Lipid nanoparticle, and preparation method therefor and use thereof

Abstract

A lipid nanoparticle, and a preparation method therefor and the use thereof. The lipid nanoparticle comprises packaging materials, namely, cationic lipids, neutral lipids, PEG or PEGylated lipids, and cholesterol or derivatives thereof at a molar percentage of (33-50)%:(10-13.33)%:(1.5-2)%:(38.5-51.33)%. The synthesis of a targeted LNP within the molar percentage range has a higher encapsulation efficiency, and can significantly improve the transduction efficiency of the targeted LNP, which can not only improve the encapsulation efficiency and loading capacity of the particle, but also enhances the targeting function of T cells, thereby realizing highly efficient transfection of T cells.

Description

Lipid nanoparticles, their preparation methods and applications

[0001] This application claims priority to Chinese Patent Application No. 202510186021.5, filed on February 19, 2025, entitled "Lipid Nanoparticles and Preparation Methods Thereof and Applications Thereof", and Chinese Patent Application No. 202510186026.8, filed on February 19, 2025, entitled "Fusion Proteins and Their Applications in the Preparation of T-cell Targeting LNPs Thereof", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of nucleic acid gene therapy technology, and in particular to lipid nanoparticles, their preparation methods, and applications. Background Technology

[0003] Lipid nanoparticles (LNPs) are lipid vesicles composed of cationic lipids, cholesterol, polyethylene glycol lipids, and auxiliary lipids (or neutral lipids) that can be used for nucleic acid delivery.

[0004] The encapsulation efficiency of LNPs is a core quality indicator for LNP formulations. Encapsulation efficiency determines the quality and efficacy of the drug and is incorporated into the core quality control standards for LNP-related products. Traditional LNPs are non-targeted applications. With technological advancements, targeted delivery applications are increasingly combining the delivery characteristics of LNPs with the targeting principles of target proteins to cells in vivo. This necessitates the insertion of exogenous target proteins into the LNP structure to form targeted LNPs.

[0005] CAR cell therapy is a promising immunotherapy, but current methods involve editing immune cells (such as T cells or NK cells) using viral or non-viral vectors, followed by expansion and reinfusion. However, this approach is costly and time-consuming (weeks to months), increasing the burden on patients and potentially leading to disease progression. Existing technologies utilize targeted LNP particles to encapsulate CAR nucleic acids, delivering them directly into the body to enable T cells to express CAR. This approach avoids the complex in vitro cell editing and culture process, potentially significantly shortening the treatment cycle and reducing costs. However, in vivo CAR-T preparation protocols typically use CD3 ScFv as the target protein, which has limited targeted transduction efficiency. This means that LNP-encapsulated CAR nucleic acids may not be efficiently delivered to the target immune cells, thus affecting the final efficacy of CAR cells.

[0006] Therefore, it is crucial to develop new LNPs to improve the in vivo editing efficiency of CAR nucleic acids. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide lipid nanoparticles, their preparation methods and applications.

[0008] The lipid nanoparticles provided by this invention have packaging materials comprising: cationic lipids, neutral lipids, PEG or PEGylated lipids, cholesterol or its derivatives; the molar percentages of the cationic lipids, neutral lipids, PEG or PEGylated lipids, cholesterol or its derivatives are (33%–50%): (10%–13.33%): (1.5%–2%): (38.5%–51.33%).

[0009] To improve the transfection ability of LNP on T cells, the PEG content in the coating was optimized. However, the results showed that simply changing the PEG content did not improve the transfection ability of the targeting particles on T cells. Therefore, this invention optimized the ratio of each component in the coating material. The results showed that, at a specific ratio, the transfection ability of LNP on cells was significantly improved. Specifically, the molar percentages of cationic lipids, neutral lipids, PEG or PEGylated lipids, cholesterol or its derivatives were (33%–35%): (10%–13.33%): (1.5%–2%): (50%–51.33%).

[0010] For example, the molar percentages of cationic lipids, neutral lipids, PEG or PEGylated lipids, cholesterol or their derivatives are (33%–34%): (13%–13.33%): (1.8%–2%): (51%–51.33%). More specifically, the molar percentages of cationic lipids, neutral lipids, PEG or PEGylated lipids, cholesterol or their derivatives are 33.33%: 13.33%: 2%: 51.33%.

[0011] In a specific embodiment, using an optimized lipid molar percentage range for targeted LNP synthesis results in higher encapsulation efficiency and significantly improved targeted LNP transduction efficiency. This not only enhances particle encapsulation efficiency and loading capacity but also improves T cell targeting function, achieving highly efficient T cell transfection.

[0012] In this invention, the cationic lipids are selected from at least one of SM102, DLin-MC3-DMA, ALC-0315, or ATX-126;

[0013] In this invention, the neutral lipids are selected from at least one of DSPC, dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylcholine (DPPC), distearate phosphatidylethanolamine (DSPE), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), or phosphatidylserine (PS).

[0014] In this invention, the cholesterol derivative includes: 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol hydrochloride (DC-CHOL).

[0015] In this invention, the PEG is a PEG with an average molecular weight of 500 to 5000; for example, at least one of PEG500, PEG1000, PEG1500, PEG2000, PEG3000, PEG4000 or PEG5000.

[0016] In this invention, the PEGylated lipid is at least one of ALC-0159, DMG-PEG, PEG2000-C-DMG, DSPE-PEG, DPPE-MPEG(2000), DSPE-PEG-Amine, DSPE-PEG-NHS,MW 2000, DSPE-PEG-Maleimide (also known as DSPE-PEG-MAL or DSPE-PEG-maleimide), DMG-PEG-MAL (DMG-PEG-maleimide), aminated-PEG-DMG, and hydroxylated-PEG-DMG, wherein the molecular weight of PEG can be selected from 500-5000, preferably PEG2000.

[0017] In some embodiments, the packaging material for the lipid nanoparticles is composed of SM102, DSPC, PEGylated lipids, and cholesterol. In this embodiment, the molar percentages of SM102, DSPC, PEGylated lipids, and cholesterol in the packaging material are 33.33%: 13.33%: 2%: 51.33%.

[0018] In the embodiments described above, the PEGylated lipids are PEG2000-DMG, PEG2000-DSPC, amino-PEG-DMG, and hydroxylated-PEG-DMG;

[0019] In this invention, the packaging material further includes PEGylated targeting proteins; the PEGylated targeting proteins include fusion proteins and PEGylated lipids, wherein the fusion proteins include antibodies targeting CD3 molecules and fusion proteins at the N-terminus of the extracellular domain of CD86.

[0020] In a specific embodiment, the PEGylated targeting protein is formed by an antibody containing a CD3 molecule, a CD86 extracellular N-terminus and a G4H12 fragment, and PEGylated lipids.

[0021] The antibody targeting CD3 molecules used in this invention is a single-chain variable region (ScFv) derived from an OKT3 antibody. In some embodiments, the CD3 ScFv has an amino acid sequence as shown in SEQ ID NO:5, or has an amino acid sequence obtained by deleting, substituting, or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO:5, or has an amino acid sequence that has more than 80% identity with the amino acid sequence shown in SEQ ID NO:5.

[0022] CD86 is a cell surface membrane protein belonging to the B7 family. It is highly expressed on the surface of antigen-presenting cells and contains two extracellular immunoglobulin domains. This invention utilizes the N-terminus of the CD86 extracellular segment, which participates in interactions with other ligands (such as CD28 or CTLA-4). In some embodiments, the N-terminus of the CD86 extracellular segment has the amino acid sequence shown in SEQ ID NO:6, or has an amino acid sequence obtained by deleting, substituting, or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO:6, or has an amino acid sequence with more than 80% identity to the amino acid sequence shown in SEQ ID NO:6.

[0023] In this invention, the role of G4H12 is to provide a disulfide bond at the tail of the fusion protein, which is then reduced by TCEP to form a free thiol group. This thiol group binds to the MAL group on DSPE-PEG-MAL. In some embodiments, the G4H12 fragment has the amino acid sequence shown in SEQ ID NO:7, or has an amino acid sequence obtained by deleting, substituting, or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO:7, or has an amino acid sequence with more than 80% identity to the amino acid sequence shown in SEQ ID NO:7.

[0024] In this invention, the "identity of more than 80%" means that the identity is not less than 80%, not less than 85%, not less than 90%, not less than 95%, not less than 98%, not less than 99%, not less than 99.5%, not less than 99.8%, or not less than 99.9%.

[0025] In this invention, the linking order of the fragments in the fusion protein is not limited. For example, from the N-terminus to the C-terminus, the sequence may be CD3 ScFv, the N-terminus of the CD86 extracellular segment, and the G4H12 fragment; or the N-terminus of the CD86 extracellular segment, CD3 ScFv, and the G4H12 fragment; or the G4H12 fragment, CD3 ScFv, and the N-terminus of the CD86 extracellular segment; or the G4H12 fragment, the N-terminus of the CD86 extracellular segment, and CD3 ScFv. In a specific embodiment, the fusion protein CD3 ScFv is linked to the N-terminus of the CD86 extracellular segment by 7 amino acids, and the C-terminus is linked to G4H12 to form the fusion protein.

[0026] In the fusion protein described in this invention, the various fragments may or may not contain linkers, and this invention does not impose any limitations on this. For example, a linker may be present between the N-terminus of the CD3 ScFv and CD86 extracellular segments.

[0027] More specifically, the fusion protein of the present invention comprises, from the N-terminus to the C-terminus, CD3 ScFv, linker, the N-terminus of the extracellular segment of CD86, and the G4H12 fragment.

[0028] As a feasible example, the linker is G4S, (G4S) n(n>1) GSTSGSGKPGSGEGSTKG, etc.

[0029] In this invention, the fusion protein is as described above (denoted as: CD3 ScFv-CD86-G4H12). Specifically, CD3 ScFv is coupled to the N-terminus of the extracellular segment of CD86 via amino acids, and G4H12 is linked to the C-terminus of the extracellular segment of CD86. The fusion protein described in this application includes a CD3-targeting ScFv (OKT3) linked to the extracellular segment of CD86 via a linker, and a G4H12 amino acid sequence is designed after CD86 to form the CD3 ScFv-CD86-G4H12 structure. This achieves targeted function, enhances T cell endocytosis without over-activating T cells.

[0030] In a specific instance, the fusion protein has the amino acid sequence shown in SEQ ID NO:1.

[0031] The aforementioned PEGylated targeting protein includes the fusion protein and PEGylated lipids as described above. The PEGylated lipids are selected from at least one of the following: ALC-0159, DMG-PEG, PEG2000-C-DMG, DSPE-PEG, DPPE-MPEG(2000), DSPE-PEG-Amine, DSPE-PEG-NHS,MW 2000, DSPE-PEG-Maleimide (also known as DSPE-PEG-MAL or DSPE-PEG-maleimide), DMG-PEG-MAL (DMG-PEG-maleimide), DMG-PEG2000, DSPE-PEG2000, DSPE-PEG2000-MAL, and DMG-PEG2000-MAL. In a specific embodiment, the average molecular weight of PEG in the PEGylated targeting protein is 2000.

[0032] The fusion protein in the PEGylated targeting protein provided by this invention is linked to PEGylated lipids via thioether bonds.

[0033] In this invention, the C-terminus of the fusion protein is reduced to form a -SH group, which then undergoes an alkylation reaction with the double bond in the PEGylated lipid to form a thioether bond.

[0034] The -SH group is formed when the target protein carrying G4H12 reduces the disulfide bond of G4H12 with the TCEP reducing agent to form -SH.

[0035] In this invention, the method for preparing a PEGylated target protein (containing CD3 ScFv, the N-terminus of the extracellular segment of CD86 and the G4H12 fragment) and PEGylated lipids includes mixing the target protein with a TCEP solution for reaction, adding a solution of PEGylated lipids, and obtaining the PEGylated target protein after the reaction.

[0036] This invention uses the TCEP method to modify the fusion protein to form a targeted protein-PEG2000-DSPE complex. The fusion protein-PEG2000-DSPE complex and other lipid components of LNP are directly synthesized into target nanoparticles through microfluidics. The synthesis process is a one-step process, which is simpler and solves the problem of dimer formation in SATS synthesis. It also improves the encapsulation efficiency, coupling efficiency and transduction efficiency of targeted LNP synthesis.

[0037] In this embodiment of the invention, the molar ratio of the fusion protein, TCEP, and PEGylated lipids as described above is 1:(25-50):(5-10). As a feasible example, the molar ratio of the fusion protein, TCEP, and PEGylated lipids as described above is 1:25:10.

