CN116323947A - A nucleic acid molecule encoding a Kras gene mutant - Google Patents

Abstract

Provided are a nucleic acid molecule encoding a Kras gene mutant, and an application of an oncolytic herpes simplex virus (oHSV) vector comprising the nucleic acid molecule in preparing antitumor drugs. Mutants include 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 One or more of mutants, Kras G12S mutants, Kras Q61R mutants, Kras A59T mutants, Kras A146T mutants, Kras Y64H mutants, and Kras A18D mutants.

Description

A nucleic acid molecule encoding a Kras gene mutant technical field

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

Background technique

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

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

Kras gene is a proto-oncogene, about 35kb in length, located on chromosome 12, one of the members of the RAS gene family, encoding Kras protein. Kras protein is a membrane-bound protein located inside the cell membrane and located in the EGFR (epidermal growth factor receptor) signaling pathway, which plays an important role in the occurrence and development of tumors. The growth, proliferation, angiogenesis and other processes of tumor cells require intracellular proteins to conduct signal transduction, and the Kras gene is the determinant of the transduction protein. Kras mutants encode abnormal proteins, which stimulate the growth and spread of malignant tumor cells and are not affected by Signaling effects of upstream EGFR.

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

Contents of the invention

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

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

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

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

In certain embodiments, the 3' end of the gene encoding the Kras G12D mutant is directly or indirectly connected 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 connected 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 connected 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 connected 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 connected 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 connected to the 5' end of the gene encoding the Kras G12C mutant.

In certain embodiments, each of said genes encoding a Kras mutant is arranged in tandem in said isolated nucleic acid molecule.

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

In certain embodiments, each of the genes encoding the Kras mutants is arranged in tandem to obtain a tandem mini-gene.

In certain embodiments, each of said 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-terminal of the mutation site and at least 10 amino acids immediately adjacent to the C-terminal 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 the amino acid sequence shown in SEQ ID NO:54.

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

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

In certain embodiments, the Kras A18D mutant comprises the amino acid sequence shown in 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 shown in SEQ ID NO:25.

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

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

In certain embodiments, the gene encoding the Kras G13A mutant comprises the nucleotide sequence shown 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 shown in SEQ ID NO:30.

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

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

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

In some embodiments, the gene encoding the Kras Q61R mutant comprises the nucleotide sequence shown in SEQ ID NO:36.

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

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

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

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

In certain embodiments, the tandem mini-genes sequentially comprise 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 from the 5' end to the 3' end. genes encoding Kras G13D mutants, genes encoding Kras Y64H mutants, genes encoding Kras G12C mutants, genes encoding Kras Q61H mutants, genes encoding Kras A18D mutants, genes encoding Kras G12A mutants gene, the gene encoding the Kras A146T mutant and the gene encoding the Kras G12S mutant.

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

In certain embodiments, the tandem mini-genes sequentially comprise 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 from the 5' end to the 3' end. The gene encoding the Kras G13D mutant, the gene encoding the Kras Y64H mutant, and the gene encoding the Kras G12C mutant.

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

In certain embodiments, the tandem mini-genes sequentially comprise 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 from the 5' end to the 3' end. genes encoding Kras A146T mutants, genes encoding Kras Q61H mutants, genes encoding Kras G13D mutants, genes encoding Kras A59T mutants, genes encoding Kras A146T mutants, and genes encoding Kras G12C mutants Gene.

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

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

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

In some embodiments, the polynucleotide encoding CD14 protein secretory peptide is located at the 5' end of the gene encoding the Kras mutant.

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

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

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

On the other hand, the present application provides a vector comprising the nucleic acid molecule.

In some embodiments, the vector comprises a viral vector.

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

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

In certain embodiments, the HSV-1 vector lacks the neurovirulence factor gamma 34.5 gene.

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

In certain embodiments, the vector includes a promoter.

In certain embodiments, the promoter comprises a CMV promoter.

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

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

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

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

In certain embodiments, said isolated nucleic acid molecule in said 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, KrasG13D mutant and KrasG12C mutant.

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

In certain embodiments, the 3' end of the gene encoding the Kras G12D mutant is directly or indirectly connected 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 connected 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 connected 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 connected 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 connected 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 connected to the 5' end of the gene encoding the Kras G12C mutant.

In certain embodiments, each of said genes encoding a Kras mutant is arranged in tandem in said isolated nucleic acid molecule.

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

In certain embodiments, each of the genes encoding the Kras mutants is arranged in tandem to obtain a tandem mini-gene.

In certain embodiments, each of said 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-terminal of the mutation site and at least 10 amino acids immediately adjacent to the C-terminal 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 the amino acid sequence shown in SEQ ID NO:54.

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

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

In certain embodiments, the Kras A18D mutant comprises the amino acid sequence shown in 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 shown in SEQ ID NO:25.

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

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

In certain embodiments, the gene encoding the Kras G13A mutant comprises the nucleotide sequence shown 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 shown in SEQ ID NO:30.

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

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

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

In some embodiments, the gene encoding the Kras Q61R mutant comprises the nucleotide sequence shown in SEQ ID NO:36.

