Recombinant protein aiming at KRAS G12D mutation and application

Abstract

The invention relates to a recombinant protein aiming at KRAS G12D mutation and application of the recombinant protein. In particular, the recombinant protein comprises an immunostimulatory domain, and an antigen domain comprising at least one KRAS G12D antigen fragment.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the field of immunotherapy, and more specifically, to a recombinant protein vaccine targeting the KRASG12D mutation, encoding a polynucleotide, a method of preparation thereof, a pharmaceutical composition comprising the vaccine, and its use in the prevention and / or treatment of cancer. Background Technology

[0002] Malignant tumors pose a significant challenge to global public health. Despite continuous advancements in cancer treatment in recent years, most patients with advanced-stage cancer still suffer from poor prognoses, and their survival rates urgently need improvement. KRAS proteins play a crucial role in cell signal transduction, and their gene mutations are common oncogenic drivers, widely present in various high-incidence cancers, such as pancreatic and colorectal cancers. Among these, the KRAS G12D mutation, due to its high frequency and well-defined pathogenic mechanism, has become a research target of significant therapeutic importance.

[0003] Several small-molecule inhibitors targeting KRAS G12D have entered clinical trials. These include GFH375, an oral inhibitor with high selectivity that can simultaneously inhibit both activated and inactivated KRAS G12D; MRTX1133, a potent, selective, non-covalent inhibitor; and JAB-23E73, a pan-KRAS oral inhibitor that can inhibit multiple KRAS mutants, including G12D. Additionally, there is ASP3082, a protein degrader developed using PROTAC technology, which induces target protein degradation by recruiting E3 ubiquitin ligases. However, these drugs still face many challenges in practical application, such as the development of drug resistance, insufficient selectivity for wild-type KRAS, and long-term safety concerns.

[0004] With the development of immunotherapy, therapeutic cancer vaccines have gradually become an important strategy for combating KRAS mutations. Currently, several KRAS G12D vaccines are in clinical evaluation, such as the mRNA-based vaccine mRNA-5671 / V941 and the synthetic peptide-based vaccine ELI-002. Preliminary results show good safety and a certain degree of immunogenicity, but significant limitations remain, including insufficient immune response strength, limited persistence, and the fact that current delivery systems and adjuvant processes are not yet optimal. Furthermore, the uncertainty of efficacy in advanced patients and the limited range of responses due to individual HLA typing differences also restrict their clinical application.

[0005] Therefore, there is an urgent need in the field to develop a novel therapeutic vaccine that aims to induce a more potent and durable specific immune response against the KRAS G12D mutation, in order to overcome key bottlenecks in existing technologies and fill the current gaps in this therapeutic field. Summary of the Invention

[0006] This invention has achieved a significant breakthrough in preclinical research, achieving for the first time the complete eradication of the KRASG12D gene mutation using vaccine immunization, successfully solving the key challenge of drug development for vaccines targeting this target. In a mouse model of colon cancer, this invention has demonstrated for the first time a significant effect in inducing complete tumor regression, achieving a breakthrough in curative efficacy from zero to one.

[0007] The present invention provides a recombinant protein comprising: (1) an immunostimulatory domain, and (2) an antigen domain comprising a KRAS G12D antigen fragment.

[0008] In one or more embodiments, the immunostimulatory domain is a diphtheria toxin T domain.

[0009] In one or more embodiments, the sequence of the immunostimulatory domain is the sequence after inserting one or more KRAS G12D antigen fragments into the sequence shown in SEQ ID NO:17, preferably inserted between amino acids 88 and 89. In one or more embodiments, the sequence of the immunostimulatory domain is the sequence after inserting a KRAS G12D antigen fragment between positions 88 and 89 of the sequence shown in SEQ ID NO:17. In one or more embodiments, either end of the KRAS G12D antigen fragment is directly connected to SEQ ID NO:17 or connected via a adapter. In one or more embodiments, the adapter contains any number of one or more of G, S, and H. In one or more embodiments, the adapter contains G. m H n G o Where m is an integer from 1 to 5, n is an integer from 0 to 5, and o is an integer from 0 to 5. Preferably, the connector is GG, GGHH, HHGG, GGHGG, or GGHHGG.

[0010] In one or more embodiments, the amino acid sequence of the immunostimulatory domain is as shown in SEQ ID NO: 2, or is a sequence that has at least 95% sequence identity with SEQ ID NO: 2 and retains the same biological function.

[0011] In one or more embodiments, the antigen domain comprises at least one KRAS G12D antigen fragment, such as one, two, three, four, or five KRAS G12D antigen fragments. In one or more embodiments, the antigen domain comprises four KRAS G12D antigen fragments.

[0012] In one or more embodiments, the KRAS G12D antigen fragment is a tandem repeat.

[0013] In one or more embodiments, the tandemly repeated KRAS G12D antigen fragments are directly linked or linked via a linker. In one or more embodiments, the linker comprises any number of one or more of G, S, and H. In one or more embodiments, the linker comprises G. m H n G o Where m is an integer from 1 to 5, n is an integer from 0 to 5, and o is an integer from 0 to 5. Preferably, the connector is GG, GGHH, HHGG, GGHGG, or GGHHGG.

[0014] In one or more embodiments, the KRAS G12D antigen fragment is located at the N-terminus and / or C-terminus of the recombinant protein.

[0015] In one or more embodiments, the amino acid sequence of the KRAS G12D antigen fragment is as shown in SEQ ID NO: 1, or a sequence that has at least 95% sequence identity with SEQ ID NO: 1 and retains the function of inducing a specific immune response against KRAS G12D.

[0016] In one or more embodiments, the recombinant protein further includes one or more of the following structures: (3) a transmembrane enhancement domain, (4) an immunomodulatory domain, (5) a protease cleavage site, and (6) a hydrophobic anchoring sequence.

[0017] In one or more embodiments, the transmembrane enhancement domain is a histidine-rich amphiphilic α-helical peptide.

[0018] In one or more embodiments, the amino acid sequence of the α-helical peptide is as shown in SEQ ID NO: 3, or a sequence that has at least 95% sequence identity with SEQ ID NO: 3 and retains the same biological function.

[0019] In one or more embodiments, the immunomodulatory domain is selected from one or more of the following domains: CD40L extracellular domain, IL-36β cytokine, and HMGB1 Box A domain.

[0020] In one or more embodiments, the amino acid sequence of the extracellular functional domain of CD40L is as shown in SEQ ID NO: 6, or is a sequence that has at least 95% sequence identity with SEQ ID NO: 6 and retains the same biological function.

[0021] In one or more embodiments, the amino acid sequence of the IL-36β cytokine is as shown in SEQ ID NO: 7, or a sequence that has at least 95% sequence identity with SEQ ID NO: 7 and retains the same biological function.

[0022] In one or more embodiments, the amino acid sequence of the HMGB1 Box A domain is as shown in SEQ ID NO: 8, or is a sequence that has at least 95% sequence identity with SEQ ID NO: 8 and retains the same biological function.