[0038] Specifically, the preparation of the complex includes:

[0039] 1) Preparation of targeted protein solutions.

[0040] 2) Add an appropriate amount of 0.5M TCEP solution to the targeted protein solution and react at room temperature in the dark.

[0041] 3) Weigh an appropriate amount of DSPE-PEG-MAL and dissolve it in DMSO to prepare a 5mM (14.7mg / ml) DSPE-PEG-MAL working solution.

[0042] 4) Add an appropriate amount of 5mM DSPE-PEG-MAL working solution to the solution in step 2), with the target protein:TCEP:DSPE-PEG-MAL = 1:25:10. React at room temperature in the dark.

[0043] 5) Add the solution obtained in step 4) to an equilibrated desalting column to remove impurities. This yields the PEGylated target protein CD3 ScFv-CD86-PEG-DSPE linked to the target protein.

[0044] In a specific embodiment, a step can be added whereby solutions obtained from different steps are added to an equilibrated desalting column for solution displacement or impurity removal. In another specific embodiment, the TCEP method is used to modify the fusion protein, which can also be CD3 ScFv-CD86-PEG-DMG. The preparation of the complex differs from the above preparation scheme only in that DSPE-PEG-MAL is replaced with DMG-PEG-MAL.

[0045] In this invention, the lipid nanoparticles also include nucleic acids coated within the packaging material.

[0046] The nucleic acid includes, but is not limited to, at least one of mRNA, siRNA, miRNA, DNA, dsRNA, and sDNA. The mRNA includes mRNA encoding CAR, mRNA encoding fluorescent protein, siRNA, miRNA, DNA, dsRNA, sDNA, small interfering RNA, miRNA, aiRNA, shRNA, tRNA, ssDNA, dsDNA, plasmids, etc.

[0047] In some embodiments, the nucleic acid is a chimeric antigen receptor (CAR) nucleic acid sequence that recognizes CD19-positive tumor cells; in other embodiments, the chimeric antigen receptor (CAR) nucleic acid sequence that recognizes CD19-positive tumor cells can be replaced with a target molecule nucleic acid sequence that recognizes at least one surface-expressed target molecule, including solid tumors, hematologic malignancies, and / or tissues, wherein the target molecule includes any one or more of the following combinations: CD19, CD20, CD22, CD33, CLL-1 (CLEC12A), CD7, CD5, CD70, CD123, CEA, CEACAM5, CEACAM6, CE ACAM7, Mesothelin, MUC1, CLDN18.2, CDH17, Trop2, BCMA, NKG2D, PDL1, EGFR, EGFRVIII, PSCA, PSMA, MUC16, CD133, GD2, IL13R2, B7H3, Her2, CD30, SLAMF7, CD38, GPC3, WT1, AFP, FOLR1, c-Met, LeY (Lewis-Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), MAGE-A1 (melanoma-associated antigen 1), and TAG-72. It can be used to treat tumors such as colon cancer, rectal cancer, small intestine cancer, anal cancer, bile duct cancer, stomach cancer, esophageal cancer, gallbladder cancer, lung cancer, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, kidney cancer, bladder cancer, cancers of the central nervous system, glioblastoma, skin cancer, melanoma, lymphoma, head and neck cancer, multiple myeloma, and leukemia.

[0048] In some embodiments, the chimeric antigen receptor (CAR) nucleic acid sequence that recognizes CD19-positive tumor cells can be replaced with an antigenic peptide nucleic acid sequence that can elicit an immune response in the organism, such as a combination of at least one or more nucleic acid fragments of HPV genome E1-E7, L1-L2, or antigenic peptide nucleic acid sequences or sequences of viruses or bacteria related to diseases such as hepatitis A, hepatitis B, poliomyelitis, influenza, and pneumonia, thereby obtaining a vaccine drug for treating the above-mentioned diseases or as a component of a drug combination.

[0049] In some embodiments, the chimeric antigen receptor (CAR) nucleic acid sequence that recognizes CD19-positive tumor cells can be replaced with active agents or therapeutic agents related to ophthalmic diseases, metabolic diseases, hyperlipidemia, hepatitis B, pseudohypertrophic muscular dystrophy, non-alcoholic fatty liver disease, heart disease, amyotrophic lateral sclerosis, cystic fibrosis, etc., such as siRNA, miRNA, ASO, small activating RNA (saRNA), aptamers, transfer RNA (tRNA) fragments, etc., as drugs or drug combinations to treat ophthalmic diseases, metabolic diseases, hyperlipidemia, hepatitis B, pseudohypertrophic muscular dystrophy, non-alcoholic fatty liver disease, heart disease, amyotrophic lateral sclerosis, cystic fibrosis, etc.

[0050] Furthermore, the present invention also provides a method for preparing the lipid nanoparticles, comprising:

[0051] The lipid nanoparticle packaging material described above is dissolved in ethanol to obtain the alcohol phase;

[0052] The PEG-conjugated fusion protein and the nucleic acid as described above were dissolved in citrate buffer to obtain the citrate phase; the alcohol phase and the citrate phase were then prepared into lipid nanoparticles via microfluidic control.

[0053] In a specific embodiment, the citrate buffer solution has a pH of 3-5 and a concentration of 0.01M-0.03M. For example, the citrate buffer solution has a pH of 3.0, 3.5, 4.0, 4.5, or 5.0; and a concentration of 0.01M, 0.015M, 0.02M, 0.025M, or 0.03M. Preferably, the citrate buffer solution has a pH of 4.0 and a concentration of 0.02M.

[0054] In the microfluidic step, the ratio of alcohol phase flow rate to citric acid phase flow rate is 1:(1-5). For example, the ratio of alcohol phase flow rate to citric acid phase flow rate is 1:1, 1:2, 1:3, 1:4, or 1:5. Preferably, the ratio of alcohol phase flow rate to citric acid phase flow rate is 1:3.

[0055] Furthermore, the present invention also provides the use of the lipid nanoparticles in the preparation of products for in vivo delivery of nucleic acids to mammalian subjects.

[0056] Furthermore, the present invention also provides a nucleic acid delivery reagent comprising the aforementioned lipid nanoparticles and buffer solution.

[0057] Furthermore, the present invention also provides a pharmaceutical composition comprising the lipid nanoparticles as described above.

[0058] Furthermore, the present invention also provides the use of the lipid nanoparticles as described above in the preparation of products that introduce nucleic acids into cells.

[0059] Furthermore, the present invention also provides the use of the lipid nanoparticles as described above in the preparation of medicaments for treating diseases or functional disorders in mammalian subjects requiring treatment.

[0060] In this invention, the disease includes at least one of the following: disease caused by infection, metabolic disease, hyperlipidemia, hepatitis B, pseudohypertrophic muscular dystrophy, non-alcoholic fatty liver disease, heart disease, amyotrophic lateral sclerosis, cystic fibrosis, genetic defect disease, or tumor.

[0061] Furthermore, the present invention also provides a cell transfection method, which includes incubating cells after contacting them with the cell transfection reagent as described above. The cell transfection reagent of the present invention can be used for cell transfection, such as animal cells, human cells, plant cells, or microorganisms. When the PEGylated targeting protein therein is CD3ScFv-CD86-PEG-DSPE or CD3ScFv-CD86-PEG-DMG, it targets T cells, and the cell transfection reagent is a T cell-targeting transfection reagent.

[0062] In some embodiments, the cells are in vivo cells, such as those that recognize targeting molecules that target B cells, such as CD19, CD56, CD141, CD1c, CD11b, and CD14, or APC cells such as B cells, NK cells, macrophages, and dendritic cells. In a specific embodiment, the cells are T cells.

[0063] The fusion protein provided by the present invention includes CD3 ScFv, the N-terminus of the extracellular domain of CD86, and the G4H12 fragment.

[0064] The antibody targeting CD3 molecules used in this invention is a single-chain variable region (ScFv) derived from an OKT3 antibody. In some embodiments, the CD3 ScFv has an amino acid sequence as shown in SEQ ID NO:5, or has an amino acid sequence obtained by deleting, substituting, or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO:5, or has an amino acid sequence that has more than 80% identity with the amino acid sequence shown in SEQ ID NO:5.

[0065] CD86 is a cell surface membrane protein belonging to the B7 family. It is highly expressed on the surface of antigen-presenting cells and contains two extracellular immunoglobulin domains. This invention utilizes the N-terminus of the CD86 extracellular segment, which participates in interactions with other ligands (such as CD28 or CTLA-4). In some embodiments, the N-terminus of the CD86 extracellular segment has the amino acid sequence shown in SEQ ID NO:6, or has an amino acid sequence obtained by deleting, substituting, or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO:6, or has an amino acid sequence with more than 80% identity to the amino acid sequence shown in SEQ ID NO:6.

[0066] In this invention, the role of G4H12 is to provide a disulfide bond at the tail of the fusion protein, which is then reduced by TCEP to form a free thiol group. This thiol group binds to the MAL group on DSPE-PEG-MAL. In some embodiments, the G4H12 fragment has the amino acid sequence shown in SEQ ID NO:7, or has an amino acid sequence obtained by deleting, substituting, or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO:7, or has an amino acid sequence with more than 80% identity to the amino acid sequence shown in SEQ ID NO:7.

[0067] In this invention, the "identity of more than 80%" means that the identity is not less than 80%, not less than 85%, not less than 90%, not less than 95%, not less than 98%, not less than 99%, not less than 99.5%, not less than 99.8%, or not less than 99.9%.

[0068] In this invention, the linking order of the fragments in the fusion protein is not limited. For example, from the N-terminus to the C-terminus, the sequence may be CD3 ScFv, the N-terminus of the CD86 extracellular segment, and the G4H12 fragment; or the N-terminus of the CD86 extracellular segment, CD3 ScFv, and the G4H12 fragment; or the G4H12 fragment, CD3 ScFv, and the N-terminus of the CD86 extracellular segment; or the G4H12 fragment, the N-terminus of the CD86 extracellular segment, and CD3 ScFv. In a specific embodiment, CD3 ScFv is linked to the N-terminus of the CD86 extracellular segment by 7 amino acids, and G4H12 is linked to the C-terminus to form the fusion protein.

[0069] In the fusion protein described in this invention, the various fragments may or may not contain linkers, and this invention does not impose any limitations on this. For example, a linker may be present between the N-terminus of the CD3 ScFv and CD86 extracellular segments.

[0070] More specifically, the fusion protein of the present invention comprises, from the N-terminus to the C-terminus, CD3 ScFv, linker, the N-terminus of the extracellular segment of CD86, and the G4H12 fragment.

[0071] As a feasible example, the linker is G4S, (G4S)n (n>1), GSTGSGKPGSGEGSTKG, etc.

[0072] In some specific embodiments, the fusion protein has the amino acid sequence shown in SEQ ID NO:1. In this invention, the fusion protein as described above is denoted as: CD3 ScFv-CD86-G4H12. Specifically, CD3 ScFv is coupled to the N-terminus of the extracellular segment of CD86 via amino acids, and G4H12 is linked to the C-terminus of the extracellular segment of CD86. The fusion protein described in this application includes a CD3-targeting ScFv (OKT3) linked to the extracellular segment of CD86 via a linker, and a G4H12 amino acid sequence is designed after CD86 to form the CD3 ScFv-CD86-G4H12 structure, with the amino acid sequence shown in SEQ ID NO:1. This achieves targeted function, enhances T cell endocytosis without over-activating T cells.

[0073] This invention also provides some biological materials, including at least one of the following:

[0074] I) Nucleic acid encoding the fusion protein as described above;

[0075] II) Expression units containing the nucleic acid described in I);

[0076] III) A recombinant vector containing the nucleic acid described in I) or the expression unit described in II);

[0077] IV) Transformants that have been transformed or transfected with the expression vector described in III);

[0078] The culture products of the transformants described in (V) and (IV).

[0079] The method for preparing the fusion protein described in this invention employs genetic engineering techniques, specifically including culturing the transformants as described above to obtain a product containing the fusion protein as described above.

[0080] This invention also provides a PEGylated targeting protein, comprising the fusion protein as described above and PEGylated lipids, wherein the PEGylated lipids are selected from at least one of the following: ALC-0159, DMG-PEG, PEG2000-C-DMG, DSPE-PEG, DPPE-MPEG(2000), DSPE-PEG-Amine, DSPE-PEG-NHS,MW 2000, DSPE-PEG-Maleimide (also known as DSPE-PEG-MAL or DSPE-PEG-maleimide), DMG-PEG-MAL (DMG-PEG-maleimide), DMG-PEG2000, DSPE-PEG2000, DSPE-PEG2000-MAL, and DMG-PEG2000-MAL. In a specific embodiment, the average molecular weight of PEG in the PEGylated targeting protein is 2000.

