CN115715803A

CN115715803A - Application of oncolytic virus and immune-related drugs in synergistic inhibition of solid tumors

Info

Publication number: CN115715803A
Application number: CN202110976980.9A
Authority: CN
Inventors: 徐贤斌; 王爱霞; 刘建飞; 吴丹; 伊萌; 顾玮蕾
Original assignee: Shanghai Sunway Biotech Co Ltd
Current assignee: Shanghai Sunway Biotech Co Ltd
Priority date: 2021-08-24
Filing date: 2021-08-24
Publication date: 2023-02-28

Abstract

The invention discloses application of oncolytic virus and immune-related drugs in synergistic treatment of solid tumors. In particular, the invention provides the use of an active ingredient combination comprising a recombinant human adenovirus type 5 and an immune-related drug for the preparation of a pharmaceutical composition for the synergistic treatment of tumors. The active ingredient combination can effectively inhibit tumors synergistically, and can obviously reduce toxic and side effects, so that the active ingredient combination can be widely applied to targeted treatment of solid tumors.

Description

Application of oncolytic virus and immune-related drugs in synergistic inhibition of solid tumors

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of oncolytic virus and immune-related medicine in synergistic inhibition of solid tumors and related medicine thereof.

Background

Recombinant human adenovirus type 5 (H101, an Kerui,

) Is a recombinant human adenovirus type 5 oncolytic virus medicine marketed by Shanghai three-dimensional biotechnology limited company in 2006, and currently, an Kerui already produces 12 thousands of products, which are mainly used for treating head and neck cancer. In clinical studies, an Kerui was found to produce therapeutic effects on distal tumors under conditions of intratumoral injection and local heating of tumor tissue. This is caused by An Kerui which presents tumor specific antigens on the surface of tumor cells or inside the cells to immune cells during the process of oncolytic, triggering the body's anti-tumor immune response.

The immune response of the body against tumors can also be generated by the body, and the immune response comprises an immune stimulating factor, an immune checkpoint inhibitor, a tumor specific antigen and the like. Wherein, the human Granulocyte-macrophage colony stimulating factor (GM-CSF) as an immune stimulating factor can activate Antigen Presenting Cells (APCs), mainly Dendritic Cells (DC) and macrophages, and the APC activates T cells by processing and presenting tumor antigens to achieve the effect of killing tumors; immune checkpoint inhibitors have achieved success in clinical applications as an important therapeutic approach to the treatment of various cancers; tumor specific antigens are capable of inducing an immune response and may be presented in a variety of ways, including adenovirus, adeno-associated virus, DNA or RNA vaccines, and the like.

At present, because the deep part and the interior of the solid tumor are lack of lymphocyte infiltration, the infiltration lymphocytes for resisting the tumor can be limited by various immunosuppressive mechanisms, and the immunotherapy effect is influenced.

Therefore, there is an urgent need in the art to develop a new method for treating solid tumors.

Disclosure of Invention

The invention aims to provide a treatment method and a medicament for effectively treating tumors, and particularly provides application of recombinant human adenovirus type 5 and an immune-related medicament in synergistic treatment of tumors.

According to a first aspect of the present invention, there is provided the use of an active ingredient combination, characterized in that it comprises a first active ingredient oncolytic virus and a second active ingredient immune-related drug, and in that it is used for the preparation of a pharmaceutical composition or a kit for the synergistic treatment of solid tumors.

In another preferred embodiment, the oncolytic virus is a recombinant human adenovirus type 5.

In another preferred embodiment, the oncolytic virus is recombinant human adenovirus type 5 injection with the trade name of An Kerui.

In another preferred embodiment, the E1B-55kDa and E3 region gene segments of human adenovirus type 5 are deleted from the genome of said oncolytic virus.

In another preferred embodiment, the immune-related drug is selected from the group consisting of: an immune stimulating factor, an immune checkpoint inhibitor, a tumor specific antigen, or a combination thereof.

In another preferred embodiment, the immunostimulatory factor is granulocyte-macrophage colony-stimulating factor (GM-CSF).