[0023] In one or more embodiments, the protease cleavage site is a Furin protease recognition site.

[0024] In one or more embodiments, the amino acid sequence of the Furin protease recognition site is as shown in SEQ ID NO: 5, or a sequence that has at least 95% sequence identity with SEQ ID NO: 5 and retains the same biological function.

[0025] In one or more embodiments, the hydrophobic anchoring sequence is as shown in SEQ ID NO:4, or is a sequence that has at least 95% sequence identity with SEQ ID NO:4 and retains the same biological function.

[0026] In one or more embodiments, the recombinant protein may further include (7) a purification tag.

[0027] In one or more embodiments, the purification tag is a SUMO tag and / or a histidine tag. Preferably, the SUMO tag is a SUMO3 tag.

[0028] In one or more embodiments, the purification tag is located at the N-terminus of the recombinant protein.

[0029] In one or more embodiments, the purification label is removed.

[0030] In one or more embodiments, the recombinant protein comprises, from the N-terminus to the C-terminus, an immunostimulatory domain and one or more KRAS G12D antigen fragments.

[0031] In one or more embodiments, the recombinant protein comprises, from the N-terminus to the C-terminus, a transmembrane enhancement domain, one or more KRAS G12D antigen fragments, an immunostimulatory domain, one or more KRAS G12D antigen fragments, and a hydrophobic anchoring sequence.

[0032] In one or more embodiments, the recombinant protein comprises, from N-terminus to C-terminus, the following in sequence: a transmembrane enhancement domain, one or more KRAS G12D antigen fragments, a hydrophobic anchoring sequence, a Furin protease recognition site, an immunostimulatory domain, one or more KRAS G12D antigen fragments, a CD40L extracellular domain, a Furin protease recognition site, an amino acid sequence of IL-36β cytokine, a Furin protease recognition site, and an HMGB1 Box A domain.

[0033] In one or more embodiments, the recombinant protein comprises, from N-terminus to C-terminus, a transmembrane enhancement domain, one or more KRAS G12D antigen fragments, a hydrophobic anchoring sequence, a Furin protease recognition site, an immunostimulatory domain, one or more KRAS G12D antigen fragments, a CD40L extracellular domain, a Furin protease recognition site, and an HMGB1 Box A domain.

[0034] In one or more embodiments, the recombinant protein comprises, from N-terminus to C-terminus, a transmembrane enhancement domain, one or more KRAS G12D antigen fragments, a hydrophobic anchoring sequence, a Furin protease recognition site, an immunostimulatory domain, one or more KRAS G12D antigen fragments, an IL-36β cytokine, a Furin protease recognition site, and an HMGB1 Box A domain.

[0035] In one or more embodiments, the domains of the recombinant protein are directly linked or linked via a linker. In one or more embodiments, the linker comprises any number of one or more of G, S, and H. In one or more embodiments, the linker comprises G. m H n G o Where m is an integer from 1 to 5, n is an integer from 0 to 5, and o is an integer from 0 to 5. Preferably, the connector is GG, GGHH, HHGG, GGHGG, or GGHHGG.

[0036] In one or more embodiments, the recombinant protein domains are linked by flexible linker peptides.

[0037] In one or more embodiments, the amino acid sequence of the recombinant protein is shown in any one of SEQ ID NO: 9-13.

[0038] The present invention also provides an isolated polynucleotide, wherein: (a) Encoding the recombinant protein as described in any embodiment herein, (b) A nucleotide sequence that has at least 80% (preferably at least 90%) identity with the sequence shown in (a), and (c) The complementary sequence of (a) or (b).

[0039] In some implementations, the polynucleotide is codon-optimized to suit expression in selected host cells.

[0040] In one or more embodiments, the codon optimization is performed on a prokaryotic expression system. Preferably, the prokaryotic expression system is an *E. coli* expression system.

[0041] In one or more embodiments, the sequence of the polynucleotide is shown in any one of SEQ ID NO: 14-16.

[0042] The present invention also provides a nucleic acid construct comprising the polynucleotides described in any of the embodiments herein.

[0043] In one or more embodiments, the nucleic acid construct is a vector. Preferably, the nucleic acid construct is an expression vector or a homologous recombination vector.

[0044] In one or more embodiments, the expression vector is a prokaryotic expression vector.

[0045] In one or more embodiments, the expression vector is selected from the pET series, pGEX series, pBAD series, or pCOLD series.

[0046] In one or more embodiments, the expression vector is the pET21M-SUMO3 vector.

[0047] The present invention also provides a host cell, wherein: (1) Contains, expresses, or secretes the recombinant protein described herein. (2) Contains the polynucleotides described herein, or (3) Contains the nucleic acid constructs described in this article.

[0048] In one or more embodiments, the host cell is a prokaryotic cell or a eukaryotic cell.

[0049] In one or more embodiments, the host cell is a prokaryotic cell, such as *Escherichia coli*, *Bacillus*, or lactic acid bacteria. In one or more embodiments, the host cell is a eukaryotic cell, such as *Saccharomyces cerevisiae*, *Bacillus subtilis*, or *Streptomyces*.

[0050] In one or more embodiments, the host cell is an Escherichia coli cell, preferably an Escherichia coli ER2566 strain.

[0051] The present invention also provides a method for preparing the recombinant protein described herein, comprising the steps of: culturing the host cells described herein under conditions suitable for expressing the recombinant protein described herein.

[0052] In one or more embodiments, the method includes the step of culturing the host cells of the present invention in a culture medium suitable for host cell growth to obtain the recombinant protein described herein.

[0053] In one or more embodiments, the method includes the steps of: (1) Culture and induction of expression: The host cells are cultured in a culture medium and culture conditions suitable for host cell growth and protein expression, and an inducer is added at an appropriate time to induce the expression of recombinant protein; (2) Harvesting and lysis: The host cells are collected and the cells are broken by physical, chemical or enzymatic methods to obtain a crude extract containing the recombinant protein; (3) Purification: The recombinant protein is purified from the crude extract.

[0054] In one or more embodiments, the appropriate timing for step (1) of the method is when the cells have grown to the logarithmic growth phase.

[0055] In one or more embodiments, the cultivation in step (1) of the method is an oscillating cultivation.

[0056] In one or more embodiments, the culture medium in step (1) of the method may be selected from: LB medium, TB medium or M9 minimum medium.

[0057] In one or more embodiments, the inducing agent in step (1) of the method includes, but is not limited to, isopropyl-β-D-thiogalactoside (IPTG) and arabinose.

[0058] In one or more embodiments, the cell disruption method in step (2) of the method may be ultrasonic disruption, high-pressure homogenization, or enzymatic dissolution. Preferably, when the recombinant protein contains a tag (such as a histidine tag), affinity chromatography (e.g., nickel column affinity chromatography) is used for capture and purification.

[0059] In one or more embodiments, the purification method in step (3) of the method includes, but is not limited to, affinity chromatography, ion exchange chromatography, hydrophobic chromatography or molecular sieve chromatography.

[0060] In one or more embodiments, the method may further include a step (4) of removing the label after step (3).

