or having at least 85% identity to SEQ ID NO: 253, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 253.

[0628] In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 254, or having at least 85% identity to SEQ ID NO: 254, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 254; and a nucleic acid having SEQ ID NO: 5 operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 254, or having at least 85% identity to SEQ ID NO: 254, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 254. In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 254, or having at least 85% identity to SEQ ID NO: 254, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 254; and a nucleic acid having SEQ ID NO: 722 operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 254, or having at least 85% identity to SEQ ID NO: 254, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 254. In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 254, or having at least 85% identity to SEQ ID NO: 254, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 254; and a nucleic acid having SEQ ID NO: 3 operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 254, or having at least 85% identity to SEQ ID NO: 254, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 254.

[0629] In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 255, or having at least 85% identity to SEQ ID NO: 255, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 255; and a nucleic acid having SEQ ID NO: 5 operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 255, or having at least 85% identity to SEQ ID NO: 255, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 255. In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 255, or having at least 85% identity to SEQ ID NO: 255, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 255; and a nucleic acid having SEQ ID NO: 722 operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 255, or having at least 85% identity to SEQ ID NO: 255, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 255. In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 255, or having at least 85% identity to SEQ ID NO: 255, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 255; and a nucleic acid having SEQ ID NO: 3 operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 255, or having at least 85% identity to SEQ ID NO: 255, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 255.

[0630] In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 256, or having at least 85% identity to SEQ ID NO: 256, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 256; and a nucleic acid having SEQ ID NO: 5, operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 256, or having at least 85% identity to SEQ ID NO: 256, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 256. In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 256, or having at least 85% identity to SEQ ID NO: 256, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 256; and a nucleic acid having SEQ ID NO: 722, operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 256, or having at least 85% identity to SEQ ID NO: 256, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 256. In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 256, or having at least 85% identity to SEQ ID NO: 256, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 256; and a nucleic acid having SEQ ID NO: 3, operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 256, or having at least 85% identity to SEQ ID NO: 256, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 256.

[0631] In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 257, or having at least 85% identity to SEQ ID NO: 257, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 257; and a nucleic acid having SEQ ID NO: 5 operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 257, or having at least 85% identity to SEQ ID NO: 257, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 257. In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 257, or having at least 85% identity to SEQ ID NO: 257, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 257; and a nucleic acid having SEQ ID NO: 722 operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 257, or having at least 85% identity to SEQ ID NO: 257, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 257. In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 257, or having at least 85% identity to SEQ ID NO: 257, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 257; and a nucleic acid having SEQ ID NO: 3 operably linked to the nucleic acid having a nucleic acid sequence of SEQ ID NO: 257, or having at least 85% identity to SEQ ID NO: 257, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 257.

[0632] In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 258, or at least 85% identity to SEQ ID NO: 258, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 258; and a nucleic acid having SEQ ID NO: 5 operably linked to the nucleic acid sequence having SEQ ID NO: 258, or at least 85% identity to SEQ ID NO: 258, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 258. In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 258, or at least 85% identity to SEQ ID NO: 258, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 258; and a nucleic acid having SEQ ID NO: 722 operably linked to the nucleic acid sequence having SEQ ID NO: 258, or at least 85% identity to SEQ ID NO: 258, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 258. In some embodiments, the nucleic acid sequence comprises: a nucleic acid having a nucleic acid sequence of SEQ ID NO: 258, or at least 85% identity to SEQ ID NO: 258, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 258; and a nucleic acid having SEQ ID NO: 3 operably linked to the nucleic acid sequence having SEQ ID NO: 258, or at least 85% identity to SEQ ID NO: 258, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NO: 258.

[0633] Pharmaceutical compositions

[0634] In another aspect, the present disclosure also provides a pharmaceutical composition comprising the lipid nanoparticle composition, IL-15 variant, fusion protein, or nucleic acid provided herein, and a pharmaceutically acceptable excipient.

[0635] The pharmaceutically acceptable excipients are conventional pharmaceutical excipients in the art, which can be prepared in a manner known in the pharmaceutical art. Some examples of materials which can serve as pharmaceutically acceptable excipients or carriers include: (1) sugars, such as lactose, dextrose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter or suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) alcohols, such as ethyl alcohol and propyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations, such as acetone.

[0636] The pharmaceutical composition can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, etc., for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.

[0637] The form of the pharmaceutical composition depends on a number of standards, including but not limited to, the route of administration, the degree of disease, or the dose of administration.

[0638] The pharmaceutical composition can be formulated for oral, nasal, rectal, transdermal, intravenous, intradermal, intramuscular, intranasal, and / or subcutaneous administration. Depending on the desired route of administration, the pharmaceutical composition can be formulated into the following forms: tablets, capsules, pills, dragees, powders, granules, sachets, cachets, lozenges, suspensions, emulsions, solutions, syrups, aerosols (in the form of a solid or liquid medium), sprays, ointments, pastes, creams, lotions, gels, patches, inhalants, or suppositories.

[0639] The pharmaceutical compositions can be formulated to provide rapid, sustained, or delayed release of the active ingredient after administration to the patient using e.g., procedures known in the art. In some embodiments, the pharmaceutical compositions are formulated in sustained release form. In some embodiments, the extended time can be about 1 hour to 24 hours, 2 hours to 12 hours, 3 hours to 8 hours, 4 hours to 6 hours, 1 to 2 days or more. In certain embodiments, the extended time is at least about 4 hours, at least about 8 hours, at least about 12 hours, or at least about 24 hours. The pharmaceutical compositions can be formulated in the form of tablets. For example, not only can the release rate of the active agent be controlled by its dissolution in the gastrointestinal fluid and subsequent diffusion from the tablet or pill independent of pH, the release rate of the active agent can also be influenced by the physical processes of tablet disintegration and erosion. In some embodiments, polymeric materials as disclosed in the following references can be used for sustained release: Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J Macromol. Sci. Rev. Macromol Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71 :105. The above references are incorporated herein by reference in their entirety.

[0640] In certain embodiments, the pharmaceutical compositions of the present disclosure can be administered at a dosage level sufficient to deliver from about 0.0001 mg / kg to about 10 mg / kg (e.g., from about 0.0001 mg / kg to about 10 mg / kg, from about 0.001 mg / kg to about 10 mg / kg, from about 0.005 mg / kg to about 10 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.05 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, from about 1 mg / kg to about 10 mg / kg, from about 2 mg / kg to about 10 mg / kg, from about 5 mg / kg to about 10 mg / kg, from about 0.0001 mg / kg to about 5 mg / kg, from about 0.001 mg / kg to about 5 mg / kg, from about 0.005 mg / kg to about 5 mg / kg, from about 0.01 mg / kg to about 5 mg / kg, from about 0.05 mg / kg to about 5 mg / kg, from about 0.1 mg / kg to about 5 mg / kg, from about 1 mg / kg to about 5 mg / kg, from about 2 mg / kg to about 5 mg / kg, from about 0.0001 mg / kg to about 2.5 mg / kg, from about 0.001 mg / kg to about 2.5 mg / kg, from about 0.005 mg / kg to about 2.5 mg / kg, from about 0.01 mg / kg to about 2.5 mg / kg, from about 0.05 mg / kg to about 2.5 mg / kg, from about 0.1 mg / kg to about 2.5 mg / kg, from about 1 mg / kg to about 2.5 mg / kg, from about 2 mg / kg to about 2.5 mg / kg, from about 0.0001 mg / kg to about 1 mg / kg, from about 0.001 mg / kg to about 1 mg / kg, from about 0.005 mg / kg to about 1 mg / kg, from about 0.01 mg / kg to about 1 mg / kg, from about 0.05 mg / kg to about 1 mg / kg, from about 0.1 mg / kg to about 1 mg / kg, from about 0.0001 mg / kg to about 0.25 mg / kg, from about 0.001 mg / kg to about 0.25 mg / kg, from about 0.005 mg / kg to about 0.25 mg / kg, from about 0.01 mg / kg to about 0.25 mg / kg, from about 0.05 mg / kg to about 0.25 mg / kg, or from about 0.1 mg / kg to about 0.25 mg / kg) of the therapeutic agent in a given dose, wherein a dose of 1 mg / kg provides 1 mg of the therapeutic agent and / or prophylactic agent per 1 kg of the subject’s body weight.

[0641] In certain embodiments, the pharmaceutical compositions can be formulated in unit dosage form. The term "unit dosage form" refers to physically discrete units suitable for unitary dosing to human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical carrier. In some embodiments, the dose of therapeutic agent that can be administered is from about 0.001 mg / kg to about 10 mg / kg. In other embodiments, the dose of therapeutic agent that can be administered is from about 0.005 mg / kg to about 2.5 mg / kg. In certain embodiments, the dose of therapeutic agent that can be administered is from about 0.1 mg / kg to about 1 mg / kg. In other embodiments, the dose of therapeutic agent that can be administered is from about 0.05 mg / kg to about 0.25 mg / kg. The dose can be administered in one or more portions, at the same or different amounts, per day to achieve the desired level of therapeutic, diagnostic, prophylactic, or imaging effect. The desired dose can be delivered, for example, three times per day, twice per day, once per day, every other day, every third day, weekly, biweekly, triweekly, or quarterly.

[0642] Methods of enhancing immune cell activation and methods of treating disease

[0643] In another aspect, the present disclosure provides a method of enhancing immune cell activation in a subject, comprising administering to the subject an effective amount of a lipid nanoparticle composition of the present disclosure, an effective amount of an IL-15 polypeptide variant of the present disclosure, an effective amount of a fusion protein of the present disclosure, or an effective amount of an isolated nucleic acid sequence of the present disclosure.

[0644] In another aspect, the present disclosure provides a method of treating a disease or disorder in a subject, comprising administering to the subject an effective amount of a lipid nanoparticle composition of the present disclosure, an effective amount of an IL-15 polypeptide variant of the present disclosure, an effective amount of a fusion protein of the present disclosure, or an effective amount of an isolated nucleic acid sequence of the present disclosure.

[0645] In another aspect, the present disclosure provides use of a lipid nanoparticle of the present disclosure, a lipid nanoparticle composition of the present disclosure, an IL-15 polypeptide variant of the present disclosure, a fusion protein of the present disclosure, or an isolated nucleic acid sequence of the present disclosure, or a pharmaceutical composition comprising the lipid nanoparticle, the lipid nanoparticle composition, the fusion protein, or the nucleic acid sequence, for treating a disease or disorder.

[0646] In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is an epithelial tumor, Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma, a prostate tumor, an ovarian tumor, a renal cell tumor, a gastrointestinal tumor, a liver tumor, a colorectal tumor, an angioma, a mesothelioma, a pancreatic tumor, a breast tumor, a sarcoma, a lung tumor, a colon tumor, a brain tumor, a melanoma, a small cell lung tumor, a neuroblastoma, a testicular tumor, a carcinoma, an adenocarcinoma, a glioma, a seminoma, a retinoblastoma, or an osteosarcoma.

[0647] As used herein, the term "treatment" is intended to have its normal meaning, i.e., to cope with a disease so as to completely or partially relieve one, some or all of its symptoms, or to correct or compensate for the underlying pathology, so as to achieve a beneficial or desired clinical outcome. For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether or not detectable. "Treatment" can also mean prolonging survival as compared with expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those in which the condition or disorder is to be prevented. Unless there are specific contrary indications, the term "treatment" also encompasses prophylaxis. The terms "therapeutic" and "therapeutically" are to be construed correspondingly.

[0648] As used herein, "prevention" or "preventive" is intended to have its normal meaning and includes primary prevention, to prevent development of a disease, and secondary prevention, i.e., temporary or permanent protection of a patient from a disease from worsening or from developing new symptoms associated with the disease, once the disease has occurred.

[0649] The term "treatment" or "treating" is used synonymously with "therapy". Similarly, the term "treat" can be considered as "apply a therapy", wherein "therapy" is as defined herein.

[0650] In some embodiments, the lipid nanoparticle compositions of the present disclosure, or pharmaceutical compositions comprising the lipid nanoparticle compositions, can be further used in combination with other biologically active ingredients, such as but not limited to a second and different anti-neoplastic agent, and non-pharmaceutical therapies, such as but not limited to surgery or radiation therapy. For example, the lipid nanoparticle compositions of the present disclosure, or pharmaceutical compositions comprising the lipid nanoparticle compositions, can be used in combination with other pharmaceutically active compounds, or non-pharmaceutical therapies, preferably compounds or pharmaceutical compositions comprising the lipid nanoparticle compositions of the present disclosure that are capable of enhancing the effect of the lipid nanoparticle compositions of the present disclosure. The lipid nanoparticle compositions of the present disclosure, or pharmaceutical compositions comprising the lipid nanoparticle compositions, can be administered simultaneously (as a single preparation or separate preparations) or sequentially with the other therapies. Generally, combination therapy contemplates administration of two or more pharmaceuticals / treatments during a single cycle or course of therapy.

[0651] In some embodiments, the lipid nanoparticle compositions of the present disclosure, the lipid nanoparticle compositions of the present disclosure, the IL-15 polypeptide variants of the present disclosure, the fusion proteins of the present disclosure, the isolated nucleic acid sequences of the present disclosure, or the pharmaceutical compositions are used in combination with one or more traditional chemotherapeutic agents, which encompass a broad range of therapeutic treatments in the field of oncology. These agents are administered at various stages of the disease in order to shrink tumors, kill residual cancer cells after surgery, induce remission, maintain remission, and / or alleviate symptoms associated with cancer or its treatment.

