A lipid nanoparticle for in vivo production of chimeric antigen receptor neutrophils, methods of making and uses thereof

Abstract

The application belongs to the field of biological medicine, and relates to a lipid component of a chimeric antigen receptor neutrophil targeting Her2 generated in vivo, a preparation method thereof and application in solid tumor treatment. The lipid nanoparticle is a double-targeted lipid nanoparticle, and the double targeting is realized by Ly6G antibody fragments and NEBP polypeptides. The lipid nanoparticle comprises a lipid component and mRNA encoding Her2-CAR and IFN-gamma. The mRNA encoding Her2-CAR and IFN-gamma is delivered by the lipid nanoparticle LNP, and is delivered to neutrophils in vivo and expresses Her2-CAR and IFN-gamma to generate CAR-neutrophils, so that the cumbersome step of editing immune cells in vitro is omitted, and potential adverse reactions caused by reinfusion of CAR-immune cells prepared in vitro into the body are avoided.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a lipid nanoparticle that generates chimeric antigen receptor neutrophils targeting Her2 in vivo, its preparation method, and its application in the treatment of solid tumors. Background Technology

[0002] Chimeric antigen receptor (CAR) is a new technology developed based on cancer immunotherapy. It combines the capabilities of antibody binding and immune activation, and has the ability to modify immune cells and enhance their anti-tumor effects. Clinically, CAR technology is widely used to treat hematologic malignancies, especially chimeric antigen receptor-T cells (CAR-T), which has achieved numerous successes. However, CAR-T therapy also has certain limitations. For example, CAR-T cells have difficulty infiltrating solid tumors and are affected by the immunosuppressive effects of the tumor microenvironment. In addition, CAR-T therapy often causes adverse reactions such as cytokine release syndrome (CRS) and neurotoxicity in clinical use.

[0003] Chimeric antigen receptor neutrophil (CAR-NE) therapy is a novel therapy that uses gene editing technology to engineer neutrophils to express specific chimeric antigen receptors, thereby enhancing their ability to inhibit tumor cells. Neutrophils have the advantages of high blood content and rapid recruitment, allowing them to infiltrate and accumulate in various types of tumors, exhibiting invasiveness and plasticity within tumor tissue. Tumor-associated neutrophils (TANs) can promote or inhibit tumor growth depending on their polarization state, such as exhibiting anti-tumor TAN1 or pro-tumor TAN2 neutrophils. Simultaneously, the chemokines and pro-inflammatory cytokines produced by TANs can recruit and activate CD8+ T cells. However, the immune cells required for current CAR-immunotherapy still need to be isolated from the patient's own body, modified in vitro, and then infused back into the patient. Therefore, CAR-neutrophil therapy still faces the challenge of complex in vitro preparation processes.

[0004] Nucleic acid drugs are widely used in the treatment of various diseases, especially mRNA, which has relatively high translation efficiency and safety. Lipid nanoparticle (LNP) delivery systems have shown great advantages in nucleic acid drug delivery, enabling safe and efficient targeted delivery to a certain extent. LNP delivery systems mainly consist of ionizable lipids, cofactor phospholipids, cholesterol, PEGylated lipids, and nucleic acid drugs. They can protect the encapsulated nucleic acid drugs from degradation by nucleases in vivo and promote the transport of nucleic acid drugs across cell membranes to the target site.

[0005] Interferon-γ (IFN-γ) is a pleiotropic cytokine with antiviral, antitumor, and immunomodulatory functions, playing a crucial role in activating cellular immunity and subsequently stimulating antitumor immune responses. Based on its cell-inhibiting, apoptosis-promoting, and antiproliferative functions, IFN-γ is considered potentially helpful in adjuvant immunotherapy for various types of cancer. In the tumor microenvironment, IFN-γ not only promotes neutrophil polarization to the TAN1 (antitumor) phenotype but also regulates the function of T and NK cells, enabling them to jointly exert antitumor effects. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and solve the following technical problems: 1) To provide a lipid nanoparticle delivery system, its preparation method, and its application, which can directly generate chimeric antigen receptor (CAR) neutrophils in vivo and be used to treat solid tumors (experiments targeting breast cancer with high Her2 expression). Addressing the limitations of current in vitro CAR immune cell preparation methods, this invention uses a dual-targeting LNP vector to encapsulate CAR-encoding mRNA, directly targeting and modifying neutrophils in vivo to form CAR-neutrophils. 2) Simultaneously addressing the immunosuppression problem in the tumor microenvironment, this invention increases the in vivo IFN-γ content by enabling neutrophils to express IFN-γ, thereby enhancing the neutrophil-killing ability and promoting tumor cell apoptosis.

[0007] The technical solution of this invention to solve the technical problem is as follows:

[0008] In a first aspect of the present invention, a lipid nanoparticle for generating chimeric antigen receptor neutrophils in vivo is provided, wherein the lipid nanoparticle is a dual-targeting lipid nanoparticle, and the dual targeting is achieved through a Ly6G antibody fragment and a NEBP peptide.

[0009] The lipid nanoparticles contain lipid components and mRNA encoding Her2-CAR and IFN-γ;

[0010] The mRNA is obtained by in vitro transcription synthesis of DNA with nucleotide sequences as shown in SEQ ID NO: 3 or SEQ ID NO: 4;

[0011] The lipid component comprises ionizable lipid SM-102, cofactor phospholipids, cholesterol, DMG-PEG2000, and targeted lipid molecules DSPE-PEG-NEBP and DSPE-PEG-MAL, in a molar ratio of 40–50 : 5–15 : 19–38 : 1–10 : 2 : 1; the cofactor phospholipids are one or both of DSPC or DOPE.

[0012] The lipid nanoparticles have a nitrogen-to-phosphorus ratio (N / P) of 5–8; the preferred N / P ratio is 6.

[0013] The DSPE-PEG-NEBP is prepared by linking the NEBP polypeptide with the sequence shown in SEQ ID NO: 5 to DSPE-PEG-MAL;

[0014] The lipid nanoparticles are attached to a targeting molecule Ly6G-SH, which is covalently linked to the lipid nanoparticles via a coupling reaction between the thiol group and the maleimide group on the lipid component DSPE-PEG-MAL.

