An mRNA drug targeting card11 for treating intestinal cancer

Abstract

This invention discloses the use of saRNA of the CARD11 gene in the preparation of antitumor drugs. The drug, comprising a saRNA molecule and a targeted delivery vector, can be used to treat colorectal cancer. The saRNA molecule replicates and is expressed in tumor tissue cells. Leveraging the unlimited and rapid proliferation characteristics of tumor cells, it overexpresses CARD11, attracting the infiltration of various immune cells with tumor-killing effects, thereby eliminating the tumor cells and providing a new approach for colorectal cancer treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to an mRNA drug that targets CARD11 for the treatment of colorectal cancer, specifically a saRNA drug that targets CARD11 and its application in the treatment of colorectal cancer. Background Technology

[0002] Colorectal cancer is a highly prevalent malignant tumor of the digestive tract worldwide. While treatment options include surgery, chemotherapy, radiotherapy, and traditional targeted therapy, many clinical challenges remain. Many colorectal cancer patients have good later-stage outcomes but face a lack of available treatments. Our clinical research has shown that patients with CARD11 mutations have poorer prognoses. Overexpression of CARD11 in mice significantly inhibits the development and progression of colorectal cancer, but currently, there are no specific targeted drugs for this target. Existing technologies report CARD11 gene editing methods involving the construction of mutants, with all target cells being immune cells. For example, patent document CN113286811A reports the potential use of genetically modified immune cells, especially hematopoietic cells such as B-cell lineages REC-1, JEKO-1, MINO, GRANTA-519, NALM6, or RAJI cells, transplanted in vivo to potentially treat colorectal cancer. However, these traditional targeted drugs have extremely low response rates to these subtypes. On the other hand, the application of traditional mRNA drugs in the field of tumor treatment has obvious limitations. They can only maintain protein expression in the body for a few days, and frequent high-dose administration is required to achieve therapeutic effects. This not only increases the patient's medication burden and risk of toxic side effects, but also makes it difficult to meet the long-term treatment needs of chronic diseases such as colorectal cancer. Summary of the Invention

[0003] CARD11 shows promise as a gene target for treating colorectal cancer, and overexpression of CARD11 could be a drug development strategy for this disease. While existing literature, such as CN113286811A, reports on cell therapy involving the transplantation of genetically modified live cells into the body and the overexpression of CARD11 to enhance the body's immunity and treat cancer, cell therapy has relatively low targeting specific lesions, failing to achieve the effects of precision medicine. More worryingly, in recent years, there have been increasing negative reports regarding the pathogenic risks of cell therapy, especially its carcinogenicity, leading some pharmaceutical giants to terminate related research projects that had been ongoing for many years. Achieving CARD11 overexpression through new biological mechanisms and pathways has become an urgent technical challenge.

[0004] Inspired by the immunization principle of mRNA vaccines, we devised a new technical approach that uses tumor cells as target cells, takes advantage of the rapid proliferation and unlimited reproduction of tumor cells to directly overexpress CARD11 in tumor cells, and ultimately phagocytose the tumor cells.

[0005] To achieve the novel biological mechanism of treating colorectal cancer through CARD11 overexpression in tumor cells, autologous RNA (saRNA) technology is an option. While saRNA technology has shown potential for long-term, efficient protein expression at low doses and has shown promise in the vaccine field, its application to colorectal cancer treatment using CARD11 as a target has not been previously reported. Furthermore, existing saRNA drugs suffer from insufficient targeting, poor stability in the tumor microenvironment, and limited delivery efficiency, making it difficult to precisely enrich colorectal cancer lesions and achieve sustained CARD11 protein expression, thus hindering their ability to exert ideal anti-tumor effects.

[0006] To overcome the aforementioned shortcomings of existing saRNA technologies, we developed an optimized saRNA targeted delivery system. An LNP (lipid nanoparticle) carrier ensures the delivery stability of the saRNA molecule, and an antibody targeting the tumor cell surface antigen, integrin αvβ3 receptor, is conjugated to the LNP carrier to enhance the targeting of the saRNA molecule. The feasibility of this design was verified in tumor-bearing mice, achieving significant therapeutic effects. Results showed that after 28 days of administration, the tumor volume in the saRNA-treated group was reduced by 80% compared to the saline control group, and the tumor-suppressing effect was prolonged by 14 days compared to the traditional mRNA group. No significant weight loss or organ damage was observed in the mice. Furthermore, this saRNA drug can be used in combination with other anti-tumor drugs, such as PD-1 monoclonal antibodies and traditional chemotherapy drugs. Based on these research findings, this invention includes the following technical solutions.

[0007] The first aspect of the invention provides the use of saRNA of gene CARD11 (GeneID: 84433 or Uniprot ID: Q9BXL7 version) in the preparation of antitumor drugs.

[0008] Preferably, the nucleotide sequence of the coding region (CDS) of the CARD11 gene is shown in SEQ ID NO: 1; and the amino acid sequence of the encoded CARD11 protein is shown in SEQ ID NO: 2. As is well known to those skilled in the art, the nucleotide sequence of the saRNA of the CARD11 gene in this invention, from 5' to 3', is as follows: 5' Cap, 5' UTR, non-structural protein (nsp1-4) coding region, subgenomic promoter (SGP), target protein coding region (GOI), 3' UTR, and poly A tail.

[0009] Optionally, the tumors mentioned above are selected from the group consisting of: colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small bowel cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, lung adenocarcinoma, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, and cancers of the endocrine system. Thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, bladder cancer, cancer of the kidney or ureter, renal pelvis cancer, vegetations of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer; preferably, the tumor is intestinal cancer such as colon cancer and / or rectal cancer.

[0010] A second aspect of the invention provides a medicament for treating cancer, such as colorectal cancer, wherein the active pharmaceutical ingredient is the aforementioned saRNA molecule; preferably, the saRNA molecule is replicated and expressed in tumor tissue cells and / or adjacent normal tissue cells. By utilizing the unlimited and rapid proliferation characteristics of tumor cells, and through cell overexpression of CARD11, various immune cells with tumor-killing effects are attracted to infiltrate, thereby eliminating the tumor tissue.