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

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

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

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

In certain embodiments, the tandem mini-genes sequentially comprise 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 from the 5' end to the 3' end. genes encoding Kras G13D mutants, genes encoding Kras Y64H mutants, genes encoding Kras G12C mutants, genes encoding Kras Q61H mutants, genes encoding Kras A18D mutants, genes encoding Kras G12A mutants gene, the gene encoding the Kras A146T mutant and the gene encoding the Kras G12S mutant.

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

In certain embodiments, the tandem mini-genes sequentially comprise 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 from the 5' end to the 3' end. The gene encoding the Kras G13D mutant, the gene encoding the Kras Y64H mutant, and the gene encoding the Kras G12C mutant.

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

In certain embodiments, the tandem mini-genes sequentially comprise 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 from the 5' end to the 3' end. genes encoding Kras A146T mutants, genes encoding Kras Q61H mutants, genes encoding Kras G13D mutants, genes encoding Kras A59T mutants, genes encoding Kras A146T mutants, and genes encoding Kras G12C mutants Gene.

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, said isolated nucleic acid molecule in said composition further comprises a polynucleotide encoding a secreted peptide.

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

In some embodiments, the polynucleotide encoding CD14 protein secretory peptide is located at the 5' end of the gene encoding the Kras mutant.

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

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

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

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

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

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

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

In certain embodiments, the HSV-1 vector lacks the neurovirulence factor gamma 34.5 gene.

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

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

In certain embodiments, the promoter comprises a CMV promoter.

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

In another aspect, the present application provides the nucleic acid molecule, the carrier, the pharmaceutical composition and/or the application of the composition in the preparation of a drug for treating tumors.

In certain embodiments, the tumor comprises a solid tumor.

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

In certain embodiments, the tumor comprises colorectal cancer.

In certain embodiments, the tumor comprises breast cancer.

In certain embodiments, the tumor comprises pancreatic cancer.

Those skilled in the art can easily perceive other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will appreciate, the content of the present application enables those skilled in the art to make changes to the specific embodiments which are disclosed without departing from the spirit and scope of the invention to which this application relates. Correspondingly, the drawings and descriptions in the specification of the present application are only exemplary rather than restrictive.

Description of drawings

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

Figure 1 shows the structure of the Kras mutant tandem mini-gene described in this application.

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

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

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

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

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

Fig. 4A shows the change of tumor volume in CT26.WT subcutaneously transplanted tumor mice described in this application.

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

Fig. 5 shows the statistical graph of tumor weight in mice with CT26.WT subcutaneously transplanted tumor described in the present application.

Figures 6A-6B show photographs of tumors in the CT26.WT model described in this application after euthanasia.

Figure 7 shows the change in tumor volume of animals described in this application (#5203, #5204).

Fig. 8 shows the photos of the end point of the tumor rechallenge model animal experiment described in this application.

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

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

Figure 11 shows the changes in the tumor volumes of the various groups of animals described in this application.

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

Figure 13 shows the photographs of tumors in the A549 model described in this application after euthanasia.

Detailed ways

The implementation of the invention of the present application will be described in the following specific examples, and those skilled in the art can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.

Definition of Terms

The implementation of the invention of the present application will be described in the following specific examples, and those skilled in the art can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.

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

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

In this application, the term "tagged protein" usually refers to a protein that is fused with the target protein to facilitate detection of the target protein. The tag protein used in this application is a 6xHis tag protein, also known as a polyhistidine tag protein, which utilizes the sequence of histidine residues to bind to several types of fixed ions (such as nickel , copper and cobalt), and has almost no effect on the characteristics of the target protein itself, so as to achieve the purpose of easy detection and purification of His-tagged proteins.

In this application, the term "vector" generally refers to a tool capable of carrying an exogenous target gene or DNA fragment into a host cell for replication and expression. According to the source, they can be divided 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 linking with the nucleic acid molecule for replication and expression in host cells.

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

In this application, the term "γ34.5 gene" generally refers to the apoptosis suppressing gene in the HSV-1 genome, which is also an important neurotoxic gene, GeneBank No: GU734771.1. Knockout of the γ34.5 gene is currently the most common attenuation strategy for oncolytic viruses based on the HSV backbone. HSV-1 with defective γ34.5 gene function not only greatly reduces the toxicity, but also allows the virus to selectively replicate in tumor cells by using the characteristics of tumor cell IFN-PKR signal transduction defects. The host PKR phosphorylation induced by virus infection can inhibit the replication of the virus, but the gene product of HSV-1γ34.5 can resist the inhibitory effect of PKR, so that the virus can replicate in normal cells, and many tumor cells have defects in the PKR system, resulting in The HSV virus knocked out of the γ34.5 gene can effectively replicate and kill tumors in tumor cells but cannot replicate in normal cells, which provides conditions for the transformation of HSV viruses to specifically kill tumors.

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 the target gene, capable of transcribing the target gene, which can be recognized by RNA polymerase , and start to transcribe and synthesize RNA. In the present application, the promoter refers to a DNA sequence that is located upstream of the 5' end of the transcription start site of the gene encoding the Kras mutant and controls its transcription.

