RECOMBINANT ONCOLYTIC VIRUS, SYNTHETIC DNA SEQUENCE, AND APPLICATION THEREOF

Abstract

The disclosure provides a recombinant oncolytic virus, a synthetic DNA sequence and applications of the virus. The recombinant oncolytic virus includes a genome and an exogenous DNA sequence inserted in the genome. The exogenous DNA sequence adapts to express a basic peptide fragment, to increase the environmental pH in a host infected by the recombinant oncolytic virus. The basic peptide fragment includes more than 60% of basic amino acids. The recombinant oncolytic virus and the synthetic DNA sequence of the disclosure are used to prepare an anti-tumor drug.

Claims

1. A recombinant oncolytic virus, comprising: an oncolytic virus genome and an exogenous DNA sequence inserted in the oncolytic virus genome, the exogenous DNA sequence being adapted to express a basic peptide fragment and to increase an environmental pH in a host infected by the recombinant oncolytic virus.

2. The virus of claim 1, wherein the oncolytic virus genome is a genome of herpes virus, Coxsackie viruses, adenovirus, cowpox virus, measles virus, poliomyelitis virus, retroviruses, reovirus, respiratory syncytial virus, parvovirus H1, vesicular stomatitis virus, or Newcastle disease virus, and preferably adenovirus, Newcastle disease virus, or Coxsackie virus.

3. The virus of claim 1, wherein the environmental pH in the host infected by the recombinant oncolytic virus is increased by about 0.4 to about 0.6.

4. The virus of claim 1, wherein the basic peptide fragment comprises 4 to 10 amino acids.

5. The virus of claim 4, wherein the basic peptide fragment comprises more than 60% of basic amino acids.

6. The virus of claim 5, wherein the basic peptide fragment comprises more than 80% of the basic amino acids.

7. The virus of claim 5, wherein the basic amino acids are selected from arginine, lysine, and histidine.

8. The virus of claim 7, wherein the basic amino acids are selected from arginine and lysine.

9. The virus of claim 1, wherein the basic peptide fragment is selected from: TABLE-US-00017 (SEQ ID NO. 3) Arg-Lys-Arg-Lys; (SEQ ID NO. 5) Lys-Arg-Lys-Arg; (SEQ ID NO. 7) Arg-Arg-Lys-Lys; (SEQ ID NO. 9) Lys-Lys-Arg-Arg; (SEQ ID NO. 11) Lys-Arg-Arg-Lys; (SEQ ID NO. 13) Arg-Lys-Lys-Arg; (SEQ ID NO. 15) Arg-Arg-His-Lys-Lys; (SEQ ID NO. 17) Lys-His-Arg-Lys-His-Arg; (SEQ ID NO. 19) Lys-His-Arg-Cys-Lys-Pro; (SEQ ID NO. 21) Arg-Arg-His-Lys-Met-Lys; (SEQ ID NO. 23) His-Arg-Lys-Cys-Arg-Lys; (SEQ ID NO. 25) Lys-Arg-Trp-Arg-Lys-His-Arg; (SEQ ID NO. 27) His-Lys-Gly-Arg-Lys-Cys-Arg-Val; (SEQ ID NO. 29) Lys-Arg-Trp-His-Lys-Met-Arg-Lys-His; (SEQ ID NO. 31) His-Phe-Trp-Arg-Gln-Cys-Ala-Met-Lys; (SEQ ID NO. 33) Tyr-Phe-Pro-Arg-His-Gln-Lys-Trp-Lys; (SEQ ID NO. 35) Trp-Lys-Tyr-Arg-Gln-Ile-Ser-Thr-Cys; and (SEQ ID NO. 37) Arg-Lys-His-Lys-Met-Arg-Lys-Cys-His-Lys.

10. The virus of claim 1, wherein the recombinant oncolytic virus is Coxsackie virus B3 strain.

11. The virus of claim 10, wherein the basic peptide fragment is selected from: TABLE-US-00018 (SEQ ID NO. 11) Lys-Arg-Arg-Lys; (SEQ ID NO. 29) Lys-Arg-Trp-His-Lys-Met-Arg-Lys-His; and (SEQ ID NO. 31) His-Phe-Trp-Arg-Gln-Cys-Ala-Met-Lys.

12. The virus of claim 11, wherein the recombinant oncolytic virus is a variant attenuated Coxsackie virus B3 strain comprising base mutations of T97C, G1180A, T1654C, T1756C, G2276A, A2685C, G2690A, C3120A, A3231G, G4327A, T5088C, A5270G, C7026T, and/or G7192A.

