Lipid nanoparticle used for delivering nucleic acid molecule encoding secretory protein and use thereof

Abstract

Provided in the present invention are a lipid nanoparticle used for delivering a nucleic acid molecule that encodes a secretory protein and the use thereof. The lipid nanoparticle can deliver the nucleic acid molecule to the liver for the treatment of liver diseases and other diseases. Also provided in the present invention are a preparation method for the lipid nanoparticle, a pharmaceutical composition comprising the lipid nanoparticle, and the use of the lipid nanoparticle and the pharmaceutical composition in the treatment of diseases.

Description

Lipid nanoparticles for delivery of nucleic acid molecules encoding secreted proteins and uses thereof

[0001] This application claims priority to Chinese Patent Application No. CN202411303127.0, filed on September 18, 2024, and Chinese Patent Application No. CN202411433393.5, filed on October 14, 2024, which are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0002] The present application relates to the field of biological medicine, in particular to lipid nanoparticles for delivery of nucleic acid molecules encoding secreted proteins, compositions comprising the same and uses thereof in the treatment of diseases. BACKGROUND

[0003] In recent years, the rapid development of mRNA technology and lipid nanoparticle (LNP) delivery systems has enabled mRNA therapy to be applied to the prevention and treatment of various diseases, with a very broad application scenario, including vaccines, protein replacement therapy, tumor immunotherapy, cell reprogramming, and gene editing, etc.

[0004] mRNA therapy can achieve higher protein expression efficiency and longer protein expression time, and has obvious advantages over DNA therapy, including: (1) mRNA begins to translate proteins after entering the cytoplasm, and does not need to enter the nucleus to function, which is more efficient than DNA entering the nucleus to transcribe mRNA. (2) Unlike DNA and viral vectors, mRNA cannot be inserted into the genome and can only express the encoded protein transiently, with a lower risk of insertion mutagenesis. (3) mRNA is easy to synthesize in vitro and relatively inexpensive, allowing for rapid application. (4) mRNA can theoretically express any protein and can be used to treat almost any disease. To achieve clinical application of mRNA, efficient and safe delivery of mRNA is one of the biggest challenges of mRNA therapy. Currently, LNP is one of the most widely used in vivo nucleic acid delivery platforms, with the advantages of simple preparation, good biodegradability, no immunogenicity, and good safety. The main components of LNP include ionizable lipid molecules, cholesterol, neutral lipids, and PEGylated lipids (PEG lipids). Among them, ionizable lipids are the core part of the LNP structure, and their molecular structure plays a decisive role in the delivery efficiency, targeting, and formulation stability of the entire liposome nanoparticle. The latter three determine the structure and stability of the LNP.

[0005] GPC3 (Glypican-3) is a cell surface glycoprotein mainly involved in cell proliferation, differentiation, migration and apoptosis. GPC3 is expressed in the liver and kidney of fetuses and is almost not expressed in healthy human tissues, but GPC3 is highly expressed in 70-80% of hepatocellular carcinoma (HCC) tissues and is also expressed in small amounts in other tumors such as ovarian clear cell carcinoma, melanoma, lung squamous cell carcinoma, etc. Although the function of GPC3 is not fully understood, a large number of studies have shown that GPC3 is closely related to the occurrence, metastasis and prognosis of HCC. Therefore, GPC3 has become a new target for the diagnosis and immunotherapy of HCC. Immunotherapy targeting GPC3 protein mainly includes monoclonal antibodies, antibody-drug conjugates, tumor vaccines, chimeric antigen receptor T cells (CAR-T) and bispecific antibodies, etc. Currently, there is no GPC3 targeted drug on the market worldwide. Among them, the bispecific antibody targeting GPC3 and CD3 can form an immunological synapse between T cells and tumor cells, summon T cells to the tumor site, activate T cell immune mechanisms, and mediate T cell killing of GPC3 overexpressing tumor cells. As a powerful expansion of monoclonal antibody therapy, multiple bispecific antibody projects are undergoing preclinical and clinical research, showing good development prospects.

[0006] On this basis, there has also been an urgent need in the art for nucleic acid molecules for the treatment of tumors and lipid nanoparticles for delivering such therapeutic nucleic acid molecules, and the present invention meets this need. SUMMARY

[0007] In one aspect, the present invention provides a lipid nanoparticle for delivering a nucleic acid molecule encoding a secreted protein, comprising an ionizable lipid, a structural lipid, a neutral lipid, and a polymeric lipid, wherein the ionizable lipid is a compound of Formula (II), or a pharmaceutically acceptable salt, isotopologue, tautomer, or stereoisomer thereof,

[0008] wherein,

[0009] a = 1, 2, 3, 4, 5, or 6;

[0010] b = 4, 5, 6, 7, 8, 9, or 10;

[0011] c = 1, 2, 3, 4, 5, or 6;

[0012] d = 0, 1, 2, 3, or 4;

[0013] c + d = 3, 4, 5, 6, 7, 8, or 9;

[0014] M1and M2are independently selected from the group consisting of -C(O)O-, -OC(O)-, -O-, -SC(O)O-, -OC(O)NR-, -NRC(O)NR-, -OC(O)S-, -OC(O)O-, -NRC(O)O-, -SC(O)-, -C(O)S-, -NR-, -C(O)NR-, -NRC(O)-, -NRC(O)S-, -SC(O)NR-, -C(O)-, -OC(S)-, -C(S)O-, -OC(S)NR-, -NRC(S)O-, -S-S-, and -S(O) 0-2 -;

[0015] R1and R2are independently selected from the group consisting of C 4-20 alkyl, C 4-20 alkenyl, and C 4-20 alkynyl, which is optionally substituted with one or more R 1s , and wherein one or more methylene units are optionally and independently replaced with -NR'-;

[0016] R 1s is independently selected from the group consisting of H, C 1-20 alkyl, -L c -OR c , -L c -SR c , and -L c -NR c R' c ;

[0017] R and R' are each independently selected from the group consisting of H and C 1-20 alkyl;

[0018] L c is independently selected from the group consisting of a bond and C 1-20 alkylene;

[0019] R c and R' c are independently selected from the group consisting of H, C 1-20 alkyl, C 3-14 cycloalkyl, and 3- to 14-membered heterocyclyl;

[0020] R4and R5are independently selected from the group consisting of C 1-8 alkyl, which is optionally substituted with one or more R 4s ;

[0021] or R4, R5, together with the carbon atom to which they are attached, form a C 3-14 cycloalkylene or 3- to 14-membered heterocyclylene, which is optionally substituted with one or more R 4s ;

[0022] R 4s is independently selected from the group consisting of H, halogen, cyano, C1-8 Alkyl, C 1-8 Halogenated alkyl groups, -L d -OR d -L d -SR d and -L d -NR d R' d ;

[0023] L d Independently selected from chemical bonds and C 1-8 Alkylene;

[0024] R d and R' d Independently selected from H and C 1-8 Alkyl, C 3-14 Cycloalkyl and 3 to 14-membered heterocyclic groups.

[0025] In a preferred embodiment, M1 and M2 are independently selected from -C(O)O- or -OC(O)-, preferably, where M1 is -OC(O)- and M2 is -C(O)O-.

[0026] In another preferred embodiment, R4 and R5 are independently C 1-8 Alkyl, preferably C 1-3 Alkyl groups, more preferably methyl groups; or R4, R5 together with the carbon atoms they are attached to form C4+. 3-6 Cycloalkylene or 3-6 membered heterocyclic alkylene, preferably forming C 3-4 Cycloalkylene compounds, preferably cyclopropylene compounds.

[0027] In another preferred embodiment, R1 and R2 are independently selected from C. 4-20 Alkyl group, preferably, R1 is C 6-14 Alkyl and R2 is C 8-20 Alkyl, more preferably, R1 is C 8-12 Straight-chain alkyl, R2 is C 8-20 Branched alkyl groups.

[0028] In another preferred embodiment, a is 2, b is 7, c is 5, and d is 1.

[0029] In a particularly preferred embodiment, the compound of formula (II) is selected from the compounds in Table (I) below, or their pharmaceutically acceptable salts, isotopic variants, tautomers, or stereoisomers:

[0030] Table (I)

[0031] In some embodiments, the present application provides a lipid nanoparticle as described above, comprising the following molar percentages of components:

[0032] ionizable lipid: 35-65%, preferably 40-55%;

[0033] structural lipid: 30-50%, preferably 35-50%;

[0034] neutral lipid: 5-30%, preferably 8-20%;

[0035] polymeric lipid: 0.5-3.5%, preferably 1-3%, more preferably 1-2%.

[0036] In some embodiments, the present application provides a lipid nanoparticle as described above, comprising the following molar percentages of components:

[0037] ionizable lipid: 40-65%, preferably 45-55%, more preferably 50%;

[0038] structural lipid: 30-50%, preferably 35-40%, more preferably 38.5%;

[0039] neutral lipid: 5-30%, preferably 8-15%, more preferably 10%;

[0040] polymeric lipid: 0.5-3.5%, preferably 1-3%, more preferably 1-2%, more preferably 1.5%.

[0041] In one preferred embodiment, the lipid nanoparticle, comprises the following molar percentages of components:

[0042] ionizable lipid: 40%;

[0043] structural lipid: 43.5%;

[0044] neutral lipid: 15%;

[0045] polymeric lipid: 1.5%.

[0046] In some embodiments, the present application provides a lipid nanoparticle as described above, further comprising at least one cargo selected from a nucleic acid molecule encoding a secreted protein.

[0047] In some embodiments, the present application provides a lipid nanoparticle as described above, wherein the secreted protein is selected from any one of a cytokine, an antigen binding protein; preferably, the antigen binding protein is selected from an antibody or an antibody fragment, more preferably a bispecific antibody or a fragment thereof; more preferably, the bispecific antibody is selected from any one of BiTE Triomabs, Knobs-into-holes, Crossmab, Ortho-Fab, DVD-Ig, Two-in-one, IgG-scFv, scFv2-Fc, bi-Nanobody, DART or TandAb or VHH formats.

[0048] In some embodiments, the present application provides a lipid nanoparticle as described above, wherein the nucleic acid molecule encoding a secreted protein is selected from an antisense oligonucleotide (ASO), an RNA or a DNA;

[0049] Preferably, wherein the RNA is selected from at least one of a messenger RNA (mRNA), a modified mRNA (mmRNA), a circular RNA (circRNA) and a self-replicating RNA (SrRNA), preferably a mRNA or a modified mRNA, more preferably a modified mRNA; or

[0050] Preferably, wherein the DNA is selected from at least one of a plasmid DNA (pDNA) and a minicircle DNA (mcDNA).

[0051] In some embodiments, the present application provides a lipid nanoparticle as described above, wherein the payload is a mRNA or a modified mRNA molecule;

[0052] Preferably, the payload is a mRNA or a modified mRNA molecule comprising a coding sequence for an antibody against a liver tumor surface antigen; or, the payload is a mRNA or a modified mRNA molecule comprising a coding sequence for an antibody against a protein associated with a liver tumor; or, the payload is a mRNA or a modified mRNA molecule comprising a coding sequence for an antibody against a T cell surface antigen;

[0053] More preferably, the payload is a mRNA or a modified mRNA molecule comprising a coding sequence for an antibody against GPC3; or, the payload is a mRNA or a modified mRNA molecule comprising a coding sequence for an antibody against CD3;

[0054] More preferably, the payload is a mRNA or a modified mRNA molecule comprising a coding sequence for a bispecific antibody;

[0055] More preferably, the payload is an mRNA or modified mRNA molecule comprising a coding sequence for a bispecific antibody against GPC3 and CD3;

[0056] More preferably, wherein the coding sequence for the bispecific antibody against GPC3 and CD3 comprises a scFv against GPC3, the heavy chain variable region of the scFv against GPC3 comprises a VH CDR1 set forth in SEQ ID NO: 17, a VH CDR2 set forth in SEQ ID NO: 18, a VH CDR3 set forth in SEQ ID NO: 19, the light chain variable region of the scFv against GPC3 comprises a VL CDR1 set forth in SEQ ID NO: 20, a VL CDR2 set forth in SEQ ID NO: 21, a VL CDR3 set forth in SEQ ID NO: 22.

[0057] More preferably, wherein the scFv against GPC3 comprises a heavy chain variable region set forth in SEQ ID NO: 23 and a light chain variable region set forth in SEQ ID NO: 24.

[0058] More preferably, wherein the bispecific antibody against GPC3 and CD3 is in the BiTE format.

[0059] In some embodiments, the scFv against GPC3 comprises an amino acid sequence set forth in SEQ ID NO: 25.

[0060] In some embodiments, the bispecific antibody further comprises a scFv against CD3, the scFv against CD3 comprises an amino acid sequence set forth in SEQ ID NO: 26.

[0061] In some embodiments, the bispecific antibody comprises an amino acid sequence set forth in SEQ ID NO: 3.

[0062] In some embodiments, the coding sequence of the bispecific antibody is selected from the group consisting of SEQ ID NOs: 4-8. In some preferred embodiments, the coding sequence of the bispecific antibody is set forth in SEQ ID NO: 4 or SEQ ID NO: 7. In some further preferred embodiments, the coding sequence of the bispecific antibody is set forth in SEQ ID NO: 4.

[0063] In some embodiments, the bispecific antibody comprises an amino acid sequence set forth in SEQ ID NO: 14.

[0064] In some embodiments, the coding sequence of the bispecific antibody is set forth in SEQ ID NO: 15.

[0065] In some embodiments, the mRNA molecule further comprises a 5’ UTR and / or a 3’ UTR.

[0066] A variety of useful 5’ UTRs and / or 3’ UTRs are known in the art. One of skill in the art is capable of determining a 5’ UTR and / or 3’ UTR suitable for use in the present application.

[0067] In some embodiments, the 5’ UTR comprises the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the 3’ UTR comprises the nucleotide sequence set forth in SEQ ID NO: 2.

[0068] In some embodiments, the mRNA molecule further comprises a poly(A) sequence.

[0069] In some embodiments of the present application, the poly(A) sequence comprises about 20 to about 500 (e.g., contiguous) adenine nucleotides (A), for example, about 25, about 50, about 100, about 150, about 175, about 200, about 300, about 400, about 500 (e.g., contiguous) adenine nucleotides (A). In some preferred embodiments, the poly(A) sequence comprises 120 (e.g., contiguous) adenine nucleotides (A).

[0070] In some embodiments, the mRNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 9-13. In some preferred embodiments, the mRNA molecule comprises a nucleotide sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 12. In some further preferred embodiments, the mRNA molecule comprises a nucleotide sequence set forth in SEQ ID NO: 9.

[0071] In some embodiments, the mRNA molecule comprises a nucleotide sequence set forth in SEQ ID NO: 16.

[0072] In some embodiments, the mRNA molecule further comprises a 5’ cap structure. In some embodiments of the present application, the 5’ cap structure is a Cap1 cap structure.

[0073] In some embodiments, the mRNA molecule of the present application can further comprise at least one nucleotide modification. The at least one nucleotide modification includes, but is not limited to, a cytidine modification, a uridine modification, or an adenosine modification. In some embodiments, the at least one nucleotide modification includes, but is not limited to, 5-methylcytosine (m5C), N6-methyladenosine (m6A), pseudouridine (ψ), N1-methylpseudouridine (m1ψ), and 5-methoxyuridine (5moU).

[0074] In some embodiments, the mRNA molecule is chemically synthesized. In some embodiments, the RNA molecule is obtained by in vitro transcription.

[0075] In addition, the mRNA molecules of the present application have certain stability, and can tolerate insertion of certain additional sequences (such as microRNA binding sites) without affecting their ability to be translated, nor affecting the stability of the mRNA molecule. In some embodiments, the additional sequences (such as microRNA binding sites) can be inserted into the 3' UTR. The microRNA binding sites include the full-length reverse complement of a microRNA (exemplary length can be 19-25 nt) or the reverse complement of the seed sequence thereof (exemplary length can be 7-8 nt).

[0076] In another aspect, the present application provides a nucleic acid vector comprising a coding sequence of the mRNA molecule of the present application. In some embodiments, the nucleic acid vector is used to generate the mRNA molecule of the present application.

[0077] In some embodiments, the nucleic acid vector further comprises a RNA polymerase promoter sequence operably linked to the coding sequence of the mRNA molecule. The operably linked promoter allows for in vivo and / or in vitro transcription of the RNA molecule. The promoter is, for example, a T7 RNA polymerase promoter, a T6 viral RNA polymerase promoter, a SP6 viral RNA polymerase promoter, a T3 viral RNA polymerase promoter, or a T4 viral RNA polymerase promoter.

[0078] In some embodiments, the nucleic acid vector is a plasmid vector. In some embodiments, the nucleic acid vector comprises a restriction endonuclease site, such as a type IIS restriction endonuclease site, flanking the 3' of the coding sequence of the mRNA molecule. Suitable restriction endonucleases include, but are not limited to, BsmBI, BsaI, SapI, etc. The restriction endonuclease site can be used to linearize the nucleic acid vector for in vitro transcription.

[0079] Methods for in vitro transcription of mRNA molecules from nucleic acid vectors are known in the art, and can be performed using commercially available kits.

[0080] In another aspect, the present application provides a method of preparing the lipid nanoparticle of the present application, comprising: mixing the lipid components in the lipid nanoparticle, and mixing with the cargo to obtain the lipid nanoparticle.

[0081] In another aspect, the present application provides a composition comprising the lipid nanoparticle of the present application.

[0082] In another aspect, the present application provides a pharmaceutical composition comprising the lipid nanoparticle of any of the above embodiments and optionally a pharmaceutically acceptable excipient, such as a carrier, adjuvant or vehicle, preferably a liquid formulation, more preferably an injection.

[0083] In another aspect, the present application provides the use of the lipid nanoparticle or the pharmaceutical composition of the present application in the manufacture of a medicament for the treatment of a disease or for improving a patient's prognostic condition.

[0084] Preferably, the disease is a liver disease or a liver-related disease; more preferably, the liver disease or liver-related disease is a malignant tumor of the liver, more preferably hepatocellular carcinoma (HCC); or

[0085] Preferably, the disease is a GPC3-related disease; more preferably, the disease is a tumor that overexpresses GPC3, such as a liver cancer, e.g. hepatocellular carcinoma (HCC), a lung cancer, e.g. lung squamous cell carcinoma (SqCC), a gastric cancer, an ovarian cancer, e.g. ovarian clear cell carcinoma, a melanoma or a pediatric embryonal tumor.

[0086] In another aspect, the present application provides the use of the lipid nanoparticle or the pharmaceutical composition of the present application in the manufacture of a medicament for the delivery of a cargo.

[0087] In another aspect, the present application provides a method for treating a disease or for improving a patient's prognostic condition in a subject, comprising administering to said subject an effective amount of the lipid nanoparticle or the pharmaceutical composition of the present application.

[0088] Preferably, the disease is a liver disease or a liver-related disease; more preferably, the liver disease or liver-related disease is a malignant tumor of the liver, more preferably hepatocellular carcinoma (HCC); or

[0089] Preferably, the disease is a GPC3-related disease; more preferably, the disease is a tumor that overexpresses GPC3, such as a liver cancer, e.g. hepatocellular carcinoma (HCC), a lung cancer, e.g. lung squamous cell carcinoma (SqCC), a gastric cancer, an ovarian cancer, e.g. ovarian clear cell carcinoma, a melanoma or a pediatric embryonal tumor.

[0090] In one embodiment, the lipid nanoparticle or the pharmaceutical composition is administered systemically, preferably by intravenous injection, arterial injection or intraperitoneal injection, more preferably by intraperitoneal injection or intravenous injection.

[0091] In another aspect, the present application provides the lipid nanoparticle or the pharmaceutical composition of the present application for use in the treatment of a disease or for improving a patient's prognostic condition.

[0092] Preferably, the disease is a liver disease or a liver-related disease; more preferably, the liver disease or liver-related disease is a malignancy of the liver, more preferably hepatocellular carcinoma (HCC); or

[0093] Preferably, the disease is a GPC3-related disease; more preferably, the disease is a tumor that overexpresses GPC3, such as a liver cancer, e.g. hepatocellular carcinoma (HCC), a lung cancer, e.g. lung squamous cell carcinoma (SqCC), a gastric cancer, an ovarian cancer, e.g. ovarian clear cell carcinoma, a melanoma or a pediatric embryonal tumor.

[0094] In another aspect, the present application provides a method of delivering a payload in a subject, comprising administering to the subject a lipid nanoparticle or a pharmaceutical composition of the present application.