[0061] In one or more embodiments, the method may further include (5) the steps of concentrating, changing the liquid (e.g., by ultrafiltration) and / or sterilizing the resulting recombinant protein after step (3).

[0062] The present invention also provides the use of the recombinant proteins, polynucleotides, nucleic acid constructs, and / or host cells described in any embodiment herein in the preparation of kits and / or pharmaceutical compositions for the treatment and / or prevention of KRAS G12D mutation-related diseases.

[0063] In one or more embodiments, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0064] In one or more embodiments, the pharmaceutically acceptable excipients include, but are not limited to, carriers, diluents, excipients, compound adjuvants, surfactants, preservatives, and lyophilization protectants.

[0065] In one or more embodiments, the diseases associated with the KRAS G12D mutation include cancer.

[0066] In one or more embodiments, the cancer includes pancreatic cancer, colorectal cancer, lung cancer, and bile duct cancer.

[0067] The present invention also provides a reagent or kit comprising the recombinant protein, polynucleotide, nucleic acid construct, and / or host cell described in any of the embodiments herein.

[0068] In one or more embodiments, the kit may further include a negative control (e.g., water), a positive control (e.g., a standard), a buffer solution, and / or instructions for use.

[0069] The present invention also provides a pharmaceutical composition comprising (1) a recombinant protein, polynucleotide, nucleic acid construct, and / or host cell as described in any embodiment herein, and (2) a pharmaceutically acceptable excipient.

[0070] In one or more embodiments, the excipients include, but are not limited to, carriers, diluents, excipients, compound adjuvants, surfactants, preservatives, and lyophilization protectants.

[0071] In one or more embodiments, the pharmaceutical composition may be a vaccine formulation.

[0072] In one or more embodiments, the pharmaceutical composition is used to treat and / or prevent KRAS G12D mutation-related diseases.

[0073] In one or more embodiments, the diseases associated with the KRAS G12D mutation include cancer.

[0074] In one or more embodiments, the cancer includes pancreatic cancer, colorectal cancer, lung cancer, and bile duct cancer.

[0075] The beneficial effects of this invention are as follows: This invention is the first to achieve the goal of completely eradicating the KRAS G12D gene mutation using vaccine immunization in preclinical studies, solving a key problem in the drug development of vaccines targeting this target. Attached Figure Description

[0076] Figure 1 This is a schematic diagram illustrating the construction of recombinant expression plasmids.

[0077] Figure 2 The images show the SDS-PAGE results of the purified recombinant protein. A represents the first Ni column SDS-PAGE analysis; B represents the second Ni column SDS-PAGE analysis; and C represents the samples before and after buffer replacement on the desalting column. Detailed Implementation

[0078] The inventors investigated recombinant proteins that induce more potent and durable specific immune responses against KRAS G12D mutations and their applications, solving a key challenge in the drug development of vaccines targeting this target. The KRAS G12D mutant protein can be processed into a neoantigen, which is then presented by the major histocompatibility complex (MHC) and recognized by T cells, thereby inducing a specific anti-tumor immune response. In a mouse model of colon cancer, this invention demonstrated for the first time a significant effect in inducing complete tumor regression.

[0079] As used herein, the terms “containing,” “having,” or “including” include “comprising,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0080] As used herein, the term "antigen" refers to a molecule capable of inducing an immune response, including but not limited to immunogenic peptides, peptides, or polysaccharides. The immune response induced by an antigen can be B cell-driven (antibody-mediated immune response) and / or T cell-driven (cellular immune response).

[0081] As used herein, the term "KRAS G12D" refers to a mutation in the 12th codon of the KRAS gene where a G→D mutation occurs (i.e., glycine is replaced by aspartic acid). This mutation is commonly found in malignant tumors such as pancreatic cancer and colorectal cancer, and is the target antigen of the vaccine of this invention.

[0082] The present invention first provides a recombinant protein comprising: (1) an immunostimulatory domain, and (2) an antigen domain containing a fragment of the KRASG12D antigen; optionally, the recombinant protein further comprises one or more of a transmembrane enhancement domain, an immunoregulatory domain, a protease cleavage site, a purification tag, and a hydrophobic anchoring sequence.

[0083] "Immunostimulatory domain" refers to a protein domain that can enhance the immune response. In this invention, the T domain of diphtheria toxin is preferably used as the immunostimulatory domain to enhance the immunogenicity of the recombinant protein. In one or more embodiments, the immunostimulatory domain is the T domain of diphtheria toxin, whose amino acid sequence is shown in SEQ ID NO: 2, or a sequence that has at least 95% sequence identity with SEQ ID NO: 2 and retains the same biological function.

[0084] In one or more embodiments, the antigen domain comprises at least one KRAS G12D antigen fragment, such as one, two, three, four, or five KRAS G12D antigen fragments, and if the number of antigen fragments is greater than one, the antigen fragments are tandem repeats. A “tandem repeat” refers to two or more identical or highly similar (e.g., having at least 95% sequence identity) amino acid sequences (such as SEQ ID NO: 1) linked head-to-tail directly or via linker peptides to form a continuous polypeptide chain. The KRAS G12D antigen fragment is located at the N-terminus and / or C-terminus of the recombinant protein. The amino acid sequence of the KRAS G12D antigen fragment is as shown in SEQ ID NO: 1, or a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO: 1 and retaining the function of inducing a specific immune response against KRAS G12D.

[0085] "Transmembrane domain (T domain)" refers to the functional domain in diphtheria toxin responsible for transmembrane and oligomerization. In one or more embodiments, the transmembrane enhancing domain is a histidine-rich amphiphilic α-helical peptide with the sequence shown in SEQ ID NO: 3, or a sequence having at least 95% sequence identity with SEQ ID NO: 3 and retaining the same biological function.

[0086] In one or more embodiments, the immunomodulatory domain is selected from one or more of the following domains: CD40L extracellular domain, IL-36β cytokine, and HMGB1 Box A domain. The amino acid sequence of the CD40L extracellular domain is as shown in SEQ ID NO: 6, or a sequence having at least 95% sequence identity with SEQ ID NO: 6 and retaining the same biological function; the amino acid sequence of the IL-36β cytokine is as shown in SEQ ID NO: 7, or a sequence having at least 95% sequence identity with SEQ ID NO: 7 and retaining the same biological function; the amino acid sequence of the HMGB1 Box A domain is as shown in SEQ ID NO: 8, or a sequence having at least 95% sequence identity with SEQ ID NO: 8 and retaining the same biological function.

[0087] A “SUMO tag” or “His-SUMO tag” refers to a complex fusion tag that typically comprises, from N-terminus to C-terminus, an affinity purification tag (such as a polyhistidine tag / His-tag), an optional linker peptide, and a SUMO protein sequence (such as SUMO3). In this invention, the tag is used for fusion to the N-terminus of the target recombinant protein. The tag has a specific protease recognition structure, allowing precise removal of the entire tag sequence via enzymatic cleavage, thereby releasing the target recombinant protein with a native N-terminus. A “histidine tag” or “His-tag” refers to a short peptide tag consisting of a series of consecutive (typically 6) histidine residues, used for convenient purification of the recombinant protein by metal chelate affinity chromatography (such as a nickel column). In one or more embodiments, the protease cleavage site is a Furin protease recognition site, the sequence of which is shown in SEQ ID NO: 5, or a sequence having at least 95% sequence identity with SEQ ID NO: 5 and retaining the same biological function.

