Novel lipid nanoparticle system, car-t construction method, and use thereof

Abstract

Provided are a lipid nanoparticle composition and use thereof in the preparation of a composition for treating liver diseases comprising cancer. The composition comprises an ionizable cationic lipid, an auxiliary phospholipid, a polymeric phospholipid, and a sterol lipid.

Description

Novel lipid nanoparticle systems, CAR-T construction methods and their applications Technical Field

[0001] This invention relates to lipid nanoparticles for delivering nucleic acids, methods for their preparation, and uses of such lipid nanoparticles, including for constructing transiently transfected CAR-T cells in vivo and the use of said CAR-T cells in the treatment of liver diseases, particularly liver cancer. Background Technology

[0002] Lipid nanoparticles (LNPs) play a crucial role in effectively protecting oligonucleotides from degradation in vivo and transporting them into cells. Empty LNPs typically consist of ionizable lipids (~50 mol%), PEGylated lipids (~1.5 mol%), cholesterol (~20-50 mol%), and helper phospholipids (~10-20 mol%). As a key component of COVID-19 mRNA vaccines, LNPs are currently attracting significant attention. Clinical authorizations of LNP formulations demonstrate this: Patisiran, BNT162b2, and mRNA-1273. In summary, LNPs have emerged as promising carriers for various therapies across the pharmaceutical industry. A growing number of researchers are pursuing more targeted applications of LNPs.

[0003] Previous studies have shown that the particle size, charge, and component ratio of LNPs all affect their organ-specific distribution bias in vivo. Current organ-targeting formulations require a high proportion of lipid oligonucleotides to maintain their specific organ targeting, but excessively high lipid proportions can cause significant toxicity, making these formulations unsuitable for practical clinical applications. Ligand modification of LNP formulations to enhance their targeting of specific cells has also resulted in drawbacks such as excessively high lipid proportions and strong non-specific uptake.

[0004] CAR-T therapy is a novel precision-targeted therapy for treating tumors. It involves isolating peripheral blood T cells in vitro, modifying them using lentiviral vectors, and then reinfusing them into the patient. This allows the T cells to recognize and kill tumor cell antigens. CAR-T therapy has achieved great success in treating hematologic malignancies, but its efficacy in treating solid tumors is poor. Traditional CAR-T cells require viral infection and permanent gene integration, which may bring potential risks of mutational carcinogenesis and viral infection. The economic and time costs of constructing CAR-T cells are also high. Furthermore, the efficiency of T cells reinfused into the patient to migrate and infiltrate the solid tumor is low, failing to effectively kill tumor cells.

[0005] Liver cancer, especially hepatocellular carcinoma (HCC), typically exhibits complex immune escape mechanisms, including reduced antigen expression and increased secretion of immunosuppressive factors. This makes it difficult for CAR-T cells to generate a sustained response to HCC. The HCC microenvironment contains numerous immunosuppressive factors, such as PD-L1 and TGF-β, which may inhibit CAR-T cell activity and limit their tumor-killing effects. The liver is a vital organ, and CAR-T cell therapy may induce hepatotoxicity and even lead to severe liver function damage; therefore, the safety of this treatment requires special attention.

[0006] Therefore, there is an urgent need to develop liver-targeting LNPs with low toxicity and high specificity, and to develop novel CAR-T cell therapy tools based on these, which will help achieve better treatment results. Summary of the Invention

[0007] This invention provides a composition for delivering a target substance (e.g., a nucleic acid drug) to the liver. Preferably, the composition is in the form of lipid nanoparticles (LNPs) for encapsulating the target substance within the LNPs for delivery. The composition of this invention is particularly suitable for delivering nucleic acid molecules to the liver to engineer liver cells for the treatment of liver diseases, such as hepatocellular carcinoma. Specifically, this invention develops low-toxicity, in vivo liver-targeting LNPs by reducing the lipid content and altering the lipid composition. This invention further enhances the targeting of the LNPs to T cells in the liver by modifying the LNPs with ligands that recognize T cells.

[0008] This invention also provides a method for preparing CAR-T cells in vivo or in vitro, which involves delivering LNPs encapsulating nucleic acids encoding CARs to T cells to prepare engineered CAR-T cells. The method of this invention can be performed without T cell isolation, and the modified LNPs exhibit high specificity and high expression efficiency in in situ T cells, and can be used for in situ liver T cell mRNA delivery to construct CAR-T cells in vivo.

[0009] In some aspects, this disclosure provides lipid nanoparticles for delivering a target substance to the liver. In some embodiments, the target substance is a nucleic acid. When the lipid nanoparticles encapsulate nucleic acids, the lipid tails (long-chain fatty acids) contained in the LNPs are hydrophobic, enabling the formation of lipid bilayers or multilayers in an aqueous phase through hydrophobic interactions, thereby encapsulating nucleic acids such as mRNA within the nanoparticle core.

[0010] In some embodiments, the lipid nanoparticles consist of a lipid membrane formed by the non-covalent arrangement of multiple lipid molecules serving as the source material. Specifically, the lipid nanoparticles may comprise ionizable cationic lipids, cofactory phospholipids, cholesterol, and polymeric phospholipids as their lipid components. Preferably, the molar ratio between the ionizable cationic lipids, cholesterol, cofactory phospholipids, and polymeric phospholipids is in the range of approximately (51.8 ± 20%):(37.2 ± 20%):(9.7 ± 20%):(1.3 ± 20%).

[0011] The expression "X±20%" used throughout this application can be used interchangeably with "X±0.2X", meaning that the value of X can fluctuate by 20% of its value, that is, within the range of 80% to 1.2 times X. Similarly, the expressions "X±10%" or "X±5%" used throughout this application can be used interchangeably with "X±0.1X" or "X±0.05X", respectively, meaning that the value of X can fluctuate by 10% (that is, within the range of 0.9X to 1.1X) or 5% (that is, within the range of 0.95X to 1.05X).

[0012] In some aspects, this disclosure provides a liver-targeting composition comprising an ionizable cationic lipid, an auxiliary phospholipid, a polymeric phospholipid, and a sterol lipid, wherein the cationic lipid is DLin-MC3-DMA and DODAP, and the auxiliary phospholipid is DOPE.

[0013] In some embodiments, the ionizable cationic lipids and cofactory phospholipids account for about 49%-74% of the total lipids in molar percentage. Optionally, the molar ratio between the ionizable cationic lipids and cofactory phospholipids is in the range of about (51.8±20%):(9.7±20%), more preferably in the range of about (51.8±10%):(9.7±10%), and even more preferably in the range of about (51.8±5%):(9.7±5%).

[0014] In some implementations, the molar ratio between DLin-MC3-DMA and DODAP is in the range of about (40-60):(2.0-5.0). Optionally, the molar ratio of DLin-MC3-DMA, DODAP, and DOPE is in the range of about (40-60):(2.0-5.0):(8.0-12).

[0015] In some embodiments, the molar ratio between DLin-MC3-DMA and DODAP is in the range of about (48.6 ± 20%):(3.2 ± 20%). Optionally, the molar ratio of DLin-MC3-DMA, DODAP, and DOPE is in the range of about (48.6 ± 20%):(3.2 ± 20%):(9.7 ± 20%), more preferably in the range of about (48.6 ± 10%):(3.2 ± 10%):(9.7 ± 10%), and even more preferably in the range of about (48.6 ± 5%):(3.2 ± 5%):(9.7 ± 5%).

[0016] In some embodiments, the sterol lipids are selected from cholesterol, sitosterol, coccosterol, rock saponin, brassosterol, ergosterol, tomatine, ursolic acid, α-tocopherol, stigmasterol, alfalfa sterol, ergocalciferol, or campesterol, preferably cholesterol and / or β-sitosterol, more preferably cholesterol. Optionally, the sterol lipids account for about 29.76-44.64% of the total lipids in molar percentage.

[0017] In some embodiments, the polymeric phospholipid is a polyethylene glycol-modified (PEG) phospholipid, such as selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. Preferably, the PEG-modified phospholipid contains a PEG portion of about 1000 Da to about 20 kDa, and more preferably contains a PEG portion of about 1000 Da to about 5000 Da.

[0018] Preferably, the polyethylene glycol-modified phospholipid is selected from DMPE-PEG1000, DPPE-PEG1000, DSPE-PEG1000, DOPE-PEG1000, DMG-PEG2000, Ceramide-PEG2000, DMPE-PEG2000, DPPE-PEG2000, DSPE-PEG2000, Azido-PEG2000, DSPE-PEG2000-Mannose, Ceramide-PEG5000, and DSPE-PEG5000, and is more preferably DMG-PEG2000.

[0019] In some embodiments, the composition comprises DLin-MC3-DMA, DODAP, DOPE, cholesterol, and DMG-PEG2000. Optionally, DMG-PEG2000 may be modified, for example, to include an N-hydroxysuccinimide group.

[0020] In some specific embodiments, the molar ratio of DLin-MC3-DMA, DODAP, DOPE, cholesterol, and DMG-PEG2000 is in the range of about (48.6±20%):(3.2±20%):(9.7±20%):(37.2±20%):(1.3±20%). Preferably, it is in the range of about (48.6±10%):(3.2±10%):(9.7±10%):(37.2±10%):(1.3±10%). More preferably, it is in the range of about (48.6±5%):(3.2±5%):(9.7±5%):(37.2±5%):(1.3±5%). DMG-PEG2000 may be partially modified to include N-hydroxysuccinimide groups.

[0021] In some specific embodiments, the molar ratio among DLin-MC3-DMA, DODAP, DOPE, cholesterol, DMG-PEG2000, and DMG-PEG2000-NHS is in the range of approximately (48.6±20%):(3.2±20%):(9.7±20%):(37.2±20%):(0.65±20%):(0.65±20%). Preferably, it is in the range of approximately (48.6±10%):(3.2±10%):(9.7±10%):(37.2±10%):(0.65±10%):(0.65±10%). More preferably, it is in the range of approximately (48.6±5%):(3.2±5%):(9.7±5%):(37.2±5%):(0.65±5%):(0.65±5%). The molar ratio between DMG-PEG2000 and DMG-PEG2000-NHS is not limited to 1:1.

[0022] In some embodiments, the lipid component in the composition is presented or assembled in the form of lipid nanoparticles.

[0023] In some embodiments, the lipid nanoparticles have a particle size of about 50-200 nm. Preferably, the lipid nanoparticles have a particle size of less than 180 nm.

[0024] In some embodiments, the lipid nanoparticles have an average zeta potential of about -5 mV to about +10 mV, preferably about 0 mV to about +10 mV.

[0025] In some embodiments, the composition further comprises a target substance, such as one or more therapeutic or preventative agents. Preferably, the therapeutic or preventative agent is encapsulated in lipid particles formed from the lipid component.

[0026] In some embodiments, the therapeutic or preventative agent is a nucleic acid. Preferably, the nucleic acid is ASO, RNA, or DNA. For example, the RNA may be selected from interfering RNA (RNAi), small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aRNA), messenger RNA (mRNA), modified messenger RNA (mmRNA), long non-coding RNA (lncRNA), microRNA (miRNA), small activating RNA (saRNA), polymeric coding nucleic acid (MCNA), polymeric coding nucleic acid (PCNA), guide RNA (gRNA), and CRISPR RNA (crRNA), preferably mRNA.

[0027] Preferably, the mass ratio of lipids to nucleic acids in the lipid nanoparticles of the present invention is less than 20:1, more preferably not more than 15:1, for example not more than 14:1, not more than 13:1, not more than 12:1, not more than 11:1, or not more than 10:1.

[0028] In some embodiments, the nucleic acid is mRNA or DNA encoding a chimeric antigen receptor (CAR) that targets a target molecule located in the liver. Optionally, the nucleic acid also encodes an antibody or its antigen-binding portion that targets an immune checkpoint molecule or a liver tumor-associated antigen, such as an antibody or its antigen-binding fragment that targets PD-1, PD-L1, TGF-β, or CTLA-4. Optionally, the antibody or its antigen-binding fragment is Fab, Fab', F(ab')2, Fd, Fv, a single-chain variable fragment (scFv), a single-chain antibody, VHH, vNAR, or a single-domain antibody.

[0029] In some embodiments, the nucleic acid comprises a nucleic acid sequence encoding a CAR polypeptide targeting GPC3 and a nucleic acid sequence encoding an antibody or antigen-binding fragment targeting PD-1, preferably operatively linked via a cleavable peptide linker encoding a sequence. Optionally, the cleavable peptide linker is a self-cleaving peptide linker, such as a P2A, T2A, or F2A peptide linker.

[0030] In some embodiments, the CAR polypeptide comprises a GPC3 binding domain, a transmembrane domain, and one or more intracellular domains, as well as optionally a leader peptide.

[0031] Optionally, the transmembrane domain comprises a transmembrane domain of CD4, CD8α, or CD28. Optionally, the one or more intracellular domains comprise a co-stimulatory domain or a portion thereof. Optionally, the co-stimulatory domain comprises one or more of CD3ζ, CD2, CD27, CD28, 4-1BB, OX-40, ICOS, IL-2Rβ, GITR, MyD88 / CD40a co-stimulatory domains, and / or variants thereof. Optionally, the intracellular domain comprises a CD3ζ co-stimulatory domain and / or a CD28 co-stimulatory domain.

[0032] In some embodiments, the CAR further includes a hinge / spacer subdomain, optionally wherein the hinge / spacer subdomain is located between the antigen-binding domain and the transmembrane domain.

[0033] Optionally, the hinge / spacer subdomain includes an IgG1 hinge domain or a variant thereof, an IgG2 hinge domain or a variant thereof, an IgG3 hinge domain or a variant thereof, an IgG4 hinge domain or a variant thereof, a CD8 hinge domain or a variant thereof, or a CD28 hinge domain or a variant thereof.

[0034] In some implementations, the GPC3 binding domain is scFv, and the nucleic acid sequence encoding the scFv is optionally shown in SEQ ID NO:4.

[0035] In some embodiments, the antibody targeting PD-1 or its antigen-binding fragment is an scFv, and optionally, the nucleic acid sequence encoding the scFv is shown in SEQ ID NO:10.

[0036] In some embodiments, the lipid nanoparticles have ligands on their surface that can target T cells or are modified with ligands that can target T cells on their surface.

[0037] In some embodiments, the ligand comprises one or more antibodies or their antigen-binding portions selected from: anti-CD3 antibody, anti-CD2 antibody, anti-CD4 antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD28 antibody, and anti-CD127 antibody. Optionally, the ligand is a ligand comprising an anti-CD3 antibody or its antigen-binding portion.

[0038] In some aspects, this disclosure provides lipid nanoparticles comprising the compositions disclosed herein.

[0039] In some aspects, this disclosure provides a method for preparing lipid nanoparticles from compositions as disclosed herein, the method comprising:

[0040] A lipid premix was prepared by mixing the ionizable cationic lipids, sterol lipids, auxiliary phospholipids, and polymeric phospholipids in predetermined proportions; and

[0041] The lipid premix is ​​mixed with a solution containing a target substance, such as a therapeutic agent or a preventative agent.

