CN116271008A

CN116271008A - Pharmaceutical composition containing An Kerui and Carilizumab and application thereof

Info

Publication number: CN116271008A
Application number: CN202211734363.9A
Authority: CN
Inventors: 王�华; 李珂; 劳家颂; 宋建东; 琚姝; 王玮; 赵亚龙
Original assignee: Guangdong Techpool Bio Pharma Co Ltd; Shanghai Sunway Biotech Co Ltd
Current assignee: Guangdong Techpool Bio Pharma Co Ltd; Shanghai Sunway Biotech Co Ltd
Priority date: 2022-12-30
Filing date: 2022-12-30
Publication date: 2023-06-23

Abstract

The invention belongs to the field of tumor immunotherapy, and particularly relates to a pharmaceutical composition containing An Kerui and Carilizumab and application thereof. The invention provides a pharmaceutical composition containing An Kerui and Carlizumab and application thereof, wherein the pharmaceutical composition comprises An Kerui and Carlizumab Li Zhushan. Compared with single medicine, the An Kerui combined with the Carelizumab can synergistically inhibit the growth of tumors, prolong the survival time of tumor mice, and can generate more remarkable anti-tumor effect than any medicine alone. In addition, the invention further verifies the mechanism of An Kerui mediated anti-tumor immune response through experiments, provides a basis for An Kerui combined with the Carilizumab for treating tumors, has important clinical significance, and lays a foundation for further developing clinical experiments.

Description

Pharmaceutical composition containing An Kerui and Carilizumab and application thereof

Technical Field

The invention belongs to the field of tumor immunotherapy, and particularly relates to a pharmaceutical composition containing An Kerui and Carilizumab and application thereof.

Background

Malignant tumors have an increasing incidence year by year, mostly progressive or metastatic malignant tumors. Traditional malignant tumor treatment methods comprise operations, radiotherapy and chemotherapy, molecular targeted drugs and the like, but still cannot obtain better treatment effects. In recent years, immune checkpoint inhibitors have been a new method for treating malignant tumors due to their durable anti-tumor immune response, but only less than 30% of patients have their therapeutic effects, and most patients do not respond to immune checkpoint inhibitors.

The existing research shows that the high expression of PD-L1 by tumor and the number of CD8+ T cells infiltrating to the tumor are closely related to the response rate of immune checkpoint inhibitor, so that increasing the expression of PD-L1 by tumor tissue and attracting CD8+ T cells to the tumor tissue are an important strategy for improving the curative effect of immune checkpoint inhibitor. The prior researches show that oncolytic virus infection of tumors can induce the expression of tumor tissues PD-L1 and recruit CD8+ T cells to reach the tumor tissues, and the principle is mainly that the tumor cells are infected and destroyed, the whole-body anti-tumor immunity is stimulated by releasing injury related molecular patterns (DAMPs), pathogen related molecular pattern molecules (PAMPs) and cytokines, and CD4+ and CD8+ cells are recruited to destroy the tumors. Thus, by utilizing this property of oncolytic viruses, in combination with immune checkpoint inhibitors, another strategy is aimed at improving the efficacy of tumor therapy.

Carilizumab (trade name: ai Ruika Camrelizumab for Injection), an inhibitor of immunological tests, or called PD-1 inhibitor, is a human immunoglobulin G4 (IgG 4) monoclonal antibody (HuMAb) that binds to the PD-1 receptor, blocks its interaction with PD-L1 and PD-L2, and further blocks the immunosuppressive response mediated by the PD-1 pathway. An Kerui, also called H101, is an oncolytic virus with deletion of E1B55KD and E3 region gene fragments, and is also the first adenovirus approved by the national food and drug administration in 2005 for treating head and neck cancer. In the prior study, a study for treating tumors by combining the two is not found, and no report that the two have synergistic treatment on cancers exists.