[0095] In another aspect, the present application provides a lipid nanoparticle or a pharmaceutical composition of the present application for use in the delivery of a payload.

[0096] In another aspect, the present application provides the use of a lipid nanoparticle or a pharmaceutical composition of the present application in the manufacture of a medicament for preventing tumor occurrence or preventing tumor recurrence.

[0097] In another aspect, the present application provides a lipid nanoparticle or a pharmaceutical composition of the present application for use in preventing tumor occurrence or preventing tumor recurrence.

[0098] In another aspect, the present application provides a method of preventing tumor occurrence or preventing tumor recurrence in a subject, comprising administering to the subject an effective amount of a lipid nanoparticle or a pharmaceutical composition of the present application.

[0099] In another aspect, the present application provides a lipid nanoparticle or a pharmaceutical composition of the present application, wherein the payload is an mRNA encoding a secreted protein, and the half-life in vivo of the secreted protein obtained by administration of the lipid nanoparticle or the pharmaceutical composition via a systemic route of administration is longer than the half-life in vivo of the secreted protein obtained by administration of the secreted protein via a systemic route of administration, preferably 1-fold, 2-fold, 3-fold, 4-fold or 5-fold or more longer, more preferably 10-fold or more longer.

[0100] In another aspect, the present application provides a lipid nanoparticle or a pharmaceutical composition of the present application for use in improving or enhancing the efficacy of a secreted protein in the prevention or treatment of a disease, wherein the payload is an mRNA encoding the secreted protein, and the half-life in vivo of the secreted protein obtained by administration of the lipid nanoparticle or the pharmaceutical composition via a systemic route of administration is longer than the half-life in vivo of the secreted protein obtained by administration of the secreted protein via a systemic route of administration, preferably 1-fold, 2-fold, 3-fold, 4-fold or 5-fold or more longer, more preferably 10-fold or more longer.

[0101] In another aspect, the present application provides a method of improving or enhancing the efficacy of a secreted protein in preventing or treating a disease, the method comprising administering a lipid nanoparticle or a pharmaceutical composition of the present application, wherein the payload is an mRNA encoding the secreted protein, and the half-life of the secreted protein in vivo obtained by administering the lipid nanoparticle or the pharmaceutical composition via a systemic route of administration is longer than the half-life of the secreted protein in vivo obtained by administering the secreted protein via a systemic route of administration, preferably 1-fold, 2-fold, 3-fold, 4-fold or 5-fold or more, more preferably 10-fold or more.

[0102] In another aspect, the present application provides a lipid nanoparticle or a pharmaceutical composition of the present application for use in reducing side effects of a secreted protein in preventing or treating a disease, wherein the payload is an mRNA encoding the secreted protein, and the half-life of the secreted protein in vivo obtained by administering the lipid nanoparticle or the pharmaceutical composition is longer than the half-life of the secreted protein in vivo obtained by administering the secreted protein via a systemic route of administration, preferably 1-fold, 2-fold, 3-fold, 4-fold or 5-fold or more, more preferably 10-fold or more.

[0103] In another aspect, the present application provides a method of reducing side effects of a secreted protein in preventing or treating a disease, the method comprising administering a lipid nanoparticle or a pharmaceutical composition of the present application, wherein the payload is an mRNA encoding the secreted protein, and the half-life of the secreted protein in vivo obtained by administering the lipid nanoparticle or the pharmaceutical composition via a systemic route of administration is longer than the half-life of the secreted protein in vivo obtained by administering the secreted protein via a systemic route of administration, preferably 1-fold, 2-fold, 3-fold, 4-fold or 5-fold or more, more preferably 10-fold or more.

[0104] Definitions

[0105] Chemical Definitions

[0106] The definitions of specific functional groups and chemical terms are more fully described below.

[0107] When a range of values is listed, it is intended to include each value and sub-range within the range. For example, "C 1-6 "alkyl" includes C1, C2, C3, C4, C5, C6, C 1-6 , C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5 , C3-4 , C 4-6 , C 4-5 , and C 5-6 alkyl.

[0108] The term "alkyl" refers to straight- or branched-chain saturated hydrocarbon moieties. "C 1-20 alkyl" refers to straight- or branched-chain saturated hydrocarbon groups having from 1 to 20 carbon atoms. In some embodiments, C 4-20 alkyl, C 8-20 alkyl, C 6-14 alkyl, C 7-12 alkyl, C 8-12 alkyl, C 4-10 alkyl, C 7-11 alkyl, C 8-11 alkyl, C 8-10 alkyl, C 9-10 alkyl, C 8-9 alkyl, C 4-9 alkyl, C 6-9 alkyl, C 7-9 alkyl, C 2-8 alkyl, C 5-8 alkyl, C 7-8 alkyl, C 4-6 alkyl, C 1-20 alkyl, C 1-14 alkyl, C 2-14 alkyl, C 1-13 alkyl, C 1-12 alkyl, C 1-10 alkyl, C 1-9 alkyl, C 1-8 alkyl, C 1-7 alkyl, C 2-7 alkyl, C 1-6 alkyl, C 2-6 alkyl, C 1-5 alkyl, C 1-4 alkyl, C 2-4 alkyl, C 1-3 alkyl, C 2-3 alkyl, C 1-2 alkyl, and Me are preferred. Examples of C 1-6 alkyl include: methyl (Ci), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), t-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), t-amyl (C5), and n-hexyl (C6). The term "C 1-6"Alkyl" also includes heteroalkyl in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by a heteroatom (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). Alkyl groups can be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. Conventional alkyl abbreviations include: Me (-CH3), Et (-CH2CH3), iPr (-CH(CH3)2), nPr (-CH2CH2CH3), n-Bu (-CH2CH2CH2CH3), or i-Bu (-CH2CH(CH3)2). In some embodiments, alkyl is preferably straight chain alkyl.

[0109] The term "alkenyl" refers to a straight-chain or branched hydrocarbon moiety having one or more carbon-carbon double bonds. "C 4-20 "Alkenyl" refers to a straight-chain or branched hydrocarbon group having from 4 to 20 carbon atoms and at least one carbon-carbon double bond. In some embodiments, C 2-13 "Alkenyl", C 4- 14 "Alkenyl", C 6-14 "Alkenyl", C 4-10 "Alkenyl", C 2-10 "Alkenyl", C 2-9 "Alkenyl", C 2-6 "Alkenyl" and C 2-4 "Alkenyl" is preferred. C 2-6 Examples of "Alkenyl" include: ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. The term "C 2-6 "Alkenyl" also includes heteroalkenyl in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by a heteroatom (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). Alkenyl groups can be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0110] The term "alkynyl" refers to a straight-chain or branched hydrocarbon moiety having one or more carbon-carbon triple bonds. "C 4-20 "Alkynyl" refers to a straight-chain or branched hydrocarbon group having from 4 to 20 carbon atoms, at least one carbon-carbon triple bond, and optionally one or more carbon-carbon double bonds. In some embodiments, C 2-13 "Alkynyl", C 4-14 "Alkynyl", C 6-14 "Alkynyl", C 4-10 "Alkynyl", C 2-10 "Alkynyl", C 2-9 "Alkynyl", C 2-6 "Alkynyl" and C 2-4 "Alkynyl" is preferred. C 2-6Examples of alkynyl groups include, but are not limited to: ethynyl (C2), 1- propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), pentynyl (C5), hexynyl (C6), and the like. The term "C 2-6 Examples of alkynyl groups include, but are not limited to: ethynyl (C2), 1- propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), pentynyl (C5), hexynyl (C6), and the like. The term "C

[0111] The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 1-20 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 1-20 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 4-20 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 6-14 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 7-12 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 8-12 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 4-10 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 7-11 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 8-11 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 8-10 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 9-10 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 8-9 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 4-9 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 6-9 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 7-9 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 2-8 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 5-8 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 7-8 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 4-6 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 1-20 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 1-14 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 2-14 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 1-13 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 1-12 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 1-10 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 1-9 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 1-8 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 1-7 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 2-7 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 1-6 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 2-6 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 1-5 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 1-4 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 2-4 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 1-3 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 2-3 The term "alkylene" refers to a divalent radical resulting from the removal of two hydrogen atoms from an alkyl group, which can be substituted or unsubstituted. 1-2Alkylene and methylene are preferred. Unsubstituted alkylene groups include, but are not limited to, methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), butylene (-CH2CH2CH2CH2-), pentylene (-CH2CH2CH2CH2CH2-), hexylene (-CH2CH2CH2CH2CH2CH2-), and the like. Exemplary substituted alkylene groups, e.g., alkylene groups substituted with one or more alkyl (methyl) groups, include, but are not limited to, substituted methylene (-CH(CH3)-, -C(CH3)2-), substituted ethylene (-CH(CH3)CH2-, -CH2CH(CH3)-, -C(CH3)2CH2-, -CH2C(CH3)2-), substituted propylene (-CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)-, -C(CH3)2CH2CH2-, -CH2C(CH3)2CH2-, -CH2CH2C(CH3)2-), and the like. 2-

[0112] "C 0-6 Alkylene" refers to a chemical bond and the above "C 1-6 Alkylene" refers to a chemical bond and the above "C 0-4 Alkylene" refers to a chemical bond and the above "C 1-4 Alkylene" refers to a chemical bond and the above "C

[0113] The term "the total length of variable A and variable B is x carbon atoms" means the sum of the number of carbon atoms in the main chain of the group represented by variable A and the number of carbon atoms in the main chain of the group represented by variable B is x.

[0114] "Halo" or "halogen" refers to fluoro (F), chloro (Cl), bromo (Br), and iodo (I).

[0115] Thus, "C 1-10 Haloalkyl" refers to the above "C 1-10 Alkyl" groups that are substituted with one or more halo groups. In some embodiments, C 1-8 Haloalkyl, C 1-6 Haloalkyl, C 1-4 Haloalkyl, C 1-3 Haloalkyl is particularly preferred, more preferably C 1-2 ​Halogenated alkyl groups. Exemplary alkyl halogenated groups include, but are not limited to: -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCl3, -CH2Cl, -CHCl2, 2,2,2-trifluoro-1,1-dimethyl-ethyl, etc. The alkyl halogenated group can be substituted at any available connection point, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0116] “C 3-14 "Cycloalkyl" or "3- to 14-membered cycloalkyl" refers to a non-aromatic cyclic hydrocarbon group having 3 to 14 ring carbon atoms and zero heteroatoms, optionally containing 1, 2, or 3 double or triple bonds. In some embodiments, 3- to 10-membered cycloalkyl, 5- to 10-membered cycloalkyl, 3- to 8-membered cycloalkyl, 3- to 7-membered cycloalkyl, and 3- to 6-membered cycloalkyl are particularly preferred, more preferably 5- to 7-membered cycloalkyl, 4- to 6-membered cycloalkyl, 3- to 5-membered cycloalkyl, 3- to 4-membered cycloalkyl, and 5- to 6-membered cycloalkyl, more preferably 5-membered cycloalkyl, more preferably 6-membered cycloalkyl, and more preferably cyclopropyl. Cycloalkyl also includes a cyclic system in which the aforementioned cycloalkyl ring is fused with one or more aryl or heteroaryl groups, wherein the bonding point is on the cycloalkyl ring, and in such cases, the number of carbons continues to indicate the number of carbons in the cycloalkyl system. Cycloalkyl also includes a cyclic system in which the aforementioned cycloalkyl ring, Substituents on any non-adjacent carbon atoms are linked together to form a bridged ring, forming a polycyclic alkane sharing two or more carbon atoms. Cycloalkyl groups also include the aforementioned cycloalkyl rings, where substituents on the same carbon atom are linked together to form a ring, forming a polycyclic alkane sharing one carbon atom. Exemplary cycloalkyl groups include, but are not limited to: cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cyclohepttrienyl (C7), etc. The cycloalkyl group may be optionally substituted with one or more substituents, for example, substituted with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0117] “C 3-14 "Cycloalkylene" refers to the alkylene oxide that has had C removed. 3-14 The cycloalkyl group is a divalent group formed by another hydrogen atom, and can be substituted or unsubstituted. In some embodiments, C 3-10 Cycloalkylene, C 3-7 Cycloalkylene, C 3-6 Cycloalkylene, C 3-5 Cycloalkylene and C 3-4 Cycloalkylene compounds are particularly preferred, especially cyclopropylene compounds.

[0118] "3-14 membered heterocyclyl" or "3 to 14 membered heterocyclyl" refers to a saturated or unsaturated radical of a 3 to 14 membered non-aromatic ring system having ring carbon atoms and 1 to 5 ring heteroatoms, wherein each heteroatom is independently selected from the group consisting of nitrogen, oxygen, sulfur, boron, phosphorus, and silicon, wherein optionally containing 1, 2, or 3 double or triple bonds. In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valence permits. In some embodiments, 3 to 10 membered heterocyclyl is preferred, which is a 3 to 10 membered non-aromatic ring system having ring carbon atoms and 1 to 5 ring heteroatoms; in some embodiments, 5 to 10 membered heterocyclyl is preferred, which is a 5 to 10 membered non-aromatic ring system having ring carbon atoms and 1 to 5 ring heteroatoms; in some embodiments, 3 to 8 membered heterocyclyl is preferred, which is a 3 to 8 membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms; in some embodiments, 3 to 7 membered heterocyclyl is preferred, which is a 3 to 7 membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms; 5 to 7 membered heterocyclyl is preferred, which is a 5 to 7 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms; 3 to 6 membered heterocyclyl is preferred, which is a 3 to 6 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms; 4 to 6 membered heterocyclyl is preferred, which is a 4 to 6 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms; 5 to 6 membered heterocyclyl is more preferred, which is a 5 to 6 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms; 5 membered heterocyclyl is preferred, which is a 5 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms; 6 membered heterocyclyl is preferred, which is a 6 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms. Heterocyclyl also includes ring systems in which the above heterocyclyl ring is fused with one or more cycloalkyl rings, wherein the point of attachment is on the heterocyclyl ring, or with one or more aryl or heteroaryl rings, wherein the point of attachment is on the heterocyclyl ring; and in such cases the number of ring members continues to refer to the number of ring members in the heterocyclyl ring system. Heterocyclyl also includes ring systems in which the above heterocyclyl ring, wherein any non-adjacent carbon or nitrogen atoms on which substituents are attached form a bridge ring, together form a polycycloalkyl that shares two or more carbon or nitrogen atoms. Heterocyclyl also includes ring systems in which the above heterocyclyl ring, wherein substituents on the same carbon atom are connected to form a ring, together form a polycycloalkyl that shares one carbon atom. Exemplary 3 membered heterocyclyl groups containing one heteroatom include, but are not limited to: aziridinyl, oxiranyl, thiorenyl. Exemplary 4 membered heterocyclyl groups containing one heteroatom include, but are not limited to: azetidinyl, oxetanyl, and thietanyl. Exemplary 5 membered heterocyclyl groups containing one heteroatom include, but are not limited to: tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione.Exemplary 5-membered heterocyclic groups containing two heteroatoms include, but are not limited to: pyrazolyl, dioxasulfuranyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclic groups containing three heteroatoms include, but are not limited to: triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclic groups containing one heteroatom include, but are not limited to: piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclic groups containing two heteroatoms include, but are not limited to: piperazinyl, morpholinyl, disulfuranyl, and dioxalyl. Exemplary 6-membered heterocyclic groups containing three heteroatoms include, but are not limited to: triazinanyl. Exemplary 7-membered heterocyclic groups containing one heteroatom include, but are not limited to: azirheptanyl, oxasulfuranyl, and thioheptanyl. Exemplary 5-membered heterocyclic groups fused to a C6 aryl ring (also referred to herein as 5,6-bicyclic heterocyclic groups) include, but are not limited to: dihydroindolyl, isodihydroindolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, benzoxazolinoneyl, etc. Exemplary 6-membered heterocyclic groups fused to a C6 aryl ring (also referred to herein as 6,6-bicyclic heterocyclic groups) include, but are not limited to: tetrahydroquinolinyl, tetrahydroisoquinolinyl, etc. Heterocyclic groups also include those that share one or two atoms with a cycloalkyl, heterocyclic, aryl, or heteroaryl group to form a bridged or spirocyclic ring, wherein the shared atom may be a carbon or nitrogen atom, provided that the valence allows. Heterocyclic groups also include those that can be optionally substituted with one or more substituents, for example, substituted with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0119] “C 6-10 "Aryl" refers to a monocyclic or polycyclic (e.g., bicyclic) 4n+2 aromatic ring system (e.g., having 6 or 10 shared π electrons arranged in a ring) having 6-10 ring carbon atoms and zero heteroatoms. In some embodiments, the aryl group has six ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, the aryl group has ten ring carbon atoms ("C6 aryl"). 10 "Aryl"; for example, naphthyl, such as 1-naphthyl and 2-naphthyl). Aryl also includes a ring system in which the above-mentioned aryl ring is fused with one or more cycloalkyl or heterocyclic groups, and the connection point is on the aryl ring, in which case the number of carbon atoms continues to represent the number of carbon atoms in the aryl ring system. The aryl group may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0120] "5-14 membered heteroaryl" or "5 to 14 membered heteroaryl" refers to a radical of a 5-14 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur. In heteroaryl groups containing one or more nitrogen atoms, the point of attachment can be a carbon or a nitrogen atom, as valence permits. Heteroaryl bicyclic systems can include one or more heteroatoms in one or both rings. Heteroaryl also includes ring systems wherein an above-described heteroaryl ring is fused with one or more cycloalkyl or heterocyclyl rings, and the point of attachment is on the heteroaryl ring, in which case the number of carbon atoms indicates the number of carbon atoms in the heteroaryl ring system. In some embodiments, 5-10 membered heteroaryl is preferred, which is a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms. In other embodiments, 5-6 membered heteroaryl is particularly preferred, which is a 5-6 membered monocyclic or bicyclic 4n+2 aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms. Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl (e.g., 1,2,4-oxadiazolyl), and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridinyl or pyridinonyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzoimidazolyl, benzoxazolyl, benzoisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzoisothiazolyl, benzothiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. A heteroaryl group can be optionally substituted with one or more substituents, e.g., with one to five substituents, one to three substituents, or one substituent.

[0121] "Optionally substituted" means that the group can be substituted with the indicated substituents or can be unsubstituted.

[0122] The divalent radical formed by removing two hydrogens from the above defined alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups is collectively referred to as an "alkylene radical." The ring- forming radicals of cycloalkyl, heterocyclyl, aryl, and heteroaryl are collectively referred to as "ring radicals."

[0123] The alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups defined herein are optionally substituted.