In another preferred embodiment, the immunodetection site inhibitor is selected from the group consisting of: an anti-CTLA 4 antibody or antigen-binding fragment thereof that specifically binds CTLA4, an anti-PD 1 antibody or antigen-binding fragment thereof that specifically binds PD1, an anti-PD-L1 antibody or antigen-binding fragment thereof that specifically binds PD-L1, or a combination thereof.

In another preferred embodiment, the tumor specific antigen is selected from the group consisting of: SAGE1, NY-ESO-1, gp33, gp100, TX103, mucin-1, WT-1, MART-1, MAGE A3, MAGE A4, MAGE B2, PRAME, SURVIVIN, MART-1, col6A3, tyrosinase, T antigen, SLC45A2, VCX/Y, HPV, alpha fetoprotein, carcinoembryonic antigen, CA 125, her2, dopachrome tautomerase, BAGE protein, GAGE protein, SURVIVIN, tyrosinase, SSX2, cyclin-A1, KIF20A, MUC AC, meloe, lengsin, kallikrein 4, IGF2B3, glypican, or a combination thereof.

In another preferred embodiment, the oncolytic virus is 0.5 × 10 according to tumor volume ¹² VP to 2.5X 10 ¹² Dosing of VP.

In another preferred embodiment, the solid tumor is selected from the group consisting of: nasopharyngeal carcinoma, chondrosarcoma, colon cancer, dukes's D or non-small cell lung cancer, breast cancer, prostate cancer, ovarian cancer, malignant liver cancer, esophageal cancer, small cell lung cancer, colorectal cancer, gastric cancer, cervical cancer, melanoma, or a combination thereof.

In another preferred embodiment, the solid tumor is also present as a distant tumor to metastasize.

In another preferred example, the solid tumor is colorectal cancer.

In a second aspect of the invention, there is provided a combination of active ingredients consisting of an oncolytic virus and an immune-related drug.

In another preferred embodiment, the oncolytic virus is a recombinant human adenovirus type 5 or a derivative having the same function, preferably a recombinant human adenovirus type 5.

In another preferred embodiment, the immunodetection point inhibitor is selected from the group consisting of: an anti-CTLA 4 antibody or antigen-binding fragment thereof that specifically binds CTLA4, an anti-PD 1 antibody or antigen-binding fragment thereof that specifically binds PD1, an anti-PD-L1 antibody or antigen-binding fragment thereof that specifically binds PD-L1, or a combination thereof.

In another preferred embodiment, the tumor specific antigen is selected from the group consisting of: SAGE1, NY-ESO-1, gp33, gp100, TX103, mucin-1, WT-1, MART-1, MAGE A3, MAGE A4, MAGE B2, PRAME, SURVIVIN, MART-1, col6A3, tyrosinase, T antigen, SLC45A2, VCX/Y, HPV, alpha fetoprotein, carcinoembryonic antigen, CA 125, her2, dopachrome tautomerase, BAGE protein, GAGE protein, SURVIVIN, tyrosinase, SSX2, cyclin-A1, KIF20A, MUC AC, meloe, lengsin, kallikrein 4, IGF2B3, or glypican or a combination thereof.

In another preferred embodiment, the tumor-specific antigen is an adenovirus, adeno-associated virus, DNA or RNA vaccine carrying the antigen gene.

In a third aspect of the present invention, there is provided a pharmaceutical composition comprising (a) an oncolytic virus, (b) an immune-related drug, and (c) a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition comprises a recombinant human adenovirus type 5, an immune checkpoint inhibitor and an immune stimulating factor.

In another preferred embodiment, the pharmaceutical composition comprises a recombinant human adenovirus type 5 and a tumor specific antigen.

In a fourth aspect of the invention, there is provided a kit comprising:

(a) A first formulation comprising an oncolytic virus and a pharmaceutically acceptable carrier;

(b) A second formulation comprising an immune-related drug and a pharmaceutically acceptable carrier;

(c) The specification describes methods of treating solid tumors using oncolytic viruses in combination with immune-related drugs.

In another preferred embodiment, the first formulation and the second formulation are independent of each other.

In another preferred embodiment, the first formulation and the second formulation are lyophilized formulations or liquid formulations.

In another preferred embodiment, the first and second formulations are injectable formulations.

In another preferred embodiment, the first formulation is administered before, during or after the administration of the second formulation.