13. The virus of claim 11, wherein the exogenous DNA sequence is inserted onto a pVAX1 vector.

14. The virus of claim 11, wherein the basic peptide fragment is Lys-Arg-Trp-His-Lys-Met-Arg-Lys-His (SEQ ID NO. 29).

15. A method of preparing an anti-tumor drug comprising employing a recombinant oncolytic virus of claim 1.

16. The method of claim 15, wherein the anti-tumor drug is an anti-solid tumor drug.

17. The method of claim 16, wherein the anti-solid tumor drug is used for treating respiratory tract tumors, gastrointestinal tumors, endocrine tumors, or gynecological tumors.

18. An anti-tumor drug, comprising the recombinant oncolytic virus of claim 1.

19. The drug of claim 18, further comprising a checkpoint inhibitor.

20. A method for treating a malignant tumor, comprising administering the anti-tumor drug of claim 18 intravenously or locally to a patient in need thereof.

21. The method of claim 20, wherein the malignant tumor is a solid tumor.

22. The method of claim 21, wherein the malignant tumor is respiratory tract tumor, gastrointestinal tumor, endocrine tumor, or gynecological tumor.

23. A synthetic DNA sequence, the synthetic DNA sequence being adapted to express a basic peptide fragment comprising more than 60% of basic amino acids.

24. The synthetic DNA sequence of claim 23, wherein the basic peptide fragment comprises more than 80% of the basic amino acids.

25. The synthetic DNA sequence of claim 24, wherein the basic amino acids are selected from Arginine, Lysine, and Histidine.

26. The synthetic DNA sequence of claim 25, wherein the basic amino acids are selected from Arginine, and Lysine.

27. The synthetic DNA sequence of claim 24, wherein the basic peptide fragment is selected from: TABLE-US-00019 (SEQ ID NO. 3) Arg-Lys-Arg-Lys; (SEQ ID NO. 5) Lys-Arg-Lys-Arg; (SEQ ID NO. 7) Arg-Arg-Lys-Lys; (SEQ ID NO. 9) Lys-Lys-Arg-Arg; (SEQ ID NO. 11) Lys-Arg-Arg-Lys; (SEQ ID NO. 13) Arg-Lys-Lys-Arg; (SEQ ID NO. 15) Arg-Arg-His-Lys-Lys; (SEQ ID NO. 17) Lys-His-Arg-Lys-His-Arg; (SEQ ID NO. 19) Lys-His-Arg-Cys-Lys-Pro; (SEQ ID NO. 21) Arg-Arg-His-Lys-Met-Lys; (SEQ ID NO. 23) His-Arg-Lys-Cys-Arg-Lys; (SEQ ID NO. 25) Lys-Arg-Trp-Arg-Lys-His-Arg; (SEQ ID NO. 27) His-Lys-Gly-Arg-Lys-Cys-Arg-Val; (SEQ ID NO. 29) Lys-Arg-Trp-His-Lys-Met-Arg-Lys-His; (SEQ ID NO. 31) His-Phe-Trp-Arg-Gln-Cys-Ala-Met-Lys; (SEQ ID NO. 33) Tyr-Phe-Pro-Arg-His-Gln-Lys-Trp-Lys; (SEQ ID NO. 35) Trp-Lys-Tyr-Arg-Gln-Ile-Ser-Thr-Cys; and (SEQ ID NO. 37) Arg-Lys-His-Lys-Met-Arg-Lys-Cys-His-Lys.

28. The synthetic DNA sequence of claim 27, wherein the basic peptide fragment is selected from: TABLE-US-00020 (SEQ ID NO. 11) Lys-Arg-Arg-Lys; (SEQ ID NO. 29) Lys-Arg-Trp-His-Lys-Met-Arg-Lys-His; and (SEQ ID NO. 31) His-Phe-Trp-Arg-Gln-Cys-Ala-Met-Lys.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIG. 1 is a schematic gene map of Coxsackie virus carrying a eukaryotic expression vector pVAX1 provided in an example of the disclosure;

[0040] FIG. 2 is a schematic gene map of Coxsackie virus carrying a eukaryotic expression vector pVAX1 in which a synthetic DNA sequence is inserted provided in an example of the disclosure;

[0041] FIG. 3 is a simplified process chart of virus purification provided in an example of the disclosure;

[0042] FIG. 4 is a tumor volume curve provided in Example 20 of the disclosure;

[0043] FIG. 5 compares the tissue sections provided in Example 20 of the disclosure;

[0044] FIG. 6 is a tumor volume curve provided in Example 21 of the disclosure;

[0045] FIG. 7 is a tumor volume curve provided in Example 22 of the disclosure;

[0046] FIG. 8 is a tumor volume curve provided in Example 23 of the disclosure;

[0047] FIG. 9 shows the results of microscopic examination provided in Example 24 of the disclosure; and

[0048] FIG. 10 compares the pH measurements provided in Example 24 of the disclosure.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0049] For further illustrating the invention, experiments detailing a recombinant oncolytic virus, a basic peptide fragment and applications thereof are described below.