[0088] In one or more embodiments, the hydrophobic anchoring sequence is as shown in SEQ ID NO:4, or is a sequence that has at least 95% sequence identity with SEQ ID NO:4 and retains the same biological function.

[0089] In one or more embodiments, the purification tag is a SUMO tag and / or a histidine tag. Preferably, the SUMO tag is a SUMO3 tag. The purification tag is located at the N-terminus of the recombinant protein. In one or more embodiments, the purification tag is removed.

[0090] As used herein, the term "sequence identity" refers to the percentage of similarity between nucleotide or amino acid sequences calculated using a sequence alignment algorithm (such as the BLAST algorithm, using default parameters). The identity percentage represents the proportion of identical residues within the aligned region. For amino acid sequences, the identity is calculated after aligning the entire sequence and introducing gaps (if necessary) to achieve the maximum percentage of identity. The term "biological function" refers to the ability of the recombinant protein to induce a specific immune response against the KRAS G12D mutant protein, including activation of T cell and / or B cell responses, thereby generating protective immunity in animals or humans.

[0091] Specifically, in some embodiments, the recombinant protein comprises, from the N-terminus to the C-terminus, an immunostimulatory domain (such as the T domain of diphtheria toxin), at least one KRAS G12D antigen unit (e.g., a tandem repeat of the sequence shown in SEQ ID NO: 1, which may be 1, 2, 3, 4, or 5), and an optional purification tag (such as a histidine tag). It should be understood that this terminology also covers variants that have at least 95% sequence identity with the above-described structure and retain the biological function of inducing a specific immune response against KRAS G12D.

[0092] In one or more embodiments, the recombinant protein domains are linked by flexible linker peptides. A "flexible linker peptide" refers to a short peptide sequence (e.g., a Gly-Gly sequence) composed of flexible amino acids such as glycine and serine, used to link multiple protein domains, increasing structural flexibility, avoiding steric hindrance, and thus maintaining protein function.

[0093] In one or more embodiments, the amino acid sequence of the recombinant protein is shown in any one of SEQ ID NO: 9-13.

[0094] As used herein, the term "recombinant protein" refers to a protein produced by introducing the gene of a target protein into a host cell (such as a bacterial, yeast, or mammalian cell) through genetic engineering techniques, and then utilizing the cell's biosynthetic system. In this article, it specifically refers to a protein prepared through genetic engineering that contains a KRAS G12D mutant-derived antigen sequence.

[0095] In this paper, the ability to "induce an immune response against cancer cells expressing the KRAS G12D mutant protein" or "preserve biological function" can be verified by conventional immunological experiments in the art, including but not limited to: detecting the level of IFN-γ secreted by specific T cells by enzyme-linked immunospot assay; analyzing the expression of T cell activation markers by flow cytometry; and observing the tumor growth inhibitory effect on mice inoculated with KRAS G12D mutant tumor cells after immunization with the recombinant protein in vivo animal experiments.

[0096] The present invention also provides an isolated polynucleotide having a sequence encoding a recombinant protein as described herein, or a complementary sequence thereof. The polynucleotide of the present invention may be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a sense strand or an antisense strand.

[0097] "Codon optimization" refers to adjusting the use of codons in a nucleotide sequence to better suit expression in a specific host cell (such as *E. coli*), thereby improving the yield and stability of recombinant proteins. Optimization is based on the codon preferences of the host cell. In this invention, the polynucleotide is codon-optimized to adapt for expression in selected host cells. This codon optimization is performed on a prokaryotic expression system. Preferably, the prokaryotic expression system is an *E. coli* expression system.

[0098] The full-length nucleotide sequence or fragment thereof of the present invention can generally be obtained by PCR amplification, recombination, or artificial synthesis. Once the relevant sequence is obtained, it can be obtained in large quantities using recombination. This typically involves cloning it into a vector, transforming it into cells, and then isolating the relevant sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules existing in isolated forms. Currently, the DNA sequence encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors, etc.) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[0099] This invention also relates to nucleic acid constructs comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, such as expression vectors and recombinant vectors. "Expression vector" refers to a DNA molecule, such as a plasmid, viral vector, granulosome, or artificial chromosome, used to introduce recombinant genetic material into a host cell and express a target protein. Expression vectors typically contain a promoter, a multiple cloning site, and a selection marker to drive the expression of a heterologous sequence, such as a polynucleotide encoding the recombinant protein of this invention. These vectors can be used to transform appropriate host cells to enable them to express proteins. Vectors typically contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. These sequences typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splicing sites, a leader sequence encoding a polypeptide secretion, a ribosome binding site, a polyadenylated sequence, a multiple linker region encoding the nucleic acid to be expressed, and optional marker elements. In one or more embodiments, the nucleic acid construct is a vector; preferably, the nucleic acid construct is an integration vector, a cloning vector, or an expression vector. In one or more embodiments, the vector is a recombinant expression plasmid that can be used for constitutive or inducible expression of the nucleic acid sequence described in this invention.

[0100] In this invention, the expression vector is a prokaryotic expression vector. In one or more embodiments, the expression vector is selected from the pET series, pGEX series, pBAD series, or pCOLD series. In one or more embodiments, the expression vector is the pET21M-SUMO3 vector.

[0101] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. Transformation can also be performed by electroporation if desired. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0102] In this document, "host cell" refers to the cell used to express the recombinant protein, preferably a prokaryotic cell (such as *Escherichia coli*), but may also include eukaryotic cells (such as HEK293 or CHO cells). In one or more embodiments, the host cell (i) contains, expresses, or secretes the recombinant protein described herein, (ii) contains the polynucleotide described herein, or (iii) contains the nucleic acid construct described herein. In one or more embodiments, the host cell is a prokaryotic cell, such as *Escherichia coli*, *Bacillus*, or lactic acid bacteria. In one or more embodiments, the host cell is a eukaryotic cell, such as *Saccharomyces cerevisiae*, *Bacillus subtilis*, or *Streptomyces*. Preferably, the host cell is an *Escherichia coli* cell, preferably *Escherichia coli* strain ER2566.