[0015] The Ly6G-SH is prepared by a thiolization reaction of Ly6G-Fab.

[0016] Furthermore, in the lipid component, the molar ratio of ionizable lipid SM-102, cofactor phospholipid, cholesterol, DMG-PEG2000, and targeted lipid molecules DSPE-PEG-NEBP and DSPE-PEG-MAL is 40~50:10:19~38:1.5:2:1.

[0017] In a preferred embodiment of the present invention, the lipid component comprises SM-102, DSPC, cholesterol, DMG-PEG2000, and targeted lipid molecules DSPE-PEG-NEBP and DSPE-PEG-MAL, wherein the molar ratio is 50:10:38:1.5:2:1.

[0018] The Her2-CAR is a specific CAR targeting Her2 in breast cancer, comprising the following domains in sequence: CD8 signal peptide, anti-Her2 scFv fragment, CD8α hinge region, transmembrane domain, 4-1BB intracellular domain, and CD3ζ intracellular domain; the transmembrane domain is CD28 or CD32a; the Her2-CAR and IFN-γ are co-expressed by being linked through an autocracked peptide sequence.

[0019] The DNA sequence shown in SEQ ID NO: 3 is the encoding DNA of Her2-CAR1 / IFN-γ. Her2-CAR1 / IFN-γ includes, in sequence, a CD8 signal peptide, a fragment of the Her2 monoclonal antibody scFv specifically for breast cancer cells, a CD8α hinge region, a CD28 transmembrane domain, a 4-1BB intracellular domain, a CD3ζ intracellular domain, and a fragment encoding IFN-γ.

[0020] The DNA sequence shown in SEQ ID NO: 4 is the encoding DNA for Her2-CAR2 / IFN-γ. The Her2-CAR2 / IFN-γ includes, in sequence, a CD8 signal peptide, a fragment of a Her2 monoclonal antibody specific to breast cancer cells (scFv), a CD8α hinge region, a neutrophil-specific CD32a transmembrane domain, a 4-1BB intracellular domain, a CD3ζ intracellular domain, and a fragment encoding IFN-γ.

[0021] In a second aspect of the present invention, a method for preparing lipid nanoparticles as described in the first aspect is provided, comprising the following steps:

[0022] (1) Preparation of targeted lipid molecules DSPE-PEG-NEBP and Ly6G-SH:

[0023] A. NEBP peptide was synthesized by solid-phase synthesis, purified, and then added to DSPE-PEG-MAL. The thiol group on the NEBP peptide reacted specifically with the maleimide on DSPE-PEG-MAL, thus linking the NEBP peptide to DSPE-PEG-MAL to prepare DSPE-PEG-NEBP for later use. The sequence of the NEBP peptide is shown in SEQ ID NO: 5.

[0024] B. Traut's reagent and Ly6G-Fab were mixed in a 1:1 molar ratio to prepare Ly6G-SH via a thiolization reaction for later use. After purifying the reaction product, i.e., lipid nanoparticles, a post-insertion method was used to link Ly6G-SH with DSPE-PEG-MAL through the specific reaction of thiol groups with maleimide (MAL), so that the targeted molecule was inserted into the surface of the lipid nanoparticles.

[0025] (2) Preparation of lipid nanoparticles, the steps are as follows:

[0026] A. Ionizable lipids SM-102, DSPC, cholesterol, DMG-PEG2000, and targeted lipid molecules DSPE-PEG-NEBP and DSPE-PEG-MAL were dissolved in ethanol in proportion to form an oil phase lipid mixture.

[0027] B. With an N / P ratio of 6, mix the mRNA solution and lipid solution. Dissolve the mRNA encoding Her2-CAR and IFN-γ in sodium citrate buffer to obtain the mixed mRNA solution.

[0028] C. The mRNA solution and lipid mixture were added to a microfluidic device to prepare an LNP containing mRNA encoding Her2-CAR and IFN-γ.

[0029] D. Ly6G-SH and LNP prepared in the previous step were co-incubated at 37°C for 1 hour at a molar ratio of 1:1 to obtain dual-targeting lipid nanoparticles.

[0030] In a third aspect of the invention, the use of dual-targeting lipid nanoparticles as described in the first aspect is provided in the preparation of medicaments for the prevention and / or treatment of tumors.

[0031] Furthermore, the tumor is a solid tumor.

[0032] Furthermore, the tumor is a Her2-positive tumor.

[0033] In a preferred embodiment of the present invention, the tumor is Her2-positive breast cancer.

[0034] The innovations of this invention mainly include the following points:

[0035] 1. A novel technical route for in situ generation of CAR-neutrophils in vivo was proposed.

[0036] This invention utilizes LNP delivery of CAR-mRNA, bypassing the in vitro isolation, transduction, and amplification processes, to directly induce Her2-CAR expression in neutrophils in vivo. For example... Figure 11 As shown, neutrophils showed significant infiltration at the tumor site, and at the same time... Figure 6 As shown, CAR is expressed on neutrophils.

[0037] 2. Constructing a dual-targeting LNP to achieve neutrophil-specific delivery

[0038] This invention modifies LNPs with dual-targeting ligands: Ly6G-Fab for recognizing neutrophil-specific antigens on the surface; and NEBP peptide for binding to neutrophil elastase. It utilizes two neutrophil-targeting elements with completely different mechanisms of action: Ly6G-Fab for membrane surface antigen recognition (cell-specific); and NEBP peptide for selective enrichment and endocytosis mediated by functional protein (NE). A neutrophil-targeting LNP delivery system with two targets is constructed to improve targeting of the neutrophil system, reduce impact on other immune cells, and increase CAR expression on neutrophils.

[0039] like Figure 7 As shown, the expression of CAR on neutrophils and macrophages was detected after delivery of this dual-targeting LNP. Compared with LNPs without targeting, CAR was clearly expressed on neutrophils, while almost no expression was detected on macrophages, indicating that it has good targeting ability to neutrophils.

[0040] 3. Combined delivery of CAR and IFN-γ mRNA to regulate the tumor immune microenvironment.

