CN113425856A

CN113425856A - Pharmaceutical composition containing genetically modified oncolytic virus and application thereof in treating cancer

Info

Publication number: CN113425856A
Application number: CN202110764637.8A
Authority: CN
Inventors: 亓爱杰; 李少波; 韦素碧
Original assignee: Nuosa Union Beijing Biomedical Technology Co ltd
Current assignee: CARBIOGENE THERAPEUTICS Co.,Ltd.
Priority date: 2021-07-07
Filing date: 2021-07-07
Publication date: 2021-09-24
Anticipated expiration: 2041-07-07
Also published as: CN113425856B

Abstract

The present invention relates to a pharmaceutical composition comprising a genetically modified oncolytic virus and its use in the treatment of cancer. The invention prepares an oncolytic virus expressing IL-24, gefitinib and a monoclonal antibody specifically aiming at PD-1. The oncolytic virus, gefitinib and monoclonal antibody can be effectively used for treating nasopharyngeal carcinoma. The combined use of the oncolytic virus, gefitinib and a monoclonal antibody can find that the oncolytic virus has a synergistic treatment effect and has an excellent application prospect.

Description

Pharmaceutical composition containing genetically modified oncolytic virus and application thereof in treating cancer

Technical Field

The present invention relates to the field of biology, and more particularly, to pharmaceutical compositions comprising genetically modified oncolytic viruses and their use in the treatment of cancer.

Background

Oncolytic virus is a natural or genetically modified virus, can selectively infect tumor cells, can replicate in large quantities in the tumor cells and finally lyse the tumor cells, and can release a large quantity of tumor antigens simultaneously to improve the tumor microenvironment.

The oncolytic virus plays an anti-tumor role through direct oncolytic action and immunity activity induction of an organism, and due to the lack of necessary conditions for replication in normal cells, the oncolytic virus can reproduce and proliferate in a tumor cell in a targeted manner, finally, the tumor cell is cracked and killed, and the anti-oncolytic virus has no influence on healthy cells. Therefore, the oncolytic virus has good safety and has the advantage of long-acting expression of anti-tumor exogenous genes. Currently common oncolytic viruses include Ad, herpes simplex virus type 1 (HSV-1), Vesicular Stomatitis Virus (VSV), Newcastle Disease Virus (NDV), measles virus (measles virus), poliovirus (poliovirus), parvovirus (parvovirus), reovirus (reoviruses), VV, and the like.

Research has shown that many factors such as lack of co-stimulatory molecules in tumor cells seriously affect the induction and generation of tumor antigen-specific CTL and the corresponding killing activity. By inserting genes with products expressing tumor killing activity, such as immune activation genes, prodrug invertase genes, anti-tumor angiogenesis genes, apoptosis inducing genes and the like, into the genome of the oncolytic virus, the oncolytic virus can be directly used as a delivery vehicle of a target gene, so that the target gene is continuously expressed in tumor cells along with the replication of the virus, and the killing activity of the oncolytic virus is further enhanced.

The gene for expressing the immunoinflammatory mediator is inserted into the genome of the oncolytic virus, so that the local immunoinflammatory mediator in a tumor microenvironment is abnormally increased, the anti-tumor immune response of an organism is activated, and the anti-tumor effect of the oncolytic virus is enhanced. At present, various immune activation genes are applied to genetic recombination of oncolytic viruses, such as granulocyte-macrophage colony stimulating factor (GM-CSF), interleukins (IL-2, IL-12, IL-15 and IL-18), interferons (IFN-alpha and IFN-beta) and the like. Also, recombinant adenovirus RdB/IL-12/IL-18 capable of simultaneously expressing IL-12 and IL-18 has been designed, IL-12 stimulates the anti-tumor immune response of the organism by activating NK cells and cytotoxic T cells, IL-18 enhances the NK cytotoxic effect and promotes the differentiation of T cells. In mouse melanoma transplantation animal model, obvious increase of IL-12, IL-18, IFN-gamma and GM-CSF concentration was detected in tumor body after RdB/IL-12/IL-18 local injection, and in tumor tissue section, massive NK cell, CIM and CD8T cell infiltration and extensive necrosis of tumor tissue were observed, further demonstrating strong killing activity of IL-12 and IL-18 combined action on tumor cells. The same results were also verified in a mouse colon cancer animal model, and compared with the control group, the gene recombinant measles virus MV-GMCSF expressing GM-CSF can significantly delay the progression of mouse colon cancer and prolong the median survival time of mice.

