MOLECULE WITH TARGETING FUNCTION AND APPLICATION

Abstract

The present invention relates to the technical field of medicine, and relates in particular to a molecule having a targeting function. The molecule having a targeting function can specifically deliver a drug to a specific cell, which facilitates precise programming of a specific cell in the body, a therapeutic effect of the drug is produced, and consequently drug dosage and side effects are greatly reduced, and the effect of precise treatment to cells in the body is achieved.

Description

Molecules with targeting functions and their applications

[0001] Cross-references

[0002] This application claims priority to Chinese Patent Application No. 2023116272306, filed on November 30, 2023, entitled “Lipid Nanoparticles and Nanoparticle Drugs,” and Chinese Patent Application No. 2024109186851, filed on July 10, 2024, entitled “Molecules with Targeting Function and Applications,” the disclosures of which are hereby incorporated by reference in their entirety. Technical Field

[0003] The present invention relates to the field of medical technology, and in particular to a molecule with targeting function and its application. Background Art

[0004] Lipid nanoparticles (LNPs) are composed of four components: an ionizable lipid compound, cholesterol, phospholipids, and PEG lipids. LNPs are a well-established delivery system technology platform, suitable for delivering RNA drugs, vaccines, or gene editing tools. Research on LNP composition is currently in-depth.

[0005] The composition of LNPs significantly influences their drug delivery performance. Ionizable lipid compounds are the core components of LNPs, determining mRNA delivery and transfection efficiency. In practice, nucleic acid drugs are susceptible to nonspecific cellular uptake during delivery, leading to higher dosages and significant side effects.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] To solve the above technical problems, the present invention provides a molecule with targeting function and its application, which can target the delivery of pharmacological molecules to specific cells in the body, reduce the uptake of drugs by nonspecific cells, thereby reducing the dosage and side effects of drugs, and achieve more effective in vivo precision treatment effects on diseases.

[0008] Specifically, the technical solutions provided by the present invention are as follows:

[0009] In a first aspect, the present invention provides a molecule with targeting function, comprising:

[0010] (I) one or more hydrophobic chains;

[0011] (II) one or more linkers;

[0012] (III) one or more Nanobodies.

[0013] Preferably, the number of said Nanobodies is ≤20; preferably 1-7.

[0014] Preferably, an amino acid containing a reactive site is connected between the Nanobody and the linker, and the amino acid includes but is not limited to cysteine ​​and lysine.

[0015] Preferably, the Nanobodies are connected via a linker sequence, which is located between the Nanobodies and the amino acids, and which comprises (GS)n and / or (GGGS)m, wherein n and m are identical or different values, and the value of n or m is selected from 0, 1, 2, 3 or 4.

[0016] Preferably, the hydrophobic chain comprises one or more substituted or unsubstituted C12-C18 alkyl groups.

[0017] Preferably, the linker comprises one or more structural units selected from -C(=O)O-, -OC(=O)O-, -C(=O)NH-, -C(=O)S-, -S-, -SS-, a triazole group, a hydrazone bond, SMCC, Sulfo-SMCC, SATA, a polypeptide as a structural unit, PEG or a PEG derivative.

[0018] The PEG derivative can be maleimide-functionalized polyethylene glycol, such as PEG-MAL.

[0019] Preferably, the relative molecular mass of the PEG or PEG derivative is 500-5000, preferably 1000, 2000, 3000, 4000 or 5000.

[0020] Preferably, the nanobody comprises an antigen-binding domain that is specific for an antigen, and the antigen is a membrane protein molecule.

[0021] Preferably, the membrane protein molecule is an immune cell or immune-related cell membrane protein molecule, and the immune cell or immune-related cell membrane protein molecule includes but is not limited to one or more of CD3, CD4, CD5, CD7, CD8, CD25, CD38, CD61, CD42a, CD105, CD90, CD15, CD127, CD56, CD68, CD19, CD11c, CD138, F4 / 80, CD62P, CD49f, CD31, RANK, ALPL, PDPN, CD34, FcεR1α, CD203c, CD63, CD193, CD66b, CD41, CD117, and ASGPR;

[0022] Preferably, the membrane protein molecule is a central nervous system cell membrane protein molecule, and the central nervous system cell membrane protein molecule includes but is not limited to one or more of GD2, GD3, MOG, and TMEM119;

[0023] Preferably, the membrane protein molecule is a tumor-associated antigen, including but not limited to one or more of CD133, PSMA, CLDN18.2, DLL3, TROP2, EGFRVIII, CA125, MUC1, MUC16, MSLN, CA9, HER2, HER3, TGM4, PSCA, CLDN6, STEAP2, GPC3, IL-13Ra2, EGFR, STEAP1, BCMA, FRa, VEGFR2, PDGFR-β, CEA, NCAM, FAP, SLC2A2, SEZ6L2, and LRP11;

[0024] Preferably, the membrane protein molecule is an immune regulatory molecule, including but not limited to one or more of B7-H1, B7-H3, and B7-H4;

[0025] Preferably, the membrane protein molecules include but are not limited to the following targets: CD3, CD4, CD5, CD7, CD8, CD25, CD38, CD61, CD42a, CD105, CD90, CD15, CD127, CD56, CD68, CD19, CD11c, CD138, F4 / 80, CD62P, CD49f, CD31, RANK, ALPL, PDPN, CD34, FcεR1α, CD203c, CD63, CD193, CD66b, CD41, CD117, ASGPR, GD2, GD3, MOG, One or more of TMEM119, CD133, PSMA, CLDN18.2, DLL3, TROP2, EGFRVIII, CA125, MUC1, MUC16, MSLN, CA9, HER2, HER3, TGM4, PSCA, CLDN6, STEAP2, GPC3, IL-13Ra2, EGFR, STEAP1, BCMA, FRa, VEGFR2, PDGFR-β, CEA, NCAM, FAP, SLC2A2, SEZ6L2, LRP11, B7-H1, B7-H3, B7-H4.

[0026] Preferably, the nanobody is a nanobody targeting CD8, and the amino acid sequence of the nanobody is as shown in any one of SEQ ID NO: 1-3;

[0027] And / or, the Nanobody is a Nanobody targeting CD19, and the amino acid sequence of the Nanobody is as shown in any one of SEQ ID NOs: 79-81;

[0028] And / or, the nanobody is a nanobody targeting CD56;

[0029] And / or, the Nanobody is a Nanobody targeting CD5, and the amino acid sequence of the Nanobody is shown in any one of SEQ ID NO: 4-78;

[0030] And / or, the Nanobody is a PSMA-targeted Nanobody, and the amino acid sequence of the Nanobody is as shown in any one of SEQ ID NOs: 82-83;

[0031] And / or, the Nanobody is a Nanobody targeting CD133, and the amino acid sequence of the Nanobody is shown in any one of SEQ ID NOs: 84-85.

[0032] The specific sequences of the present invention are as follows:

[0033] Anti-human CD8Nb seq (corresponding sequence SEQ ID NO: 1-3)

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[0037] Anti-human CD5Nb seq (corresponding sequence SEQ ID NO: 4-78)

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[0120] In a second aspect, the present invention provides a nanobody with targeting function, whose amino acid sequence is shown in any one of SEQ ID NO: 4-78; or, the amino acid sequence of the nanobody has an identity of ≥80% with the amino acid sequence shown in any one of SEQ ID NO: 4-78; preferably ≥90%; more preferably ≥95%.

[0121] In a third aspect, the present invention provides a lipid nanoparticle, the components of which include the molecule with targeting function, wherein the content of the molecule with targeting function in the total lipid of the lipid nanoparticle is 0.01-1.0 mol%, preferably 0.01-0.5 mol%.

[0122] Preferably, the components of the lipid nanoparticles include polymer-bound lipids and ionizable amino lipids; the polymer-bound lipids are selected from lipids modified with PEG or its derivatives;

[0123] The relative molecular mass of the lipid modified with PEG or its derivatives is 2000-5000, preferably 2000, 3000, 4000 or 5000;

[0124] Preferably, the content of the polymer-bound lipid in the total lipid of the lipid nanoparticles is 0.2-5 mol%, preferably 0.5-2 mol%.

[0125] Preferably, the PEG derivative comprises maleimide-functionalized polyethylene glycol and non-maleimide-functionalized polyethylene glycol; wherein the polyethylene glycol can be selected from DMG-PEG2000; the maleimide-functionalized polyethylene glycol can be selected from PEG-MAL; the content of maleimide-functionalized polyethylene glycol in the total lipid of the lipid nanoparticles is: 0.01-1.0 mol%, preferably 0.01-0.5 mol%.

[0126] Preferably, the ionizable amino lipid accounts for 30-70 mol%, preferably 40-60 mol% of the total lipids in the lipid nanoparticles;

[0127] More preferably, the ionizable amino lipid has a structure represented by general formula (I), or is an isomer, pharmaceutically acceptable salt, prodrug or solvate of the structure represented by general formula (I);

[0128] wherein G is selected from H, OR, CN, -C(=O)OR', -OC(=O)R', -C(=O)NR'R", -NR'C(=O)R", NR'R", or a cyclic alkyl structure containing at least one heteroatom; wherein the heteroatom is O or N;

[0129] wherein R, R', and R" are the same or different from each other and are independently selected from H, C1-C 10 Alkyl, C3-C 10 Cycloalkyl, C3-C 10 Alkenyl or C3-C 10 Cycloalkenyl, C1-C 10 Alkyl, C3-C 10 Cycloalkyl, C3-C 10 an alkenyl group or a cyclic alkyl structure containing at least one heteroatom;

[0130] Preferably, R, R', and R" are the same or different from each other and are each independently selected from H, C1-C8 alkyl, C3-C8 cycloalkyl, C3-C8 alkenyl, C3-C8 cycloalkenyl, C1-C8 terminally connected tertiary amine 10 Alkyl, C3-C 10 Cycloalkyl, C3-C 10 an alkenyl group or a cyclic alkyl structure containing at least one heteroatom;

[0131] wherein the substitutable carbon atoms or heteroatoms in the cyclic alkyl structure are unsubstituted or substituted with one or more hydroxyl groups, C1-C4 alkyl groups, C2-C4 alkenyl groups, C3-C8 cycloalkyl groups, or C3-C8 cycloalkenyl groups;

[0132] Preferably, the cyclic alkyl structure is unsubstituted or substituted with one or more C1-C4 alkyl groups, C3-C8 cycloalkyl groups or hydroxyl groups;

[0133] More preferably, R' and R" are the same or different from each other and are independently selected from H, C1-C4 alkyl, C1-C4 terminally connected to a tertiary amine 10 Alkyl, C3-C 10 Cycloalkyl or a cyclic alkyl structure containing at least one heteroatom; the cyclic alkyl structure is unsubstituted or substituted by one or more C1-C4 alkyl or C3-C8 cycloalkyl groups.

[0134] And / or, M1, M2, M3, M4 are the same as or different from each other and are each independently selected from C1-C 24 Alkylene, C3-C 24 Cycloalkylene, C2-C 24 Alkenylene or C3-C 24Preferably, M1, M2, M3, M4 are the same as or different from each other, and M1 and M4 are each independently selected from C4-C 22 Alkylene, branched C4-C 22 Cycloalkylene, C4-C 22 Alkenylene or branched C4-C 22 Cycloalkenylene, M2 and M3 are each independently selected from C4-C 22 Alkylene, C4-C 22 Cycloalkylene, C4-C 22 Alkenylene or C4-C 22 Preferably, M2 and M3 are the same and are C4-C 22 Alkylene; M1 is the same as M4 and is a C4-C 22 alkylene;

[0135] and / or, R 1 、R 2 are the same as or different from each other and are independently selected from H, C1-C 24 Alkyl, C3-C 24 Cycloalkyl, C2-C 24 Alkenyl or C3-C 24 Cycloalkenyl; preferably, R 1 、R 2 are the same as or different from each other and are each independently selected from C4-C 22 Alkyl or C4-C 22 alkenyl;

[0136] and / or, L1, L2, L3, L4 are the same as or different from each other and are independently selected from -C(=O)O-, -OC(=O)-, -C(=O)S-, -SC(=O)-, -C(=O)NR-, -NRC(=O)-, -S(=O)-, -OS(=O)2-, -S(=O)2O-, -O-, -S- or -SS-; preferably, L1, L2, L3, L4 are the same as or different from each other and are independently selected from -C(=O)O-, -OC(=O)-, -C(=O)NR-, -NRC(=O)- or -SS-; when L1, L2, L3, L4 are independently selected from -C(=O)NR- or -NRC(=O)-, wherein R is independently selected from H or Cl-C 10 Alkyl; preferably, L1 and L4 are the same and are -C(=O)O- or -OC(=O)-;

[0137] And / or, M5 is selected from a single bond, C1-C 16 Alkylene, C2-C 16Preferably, M5 is selected from a single bond, a C2-C 16 Alkylene, C2-C 16 Alkenylene, C4-C8 cycloalkylene or C3-C8 cycloalkenylene; preferably, M5 is selected from a single bond, C2-C 16 Alkylene or C4-C6 cycloalkylene;

[0138] Further preferably, M5 and G are connected to form Specifically, you can choose one from A1 to A52:

[0139] Among them, the Preferably one selected from A1-A18, A22-A24, A28-A52; further preferably one selected from A15, A23, A29, A33, A39-A52;

[0140] and / or, R 1 -L1-M1-L2-M2- fragment is R 1 -C(=O)O-M1-OC(=O)-M2-、R 1 -C(=O)NR-M1-OC(=O)-M2- or R 1 -C(=O)O-M1-SS-M2-;R 2 -L4-M4-L3-M3- fragment is R 2 -C(=O)O-M4-OC(=O)-M3-、R 2 -C(=O)NR-M4-OC(=O)-M3-or R 2 -C(=O)O-M4-SS-M3-;

[0141] More preferably, the structure of general formula (I) is selected from one of the following structures:

[0142] Preferably, the lipid nanoparticles further comprise at least one of a steroid and a neutral lipid;

[0143] Preferably, the molar ratio of ionizable amino lipid, steroid, neutral lipid and polymer-bound lipid is 30-70:0-65:0-30:0.2-5; more preferably 40-60:35-60:0-20:0.5-2;

[0144] Further preferably, the steroid is one or more of cholesterol and its derivatives, cholesterol esters, steroid hormones, steroid vitamins, and phytosterols, preferably cholesterol; and / or the neutral lipid is a phospholipid; and / or the polymer-bound lipid is a pegylated lipid, preferably DMG-PEG2000;

[0145] More preferably, the molar ratio of ionizable amino lipid, cholesterol, phospholipid and PEGylated lipid is 40-50:40-45:0-15:0.5-2.

[0146] Preferably, the lipid nanoparticles are targeted to deliver nucleic acids to target cells of a subject, including immune cells, central nervous cells, tumor cells, etc. When the target cells are contacted with the lipid nanoparticles, the lipid nanoparticles provide at least one of the following benefits:

[0147] (i) increased specificity of targeted delivery to the target cells compared to a reference LNP;

[0148] (ii) the half-life of the nucleic acid or polypeptide encoded by the nucleic acid in the cell of interest is increased compared to a reference LNP;

[0149] (iii) increased transfection efficiency compared to reference LNPs; and

[0150] (iv) Low levels of dye-accessible mRNA (<15%) and high RNA encapsulation efficiency, with at least 80% of the mRNA recovered in the final formulation relative to the total RNA used in the LNP bulk preparation.

[0151] In a fourth aspect, the present invention provides a method for preparing the lipid nanoparticles, which comprises the following steps: mixing polymer-bound lipids, ionizable amino lipids, steroids and auxiliary phospholipids to obtain lipid nanoparticles without targeting function, and then connecting the molecules with targeting function to the lipid nanoparticles without targeting function through membrane fusion or chemical bonds to obtain lipid nanoparticles with targeting function.

[0152] In a fifth aspect, the present invention provides another method for preparing the lipid nanoparticles, comprising the steps of: mixing a polymer-bound lipid, an ionizable amino lipid, a steroid, and an auxiliary phospholipid to obtain lipid nanoparticles without a targeting function, and then chemically linking a nanobody to the lipid nanoparticles without a targeting function to obtain lipid nanoparticles with a targeting function;

[0153] Preferably, the polymer-binding lipid comprises maleimide-functionalized polyethylene glycol, and the Nanobody is linked to the maleimide-functionalized polyethylene glycol.

[0154] In a sixth aspect, the present invention provides another method for preparing the lipid nanoparticles, which comprises the following steps: mixing polymer-bound lipids, ionizable amino lipids, steroids, auxiliary phospholipids and the molecules with targeting function to obtain lipid nanoparticles with targeting function.

[0155] In a seventh aspect, the present invention provides a method for targeted delivery of a pharmacodynamic molecule to specific cells of a subject, the method comprising: contacting the specific cells with the lipid nanoparticles; preferably, the specific cells include but are not limited to immune cells, immune-related cells, central nervous cells, tumor cells, somatic cells, cells infected by viruses, bacteria, and fungi; preferably, the targeted cells are B cells, T cells, or NK cells.

[0156] In an eighth aspect, the present invention provides a method for expressing a target protein or polypeptide in a target cell of a subject, the method comprising: contacting the target cell with the lipid nanoparticle; preferably, the target cell includes but is not limited to immune cells, immune-related cells, central nervous cells, tumor cells, somatic cells, cells infected by viruses, bacteria, and fungi; preferably, the target cell is a B cell, a T cell, or a NK cell.