[0050] The disclosure provides a recombinant oncolytic virus, which comprises an exogenous DNA sequence inserted in its genome, and expresses the basic peptide fragment in a physiological process, such that the environmental pH in a host infected therewith is increased by about 0.4 to 0.6. After infection with the recombinant oncolytic virus comprising the exogenous gene, the basic peptide fragment is highly expressed thus changing the microenvironment of the tumor tissue, such that the tumor tissues are inhibited and killed.

[0051] The recombinant oncolytic virus can be herpes viruses, Coxsackie viruses, adenovirus, cowpox virus, measles virus, poliomyelitis virus, retroviruses, reovirus, respiratory syncytial virus, parvovirus H1, vesicular stomatitis virus, or Newcastle disease virus. The recombinant oncolytic virus is preferably recombinant oncolytic viruses that deactivate or delete specific genes in the target cells, for example, adenovirus, Newcastle disease virus or Coxsackie viruses.

[0052] The basic peptide fragment comprises 4 to 10 amino acids, in which the basic amino acids account for more than 60%, and preferably more than 80%. The basic amino acids are selected from Arginine, Lysine, or Histidine, and preferably from Arginine or Lysine. The basic peptide fragment has an N-terminal amino acid that is Lysine optimally.

[0053] The basic peptide fragment is selected from:

TABLE-US-00006 (SEQ ID NO. 3) Arg-Lys-Arg-Lys; (SEQ ID NO. 5) Lys-Arg-Lys-Arg; (SEQ ID NO. 7) Arg-Arg-Lys-Lys; (SEQ ID NO. 9) Lys-Lys-Arg-Arg; (SEQ ID NO. 11) Lys-Arg-Arg-Lys; (SEQ ID NO. 13) Arg-Lys-Lys-Arg; (SEQ ID NO. 15) Arg-Arg-His-Lys-Lys; (SEQ ID NO. 17) Lys-His-Arg-Lys-His-Arg; (SEQ ID NO. 19) Lys-His-Arg-Cys-Lys-Pro; (SEQ ID NO. 21) Arg-Arg-His-Lys-Met-Lys; (SEQ ID NO. 23) His-Arg-Lys-Cys-Arg-Lys; (SEQ ID NO. 25) Lys-Arg-Trp-Arg-Lys-His-Arg; (SEQ ID NO. 27) His-Lys-Gly-Arg-Lys-Cys-Arg-Val; (SEQ ID NO. 29) Lys-Arg-Trp-His-Lys-Met-Arg-Lys-His; (SEQ ID NO. 31) His-Phe-Trp-Arg-Gln-Cys-Ala-Met-Lys; (SEQ ID NO. 33) Tyr-Phe-Pro-Arg-His-Gln-Lys-Trp-Lys; (SEQ ID NO. 35) Trp-Lys-Tyr-Arg-Gln-Ile-Ser-Thr-Cys; and (SEQ ID NO. 37) Arg-Lys-His-Lys-Met-Arg-Lys-Cys-His-Lys

[0054] In a preferred embodiment, Coxsackie virus, particularly an attenuated variant strain of Coxsackie virus is used. In a pVAX1 vector constructed to have the viral genome, a gene is inserted that expresses a basic peptide fragment selected from:

TABLE-US-00007 (SEQ ID NO. 5) Lys-Arg-Lys-Arg; (SEQ ID NO. 29) Lys-Arg-Trp-His-Lys-Met-Arg-Lys-His; and (SEQ ID NO. 31) His-Phe-Trp-Arg-Gln-Cys-Ala-Met-Lys.

[0055] In a further preferred embodiment, the basic peptide fragment is selected from

TABLE-US-00008 (SEQ ID NO. 11) Lys-Arg-Arg-Lys; and (SEQ ID NO. 29) Lys-Arg-Trp-His-Lys-Met-Arg-Lys-His.

[0056] When the Coxsackie virus CVB 3 strain is used, the exogenous peptide fragment is Lys-Arg-Trp-His-Lys-Met-Arg-Lys-His (SEQ ID NO. 29), so excellent tumor inhibition effect and good safety are exhibited.

[0057] The Coxsackie virus CVB 3 is preferably the variant attenuated Coxsackie virus B3 strain comprising base mutations of T97C, G1180A, T1654C, T1756C, G2276A, A2685C, G2690A, C3120A, A3231G, G4327A, T5088C, A5270G, C7026T, and/or G7192A. The sequence encoding the basic peptide fragment is inserted onto the pVAX1 vector.

[0058] The DNA sequence encoding the basic peptide fragment is inserted between the 5UTR and VP4 of the recombinant vector.