In recent years, research related to oncolytic viruses has been greatly advanced, and has attracted much attention. However, strictly speaking, the marketed and approved product is only a 2015 FDA approved T-vec (Imlygic) of an Advance company, which is used for local treatment of melanoma patients and is the first drug for obtaining FDA approved oncolytic virus treatment. In 2016, were approved for marketing in Europe and Canada, respectively. In China, Cocury (H101) of three-dimensional organisms is approved by CFDA in 2005, but the clinical curative effect of the product is not internationally recognized until now due to the superposition of various factors such as imperfect domestic clinical test standards, loose examination and approval policies, the war of product patents and the like. In addition, the development of oncolytic virus is currently carried out by a plurality of companies in China, and great progress is made, so that clinical applications of the oncolytic virus are submitted to a drug administration by the plurality of companies in 2020. Meanwhile, tumor immunotherapy gradually inclines to combination therapy, and clinical evidence shows that oncolytic virus can enhance the response rate of tumors to immune therapy methods such as immune regulation checkpoint inhibitors, CAR-T cells and the like, and the oncolytic virus is an ideal object in combination therapy strategies. Making oncolytic viruses a hot direction in tumor immunization studies.

However, currently, there are also few proposals for the use of oncolytic virus products, particularly modified oncolytic virus products, in combination with immune checkpoint inhibitors, which are provided in the home.

Disclosure of Invention

In one aspect, it is an object of the present invention to provide means for improving cancer treatment.

This is achieved according to the invention by the subject-matter defined in the claims.

The present invention provides a pharmaceutical composition comprising (a) an oncolytic virus and (b) a PD-1 inhibitor and (c) gefitinib. The invention also provides the use of the pharmaceutical composition for the treatment of cancer.

The pharmaceutical composition of the present invention may include the oncolytic virus of the present invention, including pharmaceutically acceptable ingredients, buffers, excipients, adjuvants, preservatives, fillers, stabilizers, thickeners and other ingredients commonly used in formulation. In addition, in some cases, in addition to the oncolytic virus of the invention, other therapeutically effective tumor-dissolving viruses and/or therapeutically effective drugs may be included, such as anti-cancer agents, adjunct ingredients (e.g., immune checkpoint inhibitors, such as CTLA-4 blocks, and immune activators, such as GM-CSF).

Preferably, in the pharmaceutical composition, the oncolytic virus and the checkpoint inhibitor are present in effective doses and are combined with a pharmaceutically acceptable carrier.

Further, the oncolytic virus is a transgenic oncolytic virus expressing IL-24.

Further, the transgenic oncolytic virus is pAdxsi adenovirus expressing IL-24.

Further, the oncolytic virus is pAd-HS4-AFP-E1A-IL-24 virus plasmid, which is obtained by a person skilled in the art through a conventional construction method or a whole gene synthesis method, as long as the virus can express IL-24.

Furthermore, the invention also provides a specific PD-1 monoclonal antibody, which is a monoclonal antibody which is obtained by immunizing a mouse by the PD-1 protein and is specifically combined with PD-1 by a hybridoma screening technology. The variable region of the light chain of the monoclonal antibody is shown as SEQ ID NO: 1, and the heavy chain variable region is shown as SEQ ID NO: 2, the monoclonal antibody has a good binding effect with a binding constant of 5.9nM to PD-1 protein, and has good specificity and no reaction with other proteins.