[0041] This invention improves upon the CAR structure by constructing an mRNA that simultaneously expresses CAR and IFN-γ, co-expressing it on neutrophils. Neutrophils are naturally prone to N2 (pro-tumor) polarization, and CAR signaling alone is insufficient to overcome TME inhibition. IFN-γ, in autocrine mode, can stabilize the neutrophil N1 (anti-tumor) phenotype and amplify ROS / NETs / phagocytic function. The CAR2-IFNγ group is the main innovative structure. The inventors constructed a CAR containing both a CD32a domain and an IFNγ expression sequence to simultaneously achieve CAR expression on neutrophils to activate neutrophils, and IFNγ expression to assist in tumor suppression. In practical applications, the CAR2-IFNγ group has shown relatively better in vitro and in vivo therapeutic effects compared to other CAR structures.

[0042] like Figure 9 , Figure 16 As shown, IFNγ's antiviral, antitumor, and immunomodulatory functions work together to exert an antitumor effect, while CAR-neutrophils lacking IFNγ cannot achieve the same therapeutic effect. Simultaneously, it can promote neutrophil polarization within the tumor microenvironment to the TAN1 (antitumor) phenotype. Due to the different tumor-suppressive mechanism of neutrophils, they do not produce and release large amounts of cytokines, thus fundamentally avoiding adverse reactions such as cytokine syndrome.

[0043] The present invention has the following beneficial effects:

[0044] 1) This invention uses lipid nanoparticles (LNPs) to deliver mRNA encoding Her2 CAR and IFN-γ, directly editing neutrophils in vivo and generating CAR-neutrophils expressing Her2 CAR and IFN-γ. This eliminates the cumbersome steps of in vitro engineering and editing of immune cells, and avoids potential adverse reactions such as cytokine syndrome and neurotoxicity caused by in vitro preparation of CAR-immune cells and their reinfusion in vivo.

[0045] 2) By adding targeted lipid molecules to LNP, it can recognize Ly6G and neutrophil elastase (NE) on the surface of neutrophils, thereby achieving recognition and targeted delivery to neutrophils.

[0046] 3) While CAR is expressed, IFN-γ expression regulates the tumor immune microenvironment, promotes the differentiation of neutrophils into N1 type, enhances the tumor-suppressing ability of neutrophils, and plays a synergistic role in inhibiting solid tumors.

[0047] The combination of the dual-targeting structure of the present invention and IFN-γ co-expression produces a synergistic technical effect that cannot be expected by existing technologies alone or in simple combinations. Attached Figure Description

[0048] Figure 1 Schematic diagrams of four different Her2-targeted CAR structures.

[0049] Figure 2 Characterization of two lipid molecules, DSPE-PEG-MAL and DSPE-PEG-NEBP. (A) Top: 1H NMR spectrum of DSPE-PEG-MAL; Bottom: 1H NMR spectrum of DSPE-PEG-NEBP; (B) MOLDI-TOF spectrum of DSPE-PEG-MAL; (C) MOLDI-TOF spectrum of DSPE-PEG-NEBP.

[0050] Figure 3 Characterization of dual-targeting LNPs synthesized according to specific phospholipid compositions and ratios. Among them, (A) the particle size of each synthesized LNP according to the formulation; (B) the zeta potential of each LNP; and (C) the polydispersity index (PDI) of each LNP.

[0051] Figure 4 This diagram illustrates the preparation process and characterization of dual-targeted LNPs. (A) Schematic diagram of lipid nanoparticle synthesis using a microfluidic device; (B) TEM electron microscopy analysis of the synthesized LNP morphology; (C) Particle size of the synthesized LNPs; (D) Zeta potential of the LNPs; (E) Encapsulation efficiency of the LNPs.

[0052] Figure 5 To assess the in vitro transduction efficiency of dual-targeting lipid nanoparticles, LNPs loaded with mCherry were used, and their in vitro transfection of neutrophils was detected by (A) confocal microscopy and (B) flow cytometry.

[0053] Figure 6 The expression of CAR on neutrophils after Her2-CAR2 / IFN-γ@LNP-NE transduction.

[0054] Figure 7 This study investigated the in vivo targeting ability of NEBP and Ly6G targets. Specifically, (A) flow cytometry was used to compare the ability of LNPs without targets, with NEBP targets, and with both NEBP and Ly6G targets to target neutrophils in vivo; (B) flow cytometry was used to detect off-target transfection of each group of LNPs into macrophages in vivo; and (C) the results of flow cytometry analysis of in vivo neutrophil targeting and off-target macrophage transfection were quantitatively analyzed.

[0055] Figure 8 This study aimed to evaluate the in vitro function and killing ability of CAR-neutrophils. The evaluation included: (A) the formation of immune synapses after co-incubation of CAR-neutrophils with target cells; (B) confocal microscopy detection of NET production; (C) detection of ROS production after co-incubation of neutrophils treated with different methods with target cells; and (D) flow cytometry detection of the killing effect of CAR-neutrophils on target cells, assessed by the percentage of PI-positive cells in the target cell population.

[0056] Figure 9 This shows the in vitro CAR-neutrophil typing results.

[0057] Figure 10 This is a protocol for in vivo animal experiments.

[0058] Figure 11 The data represents the infiltration of neutrophils in the tumor site and the number of CAR-neutrophils. Specifically, (A) the proportion of various immune cells infiltrating the 4T1-Her2 tumor; and (B) the number of CAR-expressing neutrophils in the tumor tissue.

[0059] Figure 12 The changes in tumor volume and body weight in mice during continuous drug administration are shown. (A) Changes in tumor volume and body weight in mice; (B) The trends in tumor size changes for each mouse in the PBS, CAR1, CAR1+IFNγ, CAR2, and CAR2+IFNγ groups during drug administration.

[0060] Figure 13 The expression of IFN-γ on neutrophils after continuous in vivo drug administration. (A) Quantitative statistics of the results of in vivo IFN-γ expression detected by flow cytometry; (B) Results of in vivo IFN-γ expression detected by flow cytometry.

