A highly stable oncolytic virus composition, preparation method thereof, and application thereof

Abstract

The present invention relates to an oncolytic virus composition with high stability. The composition contains an oncolytic virus with a titer of (1-10)×10<supgt;8< / supgt> pfu / ml, 1-20% of a stabilizer (w / v), 1-150 mM of an amino acid, and water. The pH of the composition is 6-7.5. Among them, the oncolytic virus is selected from any one of herpes simplex virus type 1, herpes simplex virus type 2, oHSV-BJR virus, VA1RNA6 virus, and HER2 virus, and the stabilizer is selected from any one or a combination of glucose, sucrose, trehalose, lactose, galactose, maltose, fructan, raffinose, mannitol, dextran, polyethylene glycol, sorbitol, and glycerol. The present invention scientifically screens the components and ratios of the oncolytic virus composition, enabling the oncolytic virus to maintain its viral activity during repeated freeze-thaw cycles and under the conditions of 25°C - 37°C, ensuring the quality of the drug and the safety and effectiveness of clinical medication.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a highly stable oncolytic virus composition, a preparation method thereof, and an application thereof. Background Art

[0002] Malignant tumors are one of the major diseases that threaten human health and life, and their incidence is increasing year by year. Surgery, radiotherapy and chemotherapy are the three conventional methods of tumor treatment, but they all have the characteristics of insufficient specificity, resulting in varying degrees of damage to normal tissues and organs while clearing tumor cells. Radiotherapy and chemotherapy have been used clinically for many years, but they have potential risks and toxic side effects such as low selectivity, large side effects, high-dose lethality, vomiting, and nausea.

[0003] Oncolytic viruses have therapeutic effects on a variety of cancers, and their application prospects in the treatment of cancer are worth looking forward to. In 2017 alone, there were as many as 80 clinical trials using oncolytic viruses to treat various cancers. With the expansion of the field of oncolytic immunotherapy, the clinical application scope of oncolytic virus compositions continues to expand. CN110283794A discloses a recombinant oncolytic virus oHSV-BJR with broad-spectrum anticancer properties, which is used to inhibit the growth and proliferation of lung cancer, liver cancer, gastric cancer, rectal cancer, etc., and has relatively reliable safety for normal cell non-cancerous cells. However, oncolytic viruses are unstable, and there is a decrease in virus titer or titer when stored and transported at a temperature higher than -60 ° C. It is necessary to store and transport under harsh conditions of -80 ° C, and it is necessary to avoid a decrease in virus titer during repeated freezing and thawing. To this end, it is necessary to study the stability of oncolytic viruses and their preparations under various environmental conditions to ensure the quality of drugs and the safety and effectiveness of clinical medication. Summary of the invention

[0004] The object of the present invention is to provide a highly stable oncolytic virus composition, the composition containing a titer of (1-10)×10 8pfu / ml of oncolytic virus, 1-20% of a stabilizer (w / v), 1-150 mM of an amino acid and water, the composition has a pH of 6-7.5, wherein the oncolytic virus is selected from any one of herpes simplex virus type 1, herpes simplex virus type 2, oHSV-BJR virus, VA1RNA6 virus, and HER2 virus, the stabilizer is selected from any one of glucose, sucrose, trehalose, lactose, galactose, maltose, fructan, raffinose, mannitol, dextran, polyethylene glycol, sorbitol, and glycerol, or a combination thereof, and the amino acid is selected from alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), thiazolidinone (D), dapoxetine (D), dapoxetine (E), dapoxetine (F), dapoxetine (D), dapoxetine (E ... any one or a combination of cystine (C), glutamic acid (E), glutamine (Q), glycine (G), histidine (H), methionine (M), proline (P), serine (S), threonine (T), tyrosine (Y), and valine (V); and the pH adjuster is selected from any one or a combination of sodium hydroxide, potassium hydroxide, ammonia water, hydrochloric acid, sulfuric acid, phosphoric acid, sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium carbonate, sodium bicarbonate, and potassium bicarbonate.

[0005] In the preferred technical solution of the present invention, the titer of the oncolytic virus in the composition is (5-10)×10 8 pfu / ml, preferably (6-8)×10 8 pfu / ml.