[0059] Application of the recombinant oncolytic virus provided in the disclosure in the preparation of anti-tumor drug, particularly anti-solid tumor drugs, for examples, drugs for treating respiratory tract tumors, gastrointestinal tumors, endocrine tumors, or gynecological tumors, is further provided in the disclosure. The recombinant oncolytic virus provided in the invention, upon reaching the focal area, express the basic peptide fragment by making use of the targeting and replication ability of the virus, based on the severity of the tumor. It is experimentally confirmed that the basic peptide fragment changes the pH in the microenvironment where tumor cells resides, including the intracellular and intercellular environment. This change brings a series of comprehensive impacts on the metabolism of tumor cells, and finally results in an obvious tumor suppression effect. Therefore, the existing recombinant oncolytic viruses, in theory, can be genetically modified to carry the exogenous synthetic DNA sequence, so that in addition to the original inhibition on or killing of tumor cells, an inhibitory effect is produced by changing the microenvironment where the tumor cells grow. The two effects are synergistic, so as to inhibit the tumor more effectively. The recombinant oncolytic virus provided in the disclosure exerts an anti-tumor effect by impacting the microenvironment where the tumor cells grow, and thus has a more pronounced inhibitory effect on the solid tumor in which the tumor cells are concentrated. At the same time, for the recombinant oncolytic virus that causes serious physiological reaction, the dosage and dosage range of the virus used is reduced due to the superimposed tumor suppressive effect, so the physiological adverse effect resulting therefrom is lowered, the application of the recombinant oncolytic virus is widened and the safety of the recombinant oncolytic virus is improved.

[0060] The disclosure provides an anti-tumor drug, which comprises the recombinant oncolytic virus provided in the disclosure. Preferably, the anti-tumor drug further comprises an immune checkpoint inhibitor. The drug is administered intravenously or topically to the focus, thus exerting a good tumor suppressive effect. The immune checkpoint inhibitor (PD-1, PD-L1, and CTLA4) is not highly responsive to solid tumors (except melanoma), possibly due to the insufficient stimulation to the immune system in the patients with solid tumors after the release of the immune system by the checkpoint inhibitor. In the disclosure, the recombinant oncolytic virus and the immune checkpoint inhibitor synergize, thereby significantly enhancing the killing effect of the immune system for solid tumors, increasing the local infiltration of immune cells into the tumors, and up-regulating PD-L1. Particularly, the recombinant CVB3 virus induces the local production of specific and nonspecific immune responses in the tumor, such as exposure of calreticulin (CRT), translocation of ATP, and intracellular translocation of HGMB1 (Extracellular High Mobility Group Box 1). As the recombinant oncolytic virus proliferates, the IFN and/or cytokines are induced to produce activated NK and DC cells, whereby various mature DCs and cytotoxic CD107a+ NK cells are facilitate to enter the tumor site, resulting in the changes in the immune cellular profile in the tumor microenvironment and thus restoring the inherent antitumor immunity of an organism. The synergistic effect between the recombinant Coxsackie virus and the immune checkpoint inhibitor provided in the disclosure is particularly obvious.

[0061] The disclosure further provides a synthetic DNA sequence that expresses a basic peptide fragment. The basic amino acid in the basic peptide fragment has a content of more than 60% and preferably more than 80%. The basic amino acid is selected from Arginine, Lysine or Histidine, and preferably from Arginine or Lysine. The basic peptide fragment preferably has a N-terminal amino acid that is Lysine.

[0062] The basic peptide fragment encoded by the synthetic DNA sequence is elected from:

TABLE-US-00009 (SEQ ID NO. 3) Arg-Lys-Arg-Lys; (SEQ ID NO. 5) Lys-Arg-Lys-Arg; (SEQ ID NO. 7) Arg-Arg-Lys-Lys; (SEQ ID NO. 9) Lys-Lys-Arg-Arg; (SEQ ID NO. 11) Lys-Arg-Arg-Lys; (SEQ ID NO. 13) Arg-Lys-Lys-Arg; (SEQ ID NO. 15) Arg-Arg-His-Lys-Lys; (SEQ ID NO. 17) Lys-His-Arg-Lys-His-Arg; (SEQ ID NO. 19) Lys-His-Arg-Cys-Lys-Pro; (SEQ ID NO. 21) Arg-Arg-His-Lys-Met-Lys; (SEQ ID NO. 23) His-Arg-Lys-Cys-Arg-Lys; (SEQ ID NO. 25) Lys-Arg-Trp-Arg-Lys-His-Arg; (SEQ ID NO. 27) His-Lys-Gly-Arg-Lys-Cys-Arg-Val; (SEQ ID NO. 29) Lys-Arg-Trp-His-Lys-Met-Arg-Lys-His; (SEQ ID NO. 31) His-Phe-Trp-Arg-Gln-Cys-Ala-Met-Lys; (SEQ ID NO. 33) Tyr-Phe-Pro-Arg-His-Gln-Lys-Trp-Lys; (SEQ ID NO. 35) Trp-Lys-Tyr-Arg-Gln-Ile-Ser-Thr-Cys; and (SEQ ID NO. 37) Arg-Lys-His-Lys-Met-Arg-Lys-Cys-His-Lys.

