WO2019174610A1 - Oncolytic virus and synthetic dna sequence, and application thereof - Google Patents

Abstract

Disclosed are an oncolytic virus and synthetic DNA sequence, and an application thereof. The expression sequence of an exogenous basic peptide is inserted in the genome of the oncolytic virus, and the basic peptide is expressed in a physiological process, so that the pH value of the host environment infected thereby is increased. The synthetic DNA sequence is used for expressing a basic peptide, the content of basic amino acid in the basic peptide exceeding 60%. The provided oncolytic virus and synthetic DNA sequence are applied to the preparation of an antitumor drug and have a good antitumor effect.

Description

Oncolytic virus, synthetic DNA sequence and application thereof Technical field

The invention belongs to the field of biomedicine, and more particularly to an oncolytic virus, a basic peptide segment and an application thereof.

Background technique

The oncolytic virus has the property of infecting and proliferating in tumor cells, thereby destroying the tumor tissue. In clinical studies, the safety and therapeutic efficacy of a variety of viruses with oncolytic properties for the treatment of different cancers has been reported and confirmed.

The current working principles of oncolytic viruses include:

1) Oncolytic viruses can selectively infect tumor cells by inactivation or deletion of specific genes in target cells, and ultimately destroy tumor cells. For example, adenovirus, Newcastle virus, Coxsackie virus is a single-stranded RNA virus, and RNA replication is carried out in the cytoplasm without causing insertional gene mutation of the host cell. Coxsackie virus is divided into two subtypes A and B based on its antigenicity in mice. In most cases, patients infected with wild-type Coxsackie virus will only have mild "cold" symptoms. Currently, both subtypes of Coxsackie virus have been tried alone or in combination for tumor therapy.

2) The oncolytic virus for the purpose of "killing" tumor cells is highly toxic, and when the body is infected with the wild type virus, pathological reactions such as fever and chills may occur. With the increase of serum AMY, CK, AST, ALT and other indicators, the organs of the body may be damaged. For example, pancreatic exocrine tissue damage, myocardial tissue and liver inflammation.

Summary of the invention

In view of the above defects or improvement requirements of the prior art, the present invention provides an oncolytic virus, a synthetic DNA sequence and an application thereof, which aims to inhibit tumor cells by providing an oncolytic virus capable of changing the tumor microenvironment. This solves the technical problem that the existing oncolytic virus tumor suppression effect is poor or the toxicity is too strong.

In order to achieve the above object, according to one aspect of the present invention, there is provided an oncolytic virus characterized in that an expression sequence of an exogenous basic peptide is inserted into a genome, and the basic peptide is expressed in a physiological process. In the segment, the pH of the host environment in which it is infected is increased by about 0.4 to about 0.6.

Preferably, the oncolytic virus, the oncolytic virus is herpes virus, coxsackie virus, adenovirus, vaccinia virus, measles virus, poliovirus, retrovirus, reovirus, respiratory tract Cytovirus, parvovirus H1, vesicular stomatitis virus, or Newcastle disease virus, preferably adenovirus, Newcastle virus or Coxsackie virus.

Preferably, the oncolytic virus, wherein the exogenous basic peptide is a 4 peptide to 10 peptide.

Preferably, the oncolytic virus has a basic amino acid content of more than 60% in the exogenous basic peptide.

Preferably, the oncolytic virus has a basic amino acid content of more than 80% in the exogenous basic peptide.

Preferably, the oncolytic virus, wherein the basic amino acid is selected from the group consisting of: arginine, lysine or histidine.

Preferably, the oncolytic virus, wherein the basic amino acid is selected from the group consisting of: arginine or lysine.

Preferably, the oncolytic virus has an N-terminal amino acid of the basic peptide segment which is lysine.

Preferably, the oncolytic virus, wherein the exogenous basic peptide is selected from the group consisting of:

(1) Arg-Lys-Arg-Lys; (2) Lys-Arg-Lys-Arg; (3) Arg-Arg-Lys-Lys; (4) Lys-Lys-Arg-Arg;

(5) Lys-Arg-Arg-Lys; (6) Arg-Lys-Lys-Arg; (7) Arg-Arg-His-Lys-Lys; (8) Lys-His-Arg-Lys-His-Arg;

(9) Lys-His-Arg-Cys-Lys-Pro; (10) Arg-Arg-His-Lys-Met-Lys; (11) His-Arg-Lys-Cys-Arg-Lys;

(12) Lys-Arg-Trp-Arg-Lys-His-Arg; (13) His-Lys-Gly-Arg-Lys-Cys-Arg-Val;

(14) Lys-Arg-Trp-His-Lys-Met-Arg-Lys-His; (15) His-Phe-Trp-Arg-Gln-Cys-Ala-Met-Lys;

(16) Tyr-Phe-Pro-Arg-His-Gln-Lys-Trp-Lys; (17) Trp-Lys-Tyr-Arg-Gln-Ile-Ser-Thr-Cys;

(18) Arg-Lys-His-Lys-Met-Arg-Lys-Cys-His-Lys.

Preferably, the oncolytic virus, wherein the oncolytic virus is a Coxsackievirus B3 strain.

Preferably, the oncolytic virus, wherein the exogenous basic peptide is selected from the group consisting of:

(5) Lys-Arg-Arg-Lys; (14) Lys-Arg-Trp-His-Lys-Met-Arg-Lys-His;

(15) His-Phe-Trp-Arg-Gln-Cys-Ala-Met-Lys.

Preferably, the oncolytic virus, wherein the oncolytic virus is a variant attenuated strain Coxsackie virus B3 strain, comprises the following base mutation sites: T96C, G1180A, T1654C, T1756C, G2276A, A2685C, G2690A, C3120A , A3231G, G4327A, T5088C, A5270G, C7026T, and/or G7192A.

Preferably, the oncolytic virus, the coding sequence of the exogenous basic peptide fragment is inserted on the pVAX1 vector.

Preferably, the oncolytic virus, wherein the exogenous basic peptide is selected from the group consisting of Lys-Arg-Trp-His-Lys-Met-Arg-Lys-His.