[0061] Figure 14 To detect the efficiency of each dual-targeting LNP in vivo transduction of CAR-neutrophils after drug administration.

[0062] Figure 15 TUNEL staining was used to assess the killing effect of CAR-neutrophils within the tumor.

[0063] Figure 16 This describes the production of plasma cytokines in mice after continuous drug administration.

[0064] Figure 17 The results show the detection results of biochemical indicators related to liver and kidney function in mouse serum. Detailed Implementation

[0065] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments.

[0066] The following is a description of the sources of the experimental materials involved in the embodiments of the present invention:

[0067]

[0068] .

[0069] Ly6G-Fab preparation method: Ly6G-Fab was obtained by papain digestion under controlled conditions using InVivoMAb anti-mouse Ly6G (BE0075-1) purchased from BioXCell. The Fab digestion kit was the Pierce™ Fab Preparation Kit (44985) from Thermo Scientific.

[0070] Example 1

[0071] (I) Construction and characterization of Her2-CAR / IFN-γ@LNP-NE

[0072] 1.1 Construction of mRNAs encoding Her2-CAR and IFN-γ

[0073] Using Her2-overexpressing breast cancer-specific target Human Epidermal Growth Factor Receptor 2 (Her2) as a specific antigen, a CAR extracellular domain was designed. This chimeric antigen receptor includes a CD8 signal peptide, a fragment of a Her2-specific anti-breast cancer cell monoclonal antibody scFv, a CD8α hinge region, a CD28 transmembrane domain or a neutrophil-specific CD32a transmembrane domain, a 4-1BB intracellular domain, a CD3ζ intracellular domain, and a fragment encoding IFN-γ. Based on this, four different CAR structures were designed: Her2-CAR1, Her2-CAR2, Her2-CAR1 / IFN-γ, and Her2-CAR2 / IFN-γ, with specific structures as follows: Figure 1 As shown.

[0074] The amino acid sequences of each component were identified using NCBI, optimized, and then tandemly linked. The tandem sequence was: CD8 signal peptide, scFv fragment of a specific anti-breast cancer cell Her2 monoclonal antibody, CD8α hinge region, CD28 transmembrane domain or neutrophil-specific CD32a transmembrane domain, 4-1BB intracellular domain, CD3ζ intracellular domain, and a fragment encoding IFN-γ. This constructed a specific CAR targeting Her2 breast cancer, and its encoding DNA sequence is shown in SEQ ID NO: 1-4, as detailed below:

[0075] The encoding DNA sequence of Her2-CAR1 is shown in SEQ ID NO: 1.

[0076] The encoding DNA sequence of Her2-CAR2 is shown in SEQ ID NO: 2.

[0077] The encoding DNA sequence of Her2-CAR1 / IFN-γ is shown in SEQ ID NO: 3. Figure 1 As shown, the Her2-CAR1 / IFN-γ DNA sequence contains a linker T2A between the CD3ζ intracellular domain and the segment encoding IFN-γ.

[0078] The encoding DNA sequence of Her2-CAR2 / IFN-γ is shown in SEQ ID NO: 4. Figure 1 As shown, the Her2-CAR2 / IFN-γ DNA sequence contains a linker T2A between the CD3ζ intracellular domain and the segment encoding IFN-γ.

[0079] Four DNA sequences encoding Her2-CAR and IFN-γ fragments were synthesized and amplified using polymerase chain reaction (PCR). The amplified products were recovered by agarose gel electrophoresis, ligated into a linearized pLVX-Puro lentiviral vector, and sequenced for verification. To synthesize mRNA encoding Her2-CAR1, four mRNA sequences encoding Her2-CAR and IFN-γ fragments were synthesized via in vitro transcription (IVT), and the resulting products were purified by enzyme digestion. All four CAR mRNAs used in the experiments were synthesized by Shenzhen Ruiji Biotechnology Co., Ltd.

[0080] 1.2 Synthesis of targeted lipid molecules

[0081] NEBP peptide (sequence CGEAIPMSIPPEVK, as shown in SEQ ID NO: 5) was synthesized by solid-phase synthesis. After purification, the NEBP peptide was linked to DSPE-PEG-MAL by a specific reaction between the thiol group and maleimide (MAL) to prepare DSPE-PEG-NEBP. The structure of DSPE-PEG-NEBP was characterized by HPLC and 1H-NMR.

[0082] Traut's reagent and Ly6G-Fab were mixed at a 1:1 molar ratio, protected under nitrogen, and stirred overnight at 4°C to obtain thiolized Ly6G-SH. After purification of the reaction product (i.e., lipid nanoparticles), a post-insertion method was used to link Ly6G-SH to DSPE-PEG-MAL via the specific reaction of thiol groups with maleimide (MAL), allowing the targeted molecule to be inserted onto the surface of the lipid nanoparticles. Specifically, lipid nanoparticles prepared using a microfluidic device were mixed with purified Ly6G-SH and reacted at 37°C for 1 hour to link Ly6G-SH to DSPE-PEG-MAL.

[0083] The structure of the synthesized lipid molecules was determined by hydrogen spectroscopy, and their molecular weight was detected by MOLDI-TOF. The results are as follows: Figure 2 As shown, the MOLDI-TOF spectra indicate that the molecular weight of DSPE-PEG-MAL is approximately 4131, and the molecular weight of DSPE-PEG-NEBP is approximately 5914.

[0084] 1.3 Preparation and characterization of dual-targeting LNPs encapsulating CAR mRNA structures

[0085] 1.3.1 Screening experiment for specific LNP composition and ratio:

[0086] Accurately weigh each phospholipid material according to the proportions in the table below, dissolve them in ethanol to a total volume of 1 ml, and prepare the oil phase lipid mixture. Add 17 μg of Her2-CAR1 mRNA to a pH 4 sodium citrate buffer solution and dilute to 3 mL, then mix well to prepare the mixed mRNA solution. Add 3 mL of mRNA solution and 1 mL of lipid mixture to a microfluidic device and perform rapid mixing via microfluidics, as shown in the table below. Figure 4 As shown in Figure A, LNPs loaded with Her2-CAR1 mRNA were prepared. Finally, the thiolized Ly6G-Fab and LNPs were co-incubated at 37°C for 1 hour at a 1:1 molar ratio to obtain dual-targeting LNPs loaded with Her2-CAR1 mRNA.