[0063] The basic peptide fragment preferably has a sequence of:

TABLE-US-00010 (SEQ ID NO. 11) Lys-Arg-Arg-Lys; (SEQ ID NO. 29) Lys-Arg-Trp-His-Lys-Met-Arg-Lys-His; and (SEQ ID NO. 31) His-Phe-Trp-Arg-Gln-Cys-Ala-Met-Lys.

Examples 1 to 18: Recombinant Coxsackie Viruses Having a Synthetic DNA Sequence Inserted in Their Genome

[0064] The whole genome sequence of the Coxsackie B3 nancy strain is as shown in GeneBank ID: JX312064.1. The recombinant Coxsackie virus strain comprises the base mutations of T97C, G1180A, T1654C, T1756C, G2276A, A2685C, G2690A, C3120A, A3231G, G4327A, T5088C, A5270G, C7026T, and G7192A. The complete cDNA sequence of the recombinant Coxsackie virus strain is synthesized by Wuhan Boweid Biotechnology Co., Ltd and constructed onto a eukaryotic expression vector pVAX1 by a molecular biological method, as shown in FIG. 1.

[0065] The recombinant oncolytic virus provided in this example comprises the exogenous synthetic DNA sequence inserted between the 5UTR and VP4 of the constructed recombinant vector by a reverse genetic method. A 15 bp (SEQ ID NO. 1) and a 24 bp (SEQ ID NO. 2) DNA sequence are located at respectively the 5 and the 3 terminus of the sequence, which are provided for recognition and cleavage by the Protease C, as shown in FIG. 2.

TABLE-US-00011 TABLE 1 Basic peptide fragments and Its Encoding Genes Polypeptide Example Polypeptide name sequence Gene name Gene sequence 1 Polypeptide 1 SEQ ID NO. 3 nucleotide seque 1 SEQ ID NO. 4 2 Polypeptide 2 SEQ ID NO. 5 nucleotide seque 2 SEQ ID NO. 6 3 Polypeptide 3 SEQ ID NO. 7 nucleotide seque 3 SEQ ID NO. 8 4 Polypeptide 4 SEQ ID NO. 9 nucleotide seque 4 SEQ ID NO. 10 5 Polypeptide 5 SEQ ID NO. nucleotide seque 5 SEQ ID NO. 11 12 6 Polypeptide 6 SEQ ID NO. nucleotide seque 6 SEQ ID NO. 13 14 7 Polypeptide 7 SEQ ID NO. nucleotide seque 7 SEQ ID NO. 15 16 8 Polypeptide 8 SEQ ID NO. nucleotide seque 8 SEQ ID NO. 17 18 9 Polypeptide 9 SEQ ID NO. nucleotide seque 9 SEQ ID NO. 19 20 10 Polypeptide 10 SEQ ID NO. nucleotide seque 10 SEQ ID NO. 21 22 11 Polypeptide 11 SEQ ID NO. nucleotide seque 11 SEQ ID NO. 23 24 12 Polypeptide 12 SEQ ID NO. nucleotide seque 12 SEQ ID NO. 25 26 13 Polypeptide 13 SEQ ID NO. nucleotide seque 13 SEQ ID NO. 27 28 14 Polypeptide 14 SEQ ID NO. nucleotide seque 14 SEQ ID NO. 29 30 15 Polypeptide 15 SEQ ID NO. nucleotide seque 15 SEQ ID NO. 31 32 16 Polypeptide 16 SEQ ID NO. nucleotide seque 16 SEQ ID NO. 33 34 17 Polypeptide 17 SEQ ID NO. nucleotide seque 17 SEQ ID NO. 35 36 18 Polypeptide 18 SEQ ID NO. nucleotide seque 18 SEQ ID NO. 37 38

[0066] The sequence is specifically inserted by inserting the basic peptide fragment expressing DNA sequence between the 5UTR and VP4 of the recombinant vector. After screening, a positive clone is obtained, which is sequenced and extracted for the plasmid, to obtain a complete cDNA for viral packaging.

[0067] This example was specifically as follows.

[0068] (1) Synthesis of Coxsackie virus gene CVB3-Am

[0069] PUC57-CVB3-Am was synthesized by Suzhou Genwiz Biotechnology Co., Ltd.

[0070] (2) Mini extraction of vector pVAX1 and pUC19

[0071] The pVAX1-SalI and pUC19 plasmids were extracted using the kit available from Axygen.

