WO2022048574A1

WO2022048574A1 - Nucleic acid molecule encoding kras gene mutant

Info

Publication number: WO2022048574A1
Application number: PCT/CN2021/116075
Authority: WO
Inventors: 张骅; 华坚; 陈晓庆; 刘园园
Original assignee: 上药生物治疗（香港）有限公司
Priority date: 2020-09-01
Filing date: 2021-09-01
Publication date: 2022-03-10
Also published as: CN116323947A

Abstract

Provided are a nucleic acid molecule encoding a Kras gene mutant and an application of an oncolytic herpes simplex virus (oHSV) vector containing said nucleic acid molecule in preparing an anti-tumor drug. The mutant comprises one or more among a Kras G12D mutant, a Kras G13D mutant, a Kras G12V mutant, a Kras G13C mutant, a Kras G12C mutant, a Kras G13A mutant, a Kras G12A mutant, a Kras Q61L mutant, a Kras G12R mutant, a Kras Q61H, a Kras G12S mutant, a Kras Q61R mutant, a Kras A59T mutant, a Kras A146T mutant, a Kras Y64H mutant, and a Kras A18D mutant.

Description

A nucleic acid molecule encoding Kras gene mutant

technical field

The present application relates to the field of biomedicine, in particular to a nucleic acid molecule encoding a Kras gene mutant, and the application of an oncolytic herpes simplex virus (oHSV) vector containing the nucleic acid molecule in the preparation of antitumor drugs.

Background technique

Oncolytic virus is a kind of natural or artificially modified virus that can specifically replicate in a large amount in tumor cells and eventually eliminate tumor cells, but has no killing effect on normal tissue cells. At present, there are dozens of viruses used in oncolytic therapy, including herpes simplex virus, adenovirus, reovirus, measles virus, etc. Among them, the most concerned is herpes simplex virus (HSV).

Herpes simplex virus (HSV) is a commonly used virus in genetic engineering, which is divided into type 1 and type 2. With the development of virology and genetic engineering technology, people can modify viral genes and apply them in the treatment of tumors. As early as 1991, Martuza et al. genetically modified herpes simplex virus type 1 (HSV-1) and established an oncolytic virus strain that can inhibit the proliferation of tumor cells and has replication activity for malignant brain tumors. Treatment.

Kras gene is a proto-oncogene, about 35kb long, located on chromosome 12, is one of the members of RAS gene family, encoding Kras protein. Kras protein is a membrane-bound protein that is located on the inner side of the cell membrane and is also located in the EGFR (epidermal growth factor receptor) signaling pathway, which plays an important role in the occurrence and development of tumors. The growth, proliferation, angiogenesis and other processes of tumor cells require intracellular proteins for signal transduction, and the Kras gene is the determinant of the transduction protein. The Kras mutant encodes an abnormal protein, which stimulates the growth and spread of malignant tumor cells, and is not affected by it. Signaling effects of upstream EGFR.

At present, oncolytic viruses still have some problems in tumor treatment. For example, patients who have been attacked by viruses have residual virus antibodies in their bodies, and tumor cells cannot be completely eliminated. Therefore, it is necessary to develop novel oncolytic viruses and evaluate their antitumor activity.

SUMMARY OF THE INVENTION

In one aspect, the application provides an isolated nucleic acid molecule comprising one or more genes each independently selected from the group consisting of: Kras G12D mutant, Kras G13D mutant, Kras G12V Mutant, Kras G13C mutant, Kras G12C mutant, Kras G13A mutant, Kras G12A mutant, Kras Q61L mutant, Kras G12R mutant, Kras Q61H mutant, Kras G12S mutant, Kras Q61R mutant, Kras A59T Mutant, Kras A146T mutant, Kras Y64H mutant and Kras A18D mutant.

In another aspect, the application provides an isolated nucleic acid molecule that does not comprise a polynucleotide encoding a 6x His-tagged protein, and that comprises one or more genes each independently encoding a Kras mutant selected from the group consisting of: Kras G12D mutant, Kras G13D mutant, Kras G12V mutant, Kras G13C mutant, Kras G12C mutant, Kras G13A mutant, Kras G12A mutant, Kras Q61L mutant, Kras G12R mutant, Kras Q61H mutant, Kras G12S mutant, Kras Q61R mutant, Kras A59T mutant, Kras A146T mutant, Kras Y64H mutant and Kras A18D mutant.

In certain embodiments, the isolated nucleic acid molecule comprises a gene encoding a Kras mutant selected from the group consisting of: Kras A59T mutant, Kras G12D mutant, Kras G12V mutant, KrasA146T mutant, Kras G13D mutant, and KrasG12C mutant.

In certain embodiments, the isolated nucleic acid molecule comprises a gene encoding a Kras mutant selected from the group consisting of: Kras A59T mutant, Kras G12D mutant, Kras G12V mutant, KrasA146T mutant, Kras G13D mutant, Kras Y64H mutant and Kras G12C mutant.

In certain embodiments, the 3' end of the gene encoding the Kras G12D mutant is directly or indirectly linked to the 5' end of the gene encoding the Kras A146T mutant.

In certain embodiments, the 3' end of the gene encoding the Kras A46T mutant is directly or indirectly linked to the 5' end of the gene encoding the Kras G12V mutant.

In certain embodiments, the 3' end of the gene encoding the Kras G12V mutant is directly or indirectly linked to the 5' end of the gene encoding the Kras A59T mutant.

In certain embodiments, the 3' end of the gene encoding the Kras A59T mutant is directly or indirectly linked to the 5' end of the gene encoding the Kras G13D mutant.

In certain embodiments, the 3' end of the gene encoding the Kras G13D mutant is directly or indirectly linked to the 5' end of the gene encoding the Kras Y64H mutant.

In certain embodiments, the 3' end of the gene encoding the Kras Y64H mutant is directly or indirectly linked to the 5' end of the gene encoding the Kras G12C mutant.

In certain embodiments, each of the genes encoding the Kras mutants are arranged in tandem in the isolated nucleic acid molecule.

In certain embodiments, each of the genes encoding the Kras mutants is a minigene Minigene.

In certain embodiments, each of the genes encoding the Kras mutants is arranged in tandem to yield a tandem minigene.

In certain embodiments, each of the Kras mutants comprises at least 20 amino acids.

In certain embodiments, each of the Kras mutants comprises at least 9 amino acids immediately N-terminal to the mutation site and at least 10 amino acids C-terminal to the mutation site.

In certain embodiments, the Kras G12D mutant comprises the amino acid sequence set forth in SEQ ID NO:1.

In certain embodiments, the Kras G13D mutant comprises the amino acid sequence set forth in SEQ ID NO:4.

In certain embodiments, the Kras G12V mutant comprises the amino acid sequence set forth in SEQ ID NO:7.

In certain embodiments, the Kras G13C mutant comprises the amino acid sequence set forth in SEQ ID NO:8.

In certain embodiments, the Kras G12C mutant comprises the amino acid sequence set forth in SEQ ID NO:9.

In certain embodiments, the Kras G13A mutant comprises the amino acid sequence set forth in SEQ ID NO:10.

In certain embodiments, the Kras G12A mutant comprises the amino acid sequence set forth in SEQ ID NO:11.

In certain embodiments, the Kras Q61L mutant comprises the amino acid sequence set forth in SEQ ID NO:12.

In certain embodiments, the Kras G12R mutant comprises the amino acid sequence set forth in SEQ ID NO:15.

In certain embodiments, the Kras Q61H mutant comprises the amino acid sequence set forth in SEQ ID NO:16.

In certain embodiments, the Kras G12S mutant comprises the amino acid sequence set forth in SEQ ID NO:17.

In certain embodiments, the Kras Q61R mutant comprises the amino acid sequence set forth in SEQ ID NO:18.

In certain embodiments, the Kras A59T mutant comprises the amino acid sequence set forth in SEQ ID NO:54.

In certain embodiments, the Kras A146T mutant comprises the amino acid sequence set forth in SEQ ID NO:56.

In certain embodiments, the Kras Y64H mutant comprises the amino acid sequence set forth in SEQ ID NO:58.

In certain embodiments, the Kras A18D mutant comprises the amino acid sequence set forth in SEQ ID NO:60.

In certain embodiments, the gene encoding the Kras G12D mutant comprises the nucleotide sequence set forth in SEQ ID NO:19.

In certain embodiments, the gene encoding the Kras G13D mutant comprises the nucleotide sequence set forth in SEQ ID NO:22.

In certain embodiments, the gene encoding the Kras G12V mutant comprises the nucleotide sequence set forth in SEQ ID NO:25.

In certain embodiments, the gene encoding the Kras G13C mutant comprises the nucleotide sequence set forth in SEQ ID NO:26.

In certain embodiments, the gene encoding the Kras G12C mutant comprises the nucleotide sequence set forth in SEQ ID NO:27.

In certain embodiments, the gene encoding the Kras G13A mutant comprises the nucleotide sequence set forth in SEQ ID NO:28.

In certain embodiments, the gene encoding the Kras G12A mutant comprises the nucleotide sequence set forth in SEQ ID NO:29.

In certain embodiments, the gene encoding the Kras Q61L mutant comprises the nucleotide sequence set forth in SEQ ID NO:30.

In certain embodiments, the gene encoding the Kras G12R mutant comprises the nucleotide sequence set forth in SEQ ID NO:33.

In certain embodiments, the gene encoding the Kras Q61H mutant comprises the nucleotide sequence set forth in SEQ ID NO:34.

In certain embodiments, the gene encoding the Kras G12S mutant comprises the nucleotide sequence set forth in SEQ ID NO:35.

In certain embodiments, the gene encoding the Kras Q61R mutant comprises the nucleotide sequence set forth in SEQ ID NO:36.

In certain embodiments, the gene encoding the Kras A59T mutant comprises the nucleotide sequence set forth in SEQ ID NO:55.

In certain embodiments, the gene encoding the Kras A146T mutant comprises the nucleotide sequence set forth in SEQ ID NO:57.

In certain embodiments, the gene encoding the Kras Y64H mutant comprises the nucleotide sequence set forth in SEQ ID NO:59.

In certain embodiments, the gene encoding the Kras A18D mutant comprises the nucleotide sequence set forth in SEQ ID NO:61.

In certain embodiments, the tandem minigene comprises, from 5' end to 3' end, a gene encoding a Kras G12D mutant, a gene encoding a Kras A59T mutant, a gene encoding a Kras G12V mutant, and a gene encoding a Kras A146T mutation gene encoding Kras G13D mutant, gene encoding Kras Y64H mutant, gene encoding Kras G12C mutant, gene encoding Kras Q61H mutant, gene encoding Kras A18D mutant, gene encoding Kras G12A mutant Gene, the gene encoding the Kras A146T mutant, and the gene encoding the Kras G12S mutant.

In certain embodiments, the isolated nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO:65.

In certain embodiments, the tandem minigene comprises, from 5' end to 3' end, a gene encoding a Kras G12D mutant, a gene encoding a Kras A146T mutant, a gene encoding a Kras G12V mutant, and a gene encoding a Kras A59T mutation The gene encoding the Kras G13D mutant, the gene encoding the Kras Y64H mutant, and the gene encoding the Kras G12C mutant.

In certain embodiments, the isolated nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO:66.

In certain embodiments, the tandem minigene comprises, from the 5' end to the 3' end, the gene encoding the Kras G12D mutant, the gene encoding the Kras A59T mutant, the gene encoding the Kras A18D mutant, and the gene encoding the Kras G12V mutant The gene encoding the Kras A146T mutant, the gene encoding the Kras Q61H mutant, the gene encoding the Kras G13D mutant, the gene encoding the Kras A59T mutant, the gene encoding the Kras A146T mutant, and the gene encoding the Kras G12C mutant Gene.

In certain embodiments, the isolated nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO:67.

In certain embodiments, the isolated nucleic acid molecule further comprises a polynucleotide encoding a secreted peptide.

In certain embodiments, the polynucleotide encoding the secreted peptide is a polynucleotide encoding the secreted peptide of CD14 protein.

In certain embodiments, the polynucleotide encoding the CD14 protein secretory peptide is located 5' to the gene encoding the Kras mutant.

In certain embodiments, the polynucleotide encoding the CD14 protein secreted peptide comprises the nucleotide sequence set forth in any one of SEQ ID NO:37.

In certain embodiments, the isolated nucleic acid molecule further comprises a polynucleotide encoding a tag protein.

In certain embodiments, the isolated nucleic acid molecule comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 62-64.

In another aspect, the present application provides a vector comprising the nucleic acid molecule.

In certain embodiments, the vector comprises a viral vector.

In certain embodiments, the vector comprises an oncolytic herpes simplex virus oHSV vector.

In certain embodiments, the vector comprises a herpes simplex virus type I HSV-1 vector.

In certain embodiments, the HSV-1 vector is deficient in the neurotoxic factor gamma 34.5 gene.

In certain embodiments, in the vector, the isolated nucleic acid molecule is located between the UL3 gene and the UL4 gene of the HSV-1 vector.

In certain embodiments, the vector includes a promoter.

In certain embodiments, the promoter comprises a CMV promoter.

In certain embodiments, the vector comprises the nucleotide sequence shown in GenBank No: GU734771.1 of the NCBI database.

In another aspect, the present application provides a pharmaceutical composition comprising the nucleic acid molecule, and/or the carrier, and optionally a pharmaceutically acceptable adjuvant.

In another aspect, the present application provides a composition comprising the isolated nucleic acid molecule described herein, the carrier described herein or the pharmaceutical composition described herein, and physiological saline.

In certain embodiments, the isolated nucleic acid molecule in the composition comprises one or more genes each independently encoding a Kras mutant selected from the group consisting of: Kras G12D mutant, Kras G13D mutant, Kras G12V mutant, Kras G13C mutant, Kras G12C mutant, Kras G13A mutant, Kras G12A mutant, Kras Q61L mutant, Kras G12R mutant, Kras Q61H mutant, Kras G12S mutant, Kras Q61R mutant, Kras A59T mutant, Kras A146T mutant, Kras Y64H mutant and Kras A18D mutant.

In certain embodiments, the isolated nucleic acid molecule in the composition comprises a gene encoding a Kras mutant selected from the group consisting of: Kras A59T mutant, Kras G12D mutant, Kras G12V mutant, KrasA146T mutant, Kras G13D mutant and KrasG12C mutant.

In certain embodiments, the isolated nucleic acid molecule in the composition comprises a gene encoding a Kras mutant selected from the group consisting of: Kras A59T mutant, Kras G12D mutant, Kras G12V mutant, KrasA146T mutant, Kras G13D mutant, Kras Y64H mutant and Kras G12C mutant.

In certain embodiments, the tandem minigene comprises, from the 5' end to the 3' end, the gene encoding the Kras G12D mutant, the gene encoding the Kras A59T mutant, the gene encoding the Kras G12V mutant, and the gene encoding the Kras A146T mutation gene encoding Kras G13D mutant, gene encoding Kras Y64H mutant, gene encoding Kras G12C mutant, gene encoding Kras Q61H mutant, gene encoding Kras A18D mutant, gene encoding Kras G12A mutant Gene, the gene encoding the Kras A146T mutant, and the gene encoding the Kras G12S mutant.

In certain embodiments, the isolated nucleic acid molecule in the composition comprises the nucleotide sequence set forth in SEQ ID NO:65.

In certain embodiments, the tandem minigene comprises, from the 5' end to the 3' end, the gene encoding the Kras G12D mutant, the gene encoding the Kras A146T mutant, the gene encoding the Kras G12V mutant, and the Kras A59T mutation The gene encoding the Kras G13D mutant, the gene encoding the Kras Y64H mutant, and the gene encoding the Kras G12C mutant.

In certain embodiments, the isolated nucleic acid molecule in the composition comprises the nucleotide sequence set forth in SEQ ID NO:66.

In certain embodiments, the isolated nucleic acid molecule in the composition comprises the nucleotide sequence set forth in SEQ ID NO:67.

In certain embodiments, the isolated nucleic acid molecule in the composition further comprises a polynucleotide encoding a secreted peptide.

In certain embodiments, the isolated nucleic acid molecule in the composition further comprises a polynucleotide encoding a tag protein.

In certain embodiments, the isolated nucleic acid molecule in the composition comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 62-64.

In certain embodiments, the composition comprises a vector described herein comprising an isolated nucleic acid molecule described herein.

In certain embodiments, the vector in the composition comprises a viral vector.

In certain embodiments, the vector in the composition comprises an oncolytic herpes simplex virus oHSV vector.

In certain embodiments, the vector in the composition comprises a herpes simplex virus type I HSV-1 vector.

In certain embodiments, in the composition, in the vector, the nucleic acid molecule is located between the UL3 gene and the UL4 gene of the HSV-1 vector.

In certain embodiments, the vector in the composition includes a promoter.

In certain embodiments, the promoter comprises a CMV promoter.

In certain embodiments, the vector in the composition comprises the nucleotide sequence shown in GenBank No: GU734771.1 of the NCBI database.

On the other hand, the present application provides the nucleic acid molecule, the carrier, the pharmaceutical composition and/or the application of the composition in the preparation of a medicament for treating tumors.

In certain embodiments, the tumor comprises a solid tumor.

In certain embodiments, the tumor comprises non-small cell lung cancer.

In certain embodiments, the tumor comprises colorectal cancer.

In certain embodiments, the tumor comprises breast cancer.

In certain embodiments, the tumor comprises pancreatic cancer.