[0042] Preferably, the components of the lipid fraction are mixed with a solvent, and then mixed with a solution in which the target substance is dissolved. Preferably, the solvent is an organic solvent, preferably an alcohol solvent, and more preferably ethanol. Preferably, the target substance is nucleic acid, which is dissolved using a buffer solution, optionally an acetate or citrate solution.

[0043] In some embodiments, the lipid premix is ​​mixed with a solution containing the target substance in a microfluidic mixing system. For example, the lipid premix and the solution containing the target substance are mixed in the microfluidic mixing system at a specific flow rate and a flow ratio of about 1:3. In some examples, the flow rate is about 1-200 mL / min, for example about 10-200 mL / min, about 20-180 mL / min, or about 40-60 mL / min.

[0044] In some embodiments, the method further includes the step of modifying the lipid nanoparticles with a ligand by incubating the lipid nanoparticles with a solution containing the ligand. Preferably, the ligand comprises one or more antibodies selected from the group consisting of anti-CD3 antibodies, anti-CD2 antibodies, anti-CD4 antibodies, anti-CD7 antibodies, anti-CD8 antibodies, anti-CD28 antibodies, and anti-CD127 antibodies, and more preferably a ligand comprising an anti-CD3 antibody or an antigen-binding domain thereof.

[0045] In some aspects, this disclosure provides a method for engineering immune cells, the method comprising contacting the immune cells with a composition or lipid nanoparticles as disclosed herein, thereby introducing a target substance into the cells. Preferably, the immune cells are T cells.

[0046] In some aspects, this disclosure provides a method for engineering immune cells in a subject in vivo, the method comprising administering to the subject a composition as disclosed herein. The subject may be a mammal, such as a human or a non-human animal, preferably a human. The immune cells may be T cells, preferably liver T cells.

[0047] In some embodiments, the composition comprises a CAR encoding a tumor antigen targeting liver cancer, and optionally an antibody or antigen-binding fragment thereof targeting PD-1.

[0048] In some embodiments, the immune cells are selected from one or more of T cells, NK cells, monocytes, macrophages, dendritic cells, and NKT cells.

[0049] In some embodiments, the T cells are selected from one or more of CD4 T cells, CD8 T cells, and Gamma delta (γδ) T cells.

[0050] In some aspects, this disclosure provides a pharmaceutical composition comprising the composition disclosed herein, and a pharmaceutically acceptable carrier or excipient.

[0051] In some aspects, this disclosure provides methods for treating or preventing liver-related diseases, comprising administering compositions, lipid nanoparticles, or pharmaceutical compositions as disclosed herein. This disclosure also provides the use of compositions, lipid nanoparticles, or pharmaceutical compositions as disclosed herein in the preparation of medicaments for treating or preventing liver-related diseases. Optionally, the liver-related disease is selected from hepatocellular carcinoma, such as primary hepatocellular carcinoma or metastatic liver cancer.

[0052] In some aspects, this disclosure provides the use of compositions, lipid nanoparticles, or pharmaceutical compositions as disclosed herein in the preparation of medicaments for delivering a target substance selected from therapeutic and / or preventative agents. Preferably, the target site of delivery is the liver.

[0053] In some embodiments, the therapeutic or preventative agent is a nucleic acid, preferably selected from one or more of ASO, RNA, or DNA. More specifically, the RNA may be selected from one or more of interfering RNA (RNAi), small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aRNA), messenger RNA (mRNA), modified messenger RNA (mmRNA), long non-coding RNA (lncRNA), microRNA (miRNA), small activating RNA (saRNA), polymeric coding nucleic acid (MCNA), polymeric coding nucleic acid (PCNA), guide RNA (gRNA), CRISPR RNA (crRNA), or ribozymes, preferably mRNA, more preferably modified mRNA.

[0054] In some aspects, this disclosure provides for the use of compositions, lipid nanoparticles, or pharmaceutical compositions as disclosed herein in the preparation of vaccine compositions or in the preparation of vaccine compositions. The vaccine compositions may be used to treat or prevent liver-related diseases, such as hepatocellular carcinoma, including primary hepatocellular carcinoma and metastatic liver cancer. Attached Figure Description

[0055] Figure 1 shows (A) bioluminescence imaging of organs in ICR mice 6 h after drug treatment; (B) flow cytometry data of changes in CD3-positive T cells in the liver of ICR mice 6 h after drug treatment. Figure A shows the imaging results of major organs of mice after intraperitoneal injection of luciferase substrate following tail vein injection of tLNP, MC3LNP, and PBS. MC3LNP is a positive control, and PBS is a negative control.

[0056] Figure 2 shows (A) bioluminescence imaging of small animals 6 h after NOG treatment; (B) bioluminescence imaging of organs in NOG mice 6 h after NOG treatment. PBS served as a negative control.

[0057] Figure 3 shows (A) transmission electron microscopy image of αCD3-LNP; (B, C) particle size distribution of tLNP and αCD3-LNP; and (D) Zetal potential diagram of tLNP and αCD3-LNP.

[0058] Figure 4 shows the expression distribution of GPC3 in proteomics and transcriptomics data of different cancer cell lines.

[0059] Figure 5 shows flow cytometry data of GPC3 protein and PD-L1 expression levels in HCC cell lines and three PDX dissociated cells.

[0060] Figure 6 shows (A) a schematic diagram of the GPC3 & PD1scFv mRNA sequence; (B) a flow cytometry plot of the proportion of CAR Fab positive cells after T cells were treated with 100 ng / ml IgG-LNP / αCD3-MC3 LNP / αCD3-LNP; (C) a flow cytometry plot of the proportion of CAR Fab positive cells after T cells were treated with different doses of αCD3-LNP; (D) a quantitative plot of the proportion of CAR Fab positive cells after T cells were treated with different doses of IgG-LNP / αCD3-LNP, n=3; (E,F) flow cytometry and quantitative plots of the proportion of CAR Fab positive cells at different time points after T cells were treated with 1000 ng / ml αCD3-LNP; (G) a Western blotting plot of the cell supernatant of T cells treated with αCD3-LNP for 48 h; and (H) 100 ng / ml αCD3-LNP. Live-cell fluorescence imaging of CART cells (488 fluorescently labeled) obtained after treating T cells with GPC3&PD1scFvmRNA for 24 hours and co-cultured with Huh7 cells at a ratio of 6:1.

[0061] Figure 7 shows (A) the proportion of cancer cells killed by T cells after co-culturing with HCC cell lines for 48 h after treatment with 100 ng / ml αCD3-LNP; (B, C) flow cytometry and quantitative plots of the proportion of GPC3 CAR positive liver T cells in NOG mice after treatment with 10 μg αCD3-LNP for 2 / 5 / 8 days; (D) the proportion of CAR positive cells in liver CD3 positive cell suspension collected after tail vein injection of αCD3-LNP in HSC-NOG-EXL mice; (E) Western blotting plot of PD1scFv expression in liver cell suspension collected after tail vein injection of αCD3-LNP in HSC-NOG-EXL mice; and (F) IF slices of liver tumors in JHH7 liver-bearing NOG mice 24 h after tail vein injection of αCD3-LNP.

[0062] Figure 8 shows: (A) Time axis of anti-tumor evaluation in Huh7-luc orthotopic tumor-bearing NOG mice; (B) BLI imaging of Huh7-luc orthotopic tumor-bearing NOG mice during treatment; (C) Total BLI value quantification of Huh7-luc orthotopic tumor-bearing NOG mice on day 35; (D) Survival curve of Huh7-luc orthotopic tumor-bearing NOG mice; (E) Weight change of Huh7-luc orthotopic tumor-bearing NOG mice; (F) Time axis of anti-tumor evaluation in JHH7-luc orthotopic tumor-bearing NOG mice; (G) BLI imaging of JHH7-luc orthotopic tumor-bearing NOG mice during treatment; (H) Total BLI value quantification of JHH7-luc orthotopic tumor-bearing NOG mice on day 14; (I) Survival curve of JHH7-luc orthotopic tumor-bearing NOG mice; (J) Weight change of JHH7-luc orthotopic tumor-bearing NOG mice.

[0063] Figure 9 shows (A) the time axis of anti-tumor evaluation in Huh7-luc orthotopic tumor-bearing HSC-NOG-EXL mice; (B) BLI imaging during treatment in Huh7-luc orthotopic tumor-bearing HSC-NOG-EXL mice; (C) the total BLI value quantification of Huh7-luc orthotopic tumor-bearing HSC-NOG-EXL mice on day 33; (D) the survival curve of Huh7-luc orthotopic tumor-bearing HSC-NOG-EXL mice; and (E) the body weight change of Huh7-luc orthotopic tumor-bearing HSC-NOG-EXL mice.

[0064] Invention Details

[0065] In this invention, unless otherwise stated, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art. All patents, patent applications, and other publications cited herein are incorporated herein by reference in their entirety. Furthermore, the terms and laboratory procedures related to immunology, molecular biology, biochemistry, nucleic acid chemistry, cell and tissue culture, etc., used herein are widely used terms and routine procedures in their respective fields. If any definition presented herein conflicts with a definition presented in a patent, patent application, or other publication incorporated herein by reference, the definition presented herein shall prevail.

[0066] It should be noted that, unless the context clearly indicates otherwise, as used herein, the singular forms “a” and “the” include plural indicators. Thus, for example, a reference to “composition” includes multiple compositions and a reference to “cell” includes multiple cells, etc. Unless otherwise stated, “or” is used in an inclusive sense and means “and / or”.

[0067] Unless expressly stated in the foregoing description, embodiments in this specification that "comprise" various components are also contemplated as "composed of" or "substantially composed of" the components; embodiments in this specification that "composed of" various components are also contemplated as "comprising" or "substantially composed of" the components; embodiments in this specification that "about" various components are also contemplated as "being" the components; and embodiments in this specification that "substantially composed of" various components are also contemplated as "composed of" or "comprising" the components (this interchangeability does not apply to the use of these terms in the claims).

[0068] Numerical ranges include numbers within defined ranges. Taking into account significant figures and measurement-related errors, measured and measurable values ​​should be understood as approximate. As used herein, the term "about" has its meaning as understood in the art. Unless otherwise specified, numbers used herein, with or without modifiers such as "about," should be understood to cover normal divergence and / or fluctuations, as will be understood by one of ordinary skill in the art. In some embodiments, the term "about" refers to a range of values ​​(greater or less than) 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in any direction of the reference value, unless otherwise stated or otherwise apparent from the context (except where such a value would exceed 100% of the possible value). The expression "X±20%" as used in this application means that the value of X can fluctuate by 20%, that is, within the range of 0.8X to 1.2X. Similarly, the expressions "X±10%" or "X±5%" as used in this application mean that the value of X can fluctuate by 10% or 5%, that is, within the range of 0.9X to 1.1X, or within the range of 0.95X to 1.05X.

[0069] As used herein, “lipid nanoparticles (LNPs)” refer to particles having at least one nanometer (nm) scale size (e.g., 1 to 1000 nm). LNPs can be used to load nucleic acids, thereby protecting the nucleic acid payload from degradation or activation of RNA sensing mechanisms and subsequent innate immune responses, and enabling the delivery of the nucleic acid payload into the cytoplasm. LNPs can also be used as adjuvants in vaccination. LNPs are a versatile platform that can encapsulate various types of RNA as payloads and administer them via different routes. mRNA-LNPs have been successfully used in prophylactic vaccines against pathogens and have also been tested in oncology clinical trials.

[0070] As used in this article, "mRNA-LNP" refers to an LNP that uses mRNA (double-stranded or single-stranded) as the payload or target material. mRNA is typically synthesized via in vitro transcription and may contain an mRNA cap and a poly(A) tail, which contributes to mRNA stability and enhanced expression. mRNA-LNPs are prepared by mixing mRNA with various lipid components. mRNA-LNPs are typically 50–200 nm in diameter, uniformly distributed, and encapsulate nearly 100% of the nucleic acid payload. Under acidic pH conditions, the positive charge on the head group interacts with the negative charge on the RNA molecule, promoting the encapsulation and internalization of the nucleic acid payload within the LNP before its release into the cytoplasm.

[0071] As used herein, the terms “cationic lipid” and “ionizable cationic lipid” are used interchangeably, referring to lipids that are positively charged at any pH or hydrogen ion activity in their environment, or capable of becoming positively charged in response to the pH or hydrogen ion activity of their environment (e.g., the environment in which they are intended for use). Cationic lipids are synthetic lipids containing a lipid tail with a cationic ionizable head group. Cationic lipids have two key functions, including facilitating nucleic acid encapsulation in LNPs and mediating endosome membrane rupture to release nucleic acids into the cytoplasm. Furthermore, cationic lipids also play an important role in endosome uptake, either directly through the interaction of the positive charge on certain cationic lipids with the negatively charged cell membrane, or through binding to plasma proteins that support cellular uptake. Structurally, cationic lipids interact hydrostatically with the nucleic acid payload, forming inverted micelles around the nucleic acid. This allows helper phospholipids and sterols to spontaneously self-assemble into a nucleus, resulting in a solid nucleus LNP. Cationic lipids are considered a key component in terms of activity and have been the focus of many research attempts to optimize LNP formulations. Currently, the ionizable lipids in clinically approved mRNA-LNPs are ALC-0315, SM-102, and DLin-MC3-DMA, which are used for BNT162b2, mRNA-1273, and patisiran 2, respectively.

[0072] As used herein, the terms "non-cationic lipid" and "non-ionizable lipid" are used interchangeably and refer to lipids other than ionizable cationic lipids, and lipids that do not have a net positive charge at a selected pH, such as physiological pH. Examples of non-cationic lipids used in the lipid nanoparticle compositions of the present invention include polymeric phospholipids, accessory phospholipids, and structural lipids.

[0073] As used herein, the term "polymer lipid" refers to a molecule that contains both a lipid portion and a polymer portion. In this invention, polymer lipids generally refer to polymer phospholipids.

[0074] As used herein, “structural lipids” refers to lipids that enhance the stability of nanoparticles by filling the gaps between lipids. Sterol lipids are common structural lipids, including cholesterol or sterol analogs. Cholesterol or sterol analogs are compounds with a cyclopentane-polyhydrophenanthrene carbon skeleton.

[0075] As used herein, the terms "specific targeting" or "specific targeting" refer to a process that promotes the delivery of an agent (such as the therapeutic payload in the lipid nanoparticle composition described herein) to a specific organ, tissue, cell, and / or intracellular compartment (referred to as a target site) compared to delivery to any other organ, tissue, cell, or intracellular compartment (referred to as a non-target site). Specific targeting can be detected using methods known in the art, for example by comparing the concentration of the delivered agent in a target cell population with the concentration of the delivered agent in a non-target cell population after systemic administration. In some embodiments, specific targeting results in a concentration at the target site that is at least twice as high as the concentration at the non-target site. Accordingly, "specific targeting" means that the composition has the function of achieving specific targeting.

[0076] As used herein, the term "T cell" refers to lymphocytes derived from the thymus and playing a crucial role in cell-mediated immunity. T cells include CD4+ T cells, CD8+ T cells, memory T cells, regulatory T cells, Gamma delta (γδ) T cells, and natural killer T cells, among others. For example, T cells incorporating CARs or fusion proteins can be CD8+ T cells, CD4+ T cells, or a combination of both.