[0103] The present invention also provides a method for preparing the recombinant protein described herein, the method comprising the steps of: culturing the host cells described herein under conditions suitable for expressing the recombinant protein described herein. Specifically, the method comprises: (1) culturing and inducing expression: culturing the host cells under culture medium and culture conditions suitable for host cell growth and protein expression, and adding an inducer at an appropriate time to induce the expression of the recombinant protein; (2) harvesting and lysing: collecting the host cells and lysing the cells by physical, chemical or enzymatic methods to obtain a crude extract containing the recombinant protein; (3) purification: purifying the recombinant protein from the crude extract. In one or more embodiments, in step (1), the appropriate time is when the cells grow to the logarithmic growth phase; the culture is a shaking culture; the culture medium may be selected from: LB medium, TB medium or M9 minimum medium. In one or more embodiments, in step (2), the cell disruption method may be ultrasonic disruption, high-pressure homogenization or enzymatic lysis. Preferably, when the recombinant protein contains a tag (such as a histidine tag), affinity chromatography (e.g., nickel column affinity chromatography) is used for capture and purification. In one or more embodiments, in step (3), the purification method includes, but is not limited to, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, or molecular sieve chromatography. Furthermore, the method may also include, after step (3), a step of removing the tag and / or a step of concentrating, changing the medium (e.g., by ultrafiltration), and / or sterilizing and filtering the resulting recombinant protein.

[0104] This invention also provides the use of the recombinant proteins, polynucleotides, nucleic acid constructs, and / or host cells described in any embodiment herein in the preparation of kits and / or pharmaceutical compositions for the treatment and / or prevention of KRAS G12D mutation-related diseases. In one or more embodiments, the pharmaceutical composition further includes pharmaceutically acceptable excipients. Further, the pharmaceutically acceptable excipients include, but are not limited to, carriers, diluents, excipients, compound adjuvants, surfactants, preservatives, and lyophilization protectants. In one or more embodiments, the KRAS G12D mutation-related diseases include cancer. Further, the cancers include pancreatic cancer, colorectal cancer, lung cancer, and cholangiocarcinoma.

[0105] The term “prevention and / or treatment” refers to reducing an individual’s risk of developing cancer associated with the KRAS G12D mutation (prevention) and / or inhibiting the development of existing such cancers, alleviating their symptoms, or causing their remission (treatment) by administering the vaccine of the present invention.

[0106] Accordingly, the present invention also provides a reagent or kit comprising the recombinant protein, polynucleotide, nucleic acid construct, and / or host cell described in any embodiment herein. In one or more embodiments, the kit further comprises a negative control (e.g., water), a positive control (e.g., a standard), a buffer, and / or instructions for use.

[0107] Specifically, the kit may include one or more associated reagents, along with instructions describing the intended use and proper use of these reagents. In some embodiments, the kit may include instructions regarding mixing one or more components of the kit and / or separating and mixing samples and administering them to a subject. In some embodiments, the reagents in the kit are pharmaceutical formulations and dosages suitable for a specific application and method of reagent administration. Kits used for research purposes may contain appropriate concentrations or amounts of components for performing various experiments.

[0108] The kit may be designed to facilitate use of the methods described herein and may take many forms. Where applicable, each composition of the kit may be provided in liquid form (e.g., in solution) or in solid form (e.g., dry powder). In some cases, some compositions may be constructible or additionally processable (e.g., processed into an active form), for example by adding a suitable solvent or other substance (e.g., water or cell culture medium), which may or may not be provided with the kit. In some embodiments, the compositions may be provided in a preservation solution (e.g., a cryopreservation solution). Non-limiting examples of preservation solutions include DMSO and paraformaldehyde. In some embodiments, the preservation solution contains a certain amount of a metalloproteinase inhibitor.

[0109] In some embodiments, the kit contains any one or more of the components described herein in one or more containers. Therefore, in some embodiments, the kit may include a container holding the reagents described herein. The reagents may be in liquid, gel, or solid (powder) form. The reagents may be aseptically prepared, packaged in syringes, and transported frozen. Alternatively, they may be contained in vials or other containers for storage. A second container may contain other aseptically prepared reagents. Alternatively, the kit may include premixed active agents and be transported in syringes, vials, tubes, or other containers. The kit may have one or more components required to administer the reagents to a subject, such as syringes, topical application devices, or IV needles and bags.

[0110] Furthermore, the present invention also provides a pharmaceutical composition comprising (1) a recombinant protein, polynucleotide, nucleic acid construct, and / or host cell as described in any embodiment herein, and (2) pharmaceutically acceptable excipients. The excipients include, but are not limited to, carriers, diluents, excipients, compound adjuvants, surfactants, preservatives, and lyophilization protectants. The excipients are preferably non-toxic to the recipient at the dose and concentration used. Such excipients include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. In some embodiments, the pharmaceutical composition may contain substances for improving, maintaining, or retaining, for example, the composition's pH, permeability, viscosity, clarity, color isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or permeation. These substances are known in the art. The optimal pharmaceutical composition may be determined based on the intended route of administration, delivery method, and required dose. In one or more embodiments, the pharmaceutical composition may be a vaccine formulation.

[0111] Pharmaceutical compositions for internal administration are typically provided in sterile formulations. Sterilization is achieved by filtration through a sterile filter membrane. When the composition is lyophilized, sterilization can be performed using this method before or after lyophilization and rehydration. The pharmaceutical compositions of the present invention may be used for parenteral delivery. Compositions for parenteral administration may be lyophilized or stored in solution. They are prepared, for example, by conventional methods using physiological saline or aqueous solutions containing glucose and other excipients. Parenteral compositions are typically placed in containers with sterile access openings, such as intravenous solution bands or vials with stoppers puncturable by a hypodermic needle. Alternatively, the compositions may be used for inhalation or delivery via the digestive tract (e.g., orally). The preparation of the pharmaceutically acceptable compositions is within the scope of the art. Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations containing antibodies in sustained or controlled-release delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery methods (such as liposome carriers, bioeasily perishable microparticles or porous beads, and accumulation injection) are also known to those skilled in the art.

[0112] Once formulated, the pharmaceutical composition is stored in sterile vials as a solution, suspension, gel, emulsion, solid, crystal, or as a dehydrated or lyophilized powder. The formulation may be stored in a ready-to-use form or rehydrated before administration (e.g., lyophilized). The present invention also provides kits for generating single-dose administration units. The kits of the present invention may each contain a first container with dried protein and a second container with an aqueous formulation. In some embodiments of the invention, kits containing single-lumen and multi-lumen pre-filled syringes (e.g., liquid syringes and lyophilized syringes) are provided.

[0113] This invention also provides a method for treating patients (particularly patients with KRAS G12D mutation-related diseases) by administering the reagents or pharmaceutical compositions described in any embodiment of the invention. In this document, the terms “patient,” “subject,” “individual,” and “object” are used interchangeably and include any living organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit, etc.), and most preferably a human. “Treatment” refers to a subject employing the treatment regimen described herein to achieve at least one positive therapeutic effect (e.g., relief or elimination of symptoms of KRAS G12D mutation-related diseases). Effective treatment regimens for patients can vary depending on various factors, such as the patient’s disease status, age, weight, and the ability of the therapy to elicit an anti-disease response in the subject. In one or more embodiments, the KRAS G12D mutation-related disease includes cancer; said cancer includes pancreatic cancer, colorectal cancer, lung cancer, and cholangiocarcinoma.

[0114] The following specific embodiments further illustrate the invention. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise specified. Unless otherwise specified, percentages and parts are by weight. The experimental materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0115] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form preferred technical solutions.