Other aspects and advantages of the present application can be readily appreciated by those skilled in the art from the following detailed description. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will recognize, the content of this application enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention to which this application relates. Accordingly, the drawings and descriptions in the specification of the present application are only exemplary and not restrictive.

Description of drawings

The invention to which this application relates is set forth with particularity characteristic of the appended claims. The features and advantages of the inventions involved in this application can be better understood by reference to the exemplary embodiments described in detail hereinafter and the accompanying drawings. A brief description of the drawings is as follows:

Figure 1 shows the structure of the Kras mutant tandem minigene described in the present application.

Figure 2A shows the structural composition of wild-type HSV-1 (F) described in this application.

Figure 2B shows the structural composition of the modified HSV-1(F)-KR10 described in the present application.

Figure 2C shows the structural composition of the BAC plasmid containing the KR11 nucleotide sequence described in this application.

Figure 2D shows the structural composition of the BAC plasmid containing the KR12 nucleotide sequence described in this application.

Figure 3 shows the body weight change trend of the CT26.WT subcutaneously transplanted tumor mice described in the present application.

Figure 4A shows the changes of tumor volume in the CT26.WT subcutaneously transplanted tumor mice described in the present application.

Figure 4B shows the individual data of the tumor volume change in the CT26.WT subcutaneously transplanted tumor mice described in the present application.

Figure 5 shows the statistics of tumor weight in CT26.WT subcutaneously transplanted mice described in the present application.

Figures 6A-6B show tumor photographs after euthanasia of the CT26.WT model described herein.

Figure 7 shows the changes in tumor volume in animals described herein (#5203, #5204).

Figure 8 shows the photo of the end point of the tumor re-challenge model animal experiment described in the present application.

Figure 9 shows the body weight change trend of the A549 subcutaneously transplanted tumor mice described in the present application.

Figure 10 shows the changes of tumor volume in mice with A549 subcutaneously transplanted tumor described in the present application.

Figure 11 shows the changes in tumor volume of each group of animals described in this application.

Figure 12 shows the tumor weight statistics of each group described in this application.

Figure 13 shows a photograph of the tumor after euthanasia of the A549 model described in this application.

detailed description

The embodiments of the invention of the present application are described below with specific specific examples, and those skilled in the art can easily understand other advantages and effects of the invention of the present application from the contents disclosed in this specification.

Definition of Terms

In this application, the term "nucleic acid molecule" generally refers to polymers of nucleotides (eg, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)), including naturally occurring (adenine, guanine, cytosine, uridine) pyrimidine and thymine), non-naturally occurring and modified nucleic acids. In the present application, the nucleic acid molecule comprises the nucleotide sequence of the gene encoding each of the Kras mutants, and each of the Kras mutants comprises at least 10 amino acids N-terminal to the mutation site immediately adjacent to the site and all The C-terminal of the mutation site is immediately adjacent to at least 10 amino acids of the site; the nucleic acid molecule further comprises a polynucleotide of the CD14 protein secreted peptide located at the 5' end of the gene encoding the Kras mutant, and the Kras mutation The polynucleotide of 6x His at the 3' end of the gene of the body.

In this application, the term "secretory peptide" generally refers to short peptide chains that direct the transfer of newly synthesized proteins to the secretory pathway. In this application, the N of the Kras mutant polypeptide carries the CD14 protein secretion peptide sequence to guide the extracellular secretion of the Kras polypeptide, so as to facilitate the replication and expression of the recombinant HSV-1 in the tumor cells at a later stage, and release the Kras mutant polypeptide by antigen-presenting cells (antigen- presenting cell, APC) is effectively presented after ingestion.

In this application, the term "tagged protein" generally refers to a protein that is fused and expressed together with the target protein for the convenience of detection of the target protein. The tag protein used in this application is a 6xHis tag protein, also known as a polyhistidine tag protein, using the sequence of histidine residues to bind to several types of immobilized ions (such as nickel under specific buffer conditions) , copper and cobalt), but has little effect on the properties of the target protein itself, so as to achieve the purpose of easy detection and purification of His-tagged proteins.

In this application, the term "vector" generally refers to a tool capable of carrying an exogenous gene or DNA fragment of interest into a host cell for replication and expression. According to the source, it can be divided into plasmid vector, phage vector, viral vector and yeast artificial chromosome vector. In the present application, the vector may be an HSV-1 vector capable of being linked to the nucleic acid molecule and replicated and expressed in a host cell.

In this application, the term "HSV-1" generally refers to a neurotropic, enveloped, double-stranded DNA virus belonging to the alphavirus subfamily of the family Herpesviridae, with a genome length of 152 kb consisting of two interconnected long The segment UL and the short segment US are composed. Because of its advantages of large gene capacity, short replication cycle, high infection efficiency, and the ability to insert multiple therapeutic genes, it has become the first choice for anti-tumor drug research in genetic engineering. The term "UL3 gene" refers to the gene encoding protein_id: ADD59983.1 in human HSV [GU734771.1], and the term "UL4 gene" refers to the gene encoding protein_id: ADD60023.1 in human HSV [GU734771.1], the UL3 gene Neither the UL4 nor the UL4 gene is necessary to maintain the survival and replication of the HSV virus.

In this application, the term "γ34.5 gene" generally refers to the apoptosis suppressor gene in the HSV-1 genome, which is also an important neurotoxic gene, GeneBank No: GU734771.1. Knockout of the γ34.5 gene is also the most common attenuation strategy for oncolytic viruses based on HSV as the backbone. HSV-1 with defective γ34.5 gene function not only greatly reduces the toxicity, but also enables the virus to selectively replicate in tumor cells by exploiting the defect of IFN-PKR signaling in tumor cells. Since host PKR phosphorylation induced by virus infection can inhibit virus replication, the gene product of HSV-1γ34.5 can resist the inhibitory effect of PKR, enabling the virus to replicate in normal cells, while many tumor cells are defective in the PKR system, resulting in The HSV virus with knockout of the γ34.5 gene can effectively replicate and kill tumors in tumor cells but cannot replicate in normal cells, which provides conditions for HSV virus engineering to specifically kill tumors.

In the present application, the term "promoter" usually refers to a deoxyribonucleic acid (DNA) sequence located upstream of the 5' end of the transcription initiation site of the target gene, which can enable the transcription of the target gene, which can be recognized by RNA polymerase , and begin to transcribe synthetic RNA. In the present application, the promoter refers to a DNA sequence located upstream of the 5' end of the transcription initiation site of the gene encoding the Kras mutant and controls its transcription.

In this application, "pharmaceutical composition" generally refers to a composition suitable for administration to a patient, comprising one or more pharmaceutically effective carriers, stabilizers, excipients, diluents, solubilizers, surfactants , emulsifiers, preservatives and/or adjuvants suitable formulations. The pharmaceutical composition can be administered intravenously, intraperitoneally, subcutaneously, intramuscularly, topically or intradermally. In the present application, the pharmaceutical composition comprises a nucleic acid molecule encoding a Kras gene mutant, the oncolytic herpes simplex virus vector, and optionally a pharmaceutically acceptable adjuvant.

In this application, the term "physiological saline" can encompass any pharmaceutically acceptable concentration range. In certain embodiments, the compositions described herein may include a physiological saline component.

In this application, a "solid tumor" generally refers to an abnormal tissue growth or mass that typically does not contain cysts or fluid areas. Solid tumors can be benign (noncancerous) or malignant (cancerous). About 90% of clinical cancer cases are currently solid tumors. Different types of solid tumors are named after the cell type that forms them. For example, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocytoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, Salivary gland cancer, prostate cancer, vaginal cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, bile duct cancer, and head and neck cancer, etc.

In this application, "pancreatic tumor" generally includes pancreatic sarcoma, pancreatic cystadenoma, pancreatic cystadenocarcinoma. Pancreatic tumor is one of the most common malignant tumors in the digestive tract, and it is the most common malignant tumor, which mostly occurs in the head of the pancreas. Currently, surgical resection is usually the first choice for the treatment of pancreatic tumors.

Detailed description of the invention

nucleic acid molecule

For example, the isolated nucleic acid molecule may comprise one or more genes encoding each Kras mutant, respectively. For example, the isolated nucleic acid molecule may comprise non-repetitive one or more genes encoding each Kras mutant, respectively. For example, the isolated nucleic acid molecule may comprise duplicates of one or more genes encoding each Kras mutant, respectively. When a plurality of genes encoding each of the Kras mutants are included, the Kras mutant genes are independently selected from: Kras G12D mutant, Kras G13D mutant, Kras G12V mutant, Kras G13C mutant, Kras G12C mutant , Kras G13A mutant, Kras G12A mutant, Kras Q61L mutant, Kras G12R mutant, Kras Q61H mutant, Kras G12S mutant, Kras Q61R mutant, Kras A59T mutant, Kras A146T mutant, Kras Y64H mutant and the Kras A18D mutant.

In the present application, the isolated nucleic acid molecule may comprise a gene encoding a Kras mutant selected from the group consisting of: Kras A59T mutant, Kras G12D mutant, Kras G12V mutant, KrasA146T mutant, Kras G13D mutant and KrasG12C mutant body.

In the present application, the isolated nucleic acid molecule may comprise a gene encoding a Kras mutant selected from the group consisting of: Kras A59T mutant, Kras G12D mutant, Kras G12V mutant, KrasA146T mutant, Kras G13D mutant, Kras Y64H Mutants and Kras G12C mutants.

In the present application, the 3' end of the gene encoding the Kras G12D mutant may be directly or indirectly linked to the 5' end of the gene encoding the Kras A146T mutant.

In the present application, the 3' end of the gene encoding the Kras A46T mutant may be directly or indirectly linked to the 5' end of the gene encoding the Kras G12V mutant.

In the present application, the 3' end of the gene encoding the Kras G12V mutant may be directly or indirectly linked to the 5' end of the gene encoding the Kras A59T mutant.

In the present application, the 3' end of the gene encoding the Kras A59T mutant may be directly or indirectly linked to the 5' end of the gene encoding the Kras G13D mutant.

In the present application, the 3' end of the gene encoding the Kras G13D mutant may be directly or indirectly linked to the 5' end of the gene encoding the Kras Y64H mutant.

In the present application, the 3' end of the gene encoding the Kras Y64H mutant may be directly or indirectly linked to the 5' end of the gene encoding the Kras G12C mutant.

In the present application, each of the genes encoding the Kras mutants may be arranged in tandem in the isolated nucleic acid molecules.

In the present application, each of the genes encoding Kras mutants may be Minigene.

In the present application, tandem mini-genes can be obtained by arranging the genes encoding Kras mutants in tandem.

In the present application, each of the Kras mutants may comprise at least 20 amino acids.

In the present application, each of the Kras mutants may comprise at least 9 amino acids immediately N-terminal to the mutation site and at least 10 amino acids C-terminal to the mutation site.

In certain embodiments, the order of the Kras mutants from the 5' end to the 3' end is the gene encoding the Kras G12D mutant, the gene encoding the Kras A59T mutant, the gene encoding the Kras G12V mutant, Gene encoding Kras A146T mutant, gene encoding Kras G13D mutant, gene encoding Kras Y64H mutant, gene encoding Kras G12C mutant, gene encoding Kras Q61H mutant, gene encoding Kras A18D mutant, gene encoding Kras Gene of G12A mutant, gene encoding Kras A146T mutant, and gene encoding Kras G12S mutant. For example, the isolated nucleic acid molecule can comprise the amino acid sequence set forth in SEQ ID NO:65.

In certain embodiments, the order of the Kras mutants from the 5' end to the 3' end is the gene encoding the Kras G12D mutant, the gene encoding the Kras A146T mutant, the gene encoding the Kras G12V mutant, The gene encoding the Kras A59T mutant, the gene encoding the Kras G13D mutant, the gene encoding the Kras Y64H mutant, and the gene encoding the Kras G12C mutant. For example, the isolated nucleic acid molecule can comprise the amino acid sequence set forth in SEQ ID NO:66.

In certain embodiments, the order of the Kras mutants from the 5' end to the 3' end is the gene encoding the Kras G12D mutant, the gene encoding the Kras A59T mutant, the gene encoding the Kras A18D mutant, Gene encoding Kras G12V mutant, gene encoding Kras A146T mutant, gene encoding Kras Q61H mutant, gene encoding Kras G13D mutant, gene encoding Kras A59T mutant, gene encoding Kras A146T mutant and gene encoding Kras Gene of the G12C mutant. For example, the isolated nucleic acid molecule can comprise the amino acid sequence set forth in SEQ ID NO:67.

The nucleotide sequence encoding the wild-type Kras gene may comprise the nucleotide sequence shown in SEQ ID NO:39, and the amino acid sequence encoded by the wild-type Kras gene may comprise the amino acid sequence shown in SEQ ID NO:38.

The Kras G12D mutant refers to a segment comprising the amino acid G at position 12 cut from the amino acid sequence of the wild-type Kras polypeptide, and the amino acid G at position 12 in the cut-out sequence is replaced by D, the The sequence comprises at least 20 amino acids, such as 21, such as 22, such as 23, such as 24, such as 25, such as 26. The Kras G12D mutant may comprise at least 9 (e.g., at least 10, at least 11) amino acids immediately adjacent to the N-terminus of the mutation site G12D and at least 10 (e.g., at least 11, at least 11) amino acids immediately adjacent to the C-terminus of the mutation site G12D. at least 12, at least 13, at least 14) amino acids. The amino acid sequence adjacent to the N-terminus of the mutation site G12D is the X extending from the 11th (the sequence from the N-terminus to the C-terminus) amino acid to the N-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38). amino acids, X can be 9, 10 or 11. It may comprise the amino acid sequence shown in SEQ ID NO:2. The amino acid sequence of the C-terminus adjacent to the mutation site G12D is the Y extending from the 13th (the sequence from the N-terminus to the C-terminus) amino acid to the C-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38). amino acids, Y can be 10, 11, 12, 13 or 14. It may comprise the amino acid sequence shown in SEQ ID NO:3.

For example, the amino acid sequence of the Kras G12D mutant can sequentially comprise the amino acid sequence shown in SEQ ID NO:2, the Kras G12D site, and the amino acid sequence shown in SEQ ID NO:3 from the N-terminus to the C-terminus.

For example, the Kras G12D mutant may comprise the amino acid sequence set forth in SEQ ID NO:1.

The gene encoding the Kras G12D mutant may comprise a nucleotide sequence encoding an amino acid sequence immediately adjacent to the N-terminal of the mutation site G12D, a nucleotide sequence encoding the mutation site G12D, a nucleotide sequence encoding the immediately adjacent mutation site The nucleotide sequence of the amino acid sequence of the C-terminus of G12D.

For example, the gene encoding the Kras G12D mutant may sequentially comprise a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 20, a nucleotide sequence encoding the mutation site G12D, and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 20. : the nucleotide sequence of the amino acid sequence shown in 21.

For example, the gene encoding the Kras G12D mutant may comprise the nucleotide sequence shown in SEQ ID NO:19.

The Kras G13D mutant refers to a sequence comprising the thirteenth amino acid G cut from the amino acid sequence of the wild-type Kras polypeptide, and the amino acid G at the thirteenth position in the cut-out sequence is replaced by D, the said The sequence comprises at least 21 amino acids, such as 22, such as 23, such as 24, such as 25, such as 26, such as 27. The Kras G13D mutant may comprise at least 10 (e.g., at least 11, at least 12) amino acids immediately adjacent to the N-terminus of the mutation site G13D and at least 10 (e.g., at least 11, at least 12) amino acids immediately adjacent to the C-terminus of the mutation site G13D. at least 12, at least 13, at least 14) amino acids. The amino acid sequence adjacent to the N-terminus of the mutation site G13D is the X extending from the 12th (the sequence from the N-terminus to the C-terminus) amino acid to the N-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38). amino acids, X can be 10, 11 or 12. It may comprise the amino acid sequence shown in SEQ ID NO:5. The amino acid sequence adjacent to the C-terminus of the mutation site G13D is the Y extending from the 14th (the sequence from the N-terminus to the C-terminus) amino acid to the C-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38). amino acids, Y can be 10, 11, 12, 13 or 14. It may comprise the amino acid sequence shown in SEQ ID NO:6.

For example, the amino acid sequence of the Kras G13D mutant may comprise the amino acid sequence shown in SEQ ID NO: 5, the Kras G13D site, and the amino acid sequence shown in SEQ ID NO: 6 in order from the N-terminus to the C-terminus.

For example, the Kras G13D mutant may comprise the amino acid sequence set forth in SEQ ID NO:4.

The gene encoding the Kras G13D mutant may comprise a nucleotide sequence encoding the amino acid sequence immediately adjacent to the N-terminal of the mutation site G13D, a nucleotide sequence encoding the mutation site G13D, a nucleotide sequence encoding the immediately adjacent mutation site The nucleotide sequence of the amino acid sequence of the C-terminus of G13D.

For example, the gene encoding the Kras G13D mutant may sequentially comprise a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 23, a nucleotide sequence encoding the mutation site G13D, and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 23. : the nucleotide sequence of the amino acid sequence shown in 24.

For example, the gene encoding the Kras G13D mutant may comprise the nucleotide sequence shown in SEQ ID NO:22.