[0006] In a preferred technical solution of the present invention, the composition contains 2-8% (w / v), preferably 3-5% (w / v) of stabilizer.

[0007] In a preferred technical solution of the present invention, the composition contains 5-120 mM amino acids, preferably 10-100 mM.

[0008] In a preferred technical solution of the present invention, the pH of the composition is 6.5-7.2.

[0009] In the preferred technical solution of the present invention, the composition contains a titer of (1-10)×10 8 pfu / ml of oncolytic virus, 1-10% (w / v) of sorbitol, glycerol, any one of trehalose, 1-10% (w / v) of proline, 1-10 mM of histidine solution, and the composition has a pH of 6.5-7.2.

[0010] In the preferred technical solution of the present invention, the composition contains a titer of (2-8)×10 8 pfu / ml of oncolytic virus, 2-5% (w / v) of sorbitol, glycerol, any one of trehalose, 1-5% (w / v) of proline, 5-10 mmol / L of histidine solution, and the composition has a pH of 6.5-7.2.

[0011] In the preferred technical solution of the present invention, the composition contains a titer of 7.6×10 8 pfu / ml of oncolytic virus, 5% (w / v) sorbitol, glycerol, any one of trehalose, 1.15% (w / v) proline, 10 mmol / L histidine solution, and the composition has a pH of 6.5-7.2.

[0012] In a preferred technical solution of the present invention, the composition can be prepared into a lyophilized preparation for injection.

[0013] In a preferred technical solution of the present invention, after the composition is repeatedly frozen and thawed 5 times at 25° C., the virus titer loss is ≤30%, preferably less than ≤20%, and more preferably ≤10%.

[0014] In a preferred technical solution of the present invention, the composition can be placed at 25° C. for 1-6 days, preferably for 1-4 days.

[0015] In a preferred technical solution of the present invention, after the composition is placed at 25° C. for 2 days, the virus titer loss is ≤30%, preferably ≤20%, and more preferably ≤10%.

[0016] In a preferred technical solution of the present invention, after the composition is placed at 25° C. for 4 days, the virus titer loss is ≤30%, preferably ≤20%, and more preferably ≤10%.

[0017] In a preferred technical solution of the present invention, the composition can be placed at 37°C for 1-36 hours, preferably for 1-24 hours.

[0018] In a preferred technical solution of the present invention, after the composition is placed at 37° C. for 36 hours, the virus titer loss is ≤30%, preferably ≤20%, and more preferably ≤10%.

[0019] In a preferred technical solution of the present invention, after the composition is placed at 37° C. for 24 hours, the virus titer loss is ≤30%, preferably ≤20%, and more preferably ≤10%.

[0020] Another object of the present invention is to provide a method for preparing a highly stable oncolytic virus composition, wherein the composition contains a titer of (1-10)×10 8pfu / ml of oncolytic virus, 1-20% of a stabilizer (w / v), 1-150 mM of an amino acid and water, the composition having a pH of 6-7.5, wherein the oncolytic virus is selected from any one of herpes simplex virus type 1, herpes simplex virus type 2, oHSV-BJR virus, VA1 RNA6 virus, and HER2 virus, the stabilizer is selected from any one of glucose, sucrose, trehalose, lactose, galactose, maltose, fructan, raffinose, mannitol, dextran, polyethylene glycol, sorbitol, and glycerol, or a combination thereof, and the amino acid is selected from alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamic acid (E), glutamine (Q), glycine (G), histidine (H), methionine (M), proline (P), serine (S), threonine (T), and tyrosine (Y). , valine (V) or any one or a combination thereof, the pH adjuster is selected from any one or a combination of sodium hydroxide, potassium hydroxide, ammonia water, hydrochloric acid, sulfuric acid, phosphoric acid, sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium carbonate, sodium bicarbonate, and potassium bicarbonate, comprising the following steps: adding a required amount of stabilizer to a 1-10 mM amino acid solution, adjusting the solution to obtain a pH of 6-7.5, filtering through a filter membrane with a pore size of 0.15-0.45 um, adding a required amount of oncolytic virus to the collected filtrate, and mixing evenly to obtain.

