CN116323947A

CN116323947A - Nucleic acid molecule for encoding Kras gene mutant

Info

Publication number: CN116323947A
Application number: CN202180051207.XA
Authority: CN
Inventors: 张骅; 华坚; 陈晓庆; 刘园园
Original assignee: Shanghai Pharmaceutical Group Biotherapy Technology Co ltd; Shangyao Biotherapy Hong Kong Ltd
Current assignee: Shanghai Pharmaceutical Group Biotherapy Technology Co ltd; Shangyao Biotherapy Hong Kong Ltd
Priority date: 2020-09-01
Filing date: 2021-09-01
Publication date: 2023-06-23
Also published as: WO2022048574A1

Abstract

Provides a nucleic acid molecule for encoding a Kras gene mutant and application of an oncolytic herpes simplex virus (oHSV) vector containing the nucleic acid molecule in preparing antitumor drugs. Mutants include one or more of the 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.

Description

Nucleic acid molecule for encoding Kras gene mutant

Technical Field

The application relates to the field of biological medicine, in particular to a nucleic acid molecule for encoding a Kras gene mutant and application of an oncolytic herpes simplex virus (oHSV) vector containing the nucleic acid molecule in preparing an antitumor drug.

Background

Oncolytic viruses are a type of virus that is natural, or engineered, capable of specifically replicating in large numbers within tumor cells and eventually eliminating tumor cells, without killing normal tissue cells. Currently, there are up to tens of viruses used in oncolytic therapy, including herpes simplex virus, adenovirus, reovirus, measles virus, and the like. Among these, herpes simplex virus (herpes simplex virus, HSV) is the most interesting.

Herpes simplex virus (herpes simplex virus, HSV) is a virus commonly used in genetic engineering and is classified as type 1 and type 2. Along with the development of virology and genetic engineering technology, one can modify viral genes and apply them to the treatment of tumors. As early as 1991, martuza et al genetically engineered herpes simplex virus type 1 (herpes simplex virus type, HSV-1) to create oncolytic strains capable of inhibiting tumor cell proliferation and replication activity for the treatment of malignant brain tumors.

The Kras gene is a protooncogene, approximately 35kb long, located on chromosome 12, one of the RAS gene family members, encoding Kras protein. The Kras protein is a membrane-bound protein, is positioned on the inner side of a cell membrane and is positioned on a EGFR (epidermal growth factor receptor) signal path, and plays an important role in the occurrence and development of tumors. The processes of growth, proliferation, angiogenesis and the like of tumor cells all need intracellular proteins for signal transduction, but the Kras gene is a determinant of the transduction protein, and a Kras mutant encodes abnormal proteins, stimulates and promotes the growth and diffusion of malignant tumor cells, and is not influenced by the signals of upstream EGFR.

Currently, oncolytic viruses still have some problems in tumor therapy. For example, a patient who has been affected by a virus has residual viral antibodies, tumor cells cannot be completely eliminated, and the like. Therefore, it is necessary to develop a novel oncolytic virus and evaluate its antitumor activity.

Disclosure of Invention

In one aspect, the present application provides an isolated nucleic acid molecule comprising one or more genes each independently selected from the group consisting of the following Kras mutants: the mutants of Kras G12D, kras G13D, kras G12V, kras G13C, kras G12C, kras G13A, kras G12A, kras Q61L, kras G12R, kras Q61H, kras G12S, kras Q61R, kras a59T, kras a146T, kras Y64H and Kras a 18D.

In another aspect, the present application provides an isolated nucleic acid molecule that does not comprise a polynucleotide encoding a 6x His tag protein, and that comprises one or more genes each independently encoding a Kras mutant selected from the group consisting of: the mutants of Kras G12D, kras G13D, kras G12V, kras G13C, kras G12C, kras G13A, kras G12A, kras Q61L, kras G12R, kras Q61H, kras G12S, kras Q61R, kras a59T, kras a146T, kras Y64H and Kras a 18D.

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: the Kras A59T mutant, the Kras G12D mutant, the Kras G12V mutant, the KrasA146T mutant, the Kras G13D mutant, the Kras Y64H mutant and the 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 Kras mutants are arranged in tandem in the isolated nucleic acid molecule.

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

In certain embodiments, each of the genes encoding the Kras mutants are tandem to provide 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 adjacent to the N-terminus of the mutation site and at least 10 amino acids immediately adjacent to the C-terminus of the mutation site.

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

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

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

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

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

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

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

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

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

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

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

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

In certain embodiments, the Kras a59T mutant comprises SEQ ID NO:54, and an amino acid sequence shown in seq id no.

In certain embodiments, the Kras a146T mutant comprises SEQ ID NO:56, and an amino acid sequence shown in seq id no.

In certain embodiments, the Kras Y64H mutant comprises SEQ ID NO:58, and an amino acid sequence as set forth in seq id no.

In certain embodiments, the Kras a18D mutant comprises SEQ ID NO: 60.

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

In certain embodiments, the gene encoding the Kras G13D mutant comprises the nucleotide sequence shown 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 shown 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 SEQ ID NO: 55.

In certain embodiments, the gene encoding the Kras a146T mutant comprises SEQ ID NO: 57.

In certain embodiments, the gene encoding the Kras Y64H mutant comprises SEQ ID NO: 59.

In certain embodiments, the gene encoding the Kras a18D mutant comprises SEQ ID NO:61, and a nucleotide sequence set forth in seq id no.

In certain embodiments, 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, a gene encoding a Kras G12V mutant, a gene encoding a Kras a146T mutant, a gene encoding a Kras G13D mutant, a gene encoding a Kras Y64H mutant, a gene encoding a Kras G12C mutant, a gene encoding a Kras Q61H mutant, a gene encoding a Kras a18D mutant, a gene encoding a Kras G12A mutant, a gene encoding a Kras a146T mutant, and a gene encoding a 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, 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 G12V mutant, a gene encoding a Kras a59T mutant, a gene encoding a Kras G13D mutant, a gene encoding a Kras Y64H mutant, and a gene encoding a 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, in order from the 5 'end to the 3' end, a gene encoding a Kras G12D mutant, a gene encoding a Kras a59T mutant, a gene encoding a Kras a18D mutant, a gene encoding a Kras G12V mutant, a gene encoding a Kras a146T mutant, a gene encoding a Kras Q61H mutant, a gene encoding a Kras G13D mutant, a gene encoding a Kras a59T mutant, a gene encoding a Kras a146T mutant, and a gene encoding a Kras G12C mutant.

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 secretory peptide.

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

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

In certain embodiments, the polynucleotide encoding a CD14 protein secretion peptide comprises SEQ ID NO:37, or a nucleotide sequence set forth in any one of seq id no.

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 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 ohv vector.

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

In certain embodiments, the HSV-1 vector lacks 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 comprises a promoter.

In certain embodiments, the promoter comprises a CMV promoter.

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

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

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

In certain embodiments, the isolated nucleic acid molecules in the composition comprise one or more genes each independently encoding a Kras mutant selected from the group consisting of: the mutants of Kras G12D, kras G13D, kras G12V, kras G13C, kras G12C, kras G13A, kras G12A, kras Q61L, kras G12R, kras Q61H, kras G12S, kras Q61R, kras a59T, kras a146T, kras Y64H and Kras a 18D.

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: the Kras A59T mutant, the Kras G12D mutant, the Kras G12V mutant, the KrasA146T mutant, the Kras G13D mutant, the Kras Y64H mutant and the Kras G12C mutant.

In certain embodiments, the Kras a18D mutant comprises SEQ ID NO: 60.

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 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 secretory 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 nucleic acid sequence of SEQ ID NOs: 62-64.

In certain embodiments, the composition comprises a vector described herein, which comprises 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 ohv 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 comprises a promoter.

In certain embodiments, the promoter comprises a CMV promoter.

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

In another aspect, the present application provides the use of said nucleic acid molecule, said vector, said pharmaceutical composition and/or said composition for the preparation of a medicament for the treatment of a tumor.

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 will become readily apparent to 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 present disclosure enables one skilled in the art to make modifications to the disclosed embodiments without departing from the spirit and scope of the invention as described herein. Accordingly, the drawings and descriptions herein are to be regarded as illustrative in nature and not as restrictive.

Drawings

The specific features of the invention related to this application are set forth in the appended claims. The features and advantages of the invention that are related to the present application will be better understood by reference to the exemplary embodiments and the drawings that are described in detail below. The drawings are briefly described as follows:

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

FIG. 2A shows the structural composition of wild-type HSV-1 (F) described herein.

FIG. 2B shows the structural composition of the modified HSV-1 (F) -KR10 described herein.

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

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

FIG. 3 shows the trend of weight change in CT26.WT subcutaneously transplanted tumor mice as described herein.

FIG. 4A shows tumor volume changes in CT26.WT subcutaneously transplanted tumor mice as described herein.

FIG. 4B shows individual data of tumor volume change in CT26.WT subcutaneously transplanted tumor mice as described herein.

FIG. 5 shows tumor weight statistics of CT26.WT subcutaneously transplanted tumor mice as described herein.

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

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

FIG. 8 shows a photograph of the experimental end point of the tumor re-excitation model animal described in the present application.

Figure 9 shows the trend of body weight change in a549 subcutaneous engrafted tumor mice described herein.

Fig. 10 shows tumor volume changes in a549 subcutaneously transplanted tumor mice described herein.

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

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

Fig. 13 shows a photograph of a tumor after euthanasia of the a549 model described herein.

Detailed Description

Further advantages and effects of the invention of the present application will become apparent to those skilled in the art from the disclosure of the present application, from the following description of specific embodiments.

Definition of terms

In this application, the term "nucleic acid molecule" refers generally to polymers of nucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including naturally occurring (adenine, guanine, cytosine, uracil, and thymine), non-naturally occurring, and modified nucleic acids. In the present application, the nucleic acid molecule comprises a nucleotide sequence of a gene encoding each of the Kras mutants comprising at least 10 amino acids immediately adjacent to the N-terminus of the mutation site and at least 10 amino acids immediately adjacent to the C-terminus of the mutation site; the nucleic acid molecule further comprises a polynucleotide of a CD14 protein secretion peptide located at the 5 'end of the gene encoding the Kras mutant, and a polynucleotide of 6 xhis located at the 3' end of the gene of the Kras mutant.