[0116] Example

[0117] Example 1: Construction of the recombinant expression vector SUMO3-LsYz-1

[0118] Based on the amino acid sequence of the target recombinant protein (SEQ ID NO:9), the nucleotide sequence encoding the protein (as shown in SEQ ID NO:14) was designed through codon optimization.

[0119] The full-length nucleotide sequence described above was synthesized by a biotechnology company and cloned into the multiple cloning site of the expression vector pET21M-SUMO3 to construct the recombinant expression plasmid SUMO3-LsYz-1. Figure 1 ).

[0120] Sequencing confirmed that the inserted sequence of the recombinant plasmid was completely consistent with the designed sequence.

[0121] Example 2: Construction and Induction of Recombinant Expression Strains

[0122] Transformation: 1 μL of the constructed recombinant plasmid SUMO3-LsYz-1 was transformed into E. coli ER2566 competent cells. After heat shock at 42℃ for 90 seconds, ice bath, and recovery, the cells were plated on LB agar plates containing 100 μg / mL ampicillin and incubated overnight at 37℃ inverted.

[0123] Expanded culture: Pick a single colony from the plate and inoculate it into LB liquid medium containing Amp, and incubate overnight at 37°C with shaking. The next day, expand the inoculation to LB medium at a ratio of 1:100 (e.g., 6 L), and incubate at 37°C and 220 rpm until the bacterial OD600 reaches 0.6-0.8.

[0124] Induction of expression: Isopropyl-β-D-thiogalactoside was added to the bacterial culture to a final concentration of 0.25 mM, and the culture temperature was lowered to 16℃ to induce expression overnight.

[0125] Cell collection: After induction, centrifuge at 4000 rpm for 15 minutes to collect the cell pellet, and store at -20℃ for later use.

[0126] Example 3: Purification and preparation of recombinant protein His-SUMO3-LsYz-1

[0127] This embodiment employs a strategy combining the SUMO fusion expression system with two-step nickel column chromatography and ion exchange chromatography to obtain high-purity tag-free recombinant protein.

[0128] Lysis and clarification: The collected bacterial cells were resuspended in lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl). The cells were then lysed using a low-temperature autoclave, followed by centrifugation at 4°C and 15,000 rpm for 1 hour, and the supernatant was collected.

[0129] First nickel-column affinity chromatography: The supernatant was loaded onto a pre-equilibrated Ni-NTA affinity chromatography column. To effectively remove weakly bound contaminating proteins and degradation fragments, a gradient imidazole washing strategy was employed: washing was performed sequentially with buffers containing 20 mM, 60 mM, and 150 mM imidazole until the OD280 of the eluent returned to baseline. Finally, the target fusion protein (His-SUMO3-LsYz-1) was eluted with elution buffer containing 200-300 mM imidazole.

[0130] Tag removal and reverse purification: The eluted fusion protein fractions were combined and specifically digested with Senp2 protease to remove the N-terminal His-SUMO3 tag. Simultaneously, the sample was dialyzed into a low-salt, low-imidazolium buffer (20 mM Tris-HCl pH 7.4, 100 mM NaCl). The digested sample was then re-loaded onto a Ni-NTA chromatography column. At this point, the cleaved His-SUMO3 tag and the tagged, undigested protein bound to the column, while the tag-removed recombinant protein (LsYz-1) did not bind to the nickel column and remained in the flow-through. The flow-through was collected.

[0131] Anion exchange chromatography: To further remove trace degradation fragments and concentrate the protein, the flow-through buffer was diluted to a low salt concentration (50 mM NaCl) and loaded onto anion exchange chromatography column. The target protein bound to the column, and then eluted with high-salt buffer (1 M NaCl), and the elution peak was collected.

[0132] Formulation: The purified protein is replaced with a storage buffer (20 mM Tris-HCl pH 7.4, 100 mM NaCl) through a desalting column to obtain the final recombinant protein vaccine stock solution.

[0133] Proteins LsYz-2 to LsYz-5 (amino acid sequences are shown in SEQ ID NO: 10-13, and exemplary nucleotide sequences are shown in SEQ ID NO: 14-15) were prepared using the same method as in Examples 1-3.

[0134] Example 4: Identification and quality analysis of recombinant protein LsYz-1

[0135] SDS-PAGE analysis was performed on the samples from each step of Example 3 (see Appendix). Figure 2 ): Expression and initial purification: The results of the first nickel column purification showed ( Figure 2 A) By washing with gradient imidazole, most of the impurities were effectively removed, and a clear fusion protein band (approximately 40 kDa) was visible in the eluted fraction.

[0136] Tag removal effect: After Senp2 enzyme digestion and second nickel column reverse purification ( Figure 2 B), the molecular weight of the protein band in the flow-through fluid migrated (approximately 27.6 kDa), consistent with the theoretical molecular weight, indicating that the SUMO tag had been successfully excised and removed.

[0137] Final purity: after anion exchange chromatography and desalting treatment ( Figure 2 C) The final product appears as a single, clear band on the electrophoretic gel, without obvious degradation bands or polymers, and has a purity greater than 95%, meeting the requirements for the preparation of subsequent vaccine formulations.

[0138] The same method was used to identify and analyze proteins LsYz-2 to LsYz-5.

[0139] Example 5, Preparation of the pharmaceutical composition (vaccine)

[0140] Component preparation: Antigen: Use the stock solution of LsYz recombinant protein (LsYz-1, LsYz-2, LsYz-3, LsYz-4 or LsYz-5) prepared in Example 3.

[0141] Combined adjuvant: Aluminum hydroxide adjuvant and CpG1826 (TLR9 ​​agonist) were selected.

[0142] Preparation process: 1. Prepare according to a single immunization dose (100 μl total volume per mouse), which contains 100 μg of LsYz recombinant protein, 300 μg of aluminum hydroxide adjuvant and 30 μg of CpG1826.

[0143] 2. Mix the recombinant protein and adjuvant thoroughly under aseptic conditions.

[0144] 3. Place the mixture on ice and let it stand for 10-20 minutes to allow the antigen and adjuvant to fully bind.

[0145] 4. Once prepared, administer the vaccine within 2 hours to ensure antigen activity and vaccine stability.

[0146] Example 6, Immunogenicity Verification

[0147] To verify the immune-inducing ability of the LsYz recombinant protein vaccine in vivo, specific antibody titers were measured.

[0148] Experimental animals: 6-8 week old female BALB / c mice were selected.

[0149] Immunization program: The experimental group mice were immunized with the LsYz vaccine formulation prepared in Example 5 on days 1, 4, 8, 15, 22, and 38. The administration route was two subcutaneous injections at locations rich in lymph nodes in the groin area.

[0150] Detection method: Peripheral blood was collected from mice within a specified time after the last immunization, and serum was extracted by centrifugation. The titer of specific antibodies against the KRAS G12D antigen fragment (SEQ ID NO:1) in the serum was detected by enzyme-linked immunosorbent assay (ELISA).