[0021] In the preferred technical scheme of the present invention, the preparation method comprises the following steps: adding 1-10% (w / v) proline and 1-10% (w / v) stabilizer to a 1-10mM histidine solution, adjusting the pH of the solution to 6-7.5, filtering through a filter membrane with a pore size of 0.15-0.45um, adding the required amount of oncolytic virus to the collected filtrate, and mixing evenly to obtain.

[0022] In the preferred technical solution of the present invention, the pore size of the filter membrane is 0.22-0.30 um.

[0023] In the preferred technical solution of the present invention, the titer of the oncolytic virus in the composition is (5-10)×10 8 pfu / ml, preferably (6-8)×10 8 pfu / ml.

[0024] In a preferred technical solution of the present invention, the composition contains 2-8% (w / v), preferably 3-5% (w / v) of stabilizer.

[0025] In a preferred technical solution of the present invention, the composition contains 5-120 mM amino acids, preferably 10-100 mM.

[0026] In a preferred technical solution of the present invention, the pH of the composition is 6.5-7.2.

[0027] In the preferred technical solution of the present invention, the composition contains a titer of (1-10)×10 8 pfu / ml of oncolytic virus, 1-10% (w / v) of sorbitol, glycerol, any one of trehalose, 1-10% (w / v) of proline, 1-10 mM of histidine solution, and the composition has a pH of 6.5-7.2.

[0028] In the preferred technical solution of the present invention, the composition contains a titer of (2-8)×10 8 pfu / ml of oncolytic virus, 2-5% (w / v) of sorbitol, glycerol, any one of trehalose, 1-5% (w / v) of proline, 5-10 mmol / L of histidine solution, and the composition has a pH of 6.5-7.2.

[0029] In the preferred technical solution of the present invention, the composition contains a titer of 7.6×10 8 pfu / ml of oncolytic virus, 5% (w / v) sorbitol, glycerol, any one of trehalose, 1.15% (w / v) proline, 10 mmol / L histidine solution, and the composition has a pH of 6.5-7.2.

[0030] In a preferred technical solution of the present invention, the composition can be prepared into a lyophilized preparation for injection.

[0031] In a preferred technical solution of the present invention, after the composition is repeatedly frozen and thawed 5 times at 25° C., the virus titer loss is ≤30%, preferably less than ≤20%, and more preferably ≤10%.

[0032] In a preferred technical solution of the present invention, the composition can be placed at 25°C for 1-6 days, preferably for 2-4 days.

[0033] In a preferred technical solution of the present invention, after the composition is placed at 25° C. for 2 days, the virus titer loss is ≤30%, preferably ≤20%, and more preferably ≤10%.

[0034] In a preferred technical solution of the present invention, after the composition is placed at 25° C. for 4 days, the virus titer loss is ≤30%, preferably ≤20%, and more preferably ≤10%.

[0035] In a preferred technical solution of the present invention, the composition can be placed at 37°C for 1-36 hours, preferably for 1-24 hours.

[0036] In a preferred technical solution of the present invention, after the composition is placed at 37° C. for 36 hours, the virus titer loss is ≤30%, preferably ≤20%, and more preferably ≤10%.

[0037] In a preferred technical solution of the present invention, after the composition is placed at 37° C. for 24 hours, the virus titer loss is ≤30%, preferably ≤20%, and more preferably ≤10%.

[0038] Another object of the present invention is to provide use of the high-stability oncolytic virus composition of the present invention in the preparation of anti-tumor drugs.

[0039] In a preferred technical solution of the present invention, the tumor is selected from any one of lung cancer, gastric cancer, liver cancer, rectal cancer, kidney cancer, pancreatic cancer, esophageal cancer, prostate cancer, bladder cancer, skin cancer, breast cancer, endometrial cancer, cervical cancer, ovarian cancer, brain tumor, head and neck cancer, non-Hodgkin's lymphoma, leukemia, and melanoma.