[0081] The fusion protein in the PEGylated targeting protein provided by this invention is linked to PEGylated lipids via thioether bonds.

[0082] In this invention, the C-terminus of the fusion protein is reduced to form a -SH group, which then undergoes an alkylation reaction with the double bond in the PEGylated lipid to form a thioether bond.

[0083] The -SH group is formed when the target protein carrying G4H12 reduces the disulfide bond of G4H12 with the TCEP reducing agent to form -SH.

[0084] Furthermore, the present invention also provides the application of the fusion protein as described above or the PEGylated targeting protein as described above in the preparation of T cell-targeting LNPs.

[0085] In this invention, the T cells are either T cells or CAR-T cells, and this invention does not limit the specific type of T cells.

[0086] In this invention, the packaging material for the T-cell targeted LNP includes cationic lipids, neutral lipids, PEG or PEGylated lipids, and cholesterol.

[0087] The T-cell-targeting LNP described in this invention is used to encapsulate nucleic acids, which are biological macromolecules with nucleotides as their basic building blocks, capable of carrying and transmitting genetic information. Nucleic acid is a general term for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA); it can be mRNA, small interfering RNA, tRNA, miRNA, aiRNA, ssRNA, short hairpin RNA (shRNA), dsRNA, gene sequences, ssDNA, dsDNA, or plasmids. The mRNA can be mRNA encoding fluorescent signals, mRNA encoding chimeric antigen receptors, or mRNA sequences of antigenic polypeptides that can elicit an immune response in the body; this invention does not limit this.

[0088] The lipid nanoparticles provided by this invention have a packaging material in which the molar percentages of cationic lipids, neutral lipids, PEG or PEGylated lipids, cholesterol or its derivatives are (33%–50%): (10%–13.33%): (1.5%–2%): (38.5%–51.33%). Targeted LNP synthesis within this molar percentage range results in higher encapsulation efficiency and significantly improved targeted LNP transduction efficiency. This not only enhances the encapsulation efficiency and loading capacity of the particles but also improves the targeting function of T cells, achieving highly efficient T cell transfection. Attached Figure Description

[0089] Figure 1 shows the validation of the ability of targeted LNPs synthesized with different proportions of PEG-DMG to transduce GFP in T cells. In Figure 1, A represents the GFP positivity rate of targeted LNPs synthesized with different proportions of PEG-DMG in T cells; and B represents the GFP expression intensity (MFI) of targeted LNPs synthesized with different proportions of PEG-DMG in T cells.

[0090] Figure 2 shows the ability of targeted LNP particles with different formulations to transduce GFP in T cells, where A in Figure 2 represents the GFP positivity rate and B in Figure 2 represents the GFP expression intensity.

[0091] Figure 3 shows the detection of the encapsulation efficiency of targeted LNP particles for different formulations;

[0092] Figure 4 shows the detection of the encapsulation efficiency of targeted LNP particles for different optimized formulations;

[0093] Figure 5 shows the results of the transduction efficiency and intensity of the targeted LNP synthesized by the molar percentage described in this application for CD19 CAR, where A in Figure 5 is the transduction efficiency graph and B in Figure 5 is the transduction intensity (MFI) graph.

[0094] Figure 6 shows the molar percentage encapsulation efficiency of the targeted LNP containing CD19 CAR nucleic acid synthesized according to this application;

[0095] Figure 7 shows the encapsulation efficiency of the target protein synthesized using the TCPE method in targeted LNP synthesis;

[0096] Figure 8 illustrates the CD3ScFv-CD86-PEG2000-DSPE TCEP modification process;

[0097] Figure 9 illustrates the CD3 ScFv-CD86-PEG2000-DSPE SATA modification process;

[0098] Figure 10 shows the flowchart of the synthesis of specifically targeted LNPs;

[0099] Figure 11 shows the in vitro transduction efficiency verification of synthesis process 1; in Figure 11, A represents the GFP positivity rate of T cells transfected with different synthesis schemes, and the vertical axis represents the transduced GFP gene expression rate; in Figure 11, B represents the average fluorescence intensity of GFP in T cells transfected with different synthesis schemes, and the vertical axis represents the transduced GFP gene expression intensity represented by MFI.

[0100] Figure 12 shows the effect of different target proteins on transduction efficiency;

[0101] Figure 13 shows the effects of different proteins on T cell activation;

[0102] Figure 14 shows the encapsulation efficiency of targeted LNPs synthesized using different synthesis processes;

[0103] Figure 15 shows the in vitro transduction verification of targeted LNPs synthesized by different synthetic processes; A in Figure 15 represents the positive rate of in vitro transduction of targeted LNPs synthesized by PEGylated lipids from different processes; B in Figure 15 represents the average fluorescence intensity of in vitro transduction of targeted LNPs synthesized by PEGylated lipids from different processes.

[0104] Figure 16 shows the in vivo transduction efficiency verification of targeted LNPs synthesized by different synthetic processes;

[0105] Figure 17 shows the verification of encapsulation efficiency and transduction efficiency of different protein modification systems; Figure 17A shows the encapsulation efficiency detection, and Figure 17B shows the transduction efficiency detection.

[0106] Figure 18. In vivo efficacy validation of the targeted fusion protein LNP particles. Detailed Implementation

[0107] This invention provides lipid nanoparticles, their preparation methods, and applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0108] Unless otherwise defined in this invention, the scientific and technical terms related to this invention shall have the meanings understood by one of ordinary skill in the art.

[0109] Unless otherwise stated herein, singular terms shall include plural terms, and plural terms shall include singular terms. More specifically, as used in this specification and the appended claims, unless otherwise expressly indicated, the singular forms “a” and “this” include plural indicators.

[0110] In this invention, the terms "comprising," "including," and "having" are used interchangeably to indicate the inclusiveness of a solution, meaning that the solution may contain elements other than those listed. It should also be understood that the use of "comprising," "including," and "having" herein also provides for solutions "consisting of...".

[0111] As used herein, “and / or” includes the meaning of “and,” “or,” and “all or any other combination of elements linked by the term.”

[0112] "At least one" means one or more, and "more than" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0113] LNP (Lipid Nanoparticle), also known as lipid nanoparticle, is a lipid vesicle composed of cationic lipids, accessory lipids, PEG / PEG derivatives (also known as PEGylated lipids), and cholesterol, which can be used for nucleic acid delivery. LNPs have a well-defined composition and can be mass-produced on a large scale using ethanol hydration in specific proportions. Their main mechanism of action involves intracellular drug delivery via endocytosis or pinocytosis and binding to cellular LDLRs. LNPs dissociate based on the pH of the intracellular lysosomes, releasing the drug and allowing it to exert its function.

[0114] Antibody: Commonly referred to as "immunoglobulin," it encompasses antibodies with the structural features of natural antibodies and antibody-like molecules with structural features different from natural antibodies but exhibiting specificity for binding to antigen molecules. In this application, the term "antibody" has its broadest meaning, encompassing immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing antigen-binding sites or domains, and can be used to refer to antigen-binding structural fragments (e.g., antigen-binding fragments) or complexes of one or more antigen-binding fragments (e.g., scFv). Immunoglobulin molecules can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), type (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0115] ScFv: Single-chain antibody, a synthetic antibody formed by linking the variable regions of the antibody heavy chain and light chain through a short peptide of 15-20 amino acids (sometimes referred to as a linker). It retains the antibody's activity against the antigen, has a small molecular weight, strong penetrability, and weak immunogenicity. The anti-CD3 antibody described in this application is an anti-CD3 ScFv, referred to as CD3 ScFv in the specific structure. Anti-CD3 ScFv can be derived from publicly disclosed antibodies targeting CD3, such as OKT3 antibody, UCHT1, and SP34.

[0116] Fusion Protein: The fusion protein described in this application refers to the expression product of two recombined genes obtained through DNA recombination technology. In this application, the fusion protein can be a protein complex formed by the above-mentioned antibody binding to one or more other proteins, peptides, or protein functional domains in any manner. In some embodiments, the binding is a direct and / or indirect connection between the antibody and the other one or more proteins, peptides, or protein functional domains. In some embodiments, the carbon (C) terminus of the antibody in the fusion protein is connected to the nitrogen (N) terminus of the other protein, peptide, or protein functional domain. In some embodiments, the nitrogen (N) terminus of the antibody in the fusion protein is connected to the carbon (C) terminus of the other protein, peptide, or protein functional domain. In some embodiments, the nitrogen (N) terminus of the antibody in the fusion protein is connected to the nitrogen (N) terminus of the other protein, peptide, or protein functional domain. In some embodiments, the carbon (C) terminus of the antibody in the fusion protein is connected to the carbon (C) terminus of the other protein, peptide, or protein functional domain. In some embodiments, the antibody in the fusion protein is tandem with one or more other proteins, peptides, or protein functional domains. In some embodiments, the C-terminus and / or N-terminus of the antibody in the fusion protein is linked to at least two or more other proteins, peptides, or protein functional domains, and these other proteins, peptides, or protein functional domains are not linked in tandem. In some embodiments, the other one or more proteins, peptides, or protein functional domains bind the same or different antigens or different antigenic epitopes to the antibody.

[0117] Cationic lipids are a class of biomacromolecules containing cationic hydrophilic groups. Common cationic polymers include polyethyleneimine (PEI), polylysine (PLL), dendritic macromolecules, and cationic lipids. Cationic lipids are widely used due to their good biocompatibility, simple and well-defined structure, reproducibility, and large-scale production capabilities. Cationic lipid components mainly consist of amphiphilic organic small molecules linked by nonpolar hydrophobic tails and polar hydrophilic heads via linkages. The hydrophilic head is generally composed of a single or multiple protonated amino groups, ensuring electrostatic interaction with negatively charged nucleic acids. The hydrophobic structure is usually composed of steroidal compounds such as cholesterol and alkyl chains. Based on the different linkage methods of the hydrophilic head and hydrophobic tail, cationic lipids can be structurally classified into three types: head-tail, geminal, and dumbbell-shaped, with the head-tail structure being the most common. Cationic lipids constitute the most important part of LNP formulations; they are key components of LNPs, facilitating binding to negatively charged cell membranes and serving as a core component for nucleic acid drug delivery. Common cationic lipids include: DLin-MC3-DMA, ALC-0315, SM102, ATX-126, etc.

[0118] Neutral lipids are structural components of LNPs, also known as accessory lipids. Common examples include DOPE (dioleoylphosphatidylethanolamine), DSPC (dispalmitoylphosphatidylcholine), and DOPC (dioleoyllecithin). Accessory lipids often serve as structural lipids in LNP formulations, spontaneously forming a bilayer membrane structure. This enhances the membrane stability of LNPs, preventing their rapid metabolism and excretion in the bloodstream. Simultaneously, they can disrupt endosome stability, improving nucleic acid delivery efficiency.

[0119] PEG (polyethylene glycol) lipids are polymers in which PEG is chemically bonded to the ends of lipids. They are crucial components in lipid peroxides (LNPs), regulating half-life and cellular uptake, influencing LNP population size and dispersibility, preventing LNP aggregation, and ensuring particle stability during preparation and storage. The length and structure of the PEG chain significantly impact its role in LNPs. Longer PEG chains provide better protection but may result in slower PEG removal from the LNP surface, affecting cellular uptake. Shorter PEG chains are easier to remove but may be less stable. In LNP applications, PEG with an average molecular weight of 500-5000 is commonly chosen, with PEG2000 (average molecular weight 2000) being preferred, to ensure LNP stability and effective PEG removal from the LNP.

[0120] PEGylated target proteins: These are proteins that link polyethylene glycol (PEG) polymers to protein molecules. They are single-chain antibodies, peptides, proteins, or fusion proteins expressed through gene recombination that are chemically modified to react with PEG or PEG derivatives (such as DSPE-PEG-MAL, DMG-PEG-MAL, etc.) to form polymers, called PEGylated (fusion) proteins. In some embodiments, these proteins can be proteins or fusion proteins that target biological tissues, organs, or specific cells, and can also be called targeted PEGylated proteins.