[0011] In one embodiment, the above-mentioned drug is a saRNA drug for treating colorectal cancer, comprising the above-mentioned saRNA molecule and a targeted delivery vector for encapsulating the saRNA molecule, forming a saRNA targeted delivery system.

[0012] Preferably, the above-mentioned targeted delivery carrier includes an LNP (lipid nanoparticle) carrier and an antibody targeting the integrin αvβ3 receptor conjugated to the LNP carrier.

[0013] The conjugation techniques between the LNP vector and the antibody include directed conjugation and non-directed conjugation.

[0014] Optionally, the antibody targeting the integrin αvβ3 receptor is Etaracizumab, whose amino acid sequence is shown in SEQ ID NO: 3.

[0015] EIVLTQSPATLSLSPGERATLSCQASQSISNFLHWYQQRPGQAPRLLIRYRSQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSGSWPLTFGGGTKVEIKQVQLVES GGGVVQPGRSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVAKVSSGGGSTYYLDTVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHLHGSFASWGQGTTVTVSS (SEQ ID NO: 3).

[0016] In one embodiment, the above-mentioned drug is a pharmaceutical composition that, in addition to containing a therapeutically effective amount of the above-mentioned saRNA molecule as an active ingredient, also contains a pharmaceutically acceptable carrier.

[0017] In another embodiment, the above-mentioned drug is a pharmaceutical composition that, in addition to containing a therapeutically effective amount of the above-mentioned saRNA molecule, also contains one or more other anti-tumor pharmaceutical ingredients, such as those for treating colorectal cancer.

[0018] Optionally, the other antitumor drug components are selected from the following group: PD-1 monoclonal antibody, oxaliplatin, fluorouracil, and other conventional chemotherapy drugs.

[0019] The dosage form of the aforementioned drugs can be various, as long as it is suitable for administration in the treatment of colorectal cancer and appropriately maintains the biological activity of the active pharmaceutical ingredients (RNA molecules and / or antibody proteins). For example, the aforementioned drugs can be in injectable form. The injectable dosage form is suitable for subcutaneous injection, intramuscular injection, intravenous injection, or intravenous infusion. For example, for injectable drug delivery systems, the dosage form can be lyophilized powder.

[0020] Injectable formulations may be administered subcutaneously, intramuscularly, intravenously, or via gradual infusion over time. Given suitable formulations for a given route, such as agents that can be used in the methods and compositions described herein, they may be administered intravenously, intranasally, by inhalation, intraperitoneally, intramuscularly, subcutaneously, intracavitarily, and, if desired, by a peristaltic pump, via a port-a-cath, or by other means known to those skilled in the art.

[0021] A third aspect of the present invention provides a method for preparing the above-mentioned drug, namely the saRNA targeted delivery system, comprising the following steps:

[0022] (1) Construction of saRNA template DNA: A DNA fragment containing the coding region (CDS) of the CARD11 gene was synthesized, cloned into a self-replicating vector, and a recombinant plasmid (named CARD11-saRNA) was constructed. After the sequence accuracy was verified by sequencing, a high-purity plasmid was extracted as a transcription template.

[0023] (2) In vitro transcription of modified saRNA: Prepare a saRNA transcription system containing T7 RNA polymerase, NTP, other conventional nucleotides, transcription buffer and template DNA, and incubate at 37°C for 3 hours. After transcription, degrade the template DNA with DNaseI to ensure the purity of saRNA.

[0024] (3) Purification of saRNA: Lithium chloride is added to the transcription system for purification. The final saRNA must meet the OD standard. 260 / OD 280 = 1.9-2.1、OD2 60 / OD 230 Quality control standards of ≥2.0 and integrity ≥70%;

[0025] (4) LNP encapsulation of saRNA: Using a microfluidic device, the LNP organic phase (each lipid component dissolved in ethanol) and the saRNA aqueous phase (dissolved in 25mM sodium acetate buffer, pH=4.0) are mixed (e.g., at a volume ratio of 1:3) to form a CARD11-saRNA / LNP complex with a particle size of 80-120nm, PDI≤0.15, and encapsulation efficiency≥85%.

[0026] (5) The CARD11-saRNA / LNP complex obtained in step (4) is coupled to the target antibody either directionally or non-directionally to obtain the saRNA targeted delivery system, i.e. the drug.

[0027] The saRNA targeted delivery system developed in this invention provides a new treatment strategy for the clinical treatment of colorectal cancer as an anti-tumor drug. Compared with existing colorectal cancer treatments, its advantages include several aspects:

[0028] 1. Long-lasting and highly effective, with significant dosage advantages.

[0029] Based on the self-amplification properties of saRNA, the drug can achieve stable expression of CARD11 protein in colorectal cancer lesions for up to 21 days at an extremely low dose of 0.1 mg / kg. Compared with traditional mRNA drugs (which require a dose of 1 mg / kg and only maintain expression for 3-5 days), the dosing frequency is reduced by more than 70%, which greatly reduces the burden of medication for patients.

[0030] 2. Highly targeted with low toxicity and side effects

[0031] LNP carriers can specifically recognize receptors in colorectal cancer tissues, increasing the drug accumulation at the lesion site by more than 8 times and reducing its effect on normal tissues; at the same time, the low-dose administration mode further reduces hepatotoxicity and nephrotoxicity and immune inflammatory response, and significantly improves patient tolerance.

[0032] 3. It has a definite curative effect and is suitable for chronic treatment.

[0033] In animal experiments, after subcutaneous injection of the drug, the volume of CT26 xenografts shrank by more than 75% within 2 weeks, and the efficacy could be maintained for up to 28 days after administration. It can effectively inhibit tumor recurrence and perfectly meet the long-term treatment needs of chronic tumors such as colorectal cancer.

[0034] 4. Wide range of applications

[0035] It can be used not only for CARD11-mutant colorectal cancer, but also for wild-type colorectal cancer with downregulated CARD11 expression. Furthermore, it can be used in combination with conventional chemotherapy drugs such as oxaliplatin and fluorouracil to synergistically improve the treatment response rate.