[0040] Unless otherwise specified, the oncolytic virus titer was determined by the following method: 3.5×10 5 Complementary Cell C ICP27 Plant in a 6-well cell culture plate and culture overnight in culture medium. Dilute the oncolytic virus solution to be tested 10 times in series, and infect the cells with 0.1 ml of the virus with different dilution multiples. After 1 hour, aspirate the culture medium and add 3 ml of culture medium containing 1.25% methylcellulose to each well of cells. The cells were cultured in a CO2 incubator for 5 days, stained with 0.1% amethyst blue prepared in 50% methanol, 50% ethanol, and the virus plaques were counted. Calculate the virus titer (PFU / ml).

[0041] Unless otherwise specified, when the present invention relates to the percentage between liquids, the percentage is volume / volume percentage; when the present invention relates to the percentage between liquids and solids, the percentage is volume / weight percentage; when the present invention relates to the percentage between solids and liquids, the percentage is weight / volume percentage; the rest are weight / weight percentages.

[0042] Compared with the prior art, the present invention has the following beneficial technical effects:

[0043] 1. The present invention scientifically screens the components and ratios of the oncolytic virus composition, so that the oncolytic virus can maintain viral activity under repeated freezing and thawing and at 25°C-37°C, significantly reducing the loss of viral activity, ensuring the quality of the drug and the safety and effectiveness of clinical drug use, facilitating the preparation, clinical application, storage, and transportation of the oncolytic virus and significantly reducing its cost.

[0044] 2. The preparation method of the present invention has the advantages of simple operation, low cost, and suitability for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Study on the stability of the oncolytic virus composition of the present invention under repeated freezing and thawing conditions;

[0046] Figure 2 The stability of the oncolytic virus composition of the present invention when placed at 37°C;

[0047] Figure 3 The stability of the oncolytic virus composition of the present invention when placed at 25°C was investigated. DETAILED DESCRIPTION

[0048] The present invention is further described below with reference to the embodiments.

[0049] Example 1 Preparation of oncolytic virus composition of the present invention

[0050] Composition of oncolytic virus composition:

[0051] oHSV-BJR virus (titer 7.6×10 8 pfu / ml)

[0052] Sorbitol 5.0g

[0053] Proline 0.5770g

[0054] Histidine (10mmol / L) dilute to 50ml

[0055] Preparation of oncolytic virus composition: Add the required amount of sorbitol and proline to 40 ml of 10 mmol / L histidine solution, adjust the pH of the system to 6.5 with sodium hydroxide solution, filter through a 0.22 um filter membrane, add the required amount of oHSV-BJR virus to the filtrate, and then add the required amount of 10 mmol / L histidine solution to make the volume to 50 ml.

[0056] Example 2 Preparation of oncolytic virus composition of the present invention

[0057] Composition of oncolytic virus composition:

[0058] oHSV-BJR virus (titer 7.6×10 8 pfu / ml)

[0059] Trehalose 5g

[0060] Proline 0.5770g

[0061] Histidine (10 mmol / L) dilute to 50 ml

[0062] Preparation of oncolytic virus composition: Add the required amount of trehalose and proline to 40 ml of 10 mmol / L histidine solution, adjust the pH of the system to 6.5 with sodium hydroxide solution, filter through a 0.22 um filter membrane, add the required amount of oHSV-BJR virus to the filtrate, and then add the required amount of 10 mmol / L histidine solution to make the volume to 50 ml.

[0063] Example 3 Preparation of oncolytic virus composition of the present invention

[0064] Composition of oncolytic virus composition:

[0065] oHSV-BJR virus (titer 7.6×10 8 pfu / ml)

[0066] Glycerin 5.0g

[0067] Proline 0.5770g

[0068] Histidine (10mmol / L) dilute to 50ml

[0069] Preparation of oncolytic virus composition: Add the required amount of glycerol and proline to 40 ml of 10 mmol / L histidine solution, adjust the pH of the system to 6.5 with sodium hydroxide solution, filter through a 0.22 um filter membrane, add the required amount of oHSV-BJR virus to the filtrate, and then add the required amount of 10 mmol / L histidine solution to make the volume to 50 ml.