The Kras G12V mutant refers to a segment comprising the amino acid G at position 12 cut from the amino acid sequence of the wild-type Kras polypeptide, and the amino acid G at position 12 in the cut-out sequence is replaced by V, and the The sequence comprises at least 20 amino acids, such as 21, such as 22, such as 23, such as 24, such as 25, such as 26. The Kras G12V mutant may comprise at least 9 (e.g. at least 10, at least 11) amino acids next to the N-terminus of the mutation site G12V and at least 10 (e.g. at least 11, at least 11) amino acids immediately adjacent to the C-terminus of the mutation site G12V. at least 12, at least 13, at least 14) amino acids. The amino acid sequence adjacent to the N-terminus of the mutation site G12V is the X extending from the 11th (the sequence from the N-terminus to the C-terminus) amino acid to the N-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38). amino acids, X can be 9, 10 or 11. It may comprise the amino acid sequence shown in SEQ ID NO:2. The amino acid sequence of the C-terminus adjacent to the mutation site G12V is the Y extending from the 13th (the sequence from the N-terminus to the C-terminus) amino acid to the C-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38). amino acids, Y can be 10, 11, 12, 13 or 14. It may comprise the amino acid sequence shown in SEQ ID NO:3.

For example, the amino acid sequence of the Kras G12V mutant may comprise the amino acid sequence shown in SEQ ID NO: 2, the Kras G12V site, and the amino acid sequence shown in SEQ ID NO: 3 in order from the N-terminus to the C-terminus.

For example, the Kras G12V mutant may comprise the amino acid sequence set forth in SEQ ID NO:7.

The gene encoding the Kras G12V mutant may comprise a nucleotide sequence encoding the amino acid sequence of the N-terminus of the mutation site G12V, a nucleotide sequence encoding the mutation site G12V, and a nucleotide sequence encoding the immediately adjacent mutation site. The nucleotide sequence of the amino acid sequence of the C-terminus of G12V.

For example, the gene encoding the Kras G12V mutant may sequentially comprise a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 20, a nucleotide sequence encoding the mutation site G12V, and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 20. : the nucleotide sequence of the amino acid sequence shown in 21.

For example, the gene encoding the Kras G12V mutant may comprise the nucleotide sequence shown in SEQ ID NO:25.

The Kras G13C mutant refers to a segment comprising the amino acid G at position 13 cut from the amino acid sequence of the wild-type Kras polypeptide, and the amino acid G at position 13 in the cut-out sequence is replaced by C, the The sequence comprises at least 21 amino acids, such as 22, such as 23, such as 24, such as 25, such as 26, such as 27. The Kras G13C mutant may comprise at least 10 (e.g., at least 11, at least 12) amino acids immediately adjacent to the N-terminus of the mutation site G13C and at least 10 (e.g., at least 11, at least 12) amino acids immediately adjacent to the C-terminus of the mutation site G13C. at least 12, at least 13, at least 14) amino acids. The amino acid sequence adjacent to the N-terminus of the mutation site G13C is the X extending from the 12th position (the sequence from the N-terminus to the C-terminus) amino acid to the N-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38). amino acids, X can be 10, 11 or 12. It may comprise the amino acid sequence shown in SEQ ID NO:5. The amino acid sequence of the C-terminus adjacent to the mutation site G13C is the Y extending from the 14th (the sequence from the N-terminus to the C-terminus) amino acid to the C-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38). amino acids, Y can be 10, 11, 12, 13 or 14. It may comprise the amino acid sequence shown in SEQ ID NO:6.

For example, the amino acid sequence of the Kras G13C mutant may comprise the amino acid sequence shown in SEQ ID NO: 5, the Kras G13C site, and the amino acid sequence shown in SEQ ID NO: 6 in order from the N-terminus to the C-terminus.

For example, the Kras G13C mutant may comprise the amino acid sequence shown in SEQ ID NO:8.

The gene encoding the Kras G13C mutant may comprise a nucleotide sequence encoding the amino acid sequence of the N-terminal of the mutation site G13C, a nucleotide sequence encoding the mutation site G13C, and a nucleotide sequence encoding the immediately adjacent mutation site. The nucleotide sequence of the amino acid sequence of the C-terminus of G13C.

For example, the gene encoding the Kras G13C mutant may sequentially comprise a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 23, a nucleotide sequence encoding the mutation site G13C, and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 23. : the nucleotide sequence of the amino acid sequence shown in 24.

For example, the gene encoding the Kras G13C mutant may comprise the nucleotide sequence shown in SEQ ID NO:26.

The Kras G12C mutant refers to a sequence comprising the twelfth amino acid G cut out from the amino acid sequence of the wild-type Kras polypeptide, and the amino acid G at the twelfth position in the cut-out sequence is replaced by C, the The sequence comprises at least 20 amino acids, such as 21, such as 22, such as 23, such as 24, such as 25, such as 26. The Kras G12C mutant may comprise at least 9 (eg, at least 10, at least 11) amino acids immediately adjacent to the N-terminus of the mutation site G12C and at least 10 (eg, at least 11, at least 11) amino acids immediately adjacent to the C-terminus of the mutation site G12C. at least 12, at least 13, at least 14) amino acids. The amino acid sequence adjacent to the N-terminus of the mutation site G12C is the X extending from the 11th (the sequence from the N-terminus to the C-terminus) amino acid to the N-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38). amino acids, X can be 9, 10 or 11. It may comprise the amino acid sequence shown in SEQ ID NO:2. The amino acid sequence of the C-terminus adjacent to the mutation site G12C is the Y extending from the 13th (the sequence from the N-terminus to the C-terminus) amino acid to the C-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38). amino acids, Y can be 10, 11, 12, 13 or 14. It may comprise the amino acid sequence shown in SEQ ID NO:3.

For example, the amino acid sequence of the Kras G12C mutant can sequentially comprise the amino acid sequence shown in SEQ ID NO:2, the Kras G12C site, and the amino acid sequence shown in SEQ ID NO:3 from the N-terminus to the C-terminus.

For example, the Kras G12C mutant may comprise the amino acid sequence set forth in SEQ ID NO:9.

The gene encoding the Kras G12C mutant may comprise a nucleotide sequence encoding the amino acid sequence of the N-terminus of the mutation site G12C, a nucleotide sequence encoding the mutation site G12C, and a nucleotide sequence encoding the immediately adjacent mutation site. The nucleotide sequence of the amino acid sequence of the C-terminus of G12C.

For example, the gene encoding the Kras G12C mutant may sequentially comprise a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 20, a nucleotide sequence encoding the mutation site G12C, and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 20. : the nucleotide sequence of the amino acid sequence shown in 21.

For example, the gene encoding the Kras G12C mutant may comprise the nucleotide sequence shown in SEQ ID NO:27.

The Kras G13A mutant refers to a sequence comprising the thirteenth amino acid G cut from the amino acid sequence of the wild-type Kras polypeptide, and the amino acid G at the thirteenth position in the cut-out sequence is replaced by A, the The sequence comprises at least 21 amino acids, such as 22, such as 23, such as 24, such as 25, such as 26, such as 27. The Kras G13A mutant may comprise at least 10 (e.g., at least 11, at least 12) amino acids immediately adjacent to the N-terminus of the mutation site G13A and at least 10 (e.g., at least 11, at least 12) amino acids immediately adjacent to the C-terminus of the mutation site G13A. at least 12, at least 13, at least 14) amino acids. The amino acid sequence adjacent to the N-terminus of the mutation site G13A is the X extending from the 12th (the sequence from the N-terminus to the C-terminus) amino acid to the N-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38). amino acids, X can be 10, 11 or 12. It may comprise the amino acid sequence shown in SEQ ID NO:5. The amino acid sequence of the C-terminus adjacent to the mutation site G13A is the Y extending from the 14th position (the sequence from the N-terminus to the C-terminus) amino acid to the C-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38). amino acids, Y can be 10, 11, 12, 13 or 14. It may comprise the amino acid sequence shown in SEQ ID NO:6.

For example, the amino acid sequence of the Kras G13A mutant may comprise the amino acid sequence shown in SEQ ID NO: 5, the Kras G13A site, and the amino acid sequence shown in SEQ ID NO: 6 in order from the N-terminus to the C-terminus.

For example, the Kras G13A mutant may comprise the amino acid sequence set forth in SEQ ID NO:10.

The gene encoding the Kras G13A mutant may comprise a nucleotide sequence encoding the amino acid sequence of the N-terminus of the mutation site G13A, a nucleotide sequence encoding the mutation site G13A, encoding the immediately adjacent mutation site The nucleotide sequence of the amino acid sequence of the C-terminus of G13A.

For example, the gene encoding the Kras G13A mutant may sequentially comprise a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 23, a nucleotide sequence encoding the mutation site G13A, and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 23. : the nucleotide sequence of the amino acid sequence shown in 24.

For example, the gene encoding the Kras G13A mutant may comprise the nucleotide sequence shown in SEQ ID NO:28.

The Kras G12A mutant refers to a sequence comprising the twelfth amino acid G cut out from the amino acid sequence of the wild-type Kras polypeptide, and the amino acid G at the twelfth position in the cut-out sequence is replaced by A, the The sequence comprises at least 20 amino acids, such as 21, such as 22, such as 23, such as 24, such as 25, such as 26. The Kras G12A mutant may comprise at least 9 (eg, at least 10, at least 11) amino acids immediately adjacent to the N-terminus of the mutation site G12A and at least 10 (eg, at least 11, at least 11) amino acids immediately adjacent to the C-terminus of the mutation site G12A. at least 12, at least 13, at least 14) amino acids. The amino acid sequence adjacent to the N-terminus of the mutation site G12A is the X extending from the 11th (the sequence from the N-terminus to the C-terminus) amino acid to the N-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38). amino acids, X can be 9, 10 or 11. It may comprise the amino acid sequence shown in SEQ ID NO:2. The amino acid sequence of the C-terminus adjacent to the mutation site G12A is the Y extending from the 13th position (the sequence from the N-terminus to the C-terminus) amino acid to the C-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38). amino acids, Y can be 10, 11, 12, 13 or 14. It may comprise the amino acid sequence shown in SEQ ID NO:3.

For example, the amino acid sequence of the Kras G12A mutant may comprise the amino acid sequence shown in SEQ ID NO: 2, the Kras G12A site, and the amino acid sequence shown in SEQ ID NO: 3 in order from the N-terminus to the C-terminus.

For example, the Kras G12A mutant may comprise the amino acid sequence set forth in SEQ ID NO:11.

The gene encoding the Kras G12A mutant may comprise a nucleotide sequence encoding the amino acid sequence of the N-terminus of the mutation site G12A, a nucleotide sequence encoding the mutation site G12A, encoding the immediately adjacent mutation site The nucleotide sequence of the amino acid sequence of the C-terminus of G12A.

For example, the gene encoding the Kras G12A mutant may sequentially comprise a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 20, a nucleotide sequence encoding the mutation site G12A, and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 20. : the nucleotide sequence of the amino acid sequence shown in 21.

For example, the gene encoding the Kras G12A mutant may comprise the nucleotide sequence shown in SEQ ID NO:29.

The Kras Q61L mutant refers to a segment comprising the amino acid Q at position 61 cut out from the amino acid sequence of the wild-type Kras polypeptide, and the amino acid Q at position 61 in the cut-out sequence is replaced by L, the The sequence comprises at least 21 amino acids, such as 22, such as 23, such as 24, such as 25, such as 26, such as 27, such as 28, such as 29. The Kras Q61L mutant may comprise at least 10 (e.g., at least 11, at least 12, at least 13, at least 14) amino acids immediately N-terminal to the mutation site Q61L and at least 10 amino acids immediately C-terminal to the mutation site Q61L. 10 (eg, at least 11, at least 12, at least 13, at least 14) amino acids. The amino acid sequence adjacent to the N-terminus of the mutation site Q61L is the X extending from the 60th (the sequence from the N-terminus to the C-terminus) amino acid to the N-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38). amino acids, X can be 10, 11, 12, 13 or 14. It may comprise the amino acid sequence shown in SEQ ID NO:13. The amino acid sequence of the C-terminus adjacent to the mutation site Q61L is the Y extending from the 62nd (the sequence from the N-terminus to the C-terminus) amino acid to the C-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38). amino acids, Y can be 10, 11, 12, 13 or 14. It may comprise the amino acid sequence shown in SEQ ID NO:14.

For example, the amino acid sequence of the Kras Q61L mutant may comprise the amino acid sequence shown in SEQ ID NO: 13, the Kras Q61L site, and the amino acid sequence shown in SEQ ID NO: 14 in order from the N-terminus to the C-terminus.

For example, the Kras Q61L mutant may comprise the amino acid sequence set forth in SEQ ID NO:12.

The gene encoding the Kras Q61L mutant may comprise a nucleotide sequence encoding the amino acid sequence of the N-terminus of the mutation site Q61L, a nucleotide sequence encoding the mutation site Q61L, encoding the immediately adjacent mutation site The nucleotide sequence of the amino acid sequence of the C-terminal of Q61L.

For example, the gene encoding the Kras Q61L mutant may in turn comprise a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 31, a nucleotide sequence encoding the mutation site Q61L, and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 31. : the nucleotide sequence of the amino acid sequence shown in 32.

For example, the gene encoding the Kras Q61L mutant may comprise the nucleotide sequence shown in SEQ ID NO:30.

The Kras G12R mutant refers to a segment comprising the amino acid G at position 12 cut from the amino acid sequence of the wild-type Kras polypeptide, and the amino acid G at position 12 in the cut-out sequence is replaced by R, the The sequence comprises at least 20 amino acids, such as 21, such as 22, such as 23, such as 24, such as 25, such as 26. The Kras G12R mutant may comprise at least 9 (eg, at least 10, at least 11) amino acids immediately adjacent to the N-terminus of the mutation site G12R and at least 10 (eg, at least 11, at least 11) amino acids immediately adjacent to the C-terminus of the mutation site G12R. at least 12, at least 13, at least 14) amino acids. The amino acid sequence adjacent to the N-terminus of the mutation site G12R is the X extending from the 11th (the sequence from the N-terminus to the C-terminus) amino acid to the N-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38). amino acids, X can be 9, 10 or 11. It may comprise the amino acid sequence shown in SEQ ID NO:2. The amino acid sequence of the C-terminus adjacent to the mutation site G12R is the Y extending from the 13th (the sequence from the N-terminus to the C-terminus) amino acid to the C-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38). amino acids, Y can be 10, 11, 12, 13 or 14. It may comprise the amino acid sequence shown in SEQ ID NO:3.

For example, the amino acid sequence of the Kras G12R mutant may comprise the amino acid sequence shown in SEQ ID NO: 2, the Kras G12R site, and the amino acid sequence shown in SEQ ID NO: 3 in order from the N-terminus to the C-terminus.

For example, the Kras G12R mutant may comprise the amino acid sequence set forth in SEQ ID NO:15.

The gene encoding the Kras G12R mutant may comprise a nucleotide sequence encoding the amino acid sequence of the N-terminal of the mutation site G12R, a nucleotide sequence encoding the mutation site G12R, and a nucleotide sequence encoding the immediately adjacent mutation site. The nucleotide sequence of the amino acid sequence of the C-terminus of G12R.

For example, the gene encoding the Kras G12R mutant may sequentially comprise a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 20, a nucleotide sequence encoding the mutation site G12R, and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 20. : the nucleotide sequence of the amino acid sequence shown in 21.

For example, the gene encoding the Kras G12R mutant may comprise the nucleotide sequence shown in SEQ ID NO:33. .

The Kras Q61H mutant refers to a sequence comprising the 61st amino acid Q cut from the amino acid sequence of the wild-type Kras polypeptide, and the 61st amino acid Q in the cut-out sequence is replaced by H, the The sequence comprises at least 21 amino acids, such as 22, such as 23, such as 24, such as 25, such as 26, such as 27, such as 28, such as 29. The Kras Q61H mutant may comprise at least 10 (e.g., at least 11, at least 12, at least 13, at least 14) amino acids immediately adjacent to the N-terminus of the mutation site Q61H and at least 10 amino acids immediately adjacent to the C-terminus of the mutation site Q61H. 10 (eg, at least 11, at least 12, at least 13, at least 14) amino acids. The amino acid sequence adjacent to the N-terminus of the mutation site Q61H is the X extending from the 60th position (the sequence from the N-terminus to the C-terminus) amino acid to the N-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38). amino acids, X can be 10, 11, 12, 13 or 14. It may comprise the amino acid sequence shown in SEQ ID NO:13. The amino acid sequence of the C-terminus adjacent to the mutation site Q61H is the Y extending from the 62nd (the sequence from the N-terminus to the C-terminus) amino acid to the C-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38). amino acids, Y can be 10, 11, 12, 13 or 14. It may comprise the amino acid sequence shown in SEQ ID NO:14.

For example, the amino acid sequence of the Kras Q61H mutant may comprise the amino acid sequence shown in SEQ ID NO: 13, the Kras Q61H site, and the amino acid sequence shown in SEQ ID NO: 14 in order from the N-terminus to the C-terminus.

For example, the Kras Q61H mutant may comprise the amino acid sequence set forth in SEQ ID NO:16.

The gene encoding the Kras Q61H mutant may comprise a nucleotide sequence encoding the amino acid sequence of the N-terminal of the mutation site Q61H, a nucleotide sequence encoding the mutation site Q61H, encoding the immediately adjacent mutation site The nucleotide sequence of the amino acid sequence of the C-terminal of Q61H.

For example, the gene encoding the Kras Q61H mutant may sequentially comprise a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 31, a nucleotide sequence encoding the mutation site Q61H, and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 31. : the nucleotide sequence of the amino acid sequence shown in 32.