In the prior art, adenovirus ONYX-015 with E1B55KD deletion is injected by intratumoral injection to treat pancreatic cancer, and the results of phase I/II clinical test show that the adenovirus ONYX-015 has the characteristics of high safety and weak anti-tumor effect. Thus, although many clinical studies have shown that oncolytic viruses have strong anti-tumor effects over the past several decades, the results of their clinical trials are unsatisfactory, while immune checkpoint inhibitors have a low therapeutic response rate. Therefore, aiming at the defect of cancer treatment by using oncolytic viruses and immune checkpoint inhibitors, the method provides a combination of advantages and disadvantages, so as to achieve the aim of improving the curative effect of treating tumors, and is an important direction of tumor treatment research and development.

Disclosure of Invention

Aiming at the problem that advanced or metastatic cancers can not be cured and the problem that An Kerui infection efficiency and oncolytic effect are poor by utilizing An Kerui in combination with the traditional treatment method, the invention provides a new scheme, and the treatment effect of the Caririnotecan Li Zhushan on cancers is obviously enhanced by the combined application of An Kerui and Caririnotecan.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

it is a first object of the present invention to provide a pharmaceutical composition for the treatment of tumors comprising An Kerui and kari Li Zhushan antibodies.

Preferably, the pharmaceutical composition is in the form of an injection.

Preferably, the tumor is a tumor that is insensitive to An Kerui.

Preferably, the tumor is a solid tumor.

Preferably, the solid tumor is selected from lung cancer, breast cancer, colon cancer, ovarian cancer, pancreatic cancer, colorectal cancer, bladder cancer, prostate cancer, cervical cancer, renal cancer, and melanoma.

Preferably, the treatment of the tumor selected from surgery, chemotherapy, radiation therapy, or a combination thereof is followed by recurrent or progressive.

Preferably, the mode of administration of the pharmaceutical composition comprises simultaneous or sequential administration of the An Kerui and kari Li Zhushan antibodies to a subject.

Preferably, the An Kerui is administered at a dose of 8×10 ⁴ pfu/mm ³ Once every 2 days; the administration dosage of the carlizumab is 0.16 mug/mm ³ Once every 4 days.

A second object of the present invention is to provide the use of a pharmaceutical composition as described above for the manufacture of a medicament for treating cancer in a subject.

The invention adopts An Kerui and Carelizumab for combined application, and the design principle and conception are as follows:

firstly, the anti-tumor effect of the combination of An Kerui and the Carilizumab on cancer is researched by adopting a humanized immune system mouse tumor model, and the result shows that the An Kerui and the Carilizumab can generate more remarkable anti-tumor effect compared with the single use of any one of the medicines.

Second, to further investigate the mechanism by which An Kerui mediates anti-tumor immune responses, the present invention examined immune-related markers in An Kerui-infected tumor cells, found that virus infection of cancer cells resulted in down-regulation of CD47 in cancer cells and promoted phagocytosis of YTS-1 cells by induced THP-1 cells. Thus, an Kerui mediates the mechanism of anti-tumor immune response by inhibiting CD47 on the surface of tumor cells, thereby promoting phagocytosis of virus-infected tumor cells by macrophages.

Then, to further investigate the effect of down-regulation of CD47 and activation of macrophage phagocytosis on T cell viability, the present invention further demonstrated by experiments that An Kerui activated phagocytosis of macrophages and further promoted activation of cd8+ T cells.

Finally, since macrophage-derived IL-12 plays an important role in the process of inducing T cells to secrete IFN-gamma, the invention proves that the macrophage-derived IL-12 induces the T cells to secrete IFN-gamma by removing macrophage-derived IL-12 through experiments by adopting an anti-IL-12 neutralizing antibody, and the result shows that the T cells can not secrete IFN-gamma any more.

Taken together, the above experimental results demonstrate that An Kerui enhances the therapeutic effect of kari Li Zhushan against cancer by inhibiting the signaling pathway of CD47 in infected cells, that is, the combined use of An Kerui and karilizumab significantly enhances the therapeutic effect against bladder cancer. The invention provides a basis for An Kerui combined with the Carelizumab to treat tumors, has important clinical significance, and lays a foundation for further developing clinical experiments.