Light chain variable region (SEQ ID NO: 1)

DIVITQSPALMAASPGEKVTITCTCYSDEGYHNCNWYQQKSGISPKPWIYLPEFYQCGVPARFSGSGSGTSYSLTITSMEAEDAATYYCINLILGCERFGAGTKLELK

Heavy chain variable region (SEQ ID NO: 2)

EVQLEESGTELARPGASVKLSCKASGYIFSFWALGWIKQRPGQGLEWIGNFTTQAVERTKHDFQFGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGGAKRCLNWGLGTTLAVSS。

It is noted that in this disclosure, and in the claims and/or paragraphs particularly, the terms "comprising," "including," and the like may have the meaning attributed to it in U.S. patent statutes; for example, they can mean "including", "comprising", "including", and the like; and the terms "consisting essentially of and" consisting essentially of have their definitions in U.S. patent statutes, e.g., they allow for elements not expressly listed but exclude elements found in the prior art or that affect a basic or novel feature of the invention.

In some embodiments, the oncolytic virus is derived from a poxviridae family. In some embodiments, the oncolytic virus is derived from a chordaviridae or arbovirus subfamily.

In some embodiments, the oncolytic virus is from an adenovirus. Or other available oncolytic viruses.

In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is a sarcoma or carcinoma.

In some embodiments, the solid tumor is fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyoma, rhabdomyosarcoma, colon carcinoma, pancreatic carcinoma, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, bladder cancer, medullary carcinoma, bronchial cancer, renal cell carcinoma, liver cancer, bile duct cancer, choriocarcinoma, seminoma, embryonic cancer, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, epididymoma, spongioblastoma, acoustic neuroma, oligodendroglioma, glioma, meningioma, melanoma, neuroblastoma or retinoblastoma.

In some embodiments, the oncolytic virus, gefitinib, and the PD-1 antibody are administered together. In some embodiments, the method comprises administering the oncolytic virus and the PD-1 inhibitor for at least 1 day, at least 2 days, at least 3 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 3 months, at least 6 months, or at least 1 year. In some embodiments, the method comprises administering to the subject an oncolytic virus, a nuclear mass transport inhibitor, or both, twice weekly, biweekly, every three weeks, every four weeks, monthly, or every two months. In some embodiments, the oncolytic virus is administered locally to the cancer tissue to be treated. In some embodiments, the oncolytic virus is administered by intratumoral injection. In some embodiments, the multiple infection of the tissue by the oncolytic virus (MoI) is from about 0.01 to about 10. In some embodiments, the multiple infection (MOI) of the tissue by the oncolytic virus is from about 0.05 to about 5.

Advantageous effects

The invention prepares an oncolytic virus expressing IL-24, gefitinib and a monoclonal antibody specifically aiming at PD-1. The use of the oncolytic virus together with a monoclonal antibody may be useful for the treatment of nasopharyngeal carcinoma. The combined use of the oncolytic virus and the monoclonal antibody can find that the combined use of the oncolytic virus and the monoclonal antibody has synergistic treatment effect and excellent application prospect.

Drawings

FIG. 1 is a graph showing the effect of the use of the PD-1 antibody in combination with an oncolytic virus

FIG. 2 graph of the effect of the PD-1 antibody in combination with oncolytic virus and gefitinib

Detailed Description

Examples will be given below and further illustrate the present invention, but the examples are merely illustrative of the embodiments of the present invention and do not limit the scope of the present invention.

EXAMPLE 1 preparation of transgenic oncolytic viruses

Plasmids containing HS-4-AFP-E1A-PolyA were purchased from Shanghai three-dimensional Biotechnology, Inc. pShuttle-Basic-EGFP recombinant shuttle vector and pAdxsi recombinant adenovirus backbone vector were purchased from Nossel genome research center, Inc.

