KRAS self-replicating mRNA vaccine and preparation method and application thereof

Abstract

The application discloses a KRAS tetravalent self-replicating mRNA vaccine, which is prepared by inserting an optimized sequence encoding KRAS G12D, G13D, G12V and G12C mutants into a self-replicating mRNA carrier and encapsulating the self-replicating mRNA carrier by LNP. The vaccine can self-amplify to enhance antigen expression, induce a high-efficiency specific immune response, and is suitable for treating KRAS mutation related tumors such as pancreatic cancer, colorectal cancer and non-small cell lung cancer, and can cover a wide range of patients and realize large-scale production.

Description

Technical Field

[0001] This invention relates to the field of mRNA vaccine technology, and more particularly to tumor therapeutic vaccines based on self-replicating mRNA technology, specifically the construction and application of self-replicating mRNA vaccines targeting G12V, G13D, G12D, and G12C mutations in the KRAS gene. Background Technology

[0002] KRAS is a Kirsten RAt Sarcoma viral oncogene and a member of the RAS oncogene family. KRAS acts as a molecular switch cycling between inactive (GDP-binding) and active (GTP-binding) states, transducing upstream cellular signals received from various tyrosine kinases to downstream effectors, regulating a wide range of processes and playing a crucial regulatory role in cell proliferation, differentiation, and survival signaling pathways. The role of mutated KRAS in malignant tumors was discovered early on. Aberrant KRAS expression accounts for up to 20% of all cancers, with a particularly high incidence in pancreatic ductal adenocarcinoma, colorectal cancer, and non-small cell lung cancer. Missense mutations at codons 12, 13, and 61 of the KRAS primary amino acid sequence are the most common KRAS driver mutations, which stabilize GTP binding and lead to constitutive activation of KRAS and downstream signaling. For a long time, this target was considered "undruggable" due to the lack of pocket structures for drug binding in its mutant subtypes (Salman R. Punekar et al., Nature Reviews Clinical Oncology, Vol. 19, (2022)). The breakthrough of KRAS G12C inhibitors has changed the long-standing targeting strategy for KRAS. However, since monotherapy almost always leads to resistance, the industry still needs new therapies to seek a cure.

[0003] Research on attacking tumors with tumor neoantigens has been a hot topic in cancer research. Engineered T-cell immunotherapy has successfully treated hematologic malignancies. However, the use of these therapies to treat solid tumors has encountered significant obstacles: clinical success against solid tumors is limited due to low efficacy, dose-limited toxicity, and high cost. With the development of mRNA technology, personalized tumor vaccines using mRNA as a carrier have emerged, which can induce a selective immune response against cancer cells rather than normal cells, and have achieved good results in clinical practice (NCT03897881). Gal Cafri et al., J Clin Invest. 2020; 130(11):5976-5988.

[0004] However, due to individual differences, selecting relevant and immunogenic vaccine neoantigens remains an expensive and time-consuming process, limiting the applicability of personalized mRNA tumor vaccines. Meanwhile, while universal vaccines offer the advantage of broad population coverage, current research largely focuses on single mutation types, failing to cover patient populations carrying other high-frequency mutations.

[0005] In summary, there is an urgent need in this field for tumor vaccines that possess the following characteristics: 1) target multiple high-frequency KRAS mutations to achieve broad-spectrum population coverage; 2) utilize the self-amplification properties of saRNA to enhance antigen expression intensity and duration, thereby improving T cell response persistence; and 3) adopt standardized sequence design and production processes to support large-scale preparation and reduce the cost of personalized customization. Summary of the Invention

[0006] To address the challenges of individualized formulations in existing technologies, such as difficulty in reaching specific populations, weak immunogenicity, and high preparation costs, a multivalent KRAS mRNA cancer vaccine with self-replication capabilities is proposed to enhance the durability of the immune response.

[0007] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0008] On one hand, the present invention provides a KRAS mutant protein and its nucleotide sequence. Specifically, the KRAS mutant protein of the present invention is shown in SEQ ID NO: 1. The present invention also provides a nucleotide sequence encoding a KRAS mutant protein optimized for expression in humans. Specifically, the optimized nucleotide sequence is shown in SEQ ID NO: 2.

[0009] On the other hand, the present invention provides a self-replicating mRNA molecule comprising a nucleotide sequence encoding a KRAS mutant protein as described in the present invention. Specifically, the self-replicating mRNA molecule of the present invention further comprises sequences encoding an MHC I transport domain and a signal peptide. More specifically, the MHC I transport domain is a Homo sapiens or Homo sapiens-derived MHC I transport domain. Specifically, the nucleotide sequence of the self-replicating mRNA molecule is shown in SEQ ID NO:10.

[0010] On the other hand, the present invention provides a self-replicating mRNA vaccine comprising a nucleotide sequence encoding a KRAS mutant protein as described in the present invention, or comprising a self-replicating mRNA molecule as described in the present invention, and a pharmaceutically acceptable vector. In the self-replicating mRNA vaccine according to the present invention, the nucleotide sequence encoding the KRAS mutant protein or the self-replicating mRNA molecule is encapsulated in lipid nanoparticles (LNPs). The lipid nanoparticles have an average particle size of 80-100 nm and a polydispersity index (PDI) of less than 0.2.