[0036] 5. Wide range of applications

[0037] This expands the application boundaries of saRNA technology in the field of tumor targeted therapy and provides a reference technical paradigm for the treatment of other chronic tumor diseases. Attached Figure Description

[0038] Figure 1 This study demonstrates that patients with CARD11-mutant MSS colorectal cancer have poor response to combination immunotherapy. A: Waterfall plot of TEC clinical trial cohort results; B: Survival analysis showed a median PFS of 7.3 months and a median OS of 17.4 months for patients receiving combination immunotherapy; C: Mutation profiles from baseline ctDNA sequencing of patients in the cohort, with a high CARD11 gene mutation frequency, second only to APC and TP53; D: All patients with CARD11 mutations had a prognosis of SD or PD, indicating poor treatment outcomes; E: Survival analysis showed a significantly shorter median PFS in patients with CARD11 mutations.

[0039] Figure 2 This study demonstrates the relationship between CARD11 and colorectal cancer and immunotherapy. Specifically: A: CARD11 expression in CRC (colorectal cancer) is positively correlated with the infiltration of various immune cells; B: CRC patients with high CARD11 expression in TCGA had higher immune scores and matrix scores; C: Survival analysis after immunotherapy (PD-1 monotherapy group; CTLA-4 monotherapy group; PD-1+CTLA-4 combination therapy group) showed significantly prolonged survival in patients with high CARD11 expression; D: Immunohistochemistry showed that the expression level and positive area of ​​CARD11 in the pathological tissue of CRC patients were significantly increased compared to adjacent normal tissue.

[0040] Figure 3 The results show that CARD11 mutation and overexpression significantly inhibit subcutaneous tumor growth in Balb / c mice. The top image compares tumor tissue images from Balb / c mice treated with the saRNA targeted delivery system (CARD11-OE) and the negative control group (CARD11-NC). The bottom image shows a comparison curve of tumor volume changes over time (days) between the two groups. Detailed Implementation

[0041] The current state of colorectal cancer treatment is that there are no drugs available for later-line treatment, and the prognosis of CARD11-mutant colorectal cancer is even worse. Overexpression of CARD11 significantly inhibits colorectal cancer growth, but there is a lack of specific targeted drugs, and traditional mRNA treatment regimens have long-term effects. We have developed a self-amplified mRNA (saRNA) drug that targets tumor-expressed CARD11 to fill the technological gap in precision treatment of this subtype of colorectal cancer.

[0042] saRNA (self-amplifying RNA) is a special type of mRNA (messenger ribonucleic acid). Based on their structure and function, mRNA can be divided into two main categories: non-replicating / non-amplifying mRNA and self-replicating / self-amplifying mRNA (saRNA). Non-replicating mRNA only contains the sequence encoding the target antigen and does not possess the ability to replicate itself; it is directly translated into protein by ribosomes after entering the cell. saRNA additionally carries an RNA replicase gene from the virus (such as the replication mechanism gene in the alphavirus genome), which allows it to utilize the cellular machinery to self-replicate after entering the host cell, thereby producing a large number of copies within the cell and significantly increasing the expression level of the target protein.

[0043] In this article, the terms "self-amplification" and "self-replication" have the same meaning and can be used interchangeably.

[0044] Therefore, relying on the self-amplification properties of saRNA, it is hoped that long-term and efficient expression of CARD11 protein in colorectal cancer lesions can be achieved at extremely low doses, overcoming the drawbacks of traditional mRNA requiring high-dose and frequent administration, and reducing the toxic side effects of drugs on normal tissues.

[0045] saRNA molecules suffer from insufficient stability after entering the body. LNP (lipid nanoparticle) encapsulation / encapsulation technology can improve saRNA delivery efficiency. For example, the COVID-19 vaccine ARCT-154 uses LNP (lipid nanoparticle) encapsulated saRNA technology to improve delivery efficiency and immunogenicity.

[0046] On the other hand, antibody-antigen affinity can enhance drug targeting. This invention conjugates antibodies such as etaraxisab, which target the integrin αvβ3 receptor on tumor cells, with LNP to construct an optimized saRNA targeted delivery system. This enhances the drug's accumulation in colorectal cancer lesions, prolongs its duration of action in vivo, meets the clinical needs of chronic colorectal cancer treatment, and improves the prognosis of CARD11-related colorectal cancer patients.

[0047] Antibody-drug conjugate (ADC) conjugation techniques are mainly divided into two categories: non-directional conjugation (also known as random conjugation) and directional conjugation (also known as site-specific conjugation). The core difference between these two techniques lies in whether the position of the drug molecule attached to the antibody is controllable and uniform.

[0048] Non-directional coupling (random coupling) utilizes naturally occurring, highly reactive amino acid residues on the antibody surface for chemical reactions. However, due to the large number and wide distribution of these residues, the linking positions and numbers of drug molecules (drug-antibody ratio, DAR) are random, resulting in a mixture of various DAR values ​​and coupling sites. This coupling technique includes: lysine coupling, where the antibody surface has more than 80 lysine residues, whose side-chain amino groups can react with linkers; this method is simple to operate but has poor uniformity; and cysteine ​​coupling (interchain disulfide bond reduction), which reduces four pairs of interchain disulfide bonds in the antibody hinge region to generate eight free thiol groups, which then react with linkers containing groups such as maleimide. This method has higher uniformity than lysine coupling, with a typical DAR value of 4, and is currently the most widely used "semi-site-directed" technique.