[0124] Exemplary substituents on a carbon atom include, but are not limited to: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR aa , -ON(R bb )2, -N(R bb )2, -N(R bb )3 + X - , -N(OR cc )R bb , -SH, -SR aa , -SSR cc , -C(=O)R aa , -CO2H, -CHO, -C(OR cc )2, -CO2R aa , -OC(=O)R aa , -OCO2R aa , -C(=O)N(R bb )2, -OC(=O)N(R bb )2, -NR bb C(=O)R aa , -NR bb CO2R aa , -NR bb C(=O)N(R bb )2, -C(=NR bb )R aa , -C(=NR bb )OR aa , -OC(=NR bb )R aa , -OC(=NR bb )OR aa , -C(=NR bb )N(R bb )2, -OC(=NR bb )N(R bb )2, -NR bb C(=NR bb )N(R bb )2, -C(=O)NR bb SO2R aa , -NRbb SO2R aa , -SO2N(R bb )2, -SO2R aa , -SO2OR aa , -OSO2R aa , -S(=O)R aa , -OS(=O)R aa , -Si(R aa )3, -OSi(R aa )3, -C(=S)N(R bb )2, -C(=O)SR aa , -C(=S)SR aa , -SC(=S)SR aa , -SC(=O)SR aa , -OC(=O)SR aa , -SC(=O)OR aa , -SC(=O)R aa , -P(=O)2R aa , -OP(=O)2R aa , -P(=O)(R aa )2, -OP(=O)(R aa )2, -OP(=O)(OR cc )2, -P(=O)2N(R bb )2, -OP(=O)2N(R bb )2, -P(=O)(NR bb )2, -OP(=O)(NR bb )2, -NR bb P(=O)(OR cc )2, -NR bb P(=O)(NR bb )2, -P(R cc )2, -P(R cc )3, -OP(R cc )2, -OP(R cc )3, -B(R aa )2, -B(OR cc )2, -BR aa (OR cc ), alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R dd groups;

[0125] or two geminal hydrogens on a carbon atom are replaced with a group =O, =S, =NN(R bb)2, =NNR bb C(=O)R aa , =NNR bb C(=O)OR aa , =NNR bb S(=O)2R aa , =NR bb or =NOR cc substituted;

[0126] R aa each independently is selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R aa groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R dd groups;

[0127] R bb each independently is selected from: hydrogen, -OH, -OR aa , -N(R cc )2, -CN, -C(=O)R aa , -C(=O)N(R cc )2, -CO2R aa , -SO2R aa , -C(=NR cc )OR aa , -C(=NR cc )N(R cc )2, -SO2N(R cc )2, -SO2R cc , -SO2OR cc , -SOR aa , -C(=S)N(R cc )2, -C(=O)SR cc , -C(=S)SR cc , -P(=O)2R aa , -P(=O)(R aa )2, -P(=O)2N(R cc )2, -P(=O)(NR cc )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R bb groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R dd groups;

[0128] R cceach R is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R groups combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R groups; cc each R is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R groups combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R groups; dd each R is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R groups combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R groups;

[0129] R dd each R is independently selected from: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR ee , -ON(R ff )2, -N(R ff )2, -N(R ff )3 + X - , -N(OR ee )R ff , -SH, -SR ee , -SSR ee , -C(=O)R ee , -CO2H, -CO2R ee , -OC(=O)R ee , -OCO2R ee , -C(=O)N(R ff )2, -OC(=O)N(R ff )2, -NR ff C(=O)R ee , -NR ff CO2R ee , -NR ff C(=O)N(R ff )2, -C(=NR ff )OR ee , -OC(=NR ff )R ee , -OC(=NR ff )OR ee , -C(=NR ff )N(R ff )2, -OC(=NR ff )N(R ff )2, -NR ff C(=NR ff )N(R ff )2, -NR ff SO2R ee , -SO2N(R ff )2, -SO2R ee , -SO2OR ee , -OSO2R ee-S(=O)R ee -Si(R ee )3, -OSi(R ee )3, -C(=S)N(R ff )2, -C(=O)SR ee , -C(=S)SR ee , -SC(=S)SR ee , -P(=O)2R ee , -P(=O)(R ee )2, -OP(=O)(R ee )2, -OP(=O)(OR ee )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R gg groups, or two geminal R dd substituents can be combined to form =O or =S;

[0130] each R ee is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R gg groups;

[0131] each R ff is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R ff groups combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R gg groups;

[0132] each R gg is independently halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OC 1-6 alkyl, -ON(C 1-6 alkyl)2, -N(C 1-6 alkyl)2, -N(C 1-6 alkyl)3 + X - , -NH(C 1-6 alkyl)2 + X - , -NH2(C 1-6 alkyl) + X - , -NH3+ X - , -N(OC 1-6 alkyl)(C 1-6 alkyl), -N(OH)(C 1-6 alkyl), -NH(OH), -SH, -SC 1-6 alkyl, -SS(C 1-6 alkyl), -C(=O)(C 1-6 alkyl), -CO2H, -CO2(C 1-6 alkyl), -OC(=O)(C 1-6 alkyl), -OCO2(C 1-6 alkyl), -C(=O)NH2, -C(=O)N(C 1-6 alkyl)2, -OC(=O)NH(C 1-6 alkyl), -NHC(=O)(C 1-6 alkyl), -N(C 1-6 alkyl)C(=O)(C 1-6 alkyl), -NHCO2(C 1-6 alkyl), -NHC(=O)N(C 1-6 alkyl)2, -NHC(=O)NH(C 1-6 alkyl), -NHC(=O)NH2, -C(=NH)O(C 1-6 alkyl), -OC(=NH)(C 1-6 alkyl), -OC(=NH)OC 1-6 alkyl, -C(=NH)N(C 1-6 alkyl)2, -C(=NH)NH(C 1-6 alkyl), -C(=NH)NH2, -OC(=NH)N(C 1-6 alkyl)2, -OC(NH)NH(C 1-6 alkyl), -OC(NH)NH2, -NHC(NH)N(C 1-6 alkyl)2, -NHC(=NH)NH2, -NHSO2(C 1-6 alkyl), -SO2N(C 1-6 alkyl)2, -SO2NH(C 1-6 alkyl), -SO2NH2, -SO2C 1-6 alkyl, -SO2OC 1-6 alkyl, -OSO2C 1-6 alkyl, -SOC 1-6 alkyl, -Si(C 1-6 alkyl)3, -OSi(C 1-6 alkyl)3, -C(=S)N(C 1-6 alkyl)2, C(=S)NH(C 1-6alkyl), -C(=S)SC 1-6 alkyl, -C(=S)SC 1- 6alkyl, -SC(=S)SC 1-6 alkyl, -P(=O)2(C 1-6 alkyl), -P(=O)(C 1-6 alkyl)2, -OP(=O)(C 1-6 alkyl)2, -OP(=O)(OC 1-6 alkyl)2, C 1-6 alkyl, C 1-6 haloalkyl, C2-C6alkenyl, C2-C6alkynyl, C3-C7cycloalkyl, C6-C 10 aryl, 3-7 membered heterocyclyl, 5-10 membered heteroaryl; or two geminal R gg substituents can combine to form =O or =S; wherein X - is a counterion.

[0133] Exemplary substituents on a nitrogen atom include, but are not limited to: hydrogen, -OH, -OR aa , -N(R cc )2, -CN, -C(=O)R aa , -C(=O)N(R cc )2, -CO2R aa , -SO2R aa , -C(=NR bb )R aa , -C(=NR cc )OR aa , -C(=NR cc )N(R cc )2, -SO2N(R cc )2, -SO2R cc , -SO2OR cc , -SOR aa , -C(=S)N(R cc )2, -C(=O)SR cc , -C(=S)SR cc , -P(=O)2R aa , -P(=O)(R aa )2, -P(=O)2N(R cc )2, -P(=O)(NR cc )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R cc groups attached to a nitrogen atom combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd R is a group substituted, and wherein R aa R is a group substituted, and wherein R bb R is a group substituted, and wherein R cc R is a group substituted, and wherein R dd As described above.

[0134] “Nucleic acid molecule” refers to a single- or double-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecule and hybrid molecules thereof. Examples of nucleic acid molecules include, but are not limited to, messenger RNA (mRNA), microRNA (miRNA), small interfering RNA (siRNA), self-amplifying RNA (saRNA), and antisense oligonucleotides (ASO), etc. The nucleic acid can be further chemically modified, the chemical modification is selected from one of pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 5-methylcytosine, or a combination thereof. The mRNA molecule contains a protein coding region, and can further contain expression regulatory sequences, typical expression regulatory sequences include, but are not limited to, 5' cap, 5' untranslated region (5'UTR), 3' untranslated region (3'UTR), polyadenylation sequence (PolyA), miRNA binding site.

[0135] “Ionizable lipid” refers to lipids that are ionizable, such that they can exist in a positively charged or neutral form depending on the pH. In some embodiments, the ionizable lipid is an amino lipid.

[0136] “Neutral lipid” refers to a lipid molecule that is not charged under certain pH conditions, for example physiological pH conditions. Examples of neutral lipids include, but are not limited to, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE).

[0137] “Structural lipid” refers to a lipid that fills the gap between lipids to enhance the stability of the nanoparticle, commonly as a sterol. A sterol is a compound having a cyclopenta-perhydrophenanthrene-like carbon skeleton, in one preferred embodiment, the sterol is selected from cholesterol, sitosterol, coprostanol, stigmastanol, brassicasterol, ergosterol, tomatidine, ursolic acid, a-tocopherol, stigmasterol, avenasterol, ergocalciferol, or campesterol.

[0138] "Polymer lipid" refers to a molecule that contains a polymer moiety and a lipid moiety. In some embodiments, the polymer lipid is a polyethylene glycol (PEG) lipid. Other lipids that are capable of reducing aggregation, such as the product of coupling a compound having no charge, hydrophilicity, a steric blocking moiety to a lipid, can also be used.

[0139] "Lipid nanoparticle" refers to a particle that contains a lipid component and has a nanoscale size.

[0140] "Biodegradable group" refers to a functional group that contains a biodegradable bond, such as an ester, disulfide bond, and amide, among others. Biodegradation can affect the process of clearing a compound from the body. The direction of the biodegradable group of the present application is from the head to the tail of the ionizable lipid molecule.

[0141] Other definitions

[0142] As used herein, the terms "polynucleotide," "nucleic acid sequence," "nucleotide sequence," or "nucleic acid fragment" are used interchangeably and are single-stranded or double-stranded RNA or DNA polymers, optionally containing synthetic, non-natural, or altered nucleotide bases. Nucleotides are referred to by their single letter designation: "A" is adenine or deoxyadenosine (corresponding to RNA or DNA, respectively), "C" is cytosine or deoxycytosine, "G" is guanine or deoxyguanosine, "U" is uridine, "T" is deoxythymidine, "R" is purine (A or G), "Y" is pyrimidine (C or T), "K" is G or T, "H" is A or C or T, "I" is inosine, and "N" is any nucleotide. Although nucleotide sequences herein can be represented in DNA sequence (containing T), the corresponding RNA sequence (i.e., with U in place of T) can be readily determined by one of skill in the art when referring to RNA.

[0143] "Polypeptide," "peptide," and "protein" are used interchangeably herein in reference to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms "polypeptide," "peptide," "amino acid sequence," and "protein" can also include modified forms, including but not limited to glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation.

[0144] The word "comprising" is used herein to mean that the protein or nucleic acid can consist of the sequence recited, or can have additional amino acids or nucleotides at either or both ends, but still have the activity recited in the present application.

[0145] “Sequence identity” between two polypeptide sequences or between two polynucleotide sequences refers to the percentage of identical amino acids or nucleotides between the sequences. Methods to assess the level of sequence identity between polypeptide or polynucleotide sequences are known in the art. Sequence identity can be assessed using various sequence analysis software known. For example, sequence identity can be assessed by the online alignment tool of EMBL-EBI (https: / / www.ebi.ac.uk / Tools / psa / ). Sequence identity between two sequences can be assessed using the Needleman-Wunsch algorithm, using default parameters. Sequence identity can be over the full length of a given sequence.

[0146] “mRNA” or “messenger RNA” are used interchangeably herein to refer to a single-stranded ribonucleic acid molecule that carries genetic information capable of directing protein synthesis within a cell. An mRNA typically comprises a 5’ UTR, a protein-coding sequence (CDS), and a “3’ UTR”. An mRNA can also comprise a poly(A) sequence at the 3’ end and a “5’ cap”. An mRNA is generally linear, however the term can also encompass covalently closed circular RNA molecules.

[0147] As used herein, “5’ UTR” generally refers to the sequence of an mRNA molecule between the 5’ end and the translation initiation codon, which is capable of recruiting ribosomal complexes and initiating translation of the mRNA. The 5’ UTR regulates processes such as post-transcriptional modifications, formation and stability of the translation initiation complex, by interacting with transcription factors, ribosomes, and other transcriptional regulatory proteins. The term “5’ UTR” also encompasses ribosome entry site (IRES) sequences, in particular for circular RNA molecules.

[0148] As used herein, “3’ UTR” refers to the sequence of an mRNA between the stop codon of the polypeptide-coding sequence and the poly(A) sequence. The 3’ UTR can regulate translation of the mRNA by interacting with mRNA-binding proteins, miRNAs, and the like. Sequence and structural features of the 3’ UTR can influence mRNA stability, ribosome scanning, and formation of the translation termination complex, thereby influencing the expression level of the protein.

[0149] A poly(A) sequence typically comprises a plurality of adenine nucleotides. The addition of a poly(A) sequence contributes to the stability and transport of the mRNA, preventing its degradation, and plays an important role in the post-transcriptional modification process. The poly(A) sequence can be a continuous chain of pure adenine nucleotides, but can also be a variant comprising non-adenine nucleotides, as long as it is functionally equivalent to a conventional poly(A) sequence, i.e. is able to provide similar biological functions as a natural poly(A) sequence, such as affecting the stability, translation efficiency or ribosome binding of the mRNA. Known poly(A) sequences are, for example, the human growth hormone (hGH) poly(A) sequence and the simian virus 40 (SV40) poly(A) sequence. These variants can differ in the nucleotide composition, but are functionally identified as equivalent to a conventional poly(A) sequence.

[0150] As used herein, a "5' cap" for an RNA includes the 5' cap structure present on a natural mRNA as well as analogs thereof. The 5' cap structure on a natural mRNA refers to a methylated guanylate linked via a pyrophosphate to the 5' terminal nucleotide of the RNA, forming a 5',5'-triphosphate linkage. There are generally three types of 5' caps (m7G5'ppp5'Np, m7G5'ppp5'NmpNp, m7G5'ppp5'NmpNmpNp), referred to as Cap0, Cap1 and Cap2, respectively. Cap0 refers to the terminal nucleotide's ribose being unmethylated, Cap1 refers to the terminal one nucleotide's ribose being methylated, and Cap2 refers to both terminal nucleotides' riboses being methylated.

[0151] Methods for capping mRNA molecules are known in the art. The 5' cap structure of the mRNA molecule can be added using an enzymatic reaction after the mRNA molecule has been obtained by chemical synthesis or in vitro transcription (e.g. by using a commercial kit comprising a vaccinia capping enzyme and a mRNA cap structure 2'-0-methyltransferase). However, it is also possible to produce a mRNA with a cap structure by directly incorporating a nucleotide analogue with a cap structure as the first nucleotide into the transcript during in vitro transcription.

[0152] As used herein, "antibody" refers to immunoglobulins and immunoglobulin fragments, whether naturally occurring or produced in whole or in part synthetically (e.g., recombinantly), including any fragment thereof that retains the ability of a full-length immunoglobulin to bind with specificity to an immunoglobulin antigen. Thus, antibodies include any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen binding domain (antibody binding site). Antibodies include antibody fragments, such as anti-tumor cell antibody fragments. As used herein, the term antibody thus includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intrabodies, and antibody fragments, such as, but not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), bispecific antibodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above. Antibodies provided herein include members of any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass (e.g., IgG2a and IgG2b).

[0153] As used herein, a variable domain or variable region is a specific Ig domain of an antibody heavy or light chain that comprises an amino acid sequence that varies between different antibodies. Each light chain and each heavy chain has one variable region domain, VL and VH, respectively. The variable domains provide antigen specificity and are thus responsible for antigen recognition. Each variable region comprises CDRs and framework regions (FRs), the CDRs being part of the antigen binding site domain.

[0154] As used herein, "hypervariable region," "HV," "complementarity determining region," and "CDR," and "antibody CDR," are used interchangeably to refer to one of a plurality of portions within each variable region that together form the antigen binding site of an antibody. Each variable region domain comprises 3 CDRs, designated CDR1, CDR2, and CDR3. For example, a light chain variable region domain comprises 3 CDRs, designated VL CDR1, VL CDR2, and VL CDR3; a heavy chain variable region domain comprises 3 CDRs, designated VH CDR1, VH CDR2, and VH CDR3. The 3 CDRs in a variable region are not contiguous along the linear amino acid sequence, but are proximate in a folded polypeptide. The CDRs are located within loops connecting the beta sheets of the variable domain. As described herein, one of skill in the art knows and can identify CDRs based on Kabat or Chothia numbering (see, e.g., Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917).

[0155] As used herein, a framework region (FR) is a domain within an antibody variable region domain that lies within the beta sheets; FR regions are relatively more conserved than hypervariable regions in terms of amino acid sequence.

[0156] Bispecific antibody (BsAb) refers to an artificial antibody that can specifically bind to two antigens or antigen epitopes at the same time. "BiTE" (Bispecific T cell Engager) refers to a bispecific antibody in which the antigen binding region of an antibody specific to an antigen of a target cell and the antigen binding region of an antibody specific to CD3 are connected in series by a linker, which functions as a "bridge" between a T cell and a target cell, mediates T cell targeted recognition and specific killing of target cells. The target cell is, for example, a tumor cell. The molecular weight of "BiTE" is about 55-60 kDa.

[0157] Triomabs, Knobs-into-holes, Crossmab, Ortho-Fab, DVD-Ig, Two-in-one, IgG-scFv, scFv2-Fc, bi-Nanobody, DART or TandAb are different structural types of bispecific antibodies, each of which has a known structural definition in the prior art. As a "DART" (dual affinity retargeting) bispecific antibody is a heterodimeric antibody formed by two polypeptide chains, the structure of which is to link the VH and VL sequences of one antibody variable region with the VL and VH sequences of another antibody variable region, respectively. In addition, a cysteine is introduced at the C-terminus of the two polypeptide chains, and an interchain disulfide bond is formed by the cysteine to improve the stability of the product. Generally speaking, DART bispecific antibodies have a relatively short half-life due to their relatively small molecular weight and the absence of Fc fragments, for example, the half-life of a DART molecule such as MGD006 can be only a few hours.

[0158] "TandAb" (tandem diabody) is a class of tetravalent bispecific antibodies, which contains 4 scFv domains. TandAbs are formed by two peptide chains, in which the N-terminal to C-terminal of each peptide chain is arranged in the order of VL1-VH2-VL2-VH1, and the two peptide chains are reversely paired to form a homodimeric molecule. The relative molecular mass of TandAbs is about 110 kD, which is between that of whole molecule antibodies and BiTEs (about 50 kD), and the half-life of the product can reach 23 hr.

[0159] "VHH" is derived from the variable region of heavy chain antibodies (HcAb) of Camelidae (such as camels, llamas, etc.), which naturally lack light chains, so VHH has unique structural and functional properties. VHH bispecific antibodies are formed by genetically engineering two VHH monomers against different antigens or epitopes. The molecular weight is small and the half-life is short. Nanobodies are rapidly cleared from the body by glomerular filtration, and the limit of glomerular filtration of protein drugs is generally around 60 kDa, that is, small molecular weight antibody fragments or derivatives, such as nanobodies (15 kDa) and scFv (28 kDa) and even Fab (50 kDa), will be cleared from the body by glomerular filtration, resulting in a relatively short half-life. Among them, nanobodies have the smallest molecular weight among all antibody types, and their half-life is often only a few tens of minutes. Currently, nanobody drugs entering clinical trials or already on the market mainly use fusion Fc, HSA or HSA nanobodies to extend the half-life, for example, the half-life of Caplacizumab is about 10-30 hr.

[0160] “Triomabs” bispecific antibodies are a class of antibodies that can bind to two different antigens simultaneously, prepared by a special technique. The structural feature is that CD3 specific rat IgG2b antibody and tumor targeting mouse IgG2a antibody are hybridized by somatic cells to obtain antibody molecules with bispecificity. The half-life of Triomabs bispecific antibodies is usually longer, because it retains the Fc functional region, which can prolong the circulation time of the antibody in the body by binding to the FcRn receptor.

[0161] Half-life generally refers to the time required for the concentration of a drug in the plasma to decrease to half after the injection is completed. The half-life of the proteins described herein can be determined using any known method in the art. For example, the half-life of a protein can be determined by measuring the concentration of the protein in the serum of a mouse at different time points after the protein is injected intravenously (IV) into the tail vein of the mouse. The formula for calculating the half-life of a protein includes: half-life = 0.693 / Lambda Z, half-life = 0.693*Vss / C, etc., where Vss is the apparent distribution volume and CL is the clearance rate. The half-life of bispecific antibodies (BsAbs) can vary due to various factors, including the molecular structure of the antibody, whether it contains an Fc fragment, the administration method, species differences, etc.

[0162] The in vivo half-lives of different types of natural proteins usually vary widely. For example, the in vivo half-life of a cytokine is usually only a few minutes to a few hours (e.g., 3-5 minutes), and the in vivo half-life of an immunoglobulin (e.g., IgG) is usually 1-2 weeks or longer. In the case of the present application, when the load contained in the lipid nanoparticle is an mRNA encoding a secreted protein, the in vivo half-life of the secreted protein obtained by administering the lipid nanoparticle or a pharmaceutical composition comprising the lipid nanoparticle via a systemic administration route is longer than the in vivo half-life of the secreted protein directly administered via a systemic administration route, preferably 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold or more, more preferably 10-fold or more. This is the effect brought about by the in vivo delivery of the mRNA of the secreted protein by the lipid nanoparticle of the present application.

[0163] As used herein, “vector” refers to a piece of DNA extracted from a virus, plasmid, or cell of a higher organism into which a foreign DNA fragment can be or has been inserted for cloning and / or expression purposes. In certain embodiments, a vector can be stably maintained in an organism. A vector can comprise, for example, an origin of replication, a selectable marker or reporter gene, such as antibiotic resistance or GFP, and / or a multiple cloning site (MCS). The term includes linear DNA fragments (e.g., PCR products, linear plasmid fragments), plasmid vectors, viral vectors, cosmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and the like.