[0011] Furthermore, the present invention also provides the use of nucleotide sequences encoding KRAS mutant proteins as described in the present invention, or of self-replicating mRNA molecules as described in the present invention, in the preparation of self-replicating mRNA vaccines. Specifically, the self-replicating mRNA vaccine is used for the prevention or treatment of tumors associated with KRAS mutations. More specifically, the tumors are selected from pancreatic ductal adenocarcinoma, colorectal cancer, or non-small cell lung cancer.

[0012] This invention combines multiple KRAS mutant peptides with a saRNA vaccine to prepare an effective KRAS-saRNA vaccine. Utilizing the widespread mutation of KRAS in cancer patients, it significantly covers the cancer population. Furthermore, current experimental results indicate that it elicits high levels of cellular and humoral immune responses in mice (animal experiment results not yet shown). Moreover, this invention leverages the self-amplification properties of the vaccine in vivo to enhance antigen expression, thereby increasing the strength and persistence of the immune response, solving the key problems of low protein expression efficiency and weak immunogenicity in previous vaccines. Compared to previous personalized formulations of neoantigen vaccines, this invention enables large-scale production and reduces manufacturing costs.

[0013] A highly efficient and specific mRNA sequence targeting kRAS mutations, exhibiting durable immunogenicity and tumor-killing activity. Compared to T cells induced by DC-loaded peptides, T cells induced by this invention on DC-loaded peptides showed a greater number of spots (140 vs 80) in the Elispot assay, indicating better immunogenicity. Attached Figure Description

[0014] Figure 1 The optimized TKSV sequence and its parameter characterization results of this invention are shown.

[0015] Figure 2 A map of the plasmid psa3.1-Bethune-3 used for TKSV gene synthesis is shown.

[0016] Figure 3 The agarose gel electrophoresis results after extraction of plasmid psa3.1-Bethune-3 are shown. The inset on the right is a reference image, and the inset on the left is the electrophoresis result of the experiment.

[0017] Figure 4 The agarose gel electrophoresis results of linearized plasmid psa3.1-Bethune-3 are shown, with the right inset being a reference image and the left inset being the experimental electrophoresis results.

[0018] Figure 5 The results of the psa3.1-Bethune-3 mRNA sample identification by formaldehyde denaturing gel are shown.

[0019] Figure 6This shows the results of capillary electrophoresis to determine the integrity of the psa3.1-Bethune-3 mRNA sample.

[0020] Figure 7 The results show the mRNA concentration, encapsulation efficiency, particle size, and PDI of the TKSV-LNP formulation.

[0021] Figure 8 The particle size distribution results of the TKSV-LNP formulation samples are shown.

[0022] Figure 9 The results of RT-qPCR detection of TKSV-LNP expression in eukaryotic cells are shown.

[0023] Figure 10 The results of the enzyme-linked immunospot assay (ELISPOT) for TKSV-LNP are shown.

[0024] Figure 11 The results, as determined by CCK8 assay, show that TKSV-LNP vaccine-induced CTL killings were transfected with HLA-11:01-KRAS. G12D The effect of HCT-116 cells.

[0025] Figure 12 Fluorescence imaging of tumor-bearing mice 24 h and 7 days after immunization with TKSV-LNP vaccine is shown.

[0026] Figure 13 The tumor growth curves of tumor-bearing mice after immunization with the TKSV-LNP vaccine are shown.

[0027] Figure 14 The results show flow cytometry analysis of the tumor immune microenvironment after immunization with TKSV-LNP vaccine in a humanized mouse tumor-bearing model of B-HLA-A11.1.

[0028] Figure 15 The results show the levels of intratumoral cytokines in a humanized mouse tumor model immunized with TKSV-LNP vaccine after detection by ELISA. Detailed Implementation

[0029] Before explaining the embodiments of this disclosure in detail, it should be understood that the application of this disclosure is not limited to the details described. This disclosure can have other embodiments, or can be practiced or implemented in various ways. Therefore, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary, not exhaustive. Moreover, it should be understood that the wording and terminology used herein are for descriptive purposes and should not be considered restrictive.

[0030] This invention provides a nucleotide sequence encoding a mutant KRAS protein optimized for expression in humans. KRAS (Kirsten rat sarcoma viral oncogene homolog) is one of the most frequently altered oncogenic proteins in human solid tumors and belongs to the RAS family of small GTPases. The KRAS gene encodes a small GTPase transducer protein that acts as a molecular switch, alternating between an active GTP-bound state and an inactive GDP-bound state. Its function is regulated by guanine nucleotide exchange factor (GEF). Under physiological conditions, the KRAS protein participates in "housekeeping" functions such as cell growth, apoptosis, and protein transport. Located on the inner surface of the cell membrane, KRAS activates multiple downstream signaling pathways, including RAF-MEK-ERK and PI3K-AKT-mTOR, in response to signals from upstream growth factor receptors, thereby finely regulating cell proliferation, differentiation, and survival.