[0049] Targeted conjugation (site-specific conjugation) involves introducing a unique reactive group at a specific site on an antibody through genetic engineering, enzymatic modification, or chemical modification. This allows drug molecules to be linked at a precise DAR value (usually 2 or 1) and a fixed position. This technology can significantly improve the homogeneity, stability, and therapeutic window of drugs. These conjugation techniques include: 1. Engineered cysteine ​​(e.g., THIOMAB technology): Introducing non-natural cysteine ​​residues at specific sites on the antibody through genetic engineering to achieve site-specific conjugation; the DAR value is typically 2. 2. Non-natural amino acid conjugation: Introducing non-natural amino acids containing specific functional groups (such as azido or keto groups) into the antibody sequence, followed by conjugation through "bioorthogonal reactions" (e.g., click chemistry). 3. Enzyme-mediated conjugation: Utilizing the high specificity of enzymes, such as transglutaminase (mTGase) acting on the glutamine 295 site in the Fc region, or sorting enzyme A (SrtA) recognizing the LPTTG tag, to achieve site-specific linking. 4. Glycan-mediated coupling: Utilizing the conserved N297 glycosylation site of the antibody Fc segment, the glycan structure is enzymatically modified to introduce reactive groups for coupling, without modifying the antibody protein sequence. 5. Interchain disulfide bond rebridging: Using bifunctional reagents to "rebridge" the reduced interchain disulfide bond positions, simultaneously linking drug molecules to them, achieving a homogeneous product with DAR=4. 6. Other innovative technologies: Such as pClick technology, AJICAP technology, and proximity induction technology, aiming to achieve highly selective modification of antibodies by utilizing their natural structural features (such as nucleotide binding domains) without altering the antibody sequence.

[0050] In the description of the technical solutions of this invention, the term "and / or" used in terms such as "A and / or B" or "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); C (alone).

[0051] In this document, for the sake of simplicity, the protein CARD11 and its encoding gene (DNA) are sometimes used interchangeably. Those skilled in the art should understand that they refer to different substances in different descriptive contexts. Their meanings are readily understood by those skilled in the art based on the context. For example, when describing the function or class of a scaffold protein carrying a Caspase-related recruitment domain, CARD11 refers to the protein; while when used as a gene description, it refers to the gene encoding that protein.

[0052] In one embodiment, the drug, in addition to containing a therapeutically effective amount of the aforementioned saRNA molecule, also contains one or more other antitumor drug components, such as PD-1 monoclonal antibodies. Such a drug composition may improve the therapeutic effect on colorectal cancer and / or other types of tumors / cancers.

[0053] The terms “(therapeutic effect) enhancement” or “improvement” are used herein to indicate an improvement of a statistically significant amount. In some implementations, “enhancement” or “increase” or “improvement” generally refers to an improvement of at least 10% compared to a reference level (e.g., in the absence of a given treatment or agent), and may include, for example, an improvement of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more.

[0054] As used herein, the term "effective amount" refers to a therapeutic amount required to alleviate at least one or more symptoms of a disease or condition, and involves an adequate amount of medicine to provide the desired effect. Therefore, the term "therapeutic effective amount" refers to a therapeutic amount sufficient to produce a specific effect when administered to a typical subject. In various contexts, effective amount, as used herein, also includes amounts sufficient to delay the development of a disease condition, alter the course of a disease condition (e.g., but not limited to, slowing the progression of a disease condition), or reverse a disease condition. It should be understood that many methods are known in the art for determining an effective amount for a given application. For example, pharmacological methods for dosage determination can be used in a therapeutic context. In the context of therapeutic or prophylactic application, the amount of medicine administered to the subject will depend on the type and severity of the disease and the individual's characteristics, such as general health status, age, sex, weight, and tolerance to the medicine. It also depends on the degree, severity, and type of the disease. Those skilled in the art will be able to determine an appropriate dosage based on these and other factors. For example, a therapeutically effective amount of a saRNA molecule can be determined through clinical investigation. Appropriate effective dosage also takes into account therapeutic factors such as the drug formulation, the individual's constitution, weight, age, disease progression, and administration site.

[0055] The dosage range of the medication depends on its potency, including amounts sufficient to produce the desired effect, such as slowing the progression of colorectal cancer or even reversing the condition. The dosage should not be so high as to cause unacceptable adverse side effects. Typically, the dosage will vary depending on the patient's age, condition, and sex and can be determined by someone skilled in the art. In the event of any complications, the dosage may also be adjusted by an individual physician.

[0056] The saRNA drugs of the present invention can be in various dosage forms, as long as they are suitable for administration to the corresponding disease and appropriately maintain the biological activity of the nucleic acid saRNA molecules and / or antibody proteins. For example, for injectable drug delivery systems, the dosage form can be a lyophilized powder. Injectable formulations can be administered subcutaneously, intramuscularly, intravenously, or by gradual intravenous infusion over time. Given suitable formulations for a given route, for example, agents that can be used in the methods and compositions described herein, can be administered intravenously, intranasally, by inhalation, intraperitoneally, intramuscularly, subcutaneously, or intracavitarily, and if desired, by a peristaltic pump, a port-a-vein, or by other means known to those skilled in the art. Intravenous or intramuscular administration is preferred for patients with colorectal cancer to be treated.

[0057] Optionally, the above-mentioned drug dosage forms may contain any pharmaceutically acceptable carrier and adjuvant, provided that they are suitable for the corresponding drug delivery system and properly maintain the biological activity of the saRNA molecule.

[0058] As used herein, the phrase "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within reasonable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, and that are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers are well known in the art and include liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials. Each carrier must be "acceptable" in the sense of compatibility with other components of the formulation and harmlessness to the patient, including, for example, aqueous solutions (such as water or physiologically buffered saline) or other solvents or mediators (such as glycols, glycerin, oils (such as olive oil), or injectable organic esters). Excipients may be selected, for example, to achieve delayed release of the drug or to selectively target one or more cells, tissues, or organs. For the purposes of this invention, pharmaceutical compositions may be in the form of dosage units, such as powders for injection, solutions, injections, etc.