[0077] As used herein, the terms “antibody” and “its antigen-binding portion” refer to at least the smallest portion of an antibody capable of binding to a specified antigen targeted by the antibody, such as, in the case of typical antibodies produced by B cells, at least some complementarity-determining regions (CDRs) of the variable domains of the heavy chain (VH) and the variable domains of the light chain (VL). In some antibodies, such as naturally occurring IgG antibodies, the heavy chain constant region consists of a hinge region and three domains CH1, CH2, and CH3. In some antibodies, such as naturally occurring IgG antibodies, each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of both the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including different cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The heavy chain may or may not have a C-terminal lysine. Unless otherwise stated herein, amino acids in the variable regions are numbered using the Kabat numbering system, and amino acids in the constant regions are numbered using the EU system.

[0078] Antibodies or their antigen-binding fragments can be derived from or be derived from polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, or chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab, Fab', and F(ab')2, Fd, Fv, single-chain variable fragments (scFv), single-chain antibodies, VHH, vNAR, nanobodies (single-domain antibodies), disulfide-linked Fv (sdFv), fragments containing a single VL or VH domain or a portion thereof that binds to an opposite domain (e.g., the entire VL domain and a portion of the VH domain having one, two, or three CDRs), and fragments generated from Fab expression libraries. ScFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019. The antibody molecules covered by this disclosure can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules.

[0079] As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any one or more chains of two or more amino acids. Therefore, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to one or more chains having two or more amino acids is included in the definition of "polypeptide," and the term "polypeptide" may be used in place of any of these terms, or used interchangeably with them.

[0080] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an engineered antigen-binding polypeptide comprising an antigen-binding domain, a transmembrane domain, and one or more intracellular domains (e.g., co-stimulatory domains). In some embodiments, the CAR may optionally comprise a spacer domain and / or a flexible hinge domain to provide conformational freedom to facilitate binding to target antigens on target cells. In some embodiments, the CAR may optionally comprise an armor domain containing a nucleic acid sequence encoding an armor molecule. Expression of the CAR on the surface of cells (e.g., immune cells) allows the cells to target and bind specific antigens. In some embodiments, the CAR is expressed by immune cells (e.g., T cells). In some embodiments, the antigen-binding domain comprises Fab, Fab', F(ab')2, Fd, Fv, single-chain variable fragment (scFv), single-chain antibody, VHH, vNAR, nanobody (single-domain antibody), or any combination thereof. In some embodiments, the transmembrane domain comprises a transmembrane domain selected from CD4, CD8α, or CD28. In some embodiments, one or more intracellular domains include a co-stimulatory domain or a portion thereof. In some embodiments, the intracellular domain includes a co-stimulatory domain or a portion thereof. In some embodiments, the intracellular domain includes a co-stimulatory domain of CD3z or a variant thereof. For example, a CD3z co-stimulatory domain variant may contain only one or two functional immune receptor tyrosine-based activation motifs (ITAMs) of the three ITAMs present in wild-type CD3z. In some embodiments, the intracellular domain includes a co-stimulatory domain selected from the group consisting of: CD3ζ co-stimulatory domain, CD28 co-stimulatory domain, CD27 co-stimulatory domain, 4-1BB co-stimulatory domain, ICOS co-stimulatory domain, OX-40 co-stimulatory domain, GITR co-stimulatory domain, CD2 co-stimulatory domain, IL-2Rβ co-stimulatory domain, MyD88 / CD40 co-stimulatory domain, and any combination thereof. The CAR may further include a "hinge region" or "spacer" domain. Non-limiting examples of hinge / spacer subdomains include immunoglobulin hinge / spacer subdomains, such as IgG1 hinge domains and IgG2 hinge domains, IgG3 hinge domains, IgG4 hinge domains, IgG4P hinge domains (including IgG4 hinge domains containing the S241P mutation), or CD8a hinge domains, or CD28 hinge domains.

[0081] As used herein, the term “polynucleotide” includes both single and multiple nucleic acids, and refers to isolated nucleic acid molecules or constructs, such as messenger RNA (mRNA) or plasmid DNA (pDNA).

[0082] The term "nucleic acid" includes any type of nucleic acid, such as DNA or RNA.

[0083] "Conservative amino acid substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are defined in the art. These families include amino acids having the following: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, a predicted non-essential amino acid residue in the GPC3 binding moiety (e.g., an anti-GPC3 CAR or antibody) is substituted with another amino acid residue from the same side chain family.

[0084] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. When a position in two compared sequences is occupied by the same base or nucleotide monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. Typically, two sequences are compared to produce the maximum identity. Such comparisons can be performed using methods well known to those skilled in the art, for example, conveniently performed using computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48:443-453.

[0085] As used in this article, “immunotherapy” refers to the treatment of a subject who has a disease or is at risk of contracting or relapsing a disease by means of methods including inducing, enhancing, suppressing or otherwise altering the immune system or immune response.

[0086] In this document, "immune response" is as understood in the art and generally refers to the biological response in a vertebrate to foreign agents or abnormalities such as cancer cells, which protects the organism from these agents and the diseases they cause. An immune response is mediated by the action of one or more cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by these cells or any of them in the liver, resulting in the selective targeting, binding, damage, destruction, and / or clearance from the vertebrate body of invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues. Immune responses include, for example, the activation or suppression of T cells, such as effector T cells, Th cells, CD4+ cells, CD8+ T cells, or Treg cells, or the activation or suppression of any other cells of the immune system, such as NK cells.

[0087] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintaining agents, absorption delaying agents, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintaining agents include, but are not limited to, sugars, NaCl, and their analogues. Absorption delaying agents include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Stabilizers have the meaning commonly understood by those skilled in the art as being able to stabilize the desired properties of the active ingredient in a pharmaceutical product, including but not limited to monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate). In some exemplary embodiments, the pharmaceutically acceptable carrier or excipient includes sterile injectable liquids (such as aqueous or non-aqueous suspensions or solutions). In some exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solutions (e.g., 0.9% (w / v) NaCl), glucose solutions (e.g., 5% glucose), solutions containing surfactants (e.g., 0.01% polysorbate 20), pH buffer solutions (e.g., phosphate buffer solutions), Ringer's solutions, and any combination thereof.

[0088] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom (e.g., a tumor) in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the extent of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or alleviating the progression of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to expected survival (if no treatment was received).

[0089] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing disease (e.g., cancer) means an amount sufficient to prevent, stop, or delay the onset of disease (e.g., cancer); an effective amount for treating disease means an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and any other concurrent treatments.

[0090] The term "combination" and related terms refer to the simultaneous or sequential administration of the pharmaceutical composition of the present invention and other therapeutic agents. For example, the pharmaceutical composition of the present invention may be administered simultaneously or sequentially with other therapeutic agents in separate unit dosage forms, or simultaneously with other therapeutic agents in a single unit dosage form.

[0091] As used in this article, the term “subject” refers to both human and non-human animals, including rodents, mammals such as primates, and humans.

[0092] Lipid compositions and lipid nanoparticles

[0093] This disclosure provides lipid compositions containing one or more types of lipid molecules and lipid nanoparticles (LNPs) assembled from said lipid compositions. The LNPs can be used to deliver various types of target substances, such as nucleic acid molecules, including mRNA, DNA, sRNA, ASO, or proteins. The LNPs can be presented as substantially spherical hollow bodies capable of encapsulating the target substance in their central lumen. In some embodiments, the LNP compositions of the present invention comprise an LNP and at least one target substance encapsulated within the LNP. The lipid compositions and LNPs provided by the present invention have liver-targeting properties and are therefore particularly suitable for the specific delivery of target substances to the liver of a subject.

[0094] The lipid molecules contained in the lipid composition and LNP may include, but are not limited to, ionizable cationic lipids, accessory phospholipids, cholesterol, and polymeric phospholipids. Ionizable cationic lipids can be used to control the acid dissociation constant of the LNP. Polymeric phospholipids, also known as anti-aggregation phospholipids, contain lipids capable of preventing lipid particle aggregation, preferably PEG (polyethylene glycol) modified lipids. Cholesterol, as a structural lipid, is used to prevent leakage of encapsulating substances. Depending on their components, the lipid composition and LNP may have different characteristics, including charge, particle size distribution, zeta potential, and targeting (e.g., subject organ targeting). For example, an LNP containing cholesterol as a structural lipid may have different characteristics than an LNP containing different structural lipids. Similarly, the characteristics of the lipid composition and LNP are also affected by the absolute or relative amounts of their components. For example, a lipid nanoparticle composition containing a higher molar ratio (mol%) of accessory phospholipids may have different characteristics than a lipid nanoparticle composition containing a lower molar ratio of accessory phospholipids. These characteristics may also vary with the preparation method and conditions of the lipid nanoparticle composition.

[0095] Examples of cationic lipids that can be used in the lipid compositions and LNPs of the present invention include, but are not limited to, 3-(bisdodecylamino)-N1,N1,4-tris(dodecyl)-1-piperazine ethylamine (KL10), N1-[2-(bisdodecylamino)ethyl]-N1,N4,N4-tris(dodecyl)-1,4-piperazine diethylamine (KL22), 14,25-bistridecyl-15,18,21,24-tetraaza-octacosane (KL25), and 1,2-dilinyloxy-N,N-dimethylaminopropane (D... LinDMA), 2,2-dilinole-4-dimethylaminomethyl-[1,3]-dioxacyclopentane (DLin-K-DMA), 4-(dimethylamino)butyric acid 37-carbon-6,9,28,31-tetraen-19-yl ester (DLin-MC3-DMA), 2,2-dilinole-4-(2-dimethylaminoethyl)-[1,3]-dioxacyclopentane (DLin-KC2-DMA), 1,2-dioleoyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[( (3β)-cholesterol-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholesterol-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA(2R)), (2S)-2-({8- [(3β)-cholesterol-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z-,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA(2S)), (12Z,15Z)-N,N-dimethyl-2-nonyltetradec-12,15-dien-1-amine, N,N-dimethyl-1-{(1S,2R)-2-octylcyclopropyl}heptadecane-8-amine, and 1,2-dioleoyl-3-dimethylammonium-propane (DODAP). Other cationic lipids that can be used in this invention include those described in US Patent No. US2010324120A1 and International Patent Application No. PCT / US2012 / 068491. In some embodiments, the cationic lipid is selected from DLin-MC3-DMA, ALC-0315, SM-102, and DODAP.

[0096] In some embodiments, the lipid composition and LNP of the present invention comprise two or more cationic lipids. In some embodiments, the lipid composition and LNP of the present invention comprise a first cationic lipid and a second cationic lipid, wherein the first cationic lipid is selected from DLin-MC3-DMA, ALC-0315, and SM-102, and the second cationic lipid is selected from DODAP, DODMA, DLinDMA, KL10, KL22, and KL25. In some embodiments, the first cationic lipid is DLin-MC3-DMA and the second cationic lipid is DODAP. In some embodiments, the molar ratio of the first cationic lipid to the second cationic lipid is about (48.6 ± 10%):(3.2 ± 10%), more preferably about (48.6 ± 5%):(3.2 ± 5%), and more specifically about 48.6:3.2.

[0097] Examples of structural lipids that can be used in the lipid compositions and LNPs of the present invention are sterol analogs, such as those selected from cholesterol, sitosterol, coccosterol, rock saponin, campesterol, ergosterol, tomatine, ursolic acid, α-tocopherol, stigmasterol, alfalfa sterol, ergocalciferol, or campesterol. In some embodiments, the lipid compositions and LNPs of the present invention comprise cholesterol.

[0098] Examples of phospholipids that can be used in the lipid compositions and LNPs of the present invention include, but are not limited to, 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (POPE), and 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE). In some embodiments, the lipid composition and LNP of the present invention contain DOPE.

[0099] Examples of polymeric phospholipids that can be used in the lipid compositions and LNPs of the present invention are polyethylene glycol-modified (PEG) phospholipids. Other lipids capable of reducing aggregation, such as products of lipid coupling with compounds having uncharged, hydrophilic, sterically blocking moieties, may also be used. In some embodiments, the PEG-modified phospholipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol; preferably, the PEG-modified phospholipid contains a PEG moiety of about 1000 Da to about 20 kDa, more preferably a PEG moiety of about 1000 Da to about 5000 Da. Preferably, the polyethylene glycol-modified phospholipid is selected from one or more of DMPE-PEG1000, DPPE-PEG1000, DSPE-PEG1000, DOPE-PEG1000, DMG-PEG2000, Ceramide-PEG2000, DMPE-PEG2000, DPPE-PEG2000, DSPE-PEG2000, Azido-PEG2000, DSPE-PEG2000-Mannose, Ceramide-PEG5000, and DSPE-PEG5000, with DMG-PEG2000 being the most preferred. The polyethylene glycol-modified (PEG) lipid may also have other modifications, such as an NHS group. DMG-PEG-NHS has high reactivity and can react with molecules containing amino groups (such as -NH2) to form stable amide bonds. In some embodiments, the polyethylene glycol-modified phospholipids are DMG-PEG2000 and DMG-PEG2000-NHS; preferably, DMG-PEG2000 and DMG-PEG2000-NHS are present in the lipid composition and LNP of the present invention in the same molar percentage.

[0100] Lipid nanoparticle compositions can be characterized using a variety of methods. For example, microscopic examination (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of the lipid nanoparticle composition. Dynamic light scattering or potentiometry (e.g., potentiometric titration) can be used to measure the zeta potential. Dynamic light scattering can also be used to determine particle size. Instruments such as the Zetasizer Nano ZS (Malvem Instruments Ltd, Malvem, Worcestershire, UK) can also be used to measure multiple characteristics of the lipid nanoparticle composition, such as particle size, polydispersity index, and zeta potential.

[0101] The average size of the lipid nanoparticle composition, or LNP, can range from tens of nanometers to hundreds of nanometers. In some embodiments, the LNP composition has an average particle size of about 100 nm to about 200 nm, such as about 180 nm, 179 nm, 178 nm, 177 nm, 176 nm, 175 nm, 174 nm, 173 nm, 172 nm, 171 nm, 170 nm, or smaller. In some embodiments, the LNP composition has an average particle size of about 170 nm.

[0102] The zeta potential of a lipid nanoparticle composition can be used to indicate the potentiodynamics of the composition. For example, the zeta potential can describe the surface charge of the lipid nanoparticle composition. Lipid nanoparticle compositions with relatively low positive or negative charges are generally desirable because substances with higher charges can interact undesirably with cells, tissues, and other components in the body. In some embodiments, the average zeta potential of the LNP composition can be about -10 mV to about +20 mV, about -5 mV to about +10 mV, about 0 mV to about +10 mV, about +5 mV to about +10 mV, or about +6 mV to about +9 mV. In some embodiments, the average zeta potential of the LNP composition can be about +6 mV to about +10 mV.