In another preferred embodiment, the first formulation is administered by intratumoral injection and the second formulation is administered by intravenous, intraperitoneal or intratumoral injection.

In another preferred embodiment, the oncolytic virus is recombinant human adenovirus type 5 injection under the trade name of An Kerui.

In a fifth aspect of the invention, there is provided a method of treating a solid tumor, comprising the steps of: administering to a subject in need thereof an active ingredient combination according to claim 6 and/or a pharmaceutical composition according to claim 7, thereby treating a solid tumor.

In another preferred embodiment, the subject in need thereof is a human or non-human mammal.

In another preferred embodiment, the non-human mammal comprises a monkey, chimpanzee, cow, pig, dog, sheep, rabbit or mouse.

In another preferred embodiment, the subject in need thereof is a mouse.

In another preferred example, the subject in need thereof has a tumor.

In another preferred example, the method comprises: a) Administering an oncolytic virus to a subject in need thereof; and b) administering one or more immune-related drugs to the subject in need thereof.

In another preferred example, the method comprises: simultaneously administering to the subject an oncolytic virus and an immune-related drug.

In another preferred example, the method includes: administering an immune-related drug to the subject 1-6 days after the administration of the oncolytic virus.

In another preferred embodiment, the immune-related drug is anti-PD-1 monoclonal antibody.

In another preferred embodiment, the immune-related drug comprises anti-PD-1 monoclonal antibody and granulocyte-macrophage colony stimulating factor (GM-CSF).

In another preferred embodiment, the immune-related drug is adenovirus loaded with SAGE 1.

In another preferred embodiment, the recombinant human adenovirus type 5 is administered by intratumoral injection.

In another preferred embodiment, the administration mode of the immune-related drug can be intravenous, intraperitoneal or intratumoral injection.

In another preferred embodiment, the method further comprises administering to a subject in need thereof a supportive therapeutic agent for treating the tumor.

In another preferred embodiment, the supportive treatment comprises: antibiotics, analgesic drugs, nutraceuticals, or combinations thereof.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 growth curves of mouse colorectal carcinoma MC38 after initiation of combination therapy of An Kerui with anti-mPD-1 and/or mGM-CSF. Where data are presented as mean ± standard error, the tumor growth curve after D20 days fluctuates due to euthanasia after the tumor volume of the mice reached the end point, not shown.

FIG. 2 experimental animal survival curves after initiation of combination therapy of An Kerui with anti-mPD-1 and/or mGM-CSF.

Detailed Description

The present inventors have made extensive and intensive studies and, for the first time, have unexpectedly found that a specific recombinant human type 5 adenovirus, when combined with a specific immune-related drug, has a synergistic therapeutic effect in treating solid tumors. Experiments show that the recombinant human adenovirus type 5 and immune-related drugs are combined to effectively inhibit the growth of solid tumors, the effect is far better than the additive effect of the recombinant human adenovirus type 5 and the immune-related drugs, and the side effect of the traditional treatment scheme can be obviously reduced. On the basis of this, the present invention has been completed.

Description of the terms

As used herein, the term "first active ingredient" refers to a recombinant human adenovirus type 5, or an oncolytic virus. It is understood that the term includes derivatives that have the same function as recombinant human adenovirus type 5.

As used herein, the term "second active ingredient" refers to an immune-related drug, including an immune stimulating factor, an immune checkpoint inhibitor, or a tumor-specific antigen.

As used herein, the terms "active ingredient combination of the invention", "substance combination of the invention", "pharmaceutical ingredient combination of the invention" are used interchangeably and refer to the combination of the first and second active ingredients described above.

As used herein, the term "pharmaceutical composition of the invention" refers to a composition comprising or containing a first active ingredient (or a first formulation containing said first active ingredient) and a second active ingredient (or a second formulation containing said second active ingredient), wherein said first and second formulations may be the same or different formulations. Further, the first formulation and the second formulation may be the same formulation, or separate formulations.