Compared with the prior art, the invention has the following beneficial effects:

according to the invention, an animal model (a mouse subcutaneous tumor model) is utilized to research the anti-tumor effect of the combined use of An Kerui and the Carilizumab on cancers, and experimental results show that compared with single drugs, the combined use of An Kerui and the Carilizumab can synergistically inhibit tumor growth, prolong the survival time of tumor mice, and the combined treatment of An Kerui and the Carilizumab can generate more remarkable anti-tumor effect than the single use of any one drug.

In addition, the invention further verifies the mechanism of An Kerui mediated anti-tumor immune response through experiments, provides a basis for An Kerui combined immune checkpoint inhibitor treatment of tumors, has important clinical significance, and lays a foundation for further developing clinical experiments.

Drawings

FIG. 1 is a graph of An Kerui single drug mediated antitumor effect, wherein FIG. 1a is a graph of the result of the expression of a CAR of a western blotting detection cell strain, FIG. 1b is a graph of the result of apoptosis analysis detection An Kerui induced apoptosis, FIG. 1c is a graph of the result of a tumor growth experiment using a An Kerui intratumoral injection mouse subcutaneous tumor model, and FIG. 1d is a graph of the result of a survival time experiment using a An Kerui intratumoral injection mouse subcutaneous tumor model;

FIG. 2 is a graph showing the anti-tumor effect mediated by An Kerui and PD-1 inhibitors, wherein FIG. 2a is a graph showing the result of An Kerui and Caririnotecan inhibitor treatment on mice subcutaneous tumor growth, FIG. 2b is a graph showing the result of An Kerui and Caririnotecan inhibitor treatment on mice subcutaneous tumor survival time, and FIG. 2c is a graph showing the result of immunohistochemical detection on tumor tissue CD8+ T cells;

FIG. 3 is a graph showing experimental results of activating macrophages after adenovirus infection of tumor cells, wherein FIG. 3a is a graph showing the change of immune factor mRNA after infection of tumor cells by An Kerui, FIG. 3b is a graph showing the result of flow detection of CD47 expression in An Kerui infected cells, and FIG. 3c is a graph showing the result of flow detection of the number of phagocytized tumor cells by macrophages;

FIG. 4 is a graph showing experimental results of further activating T cells after An Kerui activating macrophages, wherein FIG. 4a is a graph showing experimental results of detecting cytokines TNF, IL-12 and IFN-gamma after co-culturing THP-1 and An Kerui infected tumor cells by ELISA method, FIG. 4b is a graph showing experimental results of detecting IFN-gamma expression (YTS-1 cell line) of CD8+ T cells after stimulation of co-culture supernatant, FIG. 4c is a graph showing experimental results of detecting TNF, IL-12 and IFN-gamma expression of tumor tissue by ELISA method, and FIG. 4d is a graph showing experimental results of detecting IFN-gamma expression of YTS-1 tumor tissue by immunohistochemistry;

FIG. 5 is a graph showing the experimental results of IL-12 in promoting IFN-gamma secretion by macrophages, wherein FIG. 5a is a graph showing the experimental results of flow detection of CD8+ IFN-gamma+ T cells, and FIG. 5b is a graph showing the expression results of PD-L1 in tumor cells infected with a virus.

Detailed Description

The above-described aspects of the present invention will be described in further detail with reference to the following embodiments. It should not be construed that the scope of the above subject matter of the present invention is limited to the following examples.

1. Cell strain for experiment

Human myometrial invasive bladder cancer cell line T24, expressing CAR, purchased from Shanghai cell bank; human glioblastoma U-87MG, high expressing CAR, purchased from Shanghai cell bank; the human myometrial invasive bladder cancer cell line YTS-1, which does not express CAR, is provided by university of North east Japan. Wherein, YTS-1 has p53 mutation and p16 methylation; t24 has a p53 mutation. YTS-1 low expressed adenovirus receptor (CAR), T24 cells medium expressed CAR, U-87MG high expressed CAR, for control.