[0652] In some embodiments, the lipid nanoparticle compositions of the present disclosure, the lipid nanoparticle compositions of the present disclosure, the IL-15 polypeptide variants of the present disclosure, the fusion proteins of the present disclosure, the isolated nucleic acid sequences of the present disclosure, or the pharmaceutical compositions are used in combination with one or more targeted anti-cancer agents that modulate protein kinases involved in various disease states. In some embodiments, the lipid nanoparticle compositions of the present disclosure, the lipid nanoparticle compositions of the present disclosure, the fusion proteins of the present disclosure, the isolated nucleic acid sequences of the present disclosure, or the pharmaceutical compositions are used in combination with one or more targeted anti-cancer agents that modulate non-kinase biological targets, pathways, or processes.

[0653] In some embodiments, the lipid nanoparticle compositions of the present disclosure, the IL-15 polypeptide variants of the present disclosure, the fusion proteins of the present disclosure, the isolated nucleic acid sequences of the present disclosure, or the pharmaceutical compositions are used in combination with one or more other anti-cancer agents, including but not limited to: gene therapy, RNAi cancer therapies, chemoprotective agents (e.g., amifostine, mesna, and dexrazoxane), drug antibody conjugates (e.g., brentuximab vedotin, ibritumomab tiuxetan), cancer immunotherapies such as interleukin 2, cancer vaccines (e.g., sipuleucel-T), or monoclonal antibodies (e.g., bevacizumab, alemtuzumab, rituximab, trastuzumab, etc.).

[0654] In some embodiments, the lipid nanoparticle compositions of the present disclosure, the IL-15 polypeptide variants of the present disclosure, the fusion proteins of the present disclosure, the isolated nucleic acid sequences of the present disclosure, or the pharmaceutical compositions are used in combination with one or more anti-inflammatory agents, including but not limited to NSAIDs, non-specific and COX-2 specific cyclooxygenase inhibitors, gold preparations, corticosteroids, methotrexate, tumor necrosis factor receptor (TNF) receptor antagonists, immunosuppressants, and methotrexate.

[0655] In some embodiments, the lipid nanoparticle compositions of the present disclosure, the IL-15 polypeptide variants of the present disclosure, the fusion proteins of the present disclosure, the isolated nucleic acid sequences of the present disclosure, or the pharmaceutical compositions are used in combination with radiation therapy or surgery. Radiation is typically delivered internally (implanting radioactive material near the site of the cancer) or externally from a machine that uses either photons (x-rays or gamma rays) or particle radiation. Where the combination therapy also includes radiation treatment, the radiation treatment can be given at any suitable time, as long as a beneficial effect is obtained from the combined action of the therapeutic agent and the radiation treatment. In some embodiments, the pharmaceutical composition comprises a second active ingredient.

[0656] In some embodiments, the second active ingredient has complementary activity to the compounds provided herein, such that they do not adversely affect each other. Such ingredients are suitably present in combination in amounts that are effective for the purposes intended.

[0657] In some embodiments, the second active ingredient can include:

[0658] (i) antiproliferative / antineoplastic drugs and combinations thereof, as used in medical oncology, such as alkylating agents (for example, cis-platin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulphan and nitrosoureas); antimetabolites (for example, antifolates such as fluoropyrimidines like 5-fluorouracil and tegafur, raltitrexed, methotrexate, cytosine arabinoside, hydroxyurea and gemcitabine); antitumour antibiotics (for example, anthracyclines like doxorubicin, bleomycin, daunomycin, daunorubicin, epirubicin, idarubicin, mitomycin C, dactinomycin and mithramycin); antimitotic agents (for example, vinca alkaloids like vincristine, vinblastine, vindesine, and vinorelbine, and taxoids like paclitaxel and docetaxel); and topoisomerase inhibitors (for example, epipodophyllotoxins like etoposide and teniposide, amsacrine, topotecan and camptothecins);

[0659] (ii) cytostatic agents such as antiestrogens (for example, tamoxifen, toremifene, raloxifene, droloxifene and iodoxyfene), estrogen receptor downregulators (for example, fulvestrant), antiandrogens (for example, bicalutamide, flutamide, nilutamide and cyproterone acetate), LHRH antagonists or LHRH agonists (for example, goserelin, leuprolide and buserelin), progestogens (for example, megestrol acetate), aromatase inhibitors (for example, anastrozole, letrozole, vorazole and exemestane) and inhibitors of 5a-reductase (for example, finasteride);

[0660] (iii) anti-invasive agents (for example, inhibitors of the c-Src kinase family such as 4-(6-chloro-2,3-methylenedioxyaniline)-7-[2-(4-methylpiperazin-l-yl)ethoxy]-5- tetrahydropyran-4-yloxyquinazoline (AZD0530) and N-(2-chloro-6-methylphenyl)-2-{6-[4-(2- hydroxyethyl)piperazin-l-yl]-2-methylpyrimidin-4-ylamino}thiazole-5-carboxamide (dasatinib, BMS-354825), and inhibitors of metalloproteinases such as marimastat and inhibitors of urokinase-type plasminogen activator receptor function);

[0661] (iv) inhibitors of growth factor function: for example, such inhibitors include growth factor antibodies and growth factor receptor antibodies (for example, the anti-erbB2 antibody trastuzumab [Herceptin®] and the anti-erbBl antibody nimotuzumab [TheraCimn®]); TM[and anti-ErbB1 antibody cetuximab [C225]); such inhibitors also include, for example, tyrosine kinase inhibitors, such as epidermal growth factor family inhibitors (e.g., EGFR family tyrosine kinase inhibitors, such as N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amine (gefitinib, ZD 1839), N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazoline-4-amine (erlotinib, OSI-774) and 6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)quinazoline-4-amine (CI1033) and erbB2 tyrosine kinase inhibitors, such as lapatinib), inhibitors of the hepatocyte growth factor family, inhibitors of the platelet-derived growth factor family (such as imatinib), inhibitors of serine / threonine kinases (e.g., Ras / Raf signaling inhibitors, such as farnesyltransferase inhibitors, such as sorafenib (BAY 43-9006)), and MEK and / or Akt kinase-mediated cell signaling inhibitors;

[0662] (v) Anti-angiogenic agents, such as those that inhibit the action of vascular endothelial growth factor [e.g., the anti-vascular endothelial growth factor antibody bevacizumab (Avastin)]. TM ) and VEGF receptor tyrosine kinase inhibitors (such as 4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline (ZD6474; Example 2 in WO 01 / 32651), 4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidine-1-ylpropoxy)quinazoline (AZD2171; Example 240 in WO 00 / 47212), vatalani (PTK787; WO 98 / 35985) and SU11248 (sunitinib; WO 01 / 60814), as well as compounds that act through other mechanisms (e.g., linolamine, integrin ανβ3 function inhibitors and angiogenesis inhibitors)];

[0663] (vi) Vascular damage agents, such as cobustatin A4 and compounds disclosed in international patent applications WO 99 / 02166, WO 00 / 40529, WO 00 / 41669, WO 01 / 92224, WO 02 / 04434 and WO 02 / 08213;

[0664] (vii) Antisense therapy, such as the antisense drug ISIS2503;

[0665] (viii) gene therapy approaches, including approaches to replace aberrant genes such as aberrant p53 or aberrant BRCA1 or BRCA2, GDEPT (gene-directed enzyme prodrug therapy) approaches such as those using cytosine deaminase, thymidine kinase or a bacterial nitroreductase enzyme, and approaches to increase patient tolerance to chemotherapy or radiotherapy (such as multi-drug resistance gene therapy); and

[0666] (ix) immunotherapy approaches, including approaches to increase immunogenicity of patient tumour cells in vivo and ex vivo (such as transfection with cytokines such as interleukin 2, interleukin 4, or granulocyte-macrophage colony stimulating factor), approaches to decrease T-cell anergy, approaches using transfected immune cells such as cytokine-transfected dendritic cells, approaches using cytokine-transfected tumour cell lines, and approaches using anti-idiotypic antibodies.

[0667] Examples

[0668] For illustrative purposes, the following examples are included. It is to be understood, however, that the examples do not limit the present disclosure and are merely intended to suggest a method of practicing the present disclosure. One skilled in the art will recognize that the chemical reactions described can readily be adapted to prepare a number of other compounds of the present disclosure, and alternative methods for preparing compounds of the present disclosure are deemed to be within the scope of the present disclosure. For example, non-exemplified compounds according to the present disclosure can be synthesized by a straightforward modification of the methods described, for example, by appropriately protecting interfering groups, by using other appropriate reagents other than those described, and / or by making routine modifications of reaction conditions. Alternatively, other reactions disclosed herein or known in the art will be recognized as being applicable to the preparation of other compounds of the present disclosure.

[0669] For illustrative purposes, the following shows general synthetic schemes for preparing compounds of the present disclosure, as well as key intermediates. One skilled in the art will understand that other synthetic schemes can be used to synthesize the compounds of the present disclosure. Although specific starting materials and reagents are depicted in the general schemes and discussed below, other starting materials and reagents can be readily substituted to provide a variety of derivatives and / or reaction conditions. In addition, many of the compounds prepared by the methods described below can be further modified in accordance with the present disclosure using conventional chemistry well known to those skilled in the art.

[0670] Example 1 : General Chemical Synthesis Scheme 1

[0671]

[0672] wherein,

[0673] each W is independently selected from O, S, and NH;

[0674] each Y is independently selected from O, S, and NH;

[0675] each n is independently 1, 2, or 3;

[0676] each m is 0 or 1;

[0677] each p is 1 or 2.

[0678] General procedure

[0679] General procedure 1: Heat an ethanolic solution of compound S1-1 to 60-80 °C. Add compound S1-2 to the mixture. After the reaction is complete, cool the mixture to room temperature, then wash and dry to obtain the selectively mono-substituted intermediate S1-3.

[0680] General procedure 2: Add a mixture of intermediate S1-3, compound S1-4 (with ACN and / or acetic acid), and BHT to a sealed vessel. Heat the mixture at 60-80 °C for 12-24 hours, then cool to room temperature. Purify the mixture by column chromatography to obtain compound S1-5.

[0681] Example 1.1: Synthesis of compound 1

[0682]

[0683] Step 1: To a vessel, add compound 1-4-1 (280 mg, 1.0 mmol), anhydrous DCM (6 mL), and compound 1-4-2 (104 mg, 1.1 mmol) sequentially at room temperature. Stir the mixture for 5 minutes, then cool in an ice bath. Add triethylamine (211 mg, 2.1 mmol) to the mixture. Allow the mixture to warm to room temperature, and stir at the same temperature for 5 hours. Thin layer chromatography (TLC) (eluent: petroleum ether / DCM = 3 / 1, KMnO4staining) indicates that the reaction is substantially complete. Purify the mixture by silica gel column chromatography (eluent: petroleum ether with 0-33% DCM, v / v), and concentrate under reduced pressure to obtain compound 11-4 as a colorless oil (220 mg, 65% yield).

[0684] Synthesis of compound 1-4 1H NMR: (400 MHz, Chloroform-d) δ 6.33 (dd, J = 17.3, 1.6 Hz, 1H), 6.05 (dd, J = 17.4, 10.4 Hz, 1H), 5.74 (dd, J = 10.4, 1.5 Hz, 1H), 5.34 - 5.20 (m, 2H), 4.08 (t, J = 6.8 Hz, 2H), 1.94 (q, J = 6.4 Hz, 4H), 1.59 (p, J = 6.8 Hz, 2H), 1.22 (q, J = 8.0, 5.5 Hz, 24H), 0.81 (t, J = 6.7 Hz, 3H).

[0685] Step 2: A solution of compound 1-1 (9.46 g, 65.13 mmol) in ethanol (600 mL) was heated at 65 °C (internal temperature) and compound 1-2 (2.0 g, 12.80 mmol) was added. The mixture was heated at 70-75 °C (external temperature) overnight. The mixture was cooled to room temperature and then concentrated under reduced pressure. The residue was diluted with DCM (500 mL) and to this was added brine (400 mL) under stirring. The mixture was heated at 35 °C (internal temperature) and stirred for 10 min, then allowed to settle and separate. The aqueous layer was extracted again with DCM (500 mL) and this was repeated 4-5 times. TLC (eluent: DCM / MeOH = 5:1 with 3 drops of NH4OH) indicated completion of extraction. The combined organic layers were dried over Na2S04and concentrated under reduced pressure to give compound 1-3 (3.47 g, yield 97%).

[0686] Step 3: Compound 1-3 (80 mg, 0.27 mmol) was taken in a 4 mL vessel and to this was added compound 1-4 (308 mg, 0.95 mmol) and BHT (5 mg) under N2atmosphere. The mixture was heated at 70 °C for 48 h. TLC indicated completion of reaction. The mixture was purified by silica gel column chromatography (eluent: DCM with 0-10% methanol, v / v) and concentrated under reduced pressure to give compound 1 (250 mg, yield 74%).