According to the construction method in 'research on the effect of oncolytic adenovirus carrying IL-24 gene for inhibiting primary liver cancer lung metastasis, Ph paper of Chack university, 2009', a target gene fragment is obtained by amplifying plasmid HS4-AFP-E1A-PolyA, cloned to pGEM-T Easy vector to obtain HS4-AFP-E1A-PolyA-T cloning vector, IL-24 gene is amplified, connected to pShuttle-Basic-EGFP vector to replace EGFP to obtain pShuttle-Basic-IL-24, then the fragment of HS4-AFP-E1A-PolyA is cut from HS4-AFP-E1A-PolyA-T cloning vector, connected to pShuttle-Basic-IL-24 vector to obtain pShuttle-Basic-IL-24-AFP-E1A, and transferred to pAdxsi adenovirus recombinant skeleton vector, the pAd-HS4-AFP-E1A-IL-24 virus plasmid is obtained, and the construction is correct through enzyme digestion identification.

Packaging and purifying the recombinant adenovirus: lipofectamine 2000 liposomes transfected 293 cells to obtain virus, stored at-80 ℃. (3) Adenovirus amplification and purification: purification by cesium chloride gradient centrifugation and titer determination was performed using TCID 50. The titers of the amplified viruses are all 2.1 multiplied by 10 by the method of TCID50¹¹PFU/ml。

EXAMPLE 2 preparation of PD-1 inhibitor

The immunogen is PD-1 recombinant protein (P1210, Okay organism). Mice were immunized by the general method. Briefly, an appropriate amount of Freund's adjuvant was taken out into a 1.5ml EP tube and mixed well with a shaker. The antigen protein solution was prepared with PBS. Mixing adjuvant and protein antigen solution according to required amount, emulsifying antigen by injector to form stable water-in-oil solution, and injecting into animal. According to the result of serum titer measurement, after the first immunization, 3 times of boosting immunization is usually required to achieve a good immune effect. And (3) selecting immune mice with high serum titer to perform cell fusion after intraperitoneal injection. Prior to cell fusion, mouse myeloma cells (SP2/0-Ag14, ATCC # CRL-1581) were cultured in logarithmic growth phase. After sacrifice of the immunization, the mice were harvested in sterile environment and splenic B lymphocytes and SP2/0 myeloma cells were fused using PEG chemistry. After the fusion, the cells were plated in 96-well cell culture plates, and the growth of viable hybridoma cells was observed under a microscope after 7 to 10 days. Two weeks after cell plating, culture supernatants from each well were collected and hybridoma screened using human PD-1-his protein antigen by ELISA. Briefly, the microplate was coated with 50ul of 2ug/ml of human PD-1-his antigen in PBS overnight at 4 ℃. After 4 additional PBST washes, the plates were blocked for 3 hours at 37 ℃ with 200. mu.l/well of 5% skim milk powder in PBST. The plate was washed again, 50. mu.l/well of hybridoma supernatant was added, incubated at 37 ℃ for 40 minutes, and then the plate was washed 5 times. Diluted horseradish peroxidase-labeled secondary goat-anti-mouse antibody was added at 100. mu.l/well, incubated at 37 ℃ for 40 minutes, and then the plate was washed 5 times and patted dry. The absorbance of each well at 450 nm was measured by adding 100. mu.l/well of TMB chromogenic substrate, developing at room temperature for 20 minutes, and then stopping with a 2M solution of sulfuric acid. And (4) selecting hybridoma hole cells with positive ELISA color development, transferring the hybridoma hole cells into a 24-hole plate, and continuing to culture. And performing a second round of re-screening by an ELISA method, screening out hybridomas which specifically recognize PD-1 antigens and can block the combination of PD-1, and performing subcloning by a limiting dilution method to obtain the target monoclonal cell strain 3F 2.

Through sequence identification, the variable region of the light chain of the monoclonal antibody is shown as SEQ ID NO: 1, and the heavy chain variable region is shown as SEQ ID NO: 1, the monoclonal antibody has a good binding effect with a binding constant of 5.9nM to PD-1 protein, and has good specificity and no reaction with other proteins.