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

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

In this application, "solid tumor" generally refers to an abnormal growth or mass of tissue, which usually does not contain cysts or fluid areas. Solid tumors can be benign (noncancerous) or malignant (cancerous). At present, about 90% of cancer cases in clinical practice are solid tumors. The different types of solid tumors are named after the type of cells that form them. 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, esophagus cancer, bile duct tumor, and head and neck cancer, etc.

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

Detailed description of the invention

nucleic acid molecule

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

In another aspect, the application provides an isolated nucleic acid molecule, which does not comprise a polynucleotide encoding a 6x His tag protein, and which comprises one or more genes each independently encoding a Kras mutant selected from the following: 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.

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

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

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

In the present application, the 3' end of the gene encoding the Kras G12D mutant may be directly or indirectly connected 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 connected 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 connected 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 connected 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 connected 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 connected to the 5' end of the gene encoding the Kras G12C mutant.

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

In the present application, each gene encoding a Kras mutant may be a minigene Minigene.

In the present application, the tandem mini-genes can be obtained after the genes encoding the Kras mutants are arranged in tandem.

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

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

In certain embodiments, the arrangement order of each of the Kras mutants from the 5' end to the 3' end is the gene encoding the Kras G12D mutant, the gene encoding the Kras A146T mutant, the gene encoding the Kras G12V mutant, 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 can comprise the amino acid sequence set forth in SEQ ID NO:66.

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

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

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

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

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

The gene encoding the Kras G12D mutant may comprise a nucleotide sequence encoding the amino acid sequence of the N-terminal of the mutation site G12D, a nucleotide sequence encoding the mutation site G12D, encoding the mutation site Nucleotide sequence of the amino acid sequence of the C-terminal of G12D.

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

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

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

For example, the amino acid sequence of the Kras G13D mutant may sequentially include 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 from the N-terminal to the C-terminal.

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

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

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

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

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

For example, the amino acid sequence of the Kras G12V mutant may sequentially include 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 from the N-terminal to the C-terminal.

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

The gene encoding the Kras G12V mutant may comprise a nucleotide sequence encoding the amino acid sequence of the N-terminal of the mutation site G12V, a nucleotide sequence encoding the mutation site G12V, encoding the mutation site Nucleotide sequence of the amino acid sequence of the C-terminal of G12V.

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

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

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

For example, the amino acid sequence of the Kras G13C mutant may sequentially include 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 from the N-terminal to the C-terminal.

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

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

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

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

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

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

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

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

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

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

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

For example, the amino acid sequence of the Kras G13A mutant may sequentially include 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 from the N-terminal to the C-terminal.

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

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

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

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

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

For example, the amino acid sequence of the Kras G12A mutant may sequentially include 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 from the N-terminal to the C-terminal.

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

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

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

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

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

For example, the amino acid sequence of the Kras Q61L mutant may sequentially include 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 from the N-terminal to the C-terminal.

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

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

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

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

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

For example, the amino acid sequence of the Kras G12R mutant may sequentially include 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 from the N-terminal to the C-terminal.

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

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

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

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

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

For example, the amino acid sequence of the Kras Q61H mutant may sequentially include 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 from the N-terminal to the C-terminal.

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

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

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

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

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

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

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

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

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

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

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

For example, the amino acid sequence of the Kras Q61R mutant may sequentially include 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 from the N-terminal to the C-terminal.

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

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

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

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

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

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

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

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

The Kras A59T mutant refers to a sequence containing the 59th amino acid A truncated from the amino acid sequence of the wild-type Kras polypeptide, and the 59th amino acid A in the truncated sequence is replaced with T, the The sequence comprises at least 20 amino acids, such as 21, such as 22, such as 23, such as 24, such as 25, such as 26. The Kras A59T mutant may comprise at least 9 (such as at least 10, at least 11) amino acids immediately adjacent to the N-terminal of the mutation site A59T and at least 10 (such as at least 11, at least 12, at least 13, at least 14) amino acids.

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

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

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

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

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

The Kras Y64H mutant refers to a sequence containing the 64th amino acid Y truncated from the amino acid sequence of the wild-type Kras polypeptide, and the 64th amino acid Y in the truncated sequence is replaced with H, the The sequence comprises at least 20 amino acids, such as 21, such as 22, such as 23, such as 24, such as 25, such as 26. The Kras Y64H mutant may comprise at least 9 (such as at least 10, at least 11) amino acids immediately adjacent to the N-terminal of the mutation site Y64H and at least 10 (such as at least 11, at least 11) amino acids adjacent to the C-terminal of the mutation site Y64H. at least 12, at least 13, at least 14) amino acids.

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

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

The Kras A18D mutant refers to a sequence containing the 18th amino acid A truncated from the amino acid sequence of the wild-type Kras polypeptide, and the 18th amino acid A in the truncated sequence is replaced with D, the The sequence comprises at least 20 amino acids, such as 21, such as 22, such as 23, such as 24, such as 25, such as 26. The Kras A18D mutant may comprise at least 9 (such as at least 10, at least 11) amino acids immediately adjacent to the N-terminal of the mutation site A18D and at least 10 (such as at least 11, at least 12, at least 13, at least 14) amino acids.