[0687] Compound 1 1 H NMR: (400 MHz, Chloroform-d) δ 5.44 - 5.26 (m, 5H), 4.05 (q, J = 6.4 Hz, 6H), 3.58 (dd, J = 6.9, 3.4 Hz, 1H), 2.97 - 2.83 (m, 1H), 2.72 (dt, J = 24.8, 7.0 Hz, 5H), 2.57 (dt, J = 14.3, 7.3 Hz, 1H), 2.01 (q, J = 6.4 Hz, 12H), 1.61 (t, J = 7.0 Hz, 10H), 1.28 (q, J = 8.2, 4.7 Hz, 91H), 0.87 (t, J = 6.6 Hz, 14H).

[0688] Example 1.2: Synthesis of compound 2

[0689]

[0690] Step 1: To a vessel was added anhydrous DCM (200 mL), NaHC03(7.43 g, 88.5 mmol), compound 2-4-1 (9.6 g, 35.7 mmol) and compound 2-4-2 (4 g, 44.2 mmol) in that order under ice bath. The mixture was allowed to warm to room temperature and stirred at the same temperature for 2 h. TLC (I2staining) indicated the completion of the reaction. The reaction was quenched with water (200 mL) and stirred for 10 min. The organic layer was washed with saturated aqueous NaHC03(200 mL) twice and then with water (200 mL). The organic layer was dried and concentrated to get crude compound 2-4 (11.4 g, 87%).

[0691] Synthesis of compound 2-4 1 H NMR: (400 MHz, Chloroform-d) δ 6.29 (dd, J = 17.0, 1.5 Hz, 1H), 6.10 (dd, J = 17.0, 10.3 Hz, 1H), 5.68 - 5.48 (m, 2H), 3.39 - 3.31 (m, 2H), 1.56 (p, J = 7.1 Hz, 2H), 1.28 (s, 29H), 0.90 (t, J = 6.7 Hz, 3H).

[0692] Step 2: A solution of compound 2-1 (9.46 g, 65.13 mmol) in ethanol (600 mL) was heated at 65 °C (internal temperature) and compound 2-2 (2.0 g, 12.97 mmol) was added. The mixture was heated at 70-75 °C (external temperature) overnight. The mixture was cooled to room temperature and then concentrated under reduced pressure. The residue was diluted with DCM (500 mL) and to this was added brine (400 mL) under stirring. The mixture was heated at 35 °C (internal temperature) and stirred for 10 min and then allowed to settle and separate. The aqueous layer was again extracted with DCM (500 mL) and repeated 4-5 times. TLC (eluent: DCM / MeOH = 5:1 with 3 drops of NH4OH) indicated the completion of the extraction. The combined organic layer was dried over Na2S04and concentrated under reduced pressure to get compound 2-3 (3.4 g, yield 95%).

[0693] Step 3: To compound 2-3 (100 mg, 0.33 mmol) was added compound 2-4 (384 mg, 1.19 mmol) and BHT (5 mg). The mixture was heated for 48 h. TLC indicated the completion of the reaction. The mixture was diluted with DCM (10 mL) and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: DCM with 0-10% methanol, v / v) and concentrated under reduced pressure to afford compound 2 as a colorless oil (140 mg, yield 45%).

[0694] Compound 2 1 H NMR: (400 MHz, Chloroform-d) δ 5.88-5.71 (m, 1H), 5.49-5.25 (m, 6H), 5.05-4.81 (m, 2H), 4.05 (q, J = 6.4 Hz, 7H), 3.58 (td, J = 7.0, 3.6 Hz, 1H), 2.90 (dt, J = 14.5, 7.4 Hz, 1H), 2.73 (dt, J = 25.2, 6.9 Hz, 6H), 2.57 (dt, J = 14.4, 7.3 Hz, 2H), 2.43 (q, J = 7.0, 6.4 Hz, 13H), 2.36-1.91 (m, 27H), 1.61 (t, J = 7.1 Hz, 12H), 1.30 (ddd, J = 17.8, 10.8, 5.4 Hz, 96H), 0.88 (t, J = 6.7 Hz, 12H).

[0695] Example 1.3: Synthesis of compound 3

[0696]

[0697] Step 1: A solution of compound 3-1 (9.46 g, 65.13 mmol) in ethanol (600 mL) was heated at 65 °C (internal temperature) and compound 3-2 (2.2 g, 11.94 mmol) was added. The mixture was heated at 70-75 °C (external temperature) overnight. The mixture was cooled to room temperature and then concentrated under reduced pressure. The residue was diluted with DCM (500 mL) and to this was added brine (400 mL) under stirring. The mixture was heated at 35 °C (internal temperature) and stirred for 10 min, then allowed to settle and separate. The aqueous layer was extracted again with DCM (500 mL) and repeated 4-5 times. TLC (eluent: DCM / MeOH = 5:1 with 3 drops of NH4OH) indicated the completion of the extraction. The combined organic layers were dried over Na2S04and concentrated under reduced pressure to afford compound 3-3 (3.5 g, yield 97%).

[0698] Step 2: To compound 3-3 (100 mg, 0.30 mmol) was added compound 3-4 (350 mg, 1.08 mmol) and BHT (5 mg). The mixture was heated for 48 h. TLC indicated the reaction was complete. The mixture was diluted with DCM (10 mL) and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: DCM with 0-5% methanol, v / v) and concentrated under reduced pressure to afford compound 3 as a colorless oil (198 mg, 70% yield).

[0699] Synthesis of compound 3 1 H NMR: (400 MHz, Chloroform-d) δ 4.05 (q, J = 6.6 Hz, 6H), 3.58 (s, 1H), 2.91 (dt, J = 14.3, 7.5 Hz, 1H), 2.72 (dt, J = 23.3, 6.7 Hz, 7H), 2.42 (h, J = 6.4 Hz, 10H), 2.24 (d, J = 48.6 Hz, 6H), 1.68 - 1.52 (m, 9H), 1.25 (s, 112H), 0.88 (t, J = 6.8 Hz, 12H).

[0700] Example 1.4: Synthesis of compound 4

[0701]

[0702] Step 1: To a vessel was added anhydrous DCM (300 mL), NaHC03(11.14 g, 132.6 mmol), compound 4-4-1 (14.15 g, 76.3 mmol) and compound 4-4-2 (6.0 g, 66.3 mmol) sequentially in an ice bath. The mixture was allowed to warm to room temperature and stirred at the same temperature for 2 h. TLC (I2staining) indicated the reaction was complete. The reaction was quenched with water (300 mL) and stirred for 10 min. The organic layer was washed with saturated aqueous NaHC03(300 mL) twice and then water (300 mL). The organic layer was dried and concentrated to give crude compound 4-4 (8.4 g, 47%).

[0703] Synthesis of compound 4-4 1 H NMR: (400 MHz, Chloroform-d) δ 6.27 (d, J = 16.9 Hz, 1H), 6.09 (dd, J = 17.0, 10.2 Hz, 1H), 5.64 (t, J = 11.1 Hz, 2H), 3.32 (q, J = 6.8 Hz, 2H), 1.53 (p, J = 7.1 Hz, 2H), 1.26 (s, 24H), 0.88 (t, J = 6.6 Hz, 3H).

[0704] Step 2: A solution of compound 4-1 (1.145 g, 9.77 mmol) in ethanol (100 mL) was heated at 65 °C (internal temperature) and compound 4-2 (0.30 g, 1.63 mmol) was added. The mixture was heated at 70-75 °C (external temperature) overnight. The mixture was cooled to room temperature and then concentrated under reduced pressure. The residue was diluted with DCM (300 mL) and to this was added brine (300 mL) under stirring. The mixture was heated at 35 °C (internal temperature) and stirred for 10 min, then allowed to settle and separate. The extraction was repeated 3-4 times. TLC (eluent: DCM / MeOH = 5:1 with 2 drops of NH4OH) indicated that the extraction was complete. The combined organic layers were dried over Na2SO4and concentrated under reduced pressure to give compound 4-3 (0.47 g, 95% yield).

[0705] Step 3: Compound 4-3 (20 mg, 0.07 mmol) was taken in a 2 mL vessel to which was added compound 4-4 (61 mg, 0.25 mmol), BHT (15 mg) and acetic acid (3 μL). The mixture was heated at 80 °C for 105 h. TLC (eluent: DCM / MeOH = 10 / 1 with NH4OH) indicated that the reaction was substantially complete. The mixture was subjected to silica gel column chromatography (eluent: DCM with 1-1.3% methanol and 0.5% NH4OH, v / v) and concentrated under reduced pressure to give compound 4 (45 mg, 66% yield).

[0706] Compound 4 1 H NMR: (400 MHz, Chloroform-d) δ 7.12-6.96 (m, 1H), 6.93 (t, J = 5.5 Hz, 1H), 3.67-3.48 (m, 1H), 3.10 (dq, J = 26.9, 6.6 Hz, 6H), 2.91-2.81 (m, 1H), 2.54 (d, J = 37.5 Hz, 9H), 2.44-2.22 (m, 11H), 2.16 (dd, J = 14.0, 6.4 Hz, 2H), 2.08-1.85 (m, 2H), 1.49-1.30 (m, 9H), 1.20 (d, J = 11.0 Hz, 80H), 0.81 (t, J = 6.8 Hz, 14H).

[0707] Example 1.5: Synthesis of compound 5

[0708]

[0709] Step 1: Compound 5-3 was prepared following the procedure of Example 1.4, Step 1.

[0710] Step 2: Compound 5-3 (19.6 mg, 0.07 mmol) was placed in a 2 mL vessel to which was added compound 5-4 (78.6 mg, 0.24 mmol) and BHT (17.2 mg). The mixture was heated at 70 °C for 67 h, then at 90 °C for 23 h. TLC (eluent: DCM / MeOH = 20 / 1) indicated that the reaction was essentially complete. The mixture was purified by flash column chromatography on silica gel (eluent: DCM with 0-5% methanol, v / v) and concentrated under reduced pressure to give compound 5 (40.8 mg, 49% yield).

[0711] Compound 5 1 H NMR: (400 MHz, Chloroform-d) δ 4.05 (q, J = 7.2 Hz, 5H), 3.62 (dt, J = 11.9, 6.5 Hz, 1H), 2.94 (dt, J = 14.2, 7.3 Hz, 2H), 2.84 (q, J = 6.6 Hz, 2H), 2.77 (t, J = 6.9 Hz, 5H), 2.65 - 2.58 (m, 1H), 2.47 (dq, J = 13.8, 7.5, 7.0 Hz, 7H), 2.40 - 2.17 (m, 2H), 1.71 - 1.53 (m, 6H), 1.25 (s, 106H), 0.88 (t, J = 6.8 Hz, 12H).

[0712] Example 1.6: Synthesis of compound 6

[0713]

[0714] Step 1: Compound 6-3 was prepared according to the method of Example 1.2, Step 2.

[0715] Step 2: Compound 6-3 (80 mg, 0.27 mmol) was placed in a 4 mL vessel to which was added compound 6-4 (310 mg, 0.96 mmol, prepared according to the method of Example 1.1, Step 1) and BHT (5 mg) under N2atmosphere. The mixture was heated at 70 °C for 48 h. TLC indicated that the reaction was complete. The mixture was purified by column chromatography on silica gel (eluent: DCM with 0-10% methanol, v / v) and concentrated under reduced pressure to give compound 6 (230 mg, 68% yield).

[0716] Compound 6 1H NMR: (400 MHz, Chloroform-d) δ 5.88 - 5.71 (m, 1H), 5.49 - 5.25 (m, 6H), 5.05 - 4.81 (m, 2H), 4.05 (q, J = 6.4 Hz, 7H), 3.58 (td, J = 7.0, 3.6 Hz, 1H), 2.90 (dt, J = 14.5, 7.4 Hz, 1H), 2.73 (dt, J = 25.2, 6.9 Hz, 6H), 2.57 (dt, J = 14.4, 7.3 Hz, 2H), 2.43 (q, J = 7.0, 6.4 Hz, 13H), 2.36 - 1.91 (m, 27H), 1.61 (t, J = 7.1 Hz, 12H), 1.30 (ddd, J = 17.8, 10.8, 5.4 Hz, 96H), 0.88 (t, J = 6.7 Hz, 12H).

[0717] Example 1.7: Synthesis of compound 7

[0718]

[0719] Step 1: A solution of compound 7-1 (1.44 g, 9.91 mmol) in ethanol (100 mL) was heated at 65 °C (internal temperature) and compound 7-2 (0.40 g, 1.75 mmol) was added. The mixture was heated at 70-75 °C (external temperature) overnight. The mixture was cooled to room temperature and then concentrated under reduced pressure. The residue was diluted with DCM (200 mL) and water (200 mL) was added to it under stirring. The mixture was heated at 35 °C (internal temperature) and stirred for 10 min, then allowed to settle and separate. The extraction was repeated 3-4 times. TLC (eluent: DCM / MeOH = 5:1 with 3 drops of NH4OH) indicated completion of extraction. The combined organic layer was dried over Na2S04and concentrated under reduced pressure to get compound 7-3 (0.43 g, yield 66%).