[0157] In a ninth aspect, the present invention provides a method for treating, ameliorating or preventing symptoms of a disease in a subject in need thereof, the method comprising: administering the lipid nanoparticles to the subject to deliver the pharmacodynamic molecule to the target cells of the subject; preferably, the target cells include but are not limited to immune cells, immune-related cells, central nervous cells, tumor cells, somatic cells, cells infected by viruses, bacteria, or fungi; preferably, the disease is an immune disease, an inflammatory disease, a central nervous system disease, a degenerative disease, a bone-related disease, a pathogen infection, a metabolic disease, an endocrine disease, aging, or cancer; preferably, the pharmacodynamic molecule includes an antigen used in a therapeutic or preventive vaccine for the treatment or prevention of an immune disease, an inflammatory disease, a central nervous system disease, a degenerative disease, a bone-related disease, a pathogen infection, a metabolic disease, an endocrine disease, aging, or cancer; preferably, the target cells are B cells, T cells, or NK cells.

[0158] In a tenth aspect, the present invention provides the use of the aforementioned lipid nanoparticles as a drug delivery carrier.

[0159] In an eleventh aspect, the present invention provides a pharmaceutical composition comprising the lipid nanoparticles, wherein the lipid nanoparticles contain pharmacodynamic molecules;

[0160] Preferably, the pharmacodynamic molecule comprises one or more of mRNA, DNA, siRNA, saRNA, shRNA, miRNA, circle RNA, lnc RNA, gRNA, polypeptide or protein.

[0161] Preferably, the pharmacodynamic molecule includes mRNA, which is used to encode a CAR molecule, a TCR molecule, an immunomodulatory molecule, a functional protein molecule, or a gene editing tool such as a base editor or a Cas9 protein.

[0162] Preferably, the CAR molecule comprises at least one of a sequence that can specifically recognize a tumor-specific antigen, a transmembrane linker sequence, and / or a sequence that assists the immune function of T cells.

[0163] Preferably, the sequence encoding the TCR molecule comprises a sequence that can recognize at least one of NY-ESO-1, AFP, HBsAg, MAGEA1, Mesothelin, MART-1, CD19, CD28, PD-1, CD8, CT83, E6, E7, E8, GPC3, H3.3-K27M, HIV Gag polyprotein, HLA-A / AFP, KRASG12 (V / D), KRASG12D, KRASG12V, LMP1, LMP2, EBNA1, MAGEA10, MAGEA3, MAGEA4, MC2R, mHag HA-1, MYO1G, PRAME, WT1, gp100, CEA, p53, HLA-A2, EGFR, DR5, RAS, LAGE-1, CMV, HCV, MCPyv, and HA-1H.

[0164] Preferably, the sequences encoding immunomodulatory molecules include CD28, PD-1, CTLA-4, RGMB, ICOS, CD28H, NKp30, HVEM, OX40, Fas, 4-1BB, CD27, CD30, APO-2, APO-3, BCMA, BAFFR, GITR, IL-2, IL-12A, IL-12B, IL-15, IL-23, IL-27, FLT3L, IL-36A, IL-36B, IL-36C, GM-CSF, CCL20, CXCL9, CXCL10, CXCL11, CXCL12, CCR7, CXCL11, CXCL12, CCR8, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CXCL18, CXCL19, CXCL20, CXCL21, CXCL22, CXCL23, CXCL24, CXCL25, CXCL26, CXCL27, CXCL28, CXCL29, CXCL30, CXCL31, CXCL32, CXCL33, CXCL34, CXCL35, CXCL36, CXCL37, CXCL38, CXCL39, CXCL31, CXCL39, CXCL31, CXCL33, CXCL34, CXCL35, CXCL37, CXCL38, CXCL39, CXCL31, CXCL33, CXCL34, CXCL35 The sequence of at least one of CR4, CCR5, CCL4, CCL5, CCL19, CXCR3, CCR6, B7-H1, B7-DC, B7-H3, B7-H4, PD-1H, IL-10, TGF-β, HGF, B7-1, B7-2, B7-H2, B7-H3, CD40, FasL, CD70, CD30L, 4-1BBL, OX40L, TRAIL, RANKL, TWEAK, APRIL, BAFF, LIGHT, GITRL, CD73, STAT1, IRF4, CCAR2, BCL6, iNOS, and CD103.

[0165] Preferably, the nucleic acid encoding the CAR molecule comprises a sequence that can recognize at least one molecule of CD19, BMCA, CD22, CD20, CD123, GD2, CD30, GPC3, CLDN18.2, Mesothelin, CD33, CD38, EGFRvlll, CD138, CEA, HER2, PSMA, CLL1, CD56, EGFR, MUC-1, EpCAM, CD7, NKG2D, PD-L1, LewisY, FAP, c-MET, ROR1, IL13Rα2, AFP, CD133, CD4, BTK, ROBO1, CD5, CD70, LILRB4, FLT3, Sigle-6, CD229, and SLAMF7.

[0166] In a twelfth aspect, the present invention provides a pharmaceutical preparation comprising the pharmaceutical composition and a pharmaceutically acceptable carrier;

[0167] Preferably, the pharmaceutical preparation is an aqueous injection, a lyophilized powder or a spray. Beneficial effects:

[0168] The present invention provides a molecule with a targeting function, which can specifically deliver drugs to specific cells, such as T cells, B cells, NK cells, NKT cells, γδT cells, regulatory T cells, eosinophils, basophils, mast cells, neutrophils, platelets, mesenchymal stem cells, macrophages, plasma cells, dendritic cells, microglia, neurons, astrocytes, endothelial cells, osteoclasts, osteoblasts, synovial cells, tumor cells, etc., such as specifically delivering the CAR molecule nucleic acid sequence to T cells. For example, by specifically delivering TCR molecule nucleic acid sequences into T cells, TCR-T cells targeting specific antigen sequences can be directly generated in the body to kill virus-infected cells or tumors. For example, by specifically delivering immune functional molecules or molecules that block cell function inactivation into immune cells, immune cell function can be altered and immune response can be precisely regulated. For example, by specifically delivering functional regulatory molecules to other types of cells, target cells can be precisely programmed in the body to achieve the desired effect of the drug molecules. Specific cell delivery can significantly improve drug efficacy and reduce side effects caused by drugs acting on other cells, achieving the following: 1. Pharmacological molecules can directly enter the target cells, even target cells that traditional drugs cannot enter, and 2. Extremely low dosages can achieve maximum therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0169] In order to more clearly illustrate the technical solutions of the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0170] Figures 1A and 1B are quality control information of LNP containing coupling sites in Example 20 of the present invention.

[0171] Figures 2A, 2B, 2C and 2D show the in vitro transfection of LNPs with different targeting molecular structures in Example 20 of the present invention.

[0172] FIG3 shows the in vivo transfection of TLNPs with different targeting molecular structures in Example 20 of the present invention.

[0173] FIG4A and FIG4B are specific characterization data of Pre-TLNP in Example 21 of the present invention.

[0174] 5A and 5B are specific characterization data of TLNP in Example 21 of the present invention.

[0175] FIG6 shows the in vivo delivery efficiency of TLNPs with different targeting molecule ratios according to Example 21 of the present invention.

[0176] 7A , 7B , 7C , 7D and 7E are flow cytometric analyses of the EGFP-mRNA expression efficiency of cLNP-1 and TLNP-2 in hPBMCs in Example 24 of the present invention.

[0177] FIG8 shows the in vivo transfection of TLNP-2 at different dosages in Example 24 of the present invention.

[0178] 9A , 9B , 9C , 9D and 9E are statistical diagrams of the in vivo transfection status of TLNP-2 with different ratios in Example 25 of the present invention.

[0179] Figures 10A, 10B, 10C, 10D, 10E and 10F are long-term stability studies of traditional LNP and targeted TLNP in Example 27 of the present invention.

[0180] Figures 11A, 11B, 11C and 11D show the biodistribution results in Balb / c mice studied by Cy5 fluorescence, Luc imaging and qPCR in Example 29 of the present invention.

[0181] Figures 12A and 12B are the results of Luc imaging and qPCR studies on the biodistribution of NGC in mice in Example 29 of the present invention.

[0182] FIG13 is the dosage safety study data of TLNP-2 at different working concentrations in hPBMC in Example 30 of the present invention.

[0183] FIG14 shows the liver toxicity index detection results of the acute toxicity experiment in mice of traditional cLNP and targeted TLNP at different doses in Example 31 of the present invention.

[0184] FIG15 shows the EGFP expression of TLNP-2 in NHP in vivo in Example 32 of the present invention.

[0185] FIG16 shows the EGFP targeting and off-target results of TLNP-2 in NHP in Example 32 of the present invention.

[0186] FIG17 shows the dynamic changes of EGFP expression of TLNP-2 in NHP in Example 32 of the present invention.

[0187] FIG18 shows the changes in body weight of NHPs after administration of TLNP-2 in Example 33 of the present invention.

[0188] FIG19 shows the changes in body temperature of NHPs after administration of TLNP-2 in Example 33 of the present invention.

[0189] FIG20 shows the changes in blood routine of NHPs after administration of TLNP-2 in Example 33 of the present invention.

[0190] FIG21 shows the changes in cytokines in NHP serum after administration of TLNP-2 in Example 33 of the present invention.

[0191] Figures 22A, 22B, 22C, 22D, 22E, 22F, 22G, 22H, 22I, and 22J show the blood biochemistry of NHPs after administration of TLNP-2 in Example 33 of the present invention.

[0192] Figures 23A, 23B, 23C and 23D show the in vitro transfection results of CD5-TLNPs containing different targeting molecules in Example 34 of the present invention.

[0193] 24A and 24B show the in vitro transfection results of CD5-TLNPs containing different targeting molecule ratios in Example 35 of the present invention.

[0194] Figures 25A and 25B show the in vivo transfection results of L2-TLNPs with different ratios in Example 36 of the present invention.

[0195] 26A and 26B are the experimental results of Luc imaging and qPCR in Example 37 of the present invention to study the biodistribution of hCD5 in humanized mice. DETAILED DESCRIPTION

[0196] The present invention provides molecules and applications with targeting capabilities. Specifically, the present invention provides lipid nanoparticles with targeting capabilities. These lipid nanoparticles with targeting capabilities comprise ionizable amino lipids, polymer-bound lipids, and nanobodies; optionally, these lipid nanoparticles with targeting capabilities also include steroids and / or phospholipids.

[0197] In order to better illustrate the method of obtaining the lipid nanoparticles with targeting function described in the present invention, the following is described by way of example.

[0198] (I) Preparation of five-component cell-targeting nanoparticles (comprising ionizable amino lipids, polymer-bound lipids, steroids, phospholipids, and nanobodies)

[0199] Method 1: Post-insertion chemical coupling

[0200] Ionizable amino lipids E12LA6B6O3, phospholipids, cholesterol, DMG-PEG 2000 The PEG lipid derivative containing the chemical coupling site was dissolved in ethanol at a specific molar ratio, and the mRNA was dissolved in an acidic buffer solution to ensure that the mRNA concentration in the aqueous phase was 0.33 mg / mL. The molar ratio of the ionizable amino lipid to the mRNA base (N / P) was 6.5.

[0201] The ethanol and aqueous phases were mixed using a microfluidic device, with the flow rate controlled to maintain a 1:3 ethanol to aqueous ratio. The resulting crude LNPs were terminated by the addition of 25 mM Tris buffer. Ultrafiltration was then performed once at 4°C, 1200 rcf, followed by two additional ultrafiltration purifications using 25 mM Tris buffer. The LNPs containing the chemical coupling sites (Pre-TLNPs) were collected. After adding 0.278 times the volume of a 400 mg / mL sucrose solution, the mixture was sterile filtered using a PES filter with a pore size of 0.22 μm in a clean bench.

[0202] A certain amount of the aforementioned pre-TLNPs was mixed with the corresponding nanobodies at a specific molar ratio and incubated overnight at 4°C. The LNP mixture was diluted with 87 mg / mL sucrose solution and purified using a 100 kDa ultrafiltration tube. After sterile filtration using a PES filter with a pore size of 0.22 μm in a clean bench, the final nanobody-modified cell-targeting nanoparticles (TLNPs) were obtained.

[0203] Method 2: Targeted functional molecule post-insertion incubation method

[0204] The PEG lipid derivative containing the chemical coupling site and the nanobody were mixed in a PBS buffer solution at a certain molar ratio and incubated overnight at 4°C. The reaction solution was purified by column to obtain a PEG lipid and nanobody conjugate with targeting function (PEG-Nb).

[0205] Ionizable amino lipids, phospholipids, cholesterol and DMG-PEG 2000 Dissolve the lipids in ethanol and mRNA in an acidic buffer solution at a specific molar ratio to ensure an mRNA concentration of 0.33 mg / mL in the aqueous phase. The molar ratio (N / P) of the ionizable amino lipid to mRNA was 6.5. The LNP preparation process was similar to that described in Method 1.

[0206] A certain amount of the prepared LNPs were mixed with a targeting functional molecule (PEG-Nb) at a specific molar ratio and incubated overnight at 4°C. The subsequent LNP purification process and quality control methods were carried out according to the procedures described in Method 1, and the nanobody-modified cell-targeting nanoparticles (TLNPs) were finally obtained.

[0207] Method 3: One-step insertion of targeted functional molecules

[0208] Ionizable amino lipids, phospholipids, cholesterol and DMG-PEG 2000The targeting functional molecule (PEG lipid and nanobody conjugate) and mRNA were dissolved in ethanol at a certain molar ratio in an acidic buffer solution to ensure that the mRNA concentration in the aqueous phase was 0.33 mg / mL. The molar ratio (N / P) of the ionizable amino lipid to mRNA was 6.5. The subsequent LNP preparation, purification, and quality control procedures were carried out as described in Method 1 to obtain nanobody-modified cell-targeting nanoparticles (TLNPs).

[0209] (II) Preparation of Four-Component Cell-Targeting Nanoparticles (Containing Ionizable Amino Lipids, Polymer-Bound Lipids, Steroids, and Nanobodies)

[0210] Ionizable amino lipids, cholesterol, DMG-PEG 2000 and DSPE-PEG 2000 -Mal lipids were dissolved in ethanol at a molar ratio of 40:58.5:1.45:0.05, and mRNA was dissolved in an acidic buffer solution to ensure an mRNA concentration of 0.33 mg / mL in the aqueous phase. The molar ratio (N / P) of ionizable amino lipid to mRNA was 6.5.

[0211] The specific preparation process of LNP is carried out according to method 1 in (I) to obtain the final four-component cell-targeted nanoparticles (TLNP).

[0212] (III) Preparation of Four-Component Cell-Targeting Nanoparticles (Containing Ionizable Amino Lipids, Polymer-Bound Lipids, Phospholipids, and Nanobodies)

[0213] Ionizable amino lipids, phospholipid DSPC, DMG-PEG 2000 and DSPE-PEG 2000 -Mal lipids were dissolved in ethanol at a molar ratio of 47.5:51.0:1.45:0.05, and mRNA was dissolved in an acidic buffer solution to ensure an mRNA concentration of 0.33 mg / mL in the aqueous phase. The molar ratio (N / P) of ionizable amino lipid to mRNA was 6.5.

[0214] The specific preparation process of LNP is carried out according to method 1 in (I) to obtain the final four-component cell-targeted nanoparticles (TLNP).

[0215] The following examples provide a detailed description of the technical solutions provided by the present invention, but they should not be construed as limiting the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are all commercially available.

[0216] Example 1:

[0217] In this example, a targeting functional molecule post-insertion incubation method was used to prepare five-component cell-targeting nanoparticles (containing ionizable amino lipids, polymer-bound lipids, steroids, phospholipids, and nanobodies).

[0218] DSPE-PEG2000-Mal lipids containing chemical coupling sites were mixed with CD8 nanobody (with or without GS-cysteine ​​at the end) at a molar ratio of 1:1 in a PBS reaction medium and incubated overnight at 4°C. The reaction solution was purified by hydrophobic interaction chromatography to obtain the conjugated product of CD8 nanobody and DSPE-PEG2000-Mal lipid, i.e., the CD8-targeting functional molecule.

[0219] Ionizable amino lipids E12LA6B6O3, phospholipids, cholesterol CHO and DMG-PEG 2000 The mRNA was dissolved in ethanol and in an acidic buffer solution according to the molar ratios shown in Table 1, ensuring that the mRNA concentration in the aqueous phase was 0.33 mg / mL. The molar ratio of ionizable amino lipid to mRNA (N / P) was 6.5.

[0220] The ethanol and aqueous phases were mixed using a microfluidic device, with the flow rate controlled to maintain a volume ratio of 1:3. The total flow rate of the two phases was 12 mL / min, with a flow rate ratio of 1:3 between the ethanol and aqueous phases, to produce crude LNPs. Immediately after LNP preparation, 10 volumes of 25 mM Tris buffer (Tris / Tris-HCl, pH 7.4-7.5) were added to terminate the reaction.

[0221] The crude LNP product, supplemented with the stop solution, was purified by ultrafiltration once at 4°C and 1200 rcf. Subsequently, the ultrafiltrate was added to 25 mM Tris buffer and concentrated twice at 4°C and 1200 rcf. After the LNP product was collected, 0.278 times the volume of 400 mg / mL sucrose solution (prepared by constant volume using 25 mM Tris buffer, pH = 7.4-7.5) was added and sterile filtered using a PES filter with a pore size of 0.22 μm in a laminar flow hood. A 30 μL sample was aspirated for particle size, dispersion coefficient, and encapsulation efficiency testing, and the remaining sample was stored in a refrigerator at 4°C.