[0103] LNPs with liver targeting

[0104] In some aspects, the present invention provides lipid compositions and LNPs with liver-targeting properties. The particle size, charge, and component ratio of LNPs affect their distribution bias in organs in vivo. Adjusting the content of lipid components therein to achieve specific transport of LNPs to certain organs is a strategy that has been preliminarily proven feasible. Preferably, the present invention provides lipid compositions and LNPs comprising cationic lipids, cholesterol, polymeric phospholipids, and helper phospholipids of specific compositions, thereby endowing them with targeting properties to the liver site or liver cells.

[0105] When LNPs are administered systemically, such as via intravenous injection, they enter the bloodstream and interact nonspecifically with serum proteins (e.g., complement proteins or immunoglobulins), leading to aggregation or opsonization. These opsonized particles are prematurely cleared from the alveolar capillaries by mechanically trapping aggregates larger than 7 μm, and / or by RES (reticuloendothelial system) macrophages residing in the liver, spleen, and bone marrow. Hepatic sinusoidal endothelial cells (SECs), part of the RES, internalize opsonized particles larger than 0.23 μm via their Fc and scavenger receptors; Kupffer cells (KCs) also internalize larger particles.

[0106] The inventors have discovered that by altering the proportions of lipid components, a novel LNP composition was obtained, resulting in LNPs with higher liver-targeting specificity and in situ liver expression efficiency, and lower toxicity. This invention also provides a method for selectively delivering LNPs, with or without a target substance, to liver tissues and / or cells. Taking mRNA-LNPs as an example, they enter liver cells via macropinocytosis and clathrin-mediated endocytosis. Once the mRNA payload is released into the cytoplasm, it is translated into the desired protein by the cell's ribosomes. In some embodiments, the target substance encapsulated by the LNP is an mRNA to be expressed in liver cells, such as an antibody encoding an aberrantly expressed tumor antigen in the liver, or its antigen-binding fragment.

[0107] In some embodiments, the lipid component in the LNP composition disclosed herein has a mass ratio of less than 20:1 to the target substance (e.g., nucleic acid), preferably less than 15:1, for example, a mass ratio of about 14:1, 13:1, 12:1, 11:1, 10:1 or lower, preferably a mass ratio of about 10:1.

[0108] In some embodiments, the lipid component of the LNP composition comprises the following molar percentages: ionizable cationic lipids (51.8 ± 10.36) mol%, preferably (51.8 ± 5.18) mol%; structural lipids (37.2 ± 7.44) mol%, preferably (37.2 ± 3.72) mol%; accessory phospholipids (9.7 ± 1.94) mol%, preferably (9.7 ± 0.97) mol%; and polymeric phospholipids (1.3 ± 0.26) mol%, preferably (1.3 ± 0.13) mol%. More preferably, the lipid component of the LNP composition comprises the following molar percentages: ionizable cationic lipids (51.8 ± 2.59) mol%, structural lipids (37.2 ± 1.86) mol%, accessory phospholipids (9.7 ± 0.485) mol%, and polymeric phospholipids (1.3 ± 0.065) mol%.

[0109] In some embodiments, the LNP composition comprises lipid components of a first cationic lipid, a second cationic lipid, a structural lipid, an accessory phospholipid, and a polymeric phospholipid, wherein the first cationic lipid is selected from DLin-MC3-DMA, ALC-0315, and SM-102, the second cationic lipid is DODAP, the structural lipid is cholesterol, the accessory phospholipid is DOPE, and the polymeric phospholipid is DMG-PEG2000, DMG-PEG2000-NHS, or a combination thereof; preferably, the lipid components have the following molar percentages: first cationic lipid (48.6±9.72) mol%, second cationic lipid (3.2±0.64) mol%, cholesterol (37.2±7.44) mol%, DOPE (9.7±1.94) mol%, and polymeric phospholipid (1.3±0.26) mol%.

[0110] In some embodiments, the LNP composition comprises lipid components including a first cationic lipid, a second cationic lipid, a structural lipid, an accessory phospholipid, and a polymeric phospholipid, wherein the first cationic lipid is DLin-MC3-DMA, the second cationic lipid is selected from DODAP, DODMA, DLinDMA, KL10, KL22, and KL25, the structural lipid is cholesterol, the accessory phospholipid is DOPE, and the polymeric phospholipid is DMG-PEG2000, DMG-PEG2000-NHS, or a combination thereof; preferably, the lipid components have the following molar percentages: first cationic lipid (48.6±9.72) mol%, second cationic lipid (3.2±0.64) mol%, cholesterol (37.2±7.44) mol%, accessory phospholipid (9.7±1.94) mol%, and polymeric phospholipid (1.3±0.26) mol%.

[0111] In some embodiments, the LNP composition comprises lipid components including a first cationic lipid, a second cationic lipid, a structural lipid, an accessory phospholipid, and a polymeric phospholipid, wherein the first cationic lipid is DLin-MC3-DMA, the second cationic lipid is DODAP, the structural lipid is selected from cholesterol, sitosterol, coccosterol, lycopene, campesterol, ergosterol, tomatine, ursolic acid, α-tocopherol, stigmasterol, alfalfa sterol, ergocalciferol, or campesterol, the accessory phospholipid is DOPE, and the polymeric phospholipid is DMG-PEG2000, DMG-PEG2000-NHS, or a combination thereof; preferably, the lipid components have the following molar percentages: first cationic lipid (48.6±9.72) mol%, second cationic lipid (3.2±0.64) mol%, structural lipid (37.2±7.44) mol%, accessory phospholipid (9.7±1.94) mol%, and polymeric phospholipid (1.3±0.26) mol%.

[0112] In some embodiments, the LNP composition comprises lipid components including a first cationic lipid, a second cationic lipid, a structural lipid, an accessory phospholipid, and a polymeric phospholipid, wherein the first cationic lipid is DLin-MC3-DMA, the second cationic lipid is DODAP, the structural lipid is cholesterol, the accessory phospholipid is selected from DSPC, DPPC, POPC, and DOPE, and the polymeric phospholipid is DMG-PEG2000, DMG-PEG2000-NHS, or a combination thereof; preferably, the lipid components have the following molar percentages: DLin-MC3-DMA (48.6±9.72) mol%, DODAP (3.2±0.64) mol%, cholesterol (37.2±7.44) mol%, accessory phospholipid (9.7±1.94) mol%, and polymeric phospholipid (1.3±0.26) mol%.

[0113] In some embodiments, the LNP composition comprises a first cationic lipid, a second cationic lipid, a structural lipid, an accessory phospholipid, and a polymeric phospholipid, wherein the first cationic lipid is DLin-MC3-DMA, the second cationic lipid is DODAP, the structural lipid is cholesterol, the accessory phospholipid is DOPE, and the polymeric phospholipid is selected from DMPE-PEG1000, DPPE-PEG1000, DSPE-PEG1000, DOPE-PEG1000, DMG-PEG2000, DMG-PEG2000-NHS, Ceramide-PEG2000, and DMPE-PEG2000. 00, DPPE-PEG2000, DSPE-PEG2000, Azido-PEG2000, DSPE-PEG2000-Mannose, Ceramide-PEG5000, DSPE-PEG5000 and their NHS modifications; preferably, the lipid components have the following molar percentages: DLin-MC3-DMA (48.6±9.72) mol%, DODAP (3.2±0.64) mol%, cholesterol (37.2±7.44) mol%, DOPE (9.7±1.94) mol%, and polymeric phospholipids (1.3±0.26) mol%.

[0114] In a specific embodiment, the lipid components included in the LNP composition are DLin-MC3-DMA, DOPE, DODAP, cholesterol, and DMG-PEG2000, and the molar ratio of DLin-MC3-DMA, DOPE, DODAP, cholesterol, and DMG-PEG2000 is (48.6±20%):(9.7±20%):(3.2±20%):(37.2±20%):(1.3±20%). More preferably, the molar ratio is (48.6±10%):(9.7±10%):(3.2±10%):(37.2±10%):(1.3±10%), and even more preferably (48.6±5%):(9.7±5%):(3.2±5%):(37.2±5%):(1.3±5%). In one specific example, the molar ratio is 48.6:9.7:3.2:37.2:1.3. Depending on whether liposome ligand modification is required, a portion (e.g., 50%) of DMG-PEG2000 may be modified with NHS.

[0115] LNPs modified to target T cells

[0116] The present invention further modifies or optimizes the distribution and pharmacokinetic properties of LNPs in vivo by modifying the surface of LNPs with ligands that specifically bind to receptors of target cells (e.g., T cells), thereby achieving more precise targeted delivery (active targeting).

[0117] In some further aspects, this disclosure provides modified LNPs with increased binding capacity to T cells. By combining LNP modification with liver-specific targeting, the LNPs provided in this disclosure can enhance the uptake of LNPs by hepatic T cells. Furthermore, the LNPs can efficiently transfect T cells at low concentrations, and when the target material encapsulated by the LNPs is a nucleic acid encoding a CAR, the LNPs can be used to prepare CAR-T cells.

[0118] As will be readily understood by those skilled in the art, the modification is typically on the surface of lipid nanoparticles. In some embodiments, the modification is a ligand capable of targeting a specific cell type, such as a ligand targeting T cells, which can specifically recognize surface molecules specifically or highly expressed in T cells. In some embodiments, the ligand is a ligand comprising an antigen-binding domain selected from one or more antibodies: anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD28 antibody, and anti-CD127 antibody. The antibody may be an antibody known to those skilled in the art, a commercially available antibody, or a laboratory-derived antibody, as long as the antibody provides targeting to the target cell. For the in vivo delivery of the LNP compositions disclosed herein to T cells, a preferred example of the ligand is an anti-CD3 antibody or its antigen-binding domain. Here, "antigen-binding domain" is synonymous with the antigen-binding domain constituting a CAR, which may take the form of common forms such as scFv, Fab, VHH (if the antibody is sdAb).

[0119] The ligand can bind to the shell of the LNP in any manner, as long as it is present on the surface of the lipid nanoparticle. For example, when a terminally reactive PEG lipid is used as a polymeric phospholipid, the ligand can be added to the end of the PEG. For example, lipid nanoparticles labeled with the ligand (antibody) can be prepared by reacting a PEG lipid with maleimide groups introduced at the ends (e.g., SUNBRIGHT DSPE-0200MA) with the thiol groups of the reduced antibody described above. In some embodiments, the LNP is modified by incubating the LNP with an antibody solution.

[0120] Target material delivered by LNP

[0121] The target substance in the LNP composition of the present invention can be a bioactive agent, such as a therapeutic or preventative agent. In some embodiments, the target substance is or comprises one or more bioactive agents, particularly bioactive agents to be expressed in the liver, such as nucleic acids such as mRNA, guide RNA, RNA-directed DNA binders, expression vectors, template nucleic acids, antibodies (e.g., monoclonal, chimeric, humanized, nanobodies and fragments thereof), cholesterol, hormones, peptides, proteins, chemotherapeutic agents and other types of antitumor agents, low molecular weight drugs, vitamins, cofactors, nucleosides, nucleotides, oligonucleotides, enzyme nucleic acids, antisense nucleic acids, triple-helical oligonucleotides, antisense DNA or RNA compositions, chimeric DNA:RNA compositions, homoenzymes, aptamers, etc. Ribozymes, baits and their analogues, plasmids and other types of vectors, as well as small nucleic acid molecules, RNAi agents, short interfering nucleic acids (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and "self-replicating RNA" molecules (encoding replicase activity and capable of guiding its own replication or amplification in vivo), peptide nucleic acids (PNA), locked ribonucleotides (LNA), morpholinonucleotides, threonine nucleic acids (TNA), glycol nucleic acids (GNA), sisiRNA (small internally fragmented interfering RNA), and iRNA (asymmetric interfering RNA). Such target substances can be purified or partially purified, and can be naturally occurring or synthetic, and can be chemically modified.

[0122] In some embodiments, the therapeutic or preventative agent is a nucleic acid, particularly a nucleic acid to be expressed in liver cells. Preferably, the nucleic acid is selected from one or more of ASO, RNA, or DNA. In some embodiments, the RNA is selected from one or more of interfering RNA (RNAi), small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aRNA), messenger RNA (mRNA), modified messenger RNA (mmRNA), long non-coding RNA (lncRNA), microRNA (miRNA), small activating RNA (saRNA), polymeric coding nucleic acid (MCNA), polymeric coding nucleic acid (PCNA), guide RNA (gRNA), CRISPR RNA (crRNA), or ribozymes, preferably mRNA, more preferably modified mRNA.

[0123] mRNA is a nucleic acid molecule with a strong negative charge (due to its phosphate backbone), while the cationic lipids or ionizable lipids such as DLin-MC3-DMA contained in LNP are positively charged under weakly acidic conditions (pH 4-6) and bind to the negative charge of mRNA to form a complex, which can bind to mRNA through electrostatic interactions.

[0124] In some embodiments, the mRNA encodes one or more of the following: chimeric antigen receptors, antibodies, and their antigen-binding fragments (e.g., Fab, scFv, VHH, etc.). Chimeric antigen receptors, antibodies, and their antigen-binding fragments can target tumor-associated antigens and immune checkpoint molecules. In some specific embodiments, the mRNA encodes a CAR targeting GPC3 and an scFv targeting PD-1.

[0125] Preparation of LNP Compositions

[0126] In one aspect, this invention provides a method for preparing the lipid compositions and LNPs disclosed herein. The preparation of LNPs depends on their self-assembly capability, i.e., the spontaneous organization of lipid components into nanostructure entities through intermolecular interactions. For nucleic acids as the target substance, firstly, negatively charged nucleic acids and positively charged lipids bind electrostatically, and then assemble through hydrophobic and van der Waals interactions between the lipid components, thereby forming encapsulated loaded LNPs. The LNP preparation scheme affects the self-assembled product in at least two ways: the uniformity of the LNP and the nucleic acid loading efficiency.

[0127] In some embodiments, LNPs with high particle size uniformity, stable properties, and high nucleic acid loading efficiency are produced by uniformly mixing the lipid premix with a solution of the target substance (e.g., mRNA dissolved in a buffer) using a microfluidic device. Specifically, the method includes premixing the components of the lipid component and then mixing it with a solution of the target substance. In some embodiments, the lipid component in the LNP composition contains the following components in molar percentages: ionizable cationic lipids (51.8 ± 10.36) mol%, preferably (51.8 ± 5.18) mol%; cholesterol (37.2 ± 7.44) mol%, preferably (37.2 ± 3.72) mol%; accessory phospholipids (9.7 ± 1.94) mol%, preferably (9.7 ± 0.97) mol%; and polymeric phospholipids (1.3 ± 0.26) mol%, preferably (1.3 ± 0.13) mol%. More preferably, in the LNP composition, the lipid component comprises the following components in molar percentages: ionizable cationic lipids (51.8 ± 2.59) mol%, cholesterol (37.2 ± 1.86) mol%, auxiliary phospholipids (9.7 ± 0.485) mol%, and polymeric phospholipids (1.3 ± 0.065) mol%. Pipettes, microfluidic mixing systems (e.g., Asia microfluidic systems (Syrris)), or microfluidic mixers (e.g., Classic Mixer or NanoAssemblr NxGen TM Mixer (Precision Nanosystems), or based on GenmixTM Nanomedicine manufacturing systems such as INano TM Platform, Genmix TM The above mixing is performed using an SDM or MDM mixer. The obtained lipid nanoparticles can be purified by ultrafiltration, dialysis, or sterile filtration.