[0720] Step 2: Compound 7-3 (100 mg, 0.27 mmol) was taken in a 4 mL vessel and compound 7-4 (270 mg, 0.94 mmol, prepared as per the method of Example 1.1, Step 1) and BHT (5 mg) were added to it under N2atmosphere. The mixture was heated at 70 °C for 48 h. TLC indicated completion of reaction. The mixture was purified by silica gel column chromatography (eluent: DCM with 0-10% methanol, v / v) and concentrated under reduced pressure to get compound 7 (220 mg, yield 67%).

[0721] Synthesis of compound 7 1H NMR: (400 MHz, Chloroform-d) δ 5.45-5.21 (m, 5H), 4.05 (td, J = 6.8, 4.3 Hz, 6H), 3.89-3.74 (m, 1H), 3.53-3.34 (m, 4H), 2.98-2.82 (m, 1H), 2.74 (td, J = 7.0, 4.5 Hz, 6H), 2.62-2.35 (m, 16H), 2.00 (hept, J = 5.9, 5.2 Hz, 12H), 1.58 (dt, J = 18.9, 7.0 Hz, 12H ), 1.47-1.07 (m, 90H), 0.87 (t, J = 6.7 Hz, 13H).

[0722] Example 1.8: Synthesis of compound 8

[0723]

[0724] Step 1: Compound 8-3 was prepared according to the procedure of Example 1.1, Step 2.

[0725] Step 2: Compound 8-3 (100 mg, 0.33 mmol) was taken in a 4 mL vessel, to which compound 8-4 (350 mg, 1.09 mmol) and BHT (10 mg) were added under N2atmosphere. The mixture was heated at 70 °C for 48 h. TLC indicated completion of the reaction. The mixture was purified by silica gel column chromatography (eluent: DCM with 0-10% methanol, v / v) and concentrated under reduced pressure to obtain compound 8 (230 mg, 55% yield).

[0726] Compound 8 1 H NMR: (400 MHz, Chloroform-d) δ 5.80 (ddt, J = 16.9, 10.2, 6.7 Hz, 1H), 5.46-5.23 (m, 13H), 5.05-4.88 (m, 2H), 4.17-3.94 (m, 6H), 3.58 (ddt, J = 10.4, 6.9, 3.4 Hz, 1H), 3.02-2.83 (m, 1H), 2.82-2.65 (m, 12H), 2.56 (q, J = 7.4, 6.8 Hz, 2H), 2.51-2.11 (m, 21H), 2.05 (q, J = 6.7 Hz, 16H), 1.61 (dd, J = 10.6, 4.3 Hz, 12H), 1.49-1.10 (m, 71H), 0.89 (t, J = 6.8 Hz, 12H).

[0727] Example 1.9: Synthesis of compound 9

[0728]

[0729] Step 1 : Compound 9-3 was prepared following the procedure of Example 1.2, Step 2.

[0730] Step 2: Compound 9-3 (100 mg, 0.33 mmol) was taken in a 4 mL vessel, to which compound 9-4 (360 mg, 1.12 mmol) and BHT (10 mg) were added under N2atmosphere. The mixture was heated at 70 °C for 48 h. TLC indicated completion of the reaction. The mixture was purified by silica gel column chromatography (eluent: DCM with 0-10% methanol, v / v) and concentrated under reduced pressure to obtain compound 9 (221 mg, 56% yield).

[0731] Compound 9 1 H NMR: (400 MHz, Chloroform-d) δ 5.45 - 5.26 (m, 12H), 4.05 (q, J=7.1 Hz, 6H), 3.60 (s, 1H), 2.93 (dt, J=13.9, 7.5 Hz, 3H), 2.75 (dt, J=17.7, 6.7 Hz, 15H), 2.54 - 2.20 (m, 14H), 2.05 (q, J=6.8 Hz, 14H), 1.88 (s, 5H), 1.62 (p, J=6.7 Hz, 9H), 1.46 - 1.19 (m, 72H), 0.88 (td, J=6.8, 4.5 Hz, 14H).

[0732] Example 1.10: Synthesis of compound 10

[0733]

[0734] Step 1 : Compound 10-3 was prepared following the procedure of Example 1.7, Step 1.

[0735] Step 2: Compound 10-3 (120 mg, 0.32 mmol) was taken in a 4 mL vessel, to which compound 10-4 (350 mg, 1.09 mmol) and BHT (10 mg) were added under N2atmosphere. The mixture was heated at 70 °C for 48 h. TLC indicated completion of the reaction. The mixture was purified by silica gel column chromatography (eluent: DCM with 0-10% methanol, v / v) and concentrated under reduced pressure to obtain compound 10 (205 mg, 50% yield).

[0736] Compound 10 1H NMR: (400 MHz, Chloroform-d) δ 5.48-5.24 (m, 14 H), 4.06 (q, J = 7.2 Hz, 6 H), 3.79 (dq, J = 9.9, 5.1 Hz, 1 H), 2.87 (dq, J = 17.0, 9.7, 8.5 Hz, 3 H), 2.77 (t, J = 6.4 Hz, 7 H), 2.68 (dt, J = 13.0, 6.3 Hz, 5 H), 2.44 (dt, J = 13.2, 7.7 Hz, 7 H), 2.29 (d, J = 6.9 Hz, 4 H), 2.13-2.03 (m, 12 H), 1.59 (dt, J = 17.9, 6.9 Hz, 18 H), 1.43-1.20 (m, 88 H), 0.88 (td, J = 6.8, 4.1 Hz, 16 H).

[0737] Example 1.11: Synthesis of compound 11

[0738]

[0739] Step 1: To a solution of compound 11-4-1 (10 g, 57.1 mmol) and compound 11-4-2 (10.3 g, 59.9 mmol) in DCM (100 mL) was added DMAP (698 mg, 5.7 mmol). To the mixture was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 21.9 g, 114.2 mmol) portion-wise at below 10 °C under N2atmosphere. The mixture was allowed to warm to room temperature and stirred at the same temperature for 16 h. The reaction was quenched with saturated aqueous NaHC03solution (100 mL). The aqueous layer was extracted with ethyl acetate (200 mL). The combined organic layers were washed with 5% aqueous citric acid solution (100 mL) and brine (100 mL), then washed with brine (100 mL). The organic layer was dried over Na2S04and concentrated to give 18.5 g of crude compound 11-4-3. Compound 11-4-3 was used directly without further purification.

[0740] Synthesis of compound 11-4-3 1 H NMR: (400 MHz, DMSO-d6) δ 7.15 (t, J = 6.2 Hz, 1 H), 4.02 (t, J = 6.5 Hz, 2 H), 3.64 (d, J = 6.2 Hz, 2 H), 1.55 (t, J = 6.9 Hz, 2 H), 1.38 (s, 8 H), 1.34 (s, 2 H), 1.24 (s, 19 H), 0.85 (t, J = 6.5 Hz, 4 H).

[0741] Step 2: To a solution of compound 11-4-4 (5 g, 18.9 mmol) and compound 11-4-5 (1.9 g, 20.7 mmol) in DCM (50 mL) was added DMAP (698 mg, 5.7 mmol) under N2atmosphere at below 10 °C. To the mixture was added a solution of N,N-diisopropylethylamine (DIEA, 4.9 g, 37.8 mmol) in DCM (5 mL) dropwise at the same temperature. The mixture was allowed to warm to room temperature and stirred at the same temperature for 3 h. TLC indicated the starting material was consumed. The reaction was quenched with saturated aqueous NaHC03solution (50 mL). The aqueous layer was extracted with ethyl acetate (100 mL). The combined organic layers were washed with 5% aqueous citric acid solution (50 mL) and brine (50 mL), then washed with brine (100 mL). The organic layer was dried over Na2S04and concentrated to give the crude product. The crude product was purified by silica gel column chromatography (eluent: petroleum ether and 20% ethyl acetate, v / v) and concentrated under reduced pressure to give compound 11-4 (4.4 g, 27% yield over 3 steps).

[0742] Compound 11-4-4 was prepared according to the following scheme: 1 H NMR: (400 MHz, DMSO-d6) δ 8.63 (s, 3H), 4.11 (t, J = 6.6 Hz, 2H), 3.73 (s, 2H), 1.57 (p, J = 6.7 Hz, 2H), 1.23 (s, 17H), 0.84 (t, J = 6.6 Hz, 3H).

[0743] Step 3: To a solution of compound 11-4-4 (5 g, 18.9 mmol) and compound 11-4-5 (1.9 g, 20.7 mmol) in DCM (50 mL) was added DMAP (698 mg, 5.7 mmol) under N2atmosphere at below 10 °C. To the mixture was added a solution of N,N-diisopropylethylamine (DIEA, 4.9 g, 37.8 mmol) in DCM (5 mL) dropwise at the same temperature. The mixture was allowed to warm to room temperature and stirred at the same temperature for 3 h. TLC indicated the starting material was consumed. The reaction was quenched with saturated aqueous NaHC03solution (50 mL). The aqueous layer was extracted with ethyl acetate (100 mL). The combined organic layers were washed with 5% aqueous citric acid solution (50 mL) and brine (50 mL), then washed with brine (100 mL). The organic layer was dried over Na2S04and concentrated to give the crude product. The crude product was purified by silica gel column chromatography (eluent: petroleum ether and 20% ethyl acetate, v / v) and concentrated under reduced pressure to give compound 11-4 (4.4 g, 27% yield over 3 steps).

[0744] Compound 11-4 was prepared according to the following scheme: 1H NMR: (400 MHz, DMSO-d6) δ 8.51 (t, J = 6.0 Hz, 1H), 6.29 (dd, J = 17.1, 10.2 Hz, 1H), 6.11 (dd, J = 17.1, 2.1 Hz, 1H), 5.63 (dd, J = 10.2, 2.1 Hz, 1H), 4.04 (t, J = 6.6 Hz, 2H), 3.90 (d, J = 5.9 Hz, 2H), 1.57 (s, 2H), 1.24 (s, 16H), 0.85 (t, J = 6.6 Hz, 3H).

[0745] Step 4: Compound 11-3 was prepared according to the procedure of Example 1.3, Step 1.

[0746] Step 5: Compound 11-3 (400 mg, 1.2 mmol), BHT (53.4 mg), and compound 11-4 (1.24 g, 4.4 mmol) were placed in a 2 mL vessel, to which acetic acid (14.6 mg) was added. The mixture was heated at 80 °C for 42 h. TLC (eluent: DCM / MeOH / NH4OH = 10 / 1 / 1) indicated that the reaction was essentially complete. The mixture was purified by silica gel flash column chromatography (eluent: DCM with 1.4-24% methanol and 0.5% NH4OH, v / v) and concentrated under reduced pressure to give compound 11 (390 mg, 27% yield).

[0747] Compound 11 1 H NMR: (400 MHz, Chloroform-d) δ 7.87 (t, J = 5.5 Hz, 1H), 7.68 (t, J = 5.5 Hz, 2H), 4.15 - 4.01 (m, 6H), 4.01 - 3.88 (m, 4H), 3.68 (dt, J = 17.8, 8.6 Hz, 1H), 3.03 - 2.91 (m, 1H), 2.74 (t, J = 6.0 Hz, 4H), 2.66 (dt, J = 14.1, 7.3 Hz, 2H), 2.59 - 2.39 (m, 9H), 2.40 - 2.19 (m, 5H), 1.81 - 1.66 (m, 4H), 1.62 (p, J = 6.9 Hz, 6H), 1.52 - 1.04 (m, 68H), 0.87 (q, J = 5.9, 5.5 Hz, 12H). HR-MS: [M+H] + = 1180.00680.

[0748] Example 1.12: Synthesis of compound 12

[0749]

[0750] Step 1: Compound 12-3 was prepared according to a procedure similar to Example 1.1, Step 2.

[0751] Step 2: Compound 12-3 (80 mg, 0.29 mmol) was taken in a 4 mL vessel, to which BHT (5 mg) and compound 12-4 (340 mg, 1.05 mmol, prepared according to the method of Example 1.1, Step 1) were added under N2atmosphere. The mixture was heated at 70 °C for 48 h. TLC indicated completion of the reaction. The mixture was purified by silica gel flash column chromatography (eluent: DCM with 0-10% methanol, v / v) and concentrated under reduced pressure to obtain compound 12 (295 mg, 81% yield).

[0752] Compound 12 1 H NMR: (400 MHz, Chloroform-d) δ 5.44 - 5.26 (m, 5H), 4.05 (q, J = 6.4 Hz, 6H), 3.58 (dd, J = 6.9, 3.4 Hz, 1H), 2.97 - 2.83 (m, 1H), 2.72 (dt, J = 24.8, 7.0 Hz, 5H), 2.57 (dt, J = 14.3, 7.3 Hz, 1H), 2.01 (q, J = 6.4 Hz, 12H), 1.61 (t, J = 7.0 Hz, 10H), 1.28 (q, J = 8.2, 4.7 Hz, 91H), 0.87 (t, J = 6.6 Hz, 14H). HR-MS: [M+H] + = 1240.15427.

[0753] Example 1.13: Synthesis of compound 13

[0754]

[0755] Step 1: Compound 13-3 was prepared according to the method of Example 1.3, Step 1.