[0222] A certain amount of the aforementioned LNPs was mixed with the CD8-targeting functional molecule according to the formula in Table 1, and then incubated overnight at 4°C. The LNP mixture was diluted with 87 mg / mL sucrose solution (prepared in 25 mM Tris buffer, pH 7.4-7.5) and purified using a 100 kDa ultrafiltration tube. After sterile filtration using a 0.22 μm pore size PES filter in a laminar flow hood, CD8-antibody-modified cell-targeting nanoparticles (CD8-TLNPs) were obtained.

[0223] Table 1: Proportional formula of lipids and targeting molecules for preparing CD8-TLNPs by post-insertion method

[0224] Example 2:

[0225] This example uses a one-step insertion method of targeting functional molecules to prepare five-component cell-targeting nanoparticles (containing ionizable amino lipids, polymer-bound lipids, steroids, phospholipids, and targeting functional molecules)

[0226] The CD8 targeting functional molecule was prepared according to the method described in Example 1.

[0227] Ionizable amino lipid E12LA6B6O3, phospholipids, cholesterol and DMG-PEG 2000 The CD8 targeting functional molecules and mRNA were dissolved in ethanol according to the molar ratios listed in Table 2. The concentration of mRNA in the aqueous phase was 0.33 mg / mL, and the molar ratio (N / P) of the ionizable amino lipid to mRNA was 6.5.

[0228] The ethanol and aqueous phases were mixed using a microfluidic device, with the flow rate controlled to maintain a volume ratio of 1:3. The total flow rate of the two phases was 12 mL / min, with a flow rate ratio of 1:3 between the ethanol and aqueous phases, to produce crude LNPs. Immediately after LNP preparation, 10 volumes of 25 mM Tris buffer (Tris / Tris-HCl, pH 7.4-7.5) were added to terminate the reaction.

[0229] The crude LNP product, after adding the stop solution, was purified by ultrafiltration once at 4°C and 1200 rcf. Subsequently, the ultrafiltrate was added with 25 mM Tris buffer and concentrated twice more at 4°C and 1200 rcf. After the LNP product was collected, 0.278 times the volume of 400 mg / mL sucrose solution (prepared by constant volume using 25 mM Tris buffer, pH = 7.4-7.5) was added. The product was sterile filtered using a PES filter with a pore size of 0.22 μm in a clean bench to obtain CD8-antibody-modified cell-targeted nanoparticles (CD8-TLNP).

[0230] Table 2: Proportional formula of lipids and targeting molecules for one-step preparation of CD8-TLNPs

[0231] Example 3:

[0232] In this example, a post-insertion chemical coupling method was used to prepare five-component cell-targeting nanoparticles (containing ionizable amino lipids, polymer-bound lipids, steroids, phospholipids, and nanobodies). The specific steps are as follows:

[0233] Ionizable amino lipids E12LA6B6O3, phospholipids, cholesterol, DMG-PEG 2000 DSPE-PEG2000-Mal and DSPE-PEG2000-Mal were dissolved in ethanol according to the molar ratios listed in Table 3. mRNA was dissolved in an acidic buffer solution to ensure that the mRNA concentration in the aqueous phase was 0.33 mg / mL. The molar ratio of ionizable amino lipid to mRNA (N / P) was 6.5.

[0234] The ethanol and aqueous phases were mixed using a microfluidic device, with the flow rate controlled to maintain a volume ratio of 1:3. The total flow rate of the two phases was 12 mL / min, with a flow rate ratio of 1:3 between the ethanol and aqueous phases, to produce crude LNPs. Immediately after LNP preparation, 10 volumes of 25 mM Tris buffer (Tris / Tris-HCl, pH 7.4-7.5) were added to terminate the reaction.

[0235] The crude LNPs, supplemented with the stop solution, were purified by ultrafiltration once at 4°C and 1200 rcf. Subsequently, the ultrafiltrate was added to 25 mM Tris buffer and concentrated twice more at 4°C and 1200 rcf. The LNPs containing the chemical coupling sites (Pre-TLNPs) were collected and then, after addition of 0.278 times the volume of a 400 mg / mL sucrose solution (prepared using 25 mM Tris buffer, pH 7.4-7.5), the mixture was sterile filtered using a 0.22 μm PES filter in a clean bench. A 30 μL sample was removed for particle size, dispersion coefficient, and encapsulation efficiency testing, and the remaining sample was stored in a refrigerator at 4°C.

[0236] A certain amount of the above-described pre-TLNPs was mixed with DSPE-PEG2000-Mal and the corresponding CD8 nanobody (with or without a terminal GS-cysteine) at a 1:1 molar ratio and incubated overnight at 4°C. The LNP mixture was diluted with 87 mg / mL sucrose solution (prepared in 25 mM Tris buffer, pH 7.4-7.5) and purified using a 100 kDa ultrafiltration tube. The mixture was sterile-filtered using a PES filter with a 0.22 μm pore size in a laminar flow hood to obtain the final nanobody-modified cell-targeting nanoparticles (TLNPs). A 30 μL sample was aspirated for particle size, dispersion coefficient, and encapsulation efficiency determination, and the remaining sample was stored in a refrigerator at 4°C. After encapsulation efficiency determination, the sample was diluted to the desired concentration with 87 mg / mL sucrose solution (prepared in 25 mM Tris buffer, pH 7.4-7.5) and stored until further use.

[0237] Table 3: Lipid ratio formula for preparation of CD8-TLNPs by post-insertion method

[0238] Example 4:

[0239] (I) Preparation of nanoparticles coupled with anti-CD19 nanoantibodies

[0240] Refer to the preparation method in Example 3. Ionizable amino lipid: DSPC: CHO: DMG-PEG2000: DSPE-PEG2000-Mal were dissolved in ethanol at a molar ratio of 47.5:10:41:1.49:0.01. mRNA was dissolved in 75 mM citric acid buffer at pH 4 to ensure an mRNA concentration of 0.33 mg / mL in the aqueous phase. The molar ratio of ionizable amino lipid to mRNA (N / P) was 6.5.

[0241] The ethanol and aqueous phases were mixed using a microfluidic device, with the flow rate controlled to maintain a volume ratio of 1:3. The total flow rate of the two phases was 12 mL / min, with a flow rate ratio of 1:3 between the ethanol and aqueous phases, to produce crude LNPs. Immediately after LNP preparation, 10 volumes of 25 mM Tris buffer (Tris / Tris-HCl, pH 7.4-7.5) were added to terminate the reaction.

[0242] The crude LNPs were purified by ultrafiltration three times at 4°C and 1200 rcf to obtain the chemically conjugated LNPs (Pre-TLNPs). These LNPs were then mixed with a cysteine-terminated anti-CD19 nanobody at a 1:1 molar ratio and incubated overnight at 4°C. The final anti-CD19 nanobody-conjugated nanoparticles (CD19-TLNPs) were then obtained after ultrafiltration and washing with 87 mg / mL sucrose solution and sterile filtration.

[0243] (II) Evaluation of the in vitro delivery efficiency of nanoparticles targeting human CD19 cells

[0244] Anti-CD3 coating plate: Add 1 μg / mL anti-human CD3 antibody to a 24-well plate one day in advance, add 200 μL to each well, and then store at 4°C for overnight coating.

[0245] Preparation before cell plating: aspirate and discard the liquid in the 24-well plate, and then wash twice with 1640 complete medium (10% New Zealand serum, 1% PS double antibody).

[0246] Cell plating: Dilute human PBMCs to 1 million / mL in 1640 complete medium (10% New Zealand serum, 1% PS dual antibody) supplemented with anti-CD28 antibody (1 μg / mL) and IL-2 (working concentration 400 U / mL). Plate 1 million cells per well in a 24-well plate. After plating, stabilize the cells at 37°C, 5% CO2 for 4 hours before adding LNPs.

[0247] CD19-TLNPs encapsulating eGFP-mRNA were added to the plate to a working concentration of 0.1 μg / mL. A negative control group containing PBS was also added. After 24 hours, the expression of eGFP-mRNA in the CD19+ cell subset of PBMCs was measured by flow cytometry.

[0248] Table 4: CD19-TLNP in vitro transfection results

[0249] The experimental results showed that CD19-TLNP exhibited obvious targeting effect on CD19+ cells and had good transfection efficiency.

[0250] Example 5:

[0251] Preparation of nanoparticles coupled with anti-CD133 nanoantibody

[0252] Refer to the preparation method in Example 3. Ionizable amino lipid: DSPE: CHO: DMG-PEG4000: DSPE-PEG4000-Mal were dissolved in ethanol at a molar ratio of 50.5:5:43:1.47:0.03. mRNA was dissolved in 75 mM citric acid buffer (pH 4) to ensure an mRNA concentration of 0.33 mg / mL in the aqueous phase. The molar ratio of ionizable amino lipid to mRNA (N / P) was 6.5.

[0253] The ethanol and aqueous phases were mixed using a microfluidic device, with the flow rate controlled to maintain a volume ratio of 1:3. The total flow rate of the two phases was 12 mL / min, with a flow rate ratio of 1:3 between the ethanol and aqueous phases, to produce crude LNPs. Immediately after LNP preparation, 10 volumes of 25 mM Tris buffer (Tris / Tris-HCl, pH 7.4-7.5) were added to terminate the reaction.

[0254] The crude LNPs were purified by ultrafiltration three times at 4°C and 1200 rcf to obtain LNPs containing chemical coupling sites (Pre-TLNPs). These LNPs were then mixed with a cysteine-terminated anti-CD133 nanobody at a molar ratio of 1:1.2 and incubated overnight at 4°C. The final anti-CD133 nanobody-coupled nanoparticles (CD133-TLNPs) were then obtained after ultrafiltration and sterile filtration with 87 mg / mL sucrose solution.

[0255] Example 6:

[0256] (1) Preparation of nanoparticles coupled with anti-PSMA nanoantibodies

[0257] Refer to the preparation method in Example 3. Ionizable amino lipid: DOPC: CHO: DMG-PEG2000: DSPE-PEG3000-Mal were dissolved in ethanol at a molar ratio of 43.5:12:43:1.2:0.3. mRNA was dissolved in 75 mM citric acid buffer (pH 4) to ensure an mRNA concentration of 0.33 mg / mL in the aqueous phase. The molar ratio of ionizable amino lipid to mRNA (N / P) was 6.5.

[0258] The ethanol and aqueous phases were mixed using a microfluidic device, with the flow rate controlled to maintain a volume ratio of 1:3. The total flow rate of the two phases was 12 mL / min, with a flow rate ratio of 1:3 between the ethanol and aqueous phases, to produce crude LNPs. Immediately after LNP preparation, 10 volumes of 25 mM Tris buffer (Tris / Tris-HCl, pH 7.4-7.5) were added to terminate the reaction.

[0259] The crude LNPs were purified by ultrafiltration three times at 4°C and 1200 rcf to obtain the chemically conjugated LNPs (Pre-TLNPs). These LNPs were then mixed with a cysteine-terminated anti-PSMA nanobody at a molar ratio of 1.2:1 and incubated overnight at 4°C. The final anti-PSMA nanobody-conjugated nanoparticles (PSMA-TLNPs) were then obtained after ultrafiltration and washing with 87 mg / mL sucrose solution and sterile filtration.

[0260] (II) Evaluation of the in vitro delivery efficiency of nanoparticles targeting human PSMA cells

[0261] After thawing and culturing LNCaP cells, PSMA-TLNPs encapsulating eGFP mRNA were added to achieve a working concentration of 0.1 μg / mL. A negative control group containing PBS was also established. After 24 hours, the expression of PSMA+ eGFP mRNA in LNCaP cells was determined by flow cytometry.

[0262] Table 5: PSMA-TLNP in vitro transfection results

[0263] The experimental results showed that PSMA-TLNP exhibited obvious targeting effect on PSMA+ cells and had good transfection efficiency.

[0264] Example 7:

[0265] (I) Preparation of nanoparticles coupled with anti-TMEM119 nanoantibody

[0266] Refer to the preparation method in Example 3. Ionizable amino lipid: DOPE: cholesterol ester: DMG-PEG2000: DSPE-PEG2000-Mal were dissolved in ethanol at a molar ratio of 55.5:0:43:1.495:0.005. mRNA was dissolved in 75 mM citric acid buffer (pH 4) to ensure an mRNA concentration of 0.33 mg / mL in the aqueous phase. The molar ratio of ionizable amino lipid to mRNA (N / P) was 6.5.

[0267] The ethanol and aqueous phases were mixed using a microfluidic device, with the flow rate controlled to maintain a volume ratio of 1:3. The total flow rate of the two phases was 12 mL / min, with a flow rate ratio of 1:3 between the ethanol and aqueous phases, to produce crude LNPs. Immediately after LNP preparation, 10 volumes of 25 mM Tris buffer (Tris / Tris-HCl, pH 7.4-7.5) were added to terminate the reaction.

[0268] The crude LNPs were purified by ultrafiltration three times at 4°C and 1200 rcf to obtain the chemically conjugated LNPs (Pre-TLNPs). These LNPs were then mixed with a cysteine-terminated anti-TMEM119 nanobody at a 1:1 molar ratio and incubated overnight at 4°C. The final anti-TMEM119 nanobody-conjugated nanoparticles (TMEM119-TLNPs) were then washed by ultrafiltration with 87 mg / mL sucrose solution and sterile filtered.

[0269] (II) Evaluation of the in vitro delivery efficiency of nanoparticles targeting human TMEM119 cells

[0270] SH-SY5Y cells were revived and cultured, and TMEM119-TLNPs encapsulating eGFP mRNA were added to achieve a working concentration of 0.1 μg / mL in the well plate. A negative control group containing PBS was also established. After 24 hours, the expression of TMEM119+ eGFP mRNA in SH-SY5Y cells was determined by flow cytometry.

[0271] Table 6: TMEM119-TLNP in vitro transfection results

[0272] The experimental results showed that TMEM119-TLNP exhibited obvious targeting effect on TMEM119+ cells and had good transfection efficiency.

[0273] Example 8:

[0274] Preparation of nanoparticles coupled with anti-B7-H1 nanobody

[0275] Refer to the preparation method in Example 3. Ionizable amino lipid: DSPC: CHO: DMG-PEG2000: DSPE-PEG2000-Mal were dissolved in ethanol at a molar ratio of 62.5:5:31:1.49:0.01. mRNA was dissolved in 75 mM citric acid buffer at pH 4 to ensure an mRNA concentration of 0.33 mg / mL in the aqueous phase. The molar ratio of ionizable amino lipid to mRNA (N / P) was 6.5.

[0276] The ethanol and aqueous phases were mixed using a microfluidic device, with the flow rate controlled to maintain a volume ratio of 1:3. The total flow rate of the two phases was 12 mL / min, with a flow rate ratio of 1:3 between the ethanol and aqueous phases, to produce crude LNPs. Immediately after LNP preparation, 10 volumes of 25 mM Tris buffer (Tris / Tris-HCl, pH 7.4-7.5) were added to terminate the reaction.

[0277] The crude LNPs were purified by ultrafiltration three times at 4°C and 1200 rcf to obtain the chemically conjugated LNPs (Pre-TLNPs). These LNPs were then mixed with a cysteine-terminated anti-B7-H1 nanobody at a 1:1 molar ratio and incubated overnight at 4°C. The final anti-B7-H1 nanobody-conjugated nanoparticles (B7-H1-TLNPs) were then obtained after ultrafiltration and washing with 87 mg / mL sucrose solution and sterile filtration.

[0278] Example 9:

[0279] (1) Preparation of nanoparticles coupled with anti-HER2 nanoantibodies

[0280] Refer to the preparation method in Example 3. Ionizable amino lipid: DOPC: CHO: DMG-PEG2000: DSPE-PEG2000-Mal were dissolved in ethanol at a molar ratio of 58.5:10:30:1.49:0.01. mRNA was dissolved in 75 mM citric acid buffer (pH 4) to ensure an mRNA concentration of 0.33 mg / mL in the aqueous phase. The molar ratio of ionizable amino lipid to mRNA (N / P) was 6.5.

[0281] The ethanol and aqueous phases were mixed using a microfluidic device, with the flow rate controlled to maintain a volume ratio of 1:3. The total flow rate of the two phases was 12 mL / min, with a flow rate ratio of 1:3 between the ethanol and aqueous phases, to produce crude LNPs. Immediately after LNP preparation, 10 volumes of 25 mM Tris buffer (Tris / Tris-HCl, pH 7.4-7.5) were added to terminate the reaction.

[0282] The crude LNPs were purified by ultrafiltration three times at 4°C and 1200 rcf to obtain the chemically conjugated LNPs (Pre-TLNPs). These LNPs were then mixed with a cysteine-terminated anti-HER2 nanobody at a 1:1 molar ratio and incubated overnight at 4°C. The final anti-HER2 nanobody-conjugated nanoparticles (HER2-TLNPs) were then obtained after ultrafiltration and washing with 87 mg / mL sucrose solution and sterile filtration.