According to another aspect of the present invention, the use of the oncolytic virus is provided, and the oncolytic virus provided by the present invention is applied to the preparation of an antitumor drug.

Preferably, the use of the oncolytic virus provided by the present invention is applied to the preparation of an anti-solid tumor drug.

Preferably, the application, the oncolytic virus provided by the present invention is applied to the preparation of a medicament against an anti-respiratory system tumor, a digestive tract system tumor, an endocrine system tumor, or a gynecological tumor.

According to another aspect of the present invention, an antitumor drug comprising the oncolytic virus provided by the present invention is provided.

Preferably, the anti-tumor drug further comprises a checkpoint inhibitor.

According to another aspect of the present invention, there is provided a method of treating a malignant tumor, which comprises administering the antitumor drug of the present invention intravenously or topically to a lesion.

Preferably, the method of treatment, wherein the malignant tumor is a solid tumor.

Preferably, the method of treatment, wherein the malignant tumor is a respiratory system tumor, a digestive system tumor, an endocrine system tumor, or a gynecological tumor.

According to another aspect of the present invention, there is provided a synthetic DNA sequence for expressing a basic peptide having a basic amino acid content of more than 60%.

Preferably, the synthetic DNA sequence has a basic amino acid content of more than 80% in the basic peptide segment.

Preferably, the synthetic DNA sequence, wherein the basic amino acid is selected from the group consisting of: arginine, lysine or histidine.

Preferably, the synthetic DNA sequence, wherein the basic amino acid is selected from arginine or lysine.

Preferably, the synthetic DNA sequence, wherein the N-terminal amino acid of the basic peptide segment is lysine.

Preferably, the synthetic DNA sequence, wherein the exogenous basic peptide is selected from the group consisting of:

(1) Arg-Lys-Arg-Lys; (2) Lys-Arg-Lys-Arg; (3) Arg-Arg-Lys-Lys; (4) Lys-Lys-Arg-Arg

(5) Lys-Arg-Arg-Lys; (6) Arg-Lys-Lys-Arg; (7) Arg-Arg-His-Lys-Lys; (8) Lys-His-Arg-Lys-His-Arg;

(9) Lys-His-Arg-Cys-Lys-Pro; (10) Arg-Arg-His-Lys-Met-Lys; (11) His-Arg-Lys-Cys-Arg-Lys;

(12) Lys-Arg-Trp-Arg-Lys-His-Arg; (13) His-Lys-Gly-Arg-Lys-Cys-Arg-Val;

(14) Lys-Arg-Trp-His-Lys-Met-Arg-Lys-His; (15) His-Phe-Trp-Arg-Gln-Cys-Ala-Met-Lys;

(16) Tyr-Phe-Pro-Arg-His-Gln-Lys-Trp-Lys; (17) Trp-Lys-Tyr-Arg-Gln-Ile-Ser-Thr-Cys;

(18) Arg-Lys-His-Lys-Met-Arg-Lys-Cys-His-Lys.

Preferably, the synthetic DNA sequence, wherein the basic peptide sequence is:

(5) Lys-Arg-Arg-Lys; (14) Lys-Arg-Trp-His-Lys-Met-Arg-Lys-His;

(15) His-Phe-Trp-Arg-Gln-Cys-Ala-Met-Lys.

In general, the above technical solutions conceived by the present invention can achieve the following beneficial effects compared with the prior art:

(1) The oncolytic virus provided by the invention can significantly change the pH value of the interstitial cells of the tumor lesion, thereby affecting the microenvironment of tumor cell growth, and finally inhibiting the growth of the tumor, and has a relatively broad broad-spectrum resistance. Tumor effect, applied to the preparation of anti-tumor drugs, has a good application prospect. The oncolytic virus provided by the present invention is a microenvironment for tumor growth, not a tumor cell itself, and thus tumor cells are not easily resistant to mutation. What is even more striking is that since the oncolytic virus proliferates with the proliferation of malignant tumor cells and continues to express the basic polypeptide, the anti-tumor effect has a sustained cumulative effect, and automatically adapts to the degree of tumor development without excessive treatment.

Preferably, the oncolytic virus provided by the present invention may be various types of oncolytic viruses currently known, and the expression of the basic peptide segment acts on the cell microenvironment, and kills the tumor cell or inhibits the expression of a specific gene of the tumor cell. The principle of action of oncolytic viruses does not conflict, and the effects can be superimposed on each other to further inhibit tumor growth and have an anti-tumor effect.

The preferred method adopts Coxsackie CVB 3 and the N-terminal lysine-like 4 peptide and 9-peptide expression gene, which can obviously change the acid-base environment of the tumor cell interstitial, and has excellent anti-solid tumor effect. And its toxicity is low, the side effects are small, and only a slight fever reaction is caused. In addition, Coxsackie virus is an RNA virus that does not integrate with host cells and has no risk of transcription.

(2) The tumor treatment method provided by the present invention is suitable for intravenous administration because of the excellent safety, targeting, specificity, and low toxic side effects of the oncolytic virus provided by the present invention. In particular, it has a good therapeutic effect on malignant tumors that do not have surgical conditions, such as metastatic type, diffusivity, and early stage tumors. In addition, the tumor treatment method provided by the invention has a certain tumor prevention effect.

(3) The expression gene for encoding a basic peptide segment provided by the present invention can express a basic peptide segment having a pH value which changes the microenvironment of the cell, thereby affecting the microenvironment of the tumor cell lesion region and inhibiting the growth of the tumor cell.