[0756] Step 2: Compound 13-3 (80 mg, 0.24 mmol) was taken in a 4 mL vessel, to which BHT (5 mg) and compound 13-4 (308 mg, 0.95 mmol, prepared according to the method of Example 1.1, Step 1) were added under N2atmosphere. The mixture was heated at 70 °C for 48 h. TLC indicated completion of the reaction. The mixture was purified by silica gel flash column chromatography (eluent: DCM with 0-10% methanol, v / v) and concentrated under reduced pressure to obtain compound 13 (250 mg, 74% yield).

[0757] Compound 13 1H NMR: (400 MHz, Chloroform-d) δ 5.49-5.22 (m, 6H), 4.05 (q, J = 6.5 Hz, 6H), 3.58 (td, J = 6.8, 3.4 Hz, 1H), 2.91 (dt, J = 14.3, 7.5 Hz, 1H), 2.72 (dt, J = 23.7, 6.9 Hz, 6H), 2.43 (q, J = 7.6 Hz, 13H), 2.34-2.12 (m, 6H), 2.00 (dh, J = 11.6, 6.5 Hz, 11H), 1.63 (s, 10H), 1.30 (dt, J = 16.3, 9.4 Hz, 91H), 0.88 (t, J = 6.7 Hz, 13H). [M+H] + = 1297.21294.

[0758] Example 1.14: Synthesis of compound 14

[0759]

[0760] Step 1: Compound 14-3 was prepared according to a similar procedure as in Example 1.3, Step 1.

[0761] Step 2: Compound 14-3 (80 mg, 0.22 mmol) was taken in a 4 mL vessel, to which BHT (5 mg) and compound 14-4 (259 mg, 0.80 mmol, prepared according to the procedure in Example 1.1, Step 1) were added under N2atmosphere. The mixture was heated at 70 °C for 48 h. TLC indicated completion of the reaction. The mixture was purified by silica gel flash column chromatography (eluent: DCM with 0-10% methanol, v / v) and concentrated under reduced pressure to obtain compound 14 (220 mg, 74% yield).

[0762] Compound 14 1 H NMR: (400 MHz, Chloroform-d) δ 5.45-5.23 (m, 6H), 4.05 (q, J = 6.5 Hz, 6H), 3.58 (td, J = 6.7, 3.4 Hz, 1H), 2.90 (dt, J = 14.3, 7.5 Hz, 1H), 2.80-2.36 (m, 19H), 2.33-1.87 (m, 20H), 1.60 (q, J = 7.0 Hz, 11H), 1.51-1.06 (m, 102H), 0.88 (t, J = 6.7 Hz, 14H). [M+H] + = 1325.24099.

[0763] Example 1.15: Synthesis of compound 15

[0764]

[0765] Step 1 : Compound 15-3 was prepared according to a similar procedure as in Example 1.3, Step 1.

[0766] Step 2: Compound 15-3 (100 mg, 0.26 mmol) was taken in a 4 mL vessel, to which BHT (5 mg) and compound 15-4 (301 mg, 0.93 mmol, prepared according to the procedure in Example 1.1, Step 1) were added under N2atmosphere. The mixture was heated at 70 °C for 48 h. TLC indicated the completion of the reaction. The mixture was purified by silica gel flash column chromatography (eluent: DCM with 0-10% methanol, v / v) and concentrated under reduced pressure to afford compound 15 (250 mg, 69% yield).

[0767] Characterization of compound 15 1 H NMR: (400 MHz, Chloroform-d) δ 5.49 - 5.12 (m, 5H), 4.05 (q, J = 6.6 Hz, 6H), 3.58 (td, J = 6.9, 3.5 Hz, 1H), 2.91 (dt, J = 14.3, 7.5 Hz, 1H), 2.72 (dt, J = 23.2, 6.8 Hz, 6H), 2.64 - 2.52 (m, 2H), 2.52 - 2.19 (m, 17H), 2.01 (q, J = 6.4 Hz, 14H), 1.61 (t, J = 7.0 Hz, 11H), 1.28 (dd, J = 18.7, 7.3 Hz, 99H), 0.88 (t, J = 6.7 Hz, 13H). [M+H] + = 1297.21294.

[0768] Example 1.16: Synthesis of compound 16

[0769]

[0770] Step 1 : Compound 16-3 was prepared according to a similar procedure as in Example 1.3, Step 1.

[0771] Step 2: Compound 16-3 (100 mg, 0.24 mmol) was taken in a 4 mL vessel, to which BHT (5 mg) and compound 16-4 (280 mg, 0.87 mmol, prepared according to the procedure in Example 1.1, Step 1) were added under N2atmosphere. The mixture was heated at 70 °C for 48 h. TLC indicated the completion of the reaction. The mixture was purified by silica gel flash column chromatography (eluent: DCM with 0-10% methanol, v / v) and concentrated under reduced pressure to afford compound 16 (197 mg, 59% yield).

[0772] Characterization of compound 16 1H NMR: (400 MHz, Chloroform-d) δ 5.48-5.23 (m, 5H), 4.05 (q, J = 6.6 Hz, 6H), 3.58 (td, J = 7.5, 7.1, 3.7 Hz, 1H), 2.91 (dt, J = 14.5, 7.5 Hz, 1H), 2.83-2.14 (m, 26H), 2.01 (q, J = 6.4 Hz, 12H), 1.80-1.53 (m, 10H), 1.28 (dd, J = 20.0, 7.6 Hz, 109H), 0.87 (t, J = 6.7 Hz, 14H). [M+H] + = 1381.31640.

[0773] Example 1.17: Synthesis of compound 17

[0774]

[0775] Step 1: Compound 17-3 was prepared following a similar procedure to Step 1 of Example 1.3.

[0776] Step 2: Compound 17-3 (30 mg, 0.09 mmol) was taken in a vial to which BHT (1 mg), compound 17-4 (137 mg, 0.32 mmol) and ACN (0.3 mL) were added. The mixture was purged and decanted thrice under N2atmosphere. The mixture was heated at 70 °C for 2-3 days. TLC indicated completion of the reaction. The mixture was purified by silica gel column chromatography (eluent: DCM with 0-3% methanol, v / v) and concentrated under reduced pressure to obtain compound 17 (110 mg, 76% yield).

[0777] Compound 17 1 H NMR: (400 MHz, Chloroform-d) δ 4.37 (q, J = 6.3 Hz, 6H), 3.57 (s, 1H), 2.91 (dt, J = 14.3, 7.4 Hz, 1H), 2.83-2.63 (m, 5H), 2.63-2.08 (m, 25H), 1.25 (s, 23H), 0.87 (t, J = 6.8 Hz, 3H).

[0778] Example 1.18: Synthesis of compound 18

[0779]

[0780] Step 1: Compound 18-3 was prepared following a similar procedure to Step 1 of Example 1.3.

[0781] Step 2: Compound 18-3 (30 mg, 0.09 mmol) was placed in a vial, and BHT (1 mg), compound 18-4 (170 mg, 0.33 mmol), and ACN (0.3 mL) were added. The mixture was subjected to three purgings and decantations under a nitrogen atmosphere. The mixture was heated at 70 °C for 2–3 days. TLC indicated the reaction was complete. The mixture was purified by silica gel column chromatography (eluent: DCM with 0–3% methanol, v / v) and concentrated under reduced pressure to give compound 18 (100 mg, 58% yield).

[0782] Compound 18 1 ¹H NMR: (400MHz, chloroform-d) δ 4.37 (q, J = 6.4Hz, 6H), 3.57 (s, 1H), 2.91 (dt, J = 14.0, 7.4Hz, 1H), 2.78–2.66 (m, 5H), 2.56–2.15 (m, 25H), 1.25 (s, 24H), 0.88–0.84 (m, 3H).

[0783] Example 1.19: Synthesis of Compound 19

[0784]

[0785] Step 1: Under a nitrogen atmosphere, compound 19-4-1 (1.6 g, 7.3 mmol) and triethylamine (1.86 g, 18.4 mmol) were added to DCM (20 mL). The mixture was cooled to 5 °C. Compound 19-4-2 (1.0 g, 6.1 mmol) was added dropwise. The mixture was warmed to room temperature and stirred at the same temperature for 16 hours. TLC (elution: DCM) indicated product formation. The mixture was purified by silica gel column chromatography to give compound 19-4 (0.7 g, 37% yield).

[0786] Compound 19-4 1 H NMR: (400MHz, chloroform-d) δ6.51(dd,J=16.5,9.9Hz,1H),6.23(d,J=16.6Hz,1H),5.93(d,J=9.9Hz,1H) ,4.44(s,1H),3.00(q,J=6.8Hz,2H),1.53(t,J=7.2Hz,2H),1.25(s,24H),0.87(t,J=6.7Hz,3H).

[0787] Step 2: Prepare compound 19-3 according to the method in step 2 of Example 1.1.

[0788] Step 3: To a solution of compound 19-3 (30 mg, 0.096 mmol) and compound 19-4 (117.3 mg, 0.386 mmol) in ACN (0.2 mL) was added BHT (15 mg) and acetic acid (7 mg) at room temperature under N2 atmosphere. The mixture was heated at 75 °C for 40 h. TLC (eluent: DCM / MeOH = 10 / 1) indicated that the starting material was largely consumed. The mixture was purified by silica gel column chromatography and concentrated under reduced pressure to give compound 19 (67 mg, 55% yield).

[0789] Synthesis of compound 19 1 H NMR: (400 MHz, Chloroform-d) δ 5.78 (t, J = 6.1 Hz, 1H), 3.65 (td, J = 8.7, 7.5, 4.0 Hz, 1H), 3.44 - 3.26 (m, 2H), 3.19 (dt, J = 19.3, 5.2 Hz, 7H), 3.07 (q, J = 7.1 Hz, 7H), 2.99 - 2.81 (m, 4H), 2.75 - 2.63 (m, 5H), 2.55 (dt, J = 28.9, 7.4 Hz, 3H), 2.46 - 2.20 (m, 2H), 2.00 (d, J = 11.9 Hz, 5H), 1.58 (dt, J = 12.5, 4.7 Hz, 6H), 1.26 (d, J = 5.9 Hz, 81H), 0.94 - 0.73 (m, 12H). [M+H] + = 1212.00789.

[0790] Example 1.20: Synthesis of compound 20

[0791]

[0792] Step 1: To methanol (3 mL) was added compound 20-4-1 (1 g, 4.38 mmol) and compound 20-4-2 (2.8 g, 43.8 mmol) under N2 atmosphere. The mixture was stirred at room temperature for 40 h. TLC (eluent: DCM / MeOH = 10 / 1) indicated that the starting material was not completely consumed. Compound 20-4-2 (2.8 g, 43.8 mmol) was added to the mixture. The mixture was heated at 60 °C for 16 h. TLC (eluent: PE) indicated that the starting material was consumed. The mixture was concentrated and diluted with water (10 mL). The mixture was stirred for 30 min and filtered. The residue was washed and dried to give compound 20-4-3 (540 mg).

[0793] Synthesis of compound 20-4-3 1H NMR: (400 MHz, Chloroform-d) δ 6.82 (s, 1H), 3.90 (s, 2H), 2.14 (t, J = 7.6 Hz, 2H), 1.62 (t, J = 7.3 Hz, 2H), 1.26 (d, J = 10.9 Hz, 18H), 0.87 (t, J = 6.7 Hz, 3H). HRMS: [M+H] + 229.22922.

[0794] Step 2: To DCM (20 mL) was added compound 20-4-3 (0.54 g, 2.4 mmol) and DIEA (0.62 g, 4.8 mmol) under N2atmosphere. The mixture was cooled to 5 °C. Compound 20-4-4 (0.26 g, 2.9 mmol) was added dropwise at 5-10 °C. The mixture was allowed to warm to room temperature and stirred at the same temperature for 2 h. TLC (eluent: DCM / MeOH = 10 / 1) indicated the starting material was consumed. The mixture was purified by silica gel column chromatography to give the crude product. The crude product was added to water (10 mL), stirred for 30 min, and dried to give compound 20-4 (510 mg). HRMS: [M+H] + 283.24978.

[0795] Step 3: Compound 20-3 was prepared according to the method of Example 1.1, Step 2.

[0796] Step 1: To a solution of compound 20-3 (30 mg, 0.096 mmol) and compound 20-4 (101.2 mg, 0.36 mmol, prepared according to the method of Example 1.16, Steps 1-2) in ACN (0.2 mL) was added BHT (15 mg) and acetic acid (10 mg) at room temperature under N2atmosphere. The mixture was heated at 80 °C for 16 h. TLC (eluent: DCM / MeOH = 10 / 1 + 0.5% NH4OH H) indicated the starting material was largely consumed. The mixture was purified by silica gel column chromatography and concentrated under reduced pressure to give compound 20 (50 mg, 43% yield).

[0797] Compound 20 1 H NMR: (400 MHz, Chloroform-d) δ 6.82 (s, 1H), 3.90 (s, 2H), 2.14 (t, J = 7.6 Hz, 2H), 1.62 (t, J = 7.3 Hz, 2H), 1.26 (d, J = 10.9 Hz, 18H), 0.87 (t, J = 6.7 Hz, 3H). HRMS: [M+H] + 1149.01684.

[0798] Other compounds of the present disclosure are synthesized using similar methods, but with modified reaction conditions and different starting materials.

[0799] Example 1.21: Synthesis of compounds 21-39

[0800] Compounds 21-39 are synthesized by the methods shown in the following scheme.