[0283] (II) Evaluation of the in vitro delivery efficiency of nanoparticles targeting human HER2 cells

[0284] Human breast cancer SK-BR-3 cells were revived and cultured, and cLNPs encapsulating eGFP mRNA and HER2-TLNPs were added to achieve a working LNP concentration of 0.1 μg / mL in the well plate. PBS was also added as a negative control. After 24 hours, flow cytometry was used to measure eGFP mRNA expression in the HER2+ cell subpopulation within PBMCs.

[0285] Table 7: HER2-TLNP in vitro transfection results

[0286] The experimental results showed that HER2-TLNP exhibited obvious targeting effect on HER2+ cells and had good transfection efficiency.

[0287] Example 10:

[0288] Preparation of nanoparticles coupled with anti-EGFR nanoantibodies

[0289] Refer to the preparation method in Example 3. Ionizable amino lipid: DSPC: CHO: DMG-PEG2000: DSPE-PEG2000-Mal were dissolved in ethanol at a molar ratio of 45.5:15:38:1.49:0.01. mRNA was dissolved in 75 mM citric acid buffer (pH 4) to ensure an mRNA concentration of 0.33 mg / mL in the aqueous phase. The molar ratio of ionizable amino lipid to mRNA (N / P) was 6.5.

[0290] The ethanol and aqueous phases were mixed using a microfluidic device, with the flow rate controlled to maintain a volume ratio of 1:3. The total flow rate of the two phases was 12 mL / min, with a flow rate ratio of 1:3 between the ethanol and aqueous phases, to produce crude LNPs. Immediately after LNP preparation, 10 volumes of 25 mM Tris buffer (Tris / Tris-HCl, pH 7.4-7.5) were added to terminate the reaction.

[0291] The crude LNPs were purified by ultrafiltration three times at 4°C and 1200 rcf to obtain the chemically conjugated LNPs (Pre-TLNPs). These LNPs were then mixed with a cysteine-terminated anti-EGFR nanobody at a 1:1 molar ratio and incubated overnight at 4°C. The final anti-EGFR nanobody-conjugated nanoparticles (EGFR-TLNPs) were then obtained after ultrafiltration and washing with 87 mg / mL sucrose solution and sterile filtration.

[0292] Example 11:

[0293] (1) Preparation of nanoparticles targeting human CD7 cells

[0294] Refer to the preparation method in Example 3. Pre-TLNPs were mixed with a cysteine-terminated anti-CD7 nanobody at a molar ratio of 1:1 and incubated overnight at 4°C. The nanoparticles were then washed by ultrafiltration with 87 mg / mL sucrose solution and sterile filtered to obtain the final anti-CD7 nanobody-conjugated nanoparticles (CD7-TLNPs).

[0295] (II) Evaluation of the in vitro delivery efficiency of nanoparticles targeting human CD7 cells

[0296] Anti-CD3 coating plate: Add 1 μg / mL anti-human CD3 antibody to a 24-well plate one day in advance, add 200 μL to each well, and then store at 4°C for overnight coating.

[0297] Preparation before cell plating: aspirate and discard the liquid in the 24-well plate, and then wash twice with 1640 complete medium (10% New Zealand serum, 1% PS double antibody).

[0298] Cell plating: Dilute human PBMCs to 1 million / mL in 1640 complete medium (10% New Zealand serum, 1% PS dual antibody) supplemented with anti-CD28 antibody (1 μg / mL) and IL-2 (working concentration 400 U / mL). Plate 1 million cells per well in a 24-well plate. After plating, stabilize the cells at 37°C, 5% CO2 for 4 hours before adding LNPs.

[0299] CD7-TLNPs encapsulating eGFP-mRNA were added to the plate to a working concentration of 0.1 μg / mL. A negative control group containing PBS was also added. After 24 hours, the expression of eGFP-mRNA in the CD7+ cell subset of PBMCs was determined by flow cytometry.

[0300] Table 8: CD7-TLNP in vitro transfection results

[0301] The experimental results showed that CD7-TLNP exhibited obvious targeting effect on CD7+ cells and had good transfection efficiency.

[0302] Example 12:

[0303] (1) Preparation of nanoparticles targeting human CD4 cells

[0304] Refer to the preparation method in Example 3. Pre-TLNPs were mixed with a cysteine-terminated anti-CD4 nanobody at a molar ratio of 1:1 and incubated overnight at 4°C. The nanoparticles were then washed by ultrafiltration with 87 mg / mL sucrose solution and sterile filtered to obtain the final anti-CD4 nanobody-conjugated nanoparticles (CD4-TLNPs).

[0305] (II) Evaluation of the in vitro delivery efficiency of nanoparticles targeting human CD4 cells

[0306] Anti-CD3 coating plate: Add 1 μg / mL anti-human CD3 antibody to a 24-well plate one day in advance, add 200 μL to each well, and then store at 4°C for overnight coating.

[0307] Preparation before cell plating: aspirate and discard the liquid in the 24-well plate, and then wash twice with 1640 complete medium (10% New Zealand serum, 1% PS double antibody).

[0308] Cell plating: Dilute human PBMCs to 1 million / mL in 1640 complete medium (10% New Zealand serum, 1% PS dual antibody) supplemented with anti-CD28 antibody (1 μg / mL) and IL-2 (working concentration 400 U / mL). Plate 1 million cells per well in a 24-well plate. After plating, stabilize the cells at 37°C, 5% CO2 for 4 hours before adding LNPs.

[0309] CD4-TLNPs encapsulating eGFP-mRNA were added to the plate to a working concentration of 0.1 μg / mL. A negative control group containing PBS was also added. After 24 hours, the expression of eGFP-mRNA in the CD4+ cell subset of PBMCs was determined by flow cytometry.

[0310] Table 9: CD4-TLNP in vitro transfection results

[0311] The experimental results showed that CD4-TLNP exhibited obvious targeting effect on CD4+ cells and had good transfection efficiency.

[0312] Example 13:

[0313] (1) Preparation of nanoparticles targeting human CD38 cells

[0314] Refer to the preparation method in Example 3. Pre-TLNPs were mixed with anti-CD38 nanobody at a molar ratio of 1:1 and incubated overnight at 4°C. The mixture was then washed by ultrafiltration with 87 mg / mL sucrose solution and sterile filtered to obtain the final anti-CD38 nanobody-conjugated nanoparticles (CD38-TLNPs).

[0315] (II) Evaluation of the in vitro delivery efficiency of nanoparticles targeting human CD38 cells

[0316] Anti-CD3 coating plate: Add 1 μg / mL anti-human CD3 antibody to a 24-well plate one day in advance, add 200 μL to each well, and then store at 4°C for overnight coating.

[0317] Preparation before cell plating: aspirate and discard the liquid in the 24-well plate, and then wash twice with 1640 complete medium (10% New Zealand serum, 1% PS double antibody).

[0318] Cell plating: Dilute human PBMCs to 1 million / mL in 1640 complete medium (10% New Zealand serum, 1% PS dual antibody) supplemented with anti-CD28 antibody (1 μg / mL) and IL-2 (working concentration 400 U / mL). Plate 1 million cells per well in a 24-well plate. After plating, stabilize the cells at 37°C, 5% CO2 for 4 hours before adding LNPs.

[0319] CD38-TLNPs encapsulating eGFP-mRNA were added to the plate to a working concentration of 0.1 μg / mL. A negative control group containing PBS was also added. After 24 hours, the expression of eGFP-mRNA in the CD38+ cell subset of PBMCs was determined by flow cytometry.

[0320] Table 10: CD38-TLNP in vitro transfection results

[0321] The experimental results showed that CD38-TLNP exhibited obvious targeting effect on CD38+ cells and had good transfection efficiency.

[0322] Example 14:

[0323] (1) Preparation of nanoparticles targeting human CD68 cells

[0324] Refer to the preparation method in Example 3. Pre-TLNPs were mixed with anti-CD68 nanobody at a molar ratio of 1:1 and incubated overnight at 4°C. The mixture was then washed by ultrafiltration with 87 mg / mL sucrose solution and sterile filtered to obtain the final anti-CD68 nanobody-conjugated nanoparticles (CD68-TLNPs).

[0325] (II) Evaluation of the in vitro delivery efficiency of nanoparticles targeting human CD68 cells

[0326] Anti-CD3 coating plate: Add 1 μg / mL anti-human CD3 antibody to a 24-well plate one day in advance, add 200 μL to each well, and then store at 4°C for overnight coating.

[0327] Preparation before cell plating: aspirate and discard the liquid in the 24-well plate, and then wash twice with 1640 complete medium (10% New Zealand serum, 1% PS double antibody).

[0328] Cell plating: Dilute human PBMCs to 1 million / mL in 1640 complete medium (10% New Zealand serum, 1% PS dual antibody) supplemented with anti-CD28 antibody (1 μg / mL) and IL-2 (working concentration 400 U / mL). Plate 1 million cells per well in a 24-well plate. After plating, stabilize the cells at 37°C, 5% CO2 for 4 hours before adding LNPs.

[0329] CD68-TLNPs encapsulating eGFP-mRNA were added to the plate to a working concentration of 0.1 μg / mL. A negative control group containing PBS was also added. After 24 hours, the expression of eGFP-mRNA in the CD68+ cell subset of PBMCs was determined by flow cytometry.

[0330] Table 11: CD68-TLNP in vitro transfection results

[0331] The experimental results showed that CD68-TLNP exhibited obvious targeting effect on CD68+ cells and had good transfection efficiency.

[0332] Example 15:

[0333] (1) Preparation of nanoparticles targeting human CD117 cells

[0334] Refer to the preparation method in Example 3. Pre-TLNPs were mixed with anti-CD117 nanoantibodies at a molar ratio of 1:1 and incubated overnight at 4°C. The mixture was then washed with 87 mg / mL sucrose solution by ultrafiltration and sterile filtration to obtain the final anti-CD117 nanoantibody-coupled nanoparticles (CD117-TLNPs).

[0335] (II) Evaluation of the in vitro delivery efficiency of nanoparticles targeting human CD117 cells

[0336] HLF cells were revived and cultured, and CD117-TLNPs encapsulating eGFP mRNA were added to achieve a working concentration of 0.1 μg / mL in the plate. A negative control group containing PBS was also established. After 24 hours, the expression of CD117+ eGFP mRNA in HLF cells was determined by flow cytometry.

[0337] Table 12: CD117-TLNP in vitro transfection results

[0338] The experimental results showed that CD117-TLNP exhibited obvious targeting effect on CD117+ cells and had good transfection efficiency.

[0339] Example 16:

[0340] (1) Preparation of nanoparticles targeting human B7H3 cells

[0341] Refer to the preparation method in Example 3. Pre-TLNPs were mixed with anti-B7H3 nanobody at a molar ratio of 1:1 and incubated overnight at 4°C. The mixture was then washed by ultrafiltration with 87 mg / mL sucrose solution and sterile filtered to obtain the final anti-B7H3 nanobody-conjugated nanoparticles (B7H3-TLNPs).

[0342] (II) Evaluation of the in vitro delivery efficiency of nanoparticles targeting human B7H3 cells

[0343] A2780 cells were revived and cultured, and B7H3-TLNPs encapsulating eGFP mRNA were added to achieve a working concentration of 0.1 μg / mL in the well plate. A negative control group containing PBS was also established. After 24 hours, the expression of B7H3+ eGFP mRNA in A2780 cells was determined by flow cytometry.

[0344] Table 13: B7H3-TLNP in vitro transfection results

[0345] The experimental results showed that B7H3-TLNP exhibited obvious targeting effect on B7H3+ cells and had good transfection efficiency.

[0346] Example 17:

[0347] (1) Preparation of nanoparticles targeting human GPC3 cells

[0348] Refer to the preparation method in Example 3. Pre-TLNPs were mixed with a cysteine-terminated anti-GPC3 nanobody at a molar ratio of 1:1 and incubated overnight at 4°C. The resulting nanoparticles (GPC3-TLNPs) were then washed by ultrafiltration with 87 mg / mL sucrose solution and sterile filtered.

[0349] (II) Evaluation of the in vitro delivery efficiency of nanoparticles targeting human GPC3 cells

[0350] HepG2 cells were revived and cultured, and GPC3-TLNPs encapsulating eGFP mRNA were added to achieve a working concentration of 0.1 μg / mL in the well plate. A negative control group containing PBS was also established. After 24 hours, the expression of GPC3+ eGFP mRNA in the HepG2 cells was measured by flow cytometry.

[0351] Table 14: GPC3-TLNP in vitro transfection results

[0352] The experimental results showed that GPC3-TLNP exhibited obvious targeting effect on GPC3+ cells and had good transfection efficiency.

[0353] Example 18:

[0354] DSPE-PEG 2000 -Lys-Mal2 lipid coupled to multiple nanoantibodies to form a targeting functional molecule targeting human CD8 + Preparation of T cell nanoparticles

[0355] Refer to the preparation method in Example 3. Ionizable amino lipid: DSPC: CHO: DMG-PEG2000: DSPE-PEG2000-Lys-Mal2 were dissolved in ethanol at a molar ratio of 47.5:10:41:1.49:0.01. mRNA was dissolved in an acidic buffer solution with a concentration of 75 mM citric acid, pH 4, to ensure that the mRNA concentration in the aqueous phase was 0.33 mg / mL. The molar ratio of ionizable amino lipid to mRNA (N / P) was 6.5.

[0356] The ethanol and aqueous phases were mixed using a microfluidic device, with the flow rate controlled to maintain a volume ratio of 1:3. The total flow rate of the two phases was 12 mL / min, with a flow rate ratio of 1:3 between the ethanol and aqueous phases, to produce crude LNPs. Immediately after LNP preparation, 10 volumes of 25 mM Tris buffer (Tris / Tris-HCl, pH 7.4-7.5) were added to terminate the reaction.

[0357] The crude LNP was purified by ultrafiltration three times at 4°C and 1200 rcf, and then added with 0.278 times the volume of a 400 mg / mL sucrose solution to obtain LNPs containing chemical coupling sites (Pre-TLNPs). This was then mixed with an anti-CD8 nanobody at a 1:1 molar ratio and incubated overnight at 4°C. The final anti-CD8 nanobody-conjugated nanoparticles (CD8-TLNPs) were then obtained after ultrafiltration washing with an 87 mg / mL sucrose solution and sterile filtration.

[0358] Example 19:

[0359] By Mal-PEG 2000 -Mal lipid coupled to multiple nanoantibodies to form a targeting functional molecule targeting human CD8 + Preparation of T cell nanoparticles

[0360] Refer to the preparation method in Example 3. Ionizable amino lipid: DSPC: CHO: DMG-PEG2000: Mal-PEG2000-Mal were dissolved in ethanol at a molar ratio of 47.5:10:41:1.49:0.01. mRNA was dissolved in 75 mM citric acid buffer at pH 4 to ensure an mRNA concentration of 0.33 mg / mL in the aqueous phase. The molar ratio of ionizable amino lipid to mRNA (N / P) was 6.5.

[0361] The ethanol and aqueous phases were mixed using a microfluidic device, with the flow rate controlled to maintain a volume ratio of 1:3. The total flow rate of the two phases was 12 mL / min, with a flow rate ratio of 1:3 between the ethanol and aqueous phases, to produce crude LNPs. Immediately after LNP preparation, 10 volumes of 25 mM Tris buffer (Tris / Tris-HCl, pH 7.4-7.5) were added to terminate the reaction.

[0362] The crude LNP was purified by ultrafiltration three times at 4°C and 1200 rcf, and then added with 0.278 times the volume of a 400 mg / mL sucrose solution to obtain LNPs containing chemical coupling sites (Pre-TLNPs). This was then mixed with an anti-CD8 nanobody at a 1:1 molar ratio and incubated overnight at 4°C. The final anti-CD8 nanobody-conjugated nanoparticles (CD8-TLNPs) were then obtained after ultrafiltration washing with an 87 mg / mL sucrose solution and sterile filtration.

[0363] Example 20:

[0364] This example is a structural screening of targeting functional molecules in nanoparticles targeting human CD8+ T cells

[0365] (1) Preparation of nanoparticles targeting human CD8 T cells

[0366] Table 15: Preparation of LNPs with different targeting molecular structures (Lkx)

[0367] 1) Preparation of LNPs containing coupling sites

[0368] Ionizable amino lipid: DSPC:CHO:DMG-PEG 2000 : Lkx (wherein Lk1 and Lk3 contain PEG segments, and Lk2, Lk4, and Lk5 do not contain PEG segments) five-component lipid molecules are dissolved in ethanol at a molar ratio of 47.5:10:41:1.49:0.01; Dissolved in 75 mM citric acid pH 4 buffer solution to ensure that the concentration of mRNA in the aqueous phase is 0.33 mg / mL. The molar ratio of ionizable amino lipid to mRNA (N / P) is 6.5.

[0369] The ethanol and aqueous phases were mixed using a microfluidic device, with the flow rate controlled to maintain a volume ratio of 1:3. The total flow rate of the two phases was 12 mL / min, with a flow rate ratio of 1:3 between the ethanol and aqueous phases, to produce crude LNPs. Immediately after LNP preparation, 10 volumes of 25 mM Tris buffer (Tris / Tris-HCl, pH 7.4-7.5) were added to terminate the reaction.