[0087] The prepared dual-targeting LNPs were analyzed using a particle size potentiometer to obtain the particle size and zeta potential of each different formulation of dual-targeting LNPs. After preparation, each group of dual-targeting LNPs was left to stand for one week to observe their stability. Subsequently, primary neutrophils were transfected with the dual-targeting LNPs carrying Her2-CAR1 mRNA to investigate their effectiveness in transfecting primary neutrophils in vitro.

[0088] The screening criteria for each dual-targeting LNP group were: particle size within 200 nm, PDI not greater than 0.2, slightly positive charge, clear and transparent solution after standing without obvious precipitate, and successful in vitro transfection of primary neutrophils. The screening results are shown in the table below. The amount of each component is recorded in the molar ratio in the table. LNP formulations that meet the screening criteria are marked with "√" in the result column, and otherwise marked with "×".

[0089] .

[0090] Note: In all formulations, the molar ratio of the targeted lipid molecules DSPE-PEG-NEBP and DSPE-PEG-MAL is 2:1, and the amount of mRNA added should maintain the LNP at a nitrogen-to-phosphorus ratio of N:P = 6:1.

[0091] As can be seen from the table above, the optimal ionizable lipid is SM-102, which is irreplaceable. SM-102's pKa is close to the neutrophil endosome environment, and its neutrophil toxicity is significantly lower than that of MC3, DOTAP, etc., and LNP, which delivers mRNA, is best expressed on neutrophils.

[0092] The table above shows the preferred LNP lipid components as follows: ionizable lipid SM-102, DSPC or DOPE, cholesterol, DMG-PEG2000, and targeted lipid molecules DSPE-PEG-NEBP and DSPE-PEG-MAL. The preferred molar ratio of each component is 40~50:5~15:19-38:1~10:2:1. A more favorable molar ratio is 40~50:10:19~38:1.5:2:1.

[0093] 1.3.2 LNP prescriptions that meet the screening criteria

[0094] 1.3.2.1: Prescription LNP13

[0095] According to the prescription LNP13 in the table above, accurately weigh SM-102, DSPC, cholesterol, DMG-PEG2000, and the targeted lipid molecules DSPE-PEG-NEBP and DSPE-PEG-MAL (in a molar ratio of 50:10:38:1.5:2:1), dissolve them in ethanol to a total volume of 1 ml, and prepare the oil phase lipid mixture. Dilute Her2-CAR1 mRNA to 3 ml in sodium citrate buffer (pH=4) at an N / P ratio of 6 and mix well to prepare the mixed mRNA solution. Add 3 ml of mRNA solution and 1 ml of lipid mixture to a microfluidic device for rapid mixing using microfluidics, as described above. Figure 4 As shown in Figure A, LNPs loaded with Her2-CAR1 mRNA were prepared. Finally, the thiolized Ly6G-Fab and LNPs were co-incubated at 37°C for 1 hour at a 1:1 molar ratio to obtain dual-targeting LNPs loaded with Her2-CAR1 mRNA.

[0096] 1.3.2.2: Prescription LNP4

[0097] According to the prescription LNP4 in the table above, accurately weigh SM-102, DOPE, cholesterol, DMG-PEG2000, and the targeted lipid molecules DSPE-PEG-NEBP and DSPE-PEG-MAL (in a molar ratio of 40:10:35:1.5:2:1), dissolve them in ethanol to a total volume of 1 ml, and prepare the oil phase lipid mixture. Dilute Her2-CAR1 mRNA to 3 ml in sodium citrate buffer (pH=4) at an N / P ratio of 6 and mix well to prepare the mixed mRNA solution. Add 3 ml of mRNA solution and 1 ml of lipid mixture to a microfluidic device for rapid mixing using microfluidics, as described above. Figure 4 As shown in Figure A, LNPs loaded with Her2-CAR1 mRNA were prepared. Finally, the thiolized Ly6G-Fab and LNPs were co-incubated at 37°C for 1 hour at a 1:1 molar ratio to obtain dual-targeting LNPs loaded with Her2-CAR1 mRNA.

[0098] 1.3.2.3: Prescription LNP11

[0099] According to the prescription LNP13 in the table above, accurately weigh SM-102, DSPC, cholesterol, DMG-PEG2000, and the targeted lipid molecules DSPE-PEG-NEBP and DSPE-PEG-MAL (in a molar ratio of 50:10:19:1.5:2:1), dissolve them in ethanol to a total volume of 1 ml, and prepare the oil phase lipid mixture. Dilute Her2-CAR1 mRNA to 3 mL in pH 4 sodium citrate buffer at an N / P ratio of 6 and mix well to prepare the mixed mRNA solution. Add 3 mL of mRNA solution and 1 mL of lipid mixture to a microfluidic device for rapid mixing using microfluidics, as described above. Figure 4 As shown in Figure A, LNPs loaded with Her2-CAR1 mRNA were prepared. Finally, the thiolized Ly6G-Fab and LNPs were co-incubated at 37°C for 1 hour at a 1:1 molar ratio to obtain dual-targeting LNPs loaded with Her2-CAR1 mRNA.

[0100] The detection results of the three dual-targeting LNPs prepared are as follows: Figure 3 As shown. Figure 3 The particle sizes of the three dual-targeted LNPs obtained by ZhongA are all within 200nm. Figure 3 B in the middle shows that it carries a slight positive charge, and Figure 3 The C-value shows that the PDI of each LNP is no greater than 0.2. Comparing the detection results of dual-targeting LNPs obtained from the three formulations, the dual-targeting LNP prepared by formulation LNP13 showed better results. Subsequent experiments will prepare dual-targeting LNPs carrying Her2-CAR1 mRNA according to formulation LNP13, namely Her2-CAR1@LNP-NE (also known as NE-LNP / Her2-CAR1 or NE-LNP / CAR1).