[0031] KRAS mutant proteins are among the most common oncogenic driver gene mutations in solid tumors. Oncogenic KRAS mutations lock KRAS into a persistently activated GTP-binding state by impairing endogenous GTPase activity, continuously triggering major oncogenic signaling pathways, thereby enhancing cell proliferation, promoting survival, and conferring drug resistance. KRAS mutations are a key initiating factor in KRAS-driven tumorigenesis and also play a central role in tumor progression, metabolic reprogramming, and immune microenvironment remodeling. KRAS mutations are widespread in common solid tumors such as pancreatic cancer, non-small cell lung cancer (NSCLC), and colorectal cancer. Common activating KRAS mutations mainly occur at codons 12, 13, and 61, with G12D, G12V, G12C, and G13D being the most prevalent mutation subtypes. Different mutation subtypes exhibit differentiated distribution characteristics across different tumor types: G12D is most common in pancreatic and colorectal cancers, G12V is dominant in uterine cancer, and G12C is most common in lung cancer. For a long time, KRAS was considered an "untreatable" target due to its smooth protein surface lacking a traditional binding pocket and its picomolar affinity for GTP. In recent years, the discovery of the Switch-II pocket in the KRAS G12C mutant has enabled the development of covalent inhibitors sotorasib and adagrasib, achieving unprecedented remission rates of 30%–40%.

[0032] This invention relates to the systematic optimization design of mRNA nucleotide sequences encoding KRAS mutant antigens. The core design principles include: codon optimization and GC content regulation: comprehensively considering the influence of codon bias, GC content, and secondary structure on protein translation and expression. Optimized coding region sequences can improve the codon adaptation index under a set GC ratio, while optimizing base pairing patterns and avoiding long stem-loop structures and GC-rich regions, reducing the risk of mRNA being recognized and degraded by the innate immune system in cells. 5'-UTR optimization: Optimized design by increasing ribosome loading (MRL) promotes more efficient translation initiation and elongation. 5'-UTR combinations have a significant impact on mRNA translation efficiency. 3'-UTR optimization: Through regulatory element annotation algorithms, the number of regulatory elements related to sequence degradation (such as ARE, CURE, etc.) is identified and reduced, thereby improving overall mRNA stability. Optimization of the 3'-UTR may also involve modifications to replication signals and regulatory motifs to regulate translation efficiency, immune escape, and RNA stability. 5'-Cap Structure and Poly-A Tail: The mRNA sequence contains the necessary 5'-cap analog structure and a 35-adenosine poly-A tail for recruiting and stabilizing ribosome-mRNA interactions. Integration of Self-Replicating Elements: A replicase-encoding sequence and a non-structural protein gene derived from a positive-sense RNA virus (VEEV) are integrated into the optimized sequence, enabling the mRNA to self-amplify within the cell. Through the above systematic optimization, a nucleotide sequence encoding a KRAS mutant protein optimized for expression in humans was obtained. The optimized nucleotide sequence is shown in SEQ ID NO: 10.

[0033] Self-replicating mRNA molecules Self-amplifying mRNA (saRNA) is an engineered RNA molecule that, in addition to containing the mRNA sequence encoding an antigenic protein, possesses unique self-amplification elements, allowing it to self-amplify the antigen sequence in vivo. Self-amplifying mRNA molecules contain two key regions: one encoding a sequence of the target antigen / therapeutic protein (consistent with conventional mRNA), and the other encoding a replicase protein sequence derived from positive-sense RNA viruses. Most saRNA constructs are derived from positive-sense single-stranded alphaviruses. In these designs, the viral gene encoding a structural protein is removed and replaced with a selected therapeutic sequence, thus enabling RNA replication but preventing the formation of infectious viral particles. Self-amplifying mRNA molecules also require structural elements such as a 5′ cap, 5′ and 3′ UTRs, and a Poly-A tail to ensure proper intracellular translation and replication. Compared to conventional mRNA, conventional mRNA constructs are shorter, simpler in structure, and easier to prepare. saRNA molecules are longer due to the inclusion of a replicase open reading frame and may be more sensitive to shearing and degradation during processing. However, saRNA provides amplified intracellular expression, which can reduce the total RNA requirement by several times without loss of activity.

[0034] Self-replicating mRNA vaccine Self-replicating mRNA vaccines are a novel type of vaccine constructed based on the saRNA technology platform. In addition to containing the mRNA sequence encoding the target antigen, they also include virus-derived self-replicating elements, enabling the antigen sequence to self-amplify within host cells, thereby inducing a strong and durable immune response at a low dose. Self-replicating mRNA vaccines have shown great potential for the prevention and treatment of diseases such as infectious diseases and tumors. Self-replicating mRNA vaccines possess inherent self-adjuvant properties. The double-stranded RNA intermediates generated during replication can be recognized by intracellular pattern recognition receptors (such as RIG-I-like receptors and TLRs), triggering innate immune signaling pathways and providing necessary co-stimulatory signals for adaptive immune responses. To balance excessive immune activation with an effective immune response, the innate immune response can be finely regulated through nucleoside modification (such as 5-methylcytidine)—inhibiting excessive type I interferon production in plasmacytoid dendritic cells while preserving innate immune activation in antigen-presenting cells such as macrophages, thus reducing the risk of adverse reactions and maintaining vaccine immunogenicity. The durability of the immune response can also be further enhanced by combining co-stimulatory molecule agonists.