[0370] The LNPs were purified by ultrafiltration at 4°C and 1200 rcf after adding the stop solution. Then, 25 mM Tris buffer was added to the ultrafiltrate and ultrafiltration was continued twice at 4°C and 1200 rcf. Add 0.278 times the volume of 400 mg / mL sucrose solution (prepared to volume in 25 mM Tris buffer, pH = 7.4-7.5) and sterile filter using a 0.22 μm PES filter in a laminar flow hood. Aspirate 30 μL of sample for particle size, dispersion dilution, and encapsulation efficiency testing, and store the remaining sample in a refrigerator at 4°C. After encapsulation efficiency testing, dilute the sample to the desired concentration with 87 mg / mL sucrose solution (prepared in 25 mM Tris buffer, pH = 7.4-7.5).

[0371] 2) Preparation of Nanobody-Modified TLNPs

[0372] In addition to cLNP-1, a certain amount of the other groups containing coupling sites were taken TLNP, according to the LNP formula, calculate each group The molar amount of DSPE-PEG2000-Mal in the lipid nanoparticles corresponding to the TLNP was then calculated. Pre-TLNPs were then mixed with the nanobody at a 1:1 molar ratio of Lkx to anti-human-CD8-nanobody (2.3 mg / mL, stored in a mixed buffer consisting of 20 mM Tris, 150 mM NaCl, and 5% glycerol at pH 8.0) and incubated overnight at 4°C.

[0373] After overnight incubation, the TLNPs were diluted with 87 mg / mL sucrose solution (prepared in 25 mM Tris buffer, pH 7.4-7.5) and purified using a 100 kDa ultrafiltration tube. After sterile filtration using a 0.22 μm PES filter in a laminar flow hood, a 30 μL sample was aspirated for particle size, dispersion dilution, and encapsulation efficiency testing. The remaining sample was stored in a refrigerator at 4°C. After encapsulation efficiency testing, the sample was diluted to the desired concentration using 87 mg / mL sucrose solution (prepared in 25 mM Tris buffer, pH 7.4-7.5).

[0374] Table 16: Quality control information of TLNPs modified with nanoantibodies containing different targeting molecular structures

[0375] Experimental results show that CD8+ T cell-targeted lipid nanoparticles prepared using targeting molecules of different structures all exhibited good characterization data, falling within the generally accepted numerical range in the LNP field. This demonstrates that the preparation process for the targeted LNPs described in this patent is stable and universally applicable.

[0376] Detailed statistical diagrams of LNP characterization data are shown in Figures 1A and 1B .

[0377] (II) Evaluation of the in vitro delivery efficiency of nanoparticles targeting human CD8+ T cells

[0378] Anti-CD3 coating plate: Add 1 μg / mL anti-human CD3 antibody to a 24-well plate one day in advance, add 200 μL to each well, and then store at 4°C for overnight coating.

[0379] Preparation before cell plating: aspirate and discard the liquid in the 24-well plate, and then wash twice with 1640 complete medium (10% New Zealand serum, 1% PS double antibody).

[0380] Cell plating: Dilute human PBMCs to 1 million / mL in 1640 complete medium (10% New Zealand serum, 1% PS dual antibody) supplemented with anti-CD28 antibody (1 μg / mL) and IL-2 (working concentration 400 U / mL). Plate 1 million cells per well in a 24-well plate. After plating, stabilize the cells at 37°C, 5% CO2 for 4 hours before adding LNPs.

[0381] Drug addition: Each of the six LNPs prepared in Table 16 was added to each well to achieve a working concentration of 0.1 μg / mL. PBS was also added as a negative control group.

[0382] Culture: After adding LNP, PBMC cells were transfected at 37°C, 5% CO2 for 24 hours.

[0383] Detection of eGFP-mRNA expression: Cells in each well plate were collected and stained with commercially available flow cytometry antibodies. The expression rate and mean fluorescence intensity (MFI) of eGFP-mRNA in CD4+ T and CD8+ T cell subsets in PBMC were detected by flow cytometry.

[0384] Table 17: In vitro transfection results of TLNPs containing different targeting molecular structures

[0385] The experimental results showed that TLNPs prepared by Lk1, Lk2 and Lk4 exhibited obvious CD8+T cell targeting effects at extremely low dosages and had good transfection efficiency.

[0386] Statistical diagrams of in vitro transfection of LNPs with different targeting molecular structures are shown in Figures 2A, 2B, 2C, and 2D.

[0387] (III) Evaluation of the in vivo delivery efficiency of nanoparticles targeting human CD8+ T cells

[0388] Twelve 6-8 week old NCG female mice that passed quarantine and met the SPF (specific pathogen free) level were selected and divided into 4 groups. Each NCG mouse was injected with 20M hPBMC cells in a volume of 200 μL through the tail vein. About 30 minutes later, the mouse venous wound healed and the TLNPs (Lk1, Lk2 and Lk4) prepared in Table 16 were injected again through the tail vein. Each NCG mouse was given a dose of 20 μg-mRNA-LNP with an injection volume of 200 μL. After 24 hours of expression, peripheral blood was collected and spleen erythrocytes were collected for flow cytometry detection of the expression rate of eGFP-mRNA in each cell subpopulation.

[0389] Table 18: Grouping of TLNPs with different targeting molecular structures in vivo

[0390] The experimental results showed that TLNPs prepared with Lk1 showed the best CD8+ T cell targeting effect and the lowest off-target rate in NCG mice. Subsequent experiments were conducted based on Lk1-TLNPs.

[0391] The statistical diagram of in vivo transfection of TLNPs with different targeting molecular structures is shown in Figure 3.

[0392] Example 21:

[0393] This example is a screening experiment for the proportion of targeting functional molecules in nanoparticles targeting human CD8+ T cells.

[0394] (1) Preparation of nanoparticles targeting human CD8 T cells

[0395] Table 19: Preparation of Pre-LNPs with different targeting molecule ratios

[0396] 1) Preparation of LNPs containing coupling sites

[0397] Ionizable amino lipid: DSPC:CHO:DMG-PEG 2000 DSPE-PEG2000-Mal was dissolved in ethanol according to the molar ratio described in Table 19; Dissolved in 75 mM citric acid pH 4 buffer solution to ensure that the concentration of mRNA in the aqueous phase is 0.33 mg / mL. The molar ratio of ionizable amino lipid to mRNA (N / P) is 6.5.

[0398] The ethanol and aqueous phases were mixed using a microfluidic device, with the flow rate controlled to maintain a volume ratio of 1:3. The total flow rate of the two phases was 12 mL / min, with a flow rate ratio of 1:3 between the ethanol and aqueous phases, to produce crude LNPs. Immediately after LNP preparation, 10 volumes of 25 mM Tris buffer (Tris / Tris-HCl, pH 7.4-7.5) were added to terminate the reaction.

[0399] The LNPs were purified by ultrafiltration at 4°C and 1200 rcf after adding the stop solution. Then, 25 mM Tris buffer was added to the ultrafiltrate and ultrafiltration was continued twice at 4°C and 1200 rcf. Add 0.278 times the volume of 400 mg / mL sucrose solution (prepared to volume in 25 mM Tris buffer, pH = 7.4-7.5) and sterile filter using a 0.22 μm PES filter in a laminar flow hood. Aspirate 30 μL of sample for particle size, dispersion dilution, and encapsulation efficiency testing, and store the remaining sample in a refrigerator at 4°C. After encapsulation efficiency testing, dilute the sample to the desired concentration with 87 mg / mL sucrose solution (prepared in 25 mM Tris buffer, pH = 7.4-7.5).

[0400] The experimental results show that: through the quality control data analysis of the seven groups of LNPs prepared, compared with the original four-component LNPs, when 0.0001mol%-0.50mol% DSPE-PEG2000-Mal lipids are introduced into the LNP formula, the particle size of the corresponding LNPs will be reduced.

[0401] Table 20: Quality control information of Pre TLNP containing coupling sites

[0402] The specific characterization data statistics of Pre-TLNP are shown in Figures 4A and 4B.

[0403] 2) Preparation of Nanobody-Modified TLNPs

[0404] In addition to cLNP-1, a certain amount of the other groups containing coupling sites were taken TLNP, according to the LNP formula, calculate each group The molar amount of DSPE-PEG2000-Mal in the lipid nanoparticles corresponding to TLNP. 2000Pre-TLNPs were mixed with the nanobody at a molar ratio of 1:1, with the anti-human-CD8-nanobody (concentration 2.3 mg / mL, stored in a mixed buffer consisting of 20 mM Tris, 150 mM NaCl and 5% glycerol at pH 8.0) linked to a cysteine ​​residue at -Mal lipid and terminally linked to a cysteine ​​residue (concentration 2.3 mg / mL, stored in a mixed buffer consisting of 20 mM Tris, 150 mM NaCl and 5% glycerol at pH 8.0), and incubated at 4°C overnight.

[0405] After overnight incubation, the TLNPs were diluted with 87 mg / mL sucrose solution (prepared in 25 mM Tris buffer, pH 7.4-7.5) and purified using a 100 kDa ultrafiltration tube. After sterile filtration using a 0.22 μm PES filter in a laminar flow hood, a 30 μL sample was aspirated for particle size, dispersion dilution, and encapsulation efficiency testing. The remaining sample was stored in a refrigerator at 4°C. After encapsulation efficiency testing, the sample was diluted to the desired concentration using 87 mg / mL sucrose solution (prepared in 25 mM Tris buffer, pH 7.4-7.5).

[0406] Table 21: Quality control information of TLNPs containing different ratios of target molecules

[0407] The experimental results show that when the Pre TLNP containing the coupling site is connected to the nanoantibody, the corresponding TLNP- shows good characterization data, which is within the numerical range recognized in the LNP field, indicating that the preparation process of the targeted LNP in this patent is stable and feasible.

[0408] The specific characterization data statistics of TLNP are shown in Figures 5A and 5B.

[0409] (II) Evaluation of the in vitro delivery efficiency of nanoparticles targeting human CD8+ T cells

[0410] Anti-CD3 coating plate: Add 1 μg / mL anti-human CD3 antibody to a 24-well plate one day in advance, add 200 μL to each well, and then store at 4°C for overnight coating.

[0411] Preparation before cell plating: aspirate and discard the liquid in the 24-well plate, and then wash twice with 1640 complete medium (10% New Zealand serum, 1% PS double antibody).

[0412] Cell plating: Dilute human PBMCs to 1 million / mL in 1640 complete medium (10% New Zealand serum, 1% PS dual antibody) supplemented with anti-CD28 antibody (1 μg / mL) and IL-2 (working concentration 400 U / mL). Plate 1 million cells per well in a 24-well plate. After plating, stabilize the cells at 37°C, 5% CO2 for 4 hours before adding LNPs.

[0413] Drug addition: Each LNP group listed in Table 21 was added to each well to achieve a working concentration of 1.0 μg / mL. PBS was also added as a negative control group.

[0414] Culture: After adding LNP, PBMC cells were transfected at 37°C, 5% CO2 for 24 hours.

[0415] Detection of eGFP-mRNA expression: Collect cells from each well plate, stain with commercially available flow cytometry antibodies, and then detect the expression rate of eGFP-mRNA in all cells in PBMCs, CD4+ T cells, and CD8+ T cell subsets by flow cytometry.

[0416] Table 22: In vitro transfection results of TLNPs containing different targeting molecule ratios

[0417] (III) Evaluation of the in vivo delivery efficiency of nanoparticles targeting human CD8+ T cells

[0418] Nine 6-8 week old NCG female mice that passed quarantine and met the SPF (specific pathogen free) level were selected and divided into 3 groups. Each NCG mouse was injected with 20M hPBMC cells in a volume of 200 μL through the tail vein. About 30 minutes later, the mouse venous wound healed and the TLNP-1 and TLNP-2 prepared in Table 20 were injected again through the tail vein. Each NCG mouse was given a dose of 20 μg-mRNA-LNP with an injection volume of 200 μL. After 24 hours of expression, peripheral blood was collected and spleen lysate was collected for flow cytometry detection of the expression rate of eGFP-mRNA in each cell subset.

[0419] Table 23: In vivo experimental groups of TLNPs with different targeting molecule ratios

[0420] The experimental results showed that TLNP-2 containing 0.01% targeting molecule ratio showed better CD8+T cell targeting delivery effect in NCG mice than TLNP-1 in the 0.0001% group.

[0421] The statistical diagram of in vivo transfection of TLNPs with different targeting molecule ratios is shown in Figure 6 .

[0422] Example 22:

[0423] This example targets human CD8 + Study on the ratio of DMG-PEG2000 lipids in nanoparticles for T cells

[0424] (1) Preparation of nanoparticles targeting human CD8 T cells

[0425] Table 24: Preparation of CD8-TLNPs with different PEG lipid ratios

[0426] The lipid nanoparticles used in this example were prepared according to the method described in Example 20.

[0427] (II) Evaluation of the in vitro delivery efficiency of nanoparticles targeting human CD8+ T cells

[0428] Anti-CD3 coating plate: Add 1 μg / mL anti-human CD3 antibody to a 24-well plate one day in advance, add 200 μL to each well, and then store at 4°C for overnight coating.

[0429] Preparation before cell plating: aspirate and discard the liquid in the 24-well plate, and then wash twice with 1640 complete medium (10% New Zealand serum, 1% PS double antibody).

[0430] Cell plating: Dilute human PBMCs to 1 million / mL in 1640 complete medium (10% New Zealand serum, 1% PS dual antibody) supplemented with anti-CD28 antibody (1 μg / mL) and IL-2 (working concentration 400 U / mL). Plate 1 million cells per well in a 24-well plate. After plating, stabilize the cells at 37°C, 5% CO2 for 4 hours before adding LNPs.

[0431] Drug addition: Each LNP group listed in Table 24 was added to each well to achieve a working concentration of 1.0 μg / mL. PBS was also added as a negative control group.

[0432] Culture: After adding LNP, PBMC cells were transfected at 37°C, 5% CO2 for 24 hours.

[0433] Detection of eGFP-mRNA expression: Collect cells from each well plate, stain with commercially available flow cytometry antibodies, and then detect the expression rate of eGFP-mRNA in all cells in PBMCs, CD4+ T cells, and CD8+ T cell subsets by flow cytometry.

[0434] Table 25: In vitro transfection results of TLNPs containing different PEG lipid ratios

[0435] The experimental results showed that TLNPs containing 1.0% and 1.5% DMG-PEG2000 exhibited better CD8+ T cell targeted delivery effect compared with PEG groups with other ratios.

[0436] Example 23:

[0437] This example targets human CD8 by ionizable amino lipids of different structures. + Study on the delivery efficiency of nanoparticles to T cells

[0438] (1) Preparation of nanoparticles targeting human CD8 T cells

[0439] Table 26: Preparation of TLNPs composed of different ionizable amino lipids

[0440] The lipid nanoparticles used in this example were prepared according to the ratio of ionizable amino lipid:DSPC:CHO:DMG-PEG2000:DSPE-PEG2000-Mal=47.5:10:41:1.5:0.01 and the method described in Reference Example 20.

[0441] (II) Evaluation of the in vitro delivery efficiency of nanoparticles targeting human CD8+ T cells

[0442] Anti-CD3 coating plate: Add 1 μg / mL anti-human CD3 antibody to a 24-well plate one day in advance, add 200 μL to each well, and then store at 4°C for overnight coating.

[0443] Preparation before cell plating: aspirate and discard the liquid in the 24-well plate, and then wash twice with 1640 complete medium (10% New Zealand serum, 1% PS double antibody).

[0444] Cell plating: Dilute human PBMCs to 1 million / mL in 1640 complete medium (10% New Zealand serum, 1% PS dual antibody) supplemented with anti-CD28 antibody (1 μg / mL) and IL-2 (working concentration 400 U / mL). Plate 1 million cells per well in a 24-well plate. After plating, stabilize the cells at 37°C, 5% CO2 for 4 hours before adding LNPs.

[0445] Drug addition: Each LNP group listed in Table 26 was added to each well to achieve a working concentration of 1.0 μg / mL. PBS was also added as a negative control group.

[0446] Culture: After adding LNP, PBMC cells were transfected at 37°C, 5% CO2 for 24 hours.

[0447] Detection of eGFP-mRNA expression: Collect cells from each well plate, stain with commercially available flow cytometry antibodies, and then detect the expression rate of eGFP-mRNA in all cells in PBMCs, CD4+ T cells, and CD8+ T cell subsets by flow cytometry.

[0448] Table 27: In vitro transfection results of CD8-TLNPs composed of different ionizable amino lipids

[0449] The experimental results showed that CD8-TLNPs composed of E12LA6B6O3, E10LA8B6O3, and the commercially available lipid SM-102 had comparable or even slightly better transfection efficiency in CD8+ T cell populations, but SM-102 exhibited a higher off-target rate in other non-CD8+ T cell populations. Comprehensive analysis showed that E12LA6B6O3 and E10LA8B6O3 had better targeted delivery to CD8+ T cell subsets.

[0450] Example 24:

[0451] This example is a study on the dose-dependence of nanoparticles targeting human CD8+ T cells in vitro and in vivo

[0452] (1) Preparation of nanoparticles targeting human CD8+ T cells

[0453] The two lipid nanoparticles used in this example were cLNP-1 and TLNP-2 (0.01% targeting molecule ratio) prepared in Example 21.

[0454] (II) Investigation of the dose-dependence of nanoparticle delivery targeting human CD8+ T cells in vitro

[0455] Anti-CD3 coating plate: Add 1 μg / mL anti-human CD3 antibody to a 24-well plate one day in advance, add 200 μL to each well, and then store at 4°C for overnight coating.

[0456] Preparation before cell plating: aspirate and discard the liquid in the 24-well plate, and then wash twice with 1640 complete medium (10% New Zealand serum, 1% PS double antibody).