For example, the gene encoding the Kras Q61H mutant may comprise the nucleotide sequence shown in SEQ ID NO:34.

The Kras G12S mutant refers to a segment comprising the amino acid G at position 12 cut from the amino acid sequence of the wild-type Kras polypeptide, and the amino acid G at position 12 in the cut-out sequence is replaced by S, the The sequence comprises at least 20 amino acids, such as 21, such as 22, such as 23, such as 24, such as 25, such as 26. The Kras G12S mutant may comprise at least 9 (eg, at least 10, at least 11) amino acids immediately adjacent to the N-terminus of the mutation site G12S and at least 10 (eg, at least 11, at least 11) amino acids immediately adjacent to the C-terminus of the mutation site G12S. at least 12, at least 13, at least 14) amino acids. The amino acid sequence adjacent to the N-terminus of the mutation site G12S is the X extending from the 11th (the sequence from the N-terminus to the C-terminus) amino acid to the N-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38). amino acids, X can be 9, 10 or 11. It may comprise the amino acid sequence shown in SEQ ID NO:2. The amino acid sequence of the C-terminus adjacent to the mutation site G12S is the Y extending from the 13th (the sequence from the N-terminus to the C-terminus) amino acid to the C-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38). amino acids, Y can be 10, 11, 12, 13 or 14. It may comprise the amino acid sequence shown in SEQ ID NO:3.

For example, the amino acid sequence of the Kras G12S mutant can sequentially comprise the amino acid sequence shown in SEQ ID NO: 2, the Kras G12S site, and the amino acid sequence shown in SEQ ID NO: 3 from the N-terminus to the C-terminus.

For example, the Kras G12S mutant may comprise the amino acid sequence set forth in SEQ ID NO:17.

The gene encoding the Kras G12S mutant may comprise a nucleotide sequence encoding the amino acid sequence of the N-terminus of the immediately adjacent mutation site G12S, a nucleotide sequence encoding the mutation site G12S, and a nucleotide sequence encoding the immediately adjacent mutation site. The nucleotide sequence of the amino acid sequence of the C-terminus of G12S.

For example, the gene encoding the Kras G12S mutant may sequentially comprise a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 20, a nucleotide sequence encoding the mutation site G12S, and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 20. : the nucleotide sequence of the amino acid sequence shown in 21.

For example, the gene encoding the Kras G12S mutant may comprise the nucleotide sequence shown in SEQ ID NO:35.

The Kras Q61R mutant refers to a sequence comprising the 61st amino acid Q cut out from the amino acid sequence of the wild-type Kras polypeptide, and the 61st amino acid Q in the cut-out sequence is replaced by R, the The sequence comprises at least 21 amino acids, such as 22, such as 23, such as 24, such as 25, such as 26, such as 27, such as 28, such as 29. The Kras Q61R mutant may comprise at least 10 (e.g., at least 11, at least 12, at least 13, at least 14) amino acids immediately N-terminal to the mutation site Q61R and at least 10 amino acids immediately C-terminal to the mutation site Q61R. 10 (eg, at least 11, at least 12, at least 13, at least 14) amino acids. The amino acid sequence adjacent to the N-terminus of the mutation site Q61R is the X extending from the 60th (the sequence from the N-terminus to the C-terminus) amino acid to the N-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38). amino acids, X can be 10, 11, 12, 13 or 14. It may comprise the amino acid sequence shown in SEQ ID NO:13. The amino acid sequence of the C-terminus adjacent to the mutation site Q61R is the Y extending from the 62nd (the sequence from the N-terminus to the C-terminus) amino acid to the C-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38). amino acids, Y can be 10, 11, 12, 13 or 14. It may comprise the amino acid sequence shown in SEQ ID NO:14.

For example, the amino acid sequence of the Kras Q61R mutant may comprise the amino acid sequence shown in SEQ ID NO: 13, the Kras Q61R site, and the amino acid sequence shown in SEQ ID NO: 14 in order from the N-terminus to the C-terminus.

For example, the Kras Q61R mutant may comprise the amino acid sequence set forth in SEQ ID NO:18.

The gene encoding the Kras Q61R mutant may comprise a nucleotide sequence encoding the amino acid sequence of the N-terminus of the mutation site Q61R, a nucleotide sequence encoding the mutation site Q61R, encoding the immediately adjacent mutation site The nucleotide sequence of the amino acid sequence of the C-terminal of Q61R.

For example, the gene encoding the Kras Q61R mutant may sequentially comprise a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 31, a nucleotide sequence encoding the mutation site Q61R, and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 31. : the nucleotide sequence of the amino acid sequence shown in 32.

For example, the gene encoding the Kras Q61R mutant may comprise the nucleotide sequence shown in SEQ ID NO:36.

In the nucleic acid molecules described in the present application, each of the genes encoding the Kras mutants is a Minigene. The minigene usually refers to a short segment in a gene, which can be used for the study of the function and expression regulation mechanism of the gene and for the construction of a more complex minigene containing multiple exons and introns. For example, in the present application, the minigene can be a fragment comprising the gene encoding each Kras mutant, and the encoding genes of each Kras mutant are arranged in series according to the sequence shown in FIG. 1 to form a Kras gene mutant. Tandem minigene (TMG).

The tandem mini-gene of the Kras gene mutant comprises, from the 5' end to the 3' end, the nucleotide sequence encoding the Kras G12D mutant, the nucleotide sequence encoding the Kras G12V mutant, the nucleotide sequence encoding the Kras G12V mutant, and the Kras G12V mutant. The nucleotide sequence of the G12V mutant, the nucleotide sequence encoding the Kras G13C mutant, the nucleotide sequence encoding the Kras G12C mutant, the nucleotide sequence encoding the Kras G13A mutant, the nucleotide sequence encoding the Kras G13A mutant, the Nucleotide sequence of the Kras G12A mutant, nucleotide sequence encoding the Kras Q61L mutant, nucleotide sequence encoding the Kras G12R mutant, nucleotide encoding the Kras Q61H mutant sequence, the nucleotide sequence encoding the Kras G12S mutant, the nucleotide sequence encoding the Kras Q61R mutant.

For example, the tandem mini-gene of the Kras gene mutant may comprise the amino acid sequence encoding SEQ ID NO:43.

For example, the tandem mini-gene of the Kras gene mutant comprises the nucleotide sequence shown in SEQ ID NO: 19, the nucleotide sequence shown in SEQ ID NO: 22, the nucleotide sequence shown in SEQ ID NO: 22 from the 5' end to the 3' end in order The nucleotide sequence shown in NO:25, the nucleotide sequence shown in SEQ ID NO:26, the nucleotide sequence shown in SEQ ID NO:27, the nucleotide sequence shown in SEQ ID NO:28, Nucleotide sequence shown in SEQ ID NO:29, nucleotide sequence shown in SEQ ID NO:30, nucleotide sequence shown in SEQ ID NO:33, nucleotide sequence shown in SEQ ID NO:34 sequence, the nucleotide sequence shown in SEQ ID NO:35, the nucleotide sequence shown in SEQ ID NO:36.

The Kras A59T mutant refers to a sequence comprising amino acid A at position 59 cut from the amino acid sequence of a wild-type Kras polypeptide, and the amino acid A at position 59 in the cut-out sequence is replaced by T, and the The sequence comprises at least 20 amino acids, such as 21, such as 22, such as 23, such as 24, such as 25, such as 26. The Kras A59T mutant may comprise at least 9 (e.g., at least 10, at least 11) amino acids immediately adjacent to the N-terminus of the mutation site A59T and at least 10 (e.g., at least 11, at least 11) amino acids immediately adjacent to the C-terminus of the mutation site A59T. at least 12, at least 13, at least 14) amino acids.

For example, the Kras A59T mutant may comprise the amino acid sequence set forth in SEQ ID NO:54.

For example, the gene encoding the Kras A59T mutant may comprise the nucleotide sequence shown in SEQ ID NO:55.

The Kras A146T mutant refers to a sequence comprising the amino acid A at position 146 cut from the amino acid sequence of the wild-type Kras polypeptide, and the amino acid A at position 146 in the cut-out sequence is replaced by T, the The sequence comprises at least 20 amino acids, such as 21, such as 22, such as 23, such as 24, such as 25, such as 26. The Kras A146T mutant may comprise at least 9 (e.g., at least 10, at least 11) amino acids immediately adjacent to the N-terminus of the mutation site A146T and at least 10 (e.g., at least 11, at least 11) amino acids immediately adjacent to the C-terminus of the mutation site A146T. at least 12, at least 13, at least 14) amino acids.

For example, the Kras A146T mutant may comprise the amino acid sequence set forth in SEQ ID NO:56.

For example, the gene encoding the Kras A146T mutant may comprise the nucleotide sequence shown in SEQ ID NO:57.

Said Kras Y64H mutant refers to a segment comprising the amino acid Y at position 64 cut out from the amino acid sequence of the wild-type Kras polypeptide, and the amino acid Y at position 64 in the cut-out sequence is replaced by H, the said The sequence comprises at least 20 amino acids, such as 21, such as 22, such as 23, such as 24, such as 25, such as 26. The Kras Y64H mutant may comprise at least 9 (e.g. at least 10, at least 11) amino acids next to the N-terminus of the mutation site Y64H and at least 10 (e.g. at least 11, at least 11) amino acids immediately adjacent to the C-terminus of the mutation site Y64H. at least 12, at least 13, at least 14) amino acids.

For example, the Kras Y64H mutant may comprise the amino acid sequence set forth in SEQ ID NO:58.

For example, the gene encoding the Kras Y64H mutant may comprise the nucleotide sequence shown in SEQ ID NO:59.

The Kras A18D mutant refers to a sequence comprising amino acid A at position 18 cut from the amino acid sequence of a wild-type Kras polypeptide, and the amino acid A at position 18 in the cut-out sequence is replaced by D, the The sequence comprises at least 20 amino acids, such as 21, such as 22, such as 23, such as 24, such as 25, such as 26. The Kras A18D mutant may comprise at least 9 (e.g., at least 10, at least 11) amino acids immediately adjacent to the N-terminus of the mutation site A18D and at least 10 (e.g., at least 11, at least 11) amino acids immediately adjacent to the C-terminus of the mutation site A18D. at least 12, at least 13, at least 14) amino acids.

For example, the Kras A18D mutant may comprise the amino acid sequence set forth in SEQ ID NO:60.

For example, the gene encoding the Kras A18D mutant may comprise the nucleotide sequence shown in SEQ ID NO:61.

For example, the tandem minigene of the Kras gene mutant may comprise the nucleotide sequence shown in SEQ ID NO:44.

In the present application, the nucleic acid molecule may also comprise mutants of other tumor antigens. For example, the nucleic acid molecule may also comprise mutants of tumor-associated driver genes. For example, the other tumor antigen can be TP53. For example, the other tumor antigen can be the Braf gene.

The nucleic acid molecule may also comprise a polynucleotide encoding a secreted peptide located at the 5' end of the tandem minigene of the Kras gene mutant. The secretory peptide can guide the secretion of Kras polypeptide outside the tumor cells, and after being taken up by antigen-presenting cells (APC), it can be effectively presented to T cells, thereby exerting an immune effect. The APC cells refer to a class of adjuvant cells that can present antigenic substances to T cells in the process of immune response, and the major histocompatibility complex (MHC) on the cell surface can interact with antigens, That is, the Kras mutant polypeptide is combined, and the combined complex of the two can be recognized by T cells. For example, the secreted peptide may include Hmm38 secreted peptide, CD14 protein secreted peptide.

For example, the polynucleotide encoding the CD14 protein secretory peptide may comprise the nucleotide sequence shown in SEQ ID NO:37. For example, the isolated nucleic acid molecule comprising a polynucleotide encoding a CD14 protein secreted peptide can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 62-64. For example, the isolated nucleic acid molecule can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 62-64.

The nucleic acid molecule may also comprise a polynucleotide encoding a tag protein located at the 3' end of the gene encoding the Kras mutant. The tag protein is used for detection of Kras mutant polypeptide.

For example, the tagged proteins may include FLAG-tagged proteins, HA-tagged proteins, C-Myc-tagged proteins, 6x His-tagged proteins.

For example, the polynucleotide encoding the 6x His-tagged protein may comprise the nucleotide sequence shown in SEQ ID NO:40.

carrier

In the present application, the vector may be a viral vector.

In the present application, the nucleic acid molecules described herein can be inserted into viral vectors. For example, the nucleotide sequence set forth in any one of SEQ ID NOs: 65-67 can be inserted into a viral vector. In certain embodiments, the viral vector may comprise the nucleotide sequence shown in GenBank No: GU734771.1 of the NCBI database.

In certain embodiments, genes described herein that do not contain a 6x His-tagged protein can be inserted into a viral vector. The vector may be an oncolytic herpes simplex virus (oHSV) vector. For example, the oncolytic herpes simplex virus (oHSV) vector can be a herpes simplex virus type I (HSV-1) vector. The HSV-1 vector is a gene deletion type, and the deleted gene may be neurotoxic factor γ34.5.

In the present application, the vector may be an oncolytic herpes simplex virus (oHSV) vector. For example, the oncolytic herpes simplex virus (oHSV) vector can be a herpes simplex virus type I (HSV-1) vector. The HSV-1 vector is a gene deletion type, and the deleted gene may be neurotoxic factor γ34.5.

For example, the HSV-1 vector lacks two copies of the neurotoxic factor γ34.5.

The vector also includes a promoter. For example, the promoter may include an elongation factor 1α short (EFS) promoter, an elongation factor 1α (EF-1α) promoter, a CMV promoter, a SV40 early or late promoter, an OPEFS promoter, or derivatives thereof.

For example, the promoter may comprise a CMV promoter. The promoter is located 5' upstream of the transcription initiation site of the gene encoding the Kras mutant and controls its transcription.

For example, the promoter sequence can include the sequence shown in SEQ ID NO:41.

In the present application, the tandem mini-gene of the Kras gene mutant may be located between the UL3 gene and the UL4 gene of the HSV-1 vector. In the present application, the viral vector may comprise the nucleotide sequence shown in GenBank No: GU734771.1 of the NCBI database.

In another aspect, the present application also provides a pharmaceutical composition comprising the nucleic acid molecule, and/or the carrier, and optionally a pharmaceutically acceptable adjuvant. The pharmaceutical composition refers to a formulation in a form that allows the biological activity of the active ingredient contained therein to be effective and free of additional ingredients that would be unacceptably toxic to a subject to whom the composition would be administered. The pharmaceutically acceptable adjuvant refers to any substance capable of assisting or improving the action of a drug. The adjuvant may be a particulate adjuvant, eg, an aluminum hydroxide adjuvant. The adjuvant may be a non-particulate adjuvant, eg, a cytokine. The adjuvant may be derived from plants such as saponins and polysaccharide extracts. The adjuvant may be derived from pathogenic microorganisms, eg, monophospholipids, cholera toxin, and the like.

combination

In another aspect, the present application also provides a composition, which may comprise the isolated nucleic acid molecule described in the present application, the carrier described in the present application or the pharmaceutical composition described in the present application, and physiological saline.

In the present application, the composition may comprise any one or more of the isolated nucleic acid molecules described herein, and physiological saline.

In the present application, the isolated nucleic acid molecule may comprise one or more genes each independently encoding a Kras mutant selected from the group consisting of: Kras G12D mutant, Kras G13D mutant, Kras G12V mutant, Kras G13C mutant Kras G12C mutant, Kras G13A mutant, Kras G12A mutant, Kras Q61L mutant, Kras G12R mutant, Kras Q61H mutant, Kras G12S mutant, Kras Q61R mutant, Kras A59T mutant, Kras A146T mutant mutant, Kras Y64H mutant and Kras A18D mutant.

In certain embodiments, the isolated nucleic acid molecule may comprise a gene encoding a Kras mutant selected from the group consisting of: Kras A59T mutant, Kras G12D mutant, Kras G12V mutant, KrasA146T mutant, Kras G13D mutant, and KrasG12C mutant.

In the present application, the isolated nucleic acid molecule in the composition comprises a gene encoding a Kras mutant selected from the group consisting of: Kras A59T mutant, Kras G12D mutant, Kras G12V mutant, KrasA146T mutant, Kras G13D Mutant, Kras Y64H mutant and Kras G12C mutant.

In the present application, in the isolated nucleic acid molecule in the composition, the 3' end of the gene encoding the Kras G12D mutant can be directly or indirectly with the 5' end of the gene encoding the Kras A146T mutant connected. In the present application, in the isolated nucleic acid molecule in the composition, the 3' end of the gene encoding the Kras A46T mutant can be directly or indirectly with the 5' end of the gene encoding the Kras G12V mutant connected. In the present application, in the isolated nucleic acid molecule in the composition, the 3' end of the gene encoding the Kras G12V mutant can be directly or indirectly with the 5' end of the gene encoding the Kras A59T mutant connected. In the present application, in the isolated nucleic acid molecule in the composition, the 3' end of the gene encoding the Kras A59T mutant can be directly or indirectly with the 5' end of the gene encoding the Kras G13D mutant connected. In the present application, in the isolated nucleic acid molecule in the composition, the 3' end of the gene encoding the Kras G13D mutant can be directly or indirectly with the 5' end of the gene encoding the Kras Y64H mutant connected. In the present application, in the isolated nucleic acid molecule in the composition, the 3' end of the gene encoding the Kras Y64H mutant can be directly or indirectly with the 5' end of the gene encoding the Kras G12C mutant connected. In the present application, each of the genes encoding the Kras mutants may be arranged in tandem in the isolated nucleic acid molecules. In the present application, each of the genes encoding Kras mutants may be Minigene. In the present application, tandem mini-genes can be obtained by arranging the genes encoding Kras mutants in tandem.