[0101] Following the LNP13 formulation, the Her2-CAR1 mRNA in the LNP13 preparation method is replaced with Her2-CAR2 mRNA, Her2-CAR1 / IFN-γ mRNA, and Her2-CAR2 / IFN-γ mRNA, respectively, while keeping other operations unchanged, to obtain the other three dual-targeting LNPs:

[0102] 1) Dual-targeting LNPs carrying Her2-CAR2 mRNA: Her2-CAR2@LNP-NE (also known as NE-LNP / Her2-CAR2 or NE-LNP / CAR2).

[0103] 2) Dual-targeting LNPs containing Her2-CAR1 and IFN-γ mRNA: Her2-CAR1 / IFN-γ@LNP-NE (also known as NE-LNP / Her2-CAR1+IFNγ or NE-LNP / CAR1+IFNγ).

[0104] 3) Dual-targeting LNPs containing Her2-CAR2 and IFN-γ mRNA: Her2-CAR2 / IFN-γ@LNP-NE (also known as NE-LNP / Her2-CAR2+IFNγ or NE-LNP / CAR2+IFNγ).

[0105] 1.4 Characterization of the above four types of dual-targeting lipid nanoparticles

[0106] Take a drop of diluted Her2-CAR2 / IFN-γ@LNP-NE and place it on a copper grid. After air drying for 3-5 minutes, add a drop of phosphotungstic acid solution for negative staining. Observe the sample under a transmission electron microscope after it is completely dry. The results are as follows: Figure 4 As shown in Figure B, Her2-CAR2 / IFN-γ@LNP-NE exhibits a near-spherical shape.

[0107] The encapsulation efficiency of four dual-targeting lipid nanoparticles was detected using the RiboGreen kit, and the characterization results are as follows: Figure 4 As shown in Figure E, the encapsulation efficiency of dual-targeting lipid nanoparticles carrying four different mRNAs, Her2-CAR1, Her2-CAR2, Her2-CAR1 / IFN-γ, and Her2-CAR2 / IFN-γ, remained between 70% and 100%.

[0108] Simultaneously, its hydrated particle size and zeta potential were determined using a Malvern particle size analyzer, and the characterization results are as follows: Figure 4 As shown in Figure C, the particle sizes of the four types of nanoparticles are all around 200 nm, exhibiting relatively uniformity and stability. Meanwhile, as... Figure 4 As shown in Figure D, the zeta potential of the four nanoparticles is close to 0 mV, indicating that they carry a slight positive charge.

[0109] (II) In vitro functional evaluation of four types of dual-targeting lipid nanoparticles

[0110] 2.1 Extraction and isolation of primary neutrophils from mouse bone marrow

[0111] After euthanizing Balb / C mice, femurs and tibias were harvested, bone marrow was rinsed, and cells were collected by centrifugation after passing through a 70 μm cell filter. Primary neutrophils were isolated using a mouse bone marrow neutrophil isolation kit for subsequent experiments.

[0112] 2.2 Detection of the effect of dual-targeting lipid nanoparticles on in vitro transduction of neutrophils

[0113] Following the preparation method described in 1.3 above and the LNP13 formulation, mRNA (sequence such as SEQ ID NO: 6) encoding an mCherry sequence was loaded into dual-targeting lipid nanoparticles prepared with the LNP13 formulation.

[0114] Primary neutrophils were extracted from mouse bone marrow. After co-incubating the extracted neutrophils with dual-targeting lipid nanoparticles coated with mCherry for 12 hours, the uptake was qualitatively analyzed using laser confocal microscopy. To determine the location of the neutrophils under confocal microscopy, DAPI was used to stain the primary neutrophils, and bright-field imaging was performed simultaneously. Results are as follows: Figure 5 As shown in Figure A, after LNP transfection, DAPI and mCherry signals significantly overlapped spatially, indicating significant expression of mCherry on the surface of neutrophils. Uptake was quantitatively analyzed by flow cytometry. Results are as follows... Figure 5 As shown in Figure B, 88.1% of the transfected neutrophils were mCherry-positive, indicating that mCherry expression was successfully detected on the surface of neutrophils.

[0115] 2.3 Detection of CAR expression on neutrophils after Her2-CAR2 / IFN-γ@LNP-NE transduction

[0116] Primary neutrophils were isolated and co-incubated with Her2-CAR2 / IFN-γ@LNP-NE. The expression of CAR on the neutrophils was then detected by flow cytometry. Results are as follows: Figure 6 As shown, compared to untreated neutrophils ( Figure 6 Left UTD group), after Her2-CAR2 / IFN-γ@LNP-NE transfection ( Figure 6 The expression of CAR (Her2-CAR2 / IFN-γ@LNP-NE) was detectable in 20.5% of neutrophils.

[0117] 2.4 Evaluation of the targeting ability of NEBP and ly6G targets in vivo.

[0118] Following the preparation method described in section 1.3 above and the formulation shown in the table below, mRNA encoding the mCherry sequence was packaged into LNPs. Dual-targeting NEBP / Ly6G-LNP / mCherry, single-targeting NEBP-LNP / mCherry, and non-targeting LNP / mCherry were prepared, respectively.

[0119] .

[0120] The three LNPs were then co-incubated with neutrophils, and the expression of mCherry on neutrophils was detected to assess the in vivo targeting of NEBP and ly6G targets. The results are as follows: Figure 7 As shown in Figure A, the statistical results are as follows: Figure 7 In the study, the expression of mCherry on neutrophils transfected with NEBP / Ly6G-LNP / mCherry was 73.8%, compared with 36.8% after transfection with NEBP-LNP / mCherry and 3.50% after transfection with LNP / mCherry. This indicates that the dual-target system significantly improved the targeting of LNP delivery system to neutrophils.

[0121] Simultaneously, the expression of mCherry on macrophages was detected to assess whether there were significant off-target effects. Results are as follows: Figure 7 As shown in Figure B, the statistical results are as follows: Figure 7 In the C-cell model, after transfection with LNP / mCherry, mCherry expression in macrophages was 12.8%, after transfection with NEBP-LNP / mCherry it was 7.96%, while after transfection with NEBP / Ly6G-LNP / mCherry only 1.18% of macrophages expressed mCherry. This indicates that the dual-target approach significantly reduced off-target effects.