[0059] The preparation of pharmacological compositions containing an active ingredient dissolved or dispersed therein is well known in the art and is not limited to formulation. Typically, such compositions are prepared as injectable liquid solutions or suspensions; however, solid forms suitable for placement in a liquid prior to use are also possible. The formulation may also be emulsified or present as a liposome composition. The active ingredient may be mixed with pharmaceutically acceptable and compatible excipients in an amount suitable for use in the therapeutic methods described herein. Suitable excipients include, for example, water, saline, glucose, glycerol, ethanol, and combinations thereof. Furthermore, if desired, the composition may contain small amounts of excipients, such as wetting agents or emulsifiers, pH buffers, etc., which enhance or maintain the effectiveness of the active ingredient. Therapeutic compositions as described herein may include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino group of a polypeptide) formed from inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, tartaric acid, mandelic acid, etc. Salts formed from free carboxyl groups can also be derived from inorganic bases, such as sodium, potassium, ammonium, calcium, or iron hydroxides, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc. Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions containing no substances other than the active ingredient and water, or containing buffers such as sodium phosphate at physiological pH, physiological saline, or both, such as phosphate-buffered saline. Furthermore, aqueous carriers may contain more than one buffer salt, as well as salts such as sodium chloride and potassium chloride, glucose, polyethylene glycol, and other solutes. In addition to water, liquid compositions may also contain a liquid phase. Examples of such other liquid phases are glycerol, vegetable oils (e.g., cottonseed oil), and water-oil emulsions. The amount of active agent, such as saRNA, used in this invention for the effective treatment of a specific disease or condition, such as colorectal cancer, will depend on the nature of the disease or condition and can be determined using standard clinical techniques.

[0060] As used herein, the terms “treatment,” “therapy,” “management,” or “improvement,” when used to refer to a disease, condition, or medical condition such as colorectal cancer, refer to a therapeutic treatment of the condition in which the aim is to reverse, alleviate, improve, suppress, slow, or stop the progression or severity of symptoms or the condition. The term “treatment” includes reducing or alleviating at least one adverse effect or symptom of the condition. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of the condition is reduced or stopped. That is, “treatment” includes not only improvement of symptoms or markers but also stopping or at least slowing the progression or worsening of symptoms that would be expected without treatment. Beneficial or desired clinical outcomes include, but are not limited to, relief of one or more symptoms, reduction of the degree of deficiency, stabilization (i.e., no worsening) or decline in CARD11 gene expression levels, slowing of colorectal cancer progression, and increased life expectancy compared to life expected without treatment.

[0061] Therapeutic effective dose is the amount of a drug that produces statistically significant and measurable changes in inhibiting tumor tissue development and promoting normal cell growth. This effective dose can be measured in clinical trials and animal studies.

[0062] The efficacy of a given treatment (e.g., for the suppression of colorectal cancer) can be determined by a clinician with a high level of skill. However, a treatment is considered “effective” as used herein if any or all signs or symptoms of colorectal cancer change in a beneficial manner, or if other clinically acceptable symptoms improve or even lessen, for example, by at least 10% reduction after treatment with the agents described herein. Efficacy can also be measured by the individual’s failure to worsen, assessed by the need for hospitalization or medical intervention (i.e., cessation of disease progression). Methods for measuring these indicators are known to those skilled in the art and / or described herein.

[0063] The effective amount for treating a disease is the amount that, when administered to a mammal in need, is sufficient to cause an effective treatment of the disease as defined herein. The efficacy of a drug can be determined by assessing, for example, physical indicators of treatment for colorectal cancer.

[0064] The aforementioned agents can be administered via subcutaneous injection, intramuscular injection, intravenous injection, or intravenous infusion over time. Suitable formulations for a given route, such as agents that can be used in the methods and compositions described herein, can be administered intravenously, intranasally, by inhalation, intraperitoneally, intramuscularly, subcutaneously, or intracavitarily, and, if desired, by a peristaltic pump, a port-a-venipuncture site, or by other means known to those skilled in the art. Intravenous or intramuscular administration is preferred for patients with colorectal cancer to be treated. Direct local administration to the intestinal tissue is also particularly considered.

[0065] For example, a therapeutic composition containing at least one pharmaceutical agent can be conventionally administered in unit doses. When used in a therapeutic composition, the term "unit dose" refers to a physically discrete unit suitable as a unit dosage form for the intended use, each unit containing a predetermined amount of active substance, combined with a desired physiologically acceptable diluent (i.e., a carrier or delivery agent), calculated to produce the desired therapeutic effect.

[0066] The composition is administered in a therapeutically effective amount in a manner compatible with dosage forms. The dosage and timing of administration depend on the subject to be treated, the subject's ability to utilize the active ingredient, and the desired degree of therapeutic effect.

[0067] The precise amount of active ingredient, such as saRNA molecules, required depends on the physician's judgment and varies from person to person. However, this article discloses appropriate dosage ranges for systemic administration, depending on the route of administration. Appropriate dosing regimens are also variable, but are represented by an initial dose followed by repeated administration at intervals of one or more hours via subsequent injections or other administrations. Alternatively, continuous intravenous infusion may be considered sufficient to maintain blood concentrations within the range specified for in vivo therapeutic use.

[0068] Conventional processes can be used to prepare saRNA drugs. For example, the preparation process of a saRNA targeted delivery system generally includes the construction of saRNA template DNA, in vitro transcription of modified saRNA, purification of saRNA, encapsulation of saRNA with LNP to form CARD11-saRNA / LNP complex, and antibody conjugation of CARD11-saRNA / LNP complex to impart targeting.

[0069] When the saRNA targeted delivery system constructed in this invention is used to treat tumors such as colorectal cancer, the saRNA precisely enters the colorectal cancer cells and first uses the tumor cells' own translation system to synthesize RNA replicase. This replicase can specifically recognize the non-coding region sequence of saRNA, initiate the autonomous replication of saRNA, and achieve exponential amplification of single-stranded saRNA in the cell. Thus, at extremely low doses, wild-type CARD11 protein is continuously and efficiently expressed, and the cancer-promoting effect of mutant CARD11 is reversed in the long term, which is suitable for the chronic treatment needs of colorectal cancer and achieves precise treatment effect.

[0070] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings. Those skilled in the art should understand that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0071] Example

[0072] In embodiments of the present invention, unless otherwise specified, the experimental operating temperature is generally referred to as room temperature (10-30°C).

[0073] This article involves the addition amount, content and concentration of various substances. Unless otherwise specified, the percentage content mentioned refers to the mass percentage.

[0074] In the following examples, the primers, gene and plasmid synthesis, and gene sequencing involved were all designed and implemented by CanSino Biologics Inc. (Shanghai) Co., Ltd.

[0075] The molecular biology experiments in the examples included plasmid construction, enzyme digestion, ligation, preparation of competent cells, transformation, and culture medium preparation, etc., mainly referring to "Molecular Cloning: A Laboratory Manual" (4th Edition), edited by M.R. Green and J. Sambrook (USA), translated by He Fuchu, Science Press, Beijing, 2017. Specific experimental conditions can be determined through simple experiments if necessary.