[0070] Example 4 Preparation of oncolytic virus composition of the present invention

[0071] Composition of oncolytic virus composition:

[0072] oHSV-BJR virus (titer 7.6×10 8 pfu / ml)

[0073] Trehalose 5.0g

[0074] Proline 0.5770g

[0075] Histidine (10mmol / L) dilute to 50ml

[0076] Preparation of oncolytic virus composition: Add the required amount of trehalose and proline to 40 ml of 10 mmol / L histidine solution, adjust the pH of the system to 7.16 with sodium hydroxide solution, filter through a 0.22 um filter membrane, add the required amount of oHSV-BJR virus to the filtrate, and then add the required amount of 10 mmol / L histidine solution to make the volume to 50 ml.

[0077] Test Example 1 Study on the stability of the oncolytic virus composition of the present invention under repeated freezing and thawing conditions

[0078] The oncolytic virus composition prepared in Example 1-4 was taken out from the -80°C storage environment, placed at 25°C for 20 min, then placed at -80°C for 21 days, and then placed at 25°C for 20 min. The virus titer was tested before and after each freeze-thaw cycle. Figure 1 .

[0079] Experimental Example 2: Stability of the oncolytic virus composition of the present invention at 37°C

[0080] The oncolytic virus composition prepared in Example 1-4 was taken out from the -80°C storage environment, placed in a 37°C water bath for 0h, 12h, 24h, and 36h, and the virus activity titer was detected. Figure 2 .

[0081] Test Example 3 Stability study of the oncolytic virus composition of the present invention at 25°C

[0082] The oncolytic virus composition prepared in Example 1-4 was taken out from the -80°C storage environment, placed at 25°C for 0, 2, and 4 days, and the virus activity titer was detected. Figure 3 .

[0083] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art may make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should all fall within the scope of protection of the claims of the present invention.

Claims

1. A highly stable oncolytic virus composition, comprising a titer of (1-10)×10 8 pfu / ml of oncolytic virus, 1-10% w / v sorbitol, 1-10% w / v proline, 1-10 mM histidine, the composition has a pH of 6.5-7.2, and the oncolytic virus is oHSV-BJR virus.

2. The composition according to claim 1, comprising a titer of (2-8)×10 8 pfu / ml of oncolytic virus, 2-5% w / v of sorbitol, 1-5% w / v of proline, 5-10 mmol / L of histidine solution, and the composition has a pH of 6.5-7.

2.

3. The composition according to claim 2, comprising a titer of 7.6×10 8 pfu / ml of oncolytic virus, 5% w / v sorbitol, 1.15% w / v proline, 50 ml of 10 mmol / L histidine solution, and the composition has a pH of 6.5-7.

2.

4. The composition according to any one of claims 1 to 3, which can be prepared into a lyophilized preparation for injection.

5. The composition according to any one of claims 1 to 3, wherein after the composition is repeatedly frozen and thawed 5 times at 25°C, the virus titer loss is ≤10%.

6. The composition according to any one of claims 1 to 3, which can be stored at 25°C for 1 to 6 days.

7. The composition according to any one of claims 1 to 3, characterized in that After the composition is placed at 25° C. for 4 days, the virus titer loss is ≤10%.

8. The composition according to any one of claims 1 to 3, characterized in that The composition can be placed at 37° C. for 1-36 hours.

9. The composition according to any one of claims 1 to 3, characterized in that After the composition is placed at 37° C. for 36 hours, the virus titer loss is ≤10%.

10. A method for preparing a highly stable oncolytic virus composition according to any one of claims 1 to 9, the preparation method comprising the following steps: adding 1-10% w / v proline and 1-10% w / v sorbitol to a 1-10 mM histidine solution, adjusting the pH of the solution to 6-7.5, filtering through a filter membrane with a pore size of 0.15-0.45 um, adding a required amount of oncolytic virus to the collected filtrate, and mixing evenly to obtain.

11. The preparation method according to claim 10, characterized in that: The pore size of the filter membrane is 0.22-0.30 um.

12. Use of the high-stability oncolytic virus composition as described in any one of claims 1 to 9 or the high-stability oncolytic virus composition prepared by the preparation method as described in any one of claims 10 to 11 for preparing anti-tumor drugs, wherein the tumor is selected from any one of lung cancer, gastric cancer, liver cancer, and rectal cancer.

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