[0457] Cell plating: Dilute human PBMCs to 1 million / mL in 1640 complete medium (10% New Zealand serum, 1% PS dual antibody) supplemented with anti-CD28 antibody (1 μg / mL) and IL-2 (working concentration 400 U / mL). Plate 1 million cells per well in a 24-well plate. After plating, stabilize the cells at 37°C, 5% CO2 for 4 hours before adding LNPs.

[0458] Drug addition: Add the corresponding targeting molecule-modified TLNP-2 to each well so that the working concentration of LNP in the well plate is as shown in the table below. Add PBS as a negative control group and add LNP-1 as a positive control group.

[0459] Table 28: Information on adding LNP to the well plate

[0460] Culture: After adding LNP, PBMC cells were transfected at 37°C, 5% CO2 for 24 hours.

[0461] Detection of eGFP-mRNA expression: Cells from each well were collected and stained with commercially available flow cytometry antibodies. Flow cytometry was then used to detect the expression of eGFP-mRNA in all cells of hPBMCs, CD3+ T cells, CD4+ T cells, and CD8+ T cell subsets.

[0462] Table 29: Summary of the in vitro targeting effect of nanobody-modified LNPs on T cells

[0463] The experimental results show that in the in vitro transfection experiment of hPBMC, when the working concentration of TLNP-2 is as low as 0.025μg / mL, it still maintains a high proportion of CD8T cell targeting effect, indicating that the TLNP developed in this patent has the ability to achieve high cell targeting at low doses.

[0464] Flow cytometric analysis of the EGFP-mRNA expression efficiency of cLNP-1 and TLNP-2 in hPBMCs is shown in Figures 7A, 7B, 7C, 7D, and 7E.

[0465] (III) Investigation of the dose-dependence of nanoparticle delivery targeting human CD8+ T cells in vivo

[0466] Twelve 6-8 week old NCG female mice that passed quarantine and met the SPF (specific pathogen free) level were selected and divided into 4 groups. Each NCG mouse was injected with 20M hPBMC cells in a volume of 200 μL through the tail vein. About 30 minutes later, the mouse's venous wound healed and different doses of TLNP-2 were injected again through the tail vein in a volume of 200 μL. After 24 hours of expression, peripheral blood was collected and spleen erythrocytes were collected for flow cytometry detection of the expression rate of eGFP-mRNA in each cell subpopulation.

[0467] Table 30: TLNP-2 in vivo experimental grouping

[0468] The experimental results show that TLNP-2 containing 0.01% targeting molecule ratio has a good CD8+T cell targeting delivery effect at a dosage of 0.5mpk in NCG mice.

[0469] The statistical diagram of in vivo transfection of TLNP-2 at different dosages is shown in FIG8 .

[0470] Example 25:

[0471] This example is a study on the formulation of nanoparticle components targeting human CD8+ T cells

[0472] (1) Preparation of nanoparticles targeting human CD8 T cells

[0473] Table 31: Preparation of TLNPs with different ratios

[0474] The two lipid nanoparticles TLNP-2-F1 and TLNP-2-F2 used in this example were prepared according to the method described in Example 20.

[0475] (II) Investigation of the in vivo delivery efficiency of nanoparticles targeting human CD8+ T cells with different formulations

[0476] Nine 6-8 week old NCG female mice that passed quarantine and met the SPF (specific pathogen free) level were selected and divided into 3 groups. Each NCG mouse was injected with 20M hPBMC cells in a volume of 200 μL through the tail vein. About 30 minutes later, the mouse's venous wound healed and the prepared TLNP-2-F1 and TLNP-2-F2 were injected again through the tail vein. Each NCG mouse was given a dose of 10 μg-mRNA-LNP with an injection volume of 200 μL. After 24 hours of expression, peripheral blood was collected and spleen erythrocytes were collected for flow cytometry detection of the expression rate of eGFP-mRNA in each cell subpopulation.

[0477] Table 32: In vivo experimental groups of TLNP with different ratios

[0478] The experimental results showed that TLNP-2-F2 prepared by formula F2 and containing 0.01% targeting molecule ratio showed better CD8+T cell targeting delivery effect in NCG mice than the F1 group at a dosage of 0.5mpk.

[0479] Statistical diagrams of the in vivo transfection status of TLNP-2 at different ratios are shown in Figures 9A, 9B, 9C, 9D, and 9E.

[0480] Example 26:

[0481] This example is an in vitro freeze-thaw stability evaluation of nanoparticles targeting human CD8+ T cells

[0482] (1) Preparation of nanoparticles targeting human CD8+ T cells

[0483] The two lipid nanoparticles used in this example are cLNP-1 and TLNP-2 prepared in Example 21.

[0484] (II) Investigation of freeze-thaw stability of nanoparticles targeting human CD8+ T cells

[0485] The prepared LNPs were frozen and stored in a -80° refrigerator for more than 24 hours, then taken out and quickly re-dissolved in a 4° refrigerator. This sample was frozen and thawed once.

[0486] The reconstituted sample was frozen again in a -80° refrigerator for more than 24 hours, and then taken out and quickly reconstituted in a 4° refrigerator. This sample was frozen and thawed twice.

[0487] The reconstituted sample was frozen again in a -80° refrigerator for more than 24 hours, and then taken out and quickly reconstituted in a 4° refrigerator. This sample was frozen and thawed three times.

[0488] The particle size, PDI and encapsulation efficiency of LNP after each reconstitution were tested to investigate the freeze-thaw stability of LNP.

[0489] Table 33: Characterization information of LNP after freeze-thaw

[0490] Example 27:

[0491] This example targets human CD8 + Evaluation of the long-term stability of nanoparticles in T cells

[0492] (1) Preparation of nanoparticles targeting human CD8+ T cells

[0493] Table 34: LNP preparation formula with different targeting molecule ratios

[0494] In this example, the preparation methods of LNPs containing coupling sites and LNPs modified with nanobodies refer to the process described in Example 21.

[0495] (II) Investigation of the stability of nanoparticles targeting human CD8+ T cells

[0496] 1. Nanoparticles prepared on day 0:

[0497] 1) Characterization of the newly prepared nanobody-modified LNPs

[0498] Table 35: Quality control information of nanobody-modified LNPs (Day 0)

[0499] 2) Detection of in vitro delivery efficiency of the newly prepared nanobody-modified LNPs

[0500] Anti-CD3 coating plate: Add 1 μg / mL anti-human CD3 antibody to a 24-well plate one day in advance, add 200 μL to each well, and then store at 4°C for overnight coating.

[0501] Preparation before cell plating: aspirate and discard the liquid in the 24-well plate, and then wash twice with 1640 complete medium (10% New Zealand serum, 1% PS double antibody).

[0502] Cell plating: Dilute PBMCs to 1 million / mL in 1640 complete medium (10% New Zealand serum, 1% PS double-antibody). Plate 1 million cells per well in a 24-well plate. After plating, allow cells to stabilize for 4 hours before adding LNPs.

[0503] Drug addition: Add the corresponding targeting molecule-modified LNP to each well to achieve a working concentration of 1 μg / mL. PBS was also added as a negative control.

[0504] Culture: After adding LNP, PBMC cells were transfected at 37°C, 5% CO2 for 24 hours.

[0505] Detection of eGFP-mRNA expression: Cells in each well plate were collected, stained with commercially available flow cytometry antibodies, and the expression rate of eGFP-mRNA in all cells in PBMC, CD4 T cells, and CD8 T cells was detected by flow cytometry.

[0506] Table 36: Summary of data on the targeting effect of nanobody-modified LNPs on T cells in vitro (Day 0)

[0507] 2. 3 days after nanoparticle preparation:

[0508] 1) Characterization of the prepared nanobody-modified LNPs (Day 3)

[0509] Table 37: Quality control information of nanobody-modified LNPs (Day 3)

[0510] 3) Testing the in vitro delivery efficiency of the prepared nanobody-modified LNPs (Day 3)

[0511] Same steps as above

[0512] Table 38: Summary of the targeting effect data of nanobody-modified LNPs on T cells in vitro (Day 3)

[0513] 3. 7 days after nanoparticle preparation:

[0514] 1) Characterization of the prepared nanobody-modified LNPs (Day 7)

[0515] Table 39: Quality control information of nanobody-modified LNPs (Day 7)

[0516] 3) Testing the in vitro delivery efficiency of the prepared nanobody-modified LNPs (Day 7)

[0517] Same steps as above

[0518] Table 40: Summary of the targeting effect data of nanobody-modified LNPs on T cells in vitro (Day 7)

[0519] 4. 14 days after nanoparticle preparation:

[0520] 1) Characterization of the prepared nanobody-modified LNPs (Day 14)

[0521] Table 41: Quality control information of nanobody-modified LNPs (Day 14)

[0522] 3) Testing the in vitro delivery efficiency of the prepared nanobody-modified LNPs (Day 14)

[0523] Same steps as above

[0524] Table 42: Summary of the targeting effect data of nanobody-modified LNPs on T cells in vitro (Day 14)

[0525] 5. One month after nanoparticle preparation:

[0526] 1) Characterization of the prepared nanobody-modified LNPs (Month 1)

[0527] Table 43: Quality control information of nanobody-modified LNPs (first month)

[0528] 2) Testing the in vitro delivery efficiency of the prepared nanobody-modified LNPs (first month)

[0529] Same steps as above

[0530] Table 44: Summary of the in vitro targeting effect of nanobody-modified LNPs on T cells (Month 1)

[0531] The experimental results show that after the nanoantibody-modified LNP was stored at 4°C for one month, although the change trend and amplitude of its physical and chemical properties were similar to those of the traditional four-component cLNP, it was still able to maintain a good CD8 T cell targeting effect, indicating that the TLNP developed in this patent has good stability and can maintain a high cell targeting ability for a long time.

[0532] Statistical graphs of the long-term stability study data of traditional LNP and targeted TLNP are detailed in Figures 10A, 10B, 10C, 10D, 10E, and 10F.

[0533] Example 28:

[0534] This example targets human CD8 + Functional evaluation of T cells after nanoparticle drug delivery

[0535] (1) Preparation of nanoparticles targeting human CD8+ T cells

[0536] The two lipid nanoparticles used in this example are cLNP-1 and TLNP-2 prepared in Example 21.

[0537] (II) Functional evaluation of nanoparticle drug delivery targeting human CD8+ T cells

[0538] Anti-CD3 coating plate: Add 1 μg / mL anti-human CD3 antibody to a 24-well plate one day in advance, add 200 μL to each well, and then store at 4°C for overnight coating.

[0539] Preparation before cell plating: aspirate and discard the liquid in the 24-well plate, and then wash twice with 1640 complete medium (10% New Zealand serum, 1% PS double antibody).

[0540] Cell plating: Dilute PBMCs to 1 million / mL in 1640 complete medium (10% New Zealand serum, 1% PS double-antibody). Plate 1 million cells per well in a 24-well plate. After plating, allow cells to stabilize for 4 hours before adding LNPs.

[0541] Drug addition: Add the corresponding targeting molecule-modified LNP to each well to achieve a working concentration of 1 μg / mL. PBS was also added as a negative control.

[0542] Culture: After adding LNP, PBMC cells were transfected at 37°C, 5% CO2 for 24 hours.

[0543] Killing experiment: Collect cells from each well plate and count them, and make them into 1 million / ml. Prepare CD19+ B lymphocyte tumor cell line, count them, and make them into 1 million / ml.

[0544] Resuscitated human PBMCs were used as a control for cells transfected with CD19CAR-LNP. Different ET ratios were added according to the table. Each numbered group was treated with 3 replicates:

[0545] Incubate at 37°C for 4-8 hours;

[0546] Prepare the dead cell dye 7-AAD at a stock concentration of 2 mg / ml and dilute it to 0.1 μg / 10 μl with water;

[0547] Add 2.5 μL of 7-AAD to the flow cytometer and immediately test on the flow cytometer. 7-AAD is detected on the PerCP-Cy5.5 channel and Violet is detected on the BV421 channel.

[0548] The killing rate is CFSE+7-AAD+ / Total CFSE+.

[0549] Table 45: Summary of data on functional evaluation of specific killing effects after nanoparticle delivery of CAR targeting human CD8+ T cells (mean of 3 replicate wells)

[0550] Example 29:

[0551] This example targets human CD8 + Study on the distribution of nanoparticles in T cells in vivo

[0552] (1) Preparation of nanoparticles targeting human CD8 T cells

[0553] The preparation method described in Example 21 was used. The two lipid nanoparticles used in this example were cLNP-1 and TLNP-2, which encapsulated Luciferase mRNA or 25% Cy5 dye-labeled Luciferase mRNA, respectively.

[0554] (II) Cy5-mRNA biodistribution of human CD8 T cell-targeted nanoparticles in Balb / c mice

[0555] Nine 6-8 week old female Balb / c mice (3 mice per group) that passed quarantine and met SPF (specific pathogen free) standards were selected. Each Balb / c mouse in the G1 group was injected with 200 μL of PBS via the tail vein. Each Balb / c mouse in the G2 and G3 groups was injected with 5 μg of 25%-Cy5- LNP, injection volume was 200 μL.

[0556] Two hours after drug administration, mice were anesthetized with isoflurane and dissected to remove the brain, heart, liver, spleen, lungs, kidneys, thymus, lymph nodes, and bone marrow for ex vivo organ imaging. Images were taken using a Perkin Elmer Living Imaging instrument from Shenzhen Bay Laboratory, using the system's default Cy5 imaging mode.

[0557] Table 46: Cy5 fluorescence labeling study of LNP in vivo distribution experimental group

[0558] The results showed that the biodistribution of conventional LNP and cell-targeted TLNP in normal Balb / c mice was similar, with Cy5 mRNA enrichment highest in the liver, followed by the spleen, kidney, and lung.

[0559] (III) Luc-mRNA biodistribution of human CD8 T cell-targeted nanoparticles in Balb / c mice

[0560] Nine 6-8 week old female Balb / c mice (3 mice per group) that passed quarantine and met SPF (specific pathogen free) standards were selected. Each Balb / c mouse in the G1 group was injected with 200 μL of PBS through the tail vein. Each Balb / c mouse in the G2 and G3 groups was injected with 5 μg of LNP, injection volume was 200 μL.

[0561] Six hours after administration, mice were anesthetized with isoflurane and intraperitoneally injected with 250 μL of luciferase substrate. Ten minutes later, the mice were dissected, and the brain, heart, liver, spleen, lungs, kidneys, thymus, lymph nodes, and bone marrow were removed for ex vivo organ imaging. The imaging instrument used was a Perkin Elmer Living Imaging instrument from Shenzhen Bay Laboratory, set to F8 mode with an exposure time of 2 seconds. After imaging, mRNA was extracted from the organs, and qPCR experiments were performed to calculate the enrichment of mRNA in each organ.

[0562] Table 47: Luc imaging study LNP in vivo distribution experimental group

[0563] The results showed that the biodistribution of conventional LNPs and cell-targeted TLNPs in normal Balb / c mice was similar. Luc protein expression and Luc mRNA enrichment were highest in the spleen, with the TLNP-2 group slightly higher than the cLNP group.

[0564] Statistical graphs of the biodistribution experimental results in Balb / c mice using Cy5 fluorescence, Luc imaging, and qPCR are shown in Figures 11A, 11B, 11C, and 11D.

[0565] (I) Study on the biodistribution of Luc-mRNA of human CD8 T cell-targeted nanoparticles in NCG mice

[0566] Nine 6-8 week old NCG female mice that passed quarantine and met SPF (Specific Pathogen Free) standards were selected and divided into three groups. Each NCG mouse was injected with 20M hPBMC cells in a volume of 200μL via the tail vein. Approximately 30 minutes later, after the venous wounds of the mice had healed, the prepared cLNPs and TLNP-2 were injected via the tail vein. Each NCG mouse was given a dose of 5μg-Luc-LNP in a volume of 200μL.

[0567] Six hours after administration, mice were anesthetized with isoflurane and intraperitoneally injected with 250 μL of luciferase substrate. Ten minutes later, the mice were dissected, and the brain, heart, liver, spleen, lungs, kidneys, and bone marrow were removed for ex vivo organ imaging. The imaging instrument used was a Perkin Elmer Living Imaging instrument from Shenzhen Bay Laboratory, with the parameters set to F8 mode and an exposure time of 2 seconds. After imaging, mRNA was extracted from the spleen, liver, and peripheral blood, and qPCR experiments were performed to calculate the enrichment of mRNA in each organ.

[0568] Table 48: Luc imaging study LNP in vivo distribution experimental grouping

[0569] The experimental results showed that in the NCG mouse model, compared with traditional LNP, CD8+T cell-targeted TLNP-2 can more selectively deliver Luc-mRNA to organs rich in hPBMCs, and it exhibits a higher spleen-liver fluorescence intensity ratio.

[0570] Statistical graphs of the experimental results of Luc imaging and qPCR to study the biodistribution of NGC in mice are shown in Figures 12A and 12B.

[0571] Example 30:

[0572] This example targets human CD8 + Evaluation of Nanoparticle Dose Toxicity in Vitro for T Cells

[0573] (1) Preparation of nanoparticles targeting human CD8 T cells

[0574] The two lipid nanoparticles used in this example are cLNP-1 and TLNP-2 prepared in Example 21.