[0128] In some embodiments, the lipid components of the LNP (e.g., ionizable cationic lipids, cholesterol, and cofactor phospholipids) are dissolved in a solvent under suitable conditions to prepare a solution and then mixed in appropriate molar ratios to prepare a lipid premix. The lipid components can be prepared as separate solutions or in the same solution and then mixed in appropriate molar ratios to prepare the lipid premix. Suitable preparation methods for solutions containing different lipid components are well known to those skilled in the art. The solvent is preferably an organic solvent, such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, tert-butanol, acetone, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, or mixtures thereof, for example, ethanol. In some embodiments, the target substance is nucleic acid, which is dissolved using a buffer solution selected from acidic buffer solutions (e.g., acetate buffer solution, citrate buffer solution) or neutral buffer solutions (e.g., 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, (HEPE) buffer solution, tris(hydroxymethyl)aminomethane (Tris) buffer solution, phosphate buffer solution, phosphate-buffered saline (PBS)).

[0129] In some embodiments, the lipid premix is ​​mixed with a solution containing the target substance in a microfluidic mixing system. Further, after thorough mixing, it is diluted with buffer, centrifuged, and concentrated to produce concentrated LNPs. In some embodiments, the lipid premix is ​​mixed with a solution containing the target substance in a microfluidic mixing system. As described above, the LNPs can also be incubated with a solution containing T-cell targeting ligands to achieve targeting of hepatic T cells. In some embodiments, the mass ratio of lipid components to nucleic acids in the lipid nanoparticle composition of the present invention obtained as described above is less than 15:1, for example, about 14:1, 13:1, 12:1, 11:1, 10:1, or lower; preferably, the mass ratio is about 10:1.

[0130] Constructing immune cells expressing CAR

[0131] This invention relates to methods for constructing immune cells expressing CARs in vivo or in vitro. The invention provides a method for engineering immune cells by introducing the target substance from the LNP disclosed herein.

[0132] In some implementations, the target substance is a nucleic acid encoding a CAR and the immune cell is a T cell, thereby generating engineered CAR-T cells. The CAR-T cells express a CAR and optionally one or more other proteins. A CAR generally comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain. The antigen-binding domain is often derived from an antibody or its antigen-binding fragment capable of recognizing and binding specific antigens, for example, in the form of scFv, VHH, or Fab. CARs recognize cell surface markers associated with specific disease states, such as tumors, by selecting appropriate antigen-binding domains. The intracellular signal transduction domain is used to transduce effector signals and functional signals and guide cells to perform specific functions (e.g., cytolytic or co-activating activities, including cytokine secretion). It typically comprises a primary signal transduction domain and a co-stimulatory signal transduction domain. The primary signal transduction domain is a protein motif capable of regulating the primary activation of the TCR complex in a stimulatory or inhibitory manner. The stimulatory primary signal transduction domain typically contains a signal transduction motif known as an immune receptor tyrosine-based activation motif (ITAM). Costimulatory signaling domains are intracellular signaling domains derived from costimulatory molecules. Costimulatory molecules are cell surface molecules, other than antigen receptors or Fc receptors, that provide the second signal required for the efficient activation and function of T lymphocytes after binding to antigens.

[0133] As used herein, "immune cell" is not particularly limited, as long as it is a cell capable of destroying target cells (pathogenic cells) such as cancer cells through some mechanism of action (i.e., immune effector cells). Examples include T cells responsible for cell-mediated immunity in acquired immunity; NK cells, monocytes, macrophages, dendritic cells, etc., responsible for innate immunity; and NKT cells, which are T cells with NK cell characteristics. In a preferred embodiment, the immune cell is a T cell.

[0134] In some embodiments, the present invention provides a method for constructing / preparing CAR-expressing immune cells (e.g., CAR-T) in vitro or in vitro, the method comprising:

[0135] The process involves contacting immune cells (e.g., T cells) with the LNP composition of the present invention and introducing nucleic acids encoding CAR (optionally, other proteins) into the cells; and collecting the CAR-expressing immune cells. The immune cells may be autologous or allogeneic.

[0136] In some embodiments, the present invention provides a method for generating CAR-expressing immune cells (e.g., CAR-T) in a subject, the method comprising:

[0137] The LNP composition of the present invention is administered to a subject, the LNP composition being delivered to the liver site of the subject through its liver-targeting capability; preferably, the surface of the LNP composition also has ligands targeting immune cells, thereby contacting immune cells (e.g., T cells) in the liver and introducing nucleic acids encoding CAR (optionally, other proteins) contained in the LNP composition into the immune cells. Unlike conventional CAR-T therapy, this method does not require prior collection of the subject's immune cells and in vitro engineering.

[0138] The nucleotide sequence encoding the CAR can be operatively linked to nucleotide sequences encoding other proteins (e.g., antigen-binding fragments) via nucleotide sequences encoding self-cleaving adapter sequences (such as 2A adapters), furin cleavage sites, or internal ribosome entry sites (IRES). The self-cleaving adapter sequence can be a 2A sequence, such as a P2A or T2A sequence. Thus, cleavage occurs during ribosome translation to generate the CAR peptide and other proteins, such as antigen-binding fragments targeting other antigens. In one specific embodiment, the other protein is an anti-PD-1 scFv.

[0139] The type of ligand on the surface of the LNP composition can be selected based on the type of immune cells that need to express CAR, so that specific immune cells in vivo express CAR. Examples of such ligands include, but are not limited to, ligands containing one or more antigen-binding domains of anti-CD2 antibodies, anti-CD3 antibodies, anti-CD4 antibodies, anti-CD7 antibodies, anti-CD8 antibodies, anti-CD28 antibodies, and anti-CD127 antibodies. In some embodiments, the ligand is a ligand containing an antigen-binding domain of an anti-CD3 antibody. In some embodiments, the immune cells are T cells, preferably liver T cells. Other T cells that can be used in the methods of the present invention can be T helper (Th) cells, such as T helper 1 (Th1) or T helper 2 (Th2) cells. T cells can be helper T cells (HTL; CD4+ T cells), cytotoxic T cells (CTL; ​​CD8+ T cells), gamma delta (γδ) T cells, or any other T cell subset. In some embodiments, T cells can include primary T cells and memory T cells.

[0140] In some embodiments, the CAR of the present invention and the antigen it is designed to bind to are antigens associated with liver-related diseases. The antigen bound to the CAR may be selected from a variety of liver tumor-associated antigens, liver tumor-specific antigens, or antigens associated with other liver immune diseases or T-cell surface antigens. For example, and not as a limitation, the CAR may be designed to recognize any of the following antigens: CD70, CD3, CD19, CD20, 4.1BB (CD137), OX40 (CD134), CD16, CD47, CD22, CD33, CD38, CD123, CD133, CEA, cdH3, EpCAM, epidermal growth factor receptor (EGFR), EGFRvIII, HER2, HER3, dLL3, BCMA, Sialyl-Lea, 5T4, ROR1, mesothelin, folate receptor 1, VEGF receptor, EpCAM, HER2 / neu, HER3 / neu, G250, CEA, MAGE, V EGF, FGFR, alphaVbeta3-integrin, HLA, HLA-DR, ASC, CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD11, CD13, CD14, CD21, CD23, CD24, CD28, CD30, CD37, CD40, CD41, CD44, CD52, CD64, c-erb-2, CALLA, MHCII, CD44v3, CD44v6, p97, gangliosides GM1, GM2, GM3, GD1a, GD1b, GD2, GD3, GT1b, GT3, GQ1, NY-ESO-1, NFX2, SSX2, SSX4 Trp2, gp100, tyrosinase, Muc-1, telomerase, survivin, G250, p53, CA125, MUC, Lewis Y antigen, HSP-27, HSP-70, HSP-72, HSP-90, Pgp, MCSP, EpHA2, GC182, GT468 or GT512, IL-17, IL-20, IL-13, IL-4, PD1, GPC3, AFP, HBV surface antigen, NKG2D, c-MET. In some embodiments, the antigen recognized by the CAR is GPC3 or PD-1.

[0141] In some embodiments, the CAR comprises an antigen-binding domain in the form of scFv. In other embodiments, the CAR comprises an antigen-binding domain in the form of VHH.

[0142] In some embodiments, the CAR further includes a sequence comprising a transmembrane domain. The transmembrane domain may include a hydrophobic α-helix across the cell membrane. The transmembrane domain may be derived from, for example, CD4, CD8α, or CD28. A wide variety of transmembrane domains known in the art can be used in the CAR disclosed herein. In some embodiments, the transmembrane domain comprises a transmembrane domain selected from CD3, CD4, CD8α, or CD28. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain.

[0143] The CAR may also include one or more sequences forming an intracellular domain and / or a co-stimulatory domain (sometimes also referred to as a signaling domain). A co-stimulatory domain is a domain capable of enhancing or modulating the response of immune effector cells (i.e., capable of initiating the response of immune effector cells). In some embodiments, the co-stimulatory domain and / or signaling domain may be derived from an intracellular T cell receptor (TCR) signaling domain (e.g., the cytoplasmic domain of CD3ζ containing a sequence motif called an immune receptor tyrosine-based activation motif (ITAM)). The co-stimulatory domain may include, for example, sequences from one or more of CD3ζ (CD3zeta), CD28, 4-1BB, OX-40, ICOS, CD27, GITR, CD2, IL-2Rβ, and MyD88 / CD40. In some embodiments, the co-stimulatory domain may include variants of one or more of CD3ζ (CD3zeta), CD28, 4-1BB, OX-40, ICOS, CD27, GITR, CD2, IL-2Rβ, and MyD88 / CD40. For example, in some embodiments, the CAR co-stimulatory domain may also include a modification of the CD3ζ domain. For instance, a CD3ζ signaling domain variant may contain one or two of the three ITAMs present in wild-type CD3ζ, representing functional immune receptor tyrosine-based activation motifs (ITAMs). The choice of co-stimulatory domain influences the phenotype and metabolic characteristics of CAR cells. For example, CD28 co-stimulation produces an effective, but transient, effector-like phenotype with high levels of cytolytic capacity, interleukin-2 (IL-2) secretion, and glycolysis. In contrast, T cells modified with CARs carrying a 4-1BB co-stimulatory domain tend to expand and persist longer in vivo, exhibit increased oxidative metabolism, are less prone to exhaustion, and have an increased capacity to generate central memory T cells. In some embodiments, the intracellular signaling domain includes a co-stimulatory domain or a portion thereof.

[0144] In some embodiments, the intracellular signal transduction domain of the CAR contains ITAM derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b and CD66d.

[0145] In some embodiments, the co-stimulatory domain of the CAR is derived from co-stimulatory molecules selected from CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD270 (HVEM), CD278 (ICOS), and DAP10.

[0146] In some embodiments, the CAR includes a spacer domain to provide conformational freedom, thereby facilitating binding to target antigens on target cells. The optimal length of the spacer domain can depend on the proximity of the binding epitope to the target cell surface. For example, proximal epitopes may require longer spacers, while distal epitopes may require shorter spacers. The CAR may have long, medium, and short spacers. Long spacers may include the CH2CH3 domain (approximately 220 amino acids) of immunoglobulin G1 (IgG1) or IgG4 (natural or with modifications common in therapeutic antibodies, such as the S228P mutation), while the CH3 region may be used alone to construct medium spacers (approximately 120 amino acids). Shorter spacers may be derived from segments of CD28, CD8α, CD3, or CD4 (<60 amino acids). Shorter spacers may also be derived from hinge regions of IgG molecules. These hinge regions may be derived from any IgG isotype and may or may not contain mutations common in therapeutic antibodies, such as the S228P mutation mentioned above. For example, the hinge domain may include an IgG1 hinge domain or a variant thereof, an IgG2 hinge domain or a variant thereof, an IgG3 hinge domain or a variant thereof, an IgG4 hinge domain or a variant thereof, a CD8 hinge domain or a variant thereof, or a CD28 hinge domain or a variant thereof. The terms "hinge" and "spacer" are often used interchangeably; for example, an IgG4 sequence may be considered as both a "hinge" sequence and a "spacer" sequence (i.e., a hinge / spacer sequence). Therefore, the CAR of the present invention may have a spacer domain and / or a hinge domain. In some embodiments, the hinge domain may include an IgG1 hinge domain or a variant thereof, an IgG2 hinge domain or a variant thereof, an IgG3 hinge domain or a variant thereof, an IgG4 hinge domain or a variant thereof (particularly an IgG4P hinge domain), a CD8 hinge domain or a variant thereof, or a CD28 hinge domain or a variant thereof.

[0147] In some embodiments, the CAR may further include a sequence comprising a signal peptide. The function of the signal peptide is to facilitate the transfer of the CAR to the cell membrane. Examples of signal peptides that can be used in the CAR of the present invention include IgG1 heavy chain signal peptides, Igκ or λ light chain signal peptides, granulocyte-macrophage colony-stimulating factor receptor 2 (GM-CSFR2 or CSFR2) signal peptides, CD8a signal peptides, or CD33 signal peptides.

[0148] Chimeric antigen receptor (CAR) construct targeting GPC3

[0149] Glypican-3 (GPC3) is a heparan sulfate proteoglycan involved in cell growth, differentiation, and migration. GPC3 is expressed in the liver and kidneys of healthy fetuses, but is almost entirely absent in adult tissues except for the placenta. Multiple studies have demonstrated that GPC3 is an attractive liver cancer-specific protein, highly expressed in hepatocellular carcinoma (HCC) but with limited expression in normal tissues. Immunotherapy strategies utilizing GPC3 antibodies or peptide vaccines have been explored for the treatment of liver cancer. Clinical data on monoclonal antibodies suggest good tolerability in advanced patients, indicating that GPC3 is a relatively safe target. Although the exact function of GPC3 remains unclear, its strong association with the malignant transformation of HCC has led to its identification as a potential target for cancer immunotherapy, and it has also been selected as a target for HCC cell immunotherapy in several studies. Furthermore, GPC3 is differentially expressed in other tumors besides HCC, such as non-small cell lung cancer, testicular and ovarian yolk sac tumors, malignant melanoma, ovarian clear cell carcinoma (OCCA), gastric cancer (GC), ESCC, testicular germ cell tumors, colon cancer, and renal rhabdoid tumors. Therefore, this invention relates to the use of CAR cell therapy to treat liver diseases, particularly liver cancer.

[0150] This invention provides a CAR construct targeting the GPC3 protein, comprising an antigen-binding domain, a spacer domain, a hinge region, a signal peptide domain, a transmembrane domain, and one or more intracellular domains (e.g., one or more co-stimulatory domains). In some embodiments, the CAR construct may optionally include an armor domain containing a nucleic acid sequence encoding an armor molecule.

[0151] In some implementations, the GPC3-targeting CAR includes an antigen-binding domain, a spacer domain, a hinge region, a signal peptide domain, a transmembrane domain, and one or more co-stimulatory / signal transduction domains.