[0801]

[0802] wherein,

[0803] each W is independently selected from O, S, and NH;

[0804] each Y is independently selected from O, S, and NH;

[0805] each n is independently 1, 2, or 3;

[0806] each m is 0 or 1;

[0807] each p is 1 or 2.

[0808] General procedure

[0809] A mixture of compound S2-1, S2-2, 2,6-di-tert-butyl-4-methylphenol (BHT), acetonitrile (ACN), and / or acetic acid is added to a sealed vessel. The mixture is heated at 60-80 °C for 18-36 hours. The mixture is then cooled to room temperature and purified by column chromatography to yield the desired compound S2-3.

[0810] The following compounds are synthesized using modified reaction conditions and different starting materials.

[0811]

[0812]

[0813]

[0814]

[0815]

[0816]

[0817] Example 2: Preparation of mRNA

[0818] mRNA can be prepared in vitro by any method known in the art. For example, the mRNA used in the illustrative examples can be prepared using the TranscriptAid T7 High Yield Transcription Kit (Thermo K0441). In vitro transcription is performed using the TranscriptAid T7 High Yield Transcription Kit using linear double stranded DNA as a template to generate the mRNA of interest, with the addition of a proportion of pseudouridine and capping reagents. In vitro transcription conditions: reactions are set up according to the kit instructions, and are incubated at 37°C for 0.5-2 hours, the transcription is digested with DNase for 30 minutes, and the transcription is purified using the Monarch RNA Cleanup kit (NEBT2040L).

[0819] Exemplary mRNA constructs are shown in Table 7 below. Each construct also comprises a signal peptide coding sequence operably linked to the 5’ end of the construct. The signal peptide can be the F7 signal peptide (SEQ ID NO: 4), which is encoded by the sequence of SEQ ID NO: 5, or by the sequence of SEQ ID NO: 722. The signal peptide can be the IL15Ra signal peptide (SEQ ID NO: 2), which is encoded by the sequence of SEQ ID NO: 3.

[0820] Table 7. Nucleic acid descriptions and sequences

[0821]

[0822]

[0823] Example 3: In vitro expression test

[0824] Test mRNA was transfected into HEK293T cells by lipofectamine 2000 (Thermo Fisher) and 3 pg RNA was added per 5 x 10 4 cells. After 24 hours of incubation, the supernatant and cell pellet were collected.

[0825] The collected supernatant was tested using the IL15 detection kit (R&D) to assess the concentration of IL15. The procedure was followed according to the kit instructions. Based on the concentration of encoded IL15, the concentration of the fusion protein encoded by the test mRNA was calculated.

[0826] The precipitated cells were lysed with RIPA lysis buffer. Western Blotting (WB) was performed to evaluate the concentration of IL15. The method is described as follows: the total protein amount was quantified by BCA test and the loading sample was prepared to load 12 pg of total protein per well on SDS-PAGE, followed by standard gel electrophoresis procedure; after electrophoresis, the gel was transferred to the membrane using eBlot standard procedure; after transfer, the membrane was blocked with 5% skim milk at room temperature for 1 hour; the membrane was washed with PBST buffer for 3 times (5 minutes each time); the membrane was incubated with 5% skim milk diluted primary antibody rabbit anti-IL15 antibody at room temperature for 1 hour; after washing, the membrane was incubated with secondary antibody rabbit antibody at room temperature for 1 hour; after washing, the desired protein on the membrane was imaged.

[0827] The concentration of the fusion protein encoded by 001 to 014 is shown in Figure 1A .

[0828] The concentration of the fusion protein encoded by 011D43Fc, 011D56Fc, 011D61Fc, 011D65Fc and 011D106Fc is shown in Figure 1B .

[0829] The concentration of the fusion protein encoded by PDL1Ab_014, PDL1Ab_014D43, PDL1Ab_014D56, PDL1Ab_014D61, PDL1Ab_014D65, PDL1Ab_014D66, PDL1Ab_014D106, PDL1Ab_P2A_011 and PDL1Ab_P2A_014 is shown in Figure 1C .

[0830] Figure 1D The in vitro expression of 011, 011D43Fc, 011D56Fc, 011D61Fc, 011D65Fc and 011D106Fc is shown.

[0831] The in vitro expression of 001D1 to 001D108 is shown in Table 8.

[0832] Table 8. In vitro expression of test nucleic acids

[0833]

[0834]

[0835]

[0836]

[0837]

[0838] Example 4: In vitro affinity test

[0839] The affinity of the expression supernatant obtained in Example 3 to human IL15Rβ / γ (IL2Rβ / γ) was determined by ELISA. IL15Rβ / γ was coated in an enzyme plate with 50 ng per well in a carbonate buffer and incubated overnight at 4°C. The supernatant was discarded and the wells were washed 3 times with PBST buffer and 100 μL of PBS with 0.5% BSA was added and incubated at 37°C for 1 h to reduce non-specific adsorption of proteins. The supernatant was discarded and the wells were washed 3 times with PBST buffer. The cell culture stock was added and diluted with 0.5% BSA-PBS and then incubated at 37°C for 2 h. The supernatant was discarded and the coated wells were washed 3 times with PBST buffer and anti-human IgG antibodies or HRP-labeled IL15 antibodies diluted with 0.5% BSA-PBS were added and then incubated at 37°C for 1 h. The supernatant was discarded and the wells were washed 3 times with PBST buffer. HRP color development substrate was added. The affinity was calculated by absorbance at 450 nm.

[0840] The affinity of the proteins encoded by 001 to 004 to IL15Rβ / γ is shown in Figure 2A and Figure 2B .

[0841] The results show that the proteins expressed in HEK293T cells from the respective test mRNA (001, 002, 003 and 004, respectively), which are indicated as “001 mRNA”, “002 mRNA”, “003 mRNA” and “004 mRNA”, have a higher binding affinity to human IL15Rβ / γ than the proteins with the same amino acid sequence, which are indicated as “001 PRO”, “002 PRO”, “003 PRO” and “004 PRO”, respectively, which were obtained from HEK293 cells and then affinity purified by a skilled and experienced external contract research organization using their own expression system.

[0842] The proteins expressed in HEK293T cells from the respective test mRNA also show a higher binding affinity to human IL15Rβ / γ than ALT-803 (“BM”, a pharmacological grade IL-15 / IL-15Rα complex fused to IgG1 Fc, wherein IL-15 is additionally mutated (N72D) to further increase the biological activity and agonism of the IL-2 and 15βγ receptors).

[0843] The affinity of the proteins encoded by 007 to 014 (i.e. STARNA15-7 to STARNA15-14) to IL15Rβ / γ is shown in Figure 2C .

[0844] The affinity of the proteins encoded by 011 (i.e., 15-11), 011D43Fc, 011D56Fc, 011D61Fc, 011D65Fc, and 011D106Fc to IL15Rβ / γ is shown in Table 9. Figure 2D

[0845] The affinity of the proteins encoded by PDL1Ab_014, PDL1Ab_014D43, PDL1Ab_014D56, PDL1Ab_014D61, PDL1Ab_014D65, PDL1Ab_014D66, PDL1Ab_014D106, PDL1Ab_P2A_011, and PDL1Ab_P2A_014 to IL15Rβ / γ is shown in Table 9. Figure 2E

[0846] 001D1 to 001D108 in vitro affinity to IL15Rβ / γ is shown in Table 9.

[0847] Table 9. Affinity of proteins encoded by test nucleic acids to IL15Rβ / γ

[0848]

[0849]

[0850]

[0851] Example 5: IL2Rβ-STAT5 signaling pathway activation test

[0852] The IL2Rβ-STAT5-LUC cell line was constructed by the following method:

[0853] 1. Repeat the STAT5 binding element (BE) sequence 6 times to obtain an enhanced STAT5 BE (TCTGGGAGTCTGAGACTCTGTGAATCTGGGAGTCTGAGACTCTGTGAA) and insert it into the 5' end sequence of the miniTATA box to construct a STAT5 promoter. Replace the STAT5 promoter with the luciferase promoter to construct a luciferase reporter gene lentiviral vector STAT5-LUC, which is activated by STAT5 signal conditions.

[0854] 2. Obtain the human IL2Rβ nucleic acid sequence (NM_000878.5) from NCBI to construct an overexpression lentiviral vector.

[0855] 3. Infect the TF-1 cell line with the STAT5-LUC lentiviral vector and the IL2Rβ lentiviral vector, respectively, and screen to obtain an IL2Rβ-STAT5-LUC stable cell line. ​​

[0856] Vector synthesis, lentivirus packaging, lentivirus infection and clone screening were all commissioned to professional companies. The IL2Rβ-STAT5 signal activation activity of nucleic acids was determined using the IL2Rβ-STAT5-LUC cell line.

[0857] The IL2Rβ-STAT5-LUC cell line was suspended using RPMI1640 complete medium, inoculated into a 96-well cell culture plate at 1.5x10 4 The cell supernatant obtained in Example 3 was added to the culture plate, and incubated in a 37°C and 5% CO2 incubator for 24 hours. 100 μL of cell lysate / luciferase substrate (Novizan) was added to the culture plate and shaken at room temperature for 10 minutes. 100 μL of the mixture was taken to a white ELISA plate to determine the chemiluminescence value.

[0858] The activation levels of the reporter cell lines of 001 to 014 are shown in Table 1. Figure 3A

[0859] 011, 011D43Fc, 011D56Fc, 011D61Fc, 011D65Fc and 011D106Fc Figure 3B

[0860] PDL1Ab_014, PDL1Ab_014D43, PDL1Ab_014D56, PDL1Ab_014D61, PDL1Ab_014D65, PDL1Ab_014D66, PDL1Ab_014D106, PDL1Ab_P2A_011 and PDL1Ab_P2A_014 Figure 3C

[0861] The activation levels of the reporter cell lines of 001D1 to 001D108 are shown in Table 10.

[0862] Table 10. Activation of STAT5 signaling pathway by nucleic acids

[0863]

[0864]

[0865]

[0866]

[0867] Example 6: PD-L1 affinity test of nucleic acids containing anti-PD-L1 antibody coding sequence

[0868] ​​​The affinity of the anti-PD-L1 antibody expressed in the supernatant obtained in Example 3 to human PD-L1 was detected by ELISA method. Human PD-L1 protein was coated in an enzyme-labeled plate at 50 ng / well with a carbonate buffer and incubated at 4°C overnight. The supernatant was discarded, the wells were washed with PBST buffer for 3 times, and 100 μL of PBS containing 0.5% BSA was added, incubated at 37°C for 1 h to reduce non-specific adsorption of proteins. Discard the supernatant, wash the coated wells with PBST buffer for 3 times. Add the cell culture stock solution and dilute with 0.5% BSA-PBS, then incubate at 37°C for 2 h. Discard the supernatant, wash the coated wells with PBST buffer for 3 times, and add HRP-labeled anti-human IgG antibody diluted with 0.5% BSA-PBS, then incubate at 37°C for 1 h. Discard the supernatant, wash the coated wells with PBST buffer for 3 times. Add HRP color developing substrate. The affinity was calculated by the absorbance at 450 nm.

[0869] The affinity of the protein encoded by PDL1Ab_014, PDL1Ab_014D43, PDL1Ab_014D56, PDL1Ab_014D61, PDL1Ab_014D65, PDL1Ab_014D66 and PDL1Ab_014D106 to PD-L1 is shown in Figure 4 . AtezoHLFc refers to a continuously expressed Fc fusion protein comprising, from N-terminus to C-terminus, an atezolizumab heavy chain variable region, a linker, an atezolizumab light chain variable region, a linker, and an atezolizumab Fc portion, wherein the amino acid sequence of the linker is GGGGSGGGGSGGGGS.

[0870] Example 7: Human PBMC activation assay

[0871] Human PBMCs were incubated with the cell supernatant obtained in Example 3, and the cells were suspended in 200 μL system and incubated in a 37°C, 5% CO2 incubator for 4 days. The cells were collected and labeled with cell surface marker antibodies (shown in the table below). 50 μL of cell suspension (about 1 x 10 5 cells) and 0.25 μL of staining antibody were added to each tube, then resuspended and mixed well, and incubated on ice for 30 minutes in the dark. The cells were washed with 1 mL of PBS buffer, centrifuged at room temperature for 3 minutes (400g), and the supernatant was discarded; repeat once. The cells were suspended with 200 mL of 4% paraformaldehyde, and the samples were analyzed by flow cytometry to obtain the level of in vitro activation of PBMC subpopulations by the test nucleic acid (in Figures 5A-5D ).

[0872] The results show that the nucleic acid expressed protein of the present disclosure exhibits comparable or higher activity in activating the proliferation of PBMC subpopulations compared to the benchmark (“BM”, i.e. ALT-803).

[0873] After PBMC activation, IL-6 levels in the cell supernatant were measured. Figure 6A (shown in) and IFNr (in Figure 6B The level of cell proliferation (as shown in the figure) was measured in the collected cells. Figure 6C (shown in) and STAT5 phosphorylation (in) Figure 6D (As shown in the diagram). All procedures were performed in accordance with the instructions for the corresponding kit.