[0072] (3) Construction of pVAX1-SalI-CVB3-Am vector

[0073] a. Double cleavage and recovery

[0074] The plasmids pVAX1 (Apa I.fwdarw.Sal I) and pUC57-CVB3-Am were cleaved with both NotIand SalI, and subjected to 1% agarose gel electrophoresis after reaction. The vector of 2999 bp and the CVB3-Am fragment of about 7500 bp were recovered, followed by gel extraction. The purification of the cleaved products was carried out following the specific steps as described in instructions for the Gel Extraction Kit from Axygen.

[0075] b. Ligation and transformation

[0076] The CVB3-Am fragment and the vector pVAX1 (Apa I.fwdarw.Sal I) that were cleaved with both NotIand SalI were ligated at a ratio by the T4DNA ligase available from TAKARA, and then transformed into Stbl3.

[0077] c. Screening and identification of positive clones

[0078] Single colonies grown on a LB+Kana plate were randomly picked up and subjected to colony PCR. The correct positive clones were sent to Suzhou Genewiz Biotechnology Co., Ltd. for sequencing.

[0079] The pVAX1 vector carrying the complete cDNA sequence of the recombinant Coxsackie virus was transfected into Cos7 packaging cells. The cells were cultured to obtain an infectious recombinant virus suspension.

[0080] A PolyA sequence ranging from 20 to 100 and preferably from 30 to 80 in length were preferably inserted after the 3UTR, to effectively ensure the stability of the basic peptide fragment encoding gene, thereby ensuring the expression thereof. The virus can be stored at 20 C. for over 1 year, and at room temperature for 2 days without decline of the titer. Therefore, the virus is highly stable, and convenient in storage and transportation.

[0081] Comparative Example 1: The synthetic DNA sequence was inserted between the VP1 and 2A elements in the pVAX1 vector to obtain the virus. The virus could not stably express the basic peptide fragment and had a limited inhibitory effect on cancer cells.

Example 19. Preparation of Test Samples for Pharmacodynamics Study

[0082] The infectious recombinant virus suspension described in Examples 1 to 18 was inoculated to the expansion cultured Vero cells, and a purified virus suspension was obtained after the production and purification processes, which was used as a test sample. The process for virus purification was as shown in FIG. 3.

[0083] The purified virus suspension needs to be inspected to satisfy the following criteria as shown in Table 2.

TABLE-US-00012 TABLE 2 Item Standard limit of inspection Method released Protein Chinese Pharmacopoeia 2015 Edition, 1 g/dose content Part VI, General rule 0731 Protein Determination Method V: Bradford method. Virus titer TCID50 Karber method 10.sup.7TCID.sub.50/mL

Example 20. In Vivo Pharmacodynamics Study on the Selective Inhibition of Recombinant Coxsackie Virus on Solid Tumors

[0084] The test samples used in this example were prepared and tested according to the protocol described in Example 19.

[0085] In this example, the recombinant Coxsackie viruses of Examples 1 to 18 were used as test samples, for example, those prepared in Example 5, Example 14, and Example 17.

[0086] The viruses above were prepared into test samples following the method as described in Example 19.

[0087] A subcutaneous A549 cell transplanted tumor model of lung cancer in nude mice was established. 30 tumor-bearing animals having uniform tumor volume were screened. The 30 animals with a tumor volume of 45-70 mm.sup.3 (average tumor volume of 56 mm.sup.3) were assigned to Groups 1-5 at random. Each group of animals were randomly numbered using Excel software and ranked according to the random number from small to large. There were a total of 5 groups, each group having 6 animals. The groups, dosage and administration mode are shown in Table 3.

TABLE-US-00013 TABLE 3 Agent administered to Dosage Concentration Administration Volume Group the animals (PFU/kg) (PFU/mL) mode dosed 1 Saline (negative) 0.1 mL/10 g Intravenous 0.1 mL/ injection 10 g 2 Cisplatin 6 mg/kg 0.6 mg/mL Intravenous 0.1 mL/ (positive) injection 10 g 3 Example 5 6 10.sup.6 6 10.sup.4 Intravenous 0.1 mL/ injection 10 g 4 Example 14 6 10.sup.5 6 10.sup.3 Intravenous 0.1 mL/ injection 10 g 5 Example 17 6 10.sup.4 6 10.sup.2 Intravenous 0.1 mL/ injection 10 g

[0088] The animals in Group 2 (Cisplatin) were administered once a week for 4 consecutive weeks. After 1-week observation, the animals were euthanized on Day 41. In the saline group, the animals were administered with saline once a week for 6 consecutive weeks, and the animals were euthanized on Day 41. The animals were observed twice a day during administration to observe the general clinical symptoms of animals, and the body weight and tumor size were measured twice a week.

[0089] Results: Throughout the experiment, the average body weight of the animals is increased, and there is no significant difference between groups (P<0.05). The curve of increase in average tumor volume of the animals in each group with time is shown in FIG. 4.