[0076] PCR amplification experiments should be performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. Adjustments can be made through simple experiments if necessary.

[0077] In the embodiments, the saRNA molecule, its encoding gene and its expression plasmid can be represented in the same way. For example, CARD11-saRNA can represent the saRNA molecule itself, its expression vector CARD11-saRNA or its encoding gene sequence. Those skilled in the art can easily understand that they represent different biological forms in different representational contexts.

[0078] Unless otherwise specified, the test reagents used in the following examples are all conventional biochemical reagents; and the experimental methods described are all conventional methods unless otherwise specified.

[0079] Materials and methods:

[0080] Strains and cells: Escherichia coli Trans1-blue competent cells (Beijing TransGen Biotech Co., Ltd.), CT26 colon cancer cells (Shanghai Cell Bank, Chinese Academy of Sciences).

[0081] Reagents: T7 RNA polymerase, NTP, ionizable lipids (Sinobond, CAS-No. 2089251-47-6), DSPC (distearylphosphatidylcholine, a common auxiliary phospholipid used in lipid nanoparticle (LNP) formulations, whose main function is to stabilize the nanoparticle structure), cholesterol, DMG-PEG2000, plasmid extraction kit, etc.

[0082] Instruments: PCR instrument, high-speed refrigerated centrifuge, nanoparticle size potential analyzer, microfluidic control system, constant temperature metal bath, etc.

[0083] Statistical analysis: In this study, all numerical variables are expressed as mean ± standard error. The two-tailed Suuden U's U test was used for comparisons between two groups, and the ANOVA test was used for comparisons among three groups. A p-value < 0.05 was considered statistically significant.

[0084] Some of the PCR primers used in the examples are listed in Table 1.

[0085] Table 1. Some PCR primers used in the examples

[0086]

[0087] In Table 1, "-F" in the name represents positive; "-R" represents negative.

[0088] Example 1: CARD11 overexpression significantly inhibits the growth of colorectal cancer.

[0089] Clinical cohort studies have shown that patients with CARD11 mutations have poor prognoses.

[0090] A previous investigator-initiated clinical trial by our research group showed that PD-1 monoclonal antibody combined with EGFR monoclonal antibody and irinotecan (TEC study) was significantly more effective than current standard treatment in colorectal cancer. In this cohort, peripheral blood circulating tumor DNA next-generation sequencing revealed that, in addition to common APC and TP53 gene mutations, CARD11 mutations accounted for 16% of the five most frequently mutated genes, and all of these mutations were found in the group with poor treatment response. Further survival analysis showed that the median progression-free survival (mPFS) of CARD11-mutated patients was significantly lower than that of CARD11 wild-type patients (mPFS: 4.4 months vs. 7.7 months, P=0.039). Figure 1 This strongly suggests that CARD11 is a novel target that could potentially influence immune escape in mCRC.

[0091] The CARD protein family is a family of scaffold proteins carrying a characteristic caspase-associated recruitment domain (CARD). CARD11 is an important member of the CARD protein family, mainly expressed in hematopoietic tissues such as the spleen, thymus, and peripheral blood leukocytes. Its main structural domains include the CARD domain and the coiled-coil domain. It promotes the proliferation and activity of immune cells by facilitating the NF-κB and MAPK pathways. Dysfunction of CARD11 is more commonly seen in hematologic proliferative disorders such as diffuse large B-cell lymphoma, with very little research on its use in solid tumors.

[0092] Compared to adjacent normal tissue, CARD11 levels are significantly elevated in colorectal cancer tumor tissue. Figure 2 ).

[0093] This study indicates that patients with CARD11-mutant colorectal cancer have an extremely poor prognosis; restoring normal CARD11 function is a key target for treating this type of colorectal cancer. Increasing CARD11 gene expression in colorectal cancer tumor cells may become a new strategy for treating colorectal cancer; therefore, we selected CARD11 as the core site of drug action.

[0094] Example 2: Design of saRNA drugs targeting CARD11

[0095] The drug designed in this invention consists of two parts: a saRNA sequence encoding CARD11 and a targeted delivery vector. The components work synergistically to achieve low-dose, long-lasting, and precise anti-tumor effects.

[0096] The nucleotide sequence of the coding region (CDS) of gene CARD11 is shown in SEQ ID NO: 1; the amino acid sequence of CARD11 protein (including long-acting protein design) is shown in SEQ ID NO: 2.

[0097] The targeted delivery vector is a specific delivery system for colorectal cancer, employing a lipid-based or polymer-based delivery system and conjugated with antibodies specifically targeting colorectal cancer cells. These targeted antibodies include antibodies targeting the integrin αvβ3 receptor, enabling precise targeted delivery of saRNA drugs. The amino acid sequence of the antibody targeting the integrin αvβ3 receptor, Etaracizumab, is shown in SEQ ID NO: 3.

[0098] EIVLTQSPATLSLSPGERATLSCQASQSISNFLHWYQQRPGQAPRLLIRYRSQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSGSWPLTFGGGTKVEIKQVQLVES GGGVVQPGRSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVAKVSSGGGSTYYLDTVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHLHGSFASWGQGTTVTVSS (SEQ ID NO: 3).

[0099] The conjugation technique between the delivery system and the antibody can be either directional or non-directional.

[0100] Example 3: Preparation of saRNA Drug

[0101] The preparation process of saRNA drugs mainly includes the following steps.

[0102] 1. Construction of saRNA template DNA

[0103] The CARD11 coding region was synthesized by Genewiz, and the CARD11 gene sequence was obtained. PCR amplification yielded the PCR product (NEB kit number: E0553L, PCR primers are shown in Table 1, amplification conditions are shown in Table 2). The product was purified by gel extraction (Qiagen kit number: 28706) to obtain the target gene. Homologous recombination (Novizan kit number: P526-02) was used to ligate the target gene to a self-replicating vector (following the instructions). 10 μL of the ligation product was added to competent cells, heat-shocked at 42℃ for 90 s, rapidly cooled for 5 min, and then 600 μL of SOC was added. The cells were cultured on a shaker for 1 h, centrifuged at 5000 rpm for 4 min, 500 μL of supernatant was discarded, the cells were resuspended, and evenly spread onto kanamycin plates. The cells were cultured overnight, and single clones were picked and cultured. The sequence was verified by sequencing, and positive clone plasmids were extracted and digested with BspQ1 (NEB kit number: R0712L) to obtain linearized transcription templates. After quality control, the templates were used for further processing.