DRAWINGS

1 is a schematic diagram showing the structure of a Coxsackie virus gene carrying the eukaryotic expression vector pVAX1 according to an embodiment of the present invention;

2 is a schematic diagram showing the structure of a Coxsackie virus gene carrying the eukaryotic expression vector pVAX1 inserted into a synthetic DNA sequence according to an embodiment of the present invention;

3 is a schematic flow chart of a virus purification process provided by an embodiment of the present invention;

Figure 4 is a graph showing the tumor volume of Example 20 of the present invention;

Figure 5 is a comparison view of a tissue section provided in Example 20 of the present invention;

Figure 6 is a graph showing a tumor volume curve according to Embodiment 21 of the present invention;

Figure 7 is a graph showing a tumor volume curve according to Embodiment 22 of the present invention;

Figure 8 is a graph showing a tumor volume curve according to Embodiment 23 of the present invention;

Figure 9 is a graph showing the inhibitory effect of tumor cells in vitro according to Example 24 of the present invention;

Figure 10 is a microscopic examination result of myocardial cytotoxicity provided in Example 25 of the present invention;

Figure 11 is a photograph of a mouse after 6 days of toxicity test on BALB/C mice provided in Example 25 of the present invention;

Figure 12 is a photomicrograph of a tissue section of a mouse myocardial tissue after 6 days of toxicity test in BALB/C mice according to Example 25 of the present invention;

Figure 13 is a photograph showing the experimental observation of toxicity of suckling mice provided in Example 25 of the present invention;

Figure 14 is a diagram showing the results of a microscopic examination provided in Embodiment 26 of the present invention;

Figure 15 is a comparison diagram of pH detection provided in Example 24 of the present invention;

Figure 16 is a graph showing the in vitro inhibitory effect on different types of tumor cells provided in Example 27 of the present invention.

detailed description

The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It is understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Further, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not constitute a conflict with each other.

The oncolytic virus provided by the invention has an expression sequence of an exogenous basic peptide inserted in the genome, and expresses the basic peptide in a physiological process, so that the pH of the infected host environment increases, and the increase is about 0.4. To 0.6. After the infection of the oncolytic virus of the exogenous peptide gene, the large expression of the exogenous alkaline peptide can make the microenvironment of the tumor tissue alkaline, so that the tumor tissue can be better inhibited and eliminated.

The oncolytic virus is herpes virus, coxsackie virus, adenovirus, vaccinia virus, measles virus, poliovirus, retrovirus, reovirus, respiratory syncytial virus, parvovirus H1, vesicular mouth Inflammatory virus, or Newcastle disease virus. The oncolytic virus is preferably an oncolytic virus such as an adenovirus, a Newcastle virus or a Coxsackie virus that inactivates or deletes a specific gene in a target cell.

The exogenous basic peptide is a 4 peptide to 10 peptide; wherein the basic amino acid content exceeds 60%, preferably exceeds 80%; the basic amino acid is selected from: arginine, lysine or histidine, preferably The basic amino acid is selected from the group consisting of: arginine or lysine; the N-terminal amino acid of the exogenous basic peptide segment is preferably lysine.

The exogenous basic peptide is selected from the following peptides:

(1) Arg-Lys-Arg-Lys; (2) Lys-Arg-Lys-Arg; (3) Arg-Arg-Lys-Lys; (4) Lys-Lys-Arg-Arg;

(5) Lys-Arg-Arg-Lys; (6) Arg-Lys-Lys-Arg; (7) Arg-Arg-His-Lys-Lys; (8) Lys-His-Arg-Lys-His-Arg;

(9) Lys-His-Arg-Cys-Lys-Pro; (10) Arg-Arg-His-Lys-Met-Lys; (11) His-Arg-Lys-Cys-Arg-Lys;

(12) Lys-Arg-Trp-Arg-Lys-His-Arg; (13) His-Lys-Gly-Arg-Lys-Cys-Arg-Val;

(14) Lys-Arg-Trp-His-Lys-Met-Arg-Lys-His; (15) His-Phe-Trp-Arg-Gln-Cys-Ala-Met-Lys;

(16) Tyr-Phe-Pro-Arg-His-Gln-Lys-Trp-Lys; (17) Trp-Lys-Tyr-Arg-Gln-Ile-Ser-Thr-Cys;

(18) Arg-Lys-His-Lys-Met-Arg-Lys-Cys-His-Lys.

Preferably, the coxsackie virus, specifically the coxsackie virus attenuating variant, is used to insert the following exogenous basic peptide on the pVAX1 vector in which the viral genome is constructed:

(5) Lys-Arg-Arg-Lys; (14) Lys-Arg-Trp-His-Lys-Met-Arg-Lys-His;

(15) His-Phe-Trp-Arg-Gln-Cys-Ala-Met-Lys.

More preferably, the exogenous basic peptide is selected from the group consisting of:

(5) Lys-Arg-Arg-Lys; (14) Lys-Arg-Trp-His-Lys-Met-Arg-Lys-His;

Wherein, when the Coxsackie virus CVB 3 virus strain is used, the exogenous peptide segment (14) Lys-Arg-Trp-His-Lys-Met-Arg-Lys-His exhibits excellent tumor suppressing effect and Good security.

The Coxsackie virus CVB 3 virus preferably adopts the mutant attenuated strain Coxsackie virus B3 strain, and includes the following base mutation sites: T96C, G1180A, T1654C, T1756C, G2276A, A2685C, G2690A, C3120A, A3231G, G4327A. , T5088C, A5270G, C7026T, and / or G7192A. The coding sequence of the exogenous basic peptide fragment was inserted on the pVAX1 vector.

The foreign basic peptide DNA sequence is inserted between the 5'UTR and VP4 fragments of the recombinant vector

The oncolytic virus provided by the invention is applied to prepare antitumor drugs, especially anti-solid tumor drugs, for example, drugs for preparing anti-respiratory system tumors, digestive system tumors, endocrine system tumors, or gynecological tumors. The oncolytic virus provided by the invention, after reaching the lesion area, utilizes the targeting and replication ability of the virus to express the basic peptide segment according to the degree of tumor. The experiment confirmed that the basic peptide segment changed the pH of the microenvironment of the tumor cells in the tumor cells and the intercellular environment. This change brings a series of combined effects to the metabolism of tumor cells, which ultimately leads to significant tumor suppression. Therefore, the current oncolytic virus can theoretically carry the expression gene of the exogenous basic peptide by genetic modification, thereby changing the microscopic presence of the tumor cell in addition to the original effect of inhibiting or killing the tumor cell. The environment produces an inhibitory effect, and the two act synergistically to inhibit the tumor more effectively. Since the oncolytic virus provided by the present invention exerts an anti-tumor effect by affecting the microenvironment in which the tumor cells are located, it has a more pronounced inhibitory effect on the solid tumor in which the tumor cells are concentrated. At the same time, for oncolytic viruses that cause severe physiological reactions, because of the superimposed tumor inhibition effect, the amount and range of viruses are relatively reduced, so the physiological side effects caused by them are relatively reduced, and the use range of the oncolytic virus is broadened and improved. The safety of the oncolytic virus.