Immunodetection point inhibitors

In a preferred embodiment of the present invention, the immune-related drug of the present invention comprises an immunodetection point inhibitor selected from the group consisting of: an anti-CTLA 4 antibody or antigen-binding fragment thereof that specifically binds CTLA4, an anti-PD-1 antibody or antigen-binding fragment thereof that specifically binds PD-1, an anti-PD-L1 antibody or antigen-binding fragment thereof that specifically binds PD-L1, or a combination thereof. CTLA4, PD-1 and PD-L1 inhibit immune response of organism by inhibiting activation of T cells, so that tumor cells can escape in immunity. By using the immunodetection point inhibitor, the inhibition on the immune system of the organism is relieved, T cells are activated, and tumor cells are killed.

In a preferred embodiment of the invention, the immunodetection point inhibitor is an anti-PD-1 monoclonal antibody.

PD-1 and PD-L1

PD-1 is a programmed death receptor 1, also known as CD279 (cluster of differentiation 279), an important immunosuppressive molecule. PD-1 modulates the immune system and promotes self-tolerance by down-regulating the immune system's response to human cells, as well as by inhibiting T cell inflammatory activity. PD-1 is an immune checkpoint, preventing autoimmunity by two mechanisms. First, it promotes apoptosis (programmed cell death) of antigen-specific T cells in lymph nodes. Second, it reduces apoptosis of regulatory T cells (anti-inflammatory, suppressor T cells). Binding of PD-1 to its ligand PD-L1 initiates programmed death of T cells, allowing tumor cells to gain immune escape.

In the invention, after the anti-PD-1 monoclonal antibody is injected into a tested mouse, the anti-PD-1 monoclonal antibody is combined with PD-1 on the surface of an immune cell of the mouse, so that the combination of PD-L1 and PD-1 is blocked, an immune system which is originally in an inhibition state is reactivated, and then an in-vivo tumor cell is attacked.

Immunostimulatory factor

In a preferred embodiment of the present invention, the immune-related drug of the present invention comprises an immunostimulant. In the present embodiment, the immunostimulatory factor is granulocyte-macrophage colony stimulating factor (GM-CSF).

In a preferred embodiment, experiments show that the mouse granulocyte-macrophage colony stimulating factor (mGM-CSF) has a synergistic and more significant inhibition effect on mouse solid tumors when used in combination with the oncolytic virus and the anti-PD-1 monoclonal antibody.

Tumor specific antigens

In a preferred embodiment of the present invention, the immune-related drug of the present invention comprises a tumor-specific antigen. The term "tumor-specific antigen" refers to a novel antigen that is expressed only on the surface of a certain tumor cell and not on normal cells, and is also called a unique tumor antigen. Most of them are mutation products of oncogenes or tumor suppressor genes, which are presented on the surface of tumor cells via MHC-I molecules and can be specifically recognized and bound by CD8+ T cells.

The tumor specific antigen used in the invention is adenovirus, adeno-associated virus, DNA or RNA vaccine which is prepared by in vitro recombination technology and carries the antigen gene, induces immune response and realizes the killing of tumor cells.

In a preferred embodiment of the invention, the tumor-specific antigen used is SAGE1 adenovirus (Ad-SAGE 1), and the SAGE1 adenovirus is an adenovirus loaded with the SAGE1 antigen gene prepared by in vitro recombination technology. In the present example, experiments have shown that when SAGE1 adenovirus (Ad-SAGE 1) is used in combination with oncolytic virus, it also has a more systemic, significant inhibitory effect on mouse tumors.

Pharmaceutical composition and mode of administration

The present invention also provides a composition useful for the synergistic treatment of solid tumors, which is useful for inhibiting tumor growth and/or metastasis.

The pharmaceutical composition of the present invention comprises: an effective amount of recombinant human adenovirus type 5, an effective amount of immune-related drugs and a pharmaceutically acceptable carrier.

In general, recombinant human adenovirus type 5, or an immune-related agent, of the invention can be formulated in a non-toxic, inert, and pharmaceutically acceptable carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8.

As used herein, "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

The term refers to such pharmaceutical carriers: they are not essential active ingredients per se and are not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in the composition may comprise liquids such as water, saline, buffers. In addition, auxiliary substances, such as fillers, lubricants, glidants, wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers. The vector may also contain a cell transfection reagent.