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

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

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

In the present application, the nucleic acid molecule may also contain mutant strains of other tumor antigens. For example, the nucleic acid molecule may also comprise a mutant strain of a tumor-associated driver gene. For example, the other tumor antigen may be TP53. For example, said other tumor antigen may be the Braf gene.

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

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

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

For example, the tagged protein may include FLAG tagged protein, HA tagged protein, C-Myc tagged protein, 6xHis tagged protein.

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

carrier

On the other hand, the present application provides a vector comprising the nucleic acid molecule.

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

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

In some embodiments, the gene described in this application that does not contain the 6x His tag protein can be inserted into a viral vector. The vector may be an oncolytic herpes simplex virus (oHSV) vector. For example, the oncolytic herpes simplex virus (oHSV) vector may be a herpes simplex virus type I (HSV-1) vector. The HSV-1 vector is a gene deletion type, and the deletion gene may be neurotoxicity factor γ34.5.

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

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

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

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

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

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

On the other hand, the present application also provides a pharmaceutical composition, which comprises the nucleic acid molecule, and/or the carrier, and optionally a pharmaceutically acceptable adjuvant. By pharmaceutical composition is meant a preparation in such a form as to allow the biological activity of the active ingredients contained therein to be effective and free of additional ingredients which would be unacceptably toxic to a subject to whom the composition would be administered. The pharmaceutically acceptable adjuvant refers to any substance that can assist or improve the action of a drug. The adjuvant may be a particulate adjuvant, eg, aluminum hydroxide adjuvant. The adjuvant may be a non-particulate adjuvant, eg, a cytokine. The adjuvants can be derived from plants, such as saponin and polysaccharide extracts. The adjuvant can be derived from pathogenic microorganisms, for example, monophospholipids, cholera toxin and the like.

combination

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

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

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

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

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

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

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

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

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

In the present application, in the isolated nucleic acid molecules in the composition, the tandem mini-genes may sequentially comprise the gene encoding the Kras G12D mutant, the gene encoding the Kras A59T mutant from the 5' end to the 3' end. gene, gene encoding Kras G12V mutant, gene encoding Kras A146T mutant, gene encoding Kras G13D mutant, gene encoding Kras Y64H mutant, gene encoding Kras G12C mutant, gene encoding Kras Q61H mutant, 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 tandem minigene may comprise the nucleotide sequence shown in SEQ ID NO:65.

In the present application, in the isolated nucleic acid molecules in the composition, the tandem mini-genes may sequentially comprise the gene encoding the Kras G12D mutant, the gene encoding the Kras A146T mutant from the 5' end to the 3' end. gene, 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 tandem minigene may comprise the nucleotide sequence shown in SEQ ID NO:66.

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

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

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

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

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

use

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

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

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

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

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

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

1. An isolated nucleic acid molecule comprising genes encoding respectively the following Kras mutants: 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, and Kras Q61R mutant.

2. The isolated nucleic acid molecule of embodiment 1, wherein each of said genes encoding a Kras mutant is arranged in tandem in said isolated nucleic acid molecule.

3. The isolated nucleic acid molecule according to any one of embodiments 1-2, wherein each of said genes encoding a Kras mutant is a Minigene.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

31. The isolated nucleic acid molecule of embodiment 30, said polynucleotide encoding a secreted peptide is a polynucleotide encoding a secreted peptide of CD14 protein.

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

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

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

35. The isolated nucleic acid molecule of embodiment 34, wherein said tag protein comprises 6x His.

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

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

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

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

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

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

43. The vector according to embodiment 42, wherein the HSV-1 vector lacks the neurotoxic factor gamma 34.5 gene.

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

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

46. The vector according to embodiment 45, wherein said promoter comprises a CMV promoter.

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

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

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

50. The use according to embodiment 49, wherein said tumor comprises a solid tumor.

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

52. The use according to 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.

Not intending to be limited by any theory, the following examples are only for explaining the nucleic acid molecules, preparation methods and uses of the present application, and are not intended to limit the scope of the present invention.

Example

Embodiment 1 constructs the BAC plasmid of recombinant virus KR10

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

After truncating the sequence from amino acid 1 to amino acid 24 (SEQ ID NO: 1) from the amino acid sequence of the wild-type Kras polypeptide, the 12th G amino acid in the truncated sequence is replaced with D amino acid , there are 11 wild-type amino acids (SEQ ID NO: 2) at the N-terminus immediately adjacent to the G12D mutation site, and 12 wild-type amino acids (SEQ ID NO: 3) at the C-terminus immediately adjacent to the G12D mutation site. The Kras G12D mutant of the nucleotides of the amino acids connected at the end and the C-terminus, its nucleotide sequence is shown in SEQ ID NO:19.