In the present application, in the isolated nucleic acid molecules in the composition, each of the Kras mutants may comprise at least 20 amino acids.

In the present application, in the isolated nucleic acid molecule in the composition, each of the Kras mutants may comprise at least 9 amino acids N-terminal to the mutation site immediately adjacent to the site and the mutation site At least 10 amino acids immediately C-terminal to this site.

In the present application, the Kras G12D mutant may comprise the amino acid sequence shown in SEQ ID NO:1. In the present application, the Kras G13D mutant may comprise the amino acid sequence shown in SEQ ID NO:4. In the present application, the Kras G12V mutant may comprise the amino acid sequence shown in SEQ ID NO:7. In the present application, the Kras G13C mutant may comprise the amino acid sequence shown in SEQ ID NO:8. In the present application, the Kras G12C mutant may comprise the amino acid sequence shown in SEQ ID NO:9. In the present application, the Kras G13A mutant may comprise the amino acid sequence shown in SEQ ID NO:10. In the present application, the Kras G12A mutant may comprise the amino acid sequence shown in SEQ ID NO: 11. In the present application, the Kras G12A mutant may comprise the amino acid sequence shown in SEQ ID NO: 11. In the present application, the Kras Q61L mutant may comprise the amino acid sequence shown in SEQ ID NO:12. In the present application, the Kras G12R mutant may comprise the amino acid sequence shown in SEQ ID NO:15. In the present application, the Kras Q61H mutant may comprise the amino acid sequence shown in SEQ ID NO:16. In the present application, the Kras G12S mutant may comprise the amino acid sequence shown in SEQ ID NO:17. In the present application, the Kras Q61R mutant may comprise the amino acid sequence shown in SEQ ID NO:18. In the present application, the Kras A59T mutant may comprise the amino acid sequence shown in SEQ ID NO:54. In the present application, the Kras A146T mutant may comprise the amino acid sequence shown in SEQ ID NO:56. In the present application, the Kras Y64H mutant may comprise the amino acid sequence shown in SEQ ID NO:58. In the present application, the Kras A18D mutant may comprise the amino acid sequence shown in SEQ ID NO:60.

In the present application, in the isolated nucleic acid molecule in the composition, the tandem mini-gene may sequentially comprise a gene encoding a Kras G12D mutant, a gene encoding a Kras A59T mutant from the 5' end to the 3' end. Gene, gene encoding Kras G12V mutant, gene encoding Kras A146T mutant, gene encoding Kras G13D mutant, gene encoding Kras Y64H mutant, gene encoding Kras G12C mutant, gene encoding Kras Q61H mutant, The gene encoding the Kras A18D mutant, the gene encoding the Kras G12A mutant, the gene encoding the Kras A146T mutant, and the gene encoding the Kras G12S mutant. For example, the tandem minigene may comprise the nucleotide sequence set forth in SEQ ID NO:65.

In the present application, in the isolated nucleic acid molecule in the composition, the tandem mini-gene may sequentially comprise a gene encoding a Kras G12D mutant, a gene encoding a Kras A146T mutant from the 5' end to the 3' end. Genes, genes encoding Kras G12V mutants, genes encoding Kras A59T mutants, genes encoding Kras G13D mutants, genes encoding Kras Y64H mutants, and genes encoding Kras G12C mutants. For example, the tandem minigene may comprise the nucleotide sequence set forth in SEQ ID NO:66.

In the present application, in the isolated nucleic acid molecule in the composition, the tandem mini-gene may sequentially comprise a gene encoding a Kras G12D mutant, a gene encoding a Kras A59T mutant from the 5' end to the 3' end. Gene, gene encoding Kras A18D mutant, gene encoding Kras G12V mutant, gene encoding Kras A146T mutant, gene encoding Kras Q61H mutant, gene encoding Kras G13D mutant, gene encoding Kras A59T mutant, The gene encoding the Kras A146T mutant and the gene encoding the Kras G12C mutant. For example, the tandem minigene may comprise the nucleotide sequence set forth in SEQ ID NO:67.

In the present application, the composition may comprise the nucleotide sequence shown in any one of SEQ ID NOs: 62-67, and physiological saline. In certain embodiments, the composition may comprise the nucleotide sequence set forth in SEQ ID NO: 63, and physiological saline.

In this application, the composition may comprise any one or more of the carriers described in this application, and physiological saline.

In the present application, the composition may comprise physiological saline, and a viral vector comprising the nucleotide sequence shown in any one of SEQ ID NOs: 62-67. In certain embodiments, the composition may comprise physiological saline, and a viral vector comprising the nucleotide sequence set forth in any one of SEQ ID NO:63.

In the present application, the composition may comprise any one or more of the pharmaceutical compositions described in the present application, and physiological saline.

use

On the other hand, the present application also provides the nucleic acid molecule, the carrier, the pharmaceutical composition and/or the application of the composition in the preparation of a medicament for treating tumors. For example, the tumor can be a solid tumor. For example, the tumor can be non-small cell lung cancer. For example, the tumor can be colorectal cancer. For example, the tumor can be pancreatic cancer. For example, the tumor can be breast cancer.

The present application provides the nucleic acid molecule, the carrier, the pharmaceutical composition and/or the composition, which can treat tumors.

The present application provides a method of treating a tumor, which may include the steps of: administering to a subject an effective amount comprising the nucleic acid molecule, the carrier, the pharmaceutical composition, and/or the composition.

In this application, the effective amount generally refers to any drug amount that promotes disease regression when used alone or in combination with another therapeutic agent.

The isolated nucleic acid molecule, the carrier, the pharmaceutical composition and/or the composition described herein can be effectively used to inhibit the proliferation and/or growth of tumor cells. The Kras mutant TMG-2, Kras mutant TMG-5, and Kras mutant TMG-7 constructed in the present application are expressed in oncolytic virus vectors and can exert good therapeutic effects.

On the other hand, the present application provides the following embodiments:

1. An isolated nucleic acid molecule comprising genes encoding the following Kras mutants, respectively: Kras G12D mutant, Kras G13D mutant, Kras G12V mutant, Kras G13C mutant, Kras G12C mutant, Kras G13A mutant, Kras G12A Mutants, Kras Q61L mutants, Kras G12R mutants, Kras Q61H mutants, Kras G12S mutants and Kras Q61R mutants.

2. The isolated nucleic acid molecule of embodiment 1, wherein each of the genes encoding the Kras mutants are arranged in tandem in the isolated nucleic acid molecule.

3. The isolated nucleic acid molecule of any one of embodiments 1-2, wherein each of the genes encoding the Kras mutants is a minigene Minigene.

4. The isolated nucleic acid molecule of any one of embodiments 1-3, wherein each of the Kras mutants comprises at least 20 amino acids.

5. The isolated nucleic acid molecule of any one of embodiments 1-4, wherein each of the Kras mutants comprises at least 9 amino acids N-terminal to the mutant site immediately adjacent to the site and the mutant site The C-terminus of the site is immediately adjacent to at least 10 amino acids of the site.

6. The isolated nucleic acid molecule of any one of embodiments 1-5, wherein the Kras G12D mutant comprises the amino acid sequence set forth in SEQ ID NO:1.

7. The isolated nucleic acid molecule of any one of embodiments 1-6, wherein the Kras G13D mutant comprises the amino acid sequence set forth in SEQ ID NO:4.

8. The isolated nucleic acid molecule of any one of embodiments 1-7, wherein the Kras G12V mutant comprises the amino acid sequence set forth in SEQ ID NO:7.

9. The isolated nucleic acid molecule of any one of embodiments 1-8, wherein the Kras G13C mutant comprises the amino acid sequence set forth in SEQ ID NO:8.

10. The isolated nucleic acid molecule of any one of embodiments 1-9, wherein the Kras G12C mutant comprises the amino acid sequence set forth in SEQ ID NO:9.

11. The isolated nucleic acid molecule of any one of embodiments 1-10, wherein the Kras G13A mutant comprises the amino acid sequence set forth in SEQ ID NO:10.

12. The isolated nucleic acid molecule of any one of embodiments 1-11, wherein the Kras G12A mutant comprises the amino acid sequence set forth in SEQ ID NO:11.

13. The isolated nucleic acid molecule of any one of embodiments 1-12, wherein the Kras Q61L mutant comprises the amino acid sequence set forth in SEQ ID NO:12.

14. The isolated nucleic acid molecule of any one of embodiments 1-13, wherein the Kras G12R mutant comprises the amino acid sequence set forth in SEQ ID NO:15.

15. The isolated nucleic acid molecule of any one of embodiments 1-14, wherein the Kras Q61H mutant comprises the amino acid sequence set forth in SEQ ID NO:16.

16. The isolated nucleic acid molecule of any one of embodiments 1-15, wherein the Kras G12S mutant comprises the amino acid sequence set forth in SEQ ID NO:17.

17. The isolated nucleic acid molecule of any one of embodiments 1-16, wherein the Kras Q61R mutant comprises the amino acid sequence set forth in SEQ ID NO:18.

18. The isolated nucleic acid molecule of any one of embodiments 1-17, wherein the gene encoding the Kras G12D mutant comprises the nucleotide sequence set forth in SEQ ID NO:19.

19. The isolated nucleic acid molecule of any one of embodiments 1-18, wherein the gene encoding the Kras G13D mutant comprises the nucleotide sequence set forth in SEQ ID NO:22.

20. The isolated nucleic acid molecule of any one of embodiments 1-19, wherein the gene encoding the Kras G12V mutant comprises the nucleotide sequence set forth in SEQ ID NO:25.

21. The isolated nucleic acid molecule of any one of embodiments 1-20, wherein the gene encoding the Kras G13C mutant comprises the nucleotide sequence set forth in SEQ ID NO:26.

22. The isolated nucleic acid molecule of any one of embodiments 1-21, wherein the gene encoding the Kras G12C mutant comprises the nucleotide sequence set forth in SEQ ID NO:27.

23. The isolated nucleic acid molecule of any one of embodiments 1-22, wherein the gene encoding the Kras G13A mutant comprises the nucleotide sequence set forth in SEQ ID NO:28.

24. The isolated nucleic acid molecule of any one of embodiments 1-23, wherein the gene encoding the Kras G12A mutant comprises the nucleotide sequence set forth in SEQ ID NO:29.

25. The isolated nucleic acid molecule of any one of embodiments 1-24, wherein the gene encoding the Kras Q61L mutant comprises the nucleotide sequence set forth in SEQ ID NO:30.

26. The isolated nucleic acid molecule of any one of embodiments 1-25, wherein the gene encoding the Kras G12R mutant comprises the nucleotide sequence set forth in SEQ ID NO:33.

27. The isolated nucleic acid molecule of any one of embodiments 1-26, wherein the gene encoding the Kras Q61H mutant comprises the nucleotide sequence set forth in SEQ ID NO:34.

28. The isolated nucleic acid molecule of any one of embodiments 1-27, wherein the gene encoding the Kras G12S mutant comprises the nucleotide sequence set forth in SEQ ID NO:35.

29. The isolated nucleic acid molecule of any one of embodiments 1-28, wherein the gene encoding the Kras Q61R mutant comprises the nucleotide sequence set forth in SEQ ID NO:36.

30. The isolated nucleic acid molecule of any one of embodiments 1-29, further comprising a polynucleotide encoding a secreted peptide.

31. The isolated nucleic acid molecule according to embodiment 30, wherein the polynucleotide encoding the secreted peptide is a polynucleotide encoding the secreted peptide of the CD14 protein.

32. The isolated nucleic acid molecule of embodiment 31, wherein the polynucleotide encoding the CD14 protein secreted peptide is located 5' to the gene encoding the Kras mutant.

33. The isolated nucleic acid molecule of any one of embodiments 31-32, wherein the polynucleotide encoding the CD14 protein secreted peptide comprises the nucleotide sequence set forth in any one of SEQ ID NO:37.

34. The isolated nucleic acid molecule of any one of embodiments 1-33, further comprising a polynucleotide encoding a tag protein.

35. The isolated nucleic acid molecule of embodiment 34, wherein the tag protein comprises 6xHis.

36. The isolated nucleic acid molecule of any one of embodiments 34-35, wherein the polynucleotide encoding the tag protein is located 3' to the gene encoding the Kras mutant.

37. The nucleic acid molecule of any one of embodiments 34-36, wherein the polynucleotide encoding the tag protein comprises the nucleotide sequence set forth in SEQ ID NO:40.

38. The isolated nucleic acid molecule of any one of embodiments 1-37, comprising the nucleotide sequence set forth in any one of SEQ ID NOs: 49-51.

39. A vector comprising the nucleic acid molecule of any one of embodiments 1-38.

40. The vector of embodiment 39, comprising a viral vector.

41. The vector of any one of embodiments 39-40, comprising an oncolytic herpes simplex virus oHSV vector.

42. The vector of any one of embodiments 39-41, comprising a herpes simplex virus type I HSV-1 vector.

43. The vector of embodiment 42, wherein the HSV-1 vector is deficient in the neurotoxic factor gamma 34.5 gene.

44. The vector of any one of embodiments 39-43, wherein the nucleic acid molecule is located between the UL3 gene and the UL4 gene of the HSV-1 vector.

45. The vector of any one of embodiments 39-44, comprising a promoter.

46. The vector of embodiment 45, wherein the promoter comprises a CMV promoter.

47. The vector of any one of embodiments 39-46, comprising the nucleotide sequence shown in GenBank No: GU734771.1 of the NCBI database.

48. A pharmaceutical composition comprising the nucleic acid molecule of any one of embodiments 1-38, and/or the carrier of any one of embodiments 39-47, and optionally a pharmaceutically acceptable adjuvant. agent.

49. Use of the nucleic acid molecule according to any one of Embodiments 1-38 and/or the vector according to any one of Embodiments 39-47 in the preparation of a medicament for treating tumors.

50. The use of embodiment 49, wherein the tumor comprises a solid tumor.

51. The use of any one of embodiments 49-50, wherein the tumor comprises a pancreatic tumor.

52. The use of any one of embodiments 49-51, wherein the tumor comprises non-small cell lung cancer.

53. The use of any one of embodiments 49-52, wherein the tumor comprises colorectal cancer.

Without intending to be limited by any theory, the following examples are only intended to illustrate the nucleic acid molecules, preparation methods and uses of the present application, and are not intended to limit the scope of the invention of the present application.

Example

Example 1 Construction of BAC plasmid of recombinant virus KR10

1.1 Nucleotide sequence of synthetic CD14 protein-Kras mutant (TMG)-6x His

After truncating the amino acid sequence from the 1st amino acid to the 24th amino acid (SEQ ID NO: 1) from the amino acid sequence of the wild-type Kras polypeptide, the G amino acid at the 12th position in the truncated sequence is replaced by a D amino acid , the N-terminus adjacent to the G12D mutation site is 11 wild-type amino acids (SEQ ID NO: 2), and the C-terminus immediately adjacent to the G12D mutation site is 12 wild-type amino acids (SEQ ID NO: 3), obtaining a sequence that contains coding N The Kras G12D mutant of the nucleotides of the amino acids connected to the terminal and C-terminus, the nucleotide sequence of which is shown in SEQ ID NO: 19.

Referring to the above-mentioned method, respectively synthesized Kras G13D mutant (as shown in SEQ ID NO: 22), Kras G12V mutant (as shown in SEQ ID NO: 25), Kras G13C mutant (as shown in SEQ ID NO: 26) , Kras G12C mutant (as shown in SEQ ID NO: 27), Kras G13A mutant (as shown in SEQ ID NO: 28), Kras G12A mutant (as shown in SEQ ID NO: 29), Kras Q61L mutant (as shown in SEQ ID NO:30), Kras G12R mutant (as shown in SEQ ID NO:33), Kras Q61H mutant (as shown in SEQ ID NO:34), Kras G12S mutant (as shown in SEQ ID NO:34) : 35) and the nucleotide sequence of the Kras Q61R mutant (as shown in SEQ ID NO: 36), and the nucleotide sequences encoding each Kras mutant were concatenated according to the sequence to obtain a concatenated mini gene (Tandem minigene, TMG) form of Kras mutant whose nucleotide sequence is shown in SEQ ID NO:44. The above mutant sequences were synthesized by Aike Biotechnology.

Referring to the above-mentioned method, the Kras G12D mutant (as shown in SEQ ID NO: 19), the Kras A59T mutant (as shown in SEQ ID NO: 55), and the Kras G12V mutant (as shown in SEQ ID NO: 25) were synthesized respectively , Kras A146T mutant (as shown in SEQ ID NO:57), Kras G13D mutant (as shown in SEQ ID NO:22), Kras Y64H mutant (as shown in SEQ ID NO:59), Kras G12C mutant (as shown in SEQ ID NO:27), Kras Q61H mutant (as shown in SEQ ID NO:34), Kras A18D mutant (as shown in SEQ ID NO:61), Kras G12A mutant (as shown in SEQ ID NO:61) : 29), the Kras A146T mutant (as shown in SEQ ID NO: 57) and the Kras G12S mutant (as shown in SEQ ID NO: 35), and will encode each Kras in that order The nucleotide sequences of the mutants were concatenated to obtain a Kras mutant in the form of a Tandem minigene (TMG), called TMG-2. The above mutant sequences were synthesized by Aike Biotechnology.