As used herein, the term "effective amount" or "effective dose" refers to an amount that produces a function or activity in a human and/or animal and/or a cell and is acceptable to a human and/or animal.

As used herein, a "pharmaceutically acceptable" component is one that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents. Such vectors include (but are not limited to): saline, buffer, dextrose, water, glycerol, polysorbate, ethanol, and combinations thereof. The pharmaceutical preparation is usually adapted to the administration mode, and the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. The pharmaceutical preparation of the invention can also be prepared into a sustained release preparation.

Furthermore, the active ingredient combinations according to the invention can also be used together with other therapeutic agents, such as antineoplastic or chemotherapeutic agents.

When using the pharmaceutical composition, a safe and effective amount of the active ingredient combination, including the first active ingredient (or formulation thereof) and/or the second active ingredient (or formulation thereof), is administered to the mammal.

It will be appreciated that the effective amount of the first active ingredient (or a formulation thereof) and/or the second active ingredient (or a formulation thereof) in the active ingredient combinations of the invention may vary with the mode of administration and the severity of the tumour, etc. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc.; the severity of the tumor, the weight of the patient, the immune status of the patient, the route of administration, etc.

Typically, for the first active ingredient, the therapeutically effective amount will generally be at least 0.5 x10 ¹² VP to 2.5X 10 ¹² VP, of course, the specific dose will also take into account factors such as tumor volume, patient health, etc., which are within the skill of the skilled practitioner.

For the second active ingredient, a typical example is an immune checkpoint inhibitor, which is a safe and effective amount, usually at least about 1mg/kg body weight, and in most cases no more than about 20mg/kg body weight, preferably the dose is from about 2mg/kg body weight to about 10mg/kg body weight. Of course, the particular dosage will also take into account such factors as the type of drug employed, the dosage form, the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

As the second active ingredient, another typical example is an immunostimulating factor, which is generally used in a safe and effective amount of at least about 2 to 10. Mu.g/kg body weight, preferably in a dose of about 3 to 5. Mu.g/kg body weight. Of course, the specific dosage will depend upon such factors as the type of drug employed, the dosage form, the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

Another typical example of a second active ingredient is a tumor-specific antigen, the particular dose of which will depend on the tumor volume employed, the health of the patient, etc., and is within the skill of the skilled practitioner.

The mode of administration of the pharmaceutical composition of the present invention is not particularly limited, and representative examples include (but are not limited to): intratumoral injection, intraperitoneal injection and intravenous injection.

Medicine box

The present invention provides a kit comprising:

a component (1): a formulation comprising a recombinant human adenovirus type 5;

a component (2): formulations containing immune-related drugs;

a component (3): and (6) instructions.

The preparation containing the recombinant human adenovirus type 5 comprises (but is not limited to): lyophilized preparation, liquid preparation or injection.

The preparation of the immune-related medicine comprises (but is not limited to): a lyophilized preparation, a liquid preparation, or an injection.

Typically, the kit contains one or more (e.g., at least two) unit dosage forms comprising recombinant human adenovirus type 5 and one or more (e.g., at least two) unit dosage forms comprising an immune-related drug; preferably 4-10 each.

As used herein, the term "unit dosage form" refers to a composition that is prepared for ease of administration into a dosage form required for a single administration, including, but not limited to, various solid agents (e.g., tablets), liquid agents, capsules, sustained release agents.

In the present invention, the unit dosage form of the recombinant human type 5 adenovirus is usually a liquid agent.

The description provided by the invention can be as follows: the use method of the kit is to use a unit dosage form containing the recombinant human adenovirus type 5 and a unit dosage form containing the immune-related medicine at the same time.

The medicine box provided by the invention is prepared by the following steps: the preparation containing the recombinant human adenovirus type 5 and the preparation containing the immune-related drugs are placed together with the instructions to form a kit.

The preparation containing the recombinant human adenovirus 5 preferably contains a unit dosage form of the recombinant human adenovirus 5, and the preparation containing the immune-related drugs preferably contains a unit dosage form of the immune-related drugs.

Preferably, the steps include placing at least one unit dosage form containing recombinant human adenovirus type 5 and at least one unit dosage form containing an immune-related drug together with instructions to form a kit.