Wherein T24 and YTS-1 cells were cultured in RPMI-1640 medium supplemented with 10% (vol/vol) Fetal Bovine Serum (FBS) and 1% antibiotics (100U/mL penicillin, 100. Mu.g/mL streptomycin sulfate); U87-MG cells were cultured in DMEM supplemented with 10% Fetal Bovine Serum (FBS) and 1% antibiotics; THP-1 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 1% antibiotics and beta-mercaptoethanol (0.05 mM).

All cells were placed at 37℃plus 5% (vol/vol) CO ₂ Is contained in a humidified incubator.

2. Oncolytic adenoviruses for experiments

An Kerui: an Kerui, also known as H101, is an oncolytic adenovirus deleted for E1B55KD and E3. Viruses were amplified and titered prior to the experiment as follows: 293 cells are cultured in a 10cm culture dish, adenovirus is added when the culture is carried out to a proper density, after the cells are infected for 3 to 5 days until floating, the cells are collected and are cracked by a freeze thawing method, the cells are centrifuged at a high speed, and the supernatant, namely virus liquid, is collected and stored at the temperature of minus 80 ℃. Viral titers were determined using 293T cells.

3. Experimental animal

NSG mice (female, 6-8 weeks old) were purchased from the collection of extraction medicine kang (Jiangsu, china), and the mice were kept in SPF environment, and the experimental protocol was approved by the institutional animal care and use committee of the university of Zhejiang, medical college.

4. Data analysis

The experimental results of the invention are all analyzed by SPSS software, the results are represented by mean value + -standard error, the statistical analysis is carried out by unpaired t test, and P <0.05 represents statistically significant difference.

Example 1 An Kerui Single drug-mediated antitumor Effect

1.1 experiment of the Effect of different cells on An Kerui-mediated apoptosis and oncolytic Effect

In order to determine whether the susceptibility of different cells to viruses would affect the toxic effect and oncolytic effect of An Kerui on cells and further affect the anti-tumor immune effect, the invention uses cell lines expressing different levels to verify An Kerui-mediated apoptosis and oncolytic effects.

The expression of cell lines CAR at different levels was detected by western blotting, and the experimental procedure was as follows: cells were collected and washed 2 times with PBS. Cells were then lysed on ice for 30 min with RIPA lysis buffer (bi yun day, P0013B) added with 1mM PMSF. Cell lysates were separated by SDS-PAGE (12%) and transferred to PVDF membrane (Millipore, ISEQ 00010). Blocking with 5% BSA and incubation with anti-CAR (Proteintech, 11777-1-AP) or anti-GAPDH (Abways Technology, AB 0036) antibodies was carried out overnight at 4 ℃. The next day, membranes were rinsed 3 times with PBST and incubated with goat anti-rabbit IgG (h+l) HRP antibody (co-product, GAR 007) for 1H at room temperature. UltraSignal ECL Western Blotting Detection Reagent (Sizhengbai, 4AW 011-1000) was applied to the film, and the film was then scanned using a Tanon 4500 gel imaging system (tan 4500, tianenergy Co.).

Apoptosis induction by apoptosis assay detection An Kerui: apoptosis analysis was performed by Annexin V/PI double staining, and cultured cells were collected, washed, centrifuged, resuspended, incubated at 4 ℃ for 20min with fluorescent (SA-flow) solution, protected from light and sometimes vibrated. Apoptotic cells were detected by flow cytometry, specifically, U87-MG, YTS-1 or T24 cells were collected, washed with PBS, and detected according to instructions using an Annexin V-APC/PI apoptosis kit (Union, AP 107).

As shown in fig. 1a and 1b, as can be seen from fig. 1a, apoptosis of the cell line U87-MG with high CAR expression is remarkable after being infected by An Kerui, and the apoptosis proportion is closely related to the virus titer; from FIG. 1b, it can be seen that there is no apparent apoptosis in the YTS-1 cell line that underexpresses the CAR.