[0152] In some embodiments, the antigen-binding domain includes an antibody targeting GPC3 or one or more antigen-binding fragments thereof. The antibody may be an antibody known to those skilled in the art, a commercially available antibody, or a laboratory-derived antibody, as long as it provides targeting for GPC3. In some embodiments, the GPC3-targeting CAR construct comprises an scFv containing a light chain variable region (VL) and a heavy chain variable region (VH) from one or more antibodies specific to GPC3, wherein the VL and VH are directly linked or operatively linked together via a linker sequence (e.g., a flexible linker such as (GGGGS)n, n = 1, 2, 3, 4).

[0153] In some embodiments, the signal peptide domain is selected from IgG1 heavy chain signal peptide, Igκ or λ light chain signal peptide, granulocyte-macrophage colony-stimulating factor receptor 2 (GM-CSFR2 or CSFR2) signal peptide, CD8a signal peptide, or CD33 signal peptide.

[0154] In some embodiments, the transmembrane domain is selected from transmembrane domains derived from CD4, CD8α, or CD28.

[0155] In some embodiments, the co-stimulatory domain is selected from the group consisting of: the CD28 co-stimulatory domain, the CD27 co-stimulatory domain, the 4-1BB co-stimulatory domain, the ICOS co-stimulatory domain, the OX-40 co-stimulatory domain, the GITR co-stimulatory domain, the CD2 co-stimulatory domain, the IL-2Rβ co-stimulatory domain, the intracellular domain of the MyD88 / CD40 co-stimulatory domain, and any combination thereof. In some embodiments, the co-stimulatory domain comprises a portion of the intracellular T cell receptor (TCR) signaling domain CD3ζ (the CD3ζ signaling domain is also referred to herein as the "CD3ζ co-stimulatory domain").

[0156] In some embodiments, the antigen-binding domain of the CAR targeting GPC3 may comprise or consist of an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:1.

[0157] In some implementations, the GPC3-targeting CAR construct may further include one or more armored domains encoding one or more armored molecules, which, when used to transform cells, can cause the transformed cells to express a GPC3-targeting CAR protein (also referred to herein as GPC3CAR) and one or more armored molecules, such as cytokines that regulate the tissue microenvironment.

[0158] In this document, "armor molecule" refers to a protein that, when expressed on the cell surface or secreted in the tumor microenvironment, resists immunosuppression of cells in the tumor microenvironment and can provide many additional benefits not described herein, thereby allowing T cells to survive in the immunosuppressive tumor microenvironment (TME). In some embodiments, the expression of the armor molecule can be inducible or constitutive. In some embodiments, the armor molecule is expressed on the cell surface. In some embodiments, the armor molecule is secreted extracellularly to armor CAR T cells. Expression of the armor molecule on the cell surface and / or secretion into the TME can improve the efficacy and persistence of CAR T cells. In this document, such CAR T cells are also referred to as "armored CAR T cells". The armor molecule can be selected based on the tumor microenvironment and other elements of the innate and adaptive immune system. In some embodiments, the armor molecule comprises an antibody against TGF-β, CTLA-4, or PD-1, or an antigen-binding fragment thereof; preferably, the armor molecule comprises an antibody against PD-1, or an antigen-binding fragment thereof, such as PD-1 scFv. In some embodiments, the armor molecule comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO:2. In some embodiments, the armor molecule comprises the amino acid sequence shown in SEQ ID NO:2.

[0159] In some embodiments, the GPC3CAR nucleic acid construct may include an armor domain containing a nucleic acid sequence encoding an armor molecule (e.g., SEQ ID NO: 10). In some embodiments, the armor domain is located at the 3' end of the nucleic acid encoding GPC3CAR or the 5' end of the nucleic acid encoding CAR. In some embodiments, GPC3CAR and the armor domain are operatively linked under the control of a single promoter. In some embodiments, GPC3CAR and the armor domain are operatively linked via an internal ribosome entry site (IRES). In some embodiments, GPC3CAR and the armor domain are linked via a nucleotide sequence encoding a cleavable peptide linker (e.g., a self-cleaving peptide linker). In some embodiments, the cleavable peptide linker comprises a P2A peptide. As described herein, a 2A self-cleaving peptide or a 2A peptide is a class of peptides 18-22 amino acids long that can induce ribosome jumping during protein translation in cells. Examples may include, but are not limited to, P2A (e.g., SEQ ID NO: 8), E2A, F2A, and T2A. Therefore, in such implementations, the GPC3CAR nucleic acid construct containing the armor domain can express both the GPC3CAR as an independent protein and the armor molecule (encoded by the armor domain) during transcription and translation.

[0160] In some embodiments, the antigen-binding domain in GPC3CAR may contain or consist of an amino acid sequence as shown in SEQ ID NO:1, and the armor molecule may contain or consist of an amino acid sequence as shown in SEQ ID NO:2.

[0161] In a specific implementation, the GPC3CAR construct may contain or consist of a nucleic acid sequence as shown in SEQ ID NO:12.

[0162] In some embodiments, the antigen-binding domain of the GPC3CAR includes an antibody or its antigen-binding moiety that specifically binds to human GPC3, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH includes VH complementarity-determining region (CDR)1, VH-CDR2, and VH-CDR3; and wherein the VL includes VL-CDR1, VL-CDR2, and VL-CDR3.

[0163] In some embodiments, the antibody that specifically binds to GPC3 or its antigen-binding portion comprises VH and VL, wherein VH comprises VH-CDR1, VH-CDR2, and VH-CDR3; and wherein the VL comprises VL-CDR1, VL-CDR2, and VL-CDR3, wherein (a) the VH comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:14; and (b) the VL comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:13.

[0164] In some embodiments, the armor molecule comprises an anti-PD-1 antibody or antigen-binding moiety comprising VH and VL, wherein: the VH comprises the amino acid sequence shown in SEQ ID NO:14 and the VL comprises the amino acid sequence shown in SEQ ID NO:13, optionally wherein the antibody or antigen-binding moiety is operatively linked to an scFv containing the amino acid sequence shown in SEQ ID NO:2.

[0165] Applications of LNP and CAR-T

[0166] In one aspect, this invention provides LNPs for engineered T cells in vivo. Traditional CAR-T therapy relies on viral vectors (such as lentiviruses or retroviruses) to transfer the CAR gene into T cells. This invention's in vivo CAR-T therapy explores the direct delivery of mRNA to T cells via LNPs. This technology reduces the safety risks and production costs associated with viral vectors. Existing organ-targeting LNPs require a high lipid ratio (lipid:mRNA = 40:1 to 20:1), resulting in high cytotoxicity and making them unsuitable for T cell modification. The αCD3-LNP developed in this invention (lipid:mRNA ratio can be reduced to 10:1) further improves its biocompatibility and is more suitable for in vivo T cell modification into CAR-T cells.

[0167] In one aspect, the present invention provides CAR-T cells resistant to immunosuppression. Specifically, by designing specific signal transduction domains, CAR-T cells in vivo can simultaneously and locally secrete PD1 scFv to resist immunosuppressive signals in the tumor microenvironment, thereby enhancing the anti-tumor effect.

[0168] On one hand, this invention also enables personalized and broad-spectrum applications. The CAR-T therapy of this invention eliminates the need for peripheral blood cell separation: traditional CAR-T therapy requires the separation of T cells from the patient, which is challenging for some patients. In vivo CAR-T therapy avoids this step, allowing the therapy to be applied more broadly to different patient populations. In the case of personalized treatment, the flexibility of the in vivo delivery system allows for easier customization based on the patient's specific tumor characteristics and immune status, improving the specificity and effectiveness of the treatment.

[0169] Pharmaceutical Compositions and Kits

[0170] In another aspect, the present invention provides a pharmaceutical composition comprising the lipid nanoparticle composition of the present invention and a pharmaceutically acceptable excipient.

[0171] Pharmaceutically acceptable excipients used in this invention refer to non-toxic carriers, adjuvants, or mediators that do not impair the pharmacological activity of the compounds formulated together. Pharmaceutically acceptable carriers, adjuvants, or mediators that can be used in the compositions of this invention include (but are not limited to) ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffering substances (such as phosphates), glycine, sorbic acid, potassium sorbate, mixtures of saturated vegetable fatty acid metaglycerides, water, salts or electrolytes (such as protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, silica gel, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and lanolin.

[0172] The present invention also includes kits (e.g., pharmaceutical packaging). The provided kits may include the lipid nanoparticle composition of the present invention and other therapeutic or preventative agents, and first and second containers (e.g., vials, ampoules, bottles, syringes, and / or dispersible packaging or other suitable containers) containing the lipid nanoparticle composition of the present invention and other therapeutic or preventative agents. In some embodiments, the provided kits may optionally include a third container containing pharmaceutical excipients for diluting or suspending the lipid nanoparticle composition of the present invention and / or other therapeutic or preventative agents.

[0173] In some embodiments, the lipid nanoparticle composition of the present invention, provided in a first container and a second container, is combined with other therapeutic or preventative agents to form a unit dosage form.

[0174] The pharmaceutical compositions provided by this invention can be administered via a variety of routes, including but not limited to: oral administration, parenteral administration, inhalation administration, topical administration, rectal administration, nasal administration, oral administration, vaginal administration, administration via implantation, or other routes of administration. For example, parenteral administration as used herein includes subcutaneous administration, intradermal administration, intravenous administration (e.g., intravenous injection), intramuscular administration, intra-articular administration, intra-arterial administration, intra-synovial administration, intrasternal administration, intramenstrual administration, intralesional administration, and intracranial injection or infusion techniques.

[0175] Typically, an effective amount of the pharmaceutical composition of the present invention is administered. The dosage of the pharmaceutical composition is, for example, in the range of 0.001 mg to 10 mg of the nucleic acid encoding the CAR per kg of body weight. For example, when administered to a human patient, the dosage for a patient weighing 60 kg is in the range of 0.0001 to 50 mg. The above dosage is an example, and the dosage may be appropriately selected by a physician based on the condition being treated or prevented, the type of nucleic acid used, the route of administration, the pharmaceutical composition actually administered, the age, weight, and severity of symptoms of the subject or patient.

[0176] When used to prevent the conditions described in this invention, the pharmaceutical composition provided herein is administered to subjects at risk of developing the condition, typically based on a physician's advice and under physician supervision, at the dosage levels described above. Subjects at risk of developing a specific condition generally include subjects with a family history of the condition, or those identified through genetic testing or screening as particularly susceptible to developing the condition.

[0177] Long-term administration of the pharmaceutical compositions provided herein is also permitted (“long-term administration”). Long-term administration means administering the compound or a pharmaceutical composition thereof over a prolonged period, such as 3 months, 6 months, 1 year, 2 years, 3 years, 5 years, etc., or may be administered indefinitely, such as for the remainder of the subject’s life. In some embodiments, long-term administration is intended to provide a constant level of said compound in the blood over a prolonged period, such as within a therapeutic window.

[0178] Various methods of administration can be used to further deliver the pharmaceutical composition of the present invention. For example, in some embodiments, the pharmaceutical composition can be administered by bolus injection, for instance, to increase the concentration of the compound in the blood to an effective level. The bolus dose depends on the target systemic level of the active component through the body; for example, an intramuscular or subcutaneous bolus dose results in a slow release of the active component, while a bolus dose delivered directly to a vein (e.g., via IV intravenous infusion) allows for a more rapid delivery, causing the concentration of the active component in the blood to rapidly increase to an effective level. In other embodiments, the pharmaceutical composition can be administered in the form of a continuous infusion, for example, via IV intravenous infusion, thereby providing a steady-state concentration of the active component in the subject's body. Furthermore, in other embodiments, a bolus dose of the pharmaceutical composition can be administered first, followed by a continuous infusion.

[0179] The pharmaceutical compositions of the present invention may be vaccine compositions, wherein the lipid nanoparticle composition comprises at least one nucleic acid molecule having a therapeutic or prophylactic effect, the nucleic acid molecule encoding an antigen associated with a target disease (e.g., an infectious disease or a neoplastic disease). Administration of the vaccine composition to a subject (“vaccination”) allows the production of the encoded peptide or protein, thereby evoking an immune response against the target disease in the subject. In some embodiments, the immune response includes adaptive immune responses, such as the production of antibodies against the encoded antigen, and / or the activation and proliferation of immune cells capable of specifically eliminating diseased cells expressing said antigen. In some embodiments, the immune response further includes an innate immune response. According to this disclosure, the vaccine may be administered to a subject before or after the onset of clinical symptoms of the target disease. In some embodiments, vaccination of healthy or asymptomatic subjects renders the vaccinated subject immune or less susceptible to the development of the target disease. In some embodiments, vaccination of subjects exhibiting disease symptoms improves the disease status of the vaccinated subject or treats said disease.

[0180] The pharmaceutical compositions of the present invention can be used to treat, but are not limited to, autoimmune diseases (e.g., systemic lupus erythematosus, psoriasis, rheumatoid arthritis, autoimmune hepatitis, etc.), B-cell lymphomas (e.g., diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, etc.), multiple myeloma, epithelial tumors (e.g., ovarian epithelial tumors, thymic epithelial tumors, etc.), hepatocellular carcinoma, metastatic liver cancer, primary liver cancer, etc.

[0181] By administration to mammals (e.g., humans or other mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cattle, sheep, monkeys), preferably humans), the pharmaceutical composition containing the invention can induce CAR expression in immune cells such as T cells (also referred to herein as "in vivo immune cells" or "in vivo T cells") in the animal's body. In vivo immune cells specifically recognize cancer cells, etc., expressing surface antigens (e.g., GPC3) targeted by the CAR, and kill diseased cells, thereby exhibiting a preventive or therapeutic effect on the disease.

[0182] The types of cancers that can be treated with the pharmaceutical compositions of the present invention include any cancer in which GPC3 is expressed on the cell surface of cancer cells. These cancers include, but are not limited to, hepatocellular carcinoma (HCC), non-small cell lung cancer, testicular and ovarian yolk sac tumors, malignant melanoma, ovarian clear cell carcinoma (OCCA), gastric cancer (GC), ESCC, testicular germ cell tumors, colon cancer, and renal rhabdomyosarcoma.

[0183] Beneficial effects of the present invention

[0184] In vivo CAR-T therapy constructed using LNP-mRNA technology has significant advantages over traditional in vitro CAR-T therapy, as detailed below:

[0185] A. Liver targeting and expression efficiency

[0186] Commercially available LNPs typically have a lipid-to-nucleic acid mass ratio of at least 20:1. By halving the lipid ratio (10:1), our formulation outperforms published liver-targeting LNP formulations in terms of liver-targeting specificity and in situ liver expression efficiency. Furthermore, this invention exhibits significantly lower toxicity to mouse hepatic T cells compared to the known liver-targeting formulation MC3 LNP (patisiran).

[0187] B. Highly efficient and specific T-cell delivery

[0188] By modifying the LNP of this invention with a CD3 antibody (αCD3-LNP), the uptake by CD3-positive cells (T cells) in situ in the liver was significantly improved without altering its in vivo biodistribution. In vitro experiments showed that αCD3-LNP could efficiently transfect human primary T cells at low concentrations (0.05 pg mRNA / cell), with an average CAR positivity rate of 92.6% (n=3). The CAR positivity rate peaked around Day 1, and then gradually declined over time, with some positive cells maintaining expression for more than 10 days.