[0122] 2.5 Evaluation of the in vitro function and target cell killing ability of CAR-neutrophils

[0123] (1) Formation of immune synapses

[0124] The formation of immune synapses is a key step in CAR-neutrophil killing of tumor cells. A stable 4T1-Her2 transgenic cell line was constructed as the target cell line. CAR-neutrophils transfected with LNPs carrying Her2-CAR1 / IFN-γ and Her2-CAR2 / IFN-γ were co-incubated with the target cells at a ratio of 5:1. The formation of immune synapses was observed using confocal microscopy. The results are as follows: Figure 8 As shown in Figure A, an immune synaptic structure appears between CAR-neutrophils and target cells after co-incubation.

[0125] (2) NETs generated

[0126] Similarly, after co-incubating transfected CAR-neutrophils with target cells, the cells were stained with DAPI, and NET formation was detected by confocal microscopy. Neutrophils treated with phorbol ester (PMA) served as a positive control, while untreated wild-type neutrophils co-incubated with target cells served as the untreated group (UTD). Results are as follows: Figure 8In the middle B, compared with untransfected neutrophils (UTD) and phorbol ester (PMA) as a positive control, Her2-CAR1 / IFN-γ and Her2-CAR2 / IFN-γ showed a certain degree of NET production.

[0127] (3) ROS generation

[0128] Meanwhile, flow cytometry was used to detect the production of reactive oxygen species (ROS) after co-incubation of transfected CAR-neutrophils with target cells. The results were as follows: Figure 8 As shown in Figure C, 57.36% of untransfected neutrophils (UTD) were ROS positive, while the ROS positive rate of CAR-neutrophils obtained by transfection with LNPs carrying different mRNAs was over 85%, indicating that ROS production in CAR-neutrophils was significantly increased.

[0129] (4) External killing ability

[0130] After CFSE-stained 4T1-Her2 target cells were co-incubated with CAR-neutrophils for 12 hours, the suspended neutrophils were removed, and the adherent target cells were stained with PI. The killing effect of CAR-neutrophils on target cells was detected by flow cytometry. The results are as follows: Figure 8 As shown in Figure D, 23.9% of target cells died after co-incubation with untransfected neutrophils (UTDs), while more than 30% of target cells were killed after co-incubation with different groups of CAR-neutrophils. CAR-neutrophils in target cells have a certain killing ability against Her2-overexpressing target cells.

[0131] 2.6 Detection of in vitro CAR-neutrophil typing

[0132] Neutrophils were transfected with dual-targeting LNPs loaded with Her2-CAR2 / IFN-γ to obtain CAR-neutrophils. RNA seq and qPCR were used to detect whether, under stimulation by 4T1-Her2 target cells, CAR-neutrophils differentiated to some extent into the N1 type, which has tumor-suppressive function. The relative expression levels of ICAM-1 and iNOS were used as markers for N1-type neutrophils, and the relative expression levels of VEGF, ARG-1, and CCL2 were used as markers for N2-type neutrophils. Results are as follows: Figure 9 As shown, transfected tumor-associated neutrophils differentiated more into N1 tumor suppressor neutrophils compared to untreated wild-type neutrophils.

[0133] (III) Detection of in vivo functions of four dual-targeting lipid nanoparticles

[0134] NSG mice orally inoculated with 4T1-Her2 were randomly divided into 5 groups, with 5 mice in each group. Starting on day 7 post-inoculation, each group of NSG mice was injected with NE-LNP / Her2-CAR1, NE-LNP / Her2-CAR2, NE-LNP / Her2-CAR1+IFNγ, NE-LNP / Her2-CAR2+IFNγ, and PBS as a control, respectively. Injections were administered twice weekly, following the specific protocol described below. Figure 10 As shown. The following indicators were measured during the administration period:

[0135] 3.1 Detect the infiltration of immune cells in tumor tissue and the amount of CAR expressed by neutrophils in the tumor site.

[0136] On day 10, tumors were removed from mice and prepared into single-cell suspensions. Labeled flow cytometry antibodies were used to characterize immune cells: T cells (CD45+CD3+), B cells (CD45+CD19+), NK cells (CD45+CD3-CD49b+), macrophages (CD45+CD11b+F4 / 80+), and neutrophils (CD45+CD11b+Ly6G+), and the percentage of each immune cell type was detected. The infiltration status of T cells (T), B cells (B), macrophages (M), NK cells (NK), and neutrophils (neu) is shown below. Figure 11 As shown in Figure A, neutrophil infiltration was most abundant in the tumor site. Simultaneously, flow cytometry was used to detect CAR expression on neutrophils (CD45+CD11b+Ly6G+) in the tumor site, and the results are as follows. Figure 11 As shown in Figure B, neutrophils at the tumor site after drug administration ( Figure 11 The expression rate of B (left) was 8.84%. Figure 11 (Middle B right).

[0137] 3.2 In vivo pharmacodynamic evaluation was performed by observing changes in tumor size and body weight in mice after drug administration.

[0138] During the drug administration period, the vital signs of mice in each group were continuously monitored according to the protocol. The changes in tumor volume in mice over time were as follows: Figure 12 As shown in the left image of A, the change in weight over time is as follows: Figure 12 As shown in Figure A on the right. Simultaneously, the tumor sizes of the LNP groups treated with CAR1 mRNA, CAR2 mRNA, CAR1+IFNγ mRNA, CAR2+IFNγ mRNA, and the control group (PBS) were statistically analyzed. Figure 12 As shown in Figure B, the development trend of tumor size in each group of mice is presented. From the perspective of tumor size and mouse weight, among the four different CAR structures, the CAR1+IFNγ group and the CAR2+IFNγ group showed relatively better results.

[0139] 3.3 Her2-CAR / IFN-γ@LNP-NE produces IFN-γ in vivo

[0140] Peripheral blood was collected from mice after continuous drug administration, and single-cell suspensions were prepared. The expression of IFN-γ on neutrophils was detected by flow cytometry. The results are as follows: Figure 13 As shown, IFN-γ expression was detected in mouse neutrophils after administration of NE-LNP / CAR1+IFNγ and NE-LNP / CAR2+IFNγ.