In this application, the term "secretory peptide" generally refers to a short peptide chain that directs the transfer of a newly synthesized protein to the secretory pathway. In the application, the N of the Kras mutant polypeptide carries a CD14 protein secretion peptide sequence to guide the extracellular secretion of the Kras polypeptide, so that the Kras mutant polypeptide can be effectively presented after being replication expressed in tumor cells and released by antigen-presenting cells (APC) in the later period.

In the present application, the term "tag protein" generally refers to a protein that is fusion expressed together with a protein of interest in order to facilitate detection of the protein of interest. The tag protein used in the application is a 6XHis tag protein, also called as poly-histidine tag protein, and the sequence of histidine residues can be combined to several types of fixed ions (such as nickel, copper and cobalt) under specific buffer conditions, so that the purpose of easily detecting and purifying the His tag protein is achieved.

In this application, the term "vector" generally refers to a tool capable of carrying a foreign gene or DNA fragment of interest into a host cell for replication and expression. According to the origin, they can be classified into plasmid vectors, phage vectors, viral vectors and yeast artificial chromosome vectors. In the present application, the vector may be an HSV-1 vector capable of being linked to the nucleic acid molecule and replicating and expressing in a host cell.

In the present application, the term "HSV-1" generally refers to a neurotropic, enveloped double stranded DNA virus belonging to the subfamily alpha virus of the family Herpesviridae, having a genome length of 152kb, consisting of 2 interconnected long segments UL and short segments US. The method has the advantages of large gene capacity, short replication cycle, high infection efficiency, capability of inserting a plurality of therapeutic genes and the like, and becomes the first choice for researching anti-tumor drugs 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], neither the UL3 gene nor the UL4 gene being necessary for maintaining HSV virus survival and replication.

In the present application, the term "γ34.5 gene" generally refers to the apoptosis-inhibiting gene in the HSV-1 genome, which is also an important neurotoxic gene, geneBank No: GU734771.1. The knockout of the gamma 34.5 gene is also the most common attenuation strategy based on the oncolytic virus with HSV as a framework at present. HSV-1, which has defective function of the gamma 34.5 gene, has greatly reduced toxicity and can make virus replicate in tumor cells selectively by utilizing the characteristic of defective IFN-PKR signal transduction of the tumor cells. Because host PKR phosphorylation induced during virus infection can inhibit virus replication, but HSV-1 gamma 34.5 gene product can resist PKR inhibition, so that virus can replicate in normal cells, and many tumor cell PKR system defects lead to the fact that HSV virus with gamma 34.5 gene knocked out can effectively replicate in tumor cells to kill tumors but cannot replicate in normal cells, thereby providing conditions for HSV virus modification specific tumor killing.

In this application, the term "promoter" generally refers to a deoxyribonucleic acid (DNA) sequence located upstream of the 5' end of the transcription initiation site of a gene of interest, which enables transcription of the gene of interest, which can be recognized by RNA polymerase and initiates transcription of synthetic RNA. In the present application, the promoter means a DNA sequence located upstream of the 5' end of the transcription initiation site of the gene encoding the Kras mutant and controlling the transcription thereof.

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 in a suitable formulation. The pharmaceutical composition may be administered intravenously, intraperitoneally, subcutaneously, intramuscularly, topically, intradermally, or the like. In the present application, the pharmaceutical composition comprises a nucleic acid molecule encoding a Kras gene mutant, the simple oncolytic herpes virus vector, and optionally a pharmaceutically acceptable adjuvant.

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

In this application, "solid tumor" generally refers to abnormal tissue growth or mass, which generally does not contain cysts or liquid areas. Solid tumors may be benign (non-cancerous) or malignant (cancerous). About 90% of cancer cases clinically today are solid tumors. Different types of solid tumors are named for the cell type from which they are formed. For example, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, 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 tumor, head and neck cancer, and the like.

In this application, "pancreatic tumor" generally includes pancreatic sarcoma, pancreatic cyst adenoma. Pancreatic tumors are one of the common malignant tumors of the digestive tract, the most common of which, frequently occurring in the head of pancreas. Surgical resection is generally preferred for current clinical treatment of pancreatic tumors.

Detailed Description

Nucleic acid molecules

For example, the isolated nucleic acid molecule may comprise one or more genes encoding each Kras mutant separately. For example, the isolated nucleic acid molecule may comprise one or more genes encoding each Kras mutant separately that are not duplicated. For example, the isolated nucleic acid molecule can comprise a repeat of one or more genes encoding each Kras mutant separately. When comprising a plurality of genes encoding each Kras mutant, the genes of the Kras mutants are independently selected from: the mutants of Kras G12D, kras G13D, kras G12V, kras G13C, kras G12C, kras G13A, kras G12A, kras Q61L, kras G12R, kras Q61H, kras G12S, kras Q61R, kras a59T, kras a146T, kras Y64H and Kras a 18D.

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, kras a146T mutant, kras G13D mutant and Kras G12C mutant.

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

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 molecule.

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

In the present application, each of the genes encoding the Kras mutants can be tandem-arranged to obtain a tandem minigene.

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 adjacent to the N-terminus of the mutation site and at least 10 amino acids immediately adjacent to the C-terminus of the mutation site.

In certain embodiments, the sequence of each of the Kras mutants from 5 'to 3' is, in order, a gene encoding a Kras G12D mutant, a gene encoding a Kras a59T mutant, a gene encoding a Kras G12V mutant, a gene encoding a Kras a146T mutant, a gene encoding a Kras G13D mutant, a gene encoding a Kras Y64H mutant, a gene encoding a Kras G12C mutant, a gene encoding a Kras Q61H mutant, a gene encoding a Kras a18D mutant, a gene encoding a Kras G12A mutant, a gene encoding a Kras a146T mutant, and a gene encoding a Kras G12S mutant. For example, the isolated nucleic acid molecule may comprise the amino acid sequence shown in SEQ ID NO. 65.

In certain embodiments, the sequence of each of the Kras mutants from 5 'to 3' is, in order, a gene encoding a Kras G12D mutant, a gene encoding a Kras a146T mutant, a gene encoding a Kras G12V mutant, a gene encoding a Kras a59T mutant, a gene encoding a Kras G13D mutant, a gene encoding a Kras Y64H mutant, and a gene encoding a Kras G12C mutant. For example, the isolated nucleic acid molecule may comprise the amino acid sequence shown as SEQ ID NO. 66.

In certain embodiments, each of the Kras mutants is sequenced from the 5 'end to the 3' end as a gene encoding a Kras G12D mutant, a gene encoding a Kras a59T mutant, a gene encoding a Kras a18D mutant, a gene encoding a Kras G12V mutant, a gene encoding a Kras a146T mutant, a gene encoding a Kras Q61H mutant, a gene encoding a Kras G13D mutant, a gene encoding a Kras a59T mutant, a gene encoding a Kras a146T mutant, and a gene encoding a Kras G12C mutant. For example, the isolated nucleic acid molecule may comprise the amino acid sequence shown in SEQ ID NO. 67.

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

The Kras G12D mutant refers to a sequence comprising amino acid G at position 12, which is truncated from the amino acid sequence of a wild-type Kras polypeptide, wherein the amino acid G at position 12 in the truncated sequence is replaced by D, and 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 can 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 12, at least 13, at least 14) amino acids immediately adjacent to the C-terminus of the mutation site G12D. The amino acid sequence of the N end of the mutation site G12D is X amino acids extending from the 11 th amino acid to the N end (from the N end to the C end) in a wild-type Kras polypeptide sequence (shown as SEQ ID NO: 38), and the number of 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-terminal of the immediately adjacent mutation site G12D is Y amino acids extending from the 13 th amino acid (the sequence from the N end to the C end) to the C end in a wild-type Kras polypeptide sequence (shown as SEQ ID NO: 38), and 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 may 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 in that order from the N-terminus to the C-terminus.

For example, the Kras G12D mutant may comprise the amino acid sequence shown as SEQ ID NO. 1.

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

For example, the gene encoding the Kras G12D mutant may comprise, in order, a nucleotide sequence encoding the amino acid sequence shown as SEQ ID NO. 20, a nucleotide sequence encoding the mutation site G12D, and a nucleotide sequence encoding the amino acid sequence shown as SEQ ID NO. 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 amino acid G at position 13, truncated from the amino acid sequence of a wild-type Kras polypeptide, wherein the amino acid G at position 13 in the truncated sequence is replaced with D, and the sequence comprises at least 21 amino acids, such as 22, e.g. 23, such as 24, e.g. 25, e.g. 26, e.g. 27. The Kras G13D mutant can 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, at least 13, at least 14) amino acids immediately adjacent to the C-terminus of the mutation site G13D. The amino acid sequence of the N end of the mutation site G13D is X amino acids extending from the 12 th amino acid (the sequence from the N end to the C end) to the N end in a wild-type Kras polypeptide sequence (shown as SEQ ID NO: 38), and the 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 terminal of the immediately adjacent mutation site G13D is Y amino acids extending from the 14 th amino acid to the C terminal (from the N terminal to the C terminal) in a wild-type Kras polypeptide sequence (shown as SEQ ID NO: 38), and 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 that order from the N-terminus to the C-terminus.

For example, the Kras G13D mutant may comprise the amino acid sequence shown as 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-terminus of mutation site G13D, a nucleotide sequence encoding the amino acid sequence immediately adjacent to the C-terminus of mutation site G13D.