[0090] On Day 41, the tissue sections sampled from the animals in the negative control group and in the treatment group with the test sample prepared in Example 5 were examined, and compared, as shown in FIG. 5.

[0091] As can be seen from FIG. 5, the treatment group with the test sample prepared in Example 5 on the right has obviously damaged tumor cells and much darker eosin staining, compared with the negative control group on the left.

Example 21. In Vivo Pharmacodynamics Study on the Selective Inhibition of Recombinant Coxsackie Virus on Solid Tumors

[0092] The test samples used in this example were prepared and tested according to the protocol described in Example 19.

[0093] In this example, three recombinant Coxsackie viruses having a synthetic DNA sequence inserted in their genomes were used as test samples, which were respectively those prepared in Example 1, Example 2, Example 4, and Example 5.

[0094] The viruses above were prepared into test samples following the method as described in Example 19.

[0095] A subcutaneous A549 cell transplanted tumor model of lung cancer in nude mice was established. 30 tumor-bearing animals having uniform tumor volume were screened. The 30 animals with a tumor volume of 45-72 mm.sup.3 (average tumor volume of 57 mm.sup.3) were assigned to Groups 1-6 at random. Each group of animals were randomly numbered using Excel software and ranked according to the random number from small to large. There were a total of 6 groups, each group having 5 animals. The groups, dosage and administration mode are shown in Table 4.

TABLE-US-00014 TABLE 4 Agent administered to Dosage Concentration Administration Volume Group the animals (PFU/kg) (PFU/mL) mode dosed 1 Saline (negative) 0.1 mL/10 g Intravenous 0.1 mL/ injection 10 g 2 Cisplatin 6 mg/kg 0.6 mg/mL Intravenous 0.1 mL/ (positive) injection 10 g 3 Example 1 6 10.sup.6 6 10.sup.4 Intravenous 0.1 mL/ injection 10 g 4 Example 2 6 10.sup.5 6 10.sup.3 Intravenous 0.1 mL/ injection 10 g 5 Example 4 6 10.sup.4 6 10.sup.2 Intravenous 0.1 mL/ injection 10 g 6 Example 5 6 10.sup.4 6 10.sup.2 Intravenous 0.1 mL/ injection 10 g

[0096] The animals in Group 2 (Cisplatin) were administered once a week for 4 consecutive weeks. After 1-week observation, the animals were euthanized on Day 48. In the saline group, the animals were administered with saline once a week for 7 consecutive weeks, and the animals were euthanized on Day 48. The animals were observed twice a day during administration to observe the general clinical symptoms of animals, and the body weight and tumor size were measured twice a week.

[0097] Results: Throughout the experiment, the average body weight of the animals is increased, and there is no significant difference between groups (P<0.05). The curve of increase in average tumor volume of the animals in each group with time is shown in FIG. 6.

[0098] It can be seen that the test samples prepared in Examples 1, 2, 4, and 5 all have anti-tumor effects, of which the test sample prepared in Example 5 can significantly inhibits tumor growth.

Example 22. In Vivo Pharmacodynamics Study on the Selective Inhibition of Recombinant Coxsackie Virus on Solid Tumors

[0099] The test samples used in this example were prepared and tested according to the protocol described in Example 19.

[0100] In this example, two recombinant Coxsackie viruses having a synthetic DNA sequence inserted in their genomes were used as test samples, which were respectively those prepared in Examples 18, and Example 13.

[0101] The viruses above were prepared into test samples following the method as described in Example 19.

[0102] A subcutaneous A549 cell transplanted tumor model of lung cancer in nude mice was established. 20 tumor-bearing animals having uniform tumor volume were screened. The 20 animals with a tumor volume of 62-92 mm.sup.3 (average tumor volume of 79 mm.sup.3) were assigned to Groups 1-4 at random. Each group of animals were randomly numbered using Excel software and ranked according to the random number from small to large. There were a total of 4 groups, each group having 5 animals. The groups, dosage and administration mode are shown in Table 5.

TABLE-US-00015 TABLE 5 Agent administered to Dosage Concentration Administration Volume Group the animals (PFU/kg) (PFU/mL) mode dosed 1 Saline (negative) 0.1 mL/10 g Intravenous 0.1 mL/ injection 10 g 2 Cisplatin 6 mg/kg 0.6 mg/mL Intravenous 0.1 mL/ (positive) injection 10 g 3 Example 18 6 10.sup.6 6 10.sup.4 Intravenous 0.1 mL/ injection 10 g 4 Example 13 6 10.sup.5 6 10.sup.3 Intravenous 0.1 mL/ injection 10 g

[0103] The animals in Group 2 (Cisplatin) were administered once a week for 4 consecutive weeks. After 1-week observation, the animals were euthanized on Day 42. In the saline group, the animals were administered with saline once a week for 6 consecutive weeks, and the animals were euthanized on Day 42. The animals were observed twice a day during administration to observe the general clinical symptoms of animals, and the body weight and tumor size were measured twice a week.