[0164] The term "treatment" as used herein refers to reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. The term "treatment" as used herein refers to the act of treating as defined immediately above.

[0165] The term "improving the prognosis of a patient" as used herein refers to preventing and / or treating the progression of a disease disorder or condition that has been treated, the recurrence of such, further treatment of residual lesions or improving the quality of life of the patient, etc. The term "pharmaceutically acceptable salt" as used herein refers to those organic or inorganic salts of the compounds of the present application which are adapted for use in contact with the tissues of patients without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio, and effective for their intended use, including, where possible, the zwitterionic forms of the compounds of the present application.

[0166] Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals' hydroxides or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, and procaine.

[0167] Base addition salts of acidic compounds can be prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt. The free acid form can be regenerated by contacting the salt form with a sufficient amount of an acid to produce the free acid form. The free acid and base forms can be regenerated by the application of standard techniques of the art, by in situ salt formation. The free acid form differs from the salt form somewhat in certain physical properties, such as solubility in polar solvents, but otherwise the salt and free acid forms are equivalent for the purposes of the present application.

[0168] The salt can be a sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide salt prepared from inorganic acids such as hydrochloric acid, nitric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, and the like. Representative salts include: hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthoate, mesylate, glucoheptonate, lactobionate, laurylsulphonate, and isethionate, and the like. The salt can also be prepared from organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkyldioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Representative salts include acetate, propionate, caprylate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, naphthoate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate, mesylate, and the like. Pharmaceutically acceptable salts can include cations based on alkali and alkaline earth metals, for example, sodium, lithium, potassium, calcium, magnesium, and aluminum, as well as ammonium, quaternary ammonium, and amine cations such as, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Salts of amino acids can also be encompassed (e.g., arginate, gluconate, galacturonate, and the like (see, e.g., Berge S.M. et al., "Pharmaceutical Salts", J. Pharm. Sci., 1977; 66: 1-19, which is incorporated herein by reference).

[0169] Pharmaceutically acceptable excipient refers to a substance that is adjuvant to the active ingredients in a pharmaceutical formulation, also known as an auxiliary agent.

[0170] A "subject" includes, but is not limited to, a human (i.e., a male or female of any age group, 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 a non-human animal, e.g., a mammal, e.g., a primate (e.g., a cynomolgus monkey, a rhesus monkey), a cow, a pig, a horse, a sheep, a goat, a rodent, a cat, and / or a dog. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. The terms "human," "patient," and "subject" are used interchangeably herein.

[0171] "Disease," "disorder," and "condition" are used interchangeably herein.

[0172] The term "treatment" as used herein includes an action that occurs while the subject is suffering from a particular disease, disorder or condition, that reduces the severity of the disease, disorder or condition, or retards or slows the progression of the disease, disorder or condition ("therapeutic treatment"), and also includes an action that occurs before the subject begins to suffer from a particular disease, disorder or condition ("prophylactic treatment").

[0173] Generally, an "effective amount" of a pharmaceutical composition refers to a quantity sufficient to achieve a desired biological result. As will be understood by those of ordinary skill in the art, the effective amount of a pharmaceutical composition of the present application can vary depending on such factors as the biological endpoint, the pharmacokinetics of the pharmaceutical composition, the disease being treated, the mode of administration, and the age, health, and symptoms of the subject. An effective amount includes both a therapeutically effective amount and a prophylactically effective amount.

[0174] The term "therapeutically effective amount" of a pharmaceutical composition as used herein, unless otherwise indicated, is an amount that is sufficient to provide a therapeutic benefit in the treatment of a disease, disorder, or condition, or to delay or minimize one or more symptoms associated with the disease, disorder, or condition. A therapeutically effective amount of a pharmaceutical composition refers to the amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment of a disease, disorder, or condition. The term "therapeutically effective amount" can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of disease or condition, or enhances the therapeutic effect of another therapeutic agent.

[0175] The term "prophylactically effective amount" of a pharmaceutical composition as used herein, unless otherwise indicated, is an amount that is sufficient to prevent a disease, disorder, or condition, or to prevent one or more symptoms that are associated with the disease, disorder, or condition, or to prevent the recurrence of a disease, disorder, or condition. A prophylactically effective amount of a pharmaceutical composition refers to the amount of therapeutic agent alone, or in combination with other agents, that provides a prophylactic benefit in the prevention of a disease, disorder, or condition. The term "prophylactically effective amount" can encompass an amount that improves overall prophylaxis, or enhances the prophylactic effect of another prophylactic agent.

[0176] "Combination" and related terms refer to administration of a pharmaceutical composition of the present application and another therapeutic agent simultaneously or sequentially. For example, a pharmaceutical composition of the present application can be administered simultaneously or sequentially with another therapeutic agent in separate unit dosage forms, or administered simultaneously with another therapeutic agent in a single unit dosage form.

[0177] The code of each experimental group in this paper, such as "Lipid5-LNP" represents the LNP prepared by using ionizable lipid Lipid5; "Lipid5-15#bispecific antibody mRNA" or "Lipid5-15#mRNA" both represent that the ionizable lipid used for preparing LNP in this group is Lipid5, and the 15#bispecific antibody mRNA is loaded, and the sequence of the 15#bispecific antibody mRNA corresponds to SEQ ID NO: 16 in the sequence listing; "MTS001-LNP Empty" represents that the ionizable lipid used for preparing LNP is MTS001, and the LNP does not load nucleic acid molecules.

[0178] The skilled person will understand that numerical values or numerical ranges herein generally include all values and ranges of values between the stated numerical values, e.g. 15% means all values within the range of 12% to 18%.

[0179] Embodiments

[0180] In one aspect, the present application provides a lipid nanoparticle for delivering a nucleic acid molecule encoding a secreted protein, comprising an ionizable lipid, a structural lipid, a neutral lipid, a polymeric lipid, wherein the ionizable lipid is a compound of Formula (II), or a pharmaceutically acceptable salt, isotopologue, tautomer, or stereoisomer thereof,

[0181] wherein,

[0182] a = 1, 2, 3, 4, 5, or 6;

[0183] b = 4, 5, 6, 7, 8, 9, or 10;

[0184] c = 1, 2, 3, 4, 5, or 6;

[0185] d = 0, 1, 2, 3, or 4;

[0186] c + d = 3, 4, 5, 6, 7, 8, or 9;

[0187] M1and M2are independently selected from -C(O)O-, -OC(O)-, -O-, -SC(O)O-, -OC(O)NR-, -NRC(O)NR-, -OC(O)S-, -OC(O)O-, -NRC(O)O-, -SC(O)-, -C(O)S-, -NR-, -C(O)NR-, -NRC(O)-, -NRC(O)S-, -SC(O)NR-, -C(O)-, -OC(S)-, -C(S)O-, -OC(S)NR-, -NRC(S)O-, -S-S-, and -S(O) 0-2 -;

[0188] R1 and R2 are independently selected from C 4-20 Alkyl, C 4-20 alkenyl and C 4-20 Alkyne group, which is optionally surrounded by one or more R groups 1s Substitution, and one or more methylene units therein are optionally and independently substituted with -NR'-;

[0189] R 1s Independently selected from H and C 1-20 Alkyl, -L c -OR c -L c -SR c and -L c -NR c R' c ;

[0190] R and R' are each independently selected from H and C. 1-20 alkyl;

[0191] L c Independently selected from chemical bonds and C 1-20 Alkylene;

[0192] R c and R' c Independently selected from H and C 1-20 Alkyl, C 3-14 cycloalkyl groups and 3 to 14-membered heterocyclic groups;

[0193] R4 and R5 are independently selected from C 1-8 Alkyl groups, which are optionally composed of one or more R groups 4s replace;

[0194] Or R4, R5, together with the carbon atoms they are attached to, form C. 3-14 Cycloalkyl or 3 to 14-membered heterocyclic group, optionally surrounded by one or more R 4s replace;

[0195] R 4s Independently selected from H, halogen, cyano, C 1-8 Alkyl, C 1-8 Halogenated alkyl, -L d -OR d -L d -SR d and -L d -NR d R' d ;

[0196] L d Independently selected from chemical bonds and C 1-8 Alkylene;

[0197] Rd and R' d are independently selected from H, C 1-8 alkyl, C 3-14 cycloalkyl and 3- to 14-membered heterocyclyl.

[0198] In some embodiments, R1and R2are independently selected from the following groups: -(CH2)5CH3, -(CH2)6CH3, -(CH2)7CH3, -(CH2)8CH3, -(CH2)9CH3, -(CH2) 10 CH3, -(CH2) 11 CH3, -CH2-C≡C-(CH2)5CH3, -CH2-C≡C-(CH2)6CH3, -(CH2)2-C≡C-(CH2)5CH3, -(CH2)4-C≡C-(CH2)3CH3, -CH2-CH=CH-(CH2)5CH3, -CH2-CH=CH-(CH2)6CH3, -(CH2)2-CH=CH-(CH2)5CH3, -(CH2)4-CH=CH-(CH2)3CH3, -(CH2)5-CH=CH-CH2CH3,

[0199] In some embodiments, the above-mentioned compound of formula (II), or a pharmaceutically acceptable salt, isotopically enriched variant, tautomer, or stereoisomer thereof, wherein,

[0200] a = 2, 3 or 4; preferably a = 2;

[0201] b = 7;

[0202] c = 4, 5 or 6; preferably c = 5;

[0203] d = 0, 1, 2, 3 or 4; preferably d = 1;

[0204] c + d = 5 or 6; preferably 6;

[0205] M1and M2are independently selected from -C(O)O- and -OC(O)-; or one of M1and M2is -C(O)O- and the other is -OC(O)-; preferably M1is -OC(O)- and M2is -C(O)O-;

[0206] R1and R2are independently selected from C 7-12 alkyl, preferably C 8-12 alkyl, optionally substituted with 1 R 1s ;

[0207] R 1s are independently selected from H and C 1-10 alkyl; preferably from H and C1-9 alkyl;

[0208] R4 and R5 are independently selected from C 1-3 Alkyl group, preferably methyl group.

[0209] In a preferred embodiment, M1 and M2 are independently selected from -C(O)O- or -OC(O)-, preferably, where M1 is -OC(O)- and M2 is -C(O)O-.

[0210] In another preferred embodiment, R4 and R5 are independently C 1-8 Alkyl, preferably C 1-3 Alkyl, more preferably methyl; or R4, R5 together with the carbon atoms they are attached to form C 3-6 Cycloalkylene or 3-6 membered heterocyclic alkylene, preferably forming C 3-4 Cycloalkylene compounds, preferably cyclopropylene compounds.

[0211] In another preferred embodiment, R1 and R2 are independently selected from C. 4-20 Alkyl group, preferably, R1 is C 6-14 Alkyl and R2 is C 8-20 Alkyl, more preferably, R1 is C 8-12 Straight-chain alkyl, R2 is C 8-20 Branched alkyl groups.

[0212] In another preferred embodiment, R1 is selected from C 8-11 Straight-chain alkyl, preferably C 9-10 Straight-chain alkyl groups.

[0213] In another preferred embodiment, R2 is selected from... Preferred

[0214] In another preferred embodiment, R2 is selected from... Preferred

[0215] In another preferred embodiment, a is 2, b is 7, c is 5, and d is 1.

[0216] In another preferred embodiment, R1 is selected from C 8-11 Straight-chain alkyl, preferably C 9-10 Straight-chain alkyl, preferably C9 straight-chain alkyl;

[0217] R2 is selected from C 8-11 Alkyl, preferably C 9-10 Alkyl groups, preferably C9 alkyl groups, optionally marked with one R 1s replace;

[0218] R1s independently selected from C 7-9 alkyl, preferably C7alkyl;

[0219] More preferably, R2is selected from preferably

[0220] preferably,

[0221] R1is C 10 straight chain alkyl;

[0222] R2is selected from C 8-11 alkyl, preferably C 9-10 alkyl, preferably C 10 alkyl, optionally substituted with 1 R 1s substituent;

[0223] R 1s independently selected from C 7-9 alkyl, preferably C9alkyl;

[0224] preferably R 1s is selected from C 8-9 alkyl, preferably C9alkyl;

[0225] More preferably, R2is selected from preferably

[0226] In some embodiments, the ionizable lipid is a compound of Formula (II-1), or a pharmaceutically acceptable salt, isotopically enriched variant, tautomer, or stereoisomer thereof:

[0227] M1and M2are independently selected from -C(O)O- and -OC(O)-; preferably one of M1and M2is -C(O)O- and the other is -OC(O)-; preferably M1is -OC(O)- and M2is -C(O)O-;

[0228] R1is selected from C 8-11 straight chain alkyl, preferably C 9-10 straight chain alkyl, preferably C9straight chain alkyl, preferably C 10 straight chain alkyl;

[0229] R2is selected from C 7-12 alkyl, preferably C 8-12 alkyl, preferably C 9-10 alkyl, optionally substituted with 1 R 1s substituent;

[0230] R 1s is independently selected from H and C 1-10 alkyl; preferably selected from H and C 1-9alkyl; preferably selected from C 6-9 alkyl; preferably selected from C 7-9 alkyl;

[0231] R4and R5are independently selected from C 1-3 alkyl, preferably methyl;

[0232] or R4, R5together with the carbon atom to which they are attached form a C 3-4 cycloalkylene, preferably cyclopropylene.

[0233] In a particularly preferred embodiment, the compound of formula (II) is selected from the following compounds of Table (I) or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof:

[0234] Table (I)

[0235] In some embodiments, the ionizable lipid is present in a molar percentage of 35-65% of the total lipids, preferably 40-55%.

[0236] In some embodiments, the ionizable lipid is present in a molar percentage of 40-65% of the total lipids, preferably 45-55%. In some embodiments, the ionizable lipid is present in a molar percentage of 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, or 55% of the total lipids.

[0237] In some embodiments, the structural lipid is selected from one or more of cholesterols, sitosterols, coprostanols, fecosterols, brassicasterols, ergosterols, tomatidine, ursolic acid, a-tocopherol, stigmasterol, avenasterol, ergocalciferol, and campesterol; preferably, the structural lipid is selected from cholesterols and / or b-sitosterols; more preferably, the structural lipid is cholesterols.

[0238] In some embodiments, the structural lipid is present in a molar percentage of 30-50% of the total lipids, preferably 35-50%, preferably 30-45%, more preferably 35-40%.

[0239] In a preferred embodiment, the structural lipid is present in a molar percentage of 30%, 32%, 35%, 35.5%, 36%, 36.5%, 37%, 37.5%, 38%, 38.5%, 39%, 39.5%, 40%, 40.5%, 41%, 41.5%, 42%, 42.5%, 43%, 43.5%, 44%, 44.5%, or 45% of the total lipids.

[0240] In some embodiments, the neutral lipid is selected from phosphatidylcholine and / or phosphatidylethanolamine.

[0241] Preferably, the phosphatidylcholine is selected from one or more of: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1.2-dioleoyl-sn-glycero-3-phosphocholine (DLPC), 1.2-ditridecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 2-((2,3-bis(oleoyloxy)propyl))dimethylammonio)ethyl hydrogen phosphate (DOCP), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), dimyristoyl phosphatidylethanolamine (DMPE), 1-palmitoyl-2-oleoyl phosphatidyl ethanolamine (POPE), and dipalmitoyl phosphatidyl ethanolamine (DPPE), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1.2-diolacyl-sn-glycero-3-phospho-(1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof, preferably DSPC.

[0242] In some embodiments, the neutral lipid is present in a molar percentage of 5-30% of the total lipid, preferably 5-20% or 5-15%, more preferably 8-15%.

[0243] In some embodiments, the neutral lipid is present in a molar percentage of 5.0%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 16%, 17%, 18%, 19%, or 20% of the total lipid.

[0244] In some embodiments, the polymeric lipid is a PEGylated lipid.

[0245] Preferably, the PEGylated lipid is selected from one or more of: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol;

[0246] Preferably, the pegylated lipid comprises a PEG moiety of 1000 Da to 20 kDa, preferably a PEG moiety of about 1000 Da to about 5000 Da;

[0247] Preferably, the pegylated lipid is selected from one or more of: DMPE-PEG1000, DPPE-PEG1000, DSPE-PEG1000, DOPE-PEG1000, DMG-PEG2000, Ceramide-PEG2000, DMPE-PEG2000, DPPE-PEG2000, DSPE-PEG2000, Azido-PEG2000, DSPE-PEG2000, DSPE-PEG2000-Mannose, DSPE-PEG2000 amine, DSPE-PEG3350, DSPE-PEG3500, DMG-PEG3500, DPPE-PEG3500, DSPE-PEG4000, DMG-PEG4000, Ceramide-PEG5000, DSPE-PEG5000, DMG-PEG5000, and ALC-0159, preferably DMG-PEG2000, DSPE-PEG2000, DMG-PEG3500, DMG-PEG4000, and / or DMG-PEG5000.

[0248] In some embodiments, the pegylated lipid is present in a molar percentage of 0.5-3.5% of the total lipid, preferably 1-3%, more preferably 1-2%.

[0249] In some embodiments, the pegylated lipid is present in a molar percentage of 0.5%, 0.75%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.2%, 2.4%, 2.6%, 2.8%, 3%, 3.25%, or 3.5% of the total lipid. In some embodiments, the lipid nanoparticle comprises the following molar percentages of components:

[0250] Ionizable lipid: 35-65%;

[0251] Structural lipid: 30-50%;

[0252] Neutral lipid: 5-30%;

[0253] Polymer lipid: 0.5-3.5%;

[0254] In some embodiments, the lipid nanoparticle comprises the following molar percentages of components: In some embodiments, the lipid nanoparticle comprises the following molar percentages of components:

[0255] In some embodiments, the lipid nanoparticle comprises the following molar percentages of components:

[0256] In a more particular embodiment, the lipid nanoparticle comprises the following molar percentages of components:

[0257] In a more particular embodiment, the lipid nanoparticle comprises the following molar percentages of components:

[0258] In a more particular embodiment, the lipid nanoparticle comprises the following molar percentages of components:

[0259] In some embodiments, the lipid nanoparticle contains 35-65 mol% of an ionizable lipid compound, 30-50 mol% of cholesterol, 5-30 mol% of DSPC or DOPE, and 0.5-3.5 mol% of DMG-PEG2000.

[0260] In some embodiments, the lipid nanoparticle contains 40-65 mol% of an ionizable lipid compound, 30-50 mol% of cholesterol, 5-30 mol% of DSPC or DOPE, and 0.5-3.5 mol% of DMG-PEG2000.

[0261] In some embodiments, the lipid nanoparticle contains 45-55 mol% of an ionizable lipid compound, 30-50 mol% of cholesterol, 8-20 mol% of DSPC or DOPE, and 1-3 mol% of DMG-PEG2000, preferably 1-2 mol% of DMG-PEG2000.

[0262] In some embodiments, the lipid nanoparticle contains 45-55 mol% of an ionizable lipid compound, 30-45 mol% of cholesterol, 5-20 mol% of DSPC or DOPE, and 1-3 mol% of DMG-PEG2000.

[0263] In some embodiments, the lipid nanoparticle contains 45-55 mol% of an ionizable lipid compound, 30-45 mol% of cholesterol, 5-20 mol% of DSPC or DOPE, and 1-2 mol% of DMG-PEG2000.

[0264] In some embodiments, the lipid nanoparticle contains 45-55 mol% of an ionizable lipid compound, 35-43.5 mol% of cholesterol, 8-15 mol% of DSPC or DOPE, and 1-2 mol% of DMG-PEG2000.

[0265] In some specific embodiments, the lipid nanoparticle contains 50 mol% of compound MTS001 or MTS002, 38.5 mol% of cholesterol, 10 mol% of DSPC, and 1.5 mol% of DMG-PEG2000.

[0266] In some specific embodiments, the lipid nanoparticle contains 40 mol% of compound MTS001 or MTS002, 43.5 mol% of cholesterol, 15 mol% of DOPE, and 1.5 mol% of DMG-PEG2000.

[0267] In some embodiments, the above-mentioned lipid nanoparticle further comprises at least one cargo selected from a nucleic acid molecule encoding a secreted protein.