The present invention provides an antitumor drug comprising the oncolytic virus provided by the present invention. Preferably, an immunoassay point inhibitor is also included. The drug can exert a good tumor suppressing effect by intravenous administration or topical administration to the lesion. Immunological checkpoint inhibitors (PD-1, PD-L1, CTLA4), the response rate to solid tumors is not high (except melanoma), may be due to checkpoint inhibitors released after the immune system, solid tumor patients The stimulation of the immune system is insufficient. The synergistic effect of the oncolytic virus immunoassay inhibitor provided by the invention significantly improves the killing effect of the immune system on the solid tumor, enhances the permeability of the local immune cells of the tumor, and up-regulates the PD-L1. Especially for recombinant CVB 3 virus, it induces local specific and non-specific immune responses in tumors, leading to some immune changes such as: calreticulin (CRT) exposure, ATP valgus, HGMB1 (Extracellular High Mobility Group Box1) Translocation within the cell. As the oncolytic virus proliferates, by inducing IFN or/and cytokine production to activate NK cells and DC cells, various mature DCs and cytotoxic CD107a+ NK cells are promoted into the tumor site, resulting in an immune cell spectrum in the tumor microenvironment. Changes, while restoring the body's inherent anti-tumor immunity. The synergistic effect of the recombinant Coxsackie virus provided by the present invention and an immunoassay inhibitor is particularly remarkable.

The present invention also provides an expression gene of a basic peptide fragment for expressing a basic peptide having a basic amino acid content of more than 60%, preferably more than 80%; The basic amino acid is selected from the group consisting of arginine, lysine or histidine, preferably the basic amino acid is selected from arginine or lysine. The N-terminal amino acid of the basic peptide segment is preferably lysine.

The synthetic DNA sequence encoding the exogenous basic peptide is selected from the following peptides:

(1) Arg-Lys-Arg-Lys; (2) Lys-Arg-Lys-Arg; (3) Arg-Arg-Lys-Lys; (4) Lys-Lys-Arg-Arg;

(5) Lys-Arg-Arg-Lys; (6) Arg-Lys-Lys-Arg; (7) Arg-Arg-His-Lys-Lys; (8) Lys-His-Arg-Lys-His-Arg;

(9) Lys-His-Arg-Cys-Lys-Pro; (10) Arg-Arg-His-Lys-Met-Lys; (11) His-Arg-Lys-Cys-Arg-Lys;

(12) Lys-Arg-Trp-Arg-Lys-His-Arg; (13) His-Lys-Gly-Arg-Lys-Cys-Arg-Val;

(14) Lys-Arg-Trp-His-Lys-Met-Arg-Lys-His; (15) His-Phe-Trp-Arg-Gln-Cys-Ala-Met-Lys;

(16) Tyr-Phe-Pro-Arg-His-Gln-Lys-Trp-Lys; (17) Trp-Lys-Tyr-Arg-Gln-Ile-Ser-Thr-Cys;

(18) Arg-Lys-His-Lys-Met-Arg-Lys-Cys-His-Lys.

The basic peptide sequence is preferably:

(5) Lys-Arg-Arg-Lys; (14) Lys-Arg-Trp-His-Lys-Met-Arg-Lys-His;

(15) His-Phe-Trp-Arg-Gln-Cys-Ala-Met-Lys.

The following are examples:

Recombinant Coxsackie virus in the genome of Examples 1 to 18 inserted into the basic peptide gene sequence

The full-length sequence of the Coxsackie B3 nancy strain is shown in GeneBank ID: JX312064.1 (presented by Tongji Medical College). The recombinant Coxsackie virus strain (rCVB3) used in the examples contained the following base mutation sites: T96C, G1180A, T1654C, T1756C, G2276A, A2685C, G2690A, C3120A, A3231G, G4327A, T5088C, A5270G, C7026T, G7192A. The complete cDNA sequence of the recombinant Coxsackie virus strain was synthesized by Wuhan Boweed Biotechnology Co., Ltd. and constructed by molecular biology to the eukaryotic expression vector pVAX1, as shown in Figure 1:

The oncolytic virus provided in the present example was inserted into the exogenous basic peptide gene sequence by reverse genetics between the 5' UTR and VP4 fragments of the above constructed recombinant vector. A 15 bp (SEQ NO. 1) and 24 bp (SEQ NO. 2) DNA sequences were used at the 5' and 3' ends of the sequence for recognition and cleavage of protease C, as shown in Figure 2.

The exogenous alkaline peptide and coding gene are shown in the following table:

Example	Peptide name	Polypeptide sequence	Gene name		gene sequence
1	Polypeptide 1	SEQ NO.3	Nucleotide seque 1	SEQ NO.4
2	Polypeptide 2	SEQ NO.5	Nucleotide seque 2	SEQ NO.6
3	Polypeptide 3	SEQ NO.7	Nucleotide seque 3	SEQ NO.8
4	Polypeptide 4	SEQ NO.9	Nucleotide seque 4	SEQ NO.10
5	Polypeptide 5	SEQ NO.11	Nucleotide seque 5	SEQ NO.12
6	Polypeptide 6	SEQ NO.13	Nucleotide seque 6	SEQ NO.14
7	Polypeptide 7	SEQ NO.15	Nucleotide seque 7	SEQ NO.16
8	Polypeptide 8	SEQ NO.17	Nucleotide seque 8	SEQ NO.18
9	Polypeptide 9	SEQ NO.19	Nucleotide seque 9	SEQ NO.20
10	Polypeptide 10	SEQ NO.21	Nucleotide seque 10	SEQ NO.22
11	Polypeptide 11	SEQ NO.23	Nucleotide seque 11	SEQ NO.24
12	Polypeptide 12	SEQ NO.25	Nucleotide seque 12	SEQ NO.26
13	Polypeptide 13	SEQ NO.27	Nucleotide seque 13	SEQ NO.28