Light chain variable region (SEQ ID NO: 1)

Heavy chain variable region (SEQ ID NO: 2)

Example 3 Effect of transgenic oncolytic tumor vaccine on CTL cells

The high titer virus prepared in example 1 was selected to infect human nasopharyngeal carcinoma cells CNE2, while the empty virus was transfected as a control. G418 (250. mu.g/ml) was screened for 2 weeks to obtain positive clones. The integration of the IL24 gene into CEN2 cells was confirmed by PCR detection. The double-antibody sandwich ELISA detects IL24 transfection positive cells, and the cell amount detected is 10⁶And (4) respectively. The results are shown in table 1 below.

TABLE 1 secretion of IL-24

Type (B)	Amount of secretion
		IL24 positive	945.3±23.4pg/24h

As can be seen from the results in Table 1, the secretion of IL24 in the transgenic cells was 945.3. + -. 23.4pg/24h, and the secretion of IL24 in the control cells was low, indicating that the transgene was successful.

Mitomycin C (40 mug.10)⁶Cells/ml, 37 ℃,1h) treated wild type tumor cells, empty vector and IL24 gene transfected tumor cells were stimulator cells (5X 10)⁵) Peripheral blood mononuclear cells (PBMC, 10) from healthy donors⁷) Mixed culture at 37 deg.C and 5% CO₂After 1 week of incubation, the reaction cells were collected and restimulated at 2X 10⁶Separately adding stimulating cells (2X 10) to the reaction cells⁵) Autologous feeder cells (mitomycin C-treated PBMC, 10)⁵) Culturing for 1 week, collecting the reaction cells, and culturing for 4 hr⁵¹The CTL killing activity was detected by Cr release assay. The results are shown in Table 2.

TABLE 2CTL killing Activity results

Type (B)	Killing activity (%)
		Mitomycin-treated wild-type tumor cells	12.3±1.9
Empty virus tumor cells	12.6±1.8
		Tumor cells transformed with IL-24 virus	76.5±2.3

As can be seen from Table 2, the IL-24 gene modified tumor vaccine can induce strong CTL response, and the killing power of the tumor vaccine reaches (76.5 +/-2.3)%, so that the tumor vaccine has a better effect.

Example 4 verification of tumor-inhibiting Effect of oncolytic Virus

Balb-c nude mice were purchased from Shanghai Slek laboratory animal center, female, 4 weeks old, and had a body mass of 15-20 g. Conventionally culturing human nasopharyngeal carcinoma cell CNE2, amplifying, collecting cells in logarithmic growth phase, digesting, and making into single cell suspension. And (5) carrying out ear-cutting marking on the experimental animal, and recording the quality of the experimental animal. 0.3 percent sodium pentobarbital is injected into the abdominal cavity of an anesthesia experimental animal according to the dose of 30-40 mg/kg. 100 μ l of CNE2 cell suspension was injected subcutaneously into bilateral flank of nude mice, and the amount of cells injected into each side of each nude mouse was 1X 10⁶And (4) respectively. Animal grouping and treatment: nude mice were randomly divided into 3 groups: 10 oncolytic virus treated groups and oncolytic virus + PD-1 mab treated groups as well as control groups. After injecting the tumor into the experimental animal for 4 days, measuring the size of the subcutaneous tumor by using a vernier caliper, and when the subcutaneous tumor is ill, measuring the subcutaneous tumor by using the vernier caliperWhen the tumor on the side of the virus group was grown to 5mm (about day 8), the virus was injected intratumorally. Virome treatment the virus of example 1 was injected twice a week at 5X 10⁵Mu.l for 4 weeks, and the control group was injected twice weekly with 100. mu.l of phosphate buffered saline for 4 weeks. Oncolytic Virus + PD-1 monoclonal antibody treatment group twice weekly injections of the virus of example 1 at 5X 10⁵Mu.l 50. mu.l and PD-1 mAb 1mg/kg, injected for 4 weeks. Tumor size measurement: the maximum major diameter (a) and maximum width (b) of the tumor were measured after each inoculation, and the tumor volume V was calculated according to the formula (a × b2/2) to plot the tumor volume growth curve. The results are shown in FIG. 1.