[0575] (II) Investigation of the toxicity of nanoparticle delivery targeting human CD8+ T cells

[0576] Anti-CD3 coating plate: Add 1 μg / mL anti-human CD3 antibody to a 24-well plate one day in advance, add 200 μL to each well, and then store at 4°C for overnight coating.

[0577] Preparation before cell plating: aspirate and discard the liquid in the 24-well plate, and then wash twice with 1640 complete medium (10% New Zealand serum, 1% PS double antibody).

[0578] Cell plating: Dilute PBMCs to 1 million / mL in 1640 complete medium (10% New Zealand serum, 1% PS double-antibody). Plate 0.05 million cells per well of a 96-well plate in duplicate. After plating, allow cells to stabilize for 4 hours before adding LNPs.

[0579] Drug addition: TLNP-2 modified with the corresponding targeting molecule was added to each well so that the working concentration of LNP in the well plate was as shown in the table below. PBS was added as a negative control group.

[0580] Table 49: Information on adding LNP to the well plate

[0581] Culture: After adding LNP, PBMC cells were transfected at 37°C, 5% CO2 for 24 hours.

[0582] Cytotoxicity detection: Add 20 μL of CCK8 reagent into a 96-well plate and incubate in a 37° incubator in the dark. Detect the OD value. The calculation formula is: Cell survival rate = [(As-Ab) / (Ac-Ab)] × 100%

[0583] Wherein, As is the absorbance of the experimental well (containing cells, culture medium, CCK8 solution, and drug solution);

[0584] Ac is the absorbance of the control well (containing cells, culture medium, and CCK8 solution, but no drug);

[0585] Ab is the absorbance of blank wells (containing culture medium and CCK8 solution, but no cells or drugs).

[0586] Analyze the test data to confirm the toxicity of LNP to cells.

[0587] The experimental results showed that TLNP-2 at different working concentrations showed no cytotoxicity, indicating that the nanoantibody-modified TLNP is safe and cells have a very high dose tolerance to this LNP.

[0588] The dose safety study data of TLNP-2 at different working concentrations in hPBMC are detailed in Figure 13.

[0589] Example 31:

[0590] This example targets human CD8 + Evaluation of the safety of T cell nanoparticles in mice

[0591] (1) Preparation of nanoparticles targeting human CD8+ T cells

[0592] The two lipid nanoparticles used in this example are cLNP-1 and TLNP-2 prepared in Example 21.

[0593] (II) Investigation of the in vivo safety of nanoparticles targeting human CD8+ T cells

[0594] The prepared LNPs were injected into mice via the tail vein at 80 μg per mouse (acute toxicity) or 20 μg per mouse for 6 consecutive injections, once a week (chronic toxicity);

[0595] Twenty-four hours after the onset of acute poisoning, blood was collected from mice to separate serum, and liver and kidney function and cytokine levels were tested. The main tissues and organs of the mice were dissected and subjected to histopathological examination.

[0596] One week after the last injection, blood was collected from mice in the long-term poisoning group to separate serum and test liver and kidney function and cytokine levels. The main tissues and organs of the mice were dissected and subjected to histopathological examination.

[0597] Analyze the test data and confirm the toxicity of LNP.

[0598] The experimental results showed that the liver and kidney functions, cytokine levels and tissue sections of mice in both the acute poisoning group and the chronic poisoning group were basically consistent with those in the non-drug group, indicating that TLNP did not show obvious toxicity and was relatively safe.

[0599] The liver toxicity index detection results of the acute toxicity experiment of traditional cLNP and targeted TLNP in mice at different doses are shown in Figure 14.

[0600] Example 32:

[0601] This example targets human CD8 + Evaluation of the in vivo delivery efficiency of nanoparticles for T cells in NHPs

[0602] (1) Preparation of nanoparticles targeting human CD8+ T cells

[0603] The lipid nanoparticles used in this example are the TLNP-2 prepared in Example 21.

[0604] (II) Evaluation of nanoparticle delivery efficiency targeting human CD8+ T cells

[0605] Two cynomolgus macaques (NHPs) that had passed quarantine and met the required weight were divided into two groups. They received intravenous injections of the prepared TLNP-EGFP at a concentration of 100 μg / mL. One group received a high-dose dose of 0.2 mpk, while the other group received a low-dose dose of 0.1 mpk. Peripheral blood was collected before dosing and on days 1-7, 10, and 16 after dosing, and flow cytometry was used to measure the expression of eGFP mRNA in various cell subsets.

[0606] Experimental results showed that compared with the control group, TLNP administration demonstrated significant targeted delivery in CD8 cells in NHPs, with the high-dose group demonstrating higher targeted delivery than the low-dose group. Both the high-dose and low-dose groups demonstrated high on-target activity and low off-target effects. Dynamic changes in EGFP expression after TLNP administration demonstrated that eGFP mRNA expression remained high in NHPs for one week.

[0607] The EGFP expression of TLNP-2 in NHP is shown in Figure 15 .

[0608] The EGFP targeting and off-target effects of TLNP-2 in NHPs are detailed in Figure 16 .

[0609] The dynamic changes of EGFP expression in TLNP-2NHP in vivo are shown in Figure 17 .

[0610] Example 33:

[0611] This example targets human CD8 + In vivo safety evaluation of T cell-derived nanoparticles for NHPs

[0612] (1) Preparation of nanoparticles targeting human CD8+ T cells

[0613] The lipid nanoparticles used in this example are TLNP-2 prepared in Example 21.

[0614] (II) Safety evaluation of nanoparticles targeting human CD8+ T cells

[0615] The NHP dosage was the same as in Example 32. The safety evaluation of nanoparticles targeting human CD8+ T cells in NHP included: measuring the body weight of NHP before administration and on days 25, 37, 39, 43, and 46 after administration; measuring the body temperature of NHP before administration and on days 1-7, 14, 21, and 25 after administration; performing a routine blood test before administration and on day 1 after administration; collecting serum for solid phase chip cytokine detection before administration and on days 1-6 after administration; and collecting plasma for blood biochemical analysis before administration and on days 1-7, 10, and 16 after administration.

[0616] Experimental results showed that continuous monitoring of NHP weight and temperature after TLNP administration maintained pre-dose levels. Routine blood tests revealed a slight increase in granulocytes after administration, with minimal changes in other routine blood tests. Continuous cytokine analysis revealed minimal fluctuations in cytokine levels. Blood biochemistry analysis revealed only transient changes in CK and AST levels after administration, which quickly returned to pre-dose levels. These findings demonstrate the high safety of the human CD8+ T cell-targeting nanoparticles in NHPs.

[0617] The changes in NHP body weight after TLNP-2 administration are shown in Figure 18.

[0618] The changes in NHP body temperature after TLNP-2 administration are shown in Figure 19 .

[0619] The changes in NHP blood routine after TLNP-2 administration are shown in Figure 20.

[0620] The changes in NHP serum factors after TLNP-2 administration are shown in Figure 21.

[0621] The blood biochemical profiles of NHPs after TLNP-2 administration are detailed in Figures 22A, 22B, 22C, 22D, 22E, 22F, 22G, 22H, 22I, and 22J.

[0622] Example 34:

[0623] This example targets human CD5 + Screening of T cell targeting molecules in nanoparticles

[0624] (1) Preparation of nanoparticles targeting human CD5+ T cells

[0625] Table 50: Preparation of LNPs with different targeting molecules (Lx)

[0626] The five groups of TLNPs required in this embodiment were prepared by the method described in Example 20. 2000 DSPE-PEG2000-Mal five-component lipid molecules were dissolved in ethanol at a molar ratio of 47.5:10:41:1.49:0.01; Dissolved in 75 mM citric acid pH 4 buffer solution to ensure that the concentration of mRNA in the aqueous phase is 0.33 mg / mL. The molar ratio of ionizable amino lipid to mRNA (N / P) is 6.5.

[0627] Table 51: Quality control information of Lx-TLNP containing different targeting molecules modified with nanoantibodies

[0628] (2) Targeting human CD5 + Evaluation of the in vitro delivery efficiency of nanoparticles to T cells

[0629] Anti-CD3 coating plate: Add 1 μg / mL anti-human CD3 antibody to a 24-well plate one day in advance, add 200 μL to each well, and then store at 4°C for overnight coating.

[0630] Preparation before cell plating: aspirate and discard the liquid in the 24-well plate, and then wash twice with 1640 complete medium (10% New Zealand serum, 1% PS double antibody).

[0631] Cell plating: Dilute human PBMCs to 1 million / mL in 1640 complete medium (10% New Zealand serum, 1% PS dual antibody) supplemented with anti-CD28 antibody (1 μg / mL) and IL-2 (working concentration 400 U / mL). Plate 1 million cells per well in a 24-well plate. After plating, stabilize the cells at 37°C, 5% CO2 for 4 hours before adding LNPs.

[0632] Drug addition: Each well was added with the five groups of LNPs prepared in Table 50, so that the working concentration of LNPs in the well plate was 0.5 μg / mL. In addition, PBS was added as a negative control group.

[0633] Culture: After adding LNP, PBMC cells were transfected at 37°C, 5% CO2 for 24 hours.

[0634] Detection of eGFP-mRNA expression: Cells in each well plate were collected and stained with commercially available flow cytometry antibodies. The expression rate and mean fluorescence intensity (MFI) of eGFP-mRNA in CD5+ T and CD3+ T cell subsets in PBMC were detected by flow cytometry.

[0635] Table 52: In vitro transfection results of TLNPs containing different targeting molecules

[0636] The experimental results showed that TLNPs prepared by L1, L2 and L3 showed obvious CD5+T cell targeting effect at a lower dosage and had good transfection efficiency.

[0637] The in vitro transfection results of CD5-TLNPs containing different targeting molecules are shown in Figures 23A, 23B, 23C and 23D.

[0638] Example 35:

[0639] This example targets human CD5 + Screening of the ratio of T cell targeting molecules in nanoparticles

[0640] (1) Preparation of targeted human CD5 + Nanoparticles for T cells

[0641] Table 53: Preparation of TLNPs with different targeting molecule ratios

[0642] The twelve groups of TLNPs required in this embodiment were prepared by the method described in Example 20. 2000 DSPE-PEG2000-Mal five-component lipid molecules were dissolved in ethanol at a molar ratio of 47.5:10:41:X:Y (the sum of X and Y was 1.5); Dissolved in 75 mM citric acid pH 4 buffer solution to ensure that the concentration of mRNA in the aqueous phase is 0.33 mg / mL. The molar ratio of ionizable amino lipid to mRNA (N / P) is 6.5.

[0643] Table 54: TLNP quality control information with different targeting molecule ratios

[0644] (2) Targeting human CD5 + Evaluation of the in vitro delivery efficiency of nanoparticles to T cells

[0645] Anti-CD3 coating plate: Add 1 μg / mL anti-human CD3 antibody to a 24-well plate one day in advance, add 200 μL to each well, and then store at 4°C for overnight coating.

[0646] Preparation before cell plating: aspirate and discard the liquid in the 24-well plate, and then wash twice with 1640 complete medium (10% New Zealand serum, 1% PS double antibody).

[0647] Cell plating: Dilute human PBMCs to 1 million / mL in 1640 complete medium (10% New Zealand serum, 1% PS dual antibody) supplemented with anti-CD28 antibody (1 μg / mL) and IL-2 (working concentration 400 U / mL). Plate 1 million cells per well in a 24-well plate. After plating, stabilize the cells at 37°C, 5% CO2 for 4 hours before adding LNPs.

[0648] Drug addition: Each well was added with 12 sets of LNPs prepared in Table 53, so that the working concentration of LNPs in the well plate was 0.5 μg / mL. In addition, PBS was added as a negative control group.

[0649] Culture: After adding LNP, PBMC cells were transfected at 37°C, 5% CO2 for 24 hours.

[0650] Detection of eGFP-mRNA expression: Cells in each well plate were collected, stained with commercially available flow cytometry antibodies, and the expression rate and mean fluorescence intensity (MFI) of eGFP-mRNA in the CD5+ T cell subsets in PBMC were detected by flow cytometry.

[0651] Table 55: In vitro transfection results of TLNPs containing different targeting molecule ratios

[0652] Experimental results showed that L2-TLNPs prepared with L2 exhibited the best CD5+ T cell targeting and delivery efficiency at lower doses. When the targeting molecule ratio was higher than 0.05%, no significant differences in delivery efficacy were observed among the different TLNP ratios. Considering the physical and chemical properties of LNPs and material cost factors, a 0.05% targeting molecule ratio was used for subsequent studies.

[0653] The in vitro transfection results of CD5-TLNPs containing different targeting molecule ratios are detailed in Figures 24A and 24B.

[0654] Example 36:

[0655] This example targets human CD5 + Screening of nanoparticle formulations for T cell therapy

[0656] (1) Preparation of targeted human CD5 + Nanoparticles for T cells

[0657] Table 56: Preparation of CD5-TLNPs with different ratios

[0658] The two lipid nanoparticles L2-TLNP-F1 and L2-TLNP-F2 used in this example were prepared by the method described in Example 21.

[0659] (II) Investigation of the in vivo delivery efficiency of nanoparticles targeting human CD5+ T cells with different formulations

[0660] Nine 6-8-week-old hCD5 humanized female mice that passed quarantine and met SPF (Specific Pathogen Free) standards were selected and divided into three groups. The prepared L2-TLNP-F1 and L2-TLNP-F2 were injected into the tail vein of the mice. Each mouse received a dose of 20 μg mRNA-LNP in a 200 μL injection volume. After 24 hours of expression, peripheral blood, spleen, lymph nodes, and tibia were collected for flow cytometry analysis of eGFP mRNA expression in cell subsets in each organ.

[0661] Table 57: In vivo experimental groups of L2-TLNP with different ratios

[0662] The experimental results showed that L2-TLNP-F2 prepared by formula F2 containing 0.05% targeting molecule ratio showed better CD5 expression in various organs of hCD5 humanized mice than F1 group at a dose of 1.0 mpk. + T cell targeted delivery effect.

[0663] Statistical diagrams of in vivo transfection of L2-TLNPs with different ratios are shown in Figures 25A and 25B.

[0664] Example 37:

[0665] This example targets human CD5 + Study on the distribution of nanoparticles in T cells in vivo

[0666] (1) Preparation of nanoparticles targeting human CD5 T cells

[0667] The preparation method described in Example 21 was used. The two lipid nanoparticles used in this example were cLNP-1 and L2-TLNP-F2, which encapsulated Luciferase mRNA, respectively.

[0668] (II) Luc-mRNA biodistribution of human CD5 T cell-targeted nanoparticles in hCD5 humanized mice

[0669] Nine hCD5 humanized female mice aged 6-8 weeks that passed quarantine and met the SPF (specific pathogen-free) level were selected (3 mice per group). Each mouse in the G1 group was injected with 200 μL of PBS through the tail vein. Each mouse in the G2 and G3 groups was injected with 2 μg of LNP, injection volume was 200 μL.

[0670] Six hours after administration, mice were anesthetized with isoflurane and intraperitoneally injected with 250 μL of luciferase substrate. Ten minutes later, the mice were dissected, and the brain, heart, liver, spleen, lungs, kidneys, thymus, lymph nodes, and bone marrow were removed for ex vivo organ imaging. The imaging instrument used was a Perkin Elmer Living Imaging instrument from Shenzhen Bay Laboratory, set to F8 mode with an exposure time of 2 seconds. After imaging, mRNA was extracted from the organs, and qPCR experiments were performed to calculate the enrichment of mRNA in each organ.

[0671] Table 58: Luc imaging study LNP in vivo distribution experimental grouping

[0672] The experimental results showed that in the hCD5 humanized mouse model, compared with traditional LNP, CD5+T cell-targeted L2-TLNP-F2 can more selectively deliver Luc-mRNA to T cell-rich organs, which shows a higher spleen-liver fluorescence intensity ratio.

[0673] Statistical graphs of the experimental results of Luc imaging and qPCR to study the biodistribution of hCD5 in humanized mice are shown in Figures 26A and 26B.

[0674] The above-described embodiments merely illustrate several implementations of the present invention, and are provided to facilitate a specific and detailed understanding of the technical solutions of the present invention. They should not be construed as limiting the scope of protection of the present invention. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, and all such modifications and improvements fall within the scope of protection of the present invention. Industrial Applicability

[0675] The present invention provides a molecule with targeting function, which can specifically deliver drugs to specific cells, help to accurately program specific cells in the body, exert the therapeutic effect of the drug, and thus greatly reduce the drug dosage and side effects, achieving the effect of precise cell treatment in the body, and has good economic value and application prospects.

Claims

1. A molecule having a targeting function, containing (I) one or more hydrophobic chains; (II) one or more linkers; (III) one or more nanobodies.

2. A molecule having a targeting function according to claim 1, characterized in that the number of nanobodies is 20 or less.

3. A molecule having a targeting function according to claim 1 or 2, characterized in that the number of nanobodies is from 1 to 7.

4. A molecule having a targeting function according to any one of paragraphs 1-3, characterized in that between the nanobody and the linker there is one amino acid containing a reactive center, wherein the amino acid contains, but is not limited to, cysteine ​​and lysine.

5. A molecule having a targeting function according to claim 4, characterized in that the nanobody is connected by means of a connecting sequence, which is located between the nanobody and the amino acid, wherein the connecting sequence contains (GS)n and / or (GGGS)m, where n and m are the same or different values, and the values ​​of n or m are selected from 0, 1, 2, 3 or 4.