[0035] Lipid nanoparticles (LNP) In one specific embodiment, the nucleotide sequence encoding the KRAS mutant protein or the self-replicating mRNA molecule of the present invention is encapsulated in lipid nanoparticles (LNPs). Lipid nanoparticles (LNPs) are nanoscale delivery carriers formed by the self-assembly of lipid components, used to encapsulate and protect nucleic acid drugs (such as mRNA and saRNA) and deliver them efficiently to target cells. In some cases, at least one lipid may be a cationic lipid. In some cases, at least one lipid may be an ionizable lipid, such as distearate phosphatidylcholine (DSPC), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), C12-200 (C12), D-Lin-MC3-DMA (MC3), or cKK-E12 (E12). The cationic lipids that can be used in this invention are commercially available, such as the SM-102 formulation available from Cayman Chemical, TargetMol, InvivoChem, etc., wherein the molar ratio of SM102, DSPC, cholesterol, and DMG-PEG 2000 is 50:38.5:10:1.5.

[0036] Self-replicating mRNA vaccine In addition to utilizing the intrinsic self-adjuvant activity conferred by the double-stranded RNA intermediates generated during the replication process, self-replicating mRNA vaccines often require the combined use of external adjuvants to further enhance the immune response or regulate the immune response type. These adjuvants mainly include monophospholipid A (MPLA) and CpG oligodeoxynucleotides that can activate Toll-like receptors, agonists that can activate the STING pathway (such as LNPs modified with SitoC7A for synergistic delivery and activation), co-stimulatory molecule agonists that can prolong the persistence of the immune response (such as OX40 and 4-1BB agonists), and cytokine adjuvants that can enhance cellular immune responses (such as IL-12). At the same time, the introduction of nucleoside chemical modifications (such as N1-methylpseuuridine, 5-methylcytidine, etc.) during the in vitro synthesis of mRNA is also an important means of regulating vaccine immunogenicity and stability. Among them, 5-methylcytidine modification can inhibit the excessive production of type I interferon by plasmacytoid dendritic cells while preserving the innate immune activation in antigen-presenting cells, thereby achieving a balance between reducing the risk of adverse reactions and maintaining vaccine efficacy.

[0037] Self-replicating mRNA vaccine formulations also contain various excipients and formulations to ensure product stability and injectability. Commonly used excipients include Tris-HCl or citrate buffers to maintain pH stability, cryoprotectants such as sucrose or trehalose to protect the structural integrity of LNP-mRNA during freeze-drying, isotonic regulators such as sodium chloride to adjust osmotic pressure, and chelating agents such as EDTA to chelate metal ions to inhibit RNase activity. In addition to lipid nanoparticles as the mainstream delivery system, the delivery methods of self-replicating mRNA vaccines are constantly expanding. These include protein nanocage technology that utilizes RNase-resistant globular protein shells to achieve high delivery efficiency and expression rates, cytoplasmic transcription systems based on T7 autogen DNA vaccine platforms that enable self-amplified T7 RNA polymerase to directly transcribe mRNA in the cytoplasm, bypassing the nuclear translocation step, and amphiphilic molecular delivery systems that efficiently transport vaccines to lymph nodes via albumin "hitchhiking" mechanisms.

[0038] Tumors associated with KRAS mutations and their treatment KRAS mutations are among the most common driver gene mutations in a variety of highly lethal solid tumors, and their distribution varies significantly across different tumor types: approximately 90% of pancreatic cancers, 40%–45% of colorectal cancers, and approximately 30% of non-small cell lung cancers carry KRAS mutations. Among these, pancreatic cancers are predominantly G12D, colorectal cancers are also predominantly G12D, while non-small cell lung cancers are predominantly G12C. In addition, a certain proportion of uterine cancers, gastric cancers, and bile duct cancers also have KRAS mutations. For a long time, KRAS has been considered an "untreatable" target due to its smooth protein surface lacking traditional binding pockets and its picomolar affinity for GTP. The first breakthrough was achieved in recent years with the approval of covalent inhibitors Sotorasib and Adagrasib targeting the Switch-II pocket of the KRAS G12C mutant. However, their remission rate is only 30%–40% and drug resistance is inevitable. To date, there are no approved targeted drugs for the more common KRAS G12D mutation, with only a few small molecule inhibitors and protein degraders in clinical development.