As can be seen from the results in FIG. 1, the addition of oncolytic virus effectively inhibited tumor growth, and in particular, reduced tumor volume after 3 weeks. After the PD-1 antibody is added and the oncolytic virus is used together, the tumor volume can be more obviously shortened, and in the 4 th week, the tumor volume is only 80mm³And the inhibitor has obvious inhibiting effect.

Example 5 verification of tumor-inhibiting Effect of oncolytic Virus in combination with antitumor drug

Balb-c nude mice were purchased from Shanghai Slek laboratory animal center, female, 4 weeks old, and had a body mass of 15-20 g. Conventionally culturing human nasopharyngeal carcinoma cell CNE2, amplifying, collecting cells in logarithmic growth phase, digesting, and making into single cell suspension. And (5) carrying out ear-cutting marking on the experimental animal, and recording the quality of the experimental animal. 0.3 percent sodium pentobarbital is injected into the abdominal cavity of an anesthesia experimental animal according to the dose of 30-40 mg/kg. 100 μ l of CNE2 cell suspension was injected subcutaneously into bilateral flank of nude mice, and the amount of cells injected into each side of each nude mouse was 1X 10⁶And (4) respectively. Animal grouping and treatment: nude mice were randomly divided into 3 groups: 10 oncolytic virus + PD-1 monoclonal antibody treatment groups, oncolytic virus + PD-1 monoclonal antibody + gefitinib treatment groups and control groups. Subcutaneous tumor size was measured with a vernier caliper starting 4d after tumor injection into the experimental animals, and intratumoral virus injection treatment was performed when the tumor on the virus-treated side grew to 5mm (about day 8). The control group was injected twice weekly with 50 μ l of phosphate buffered saline for a total of 4 weeks. Oncolytic virus + PD-1 monoclonal antibody treatment group twice weeklyVirus 5X 10 of example 1⁵Mu.l 50. mu.l and PD-1 mAb 1mg/kg, injected for 4 weeks. Oncolytic virus + PD-1 monoclonal antibody treatment group and oncolytic virus + PD-1 monoclonal antibody + gefitinib treatment group were injected twice weekly with 5X 10 of the virus of example 1⁵Mu.l 50. mu.l and PD-1 mAb 1mg/kg + gefitinib 2mg/kg for a total of 4 weeks. Tumor size measurement: the maximum major diameter (a) and maximum width (b) of the tumor were measured after each inoculation, and the tumor volume V was calculated according to the formula (a × b2/2) to plot the tumor volume growth curve. The results are shown in FIG. 2.

As can be seen from the results in FIG. 2, the addition of oncolytic virus effectively inhibited tumor growth, and in particular, reduced tumor volume after 3 weeks. After the PD-1 antibody is added and the oncolytic virus and gefitinib are used together, the tumor volume can be more remarkably shortened, and in the 4 th week, the tumor volume is only 30mm³About 80mm in comparison with the addition of PD-1 antibody and oncolytic virus³Has obviously improved inhibiting effect.

The invention has been described in detail with reference to specific embodiments and illustrative examples, but the description is not intended to be construed in a limiting sense. Those skilled in the art will appreciate that various equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its embodiments without departing from the spirit and scope of the present invention, which fall within the scope of the present invention. The scope of the invention is defined by the appended claims.

Sequence listing

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Claims

1. A pharmaceutical combination for use in the treatment of nasopharyngeal carcinoma, characterized in that said combination consists of oncolytic virus, gefitinib and PD-1 monoclonal antibody; wherein, the oncolytic virus is pAdxsi adenovirus for specifically expressing IL-24; the variable region of the light chain of the monoclonal antibody is shown as SEQ ID NO: 1, and the heavy chain variable region is shown as SEQ ID NO: 2, respectively.

2. Use of a combination of an oncolytic virus, gefitinib and a PD-1 monoclonal antibody for the preparation of a pharmaceutical composition for the treatment of nasopharyngeal carcinoma, wherein the oncolytic virus is a pAdxsi adenovirus specifically expressing IL-24; the variable region of the light chain of the monoclonal antibody is shown as SEQ ID NO: 1, and the heavy chain variable region is shown as SEQ ID NO: 2, respectively.