The main advantages of the invention are:

(1) The invention discovers for the first time that the recombinant human adenovirus type 5 combined with immune-related drugs can better inhibit the growth of solid tumors, and provides a pharmaceutical composition for treating the solid tumors by combining oncolytic viruses with the immune-related drugs based on the discovery.

(2) The drug combination provided by the invention does not cause other drug adverse reactions and toxic and side effects under the condition of obviously inhibiting the growth of tumors.

(3) The invention provides a new idea for treating solid tumors by combining oncolytic virus with immune-related medicaments.

(4) The recombinant human adenovirus type 5 has the oncolytic effect on the interior of tumor tissues, can increase lymphocyte infiltration, and can become a novel method for treating solid tumors by jointly administering an immunostimulant, an immune checkpoint inhibitor and/or a tumor specific antigen.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise specified, materials and reagents used in examples of the present invention are commercially available products. Experimental procedures without specific conditions noted in the following examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Experimental materials

The recombinant human adenovirus type 5 injection is the product name An Kerui, can be purchased from Shanghai three-dimensional biotechnology Limited company, and has the specification of 1.87 multiplied by 10 ¹² vp/ml；

anti-mPD-1 (InVivoMAb anti-mouse PD-1) was supplied by BioXcell and was specified at 6.78mg/ml;

mGM-CSF (Recombinant Mouse GM-CSF, recombinant Mouse granulocyte-macrophage colony stimulating factor) was provided by Novoprotein at a specification of 50. Mu.g/count.

Phosphate buffer, fetal bovine serum, DMEM medium were purchased from Gibco company;

trypsin-EDTA digest (0.25%) and penicillin-streptomycin mixture (100 ×) were purchased from Solarbio;

mouse colorectal cancer MC38 tumor cells were purchased from pugliptin biotechnology (beijing) limited (Biovector NTCC inc.).

The experimental animal information is as follows:

species lines: mus Musculus, C57BL/6J

The week age is as follows: 8 weeks

Sex: male sex

Weight: 20-23g

Quantity: 75 are provided with

Experimental animal providers: nanjing university-Nanjing biomedical research institute

Producing license numbers: SCXK (Su) 2015-0001

Quality certification number: 201802616

Experimental animals were housed in an SPF-level constant temperature and humidity laminar flow clean room using an independent ventilation cage IVC, one cage per 5 rats. The temperature/humidity is controlled within the range of (23 + -3) DEG C/40-70%.

Method for testing drug effect in animal body

(1) Cell culture

Mouse colorectal cancer MC38 tumor cells were cultured in a 5% CO2 incubator at 37 ℃ in DMEM medium containing inactivated 10% fetal bovine serum, 100U/ml penicillin and 100. Mu.g/ml streptomycin and 2mM glutamine, and after the cells were overgrown every 3 days or so, they were passaged in flasks, and the tumor cells in the logarithmic growth phase were used for in vivo tumor inoculation.

(2) Tumor cell inoculation and grouping

PBS resuspended MC38 tumor cells at a concentration of 3X 10 ⁷ One/ml of the cells were inoculated subcutaneously into the right flank of the experimental animal at 100. Mu.l/cell, and the mean tumor volume reached 77mm 5 days after inoculation (D5) ³ The left and right administration is divided into groups.

(3) Mouse experiment detection index

a. Tumor volume: tumor volume was measured 3 times a week using a vernier caliper, and the long and short diameters of the tumor were measured, and the volume calculation formula was: volume =0.5 × major diameter × minor diameter ² 。

b. Response after animal dosing: mice were weighed while tumor volume measurements were being made. Changes in mouse body weight were recorded as a function of time of administration. The survival and health of the mice were also observed as a general state of the animals during administration, such as activity, food intake, etc.

c. The life cycle is as follows: record that tumor volume exceeded 3000mm in a single mouse after tumor inoculation ³ Days of the day, defined as its survival time.

d. Tumor weighing and photographing: at the end of the experiment, the tumors were stripped and weighed and photographed.

Statistical analysis

Statistical analysis between groups was performed on tumor volume and survival using One-Way ANOVA test and Log Rank test, and p <0.05 was considered significantly different.