Referring to the above-mentioned method, the Kras G12D mutant (as shown in SEQ ID NO: 19), the Kras A146T mutant (as shown in SEQ ID NO: 57), and the Kras G12V mutant (as shown in SEQ ID NO: 25) were synthesized respectively. , Kras A59T mutant (as shown in SEQ ID NO:55), Kras G13D mutant (as shown in SEQ ID NO:22), Kras Y64H mutant (as shown in SEQ ID NO:59) and Kras G12C mutant (as shown in SEQ ID NO: 27), and concatenate the nucleotide sequences encoding each Kras mutant according to the sequence to obtain a Kras mutant in the form of a tandem minigene (Tandem minigene, TMG), Called TMG-5. The above mutant sequences were synthesized by Aike Biotechnology.

With reference to the above method, Kras G12D mutant, Kras A59T mutant, Kras A18D mutant, Kras G12V mutant, Kras A146T mutant, Kras Q61H, Kras G13D mutant, Kras A59T mutant, Kras A146T mutant and Kras were synthesized respectively The nucleotide sequence of the G12C mutant, and the nucleotide sequences encoding each Kras mutant were concatenated according to the stated sequence to obtain a Kras mutant in the form of a tandem minigene (TMG), named TMG-7. The above mutant sequences were synthesized by Aike Biotechnology.

The CD14 protein secretory peptide (synthesized in Aike Biotechnology, nucleotide sequence SEQ ID NO: 37) was introduced at the 5' end of the gene encoding the above-mentioned Kras mutant in the form of TMG, and a 6x His tag (synthesized at the 3' end) was introduced Yu Aike Biotechnology Company, nucleotide sequence SEQ ID NO: 40), obtain the nucleotide sequence encoding CD14 protein-Kras mutant (TMG)-6x His (as shown in SEQ ID NO: 47-49), The structural form of CD14 protein-Kras mutant (TMG)-6x His is shown in Figure 1.

1.2 Insertion of Kras mutant gene

Two copies of the neurovirulence factor γ34.5 gene were deleted on wild-type HSV-1 (F) (shown in Figure 2A), while the CD14 protein-Kras described in Example 1.1 was inserted between the UL3 and UL4 genes The nucleotide sequence of the mutant (TMG)-6xHis, resulting in the nucleotide sequence of UL3-CD14 protein-Kras-6xHis-UL4 (as shown in SEQ ID NOs: 50-52).

1.3 Construction of BAC plasmid of recombinant virus

Through molecular cloning, an intermediate plasmid pKO5.1 for BAC recombination (gifted by Prof. Dr. Bernard Roizman, University of Chicago) was constructed, and the nucleotide sequence of UL3-CD14 protein-Kras-6xHis-UL4 described in Example 1.2 Inserted into pKO5.1, it was transformed into E. coli of HSV BAC with two copies of neurovirulence factor γ34.5 gene (shown in SEQ ID NO: 53) deleted by electroporation to obtain recombinant virus KR10 the BAC plasmid. The nucleic acid sequence structure of KR10 is shown in Figure 2B.

Example 2 Construction of BAC plasmid of recombinant virus KR11

Referring to the method of Example 1.1, a CD14 protein secretion peptide (Aike Biotechnology, SEQ ID NO: 37) was introduced into the N-terminus of the GFP protein, and a 6x His tag was introduced at the C-terminus (Aike Biotechnology, SEQ ID NO: 40) . The negative control virus UL3-CD14 protein-GFP-6xHis-UL4 nucleotide sequence (SEQ ID NO:45) was synthesized with reference to the method of Example 1.2, and the BAC plasmid containing the KR11 nucleotide sequence was constructed with reference to the method of Example 1.3. The nucleic acid sequence structure of KR11 is shown in Figure 2C.

Example 3 Construction of the BAC plasmid of positive control virus KR12

Known as the Flu A Mp antigenic peptide shown in SEQ ID NO: 42, obtain its nucleotide sequence according to its amino acid sequence, and introduce the CD14 protein secreted peptide (Aike Biotechnology) at its N-terminus with reference to the method of Example 1.1. technology company, SEQ ID NO: 37), and a 6x His tag was introduced into the C-terminal (Aike Biotechnology, SEQ ID NO: 40). The nucleotide sequence (SEQ ID NO: 46) of the positive control virus UL3-CD14 protein-FLU-6xHis-UL4 was synthesized with reference to the method of Example 1.2, and the BAC plasmid containing the nucleotide sequence of KR12 was constructed with reference to the method of Example 1.3 . The nucleic acid sequence structure of KR12 is shown in Figure 2D.

Example 4 Cell experiment

4.1 Packaging of recombinant virus and TK gene repair

The BAC plasmid containing the KR10 nucleotide sequence, the BAC plasmid containing the KR11 nucleotide sequence, and the BAC plasmid containing the KR12 nucleotide sequence constructed in the above Example 1-3 were respectively mixed with the pRB103 plasmid (containing the HSV-1F virus TK gene). ) were co-transfected into Vero cells, incubated in a 37°C, 5% CO ₂ incubator for 4 hours, then the medium was changed, fresh complete growth medium (5% NBCS/DMEM) was added, and the culture was continued until the virus was packaged successfully. Plaques appear. After the cells were repeatedly frozen and thawed, a virus stock solution was obtained, and Vero-ΔTK cells (Vero cells lacking TK gene) were infected with the virus stock solution, and HAT was added for screening to pick out monoclonal viruses. Vero-ΔTK cells were re-infected with monoclonal virus, and HAT was added to select the monoclonal virus. The monoclonal virus picked after repeated screening by HAT infects Vero cells and amplifies the virus to obtain the recombinant virus with the final TK gene repaired. The expressions of Kras polypeptide, GFP protein and Flu A MP antigen peptide were detected by western blotting method. The antibody used in WB was HRP-labeled 6*His monoclonal antibody purchased from Proteintech. The results of WB detection showed the expression of Kras polypeptide, GFP protein and Flu A MP antigen peptide.

4.2 In vitro killing of tumor cells by recombinant virus

Tumor cell plates were laid, and the recombinant viruses KR10, KR11, and KR12 prepared in Example 4.1 were respectively infected with tumor cells at different titers. After 72 hours of virus exposure, CCK8 was used to detect the cell proliferation toxicity, so as to detect that the recombinant viruses KR10, KR11, and KR12 killed tumors. Ability. The results showed that KR10, KR11 and KR12 had the ability to kill tumor cells.

Example 5 In vivo experiments in mice

Thymidine kinase gene repair (TK repair), after the repaired virus is successfully repaired by protein detection, in vivo experiments in mice are carried out. The results showed that KR10 and KR11 had good tumor suppressing effect.

5.1 Antitumor efficacy of KR10 and KR11 on subcutaneous xenografts of CT26.WT mice with colorectal cancer BALB/c mice

KR10 is a genetically engineered oncolytic virus. KR10 herpes virus is based on the wild-type herpes virus HSV-1 (F strain), knocking out one copy of the γ34.5 gene in the IR region and TR region, resulting in the simultaneous knockout of two copies of the γ34.5 gene. The virus is less virulent. In addition, KRAS mutant polypeptide is inserted into the virus, and the TMG form of the mutant polypeptide is G12D-A146T-G12V-A59T-G13D-Y64H-G12C. KR11 is GFP inserted into the viral backbone of KR10.

The experimental animals used in this experiment were BALB/c mice, SPF grade, female, 80, 5-6 weeks old. Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd., animal certificate number: 20201214Abzz0619000226.

CT26.WT mouse colorectal cancer cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin. Incubate at 37°C with 5% CO2.

Cells in good growth state were taken for experiments, cells were collected and centrifuged, the original medium was discarded, an appropriate amount of PBS was added to resuspend, count, and the cell density was adjusted to 2×10 ⁷ cells/mL, and placed on ice for later use.

A subcutaneous xenograft model of mouse colon cancer (CT26.WT) in BALB/c mice was established. The tumor cell suspension was subcutaneously inoculated in the right middle wing of BALB/c mice. When the average tumor volume in the mice grew to about 120 mm ³ , 35 tumor-bearing mice were selected and randomly divided into 5 groups according to the tumor volume, with 7 animals /group, the day of group administration is D1, vehicle control group (the vehicle is DPBS containing 10% (w/v) glycerol), KR11 low-dose group 1×10 ⁶ PFU/only (virus backbone control group), KR11 High-dose group 1×10 ⁷ PFU/donor (virus backbone control group), KR10 low-dose group (1×10 ⁶ PFU/shot) and KR10 high-dose group (1×10 ⁷ PFU/shot). Administered once a week (QW×3), and administered 3 times in total. The experimental animal groups and dosing schedules are shown in Table 1. The test product was administered by intratumoral injection, the administration volume was 50 μL/animal, and when the tumor volume was less than 80 mm, single ^- point injection (the syringe entered the lesion area through a single needle inlet, and the injection point was the middle of the tumor tissue); When the tumor volume is 80mm ³ to 140mm ³ , inject at 2 points (the syringe enters the lesion area through a single needle inlet, and the injection point is 1/3 and 2/3 of the long diameter of the tumor tissue); when the tumor volume is greater than 140mm ³ , Injection at 3 points (the syringe enters the lesion area through one needle inlet, the injection point is 1/3 and 2/3 of the long diameter of the tumor tissue, the second needle enters the lesion area from the other needle inlet, and the injection point is the tumor tissue The middle is on the outside.

Table 1 Grouping of experimental animals

The animal state, animal death and clinical symptoms were observed every day, and the observation content included but not limited to: mental state, behavioral activities, tumor rupture, etc. At the same time, the tumor diameter was measured and the body weight of the animals was weighed twice a week. At the end of the experiment, the tumors of the surviving animals were stripped and weighed. The relative tumor proliferation rate T/C%, tumor growth inhibition rate TGI% and tumor weight inhibition rate IR _TW % were calculated.

Tumor volume calculation formula: tumor volume (mm ³ )=½×long diameter×short diameter ² .

The relative tumor proliferation rate T/C (%) and tumor growth inhibition rate TGI (%) were used as experimental evaluation indicators. T/C(%)=(T/T0)/(C/C0)×100%, where T and C are the tumor volumes of the administration group and control group at the end of the experiment; T0 and C0 are the administration group and the control group at the beginning of the experiment. Tumor volume in the control group. If T>T0, tumor growth inhibition rate (TGI)%=[1-T/C]×100%; if T<T0, tumor growth inhibition rate (TGI)%=[1-(T-T0)/T0] ×100%.

Complete tumor remission CR: tumor volume less than 50 mm ³ .

Tumor weight inhibition rate IR _TW (%)=(W _{control group-} W _{administration group} )/W _{control group} ×100%

All experimental data are expressed as mean ± standard error (Mean ± SEM). Statistics were performed according to the following methods: pairwise comparisons were performed using T test, if P>0.05, the test was not significant; if P≤0.05, the test was significant.

result:

During the whole experimental period, the main clinical symptom of the animals in the vehicle control group and each experimental group was tumor crusting. During the experiment, no abnormality was found in the clinical observation of the animals, and no abnormality was found in the gross anatomy. Animal clinical observations are shown in Table 2-1 and Table 2-2.

On the 20th day, the average weight of mice in the vehicle control group was 24.13±0.48g, the average weights of the KR11 low and high dose groups were 23.03±0.38g and 22.23±0.89g, respectively, and the average weights of the KR10 low and high dose groups were 21.83±0.59g and 22.39±0.49g. Compared with the time of group administration, the body weight of animals in each group increased steadily. No other obvious drug-related side effects were observed during the experiment. The weight statistics are shown in Table 3-1 and Table 3-2. The individual data of body weight are shown in Table 4-1 and Table 4-2. The body weight increase trend is shown in Figure 3 (the red triangle symbol indicates the time point of administration, and D1 is the day of the grouping).

On the 20th day, the average tumor volume of the mice in the vehicle control group was 3968.41±653.80mm ³ , the average tumor volume of the KR11 low and high dose groups were 3093.03±886.41mm ³ and 2308.04±789.41mm ³ , respectively, and the KR10 low and high dose groups were The mean tumor volumes were 1701.34 ± 512.96 mm ³ and 1309.76 ± 628.21 mm ³ , respectively. Compared with the vehicle control group, the average tumor volume in the KR11 low and high dose groups decreased, but there was no significant difference (P>0.05), and the average tumor volume in the KR10 low and high dose groups decreased significantly (P<0.05); Compared with the same dose of KR11, the tumor volume in the high-dose group had a decreasing trend, but there was no statistical difference (P>0.05). The relative tumor proliferation rates T/C% of KR11 low and high dose groups were 62.23% and 53.22%, respectively, and the relative tumor proliferation rates T/C% of KR10 low and high dose groups were 35.97% and 20.99%, respectively. The tumor growth inhibition rates TGI% of KR11 low and high dose groups were 37.77% and 46.78%, respectively, and the tumor growth inhibition rates of KR10 low and high dose groups were 64.03% and 79.01%, respectively. At the same dose, the inhibition rate of KR10 on tumor was higher than that of KR11. During the test, a total of 2 animals with high dose of KR10 had complete tumor remission (tumor disappeared); in other groups, no tumor disappeared. The statistical results of tumor volume are shown in Table 5. The statistical data of tumor volume are shown in Table 6-1 and Table 6-2, the individual data are shown in Table 7-1 and Table 7-2, and the change trend of tumor volume is shown in Figure 4A-4B.

On the 20th day, the average tumor weight of the vehicle control group was 3.914±0.617g, the average tumor weight of the KR11 low-dose and high-dose groups were 2.788±0.628g and 2.353±0.804g, respectively; the average tumor weight of the KR10 low-dose and high-dose groups was 1.615, respectively ±0.508g and 1.226±0.526g. Compared with the vehicle control group, the average tumor weight of the KR11 low and high dose groups decreased, but there was no significant difference (P>0.05). The mean tumor weight of KR10 low and high dose groups decreased significantly (P<0.05). The tumor weight inhibition rate IR _TW (%) of KR11 low and high dose groups were 28.77% and 39.88%, respectively. The tumor weight inhibition rate IR _TW (%) of KR10 low-dose and high-dose groups was 58.74% and 68.68%, respectively. The statistical results are shown in Table 8. The tumor weight statistics are shown in Table 9. Individual data are shown in Table 10. Tumor weight statistics are shown in Figure 5. Euthanasia photographs are shown in Figures 6A-6B.

On the 25th day, CT26.WT tumor was re-challenged in 2 animals in the KR10 high-dose group whose tumors had completely disappeared, that is, the same amount of CT26.WT tumor cells was re-inoculated on the contralateral side of the mice to observe the tumor growth. Thirty days after rechallenge, none of the animals developed tumors. It was proved that after administration of KR10, the long-term anti-CT26.WT tumor immune memory function was established in the above two animals. The changes in tumor volume after tumor re-challenge are shown in Figure 7. The individual data of tumor volume are shown in Table 7-3. Figure 8 shows the photos of the end point of the tumor re-challenge model animal experiment.

Conclusion: The experimental results show that under the experimental conditions, in the mouse model of colorectal cancer CT26.WT cells subcutaneously transplanted in mice, KR10 can significantly inhibit the growth of tumors at the same dose. The inhibition rates were higher than those of KR11, and the tumors of 2 animals in the KR10 high-dose group completely regressed. The above results show that the antitumor effect of KR10 is better than that of the viral backbone KR11. The re-challenge model demonstrated that KR10 could stimulate mice to establish long-term anti-tumor immune memory.

Table 2-1 Observation statistics of clinical symptoms of experimental animals

Notes: - normal, 1 dead, 2 apathetic, 3 decreased activity, 4 trembling, 5 piloerection, 6 tumor crusted, 7 arched back, NA dead/euthanized.

Table 2-2 Observation statistics of clinical symptoms of experimental animals

Table 3-1 Statistical table of body weight of experimental animals (g, Mean±SEM)

Table 3-2 Statistical table of body weight of experimental animals (g, Mean±SEM)

Note: * indicates p<0.05 compared with the vehicle control group.

Table 4-1 Individual data table of experimental animal body weight (g)

Table 4-2 Individual data table of experimental animal body weight (g)

Table 5 Statistical table of tumor volume in each group (Mean±SEM)

Note: #: indicates the P value compared with the control group. ▲: P value compared with the same dose of KR11.

Table 6-1 Statistical table of tumor volume in experimental animals (mm3, Mean±SEM)

Table 6-2 Statistical table of tumor volume in experimental animals (mm3, Mean±SEM)

Note: * indicates p<0.05 compared with the vehicle control group.