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

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

The Kras G12V mutant refers to a sequence comprising amino acid G at position 12, which is truncated from the amino acid sequence of a wild-type Kras polypeptide, wherein the amino acid G at position 12 in the truncated sequence is replaced by V, and 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 can comprise at least 9 (e.g., at least 10, at least 11) amino acids immediately adjacent to the N-terminus of the mutation site G12V, and at least 10 (e.g., at least 11, at least 12, at least 13, at least 14) amino acids immediately adjacent to the C-terminus of the mutation site G12V. The amino acid sequence of the N end of the G12V adjacent mutation site is X amino acids extending from the 11 th amino acid to the N end (from the N end to the C end) in a wild-type Kras polypeptide sequence (shown as SEQ ID NO: 38), and the 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 terminal of the immediately adjacent mutation site G12V is Y amino acids extending from the 13 th amino acid (the sequence from the N terminal to the C terminal) to the C terminal in a wild-type Kras polypeptide sequence (shown as SEQ ID NO: 38), and 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 this order from the N-terminus to the C-terminus.

For example, the Kras G12V mutant may comprise the amino acid sequence shown as SEQ ID NO. 7.

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

For example, the gene encoding the Kras G12V mutant may comprise, in order, a nucleotide sequence encoding the amino acid sequence shown as SEQ ID NO:20, a nucleotide sequence encoding the mutation site G12V, and a nucleotide sequence encoding the amino acid sequence shown as SEQ ID NO: 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 sequence which is intercepted from the amino acid sequence of wild-type Kras polypeptide and contains 13 th amino acid G, wherein the 13 th amino acid G in the intercepted sequence is replaced by C, and the sequence contains at least 21 amino acids, such as 22, for example 23, for example 24, for example 25, for example 26, for example 27. The Kras G13C mutant can 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, at least 13, at least 14) amino acids immediately adjacent to the C-terminus of the mutation site G13C. The amino acid sequence of the N end of the G13C immediate vicinity mutation site is X amino acids extending from the 12 th amino acid to the N end (from the N end to the C end) in a wild-type Kras polypeptide sequence (shown as SEQ ID NO: 38), and 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 terminal of the immediately adjacent mutation site G13C is Y amino acids extending from the 14 th amino acid to the C terminal (from the N terminal to the C terminal) in a wild-type Kras polypeptide sequence (shown as SEQ ID NO: 38), and 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 that order from the N-terminus to the C-terminus.

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

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

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

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

The Kras G12C mutant refers to a sequence comprising amino acid G at position 12, which is truncated from the amino acid sequence of a wild-type Kras polypeptide, wherein the amino acid G at position 12 in the truncated sequence is replaced by C, and 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 can comprise at least 9 (e.g., at least 10, at least 11) amino acids immediately adjacent to the N-terminus of the mutation site G12C, and at least 10 (e.g., at least 11, at least 12, at least 13, at least 14) amino acids immediately adjacent to the C-terminus of the mutation site G12C. The amino acid sequence of the N end of the G12C immediate vicinity mutation site is X amino acids extending from the 11 th amino acid to the N end (the sequence from the N end to the C end) in a wild-type Kras polypeptide sequence (shown as SEQ ID NO: 38), and the 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 terminal of the immediately adjacent mutation site G12C is Y amino acids extending from the 13 th amino acid (the sequence from the N terminal to the C terminal) to the C terminal in a wild-type Kras polypeptide sequence (shown as SEQ ID NO: 38), and 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 may 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 in that order from the N-terminus to the C-terminus.

For example, the Kras G12C mutant may comprise the amino acid sequence shown as SEQ ID NO. 9.

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

For example, the gene encoding the Kras G12C mutant may comprise, in order, a nucleotide sequence encoding the amino acid sequence shown as SEQ ID NO:20, a nucleotide sequence encoding the mutation site G12C, and a nucleotide sequence encoding the amino acid sequence shown as SEQ ID NO: 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 which is intercepted from the amino acid sequence of wild-type Kras polypeptide and contains 13 th amino acid G, wherein the 13 th amino acid G in the intercepted sequence is replaced by A, and the sequence contains at least 21 amino acids, such as 22, for example 23, for example 24, for example 25, for example 26, for example 27. The Kras G13A mutant can 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, at least 13, at least 14) amino acids immediately adjacent to the C-terminus of the mutation site G13A. The amino acid sequence of the N end of the immediately adjacent mutation site G13A is X amino acids extending from the 12 th amino acid to the N end (from the N end to the C end) in a wild-type Kras polypeptide sequence (shown as SEQ ID NO: 38), and the 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 terminal of the immediately adjacent mutation site G13A is Y amino acids extending from the 14 th amino acid to the C terminal (from the N terminal to the C terminal) in a wild-type Kras polypeptide sequence (shown as SEQ ID NO: 38), and 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 that order from the N-terminus to the C-terminus.

For example, the Kras G13A mutant may comprise the amino acid sequence shown as SEQ ID NO. 10.

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

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

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

The Kras G12A mutant refers to a sequence comprising amino acid G at position 12, which is truncated from the amino acid sequence of a wild-type Kras polypeptide, wherein the amino acid G at position 12 in the truncated sequence is replaced by a, and 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 can comprise at least 9 (e.g., at least 10, at least 11) amino acids immediately adjacent to the N-terminus of the mutation site G12A, and at least 10 (e.g., at least 11, at least 12, at least 13, at least 14) amino acids immediately adjacent to the C-terminus of the mutation site G12A. The amino acid sequence of the N end of the immediately adjacent mutation site G12A is X amino acids extending from the 11 th amino acid to the N end (from the N end to the C end) in a wild-type Kras polypeptide sequence (shown as SEQ ID NO: 38), and the number of 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 terminal of the immediately adjacent mutation site G12A is Y amino acids extending from the 13 th amino acid (the sequence from the N terminal to the C terminal) to the C terminal in a wild-type Kras polypeptide sequence (shown as SEQ ID NO: 38), and 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 that order from the N-terminus to the C-terminus.

For example, the Kras G12A mutant may comprise the amino acid sequence shown as SEQ ID NO. 11.

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

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

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

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

For example, the Kras Q61L mutant may comprise the amino acid sequence shown as SEQ ID NO. 12.

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

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

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

The Kras G12R mutant refers to a sequence comprising amino acid G at position 12, which is truncated from the amino acid sequence of a wild-type Kras polypeptide, wherein the amino acid G at position 12 in the truncated sequence is replaced by R, and 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 can comprise at least 9 (e.g., at least 10, at least 11) amino acids immediately adjacent to the N-terminus of the mutation site G12R, and at least 10 (e.g., at least 11, at least 12, at least 13, at least 14) amino acids immediately adjacent to the C-terminus of the mutation site G12R. The amino acid sequence of the N end of the G12R adjacent to the mutation site is X amino acids extending from the 11 th amino acid to the N end (the sequence from the N end to the C end) in a wild-type Kras polypeptide sequence (shown as SEQ ID NO: 38), and the number of the X amino acids 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 terminal of the immediately adjacent mutation site G12R is Y amino acids extending from the 13 th amino acid (the sequence from the N terminal to the C terminal) to the C terminal in a wild-type Kras polypeptide sequence (shown as SEQ ID NO: 38), and 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 that order from the N-terminus to the C-terminus.

For example, the Kras G12R mutant may comprise the amino acid sequence shown as SEQ ID NO. 15.

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

For example, the gene encoding the Kras G12R mutant may comprise, in order, a nucleotide sequence encoding the amino acid sequence shown as SEQ ID NO:20, a nucleotide sequence encoding the mutation site G12R, and a nucleotide sequence encoding the amino acid sequence shown as SEQ ID NO: 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 amino acid Q at position 61, truncated from the amino acid sequence of a wild-type Kras polypeptide, wherein amino acid Q at position 61 in the truncated sequence is replaced with H, and the sequence comprises at least 21 amino acids, e.g. 22, e.g. 23, e.g. 24, e.g. 25, e.g. 26, e.g. 27, e.g. 28, e.g. 29. The Kras Q61H mutant can 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 mutation site Q61H, and at least 10 (e.g., at least 11, at least 12, at least 13, at least 14) amino acids immediately adjacent to the C-terminus of mutation site Q61H. The amino acid sequence of the N end of the immediate vicinity mutation site Q61H is X amino acids extending from the 60 th amino acid to the N end (from the N end to the C end) in a wild-type Kras polypeptide sequence (shown as SEQ ID NO: 38), and 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 terminal of the immediate vicinity mutation site Q61H is Y amino acids extending from the 62 th amino acid (the sequence from the N terminal to the C terminal) to the C terminal in a wild-type Kras polypeptide sequence (shown as SEQ ID NO: 38), and 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 that order from the N-terminus to the C-terminus.

For example, the Kras Q61H mutant may comprise the amino acid sequence shown as SEQ ID NO. 16.

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

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

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

The Kras G12S mutant refers to a sequence comprising amino acid G at position 12, which is truncated from the amino acid sequence of a wild-type Kras polypeptide, wherein the amino acid G at position 12 in the truncated sequence is replaced by S, and 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 can comprise at least 9 (e.g., at least 10, at least 11) amino acids immediately adjacent to the N-terminus of the mutation site G12S and at least 10 (e.g., at least 11, at least 12, at least 13, at least 14) amino acids immediately adjacent to the C-terminus of the mutation site G12S. The amino acid sequence of the N end of the G12S adjacent mutation site is X amino acids extending from the 11 th amino acid to the N end (from the N end to the C end) in a wild-type Kras polypeptide sequence (shown as SEQ ID NO: 38), and the number of the X amino acids 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 terminal of the immediately adjacent mutation site G12S is Y amino acids extending from the 13 th amino acid (the sequence from the N terminal to the C terminal) to the C terminal in a wild-type Kras polypeptide sequence (shown as SEQ ID NO: 38), and 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 may 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 in this order from the N-terminus to the C-terminus.

For example, the Kras G12S mutant may comprise the amino acid sequence shown as SEQ ID NO. 17.