Example 1

3238 Zxft 3238 in combination with anti-mPD-1 and/or mGM-CSF for treatment of MC38 subcutaneous tumor bearing mice

(1) The MC38 subcutaneous tumor-bearing mouse model was established as described above, and the mice were dosed with the placebo controls (vehicle control, an Kerui, anti-mPD-1 (InVivoMAb anti-mouse PD-1), an Kerui + anti-mPD-1, mGM-CSF + An Kerui + anti-mPD-1, respectively, according to the dosing schedule shown in Table 1.

TABLE 1 combination dosing regimen of An Kerui with anti-mPD-1 and/or mGM-CSF

Note: the administration volume of anti-mPD-1 is 10 μ l/g body weight, the administration volume of mGM-CSF is 100 μ l/mouse, and the administration volume of An Kerui is 10 ⁹ vp/mm ³ Adjusted according to the tumor volume range before treatment (see table 2):

TABLE 2 administration volume adjustment Range 2 An Kerui

(2) Test results

The T/C (%) of each treatment group and vehicle control group was calculated (as shown in table 3). At the end of the experiment in the vehicle control group (D20), the tumor volumes of the vehicle control group, an Kerui group, anti-mPD-1 group, an Kerui + anti-mPD-1 group, mGM-CSF + An Kerui + anti-mPD-1 group were 4870mm ³ 、2537mm ³ 、3942mm ³ 、1848mm ³ 、1756mm ³ Compared with the vehicle control group, the tumor volume of each drug treatment group has very significant difference (average p)<0.001 48%, 19%, 62% and 64% of the tumor growth inhibition rate respectively; the An Kerui + anti-mPD-1 group and the mGM-CSF + An Kerui + anti-mPD-1 group have significant differences in mean survival (mean p is compared with An Kerui or with anti-mPD-1 group)<0.05)。

Survival data for each group of mice is shown in table 4, and survival curves are shown in fig. 2. The mean survival periods of the vehicle control group, the An Kerui group, the anti-mPD-1 group, the An Kerui + anti-mPD-1 group, the mGM-CSF + An Kerui + anti-mPD-1 group are respectively 18 days, 21.5 days, 20 days, 25 days and 25 days, the mean survival periods of the drug treatment groups are respectively very significant different (p is less than 0.01) compared with the vehicle control group, and the mean survival periods of the An Kerui + anti-mPD-1 group and the mGM-CSF + An Kerui + anti-mPD-1 group are respectively significant different (p is less than 0.05) compared with the An Kerui group or the anti-mPD-1 group.

TABLE 3 tumor suppression of mouse colorectal carcinoma MC38 by test substances (tumor volume)

Note: * The experiment was terminated at D20 in the vehicle control group, the remaining groups were observed, and the tumor volume data after treatment were compared by taking the measurement at D20.

a. Mean ± standard error;

b. p <0.05 compared to An Kerui or to anti-mPD-1.

TABLE 4 tumor-inhibiting action of test substances on mouse colorectal carcinoma MC38 (survival time)

Group of	Treatment of	Animal number (only)	Mean time to live (day)	Pb
					1	Solvent	12	18	--
2	An Kerui	12	21.5	--
					3	anti-mPD-1	12	20	--
4	An Kerui + anti-mPD-1	10	25	＜0.05
					5	mGM-CSF + An Kerui + anti-mPD-1	10	25	＜0.05

Note: the tumor volume of a single mouse exceeds 3000mm ³ The end of life cycle is reached;

b. p <0.05 compared to An Kerui or to anti-mPD-1.

The results show that:

(i) An Kerui shows antitumor effect on mouse colorectal cancer MC38 subcutaneous tumor model by using alone or combining with anti-mPD-1, mGM-CSF + anti-mPD-1;

(ii) 5363 the combination of An Kerui + anti-mPD-1, mGM-CSF + An Kerui + anti-mPD-1 is effective in inhibiting MC38 tumor growth and significantly better than either anti-mPD-1 or An Kerui alone.

(iii) mGM-CSF + An Kerui + anti-mPD-1 combination also has better tumor growth inhibition than An Kerui + anti-mPD-1 combination.