[0121] Cholesterol is an abundant cell membrane component and is often used as a structural lipid in LNP formulations. Cholesterol is primarily found in the outer shell of LNPs, and modifications to its sterol structure can cause tissue changes on the LNP surface. When bound to high Tm (low gel-liquid crystal phase transition) lipids, cholesterol increases membrane fluidity and narrows the bilayer. In both cases, cholesterol pulls the lipids towards a liquid state.

[0122] Molar ratio: Under specific preparation conditions, cationic lipids and nucleic acids bind effectively via electrostatics. The molar concentration of protonated nitrogen ions in the cationic lipids and the molar concentration of phosphorus atoms in the nucleic acids are in a specific ratio, such as an N / P ratio ranging from 2:1 to 10:1. LNPs are synthesized simultaneously through ethanol hydration in specific molar ratios. Different lipid proportions result in different LNP assemblies, such as a bilayer membrane structure formed on the surface of PEG polymerization, or, when the cationic lipid proportion is high, a bilayer membrane structure encapsulating cationic lipids and nucleic acid polymer particles. Molar percentage refers to the percentage of an element in a compound. Within a certain range of lipid molar percentages, lipid nanoparticles can be formed and effectively packaged into LNPs.

[0123] SATA protein modification method: also known as SATA-maleimide chemical coupling method, its working principle is as follows: SATA (N-succinimide-S-acetylthioacetate) is used to modify the primary amine group of the target protein to introduce an active group. Then, the reactant reacts with hydroxylamine to give the antibody free thiol groups. Finally, a thioether coupling chemical reaction is used to link the active thiol groups on the target protein to the MAL groups on the MAL-PEG-DSPE lipid to form a PEGylated protein complex.

[0124] TCEP protein modification method: also known as antibody site-directed modification of thiol-maleimide coupling method, its working principle is as follows: disulfide bonds are designed at specific sites of the target protein to be modified, and the disulfide bonds are reduced by TCEP reducing agent, so that the modified protein has free thiol groups. Then, the free thiol groups on the antibody are linked to the MAL groups on the MAL-PEG-DSPE lipid through a thioether coupling chemical reaction to form a PEGylated protein complex.

[0125] Nucleic acids: Biological macromolecules with nucleotides as their basic building blocks, capable of carrying and transmitting genetic information. They are a general term for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and can be mRNA, small interfering RNA, tRNA, miRNA, aiRNA, ssRNA, short hairpin RNA (shRNA), dsRNA, gene sequences, ssDNA, dsDNA, or plasmids.

[0126] mRNA, also known as messenger RNA, is a type of single-stranded ribonucleic acid transcribed from one strand of DNA as a template. It carries genetic information and can guide protein synthesis.

[0127] siRNA: Interfering RNA, also known as small interfering RNA, short interfering RNA, or silent RNA, is a double-stranded RNA of 20 to 25 nucleotides in length. It can be used to regulate gene expression and participate in some RNAi-related response pathways, such as antiviral mechanisms or changes in chromatin structure.

[0128] Delivery: The delivery described in this application refers to the delivery of the lipid nanoparticles described in this application, which encapsulate nucleic acids such as mRNA or interfering RNA, or other active agents or therapeutic agents, to the whole body or target sites within an organism. This is achieved through the fusion of the lipid nanoparticles with the cell membrane at the delivery site, endocytosis by relevant cells, or degradation by the environment at the delivery site, thereby releasing the nucleic acid molecule at the delivery site, cell, or tissue. In some embodiments, the target mRNA sequence is encapsulated in the lipid nanoparticles described in this application, which can be widely distributed within the organism, thereby delivering the target RNA throughout the body. In some embodiments, the target mRNA sequence is encapsulated in the targeted lipid nanoparticles described in this application, which can be distributed at target sites within the organism depending on the target molecules they carry, thereby delivering the target RNA to the target site or target cells. In some embodiments, the target mRNA can be a nucleic acid of a marker molecule such as GFP described in this application, or a chimeric antigen receptor (CAR) molecule mRNA, or an mRNA sequence of an antigenic polypeptide that can elicit an immune response. In some embodiments, the nucleic acid can also be small interfering RNA. The target molecules may include tRNA, miRNA, aiRNA, ssRNA, short hairpin RNA (shRNA), dsRNA, gene sequences, ssDNA, dsDNA, or plasmids. In some embodiments, the target molecules may be T-cell targeting molecules that recognize CD3, CD4, CD8, CD5, CD7, etc., or B-cell targeting molecules that recognize CD19, etc., or APC cells such as macrophages and DC cells. In some embodiments, the organism refers to mammals, such as mice, monkeys, dogs, cats, and humans. In some embodiments, lipid nanoparticles encapsulating nucleic acids such as mRNA or interfering RNA, or other active agents or therapeutic agents, are provided to the organism via any of the following methods: intravenous, subcutaneous, or intraperitoneal administration.

[0129] Preparation of 0.02M citric acid reagent:

[0130] 1) Weigh 19.213g of citric acid (manufacturer SPECTRUM Lot No.1IE0871 FW192.13) and dissolve it in a certain amount of ultrapure water. After it is completely dissolved, make up to 1L in a 1L volumetric flask to obtain 0.1M / L stock solution.

[0131] 2) Weigh 25.807g of sodium citrate (manufacturer SPECTRUM Lot No.1HK0873 FW258.07) and dissolve it in a certain amount of ultrapure water. After it is completely dissolved, make up to 1L in a 1L volumetric flask to obtain 0.1M / L stock solution.

[0132] 3) Prepare 50ml of 0.02M citrate buffer: 40ml ultrapure water + 6.55ml 0.1M / L citrate stock solution + 3.45ml 0.1M / L sodium citrate stock solution.

[0133] The LNP synthesis process involved in this case includes:

[0134] The current industry-standard LNP synthesis process involves dissolving ionizable cationic lipids such as SM102, neutral lipids, PEG or PEGylated lipids, and cholesterol or other modified cholesterol in ethanol at a molar ratio of 50%:10%:1.5%:38.5%. The target mRNA is dissolved in 0.02M citrate buffer at pH 4.0 at a P:N ratio of 1:3 with the ionizable cationic lipids. The LNP solution is synthesized using a microfluidic system (Aitesen) with the ethanol-to-citrate phase flow rate ratio at 1:3. The solution is then dialyzed using PBS buffer or TFF tangential flow (Replekin). The solution was replaced using the Research 2i[KR2i]TFF system, and the LNP was finally stored in 0.01M PBS buffer.

[0135] Current targeted LNP synthesis process: The final LNP synthesis process of the above LNP synthesis process is a solution containing LNP particles. A targeting protein or targeting protein-PEG-DSPE is added to the solution, and a targeted LNP is formed by shaking or sonication.

[0136] Precision biologically targeted LNP synthesis process (synthesis process 1) (see Figure 10): Ionizable cationic lipids such as SM102, neutral lipids, PEGylated lipids, and cholesterol or other modified cholesterol are dissolved in ethanol at a molar ratio of 50%:10%:1.5%:38.5%. PEGylated target proteins (CD3ScFv-CD86-PEG-DSPE or CD3ScFv-PEG-DSPE synthesized using SATA or TCEP protein modification methods) and target nucleic acids are dissolved in citrate buffer, with the target nucleic acid dissolved at a P:N ratio of 1:3 to the ionizable cationic lipids. The citrate buffer is pH 4.0 with a concentration of 0.02M. Microfluidics (Aitesen) is used, with the equipment settings being an ethanol-citric acid phase flow rate ratio of 1:3. The targeted LNP solution is synthesized through the microfluidic device and dialyzed using PBS buffer or TFF tangential flow (Replekin). The solution was replaced using the Research 2i[KR2i]TFF system, and the LNP was finally stored in 0.01M PBS buffer.

[0137] Precision biological targeted LNP synthesis process (synthesis process 2) (see Figure 10): Ionizable cationic lipids such as SM102, neutral lipids, PEG or PEGylated lipids, and cholesterol or other modified cholesterol are dissolved in ethanol in a molar ratio of 33%:13%:2%:51%. PEGylated target proteins (CD3ScFv-CD86-PEG-DSPE or CD3ScFv-PEG-DSPE synthesized using SATA or TCEP protein modification methods) and target nucleic acids are dissolved in citrate buffer. The target nucleic acid is dissolved in citrate buffer at a P:N ratio of 1:3 with the ionizable cationic lipids. The citrate buffer is pH 4.0 with a concentration of 0.02M. Microfluidics (Aitesen) is used with the alcohol phase flow rate:citrate phase flow rate ratio of 1:3. The targeted LNP solution is synthesized through the microfluidic device. Solution replacement is performed by dialysis with PBS buffer or TFF tangential flow. Finally, the LNP is stored in 0.01M PBS buffer.

[0138] In vitro transduction efficiency verification: T cells or peripheral blood mononuclear cells (PBMCs) 3e5 cells were placed in 48-well plates. Synthetic LNPs or protein-targeted LNPs (targeted LNPs) were taken and, according to mRNA quantification (mRNA quantification was performed using an RNA nucleic acid quantification kit (Thermo, catalog number: R11490) (probe method)), 1 μg of mRNA particles were added to the wells. The wells were then brought to a final volume of 200 μL with 1640 medium + 10% FBS. After culturing at 37°C and 5% CO2 for 24 h, cells were collected, centrifuged at 350g for 5 min, and resuspended in 100 μL of PBS. GFP did not require labeling; the positivity rate and mean fluorescence intensity (MFI) of GFP were directly detected using flow cytometry. CD19 CAR was detected using anti-antibody B937D4-AF647. 1 μg of antibody was added, and the cells were labeled at 4°C for 30 min. After washing with 1 mL of PBS, the cells were centrifuged at 350g for 5 min and then resuspended in 100 μL of PBS. The CAR positivity rate and MFI were then detected.

[0139] The existing lipid ratio range is as follows: ionizable cationic lipids such as SM102, neutral lipids, PEG or PEGylated lipids, and cholesterol or other modified cholesterol in a molar ratio range of 50%:10%:1.5%:38.5%.

[0140] The lipid ratio range described in this application is as follows: ionizable cationic lipids such as SM102, neutral lipids, PEG or PEGylated lipids, and cholesterol or other modified cholesterol in a molar ratio range of 33.33%: 13.33%: 2%: 51.33%.

[0141] The fusion protein used in this application is CD3 ScFv-CD86-G4H12, with the amino acid sequence shown in SEQ ID NO.1. The PEGylated targeting protein ultimately used as the LNP component is CD3 ScFv-CD86-PEG-DSPE or CD3 ScFv-CD86-PEG-DMG, where PEG is PEG2000. The PEGylated targeting protein linked to the targeting protein can be synthesized using the SATA protein modification method or the TCEP protein modification method.

[0142] PEGylated targeting proteins linked to the target protein were synthesized using the SATA protein modification method (see Figure 9):

[0143] 1) After removing the desalting column preservative solution from the Thermofisher 89889 desalting column, equilibrate the desalting column with 1 ml of 0.1M PB, 0.15M NaCl pH7.4 solution.

[0144] 2) 512 μl of aqueous solution of CD3 ScFv-CD86-G4H12 fusion protein, with a concentration of 3.90 mg / ml.

[0145] 3) Weigh 6.3 mg SATA (sigmaaldrich A9043) and dissolve it in 1364 μl DMSO (Origen CP-70) to prepare a 20 mM (4.62 mg / ml) SATA working solution.

[0146] 4) Add 4.5 μl of 20 mM SATA working solution to 256 μl of fusion protein solution (the molar ratio of fusion protein to SATA is 1:5), and react at room temperature in the dark for 0.5 h.

[0147] 5) Add the solutions obtained in step 4) to the desalting column after equilibration in step 1), centrifuge at 1000g for 2 min, and collect the flow-through liquid. Collect 340 μl of each solution.

[0148] 6) Add 34 μl of 0.5 M hydroxylamine, 25 mM EDTA, 0.1 M PB, and 0.15 M NaCl pH 7.4 solution (1 / 10 of the liquid volume) to the above solution and react at room temperature in the dark for 2 h.

[0149] 7) Weigh 6.0 mg of DSPE-PEG-MAL (WH-0010205 from Huasheng Biotechnology) and dissolve it in 408 μl of DMSO (Origen CP-70) to prepare a 5 mM (14.7 mg / ml) DSPE-PEG-MAL working solution.

[0150] 8) Add 17.8 μl of 5 mM DSPE-PEG-MAL working solution to each of the solutions in 6), wherein the fusion protein:SATA:DSPE-PEG-MAL = 1:5:5. Incubate at room temperature in the dark for 0.5 h.