[0268] In some embodiments, the secreted protein is selected from any one of a cytokine, an antigen binding protein; preferably, the antigen binding protein is selected from an antibody or an antibody fragment, more preferably a bispecific antibody or a fragment thereof; more preferably, the bispecific antibody is selected from any one of BiTE Triomabs, Knobs-into-holes, Crossmab, Ortho-Fab, DVD-Ig, Two-in-one, IgG-scFv, scFv2-Fc, bi-Nanobody, DART, or TandAb or VHH formats.

[0269] In some embodiments, the nucleic acid molecule encoding a secreted protein is selected from an antisense oligonucleotide (ASO), an RNA or a DNA;

[0270] Preferably, wherein the RNA is selected from at least one of a messenger RNA (mRNA), a modified mRNA (mmRNA), a circular RNA (circRNA), and a self-replicating RNA (SrRNA), preferably a mRNA, a modified mRNA, more preferably a modified mRNA;

[0271] Preferably, wherein the DNA is selected from at least one of a plasmid DNA (pDNA) and a minicircle DNA (mcDNA).

[0272] In one embodiment, the cargo is an mRNA or modified mRNA molecule comprising a coding sequence for an antibody against a surface antigen of a liver tumor.

[0273] In one embodiment, the payload is an mRNA or modified mRNA molecule comprising a coding sequence for an antibody against a protein associated with a liver tumor.

[0274] In one embodiment, the payload is an mRNA or modified mRNA molecule comprising a coding sequence for an antibody against a T cell surface antigen.

[0275] In one embodiment, the payload is an mRNA or modified mRNA molecule comprising a coding sequence for an antibody against GPC3.

[0276] In one embodiment, the payload is an mRNA or modified mRNA molecule comprising a coding sequence for an antibody against CD3.

[0277] In one embodiment, the payload is an mRNA or modified mRNA molecule comprising a coding sequence for a bi-specific antibody.

[0278] In one embodiment, the payload is an mRNA or modified mRNA molecule comprising a coding sequence for a bi-specific antibody against GPC3 and CD3.

[0279] In one embodiment, the payload is an mRNA or modified mRNA molecule comprising a coding sequence for a bi-specific T cell binding engager (BiTE) against GPC3 and CD3.

[0280] In some preferred embodiments, the payload is an mRNA molecule comprising a coding sequence (CDS) for a bi-specific antibody against GPC3 and CD3, wherein the bi-specific antibody comprises a scFv against GPC3, the heavy chain variable region of the scFv against GPC3 comprises a VH CDR1 of SEQ ID NO: 17, a VH CDR2 of SEQ ID NO: 18, a VH CDR3 of SEQ ID NO: 19, the light chain variable region of the scFv against GPC3 comprises a VL CDR1 of SEQ ID NO: 20, a VL CDR2 of SEQ ID NO: 21, a VL CDR3 of SEQ ID NO: 22.

[0281] In some preferred embodiments, the scFv against GPC3 comprises a heavy chain variable region of SEQ ID NO: 23 and a light chain variable region of SEQ ID NO: 24.

[0282] In some preferred embodiments, the bi-specific antibody against GPC3 and CD3 is in the form of a BiTE.

[0283] In some embodiments, the scFv against GPC3 comprises the amino acid sequence set forth in SEQ ID NO: 25.

[0284] In some embodiments, the bispecific antibody further comprises an scFv against CD3, the scFv against CD3 comprising the amino acid sequence set forth in SEQ ID NO: 26.

[0285] In some embodiments, the bispecific antibody comprises the amino acid sequence set forth in SEQ ID NO: 3.

[0286] In some embodiments, the coding sequence of the bispecific antibody is selected from the group consisting of SEQ ID NOs: 4-8. In some preferred embodiments, the coding sequence of the bispecific antibody is set forth in SEQ ID NO: 4 or SEQ ID NO: 7. In some further preferred embodiments, the coding sequence of the bispecific antibody is set forth in SEQ ID NO: 4.

[0287] In some embodiments, the bispecific antibody comprises the amino acid sequence set forth in SEQ ID NO: 14.

[0288] In some embodiments, the coding sequence of the bispecific antibody is set forth in SEQ ID NO: 15.

[0289] In some embodiments, the mRNA molecule further comprises a 5’ UTR and / or a 3’ UTR.

[0290] A variety of useful 5’ UTRs and / or 3’ UTRs are known in the art. One of skill in the art is capable of determining a 5’ UTR and / or 3’ UTR suitable for use in the present application.

[0291] In some embodiments, the 5’ UTR comprises the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the 3’ UTR comprises the nucleotide sequence set forth in SEQ ID NO: 2.

[0292] In some embodiments, the mRNA molecule further comprises a poly(A) sequence.

[0293] In some embodiments of the application, the poly(A) sequence comprises about 20 to about 500 (e.g., contiguous) adenine nucleotides (A), for example, about 25, about 50, about 100, about 150, about 175, about 200, about 300, about 400, about 500 (e.g., contiguous) adenine nucleotides (A). In some preferred embodiments, the poly(A) sequence comprises 120 (e.g., contiguous) adenine nucleotides (A). The addition of a poly(A) sequence aids in the stability and transport of the mRNA, preventing its degradation, and plays an important role in the post-transcriptional modification process. The poly(A) sequence can be a continuous chain of pure adenine nucleotides, but can also be a variant comprising nucleotides other than adenine, as long as it is functionally equivalent to a conventional poly(A) sequence, i.e., capable of providing similar biological functions as a natural poly(A) sequence, such as affecting the stability, translation efficiency or ribosome binding of the mRNA. Known poly(A) sequences are, for example, the human growth hormone (hGH) poly(A) sequence and the monkey virus 40 (SV40) poly(A) sequence. These variants can differ in the nucleotide composition, but are functionally identified as equivalent to a conventional poly(A) sequence.

[0294] In some embodiments, the mRNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 9-13. In some preferred embodiments, the mRNA molecule comprises a nucleotide sequence as set forth in SEQ ID NO: 9 or SEQ ID NO: 12. In some further preferred embodiments, the mRNA molecule comprises a nucleotide sequence as set forth in SEQ ID NO: 9.

[0295] In some embodiments, the mRNA molecule comprises a nucleotide sequence as set forth in SEQ ID NO: 16.

[0296] In some embodiments, the mRNA molecule further comprises a 5' cap structure. In some embodiments of the application, the 5' cap structure is a Capl cap structure.

[0297] In some embodiments, the mRNA molecule of the application can further comprise at least one nucleotide modification. The at least one nucleotide modification includes, but is not limited to, a cytidine modification, a uridine modification, or an adenosine modification. In some embodiments, the at least one nucleotide modification includes, but is not limited to, 5-methylcytosine (m5C), N6-methyladenosine (m6A), pseudouridine (ψ), N1-methylpseudouridine (m1ψ), and 5-methoxyuridine (5moU).

[0298] In some embodiments, the mRNA molecule is chemically synthesized. In some embodiments, the RNA molecule is obtained by in vitro transcription.

[0299] In addition, the mRNA molecules of the present application have a certain stability, and can tolerate the insertion of certain additional sequences (such as microRNA binding sites) without affecting their ability to be translated, nor affecting the stability of the mRNA molecule. In some embodiments, additional sequences (such as microRNA binding sites) can be inserted into the 3’ UTR. The microRNA binding sites include the full-length reverse complement of a microRNA (exemplary length can be 19-25 nt) or the reverse complement of the seed sequence thereof (exemplary length can be 7-8 nt).

[0300] In one aspect, the present application provides a nucleic acid vector comprising a coding sequence of the mRNA molecule of the present application. In some embodiments, the nucleic acid vector is used to generate the mRNA molecule of the present application.

[0301] As used herein, “vector” refers to a piece of DNA extracted from a virus, plasmid, or cell of a higher organism into which a foreign DNA segment can be or has been inserted for cloning and / or expression purposes. In certain embodiments, a vector can be stably maintained in an organism. A vector can comprise, for example, an origin of replication, a selectable marker or reporter gene, such as antibiotic resistance or GFP, and / or a multiple cloning site (MCS). The term includes linear DNA fragments (e.g., PCR products, linear plasmid fragments), plasmid vectors, viral vectors, cosmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and the like.

[0302] In some embodiments, the nucleic acid vector further comprises an RNA polymerase promoter sequence operably linked to the coding sequence of the mRNA molecule. The operably linked promoter allows for the in vivo and / or in vitro transcription of the RNA molecule. The promoter is, for example, a T7 RNA polymerase promoter, a T6 viral RNA polymerase promoter, a SP6 viral RNA polymerase promoter, a T3 viral RNA polymerase promoter, or a T4 viral RNA polymerase promoter.

[0303] In some embodiments, the nucleic acid vector is a plasmid vector. In some embodiments, the nucleic acid vector comprises a restriction endonuclease site, such as a type IIS restriction endonuclease site, flanking the 3’ of the coding sequence of the mRNA molecule. Suitable restriction endonucleases include, but are not limited to, BsmBI, BsaI, SapI, and the like. The restriction endonuclease site can be used to linearize the nucleic acid vector for in vitro transcription.

[0304] Methods for in vitro transcription of mRNA molecules from nucleic acid vectors are known in the art, for example, in vitro transcription can be performed using commercial kits.

[0305] The present application provides the above-mentioned lipid nanoparticle, wherein the N:P molar ratio of the N atoms in the ionizable lipid to the P atoms in the payload molecule is 1-15:1, preferably 2-12:1, preferably 2-9:1.

[0306] In some embodiments, the N:P molar ratio of the N atoms in the ionizable lipid to the P atoms in the payload molecule is 2-15:1, preferably 2-10:1, more preferably 4-6:1.

[0307] In some embodiments, the N:P molar ratio of the N atoms in the ionizable lipid to the P atoms in the payload molecule is 2-12:1, preferably 2-8:1, more preferably 4.5-6:1.

[0308] In some embodiments, the N:P molar ratio of the N atoms in the ionizable lipid to the P atoms in the payload molecule is 1-12:1, preferably 4-10:1, more preferably 4-9:1.

[0309] In some embodiments, the present application provides the above-mentioned lipid nanoparticle, wherein the particle size of the particle is 40-500 nm, preferably 50-250 nm, preferably 50-200 nm, more preferably 70-150 nm.

[0310] In some embodiments, the particle size of the particle is 50-300 nm, preferably 50-180 nm, more preferably 50-120 nm.

[0311] In some embodiments, the particle size of the particle is 50-200 nm, preferably 50-160 nm, preferably 60-120 nm, more preferably 60-100 nm.

[0312] In some embodiments, the particle size of the particle is 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, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm.

[0313] In some embodiments, the lipid nanoparticle comprises the following molar percentages of components and nucleic acid:

[0314] Ionizable lipid: 35-65%, preferably 40-55%;

[0315] Structural lipid: 30-50%, preferably 35-50%;

[0316] Neutral lipid: 5-30%, preferably 8-20%;

[0317] Polymeric lipid: 0.5-3.5%, preferably 1-3%, more preferably 1-2%.

[0318] In some more specific embodiments, the lipid nanoparticle comprises the following molar percentages of components and nucleic acid:

[0319] Ionizable lipid: 40-65%, preferably 45-55%;

[0320] Structural lipid: 30-50%, preferably 30-45%, more preferably 35-40%;

[0321] Neutral lipid: 5-30%, preferably 5-20%, more preferably 8-15%;

[0322] Polymeric lipid: 0.5-3.5%, preferably 1-3%, more preferably 1-2%.

[0323] In another aspect, the present application provides a method for preparing the lipid nanoparticle, comprising mixing the lipid components in the lipid nanoparticle, and mixing with the cargo to obtain the lipid nanoparticle.

[0324] In another aspect, the present application provides a pharmaceutical composition comprising the lipid nanoparticle of the present application and optionally a pharmaceutically acceptable excipient, such as a carrier, adjuvant or vehicle, preferably a liquid preparation, more preferably an injection.

[0325] In another aspect, the present application provides use of the lipid nanoparticle of the present application or the pharmaceutical composition of the present application in the manufacture of a medicament for treating a disease or improving a patient's prognosis.

[0326] In another aspect, the present application provides use of the lipid nanoparticle of the present application or the pharmaceutical composition of the present application in the manufacture of a medicament for delivering a cargo.

[0327] In another aspect, the present application provides a method for treating a disease or improving a patient's prognosis in a subject, comprising administering to the subject an effective amount of the lipid nanoparticle of the present application or the pharmaceutical composition of the present application.

[0328] Preferably, the lipid nanoparticle or the pharmaceutical composition is administered systemically, preferably intravenously, intra-arterially or intraperitoneally, more preferably intraperitoneally or intravenously.

[0329] In another aspect, the present application provides the lipid nanoparticle of the present application or the pharmaceutical composition of the present application for use in treating a disease or improving a patient's prognosis.

[0330] In a preferred embodiment of each of the above aspects, the disease is a malignant tumor.

[0331] In a preferred embodiment of each of the above aspects, the disease is a liver disease or a liver-related disease.

[0332] In a preferred embodiment of each of the above aspects, the liver disease or liver-related disease is a malignant tumor of the liver, more preferably hepatocellular carcinoma (HCC).

[0333] In a preferred embodiment of each of the above aspects, the disease is a GPC3- related disease; more preferably, the disease is a tumor that overexpresses GPC3, such as a liver cancer, e.g., hepatocellular carcinoma (HCC), a lung cancer, e.g., lung squamous cell carcinoma (SqCC), a gastric cancer, an ovarian cancer, e.g., ovarian clear cell carcinoma, a melanoma, or a pediatric embryonal tumor.

[0334] In another aspect, the present application provides a method of delivering a payload in a subject, comprising administering to the subject a lipid nanoparticle of the present application or a pharmaceutical composition of the present application.

[0335] In another aspect, the present application provides a lipid nanoparticle of the present application or a pharmaceutical composition of the present application for use in delivering a payload.

[0336] In some embodiments of each of the above aspects, the payload is as defined above.

[0337] It will be understood by a person of ordinary skill in the art that actual dosages of an active ingredient in a pharmaceutical composition of the present application can vary according to the requirements of the particular subject, composition, and mode of administration, and that the amount of active ingredient actually administered will depend on the judgment of the practitioner and the condition of the subject. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the present application applied, the route of administration, the time of administration, the rate of excretion of the particular compound being applied, the duration of the treatment, other drugs, compounds, and / or materials used in combination with the particular composition applied, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well known in the medical arts.

[0338] The LNP of the present application or the pharmaceutical composition of the present application can be administered by one or more routes of administration using one or more methods well known in the art. It will be understood by a person of ordinary skill in the art that the route and / or mode of administration will vary depending upon the desired results. Preferred routes of administration of the LNP of the present application or the pharmaceutical composition of the present application include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase "parenteral administration" means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0339] A GPC3-related disease that can be treated and / or prevented by the LNP of the present application, the pharmaceutical composition of the present application, or the method of the present application is, for example, a tumor that highly expresses (e.g., relative to healthy tissue) GPC3, including but not limited to, liver cancer such as hepatocellular carcinoma (HCC), lung cancer such as lung squamous cell carcinoma (SqCC), gastric cancer, ovarian cancer such as ovarian clear cell carcinoma, melanoma, or pediatric embryonal tumor.

[0340] In some embodiments, the LNP of the present application, the pharmaceutical composition of the present application, or the method of the present application can be used in combination with a chemotherapeutic agent, an immune checkpoint inhibitor, an antibody targeting other tumor-specific antigens, or radiotherapy.

[0341] The chemotherapeutic agent, the immune checkpoint inhibitor, or the antibody targeting other tumor antigens that can be used in combination with the LNP of the present application, the pharmaceutical composition of the present application is not particularly limited. Examples of the chemotherapeutic agent, the immune checkpoint inhibitor, and the antibody targeting other tumor antigens include, but are not limited to, ifosfamide, cyclophosphamide, dacarbazine, temozolomide, nimustine, busulfan, melphalan, enocitabine, capecitabine, carmofur, cladrabine, gemcitabine, cytarabine, tegafur, tegafur-uracil, TS-1, doxifluridine, nelarabine, hydroxyurea, fluorouracil, fludarabine, pemetrexed, pentostatin, mercaptopurine, methotrexate, irinotecan, etoposide, eribulin, sobuzoxane, docetaxel, paclitaxel, vinorelbine, vincristine, vindesine, vinblastine, actinomycin D, aclarubicin, amrubicin, idarubicin, epirubicin, zinostatin stimalamer, daunorubicin, doxorubicin, pirarubicin, bleomycin, peplomycin, mitomycin C, mitoxantrone, oxaliplatin, carboplatin, cisplatin, nedaplatin, anastrozole, exemestane, ethinyl estradiol, chlormadinone, goserelin, tamoxifen, dexamethasone, bicalutamide, toremifene, flutamide, prednisolone, fosfestrol, mitotane, methyltestosterone, leuprolide, letrozole, megestrol, ibritumomab, imatinib, everolimus, erlotinib, gefitinib, sunitinib, cetuximab, sorafenib, dasatinib, tamibarotene, trastuzumab, tretinoin, panitumumab, bevacizumab, bortezomib, lapatinib, atezolizumab, pembrolizumab, tislelizumab, camrelizumab, sugemalimab, and nivolumab, etc.

[0342] The LNP of the present application or the pharmaceutical composition of the present application and the chemotherapeutic agent, the immune checkpoint inhibitor, or the antibody targeting other tumor antigens can be all administered at once or administered separately. When administered separately (in the case of using different administration regimens from each other), they can be administered consecutively without interruption or at predetermined intervals.

[0343] The dose of the LNP of the present application or the pharmaceutical composition of the present application and the chemotherapeutic agent, immune checkpoint inhibitor or antibody targeting other tumor antigens in the combination of the present application is not particularly limited. As described above, the dose of the LNP of the present application or the pharmaceutical composition of the present application can be determined by referring to the dose when the antibody is used alone. The chemotherapeutic agent, immune checkpoint inhibitor and antibody targeting other tumor antigens can be used according to the dose indicated for each drug or can be reduced (taking into account the combined effect with the antibody of the present application).

[0344] The LNP of the present application or the pharmaceutical composition of the present application can also be combined with radiotherapy, for example, including administration of ionizing radiation to the subject, which is earlier, during and / or later than the administration of the antibody or pharmaceutical composition of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0345] Figure 1 is the expression of different LNP delivered 15# bispecific antibody mRNA encoded bispecific antibody protein in mouse serum, showing that the concentration of the tested different LNP delivered 15# bispecific antibody mRNA encoded bispecific antibody in serum is higher compared with the control Lipid5-LNP;

[0346] Figure 2 is the expression of different LNP delivered 15# bispecific antibody mRNA encoded bispecific antibody protein in mouse liver, showing that the expression amount of the tested different LNP delivered 15# bispecific antibody mRNA encoded bispecific antibody in liver is higher compared with the control Lipid5-LNP;

[0347] Figure 3 is the anti-tumor effect of different LNP delivered 15# bispecific antibody mRNA, indicating that the LNP delivered bispecific antibody mRNA of the present application inhibits the growth of tumor and presents a dose-dependent trend;

[0348] Figure 4 is the effect of different LNP delivered 15# bispecific antibody mRNA on the body weight of mice in a mouse orthotopic liver cancer model, indicating that the animals do not have body weight loss after multiple administrations of 15# bispecific antibody mRNA delivered by LNP of MTS001 and MTS002;

[0349] Figure 5 is a comparison of the concentration of bispecific antibody protein expressed by different LNP delivered 15# bispecific antibody mRNA in serum after the first administration, in which the concentration of bispecific antibody protein expressed by 15# bispecific antibody mRNA delivered by LNP of MTS001 in serum is higher than that of Lipid5-15# bispecific antibody mRNA group, although it is lower than that of MTS002-bispecific antibody mRNA group, but it shows better efficacy;

[0350] Figure 6 shows the anti-tumor effect of MTS001-LNP delivered 4# bispecific antibody mRNA, wherein it is shown that MTS001-LNP delivered 0.025 mg / kg 4# bispecific antibody mRNA resulted in a significant decrease in tumor size represented by luciferase fluorescence intensity;

[0351] Figure 7 shows the effect of MTS001-LNP delivered 4# bispecific antibody mRNA on body weight in a mouse orthotopic liver cancer model, wherein it is shown that there is no significant difference in the effect of each treatment group on body weight;

[0352] Figure 8 shows the expression of MTS001-LNP delivered 4# bispecific antibody mRNA in mouse serum and the blood drug concentration of ERY974, wherein it is shown that the protein concentration of ERY974 in serum is significantly higher than the protein concentration of 4# bispecific antibody mRNA expressed in mouse serum, proving that compared with bispecific antibodies with IgG structure, MTS001-LNP delivered 4# bispecific antibody mRNA has lower serum exposure while exerting strong efficacy, and has better safety;

[0353] Figures 9 and 10 show the results of in vivo imaging and body weight measurement of mice twice a week after tail vein injection of Tris solvent control group, LNP empty control group, MTS-LNP1 high, medium and low dose groups and ERY974 control group, the change of imaging signal intensity of animals in each group is shown in Figure 9, and the change of body weight of animals in each group is shown in Figure 10;

[0354] Figure 11 shows that the MTS-LNP1 cured group of mice did not show tumor growth after the second tumor inoculation, while the control group of CD3EDG humanized mice showed a typical tumor growth curve after tumor inoculation;

[0355] Figures 12 and 13 show the results of in vivo imaging and body weight measurement of severely immunodeficient mice twice a week after tail vein injection of Tris solvent control group, LNP empty control group, MTS-LNP1 high and low dose groups and ERY974 control group, the change of imaging signal intensity of animals in each group is shown in Figure 12, and the change of body weight of animals in each group is shown in Figure 13;

[0356] Figures 14 and 15 show the results of in vivo imaging and body weight measurement of severely immunodeficient mice twice a week after tail vein injection of PBS solvent control group, LNP empty control group, ERY974 control group and MTS-LNP1 high and low dose groups. The change of imaging signal intensity of animals in each group is shown in Figure 14, and the change of body weight of animals in each group is shown in Figure 15;

[0357] Figure 16 shows the pharmacokinetic study data of MTS-LNP1 in cynomolgus monkeys at the dose of 15, 50 and 100 μg / kg, respectively, two monkeys, one male and one female, for each dose, each line represents one monkey cynomolgus monkeys. DETAILED DESCRIPTION

[0358] EMBODIMENT

[0359] In order to make the technical solutions of the present application clearer and more explicit, the present application is further described in detail through the following embodiments. The following embodiments are only used to illustrate the specific embodiments of the present application, so that those skilled in the art can understand the present application, but are not used to limit the protection scope of the present application. In the specific embodiments of the present application, the technical means or methods not specifically described are the conventional technical means or methods in the art. The materials, reagents, etc. used in the embodiments, if not specifically described, can be obtained from commercial channels.