1.2 experiment of the anti-tumor Effect of An Kerui on subcutaneous tumors

To further determine the therapeutic effect of Ke Rui on the mouse subcutaneous tumor model, the invention adopts the mouse subcutaneous tumor model, and human Peripheral Blood Mononuclear Cells (PBMC) are injected intratumorally after the mouse subcutaneous tumor is formed, and An Kerui is injected intratumorally. The tumor volume of the mice was measured and the survival rate of the mice was observed to evaluate the effect of the treatment. Histopathological examination and immune related indices were taken.

1.2.1 construction of humanized mouse tumor model

Single NSG mice were subcutaneously injected 3X 10 ⁶ U87-MG，5×10 ⁶ T24 or YTS-1 cells. When the tumor volume reaches 100mm ³ At this time, an Kerui (2.5X10 per tumor) was injected intratumorally every 2 days ⁷ pfu). When tumor volume reaches 300mm in combination therapy ³ At this time, an Kerui (2.5X10 per tumor) was injected intratumorally every 2 days ⁷ pfu) and/or 50 μg Camrelizumab treatment every 4 days. Intratumoral injection of 1X 10 in NSG mice ⁶ PBMCs were isolated from healthy donor peripheral blood. Wherein the PBMCs are isolated according to the specification using lymphocyte separation medium (TBDsciences, LTS 1077). Tumor size was measured every 2 or 3 days with vernier calipers and tumor volume was calculated as follows: volume = maximum diameter x minimum diameter ² ×0.5。

Extraction of 1.2.2PBMC

And (5) drawing blood of a healthy person, and then adding physiological saline to dilute for later use. The centrifuge tube was added to lymphocyte separation liquid (LTS 1077, tianjin), and the diluted blood was added to the lymphocyte separation liquid and centrifuged. Sucking out PBMC, centrifuging, adding T cell culture solution, re-centrifuging, re-suspending with T cell culture solution, adding CD3 antibody, incubating in 6-well plate for 72 hr

1.2.3 histopathological and immune related index detection

Tumor tissues are taken, and the degree of CD8+T infiltration is detected by immunofluorescence. ELISA (BioLegend) the relevant cytokines TNF- α (430204), IL-12 (431704) and IFN- γ (430104) were detected in each group of Tumor lysates (Tumor Lysate).

The immunofluorescence detection process is as follows: tumor tissue was isolated on day 7 post-treatment. Placing the Tissue into Tissue-Tek

O.c.t. compounds. Then, frozen sections of 10 μm thickness were cut using CryoStar NX50 (Thermo Fisher). The sections were then fixed with pre-chilled methanol at-20℃for 10 minutes, 0.1% Triton-X-100 infiltration. After blocking with 5% bsa and 3% goat serum PBS. anti-CD 8 (proteontech, 60181-1-Ig) and anti-IFN-gamma (proteontech, 15365-1-AP) were incubated overnight at 4 ℃. The next day, cells were washed 3 times with PBS, and with IFLUOR ^TM 488- (Huaan, HA 1211) and IFLUOR ^TM 549- (Huaan, HA 1126) labeled secondary antibody, incubated for 30 min at room temperature. After 3 washes in PBS, nuclei were stained with DAPI (Invitrogen, D3571). Fluorescence signals were detected with an Olympus IX83FV3000 confocal microscope.

1.2.4 experimental results

As shown in fig. 1c and 1d, as can be seen from fig. 1c and 1d, the subcutaneous tumor model established by the U87-MG and T24 cells of the medium and high expression CAR shows a significant tumor growth inhibition effect after intratumoral injection An Kerui treatment, and the survival time of mice is significantly prolonged; whereas the subcutaneous tumor model established by the YTS-1 cell line with low expression of CAR had only a slight tumor growth inhibition effect after intratumoral injection An Kerui treatment.