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

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

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

Referring to the above method, Kras G12D mutant, Kras A59T mutant, Kras A18D mutant, Kras G12V mutant, Kras A146T mutant, Kras Q61H, Kras G13D mutant, Kras A59T mutant, Kras A146T mutant and Kras The nucleotide sequence of the G12C mutant, and the nucleotide sequences encoding each Kras mutant were concatenated according to the order to obtain the Kras mutant in the form of a tandem minigene (Tandem minigene, TMG), which is called TMG-7. The above mutant sequences were synthesized by Aiji Biotechnology Company.

At the 5' end of the gene encoding the above-mentioned Kras mutant of the TMG form, a CD14 protein secretory peptide (synthesized at Aiji Biotechnology Co., Ltd., nucleotide sequence SEQ ID NO: 37) was introduced, and at the 3' end a 6x His tag was introduced (synthesized From Aiji Biotechnology Co., Ltd., nucleotide sequence (SEQ ID NO: 40), the nucleotide sequence (as shown in SEQ ID NO: 47-49) of encoding CD14 protein-Kras mutant (TMG)-6xHis was obtained, The structural form of CD14 protein-Kras mutant (TMG)-6xHis is shown in FIG. 1 .

1.2 Insertion of Kras mutant gene

On wild-type HSV-1 (F) (as shown in Figure 2A), delete two copies of the neurovirulence factor γ34.5 gene, insert CD14 protein-Kras described in embodiment 1.1 between UL3 and UL4 gene simultaneously The nucleotide sequence of the mutant (TMG)-6xHis was obtained to obtain the nucleotide sequence of UL3-CD14 protein-Kras-6xHis-UL4 (as shown in SEQ ID NO: 50-52).

1.3 Construction of the BAC plasmid of the recombinant virus

By molecular cloning, construct the intermediate plasmid pKO5.1 (gifted by Professor Dr.Bernard Roizman, University of Chicago) for BAC recombination, the nucleotide sequence of the UL3-CD14 protein-Kras-6x His-UL4 described in embodiment 1.2 Inserted into pKO5.1, it was transformed into Escherichia coli of HSV BAC that deleted two copies of the neurovirulence factor γ34.5 gene (as shown in SEQ ID NO:53) by electroporation to obtain the recombinant virus KR10 BAC plasmid. The nucleic acid sequence structure of KR10 is shown in Figure 2B.

Embodiment 2 constructs the BAC plasmid of recombinant virus KR11

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

Embodiment 3 constructs the BAC plasmid of positive control virus KR12

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

Example 4 Cell Experiment

4.1 Packaging of recombinant virus and TK gene repair

The BAC plasmid containing the KR10 nucleotide sequence, the BAC plasmid containing the KR11 nucleotide sequence, the BAC plasmid containing the KR12 nucleotide sequence constructed in the above-mentioned embodiments 1-3 were respectively combined with the pRB103 plasmid (containing the HSV-1F virus TK gene ) were co-transfected into Vero cells, placed in a 37°C, 5% CO ₂ incubator and incubated for 4 hours, then the medium was changed, and fresh complete growth medium (5% NBCS/DMEM) was added, and the culture was continued until the virus packaging was successful. Plaques appear. Cells were harvested and frozen and thawed repeatedly to obtain the virus stock solution, and the virus stock solution was used to infect Vero-ΔTK cells (Vero cells lacking the TK gene), added to HAT for screening, and monoclonal virus was picked. The monoclonal virus was re-infected into Vero-ΔTK cells, added to HAT for screening, and the monoclonal virus was picked. The monoclonal virus selected after multiple HAT screenings is used to infect Vero cells, and the virus is amplified to obtain the final TK gene-repaired recombinant virus. The expression of Kras polypeptide, GFP protein and Flu A MP antigenic peptide were detected by Western blot WB method. The antibody used in WB was HRP-labeled 6*His monoclonal antibody purchased from Proteintech. The results of WB detection showed the expression of Kras polypeptide, GFP protein and Flu A MP antigenic peptide.

4.2 In vitro tumor cell killing test of recombinant virus

Lay tumor cell plates, infect tumor cells with the recombinant viruses KR10, KR11, and KR12 prepared in Example 4.1 at different titers, and use CCK8 to detect cell proliferation toxicity after 72 hours of virus action, so as to detect tumor killing by recombinant viruses KR10, KR11, and KR12 Ability. The results showed that KR10, KR11 and KR12 have the ability to kill tumor cells.

Example 5 In vivo experiments in mice

Thymidine kinase gene repair (TK repair), after the repaired virus was successfully repaired by protein detection, it was tested in vivo in mice. The results showed that KR10 and KR11 had good antitumor effects.

5.1 Study on the antitumor effect of KR10 and KR11 on CT26.WT mouse colorectal cancer BALB/c mouse subcutaneous xenograft tumor

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

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

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

Take cells in good growth state for experiments, collect cells, centrifuge, discard the original medium, add appropriate amount of PBS to resuspend, count, adjust cell density to 2×10 ⁷ cells/mL, and place on ice for later use.