[0151] Experimental results: The test results showed that mice vaccinated with the LsYz vaccine produced high titers of specific antibodies, indicating that the recombinant protein has good immunogenicity and can effectively activate the body's humoral immune response.

[0152] Example 7, Recombinant Protein Tumor Therapy Experiment

[0153] Tumor model establishment: (1) Cell preparation: The CT26 colon cancer cell line carrying the KRAS G12D mutation was selected. Cells were cultured to the late logarithmic growth phase and then the density was adjusted to 5. 10 5 per ml.

[0154] (2) Inoculation: 100 μl of cell suspension (approximately 5 μL) was subcutaneously injected into the axilla of each BALB / c mouse. 10 4 (One tumor cell).

[0155] Experimental grouping and drug administration: LsYz treatment group: After tumor inoculation, mice were subcutaneously injected with the LsYz vaccine (100 μg protein / mouse) as described in Example 5 according to the schedule of D1, D4, D8, D15, D22, and D38.

[0156] Negative control group: Inoculated with the same volume of physiological saline.

[0157] Positive control group: PD-1 immune checkpoint antibody (200 μg / animal) was injected intraperitoneally once every 3 days.

[0158] Observation indicators: Tumor growth: Measure the long and short diameters of the tumor every two days using calipers, and calculate the tumor volume = long diameter. Short diameter^2 0.5.

[0159] Survival status: The survival time of mice was recorded when the tumor volume exceeded 2000 mm. 3 At that time, it was considered death.

[0160] Experimental results: In a mouse model of colon cancer, the LsYz treatment group showed a significant tumor-suppressing effect. Compared with the control group, the LsYz vaccine significantly inhibited tumor growth and induced complete regression of tumors in some mice, achieving a curative effect, and significantly prolonging the survival of mice.

[0161] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments are given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

[0162] Partial sequence of this article: SEQ ID NO:1: KRAS G12D antigen fragment KLVMVGADGVGKSALTI SEQ ID NO:2: Amino acid sequence of the diphtheria toxin T domain interspersed with antigen units INLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIGGKLVMVGADGVGKSALTIGGEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRP SEQ ID NO:3: Amino acid sequence of the transmembrane enhancement domain KKALLALALHHLAHLALHLALALKKA SEQ ID NO:4: Amino acid sequence of the hydrophobic anchoring sequence SYWKPFLVNMCVATVLTAGAYLCYRFLFN SEQ ID NO:5: Amino acid sequence of the Furin protease recognition site CAGNRVRRSVGSSLSC SEQ ID NO:6: Amino acid sequence of the extracellular functional domain of CD40L SSSPAKDPPIQRLRGAVTRCEDGQLFISSYKNEYQTMEVQNNSVVIKCDGLYIIYLKGSFFQEVKIDLHFREDHNPISIPMLNDGRRIVFTVVASLAFKDKVYLTVNAPDTLCEHLQINDGELIVVQLTPGYCAPEGSYHSTVNQVPL SEQ ID NO:7:IL-36β celluloid sequence of amino acid sequence VGTEVLEESSCVNLQTQRLPCQKIKTYIIWEGAMRAVIFVTKRGLKICADPEAKWVKACIKTVDGRASTRKNMAETVPTGAQRSTSTAITLTG SEQ ID NO:8:HMGB1 Box A structure of amino acid sequence PKGETKKKFKDPNAPKRPPSAFFLFCSEYRPKIKGEHPGL SEQ ID NO:9: amino acid sequence of the protein LsYz-1 INLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIGGKLVMVGADGVGKSALTIGGEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPGGKLVMVGADGVGKSALTIGG SEQ ID NO:10: amino acid sequence of protein LsYz-2 KKALLALHLLAHLALALLKKAGGKLVMVGADGVGKSALTIGGINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIGGKLVMVGADGVGKSALTIGGEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPGGKLVMVGADGVGKSALTIGGKLVMVGADGVGKSALTIGGKLVMVGADGVGKSALTIGGKLVMVGADGVGKSALTIGGSGGSYWKPFLVNMCVATVLTAGAYLYCYRFLFN SEQ ID NO:11: amino acid sequence of protein LsYz-3 KKALLALALHHLAHLALHLALALKKAGGHHGGKLVMVGADGVGKSALTIGGHHGGKLVMVGADGVGKSALTIGGHHGGKLVMVGADGVGKSALTIGGHHGGSYWKPFLVNMCVATVLTAGAYLCYRFLFNCAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIGGKLVMVGADGVGKSALTIGGEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPGGKLVMVGADGVGKSALTIGGSSSPAKDPPIQRLRGAVTRCEDGQLFISSYKNEYQTMEVQNNSVVIKCDGLYIIYLKGSFFQEVKIDLHFREDHNPISIPMLNDGRRIVFTVVASLAFKDKVYLTVNAPDTLCEHLQINDGELIVVQLTPGYCAPEGSYHSTVNQVPLCAGNRVRRSVGSSLSCVGTEVLEESSCVNLQTQRLPCQKIKTYIIWEGAMRAVIFVTKRGLKICADPEAKWVKACIKTVDGRASTRKNMAETVPTGAQRSTSTAITLTGCAGNRVRRSVGSSLSCPKGETKKKFKDPNAPKRPPSAFFLFCSEYRPKIKGEHPGL SEQ ID NO:12: Amino acid sequence of protein LsYz-4 KKALLALALHHLAHLALHLALALKKAHHGGKLVMVGADGVGKSALTIGGHHGGKLVMVGADGVGKSALTIGGHHGGKLVMVGADGVGKSALTIGGHHSYWKPFLVNMCVATVLTAGAYLCYRFLFNCAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIGGKLVMVGADGVGKSALTIGGEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPGGKLVMVGADGVGKSALTIGGSSSPAKDPPIQRLRGAVTRCEDGQLFISSYKNEYQTMEVQNNSVVIKCDGLYIIYLKGSFFQEVKIDLHFREDHNPISIPMLNDGRRIVFTVVASLAFKDKVYLTVNAPDTLCEHLQINDGELIVVQLTPGYCAPEGSYHSTVNQVPLCAGNRVRRSVGSSLSCPKGETKKKFKDPNAPKRPPSAFFLFCSEYRPKIKGEHPGL SEQ ID NO:13: Amino acid sequence of protein LsYz-5 KKALLALALHHLAHLALHLALALKKAGGHHGGKLVMVGADGVGKSALTIGGHHGGKLVMVGADGVGKSALTIGGHHGGKLVMVGADGVGKSALTIGGHHGGSYWKPFLVNMCVATVLTAGAYLCYRFLFNCAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIGGKLVMVGADGVGKSALTIGGEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPGGKLVMVGADGVGKSALTIGGVGTEVLEESSCVNLQTQRLPCQKIKTYIIWEGAMRAVIFVTKRGLKICADPEAKWVKACIKTVDGRASTRKNMAETVPTGAQRSTSTAITLTGCAGNRVRRSVGSSLSCPKGETKKKFKDPNAPKRPPSAFFLFCSEYRPKIKGEHPGL SEQ ID NO:14: DNA sequence of the LsYz-1 protein constructed into the pET21M-SUMO3 vector and optimized atcaacctggattgggatgtgattcgcgataaaaccaaaaccaagattgaaagcctgaaagaacatggcccgattaaaaataaaatgagcgaaagcccgaataaaaccgttagcgaagaaaaagcgaaacagtatctggaagaatttcatcagaccgccctggaacacccggaactgagcgaactgaaaaccgtgaccggcaccaatccggtgtttgccggcgccaactatgccgcgtgggcggtgaacgtggcccaggttatcggcggcaaactggtgatggtgggcgccgatggtgtgggcaaaagcgcgctgaccattggcggtgaaaaaaccaccgcggcgctgagcattttaccgggcattggcagcgtgatgggaattgccgatggcgccgttcaccataataccgaagaaattgtggcgcagagcattgcgctgagcagcctgatggtggcccaggccattccgctggtgggcgaactggtggatattggctttgccgcctataactttgtggaatctattattaatctgtttcaggtggtgcacaacagctacaatcgcccgggcggcaaactggtgatggtaggcgcggatggcgtgggcaaaagcgccctgaccattggcggcaaactggtgatggttggcgcggatggcgtgggaaaatcggcgctgacgatcggcggtaaactggtgatggtgggcgccgatggcgtggggaaaagcgccctgaccattggcggtaaactggtgatggtgggcgcggatggcgtgggcaaaagcgcgctgaccattggcggctaa SEQ ID NO:15: DNA sequence of the LsYz-2 protein constructed into the pET21M-SUMO3 vector and optimized SEQ ID NO:16: The optimized DNA sequence of LsYz-3 protein constructed into the pET21M-SUMO3 vector. SEQ ID NO:17 INLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRP。