[0151] 9) Add 370 μl of the solution obtained in step 8) to the equilibrated desalting column, centrifuge at 1000 g for 2 min, and collect the flow-through. This yields the PEGylated target protein CD3 ScFv-CD86-PEG-DSPE.

[0152] In some embodiments, step 4 is: take 1.8 μl of 20 mM SATA working solution and add it to 256 μl of fusion protein solution (the molar ratio of fusion protein to SATA is 1:2), react at room temperature in the dark for 0.5 h, and the fusion protein:SATA:DSPE-PEG-MAL in step 8 is 1:2:5.

[0153] The PEGylated targeting protein CD3ScFv-CD86-PEG-DSPE, linked to the target protein, was synthesized using the TCEP protein modification method (see Figure 8).

[0154] 1) After removing the desalting column preservative solution from the Thermofisher 89889 desalting column, equilibrate the desalting column with 1 ml of 0.1M PB, 0.15M NaCl pH7.4 solution.

[0155] 2) 660 μl of aqueous solution of CD3 ScFv-CD86-G4H12 fusion protein, with a concentration of 5.05 mg / ml.

[0156] 3) Add 1.6 μl of 0.5 M TCEP (Aladdin T107252-25g) aqueous solution to 350 μl of fusion protein solution, and react at room temperature in the dark for 1 h. (The molar ratio of fusion protein to TCEP is 1:25).

[0157] 4) Add the solution obtained in step 3) to the equilibrated desalting column, centrifuge at 1000g for 2 min, and collect the flow-through liquid.

[0158] 5) Weigh 13.3 mg of DSPE-PEG-MAL (WH-0010205 from Huasheng Biotechnology) and dissolve it in 905 μl of DMSO (Origen CP-70) to prepare a 5 mM (14.7 mg / ml) DSPE-PEG-MAL working solution.

[0159] 6) Add 63.2 μl of 5 mM DSPE-PEG-MAL working solution to the flow-through solution obtained in step 4), with the fusion protein:TCEP:DSPE-PEG-MAL ratio being 1:25:10. Incubate at room temperature in the dark for 0.5 h.

[0160] 7) Add 400 μl of the solution obtained in step 6) to the equilibrated desalting column, centrifuge at 1000g for 2 min, and collect the flow-through. This yields the PEGylated target protein CD3 ScFv-CD86-PEG-DSPE.

[0161] CD3 ScFv-CD86-PEG-DMG was synthesized according to the above scheme, except that DSPE-PEG-MAL was replaced with DMG-PEG-MAL (manufacturer: Aivito, product number 160743-62-4).

[0162] Encapsulation rate detection principle:

[0163] Assessing the encapsulation effect of LNP on mRNA by measuring total mRNA and free mRNA in the formulation is a key indicator of the biological activity of nucleic acid drugs. Encapsulation efficiency is the core indicator of this. Encapsulation efficiency is measured using the RiboGreen fluorescent dye quantitative detection kit (manufacturer: Invitrogen, catalog number R11490). After the fluorescent dye binds to nucleic acid, it excites fluorescence at a specific wavelength, and the signal values ​​are analyzed and compared. First, the concentration of free RNA in the LNP-RNA solution is measured. Then, the LNP structure is destroyed using Triton-100, and the concentration of all RNA in the solution is measured. The fluorescence value of the test sample is assigned according to the concentration of the standard. The encapsulation efficiency is calculated as: (Total RNA amount - Free RNA amount) / Total RNA amount * 100%.

[0164] The specific steps are as follows:

[0165] Preparation of 1X TF Buffer: Stock solution: 1M Tris-HCl (pH 7.4), 0.5M EDTA (pH 8.0), prepare 100mL.

[0166] The 50ul sample to be tested was gradually diluted 100-fold using 1X TF Buffer;

[0167] Preparation of 0.5% Triton 100: Add 5ml of Triton 100 to 995ml of TE buffer (V / V) and shake gently.

[0168] Total LNP-RNA assay: Add 100 μL of a 100-fold diluted sample to 900 μL of 0.5% Triton 100 (named Sample 1). Free LNP-RNA assay: Add 100 μL of a 100-fold diluted sample to 900 μL of 1*TE buffer (named Sample 2).

[0169] Preparation of standard: Add 10 μL of standard to 990 μL of 1*TE buffer (the original concentration of the standard is 1 mg / mL), and then dilute the above sample with TE buffer to 200, 100, 20, 4, 0 ng / mL;

[0170] Add 100 μL of standard, sample 1, or sample 2 to each well, and repeat for three replicates.

[0171] Fluorescent dye preparation (2000-fold dilution): Add 5 μL of fluorescent dye to 10m LTE buffer;

[0172] After adding 100 μL of dye to each well, the OD value was measured in the dark under excitation light of 485 nm and emission light of 520 nm.

[0173] A standard curve was constructed based on the standard curve. Sample 1, representing the OD value of total RNA, was substituted into the standard curve. The value was multiplied by 1000 (the original solution was diluted 1000 times) to obtain the total RNA OD value. Similarly, Sample 2 was recorded as the free RNA OD value. Encapsulation efficiency = (Total RNA OD value - Free RNA OD value) / Total RNA OD value * 100%

[0174] mRNA quantification was performed using an RNA nucleic acid quantification kit (manufacturer: Thermo, catalog number: R11490) (probe method).

[0175] The sequences involved in this invention:

[0176] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0177] The present invention will be further illustrated below with reference to the embodiments:

[0178] Example 1: Optimizing PEG content alone cannot improve the transduction ability of T cells to LNP particles.

[0179] Ionizable cationic lipids such as SM102, neutral lipids, PEGylated lipids, and cholesterol or other modified cholesterol were dissolved in ethanol at a molar percentage ratio of 50%:10%:1.5%:38.5%. mRNA (target mRNA was GFP RNA, RNA sequence as shown in SEQ ID NO.3) was dissolved in 0.02M citrate buffer at pH 4.0 at a P:N ratio of 1:3 with the ionizable cationic lipids. The targeted PEGylated protein CD3ScFv-CD86-PEG-DSPE was also dissolved in the above citrate buffer. Using a microfluidic system (Aitesen), the device was set to an ethanol-to-citric acid phase flow rate ratio of 1:3. LNP solutions were synthesized via microfluidic equipment and then dialyzed using PBS buffer or TFF tangential flow (Replekin). The solution was replaced using the Research2i [KR2i] TFF system, and the LNPs were finally stored in 0.01M PBS buffer. mRNA quantification was performed using an RNA nucleic acid quantification kit (probe method, manufacturer: Thermo, catalog number: R11490). The molar percentage of PEGylated lipids was determined, and the specific molar percentage is as follows:

[0180] The current formulation is PEG-DMG 1.5%: SM102 (manufacturer: Senobeng, item number 2089251-47-6) molar percentage 50%, DSPC (manufacturer: Aveta, item number 816-94-4) molar percentage 10%, DMG-PEG-MAL molar percentage 1.5% (manufacturer: Aveta, item number 160743-62-4), and CHOL (manufacturer: Aveta, item number 57-88-5) molar percentage 38.85%.

[0181] PEG-DMG 1.8%: SM102 (manufacturer: Senobeng, item number 2089251-47-6) molar percentage 49.85%, DSPC (manufacturer: Aveta, item number 816-94-4) molar percentage 9.97%, DMG-PEG-MAL molar percentage 1.8% (manufacturer: Aveta, item number 160743-62-4), CHOL (manufacturer: Aveta, item number 57-88-5) molar percentage 38.38%.

[0182] PEG-DMG 2%: SM102 (manufacturer: Senobeng, item number 2089251-47-6) molar percentage 49.75%, DSPC (manufacturer: Aveta, item number 816-94-4) molar percentage 9.94%, DMG-PEG-MAL molar percentage 2% (manufacturer: Aveta, item number 160743-62-4), CHOL (manufacturer: Aveta, item number 57-88-5) molar percentage 38.31%.

[0183] In the targeted LNP synthesis process, the targeted PEGylated protein is prepared by modifying the PEGylated lipid DSPE-PEG-MAL and the target protein (CD3 ScFv-CD86-G4H12 in this example) using different protein modification methods, such as SATA or TCEP.

[0184] The transduction efficiency of the target particles was validated. Three batches of T cells were randomly selected, and the positivity rate of GFP transfection in T cells and the average fluorescence intensity were analyzed using a paired t-test. The results are shown in Figure 1. Figure 1A shows the GFP positivity rate of T cells transduced with targeted LNPs synthesized at different proportions of PEG-DMG; Figure 1B shows the GFP expression intensity (MFI) of T cells transduced with targeted LNPs synthesized at different proportions of PEG-DMG. The results show that the transduction efficiency is better with a PEG-DMG content of 1.5% in the current protocol. The results indicate that increasing the molar percentage of PEG-DMG alone did not improve the transduction efficiency or expression intensity of the target particles on T cells.

[0185] Example 2: Multi-component optimization of LNP components to enhance the transduction ability of targeted LNP particles to T cells.

[0186] Table 1: LNP components and proportions

[0187] The components were proportioned according to the ratios in Table 1, and the targeted LNP was synthesized according to the scheme in Example 1. The PEGylated protein linked to the target protein was either CD3ScFv-CD86-PEG-DMG or CD3ScFv-CD86-PEG-DSPE (synthesized using the SATA protein modification method).

[0188] Simultaneously, different batches of T cells were transfected to verify the transfection efficiency and encapsulation rate of the targeting particles. The results are shown in Figures 2 and 3. Figure 2 shows the ability of different formulations of targeting LNP particles to transduce GFP in T cells, where A in Figure 2 represents the GFP positivity rate and B in Figure 2 represents the GFP expression intensity.

[0189] Analysis using paired t-tests showed that the 1.3x and 2x optimized formulations were significantly superior to the current process and the 1.6x formulation. Specifically, the formulations containing SM102 (33.33%), DSPC (13.33%), PEG-DMG (2%), and CHOL (51.33%) exhibited better targeting of LNPs to enhance T cell transduction and also demonstrated superior encapsulation efficiency.

[0190] Furthermore, the ratio was increased by 2 times, and the results are shown in Figure 4. The results show that the encapsulation efficiency of the targeted particles did not increase. At the same time, increasing the ratio of cationic lipids in the overall formula is not conducive to particle stability, and therefore not conducive to product development.

[0191] SM102 is a cationic lipid. Other cationic lipids such as DLin-MC3-DMA, ALC-0315, and ATX-126 can replace SM102 to achieve the same effect. DSPC is a neutral lipid. Other neutral lipids such as dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylcholine (DPPC), distearate phosphatidylethanolamine (DSPE), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), and phosphatidylserine (PS) can all achieve the same effect. PEG-DMG is a structural component. Other PEG-DMG derivatives include, but are not limited to, aminated-PEG-DMG, hydroxylated-PEG-DMG, or other active groups. Other PEGs include PEG3000-DMG with different chain lengths. Cholesterol is the main membrane component of LNP, and other cholesterol derivatives such as 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol hydrochloride (DC-CHOL) can achieve the same effect.

[0192] In summary, targeted LNPs synthesized from ionizable cationic lipids such as SM102, neutral lipids, PEGylated lipids, and cholesterol or other modified cholesterol in a molar percentage range of 33.33%: 13.33%: 2%: 51.33% exhibit superior transduction efficiency and encapsulation rate.

[0193] Example 3: The LNP component of this application exhibits superior transduction efficiency and encapsulation efficiency in transducing CAR nucleic acids.

[0194] The targeted LNP was prepared according to the steps in Example 1, wherein the target nucleic acid molecule was a CAR RNA sequence targeting CD19, and the RNA sequence is shown in SEQ ID NO.2. The transduction ability of the targeted LNP encapsulating the target nucleic acid to target cells was verified by in vitro transduction.

[0195] In vitro transduction was validated according to the above-described in vitro transduction efficiency verification method. T cells from different donors were repeatedly transfected and statistically analyzed. 1 μg of CD19 CAR antibody was added, labeled at 4°C for 30 min, washed with 1 mL of PBS, centrifuged at 350g for 5 min, and resuspended in 100 μL of PBS. CAR positivity and MFI were then detected. The results are shown in Figure 5. Figure 5 shows the transduction efficiency and intensity of the targeted LNP synthesized according to the molar percentage described in this application for CD19 CAR, where A in Figure 5 is the transduction efficiency graph and B is the transduction intensity (MFI) graph.