[0189] C. Transient Expression and Security

[0190] The transient CAR-T cell construction process does not involve the risk of gene integration and insertion, and is carried out directly in vivo. The economic and time costs and long-term toxicity risks are significantly lower than those of traditional CAR-T therapy.

[0191] D. Wide applicability

[0192] The LNP of this invention, as a low-toxicity and highly efficient liver-targeted delivery vector, has a wide range of applications. Constructing in situ CAR-T cells is just one potential application; in the future, it can also be used for targeted liver delivery of other therapeutic mRNAs, such as cytokines and chemokines (IL-2, IL-10, IL-12, IL-13), gene-editing enzymes (CRISRP-Cas9, Base editors), and CARs targeting tumor antigens (GPC3, CD19, EpCAM, EGFR, EGFRvIII, BCMA, GD2, AFP, NKG2D, c-MET), etc. The DMG-PEG2000-NHS component retained in the formulation allows for modification of different proteins to further improve the precision of targeting cell types, such as CD3 antibody modification (targeting T cells). Other antibodies can also be used to modify and target other cell types, such as CD2 antibodies, CD3 antibodies, CD4 antibodies, CD7 antibodies, CD8 antibodies, CD28 antibodies, etc.

[0193] The present invention will be further illustrated below with reference to the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Example

[0194] Example 1. Screening liver-targeting LNP formulations and modifying them with CD3 antibodies.

[0195] In this embodiment, Luciferase mRNA-encapsulated LNPs were injected into mice via tail vein injection using various formulations. BLI in vivo imaging analysis was then used to screen for LNP formulations that enriched and expressed luciferase in the liver. The LNPs obtained from the initial screening (referred to as tLNPs) were modified with CD3 antibodies, and a second screening was performed in the same manner, followed by toxicity testing.

[0196] Using PBS as a negative control and MC3-LNP as a positive control, mice were injected intravenously via tail vein with 10 μg of LNP loaded with Luciferase mRNA (by weight) to screen for the liver-targeting formulation tLNP. MC3-LNP is composed of DLin-MC3-DMA, DSPC, cholesterol, and DMG-PEG2000 in a molar ratio of 50:10:38.5:1.5. Six hours after injection, each mouse was intraperitoneally injected with 100 μL of 15 mg / mL luciferase substrate. Ten minutes after injection, the mice were sacrificed, and the heart, liver, spleen, lung, and kidney were harvested for bioluminescence imaging (BLI). As shown in Figure 1A, luciferase was significantly expressed in the mouse liver, indicating that tLNP has strong liver targeting.

[0197] Furthermore, flow cytometry was used to detect changes in liver T cells in ICR mice after treatment with MC3-LNP, tLNP, and the same volume of PBS for 6 hours, as shown in Figure 1B. The number of T cells in the MC3-LNP treatment group was reduced by approximately 50% compared to the PBS treatment group, indicating potential significant T cell toxicity. In contrast, the number of T cells in the tLNP treatment group was close to that in the PBS treatment group, indicating that tLNP has good safety for T cells.

[0198] In the selected formulation tLNP, the molar ratio of lipid components DLin-MC3-DMA, DOPE, DODAP, cholesterol, and DMG-PEG2000 is approximately 48.6:9.7:3.2:37.2:1.3. The preparation process is as follows:

[0199] 1) Solution preparation

[0200] Solution 1: Weigh each component from the table below and dissolve it in 10 mL of ethanol. After complete dissolution, store at 4 °C.

[0201] Solution 2: Weigh 2.25 mg of DMG-PEG2000-NHS and dissolve it in 10 mL of ethanol. After complete dissolution, store at 4 °C.

[0202] Solution 3: Prepare a 10 mM DODAP solution using ethanol as the solvent and store at -80°C.

[0203] Lipid premix (prepared fresh for use): 200 μL solution 1, 200 μL solution 2 (for preparing CD3-modified LNPs), 8 μL solution 3, 267 μL ethanol.

[0204] mRNA solution (prepared fresh for use): 100ug mRNA dissolved in 1.5mL 100nM sodium citrate buffer. Luciferase mRNA was used in the screening stage.

[0205] 2) tLNP preparation (taking 100ug mRNA as an example)

[0206] (1) Take 500uL of lipid premix and 1.5mL of mRNA solution (volume ratio of 1:3, mass ratio of approximately 10:1) and put them into syringes respectively;

[0207] (2) Using a microfluidic device, mix the solution at a total flow rate of 20 mL / min and a flow rate ratio of 1:3 (lipid premix: mRNA solution = 1:3) and immediately dilute with more than 10 times the volume of PBS.

[0208] (3) After transferring the diluted solution to a 100kDa ultrafiltration tube, centrifuge at 3000rpm-4000rpm at 4℃ to concentrate it to 1mL-2mL.

[0209] After screening out tLNPs, 50% of DMG-PEG2000 was replaced with DMG-PEG2000-NHS to prepare LNPs, which were then modified with CD3 antibody (αCD3-LNP) and IgG control antibody (IgG-LNP): 5 ng of anti-human CD3 antibody (Purified anti-human CD3 Antibody OKT3clone, BioLegend, catalog number 317301) or IgG control antibody was added to the concentrated LNPs, mixed well, incubated at room temperature in the dark for 2 hours, and then stored at 4°C in the dark.

[0210] NOG mice were injected via tail vein with 10 μg (based on mRNA weight) of IgG-LNP LUC, 10 μg of αCD3-LNP LUC, and an equal volume of PBS as a negative control. Six hours later, each mouse was intraperitoneally injected with 100 μL of 15 mg / mL luciferase substrate. In vivo imaging was performed ten minutes after injection, and the mice were immediately euthanized after in vivo imaging. The heart, liver, spleen, lung, and kidney were harvested for individual bioluminescence imaging. As shown in Figure 2, the results indicate that CD3 antibody modification does not affect the in vivo liver targeting of tLNP, and the organ targeting and the in vivo expression of its encapsulated mRNA show good reproducibility.

[0211] The tLNP and αCD3-LNP liposomes were characterized separately. The characterization data of αCD3-LNP is shown in Figure 3A, where uniformly sized and well-packaged LNPs can be observed. The particle size of tLNP and αCD3-LNP is approximately 100-200 nm (Figures 3B-3C), and the average zeta potential is approximately +8 mV (Figure 3D, n=3).

[0212] Example 2. Target selection and validation

[0213] In this embodiment, GPC3 was first investigated and verified as a suitable target through statistical analysis of proteomics data, flow cytometry analysis of HCC cell lines, and flow cytometry analysis of dissociated single cells from xenograft tumors derived from patient samples.

[0214] First, the expression distribution of GPC3 was analyzed from transcriptomic (Figure 4A) and proteomic (Figures 4B-4C) data of 949 different cancer cell lines obtained from publicly available data (see Gao, Qiang et al., Integrated Proteogenomic Characterization of HBV-Related Hepatocellular Carcinoma. Cell vol.179,2(2019):561-577.e22.). The results showed that the overall expression of GPC3 protein in hepatocellular carcinoma cell lines was relatively high (Figure 4). Then, through cooperation with the hospital, three xenograft tumor models (PDX) derived from clinical samples of HCC patients were obtained, numbered PDX22, PDX34, and PDX26, respectively. The expression levels of GPC3 and PD-L1 in human hepatocellular carcinoma cell lines JHH7 and Huh7, as well as the three PDX cells, were detected by flow cytometry in the F2 generation. As shown in Figure 5, flow cytometry results revealed high expression of GPC3 and PDL1 in JHH7, Huh7 cells, and these three PDX cases. These results indicate that targeting GPC3 and combining it with immune checkpoint inhibitors is effective and reasonable. Therefore, constructing GPC3-targeted CAR-T cells with PD1 scFv secretion function using mRNA-LNP technology holds promise for improving the treatment challenges of HCC solid tumors.

[0215] Example 3. Detection of CD3-LNP-constructed CAR-T cells

[0216] In this embodiment, nucleic acid sequences encoding GPC3 CAR and anti-PD1 scFv were constructed. Taking GPC3CAR & PD1 scFv mRNA as an example, human primary T cells were transfected in vitro with mRNA loaded with αCD3-LNP, and the expression of GPC3 CAR and PD1 scFv in T cells was detected.

[0217] The composition of the GPC3 CAR & PD1scFv mRNA sequence (SEQ ID NO: 12) is shown in Figure 6A, wherein the nucleic acid sequence encoding the GPC3 CAR polypeptide and the nucleic acid sequence encoding anti-PD1 scFv are linked by a P2A adapter coding sequence. The solution was prepared according to step 1) of the LNP preparation procedure in Example 1, and then LNPs loaded with GPC3 & αPD1scFv mRNA, modified with IgG antibody or anti-human CD3 antibody respectively, were prepared according to the antibody-modified LNP preparation procedure, referred to as IgG-LNP and αCD3-LNP. αCD3-MC3 LNP loaded with GPC3 & αPD1scFv mRNA was used as a positive control.

[0218] Human primary T cells (2 x 10⁻⁶ cells) were treated with IgG-LNP, αCD3-MC3 LNP, and αCD3-LNP loaded with 100 μg (based on the weight of GPC3 & αPD1scFv mRNA). 6 The proportion of GPC3 CAR-positive cells was detected by flow cytometry 24 h after treatment (cells / mL). The results are shown in Figure 6B. The αCD3-LNP treatment group showed the highest CAR positivity rate.

[0219] T cells were treated with different doses (10, 50, 100, 200, 500, 1000 ng / mL) of αCD3-LNP and analyzed by flow cytometry. The results showed that the proportion of CAR Fab positive cells in the treated T cells increased with increasing incubation dose. When the dose reached 100 ng / mL, the CAR Fab positive cell population reached more than 80% (Fig. 6C, 6D). When T cells were treated with 1000 ng / mL of αCD3-LNP, the proportion of CAR Fab positive cells gradually decreased with time, but still maintained a positivity rate of 72% on day 7 (Fig. 6E, 6F). The cell supernatant of T cells treated with αCD3-LNP for 48 h was further collected, and the secretion level of PD1 scFv was detected by Western blotting. The results are shown in Fig. 6G, with obvious PD1 scFv bands detected only in αCD3-LNP treatment. After treating T cells with 100 μg αCD3-LNP GPC3 & αPD1scFv mRNA for 24 h, CAR-T cells labeled with 488 nm fluorescence were co-cultured with Huh7 cells at a 6:1 ratio, and live-cell fluorescence imaging was performed. As shown in Figure 6H, with the extension of co-culture time, more and more CAR-T cells accumulated around Huh7 cells and stably bound together, which is beneficial for CAR-T cells to better exert their cancer cell killing effect in the future.

[0220] Example 4: In vitro killing and penetration detection of CAR-T cells constructed with αCD3-LNP

[0221] After treating T cells with 100 ng / ml αCD3-LNP for 24 h, the treated T cells were co-cultured with three HCC cell lines at different ratios (1:3, 1:1, 3:1, 9:1). After 48 h, flow cytometry was used to detect the killing effect on cancer cells. The quantitative results are shown in Figure 7A. For the GPC3-high expressing HCC cell lines JHH7 and Huh7, the T cells treated with αCD3-LNP showed a significant killing effect on cancer cells, and the killing efficiency increased with the increase of the proportion of T cells. A similar phenomenon was observed for SKHep1 cells with low GPC3 expression, but the killing efficiency was significantly lower than that of the other two GPC3-high expressing cell lines.

[0222] Further injection of 5×10 into the tail vein of NOG mice 6 T cells were injected with 10 μg αCD3-LNP via the tail vein 24 hours later. The proportion of GPC3 CAR-positive liver T cells was detected by flow cytometry 2, 5 and 8 days after administration. The results are shown in Figures 7B and 7C. The proportion of GPC3 CAR-positive T cells in mouse liver first increased in the first 5 days after administration and then decreased. T cells successfully modified by αCD3-LNP in vivo can maintain GPC3 CAR positivity for more than 8 days, which is crucial for in vivo anti-tumor activity.

[0223] HSC-NOG-EXL mice were reconstituted with human immune cells and injected with αCD3-LNP via the tail vein. Liver cell suspensions were collected 24 hours later. Flow cytometry and Western blotting were used to detect whether the CAR sequence was successfully expressed in the mouse liver. As shown in Figures 7D and 7E, obvious CAR Fab positive cells and PD1scFv expression were successfully detected in the mouse liver cell suspension.

[0224] NOG mice were injected orally with 5 × 10⁵ liver cells. 5 5 × 10 JHH7-Luc cells were injected via tail vein after successful transplantation of the orthotopic liver tumor was confirmed. 6 T cells were injected 24 hours later, followed by a tail vein injection of 10 μg αCD3-LNP. 24 hours later, liver tissue sections were taken from tumor-bearing mice and IF staining was performed. The results are shown in Figure 7F. Obvious CD3 and GPC3 CAR double-positive T cells were also detected in the tumor tissue, indicating that GPC3 positive T cells were present inside the tumor tissue.

[0225] Example 5: In vivo antitumor study of CAR-T cells constructed from αCD3-LNP

[0226] In this embodiment, two immunodeficient mouse orthotopic liver tumor-bearing models were constructed using NOG mice. T cells and αCD3-LNP were injected via the tail vein to examine whether αCD3-LNP could improve liver cancer progression in NOG tumor-bearing mice, thus evaluating the anti-tumor efficacy of this treatment strategy against HCC. Furthermore, using a humanized immune-reconstituted mouse orthotopic liver tumor-bearing model, the anti-tumor effect of combination therapy involving GPC3CAR cells co-secreting PD1 scFv was evaluated.

[0227] 5.1 Tumor model of orthotopic transplantation of Huh7 or JHH7 cells into the liver of NOG mice

[0228] 10⁻¹⁰ Huh7-luc cells were transplanted into the liver of NOG mice. 6 Cells / mouse: Following the timeline in Figure 8A, T cells were injected on Day 0, and bioluminescence imaging (BLI) was performed on Day 5. Mice were randomly divided into two groups of six mice each based on the bioluminescence signal intensity. The experimental group received αCD3-LNP treatment, while the control group received the same volume of PBS, and BLI imaging was performed (Figure 8B). As shown in Figure 8C, on Day 35, the total BLI intensity of Huh7-luc orthotopic liver tumor-bearing NOG mice treated with αCD3-LNP was significantly lower than that of mice treated with PBS. The survival curve in Figure 8D also shows that αCD3-LNP treatment significantly prolonged the survival time of Huh7-luc orthotopic liver tumor-bearing NOG mice. Figure 8E shows the body weight changes in Huh7-luc orthotopic liver tumor-bearing NOG mice treated with αCD3-LNP or PBS; there was no significant difference between the two groups, and the mouse body weight did not change significantly.