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

For example, the gene encoding the Kras G12S mutant may comprise, in order, a nucleotide sequence encoding the amino acid sequence shown as SEQ ID NO:20, a nucleotide sequence encoding the mutation site G12S, and a nucleotide sequence encoding the amino acid sequence shown as SEQ ID NO: 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 amino acid Q at position 61, truncated from the amino acid sequence of a wild-type Kras polypeptide, wherein amino acid Q at position 61 in the truncated sequence is replaced with R, and the sequence comprises at least 21 amino acids, e.g., 22, e.g., 23, e.g., 24, e.g., 25, e.g., 26, e.g., 27, e.g., 28, e.g., 29. The Kras Q61R mutant can 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 mutation site Q61R, and at least 10 (e.g., at least 11, at least 12, at least 13, at least 14) amino acids immediately adjacent to the C-terminus of mutation site Q61R. The amino acid sequence of the N end of the immediate vicinity mutation site Q61R is X amino acids extending from the 60 th amino acid to the N end (from the N end to the C end) in a wild-type Kras polypeptide sequence (shown as SEQ ID NO: 38), and 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 terminal of the immediate vicinity mutation site Q61R is Y amino acids extending from the 62 th amino acid (the sequence from the N terminal to the C terminal) to the C terminal in a wild-type Kras polypeptide sequence (shown as SEQ ID NO: 38), and 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 that order from the N-terminus to the C-terminus.

For example, the Kras Q61R mutant may comprise the amino acid sequence shown as SEQ ID NO. 18.

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

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

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

In the nucleic acid molecules described herein, each of the genes encoding the Kras mutants is a Minigene (Minigene). The minigene (minigene) generally refers to a short segment of a gene that can be used for functional and expression regulatory mechanism studies of genes and to construct more complex minigenes comprising multiple exons and introns. For example, in the present application, the minigene (minigene) may be a fragment comprising a gene encoding each of the Kras mutants, the genes encoding each of the Kras mutants being arranged in tandem in the order shown in fig. 1, forming a Tandem Minigene (TMG) of the Kras gene mutant.

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

For example, the tandem minigene of the Kras gene mutant may comprise a sequence encoding SEQ ID NO: 43.

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

The Kras a59T mutant refers to a sequence comprising amino acid a at position 59, truncated from the amino acid sequence of a wild-type Kras polypeptide, wherein the amino acid a at position 59 in the sequence is replaced with T, and 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 can comprise at least 9 (e.g., at least 10, at least 11) amino acids immediately adjacent to the N-terminus of mutation site a59T and at least 10 (e.g., at least 11, at least 12, at least 13, at least 14) amino acids immediately adjacent to the C-terminus of mutation site a 59T.

For example, the Kras A59T mutant may comprise the amino acid sequence shown as SEQ ID NO. 54.

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

The Kras A146T mutant refers to a sequence which is intercepted from the amino acid sequence of wild-type Kras polypeptide and contains 146 th amino acid A, the 146 th amino acid A in the intercepted sequence is replaced by T, and the sequence contains at least 20 amino acids, such as 21, for example 22, such as 23, for example 24, for example 25, for example 26. The Kras a146T mutant can comprise at least 9 (e.g., at least 10, at least 11) amino acids immediately adjacent to the N-terminus of mutation site a146T and at least 10 (e.g., at least 11, at least 12, at least 13, at least 14) amino acids immediately adjacent to the C-terminus of mutation site a 146T.

For example, the Kras A146T mutant may comprise the amino acid sequence shown as SEQ ID NO. 56.

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

The Kras Y64H mutant refers to a sequence comprising amino acid Y at position 64, which is truncated from the amino acid sequence of a wild-type Kras polypeptide, and the amino acid Y at position 64 in the truncated sequence is replaced by H, wherein 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 can comprise at least 9 (e.g., at least 10, at least 11) amino acids immediately adjacent to the N-terminus of mutation site Y64H and at least 10 (e.g., at least 11, at least 12, at least 13, at least 14) amino acids immediately adjacent to the C-terminus of mutation site Y64H.

For example, the Kras Y64H mutant may comprise the amino acid sequence shown as SEQ ID NO. 58.

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

The Kras a18D mutant refers to a sequence comprising amino acid a at position 18, which is truncated from the amino acid sequence of a wild-type Kras polypeptide, wherein the amino acid a at position 18 in the truncated sequence is replaced by D, and 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 can comprise at least 9 (e.g., at least 10, at least 11) amino acids immediately adjacent to the N-terminus of mutation site a18D and at least 10 (e.g., at least 11, at least 12, at least 13, at least 14) amino acids immediately adjacent to the C-terminus of mutation site a 18D.

For example, the Kras A18D mutant may comprise the amino acid sequence shown as SEQ ID NO. 60.

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

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

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 a mutant of a tumor-associated driver gene. For example, the other tumor antigen may be TP53. For example, the other tumor antigen may be the Braf gene.

The nucleic acid molecule may further comprise a polynucleotide encoding a secretory peptide located 5' to the tandem minigene of the Kras gene mutant. The secretion peptide can guide the secretion of the Kras polypeptide outside tumor cells, and can be effectively presented to T cells after being ingested by antigen-presenting cells (APC), thereby playing an immune role. The APC cells are accessory cells which can present antigen substances to T cells in the immune response process, and the major histocompatibility complex (major histocompatibility complex, MHC) on the cell surface can be combined with antigen, namely Kras mutant polypeptide, and the combined complex can be recognized by the T cells. For example, the secretory peptide may include Hmm secretory peptide, CD14 protein secretory peptide.

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

The nucleic acid molecule may further 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 detecting Kras mutant polypeptide.

For example, the tag protein may include FLAG tag protein, HA tag protein, C-Myc tag protein, 6 XHis tag protein.

For example, the polynucleotide encoding the 6x His tag protein may comprise the sequence of SEQ ID NO:40, and a nucleotide sequence shown in seq id no.

Carrier body

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

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

In certain embodiments, genes described herein that do not comprise a protein encoding a 6x His tag may be inserted into a viral vector. The vector may be an oncolytic herpes simplex virus (ohv) vector. For example, the oncolytic herpes simplex virus (ohv) vector may be a herpes simplex virus type I (HSV-1) vector. The HSV-1 vector is a gene deleted, which may be neurotoxic factor gamma 34.5.

In the present application, the vector may be an oncolytic herpes simplex virus (ohv) vector. For example, the oncolytic herpes simplex virus (ohv) vector may be a herpes simplex virus type I (HSV-1) vector. The HSV-1 vector is a gene deleted, which may be neurotoxic factor gamma 34.5.

For example, the HSV-1 vector lacks two copies of neurotoxic factor gamma 34.5.

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

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

For example, the promoter sequence may comprise the sequence as set forth in SEQ ID NO: 41.

In this application, the tandem minigene of the Kras gene mutant may be located between the UL3 gene and 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 NCBI database.

In another aspect, the present application also provides a pharmaceutical composition comprising said nucleic acid molecule, and/or said vector, and optionally a pharmaceutically acceptable adjuvant. The pharmaceutical composition refers to a formulation in a form such that the biological activity of the active ingredient contained therein is tolerated and is free of additional ingredients having unacceptable toxicity to the subject to whom the composition is to be administered. By pharmaceutically acceptable adjuvant is meant any substance capable of assisting or improving the action of a drug. The adjuvant may be a particulate adjuvant, for example, an aluminium hydroxide adjuvant. The adjuvant may be a non-particulate adjuvant, for example, a cytokine. The adjuvants may be derived from plants such as saponins and polysaccharide extracts. The adjuvant may be derived from pathogenic microorganisms, e.g., monophosphoryl lipid, cholera toxin, etc.

Composition and method for producing the same

In another aspect, the present application also provides a composition that may comprise an isolated nucleic acid molecule as described herein, a vector as described herein or a pharmaceutical composition as described herein, and physiological saline.

In the present application, the composition may comprise any one or more of the isolated nucleic acid molecules described herein, as well as 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: the mutants of Kras G12D, kras G13D, kras G12V, kras G13C, kras G12C, kras G13A, kras G12A, kras Q61L, kras G12R, kras Q61H, kras G12S, kras Q61R, kras a59T, kras a146T, kras Y64H and Kras a 18D.

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: the Kras A59T mutant, the Kras G12D mutant, the Kras G12V mutant, the KrasA146T mutant, the Kras G13D mutant, the Kras Y64H mutant and the Kras G12C mutant.

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 isolated nucleic acid molecule in the composition. 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 isolated nucleic acid molecule in the composition. 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 isolated nucleic acid molecule in the composition. 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 isolated nucleic acid molecule in the composition. 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 isolated nucleic acid molecule in the composition. 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 isolated nucleic acid molecule in the composition. In the present application, each of the genes encoding the Kras mutants may be arranged in tandem in the isolated nucleic acid molecule. In the present application, each of the genes encoding the Kras mutants may be Minigene. In the present application, each of the genes encoding the Kras mutants can be tandem-arranged to obtain a tandem minigene.

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

In the present application, each of the Kras mutants in the isolated nucleic acid molecules in the composition may comprise at least 9 amino acids immediately adjacent to the N-terminus of the mutation site and at least 10 amino acids immediately adjacent to the C-terminus of the mutation 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 of SEQ ID NO:54, and an amino acid sequence shown in seq id no. In the present application, the Kras a146T mutant may comprise the amino acid sequence of SEQ ID NO:56, and an amino acid sequence shown in seq id no. In the present application, the Kras Y64H mutant may comprise the amino acid sequence of SEQ ID NO:58, and an amino acid sequence as set forth in seq id no. In the present application, the Kras a18D mutant may comprise the amino acid sequence of SEQ ID NO: 60.

In the present application, the tandem minigene may comprise, from the 5 'end to the 3' end, a gene encoding a Kras G12D mutant, a gene encoding a Kras a59T mutant, a gene encoding a Kras G12V mutant, a gene encoding a Kras a146T mutant, a gene encoding a Kras G13D mutant, a gene encoding a Kras Y64H mutant, a gene encoding a Kras G12C mutant, a gene encoding a Kras Q61H mutant, a gene encoding a Kras a18D mutant, a gene encoding a Kras G12A mutant, a gene encoding a Kras a146T mutant, and a gene encoding a Kras G12S mutant, in this order, in the isolated nucleic acid molecule in the composition. For example, the tandem minigene may comprise the nucleotide sequence set forth in SEQ ID NO. 65.