[0039] Against this backdrop, mRNA-based KRAS-targeted vaccines have opened up new avenues for the treatment of KRAS-mutant tumors. Their core strategy involves using self-replicating mRNA or conventional mRNA encoding KRAS-mutant antigenic epitopes (such as G12D, G12V, G13D, and G12C), which are then delivered into the body via lipid nanoparticle delivery systems. There, the mutant antigen proteins are expressed in antigen-presenting cells, thereby activating specific CD8+ cells targeting KRAS mutants. + Cytotoxic T cell response and CD4 +It assists T-cell responses and establishes long-term immune memory to exert anti-tumor immune effects. Currently, several KRAS mRNA vaccines are in clinical development globally, including Moderna's mRNA-5671 / V941 encoding four common KRAS mutations; XP001, a single-target mRNA vaccine targeting KRAS G12V independently developed in China; LK101, a personalized mRNA-DC vaccine developed in China; YKYY031, a universal public target mRNA vaccine; ELI-002, delivered using amphiphilic molecules; ABO-2102, a KRAS neoantigen mRNA vaccine from Abogen Biosciences; and a vaccine that simultaneously activates CD4+ through a "helper-killer axis" strategy. + and CD8 + T-cell responses, such as AST-1138, are potential advantages of mRNA vaccines in treating KRAS-mutant tumors compared to traditional targeted drugs. These advantages include the ability to simultaneously cover multiple mutation subtypes, overcoming the incomplete mutation coverage of single mutation inhibitors; reducing relapse risk by inducing durable immune memory; circumventing common targeted drug resistance issues through immune mechanisms rather than direct signaling pathway inhibition; and compatibility with multiple treatment modalities such as PD-1 / PD-L1 immune checkpoint inhibitors, targeted drugs, chemotherapy, and radiotherapy. It is expected that the dual mechanism of "mRNA vaccine activating specific T-cell responses combined with PD-1 inhibitors relieving immunosuppression" will break through the treatment bottleneck of KRAS-mutant tumors, becoming an important component of future comprehensive treatment strategies for KRAS-driven tumors.

[0040] The following non-limiting examples are provided for illustrative purposes and are not intended to limit the scope of the invention. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or according to the kit instructions; all reagents and instruments used are commercially available products, and unless otherwise specified, their concentrations, volumes, temperatures, times, and other parameters are clearly described in the examples.

[0041] Example Example 1: Design and characterization of self-replicating mRNA molecules For each KRAS mutation target (G13D, G12V, G12C, G12D), ORF optimization was performed using the NovoPro sequence optimization tool (available at www.novopro.cn). Minimum free energy (MFE), codon fitness index (CAI), and GC content were used as key parameters for comprehensive evaluation. The goal was to minimize MFE and maximize CAI to ensure secondary structural stability and a high degree of matching with human codon preferences, thus facilitating efficient translation and expression. After multi-target optimization screening, the Pareto optimal sequence shown below was selected as the mutant peptide TKSV (SEQ ID NO: 1) for subsequent synthesis.

[0042] MTEYKLVVVGAVGVGKSALTIQLIQ GGSGGGGSGG MTEYKLVVVGAGDVGKSALTIQLIQ GGSGGGGS GG MTEYKLVVVGADGVGKSALTIQLIQ GGSGGGGSGG MTEYKLVVVGACGVGKSALTIQLIQ (SEQ ID NO:1), where the underlined part is a connector.

[0043] The nucleotide sequence encoding SEQ ID NO: 1 is shown in SEQ ID NO: 2. The amino acid sequences of each mutant peptide, the MHC-I class molecule transport signal (MITD) sequence (SEQ ID NO: 3; coding sequence SEQ ID NO: 4), the signal peptide sequence (SEQ ID NO: 5; coding sequence SEQ ID NO: 6), and the F-LUC reporter gene sequence (SEQ ID NO: 7; coding sequence SEQ ID NO: 8) are tandemly linked by a glycine-serine linker (GS linker). The amino acid sequence and DNA sequence of the complete mutant peptide TKSV-LUC are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively. Figure 1 As shown, the coding sequence has a GC content of 63.72%, an MFE value of 1009.80 kcal / mol, and a CAI value of 0.94.

[0044] Example 2: Vector construction and synthesis based on psa3.1-1 vector The construct from Example 1 was inserted into the self-replicating RNA (saRNA) backbone vector psa3.1-1. Figure 2 A map of the plasmid psa3.1-Bethune-3 (12,026 bp) used for TKSV gene synthesis is shown. A recombinant plasmid carrying an optimized saRNA sequence (named psa3.1-Bethune-3) was constructed based on the psa3.1-1 vector, and its synthesis was commissioned to Wuhan HanHai New Enzyme Biotechnology Co., Ltd.

[0045] The synthesized plasmid was transformed into competent Escherichia coli and inoculated into LB medium containing kanamycin. Amplification was performed by fermentation at 37°C for 16 h in a shaker. The plasmid was extracted using the FastPure Enhanced EndoFree Plasmid Maxi (Plus) Kit and verified by 0.8% Tris-acetic acid-EDTA (TAE) agarose gel electrophoresis. Figure 3The agarose gel electrophoresis results after extraction of plasmid psa3.1-Bethune-3 are shown. The results show that the electrophoretic bands of the extracted plasmid (psa3.1-Bethune-3) correspond to the marker and the gene synthesis plasmid, and are consistent in size and morphology, indicating that the plasmid was successfully transformed and amplified without genomic DNA contamination.

[0046] The purified plasmid was linearized by adding BspQI restriction endonuclease at a concentration of 10 U / μL and digesting in a water bath at 50°C for 0.5 h. The digestion products were detected by 0.8% TAE agarose gel electrophoresis. Figure 4 The agarose gel electrophoresis results of the linearized plasmid psa3.1-Bethune-3 are shown. The results show that the digested plasmid presents a single, clear band, located in the same position as the linearized control, with no circular plasmid residue, indicating complete linearization and direct use for in vitro transcription.