[0229] 2 × 10⁻⁶ JHH7-luc cells were orthotopically transplanted into the livers of NOG mice. 5 Cells / mouse: Following the timeline in Figure 8F, T cells were injected on Day 0, and BLI imaging was performed on Day 4. Mice were randomly divided into two groups of eight mice each based on bioluminescence signal intensity. The experimental group received αCD3-LNP treatment on Days 4, 8, and 14, while the control group received the same volume of PBS. BLI imaging was performed on Days 4 and 14 (Figure 8G). As shown in Figure 8H, on Day 14, the total BLI intensity of JHH7-luc orthotopic liver tumor-bearing NOG mice treated with αCD3-LNP was significantly lower than that of mice treated with PBS. The survival curves in Figure 8I show that αCD3-LNP treatment significantly prolonged the survival time of JHH7-luc orthotopic liver tumor-bearing NOG mice. Figure 8J shows the body weight changes of JHH7-luc orthotopic liver tumor-bearing NOG mice treated with αCD3-LNP or PBS. There was no significant difference between the two groups, and the mouse body weight did not change significantly.

[0230] The above results indicate that the combined treatment with αCD3-LNP improved the progression of two types of orthotopic liver cancer in NOG tumor-bearing mice to some extent, with JHH7 tumor-bearing mice showing a more significant response to αCD3-LNP.

[0231] 5.2 Tumor model of orthotopic transplantation of Huh7 cells into the liver of HSC-NOG-EXL mice

[0232] HSC-NOG-EXL mice liver orthotopic transplantation of Huh7-luc cells 1×10⁻⁶ 6 One week after tumor implantation, mice were subjected to bioluminescence imaging. Based on bioluminescence signal intensity, mice were randomly divided into four groups of seven mice each. Over the following three weeks, treatment was administered at the time intervals shown in Figure 9A (Days 1, 3, 5, 7, 9, 13, 17, 21), and BLI imaging was performed (Days 7, 21, 33). The control group was treated with PBS, the experimental group was treated with αCD3-LNP loaded with GPC3CAR & PD1scFv mRNA, and the positive control group was treated with αCD3-LNP loaded with GPC3CAR mRNA and PD1 mAb, respectively. Figure 9B shows BLI imaging of each group of mice on Day 0 and Day 33. As shown in Figure 9C, on Day 33, the BLI intensity of Huh7-luc liver orthotopic tumor-bearing HSC-NOG-EXL mice treated with αCD3-LNP GPC3CAR & PD1scFv was significantly lower than that of the other groups. Figure 9D shows that αCD3-LNP treatment significantly prolonged the survival time of Huh7-luc liver tumor-bearing HSC-NOG-EXL mice. Figure 9E shows the body weight change curves of Huh7-luc liver tumor-bearing HSC-NOG-EXL mice after drug treatment; there were no significant differences between the groups, and the body weight of the mice did not change significantly.

[0233] The above results indicate that the combined treatment of CAR-T cell co-secretion of PD-1scFv provided by the αCD3-LNP of the present invention significantly improves the progression of orthotopic liver cancer in humanized immune reconstituted tumor-bearing mice.

[0234] Sequence Summary

[0235] Information about the sequences involved in this invention is provided in the table below.

[0236] Those skilled in the art will further recognize that the invention can be embodied in other specific forms without departing from its spirit or central characteristics. Since the foregoing description of the invention discloses only exemplary embodiments thereunder, it should be understood that other variations are considered to be within the scope of the invention. Therefore, the invention is not limited to the specific embodiments described in detail herein. Rather, reference should be made to the appended claims to indicate the scope and content of the invention.

Claims

1. A liver-targeting composition comprising an ionizable cationic lipid, an auxiliary phospholipid, a polymeric phospholipid, and a sterol lipid, wherein the cationic lipid is DLin-MC3-DMA and DODAP, and the auxiliary phospholipid is DOPE.

2. The composition according to claim 1, wherein the ionizable cationic lipids and cofactor phospholipids constitute about 49%-74% of the total lipids in molar percentage. Optionally, the molar ratio between the ionizable cationic lipid and the auxiliary phospholipid is in the range of about (51.8 ± 20%):(9.7 ± 20%), more preferably in the range of about (51.8 ± 10%):(9.7 ± 10%).

3. The composition according to claim 1 or 2, wherein the molar ratio between DLin-MC3-DMA and DODAP is in the range of about (48.6 ± 20%) : (3.2 ± 20%). Optionally, the molar ratio of DLin-MC3-DMA, DODAP and DOPE is in the range of approximately (48.6 ± 20%):(3.2 ± 20%):(9.7 ± 20%).

4. The composition according to any one of claims 1-3, wherein the sterol lipids are selected from cholesterol, sitosterol, coccosterol, rock saponin, brassosterol, ergosterol, tomatine, ursolic acid, α-tocopherol, stigmasterol, alfalfa sterol, ergocalciferol, or campesterol, preferably cholesterol and / or β-sitosterol, more preferably cholesterol. Optionally, the sterol lipids account for about 29.76-44.64% of the total lipids in molar percentage.

5. The composition according to any one of claims 1-4, wherein the polymeric phospholipid is a polyethylene glycol-modified (PEG) phospholipid, for example selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol; Preferably, the polyethylene glycol-modified phospholipid contains a PEG portion of about 1000 Da to about 20 kDa, and more preferably contains a PEG portion of about 1000 Da to about 5000 Da. Preferably, the polyethylene glycol-modified phospholipid is selected from DMPE-PEG1000, DPPE-PEG1000, DSPE-PEG1000, DOPE-PEG1000, DMG-PEG2000, Ceramide-PEG2000, DMPE-PEG2000, DPPE-PEG2000, DSPE-PEG2000, Azido-PEG2000, DSPE-PEG2000-Mannose, Ceramide-PEG5000, and DSPE-PEG5000, and is more preferably DMG-PEG2000.

6. The composition according to any one of claims 1-5, comprising DLin-MC3-DMA, DODAP, DOPE, cholesterol, and DMG-PEG2000. Optionally, DMG-PEG2000 is modified, for example, to include an N-hydroxysuccinimide (NHS) group.

7. The composition according to claim 6, wherein the molar ratio of DLin-MC3-DMA, DODAP, DOPE, cholesterol, and DMG-PEG2000 is in the range of about (48.6±20%):(3.2±20%):(9.7±20%):(37.2±20%):(1.3±20%). Preferably, within the range of approximately (48.6±10%):(3.2±10%):(9.7±10%):(37.2±10%):(1.3±10%), Optionally, DMG-PEG2000 is partially NHS modified.

8. The composition according to claim 7, wherein the molar ratio among DLin-MC3-DMA, DODAP, DOPE, cholesterol, DMG-PEG2000 and DMG-PEG2000-NHS is in the range of about (48.6±20%):(3.2±20%):(9.7±20%):(37.2±20%):(0.65±20%):(0.65±20%). Preferably, it is in the range of approximately (48.6±10%):(3.2±10%):(9.7±10%):(37.2±10%):(0.65±10%):(0.65±10%).

9. The composition according to any one of claims 1-8, wherein the lipid component is in the form of lipid nanoparticles.

10. The composition according to claim 9, wherein the lipid nanoparticles have a particle size of about 50-200 nm.

11. The composition according to claim 9 or 10, wherein the lipid nanoparticles have an average zeta potential of about -5 mV to about +10 mV, preferably about 0 mV to about +10 mV.

12. The composition according to any one of claims 1-11, further comprising a target substance, such as one or more therapeutic or preventative agents. Preferably, the therapeutic or preventative agent is encapsulated in lipid particles formed from the lipid components.

13. The composition according to claim 12, wherein the therapeutic or preventative agent is a nucleic acid; Preferably, the nucleic acid is ASO, RNA, or DNA; Preferably, the RNA is selected from interfering RNA (RNAi), small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aRNA), messenger RNA (mRNA), modified messenger RNA (mmRNA), long non-coding RNA (lncRNA), microRNA (miRNA), small activating RNA (saRNA), polymeric coding nucleic acid (MCNA), polymeric coding nucleic acid (PCNA), guide RNA (gRNA), and CRISPR RNA (crRNA), preferably mRNA. Preferably, the mass ratio of lipids to nucleic acids is less than 20:1, more preferably not more than 15:1, for example not more than 14:1, not more than 13:1, not more than 12:1, not more than 11:1, or not more than 10:

1.

14. The composition of claim 13, wherein the nucleic acid is mRNA or DNA encoding a chimeric antigen receptor (CAR) that targets a target molecule located in the liver. Optionally, the nucleic acid also encodes an antibody or antigen-binding portion thereof that targets immune checkpoint molecules or liver tumor-associated antigens, such as an antibody or antigen-binding fragment thereof that targets PD-1, PD-L1, TGF-b, or CTLA-4. Optionally, the antibody or its antigen-binding fragment is Fab, Fab', F(ab')2, Fd, Fv, single-chain variable fragment (scFv), single-chain antibody, VHH, vNAR, or single-domain antibody.

15. The composition of claim 14, wherein the nucleic acid comprises a nucleic acid sequence encoding a CAR polypeptide targeting GPC3 and a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof targeting PD-1, preferably, the two being operatively linked via a cleavable peptide linker encoding a sequence. Optionally, the cleavable peptide linker is a self-cleaving peptide linker; Optionally, the self-cleaving peptide linker comprises a P2A, T2A, or F2A peptide linker.

16. The composition of claim 15, wherein the CAR polypeptide comprises a GPC3 binding domain, a transmembrane domain, and one or more intracellular domains, and optionally a leader peptide. Optionally, the transmembrane domain comprises a CD4, CD8α, or CD28 transmembrane domain. Optionally, the one or more intracellular domains include a co-stimulatory domain or a portion thereof. Optionally, the co-stimulatory domain comprises one or more of the following: CD3ζ, CD2, CD27, CD28, 4-1BB, OX-40, ICOS, IL-2Rβ, GITR, MyD88 / CD40a co-stimulatory domains and / or variants thereof. Optionally, the intracellular domain includes a CD3ζ co-stimulatory domain and a CD28 co-stimulatory domain.

17. The composition of claim 16, wherein the CAR further comprises a hinge / spacer subdomain, optionally wherein the hinge / spacer subdomain is located between the antigen-binding domain and the transmembrane domain. Optionally, the hinge / spacer subdomain includes an IgG1 hinge domain or a variant thereof, an IgG2 hinge domain or a variant thereof, an IgG3 hinge domain or a variant thereof, an IgG4 hinge domain or a variant thereof, a CD8 hinge domain or a variant thereof, or a CD28 hinge domain or a variant thereof.

18. The composition according to any one of claims 16-17, wherein the GPC3 binding domain is scFv, and optionally the nucleic acid sequence encoding the scFv is shown in SEQ ID NO:

4.

19. The composition according to any one of claims 16-18, wherein the antibody targeting PD-1 or its antigen-binding fragment is scFv, optionally, the nucleic acid sequence encoding the scFv is shown in SEQ ID NO:

10.

20. The composition according to any one of claims 9-19, wherein the lipid nanoparticles have a ligand capable of targeting T cells on their surface or are modified with a ligand capable of targeting T cells on their surface.

21. The composition of claim 20, wherein the ligand is a ligand comprising one or more antibodies selected from the group consisting of: anti-CD3 antibody, anti-CD2 antibody, anti-CD4 antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD28 antibody, and anti-CD127 antibody. Optionally, the ligand is a ligand comprising an anti-CD3 antibody or its antigen-binding moiety.

22. A lipid nanoparticle comprising the composition of any one of claims 1-21.

23. A method for preparing lipid nanoparticles from the composition of any one of claims 1-21, the method comprising: A lipid premix is ​​prepared by mixing the ionizable cationic lipids, sterol lipids, auxiliary phospholipids and polymeric phospholipids in a predetermined ratio. The lipid premix is ​​mixed with a solution containing a target substance, such as a therapeutic agent or a preventative agent; Preferably, the lipid components are mixed together or separately with a solvent to prepare a lipid premix; Preferably, the solvent is an organic solvent, more preferably an alcohol solvent, and more preferably ethanol; Preferably, the target substance is nucleic acid, which is dissolved using a buffer solution; Optionally, the buffer solution is an acetate or citrate solution.

24. The method of claim 23, wherein the lipid premix is ​​mixed with the solution containing the target substance in a microfluidic mixing system.

25. The method according to any one of claims 23-24, further comprising the step of modifying the lipid nanoparticles with a ligand by incubating the lipid nanoparticles with a solution containing the ligand, preferably, the ligand comprising one or more antibodies selected from the group consisting of anti-CD3 antibody, anti-CD2 antibody, anti-CD4 antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD28 antibody, and anti-CD127 antibody, preferably a ligand comprising an anti-CD3 antibody or an antigen-binding domain thereof.

26. A method for engineering immune cells, the method comprising contacting the immune cells with a composition according to any one of claims 9-21 or a lipid nanoparticle according to claim 22, thereby introducing a target substance into the cells; Preferably, the immune cells are T cells.

27. A method for engineering immune cells in a subject in vivo, the method comprising administering to the subject the composition according to any one of claims 1-21; Optionally, the subject is a mammal, such as a human or a non-human animal, preferably a human; Optionally, the immune cells are T cells, preferably liver T cells.

28. The method according to any one of claims 26-27, wherein the composition comprises a CAR encoding a tumor antigen targeting liver cancer, and optionally an antibody or antigen-binding fragment thereof targeting PD-1.

29. The method according to any one of claims 26-28, wherein the immune cells are selected from T cells, NK cells, monocytes, macrophages and dendritic cells.

30. The method of claim 29, wherein the T cells are selected from CD4 T cells, CD8 T cells, and γδ T cells.

31. A pharmaceutical composition comprising the composition according to any one of claims 1-21, and a pharmaceutically acceptable carrier or excipient.

32. A method for treating or preventing liver-related diseases, comprising administering a composition according to any one of claims 1-21, lipid nanoparticles according to claim 22, or a pharmaceutical composition according to claim 31. Optionally, the disease is selected from hepatocellular carcinoma, such as primary hepatocellular carcinoma or metastatic liver cancer.

33. Use of the composition according to any one of claims 1-21, the lipid nanoparticle according to claim 22, or the pharmaceutical composition according to claim 31 in the preparation of a medicament for delivering a target substance, wherein the target substance is selected from therapeutic agents and / or preventive agents.

34. The use according to claim 33, wherein, The therapeutic or preventative agent is a nucleic acid; Preferably, the nucleic acid is selected from one or more of ASO, RNA, or DNA; Preferably, the RNA is selected from interfering RNA (RNAi), small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aRNA), messenger RNA (mRNA), modified messenger RNA (mmRNA), long non-coding RNA (lncRNA), microRNA (miRNA), small activating RNA (saRNA), multi-coding nucleic acid (MCNA), polymeric coding nucleic acid (PCNA), guide RNA (gRNA), and CRISPR RNA (crRNA), and is preferably mRNA.

35. Use of the composition according to any one of claims 1-21, the lipid nanoparticles according to claim 22, or the pharmaceutical composition according to claim 31 in the preparation of a vaccine composition for the prevention or treatment of liver-related diseases. Optionally, the liver-related disease is selected from hepatocellular carcinoma, such as primary hepatocellular carcinoma or metastatic liver cancer.

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