Claims

1. A recombinant protein comprising: (1) an immunostimulatory domain, and (2) an antigenic domain comprising at least one KRAS G12D antigen fragment, Preferably, the recombinant protein further comprises one or more of the following structures: (3) a transmembrane enhancement domain, (4) an immunomodulatory domain, (5) a protease cleavage site, (6) a hydrophobic anchoring sequence, and (7) a purification tag.

2. The recombinant protein as described in claim 1, characterized in that, The recombinant protein contains one or more of the following characteristics: (a) The immunostimulatory domain is the diphtheria toxin T domain. Preferably, the amino acid sequence of the immunostimulatory domain is as shown in SEQ ID NO: 2, or is a sequence that has at least 95% sequence identity with SEQ ID NO: 2 and retains the same biological function; (b) The antigen domain is a tandemly repeated KRAS G12D antigen fragment. Preferably, the amino acid sequence of the KRAS G12D antigen fragment is as shown in SEQ ID NO: 1, or is a sequence that has at least 95% sequence identity with the sequence shown in SEQ ID NO: 1 and retains the function of inducing a specific immune response against KRAS G12D. (c) The transmembrane enhancement domain is a histidine-rich amphiphilic α-helical peptide. Preferably, the amino acid sequence of the α-helical peptide is as shown in SEQ ID NO: 3, or is a sequence that has at least 95% sequence identity with SEQ ID NO: 3 and retains the same biological function; (d) The immunomodulatory domain is selected from one or more of the following domains: CD40L extracellular domain, IL-36β cytokine, HMGB1 Box A domain, Preferably, The amino acid sequence of the extracellular functional domain of CD40L is shown in SEQ ID NO: 6, or is a sequence that has at least 95% sequence identity with SEQ ID NO: 6 and retains the same biological function. The amino acid sequence of the IL-36β cytokine is shown in SEQ ID NO: 7, or a sequence that has at least 95% sequence identity with SEQ ID NO: 7 and retains the same biological function. The amino acid sequence of the HMGB1 Box A domain is as shown in SEQ ID NO: 8, or is a sequence that has at least 95% sequence identity with SEQ ID NO: 8 and retains the same biological function. (e) The protease cleavage site is a Furin protease recognition site. Preferably, the amino acid sequence of the Furin protease recognition site is as shown in SEQ ID NO: 5, or is a sequence that has at least 95% sequence identity with SEQ ID NO: 5 and retains the same biological function; (f) The hydrophobic anchoring sequence is as shown in SEQ ID NO:4, or is a sequence that has at least 95% sequence identity with SEQ ID NO:4 and retains the same biological function; (g) The purification tag is a SUMO tag and / or a histidine tag, preferably a SUMO3 tag and / or histidine.

3. The recombinant protein as described in claim 1 or 2, characterized in that, The amino acid sequence of the recombinant protein is shown in any one of SEQ ID NO: 9-13.

4. An isolated polynucleotide, which (a) Encoding the recombinant protein as described in any one of claims 1-3, (b) A nucleotide sequence that has at least 80% (preferably at least 90%) identity with the sequence shown in (a), and (c) The complementary sequence of (a) or (b), Preferably, the sequence of the polynucleotide is as shown in any one of SEQ ID NO: 14-16.

5. A nucleic acid construct comprising the polynucleotide as described in claim 4, Preferably, the nucleic acid construct is a vector.

6. A host cell, wherein: (1) Containing, expressing, or secreting the recombinant protein as described in any one of claims 1-3, (2) Contains the polynucleotide as described in claim 4, or (3) Contains the nucleic acid construct as described in claim 5. Preferably, the host cell is a prokaryotic cell or a eukaryotic cell; more preferably, the host cell is an Escherichia coli cell.

7. A method for preparing the recombinant protein according to any one of claims 1-3, comprising the step of: culturing the host cell as described in claim 6 under conditions suitable for culturing and inducing expression of the recombinant protein as described in any one of claims 1-3. Preferably, the culture medium used for the culture includes: LB medium, TB medium, or M9 minimum medium Preferably, the inducing agent used in the induction includes: isopropyl-β-D-thiogalactoside and arabinose.

8. Use of the recombinant protein as described in any one of claims 1-3, the polynucleotide as described in claim 4, the nucleic acid construct as described in claim 5, and / or the host cell as described in claim 6 in the preparation of reagents and / or kits for the treatment and / or prevention of KRASG12D mutation-related diseases. Preferably, the diseases associated with the KRAS G12D mutation include cancer; more preferably, the cancers include pancreatic cancer, colorectal cancer, lung cancer, and bile duct cancer.

9. A reagent or kit comprising the recombinant protein as described in any one of claims 1-3, the polynucleotide as described in claim 4, the nucleic acid construct as described in claim 5, and / or the host cell as described in claim 6. Preferably, the kit further includes a negative control (e.g., water), a positive control (e.g., a standard), a buffer solution, and / or instructions for use.

10. A pharmaceutical composition comprising (1) a recombinant protein as claimed in any one of claims 1-3, a polynucleotide as claimed in claim 4, a nucleic acid construct as claimed in claim 5, and / or a host cell as claimed in claim 6, and (2) pharmaceutically acceptable excipients, Preferably, the pharmaceutical composition may be a vaccine formulation.

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