Establish a BALB/c mouse subcutaneous xenograft model of mouse colon cancer (CT26.WT). Inoculate tumor cell suspension subcutaneously in the right middle wing of BALB/c mice. After the average tumor volume in the mice grows to about 120mm ³ , select 35 tumor-bearing mice and randomly divide them into 5 groups according to the tumor volume, 7 animals D1 on the day of group administration, respectively vehicle control group (vehicle is DPBS containing 10% (w/v) glycerol), KR11 low-dose group 1×10 ⁶ PFU/only (viral skeleton control group), KR11 High-dose group 1×10 ⁷ PFU/bird (viral skeleton control group), KR10 low-dose group (1×10 ⁶ PFU/bird) and KR10 high-dose group (1×10 ⁷ PFU/bird). Administered once a week (QW×3), a total of 3 administrations. The experimental animal grouping and dosing regimen are shown in Table 1. Intratumoral injection is used for the administration of the test product, the administration volume is 50 μL/animal, and when the tumor volume is less than 80mm ³ , single-point injection (the syringe enters the lesion area through a single needle inlet, and the injection point is the middle of the tumor tissue); When the tumor volume is ^80mm3-140mm3 , inject at 2 points (the syringe 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); when the tumor volume is greater than ^140mm3 , Inject at 3 points (the syringe enters the lesion area through one needle inlet, and the injection points are 1/3 and 2/3 of the long diameter of the tumor tissue, and the second needle enters the lesion area from the other needle inlet, and the injection point is the tumor tissue The middle is on the outside.

Table 1 Grouping of experimental animals

Animal status, animal death and clinical symptoms were observed every day, including but not limited to: mental state, behavioral activities, tumor ulceration, etc. At the same time, the tumor diameter was measured and the body weight of the animals was weighed twice a week. At the end of the experiment period, the tumors of the surviving animals were stripped and weighed. Calculate relative tumor 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 .

Relative tumor proliferation rate T/C (%) and tumor growth inhibition rate TGI (%) were used as experimental evaluation indexes. T/C (%)=(T/T0)/(C/C0)×100% Wherein T, C are the tumor volumes of the administration group and the control group when the experiment ends; T0, C0 are the administration groups, Tumor volume in the control group. If T>T0, tumor growth inhibition rate (TGI)%=[1-T/C]×100%; if T<T0, tumor growth inhibition rate (TGI)%=[1-(T-T0)/T0] ×100%.

Complete tumor remission CR: the tumor volume is 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 as mean ± standard error (Mean ± SEM). Statistics were performed according to the following method: T test was used for pairwise comparison. If P>0.05, the test was not significant; if P≤0.05, the test was significant.

result:

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

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

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

On the 20th day, the average tumor weight of the vehicle control group was 3.914±0.617g, the average tumor weights of the KR11 low-dose and high-dose groups were 2.788±0.628g and 2.353±0.804g respectively; the average tumor weights of the KR10 low-dose and high-dose groups were 1.615g ±0.508g and 1.226±0.526g. Compared with the vehicle control group, the average tumor weight of KR11 low-dose and high-dose groups showed a tendency to decrease, but no significant difference was found (P>0.05). The average tumor weight of KR10 low-dose and high-dose groups decreased significantly (P<0.05). The tumor weight inhibition rates IR _TW (%) of KR11 low-dose and high-dose groups were 28.77% and 39.88%, respectively. The tumor weight inhibition rates IR _TW (%) of KR10 low-dose and high-dose groups were 58.74% and 68.68%, respectively. The statistical results are shown in Table 8. The statistical data of tumor weight are shown in Table 9. See Table 10 for individual data. The statistical chart of tumor weight is shown in Figure 5. Euthanasia photos are shown in Figures 6A-6B.

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

Conclusion: The experimental results show that under the conditions of this experiment, KR10 can significantly inhibit the growth of the tumor in the mouse subcutaneous xenograft tumor model of colorectal cancer CT26.WT cells under the same dose. The inhibition rates were higher than those of KR11, and the tumors of 2 animals in the high-dose KR10 group completely regressed. The above results showed that the anti-tumor effect of KR10 was better than that of virus backbone KR11. The rechallenge model proved that KR10 can stimulate the establishment of long-term anti-tumor immune memory in mice.

Table 2-1 Statistical Table of Observation of Clinical Symptoms of Experimental Animals

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

Table 2-2 Statistical Table of Observation of Clinical Symptoms of Experimental Animals

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

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

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

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

Table 4-1 Experimental animal weight individual data table (g)

Table 4-2 Experimental animal weight individual data table (g)

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

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

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

Table 6-2 Statistical Table of Tumor Volume of Experimental Animals (mm3, Mean±SEM)

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

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

Table 7-2 Individual data table of tumor volume (mm ³ ) of experimental animals

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

Table 8. Animal tumor weights in each group (g, Mean±SEM)

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

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

Table 9 Statistical Table of Tumor Weight of Experimental Animals

group Solvent/test article dose Tumor weight (g, Mean±SEM) 1 Vehicle control group - 3.914±0.617 2 KR11 low dose group 1×10 ⁶ PFU/piece 2.788±0.628 3 KR11 high dose group 1×10 ⁷ PFU/piece 2.353±0.804 4 KR10 low dose group 1×10 ⁶ PFU/piece 1.615±0.508* 5 KR10 high dose group 1×10 ⁷ PFU/piece 1.226±0.526**

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

Table 10 Experimental animal tumor weight individual data table

5.2 Anti-tumor effect of intratumoral administration of KR10 and KR11 herpes virus on BALB/c nude mice subcutaneously transplanted with human non-small cell lung cancer cells (A549)

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

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

Take cells in good growth state for experiments, collect cells, centrifuge, discard the original medium, add appropriate amount of PBS to resuspend, count, adjust the cell density to 2.0×10 ⁷ cells/mL, and place on ice for later use.