1.3 analysis of results

From the above experimental results, it was found that the apoptosis and oncolytic effects caused by An Kerui are closely related to the expression of CAR.

Example 2 An Kerui anti-tumor Effect mediated by Carelizumab

Establishes a humanized mouse subcutaneous tumor model of an immune system, and adopts intratumoral injection An Kerui or intraperitoneal injection of the Carilizumab for treatment. The An Kerui combined karellizumab-mediated anti-tumor effect was detected and evaluated by the method described in example 1, and tumor tissue cd8+ T cells were detected by immunohistochemistry.

As shown in fig. 2, as can be seen from fig. 2a, an Kerui alone significantly inhibited the subcutaneous tumor model established by the CAR positive cell line, while no significant inhibition effect was observed on the subcutaneous tumor model established by the CAR negative cell line YTS-1; carRui Li Zhushan alone also inhibited tumor growth to varying degrees; the subcutaneous tumor established by the two-drug combined treatment group on the CAR positive cell line or the CAR negative cell line shows more remarkable tumor growth inhibition effect than that of the single-drug group; from this, an Kerui combined with the carlizumab treatment significantly inhibited the growth of subcutaneous tumors in mice. As can be seen from fig. 2b, an Kerui combined with the carlizumab significantly prolonged survival time of tumor-bearing mice. As can be seen from fig. 2c, immunohistochemical detection of cd8+ T cells of tumor tissue revealed that the number of cd8+ T cells expressing IFN- γ infiltrated into tumor tissue in the two-drug combination treatment group was significantly increased, significantly higher than in the single-drug group, confirming that the combination treatment enhanced the anti-tumor response of the cd8+ T cells expressing IFN- γ. Thus, as shown in the experimental results of fig. 2, the An Kerui combined use of the carlizumab can synergistically inhibit the tumor growth and prolong the survival time of tumor-bearing mice compared with single drug, and the immunohistochemical experimental results prove that the combined treatment enhances the anti-tumor response of the IFN-gamma expressing CD8+ T cells.

Existing studies indicate that tumor-infiltrating T cells are associated with the response rate to anti-PD-1 therapy, and patients who do not respond to PD-1 blocking therapy are more likely to lack cd8+ T cells within the tumor lesion. The anti-tumor effect of PD-1 blocking treatment is further improved by the Li Yongan Ke Rui strategy of recruiting CD8+ T cells into tumors through the combined application of An Kerui and the Carilizumab, and the method is a promising strategy of cancer immunotherapy.

Example 3 An Kerui mechanism of combining Carelizumab to mediate anti-tumor immune response

From the experimental results An Kerui of example 2, which induced up-regulation of PD-L1 in Tumor Microenvironment (TME), combined anti-PD-L1 treatment resulted in synergy, thus obtaining better therapeutic effects, it could be initially inferred that An Kerui may mediate anti-tumor immune response by: an Kerui recruiting CD8+ T cells into the Tumor Microenvironment (TME) following infection of the tumor, PD-1 blockade further activates CD8+ T cells to attack the tumor.

To further investigate and verify the mechanism by which An Kerui mediates anti-tumor immune responses, the present invention detected immune-related markers in An Kerui infected tumor cells.

3.1 An Kerui changes in immune factor mRNA after infection of tumor cells

Total RNA was extracted from tumor cells using RNAiso plus (TAKARA, 9109) and expressed in HiScript

II Q RT Supermix (Norpran, R223-01) reverse transcribes 600ng RNA into cDNA. Quantitative real-time PCR was performed using ChamQ Universal SYBR qPCR Master Mix (Northenzan, Q711-02) using the CFX96PCR detection system (Bio-Rad), wherein the PCR was performed using the following thermal cycling conditions: 95 ℃,90s,1 cycle, followed by 95 ℃,5s,40 cycles, 60 ℃,34s,40 cycles, the reaction system is shown in table 1 below.