14	Polypeptide 14	SEQ NO.29	Nucleotide seque 14	SEQ NO.30
15	Polypeptide 15	SEQ NO.31	Nucleotide seque 15	SEQ NO.32
16	Polypeptide 16	SEQ NO.33	Nucleotide seque 16	SEQ NO.34
17	Polypeptide 17	SEQ NO.35	Nucleotide seque 17	SEQ NO.36
18	Polypeptide 18	SEQ NO.37	Nucleotide seque 18	SEQ NO.38

In this embodiment, the insertion method is specifically as follows: the DNA sequence carrying the above-mentioned exogenous basic peptide is inserted between the 5'UTR and VP4 fragments of the recombinant vector, and the positive clone is screened and identified, and the plasmid is extracted and obtained for virus. The complete cDNA of the package.

details as follows:

(1) Coxsackie virus gene CVB3-Am synthesis

Gene synthesis pUC57-CVB3-Am by Suzhou Jinweizhi Biotechnology Co., Ltd.

(2) Small-scale extraction of vectors pVAX1 and pUC19

The plasmid containing pVAX1-SalI and pUC19 was extracted using a kit from Axygen.

(3) Construction of pVAX1-SalI-CVB3-Am vector

a double digestion and recovery

The plasmid pVAX1 (ApaI→SalI) and plasmid pUC57-CVB3-Am were double digested with NotI and SalI. After the reaction, 1% agarose gel electrophoresis was carried out, and 2999 bp vector and about 7500 bp CVB3-Am fragment were separately collected and gelatinized. Recovery, purification of the digested product according to the specific steps of Axygen's gel recovery kit.

b connection and conversion

The CVB3-Am fragment digested with NotI and SalI was ligated to the vector pVAX1 (ApaI→SalI) in a certain ratio using TKDNA ligase of TAKARA, and transformed into Stbl3.

Screening and identification of c positive clones

Single colonies grown on LB+Kana plates were randomly picked for colony PCR, and the correct positive clones were sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing.

The pVAX1 vector carrying the complete cDNA sequence of the recombinant Coxsackie virus was transfected into Cos7 packaging cells, and the recombinant virus solution having infectious ability was cultured and obtained.

Preferably, a PolyA sequence is inserted after the 3' UTR, and the length is between 20 and 100, preferably between 30 and 80, which can effectively ensure the stability of the gene encoding the exogenous alkaline peptide, thereby ensuring the expression effect. The virus can be stored at -20 ° C for more than one year, can be placed at room temperature for 2 days without dropping the titer, and has strong stability, which is convenient for storage and transportation.

Comparative Example 1: Insertion of the above synthetic DNA sequence between the pVAX1 vector VP1 and 2A elements to obtain a virus, the expression of the basic peptide was not stably expressed, and the inhibitory effect on cancer cells was limited.

Example 19 Preparation of a test article for pharmacodynamic research

The infectious virus-containing recombinant virus solution described in Examples 1 to 18 was inoculated into the expanded and cultured Vero cells, and a virus purification liquid was obtained as a test product by a production and purification process. The virus purification process diagram is shown in Figure 3:

The virus purification solution is tested and should meet the following criteria:

Example 20 In Vivo Efficacy of Recombinant Coxsackie Virus for Selective Inhibition of Solid Tumors

The test article used in this example was prepared and tested according to the protocol described in Example 19.

In the present example, the recombinant Coxsackie viruses of Examples 1 to 18 were used as test articles, which were prepared in Example 5, Example 14, and Example 17, respectively.

The above virus was prepared into the test sample according to the method described in Example 19.

A nude mouse model of lung cancer A549 cells was established, and 30 tumor-forming animals with uniform tumor volume were screened. 30 animals with a tumor volume of 45-70 mm ³ were selected and divided into 1 to 5 groups. The average tumor volume was about 56 mm ³ . Randomized grouping, each group of animals was given a random number by Excel software, and the random numbers were sorted in ascending order. They were divided into 5 groups of 6 animals each.

The grouping, dosage and method of administration are as follows:

Group 2 (cisplatin) was administered once a week for 4 weeks, and for 1 week, D41 animals were euthanized; physiological saline was administered once a day for 6 weeks, and D41 animals were euthanized. Observe 2 times a day during the administration, observe the general clinical symptoms of the animals, and measure the body weight and tumor diameter twice a week.

RESULTS: During the whole experiment, the average body weight of the animals increased, and there was no significant difference between the groups (P<0.05). The graph of the average tumor volume growth with time in each group is shown in Fig. 4.

On the 41st day, the negative test group and the test sample group prepared in Example 5 were separately sampled for tissue section detection, and the comparison chart is shown in Fig. 5:

It can be seen from the figure that the test group prepared in the right example 5 has obvious tumor cell damage and deeper eosin staining than the left negative control group.

Example 21 In Vivo Efficacy Study of Selective Inhibition of Solid Tumor by Recombinant Coxsackie Virus

The test article used in this example was prepared and tested according to the protocol described in Example 19.

In the present example, recombinant coxsackie virus in which three kinds of genomes were inserted with a basic peptide segment was used as a test article, which were prepared in Example 1, Example 2, Example 4, and Example 5, respectively.

The above virus was prepared into the test sample according to the method described in Example 19.

A nude mouse model of lung cancer A549 cells was established, and 30 tumor-forming animals with uniform tumor volume were screened. 30 animals with a tumor volume of 45-72 mm ³ were selected and divided into 1 to 6 groups. The average tumor volume was about 57 mm ³ . Randomized grouping, each group of animals was given a random number by Excel software, and the random numbers were sorted in ascending order. They were divided into 6 groups of 5 animals each.