In the present application, the tandem minigene may comprise, in the isolated nucleic acid molecule in the composition, 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 G12V mutant, a gene encoding a Kras a59T mutant, a gene encoding a Kras G13D mutant, a gene encoding a Kras Y64H mutant, and a gene encoding a Kras G12C mutant, in this order. For example, the tandem minigene may comprise the nucleotide sequence set forth in SEQ ID NO. 66.

In the present application, the tandem minigene may comprise, from the 5 'end to the 3' end, a gene encoding a Kras G12D mutant, a gene encoding a Kras a59T mutant, a gene encoding a Kras a18D mutant, a gene encoding a Kras G12V mutant, a gene encoding a Kras a146T mutant, a gene encoding a Kras Q61H mutant, a gene encoding a Kras G13D mutant, a gene encoding a Kras a59T mutant, a gene encoding a Kras a146T mutant, and a gene encoding a Kras G12C mutant, in this order, in the isolated nucleic acid molecule in the composition. 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 to 67, and physiological saline. In certain embodiments, the composition may comprise the nucleotide sequence set forth in SEQ ID NO. 63, as well as physiological saline.

In the present application, the composition may comprise any one or more of the carriers described herein, as well as 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 to 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 NOs 63.

In the present application, the composition may comprise any one or more of the pharmaceutical compositions described herein, as well as physiological saline.

Use of the same

In another aspect, the application also provides the nucleic acid molecule, the vector, the pharmaceutical composition and/or the application of the composition in preparing medicines for treating tumors. For example, the tumor may be a solid tumor. For example, the tumor may be non-small cell lung cancer. For example, the tumor may be colorectal cancer. For example, the tumor may be pancreatic cancer. For example, the tumor may be breast cancer.

The present application provides said nucleic acid molecules, said vectors, said pharmaceutical compositions and/or said compositions, which can treat tumors.

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

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

The isolated nucleic acid molecules, the vectors, the pharmaceutical compositions and/or the compositions described herein can be effective for inhibiting proliferation and/or growth of tumor cells. The Kras mutant TMG-2, the Kras mutant TMG-5 and the Kras mutant TMG-7 constructed by the application are expressed in an oncolytic virus vector, and can exert good therapeutic effect.

In another aspect, the present application provides the following embodiments:

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

2. The isolated nucleic acid molecule of embodiment 1, wherein each of the genes encoding Kras mutants is 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 Kras mutants is 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 immediately adjacent to the N-terminus of the mutation site and at least 10 amino acids immediately adjacent to the C-terminus of the mutation 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.

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 of embodiments 1-29, further comprising a polynucleotide encoding a secretory peptide.

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

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

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

34. The isolated nucleic acid molecule of any 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 6 xhis.

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 of SEQ ID NO:40, and a nucleotide sequence shown in seq id no.

38. The isolated nucleic acid molecule of any one of embodiments 1-37, comprising the nucleic acid sequence 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 ohv 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 lacks the neurotoxic factor γ34.5 gene.

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

45. The vector according to 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 according to any of embodiments 39-46, comprising a nucleotide sequence set forth 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 vector of any one of embodiments 39-47, and optionally a pharmaceutically acceptable adjuvant.

49. Use of a nucleic acid molecule according to any one of embodiments 1 to 38 and/or a vector according to any one of embodiments 39 to 47 for the preparation of a medicament for the treatment of a tumor.

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

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

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

Examples

EXAMPLE 1 construction of BAC plasmid of recombinant Virus KR10

1.1 Synthesis of nucleotide sequence of CD14 protein-Kras mutant (TMG) -6 xHis

After the sequence from amino acid 1 to amino acid 24 (SEQ ID NO: 1) of the wild-type Kras polypeptide is intercepted, the G amino acid 12 in the intercepted sequence is replaced by D amino acid, the N end of the adjacent G12D mutation site is 11 wild-type amino acids (SEQ ID NO: 2), the C end of the adjacent G12D mutation site is 12 wild-type amino acids (SEQ ID NO: 3), and a Kras G12D mutant comprising nucleotides encoding the amino acids of which the N end and the C end are connected is obtained, wherein the nucleotide sequence is shown as SEQ ID NO: 19.

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

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

Referring to the above method, nucleotide sequences of a Kras G12D mutant (shown as SEQ ID NO: 19), a Kras A146T mutant (shown as SEQ ID NO: 57), a Kras G12V mutant (shown as SEQ ID NO: 25), a Kras A59T mutant (shown as SEQ ID NO: 55), a Kras G13D mutant (shown as SEQ ID NO: 22), a Kras Y64H mutant (shown as SEQ ID NO: 59) and a Kras G12C mutant (shown as SEQ ID NO: 27) were synthesized, respectively, and the nucleotide sequences encoding each Kras mutant were concatenated in the order described to obtain a Kras mutant in the form of a Tandem Minigene (TMG), which is called TMG-5. The above mutant sequences were synthesized by Ai Ji Biotechnology.

With reference to the above method, nucleotide sequences of the Kras G12D mutant, the Kras a59T mutant, the Kras a18D mutant, the Kras G12V mutant, the Kras a146T mutant, the Kras Q61H, the Kras G13D mutant, the Kras a59T mutant, the Kras a146T mutant and the Kras G12C mutant are synthesized, respectively, and the nucleotide sequences encoding the respective Kras mutants are concatenated in the order described to obtain a Kras mutant in the form of a Tandem Minigene (TMG), which is called TMG-7. The above mutant sequences were synthesized by Ai Ji Biotechnology.

A CD14 protein secretion peptide (synthesized Yu Aiji Biotechnology Co., nucleotide sequence SEQ ID NO: 37) is introduced into the 5 'end of the gene encoding the TMG form Kras mutant, and a 6 xHis tag (synthesized Yu Aiji Biotechnology Co., nucleotide sequence SEQ ID NO: 40) is introduced into the 3' end, so that a nucleotide sequence encoding the CD14 protein-Kras mutant (TMG) -6 xHis (shown as SEQ ID NO: 47-49) is obtained, and the structural form of the CD14 protein-Kras mutant (TMG) -6 xHis is shown in FIG. 1.

1.2 insertion of Kras mutant Gene

Two copies of the neurovirulence factor gamma 34.5 gene were deleted on wild-type HSV-1 (F) (shown in FIG. 2A), and the nucleotide sequence of the CD14 protein-Kras mutant (TMG) -6 xHis described in example 1.1 was inserted between the UL3 and UL4 genes, to obtain the nucleotide sequence of UL3-CD14 protein-Kras-6 xHis-UL 4 (shown in SEQ ID NO: 50-52).

1.3 construction of BAC plasmids of recombinant viruses

The intermediate plasmid pKO5.1 for BAC recombination (taught by Dr. Bernard Roizman, university of Chicago) was constructed by molecular cloning, the nucleotide sequence of UL3-CD14 protein-Kras-6 x His-UL4 described in example 1.2 was inserted into pKO5.1, which was transformed by electroporation into E.coli harboring HSV BAC deleted two copies of the neurovirulence factor γ34.5 gene (as shown in SEQ ID NO: 53), to obtain the BAC plasmid of recombinant virus KR 10. The nucleic acid sequence structure of KR10 is shown in FIG. 2B.

EXAMPLE 2 construction of BAC plasmid of recombinant Virus KR11

A CD14 protein secretion peptide (Ai Ji Biotechnology Co., SEQ ID NO: 37) was introduced at the N-terminus and a 6 XHis tag (Ai Ji Biotechnology Co., SEQ ID NO: 40) was introduced at the C-terminus of GFP protein by the method of reference example 1.1. A BAC plasmid containing the KR11 nucleotide sequence was constructed by the method of reference example 1.3, by synthesizing the negative control virus UL3-CD14 protein-GFP-6 XHis-UL 4 nucleotide sequence (SEQ ID NO: 45) by the method of reference example 1.2. The nucleic acid sequence structure of KR11 is shown in FIG. 2C.

Example 3 construction of BAC plasmid of positive control Virus KR12

A Flu A Mp antigen peptide having an amino acid sequence shown as SEQ ID NO. 42 was known, and its nucleotide sequence was obtained based on its amino acid sequence, by introducing a CD14 protein secretion peptide (Ai Ji Biotechnology Co., SEQ ID NO. 37) at its N-terminus and a 6 XHis tag (Ai Ji Biotechnology Co., SEQ ID NO. 40) at its C-terminus in accordance with the method of example 1.1. A BAC plasmid containing the KR12 nucleotide sequence was constructed by the method of reference example 1.3, by synthesizing the nucleotide sequence of positive control virus UL3-CD14 protein-FLU-6 xHis-UL 4 (SEQ ID NO: 46) by the method of reference example 1.2. The nucleic acid sequence structure of KR12 is shown in FIG. 2D.

Example 4 cell experiment

4.1 packaging of recombinant viruses and TK Gene repair

The BAC plasmid containing KR10 nucleotide sequence, the BAC plasmid containing KR11 nucleotide sequence and the BAC plasmid containing KR12 nucleotide sequence constructed in the above examples 1-3 were respectively co-transfected with pRB103 plasmid (containing HSV-1F virus TK gene) into Vero cells, and placed at 37℃and 5% CO ₂ After 4 hours incubation in incubator, the medium was changed, fresh complete growth medium (5% NBCS/DMEM) was added, and the culture was continued until virus was successfully packaged and plaque appeared. And (3) collecting cells, repeatedly freezing and thawing to obtain virus stock solution, infecting Vero-delta TK cells (Vero cells deleted of TK genes) with the virus stock solution, adding HAT for screening, and selecting monoclonal viruses. The monoclonal virus was again infected with Vero- Δtk cells, HAT screened and picked. The monoclonal virus selected after HAT multiple screening is used for infecting Vero cells and amplifying virus so as to obtain the recombinant virus with final TK gene repair. The expression of Kras polypeptide, GFP protein and Flu A MP antigen peptide was detected by Western blotting WB method. The antibody used in WB was HRP-labeled 6*His monoclonal antibody from Proteintech. The WB assay showed expression of Kras polypeptide, GFP protein, flu A MP antigen peptide.

4.2 in vitro killing tumor cell test by recombinant Virus

Paving a tumor cell plate, respectively infecting tumor cells with recombinant viruses KR10, KR11 and KR12 prepared in example 4.1 at different titers, and detecting cell proliferation toxicity by using CCK8 after virus action for 72 hours, thereby detecting the capability of killing tumors by the recombinant viruses KR10, KR11 and KR 12. The results show that KR10, KR11, KR12 have the ability to kill tumor cells.

Example 5 in vivo experiments in mice

Thymidine kinase gene repair (TK repair), and the repaired virus is subjected to in vivo mouse experiment after being successfully repaired by protein detection. The results show that KR10 and KR11 have good tumor inhibiting effect.

5.1 Anti-tumor efficacy study of KR10 and KR11 on CT26.WT mouse colorectal cancer BALB/c mouse subcutaneous transplantation tumor

KR10 is a genetically engineered oncolytic virus. KR10 herpesvirus is based on wild type herpesvirus HSV-1 (F strain), and the gamma 34.5 gene of one copy of each of the IR region and the TR region is knocked out, so that the gamma 34.5 gene of two copies is knocked out simultaneously, and virus toxicity is weakened. In addition, KRAS mutant polypeptides were inserted into the virus in TMG form of G12D-A146T-G12V-A59T-G13D-Y64H-G12C. KR11 is a GFP inserted into the viral backbone of KR 10.

The experimental animals used in this experiment were BALB/c mice, SPF grade, female, 80, 5-6 weeks old. Zhejiang Vitolihua laboratory animal technologies Co., ltd., animal eligibility number: 20201214Abzz0619000226.

WT mouse colorectal cancer cells were cultured in RPMI-1640 medium containing 10% Fetal Bovine Serum (FBS) and 100U/mL penicillin and 100. Mu.g/mL streptomycin were added. 5% CO2 at 37 ℃.

Taking cells with good growth state for experiment, collecting and centrifuging the cells, and discarding the original culture mediumAdding appropriate amount of PBS, resuspending, counting, and adjusting cell density to 2×10 ⁷ cells/mL were placed on ice for use.

A BALB/c mouse subcutaneous engraftment tumor model of mouse colon cancer (CT26. WT) was established. Tumor cell suspensions were inoculated subcutaneously in the right middle wing of BALB/c mice until the average tumor volume in the mice was as long as about 120mm ³ 35 tumor-bearing mice were selected, randomly divided into 5 groups, 7 animals/group according to tumor volume, and D1 was used on the day of group administration, which was vehicle control group (vehicle was DPBS containing 10% (w/v) glycerol), KR11 low dose group 1×10 ⁶ PFU/alone (viral backbone control group), KR11 high dose group 1X 10 ⁷ PFU/alone (viral backbone control), KR10 low dose group (1X 10) ⁶ PFU/alone) and KR10 high dose group (1X 10) ⁷ PFU/PFU only). The administration was 1 time per week (qw×3) and 3 times in total. The experimental animal groupings and dosing regimens are shown in table 1. The test sample is injected in tumor with the administration volume of 50 μl/animal and tumor volume of less than 80mm ³ When in single-point injection (the injector enters a lesion area through a single needle inlet, and the injection point is the middle part of tumor tissue); tumor volume is 80mm ³ ～140mm ³ At the time, the injection is divided into 2 points (the injector enters the lesion area through a single needle inlet, and the injection points are 1/3 and 2/3 of the long diameter of the tumor tissue); tumor volume of more than 140mm ³ At the same time, the injection is carried out at 3 points (the injector enters the lesion area through one needle inlet, the injection points are 1/3 and 2/3 of the long diameter of the tumor tissue, the second needle enters the lesion area through the other needle inlet, and the injection points are positioned outside the middle part of the tumor tissue.

Table 1 experimental animal groups

Animal status, animal mortality, and clinical symptoms were observed daily, including but not limited to: mental state, behavioral activity, tumor ulceration, etc. Tumor diameter was measured weekly, animals were weighed 2 times, and swelling of surviving animals was removed at the end of experimental periodTumors were weighed. Calculating relative proliferation rate T/C%, tumor growth inhibition rate TGI% and tumor weight inhibition rate IR _TW ％。

Tumor volume calculation formula: tumor volume (mm) ³ ) =1/2×long diameter×short diameter ² 。

The relative tumor proliferation rate T/C (%) and the tumor growth inhibition rate TGI (%) were used as experimental evaluation indexes. T/C (%) = (T/T0)/(C/C0) ×100% where T, C is tumor volume of the dosing group, control group at the end of the experiment; t0 and C0 are tumor volumes of the administration group and the control group at the beginning of the experiment. If T > T0, tumor growth inhibition rate (TGI)% = [1-T/C ] ×100%; if T < T0, tumor Growth Inhibition (TGI)% = [1- (T-T0)/T0 ] ×100%.

Tumor complete remission CR: tumor volume less than 50mm ³ 。

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

All experimental data are expressed in Mean ± standard error (Mean ± SEM). The statistics are carried out according to the following method: comparing every two by using T test, if P is more than 0.05, the test is not obvious; if P is less than or equal to 0.05, the test is obvious.

Results:

the major clinical symptom of the vehicle control group and animals of each experimental group was tumor crusting throughout the experiment. During the experiment, no abnormality was seen in the clinical observation of the animals, and no abnormality was seen in the general anatomy. The animals were observed clinically in Table 2-1 and Table 2-2.

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

On day 20, the average tumor volume of mice in vehicle control group was 3968.41 + -653.80 mm ³ Average tumor volumes of KR11 low and high dose groups were 3093.03 + -886.41 mm, respectively ³ And 2308.04 + -789.41 mm ³ Average tumor volumes of KR10 low and high dose groups were 1701.34 + -512.96 mm, respectively ³ And 1309.76 + -628.21 mm ³ . The average tumor volume of the KR11 low and high dose groups showed a reduced trend compared to the vehicle control group, but no significant difference was seen (P>0.05 With significantly reduced average tumor volume in the KR10 low and high dose group (P)<0.05 A) is provided; the tumor volumes of the KR10 low and high dose groups were decreased compared to the same dose of KR11, but there was no statistical difference (P>0.05). The relative tumor proliferation rates T/C% of the KR11 low-dose group and the KR10 high-dose group are 62.23% and 53.22%, respectively, and 35.97% and 20.99% respectively. Tumor growth inhibition TGI% for KR11 low and high dose groups were 37.77% and 46.78%, respectively, and for KR10 low and high dose groups were 64.03% and 79.01%, respectively. At the same dose, the inhibition rate of KR10 on tumors is higher than that of KR11. During the test, the tumor of 2 animals is completely relieved (tumor disappears) in total with high KR10 dosage; in the other groups, no tumor disappeared. Tumor volume statistics are shown in table 5. Tumor volume statistics are shown in Table 6-1 and Table 6-2, individual data are shown in Table 7-1 and Table 7-2, and tumor volume change trend is shown in FIGS. 4A-4B.

On day 20, the average tumor weight of the vehicle control group was 3.914 + -0.617 g, and the average tumor weight of the KR11 low and high dose groups was 2.788 + -0.628 g and 2.353+ -0.804 g, respectively; the average tumor weights of the KR10 low and high dose groups are 1.615+/-0.508 g and 1.226+/-0.526 g respectively. The average tumor weights in the KR11 low and high dose groups showed a reduced trend compared to the vehicle control group, but no significant difference was seen (P>0.05). KR10 low and high dose group had significantly reduced average tumor weight (P<0.05). Tumor weight inhibition rate IR of KR11 low and high dose group _TW (%) was 28.77% and 39.88%, respectively. Tumor weight inhibition rate IR of KR10 low and high dose group _TW (%) 58.74% and 68.68%, respectively. The statistical results are shown in Table 8. Tumor weight statistics are shown in table 9. Individual data list10. The tumor weight statistical chart is shown in fig. 5. The euthanized photographs are shown in FIGS. 6A-6B.

On day 25, animals with complete disappearance of 2 tumors in the KR10 high dose group were ct26.wt tumor re-challenged, i.e. the mice were vaccinated on opposite sides with the same amount of ct26.wt tumor cells, and tumor growth was observed. 30 days after re-challenge, all animals did not develop tumors. It was demonstrated that after KR10 administration, long-term anti-ct26.wt tumor immune memory function was established in the above 2 animals. The change in tumor volume after tumor re-excitation is shown in figure 7. Tumor volume individual data are shown in Table 7-3. The end point photograph of the tumor re-excitation model animal experiment is shown in fig. 8.

Conclusion: experimental results show that in the mouse colorectal cancer CT26.WT cell mouse subcutaneous transplantation tumor model, KR10 can obviously inhibit the growth of tumors under the condition of the experiment, the inhibition rate of KR10 on the tumors is higher than KR11 under the same dose, and 2 animals in the KR10 high-dose group completely regress the tumors. The results show that the anti-tumor effect of KR10 is superior to that of the viral backbone KR11. The re-excitation model proves that KR10 can stimulate mice to establish long-term anti-tumor immune memory function.

TABLE 2-1 statistical table for observation of clinical symptoms of experimental animals

Note that: normal, 1 death, 2 listlessness, 3 reduced activity, 4 tremors, 5 erectile hairs, 6 tumor crusting, 7 bow backs, NA dead/euthanized.

TABLE 2-2 statistical table for observation of clinical symptoms of experimental animals

Table 3-1 Experimental animal weight statistics (g, mean.+ -. SEM)

Table 3-2 Experimental animal weight statistics (g, mean.+ -. SEM)

Note that: * Indicating p <0.05 compared to vehicle control.

TABLE 4-1 Experimental animal weight individual data sheet (g)

TABLE 4-2 Experimental animal weight individual data sheet (g)

Table 5 statistics of tumor volumes for each group (mean.+ -. SEM)

Note that: the # represents the P value compared to the control group. And (2) the following steps: p-value compared to KR11 at the same dose.

Table 6-1 Experimental animal tumor volume statistics (mm 3, mean.+ -. SEM)

Table 6-2 Experimental animal tumor volume statistics (mm 3, mean.+ -. SEM)

Note that: * Indicating p <0.05 compared to vehicle control.

TABLE 7-1 Experimental animal tumor volume (mm) ³ ) Individual data sheet

TABLE 7-2 Experimental animal tumor volume (mm) ³ ) Individual data sheet

TABLE 7-3 Experimental animal tumor volume (mm) ³ ) Individual data sheet

Table 8 tumor weights (g, mean.+ -. SEM) for animals of each group

Group of	Solvent/test article	Dosage of	Tumor weight (g)	P < number > -value	P < plus > $value	IR _TW (％)
1	Vehicle control group	-	3.914±0.617	-	-	-
2	KR11 low dose group	1×10 < 6 > PFU/only	2.788±0.628	0.225	-	28.77
3	KR11 high dose group	1×10 < 7 > PFU/only	2.353±0.804	0.150	-	39.88
4	KR10 low dose group	1×10 < 6 > PFU/only	1.615±0.508*	0.014	0.172	58.74
5	KR10 high dose group	1×10 < 7 > PFU/only	1.226±0.526**	0.006	0.263	68.68

Note that: * Indicating p <0.05 compared to vehicle control. * P <0.01 compared to vehicle control group. The # represents the P value compared to the control group. The following steps: compared with the same dosage of KR 11.

Table 9 experimental animal tumor weight statistics table

Group of	Solvent/test article	Dosage of	Tumor weight (g, mean.+ -. SEM)
1	Vehicle control group	-	3.914±0.617
2	KR11 low dose group	1×10 < 6 > PFU/only	2.788±0.628
3	KR11 high dose group	1×10 < 7 > PFU/only	2.353±0.804
4	KR10 low dose group	1×10 < 6 > PFU/only	1.615±0.508*
5	KR10 high dose group	1×10 < 7 > PFU/only	1.226±0.526**

Note that: * Indicating p <0.05 compared to vehicle control. * P <0.01 compared to vehicle control group.

Table 10 data sheet of experimental animal tumor weight individuals

5.2 Antitumor effect of KR10 and KR11 herpesvirus intratumoral administration on BALB/c nude mice subcutaneously transplanted human non-small cell lung cancer cell (A549) model

The experimental animals used in this experiment were BALB/C nude mice, SPF grade, female, 50 animals, 5-6 weeks old. Animal pass number from Zhejiang Vitolihua laboratory animal technologies Co., ltd: 20201214Abzz0619000711.

A549 human non-small cell lung cancer cells were cultured in DMEM medium containing 10% Fetal Bovine Serum (FBS) and 100U/mL penicillin and 100 μg/mL streptomycin were added. 37 ℃ 5% CO ₂ Culturing.

Taking cells with good growth state, performing experiment, collecting cells, centrifuging, discarding original culture medium, adding appropriate amount of PBS, resuspending, counting, and regulating cell density to 2.0X10 ⁷ cells/mL were placed on ice for use.

BALB/c nude mice establishing human non-small cell lung cancer cells (A549)Subcutaneous transplantation tumor model. Tumor cell suspensions were inoculated subcutaneously in the right middle wing of BALB/c nude mice, and on day 11 post-subcutaneous inoculation, the average tumor volume in mice was kept to about 75 mm ³ Selecting 42 tumor-bearing mice, wherein the tumor volume range is 49.94-99.10 mm ³ Animals weighing 17.8-21.5g, randomly dividing into 5 groups according to tumor volume, and treating 10 animals in vehicle group, wherein 8 animals in each treatment group are respectively: vehicle control (vehicle was DPBS with 10% (w/v) glycerol), KR11 low dose control (viral backbone) (1X 10) ⁵ PFU/alone, QW. Times.3), KR11 high dose control group (1X 10) ⁶ PFU/QW X3), KR10 low dose group (1X 10) ⁵ PFU/QW X3), KR10 high dose group (1X 10) ⁶ PFU/qw×3). The administration was carried out 3 times per week 1 time. The day of first administration was defined as D1. The experimental animal groupings and dosing regimens are shown in table 11. The test sample is injected in tumor with the administration volume of 50 μl/animal and tumor volume of less than 80mm ³ When in single-point injection (the injector enters a lesion area through a single needle inlet, and the injection point is the middle part of tumor tissue); tumor volume is 80mm ³ ～140mm ³ At the time, the injection is divided into 2 points (the injector enters the lesion area through a single needle inlet, and the injection points are 1/3 and 2/3 of the long diameter of the tumor tissue); tumor volume of more than 140mm ³ At the time, the injection is carried out at 3 points (the injector enters the lesion area through one needle inlet, the injection points are 1/3 and 2/3 of the long diameter of the tumor tissue, the second needle enters the lesion area through the other needle inlet, and the injection points are positioned outside the middle part of the tumor tissue).

Table 11 experimental animal groups

Animal status is observed daily, including but not limited to: mental state, behavioral activity, tumor ulceration, etc. Tumor diameters were measured simultaneously weekly, animals were weighed 2 times, and tumors from surviving animals were dissected at the end of the experimental period and weighed. Calculation of relative proliferation rate T/C%, inhibition rate TGI% and tumorHeavy inhibition rate IR _TW ％。

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

T/C (%) = (T/T0)/(C/C0) ×100% where T, C is tumor volume of the dosing group, control group at the end of the experiment; t0 and C0 are tumor volumes of the administration group and the control group at the beginning of the experiment. If T > T0, tumor growth inhibition rate (TGI)% = [1-T/C ] ×100%; if T < T0, tumor Growth Inhibition (TGI)% = [1- (T-T0)/T0 ] ×100%.

Tumor weight inhibition rate IR _TW (％)＝(W _{Control group-} W _{Administration group} ) Control group/W. Times.100%

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

All experimental data are expressed in Mean ± standard error (Mean ± SEM). The statistics are carried out according to the following method: checking the variance alignment by using level's, if there is no statistical significance (P > 0.05), performing statistical analysis by using one-way analysis of variance (ANOVA), and if the ANOVA has statistical significance (P < 0.05), performing overall comparison by using LSD method; if the variances were not uniform (P < 0.05), the Kruskal-Wallis test was used, and if the Kruskal-Wallis test was statistically significant (P < 0.05), the Mann-Whitney method was used to make pairwise comparisons between the averages.

Results:

during the experiment, the main clinical symptoms of animals in the vehicle control group and the administration groups are tumor crusting, and the animal state is not abnormal. The animal clinical symptoms are observed in tables 12-1, 12-2 and 12-3.

On day 21 of the end of the experiment, the average body weight of the vehicle control animals was 21.77 + -0.31 g, and the average body weights of the KR11 low and high dose animals, KR10 low and high dose animals were 22.05+ -0.56 g, 22.05+ -0.36 g, 21.58+ -0.45 g and 21.49 + -0.40 g, respectively. The body weight of each treatment group was not significantly different from that of the vehicle control group. Body weight statistics are shown in Table 13. Individual body weight data are shown in table 14. The trend of body weight gain is shown in figure 9. Red triangle symbols indicate dosing time points, D1 being the day of grouping.

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

Experimental endpoint (D21, day 21) all surviving animals were euthanized, dissected, tumor weighed and photographed. The average tumor weight of the vehicle control group was 0.47.+ -. 0.05g, the average tumor weight of the animals in the KR11 low and high dose groups and the KR10 low and high dose groups was 0.27.+ -. 0.04g, 0.19.+ -. 0.04g, 0.23.+ -. 0.05g and 0.13.+ -. 0.04g, respectively, which were significantly lower than the vehicle control group (vs vehicle control group, P<0.01). KR10 and KR11 showed no significant difference in average tumor weights in each group at the same administration level (P >0.05). Tumor weight inhibition rate IR _TW (%) were 43.38%, 58.87%, 50.32% and 72.76%, respectively. The statistical results are shown in Table 18. Tumor weight statistics are shown in Table 19, individual data are shown in Table 20, and tumor weight statistics for each group are shown in FIG. 12. The photograph of euthanasia tumors is shown in FIG. 13.

Conclusion: under the experimental conditions, in a human non-small cell lung cancer (A549) subcutaneous transplantation tumor model, the samples KR11 and KR10 have remarkable inhibition effect on tumors, have dose dependence and are well tolerated by animals. Since only the oncolytic viral backbone exerts an antitumor effect in immunodeficient mice, KR10 and KR11 have the same oncolytic viral backbone, the tumor inhibiting effect of each group is similar at the same administration level, without statistical differences.

Table 12-1 table of statistics of clinical symptom observations of experimental animals

Table 12-2 table of statistics of clinical symptom observations of experimental animals

Table 12-3 table of statistics of clinical symptom observations of experimental animals

And (3) injection: normal, 1 death, 2 listlessness, 3 reduced activity, 4 tremors, 5 erectile hairs, 6 tumor crusting, 7 bow backs, NA dead/euthanized.

Table 13 statistical table of animal weights

Table 14 table of individual body weight data of experimental animals

Table 15 experimental animal tumor volume statistics table

Note that: * Represents p <0.05 compared to vehicle control; * Represents p <0.01 compared to vehicle control group; * P <0.001 compared to vehicle control.

Table 16 Experimental animal tumor volume (mm) ³ ) Individual data sheet

Table 17 statistics of tumor volumes for each group (mean.+ -. SEM)

Group order

Group of

Dosage of

Tumor volume (mm < 3 >)

T/C(％

TGI(

The # represents the P value compared to the control group. And (2) the following steps: p-value compared to KR11 at the same dose.

Table 18 tumor weights (mean.+ -. SEM) for animals of each group

Table 19 Experimental animal tumor weight statistics (g, mean.+ -. SEM)

Note that: * Represents p <0.01 compared to vehicle control group; * P <0.001 compared to vehicle control.