[0104] Results: Throughout the experiment, the average body weight of the animals is increased, and there is no significant difference between groups (P<0.05). The curve of increase in average tumor volume of the animals in each group with time is shown in FIG. 7.

[0105] It can be seen that the test samples prepared in Examples 18 and 13 both have anti-tumor effects.

Example 23. In Vivo Pharmacodynamics Study on the Selective Inhibition of Recombinant Coxsackie Virus on Solid Tumors

[0106] The test samples used in this example were prepared and tested according to the protocol described in Example 19.

[0107] In this example, three recombinant Coxsackie viruses having a synthetic DNA sequence inserted in their genomes were used as test samples, which were respectively those prepared in Examples 8, 9 and 10.

[0108] The viruses above were prepared into test samples following the method as described in Example 19.

[0109] A subcutaneous A549 cell transplanted tumor model of lung cancer in nude mice was established. 25 tumor-bearing animals having uniform tumor volume were screened. The 25 animals with a tumor volume of 65-90 mm.sup.3 (average tumor volume of 79 mm.sup.3) were assigned to Groups 1-5 at random. Each group of animals were randomly numbered using Excel software and ranked according to the random number from small to large. There were a total of 5 groups, each group having 5 animals. The groups, dosage and administration mode are shown in Table 6.

TABLE-US-00016 TABLE 6 Agent administered to Dosage Concentration Administration Volume Group the animals (PFU/kg) (PFU/mL) mode dosed 1 Saline (negative) 0.1 mL/10 g Intravenous 0.1 mL/ injection 10 g 2 Cisplatin 6 mg/kg 0.6 mg/mL Intravenous 0.1 mL/ (positive) injection 10 g 3 Example 8 6 10.sup.6 6 10.sup.4 Intravenous 0.1 mL/ injection 10 g 4 Example 9 6 10.sup.5 6 10.sup.3 Intravenous 0.1 mL/ injection 10 g 5 Example 10 6 10.sup.4 6 10.sup.2 Intravenous 0.1 mL/ injection 10 g

[0110] The animals in Group 2 (Cisplatin) were administered once a week for 4 consecutive weeks. After 1-week observation, the animals were euthanized on Day 33. In the saline group, the animals were administered with saline once a week for 5 consecutive weeks, and the animals were euthanized on Day 33. The animals were observed twice a day during administration to observe the general clinical symptoms of animals, and the body weight and tumor size were measured twice a week.

[0111] Results: Throughout the experiment, the average body weight of the animals is increased, and there is no significant difference between groups (P<0.05). The curve of increase in average tumor volume of the animals in each group with time is shown in FIG. 8.

[0112] It can be seen that the test samples prepared in Examples 8, 9 and 10 both have anti-tumor effects.

[0113] The test samples prepared in Examples 1 to 18 all have anti-tumor effects, of which the test samples prepared in Examples 5 and 14 can significantly inhibits tumor growth.

Example 24. Study on Change of Interstitial pH in Tumors by Recombinant Coxsackie Viruses

[0114] In this example, two recombinant Coxsackie viruses having a synthetic DNA sequence inserted in their genomes were used as test samples, which were respectively those prepared in Examples 5, and Example 14.

[0115] The two viruses were prepared into test samples following the method as described in Example 19.

[0116] The two test samples were used to infect Vero cells, respectively. The Vero cells infected with the viruses prepared in Example 5 and Example 14 were designated as 4p5 and 9pep groups, respectively. There was another group of cells that was used as a negative control. Each group of cells included two replicates, which were cultured and tested under the same conditions. 3 hrs after infection, one replicate in each group of cells was stained with Eosin and examined microscopically. The result is as shown in FIG. 9.

[0117] As can be seen from FIG. 9, the two group of cells infected with the cDNA of recombinant Coxsackie viruses have obvious pathological changes. It can be seen from the staining results that the infection group is stained darker than the negative control group, indicating that its cytoplasm and interstitial space are more acidophilic.

[0118] Samples were taken randomly from 3 animals in each test group in Example 20 and the in vivo pH at the tumor site were measured on Day 41 using a Bench-top pH/mV CL-9D02 Meter. The arithmetic mean values of the measurement results for each group are taken, and shown in FIG. 10. The pH value measured with the samples taken from the animals in each test group in Example 20 were found to be increased, by a value of 0.4 to 0.6, as shown in FIG. 10.

[0119] It will be obvious to those skilled in the art that changes and modifications may be made, and therefore, the aim in the appended claims is to cover all such changes and modifications.