The grouping, dosage and method of administration are as follows:

Group 2 (cisplatin) was administered once a week for 4 weeks, and for 1 week, D48 animals were euthanized; physiological saline was administered once a day for 7 weeks, and D48 animals were euthanized. The drug was observed twice a day during the administration, and the general clinical symptoms of the animals were observed, and the body weight and tumor diameter were measured twice a week.

RESULTS: During the whole experiment, the average body weight of the animals increased, and there was no significant difference between the groups (P<0.05). The graph of the average tumor volume growth with time in each group is shown in Fig. 6.

It can be seen that the test articles prepared in Example 1, Example 2, Example 4, and Example 5 all have an anti-tumor effect, wherein the test sample of Example 5 has a significant inhibitory effect on tumor growth.

Example 22 In Vivo Pharmacodynamic Study of Selective Inhibition of Solid Tumor by Recombinant Coxsackie Virus

The test article used in this example was prepared and tested according to the protocol described in Example 19.

In the present example, recombinant coxsackie viruses in which two genomes were inserted with a basic peptide were used as the test articles, which were prepared in Example 18 and Example 13, respectively.

The above virus was prepared into the test sample according to the method described in Example 19.

A549 cells established subcutaneous tumor nude mouse model of lung cancer, 20 tumor-bearing animals screened into homogeneous tumor volume, tumor volume 62-92mm 20 to select animals are divided into ³ groups of 1 to 4, the mean tumor volume of about 79mm ^3, a fully Randomized grouping, each group of animals was given a random number by Excel software, and the random numbers were sorted in ascending order. They were divided into 4 groups of 5 animals each.

The grouping, dosage and method of administration are as follows:

Group 2 (cisplatin) was administered once a week for 4 weeks, and for 1 week, D42 animals were euthanized; physiological saline was administered once a day for 6 weeks, and D42 animals were euthanized. Observe 2 times a day during the administration, observe the general clinical symptoms of the animals, and measure the body weight and tumor diameter twice a week.

RESULTS: During the whole experiment, the average body weight of the animals increased, and there was no significant difference between the groups (P<0.05). The graph of the average tumor volume growth with time in each group is shown in Fig. 7.

It can be seen that the test samples prepared in Example 18 and Example 13 all have certain anti-tumor effects.

Example 23 In Vivo Pharmacodynamic Study of Selective Inhibition of Solid Tumor by Recombinant Coxsackie Virus

The test article used in this example was prepared and tested according to the protocol described in Example 19.

In the present example, recombinant coxsackie virus in which three kinds of genomes were inserted with a basic peptide segment was used as a test article, which were prepared in Example 8, Example 9, and Example 10, respectively.

The above virus was prepared into the test sample according to the method described in Example 19.

Calu established cell lung carcinoma subcutaneous tumor model in nude mice, tumor screening animal tumor volume 25 uniform, first select tumor volume 65-90mm 25 animals were divided into ³ groups of 1 to 5, the mean tumor volume of about 79mm ^3, a fully Randomized grouping, each group of animals was given a random number by Excel software, and the random numbers were sorted in ascending order. They were divided into 5 groups of 5 animals each.

The grouping, dosage and method of administration are as follows:

Group 2 (cisplatin) was administered once a week for 4 weeks, and for 1 week, D33 animals were euthanized; physiological saline was administered once a day for 5 weeks, and D33 animals were euthanized. Observe 2 times a day during the administration, observe the general clinical symptoms of the animals, and measure the body weight and tumor diameter twice a week.

RESULTS: During the whole experiment, the average body weight of the animals increased, and there was no significant difference between the groups (P<0.05). The graph of the average tumor volume growth with time in each group is shown in Fig. 8.

It can be seen that the test samples prepared in Example 8, Example 9, and Example 10 all have certain anti-tumor effects.

The test articles prepared in Examples 1 to 18 all had antitumor effects, and the test articles prepared in Example 5 and the test samples prepared in Example 14 had a significant inhibitory effect on tumor growth.

Example 24 In vitro pharmacodynamic study of selective inhibition of solid coxsackie virus on solid tumors

To determine the in vitro cell viability, the human lung cancer cell line A549 was assayed for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl 2H-tetrazolium bromide (MTT). Cells were seeded in 96-well plates 24 hours prior to treatment and grown to approximately 80% confluence. Recombinant CVB3 with different concentrations (1 PFU/mL, 1 x 101 PFU/mL, 1 x 102 PFU/mL, 1 x 103 PFU/mL, 1 x 104 PFU/mL, 1 x 105 PFU/mL, 1 x 106 PFU/mL, 1 x 107 PFU/mL or 1 x 108 PFU/mL), and Example 5, implementation Example 14, Example 17 Infected cells, or normal saline (NS) was used as a negative control, and cisplatin was used as a positive control. After 72 hours, the MTT assay was performed according to the manufacturer's protocol (VWR Life Sciences Amresco, Radnor, PA, USA). Briefly, 200 μL of MTT (0.5 mg/mL) was used instead of the cell culture medium, and culture was continued for 1 h at 37 ° C in 10% FBS cell culture medium, and the supernatant of each group was removed, and 200 μL of dimethyl sulfoxide (200 μL) was added. DMSO) dissolves each well of the MTT dye. The absorption spectrum was read at a wavelength of 570 nm on a microplate reader. In each case, 6 replicates were tested and all assays were performed in triplicate. The half-maximal inhibitory concentrations (IC50) of each group were calculated to be 10,479,7.1, 329, 0.5, 2,051, and 4,190,4.4, respectively.

The in vitro inhibition rate of each group on A549 cells is shown in Fig. 9. 9 Comparison of recombinant CVB3, in vitro inhibition of A549 by Examples 5, 14, and 17. The results show that the in vitro inhibitory effect of Example 5 is better than that of recombinant CVB3.