TABLE 1 quantitative real-time PCR reaction System

As shown in FIG. 3a, it is clear from FIG. 3a that An Kerui infection of tumor cells resulted in a significant decrease in CD47mRNA expression of tumor cells.

3.2 flow assay for CD47 expression in An Kerui infected cells

Cells were collected and washed 3 times with PBS. After staining with the surface marker antibody, the cells were permeabilized with IC fixation buffer (Thermo Fisher Scientific) and the intracellular cytokines were detected by staining. Flow cytometry was performed using a CytoFlex flow cytometer (Beckman Coulter, break, CA, USA), and data analysis was performed using FlowJo software (TreeStar Ashland, OR, USA). The following antibodies were used in staining cells: fixable Viable Dye eFluorTM 520 (Invitrogen, 65-0867-14), anti-human PE-CD8a (BioLegend, 301007) and anti-human APC-IFN-gamma (BioLegend, 502511), anti-human APC-CD45 ((BioLegend, 982304).

As shown in fig. 3b, as can be seen from fig. 3b, an Kerui infection of tumor cells resulted in a decrease in CD47 protein levels in tumor cells.

3.3 flow detection of the number of phagocytic tumor cells by macrophages

Since CD47 plays an important role in inhibiting macrophages from phagocytosing tumor cells, tumor-expressing CD47 is not phagocytosed by macrophages, resulting in immune escape. It was demonstrated whether virus-mediated down-regulation of CD47 would promote phagocytosis, and the invention detects changes in cytokines TNF, IL-12, IFN-gamma by inducing differentiation of THP-1 cells into M0 macrophages, co-culturing An Kerui-infected tumor cells with the induced THP-1 cells.

Stimulation of THP-1 with 50ng/mL PMA for 12 hours induced M0 macrophages. According to the instructions, the induced levels of cytokines in THP-1 and tumor tissues were analyzed using ELISA kit. ELISA kits for human IFN-. Gamma. 430104, IL-12 431704 and TNF-. Alpha. 430204 were all purchased from BioLegend.

Tumor cells were stimulated with An Kerui (moi=1.5) for 24 hours before tumor cells were collected. According to the instructions, cells were stained with CFSE (Invitrogen, C34570). CFSE-labeled cells were seeded into 6-well plates and co-cultured with induced THP-1 cells at a ratio of 3:1. After 8 hours, the induced THP-1 cells were collected and detected by a CytoFlex flow cytometer (Beckman Coulter, brea, calif., USA).

As shown in fig. 3c, it can be seen from fig. 3c that An Kerui infection resulted in down-regulation of bladder cancer cells CD47 and promoted phagocytosis of YTS-1 cells by induced THP-1 cells, and that infection of An Kerui significantly enhanced phagocytosis of tumor cells by phagocytes.

3.4 analysis of results

From the above experimental results, it can be deduced that An Kerui could potentially cause down-regulation of CD47 expression by infected tumor cells, thereby enhancing phagocytosis of virus-infected tumor cells by macrophages.

Example 4 Effect of macrophage activation on T cell viability

4.1 ELISA method was used to detect the expression of cytokines TNF, IL-12 and IFN-gamma after co-cultivation of THP-1 with An Kerui infected tumor cells. As shown in FIG. 4a, the co-culture of THP-1 cells with An Kerui-infected bladder cancer cells resulted in increased expression of cytokines TNF, IL-12 and IFN-gamma.

4.2 addition of macrophage supernatant to CD8+ T cells, co-culture of THP-1 cells with An Kerui infected cancer cells supernatant stimulation followed by flow detection of CD8+ T cell IFN- γ expression (YTS-1 cell line). The experimental results are shown in FIG. 4b, where IFN-y expression is elevated.

4.3 in the tumor tissue treated by the combination of An Kerui and Carilizumab, detecting the expression of TNF, IL-12 and IFN-gamma in the tumor tissue by ELISA method; immunohistochemical detection of IFN-gamma expression in YTS-1 tumor tissue. The experimental results are shown in FIGS. 4c and 4d, where the levels of TNF, IL-12 and IFN-gamma were increased in tumor tissue treated with the combination of An Kerui and Carilizumab.