[0196] The encapsulation efficiency of the prepared targeted LNPs was tested according to the method described above, as shown in Figure 6: Encapsulation efficiency of the targeted LNPs containing CD19 CAR nucleic acid synthesized using the molar percentages of the LNP synthesis components described in this application. The results indicate that the targeted LNPs containing CD19 CAR nucleic acid synthesized using the different molar percentages of the LNP synthesis components described in this application have a better encapsulation efficiency than LNPs with traditional ratios.

[0197] In summary, the targeted LNP synthesized from the components described in this application can better transduce target cells after encapsulating the target mRNA, thus achieving targeted delivery capability.

[0198] Example 4: PEGylated lipids synthesized using the TCEP method resulted in better encapsulation of targeted LNPs.

[0199] The formulation components SM102 (33.33%), DSPC (13.33%), PEG-DMG (2%), and CHOL (51.33%) were dissolved in proportion. CD3ScFv-CD86-PEG-DSPE, synthesized using either the SATA or TCEP protein modification method, was used as the PEGylation target protein, and CD19 CAR mRNA was dissolved in 0.02M citrate buffer at pH 4.0. The remaining components were dissolved in ethanol. CD19 CAR mRNA was dissolved in ethanol at a P:N ratio of 1:3 with ionizable cationic lipids. LNP solutions were synthesized using a microfluidic system (Aitesen) with the ethanol-to-citric acid phase flow rate ratio at 1:3. The solutions were then dialyzed using PBS buffer or TFF tangential flow (Replekin). The solution was replaced using the Research 2i [KR2i]TFF system, and the LNPs were finally stored in 0.01M PBS buffer. mRNA quantification was performed using an RNA nucleic acid quantification kit (probe method).

[0200] Encapsulation efficiency was detected using an RNA quantification kit (Thermo, catalog number R11490), and the detection method was the same as described above. The results are shown in Figure 7. The results indicate that, through a non-paired t-test, the combination of the LNP formulation described in this application and the targeted PEGylated protein modified by TCEP significantly improved the particle encapsulation efficiency, meeting the particle encapsulation efficiency requirements for industrial-scale production.

[0201] In summary, the targeted LNPs prepared using the ionizable cationic lipids described in this application, such as SM102, neutral lipids, PEGylated lipids, and cholesterol or other modified cholesterol in a molar percentage ratio of 33.33%:13.33%:2%:51.33% in the presence of target nucleic acid molecules can improve the encapsulation efficiency of LNP particles and enhance the targeted transduction efficiency; preferably, the targeting protein is modified using TCEP.

[0202] Example 5: Application of LNP component molar ratios in non-targeted LNP synthesis

[0203] LNP Synthesis: Ionizable cationic lipids SM102, DPSC, PEG-DMG (manufacturer: Avitol, catalog number 160743-62-4), and cholesterol (CHOL) were dissolved in ethanol at a molar percentage ratio of 33.33%:13.33%:2%:51.33%. mRNA was dissolved in 0.02M citrate buffer (pH 4.0) at a P:N ratio of 1:3 with the ionizable cationic lipids. The LNP solution was synthesized using a microfluidic device with the ethanol-to-citrate phase flow rate ratio of 1:3. Solution replacement was performed using PBS buffer or TFF tangential flow, and the LNPs were finally stored in 0.01M PBS buffer. In this embodiment, the PEG-DMG is a PEGylated lipid without any protein components. The PEG-DMG in this embodiment is a pure PEG-DMG compound without modification of the target protein.

[0204] The synthesized LNPs were subjected to encapsulation efficiency and loading capacity assays. The encapsulation efficiency procedure was described above. The loading capacity was determined by the amount of mRNA contained in the LNP. mRNA quantification was performed using an RNA nucleic acid quantification kit (probe method, manufacturer: Thermo, catalog number: R11490). The results are shown in Table 2 below.

[0205] Table 2: LNP Encapsulation Efficiency, Loading Capacity, and Transduction Efficiency Detection

[0206] As can be seen from Table 2 above, the LNP synthesis formulation ratio described in this application is not only applicable to the one-step synthesis of lipid components of targeted LNPs, but also applicable to the synthesis of non-targeted conventional LNPs.

[0207] In some embodiments, the LNPs described in this application can also be synthesized first using ionizable cationic lipids such as SM102, neutral lipids, PEG or PEGylated lipids, cholesterol or other modified cholesterol in a molar percentage ratio of 33.33%:13.33%:2%:51.33%, and then a targeting protein or targeting protein-PEG or targeting protein-PEG derivative is added to it, and a targeted LNP is formed by shaking or sonication.

[0208] Example 6 shows that synthesis process 1, compared with the current targeted LNP synthesis process, improves the transfection efficiency of T cells.

[0209] CD3 ScFv-CD86-PEG-DSPE is a PEGylated target protein synthesized from the CD3 ScFv-CD86-G4H12 fusion protein described in this application using a SATA modification process (synthesis process described above and Figure 9), where PEG is PEG2000. The molar ratio of protein:SATA:PEG2000-DSPE-MAL is 1:5:5. The target mRNA is GFP mRNA, with the sequence shown in SEQ ID NO.3. The GFP gene was transduced in vitro using the precise biosynthesis process 1 described above, with 150 μg of CD3ScFv-CD86-PEG2000-DSPE added to citrate buffer. The synthesis system consisted of 1.5 mL of alcohol phase and 4.5 mL of citrate. The synthesis rate was 12 mL / min for the alcohol phase and 36 mL / min for the citrate phase. After microfluidic synthesis, the particles were dialyzed, with PBS replaced every 1 h for a total of 4 times. Samples were collected and quantitatively analyzed using an RNA quantification kit. Labeled as Precise Biosynthesis Process 1. The ionizable cationic lipids are SM102, the neutral lipids are DSPC, and the PEGylated lipids are DSPE-PEG and cholesterol (CHOL). In the targeted LNP synthesis process, DSPE-PEG and the target protein (CD3 ScFv-CD86-G4H12 in this example) are modified by different protein modification methods, SATA or TCEP, to form a PEGylated target protein (CD3 ScFv-CD86-PEG-DSPE in this example).

[0210] The targeted LNP was synthesized according to the current targeted LNP synthesis process (first synthesizing LNP according to the aforementioned industry-standard LNP synthesis process and then synthesizing the targeted LNP with the targeted PEGylated protein), wherein the targeted PEGylated protein was CD3 ScFv-CD86-PEG-DSPE, labeled with the current process in the literature.

[0211] Synthesis process 1 is a one-step synthesis method, where the target protein and LNP synthetic particles are synthesized in one step. Current literature processes involve a two-step synthesis, first synthesizing LNP particles and then linking the target protein to them. In this embodiment, synthesis process 1 and the current literature process use the same LNP lipid ratio; the difference lies only in the targeted synthesis step. Validation was performed according to the aforementioned in vitro transduction efficiency verification method. T cells from different donors were transfected multiple times, and statistical analysis was conducted. The target mRNA was GFP-expressing RNA, and detection was performed directly by flow cytometry. The results are shown in Figure 11: Paired t-test analysis showed that the particles synthesized using the precise biosynthesis process 1 had significantly better in vitro transfection efficiency than the reported process. In conclusion, the one-step synthesis method described in this application is a superior targeted LNP synthesis scheme.

[0212] Example 7: The fusion protein CD3ScFv-CD86 improves T cell transfection efficiency compared to CD3ScFv.

[0213] The ability of a targeted LNP to transduce the target gene into the target cell is inseparable from the design of its target protein. Currently, CD3 antibodies are used as the source of the targeted protein in the field. This application optimizes the targeted protein and designs a CD3 ScFv-CD86 fusion protein. This embodiment verifies the fusion protein described in this application. Using the target protein CD3 ScFv-CD86-G4H12 (amino acid sequence as shown in SEQ ID NO.1) or CD3ScFv-G4H12 (amino acid sequence as shown in SEQ ID NO.4), PEGylated target proteins CD3 ScFv-CD86-PEG-DSPE or CD3 ScFv-PEG-DSPE were synthesized using the SATA modification process, where PEG was PEG2000. The targeted LNP was synthesized using synthesis process 1, with the target mRNA being an mRNA expressing GFP, the sequence of which is shown in SEQ ID NO.3. The specific steps were the same as in Example 6, where the ionizable cationic lipid was SM102, the neutral lipid was DSPC, and the PEGylated lipids were DMG-PEG and cholesterol (CHOL). Non-targeted LNP particles, or LNP groups, were synthesized using synthesis process 1. Ionizable cationic lipids SM102, neutral lipids DSPC, PEGylated lipids DMG-PEG, and cholesterol CHOL were dissolved in the alcohol phase at a molar ratio of 50%:10%:1.5%:38.5%. The target mRNA and PEGylated target protein were dissolved in citrate buffer, with the target mRNA dissolved at a P:N ratio of 1:3 to the ionizable cationic lipids. The citrate buffer was pH 4.0 and had a concentration of 0.02 M. A microfluidic system (Aitesen) was used, with the alcohol phase flow rate to citrate phase flow rate ratio at 1:3. Targeted LNP solutions were synthesized using the microfluidic system and then dialyzed using PBS buffer or TFF tangential flow (Replekin). The solution was replaced using the Research 2i[KR2i]TFF system, and the LNP was finally stored in 0.01M PBS buffer.

[0214] In the non-targeted LNP synthesis component, DMG-PEG is a simple DMG-PEG compound without the target protein, manufactured by: Weihua Biotechnology WH-0010205; in the targeted LNP synthesis process, the PEGylated lipid DSPE-PEG and the target protein CD3 ScFv-CD86-G4H12 or CD3 ScFv-G4H12 are modified by different protein modification methods, SATA method or TCEP method, to obtain the PEGylated target protein: CD3ScFv-CD86-PEG-DSPE or CD3 ScFv-PEG-DSPE.

[0215] GFP gene expression efficiency was detected using an in vitro transduction efficiency verification method, as shown in Figure 12 and Table 3. In Figure 12, the horizontal axis represents different fusion proteins, and the vertical axis represents the GFP positivity rate. Specifically, the horizontal axis represents: LNP group (without the target protein), CD3-LNP group (containing the CD3 ScFv target protein), and CD3-CD86-LNP group (containing the CD3 ScFv-CD86 fusion protein as the target protein).

[0216] Table 3. GFP gene expression efficiency transduced by different target proteins.

[0217] The results showed that, using the unpaired T assay, there was no significant difference between the LNP group and the CD3ScFv-LNP group. Compared with the CD3ScFv-CD86-LNP group (hereinafter referred to as CD3-CD86-LNP), the CD3-CD86-LNP group containing the CD3ScFv-CD86-G4H12 fusion protein described in this application had a significantly higher GFP transduction efficiency (P < 0.0001). These results indicate that the designed fusion protein significantly improved T cell transduction efficiency.

[0218] Example 8: CD3 ScFv-CD86 fusion protein CD86 signaling does not activate T cells

[0219] CD3 ScFv is a primary signal for T cell activation, activating the T cell receptor (TCR). In the human body, when infected by cells or viruses, T cells are stimulated to activate the CD3 primary activation signal. Simultaneously, the binding of CD86 on APC cells to the CD28 ligand on T cells activates the secondary activation signal, thereby enhancing the T cell's cytotoxic and cytokine secretion functions to clear viruses and other pathogens. The CD28 signaling pathway activates NF-κB signaling; the expression level of NF-κB is typically used to assess the activation status of CD28 signaling.

[0220] Theoretically, CD86 can promote T cell activation through the CD28 signaling pathway, thereby enhancing T cell transduction efficiency. However, this activation requires a functional CD86 sequence structure. The fusion protein CD3ScFv-CD86 in this application recombines CD3ScFv and CD86 proteins into a single expression cassette. In this structure, CD86 functions more like an extracellular support structure and does not perform CD28 signaling activation. To verify whether the increased transduction efficiency of the fusion protein CD3ScFv-CD86 is related to T cell activation by the CD86-CD28 signaling pathway, Jukart cells were used. Jukart cells were transfected with a lentiviral vector expressing the NF-Kb-Luc-GFP gene at a multiplicity of infection (MOI) of 3. Flow cytometry analysis confirmed successful gene construction. Jukart-NF-Kb-Luc-GFP cells, constructed from 1e6 cells, were seeded in 24-well plates. Different amounts of CD3 ScFv and the fusion protein CD3 ScFv-CD86-G4H12 were added, and after 48 h of culture, the cells were lysed, and Luc expression was detected at 450 nm using a microplate reader. The expression of the NF-Kb-Luc-GFP gene in T cells was compared after stimulation with CD3 ScFv and the fusion protein CD3 ScFv-CD86-G4H12 to verify whether the CD86 molecule functions. The results are shown in Figure 13, with the ordinate representing the absorbance of Luc expression; higher absorbance indicates higher NF-Kb gene expression.