[0047] Example 3: Production based on PSA 3.1-1 carrier Co-transcriptionalization and capping in vitro transcription (IVT) was performed using the mMESSAGE mMACHINE™ T7 mRNA Kit and CleanCap™ Reagent AG (ThermoFisher, catalog number A57620). The reaction system was as follows: 20 μL 10× reaction buffer, 15 μL each of 100 mM ATP / CTP / GTP / UTP, 12 μL 100 mM CleanCap™ Reagent AG, 10 μg linearized template DNA, and 20 μL T7 enzyme mixture. After reacting at 37°C for 2 h, the mRNA was purified using 7.5 M LiCl precipitation according to the kit instructions. The purified product was dissolved in nuclease-free water and detected by formaldehyde denaturing gel electrophoresis and capillary electrophoresis.

[0048] Figure 5 This shows the results of formaldehyde denaturing gel electrophoresis of the psa3.1-Bethune-3 mRNA sample. The electrophoresis results showed a clear and uniform saRNA band. Compared with the marker (labeled with a gradient of 6000-200bp), the band position corresponded to the expected molecular weight, and there were no obvious degradation bands, indicating that the mRNA sample was of good integrity. Figure 6 The results of capillary electrophoresis show the integrity of the psa3.1-Bethune-3 mRNA sample. The resulting saRNA stock solution was named TKSV stock solution.

[0049] Example 4: LNP Encapsulation Production The encapsulation of lipid nanoparticles was outsourced to HanHai New Enzyme Co., Ltd. In short, microfluidic mixing technology was used to encapsulate the TKSV stock solution within lipid nanoparticles (LNPs), with the lipid components having a molar ratio of SM102:DSPC:cholesterol:DMG-PEG2000 = 50:10:38.5:1.5. The aqueous and lipid phases of saRNA were injected into the microfluidic chip at a specific flow rate ratio, resulting in immediate self-assembly of LNPs. After dialysis to replace the buffer, the TKSV-LNP formulation (2 mg saRNA) was obtained.

[0050] Encapsulation efficiency and concentration were determined using a fluorescent dye method. Figure 7 The results show the mRNA concentration, encapsulation efficiency, particle size, and polydispersity index (PDI) of the TKSV-LNP formulation. Dynamic light scattering particle size analyzer analysis showed an average particle size of 86.39 nm and a polydispersity index (PDI) of 0.1523. Figure 8 The particle size distribution of the TKSV-LNP formulation samples is shown. The results indicate uniform particle size distribution and stable structure, meeting the quality requirements for in vivo delivery. Based on these results, the key indicators of the scaled-up formulation, such as mRNA concentration, encapsulation efficiency, particle size, and PDI, are satisfactory, meeting the quality requirements for LNP formulations in subsequent research and applications.

[0051] Example 5: Expression of TKSV-LNP in eukaryotic cells Peripheral blood mononuclear cells (PBMCs) were collected from healthy volunteers at a rate of 1×10⁻⁶. 6 Cells were seeded per well. The experimental group received 1 μg of TKSV-LNP per well, while the control group received an equal volume of LNP blank vector or PBS. After incubation at 37°C and 5% CO2 for 24 h, cells were collected, and total RNA was extracted using an animal cell / tissue total RNA rapid extraction kit (DNase I-free; supplier: Yali, YL23017) according to the manufacturer's instructions.

[0052] Reverse transcription system: Total RNA 1 pg ~ 1 μg, YALEPIC All-in-RT MasterMix 3 μL, 5×YALEPIC All-in-RT Reaction Mix 4 μL, ddH2O added to 20 μL. Reaction conditions: 25℃ for 5 minutes, 55℃ for 15 minutes, 85℃ for 5 seconds, and hold at 4℃.

[0053] qPCR system: 10 μL of 2× YALEPiC Universal SYBR Green qPCR MasterMix, 0.4 μL each of forward and reverse primers SEQ ID NO: 11 and 12 (10 μM), 100 ng of cDNA, and ddH2O to a final volume of 20 μL; cycling conditions: 95℃ for 3 minutes; 95℃ for 5 seconds, 60℃ for 30 seconds (40 cycles). GAPDH was used as an internal control. -ΔΔCt The relative expression level is calculated using this method.

[0054] Figure 9 The results of RT-qPCR detection of TKSV-LNP expression in PBMC cells are shown. The results showed that the expression level of the target gene in the TKSV-LNP group was significantly higher than that in the control group (P<0.05), demonstrating that this preparation can effectively penetrate the cell membrane and achieve efficient transcription and translation.

[0055] Example 6: DC cell isolation, maturation, and antigen-loaded induction of T cell activation PBMC separation: Mix 30 mL of EDTA-anticoagulated peripheral blood with PBS at a 1:1 ratio, slowly spread it onto the top layer of Ficoll separation solution, and centrifuge at 1500 rpm and 20°C for 15 min. Collect the intermediate PBMC layer, wash twice with PBS (1500 rpm, 10 min), and obtain the PBMCs.