[0141] 3.4 Each dual-targeting LNP generates Her2-targeting CAR-neutrophils in vivo.

[0142] Peripheral blood was collected from mice after continuous drug administration and prepared into single-cell suspensions. The number of CAR-neutrophils in the suspensions was detected by flow cytometry to examine the ability of each dual-targeting LNP to generate Her2-targeting CAR-neutrophils in vivo. The results are as follows: Figure 14 As shown, after drug administration, NE-LNP / CAR1 group, NE-LNP / CAR2 group, NE-LNP / CAR1+IFNγ group and NE-LNP / CAR2+IFNγ group were able to successfully produce CAR-neutrophils in vivo.

[0143] 3.5 Assessing the killing effect of CAR-neutrophils within the tumor using tumor TUNEL staining

[0144] After 20 days of continuous administration according to the method described in 3.4, the experiment was terminated. Frozen sections of tumor tissue were collected for TUNEL staining to observe the killing effect of neutrophils on tumor tissue in mice, and the fluorescence was quantified. The results are as follows: Figure 15 As shown, based on TUNEL fluorescence and quantitative results, NE-LNP / CAR1+IFNγ and NE-LNP / CAR2+IFNγ exhibit significant killing abilities.

[0145] 3.6 Detection of cytokine production in vivo by Her2-CAR / IFN-γ@LNP-NE

[0146] Tumor-bearing mice were treated with PBS for NE-LNP / CAR1, NE-LNP / CAR2, NE-LNP / CAR1+IFNγ, NE-LNP / CAR2+IFNγ, and a control group, respectively. Peripheral blood was collected from the mice, centrifuged, and cytokine expression levels were detected using ELISA. The results are as follows: Figure 16 As shown, the overall cytokine levels are low, resulting in a lower risk of cytokine syndrome.

[0147] 3.7 Liver and kidney function in mice were assessed after continuous administration to evaluate drug safety.

[0148] After administering the medication as described in 3.4 for 20 days, the experiment was terminated. Peripheral blood from mice was collected and centrifuged to obtain serum. Biochemical indicators related to liver and kidney function were then detected. The results are as follows: Figure 17 All relevant indicators were within the normal range, indicating that continuous administration had no significant effect on liver and kidney function in mice.

[0149] The above are merely embodiments of the present invention and do not limit the scope of the patent. Any equivalent modifications made based on the content of this specification, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A lipid nanoparticle for generating chimeric antigen receptor neutrophils in vivo, characterized in that, The lipid nanoparticles are dual-targeting lipid nanoparticles, and the dual targeting is achieved through Ly6G antibody fragments and NEBP peptides; The lipid nanoparticles contain lipid components and mRNA encoding Her2-CAR and IFN-γ; The mRNA is obtained by in vitro transcription synthesis of DNA with nucleotide sequences as shown in SEQ ID NO: 3 or SEQ ID NO: 4; The lipid component comprises ionizable lipid SM-102, helper phospholipid DSPC, cholesterol, DMG-PEG2000, and targeted lipid molecules DSPE-PEG-NEBP and DSPE-PEG-MAL, with a molar ratio of 50:10:38:1.5:2:

1. The nitrogen / phosphorus ratio of the lipid nanoparticles is 6; The DSPE-PEG-NEBP is prepared by linking the NEBP polypeptide with the sequence shown in SEQ ID NO: 5 to DSPE-PEG-MAL; The lipid nanoparticles are attached to a targeting molecule Ly6G-SH, which is covalently linked to the lipid nanoparticles via a coupling reaction between the thiol group and the maleimide group on the lipid component DSPE-PEG-MAL. The Ly6G-SH is prepared by a thiolization reaction of Ly6G-Fab.

2. The lipid nanoparticles for generating chimeric antigen receptor neutrophils in vivo according to claim 1, characterized in that, The Her2-CAR is a specific CAR targeting Her2 in breast cancer, comprising the following domains in sequence: CD8 signal peptide, anti-Her2 scFv fragment, CD8α hinge region, transmembrane domain, 4-1BB intracellular domain, and CD3ζ intracellular domain; the transmembrane domain is CD28 or CD32a; the Her2-CAR and IFN-γ are co-expressed by being linked through an autocracked peptide sequence.

3. The method for preparing lipid nanoparticles according to any one of claims 1-2, characterized in that, Includes the following steps: (1) Preparation of targeted lipid molecules DSPE-PEG-NEBP and Ly6G-SH: A. NEBP peptide was synthesized by solid-phase synthesis, purified, and then added to DSPE-PEG-MAL. The thiol group on the NEBP peptide reacted specifically with the maleimide on DSPE-PEG-MAL, thus linking the NEBP peptide to DSPE-PEG-MAL to prepare DSPE-PEG-NEBP for later use. The sequence of the NEBP peptide is shown in SEQ ID NO:

5. B. Mix Traut's reagent with Ly6G-Fab at a molar ratio of 1:1 and prepare Ly6G-SH by thiolization reaction for later use; (2) Preparation of lipid nanoparticles, the steps are as follows: A. Ionizable lipids SM-102, DSPC, cholesterol, DMG-PEG2000, and targeted lipid molecules DSPE-PEG-NEBP and DSPE-PEG-MAL were dissolved in ethanol in proportion to form an oil phase lipid mixture. B. With an N / P ratio of 6, mix the mRNA solution and lipid solution. Dissolve the mRNA encoding Her2-CAR and IFN-γ in sodium citrate buffer to obtain the mixed mRNA solution. C. The mRNA solution and lipid mixture were added to a microfluidic device to prepare an LNP containing mRNA encoding Her2-CAR and IFN-γ. D. Ly6G-SH and LNP prepared in the previous step were co-incubated at 37°C for 1 hour at a molar ratio of 1:1 to obtain dual-targeting lipid nanoparticles.

4. The use of the lipid nanoparticles according to any one of claims 1-2 in the preparation of a medicament for treating tumors, wherein the tumor is Her2-positive breast cancer.

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