From the results of the in vitro cell inhibition rate experiment, Example 5 and Example 14 (peptide amino acid over 60%) inhibited tumor cells by more than 95% at a viral concentration of 10 ⁷ ; In the peptide example of Example 5, the inhibitory effect was particularly remarkable.

Example 25 Safety Experiment

To evaluate the safety of the oncolytic virus provided by the present invention by using a toxicity test of cardiomyocytes, the specific steps are as follows:

The oncolytic viruses of Example 5 (rCVB3-4pep5) and Example 14 (rCVB3-9pep) were evaluated using rCVB3 and CVB3Nancy strains as positive controls.

Cardiomyocyte toxicity test: The viruses of Example 5, Example 14 and the positive control group were respectively infected with human cardiomyocytes (purchased from Suzhou Bei Na Chuanglian Biotechnology Co., Ltd.), and the final concentration of the virus was 10 ⁷ PFU/ml. The group used saline. Microscopic examination after 72 hours. As a result, as shown in Fig. 10, the CVB3Nancy strain caused cardiomyocyte lesions, while the rCVB3, Example 5 and Example 14 administration groups had no lesions.

Toxicity test on BALB/C mice: The viruses of Example 5, Example 14 and the positive control group were injected intraperitoneally into BALB/C mice (license No. 42000600028329) in a dosage form of 10 ⁸ PFU/ml, 0.3 ml per day. The negative control group used physiological saline. Observation was performed every day, and after 6 days, as shown in Fig. 11, mouse myocardial tissue was taken for tissue sectioning, and the results are shown in Fig. 12. The experimental results showed that the mice in the CVB3Nancy strain administration group were in a bad state. Myocardial tissue section results showed that the CVB3Nancy administration group caused significant myocardial damage, while the recombinant CVB3 administration group was normal.

Toxicity test on suckling rats: The viruses of Example 5, Example 14 and the positive control group were injected intraperitoneally into the suckling mice (license No. 42816300002647) at a dose of 10 ⁸ PFU/ml, each 0.1ml. The negative control group used physiological saline. Observe every day. As a result, as shown in Fig. 13, the CVB3Nancy strain administration group died on the sixth day, and the rCVB3, Example 5, and Example 14 administration groups were normal.

Through the above in vitro in vitro comparative experiments, rCVB3, Example 5 and Example 14 were significantly weakly toxic compared with the CVB3Nancy strain, and the clinical safety was high.

Example 26 Study on the changes of tumor cell cytoplasmic PH by recombinant Coxsackie virus

In the present example, two kinds of recombinant Coxsackie viruses in which the basic peptide was inserted into the genome were used as the test samples, which were Example 5 and Example 14, respectively.

Two viruses were prepared as test samples as described in Example 19.

Two test samples were used to infect Vero cells, respectively, and Example 5 and Example 14 were designated as 4p5 group and 9pep group, respectively. Another group of cells served as a negative control. Each group of cells was divided into two parts, cultured under the same conditions and subjected to test operations. One of each group of cells was stained with eosin and microscopically after 3 hours of infection. The result is shown in Figure 14.

As can be seen from the figure, the two groups of cells infected with the recombinant Coxsackie virus cDNA showed significant lesions. From the staining results, the infected group was more deeply stained than the negative control group, indicating that the cytoplasm and interstitial cells were more eosinophilic.

Each of the experimental groups in Example 20 was randomly selected for 3 samples, and the live pH measurement of the tumor site was performed on a D41 using a CL-9D02 benchtop pH/mV instrument. The measurement results of each group were taken as arithmetic mean values. As shown in Fig. 10, the pH values measured by the sampling of each of the examples were improved, and the lift value was 0.4 to 0.6, as shown in Fig. 15.

Example 27 In vitro inhibitory effect of different kinds of tumor cells in vitro

In order to determine the inhibitory effect on different types of tumor cells, four human lung cancer cell lines A549, GLC-82, NCI-H460, NCI-H1299, liver cancer SNU-398, and human lung fibroblasts were 3-(4,5- Determination of dimethylthiazol-2-yl)-2,5-diphenyl 2H-tetrazole ammonium bromide (MTT). Cells were seeded in 96-well plates 24 hours prior to treatment and grown to approximately 80% confluence. Infect cells with recombinant CVB3 at different concentrations (1 PFU/mL, 1 x 101 PFU/mL, 1 x 102 PFU/mL, 1 x 103 PFU/mL, 1 x 104 PFU/mL, 1 x 105 PFU/mL, 1 x 106 PFU/mL, 1 x 107 PFU/mL or 1 x 108 PFU/mL), or with saline (NS) As a negative control, cisplatin was used as a positive control. After 72 hours, the MTT assay was performed according to the manufacturer's protocol (VWR Life Sciences Amresco, Radnor, PA, USA). Briefly, 200 μL of MTT (0.5 mg/mL) was used instead of the cell culture medium, and culture was continued for 1 h at 37 ° C in 10% FBS cell culture medium, and the supernatant of each group was removed, and 200 μL of dimethyl sulfoxide (200 μL) was added. DMSO) dissolves each well of the MTT dye. The absorption spectrum was read at a wavelength of 570 nm on a microplate reader. In each case, 6 replicates were tested and all assays were performed in triplicate. The half maximal inhibitory concentrations (IC50) for A549, GLC-82, NCI-H460, NCI-H1299 and SNU-398 were calculated to be 10,417,7.11, 4,210,6.1, 4,575,5.4, 48.0 and 139.1, respectively. The inhibition rate results are shown in Figure 16. Figure 16 compares the in vitro inhibition of recombinant CVB3 on different cells, indicating safety against normal somatic cells.

Experiments have shown that the oncolytic cells of Example 27 have broad-spectrum inhibitory effects on different types of lung cancer, liver cancer cells, and have little lethality against normal cells.

Those skilled in the art will appreciate that the above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and scope of the present invention, All should be included in the scope of protection of the present invention.