[0056] DC precursor enrichment: PBMCs were resuspended in MACS BSA buffer (Miltenyi, catalog number 130-091-376) (10 7 Add CD8 MicroBeads (Miltenyi, catalog number 130-045-201) (20 μL / 100000 cells / 80 μL buffer) to 80 μL of buffer. 7 Cells were incubated at 4°C for 15 min. CD8 cells were then sorted using an MS magnetic bead column (Miltenyi, catalog number 130-042-201) to obtain CD8. - cell.

[0057] DC induction and maturation: CD8 - Cells were resuspended in AIM V medium (2 ml / well) containing 1% human AB serum (Gibco, catalog number 12055091) at a concentration of 1.5–2.5 × 10⁻⁶ cells / well. 6 pcs / cm 2Cells were cultured at high density and adhered to the culture medium. After 2.5 h, non-adherent cells were collected. AIM V medium containing 80 ng / mL granulocyte-macrophage colony-stimulating factor (GM-CSF), 100 ng / mL interleukin-4 (IL-4), and 5% serum was added and incubated at 37°C in a 5% CO2 incubator for 48 h. On day 3, half the medium was replaced, and the same cytokines were added, and the cells were cultured for another 48 h. On day 5, 200 ng / mL LPS and 20 ng / mL IFN-γ were added to induce maturation for 24 h.

[0058] Antigen-loaded T cell co-culture: Add 1 μg / mL TKSV-LNP or 20 μg / mL antigen peptide (KRAS G13D, G12V, G12C, or G12D) as a control to mature DCs and incubate at 37°C for 16 h. Thaw and cryopreserve CD8 cells. + T cells were co-cultured with antigen-loaded dendritic cells at a ratio of 1:10 in AIM V medium containing 5% human AB serum, with IL-21 (30 ng / mL) added. The next day, IL-2 (2 ng / mL), IL-7 (5 ng / mL), and IL-15 (5 ng / mL) were added to the mixed culture cells, and the cells were stimulated for 3 days. This stimulation was repeated 3 times.

[0059] Enzyme-linked immunospot assay (ELISPOT): Collect co-cultured T cells, 10 5 Spots were seeded per well in an ELISpot plate and stimulated with exogenous lectin (PHA) or antigenic peptide (20 μg / mL) for 24 h. After development, the number of spots was read using an ELISpot reader.

[0060] Figure 10 The results of the ELISPOT experiment for TKSV-LNP are shown. The results indicate that the number of IFN-γ secretion spots induced by the TKSV-LNP loading group was approximately 140 / 10. 5 The number of cells was significantly higher than that of the antigen peptide-loaded control group (approximately 80 cells / 10). 5 Cells (P<0.05), confirming that TKSV-LNP has stronger DC sensitization and T cell activation capabilities.

[0061] Example 7: Immunoactivation and Immunotherapy of mRNA Vaccines TKSV-LNP vaccine-induced cytotoxic T lymphocytes (CTLs) killing HLA-A*11:01 / KRAS G12DEffects of CTLs on HCT-116 cells. The CCK8 assay was used to evaluate the cytotoxic killing effect of CTLs on tumor cells.

[0062] HCT-116-HLA-A*11:01 / KRAS G12D Target cells were seeded at 5000 cells / well in 96-well plates and cultured at 37°C and 5% CO2 for 24 h to allow cell adhesion. Effector-to-target (E:T) ratios of 10:1 and 5:1 were set, and specific CTL effector cells induced in Example 6 were added to each well. Cells were co-cultured at 37°C and 5% CO2 for 48 h. The supernatant was discarded, and the cells were gently washed once with PBS. 100 μL / well of a 1:10 diluted CCK-8 working solution (Cell Counting Kit-8, CCK-8; Mellen; catalog number MA0218-2) was added, and the cells were incubated for another 0.5 h. OD values ​​were measured at 450 nm to calculate cell viability. Cell viability (%) = [(experimental wells - blank wells) / (control wells - blank wells)] × 100%.

[0063] Figure 11 The results, as determined by CCK8 assay, show that TKSV-LNP vaccine-induced CTL killings were transfected with HLA-11:01-KRAS. G12D The effect of TKSV-LNP on HCT-116 cells was investigated. The results showed that the survival rate of target cells decreased significantly with increasing effector-to-target ratio. At effector-to-target ratios of 10:1 and 5:1, the killing rate of the experimental group was significantly higher than that of the negative control group (P<0.05), indicating that TKSV-LNP can efficiently induce CTLs with specific cytotoxic activity.

[0064] Example 8: In vivo antitumor efficacy and tumor immune microenvironment analysis B-HLA-A11.1 / hKRAS resuspended in PBS G12D MC38 cells were grown at a concentration of 1.00 × 10⁻⁶. 6 / 0.1 mL / was subcutaneously injected into the right back of 32 B-HLA-A11.1 mice. When the average tumor volume reached 100 mm... 3 Sixteen mice were selected based on tumor volume and body weight and randomly assigned to the experimental group and the control group, with eight mice in each group.