Table 7-1 Individual data table of experimental animal tumor volume (mm ³ )

Table 7-2 Experimental animal tumor volume (mm ³ ) individual data table

Table 7-3 Individual data table of experimental animal tumor volume (mm ³ )

Table 8 Tumor weight of animals in each group (g, Mean±SEM)

组别group	溶媒/供试品Solvent/Test Article	剂量dose	肿瘤重量(g)Tumor weight (g)	P ^#值 P ^# value	P ^$值 P ^$ value	IR _TW(％) IR _TW (%)
11	溶媒对照组vehicle control group	--	3.914±0.6173.914±0.617	--	--	--
22	KR11低剂量组KR11 low-dose group	1×10 ⁶PFU/只 1×10 ⁶ PFU/pc	2.788±0.6282.788±0.628	0.2250.225	--	28.7728.77
33	KR11高剂量组KR11 high-dose group	1×10 ⁷PFU/只 1×10 ⁷ PFU/pc	2.353±0.8042.353±0.804	0.1500.150	--	39.8839.88
44	KR10低剂量组KR10 low-dose group	1×10 ⁶PFU/只 1×10 ⁶ PFU/pc	1.615±0.5081.615±0.508	0.0140.014	0.1720.172	58.7458.74
55	KR10高剂量组KR10 high-dose group	1×10 ⁷PFU/只 1×10 ⁷ PFU/pc	1.226±0.5261.226±0.526	0.0060.006	0.2630.263	68.6868.68

Note: * indicates p<0.05 compared with the vehicle control group. ** indicates p<0.01 compared to the vehicle control group. #: indicates the P value compared with the control group. $: Compared with the same dose of KR11.

Table 9 Experimental animal tumor weight statistics table

组别group	溶媒/供试品Solvent/Test Article	剂量dose	瘤重(g，Mean±SEM)Tumor weight (g, Mean±SEM)
11	溶媒对照组vehicle control group	--	3.914±0.6173.914±0.617
22	KR11低剂量组KR11 low-dose group	1×10 ⁶PFU/只 1×10 ⁶ PFU/pc	2.788±0.6282.788±0.628
33	KR11高剂量组KR11 high-dose group	1×10 ⁷PFU/只 1×10 ⁷ PFU/pc	2.353±0.8042.353±0.804
44	KR10低剂量组KR10 low-dose group	1×10 ⁶PFU/只 1×10 ⁶ PFU/pc	1.615±0.5081.615±0.508
55	KR10高剂量组KR10 high-dose group	1×10 ⁷PFU/只 1×10 ⁷ PFU/pc	1.226±0.5261.226±0.526

Note: * indicates p<0.05 compared with the vehicle control group. ** indicates p<0.01 compared to the vehicle control group.

Table 10 Individual data table of tumor weight of experimental animals

5.2 The antitumor effects of intratumoral administration of KR10 and KR11 herpesviruses on BALB/c nude mice subcutaneously transplanted with human non-small cell lung cancer cells (A549)

The experimental animals used in this experiment were BALB/C nude mice, SPF grade, female, 50, 5-6 weeks old. From Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd., animal certificate number: 20201214Abzz0619000711.

A549 human non-small cell lung cancer cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin. Incubate at 37°C with 5% CO ₂ .

Cells in good growth state were taken for experiments, cells were collected and centrifuged, the original medium was discarded, an appropriate amount of PBS was added to resuspend and counted, the cell density was adjusted to 2.0×10 ⁷ cells/mL, and placed on ice for later use.

A BALB/c nude mouse subcutaneous xenograft model of human non-small cell lung cancer cells (A549) was established. Tumor cell suspensions were subcutaneously inoculated in the right middle wing of BALB/c nude mice. On the 11th day after subcutaneous inoculation, when the average tumor volume in the mice grew to about 75 mm ³ , 42 tumor-bearing mice were selected. The tumor volume ranged from 49.94-99.10mm ³ , the weight of the animals was 17.8-21.5g, they were randomly divided into 5 groups according to the tumor volume, 10 in the vehicle group, 8 animals/group in each treatment group, respectively: the vehicle control group (the vehicle contains 10% ( w/v) glycerol in DPBS), KR11 low-dose control group (viral backbone) (1×10 ⁵ PFU/piece, QW×3), KR11 high-dose control group (1×10 ⁶ PFU/piece, QW×3) , KR10 low-dose group (1×10 ⁵ PFU/only, QW×3), KR10 high-dose group (1×10 ⁶ PFU/only, QW×3). 1 time a week, a total of 3 doses. The day of the first dose was defined as D1. The experimental animal groups and dosing schedules are shown in Table 11. The test product was administered by intratumoral injection, the administration volume was 50 μL/animal, and when the tumor volume was less than 80 mm, single ^- point injection (the syringe entered the lesion area through a single needle inlet, and the injection point was the middle of the tumor tissue); When the tumor volume is 80mm ³ to 140mm ³ , inject at 2 points (the syringe enters the lesion area through a single needle inlet, and the injection point is 1/3 and 2/3 of the long diameter of the tumor tissue); when the tumor volume is greater than 140mm ³ , Injection at 3 points (the syringe enters the lesion area through one needle inlet, the injection point is 1/3 and 2/3 of the long diameter of the tumor tissue, the second needle enters the lesion area from the other needle inlet, and the injection point is the tumor tissue middle to the outside).

Table 11 Grouping of experimental animals

The animal state was observed every day, and the observation content included but not limited to: mental state, behavioral activities, tumor rupture, etc. At the same time, the tumor diameter was measured and the body weight of the animals was weighed twice a week. At the end of the experiment, the tumors of the surviving animals were stripped and weighed. The relative tumor proliferation rate T/C%, tumor growth inhibition rate TGI% and tumor weight inhibition rate IR _TW % were calculated.

The relative tumor proliferation rate T/C (%) and tumor growth inhibition rate TGI (%) were used as experimental evaluation indicators.

T/C(%)=(T/T0)/(C/C0)×100%, where T and C are the tumor volumes of the administration group and control group at the end of the experiment; T0 and C0 are the administration group and the control group at the beginning of the experiment. Tumor volume in the control group. If T>T0, tumor growth inhibition rate (TGI)%=[1-T/C]×100%; if T<T0, tumor growth inhibition rate (TGI)%=[1-(T-T0)/T0] ×100%.

Tumor weight inhibition rate IR _TW (%) = (W _{control group -} W _{administration group} )/W control group × 100%

The observation time was 21 days, and the experiment was terminated.

All experimental data are expressed as mean ± standard error (Mean ± SEM). According to the following methods: use Levene's test for homogeneity of variance, if there is no statistical significance (P>0.05), use one-way analysis of variance (ANOVA) for statistical analysis, if ANOVA is statistically significant (P<0.05), carry out LSD method Comprehensive comparisons; if the variances are unequal (P<0.05), the Kruskal-Wallis test is used, and if the Kruskal-Wallis test is statistically significant (P<0.05), the Mann-Whitney method is used for pairwise comparisons between means .

result:

During the experiment, the main clinical symptoms of the animals in the vehicle control group and each administration group were tumor scabs, and there was no abnormality in the animal state. The observation of clinical symptoms of animals is shown in Table 12-1, Table 12-2 and Table 12-3.

On the 21st day at the end of the experiment, the average body weight of the animals in the vehicle control group was 21.77±0.31g, and the average weights of the animals in the KR11 low and high dose groups, KR10 low and high dose groups were 22.05±0.56g, 22.05±0.36g, 21.58±0.45 g and 21.49 ± 0.40 g. There was no significant difference in the body weight of animals in each treatment group compared with the vehicle control group. Weight statistics are shown in Table 13. Body weight individual data are shown in Table 14. The weight gain trend is shown in Figure 9. The red triangle symbol indicates the time point of administration, and D1 is the day of grouping.

On the 21st day, the mean tumor volume of the animals in the vehicle control group was 506.16±51.47mm ³ , the mean tumor volumes of the animals in the KR11 low and high dose groups, KR10 low and high dose groups were 242.17±42.89mm ³ , 138.75±39.51mm ³ , 161.33±47.50mm ³ , 66.12±26.42mm ³ . There were significant differences in mean tumor volume between each treatment group and the vehicle control group (P<0.001). There was no significant difference in the mean tumor volume of each group under the same administration level of KR10 and KR11 (P>0.05). The relative tumor proliferation rates T/C% of each treatment group were 48.57%, 30.02%, 34.20% and 14.95%, respectively. The tumor growth inhibition rate TGI% was 51.43%, 69.98%, 65.80% and 85.05%, respectively. Tumor volume statistics are shown in Table 15. Individual data are shown in Table 16. The statistical table of tumor volume in each group is shown in Table 17. The trend of tumor volume change is shown in Figure 10. Individual data are shown in Figure 11.

At the end of the experiment (D21, day 21), all surviving animals were euthanized, and tumors were dissected, weighed, and photographed. The mean tumor weight of the vehicle control group was 0.47±0.05g, and the mean tumor weights of the animals in the KR11 low and high dose groups, KR10 low and high dose groups were 0.27±0.04g, 0.19±0.04g, 0.23±0.05g and 0.13±0.13±0.05g, respectively. 0.04g, which was significantly lower than that of the vehicle control group (vs the vehicle control group, P<0.01). There was no significant difference in the mean tumor weight of each group under the same administration level of KR10 and KR11 (P>0.05). The tumor weight inhibition rates IR _TW (%) were 43.38%, 58.87%, 50.32% and 72.76%, respectively. The statistical results are shown in Table 18. The tumor weight statistics are shown in Table 19, the individual data are shown in Table 20, and the tumor weight statistics of each group are shown in Figure 12. Photos of euthanized tumors are shown in Figure 13.

Conclusion: Under the experimental conditions, in the subcutaneous xenograft model of human non-small cell lung cancer (A549), the test articles KR11 and KR10 have significant inhibitory effects on the tumor in a dose-dependent manner, and the animals tolerated well. Since only the oncolytic virus backbone plays an anti-tumor effect in immunodeficient mice, KR10 and KR11 have the same oncolytic virus backbone. Therefore, at the same administration level, the tumor-inhibiting effects of each group are similar, and there is no statistical difference.

Table 12-1 Observation statistics of clinical symptoms of experimental animals

Table 12-2 Observation statistics of clinical symptoms of experimental animals

Table 12-3 Observation statistics of clinical symptoms of experimental animals

Table 13 Experimental animal body weight statistics

Table 14 Individual data table of experimental animal body weight

Table 15 Statistical table of tumor volume in experimental animals

Note: * means p<0.05 compared with the vehicle control group; ** means p<0.01 compared with the vehicle control group; *** means p<0.001 compared with the vehicle control group.

Table 16 Experimental animal tumor volume (mm ³ ) individual data table

Table 17 Statistics of tumor volume in each group (Mean±SEM)

group order

group

dose

Tumor volume (mm ³ )

T/C(%

TGI(

#: indicates the P value compared with the control group. ▲: P value compared with the same dose of KR11.

Table 18 Animal tumor weight in each group (Mean±SEM)

Table 19 Statistical table of tumor weight of experimental animals (g, Mean±SEM)

Note: ** means p<0.01 compared with the vehicle control group; *** means p<0.001 compared with the vehicle control group.

Table 20 Individual data table of tumor weight of experimental animals

The foregoing detailed description has been presented by way of explanation and example, and is not intended to limit the scope of the appended claims. Various modifications to the embodiments presently enumerated in this application will be apparent to those of ordinary skill in the art and remain within the scope of the appended claims and their equivalents.