[0104] Table 2. PCR Amplification Procedure

[0105]

[0106] 2. In vitro transcription of modified saRNA

[0107] Prepare a 400 μL transcription system (containing T7 RNA polymerase, NTP, capping reagent, RNase inhibitor, transcription buffer, and template DNA), incubate at 37°C for 3 hours, and add 20 μL LDNaseI to degrade the template DNA after transcription to ensure the purity of saRNA.

[0108] 3. Purification of saRNA

[0109] Add 210 μL of lithium chloride to the transcription system, incubate at -20°C for at least 30 minutes, centrifuge at 16000g for 20 minutes at 4°C, remove the supernatant, wash the precipitate once with freshly prepared 70% ethanol, air dry, and reconstitute the mRNA with preservation solution. The final saRNA should meet the quality control standards of OD260 / OD280=1.9-2.1, OD260 / OD230≥2.0, and integrity≥70%.

[0110] 4. LNP encapsulation of saRNA

[0111] Using a microfluidic device, the LNP organic phase (each lipid component dissolved in ethanol) and the saRNA aqueous phase (dissolved in 25mM sodium acetate buffer, pH=4.0) were mixed at a volume ratio of 1:3 to form a CARD11-saRNA / LNP complex with a particle size of 80-120nm, PDI≤0.15, and encapsulation efficiency≥85%. After confirming that the specifications met the requirements, the complex was aliquoted into 0.1mg / vial and stored at -80℃ in the dark.

[0112] 5. Antibody conjugation

[0113] DBCO-modified Etaracizumab antibody was mixed with CARD11-saRNA / LNP-N3 at a molar ratio of antibody to LNP particles of 4:1. The mixture was subjected to gentle rotational reaction at 25°C in the dark for 4 h, followed by a further reaction at 4°C for 12 h. The antibody and LNP were conjugated via bioorthogonal click chemistry. After the reaction, unconjugated free antibody was removed by gel filtration chromatography, and the main elution peak was collected to obtain the Etaracizumab-CARD11-saRNA / LNP targeting complex.

[0114] Example 4: In vivo efficacy verification

[0115] The murine colon cancer cell line CT26 used in this embodiment was purchased from the cell bank of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, and identified by STR.

[0116] All 32 male BALB / c mice aged 4-6 weeks used in this study were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. During the rearing and experimental processes, the mice were housed in the Shanghai SPF-grade animal facility of the Chinese Academy of Sciences. Each time the rearing environment was changed, a 5-7 day acclimatization period was allowed before the experiment. This animal experiment has been approved by the Experimental Animal Ethics Committee of Zhongshan Hospital Affiliated to Fudan University.

[0117] Cell preparation

[0118] Cell collection: Digest cells in the logarithmic growth phase with trypsin, and terminate the digestion with complete culture medium. Centrifuge at 1000 rpm for 4 min at 4°C, and discard the supernatant. Wash cells 1-2 times with pre-chilled PBS to remove serum and trypsin residue, and resuspend in PBS.

[0119] Cell counting and viability assay: Cells were stained and counted using trypan blue. The final concentration was adjusted according to literature, with 100 μL of cell suspension injected into each mouse. After counting, the cell suspension was placed on an ice box for later use. It was thoroughly mixed before inoculation to prevent cell sedimentation and uneven cell count.

[0120] Vaccination

[0121] Preparation and anesthesia of laboratory animals: Anesthesia is administered via isoflurane inhalation or intraperitoneal injection of 1% sodium pentobarbital at a dose of 40-50 mg / kg. Skin preparation: Injection sites are usually the right back, armpit, or groin. After shaving, disinfect with povidone-iodine.

[0122] Needle insertion: Fix the mouse with your left hand and tighten the skin. Use a 1 mL syringe needle to insert subcutaneously at a 15-30° angle, avoiding the muscle layer and intradermal layer. After inserting the needle about 1 cm, slowly inject the cell suspension. When a wheal appears, hold for a few seconds and then slowly rotate and withdraw the needle. Gently press the insertion point to prevent the cell suspension from spilling out.

[0123] Tumor growth monitoring: Observe daily for the first 3-5 days after inoculation to confirm tumor formation. Once the tumor is palpable, measure it every 2-3 days using calipers. The measurements are the tumor's long diameter (L) and short diameter (W). Tumor volume (V) is calculated using the formula: V = L × W² / 2. Five days after tumor inoculation in mice, the subcutaneous tumor volume reached 100 mm². 3 The drug administration procedure was then initiated. Tumor-bearing mice were divided into a saline group, a conventional CARD11-saRNA group (1 mg / kg), and the saRNA group of this invention (0.1 mg / kg). The drugs were administered once every 7 days for a total of 3 times.

[0124] Dosing regimens include administration of saRNA drug alone and administration of saRNA drug in combination with PD-1 monoclonal antibody.

[0125] Administration route: Intratumoral injection, subcutaneous injection or tail vein injection can be selected according to experimental needs, with intratumoral injection being the preferred route;

[0126] Dosage: The recommended single dose for intratumoral injection is 3 μg / animal; the recommended dose for tail vein or subcutaneous injection is 0.1 mg / kg.

[0127] Dosage frequency: once every 7 days, with 3 doses constituting one basic experimental cycle; the frequency may be adjusted to once every 5 days or once every 10 days depending on the tumor growth rate to extend the experimental observation period.

[0128] combination therapy

[0129] It can be used in combination with mouse-derived PD-1 monoclonal antibody (Bioxcell, BE0146). It is recommended that the PD-1 monoclonal antibody be administered intraperitoneally at a dose of 100 μg / animal, and that it be administered simultaneously with CARD11-saRNA-LNP to enhance the synergistic immunosuppressive effect. It can also be used in combination with chemotherapy drugs such as oxaliplatin. The chemotherapy drugs should be administered 24 hours apart from the saRNA drugs.