[0065] Drug administration began on Day 0 (the day of grouping). The experimental group received TKSV-LNP, while the solvent control group received LNP, both at a dose of 10 µg / animal. Animal body weight was measured three times per week after grouping; tumor volume was measured three times per week using calipers. Tumor volume was measured before euthanasia, including both the major and minor diameters. The volume was calculated using the formula: Tumor volume = 0.5 × major diameter × minor diameter. 2 .

[0066] Animals were weighed before euthanasia. At the end of the experiment, mouse tumors were removed and weighed, and tissues were collected for flow cytometry, ELISA, and related molecular assays to evaluate the antitumor and immunomodulatory effects of TKSV-LNP.

[0067] Tumor growth inhibition rate (TGI) TV ): TGI TV (%) = [1-(Ti-T0) / (Vi-V0)]×100% (Ti: mean tumor volume in the treatment group on day i of administration, T0: mean tumor volume in the treatment group on day 0 of administration; Vi: mean tumor volume in the solvent control group on day i of administration, V0: mean tumor volume in the solvent control group on day 0 of administration). Figure 13 The tumor growth curves of tumor-bearing mice after immunization with the TKSV-LNP vaccine are shown.

[0068] Fluorescence imaging data acquisition: Whole-body imaging of mice in the experimental group was performed using an in vivo imaging (IVIS) system at 24 h after drug administration and on Day 7 (Fluc was detected on a small animal in vivo imaging system); the region of interest (ROI) was plotted in Living Image software, and the whole-body fluorescence intensity (radiative efficiency: (p / s / cm² / sr) / (μW / cm²)) was calculated. Figure 12 Fluorescence imaging of tumor-bearing mice 24 h and 7 days after immunization with TKSV-LNP vaccine is shown.

[0069] Flow cytometry: Fresh tumor tissue was mechanically minced and enzymatically digested to prepare a single-cell suspension. A portion of the suspension was used for analysis of immune cell infiltration in tumor tissue. The detection groups were: Live / Dead, mCD45, mCD3, mCD4, mCD8, mFoxP3, mIFN-γ, mCD279 (mPD-1), mCD137 (m4-1BB), and mCD366 (mTIM-3). Figure 14 The results of flow cytometry analysis of the tumor immune microenvironment after immunization of a humanized mouse tumor-bearing model with TKSV-LNP vaccine are shown.

[0070] Results: Tumor growth was significantly inhibited in the TKSV-LNP group, with a significant TGI value (P<0.05); in vivo imaging showed that the Fluc signal decayed over time after drug administration, indicating that target cells were specifically cleared. Flow cytometry analysis showed that CD8+ cells were present in the tumor. + T cells and IFN-γ +The proportion of effector T cells was significantly increased, and the expression of PD-1 / TIM-3 depletion markers was downregulated; ELISA showed that the levels of intratumoral IFN-γ, IL-2, and TNF-α were significantly upregulated (all P<0.05).

[0071] In summary, the TKSV-LNP vaccine of this invention significantly enhances the intensity and duration of antigen expression, and can efficiently activate dendritic cells and induce specific cytotoxic T cells at the cellular level. Experimental results confirm that the immunogenicity of the TKSV-LNP vaccine of this invention is significantly superior to that of traditional peptide loading regimens; in a humanized mouse tumor-bearing model, it can significantly inhibit tumor growth, promote the infiltration of effector T cells within the tumor and the secretion of anti-tumor cytokines such as IFN-γ, IL-2, and TNF-α, while downregulating T cell exhaustion markers and reshaping the tumor immune microenvironment.

[0072] Although the present disclosure has been described in detail by way of illustration and example for the purpose of clarity, it should be understood that the scope of the invention is not limited thereto, but is defined only by the appended claims and their equivalents.

Claims

1. A KRAS mutant protein, the amino acid sequence of which is shown in SEQ ID NO:

1.

2. A nucleotide sequence encoding the KRAS mutant protein of claim 1, as shown in SEQ ID NO:

2.

3. A self-replicating mRNA molecule comprising the nucleotide sequence encoding the KRAS mutant protein as described in claim 2.

4. The self-replicating mRNA molecule according to claim 3, further comprising a sequence encoding an MHC I transport domain and a signal peptide.

5. The self-replicating mRNA molecule according to claim 3 or 4, wherein the nucleotide sequence is shown in SEQ ID NO:

10.

6. A self-replicating mRNA vaccine comprising the nucleotide sequence according to claim 2, or the self-replicating mRNA molecule according to any one of claims 3-5, and a pharmaceutically acceptable vector.

7. The self-replicating mRNA vaccine according to claim 6, wherein the nucleotide sequence encoding the KRAS mutant protein or the self-replicating mRNA molecule is encapsulated in lipid nanoparticles (LNPs).

8. The self-replicating mRNA vaccine according to claim 7, wherein the lipid nanoparticles have an average particle size of 80-100 nm and a polydispersity index (PDI) of less than 0.

2.

9. Use of the nucleotide sequence according to claim 2 or the self-replicating mRNA molecule according to any one of claims 3-5 in the preparation of a self-replicating mRNA vaccine for the prevention or treatment of tumors associated with KRAS mutations.

10. The use according to claim 9, wherein the tumor is selected from pancreatic ductal adenocarcinoma, colorectal cancer, or non-small cell lung cancer.

Images