Table 20 data sheet for experimental animal tumor weight individuals

The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Numerous variations of the presently exemplified embodiments of the present application will be apparent to those of ordinary skill in the art and remain within the scope of the appended claims and equivalents thereof.

Claims

An isolated nucleic acid molecule comprising one or more genes each independently encoding a Kras mutant selected from the group consisting of: the mutants of Kras G12D, kras G13D, kras G12V, kras G13C, kras G12C, kras G13A, kras G12A, kras Q61L, kras G12R, kras Q61H, kras G12S, kras Q61R, kras a59T, kras a146T, kras Y64H and Kras a 18D.
An isolated nucleic acid molecule that does not comprise a polynucleotide encoding a 6x His tag protein, and that comprises one or more genes each independently encoding a Kras mutant selected from the group consisting of: the mutants of Kras G12D, kras G13D, kras G12V, kras G13C, kras G12C, kras G13A, kras G12A, kras Q61L, kras G12R, kras Q61H, kras G12S, kras Q61R, kras a59T, kras a146T, kras Y64H and Kras a 18D.
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: the Kras A59T mutant, the Kras G12D mutant, the Kras G12V mutant, the KrasA146T mutant, the Kras G13D mutant, the Kras Y64H mutant and the 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 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 of the genes encoding Kras mutants is Minigene.
The isolated nucleic acid molecule of any one of claims 1-12, wherein each of the genes encoding Kras mutants are 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 immediately adjacent to the N-terminus of the mutation site and at least 10 amino acids immediately adjacent to the C-terminus of the mutation 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 set forth 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 set forth 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 of SEQ ID NO:54, and an amino acid sequence shown in seq id no.
The isolated nucleic acid molecule of any one of claims 1-28, wherein the Kras a146T mutant comprises the amino acid sequence of SEQ ID NO:56, and an amino acid sequence shown in seq id no.
The isolated nucleic acid molecule of any one of claims 1-29, wherein the Kras Y64H mutant comprises the amino acid sequence of SEQ ID NO:58, and an amino acid sequence as set forth in seq id no.
The isolated nucleic acid molecule of any one of claims 1-30, wherein the Kras a18D mutant comprises the amino acid sequence of 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth 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 amino acid sequence of 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 amino acid sequence of 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 amino acid sequence of 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 amino acid sequence of SEQ ID NO:61, and a nucleotide sequence set forth in seq id no.
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, a gene encoding a Kras G12V mutant, a gene encoding a Kras a146T mutant, a gene encoding a Kras G13D mutant, a gene encoding a Kras Y64H mutant, a gene encoding a Kras G12C mutant, a gene encoding a Kras Q61H mutant, a gene encoding a Kras a18D mutant, a gene encoding a Kras G12A mutant, a gene encoding a Kras a146T mutant, and a gene encoding a Kras G12S mutant.
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, a gene encoding a Kras G12V mutant, a gene encoding a Kras a59T mutant, a gene encoding a Kras G13D mutant, a gene encoding a Kras Y64H mutant, and a gene encoding a 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, a gene encoding a Kras a18D mutant, a gene encoding a Kras G12V mutant, a gene encoding a Kras a146T mutant, a gene encoding a Kras Q61H mutant, a gene encoding a Kras G13D mutant, a gene encoding a Kras a59T mutant, a gene encoding a Kras a146T mutant, and a gene encoding a Kras G12C mutant.
The isolated nucleic acid molecule of any one of claims 1-52, comprising the nucleotide sequence set forth in SEQ ID No. 67.
The isolated nucleic acid molecule of any one of claims 1-53, further comprising a polynucleotide encoding a secretory peptide.
The isolated nucleic acid molecule of claim 54, wherein said polynucleotide encoding a secretory peptide is a polynucleotide encoding a CD14 protein secretory peptide.
The isolated nucleic acid molecule of claim 55, wherein the polynucleotide encoding a CD14 protein secretion 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 a CD14 protein secretion peptide comprises the nucleotide sequence of SEQ ID NO:37, or a nucleotide sequence set forth in any one of seq id no.
A vector comprising the nucleic acid molecule of any one of claims 1-57.
The vector of claim 58, comprising a viral vector.
The vector of any one of claims 58-59, comprising an oncolytic herpes simplex virus ohv 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 lacks a neurotoxic factor γ34.5 gene.
The vector of any of claims 58-62, wherein the nucleic acid molecule is located between the UL3 gene and UL4 gene of the HSV-1 vector.
The vector of any one of claims 58-63, comprising a promoter.
The vector of claim 64, wherein the promoter comprises a CMV promoter.
The vector of any one of claims 58-65, comprising a nucleotide sequence set forth 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 vector 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 vector of any one of claims 58-69 or the pharmaceutical composition of claim 67, and physiological saline.
The composition of claim 68, wherein said isolated nucleic acid molecules comprise one or more genes each independently encoding a Kras mutant selected from the group consisting of: the mutants of Kras G12D, kras G13D, kras G12V, kras G13C, kras G12C, kras G13A, kras G12A, kras Q61L, kras G12R, kras Q61H, kras G12S, kras Q61R, kras a59T, kras a146T, kras Y64H and Kras a 18D.
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 said isolated nucleic acid molecule comprises a gene encoding a Kras mutant selected from the group consisting of: the Kras A59T mutant, the Kras G12D mutant, the Kras G12V mutant, the KrasA146T mutant, the Kras G13D mutant, the Kras Y64H mutant and the Kras G12C mutant.
The composition of any one of claims 69-71, wherein the 3 'end of the gene encoding a Kras G12D mutant is directly or indirectly linked to the 5' end of the gene encoding a Kras a146T mutant.
The composition of any one of claims 69-72, wherein the 3 'end of the gene encoding a Kras a46T mutant is directly or indirectly linked to the 5' end of the gene encoding a Kras G12V mutant.
The composition of any one of claims 69-73, wherein the 3 'end of the gene encoding a Kras G12V mutant is directly or indirectly linked to the 5' end of the gene encoding a 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 a Kras Y64H mutant is directly or indirectly linked to the 5' end of the gene encoding a Kras G12C mutant.
The composition of any one of claims 69-77, wherein each of said genes encoding a Kras mutant is tandem in said isolated nucleic acid molecule.
The composition of any one of claims 69-78, wherein each of said genes encoding a Kras mutant is Minigene.
The composition of any one of claims 69-79, wherein each of the genes encoding Kras mutants is tandem to provide 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 adjacent to the N-terminus of the mutation site and at least 10 amino acids immediately adjacent to the C-terminus of the mutation 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 set forth 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 of SEQ ID NO:54, and an amino acid sequence shown in seq id no.
The composition of any one of claims 69-95, wherein the Kras a146T mutant comprises the amino acid sequence of SEQ ID NO:56, and an amino acid sequence shown in seq id no.
The composition of any one of claims 69-96, wherein the Kras Y64H mutant comprises the amino acid sequence of SEQ ID NO:58, and an amino acid sequence as set forth in seq id no.
The composition of any one of claims 69-97, wherein the Kras a18D mutant comprises the amino acid sequence of 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth in SEQ ID No. 36.
The composition of any one of claims 69-110, wherein the gene encoding the Kras a59T mutant comprises the amino acid sequence of SEQ ID NO: 55.
The composition of any one of claims 69-111, wherein the gene encoding the Kras a146T mutant comprises the amino acid sequence of SEQ ID NO: 57.
The composition of any one of claims 69-112, wherein the gene encoding the Kras Y64H mutant comprises the amino acid sequence of SEQ ID NO: 59.
The composition of any one of claims 69-113, wherein the gene encoding the Kras a18D mutant comprises the amino acid sequence of SEQ ID NO:61, and a nucleotide sequence set forth in seq id no.
The composition of any one of claims 60-114, 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, a gene encoding a Kras G12V mutant, a gene encoding a Kras a146T mutant, a gene encoding a Kras G13D mutant, a gene encoding a Kras Y64H mutant, a gene encoding a Kras G12C mutant, a gene encoding a Kras Q61H mutant, a gene encoding a Kras a18D mutant, a gene encoding a Kras G12A mutant, a gene encoding a Kras a146T mutant, and a gene encoding a 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 G12V mutant, a gene encoding a Kras a59T mutant, a gene encoding a Kras G13D mutant, a gene encoding a Kras Y64H mutant, and a gene encoding a 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 of any one of claims 80-118, 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, a gene encoding a Kras a18D mutant, a gene encoding a Kras G12V mutant, a gene encoding a Kras a146T mutant, a gene encoding a Kras Q61H mutant, a gene encoding a Kras G13D mutant, a gene encoding a Kras a59T mutant, a gene encoding a Kras a146T mutant, and a gene encoding a 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 secretory peptide.
The composition of claim 121, wherein the polynucleotide encoding a secretory peptide is a polynucleotide encoding a CD14 protein secretory peptide.
The composition of claim 122, wherein the polynucleotide encoding a CD14 protein secretion peptide is located at the 5' end of the gene encoding the Kras mutant.
The composition of any one of claims 122-123, wherein the polynucleotide encoding a CD14 protein secretion peptide comprises the amino acid sequence of SEQ ID NO:37, or a nucleotide sequence set forth in any one of seq id no.
The composition of any one of claims 68-124, wherein the vector 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 ohv 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 lacks a neurotoxic factor γ34.5 gene.
The composition of any of claims 128-129, wherein in the vector the nucleic acid molecule is located between the UL3 gene and 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 a nucleotide sequence set forth in GenBank No. GU734771.1 of the NCBI database.
Use of the nucleic acid molecule of any one of claims 1-57, the vector of any one of claims 58-66, the pharmaceutical composition of claim 67 and/or the composition of any one of claims 68-133 in the manufacture of a medicament for treating a tumor.
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 of claims 134-137, wherein the tumor comprises breast cancer.
The use of any of claims 134-138, wherein the tumor comprises pancreatic cancer.