[0360] Example 1: Synthesis of compound

[0361] Synthesis of compound MTS001

[0362] Under the inert atmosphere of nitrogen, sodium hydride (2.0 g, 50.64 mmol, 1.6 eq.) was added to a solution of nonanoic acid (5.0 g, 31.65 mmol, 1.0 eq.) in tetrahydrofuran (50.0 ml), and the resulting mixture was stirred at 0°C for 10 minutes. Lithium diisopropylamide (2 mol / L, 28.50 mL, 56.97 mmol, 1.8 eq.) was added to it at 0°C. The resulting mixture was stirred at 0°C for 10 minutes, 1-iodoheptane (10.7 g, 47.48 mmol, 1.5 eq.) was added to it, and the resulting mixture was stirred at 40°C for 12 hours, quenched with saturated ammonium chloride solution (10.0 mL), diluted with 500 mL of water and extracted with 3x500 mL of dichloromethane, the organic layers were combined, washed with 3x500 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography eluted with petroleum ether / ethyl acetate (3:1) to obtain 5 g (62%) of 1-1 as a yellow oil; 1 H NMR (300 MHz, CDCl3) δ: 0.85-0.89 (m, 6H), 1.25-1.32 (m, 19H), 1.41-1.67-2.27 (m, 4H), 2.29-2.33 (m, 1H), 10.29 (s, 1H);

[0363] To a stirred solution of 1-1 (2.0 g, 7.81 mmol, 1.0 eq.) in tetrahydrofuran (5.0 mL) was added borane-tetrahydrofuran complex solution (1 mol / L, 23.4 mL, 23.43 mmol, 3.0 eq.) at 0 °C, the resulting solution was warmed to 75 °C and stirred for 3 h, the mixture was cooled to room temperature, quenched the reaction with methanol (10.0 mL), the mixture was diluted with 100 ml of water, the organic layer was combined, washed with 3 x 200 ml of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (20:1) to afford 1.6 g (85%) of 1-2 as a colorless oil;

[0364] To a stirred solution of 1-2 (1.0 g, 4.13 mmol, 1.0 eq.) in dichloromethane (10.0 ml) was added 8-bromooctanoic acid (1.11 g, 4.96 mmol, 1.2 eq.), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.19 g, 6.20 mmol, 1.5 eq.) and 4-dimethylaminopyridine (756.0 mg, 6.20 mmol, 1.5 mol) under inert nitrogen atmosphere. The resulting mixture was stirred at 25 °C for 6 h, diluted with 100 mL of water, the organic layer was combined, washed with 3 x 300 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (50:1). Obtained 1 g (56%) of 1-3 as a yellow oil;

[0365] To a stirred solution of methyl isobutyrate (4.4 g, 43.0 mmol, 1.0 eq.) in tetrahydrofuran (100.0 mL) was added lithium diisopropylamide (43.0 mL, 86.00 mmol, 2.0 eq) at 0 °C. The resulting mixture was stirred at 0 °C for 30 min. To the above solution was added 1,5-dibromo-pentane (20.0 g, 86.0 mmol, 1.0 eq.) at 0 °C. The resulting mixture was stirred at 25 °C for 5 h, quenched the reaction with saturated ammonium chloride solution (1.0 mL), diluted with 300 mL of water, the organic layer was combined, washed with 3 x 500 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (50:1). Obtained 10 g (46%) of 1-4 as a light yellow oil;

[0366] To a stirred solution of 1-4 (10.0 g, 40.0 mmol, 1.0 eq.) in tetrahydrofuran (20.0 mL) was added borane-tetrahydrofuran complex solution (100.0 mL) at 0 °C. The resulting solution was stirred at 75 °C for 3 h. The mixture was cooled to 25 °C, diluted with 100 mL of water and extracted with 3 x 500 mL of dichloromethane, the organic layers were combined, washed with 3 x 500 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. This resulted in 8 g (90%) of 1-5 as a colorless oil; the preparation process was repeated to obtain sufficient product;

[0367] To a stirred solution of 1-5 (10.0 g, 44.00 mmol, 1.0 eq.) in dichloromethane (100.0 mL) was added triethylamine (13.5 g, 134.0 mmol, 3.0 eq.), decanoyl chloride (11.0 g, 58.0 mmol, 1.3 eq.) at 0 °C, the resulting mixture was stirred at 25 °C for 3 h, diluted with 100 mL of water and extracted with 3 x 300 mL of dichloromethane, the organic layers were combined, washed with 3 x 300 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (10:1) to obtain 10 g (60%) of 1-6 as a colorless oil;

[0368] To a stirred solution of ethanolamine (2.3 g, 37.2 mmol, 10.0 eq.) in acetonitrile (15.0 ml) was added potassium carbonate (1.5 g, 11.1 mmol, 3.0 eq.), 1-6 (1.4 g, 3.70 mmol, 1.0 eq.) under an inert atmosphere of nitrogen, the resulting solution was stirred at 70 °C for 3 h, the mixture was cooled to 25 °C, diluted with 10 mL of water and extracted with 3 x 50 mL of dichloromethane, the organic layers were combined, washed with 3 x 50 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with dichloromethane / methanol (10:1) to obtain 1 g (76%) of 1-7 as a colorless oil; 1 H NMR (300 MHz, CD3Cl) δ: 0.85-0.89 (m, 9H), 1.20-1.26 (m, 21H), 1.59-1.62 (m, 4H), 2.29-2.34 (m, 2H), 2.73-2.76 (m, 2H), 2.87-2.91 (m, 2H), 3.72-3.77 (m, 3H);

[0369] To a solution of 1-3 (202.8 mg, 0.45 mmol, 1.2 eq.) in N,N-dimethylformamide (2.0 mL) was added potassium carbonate (161.5 mg, 1.17 mmol, 3.0 eq.), sodium iodide (141.5 mg, 0.95 mmol, 2.5 eq.) and 1-7 (135.0 mg, 0.39 mmol, 1.0 eq.) under nitrogen inert atmosphere, the reaction mixture was stirred at 70 °C for 6 h, the mixture was cooled to 25 °C, diluted with 10 mL of water, extracted with 3 x 50 mL of dichloromethane, the organic layers were combined, washed with 3 x 50 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column: Xselect CSH F-phenyl OBD column 19 x 250 mm, 5 pm; mobile phase A: water, mobile phase B: acetonitrile; flow rate: 20 mL / min; gradient: from 75% B to 95% B in 9 min, to give 83 mg (30%) of MTS001 as a yellow oil. 1 H NMR (300 MHz, CDC13) δ: 0.80-0.90 (m, 15H), 1.13-1.72 (m, 62H), 2.22-2.27 (m, 4H), 2.64-2.79 (m, 4H), 3.71 (s, 2H), 3.88-3.90 (m, 2H); MS m / z [M+H] + (ESI): 724.80.

[0370] Synthesis of compound MTS002:

[0371] Nineteen-10-ol (4 g, 14.06 mmol, 1.0 equiv.) was dissolved in dichloromethane (40.0 mL) solution, 8-bromooctanoic acid (3.76 g, 16.87 mmol, 1.2 equiv.), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.04 g, 21.08 mmol, 1.5 equiv.) and N,N-dimethylaminopyridine (2.58 g, 21.12 mmol, 1.5 equiv.) were added successively under nitrogen protection. Stirring at 25 °C for 6 hours. After the reaction was completed, water (100 mL) was added to dilute the reaction solution, dichloromethane (3 x 100 mL) was extracted, the organic phase was combined, washed with saturated aqueous sodium chloride solution (3 x 100 mL), dried over anhydrous sodium sulfate. The organic phase was collected by filtration, and the organic solvent was removed by rotary evaporator to obtain the crude product, which was purified by silica gel column (petroleum ether / ethyl acetate = 50 / 1) to obtain compound 2-1 (3.9 g, 57%) as a yellow oil;

[0372] To a solution of 1-5 (1 g, 4.48 mmol, 1.0 equiv.) in dichloromethane (10.0 mL) was added triethylamine (0.91 g, 8.96 mmol, 2.0 equiv.) and undecanoyl chloride (1.38 g, 6.72 mmol, 1.5 equiv.) successively at 0 °C under nitrogen protection. The mixture was stirred at 25 °C for 3 h. After the reaction was completed, water (100 mL) was added to dilute the reaction solution, and dichloromethane (3 x 50 mL) was used to extract the mixture. The combined organic phase was washed with saturated aqueous sodium chloride solution (3 x 100 mL) and dried over anhydrous sodium sulfate. The organic phase was collected by filtration, and the organic solvent was removed by a rotary evaporator to obtain a crude product. The compound 2-2 (1 g, 57%) was obtained as a yellow oil by purification on a silica gel column (petroleum ether / ethyl acetate = 50 / 1);

[0373] To a solution of 2-2 (1 g, 2.56 mmol, 1.0 equiv.) and ethanolamine (1.56 g, 25.55 mmol, 10.0 equiv.) in acetonitrile (15 mL) was added potassium carbonate (1.06 g, 7.67 mmol, 3.0 equiv.) under nitrogen protection. The mixture was stirred at 70 °C for 6 h. After the reaction was completed, the mixture was cooled to 25 °C and diluted with 100 mL of water. The mixture was extracted with dichloromethane (3 x 50 mL), and the combined organic phase was washed with saturated aqueous sodium chloride solution (3 x 100 mL) and dried over anhydrous sodium sulfate. The organic phase was collected by filtration, and the organic solvent was removed by a rotary evaporator to obtain a crude product. The compound 2-3 (400 mg, 42%) was obtained as a yellow oil by purification on a silica gel column (dichloromethane / methanol = 10 / 1);

[0374] To a solution of 2-3 (150 mg, 0.40 mmol, 1.0 equiv.) and 2-1 (234.2 mg, 0.48 mmol, 1.2 equiv.) in N,N-dimethylformamide (2 mL) was added potassium carbonate (165.6 mg, 1.20 mmol, 3.0 equiv.) and sodium iodide (150.0 mg, 1.00 mmol, 2.5 equiv.) under nitrogen protection. The reaction was stirred at 70 °C for 6 h. After the reaction was completed, it was cooled to 25 °C, diluted with 10 mL of water, and then extracted with dichloromethane (3 x 20 mL). The organic phases were combined, washed with saturated aqueous sodium chloride (3 x 50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by rotary evaporation to give a crude product. Purification by high performance liquid chromatography (column: Xselect CSH F-Phenyl OBD column 19 x 250 mm, 5 μm; phase A: acetonitrile / water (10 mmol / L ammonium bicarbonate + 0.05% ammonia water), phase B: isopropanol / acetonitrile; flow rate: 20 mL / min; gradient: 75% B to 95% B, 9 min) gave compound MTS002 (99 mg, 31%) as a yellow oil.

[0375] 1 H NMR (300 MHz, CD3OD) δ: 0.80-0.90 (m, 15H), 1.29-1.34 (m, 54H), 1.52-1.64 (m, 12H), 2.28-2.35 (m, 4H), 2.52-2.57 (m, 4H), 2.63-2.68 (m, 2H), 3.60-3.64 (m, 2H), 3.80 (s, 2H), 4.87-4.89 (m, 1H); MS m / z [M+H] + (ESI): 780.80.

[0376] Synthesis of compound MTS004

[0377] To a solution of 2-3 (150 mg, 0.40 mmol, 1.0 equiv.) and 2-1 (234.2 mg, 0.48 mmol, 1.2 equiv.) in N,N-dimethylformamide (2 mL) was added potassium carbonate (165.6 mg, 1.20 mmol, 3.0 equiv.) and sodium iodide (150.0 mg, 1.00 mmol, 2.5 equiv.) under nitrogen protection. The reaction was stirred at 70 °C for 6 h. After the reaction was completed, it was cooled to 25 °C, diluted with 10 mL of water, and then extracted with dichloromethane (3 x 20 mL). The organic phases were combined, washed with saturated aqueous sodium chloride (3 x 50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by rotary evaporation to give a crude product. Purification by high performance liquid chromatography (column: Xselect CSH F-Phenyl OBD column 19 x 250 mm, 5 μm; phase A: acetonitrile / water (10 mmol / L ammonium bicarbonate + 0.05% ammonia water), phase B: isopropanol / acetonitrile; flow rate: 20 mL / min; gradient: 75% B to 95% B, 9 min) gave compound MTS002 (99 mg, 31%) as a yellow oil.

[0378] In a 40 mL sealed tube, compound 4-2 (1 g, 2.48 mmol, 1.0 eq.) was dissolved in ethanol (10 mL) at room temperature, then ethanolamine (3.03 g, 49.60 mmol, 20 eq.) was added, and the reaction system was heated to 70 °C for 3 h. LC-MS was used to monitor the reaction. When the reaction was completed, the reaction system was cooled to room temperature, and the solvent was removed by concentration under reduced pressure. Ethyl acetate (50 mL) was added to dissolve the residue, and the organic phase was washed with water (30 mL x 5). The organic phase was dried over anhydrous Na2SO4, filtered, concentrated, and the crude product was purified by silica gel column chromatography to obtain compound 4-3 (652 mg) as a yellow oil.

[0379] In a 100 mL three-necked flask, 9-bromononanoic acid (1.53 g, 6.44 mmol, 1.1 eq.), 3-heptyl-1-decanol 4-4 (1.5 g, 5.85 mmol, 1.0 eq.), EDCI (1.68 g, 8.78 mmol, 1.5 eq.), DMAP (0.71 g, 5.85 mmol, 1.0 eq.) and DCM (15 mL) were added at room temperature. The system was stirred at room temperature for 1 h. LC-MS was used to monitor the reaction. When the starting material was basically converted, the reaction was quenched by pouring into water. The organic phase was extracted with dichloromethane, combined and dried over anhydrous sodium sulfate. The filtrate was collected by filtration, and the solvent was removed by rotary evaporation under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography to obtain compound 4-5 (2.45 g) as a yellow oil.

[0380] In a 40 mL sealed tube, compound 4-3 (150 mg, 0.39 mmol, 1.0 eq.) and 4-5 (278.95 mg, 0.59 mmol, 1.5 eq.) were dissolved in a mixed solvent of acetonitrile (1 mL) and CPME (3 mL), then K2CO3 (162.12 mg, 1.17 mmol, 3.0 eq.) and KI (77.89 mg, 0.47 mmol, 1.2 eq.) were added. The reaction system was heated to 80 °C and stirred for 18 h. LC-MS was used to monitor the reaction. When the reaction was completed, the reaction system was cooled to room temperature, filtered, and the filter cake was washed with ethyl acetate (5 mL x 3). The filtrate was combined and concentrated to obtain a crude product. The crude product was purified by Prep-HPLC. The preparation conditions were as follows (Column: XBridge Shield RP18 OBD, 30*150 mm, 5 μm; Eluent A: H2O / CH3CN 60 / 40, 10 mM NH4HCO3+1% NH3﹒H2O; Eluent B: IPA / CH3CN 90 / 10; Flow rate: 60 mL / min; Gradient program: 60%-85% B in 0-12 min). Compound MTS004 (147.3 mg) was obtained as a yellow oil.

[0381] 1 H NMR (300 MHz, CDC13) δ: 4.16-4.05 (m, 4H), 3.55 (t, J = 5.1 Hz, 2H), 2.60 (t, J = 5.1 Hz, 2H), 2.47 (t, J = 6.0 Hz, 4H), 2.28 (t, J = 7.5 Hz, 4H), 1.64-1.54 (m, 8H), 1.45-1.27 (m, 58H), 0.93-0.86 (m, 9H), 0.35-0.21 (m, 4H); ESI-MS m / z: 778.75 [M+H] + .

[0382] Other ionizable lipid compounds were prepared according to the above synthesis method or methods known in the art, the structures and characterization data of which are as follows:

[0383] Example 2: Preparation of mRNA molecules encoding GPC3xCD3 bispecific antibodies

[0384] A nucleic acid fragment comprising a T7 promoter, an mRNA molecule encoding GPC3xCD3 bispecific antibodies (SEQ ID NO: 9-13) and a Type IIS restriction endonuclease cleavage site was synthesized in vitro and cloned into an in vitro transcription vector (pIVTRup, Addgene plasmid #101362). The mRNA molecule transcribed from the vector comprises a 5’ UTR as set forth in SEQ ID NO: 1, a 3’ UTR as set forth in SEQ ID NO: 2, and a polyA tail.

[0385] The obtained vector was linearized and subjected to in vitro transcription to produce mRNA molecules using T7-RNA polymerase, and a 5’-cap structure was added simultaneously. The 5’-cap structure was added by co-transcriptional capping, in which a cap analog was incorporated as the first nucleotide into the transcript during in vitro transcription, directly producing mRNA molecules with Cap1 structure. The mRNA molecules thus obtained were purified and resuspended in water. The mRNA molecules were subjected to quality control using a 5200 Fragment Analyzer (Agilent) to detect that the length and integrity of the mRNA molecules met the requirements (the numerical value was derived from the area percentage of the curve of the expected length fragment).

[0386] Example 3: Expression of mRNA encoding GPC3xCD3 bispecific antibodies in cells

[0387] 293T cells were seeded at 1 x 10 5The cells were seeded at a density of 0.5 μg / well in 24-well plates and incubated overnight at 37°C with 5% CO2. The next day, mRNA was transfected using Lipofectamine MessengerMAX (Invitrogen) at a dose of 0.5 μg / well. The supernatant was collected 24 hours after transfection. The negative control consisted of untransfected wells. The concentration of bispecific antibody protein in the cell supernatant was detected by ELISA: GPC3-Fc (Nearshore Technology) was diluted to 2 μg / mL with ELISA coating buffer, and 100 μL was added to each well. The plates were incubated overnight at 4°C. The next day, the plates were washed with ELISA washing buffer (Sigma), and then 200 μL of ELISA blocking buffer (Solepro) was added to each well. The plates were blocked at room temperature for 2 hours and then washed with ELISA washing buffer. The standard curve was prepared by diluting the GPC3-CD3 bispecific antibody standard protein with blank medium at a concentration of 250 ng / mL as the highest dose well, serially diluting six times. Blank medium diluent was added to the blank wells, followed by cell supernatant. The plates were incubated at room temperature for 2 hours, and then washed with ELISA washing buffer. The detection antibody, Biotinylated Human CD3 epsilon Protein (His Tag, ultra-sensitivity, primary amine labeling) (Acro), was diluted to 0.2 μg / mL with sample diluent, and 100 μL was added to each well. The plates were incubated at room temperature for 90 minutes, washed with ELISA washing buffer, and 100 μL of TMB chromogenic solution (Solepro) was added to each well. The reaction was allowed to proceed for 15 minutes at room temperature, followed by 100 μL of stop solution (Solepro). The OD was measured at 450 nm using a microplate reader.