Claims (23)

1. An oncolytic virus characterized in that an expression sequence of an exogenous basic peptide is inserted into a genome, and the basic peptide is expressed in a physiological process such that the pH of the infected host environment is increased.
2. The oncolytic virus according to claim 1, wherein the oncolytic virus increases the pH of the host environment in which it is infected by 0.4 to 0.6.
3. The oncolytic virus according to claim 1, wherein the exogenous basic peptide is a 4 peptide to 10 peptide.
4. The oncolytic virus according to claim 3, wherein the exogenous basic peptide has a basic amino acid content of more than 60%.
5. The oncolytic virus according to claim 4, wherein the exogenous basic peptide has a basic amino acid content of more than 80%.
6. The oncolytic virus according to claim 3 or 4, wherein the basic amino acid is selected from the group consisting of arginine, lysine or histidine.
7. The oncolytic virus according to claim 6, wherein the basic amino acid is selected from the group consisting of arginine or lysine.
8. The oncolytic virus according to claim 1, wherein the exogenous basic peptide is selected from the group consisting of the following peptides:

   (1) Arg-Lys-Arg-Lys; (2) Lys-Arg-Lys-Arg; (3) Arg-Arg-Lys-Lys; (4) Lys-Lys-Arg-Arg;

   (5) Lys-Arg-Arg-Lys; (6) Arg-Lys-Lys-Arg; (7) Arg-Arg-His-Lys-Lys; (8) Lys-His-Arg-Lys-His-Arg;

   (9) Lys-His-Arg-Cys-Lys-Pro; (10) Arg-Arg-His-Lys-Met-Lys; (11) His-Arg-Lys-Cys-Arg-Lys;

   (12) Lys-Arg-Trp-Arg-Lys-His-Arg; (13) His-Lys-Gly-Arg-Lys-Cys-Arg-Val;

   (14) Lys-Arg-Trp-His-Lys-Met-Arg-Lys-His; (15) His-Phe-Trp-Arg-Gln-Cys-Ala-Met-Lys;

   (16) Tyr-Phe-Pro-Arg-His-Gln-Lys-Trp-Lys; (17) Trp-Lys-Tyr-Arg-Gln-Ile-Ser-Thr-Cys;

   (18) Arg-Lys-His-Lys-Met-Arg-Lys-Cys-His-Lys.
9. The oncolytic virus according to any one of claims 1 to 8, wherein the oncolytic virus is a Coxsackievirus B3 strain.
10. The oncolytic virus according to claim 9, wherein the exogenous basic peptide is selected from the group consisting of:

    (5) Lys-Arg-Arg-Lys; (14) Lys-Arg-Trp-His-Lys-Met-Arg-Lys-His;

    (15) His-Phe-Trp-Arg-Gln-Cys-Ala-Met-Lys.
11. The oncolytic virus according to claim 10, wherein the oncolytic virus is a variant attenuated strain Coxsackie virus B3 strain, comprising the following base mutation sites: T96C, G1180A, T1654C, T1756C, G2276A, A2685C, G2690A, C3120A, A3231G, G4327A, T5088C, A5270G, C7026T, and/or G7192A.
12. The oncolytic virus according to claim 10, wherein the coding sequence of the exogenous basic peptide is inserted on the pVAX1 vector.
13. The oncolytic virus according to claim 10, wherein the exogenous basic peptide is selected from the group consisting of Lys-Arg-Trp-His-Lys-Met-Arg-Lys-His or Lys-Arg -Arg-Lys.
14. Use of an oncolytic virus, characterized in that the oncolytic virus according to any one of claims 1 to 13 is used for the preparation of an antitumor drug.
15. The use according to claim 14, wherein the oncolytic virus according to any one of claims 1 to 13 is used for the preparation of an anti-solid tumor drug.
16. The use according to claim 15, wherein the oncolytic virus according to any one of claims 1 to 13 is used for the preparation of a medicament for anti-respiratory system tumor, digestive system tumor, endocrine system tumor, or gynecological tumor. .
17. An antitumor drug comprising the oncolytic virus according to any one of claims 1 to 13.
18. A synthetic DNA sequence, characterized in that the synthetic DNA sequence is used to express a basic peptide having a basic amino acid content of more than 60%.
19. The synthetic DNA sequence according to claim 18, wherein the basic peptide has a basic amino acid content of more than 80%.
20. The synthetic DNA sequence according to claim 18 or 19, wherein the basic amino acid is selected from the group consisting of arginine, lysine or histidine.
21. The synthetic DNA sequence according to claim 20, wherein the basic amino acid is selected from the group consisting of arginine or lysine.
22. The synthetic DNA sequence according to claim 19, wherein said exogenous basic peptide is selected from the group consisting of:

    (1) Arg-Lys-Arg-Lys; (2) Lys-Arg-Lys-Arg; (3) Arg-Arg-Lys-Lys; (4) Lys-Lys-Arg-Arg;

    (5) Lys-Arg-Arg-Lys; (6) Arg-Lys-Lys-Arg; (7) Arg-Arg-His-Lys-Lys; (8) Lys-His-Arg-Lys-His-Arg;

    (9) Lys-His-Arg-Cys-Lys-Pro; (10) Arg-Arg-His-Lys-Met-Lys; (11) His-Arg-Lys-Cys-Arg-Lys;

    (12) Lys-Arg-Trp-Arg-Lys-His-Arg; (13) His-Lys-Gly-Arg-Lys-Cys-Arg-Val;

    (14) Lys-Arg-Trp-His-Lys-Met-Arg-Lys-His; (15) His-Phe-Trp-Arg-Gln-Cys-Ala-Met-Lys;

    (16) Tyr-Phe-Pro-Arg-His-Gln-Lys-Trp-Lys; (17) Trp-Lys-Tyr-Arg-Gln-Ile-Ser-Thr-Cys;

    (18) Arg-Lys-His-Lys-Met-Arg-Lys-Cys-His-Lys.
23. The basic peptide expression gene according to claim 22, wherein the basic peptide sequence is:

    (5) Lys-Arg-Arg-Lys; (14) Lys-Arg-Trp-His-Lys-Met-Arg-Lys-His;

    (15) His-Phe-Trp-Arg-Gln-Cys-Ala-Met-Lys.

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