Claims

An isolated nucleic acid molecule comprising one or more genes each independently encoding a Kras mutant selected from the group consisting of: Kras G12D mutant, Kras G13D mutant, Kras G12V mutant, Kras G13C mutant, KrasG12C mutant, Kras G13A mutant, Kras G12A mutant, Kras Q61L mutant, Kras G12R mutant, Kras Q61H mutant, Kras G12S mutant, Kras Q61R mutant, Kras A59T mutant, Kras A146T mutant, Kras Y64H mutant and Kras A18D mutant.
An isolated nucleic acid molecule that does not comprise a polynucleotide encoding a 6x His-tagged protein and that comprises one or more genes each independently encoding a Kras mutant selected from the group consisting of: Kras G12D mutant, Kras G13D mutant, Kras G12V mutant, Kras G13C mutant, Kras G12C mutant, Kras G13A mutant, Kras G12A mutant, Kras Q61L mutant, Kras G12R mutant, Kras Q61H mutant, Kras G12S mutant, Kras Q61R mutant, Kras A59T mutant, Kras A146T mutant, Kras Y64H mutant and Kras A18D mutant.
The isolated nucleic acid molecule of any one of claims 1-2, comprising a gene encoding a Kras mutant selected from the group consisting of: Kras A59T mutant, Kras G12D mutant, Kras G12V mutant, KrasA146T mutant, Kras G13D mutant and KrasG12C mutant.
The isolated nucleic acid molecule of any one of claims 1-3, comprising a gene encoding a Kras mutant selected from the group consisting of: Kras A59T mutant, Kras G12D mutant, Kras G12V mutant, KrasA146T mutant, Kras G13D mutant, Kras Y64H mutant and Kras G12C mutant.
The isolated nucleic acid molecule of any one of claims 1-4, wherein the 3' end of the gene encoding the Kras G12D mutant is directly or indirectly linked to the 5' end of the gene encoding the Kras A146T mutant .
The isolated nucleic acid molecule of any one of claims 1-5, wherein the 3' end of the gene encoding the Kras A46T mutant is directly or indirectly linked to the 5' end of the gene encoding the Kras G12V mutant .
The isolated nucleic acid molecule of any one of claims 1-6, wherein the 3' end of the gene encoding the Kras G12V mutant is directly or indirectly linked to the 5' end of the gene encoding the Kras A59T mutant .
The isolated nucleic acid molecule of any one of claims 1-7, wherein the 3' end of the gene encoding the Kras A59T mutant is directly or indirectly linked to the 5' end of the gene encoding the Kras G13D mutant .
The isolated nucleic acid molecule of any one of claims 1-8, wherein the 3' end of the gene encoding the Kras G13D mutant is directly or indirectly linked to the 5' end of the gene encoding the Kras Y64H mutant .
The isolated nucleic acid molecule of any one of claims 1-9, wherein the 3' end of the gene encoding the Kras Y64H mutant is directly or indirectly linked to the 5' end of the gene encoding the Kras G12C mutant .
The isolated nucleic acid molecule of any one of claims 1-10, wherein each of the genes encoding the Kras mutants are arranged in tandem in the isolated nucleic acid molecule.
The isolated nucleic acid molecule of any one of claims 1-11, wherein each said gene encoding a Kras mutant is a minigene.
The isolated nucleic acid molecule of any one of claims 1-12, wherein each of the genes encoding the Kras mutants is arranged in tandem to yield a tandem minigene.
The isolated nucleic acid molecule of any one of claims 1-13, wherein each of the Kras mutants comprises at least 20 amino acids.
The isolated nucleic acid molecule of any one of claims 1-14, wherein each of the Kras mutants comprises at least 9 amino acids N-terminal to the mutation site immediately adjacent to the site and the mutation site C The end is immediately adjacent to at least 10 amino acids of the site.
The isolated nucleic acid molecule of any one of claims 1-15, wherein the Kras G12D mutant comprises the amino acid sequence set forth in SEQ ID NO:1.
The isolated nucleic acid molecule of any one of claims 1-16, wherein the Kras G13D mutant comprises the amino acid sequence set forth in SEQ ID NO:4.
The isolated nucleic acid molecule of any one of claims 1-17, wherein the Kras G12V mutant comprises the amino acid sequence set forth in SEQ ID NO:7.
The isolated nucleic acid molecule of any one of claims 1-18, wherein the Kras G13C mutant comprises the amino acid sequence set forth in SEQ ID NO:8.
The isolated nucleic acid molecule of any one of claims 1-19, wherein the Kras G12C mutant comprises the amino acid sequence set forth in SEQ ID NO:9.
The isolated nucleic acid molecule of any one of claims 1-20, wherein the Kras G13A mutant comprises the amino acid sequence set forth in SEQ ID NO:10.
The isolated nucleic acid molecule of any one of claims 1-21, wherein the Kras G12A mutant comprises the amino acid sequence shown in SEQ ID NO:11.
The isolated nucleic acid molecule of any one of claims 1-22, wherein the Kras Q61L mutant comprises the amino acid sequence shown in SEQ ID NO:12.
The isolated nucleic acid molecule of any one of claims 1-23, wherein the Kras G12R mutant comprises the amino acid sequence set forth in SEQ ID NO:15.
The isolated nucleic acid molecule of any one of claims 1-24, wherein the Kras Q61H mutant comprises the amino acid sequence set forth in SEQ ID NO:16.
The isolated nucleic acid molecule of any one of claims 1-25, wherein the Kras G12S mutant comprises the amino acid sequence set forth in SEQ ID NO:17.
The isolated nucleic acid molecule of any one of claims 1-26, wherein the Kras Q61R mutant comprises the amino acid sequence set forth in SEQ ID NO:18.
The isolated nucleic acid molecule of any one of claims 1-27, wherein the Kras A59T mutant comprises the amino acid sequence set forth in SEQ ID NO:54.
The isolated nucleic acid molecule of any one of claims 1-28, wherein the Kras A146T mutant comprises the amino acid sequence set forth in SEQ ID NO:56.
The isolated nucleic acid molecule of any one of claims 1-29, wherein the Kras Y64H mutant comprises the amino acid sequence set forth in SEQ ID NO:58.
The isolated nucleic acid molecule of any one of claims 1-30, wherein the Kras A18D mutant comprises the amino acid sequence set forth in SEQ ID NO:60.
The isolated nucleic acid molecule of any one of claims 1-31, wherein the gene encoding the Kras G12D mutant comprises the nucleotide sequence shown in SEQ ID NO:19.
The isolated nucleic acid molecule of any one of claims 1-32, wherein the gene encoding the Kras G13D mutant comprises the nucleotide sequence shown in SEQ ID NO:22.
The isolated nucleic acid molecule of any one of claims 1-33, wherein the gene encoding the Kras G12V mutant comprises the nucleotide sequence shown in SEQ ID NO:25.
The isolated nucleic acid molecule of any one of claims 1-34, wherein the gene encoding the Kras G13C mutant comprises the nucleotide sequence shown in SEQ ID NO:26.
The isolated nucleic acid molecule of any one of claims 1-35, wherein the gene encoding the Kras G12C mutant comprises the nucleotide sequence shown in SEQ ID NO:27.
The isolated nucleic acid molecule of any one of claims 1-36, wherein the gene encoding the Kras G13A mutant comprises the nucleotide sequence shown in SEQ ID NO:28.
The isolated nucleic acid molecule of any one of claims 1-37, wherein the gene encoding the Kras G12A mutant comprises the nucleotide sequence shown in SEQ ID NO:29.
The isolated nucleic acid molecule of any one of claims 1-38, wherein the gene encoding the Kras Q61L mutant comprises the nucleotide sequence shown in SEQ ID NO:30.
The isolated nucleic acid molecule of any one of claims 1-39, wherein the gene encoding the Kras G12R mutant comprises the nucleotide sequence shown in SEQ ID NO:33.
The isolated nucleic acid molecule of any one of claims 1-40, wherein the gene encoding the Kras Q61H mutant comprises the nucleotide sequence shown in SEQ ID NO:34.
The isolated nucleic acid molecule of any one of claims 1-41, wherein the gene encoding the Kras G12S mutant comprises the nucleotide sequence shown in SEQ ID NO:35.
The isolated nucleic acid molecule of any one of claims 1-42, wherein the gene encoding the Kras Q61R mutant comprises the nucleotide sequence shown in SEQ ID NO:36.
The isolated nucleic acid molecule of any one of claims 1-43, wherein the gene encoding the Kras A59T mutant comprises the nucleotide sequence set forth in SEQ ID NO:55.
The isolated nucleic acid molecule of any one of claims 1-44, wherein the gene encoding the Kras A146T mutant comprises the nucleotide sequence set forth in SEQ ID NO:57.
The isolated nucleic acid molecule of any one of claims 1-45, wherein the gene encoding the Kras Y64H mutant comprises the nucleotide sequence set forth in SEQ ID NO:59.
The isolated nucleic acid molecule of any one of claims 1-46, wherein the gene encoding the Kras A18D mutant comprises the nucleotide sequence set forth in SEQ ID NO:61.
The isolated nucleic acid molecule of any one of claims 13-47, wherein the tandem minigene comprises, in order from the 5' end to the 3' end, a gene encoding a Kras G12D mutant, a gene encoding a Kras A59T mutant, Gene encoding KrasG12V mutant, gene encoding Kras A146T mutant, gene encoding Kras G13D mutant, gene encoding Kras Y64H mutant, gene encoding Kras G12C mutant, gene encoding Kras Q61H mutant, gene encoding Kras A18D Mutant genes, genes encoding Kras G12A mutants, genes encoding Kras A146T mutants, and genes encoding Kras G12S mutants.
The isolated nucleic acid molecule of any one of claims 1-48, comprising the nucleotide sequence set forth in SEQ ID NO:65.
The isolated nucleic acid molecule of any one of claims 13-49, wherein the tandem minigene comprises, in order from the 5' end to the 3' end, a gene encoding a Kras G12D mutant, a gene encoding a Kras A146T mutant, The gene encoding the Kras G12V mutant, the gene encoding the Kras A59T mutant, the gene encoding the Kras G13D mutant, the gene encoding the Kras Y64H mutant, and the gene encoding the Kras G12C mutant.
The isolated nucleic acid molecule of any one of claims 1-50, comprising the nucleotide sequence set forth in SEQ ID NO:66.
The isolated nucleic acid molecule of any one of claims 13-51, wherein the tandem minigene comprises, in order from the 5' end to the 3' end, a gene encoding a Kras G12D mutant, a gene encoding a Kras A59T mutant, Gene encoding Kras A18D mutant, gene encoding Kras G12V mutant, gene encoding Kras A146T mutant, gene encoding Kras Q61H mutant, gene encoding Kras G13D mutant, gene encoding Kras A59T mutant, gene encoding Kras The gene of the A146T mutant and the gene encoding the Kras G12C mutant.
The isolated nucleic acid molecule of any one of claims 1-52, comprising the nucleotide sequence shown in SEQ ID NO:67.
The isolated nucleic acid molecule of any one of claims 1-53, further comprising a polynucleotide encoding a secreted peptide.
According to the isolated nucleic acid molecule of claim 54, the polynucleotide encoding the secreted peptide is a polynucleotide encoding the secreted peptide of the CD14 protein.
The isolated nucleic acid molecule of claim 55, wherein the polynucleotide encoding the CD14 protein secreted peptide is located 5' to the gene encoding the Kras mutant.
The isolated nucleic acid molecule of any one of claims 55-56, wherein the polynucleotide encoding the CD14 protein secreted peptide comprises the nucleotide sequence set forth in any one of SEQ ID NO:37.
A vector comprising the nucleic acid molecule of any one of claims 1-57.
The vector of claim 58, which comprises a viral vector.
The vector of any one of claims 58-59, comprising an oncolytic herpes simplex virus oHSV vector.
The vector of any one of claims 58-60, comprising a herpes simplex virus type I HSV-1 vector.
The vector of claim 61, wherein the HSV-1 vector is deficient in the neurotoxic factor gamma 34.5 gene.
The vector of any one of claims 58-62, wherein the nucleic acid molecule is located between the UL3 gene and the UL4 gene of the HSV-1 vector.
The vector of any one of claims 58-63, which comprises a promoter.
The vector of claim 64, wherein the promoter comprises a CMV promoter.
The vector of any one of claims 58-65, comprising the nucleotide sequence shown in GenBank No: GU734771.1 of the NCBI database.
A pharmaceutical composition comprising the nucleic acid molecule of any one of claims 1-57, and/or the carrier of any one of claims 58-66, and optionally a pharmaceutically acceptable adjuvant.
A composition comprising the isolated nucleic acid molecule of any one of claims 1-57, the carrier of any one of claims 58-69 or the pharmaceutical composition of claim 67, and physiological saline.
The composition of claim 68, wherein the isolated nucleic acid molecule comprises one or more genes each independently encoding a Kras mutant selected from the group consisting of: Kras G12D mutant, Kras G13D mutant, Kras G12V mutant Kras G13C mutant, Kras G12C mutant, Kras G13A mutant, Kras G12A mutant, Kras Q61L mutant, Kras G12R mutant, Kras Q61H mutant, Kras G12S mutant, Kras Q61R mutant, Kras A59T mutant mutant, Kras A146T mutant, Kras Y64H mutant and Kras A18D mutant.
The composition of any one of claims 68-69, wherein the isolated nucleic acid molecule comprises a gene encoding a Kras mutant selected from the group consisting of: Kras A59T mutant, Kras G12D mutant, Kras G12V mutant, KrasA146T mutant, Kras G13D mutant and KrasG12C mutant.
The composition of any one of claims 68-70, wherein the isolated nucleic acid molecule comprises a gene encoding a Kras mutant selected from the group consisting of: Kras A59T mutant, Kras G12D mutant, Kras G12V mutant, KrasA146T mutant, Kras G13D mutant, Kras Y64H mutant and Kras G12C mutant.
The composition of any one of claims 69-71, wherein the 3' end of the gene encoding the Kras G12D mutant is directly or indirectly linked to the 5' end of the gene encoding the Kras A146T mutant.
The composition of any one of claims 69-72, wherein the 3' end of the gene encoding the Kras A46T mutant is directly or indirectly linked to the 5' end of the gene encoding the Kras G12V mutant.
The composition of any one of claims 69-73, wherein the 3' end of the gene encoding the Kras G12V mutant is directly or indirectly linked to the 5' end of the gene encoding the Kras A59T mutant.
The composition of any one of claims 69-74, wherein the 3' end of the gene encoding the Kras A59T mutant is directly or indirectly linked to the 5' end of the gene encoding the Kras G13D mutant.
The composition of any one of claims 69-75, wherein the 3' end of the gene encoding the Kras G13D mutant is directly or indirectly linked to the 5' end of the gene encoding the Kras Y64H mutant.
The composition of any one of claims 69-76, wherein the 3' end of the gene encoding the Kras Y64H mutant is directly or indirectly linked to the 5' end of the gene encoding the Kras G12C mutant.
77. The composition of any one of claims 69-77, wherein each of the genes encoding the Kras mutants are arranged in tandem in the isolated nucleic acid molecules.
The composition of any one of claims 69-78, wherein each of the genes encoding the Kras mutants is a minigene Minigene.
The composition of any one of claims 69-79, wherein each of the genes encoding the Kras mutants is arranged in tandem to obtain a tandem minigene.
The composition of any one of claims 69-80, wherein each of the Kras mutants comprises at least 20 amino acids.
The composition of any one of claims 69-81, wherein each of the Kras mutants comprises at least 9 amino acids immediately N-terminal to the site of the mutation and immediately C-terminal to the mutation site at least 10 amino acids of this site.
The composition of any one of claims 69-82, wherein the Kras G12D mutant comprises the amino acid sequence set forth in SEQ ID NO:1.
The composition of any one of claims 69-83, wherein the Kras G13D mutant comprises the amino acid sequence set forth in SEQ ID NO:4.
The composition of any one of claims 69-84, wherein the Kras G12V mutant comprises the amino acid sequence set forth in SEQ ID NO:7.
The composition of any one of claims 69-85, wherein the Kras G13C mutant comprises the amino acid sequence set forth in SEQ ID NO:8.
The composition of any one of claims 69-86, wherein the Kras G12C mutant comprises the amino acid sequence set forth in SEQ ID NO:9.
The composition of any one of claims 69-87, wherein the Kras G13A mutant comprises the amino acid sequence set forth in SEQ ID NO:10.
The composition of any one of claims 69-88, wherein the Kras G12A mutant comprises the amino acid sequence shown in SEQ ID NO:11.
The composition of any one of claims 69-89, wherein the Kras Q61L mutant comprises the amino acid sequence set forth in SEQ ID NO:12.
The composition of any one of claims 69-90, wherein the Kras G12R mutant comprises the amino acid sequence set forth in SEQ ID NO:15.
The composition of any one of claims 69-91, wherein the Kras Q61H mutant comprises the amino acid sequence set forth in SEQ ID NO:16.
The composition of any one of claims 69-92, wherein the Kras G12S mutant comprises the amino acid sequence set forth in SEQ ID NO:17.
The composition of any one of claims 69-93, wherein the Kras Q61R mutant comprises the amino acid sequence set forth in SEQ ID NO:18.
The composition of any one of claims 69-94, wherein the Kras A59T mutant comprises the amino acid sequence set forth in SEQ ID NO:54.
The composition of any one of claims 69-95, wherein the Kras A146T mutant comprises the amino acid sequence set forth in SEQ ID NO:56.
The composition of any one of claims 69-96, wherein the Kras Y64H mutant comprises the amino acid sequence set forth in SEQ ID NO:58.
The composition of any one of claims 69-97, wherein the Kras A18D mutant comprises the amino acid sequence set forth in SEQ ID NO:60.
The composition of any one of claims 69-98, wherein the gene encoding the Kras G12D mutant comprises the nucleotide sequence shown in SEQ ID NO:19.
The composition of any one of claims 69-99, wherein the gene encoding the Kras G13D mutant comprises the nucleotide sequence shown in SEQ ID NO:22.
The composition of any one of claims 69-100, wherein the gene encoding the Kras G12V mutant comprises the nucleotide sequence shown in SEQ ID NO:25.
The composition of any one of claims 69-101, wherein the gene encoding the Kras G13C mutant comprises the nucleotide sequence shown in SEQ ID NO:26.
The composition of any one of claims 69-102, wherein the gene encoding the Kras G12C mutant comprises the nucleotide sequence shown in SEQ ID NO:27.
The composition of any one of claims 69-103, wherein the gene encoding the Kras G13A mutant comprises the nucleotide sequence shown in SEQ ID NO:28.
The composition of any one of claims 69-104, wherein the gene encoding the Kras G12A mutant comprises the nucleotide sequence shown in SEQ ID NO:29.
The composition of any one of claims 69-105, wherein the gene encoding the Kras Q61L mutant comprises the nucleotide sequence shown in SEQ ID NO:30.
The composition of any one of claims 69-106, wherein the gene encoding the Kras G12R mutant comprises the nucleotide sequence shown in SEQ ID NO:33.
The composition of any one of claims 69-107, wherein the gene encoding the Kras Q61H mutant comprises the nucleotide sequence shown in SEQ ID NO:34.
The composition of any one of claims 69-108, wherein the gene encoding the Kras G12S mutant comprises the nucleotide sequence shown in SEQ ID NO:35.
The composition of any one of claims 69-109, wherein the gene encoding the Kras Q61R mutant comprises the nucleotide sequence shown in SEQ ID NO:36.
The composition of any one of claims 69-110, wherein the gene encoding the Kras A59T mutant comprises the nucleotide sequence shown in SEQ ID NO:55.
The composition of any one of claims 69-111, wherein the gene encoding the Kras A146T mutant comprises the nucleotide sequence shown in SEQ ID NO:57.
The composition of any one of claims 69-112, wherein the gene encoding the Kras Y64H mutant comprises the nucleotide sequence shown in SEQ ID NO:59.
The composition of any one of claims 69-113, wherein the gene encoding the Kras A18D mutant comprises the nucleotide sequence shown in SEQ ID NO:61.
The composition of any one of claims 60-114, wherein the tandem minigene comprises, in order from the 5' end to the 3' end, the gene encoding the Kras G12D mutant, the gene encoding the Kras A59T mutant, the gene encoding the Kras A59T mutant Gene of G12V mutant, gene encoding Kras A146T mutant, gene encoding Kras G13D mutant, gene encoding Kras Y64H mutant, gene encoding Kras G12C mutant, gene encoding Kras Q61H mutant, gene encoding Kras A18D mutant The gene encoding the Kras G12A mutant, the gene encoding the Kras A146T mutant, and the gene encoding the Kras G12S mutant.
The composition of any one of claims 68-115, comprising the nucleotide sequence set forth in SEQ ID NO:65.
The composition of any one of claims 80-116, wherein the tandem minigene comprises, in order from the 5' end to the 3' end, a gene encoding a Kras G12D mutant, a gene encoding a Kras A146T mutant, a gene encoding a Kras A146T mutant The gene encoding the G12V mutant, the gene encoding the Kras A59T mutant, the gene encoding the Kras G13D mutant, the gene encoding the Kras Y64H mutant, and the gene encoding the Kras G12C mutant.
The composition of any one of claims 68-117, comprising the nucleotide sequence set forth in SEQ ID NO:66.
The composition according to any one of claims 80-118, wherein the tandem minigene comprises, from 5' end to 3' end, a gene encoding a Kras G12D mutant, a gene encoding a Kras A59T mutant, a gene encoding a Kras A59T mutant, and a gene encoding a Kras A59T mutant. A18D mutant gene, gene encoding Kras G12V mutant, gene encoding Kras A146T mutant, gene encoding Kras Q61H mutant, gene encoding Kras G13D mutant, gene encoding Kras A59T mutant, Kras A146T mutant and the gene encoding the Kras G12C mutant.
The composition of any one of claims 68-119, comprising the nucleotide sequence set forth in SEQ ID NO:67.
The composition of any one of claims 68-120, further comprising a polynucleotide encoding a secreted peptide.
The composition of claim 121, wherein the polynucleotide encoding a secreted peptide is a polynucleotide encoding a secreted peptide of CD14 protein.
The composition of claim 122, wherein the polynucleotide encoding the CD14 protein secretory peptide is located 5' to the gene encoding the Kras mutant.
The composition of any one of claims 122-123, wherein the polynucleotide encoding the CD14 protein secreted peptide comprises the nucleotide sequence set forth in any one of SEQ ID NO:37.
The composition of any one of claims 68-124, wherein the carrier comprises the isolated nucleic acid molecule of any one of claims 1-57.
The composition of any one of claims 68-125, wherein the vector comprises a viral vector.
The composition of any one of claims 68-126, wherein the vector comprises an oncolytic herpes simplex virus oHSV vector.
The composition of any one of claims 68-127, wherein the vector comprises a herpes simplex virus type I HSV-1 vector.
The composition of any one of claims 128, wherein the HSV-1 vector is deficient in the neurotoxic factor gamma 34.5 gene.
The composition of any one of claims 128-129, wherein, in the vector, the nucleic acid molecule is located between the UL3 gene and the UL4 gene of the HSV-1 vector.
The composition of any one of claims 68-130, wherein the vector comprises a promoter.
The composition of claim 131, wherein the promoter comprises a CMV promoter.
The composition of any one of claims 68-132, wherein the vector comprises the nucleotide sequence shown in GenBank No: GU734771.1 of the NCBI database.
The nucleic acid molecule of any one of claims 1-57, the carrier of any one of claims 58-66, the pharmaceutical composition of claim 67 and/or any one of claims 68-133 The application of the composition in the preparation of a medicament for treating tumors.
The use of claim 134, wherein the tumor comprises a solid tumor.
The use of any one of claims 134-135, wherein the tumor comprises non-small cell lung cancer.
The use of any one of claims 134-136, wherein the tumor comprises colorectal cancer.
The use of any one of claims 134-137, wherein the tumor comprises breast cancer.
The use of any one of claims 134-138, wherein the tumor comprises pancreatic cancer.