From the above experimental results, an Kerui activated phagocytosis of macrophages and further promoted activation of cd8+ T cells. I.e., an Kerui activates macrophages, further activating T cells.

Example 5 further validation of the mechanism by which the combination of An Kerui and carlizumab mediates an anti-tumor immune response

Since IL-12 plays an important role in inducing T cells to secrete IFN-gamma, in order to verify the role of IL-12 in the secretion of IFN-gamma by T cells, the present invention treats the supernatant of the co-culture with an anti-IL-12 neutralizing antibody to eliminate IL-12, and detects CD8+ IFN-gamma+ T cells by flow. The experimental results are shown in FIG. 5a, and the experimental results show that the T cells do not produce IFN-gamma any more, which indicates that the virus infection promotes macrophages to phagocytose tumor cells and produce IL-12 so as to induce the T cells to secrete IFN-gamma.

Additional studies have shown that IFN-gamma can induce up-regulation of tumor PD-L1, thereby affecting T cell PD-1 signaling pathways. The invention detects the PD-L1 expression of tumor cells infected by virus through a flow, and the experimental result is shown in figure 5b, and An Kerui induces the PD-L1 expression to be increased after the YTS-1 cells are infected, so that the PD-1 signal path of T cells is activated, and the anti-tumor immune response is enhanced.

From the above, it is known from the experimental results of the present invention that An Kerui H101 in combination with garelizumab Camrelizumab for Injection showed stronger anti-tumor effect than single drug treatment in the mouse subcutaneous tumor model. By analyzing the treated tumors, it was found that tumor infiltrating T cells, especially cd8+ T cells expressing IFN- γ, were increased in the combination treatment group; in addition, cytokine expression, including TNF, IL-12 and IFN-gamma, is increased in H101-treated or combination-treated tumor tissue. These results indicate that An Kerui H101 indirectly activates macrophages and then induces T cell activation, supporting combination therapy with a kari Li Zhushan anti-block.

In combination with An Kerui mediated anti-tumor immune response mechanism, an Kerui and Carelimumab are combined for application, an Kerui can further enhance the therapeutic effect of Carelin Li Zhushan on bladder cancer by inhibiting the signal path of infected cells CD47, so that the invention provides basis for treating tumors by combining An Kerui with immune checkpoint inhibitors, has important clinical significance, and lays a foundation for further developing clinical experiments.

The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.

Claims

1. A pharmaceutical composition for treating a tumor, comprising An Kerui and kari Li Zhushan antibodies.

2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is in the form of an injection.

3. The pharmaceutical composition of claim 1, wherein the tumor is a tumor that is insensitive to An Kerui.

4. The pharmaceutical composition of claim 1, wherein the tumor is a solid tumor.

5. The pharmaceutical composition of claim 5, wherein the solid tumor is selected from the group consisting of lung cancer, breast cancer, colon cancer, ovarian cancer, pancreatic cancer, colorectal cancer, bladder cancer, prostate cancer, cervical cancer, renal cancer, and melanoma.

6. The pharmaceutical composition of claim 1, wherein the treatment of the tumor selected from surgery, chemotherapy, radiation therapy, or a combination thereof is followed by recurrent or progressive.

7. The pharmaceutical composition of claim 1, wherein the mode of administration of the pharmaceutical composition comprises simultaneous or sequential administration of the An Kerui and kari Li Zhushan antibodies to a subject.

8. The pharmaceutical composition of claim 1, wherein the An Kerui is administered at a dose of 8 x 10 ⁴ pfu/mm ³ Once every 2 days; the administration dosage of the carlizumab is 0.16 mug/mm ³ Once every 4 days.

9. Use of the pharmaceutical composition of claim 1 in the manufacture of a medicament for treating cancer in a subject.