[0388] The results showed that the expression of the 4# and 7# GPC3×CD3 bispecific antibody mRNAs was detectable in the cell supernatant, and their expression levels are shown in Table 1a below.

[0389] Table 1a: Expression levels of mRNA encoding GPC3×CD3 bispecific antibody in cells

[0390] Example 4: Preparation of lipid nanoparticles (LNP)

[0391] Materials for lipid nanoparticle assembly: (1) ionizable lipid compound: ionizable lipid (MTS001 or MTS002) designed and synthesized in this application or Lipid5 (commercially available); (2) structural lipid: Cholesterol; (3) neutral lipid (phospholipid): DSPC (Distearoylphosphatidylcholine, commercially available from AVT); (4) polymer lipid: DMG-PEG2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000, commercially available from AVT); (5) nucleic acid (or fragment): (as shown in Table 1) etc. The structure of the lipid nanoparticle assembly material and the nucleic acid sequence are shown in Table 1:

[0392] Table 1

[0393] Method for preparing lipid nanoparticle:

[0394] 1. Lipid Mix preparation

[0395] (1) Ionizable lipid (MTS001, MTS002 or Lipid5), structural lipid (CHO), neutral lipid (DSPC) and polymer lipid (DMG-PEG2000) were respectively dissolved and mixed in anhydrous ethanol according to the molar percentage of 50%, 38.5%, 10% and 1.5%;

[0396] 2. mRNA Mix preparation

[0397] 0.1M Citrate Buffer (pH = 4) was prepared using trisodium citrate (SIGMA-ALDRICH) and citric acid (ACROS), then 10mL 0.1M Citrate Buffer was taken and 30mL Nuclease-Free Water (Invitrogen) was added to obtain 25mM Citrate Buffer, and the above mRNA was configured into 0.2mg / mL mRNA Mix using 25mM Citrate Buffer;

[0398] 3. Preparation of LNP

[0399] 1) Use an automated high-throughput microfluidic system to mix Lipid Mix dissolved with a lipid mixture and mRNA Mix dissolved with mRNA components at a flow rate ratio of volume ratio (Lipid Mix:mRNA Mix) = 1:3 (1:1 to 1:4 range), mixing speed in the range of 5 mL / min to 18 mL / min (for example, total flow rate is 5 mL / min), N / P = 6;

[0400] 2) The prepared lipid nanoparticles are diluted with phosphate buffered saline solution (1X DPBS), centrifuged with ultrafiltration tube (Merck Millipore), and then replaced with Tris buffer (Dharmacon) to obtain LNP Tris solution;

[0401] 3) Prepare 10% and 30% sucrose (AVT) Tris buffer, respectively, and add 30% sucrose Tris buffer to make LNP Tris solution 10% sucrose Tris buffer, store at low temperature (for example, 4°C), and use Malvern Nanometer particle size potentiometer instrument to detect Size, PDI;

[0402] 4) Use Ribogreen RNA kit to detect API and EE%, and use 10% sucrose Tris buffer to dilute LNP to 0.1 mg / mL according to the API result;

[0403] 6) According to the amount of administration, LNP is divided and stored at low temperature (for example, 8°C to -80°C).

[0404] The preparation method of the lipid nanoparticles includes a microfluidic mixing system, but is not limited to this method, and also includes a T-type mixer and an ethanol injection method.

[0405] Example 5: Animal PK experiment

[0406] 8-10 week old C57BL6 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were divided into 3 groups, 20 mice per group. The specific administration scheme is shown in Table 2 (the average body weight of each mouse is about 20 g, that is, the single administration dose is 5 μg per mouse):

[0407] Table 2

[0408] 15# bispecific antibody mRNA is mRNA encoding GPC3xCD3 bispecific antibody. The concentration of bispecific antibody protein in mouse serum and liver homogenate was detected by ELISA method. First, GPC3-Fc (Coastal Technology) was diluted to 2 pg / mL with ELISA coating solution (Solebo), 100 pL per well was added to the enzyme-labeled plate, and it was coated at 4°C overnight. The next day, the plate was washed with ELISA washing solution (Sigma), then 200 pL of ELISA blocking solution (Solebo) was added to each well, and it was blocked at room temperature for 2 hours, and then the plate was washed with ELISA washing solution. The standard curve was prepared by diluting blank serum or blank liver homogenate supernatant with diluent (Solebo), and then diluting GPC3-CD3 bispecific antibody standard protein to 250 ng / mL as the highest dose well, and diluting by 6 times. The blank well was added with blank serum diluent or blank liver diluent, and then the serum or liver homogenate supernatant sample was added, and it was incubated at room temperature for 2 hours, and then the plate was washed with ELISA washing solution. Dilute the detection antibody Biotinylated Human CD3 epsilon Protein (His Tag, ultra sensitivity, primary amine labeling) (Acro) to 0.2 pg / mL, add 100 pL per well, and incubate at room temperature for 90 min. Wash the plate with ELISA washing solution, add 100 pL of TMB color developing solution (Solebo) per well, and react at room temperature for 15 min. Add 100 pL of stop solution (Solebo). The OD450 nm was detected by enzyme-labeled instrument.

[0409] Fig. 1, Fig. 2 show that the 15# bispecific antibody mRNA encoded bispecific antibody delivered by different LNP tested has higher expression in mouse serum and liver compared with Lipid5-LNP, and reaches peak at 3-6 hr after administration, and decreases to baseline level at 48 hr.

[0410] Example 6: Pharmacodynamics of MTS001-LNP delivered mRNA encoding GPC3xCD3 bispecific antibody in a mouse model of liver cancer

[0411] The LNP formulation is the same as Example 4, and the LNP empty group is a lipid nanoparticle without loaded mRNA. The preparation method and LNP physical property characterization method are the same as Example 4, and the LNP characterization data are shown in Table 3:

[0412] Table 3

[0413] Hepa1-6 mouse hepatoma cells overexpressing human GPC3 and luciferase luciferase (constructed by Boster Biological Technology Co., Ltd.) were inoculated in situ in the liver of 8-10 week old CD3EDG humanized mice (Southern Model Organism). The specific experimental process is as follows: the animals were injected intraperitoneally with anesthetic (Avertin), and after the mice were completely anesthetized, the mouse liver lobe was squeezed out, the liver lobe was laid on alcohol-soaked gauze, and the matrix glue resuspended Hepa1-6 mouse hepatoma cells overexpressing human GPC3 and luciferase luciferase were slowly injected into the left lobe of the mouse liver at a dose of 1 x 10 6 The syringe was removed, and a cotton swab was used to gently press to stop bleeding, and then the liver lobe was inserted back into the mouse. The mouse was sutured and placed on a hot stage, and after the mouse woke up, it was returned to the cage for continued feeding and daily observation. Seven days after cell inoculation, the mice were analyzed by luciferase live imaging (Lumina II small animal live imaging system, PerkinElmer, USA), and according to the imaging fluorescence value and body weight, appropriate mice were selected for grouping (tumor average imaging signal intensity about 4.4E+07 p / s), and were evenly distributed into 10 experimental groups, 5 mice per group, a total of 50 mice. Drug administration began the day after grouping, denoted as D0, and the specific drug administration regimen is shown in Table 4:

[0414] Table 4

[0415] The experiment used Lipid5-LNP, MTS001-LNP and MTS002-LNP to deliver 15# bispecific antibody mRNA through the tail vein injection for liver targeting. After administration, the mice were imaged in vivo and weighed twice a week. Figure 3 shows that the LNP delivery of 15# bispecific antibody mRNA according to the application inhibits the growth of tumors and shows a dose-dependent trend. Figure 4 shows that after multiple administrations of 15# bispecific antibody mRNA delivered by different LNPs, the animals did not show weight loss. The serum of mice in each administration group was collected 6 hours after the first administration, and the concentration of bispecific antibody protein in the serum was detected by the above-mentioned ELISA method. As shown in Figure 5, the concentration of bispecific antibody protein expressed by MTS001-LNP delivered high-dose 15# bispecific antibody mRNA in the serum is higher than that of Lipid5-15# bispecific antibody mRNA high-dose group and lower than that of MTS002-15# bispecific antibody mRNA high-dose group; the concentration of bispecific antibody protein expressed by each LNP-bispecific antibody mRNA low-dose group in the serum has no great difference, and MTS001-LNP shows better efficacy whether it is high-dose or low-dose in delivering bispecific antibody mRNA compared with Lipid5-LNP and MTS002-LNP. Because the tumor model used is a liver orthotopic tumor model, the tumor cells are stably transfected with luciferase, so Total Flux represents the luciferase fluorescence intensity. By monitoring the luciferase fluorescence intensity by IVIS, the size of the tumor is observed. The lower the Total Flux value, the smaller the tumor volume.

[0416] Example 7: Comparison of the efficacy of MTS001-LNP delivered 4# bispecific antibody mRNA encoding GPC3xCD3 with ERY974 in a syngeneic transplanted mouse liver cancer model

[0417] According to the method of Example 6, an 8-10 week old CD3EDG humanized mouse (Southern Model Organism) was used to construct a Hepa1-6 mouse orthotopic liver cancer model overexpressing human GPC3 and luciferase. After 7 days of cell inoculation, luciferase in vivo imaging analysis was performed on the mice, and according to the imaging fluorescence value and body weight, appropriate mice were selected for grouping (the average imaging signal intensity of the tumor was about 3E+08 p / s), and were evenly distributed into 5 experimental groups, 8 in each group, a total of 40. The next day of grouping, administration was started, recorded as D0, and the specific administration scheme is shown in Table 5.

[0418] Table 5

[0419] The experimental MTS001-LNP was used to deliver 4# bispecific antibody mRNA to the liver by tail vein injection. ERY974 is a CD3 x GPC3 bispecific antibody with IgG structure (Circlegene, positive control). After administration, the mice were imaged in vivo and weighed twice a week. As shown in FIG. 6, the MTS001-LNP delivered 4# bispecific antibody mRNA effectively inhibited tumor growth and showed a dose-dependent trend. As shown in FIG. 7, after multiple administrations, the animals in all administration groups did not show weight loss. The serum of mice in each group was collected 6 hours after the 1st, 3rd and 4th administration, and the bispecific antibody protein and ERY974 blood drug concentration were detected by the ELISA method described above. As shown in FIG. 8, the concentration of bispecific antibody protein expressed by MTS001-LNP delivered 4# bispecific antibody mRNA in serum was proportional to the administration dose. The serum exposure of bispecific antibody protein expressed by high-dose 4# bispecific antibody mRNA delivered by MTS001-LNP was much lower than that of ERY974, but it showed better efficacy.

[0420] Example 8: Lipid nanoparticles prepared using ionizable lipid compounds of the present application have good in vivo delivery efficiency

[0421] S1: Nanoparticle preparation

[0422] The materials used for the assembly of lipid nanoparticles are: (1) ionizable lipid compounds: ionizable lipids designed and synthesized in the present application or DLin-MC3-DMA (MC3, purchased from AVT) as a control group; (2) structural lipids: such as Cholesterol (purchased from Sigma-Aldrich); (3) phospholipids: such as DSPC, which is 1,2-distearoyl-SN-glycero-3-phosphocholine (purchased from AVT); (4) PEGylated lipid compounds: such as DMG-PEG2000, which is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (purchased from AVT); (5) nucleic acid fragment active ingredients: such as Luciferase mRNA, siRNA, CRISPR Cas 9 mRNA, etc. The names and structural formulas of the materials used for the assembly of lipid nanoparticles are shown in Table 6.

[0423] Table 6

[0424] Preparation method of lipid nanoparticles: (1) Ionizable lipid compounds (MTS001-MTS027), cholesterol, phospholipids and polyethylene glycol-modified lipids were dissolved and mixed in ethanol at (molar percentage) 50%, 38.5%, 10% and 1.5% respectively; (2) mRNA active ingredient was dissolved in 25mM sodium acetate solution (pH=4.5); (3) The organic phase containing the lipid mixture and the aqueous phase containing the mRNA were mixed at a flow rate ratio of 1:1 to 1:4 using an automated high-throughput microfluidic system, with a mixing speed of 10mL / min to 18mL / min; (4) The prepared lipid nanoparticles (N / P ratio of 6) were diluted with phosphate buffer solution and ultrafiltered to the original preparation volume using an ultrafiltration tube with a molecular weight cutoff of 30kDa (purchased from Millipore); (5) The obtained nanoparticles were sterilized by filtration through a 0.2μm sterile filter membrane and then stored at low temperature in a sealed glass bottle.

[0425] Lipid nanoparticles can be prepared using microfluidic mixing systems, but are not limited to this method; other methods include T-type mixers and ethanol injection.

[0426] S2: Physical property characterization of lipid nanoparticles

[0427] The particle size and particle size distribution index (PDI) of the prepared lipid nanoparticles were measured using a Zetasizer Pro (purchased from Malvern Instruments Ltd) and a DynaPro NanoStar (purchased from Wyatt) dynamic light scattering instrument. The degree of RNA encapsulation by the lipid nanoparticles was characterized by the encapsulation efficiency (%), which reflects the degree of binding between the lipid nanoparticles and the RNA fragments. This coefficient was obtained from Quant-it... TM The RiboGreen RNAAssay (purchased from Invitrogen) method was used for measurement. Lipid nanoparticle samples were diluted in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5), and a portion of the sample solution was added to 0.5% Triton X-100 and incubated at 37°C for 30 minutes. Immediately after the reaction, the fluorescence values ​​were read using a Varioskan LUX multi-functional microplate reader (purchased from Thermofisher) at an absorption wavelength of 485 nm and an emission wavelength of 528 nm to obtain the encapsulation loading rate.

[0428] S3: Animal Experiments

[0429] The delivery effect and safety of the nanoparticles loaded with luciferase mRNA (Trilink, L-7202) in mice were evaluated. The test mice were SPF C57BL / 6 mice, female, 6-8 weeks old, weighing 18-22 g, purchased from Beijing Sbielfo Biotechnology Co., Ltd. All animals were adaptively fed for more than 7 days before the test, and free access to food and water during the test, 12 / 12h light and dark alternation, indoor temperature of 20-26℃, humidity of 40-70%. The mice were randomly divided into groups, n=3. The above prepared lipid nanoparticles loaded with luciferase mRNA were injected into the mice at a single dose of 0.5 mg / kg mRNA by intravenous administration, and the mice were detected by small animal living imaging system (IVIS LUMINA III, purchased from PerkinElmer) at 6 hours after administration. The specific operation steps of detection are as follows: prepare a D-luciferin solution with a concentration of 15 mg / mL with normal saline, and give each mouse a intraperitoneal injection of substrate. After 10 minutes of substrate administration, the mice were placed in a narcotizing box for anesthesia with a concentration of 2.5% isoflurane. The anesthetized mice were placed in IVIS for fluorescence imaging and data collection and analysis of the fluorescence concentrated distribution site.

[0430] The in vivo delivery efficiency of the lipid nanoparticle carrier was represented by the average value of the fluorescence intensity and the total photon number of different animals in the same test group, as shown in Table 7. The higher the fluorescence intensity and total photon number, the higher the in vivo delivery efficiency of the lipid nanoparticle for the mRNA fragment. The lipid nanoparticles containing the ionizable lipids provided by the present application have good in vivo delivery efficiency.

[0431] Table 7

[0432] Example 9: Stability of the lipid nanoparticles provided by the present application

[0433] MTS001 lipid prepared LNP encapsulated luciferase mRNA (Trilink, L-7202) was used for LNP stability study, LNP formulation was shown in Table 8, N:P=6, Lipid 5, DOPE was purchased from AVT. The preparation of lipid nanoparticles and physical property characterization method was referred to Example 4, and the LNP was diluted to 0.1 mg / mL. The prepared lipid nanoparticles were subjected to freeze-thaw once, repeated freeze-thaw 2 times, repeated freeze-thaw 3 times of physical property characterization detection, the specific steps were as follows: the freshly prepared lipid nanoparticles were detected for physical characterization, then the lipid nanoparticles were frozen at-20℃, after 48h, they were taken out and thawed at room temperature, the thawed freeze-thaw 1 time batch of lipid nanoparticles was subjected to physical property characterization, the rest of the lipid nanoparticles were returned to-20℃ freezing, after 48h, they were taken out and thawed at room temperature, the thawed repeated freeze-thaw 2 times batch of lipid nanoparticles was subjected to physical property characterization, the rest of the lipid nanoparticles were returned to-20℃ freezing, after 48h, they were taken out and thawed at room temperature, the thawed repeated freeze-thaw 3 times batch of lipid nanoparticles was subjected to physical property characterization, the characterization results of the freshly prepared LNP (stored at 2-8℃) and the LNP subjected to repeated freeze-thaw 3 times were shown in Table 9. At the same time of carrying out the characterization stability test, the mouse C57BL / 6 in vivo small animal imaging detection of Luciferase expression was carried out, to further study the mRNA expression stability. After each freeze-thaw, a single dose of 0.5 mg / kg mRNA was injected into the mouse body by intravenous administration, and the mouse was subjected to in vivo bioluminescence detection by small animal in vivo imaging system (IVIS LUMINA III, purchased from PerkinElmer) at 6h after administration, the specific experimental method was referred to Example 8, S3 animal experiment part, the results were shown in Table 10; the prepared fresh LNP was frozen at-20℃ for 30d, then it was taken out and thawed at room temperature, the physical property and mouse in vivo bioluminescence detection data were shown in Table 11.

[0434] Table 8

[0435] Table 9

[0436] Table 10

[0437] Table 11

[0438] Example 10: Effect of the lipid nanoparticles provided by the present application for preventing tumor recurrence

[0439] Hepa1-6 mouse hepatocarcinoma cells overexpressing human GPC3 and luciferase fluorescent enzyme were inoculated in situ in the liver of 8-10 week old CD3EDG humanized mice (Shanghai South Model Organisms Technology Co., Ltd.), and the specific experimental process is described in Example 6. Eight days after cell inoculation, luciferase live imaging analysis was performed on the mice, and appropriate mice were selected for grouping according to the imaging fluorescence value and body weight (average imaging signal intensity of tumor about 2E+08 p / s), with an average of 5 mice in each of the 6 experimental groups, a total of 30 mice. LNP formulation: the molar percentage of ionizable lipid (MTS001), structural lipid (CHO), neutral lipid (DSPC) and polymer lipid (DMG-PEG2000) in total lipid is 50%, 38.5%, 10% and 1.5% respectively, loaded with 4# bispecific antibody mRNA, N / P ratio is 6, and the preparation of lipid nanoparticles refers to Example 4, and the LNP is diluted to 0.1 mg / mL, hereinafter referred to as MTS-LNP1. The next day after grouping, drug administration began, recorded as D0, and the specific administration regimen is shown in Table 12.

[0440] Table 12

[0441] Tris solvent control group, LNP empty control group, MTS-LNP1 high, medium and low dose groups and ERY974 control group were set up in the experiment, and the mice were injected with drugs through the tail vein, once a week, for a total of 3 times. The mice were subjected to live imaging and body weight measurement twice a week after drug administration. The change in imaging signal intensity of animals in each group is shown in Figure 9, and the results show that the MTS-LNP1 high dose group (0.025 mg / kg) shows a significant tumor growth inhibition effect, and the mouse tumors are completely regressed after two weeks of drug administration, and shows a dose-dependent trend, and the treatment dose is much lower than that of the control group ERY97. Figure 10 shows that after multiple administrations of MTS-LNP1, the animals did not show weight loss and tolerated well.

[0442] The ability of MTS-LNP1 to induce immune memory in an immune-competent mouse model was further evaluated. The above-mentioned 5 mice that achieved complete tumor regression after treatment with 0.025 mg / kg dose of MTS-LNP1 were again inoculated with Hepa1-6-hGPC3-luc tumor cells (inoculation method same as Example 6) after 84 days of drug withdrawal observation; at the same time, 6 CD3EDG humanized mice that had not received treatment and were inoculated with tumors were used as blank controls. As shown in Figure 11, the MTS-LNP1 cured group of mice did not show tumor growth after the second tumor inoculation, while the control group of CD3EDG humanized mice showed a typical tumor growth curve.