6. A molecule having a targeting function according to any one of claims 1 to 5, characterized in that the hydrophobic chain contains one or more substituted or unsubstituted C12-C18 alkyls.

7. A molecule having a targeting function according to any one of claims 1 to 6, characterized in that the linker comprises one or more structural units selected from -C(=O)O-, -OC(=O)O-, -C(=O)NH-, -C(=O)S-, -S-, -SS-, triazole, hydrazone bond, SMCC, sulfo-SMCC, SATA, polypeptides as structural units, PEG or PEG derivatives; wherein the PEG derivatives may be selected from maleimide-functionalized polyethylene glycol, including, but not limited to, PEG-MAL.

8. A molecule having a targeting function according to claim 7, characterized in that the relative molecular weight of PEG or PEG derivatives is 500-5000.

9. A molecule having a targeting function according to claim 8, characterized in that the relative molecular weight of PEG or PEG derivatives is 1000, 2000, 3000, 4000 or 5000.

10. A molecule having a targeting function according to any one of paragraphs 1-9, characterized in that the nanobody contains an antigen-binding domain specific to an antigen, wherein the antigen is a membrane protein molecule.

11. A molecule having a targeting function according to claim 10, characterized in that the membrane protein molecule is a cell membrane protein molecule associated with an immune cell or a cell membrane protein molecule associated with immunity, including, but not limited to, one or more of CD3, CD4, CD5, CD7, CD8, CD25, CD38, CD61, CD42a, CD105, CD90, CD15, CD127, CD56, CD68, CD19, CD11c, CD138, F4 / 80, CD62P, CD49f, CD31, RANK, ALPL, PDPN, CD34, FcεR1α, CD203c, CD63, CD193, CD66b, CD41, CD117 and ASGPR.

12. A molecule having a targeting function according to claim 10, characterized in that the membrane protein molecule is a molecule of a cell membrane protein of a cell of the central nervous system, including, but not limited to, one or more of GD2, GD3, MOG and TMEM119.

13. The targeting molecule of claim 10, wherein the membrane protein molecule is a tumor-associated antigen, including, but not limited to, one or more of CD133, PSMA, CLDN18.2, DLL3, TROP2, EGFRVIII, CA125, MUC1, MUC16, MSLN, CA9, HER2, HER3, TGM4, PSCA, CLDN6, STEAP2, GPC3, IL-13Ra2, EGFR, STEAP1, BCMA, FRa, VEGFR2, PDGFR-β, CEA, NCAM, FAP, SLC2A2, SEZ6L2, and LRP11.

14. The molecule having a targeting function according to claim 10, characterized in that the membrane protein molecule is an immunoregulatory molecule, including, but not limited to, one or more of B7-H1, B7-H3 and B7-H4.

15. The targeting molecule of claim 10, wherein the membrane protein molecule comprises, but is not limited to, one or more of the following targets: CD3, CD4, CD5, CD7, CD8, CD25, CD38, CD61, CD42a, CD105, CD90, CD15, CD127, CD56, CD68, CD19, CD11c, CD138, F4 / 80, CD62P, CD49f, CD31, RANK, ALPL, PDPN, CD34, FcεR1α, CD203c, CD63, CD193, CD66b, CD41, CD117, ASGPR, GD2, GD3, MOG, TMEM119, CD133, PSMA, CLDN18.2, DLL3, TROP2, EGFRVIII, CA125, MUC1, MUC16, MSLN, CA9, HER2, HER3, TGM4, PSCA, CLDN6, STEAP2, GPC3, IL-13Ra2, EGFR, STEAP1, BCMA, FRa, VEGFR2, PDGFR-β, CEA, NCAM, FAP, SLC2A2, SEZ6L2, LRP11, B7-H1, B7-H3 and B7-H4.

16. A molecule having a targeting function according to any one of paragraphs 1-15, characterized in that the nanobody is a nanobody targeting CD8, wherein the amino acid sequence of the nanobody is represented by any one of SEQ ID NO: 1-3; and / or the nanobody is a nanobody targeting CD19, wherein the amino acid sequence of the nanobody is represented by any one of SEQ ID NO:79-81; and / or the nanobody is a nanobody targeting CD56; and / or the nanobody is a nanobody targeting CD5, wherein the amino acid sequence of the nanobody is represented by any one of SEQ ID NO:4-78; and / or the nanobody is a nanobody targeting PSMA, wherein the amino acid sequence of the nanobody is represented by any one of SEQ ID NO: 82-83; and / or the nanobody is a nanobody targeted to CD133, wherein the amino acid sequence of the nanobody is represented by any one of SEQ ID NO:84-85.

17. A nanobody having a targeting function, wherein the amino acid sequence of the nanobody is represented by any one of SEQ ID NOs:4-78; or the amino acid sequence of the nanobody has a homology of 80% or more with the amino acid sequence represented by any one of SEQ ID NOs:4-78.

18. A lipid nanoparticle, characterized in that its components contain a molecule having a targeting function, according to any one of paragraphs 1-16, wherein the content of the molecule having a targeting function is 0.0001-1.0 mol.% of the total amount of lipids of the lipid nanoparticle.

19. A lipid nanoparticle according to claim 18, characterized in that the content of the molecule having a targeting function is 0.01-0.5 mol.% of the total amount of lipids of the lipid nanoparticle.

20. A lipid nanoparticle according to claim 18 or claim 19, characterized in that the components of the lipid nanoparticle comprise a lipid conjugated to a polymer and an ionizable aminolipid; wherein the lipid conjugated to the polymer is selected from lipids modified with PEG or PEG derivatives; wherein the relative molecular weight of the lipids modified with PEG or PEG derivatives is 2000-5000.

21. A lipid nanoparticle according to claim 20, characterized in that the relative molecular weight of the lipids modified with PEG or PEG derivatives is 2000, 3000, 4000 or 5000.

22. A lipid nanoparticle according to claim 20 or 21, characterized in that the content of the lipid conjugated with the polymer is 0.2-5 mol.% of the total amount of lipids in the lipid nanoparticle.

23. A lipid nanoparticle according to claim 22, characterized in that the content of the lipid conjugated with the polymer is 0.5-2 mol.% of the total amount of lipids in the lipid nanoparticle.

24. A lipid nanoparticle according to any one of claims 20-23, characterized in that the PEG derivatives include maleimide-functionalized polyethylene glycol and non-maleimide-functionalized polyethylene glycol; wherein the maleimide-functionalized polyethylene glycol can be selected from PEG-MAL; and the content of maleimide-functionalized polyethylene glycol is 0.0001-1.0 mol.% of the total amount of lipids in the lipid nanoparticle.

25. A lipid nanoparticle according to claim 24, characterized in that the content of polyethylene glycol functionalized with maleimide is 0.01-0.5 mol.% of the total amount of lipids in the lipid nanoparticle.

26. A lipid nanoparticle according to any one of paragraphs 20-25, characterized in that the content of the ionizable aminolipid is 30-70 mol.% of the total amount of lipids of the lipid nanoparticle.

27. A lipid nanoparticle according to claim 26, characterized in that the content of the ionizable aminolipid is 40-60 mol.% of the total amount of lipids in the lipid nanoparticle.

28. A lipid nanoparticle according to any one of paragraphs 20-27, characterized in that the ionizable aminolipid has a structure represented by the general formula (I), or is an isomer, pharmaceutically acceptable salt, prodrug or solvate of a structure represented by the general formula (I) (I), wherein G is selected from H, OR, CN, -C(=O)OR', -OC(=O)R', -C(=O)NR'R'', -NR'C(=O)R'', NR'R'' or a cycloalkyl structure containing at least one heteroatom; the carbon atom or heteroatom that may be substituted in the cycloalkyl structure is unsubstituted or substituted by one or more hydroxyl, C1-C4 alkyl, C2-C4 alkenyl, C3-C8 cycloalkyl or C3-C8 cycloalkenyl; M1, M2, M3, and M4 are the same or different from each other and each is independently selected from C1-C 24 alkylene, C3-C 24 cycloalkylene, C2-C 24 alkenylene or C3-C 24 cycloalkenylene; R 1 and R 2 are the same or different from each other, and each of them is independently selected from H, C1-C 24 alkyl, C3-C 24 cycloalkyl, C2-C 24 alkenyl or C3-C 24 cycloalkenyl; L1, L2, L3, and L4 are the same or different from each other and each is independently selected from -C(=O)O-, -OC(=O)-, -C(=O)S-, -SC(=O)-, -C(=O)NR-, -NRC(=O)-, -S(=O)-, -OS(=O)2-, -S(=O)2O-, -O-, -S-, or -SS-; R, R' and R'' are the same or different from each other and each is independently selected from H, C1-C 10 alkyl, C3-C 10 cycloalkyl, C3-C 10 alkenyl or C3-C 10 cycloalkenyl, C1-C 10 alkyl with a terminal tertiary amino group, C3-C10 cycloalkyl with a terminal tertiary amino group, C3-C 10 alkenyl with a terminal tertiary amino group or a cycloalkyl structure containing at least one heteroatom; the cycloalkyl structure is unsubstituted or substituted by one or more C1-C4 alkyl, C2-C4 alkenyl, C3-C8 cycloalkyl or C3-C8 cycloalkenyl; M5 is selected from a single bond, C1-C 16 alkylene, C2-C 16 alkenylene, C3-C8cycloalkylene or C3-C8cycloalkenylene.

29. A lipid nanoparticle according to any one of claims 20-28, characterized in that the components of the lipid nanoparticle additionally contain at least one of a steroid and a neutral lipid.

30. The lipid nanoparticle of claim 29, wherein the steroid is one or more of cholesterol and its derivatives, cholesterol ester, steroid hormone, steroid vitamin, and phytosterol; and / or the neutral lipid is a phospholipid; and / or the lipid conjugated to the polymer is a PEGylated lipid.

31. A lipid nanoparticle according to claim 30, characterized in that the steroid is cholesterol.

32. The lipid nanoparticle of claim 30, wherein the PEGylated lipid is DMG-PEG2000.

33. A lipid nanoparticle according to any one of paragraphs 29-32, characterized in that the molar ratio of the ionizable aminolipid, steroid, neutral lipid and lipid conjugated with the polymer is (30-70):(0-65):(0-30):(0.2-5).

34. A lipid nanoparticle according to claim 33, characterized in that the molar ratio of the ionizable aminolipid, steroid, neutral lipid, and lipid conjugated with the polymer is (40-60):(35-60):(0-20):(0.5-2).

35. A lipid nanoparticle according to claim 34, characterized in that the molar ratio of the ionizable aminolipid, steroid, neutral lipid, and lipid conjugated with the polymer is (40-55):(30-50):(0-20):(0.5-2).

36. A lipid nanoparticle according to claim 35, characterized in that the molar ratio of the ionizable aminolipid, steroid, neutral lipid, and lipid conjugated with the polymer is (40-50):(30-45):(0-15):(0.5-2).

37. A lipid nanoparticle according to claim 36, characterized in that the molar ratio of the ionizable aminolipid, steroid, neutral lipid, and lipid conjugated with the polymer is (47-50):(34-36):(13-16):(1.0-1.1).

38. A lipid nanoparticle according to claim 33, characterized in that the molar ratio of the ionizable aminolipid, steroid, neutral lipid, and lipid conjugated with the polymer is 47.5:36:15:1.

0.

39. A method for producing a lipid nanoparticle according to any one of claims 18-38, characterized in that the method comprises the following steps: mixing a lipid conjugated to a polymer, an ionizable aminolipid, a steroid and an auxiliary phospholipid to obtain a lipid nanoparticle without a targeting function, and then connecting a molecule having a targeting function according to any one of claims 1-16 to a lipid nanoparticle without a targeting function by membrane fusion or chemical bonding to obtain a lipid nanoparticle having a targeting function.

40. A method for producing a lipid nanoparticle according to any one of claims 18-38, characterized in that the method comprises the following steps: mixing a lipid conjugated with a polymer, an ionizable aminolipid, a steroid, and an auxiliary phospholipid to obtain a lipid nanoparticle without a targeting function, and then connecting the nanobody to the lipid nanoparticle without a targeting function by means of a chemical bond to obtain a lipid nanoparticle having a targeting function.

41. The method according to claim 40, characterized in that the lipid conjugated to the polymer contains polyethylene glycol functionalized with maleimide, and the nanobody is connected to the polyethylene glycol functionalized with maleimide.

42. A method for producing a lipid nanoparticle according to any one of claims 18-38, characterized in that the method comprises the following steps: mixing a lipid conjugated with a polymer, an ionizable aminolipid, a steroid, an auxiliary phospholipid and a molecule having a targeting function, according to any one of claims 1-16, to obtain a lipid nanoparticle having a targeting function.

43. A method for targeted delivery of pharmacologically active molecules to specific cells of a subject, comprising the step of bringing the specific cells into contact with a lipid nanoparticle according to any one of claims 18-38.

44. The method according to claim 43, characterized in that the specific cells comprise, but are not limited to, immune cells, immune-associated cells, cells of the central nervous system, tumor cells, somatic cells, and cells infected with viruses, bacteria, or fungi.

45. The method according to claim 43 or 44, characterized in that the target cells are B cells, T cells or NK cells.

46. ​​A method for expressing a target protein or polypeptide in target cells of a subject, comprising the step of contacting the target cells with a lipid nanoparticle according to any one of claims 18-38.

47. The method of claim 46, wherein the target cells comprise, but are not limited to, immune cells, immune-associated cells, central nervous system cells, tumor cells, somatic cells, and cells infected with viruses, bacteria, or fungi.

48. The method according to claim 46 or 47, wherein the target cells are B cells, T cells or NK cells.

49. Use of a lipid nanoparticle according to any one of claims 18-38 as a carrier for drug delivery.

50. A pharmaceutical composition characterized in that it contains a lipid nanoparticle according to any one of claims 18-38, wherein the lipid nanoparticle contains a pharmacologically active molecule.

51. The pharmaceutical composition of claim 50, wherein the pharmacologically active molecule comprises one or more of mRNA, DNA, siRNA, saRNA, shRNA, miRNA, circular RNA, long non-coding RNA, gRNA, polypeptide, or protein.

52. A pharmaceutical composition according to claim 50 or 51, wherein the pharmacologically active molecule comprises mRNA, wherein the mRNA is used to encode a CAR molecule, a TCR molecule, an immunoregulatory molecule, a functional protein molecule, or a gene editing tool such as a base editor or Cas9 protein.

53. A pharmaceutical composition according to any one of paragraphs 50-52, characterized in that the CAR molecule contains at least one sequence from the following sequences: a sequence that can specifically recognize a tumor-specific antigen, a transmembrane junction sequence and / or a sequence that promotes the immune function of T cells.

54. A pharmaceutical composition according to any one of claims. 50-53, characterized in that the sequence encoding the TCR molecule contains a sequence capable of recognizing at least one molecule from NY-ESO-1, AFP, HBsAg, MAGEA1, mesothelin, MART-1, CD19, CD28, PD-1, CD8, CT83, E6, E7, E8, GPC3, H3.3-K27M, HIV Gag polyprotein, HLA-A / AFP, KRASG12(V / D), KRASG12D, KRASG12V, LMP1, LMP2, EBNA1, MAGEA10, MAGEA3, MAGEA4, MC2R, mHag HA-1, MYO1G, PRAME, WT1, gp100, CEA, p53, HLA-A2, EGFR, DR5, RAS, LAGE-1, CMV, HCV, MCPyv and HA-1H.

55. A pharmaceutical composition according to any one of claims. 50-54, characterized in that the sequence encoding the immunoregulatory molecule comprises a sequence of at least one molecule from CD28, PD-1, CTLA-4, RGMB, ICOS, CD28H, NKp30, HVEM, OX40, Fas, 4-1BB, CD27, CD30, APO-2, APO-3, BCMA, BAFFR, GITR, IL-2, IL-12A, IL-12B, IL-15, IL-23, IL-27, FLT3L, IL-36A, IL-36B, IL-36C, GM-CSF, CCL20, CXCL9, CXCL10, CXCL11, CXCL12, CCR7, CXCR4, CCR5, CCL4, CCL5, CCL19, CXCR3, CCR6, B7-H1, B7-DC, B7-H3, B7-H4, PD-1H, IL-10, TGF-β, HGF, B7-1, B7-2, B7-H2, B7-H3, CD40, FasL, CD70, CD30L, 4-1BBL, OX40L, TRAIL, RANKL, TWEAK, APRIL, BAFF, LIGHT, GITRL, CD73, STAT1, IRF4, CCAR2, BCL6, iNOS and CD103.

56. A pharmaceutical composition according to any one of claims. 50-55, characterized in that the nucleic acid encoding the CAR molecule comprises a sequence capable of recognizing at least one molecule of CD19, BMCA, CD22, CD20, CD123, GD2, CD30, GPC3, CLDN18.2, mesothelin, CD33, CD38, EGFRvlll, CD138, CEA, HER2, PSMA, CLL1, CD56, EGFR, MUC-1, EpCAM, CD7, NKG2D, PD-L1, LewisY, FAP, c-MET, ROR1, IL13Rα2, AFP, CD133, CD4, BTK, ROBO1, CD5, CD70, LILRB4, FLT3, Sigle-6, CD229 and SLAMF7.

57. A pharmaceutical composition containing the pharmaceutical composition according to claim 50 and a pharmaceutically acceptable carrier.

58. The pharmaceutical composition according to paragraph 57, characterized in that the pharmaceutical composition is an aqueous injection, lyophilized powder or spray.