[0874]

[0875]

[0876] Example 8: Preparation of lipid nanoparticle composition

[0877] Step 1: Dissolve the mRNA from Example 2 in citrate buffer (pH 4) and adjust the mRNA concentration to 0.2 mg / mL to obtain an aqueous layer.

[0878] Step 2: Dissolve the test compound, 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC), cholesterol, and DMG-PEG2000 in anhydrous ethanol at the desired molar fractions (shown in Table 11 below), and adjust the total lipid concentration to 10 mg / mL to obtain the organic layer.

[0879] Table 11. Mole fraction of each component in lipid particles

[0880]

[0881] Step 3: Using a microfluidic device ( Ignite TM The aqueous and organic layers were mixed at a total flow rate of 12 mL / min at a ratio of 3:1 (v / v). The mixture was diluted 10-fold with PBS buffer (pH 7.4). Ethanol was separated by tangential flow filtration (Repligen, TFF). The solution was concentrated to 0.5 mg / mL (mRNA concentration) and filtered through a 0.22 μm microporous filter to obtain lipid particles containing mRNA.

[0882] The average particle size and polymer dispersibility index (PDI) of exemplary lipid particles were measured using a Malvern Zetasizer with DLS.

[0883] Based on utilization Fluorescence measurements were performed by ThermoFisher Scientific to measure total RNA and free RNA concentrations. Encapsulation efficiency (EE) was calculated as follows:

[0884]

[0885] The total RNA concentration was measured by appropriately diluting the test lipid particles in lx TE buffer containing 0.2% Triton-X 100. The free RNA concentration was measured by appropriately diluting the test lipid particles in lx TE buffer.

[0886] Table 12. Physical properties of test lipid particles

[0887]

[0888]

[0889] Example 9: In vitro delivery assay

[0890] OVCAR3 cells in good growth condition were trypsinized with 0.25% trypsin. The culture medium was resuspended and mixed 5 times with a pipette. The cell suspension (20 μL) was placed on a counting plate and counted with a Countstar cell counter. The cells were seeded in a 96-well plate at 10,000 cells per well and 100 μL of culture medium. The cells were cultured overnight and transfected with test lipid particles containing 25 ng of FLuc mRNA. The test lipid particles and controls were diluted with culture medium to a total volume of 100 μL. The culture medium in the cell culture plate was removed and 100 μL of test lipid particles was added. Each test lipid particle transfected 2-3 wells. A blank control group of 3 wells was set up with 100 μL of culture medium added to each well. The culture plate was placed in a cell culture incubator. After 24 hours of cell transfection, ONEGLO TM reagent kit (Promega, item E7120) was thawed at room temperature for 1 hour and 5X CellTiter-Fluor TM reagent (20 μL per well) was added to the cell culture plate. The cell culture plate was placed in a shaker and incubated at 37°C at 300 rpm for 30 minutes. The fluorescence was measured by a microplate reader (EX 390 nm / EM 505 nm). Then, ONEGLO TM reagent (100 μL per well) was added at room temperature for 3 minutes and the luminescence was measured by a microplate reader.

[0891] Figure 7 The in vitro delivery properties of the test lipid particles are shown.

[0892] Example 10: In vivo immune activation assay

[0893] The lipid nanoparticle composition comprising Formulation 11 and test nucleic acid resulting from the method of Example 8 was diluted to 0.2 mg / mL for subsequent use.

[0894] C57 / B6J mice were administered 50 pL of the lipid nanoparticle composition via tail vein injection. Four days post administration, mice were sacrificed and EDTA anti-coagulated whole blood was collected. Blood samples were diluted 1:1 with PBS. An equal volume of Ficoll (ThermoFisher) was added to a 5 mL centrifuge tube. The blood sample was slowly added on top of the Ficoll. After centrifugation at 400 x g for 20 minutes at room temperature, the PBS surface and PBMCs on top of the Ficoll layer were collected into a new tube. Cells were washed with PBS, centrifuged at 400 x g for 5 minutes at 4°C, and the supernatant was discarded to obtain purified mouse PBMCs. Cells were collected and fluorescently labeled with cell surface marker antibodies (shown in the table below). 50 pL of cell suspension (about 1 x 10 5 cells) and 0.25 pL of staining antibodies were added per tube, then resuspended and incubated on ice for 30 minutes in the dark. Blood cells were washed with 1 mL of PBS buffer, centrifuged at 400 g for 3 minutes at room temperature, and the supernatant was discarded. This procedure was repeated once. Cells were suspended in 200 mL of 4% paraformaldehyde and samples were analyzed by flow cytometry.

[0895] Antibodies Brand Catalogue Ms CD3e FITC 145-2C11 100 μg BD 553061 Ms CD8a PE-Cy7 53-6.7 100 μg BD 552877 Ms NK-1.1 APC PK136 100 μg BD 550627

[0896] The level of in vivo immune activation activity of the lipid nanoparticle composition comprising the test nucleic acid is shown in Figures 8A-8C . Figures 8A-8C The lipid nanoparticle composition of the present disclosure was shown to activate immune cells and to elevate the proliferation of T cells, especially CD8+ T cells and NKT cells at comparable or higher levels compared to ALT-803 (BM).

[0897] Example 11: In vivo anti-cancer test

[0898] The lipid nanoparticle composition comprising Formulation 11 and the test nucleic acid was prepared according to the method of Example 8 for subsequent use.

[0899] Example 11.1 : B16F10 ectopic tumor model

[0900] A cell suspension containing 1 x 10 6 B16F10 cells was injected into the abdominal cavity of C57 / B6J mice to construct an ectopic tumor model. When the tumor size was 80 mm 3 -120 mm 3Mice were randomly assigned to groups on day 0 (referred to as day 0). On days 0, 8, and 15, each mouse received an intratumoral (IT) administration of 50 μL of a diluted lipid nanoparticle composition (1 mg / mL). Tumor size was measured three times weekly. Tumor growth curves following administration of the lipid nanoparticle composition containing 001 to 004 were plotted on [the graph / chart]. Figure 9A The results show that tumor growth was effectively inhibited. The body weight curves of mice after administration of the lipid nanoparticle composition containing 001 to 004 are shown in [the figure / image / etc.]. Figure 9B As shown in the figure, its weight is basically the same as that of a normal mouse.

[0901] Example 11.2: CT26 ectopic tumor model

[0902] Will contain 1×10 6 A suspension of CT26 cells was injected into the peritoneal cavity of BALB / c mice to construct an ectopic tumor model. When the tumor size was 80 mm... 3 -120mm 3 On day 0, mice were randomly assigned to groups. On days 0, 4, 7, 11, 14, and 18, each mouse was intratumorally administered 50 μL of a diluted lipid nanoparticle composition (1 mg / mL, 0.2 mg / mL, and 0.04 mg / mL, respectively). Tumor size was measured three times a week.

[0903] Tumor growth curves after application of a lipid nanoparticle composition containing 001 to 004 Figure 10A The figure shows the tumor growth inhibition rate (TGI). The body weight curves of mice after administration of a lipid nanoparticle composition containing 001 to 004 are shown in [the figure]. Figure 10B As shown in the image. Figure 10C The white blood cell portion of the mouse's complete blood cell count at the end of the experiment is shown. Figure 10D The red blood cell portion of the mouse's complete blood cell count at the end of the experiment is shown. Figure 10E The platelet fraction of the mouse's complete blood cell count at the end of the experiment is shown. Figure 10F The results of liver function tests in mouse blood chemistry at the end of the experiment are shown. Figure 10G The results of renal function tests in mice by blood chemistry at the end of the experiment are shown, in which no statistically significant results were found for any of the indicators related to hematologic toxicity, hepatotoxicity, and nephrotoxicity associated with the formulation or therapeutic target.

[0904] according to Figure 10A The results showed that, compared to a baseline at the same dosage, the nucleic acids contained in the lipid nanoparticles exhibited comparable antitumor efficiency. Meanwhile, Figures 10B-10GIt appears that a high dose of BM is very toxic for mice, leading to a significant decrease in body weight, abnormal leukocytosis, and a decrease in red blood cells and platelets, as well as a significant increase in blood urea nitrogen, which indicates early kidney damage. In contrast, the nucleic acids of the application have no significant effect on the body mass of mice, the complete blood count, and the liver / kidney function indices, which indicates that the candidate molecules have better safety compared to the benchmark.

[0905] Example 11.3: MC38 ectopic tumor model

[0906] mRNA without PDL1 antibody

[0907] A cell suspension containing 5 x 105 5 MC38 cells was injected into the peritoneal cavity of BALB / c mice to construct an ectopic tumor model. When the tumor size was 80 mm 3 - 120 mm 3 , the mice were randomly divided into groups. On days 0, 4, 7, 11, 14, and 18, each mouse was administered 50 μL of the diluted lipid nanoparticle composition (1 mg / mL) intratumorally. The tumor size was measured three times per week. The tumor growth curve after administration of the lipid nanoparticle composition containing the nucleic acid for testing is shown in Figure 11A . The body weight curve of the mice after administration of the lipid nanoparticle composition containing the nucleic acid for testing is shown in Figure 11B .

[0908] Combination of mRNA and PDL1 antibody

[0909] Each mouse was administered 125 μL of the diluted PD-L1 antibody (1 mg / mL) intraperitoneally, and 50 μL of the diluted lipid nanoparticle composition (1 mg / mL) intratumorally. The tumor growth curve after administration of the lipid nanoparticle composition containing the nucleic acid for testing and / or the PD-L1 antibody is shown in Figure 12A . The body weight curve of the mice after administration is shown in Figure 12B .

[0910] Fusion proteins

[0911] The lipid nanoparticle composition containing nucleic acids encoding fusion proteins containing PD-L1 antibodies (PDL1Ab_014, PDL1Ab_014D43h, and PDL1Ab_014D61) was administered intratumorally. The tumor growth curve after administration is shown in Figure 13A . The body weight curve of the mice after administration is shown in Figure 13B .

[0912] Example 11: Pharmacokinetic test

[0913] C57 / B6J mice were administered test lipid nanoparticle compositions via intravenous (“IV”) or intramuscular (“IM”) injection. Blood samples were collected at given times to measure blood concentrations of the corresponding protein expressed in vivo. Pharmacokinetic curves for intravenous injection or intramuscular injection are shown in FIGS. 1 and 2, respectively. Figures 14A-14C

[0914] The foregoing description is considered as illustrative only of the principles of the disclosure. Further, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the application to the exact construction and process shown. Therefore, all suitable modifications and equivalents should be considered as falling within the scope of the application as defined by the appended claims.​

Claims

1. A lipid nanoparticle composition comprising: a target polynucleotide comprising a first nucleic acid encoding an interleukin 15 polypeptide or a variant thereof, and a lipid nanoparticle comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof, wherein, R a selected from the group consisting of hydrogen, R 5 , alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl are optionally substituted with one or more groups independently selected from halogen, hydroxyl, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, and R 6 ; R 1 For R 2 For R 3 For R 4 For R 5 If present, then R 6 If present, then each W is independently selected from O, S, or NR b and each R b is independently selected from hydrogen, alkyl, alkoxycarbonyl, acyl, or sulfonyl; each Y is independently selected from O, S, NR c , N(R c )Z(W), N(R c )N(R c ) or N(R c )N(R c )Z(W), and each R c is independently selected from hydrogen, alkyl, alkoxycarbonyl, acyl or sulfonyl; each Z is independently selected from C, S, or S(O); each n is independently 0, 1, 2, 3, 4, or 5; each m is independently 0, 1, 2, or 3; each p is independently 1, 2, 3, or 4; and R 1c , R 2c , R 3c , and R 4c are each independently selected from the group consisting of alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally substituted with one or more groups independently selected from the group consisting of halogen, hydroxyl, oxo, cyano, cycloalkyl, heterocyclyl, aryl, and heteroaryl, and the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally interrupted with one or more groups independently selected from the group consisting of cycloalkyl, heterocyclyl, aryl, and heteroaryl.

2. The lipid nanoparticle composition of claim 1, wherein, the compound is of Formula (A) or Formula (B):

3. The lipid nanoparticle composition of claim 1 or 2, wherein, R 2 , R 3 , and R 4 are the same.

4. The lipid nanoparticle composition of any one of claims 1-3, wherein, R 2 , R 3 , and R 4 are the same.

5. The lipid nanoparticle composition of any one of claims 1-4, wherein, one or more of Y is O, NR c or N(R c )N(R c )Z(W).

6. The lipid nanoparticle composition of any one of claims 1-5, wherein, one or more of W is O.

7. The lipid nanoparticle composition of claim 5, wherein, R c one or more of R1, R2, R3, R4, R5, R6, R7, R8 8. The lipid nanoparticle composition of any one of claims 1-7, wherein, one or more of Z is C or S(O).

9. The lipid nanoparticle composition of any one of claims 1-8, wherein, R 2c , R 3c , and R 4c are each independently hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, halo, -OR, -CN, -N(R)2, -COR 10. The lipid nanoparticle composition of any one of claims 1-9, wherein, R 2c , R 3c , and R 4c are each independently H, C 8-24 alkyl or alkenyl.

11. The lipid nanoparticle composition of any one of claims 1-10, wherein, R 2c , R 3c , and R 4c are each independently H, C 10-24 alkyl or alkenyl.

