A method for increasing the anti-tumor response by enhancing the cellular immune response

Abstract

The application discloses a method for improving anti-tumor response by enhancing cellular immune response, comprising administering a nucleic acid molecule encoding an antigen membrane protein to a subject in need, wherein the antigen membrane protein is an antigen membrane protein with a front signal peptide segment removed, and belongs to the technical field of biotechnology; the method disclosed by the application enhances and accelerates the cellular immune response of a receptor, thereby improving the anti-tumor activity, and has the potential for clinical application.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically, it relates to a method for enhancing anti-tumor response by strengthening cellular immune response. Background Technology

[0002] A tumor is a new growth formed by the proliferation of local tissue cells. Tumors are broadly classified into benign and malignant tumors. Malignant tumors can be further divided into carcinomas and sarcomas; carcinomas are malignant tumors originating from epithelial tissue. Cancer immunotherapy is a treatment method that utilizes the power of the immune system to fight cancer. It enhances or modifies the patient's immune response to more effectively recognize and attack cancer cells. Cancer immunotherapy includes cancer vaccines, adoptive cell transfer (ACT), immune checkpoint inhibitors, and cytokine therapy. Cancer vaccines fight cancer by activating specific anti-tumor immune responses. Cancer vaccines can also generate durable memory T cells, providing long-term protection; therefore, cancer vaccines have broad application prospects in cancer immunotherapy.

[0003] Vaccines are an important method for cancer immunotherapy, and selecting and optimizing the optimal antigen is crucial. Currently, when using vaccines to treat cancer, there are technical limitations in antigen expression and guiding cellular immune responses, leading to poor immune responses, low anti-tumor effects, and ultimately, inadequate anti-tumor efficacy. Therefore, there is a need to optimize antigens and enhance immune responses in anti-tumor immunotherapy. Summary of the Invention

[0004] The present invention aims to at least partially solve the technical problems of poor immune response and unsatisfactory anti-tumor effects of existing anti-tumor immunotherapy vaccines.

[0005] The present invention provides a method for enhancing anti-tumor response by enhancing cellular immune response, comprising administering a nucleic acid molecule encoding an antigen membrane protein to a subject in need, wherein the antigen membrane protein is an antigen membrane protein with a front signal peptide removed; In this invention, the antigen membrane protein includes one or more of PD-L1, HER2, HER3, Cripto-1, Trop2, CD24, CD20, BCMA, EGFR, VEGFR, VEGFR2, KIR, TIGIT, ALK, MET, B7H3, B7H4, CD38, FGFR2b, c-Met, FRα, TREM2, CD19, CDH6, DLL3, CLDN-18.2, CLDN-6, FAP-α, BAFF, Nectin-4, MUC1, MUC16, GPRC5D, ROR1, CCN2, EpCAM, DR5, and TpoR. In this invention, the nucleic acid molecule is DNA or RNA; preferably RNA; Furthermore, the RNA includes one or more of mRNA, saRNA, and circRNA; In this invention, the nucleic acid molecules are delivered using viral vectors and non-viral vectors; Furthermore, the viral vector is one or more of a retroviral vector, adenovirus vector, and adeno-associated virus (AAV) vector; Furthermore, the non-viral carrier is one or more of liposomes, lipid nanoparticles, micelles, cationic polymers, and core-shell nanoparticles; In this invention, the nucleic acid molecules are delivered using lipid nanoparticles. The lipid nanoparticles contain the nucleic acid molecules and also contain one or more of ionizable lipids, phospholipids, sterols, and PEG-modified lipids; or may contain pharmaceutically acceptable carriers, excipients, or adjuvants. In this invention, the mRNA comprises an open reading frame (ORF), a 5'UTR, and a 3'UTR encoding an antigen membrane protein: 5'UTR-ORF-3'UTR; wherein the antigen membrane protein is an antigen membrane protein with its leading signal peptide removed; In a specific embodiment of the present invention, the open reading frame (ORF) encoding the antigen membrane protein further includes the binding or recruitment of an E3 ubiquitinase ligase co-encoded with the antigen membrane protein. Furthermore, the E3 ubiquitinase ligase may be selected from RING type, HECT type or RBR type; Further, the E3 ubiquitinase ligase is optionally selected from one or more of VHL (Von Hippel-Lindau), MDM2, CRBN, IAPs, RNF, β-TrCP, DCAF, Keap1, or their truncated or extended forms; Furthermore, the binding or recruitment of ligands by the E3 ubiquitinase ligase to the antigen membrane protein includes insertion ligation, direct end ligation, or ligation via a linker. Furthermore, the nucleotide sequence of the binding or recruiting ligand of the E3 ubiquitinase ligase is at least 80% identical to the nucleotide sequence of SEQ ID NO:2; Furthermore, the nucleotide sequence of the binding or recruiting ligand of the E3 ubiquitinase ligase is at least 85% identical to the nucleotide sequence of SEQ ID NO:2; Furthermore, the nucleotide sequence of the binding or recruiting ligand of the E3 ubiquitinase ligase is at least 90% identical to the nucleotide sequence of SEQ ID NO:2; Furthermore, the nucleotide sequence of the binding or recruiting ligand of the E3 ubiquitinase ligase is at least 95% identical to the nucleotide sequence of SEQ ID NO:2; Furthermore, the nucleotide sequence of the binding or recruiting ligand of the E3 ubiquitinase ligase is at least 98% identical to the nucleotide sequence of SEQ ID NO:2; Furthermore, the nucleotide sequence of the binding or recruiting ligand of the E3 ubiquitinase ligase is at least 99% identical to the nucleotide sequence of SEQ ID NO:2; In a specific embodiment of the present invention, the nucleotide sequence of the E3 ubiquitinase ligase that binds to or recruits ligands is the nucleotide sequence shown in SEQ ID NO: 2. In a specific embodiment of the present invention, the binding or recruitment ligand of the E3 ubiquitinase ligase includes the amino acid sequence of SEQ ID NO: 8; In a specific embodiment of the present invention, in the open reading frame (ORF) encoding the antigen membrane protein, the nucleotide sequences of the antigen membrane protein and the binding or recruitment ligand of the E3 ubiquitinase ligase are linked by a linker. Furthermore, the nucleotide sequence of the linker is at least 80% identical to the nucleotide sequence of SEQ ID NO: 3; Furthermore, the nucleotide sequence of the linker is at least 85% identical to the nucleotide sequence of SEQ ID NO: 3; Furthermore, the nucleotide sequence of the linker is at least 90% identical to the nucleotide sequence of SEQ ID NO: 3; Furthermore, the nucleotide sequence of the linker is at least 95% identical to the nucleotide sequence of SEQ ID NO: 3; Furthermore, the nucleotide sequence of the linker is at least 98% identical to the nucleotide sequence of SEQ ID NO: 3; Furthermore, the nucleotide sequence of the linker is at least 99% identical to the nucleotide sequence of SEQ ID NO: 3; In a specific embodiment of the present invention, the nucleotide sequence of the linker is the nucleotide sequence shown in SEQ ID NO: 3; In a specific embodiment of the present invention, the amino acid sequence of the linker is (GGGGS). 1~6 or (GGS) 5~10 Or (GSG) 5~10 At least one of them; In this invention, the 5'UTR has a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 4; Furthermore, the 5'UTR has a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 4; Furthermore, the 5'UTR has a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 4; Furthermore, the 5'UTR has a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 4; Furthermore, the 5'UTR has a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 4; Furthermore, the 5'UTR has a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 4; In a specific embodiment of the present invention, the 5'UTR has the nucleotide sequence shown in SEQ ID NO: 4; In this invention, the 3'UTR has a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 5; Furthermore, the 3'UTR has a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 5; Furthermore, the 3'UTR has a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 5; Furthermore, the 3'UTR has a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 5; Furthermore, the 3'UTR has a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 5; Furthermore, the 3'UTR has a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 5; In a specific embodiment of the present invention, the 3'UTR has the nucleotide sequence shown in SEQ ID NO: 5; In this invention, the mRNA further includes a 5' end cap structure; Further, the 5' terminal cap structure is CapO, Cap1, ARCA, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-diazo-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, 5'-methylGcap or analogues thereof. In this invention, the mRNA further includes a 3' poly A tail. In this invention, the poly A has a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 6; Furthermore, the poly A has a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 6; Furthermore, the poly A has a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 6; Furthermore, the poly A has a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 6; Furthermore, the poly A has a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO: 6; Furthermore, the poly A has a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 6; In a specific embodiment of the present invention, the poly A has the nucleotide sequence shown in SEQ ID NO: 6; In this invention, the mRNA may also contain at least one chemical modification; Further, the chemical modification is selected from the following: pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deazo-pseudouridine, 2-thio-1-methylpseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methylpseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine. Furthermore, the mRNA comprises a subset selected from pseudouridine or a pseudouridine analogue; Furthermore, the mRNA is modified with N1-methylpseuuridine; In this invention, the lipid nanoparticles contain ionizable lipids in a molar ratio of 20% to 85%. Furthermore, in this invention, the lipid nanoparticles contain phospholipids in a molar ratio of 5% to 20%. Furthermore, in this invention, the lipid nanoparticles contain 30% to 60% sterols in a molar ratio. Furthermore, in this invention, the lipid nanoparticles contain PEG-modified lipids in a molar ratio of 0% to 5%; In this invention, the application further comprises a pharmaceutically acceptable carrier, excipient, or adjuvant. Furthermore, in a specific embodiment of the present invention, the excipients include one or more selected from injectable buffer media, lyophilization agents, or cryoprotectants; Furthermore, the adjuvant is one or more of Freund's incomplete adjuvant, Freund's complete adjuvant, aluminum hydroxide adjuvant, aluminum phosphate adjuvant, emulsion adjuvant, liposome adjuvant, and microbial adjuvant; In this invention, the nucleic acid molecules are encapsulated in lipid nanoparticles using a microfluidic device; In this invention, the signal peptide is a secretion signal peptide of a membrane antigen; In this invention, the tumor is a solid tumor or a hematoma; In another aspect of the invention, the invention provides the use of a nucleic acid molecule encoding an antigen membrane protein in a medicament for the prevention or treatment of cancer in an individual or for delaying the progression of cancer in an individual; by administering the antigen membrane protein described above with the front signal peptide removed, the cellular immune response of the administered subject in need is greatly enhanced, the anti-tumor response is strengthened, thereby achieving the prevention and treatment or delay of cancer progression in an individual. In this invention, the cancer is one or more of the following: stomach cancer, esophageal cancer, pancreatic cancer, ovarian cancer, breast cancer, lung cancer, colorectal cancer, prostate cancer, cervical cancer, head and neck cancer, nasopharyngeal carcinoma, leukemia, lymphoma, lung cancer, liver cancer, melanoma, and soft tissue sarcoma. Furthermore, in a specific embodiment of the invention, the treatment further includes combined administration of a second drug comprising an mRNA containing an anticancer agent or an ORF encoding other antigens and optionally a pharmaceutically acceptable carrier.

[0006] In a specific embodiment of the present invention, the anticancer agent in the second drug is one or more of the following: small molecule chemical drugs, ADC drugs, antibody drugs, protein drugs, nucleic acid drugs, and cell drugs. In this invention, by administering to a subject the nucleic acid molecule encoding the antigen membrane protein described above, or a pharmaceutical composition containing the nucleic acid molecule encoding the antigen membrane protein described above, or lipid nanoparticles containing the nucleic acid molecule encoding the antigen membrane protein described above, the antigen membrane protein is translated and expressed in the subject's body; the peptide particles generated by intracellular digestion are MHC II epitopes, which present antigens on CD8 receptors, initiate an immune response, reduce tumor size or inhibit tumor growth, and induce an anti-tumor response.

[0007] In some embodiments, the above-described pharmaceutical composition can be used to prepare liposomes in polymer form; according to embodiments of the present invention, the specific method of preparing liposomes using the above-described pharmaceutical composition is not limited, and the use of the above-described pharmaceutical composition to prepare liposomes, wherein the liposomes contain all or part of the structure of the above-described compound, is considered an application of the present invention.

[0008] For application purposes, the nucleic acid molecules and mRNA of the present invention can be directly formulated into pharmaceutical compositions. The pharmaceutical compositions of the present invention comprise one or more pharmaceutically acceptable carriers, diluents, or excipients. They may also contain ionic lipid compounds, cationic lipid compounds, etc., to effectively form liposomes and deliver the bioactive components nucleotides and mRNA, for example, in amounts present in the composition for treating a specific disease or condition. Those skilled in the art can readily determine appropriate concentrations and dosages.

[0009] In this invention, the ionic lipid compounds and cationic lipid compounds may be any of the ionic lipid compounds and cationic lipid compounds disclosed in the prior art; for example, this application may use the ionic lipid compounds disclosed in application numbers 2022800119278, PCT / CN2022 / 122626, 2023100095458, PCT / CN2023 / 084757, and 2024107508742, and incorporates all the contents disclosed in the above patent applications into this application.

[0010] In this invention, the 5'UTR can be obtained through natural occurrence, isolation and purification, or optimized synthesis; the 5'UTR related content of this application can adopt the sequences disclosed in application numbers 2023112251067 and 2023111261822, and all the content disclosed in the above patent applications is incorporated into this application.

[0011] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0012] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This is the tumor growth curve of Experiment Example 4; Figure 2 This is a cell image from Experiment 5 showing ELISpot detection of cells that induce IFN-γ secretion; Figure 3 This is a bar chart of CD3+ T cell infiltration in the tumor microenvironment as detected by flow cytometry in Experiment Example 6; Figure 4This is a bar chart of CD4+ T cell infiltration in the tumor microenvironment obtained by flow cytometry in Experiment Example 6; Figure 5 This is a bar chart of CD8+ T cell infiltration in the tumor microenvironment obtained by flow cytometry in Experiment Example 6; Figure 6 The flow cytometry analysis of Experiment 7 shows the bar graph of vaccine-induced spleen memory T cells (CD8+ T). Figure 7 This is a bar graph of vaccine-induced spleen memory T cells (CD4+ T) produced by flow cytometry analysis in Experiment 7. Detailed Implementation

[0013] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0014] It should be noted that, regarding the technical solutions disclosed in the embodiments or implementation schemes of this invention, this invention intends to cover all alternative, modified, and equivalent technical solutions, all of which are included within the scope of this invention. Those skilled in the art should recognize that many similar or equivalent methods and materials can be used to practice this invention. This invention is by no means limited to the methods and materials described herein. In the event that one or more of the cited documents, patents, and similar materials differ from or contradict this application (including but not limited to defined terminology, terminology application, described techniques, etc.), this invention shall prevail.

[0015] It should be further appreciated that certain features of the invention, for clarity, have been described in multiple independent embodiments or implementations, but may also be provided in combination in a single embodiment or implementation. Conversely, various features of the invention, for brevity, have been described in a single embodiment or implementation, but may also be provided individually or in any suitable sub-combination.

[0016] Definitions and explanations: Unless otherwise indicated, the technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and unless otherwise indicated, all patent publications cited in the entirety of this disclosure are incorporated herein by reference.

[0017] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0018] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0019] In this article, the term "(XXXX)" n~m The colon ("") represents a sequence of XXXX numbers, ranging from n to m, where X represents an amino acid. For example, "(GGGGS)". 1~6 "" indicates that 1, 2, 3, 4, 5 or 6 GGGGS are connected.

[0020] In this document, the term "pharmaceutically acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components of the formulation and / or the mammals to which it is treated.

[0021] In this document, the term "one or more" (e.g., in the definition of substituents in compounds of the general formula of the present invention) means "one, two, three, four or five, especially one, two, three or four, more especially one, two or three, and even more especially one or two".

[0022] In this article, "ionizable lipids" includes cationic lipids, ionizable lipids, and their derivatives.

[0023] In this article, "liposomes" or "liposome nanoparticles (LNPs)" refers to a drug delivery system that uses biocompatible lipid materials as carriers to dissolve or encapsulate drugs or other bioactive substances in the lipid core or adsorb and attach them to the surface of nanoparticles.

[0024] In this document, the term "pharmaceuticalally acceptable excipient" includes any solvent, dispersion medium, coating material, surfactant, antioxidant, preservative (e.g., antibacterial, antifungal), isotonic agent, salt, pharmaceutical stabilizer, binder, excipient, dispersant, lubricant, sweetener, flavoring agent, colorant, or combination thereof, all of which are known to those skilled in the art (as described in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Except in cases where any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is covered.

[0025] In this document, the term "prevention" refers to the use of a drug or measure to achieve a preventive pharmacological and / or physiological effect in healthy or high-risk individuals, preventing the onset or worsening of a disease. In this document, the term "treatment" refers to the use of a drug or measure to achieve a desired pharmacological and / or physiological effect. This effect may be preventive in terms of complete or partial prevention of the disease or its symptoms, and / or therapeutic in terms of partial or complete cure of the disease and / or adverse effects caused by the disease. As used herein, "treatment" covers diseases in mammals, particularly humans, including: (a) prevention of disease or symptoms in individuals susceptible to disease but not yet diagnosed with the disease; (b) inhibition of disease, such as blocking disease progression; or (c) relief of disease, such as reducing disease-related symptoms. As used herein, "treatment" covers any administration of a drug or compound to an individual to treat, cure, relieve, improve, reduce, or inhibit the individual's disease, including but not limited to administration of a drug containing a compound described herein to an individual in need.

[0026] The effective dose described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective dose can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the bioactive ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0027] The nucleic acid molecules and lipid nanoparticles of the present invention can be incorporated into pharmaceuticals suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These pharmaceuticals can be prepared in various forms, such as liquids, semi-solids, and solid dosage forms, including but not limited to liquid solutions (e.g., injection solutions and infusion solutions) or lyophilized powders. Typical pharmaceuticals are injection solutions. The aforementioned complexes or pharmaceutical compositions can be administered by intravenous infusion, injection, intramuscular injection, subcutaneous injection, or intratumoral injection.

[0028] According to embodiments of the present invention, the administration route of the method is intramuscular injection, intravenous injection, or intratumoral administration.

[0029] According to embodiments of the present invention, the nucleotide sequences in Table 1 and the amino acid sequences in Table 2 are disclosed below: Table 1 Nucleotide Sequences

[0030] Table 2 Amino acid sequences

[0031] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0032] Example 1: Trop2 (EL) antigen sequence design and synthesis (1) Sequence design: The full-length Trop2 antigen amino acid sequence was optimized by removing the secretion signal peptide amino acid sequence segment (△2-27), followed by codon optimization; this was used as an open reading frame (ORF) containing the membrane protein encoding the Trop2 (EL) antigen. In this embodiment, the transcription template DNA for Trop2-mRNA consists of a promoter, a 5' untranslated region, a Trop2 antigen coding region (ORF), a 3' untranslated region, and a Poly(A) linker, which are then ligated into the corresponding position on the plasmid vector. Sequencing verifies that the sequence is correct, and the mRNA transcription template DNA is obtained.

[0033] The promoter is either T7 or SP6. The nucleotide sequence of the 5' untranslated region is shown in SEQ ID NO. 4; The nucleotide sequence of the Trop2 antigen coding region is shown in SEQ ID NO. 1; the amino acid sequence of the Trop2 antigen coding region is shown in SEQ ID NO. 7. The nucleotide sequence of the 3' untranslated region is shown in SEQ ID NO. 5; The nucleotide sequence of Poly(A) is shown in SEQ ID NO. 6; Synthesis and purification: The transcribed template DNA of the Trop2-mRNA from (1) was inserted into the pUC57 vector, and the culture was fermented and expanded. Plasmid DNA was then extracted and purified. Following the instructions of the author's manual (Molecular Cloning: A Laboratory Manual, 4th Edition) and commercially available restriction endonuclease and DNA purification kits, the plasmid DNA was digested to obtain a linearized plasmid DNA template, which was then purified. The concentration and purity of the plasmid DNA and the linearized DNA template were detected by spectrophotometry and gel electrophoresis, and complete linearization was confirmed by electrophoresis.

[0034] Then, mRNA was synthesized using RNA polymerase, NTP, and cap analogues. Precursor mRNA was transcribed in vitro according to the kit (Novizan TR101-02). Specifically: Add the relevant reactants to 1.5 mL centrifuge tubes according to the dosage and order. Mix by pipetting three times or gently tapping the bottom of the tube with a finger. Centrifuge briefly to collect the reaction solution at the bottom of the tube. Incubate at 37°C for 4 h, then add 1 μL of DNase and incubate at 37°C for 15 min. Next, add 179 μL of nuclease-free water, followed by 200 μL of phenol / chloroform / isoamyl alcohol solution for mRNA extraction. After mixing, centrifuge at 15000 rpm for 10 min and collect the aqueous phase. Add 200 μL of 5 M ammonium acetate solution and incubate overnight at 4°C for mRNA precipitation. Then, centrifuge at 15000 rpm for 10 min at 4°C and carefully remove the supernatant. Wash the precipitate with 1 mL of 70% ethanol, centrifuge at 15000 rpm for 10 min at 4°C, carefully remove the supernatant, and air-dry the precipitate. Then reconstitute with 20 μL of nuclease-free water to obtain the Trop2(EL)-mRNA solution.

[0035] The purity of Trop2(EL)-mRNA was detected by agarose gel electrophoresis or a nucleic acid fragment analyzer.

[0036] Example 2: Preparation of Trop2-mRNA lipid nanoparticles (Trop2(EL)-mRNA-LNP) Trop2(EL)-mRNA lipid nanoparticles were prepared using microfluidic technology. The formulation consisted of C4 (an ionizable lipid compound; the specific compound in application number 2022800119278), DOPE, Chol, DMG-PEG2000, and Trop2-mRNA. The mass ratio of C4 to mRNA was set at 12:1 for the preparation of the organic and aqueous phases. C4, DOPE, Chol, and DMG-PEG2000 were dissolved in anhydrous ethanol and mixed in a molar ratio of 45:10:43.5:1.5 to prepare a certain volume of organic phase. Simultaneously, Trop2(EL)-mRNA was prepared in an aqueous phase using RNase-free water, with a volume ratio of aqueous phase to organic phase of 3:1. The LNP formulation loaded with Trop2(EL)-mRNA was prepared in one step using a microfluidic nanoparticle formulation instrument. The instrument parameters were set as follows: the ratio of aqueous phase to organic phase was fixed at 3:1, and the flow rate was fixed at 12 mL / min. The microfluidic initial formulation was ultrafiltered with phosphate-buffered saline (PBS) to remove ethanol, and the mRNA concentration of the final formulation was controlled to be 0.2 mg / mL, thus obtaining Trop2(EL)-mRNA lipid nanoparticles (Trop2(EL)-mRNA-LNP).

[0037] Particle size and potential detection: A certain volume of Trop2(EL)-mRNA lipid nanoparticle colloidal solution was measured and diluted 10 times with purified water. The particle size and potential of the mRNA vaccine were measured in a laser particle size analyzer (n=3, i.e., 3 measurements for each formulation). The results showed that the prepared Trop2(EL)-mRNA lipid nanoparticles had a particle size of approximately 65 nm, a PDI of approximately 0.12, and a potential of approximately 3 mV, indicating good uniformity.

[0038] Encapsulation efficiency testing: The encapsulation efficiency of the sample was 98% as determined by the Quant-iT™ RiboGreen™ kit.

[0039] The above test results show that the prepared Trop2(EL)-mRNA lipid nanoparticles have small and uniform particle size and high encapsulation efficiency.

[0040] Comparative Example 3 Using a procedure similar to that in Example 1, the full-length Trop2 antigen without the removal of the secretory signal peptide was used as the Trop2 antigen coding region of the mRNA; Trop2(FL)-mRNA was prepared; and then, using a procedure similar to that in Example 2, lipid nanoparticles of Trop2(FL)-mRNA (Trop2(FL)-mRNA-LNP) were prepared.

[0041] The nucleotide sequence of the Trop2 antigen coding region of Trop2(FL)-mRNA is shown in SEQ ID NO. 9; SEQ ID NO. 9: ATGGCCAGGGGCCCCGGCCTGGCCCCCCCCCCCCTGAGGCTGCCCCTGCTGCTGCTGGTGCTGGCCGCCGTGACCGGCCACACCGCCGCCCAGGACAACTGCACCTGCCCCACCAACAAGATGACCGTGTGCAGCCCCGACGGCCCCGGCGGCAGGTGCCAGTGCAGGGCCCTGGGCAGCGGCATGGCCGTGGACTGTAGCACCCTGACCAGCAAGTGCCTGCTGCTGAAGGCCAGGATGAGCGCCCCCAAGAACGCCAGGACCCTGGTGAGGCCCAGCGAGCACGCCCTGGTGGACAACGACGGCCTGTACGACCCCGACTGCGACCCCGAGGGCAGGTTCAAGGCCAGGCAGTGCAACCAGACCAGCGTGTGCTGGTGCGTGAACAGCGTGGGCGTGAGGAGGACCGACAAGGGCGACCTGAGCCTGAGGTGCGACGAGCTGGTGAGGACCCACCACATCCTGATCGACCTGAGGCACAGGCCCACCGCCGGCGCCTTCAACCACAGCGACCTGGACGCCGAGCTGAGGAGGCTGTTCAGGGAGAGGTACAGGCTGCACCCCAAGTTCGTGGCCGCCGTGCACTACGAGCAGCCCACCATCCAGATCGAGCTGAGGCAGAACACCAGCCAGAAGGCCGCCGGCGACGTGGACATCGGCGACGCCGCCTACTACTTCGAGAGGGACATCAAGGGCGAGAGCCTGTTCCAGGGCAGGGGCGGCCTGGACCTGAGGGTGAGGGGCGAGCCCCTCCAGGTGGAGAGGACCCTGATCTACTACCTGGACGAGATCCCCCCCAAGTTCAGCATGAAGAGGCTGACCGCCGGCCTGATCGCCGTGATCGTGGTGGTGGTGGTGGCCCTGGTGGCCGGCATGGCCGTGCTGGTGATCACCAACAGGAGGAAGAGCGGCAAGTACAAGAAGGTGGAGATCAAGGAGCTGGGCGAGCTGAGGAAGGAGCCCAGCCTG Experiment Example 4: In vivo anti-tumor effect of C4@TROP2-mRNA vaccine (in vivo mouse tumor model experiment) Test method: (1) Cell line construction The tumor-specific antigen TROP2 gene was randomly integrated into the genome of mouse colon cancer cells CT26 using a lentiviral vector, and then the cell line CT26-hTROP2, which stably expresses TROP2, was obtained by screening.

[0042] (2) Establishment of mouse tumor model CT26-hTROP2 cells in logarithmic growth phase were collected, and a cell suspension was prepared. The cells were then resuspended in pre-cooled PBS solution at 4°C, at a concentration of 1 × 10⁻⁶ cells per cell. 6 Cells were seeded into the buttocks of Balb / c mice via subcutaneous injection. The model was considered complete when the tumor diameter reached 4-5 mm.

[0043] (3) Immunotherapy: The (2) model mice were randomly divided into three groups: CTRL group, C4@FL-TROP2-mRNA group (Comparative Example 3) (abbreviated as FL-TROP2), and C4@EL-TROP2-mRNA group (Example 2) (abbreviated as EL-TROP2), with 5 mice in each group. The administration route was intravenous injection, with 10 μg injected intravenously three times (d6, d9, d14) each time. Starting from the 6th day after tumor inoculation, the tumor volume was measured every 3 days using calipers. The tumor volume was calculated using the following formula: Tumor volume = length x width x width / 2; from Figure 1 The tumor growth curves show that, compared to the Ctrl group, the TROP2-mRNA lipid nanoparticles (C4@EL-TROP2-mRNA) with the secretory signal peptide removed in this application significantly inhibited tumor growth compared to the Ctrl group and the C4@FL-TROP2-mRNA group (full length).

[0044] Test Example 5 BALB / c mice (3 mice per group) were subcutaneously injected with 1×10⁶ CT26-hTROP2 tumor cells.

[0045] The mice were divided into three groups: the CTRL group, the C4@FL-TROP2-mRNA group (full-length TROP2 mRNA sequence, comparison example 3), and the C4@EL-TROP2-mRNA group (Example 2). Six days after injection, when the tumor cells reached a diameter of 4-5 mm, either the C4@FL-TROP2-mRNA or C4@EL-TROP2-mRNA nanovaccine was administered intravenously on days 6, 9, and 14, for a total of three vaccinations. On day 15, splenic lymphocytes were collected from the mice for ELISpot assays to detect IFN-γ-secreting lymphocytes.

[0046] The results are as follows Figure 2 As shown, compared with the CTRL group and the C4@FL-TROP2-mRNA group, the C4@EL-TROP2-mRNA vaccine group can induce more IFN-γ secreting cells in the mouse spleen than the C4@FL-TROP2-mRNA group.

[0047] Cells that secrete IFN-γ play an important role in anti-tumor activity, indicating that the C4@EL-TROP2-mRNA vaccine can induce higher anti-tumor immune effects in mice.

[0048] Test Example 6 BALB / c mice (3 mice per group) were subcutaneously injected with 1×10 6 CT26-hTROP2 tumor cells were collected. The cells were divided into three groups: CTRL group, C4@FL-TROP2-mRNA group (full-length TROP2 mRNA sequence, comparison example 3), and C4@EL-TROP2-mRNA group (Example 2). Six days after injection, when the tumor cells reached a diameter of 4-5 mm, 10 μg of C4@FL-TROP2-mRNA nanovaccine and 10 μg of C4@EL-TROP2-mRNA preparation were intravenously injected on days 6, 9, and 14, respectively, for a total of three vaccinations. On day 21, the mice were dissected to remove the tumors, which were cut into small pieces. After dissolving the tumor stroma with collagenase, the tumor cells were ground into single cells, cleaved, centrifuged, washed with PBS, and then incubated with flow cytometry for CD3, CD4, and CD8 antibodies in the dark for 30 minutes. After washing twice with PBS, the cells were resuspended in 300 μL of PBS and loaded onto the flow cytometry system.

[0049] The results are as follows Figures 3-5 As shown, the C4@EL-TROP2-mRNA vaccine group exhibited increased T cell infiltration in tumor tissue. The infiltration of CD3+ T, CD4+ T, and CD8+ T cells in tumor tissue was significantly higher in the C4@EL-TROP2-mRNA vaccine group than in the CTRL and C4@FL-TROP2-mRNA groups.

[0050] The above results indicate that the C4@EL-TROP2-mRNA vaccine with the secretory signal peptide removed in this application increased the infiltration of T cells in the tumor microenvironment, especially CD8+ T cells, compared with the C4@FL-TROP2-mRNA group.

[0051] Test Example 7 BALB / c mice (3 mice per group) were subcutaneously injected with 1×10 6 CT26-hTROP2 tumor cells were collected. The cells were divided into three groups: CTRL group, C4@FL-TROP2-mRNA group (full-length TROP2 mRNA sequence, comparison example 3), and C4@EL-TROP2-mRNA group (Example 2). Six days after injection, when the tumor cells reached a diameter of 4-5 mm, 10 μg of C4@FL-TROP2-mRNA nanovaccine and 10 μg of C4@EL-TROP2-mRNA preparation were intravenously injected on days 6, 9, and 14 post-inoculation, for a total of three vaccinations. On day 21, the spleen was dissected from the mice, and the cells were ground into single cells, cleaved, centrifuged, washed with PBS, and then incubated with flow cytometry antibodies against CD3, CD4, CD8, CD44, and CD62L for 30 minutes in the dark. After washing twice with PBS, the cells were resuspended in 300 μL of PBS and loaded onto the flow cytometry machine.

[0052] The results are as follows Figures 6-7 As shown, in the C4@EL-TROP2-mRNA vaccine group, CD8+ Tem and Tcm were significantly increased, and significantly higher than those in the CTRL group and the C4@FL-TROP2-mRNA group, with significant differences in all cases. Figure 6 A and 6B).