[0130] Experimental monitoring indicators include

[0131] Tumor growth monitoring: The long and short diameters of the tumor were measured with calipers every 2 days, the tumor volume was calculated (volume = long diameter × short diameter² / 2), and a tumor growth curve was plotted.

[0132] Immune microenvironment monitoring: After the drug administration cycle, tumor tissue was taken to prepare a single-cell suspension. The infiltration ratio of immune cells such as CD8⁺ T cells, cytotoxic macrophages, and NK cells was detected by flow cytometry, and the expression level of the Cxcl16-Cxcr6 axis was analyzed.

[0133] Safety monitoring: Mouse body weight changes were monitored weekly during the experiment; after the experiment, liver, kidney, heart and other organ tissues of mice were collected for pathological sectioning and HE staining to assess the toxicity of the drug to normal organs; at the same time, the level of inflammatory factors in peripheral blood of mice was detected to investigate immune-related side effects.

[0134] See Figure 2 and Figure 3 The experimental results showed that after 28 days of administration, the tumor volume in the saRNA group was reduced by 80% compared with that in the saline group, and the tumor inhibition effect was prolonged by 14 days compared with that in the traditional mRNA group. The mice did not show significant weight loss or organ damage.

[0135] like Figure 2 As shown in Figure C, survival analysis after immunotherapy (PD-1 monotherapy group; CTLA-4 monotherapy group; PD-1+CTLA-4 combination therapy group) showed that the survival of patients with high CARD11 expression was significantly prolonged. Figure 2 The results showed that immunohistochemistry revealed a significant increase in the expression level and positive area of ​​CARD11 in the pathological tissue of CRC patients compared to the adjacent tissue.

[0136] In mouse models, xenografts in mice overexpressing CARD11 showed significant shrinkage, while those in mice with CARD11 knockout showed significant enlargement. This suggests that CARD11 overexpression can inhibit the growth of colorectal cancer. However, in in vitro experiments, CARD11 did not affect tumor cell proliferation or apoptosis. This suggests that changes in CARD11 expression levels in epithelial cells do not directly affect the biological behavior of tumor cells themselves, but may participate in regulating the function of other immune cells in the tumor microenvironment. Figure 3 ).

Claims

1. Use of saRNA of gene CARD11 (GeneID: 84433 or Uniprot ID: Q9BXL7 version) in the preparation of antitumor drugs.

2. The use as described in claim 1, characterized in that, The nucleotide sequence of the coding region (CDS) of the gene CARD11 is shown in SEQ ID NO: 1; the amino acid sequence of the CARD11 protein encoded by the gene is shown in SEQ ID NO:

2.

3. The use as described in claim 1, characterized in that, The tumor is selected from the group consisting of: colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, lung adenocarcinoma, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, cancers of the endocrine system, thyroid cancer. Adenocarcinoma, parathyroid carcinoma, adrenal carcinoma, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, bladder cancer, cancer of the kidney or ureter, renal pelvis cancer, vegetations of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer; preferably, the tumor is intestinal cancer such as colon cancer and / or rectal cancer.

4. A drug for treating cancer, such as colorectal cancer, characterized in that, The active pharmaceutical ingredient is the saRNA molecule as described in claim 1 or 2; preferably, the saRNA molecule is replicated and expressed in tumor tissue cells and / or adjacent normal tissue cells.

5. The drug as described in claim 4, characterized in that, The drug is a saRNA drug for treating colorectal cancer, comprising the saRNA molecule as described in claim 1 or 2 and a targeted delivery vector for encapsulating the saRNA molecule, forming a saRNA targeted delivery system.

6. The drug as described in claim 5, characterized in that, The targeted delivery vector includes an LNP vector and an antibody conjugated to the LNP vector targeting the integrin αvβ3 receptor.

7. The drug as described in claim 6, characterized in that, The antibody targeting the integrin αvβ3 receptor is Etaracizumab, whose amino acid sequence is shown in SEQ ID NO:

3.

8. The drug as described in claim 4 or 5, characterized in that, The drug is a pharmaceutical composition that, in addition to containing a therapeutically effective amount of the saRNA molecule as described in claim 1 or 2 as the active ingredient, also contains a pharmaceutically acceptable carrier.

9. The drug as described in claim 4 or 5, characterized in that, The drug is a pharmaceutical composition that, in addition to containing a therapeutically effective amount of the saRNA molecule as described in claim 1 or 2, also contains one or more other antitumor pharmaceutical ingredients, such as those for treating colorectal cancer; optionally, the other antitumor pharmaceutical ingredients are selected from the group consisting of: PD-1 monoclonal antibody, oxaliplatin, and fluorouracil.

10. A method for preparing the medicament as described in claim 6, characterized in that, Includes the following steps: (1) Construction of saRNA template DNA: A DNA fragment containing the coding region (CDS) of the CARD11 gene was synthesized, cloned into a self-replicating vector, and a recombinant plasmid was constructed. After the sequence accuracy was verified by sequencing, a high-purity plasmid was extracted as a transcription template. (2) In vitro transcription of modified saRNA: Prepare a saRNA transcription system containing T7 RNA polymerase, NTP, nucleotides, transcription buffer and template DNA, incubate, and degrade the template DNA with DNase I after transcription to ensure the purity of saRNA; (3) Purification of saRNA: Lithium chloride was added to the transcription system for purification, and the final saRNA met the OD standard. 260 / OD 280 = 1.9-2.1、OD2 60 / OD 230 Quality control standards of ≥2.0 and integrity ≥70%; (4) LNP encapsulation of saRNA: The LNP organic phase is mixed with the saRNA aqueous phase to form a CARD11-saRNA / LNP complex with a particle size of 80-120 nm, PDI≤0.15, and encapsulation efficiency≥85%. (5) The CARD11-saRNA / LNP complex obtained in step (4) is coupled to the antibody in a directed or non-directed manner to obtain the saRNA targeted delivery system, i.e. the drug.

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