[0443] Example 11: Pharmacodynamic evaluation of MTS-LNP1 in PBMC immune-reconstituted human orthotopic liver cancer mouse model

[0444] NOG severe immunodeficient mice (Vital River) were selected for human immune system reconstitution, and 4 days after injection of human PBMC (Shanghai Aosen), the modified Hep3B human liver cancer cells overexpressing luciferase were inoculated in situ in the liver of the mice. The specific experimental process is described in Example 6. On day 15 after inoculation of PBMC, the appropriate mice were selected for grouping according to the fluorescence signal intensity, body weight and the percentage of human CD45 positive cells in peripheral blood (hCD45%), and the average imaging signal intensity of the tumor was about 1E+09 p / s. The mice were evenly distributed into 5 experimental groups, 6 mice in each group, and a total of 30 mice. Drug administration started the day after grouping, denoted as D0, and the specific administration regimen is shown in Table 13:

[0445] Table 13

[0446] The experiment set up Tris solvent control group, LNP empty control group, MTS-LNP1 high and low dose groups and ERY974 control group. The mice were administered by tail vein injection, once a week, for a total of 3 times. The mice were subjected to live imaging and body weight measurement twice a week after administration. The change in imaging signal intensity of the animals in each group is shown in Figure 12, and the results show that the MTS-LNP1 high dose group (0.025 mg / kg) showed a significant tumor growth inhibition effect, and showed a dose-dependent trend. Figure 13 shows that compared with the control group, MTS-LNP1 did not cause body weight loss of the animals after multiple administrations, and was well tolerated.

[0447] Example 12: Pharmacodynamic evaluation of MTS-LNP1 in a PBMC immune reconstituted human orthotopic lung cancer mouse model

[0448] NOG severe immunodeficient mice (Vital River) were selected for human immune system reconstitution, and 5 days after injection of human PBMC (Shanghai Aosen), the modified NCI-H520 human lung cancer cells overexpressing luciferase were inoculated in situ in the lung of the mice. The specific experimental process is as follows: the animals were anesthetized by intraperitoneal injection of anesthetic (Avertin), and after the mice were completely anesthetized, the left side of the chest and back was prepared for skin, the mice were placed in a right lateral position, and the left chest of the mice was thoroughly disinfected with iodophor cotton ball. At the middle of the axillary line, costal margin and acromioclavicular joint, the skin was longitudinally opened about 1 cm, and the skin incision was separated with an ophthalmic forceps, and the pink lung at the chest wall was visible. A curved 4.5-5 mm 29G insulin syringe was used to inject the NCI-H520 human lung cancer cells overexpressing luciferase suspended in Matrigel at 1x10 6Administer a dose of 0.03 mL / mouse at a uniform rate into the left lung lobe of the mouse, pause for approximately 2 seconds, quickly withdraw the syringe, close the skin incision, and place the mouse on a heated table (avoid compressing the mouse's chest cavity during this process). After the mouse awakens, return it to its cage for continued feeding and daily observation. On day 13 after PBMC inoculation, suitable mice were selected for grouping based on fluorescence signal intensity, body weight, and hCD45% in peripheral blood (average tumor imaging signal intensity approximately 2E+08 p / s). These mice were evenly distributed into 5 experimental groups, with 5 mice in each group, for a total of 25 mice. Drug administration began the day after grouping, designated as D0. The specific dosing regimen is shown in Table 14.

[0449] Table 14

[0450] The experiment included a PBS solvent control group, an LNP empty vector control group, an ERY974 control group, and two MTS-LNP1 high-dose groups. Mice were administered the drug via tail vein injection twice weekly for a total of six administrations. In vivo imaging and body weight were performed twice weekly after administration. The changes in imaging signal intensity in each group are shown in Figure 14. The results indicate that both the high- and low-dose MTS-LNP1 groups showed significant tumor growth inhibition effects in a dose-dependent manner. Figure 15 shows that, compared to the control group, multiple administrations of MTS-LNP1 did not cause a decrease in body weight in the animals.

[0451] Example 13: Single or repeated administration of MTS-LNP1 in cynomolgus monkeys resulted in ideal TCE exposure and was well tolerated.

[0452] To explore the pharmacokinetic characteristics of MTS-LNP1 injection in cynomolgus monkeys, six monkeys were used. Cynomolgus monkeys were randomly divided into three groups (MTS201, MTS301, and MTS401), with two animals in each group (half male and half female). The dosages were set at 15, 50, and 100 μg / kg, respectively. All animals were administered the drug intravenously at a volume of 10 mL / kg over 30 min. Plasma samples were collected before administration and at 0.25, 1, 6, 12, 24, 48, 72, and 168 h after administration. The concentration of TCE protein expressed by MTS-LNP1 in cynomolgus monkey plasma was determined using the Electrochemiluminescence Immunoassay (ECLIA) method. Specifically, 96-well plates were coated with Human Glypican 3 / GPC3 Protein, His Tag, and premium grade reconstituted solution, incubated overnight at 2–8°C (16–20 h), blocked with 5% Milk-PBST, and then the test sample was added, followed by the addition of SULFO-TAG. TMCD3 as detection solution, MSD Read Buffer T (2x) was added to the plate and incubated at room temperature for 30 min. Sector S 600MM electrochemiluminescence instrument. The theoretical standard curve concentration is the abscissa, and the OD value difference between the average of the standard curve wells and the average of the blank wells is the ordinate. The correlation parameters of the standard curve are fitted by a four-parameter regression model (a suitable weight factor can be selected), and the sample concentration is calculated by Methodical Mind data acquisition and SoftMax Pro software. The results show that, overall, at a mRNA dose of 15-100 μg / kg, the MTS-LNP1 expressed TCE protein in vivo showed a dose-linear PK characteristic (Figure 16). The peak time (T max ) was 6-12 h, indicating that MTS-LNP1 can be rapidly expressed in vivo. The terminal elimination half-life (T 1 / 2 ) was 9.52-13.8 h, which was significantly longer than the conventional BiTE form TCE protein drug (such as Blincyto). In chimpanzees, the T 1 / 2 was 2.05 h, and in humans, the T 1 / 2 was 2.10 h (data source: Blincyto: Pharmacology Review(s). 2014; Blincyto: Clinical Pharmacology Biopharmaceutics Review(s). 2014). The peak concentration (C max ) and the area under the curve (AUC all ) showed a proportional increase with the dose at a dose of 15-100 μg / kg (Figure 16, Table 15).

[0453] Table 15

[0454] In addition, at the above-mentioned doses, it was found that the toxic reactions of MTS-LNP1 injection in cynomolgus monkeys were at an acceptable level.

[0455] Obviously, the above examples are only examples for the sake of clarity, and are not limiting of the embodiments. Based on the above description, other different forms of changes or variations can also be made by those of ordinary skill in the art. All embodiments need not and cannot be exhaustively enumerated. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

[0456] Sequence information involved in the present application

Claims

1. A lipid nanoparticle for delivering a nucleic acid molecule encoding a secreted protein, comprising an ionizable lipid, a structural lipid, a neutral lipid, and a polymeric lipid, wherein the ionizable lipid is a compound of Formula (II), or a pharmaceutically acceptable salt, isotopologue, tautomer, or stereoisomer thereof, wherein a = 1, 2, 3, 4, 5 or 6; b = 4, 5, 6, 7, 8, 9 or 10; c = 1, 2, 3, 4, 5 or 6; d = 0, 1, 2, 3 or 4; c + d = 3, 4, 5, 6, 7, 8 or 9; M1and M2are independently selected from the group consisting of -C(O)O-, -OC(O)-, -O-, -SC(O)O-, -OC(O)NR-, -NRC(O)NR-, -OC(O)S-, -OC(O)O-, -NRC(O)O-, -SC(O)-, -C(O)S-, -NR-, -C(O)NR-, -NRC(O)-, -NRC(O)S-, -SC(O)NR-, -C(O)-, -OC(S)-, -C(S)O-, -OC(S)NR-, -NRC(S)O-, -S-S-, and -S(O) 0-2 -; R1and R2are independently selected from C 4-20 alkyl, C 4-20 alkenyl and C 4-20 alkynyl, optionally substituted with one or more R 1s and wherein one or more methylene units are optionally and independently replaced with -NR'-; R 1s independently selected from H, C 1-20 alkyl, -L c -OR c , -L c -SR c and -L c -NR c R’ c ; R and R' are each independently selected from H and C 1-20 alkyl; L c independently selected from a chemical bond and C 1-20 alkylene; R c and R' c are independently selected from H, C 1-20 alkyl, C 3-14 cycloalkyl and 3- to 14-membered heterocyclyl; R4and R5are independently selected from C 1-8 alkyl, optionally substituted with one or more R 4s substituents; or R4, R5 together with the carbon atom to which they are attached form C 3-14 cycloalkylene or 3- to 14-membered heterocyclylene, which is optionally substituted by one or more R 4s substituents; R 4s independently selected from H, halogen, cyano, C 1-8 alkyl, C 1-8 haloalkyl, -L d -OR d , -L d -SR d and -L d -NR d R’ d ; L d independently selected from a chemical bond and C 1-8 alkylene; R d and R' d are independently selected from H, C 1-8 alkyl, C 3-14 cycloalkyl and 3- to 14-membered heterocyclyl.

2. The lipid nanoparticle of claim 1, wherein M1 and M2 are independently selected from -C(O)O- or -OC(O)-, preferably wherein M1 is -OC(O)- and M2 is -C(O)O-.

3. The lipid nanoparticle of any one of claims 1-2, wherein R4and R5are independently C 1-8 alkyl, preferably C 1-3 alkyl, more preferably methyl; or R4, R5, together with the carbon atom to which they are attached form C 3-6 cycloalkylene or 3-6 membered heterocyclyl, preferably form C 3-4 cycloalkylene, more preferably form cyclopropylene.

4. The lipid nanoparticle of any one of claims 1-3, wherein R1and R2are independently selected from C 4-20 alkyl, preferably R1is C 6-14 alkyl and R2is C 8-20 alkyl, more preferably R1is C 8-12 straight chain alkyl, R2is C 8-20 branched chain alkyl.

5. The lipid nanoparticle of any one of claims 1 to 4, wherein a is 2, b is 7, c is 5, and d is 1.

6. The lipid nanoparticle of any one of claims 1-5, wherein the compound of Formula (II) is selected from the following compounds, or a pharmaceutically acceptable salt, isotopically enriched variant, tautomer, or stereoisomer thereof:

7. The lipid nanoparticle of any one of claims 1 to 6, comprising the following molar percentages of components: ionizable lipid: 35-65%, preferably 40-55%; structural lipid: 30-50%, preferably 35-50%; neutral lipid: 5-30%, preferably 8-20%; polymer lipid: 0.5-3.5%, preferably 1-3%, more preferably 1-2%; preferably comprising the following molar percentages of components: ionizable lipid: 40-65%, preferably 45-55%, more preferably 50%; structural lipid: 30-50%, preferably 35-40%, more preferably 38.5%; neutral lipid: 5-30%, preferably 8-15%, more preferably 10%; polymer lipid: 0.5-3.5%, preferably 1-3%, more preferably 1-2%, more preferably 1.5%; preferably comprising the following molar percentages of components: ionizable lipid: 40%; structural lipid: 43.5%; neutral lipid: 15%; polymer lipid: 1.5%.

8. The lipid nanoparticle of any one of claims 1 to 7, further comprising at least one cargo selected from a nucleic acid molecule encoding a secreted protein.

9. The lipid nanoparticle of any one of claims 1 to 8, wherein the secreted protein is selected from any one of a cytokine, an antigen binding protein; preferably the antigen binding protein is selected from an antibody or antibody fragment, more preferably a bispecific antibody or fragment thereof; more preferably the bispecific antibody is selected from any one of BiTE Triomabs, Knobs-into-holes, Crossmab, Ortho-Fab, DVD-Ig, Two-in-one, IgG-scFv, scFv2-Fc, bi-Nanobody, DART or TandAb or VHH formats.

10. The lipid nanoparticle of any one of claims 1 to 9, wherein the nucleic acid molecule encoding a secreted protein is selected from an antisense oligonucleotide (ASO), an RNA or a DNA; preferably wherein the RNA is selected from at least one of a messenger RNA (mRNA), a modified mRNA (mmRNA), a circular RNA (circRNA) and a self-replicating RNA (SrRNA), preferably a mRNA or a modified mRNA, more preferably a modified mRNA; or Preferably, wherein the DNA is selected from at least one of the following: plasmid DNA (pDNA) and minicircle DNA (mcDNA).

11. The lipid nanoparticle of any one of claims 1-10, wherein the payload is an mRNA or modified mRNA molecule; Preferably, the payload is an mRNA or modified mRNA molecule comprising a coding sequence for an antibody against a liver tumor surface antigen; or, the payload is an mRNA or modified mRNA molecule comprising a coding sequence for an antibody against a protein associated with a liver tumor; or, the payload is an mRNA or modified mRNA molecule comprising a coding sequence for an antibody against a T cell surface antigen; More preferably, the payload is an mRNA or modified mRNA molecule comprising a coding sequence for an antibody against GPC3; or, the payload is an mRNA or modified mRNA molecule comprising a coding sequence for an antibody against CD3; More preferably, the payload is an mRNA or modified mRNA molecule comprising a coding sequence for a bispecific antibody; More preferably, the payload is an mRNA or modified mRNA molecule comprising a coding sequence for a bispecific antibody against GPC3 and CD3; More preferably, wherein the coding sequence for the bispecific antibody against CD3 and GPC3 comprises a scFv against GPC3, the heavy chain variable region of the scFv against GPC3 comprises a VH CDR1 set forth in SEQ ID NO: 17, a VH CDR2 set forth in SEQ ID NO: 18, a VH CDR3 set forth in SEQ ID NO: 19, the light chain variable region of the scFv against GPC3 comprises a VL CDR1 set forth in SEQ ID NO: 20, a VL CDR2 set forth in SEQ ID NO: 21, a VL CDR3 set forth in SEQ ID NO: 22; More preferably, wherein the scFv against GPC3 comprises a heavy chain variable region set forth in SEQ ID NO: 23 and a light chain variable region set forth in SEQ ID NO: 24; More preferably, wherein the bispecific antibody against GPC3 and CD3 is in the form of BiTE.

12. A method of preparing the lipid nanoparticle of any one of claims 1-11, comprising: Mixing the lipid components in the lipid nanoparticle, and mixing with the payload, to obtain the lipid nanoparticle.

13. A composition comprising the lipid nanoparticle of any one of claims 1-11.

14. A pharmaceutical composition containing the lipid nanoparticle of any one of claims 1-11 and optionally a pharmaceutically acceptable excipient, such as a carrier, adjuvant, or vehicle, said pharmaceutical composition preferably being a liquid formulation, more preferably an injection.

15. Use of the lipid nanoparticle of any one of claims 1-11 or the pharmaceutical composition of claim 14 in the manufacture of a medicament for the treatment of a disease or for improving a patient’s prognostic condition. i. preferably, the disease is a liver disease or a liver-related disease; more preferably, the liver disease or liver-related disease is a malignant tumor of the liver, more preferably hepatocellular carcinoma (HCC); or ii. preferably, the disease is a GPC3-related disease; more preferably, the disease is a tumor that overexpresses GPC3, such as a liver cancer, e.g. hepatocellular carcinoma (HCC), a lung cancer, e.g. lung squamous cell carcinoma (SqCC), a gastric cancer, an ovarian cancer, e.g. ovarian clear cell carcinoma, a melanoma or a pediatric embryonal tumor.

16. Use of the lipid nanoparticle of any one of claims 1-11 or the pharmaceutical composition of claim 14 for the manufacture of a medicament for the delivery of a payload.

17. A method of treating a disease or improving a prognostic condition in a patient in a subject, comprising administering to the subject an effective amount of the lipid nanoparticle of any one of claims 1-11 or the pharmaceutical composition of claim 14; i. preferably, the disease is a liver disease or a liver-related disease; more preferably, the liver disease or liver-related disease is a malignant tumor of the liver, more preferably hepatocellular carcinoma (HCC); or ii. preferably, the disease is a GPC3-related disease; more preferably, the disease is a tumor that overexpresses GPC3, such as a liver cancer, e.g. hepatocellular carcinoma (HCC), a lung cancer, e.g. lung squamous cell carcinoma (SqCC), a gastric cancer, an ovarian cancer, e.g. ovarian clear cell carcinoma, a melanoma or a pediatric embryonal tumor.

18. The method of claim 17, wherein the lipid nanoparticle or pharmaceutical composition is administered systemically, preferably intravenously, intra-arterially or intraperitoneally, more preferably intraperitoneally or intravenously.

19. The lipid nanoparticle of any one of claims 1-11 or the pharmaceutical composition of claim 14 for use in the treatment of a disease or improving a prognostic condition in a patient; i. preferably, the disease is a liver disease or a liver-related disease; more preferably, the liver disease or liver-related disease is a malignant tumor of the liver, more preferably hepatocellular carcinoma (HCC); or ii. preferably, the disease is a GPC3-related disease; more preferably, the disease is a tumor that overexpresses GPC3, such as a liver cancer, e.g. hepatocellular carcinoma (HCC), a lung cancer, e.g. lung squamous cell carcinoma (SqCC), a gastric cancer, an ovarian cancer, e.g. ovarian clear cell carcinoma, a melanoma or a pediatric embryonal tumor.

20. A method of delivering a payload in a subject, comprising administering to the subject the lipid nanoparticle of any one of claims 1-11 or the pharmaceutical composition of claim 14.

21. The lipid nanoparticle of any one of claims 1-11 or the pharmaceutical composition of claim 14 for use in the delivery of a payload.

22. Use of the lipid nanoparticle of any one of claims 1-11 or the pharmaceutical composition of claim 14 for the manufacture of a medicament for the prevention of tumor occurrence or the prevention of tumor recurrence.

23. The lipid nanoparticle of any one of claims 1-11 or the pharmaceutical composition of claim 14 for the prevention of tumor occurrence or the prevention of tumor recurrence.

24. A method of preventing tumor occurrence or preventing tumor recurrence in a subject, comprising administering to the subject an effective amount of the lipid nanoparticle of any one of claims 1-11 or the pharmaceutical composition of claim 14.

25. The lipid nanoparticle of any one of claims 1-11 or the pharmaceutical composition of claim 14, wherein the cargo is an mRNA encoding a secreted protein, and the half-life in vivo of the secreted protein obtained by administering the lipid nanoparticle or the pharmaceutical composition via a systemic route of administration is longer than the half-life in vivo of the secreted protein obtained by administering the secreted protein via a systemic route of administration, preferably 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold or more longer, more preferably 10-fold or more longer.

26. The lipid nanoparticle of any one of claims 1-11 or the pharmaceutical composition of claim 14 for use in improving or enhancing the efficacy of a secreted protein in preventing or treating a disease, wherein the cargo is an mRNA encoding the secreted protein, and the half-life in vivo of the secreted protein obtained by administering the lipid nanoparticle or the pharmaceutical composition via a systemic route of administration is longer than the half-life in vivo of the secreted protein obtained by administering the secreted protein via a systemic route of administration, preferably 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold or more longer, more preferably 10-fold or more longer.

27. A method of improving or enhancing the efficacy of a secreted protein in preventing or treating a disease, the method comprising administering the lipid nanoparticle of any one of claims 1-11 or the pharmaceutical composition of claim 14, wherein the cargo is an mRNA encoding the secreted protein, and the half-life in vivo of the secreted protein obtained by administering the lipid nanoparticle or the pharmaceutical composition via a systemic route of administration is longer than the half-life in vivo of the secreted protein obtained by administering the secreted protein via a systemic route of administration, preferably 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold or more longer, more preferably 10-fold or more longer.

28. The lipid nanoparticle of any one of claims 1-11 or the pharmaceutical composition of claim 14 for use in reducing side effects of a secreted protein in preventing or treating a disease, wherein the cargo is an mRNA encoding the secreted protein, and the half-life in vivo of the secreted protein obtained by administering the lipid nanoparticle or the pharmaceutical composition is longer than the half-life in vivo of the secreted protein obtained by administering the secreted protein via a systemic route of administration, preferably 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold or more longer, more preferably 10-fold or more longer.

29. A method of reducing side effects of a secreted protein in preventing or treating a disease, the method comprising administering the lipid nanoparticle of any one of claims 1-11 or the pharmaceutical composition of claim 14, wherein the payload is an mRNA encoding the secreted protein, and the half-life in vivo of the secreted protein obtained by administering the lipid nanoparticle or the pharmaceutical composition via a systemic route of administration is longer than the half-life in vivo of the secreted protein obtained by administering the secreted protein via a systemic route of administration, preferably 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold or more, more preferably 10-fold or more longer.

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