[0221] The results showed that, based on Luc expression, there was no significant difference between the CD3 ScFv and CD3 ScFv-CD86 fusion protein in different dose groups. This suggests that the CD3 ScFv-CD86 fusion protein is a truncated CD86 structure, lacking the complete CD86-CD28 signaling sequence, and therefore has almost no activating effect on cells. The improved transduction efficiency of the LNP containing the CD3 ScFv-CD86 fusion protein compared to the LNP using CD3 ScFv as the target protein is likely due to the novel structure formed by the truncated CD86 and CD3 ScFv, combined with its conformation after being displayed on the LNP, and cannot be explained by the known CD86-CD28 signaling mechanism in this field.

[0222] Example 9: Comparison of the performance of targeted LNPs synthesized by different synthesis processes

[0223] Example 6 demonstrates the superiority of the one-step synthesis method by comparing synthesis process 1 with the existing process in the literature. In some examples, the molar ratio of each component in LNP synthesis varies during the LNP synthesis process. The applicant designed synthesis process 2, which differs from synthesis process 1 only in the molar ratio of each LNP synthesis component. In this example, the transduction efficiency and encapsulation efficiency of the one-step synthesis process 2 with varying molar ratios of each LNP synthesis component were verified. Meanwhile, for the one-step synthesis method, PEGylated proteins with targeting proteins can be prepared using various methods. Whether PEGylated proteins with targeting proteins prepared by different methods affect the properties of LNP particles has not been studied in the prior art. In this example, we verified and compared the transduction efficiency and encapsulation efficiency of targeted LNPs synthesized using PEGylated proteins with targeting proteins prepared by the SATA method and the TCEP method as synthetic raw materials. The flowchart of the specific targeted LNP synthesis is shown in Figure 10.

[0224] 4.1 Encapsulation efficiency detection of targeted LNPs synthesized by different synthesis processes

[0225] Currently, existing technologies for synthesizing targeted LNPs all use the SATA method to modify fusion proteins, but the SATA method can lead to dimer formation. We have optimized the targeted LNP synthesis process to develop the TCEP method for fusion protein modification. The specific method is described above, and the fusion protein is CD3ScFv-CD86-G4H12 as described in this application.

[0226] Based on the aforementioned precise biosynthesis process 2, PEGylated targeting proteins synthesized using either the SATA method or the TCEP method were used as raw materials to synthesize targeted LNP particles. The specific component ratios of the targeted LNP particles were SM102 (33.33%), DSPC (13.33%), PEG-DMG (2%), and CHOL (51.33%). Targeted LNP particles synthesized using the SATA method were labeled using the SATA method, and those synthesized using the TCEP method were labeled using the TCEP method. 150 μg of ScFv or fusion protein conjugate was added to the citrate buffer. The synthesis system consisted of 1.5 mL of alcohol phase and 4.5 mL of citrate. The synthesis rate was 12 mL / min for the alcohol phase and 36 mL / min for the citrate phase. After microfluidic synthesis of the particles, dialysis was performed, with PBS replaced every 1 hour for a total of 4 times before sample collection.

[0227] The encapsulation efficiency of targeted LNP particles was detected using an RNA quantification kit, as described above. The results, shown in Figure 14, indicate that, through a non-paired t-test, the TCEP-modified fusion protein PEGylation of the targeting protein significantly improved particle encapsulation efficiency, meeting the particle encapsulation efficiency requirements for industrial-scale production.

[0228] 4.2 Validation of In Vitro Transduction of Targeted LNPs Synthesized by Different Synthetic Processes

[0229] Precision biosynthesis process 2 synthesizes targeted LNPs (see 1 for details). In vitro transfection was validated according to the above-described in vitro transduction efficiency verification method. T cells from different donors were repeatedly transfected and statistically analyzed. The target nucleic acid was RNA expressing CD19 CAR, with the sequence shown in SEQ ID NO.2. 1 μg of CAR-positive detection antibody was added, labeled at 4℃ for 30 min, washed with 1 mL of PBS, centrifuged at 350g for 5 min, and resuspended in 100 μL of PBS. The CAR positivity rate and MFI were detected by instrumentation. The results are shown in Figure 15. In Figure 15, A represents the in vitro transduction positivity rate of targeted LNPs synthesized from PEGylated target proteins from different sources, and B represents the average fluorescence intensity of targeted LNPs synthesized from PEGylated target proteins from different sources.

[0230] The results showed that the targeted PEGylated protein containing the targeted fusion protein CD3ScFv-CD86-G4H12 described in this application, synthesized using the TCEP method, as a raw material for the PEG derivative of the targeted LNP, significantly improved the mean fluorescence intensity (MFI) of the target gene expression. Paired t-test analysis showed that P = 0.0021.

[0231] 4.3 Verification of in vivo transduction efficiency for different synthesis processes

[0232] Human PBMCs were injected via tail vein into severely immunodeficient mice, such as NCG mice. The PBMC dosage ranged from 5e6 cells / mouse to 3e7 cells / mouse. Between 10 and 20 days after injection, blood samples were collected from the orbital sinus to measure the proportion of hCD45+ cells in the peripheral blood nucleated cells, which ranged from 5% to 70%. Mice were randomly assigned to groups and injected with 40 μg / mouse of PEGylated target proteins synthesized using different processes. Targeted LNPs were then synthesized using LNP synthesis process 2, with the target mRNA being the CD19 CAR gene and the target cells being T cells. Mice were euthanized 24 hours after infusion of the targeted LNPs. Spleens were harvested, and erythrocytes were lysed and counted. Cells ranging from 1e6 to 2e6 cells were used to detect the CAR positivity rate. CD19 CAR was expressed using the in-house produced anti-antibody B937D4-AF647 from Precision Biotechnology. 1 μg of antibody and 1 μg of commercially available CD45 and CD3 antibodies were added. After labeling at 4°C for 30 min, the cells were washed with 1 mL of PBS, centrifuged at 350 g for 5 min, and resuspended in 100 μL of PBS. CAR positivity and MFI were then detected. The results are shown in Figure 16, with the ordinate representing the transduced CAR gene expression efficiency.

[0233] The results indicate that the fusion protein of this application, which is modified by TCEP, significantly improves the in vivo target cell transduction efficiency of the targeted LNP by PEGylation. The results were obtained by one-way ANOVA, P = 0.0303.

[0234] Example 10: Detection of Targeted PEGylation Modification Conditions for Fusion Protein Modification

[0235] The fusion protein was modified with PEG using the TCEP method described above. Two TCEP concentrations were set, with the fusion protein:TCEP:DSPE-PEG-MAL molar ratios of 1:50:10 and 1:25:10, respectively, to compare the effects of different TCEP concentrations on the target particle encapsulation efficiency and transfection efficiency. CD3ScFv-CD86-PEG-DSPE was synthesized according to the above scheme. The formulation components SM102 (33.33%), DSPC (13.33%), DMG-PEG (2%), and CHOL (51.33%) were dissolved in ethanol to form the ethanol phase. The target nucleic acid and CD3ScFv-CD86-PEG-DSPE were dissolved in citrate buffer to form the citrate phase. The target nucleic acid was CD19 CAR mRNA, dissolved at a P:N ratio of 1:3 with ionizable cationic lipids. The citrate buffer in the citrate phase was a pH 4.0, 0.02M citrate buffer. Using a microfluidic system (Aitesen), with the device settings at an alcohol-to-citric acid flow rate ratio of 1:3, LNP solutions were synthesized via the microfluidic device and then dialyzed using PBS buffer or TFF tangential flow (Replekin). The solution was replaced using the Research 2i [KR2i]TFF system, and the LNP was finally stored in 0.01M PBS buffer. mRNA quantification was performed using an RNA nucleic acid quantification kit.

[0236] Encapsulation efficiency was assessed using an RNA quantification kit (Thermo, catalog number R11490). Different batches of T cells were simultaneously transfected to verify the transfection efficiency and encapsulation efficiency of the targeting particles. The results are shown in Figure 17, illustrating the encapsulation efficiency and transduction efficiency verification of different protein modification systems; Figure 17A represents the encapsulation efficiency detection, and Figure 17B represents the transduction efficiency detection.

[0237] The results show that the molar ratios of TCEP:DSPE-PEG-MAL of 1:50:10 and 1:25:10, respectively, do not affect the encapsulation efficiency and transduction efficiency, thus achieving the objectives of this application. Different concentrations of TCEP have no effect on the encapsulation efficiency, nor on the expression and fluorescence intensity of CAR after different batches of T transfection. Therefore, in the synthesis of PEGylated lipids modified with fusion proteins, the molar ratio of fusion protein, TCEP, or PEGylated lipids should be 1:(25-50):(5-10).

[0238] Example 11: In vivo preparation and functional validation of CAR-T cells using targeted LNP particles

[0239] Lipid nanoparticles (LNPs) encapsulated with nucleic acids can be administered to an organism via intravenous, subcutaneous, or intraperitoneal administration. This allows for the delivery of targeted nucleic acids with active or therapeutic effects to target sites throughout the body, serving as a drug or drug combination for treating diseases. To verify the ability of nucleic acid-encapsulated lipid nanoparticles (LNPs) as a drug or drug combination, a targeted LNP was synthesized using a chimeric antigen receptor (CAR) nucleic acid sequence capable of recognizing CD19-positive tumor cells such as B-cell lymphoma and B-cell leukemia, combined with the targeted LNP component described in this application. This targeted LNP was then intravenously infused into mice bearing B-cell tumors, and the effectiveness of the nucleic acid-encapsulated targeted LNP as a drug or drug combination component was tested. The preparation of the targeted LNP is described in Example 9.

[0240] Human PBMCs (1e7 cells) were injected via tail vein into severely immunodeficient mice, such as NCG mice, to construct a humanized mouse environment that mimics the human immune environment. Twenty-four hours later, 2e6 cells of Nalm6-Luc-GFP were injected via tail vein, and mice were intraperitoneally injected with 15 mg / kg of Luceferase substrate. In vivo imaging was performed using a live imaging system. After in vivo imaging, 40 μg / mouse of a targeted LNP (nucleic acid-encapsulated protein) prepared according to the protocol in Example 9 was injected via tail vein. Approximately once a week, in vivo imaging and particle injection were performed twice, and the efficacy of the particles was evaluated based on changes in fluorescence values. The results are shown in Figure 18. The experiment was divided into three groups: the Nalm6 group, the LNP group, and the targeted particle group. Compared with the LNP group, the targeted particle group showed a significant inhibitory effect on tumor proliferation. These results indicate that targeted LNPs encapsulated with nucleic acids can be used as a drug or component of a drug combination for disease treatment, and in this example, they can achieve the goal of treating tumors.

[0241] In conjunction with Examples 6, 7, and 8, the targeted fusion protein CD3ScFv-CD86-G4H12 described in this application exhibits superior targeted transduction capabilities under different modification methods (SATA or TCEP) and different synthesis processes (synthesis process 1 or synthesis process 2), promoting the transduction of target nucleic acids encapsulated by targeted LNPs into target cells. The targeted fusion protein CD3ScFv-CD86-G4H12 described in this application is used in a one-step synthesis method for targeted LNPs. The LNP components are: ionizable cationic lipids such as SM102, neutral lipids, PEG or PEGylated lipids, and cholesterol or other modified cholesterol in a molar ratio ranging from (33%-50%):(10%-13.33%):(1.5%-2%):(38.5%-51.33%), with a preferred molar ratio of 33.33%:13.33%:2%:51.33%. The synthesized targeted LNP has a more efficient ability to transduce the target gene into the target cell. Preferably, the PEGylated targeting protein linked to the target protein synthesized using the TCEP method has higher transduction efficiency and encapsulation rate.

[0242] In some embodiments, the chimeric antigen receptor (CAR) nucleic acid sequence recognizing CD19-positive tumor cells can be replaced with a target molecule nucleic acid sequence recognizing at least one surface-expressed molecule, including solid tumors, hematologic malignancies, and / or tissues. The target molecule includes any one or more of the following combinations: CD19, CD20, CD22, CD33, CLL-1 (CLEC12A), CD7, CD5, CD70, CD123, CEA, CEACAM5, CEACAM6, CEACAM7, Mesothelin, and MUC1. CLDN18.2, CDH17, Trop2, BCMA, NKG2D, PDL1, EGFR, EGFRVIII, PSCA, PSMA, MUC16, CD133, GD2, IL13R2, B7H3, Her2, CD30, SLAMF7, CD38, GPC3, WT1, AFP, FOLR1, c-Met, LeY (Lewis-Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), MAGE-A1 (melanoma-associated antigen 1), and TAG-72. It can be used to treat tumors such as colon cancer, rectal cancer, small intestine cancer, anal cancer, bile duct cancer, stomach cancer, esophageal cancer, gallbladder cancer, lung cancer, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, kidney cancer, bladder cancer, cancers of the central nervous system, glioblastoma, skin cancer, melanoma, lymphoma, head and neck cancer, multiple myeloma, and leukemia.

[0243] In some embodiments, the chimeric antigen receptor (CAR) nucleic acid sequence that recognizes CD19-positive tumor cells can be replaced with an antigenic peptide nucleic acid sequence that can elicit an immune response in the organism, such as a combination of at least one or more nucleic acid fragments of HPV genome E1-E7, L1-L2, or antigenic peptide nucleic acid sequences or sequences of viruses or bacteria related to diseases such as hepatitis A, hepatitis B, poliomyelitis, influenza, and pneumonia, thereby obtaining a vaccine drug for treating the above-mentioned diseases or as a component of a drug combination.

[0244] In some embodiments, the chimeric antigen receptor (CAR) nucleic acid sequence that recognizes CD19-positive tumor cells can be replaced with active agents or therapeutic agents related to ophthalmic diseases, metabolic diseases, hyperlipidemia, hepatitis B, pseudohypertrophic muscular dystrophy, non-alcoholic fatty liver disease, heart disease, amyotrophic lateral sclerosis, cystic fibrosis, etc., such as siRNA, miRNA, ASO, small activating RNA (saRNA), aptamers, transfer RNA (tRNA) fragments, etc., as drugs or drug combinations to treat ophthalmic diseases, metabolic diseases, hyperlipidemia, hepatitis B, pseudohypertrophic muscular dystrophy, non-alcoholic fatty liver disease, heart disease, amyotrophic lateral sclerosis, cystic fibrosis, etc.

[0245] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Lipid nanoparticles, whose packaging materials include: The ingredients include cationic lipids, neutral lipids, PEG or PEGylated lipids, cholesterol or their derivatives; wherein the molar percentages of the cationic lipids, neutral lipids, PEG or PEGylated lipids, cholesterol or their derivatives are (33%–50%): (10%–13.33%): (1.5%–2%): (38.5%–51.33%).

2. The lipid nanoparticles according to claim 1, characterized in that, The cationic lipids are selected from at least one of SM102, DLin-MC3-DMA, ALC-0315 or ATX-126; The neutral lipids are selected from at least one of DSPC, DOPE, DPPC, PC, PE, PG, PI or PS; The average molecular weight of the PEG is 500–5000; The PEGylated lipid is at least one of ALC-0159, DMG-PEG, PEG2000-C-DMG, DSPE-PEG, DPPE-MPEG(2000), DSPE-PEG-Amine, DSPE-PEG-NHS,MW 2000, DSPE-PEG-Maleimide, DMG-PEG-MAL, DMG-PEG2000, DSPE-PEG2000, DSPE-PEG2000-MAL, DMG-PEG2000-MAL, aminated-PEG-DMG, or hydroxylated-PEG-DMG.

3. The lipid nanoparticles according to claim 1 or 2, characterized in that, The packaging material is composed of SM102, DSPC, PEGylated lipids and cholesterol, wherein the molar percentages of SM102, DSPC, PEGylated lipids and cholesterol are 33.33%: 13.33%: 2%: 51.33%.

4. The lipid nanoparticles according to any one of claims 1 to 3, characterized in that, The packaging material also includes PEGylated targeting proteins; the PEGylated targeting proteins include fusion proteins and PEGylated lipids, wherein the fusion proteins include antibodies targeting CD3 molecules and the N-terminus of the extracellular domain of CD86.

5. The lipid nanoparticles according to claim 4, characterized in that, The PEGylated target protein is formed by a target protein containing CD3 ScFv, the N-terminus of the extracellular segment of CD86 and the G4H12 fragment, and PEGylated lipids. The CD3 ScFv has an amino acid sequence as shown in SEQ ID NO:5, or an amino acid sequence obtained by deleting, substituting or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO:5, or an amino acid sequence that has more than 80% identity with the amino acid sequence shown in SEQ ID NO:

5. The extracellular segment of CD86 has an N-terminus with an amino acid sequence as shown in SEQ ID NO:6, or an amino acid sequence obtained by deleting, substituting or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO:6, or an amino acid sequence that has more than 80% identity with the amino acid sequence shown in SEQ ID NO:

6. The G4H12 fragment has an amino acid sequence as shown in SEQ ID NO:7, or has an amino acid sequence obtained by deleting, substituting, or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO:7, or has an amino acid sequence that is more than 80% identical to the amino acid sequence shown in SEQ ID NO:

7.

6. The lipid nanoparticles according to claim 4 or 5, characterized in that, The fusion protein, from N-terminus to C-terminus, consists of CD3 ScFv, the N-terminus of the CD86 extracellular segment, and a G4H12 fragment; or the N-terminus of the CD86 extracellular segment, CD3 ScFv, and a G4H12 fragment; or the G4H12 fragment, CD3 ScFv, and the N-terminus of the CD86 extracellular segment; or the G4H12 fragment, the N-terminus of the CD86 extracellular segment, and CD3 ScFv. Preferably, in the fusion protein, CD3 ScFv and the extracellular segment of CD86 are linked by 7 amino acids at the N-terminus and G4H12 is linked at the C-terminus to form a fusion protein; Preferably, the fusion protein comprises, from N-terminus to C-terminus, CD3 ScFv, linker, N-terminus of CD86 extracellular segment, and G4H12 fragment; More preferably, the linker is G4S, (G4S)n (n>1), GSTGSGSGKPGSGEGSTKG; More preferably, the fusion protein has the amino acid sequence shown in SEQ ID NO:

1.

7. The lipid nanoparticles according to claim 4 or 5, characterized in that, In the PEGylated target protein, the PEGylated lipids are selected from at least one of the following: ALC-0159, DMG-PEG, PEG2000-C-DMG, DSPE-PEG, DPPE-MPEG(2000), DSPE-PEG-Amine, DSPE-PEG-NHS,MW 2000, DSPE-PEG-Maleimide, DMG-PEG-MAL, DMG-PEG2000, DSPE-PEG2000, DSPE-PEG2000-MAL, and DMG-PEG2000-MAL; preferably, the average molecular weight of PEG in the PEGylated target protein is 2000.

8. The lipid nanoparticles according to any one of claims 4 to 7, characterized in that, In the PEGylated targeting protein, the fusion protein and the PEGylated lipids are linked by a thioether bond; preferably, the C-terminus of the fusion protein is reduced to form a -SH group, and then this group undergoes an alkylation reaction with the double bond in the PEGylated lipid to form a thioether bond; preferably, the -SH group is formed by the target protein containing G4H12 reducing the disulfide bond of G4H12 with a TCEP reducing agent to form -SH.

9. The lipid nanoparticles according to any one of claims 1 to 8, characterized in that, It also includes nucleic acids encapsulated within packaging materials.

10. The lipid nanoparticles according to claim 9, characterized in that, The nucleic acid includes at least one of mRNA, siRNA, miRNA, DNA, dsRNA, sDNA, small interfering RNA, miRNA, aiRNA, shRNA, tRNA, ssDNA, dsDNA, and plasmid.

11. A method for preparing lipid nanoparticles, comprising: The lipid nanoparticle packaging material according to any one of claims 1 to 8 is dissolved in ethanol to obtain an alcohol phase; The PEG-conjugated fusion protein and the nucleic acid of claim 9 or 10 are dissolved in citrate buffer to obtain the citrate phase; the alcohol phase and the citrate phase are then prepared into lipid nanoparticles via microfluidic control.

12. The preparation method according to claim 11, characterized in that, The citrate buffer solution has a pH of 3-5 and a concentration of 0.01M-0.03M; preferably, the citrate buffer solution has a pH of 4.0 and a concentration of 0.02M. In the microfluidic step, the ratio of alcohol phase flow rate to citric acid phase flow rate is 1:(1-5); preferably, the ratio of alcohol phase flow rate to citric acid phase flow rate is 1:

3.

13. Use of the lipid nanoparticles according to any one of claims 1 to 10 in the preparation of a product for in vivo delivery of nucleic acids to be administered to mammalian subjects.

14. A nucleic acid delivery reagent comprising the lipid nanoparticles and buffer solution as described in any one of claims 1 to 10.

15. A pharmaceutical composition comprising the lipid nanoparticles according to claims 1 to 10.

16. Use of the lipid nanoparticles of claim 9 or 10 in the preparation of products that introduce nucleic acids into cells.

17. Use of the lipid nanoparticles of claim 9 or 10 in the preparation of a medicament for treating a disease or functional disorder in a mammalian subject in need of treatment.

18. The application according to claim 17, characterized in that, Diseases include at least one of the following: infectious diseases, metabolic diseases, ophthalmic diseases, hyperlipidemia, hepatitis B, pseudohypertrophic muscular dystrophy, non-alcoholic fatty liver disease, heart disease, amyotrophic lateral sclerosis, cystic fibrosis, genetic defects, or tumors.

19. A method for nucleic acid transduction, comprising lipid nanoparticles as described in any one of claims 1 to 10, wherein the lipid nanoparticles are incubated after contact with cells to transduce target nucleic acids of the target cells.

20. The transduction method according to claim 19, characterized in that, The target cells are any one or a combination of T lymphocytes, B cells, NK cells, macrophages, and DC cells.

21. Fusion protein, which includes CD3 ScFv, the N-terminus of the extracellular domain of CD86, and the G4H12 fragment.

22. The fusion protein according to claim 20, characterized in that, The CD3 ScFv has an amino acid sequence as shown in SEQ ID NO:5, or an amino acid sequence obtained by deleting, substituting, or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO:5, or an amino acid sequence that has more than 80% identity with the amino acid sequence shown in SEQ ID NO:

5.

23. The fusion protein according to claim 20, characterized in that, The extracellular segment of CD86 has an N-terminus with an amino acid sequence as shown in SEQ ID NO:6, or an amino acid sequence obtained by deleting, substituting, or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO:6, or an amino acid sequence that has more than 80% identity with the amino acid sequence shown in SEQ ID NO:

6.

24. The fusion protein according to any one of claims 20 to 22, characterized in that, It has the amino acid sequence shown in SEQ ID NO:

1.

25. Biomaterials, including at least one of the following: I) The nucleic acid encoding the fusion protein according to any one of claims 20 to 23; II) Expression units containing the nucleic acid described in I); III) A recombinant vector containing the nucleic acid described in I) or the expression unit described in II); IV) Transformants that have been transformed or transfected with the expression vector described in III); The culture products of the transformants described in (V) and (IV).

26. A PEGylated targeting protein comprising the fusion protein and PEGylated lipids as described in any one of claims 20 to 23.

27. The PEGylated targeting protein according to claim 26, characterized in that, The PEGylated lipids include at least one of ALC-0159, DMG-PEG, PEG2000-C-DMG, DSPE-PEG, DPPE-MPEG(2000), DSPE-PEG-Amine, DSPE-PEG-NHS,MW 2000, DSPE-PEG-Maleimide, DMG-PEG-MAL, DMG-PEG2000, DSPE-PEG2000, DSPE-PEG2000-MAL, and DMG-PEG2000-MAL.

28. The PEGylated targeting protein according to claim 26 or 27, characterized in that, The average molecular weight of PEG is 2000.

29. A method for preparing the PEGylated targeting protein according to any one of claims 26 to 28, comprising: The fusion protein according to any one of claims 20 to 23 is mixed with TCEP solution and reacted, PEGylated lipids are added, and after the reaction, the PEGylated target protein according to any one of claims 26 to 28 is obtained.

30. The preparation method according to claim 28, characterized in that, The molar ratio of the fusion protein, TCEP and PEGylated lipids according to any one of claims 20 to 23 is 1:(25 to 50):(5 to 10).

31. The use of the fusion protein according to any one of claims 20 to 23 or the PEGylated targeting protein according to any one of claims 26 to 28 in the preparation of T cell-targeting LNPs.

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