A BALB/c nude mouse subcutaneous xenograft model of human non-small cell lung cancer cells (A549) was established. The tumor cell suspension was inoculated subcutaneously in the right middle wing of BALB/c nude mice. On the 11th day after subcutaneous inoculation, the average tumor volume in the mice grew to about 75 mm ³ , and 42 tumor-bearing mice were selected. The tumor volume ranged from 49.94～99.10mm ³ , with a body weight of 17.8-21.5g, were randomly divided into 5 groups according to the tumor volume, 10 in the vehicle group, and 8 animals in each treatment group, respectively: vehicle control group (vehicle containing 10% ( w/v) glycerol in DPBS), KR11 low-dose control group (viral skeleton) (1×10 ⁵ PFU/monkey, QW×3), KR11 high-dose control group (1×10 ⁶ PFU/bird, QW×3) , KR10 low-dose group (1×10 ⁵ PFU/monkey, QW×3), KR10 high-dose group (1×10 ⁶ PFU/bird, QW×3). Once a week, a total of 3 administrations. The day of the first administration was defined as D1. See Table 11 for experimental animal groups and dosing regimens. Intratumoral injection is used for the administration of the test product, the administration volume is 50 μL/animal, and when the tumor volume is less than 80mm ³ , single-point injection (the syringe enters the lesion area through a single needle inlet, and the injection point is the middle of the tumor tissue); When the tumor volume is ^80mm3-140mm3 , inject at 2 points (the syringe 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); when the tumor volume is greater than ^140mm3 , Inject at 3 points (the syringe enters the lesion area through one needle inlet, and the injection points are 1/3 and 2/3 of the long diameter of the tumor tissue, and the second needle enters the lesion area from the other needle inlet, and the injection point is the tumor tissue center to outside).

Table 11 Grouping of experimental animals

The state of the animals was observed every day, and the observation contents included but not limited to: mental state, behavioral activities, tumor ulceration, etc. At the same time, the tumor diameter was measured and the body weight of the animals was weighed twice a week. At the end of the experiment period, the tumors of the surviving animals were stripped and weighed. Calculate relative tumor 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 .

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

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

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

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

All experimental data are expressed as mean ± standard error (Mean ± SEM). According to the following method of statistics: use Levene's test for homogeneity of variance, if there is no statistical significance (P>0.05), use one-way analysis of variance (ANOVA) for statistical analysis, if ANOVA has statistical significance (P<0.05), LSD method Comprehensive comparison; if the variances are not homogeneous (P<0.05), use the Kruskal-Wallis test, and if the Kruskal-Wallis test is statistically significant (P<0.05), use the Mann-Whitney method for pairwise comparisons between means .

result:

During the experiment, the main clinical symptom of the animals in the vehicle control group and each administration group was tumor scab, and no abnormality was found in the state of the animals. See Table 12-1, Table 12-2 and Table 12-3 for animal clinical symptoms.

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

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

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

Conclusion: Under the experimental conditions, in the human non-small cell lung cancer (A549) subcutaneous xenograft tumor model, the test products KR11 and KR10 both had significant inhibitory effects on tumors in a dose-dependent manner, and were well tolerated by animals. Since only the backbone of the oncolytic virus exerts anti-tumor effect in immunodeficient mice, KR10 and KR11 have the same backbone of the oncolytic virus, therefore, at the same dosage level, the tumor inhibitory effects of each group are similar without statistical difference.

Table 12-1 Statistical Table of Observation of Clinical Symptoms of Experimental Animals

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

Table 12-2 Statistical Table of Observation of Clinical Symptoms of Experimental Animals

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

Table 12-3 Statistical Table of Observation of Clinical Symptoms of Experimental Animals

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

Table 13 Statistical Table of Experimental Animal Body Weight

Table 14 Experimental animal weight individual data table

Table 15 Statistical Table of Tumor Volume of Experimental Animals

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

Table 16 Experimental Animal Tumor Volume (mm ³ ) Individual Data Sheet

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

sequence group dose Tumor volume (mm ³ ) T/C(% TGI (

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

Table 18 Tumor weight of animals in each group (Mean±SEM)

Table 19 Statistical Table of Experimental Animal Tumor Weight (g, Mean±SEM)

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

Table 20 Experimental animal tumor weight individual data table

The foregoing detailed description has been offered by way of explanation and example, not to limit the scope of the appended claims. Variations on the presently recited embodiments of this application will be apparent to those of ordinary skill in the art and remain within the scope of the appended claims and their equivalents.