12. The lipid nanoparticle composition of any one of claims 1-11, wherein, R 2c , R 3c , and R 4c one or more of R, R, and R is alkenyl comprising one, two, or three C=C double bonds.

13. The lipid nanoparticle composition of any one of claims 1-12, wherein, R 2c , R 3c , and R 4c one or more of which is an alkenyl group comprising one or more Z-alkenes.

14. The lipid nanoparticle composition of any one of claims 1-13, wherein, R 1c is alkyl.

15. The lipid nanoparticle composition of any one of claims 1-14, wherein, R 1c is C 1-12 alkyl.

16. The lipid nanoparticle composition of any one of claims 1-15, wherein, R 1c is C 4-10 alkyl.

17. The lipid nanoparticle composition of any one of claims 1-16, wherein, R a is C 1-6 alkyl optionally substituted with one or more groups independently selected from the group consisting of hydroxy, cycloalkyl, and heteroaryl.

18. The lipid nanoparticle composition of any one of claims 1-17, wherein, R a is methyl, ethyl, propyl, butyl or pentyl.

19. The lipid nanoparticle composition of any one of claims 1-4, wherein, One or more of them are independently selected from the following groups:

20. The lipid nanoparticle composition of any one of claims 1-4, wherein, R 1c , R 2c , R 3c , and R 4c are each independently selected from the group consisting of:

21. The lipid nanoparticle composition of any one of claims 1-20, wherein, R 1c , R 2c , R 3c , R 4c , R 5c and R 6c each, if present, does not contain two heteroatoms directly linked to each other.

22. The lipid nanoparticle composition of any one of claims 1-20, wherein, R 1c , R 2c , R 3c , R 4c , R 5c , and R 6c each of which is present, contains -N(R c )-N(R c )- or -S(O)2-N(R c )-.

23. The lipid nanoparticle composition of any one of claims 1-22, wherein, the compound has a structure listed in the following table:

24. The lipid nanoparticle composition of any one of claims 1-23, wherein, the lipid nanoparticle further comprises a neutral lipid, a sterol or sterol derivative, and a surfactant.

25. The lipid nanoparticle composition of claim 24, wherein, (i) the neutral lipid is selected from the group consisting of 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-glycero-3- phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2- ditridecyl-sn-glycero-3-phosphocholine (DUPC), 1,2-dioleoyl-sn-glycero-3-phospho-rac- (1-glycerol) sodium salt (DOPG), 1,2-di-O-octadecenyl-5-glycero-3-phosphocholine (18:0 diether PC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1-oleoyl-2- cholesteryl hemisuccinyl-5-glycero-3-phosphocholine (OChemsPC), and any mixture thereof; (ii) the sterol is cholesterol, beta-sitosterol, stigmasterol, ergosterol, brassicasterol, coprostanol, or campesterol; and / or (iii) the surfactant is selected from the group consisting of 1,2-dimyristoyl-rac-glycero- 3-methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-rac-glycero-3- methoxypolyethylene glycol (DSG-PEG), N-(methoxypolyethylene glycol carbonyl)- 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEG-DSPE), 1,2-dioleoyl-sn- glycero-3-phosphoethanolamine-N-[(polyethylene glycol)] (DOPE-PEG), and any mixture thereof.

26. The lipid nanoparticle composition of claim 25, wherein, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), the sterol is cholesterol, and the surfactant is DMG-PEG.

27. The lipid nanoparticle composition of any one of claims 24-26, wherein, The lipid nanoparticle comprises the following in molar ratios, based on the total amount of the lipid nanoparticle: (i) 10-65% of the compound of Formula (I); (ii) 5-30% of the neutral lipid; (iii) 15-50% of the sterol or the sterol derivative; and (iv) 0.5-5% of the surfactant.

28. The lipid nanoparticle composition of any one of claims 24-27, wherein, The lipid nanoparticle comprises the following in molar ratios, based on the total amount of the lipid nanoparticle: (i) 40-65% of the compound of Formula (I); (ii) 5-15% of the neutral lipid; (iii) 25-50% of the sterol or the sterol derivative; and (iv) 1-5% of the surfactant.

29. The lipid nanoparticle composition of any one of claims 24-28, wherein, The lipid nanoparticle comprises the following in molar ratios, based on the total amount of the lipid nanoparticle: (i) 40-65% of the compound of Formula (I); (ii) 5-15% of the DSPC; (iii) 25-50% of the cholesterol; and (iv) 1-5% of the DMG-PEG.

30. The lipid nanoparticle composition of any one of claims 24-29, wherein, The lipid nanoparticle comprises the following in molar ratios, based on the total amount of the lipid nanoparticle: (i) 40-50% of the compound of Formula (I); (ii) 5-15% of the DSPC; (iii) 25-50% of the cholesterol; and (iv) 1.5-2.55% of the DMG-PEG.

31. The lipid nanoparticle composition of any one of claims 1-30, wherein, The first nucleic acid encodes a human interleukin 15 polypeptide comprising an amino acid sequence of SEQ ID NO: 8 or 299-406, or an amino acid sequence having at least 95% identity to SEQ ID NO: 8 or 299-406.

32. The lipid nanoparticle composition of any one of claims 1-31, wherein, The first nucleic acid comprises a nucleic acid sequence of SEQ ID NO: 9 or 407-514, or a nucleic acid sequence having at least 85% identity to SEQ ID NO: 9 or 407-514, the first nucleic acid encoding a human interleukin 15 polypeptide.

33. The lipid nanoparticle composition of any one of claims 1-32, wherein, The target polynucleotide further comprises a second nucleic acid encoding an IL-15Ra sushi domain.

34. The lipid nanoparticle composition of claim 33, wherein, The first nucleic acid and the second nucleic acid are directly linked together or linked together by a linker.

35. The lipid nanoparticle composition of claim 33 or 34, wherein, The IL-15Ra sushi domain comprises an amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least 95% identity to SEQ ID NO:

6.

36. The lipid nanoparticle composition of any one of claims 33-35, the second nucleic acid comprises a nucleic acid sequence of SEQ ID NO:

7.

37. The lipid nanoparticle composition of any one of claims 1-36, wherein, The target polynucleotide further comprises a third nucleic acid encoding an Fc moiety, the third nucleic acid being directly linked or linked by a linker to the first nucleic acid and / or the second nucleic acid, optionally, the third nucleic acid is linked to the 5’ end of the first nucleic acid or the second nucleic acid, or linked to the 3’ end of the first nucleic acid or the second nucleic acid.

38. The lipid nanoparticle composition of claim 37, wherein, The Fc moiety is derived from human IgG1, IgG2, or IgG4.

39. The lipid nanoparticle composition of claim 37 or 38, wherein, The Fc portion is derived from human IgGl, optionally, the Fc portion has one or more mutations selected from the group of L234A and L235A.

40. The lipid nanoparticle composition of any one of claims 1-39, wherein, The target polynucleotide further comprises a fourth nucleic acid encoding an antibody or antigen-binding fragment thereof, the fourth nucleic acid is directly linked to the first nucleic acid and / or the second nucleic acid and / or the third nucleic acid or linked by a linker.

41. The lipid nanoparticle composition of claim 40, wherein, The antibody binds to PD-L1 or PD-1.

42. The lipid nanoparticle composition of claim 40 or 41, wherein, The anti-PD-L1 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 268, 270, 272, 274, 275, 277, 279, 281, 283, or 285, and optionally, the anti-PD-L1 antibody further comprises a light chain having the amino acid sequence of SEQ ID NO: 269, 271, 273, 276, 278, 280, 282, 284, or 286.

43. The lipid nanoparticle composition of any one of claims 1-42, wherein, The target polynucleotide encodes a polypeptide comprising an amino acid selected from the group of SEQ ID NOs: 10, 13, 15, 17, 23, 26, 28, 30-137, 287-298, and 515-617.

44. The lipid nanoparticle composition of any one of claims 1-43, wherein, The target polynucleotide encodes a polypeptide comprising an amino acid selected from the group of SEQ ID NOs: 17 and 287-298.

45. The lipid nanoparticle composition of any one of claims 1-44, wherein, The target polynucleotide comprises a nucleic acid having the sequence of SEQ ID NOs: 11-12, 14, 16, 18-19, 21-22, 24-25, 27, 29, 138-260, 618-720, or 724-728, or a nucleic acid sequence having at least 85% identity to SEQ ID NOs: 11-12, 14, 16, 18-19, 21-22, 24-25, 27, 29, 138-260, 618-720, or 724-728, which encodes a functional equivalent of the polypeptide encoded by SEQ ID NOs: 9, 11-12, 14, 16, 18-19, 21-22, 24-25, 27, 29, 138-260, 618-720, or 724-728.

46. The lipid nanoparticle composition of any one of claims 1-45, wherein, The target polynucleotide comprises a nucleic acid having the sequence of SEQ ID NO: 19, SEQ ID NOs: 246-250, SEQ ID NOs: 252-258, or a nucleic acid sequence having at least 85% identity to SEQ ID NO: 19, SEQ ID NOs: 246-250, SEQ ID NOs: 252-258, which encodes a functional equivalent thereof.

47. The lipid nanoparticle composition of any one of claims 1-46, wherein, The target polynucleotide comprises: (i) a 5’ untranslated region (UTR) comprising a nucleic acid selected from the group of SEQ ID NOs: 261-263; (ii) a 3’ untranslated region (UTR) comprising a nucleic acid selected from the group of SEQ ID NOs: 264-267; and / or (iii) a poly-A region having a length of 50-120 nucleotides.

48. An interleukin 15 (IL-15) polypeptide variant, wherein, The IL-15 polypeptide variant comprises an amino acid sequence selected from the group of SEQ ID NOs: 299-406.

49. A fusion protein comprising an interleukin 15 (IL-15) polypeptide and an IL-15 receptor alpha sushi domain (IL-15Rαsushi), wherein, The fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 13, and 30-137.

50. The fusion protein of claim 49, wherein, The fusion protein further comprises an Fc portion located at the N-terminus of the fusion protein or at the C-terminus of the fusion protein.

51. The fusion protein of claim 50, wherein, The fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 17, 23, 26, 28, 287-291, and 515-617.

52. The fusion protein of any one of claims 48-51, wherein, The fusion protein further comprises an antibody portion that binds to PD-L1 or PD-1.

53. The fusion protein of claim 52, wherein, The fusion protein comprises an amino acid selected from the group consisting of SEQ ID NOs: 292-298.

54. An isolated nucleic acid sequence encoding an interleukin 15 polypeptide or a variant thereof, the isolated nucleic acid sequence having at least 85% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 9 or 407-514.

55. An isolated nucleic acid sequence encoding a fusion protein, wherein, The fusion protein comprises an IL-15 polypeptide and an IL-15Ra sushi, and wherein the isolated nucleic acid sequence comprises: (i) a nucleic acid sequence encoding an IL-15 having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 9 or 407-514; and (ii) a nucleic acid sequence encoding an IL-15Ra sushi having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:

7.

56. The isolated nucleic acid sequence of claim 55, having at least 85% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11-12, 14, 21-22, and 138-245.

57. The isolated nucleic acid sequence of claim 55 or 56, wherein, The fusion protein further comprises an Fc portion located at the N-terminus of the fusion protein or at the C-terminus of the fusion protein.

58. The isolated nucleic acid sequence of claim 57, having at least 85% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16, 18-19, 24-25, 27, 29, 246-250, 618-720, and 724-728.

59. The isolated nucleic acid sequence of any one of claims 55-58, wherein, The fusion protein further comprises an antibody portion that binds to PD-L1 or PD-1.

60. The isolated nucleic acid sequence of claim 59, having at least 85% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 252-258.

61. The isolated nucleic acid sequence of any one of claims 55-60, wherein, The isolated nucleic acid sequence further comprises a nucleic acid encoding an isolated antibody or antigen binding fragment thereof that binds to PD-L1 or PD-1.

62. The isolated nucleic acid sequence of claim 61, having at least 85% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 259-260.

63. A method of enhancing immune cell activation in a subject, comprising administering to the subject an effective amount of the lipid nanoparticle composition of any one of claims 1-47, an effective amount of the IL-15 polypeptide variant of claim 48, an effective amount of the fusion protein of any one of claims 49-53, or an effective amount of the isolated nucleic acid sequence of any one of claims 54-62.

64. A method of treating a disease or disorder in a subject, comprising administering to the subject an effective amount of the lipid nanoparticle composition of any one of claims 1-47, an effective amount of the IL-15 polypeptide variant of claim 48, an effective amount of the fusion protein of any one of claims 49-53, or an effective amount of the isolated nucleic acid sequence of any one of claims 54-62.

65. The method of claim 64, wherein, The disease or disorder is a cancer.

66. The method of claim 65, wherein, The cancer is an epithelial tumor, Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma, a prostate tumor, an ovarian tumor, a renal cell tumor, a gastrointestinal tumor, a liver tumor, a colorectal tumor, an angioma, a mesothelioma, a pancreatic tumor, a breast tumor, a sarcoma, a lung tumor, a colon tumor, a brain tumor, a melanoma, a small cell lung tumor, a neuroblastoma, a testicular tumor, a carcinoma, an adenocarcinoma, a glioma, a seminoma, a retinoblastoma, or an osteosarcoma.

67. The method of any one of claims 63-66, further comprising a second therapeutic agent.

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