[0053] Similarly, CD4+Tem and Tcm were also significantly increased in the C4@EL-TROP2-mRNA vaccine group compared to the CTRL and C4@FL-TROP2-mRNA groups. Figure 7 A and 7B).

[0054] The results indicate that the LNP@EL-TROP2-mRNA vaccine effectively induced antigen-specific T cells, enhanced tumor-infiltrating lymphocytes (TILs), and increased memory T cells, thereby contributing to the anti-tumor immune response in mice.

[0055] In summary, by adopting the technical solution of this application, the Trop2 membrane antigen after removing the signal peptide (EL-TROP2) has a significantly enhanced cellular immune T cell response compared to the original full-length membrane antigen (FL-TROP2), which is beneficial to greatly improve the anti-tumor effect.

[0056] Lipid nanoparticles containing the mRNA of membrane antigens PD-L1, HER2, HER3, Cripto-1, CD24, CD20, BCMA, EGFR, VEGFR, VEGFR2, KIR, TIGIT, ALK, MET, B7H3, B7H4, CD38, FGFR2b, c-Met, FRα, TREM2, CD19, CDH6, DLL3, CLDN-18.2, CLDN-6, FAP-α, BAFF, Nectin-4, MUC1, MUC16, GPRC5D, ROR1, CCN2, EpCAM, DR5, and TpoR were prepared using procedures similar to those in Examples 1-2, Comparative Example 3, and Experimental Examples 4-7. The antitumor efficacy of these nanoparticles was then assessed.

[0057] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0058] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for enhancing antitumor responses by strengthening cellular immune responses, comprising administering a nucleic acid molecule encoding an antigen membrane protein to a subject in need, characterized in that, The antigen membrane protein is an antigen membrane protein with the leading signal peptide removed.

2. The method according to claim 1, characterized in that, The antigen membrane protein is one or more of Trop2, CD24, PD-L1, HER2, HER3, Cripto-1, CD20, BCMA, EGFR, VEGFR, VEGFR2, KIR, TIGIT, ALK, MET, B7H3, B7H4, CD38, FGFR2b, c-Met, FRα, TREM2, CD19, CDH6, DLL3, CLDN-18.2, CLDN-6, FAP-α, BAFF, Nectin-4, MUC1, MUC16, GPRC5D, ROR1, CCN2, EpCAM, DR5, and TpoR.

3. The method according to claim 1, characterized in that, The nucleic acid molecules are delivered using viral vectors and non-viral vectors.

4. The method according to claim 3, characterized in that, The viral vector is one or more of a retroviral vector, adenovirus vector, and adeno-associated virus (AAV) vector; the non-viral vector is one or more of liposomes, lipid nanoparticles, micelles, cationic polymers, and core-shell nanoparticles.

5. The method according to claim 1, characterized in that, The nucleic acid molecules are delivered using lipid nanoparticles.

6. The method according to claim 5, characterized in that, The lipid nanoparticles contain the nucleic acid molecules and also contain one or more of ionizable lipids, phospholipids, sterols, and PEG-modified lipids.

7. The method according to claim 5, characterized in that, The application also includes a pharmaceutically acceptable carrier, excipient, or adjuvant.

8. The method according to claim 1, characterized in that, The tumors include solid tumors and hematologic tumors.

9. The method according to claim 1, characterized in that, The nucleic acid molecules are DNA and RNA.

10. The method according to claim 9, characterized in that, The RNA includes one or more of mRNA, saRNA, and circRNA.

11. Use of a nucleic acid molecule encoding an antigen membrane protein in a medicament for the prevention or treatment of cancer in an individual, or for delaying the progression of cancer in an individual; characterized in that, As described in claim 1, the antigen membrane protein is an antigen membrane protein with the front signal peptide removed.

12. The method according to claim 11, characterized in that, The cancers mentioned include stomach cancer, esophageal cancer, pancreatic cancer, ovarian cancer, breast cancer, lung cancer, colorectal cancer, prostate cancer, cervical cancer, head and neck cancer, nasopharyngeal carcinoma, leukemia, lymphoma, liver cancer, melanoma, and soft tissue sarcoma.

13. The method according to claim 10, characterized in that, The mRNA contains an open reading frame (ORF), a 5'UTR, and a 3'UTR encoding an antigen membrane protein: 5'UTR-ORF-3'UTR; wherein the antigen membrane protein is an antigen membrane protein with its leading signal peptide removed.

14. The method according to claim 10, characterized in that, The mRNA also includes a 5' end cap structure and a 3' poly A tail.

15. The method according to claim 14, characterized in that, The 5' terminal cap structure is Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-diazo-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, 5'-methylGcap or analogues thereof.

16. The method according to claim 10, characterized in that, The mRNA contains at least one chemical modification; the chemical modification is selected from pseudouridine or pseudouridine analogues.

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