Claims (139)

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. 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.
8. 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.
9. 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.
10. 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.
11. 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.
12. The isolated nucleic acid molecule of any one of claims 1-11, wherein each of the genes encoding Kras mutants is Minigene.
13. 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.
14. The isolated nucleic acid molecule of any one of claims 1-13, wherein each of the Kras mutants comprises at least 20 amino acids.
15. 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.
16. 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.
17. 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.
18. 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.
19. 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.
20. 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.
21. 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.
22. 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.
23. 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.
24. 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.
25. 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.
26. 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.
27. 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.
28. 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.
29. 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.
30. 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.
31. 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.
32. 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.
33. 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.
34. 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.
35. 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.
36. 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.
37. 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.
38. 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.
39. 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.
40. 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.
41. 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.
42. 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.
43. 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.
44. 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.
45. 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.
46. 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.
47. 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.
48. 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.
49. The isolated nucleic acid molecule of any one of claims 1-48 comprising the nucleotide sequence set forth in SEQ ID No. 65.
50. 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.
51. The isolated nucleic acid molecule of any one of claims 1-50, comprising the nucleotide sequence set forth in SEQ ID No. 66.
52. 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.
53. The isolated nucleic acid molecule of any one of claims 1-52, comprising the nucleotide sequence set forth in SEQ ID No. 67.
54. The isolated nucleic acid molecule of any one of claims 1-53, further comprising a polynucleotide encoding a secretory peptide.
55. The isolated nucleic acid molecule of claim 54, wherein said polynucleotide encoding a secretory peptide is a polynucleotide encoding a CD14 protein secretory peptide.
56. 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.
57. 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.
58. A vector comprising the nucleic acid molecule of any one of claims 1-57.
59. The vector of claim 58, comprising a viral vector.
60. The vector of any one of claims 58-59, comprising an oncolytic herpes simplex virus ohv vector.
61. The vector of any one of claims 58-60, comprising a herpes simplex virus type I HSV-1 vector.
62. The vector of claim 61, wherein the HSV-1 vector lacks a neurotoxic factor γ34.5 gene.
63. 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.
64. The vector of any one of claims 58-63, comprising a promoter.
65. The vector of claim 64, wherein the promoter comprises a CMV promoter.
66. The vector of any one of claims 58-65, comprising a nucleotide sequence set forth in GenBank No. GU734771.1 of the NCBI database.
67. 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.
68. 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.
69. 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.
70. 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.
71. 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.
72. 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.
73. 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.
74. 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.
75. 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.
76. 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.
77. 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.
78. 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.
79. The composition of any one of claims 69-78, wherein each of said genes encoding a Kras mutant is Minigene.
80. The composition of any one of claims 69-79, wherein each of the genes encoding Kras mutants is tandem to provide a tandem minigene.
81. The composition of any one of claims 69-80, wherein each of the Kras mutants comprises at least 20 amino acids.
82. 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.
83. 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.
84. 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.
85. 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.
86. 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.
87. 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.
88. 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.
89. 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.
90. 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.
91. 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.
92. 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.
93. 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.
94. 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.
95. 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.
96. 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.
97. 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.
98. The composition of any one of claims 69-97, wherein the Kras a18D mutant comprises the amino acid sequence of SEQ ID NO: 60.
99. 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.
100. 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.
101. 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.
102. 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.
103. 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.
104. 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.
105. 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.
106. 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.
107. 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.
108. 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.
109. 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.
110. 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.
111. 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.
112. 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.
113. 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.
114. 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.
115. 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.
116. The composition of any one of claims 68-115, comprising the nucleotide sequence set forth in SEQ ID No. 65.
117. 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.
118. The composition of any one of claims 68-117, comprising the nucleotide sequence set forth in SEQ ID No. 66.
119. 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.
120. The composition of any one of claims 68-119, comprising the nucleotide sequence set forth in SEQ ID No. 67.
121. The composition of any one of claims 68-120, further comprising a polynucleotide encoding a secretory peptide.
122. The composition of claim 121, wherein the polynucleotide encoding a secretory peptide is a polynucleotide encoding a CD14 protein secretory peptide.
123. 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.
124. 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.
125. 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.
126. The composition of any one of claims 68-125, wherein the vector comprises a viral vector.
127. The composition of any one of claims 68-126, wherein the vector comprises an oncolytic herpes simplex virus ohv vector.
128. The composition of any one of claims 68-127, wherein the vector comprises a herpes simplex virus type I HSV-1 vector.
129. The composition of any one of claims 128, wherein the HSV-1 vector lacks a neurotoxic factor γ34.5 gene.
130. 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.
131. The composition of any one of claims 68-130, wherein the vector comprises a promoter.
132. The composition of claim 131, wherein the promoter comprises a CMV promoter.
133. 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.
134. 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.
135. The use of claim 134, wherein the tumor comprises a solid tumor.
136. The use of any one of claims 134-135, wherein the tumor comprises non-small cell lung cancer.
137. The use of any one of claims 134-136, wherein the tumor comprises colorectal cancer.
138. The use of any of claims 134-137, wherein the tumor comprises breast cancer.
139. The use of any of claims 134-138, wherein the tumor comprises pancreatic cancer.

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