(iv) The combined administration of An Kerui + anti-mPD-1, mGM-CSF + An Kerui + anti-mPD-1 significantly prolongs the survival of mice as compared to either anti-mPD-1 or An Kerui alone.

Example 2

An Kerui in combination with SAGE 1-loaded adenovirus (Ad-SAGE 1)

(1) An MC38 subcutaneous tumor-bearing mouse model was established using the method described above, and mice were administered according to the dosing schedule shown in table 5.

TABLE 5 Co-dosing regimen of An Kerui with SAGE1 loaded adenovirus (Ad-SAGE 1)

(2) Test results

The T/C (%) of each treatment group and vehicle control group was calculated (as shown in Table 6). The result shows that the tumor growth inhibition rate of An Kerui and Ad-SAGE1 for the colorectal cancer of the mouse is improved by about 20 percent compared with that of An Kerui or Ad-SAGE1 which is singly used, the specific and obvious synergistic effect is achieved, and no additional toxic or side effect is generated. It is demonstrated that An Kerui in combination with tumor specific antigen can enhance An Kerui tumor treatment effect.

TABLE 6 tumor suppression of mouse colorectal carcinoma MC38 by test substances (tumor volume)

Note: * The vehicle control group was terminated at D20, the rest groups were observed, and the tumor volume data after treatment were compared with the D20 measurements.

a. Mean ± standard error; b. p <0.05 compared to group An Kerui or to the Ad-SAGE1 group.

In conclusion, animal experiment results show that the combined administration of the recombinant human adenovirus type 5, the immunostimulating factor, the immune checkpoint inhibitor and the tumor specific antigen has a remarkably better inhibition effect on tumor growth than that of the recombinant human adenovirus type 5 or the immunostimulating factor, the immune checkpoint inhibitor and the tumor specific antigen which are singly used.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. Use of an active ingredient combination comprising a first active ingredient an oncolytic virus and a second active ingredient an immune-related drug, for the manufacture of a pharmaceutical composition or kit for the synergistic treatment of solid tumors.

2. The use according to claim 1, wherein the oncolytic virus is a recombinant human adenovirus type 5.

3. The use according to claim 1, wherein the E1B-55kDa and E3 region gene segments of the human adenovirus type 5 are deleted from the genome of the oncolytic virus.

4. The use of claim 1, wherein the immune-related drug is selected from the group consisting of: an immune stimulating factor, an immune checkpoint inhibitor, a tumor specific antigen, or a combination thereof.

5. The use of claim 4, wherein said immune checkpoint inhibitor is selected from the group consisting of: an anti-CTLA 4 antibody or antigen-binding fragment thereof that specifically binds CTLA4, an anti-PD 1 antibody or antigen-binding fragment thereof that specifically binds PD1, an anti-PD-L1 antibody or antigen-binding fragment thereof that specifically binds PD-L1, or a combination thereof.

6. The use according to claim 4, wherein the tumor specific antigen is selected from the group consisting of: SAGE1, NY-ESO-1, gp33, gp100, TX103, mucin-1, WT-1, MART-1, MAGE A3, MAGE A4, MAGE B2, PRAME, SURVIVIN, MART-1, col6A3, tyrosinase, T antigen, SLC45A2, VCX/Y, HPV, alpha fetoprotein, carcinoembryonic antigen, CA 125, her2, dopachrome tautomerase, BAGE protein, GAGE protein, SURVIVIN, tyrosinase, SSX2, cyclin-A1, KIF20A, MUC AC, meloe, lengsin, kallikrein 4, IGF2B3, glypican, or a combination thereof.

7. The use according to claim 1, wherein the solid tumor is selected from the group consisting of: nasopharyngeal carcinoma, chondrosarcoma, colon cancer, dukes's D or non-small cell lung cancer, breast cancer, prostate cancer, ovarian cancer, malignant liver cancer, esophageal cancer, small cell lung cancer, colorectal cancer, gastric cancer, cervical cancer, melanoma, or a combination thereof.

8. An active ingredient combination, characterized in that said combination consists of an oncolytic virus and an immune-related drug.

9. A pharmaceutical composition comprising (a) an oncolytic virus, (b) an immune-related drug, and (c) a pharmaceutically acceptable carrier.

10. A kit, comprising: