CN116139268A - Use of antibody targeting IL-1β in the preparation and treatment of castration-resistant prostate cancer - Google Patents

Abstract

The invention provides an application of an antibody targeting IL-1 beta in preparing a drug for treating castration resistant prostate cancer. The invention also provides application of IL-1 beta serving as a target spot in screening medicaments for treating castration resistant prostate cancer. The invention discovers that the anti-IL-1 beta antibody can specifically inhibit the castration resistant tumor model Pten ^Δ/Δ ；Trp53 ^Δ/Δ Growth of cells in vivo. The combination of the antibody specifically targeting IL-1 beta and the anti-PD-1 antibody has better therapeutic effect. These findings provide new ideas and advances in clinically molecularly targeted therapies.

Description

Use of antibody targeting IL-1β in the preparation and treatment of castration-resistant prostate cancer

technical field

The invention belongs to the field of biomedicine, and relates to an anti-IL-1β antibody, in particular to the use of an IL-1β-targeting antibody in preparing a drug for treating castration-resistant prostate cancer.

Background technique

Prostate cancer is a malignant tumor that occurs in the epithelial tissue of the prostate. It is one of the most threatening male malignant tumors and widely threatens the health and life of men around the world. Clinically, there are many treatment methods for prostate tumors, the most commonly used treatment methods are radical prostatectomy, radiotherapy and systemic therapy.

Among them, radical prostatectomy and radiotherapy are mainly for early in situ prostate tumors, while systemic therapy, such as androgen deprivation therapy, is required for advanced prostate tumors and metastatic prostate tumors. This is very effective at the beginning of treatment, but the therapy is only effective for tumor control for 1-4 years, over time, surgical castration and androgen deprivation therapy are no longer effective, even in the environment of low androgen production, prostate cancer cells Finally, it can also adapt to androgen deprivation and recurrence by restoring androgen receptor (AR) signaling. Most of them will develop into androgen-independent prostate cancer (castration-resistant prostate cancer, CRPC), which is also the cause of Treatment failure is the leading cause of death in patients.

AR is a major transcription factor and driver in prostate cancer. In addition to epithelial cells, several environmental cell types in prostate cancer and other solid tumors have been shown to respond to androgens. As a first-line treatment for advanced prostate cancer, Androgen Deprivation Therapy (ADT) not only has an inhibitory effect on the epithelial cells of prostate cancer, but may also affect environmental cells, especially tumor-related immune cells.

Compared with the low specificity of traditional treatment methods, molecular targeted drugs have brought new life to efficient and precise tumor targeting therapy. Molecular targeted drugs can specifically act on abnormally activated signaling pathways or biological processes in tumor cells or environmental cells. Compared with traditional radiotherapy and chemotherapy, they are more efficient and have fewer side effects. So it is very important to find a target that specifically targets castration-resistant prostate cancer and effective targeted inhibitory drugs.

Interleukin 1β (IL-1β), a member of the interleukin 1 cytokine family, is produced as a proprotein and is proteolytically processed by caspase 1 to its active form. IL-1β is an important mediator of inflammatory response, and it promotes the recruitment of monocytes at inflammatory sites by inducing the expression of adhesion molecules such as ICAM-1 and VCAM-1 on endothelial cells. IL-1β is involved in a variety of cellular activities, including cell proliferation, differentiation, apoptosis, and activation of T and B lymphocytes. Clinically, the anti-IL-1β antibody Canakinumab has been approved by the FDA for the treatment of cryopyridine-associated periodic syndrome (CAPS), tumor necrosis factor receptor-associated periodic syndrome (TRAPS), hyperimmunoglobulin D syndrome (HIDS) ) and familial Mediterranean fever (MFM).

Anti-IL-1β antibodies are neutralizing antibodies that specifically target IL-1β. In previous research reports, the function of this antibody is mainly to target IL-1β to regulate the inflammatory response.

Contents of the invention

Aiming at the above-mentioned technical problems in the prior art, the present invention provides the use of an antibody targeting IL-1β in the preparation of a drug for treating castration-resistant prostate cancer. The antibody targeting IL-1β is prepared in The use of the medicine for treating castration-resistant prostate cancer is to solve the technical problem that the medicines and treatment methods in the prior art are not effective in treating castration-resistant prostate cancer.

The invention provides the use of an antibody targeting IL-1β in the preparation of a medicament for treating castration-resistant prostate cancer.

The present invention also provides the use of IL-1β as a therapeutic target in screening drugs for treating castration-resistant prostate cancer.

The present invention also provides the use of the antibody targeting IL-1β in the preparation of a pharmaceutical composition for treating castration-resistant prostate cancer or anti-tumor.

Further, the pharmaceutical composition includes an antibody targeting PD-1.

Further, the pharmaceutical composition also includes a pharmaceutically acceptable carrier or adjuvant.

The present invention also provides the combined use of the antibody targeting IL-1β and the antibody targeting PD-1 in the preparation of a pharmaceutical composition for treating castration-resistant prostate cancer or anti-tumor.

Specifically, the mass ratio of the antibody targeting IL-1β to the antibody targeting PD-1 is (1-2): (1-2). The preferred mass ratio is 1:1.

The present invention proves through a series of experiments that antagonizing IL-1β can effectively curb the development and deterioration of prostate cancer in mice. Based on the experimental results, the present invention proposes that IL-1β can be used as a new target for treating castration-resistant prostate cancer.

Androgen deprivation therapy (ADT) is a first-line treatment for prostate cancer. Experiments of the present invention show that ADT can lead to immunosuppression, and myeloid suppressor cells MDSCs and tumor-associated macrophages (TAMs) with immunosuppressive effects are more effective after ADT. The number of cells increased; RNA-seq data analysis and experiments proved that IL-1β is the most upregulated cytokine in TAMs after ADT; mouse tumor models proved that the expression of IL-1β can promote the growth of prostate tumors; specificity Antibodies targeting IL-1β can inhibit the growth of prostate tumors in mice, resulting in a decrease in the number of immunosuppressive MDSCs and an increase in the number of immune-promoting CD8 ⁺ T cells; antibodies specifically targeting IL-1β are associated with anti-PD The combination of -1 antibody has a better therapeutic effect.

Compared with the prior art, the technical progress of the present invention is remarkable. The present invention found that androgen deprivation therapy, the first-line treatment method for prostate cancer, leads to the increase of IL-1β secreted by macrophages in prostate cancer. The present invention also found that anti-IL-1β antibody can specifically inhibit the castration-resistant tumor model Pten ^Δ/Δ ; The growth of Trp53 ^Δ/Δ cells in vivo proves that the antagonism of IL-1β can effectively curb the occurrence and development of castration-resistant prostate cancer, and can be used as an important clinical therapeutic target. Therapy provides new ideas and advances that can bring huge socioeconomic benefits.

Description of drawings

Figure 1 shows the in vivo tumor formation experiment of Pten ^Δ/Δ P53 ^Δ/Δ organoids, the tumor growth in the control group and the castration therapy group (Figure 1A is a picture of tumor growth, Figure 1B is a statistical map of tumor mass) and tumor Immune infiltration flow cytometry analysis results (Fig. 1C is the flow cytometry analysis diagram and statistical diagram of MDSC cells, and Fig. 1D is the flow cytometry analysis diagram and statistical diagram of TAM cells);

Figure 2 shows the analysis and screening results of cytokines in Pten ^Δ/Δ P53 ^Δ/Δ tumors before and after castration therapy by RNA-seq (Figure 2A) and RT-PCR (Figure 2B);

Figure 3 shows the results of the correlation analysis between AR score and IL1B expression in the human prostate cancer RNA-seq database by the ggpubr method;

Figure 4 shows the results of measuring IL-1β concentration in serum of prostate cancer patients before and after androgen deprivation therapy by ELISA experiment;

Figure 5 shows the result graph of the mRNA expression level of IL1B detected by RT-PCR in BMDM cells (Figure 5A) and TAM cells (Figure 5B) under DHT and Enzalutamide treatment;

Figure 6 shows the results of tumor growth in the in vivo tumor formation experiment of Pten ^Δ/Δ P53 ^Δ/Δ organoids overexpressed by IL-1β (Figure 6A is a picture of tumor growth, Figure 6B is a statistical map of tumor quality);

Figure 7 shows the in vivo tumorigenicity of Pten ^Δ/Δ P53 ^Δ/Δ organoids under the treatment of anti-IL-1β antibody or combined anti-IL-1β antibody and anti-PD-1 antibody (Figure 7A is a picture of tumor growth, Figure 7B is the statistical chart of tumor treatment) and flow cytometry analysis of tumor immune infiltration (Figure 7C, the upper part is the flow cytometry analysis chart and statistical chart of MDSC cells, and the lower part is the flow cytometric analysis of CD8 ⁺ T cells Technical analysis chart and statistical chart) result chart.

In the above figures, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001.

Specific implementation method

The present invention will be described in detail below in conjunction with the examples. The experimental materials of the present invention are purchased as shown below, and also prepared according to the teachings of textbooks, all of which are prior art, and will not be repeated here.

Experimental Materials:

Eight-week-old C57BL/6 male mice were purchased from Shanghai Lingchang Biotechnology Co., Ltd., castration-resistant prostate tumor Pbsn-Cre4; Pten ^fl/fl ; Trp53 ^fl/fl mouse-derived prostate cancer epithelial Pten ^Δ/Δ P53 ^Δ/Δ organoids, DMSO (purchased from Sigma, catalog number D2650), Enzalutamide (purchased from MCE, catalog number HY-70002), corn oil (purchased from Beyond, catalog number ST1177), Matrigel Matrix (purchased from Corning, catalog number 356234 ), RPMI (purchased from Yuanpei, catalog number L210KJ), collagenase I (purchased from Sangon, catalog #A004194), collagenase IV (purchased from Sangon, catalog #A004186), DNase I (purchased from STEMCELL Technologies, catalog #07900 ), Dispase (purchased from Sangon, product number A002100-0050), FBS (purchased from Gbico, product number 10270-106), PBS (purchased from Sangon, product number B548117-0500), staining solution (PBS solution containing 2% FBS). Antibodies for flow cytometry staining include: CD45.2-PerCP (purchased from Biolegend, catalog number 109826), CD11b-FITC (purchased from Biolegend, catalog number 101206), F4/80-APC (purchased from Biolegend, catalog number 123116), Gr- 1-PE (purchased from Biolegend, catalog number 108428), CD3e-FITC (purchased from Biolegend, catalog number 100306), CD8a-PE (purchased from Biolegend, catalog number 100708), CD4-AF700 (purchased from Biolegend, catalog number 100430), anti-IL -1β antibody (purchased from Bioxcell, product number BE0246), anti-PD-1 antibody (purchased from Bioxcell, product number BE0146), the gene mouse Pbsn-Cre4 used in the present invention; Pten ^fl/fl ; Trp53 ^fl/f was tested by Jackson Chamber (https://www.jax.org/) genetic mice were crossed. All mouse experiments were performed in accordance with the protocols approved by the Experimental Animal Use and Care Committee of Renji Hospital.

Example 1: Flow cytometry staining and detection of mouse prostate cancer after castration therapy

experimental method:

1) Orthotopic injection of 300 Pten ^Δ/Δ P53 ^Δ/Δ organoids into the prostate of C57BL/6 male mice, divided into two groups, 6 mice in each group.

2) On the 7th day after the orthotopic injection, the two groups of mice were treated as follows: For the castration treatment group (ADT group), an incision of about 0.4 cm was cut at the scrotum, and after the bilateral testis was ligated and removed, the The wound was sutured; for the sham operation group (Sham group), an incision of about 0.4 cm was made at the scrotum, and the wound was sutured.

3) Preparation of Enzalutamide: use DMSO as a solvent to prepare Enzalutamide with a concentration of 40uM, dilute Enzalutamide dissolved in DMSO with corn oil: DMSO = 9: 1 volume ratio; control solution with corn oil: DMSO = 9: 1 Dilute DMSO by volume.

4) On the 10th day after the in situ injection, the two groups of mice were treated as follows: for the castration treatment group, the prepared Enzalutamide was intragastrically injected at a dose of 10 mg/kg; for the sham operation group, an equal volume of Prepared control solution; intragastric administration once a day.

5) On the 21st day after the in situ injection, the mice were sacrificed by cervical dislocation, the prostate tumors were taken out, photographed and weighed for comparison.

6) Shred the prostate tumor and use digestive enzymes (based on RPMI, containing 0.2mg/mL collagenase I, 0.2mg/mL collagenase IV, 0.01mg/mL Dnase I, 0.1mg/mL Dispase and 2% FBS) was digested at 37°C for 1 hour, passed through a 45um cell sieve to make a single cell suspension, centrifuged at 400g for 5 minutes, and discarded the supernatant. Suspend the cell pellet in erythrocyte lysate, place at room temperature for 3 minutes to lyse erythrocytes, add 3 times the volume of PBS, and centrifuge to discard the supernatant. Wash twice with PBS, and discard the supernatant.

7) Suspend cells with staining solution to a concentration of 10 ⁷ /ml, add antibodies to a final concentration of 2ug/ml for each antibody, and incubate in a refrigerator at 4°C in the dark for 30 minutes. The antibodies added to the samples for analyzing MDSCs were CD45.2-PerCP, CD11b-FITC, and Gr-1-PE; the antibodies added to samples for analyzing TAMs were CD45.2-PerCP, CD11b-FITC, and F4/80-APC.

8) Wash the cells twice with PBS, resuspend the cells with 500ul PBS buffer, and then test on the machine.

The experimental results are shown in Figure 1, indicating that the castration-resistant Pten ^Δ/Δ P53 ^Δ/Δ tumor model can not be improved after androgen deprivation therapy (the picture of tumor growth is shown in Figure 1A, scale bar=1 cm; the statistical graph is shown in Figure 1B), while the immunosuppressive cells MDSCs (as shown in Figure 1C, the upper part is the flow cytometry analysis graph, and the lower part is the statistical graph) and TAMs (as shown in Figure 1D, the upper Part is the flow cytometry analysis graph, the lower part is the statistical graph) the number of cells is significantly increased.

Example 2: Screening of IL-1β as a therapeutic target for prostate cancer

experimental method:

1) The 3-month-old spontaneous prostate cancer Pbsn-Cre4; Pten ^fl/fl ; Trp53 ^fl/fl tumor mouse model was divided into a control group and a castration group, with 3 mice in each group. For mice in the control group, an incision of about 0.4 cm was made at the scrotum, and the wound was sutured; for mice in the castration treatment group, an incision of about 0.4 cm was made at the scrotum, and the bilateral testes were ligated and removed, and the wound was closed. suture.

2) After 4-6 weeks, the mice were killed by cervical dislocation, and the prostate tumors of the two groups of mice were removed, and three tumor tissues in each group were sent to Novogene for transcriptome sequencing (RNA-seq ).

3) Use the Cutadap tool to read the adapter sequence of the RNA-seq data and cut the low-quality bases at the 3' end to obtain valid data. Valid data was then mapped to the genome (mm10) using Hisat2 to generate SAM files. SAM files were converted to BAM files by SAMtools, and then gene counts were quantified using the Stringtie tool. Gene differential expression analysis was performed using DESeq2.

4) The Pbsn-Cre4 of the control group and the castration group; Pten ^fl/fl ; Trp53 ^fl/fl mice prostate tumors were shredded and then digested with digestive enzymes (using RPMI as base culture, containing 0.2mg/mL collagenase I, 0.2mg/mL Collagenase IV, 0.01mg/mL Dnase I, 0.1mg/mL Dispase and 2% FBS) were digested at 37°C for 1 hour, passed through a 45um cell sieve to make a single cell suspension, centrifuged at 400g for 5 minutes, and discarded clear. Suspend the cell pellet in erythrocyte lysate, place at room temperature for 3 minutes to lyse erythrocytes, add 3 times the volume of PBS, and centrifuge to discard the supernatant. Wash twice with PBS, and discard the supernatant.

5) Suspend cells with staining solution to a concentration of 10 ⁷ /ml, add antibodies to a final concentration of 2ug/ml for each antibody, and incubate in a refrigerator at 4°C in the dark for 30 minutes. The antibodies added to the samples for sorting TAMs were CD45.2-PerCP, CD11b-FITC, and F4/80-APC.

6) Wash the cells twice with PBS, resuspend the cells with 500ul PBS buffer, and sort them on the machine. TAMs are defined as CD45.2 ⁺ CD11b ⁺ F4/80 ⁺ cells.

7) Use Vazyme's cell/tissue total RNA extraction kit to extract the RNA in TAMs. The PrimeScript RT reagent Kit with gDNA Eraser (Takara) kit was used to reverse transcribe RNA to synthesize cDNA, and further use SYBR Green real-time quantitative RT-PCR technology (Takara RR420A) to detect the expression of differential genes obtained by RNA-seq analysis. The ΔΔct method was used to standardize the expression level of the detected gene with the expression level of the internal reference Actin.

The experimental results are shown in Figure 2, indicating that compared with the control group, the expression of IL1B in the prostate tumors of castrated Pbsn-Cre4; Pten ^fl/fl ; Trp53 ^fl/fl mice increased (as shown in Figure 2A) , and the expression levels of various immunosuppression-related cytokines in tumor-associated macrophages are the highest (as shown in Figure 2B).

Example 3: Correlation analysis between AR and IL1B in human prostate cancer

Access RNA-seq data of prostate cancer patients in dbGaP with accession numbers phs001141.v1.p1 (PROMOTE), phs000915.v2.p2 (SU2C), and phs000178.v11.p8 (TCGA). AR score was calculated by GSVA method in R (v.4.1.0), and the correlation between AR score and IL1B expression was calculated by ggpubr method (v.0.4.0).

The experimental results are shown in FIG. 3 , indicating that in human prostate cancer, the androgen receptor AR and IL1B present a negative correlation.

Embodiment 4: ELISA test measures the serum IL-1β concentration of prostate cancer patients before and after ADT treatment

1) Add 100ul standard working solution or patient serum samples before and after ADT treatment to the wells of the ELISA plate coated with IL-1β antibody, and incubate at 37°C for 90 minutes. Among them, 8 patients had serum samples before ADT treatment, and 12 patients had serum samples after ADT treatment.

2) Discard the liquid in the plate, immediately add 100ul biotinylated antibody working solution, and incubate at 37°C for 60 minutes. 3) Discard the liquid in the plate, add 350ul washing solution, soak for 1 minute, discard the liquid in the plate. Repeat plate washing 3 times.

4) Add 100ul HRP enzyme conjugate working solution to each well, incubate at 37°C for 30 minutes, discard the liquid in the plate, and wash the plate 5 times with the washing solution.

5) Add 90ul of substrate solution to each well and incubate at 37°C for 15 minutes.

6) Add 50 μL of stop solution to each well, read immediately at a wavelength of 450 nm, and obtain data for processing.

The experimental results are shown in FIG. 4 , indicating that the serum IL-1β concentration of prostate cancer patients after ADT treatment increased.

Example 5: Negative regulatory effect of AR on IL-1β in macrophages

1) Bone marrow cells were obtained by flushing the femoral marrow cavity of C57BL/6 mice with RPMI 1640 medium. Red blood cells were removed using erythrocyte lysis buffer, washed once with PBS, and centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded, and then the bone marrow cells were resuspended in RPMI 1640 containing 10% FBS and 10ng/ml M-CSF, and cultured in a 37°C incubator for 7 days to obtain bone marrow-derived macrophages (BMDMs).

2) Shred the prostate tumors of Pbsn-Cre4; Pten ^fl/fl ; Trp53 ^fl/fl mice, digest with digestive enzymes at 37°C for 1 hour, pass through a 45um cell sieve to make a single-cell suspension, and centrifuge at 400g for 5 minutes. Discard the supernatant. Suspend the cell pellet in erythrocyte lysate, place at room temperature for 3 minutes to lyse erythrocytes, add 3 times the volume of PBS, and centrifuge to discard the supernatant. Wash twice with PBS, and discard the supernatant. Suspend the cells with staining solution to a concentration of 10 ⁷ /ml, add antibodies until the final concentration of each antibody is 2ug/ml, and incubate in a refrigerator at 4°C in the dark for 30 minutes. The antibodies added to the samples for sorting TAMs were CD45.2-PerCP, CD11b-FITC, and F4/80-APC. The cells were washed twice with PBS, resuspended with 500ul PBS buffer, and sorted on the machine. TAMs were defined as CD45.2 ⁺ CD11b ⁺ F4/80 ⁺ cells.

3) BMDMs or TAMs were treated with DMSO, 10nM DHT, 10nM DHT plus 10uM Enzalutamide for 48 hours, respectively.

4) Use Vazyme's cell/tissue total RNA extraction kit to extract RNA from BMDMs or TAMs. The PrimeScript RT reagent Kit with gDNA Eraser (Takara) kit was used to reverse transcribe RNA to synthesize cDNA, and further use SYBR Green real-time quantitative RT-PCR technology (Takara RR420A) to detect the expression of differential genes obtained by RNA-seq analysis. The ΔΔct method was used to standardize the expression level of the detected gene with the expression level of the internal reference Actin.

The experimental results are shown in Figure 5, indicating that AR activation in bone marrow-derived macrophage BMDMs (as shown in Figure 5A) and tumor-associated macrophage TAMs (as shown in Figure 5B) can inhibit the expression of IL-1β, while AR Antagonism can promote the expression of IL-1β.

Example 6: Overexpression of IL-1β promotes the growth of prostate cancer

1) For the clone of secretory IL-1β, the IL-1RA secretion signal sequence is:

atggaaatctgcagaggcctccgcagtcacctaatcactctcctcctcttcctgttccattcagagacgatctgc (shown in SEQ ID NO.1)

The mature hIL1B gene sequence is: gcacctgtacgatcactgaactgcacgctccgggactcacagcaaaaaagcttggtgatgtctggtccatatgaactgaaagctctccacctccagggacaggatatggagcaacaagtggtgttctccatgtcctttgtacaaggagaagaaagtaatgacaaaatacctgtggccttggg cctcaaggaaaagaatctgtacctgtcctgcgtgttgaaagatgataagcccactctacagctggagagtgtagatcccaaaaattacccaaagaagaagatggaaaagcgatttgtcttcaacaagatagaaatcaataagctggaatttgagtctgcccagttccccaactggtacatcagcacctcaagcagaa aacatgcccgtcttcctgggagggaccaaaggcggccaggatataactgacttcaccatgcaatttgtgtcttcctaa (shown in SEQ ID NO. 2) was fused to form a secreted IL-1β sequence, which was then cloned into the plenti-CMV-GFP vector.

2) The vector construction process is as follows: Synthesize XhoI-kozac-IL-1RA peptide-mature hIL1B-EcoRI sequence. Then use XhoI and EcoRI to double digest the transformed vector and the synthesized fragment, and then purify the product, and use T4 ligase to construct the secreted IL-1β sequence into the plenti-CMV-GFP vector to obtain the target vector plenti-CMV-secreted IL-1β-GFP.

3) For lentivirus packaging secretory IL-1β overexpression vector: prepare low-passage HEK293T in a 10 cm culture dish, and replace the medium with serum-free medium before transfection. Then, 500 μL of serum-free DMEM medium was added to the EP tube, and the target vectors (plenti-CMV-GFP control plasmid and plenti-CMV-secreted IL-1B-GFP) were mixed with viral packaging helper plasmids psPAX2, pMD2.G (purchased from Addgene , No. 12260, 12259) were mixed according to the amount of DNA quality 3:2:1, and then PEI (1ug/uL) 3 times the total amount of DNA was added, and vortexed to mix. Incubate at room temperature for 15 minutes, add the DNA/PEI mixture to HEK293T, and replace it with DMEM complete medium containing 10% FBS in volume percentage for 6-8 hours, collect the cell supernatant after 48 hours of transfection, and use a 0.45uM filter (Millipore ) to remove cell debris by filtration, and the filtrate is the virus fluid.

4) Add polybrene (millipore, TR-1003-G) at a final concentration of 8 μg/ml to CTL and secreted IL1B overexpressed virus fluid, mix evenly, and add to Pten ^Δ/Δ P53 ^Δ/Δ cells. After 6 hours of virus infection, the medium was replaced with normal medium. After 48 hours of infection, the GFP-positive cells were sorted by flow cytometry, which were the CTL cells of the control group and the Pten ^Δ/Δ P53 ^Δ/Δ cells overexpressing secreted IL-1β.

5) Orthotopic injection of 5x 10 ⁵ control CTL group or Pten ^Δ/Δ P53 ^Δ/Δ cells with secreted IL-1β overexpression (IL-1βOE) into the prostate of C57BL/6 male mice, divided into two groups, Each group has 7 animals.

6) On the 18th day after the in situ injection, the mice were sacrificed by cervical dislocation, and the prostate tumors were taken out, photographed and weighed for comparison.

The experimental results are shown in FIG. 6 , indicating that the overexpression of IL-1β can promote the growth of prostate cancer (the picture of tumor growth is shown in FIG. 6A , scale bar=1 cm; the statistical graph is shown in FIG. 6B ).

Example 7: Tumor formation experiment in vivo: targeting IL-1β to treat prostate cancer

experimental method:

1) Orthotopic injection of 300 Pten ^Δ/Δ P53 ^Δ/Δ organoids into the prostate of C57BL/6 male mice, and the mice were randomly divided into four groups (IgG group, anti-IL-1β treatment group, anti-PD-1 Treatment group, combined treatment group of anti-IL-1β and anti-PD-1 (mass ratio 1:1)), 6 rats in each group.

2) On the 7th day after the orthotopic injection, the four groups of mice were treated as follows: an incision of about 0.4 cm was made in the scrotum, and the bilateral testes were ligated and removed, and the wound was sutured.

3) Starting from the 10th day after the in situ injection, the prepared Enzalutamide was intragastrically injected at a dose of 10 mg/kg every day. (Preparation method: use DMSO as a solvent to prepare Enzalutamide with a concentration of 40uM, and dilute the Enzalutamide dissolved in DMSO with a volume ratio of corn oil:DMSO=9:1.)

4) From the 10th day after in situ injection, control IgG, anti-IL-1β therapeutic antibody (intraperitoneally injected at a dose of 8 mg/kg, administered once every other day), anti-PD-1 antibody (according to 8 mg/kg kg dose intraperitoneally, administered once every other day), or a combination of anti-IL-1β antibody and anti-PD-1 antibody to treat mice.

5) On the 21st day after the in situ injection, the mice were sacrificed by cervical dislocation, the prostate tumors were taken out, photographed and weighed for comparison.

6) Shred the prostate tumor and use digestive enzymes (based on RPMI, containing 0.2mg/mL collagenase I, 0.2mg/mL collagenase IV, 0.01mg/mL Dnase I, 0.1mg/mL Dispase and 2% FBS) was digested at 37°C for 1 hour, passed through a 45um cell sieve to make a single cell suspension, centrifuged at 400g for 5 minutes, and discarded the supernatant. Suspend the cell pellet in erythrocyte lysate, place at room temperature for 3 minutes to lyse erythrocytes, add 3 times the volume of PBS, and centrifuge to discard the supernatant. Wash twice with PBS, and discard the supernatant.

7) Suspend cells with staining solution to a concentration of 10 ⁷ /ml, add antibodies to a final concentration of 2ug/ml for each antibody, and incubate in a refrigerator at 4°C in the dark for 30 minutes. Among them, the antibodies added to the samples analyzing MDSCs were CD45.2-PerCP, CD11b-FITC, Gr-1-PE; the antibodies added to the samples analyzing T cells were CD45.2-PerCP, CD3e-FITC, CD8a-PE, CD4- AF700.

8) Wash the cells twice with PBS, resuspend the cells with 500ul PBS buffer, and then test on the machine.

The experimental results are shown in Figure 7, indicating that anti-IL-1β antibody can inhibit the growth of prostate tumors, and combined with anti-PD-1 antibody has a better therapeutic effect (the picture of tumor growth is shown in Figure 7A, scale bar=1 cm ; the statistical graph is shown in Figure 7B); in addition, anti-IL-1β antibody treatment can reduce the number of suppressive immune cells MDSCs in the tumor, and increase the number of anti-tumor immune CD8 ⁺ T cells (as shown in Figure 7C, the upper part The flow cytometry analysis diagram and statistical diagram of MDSC cells, the lower part is the flow cytometry analysis diagram and statistical diagram of CD8 ⁺ T cells).

Claims (6)

1. Use of an antibody targeting IL-1 β in the manufacture of a medicament for the treatment of castration-resistant prostate cancer.

The use of il-1 beta as a therapeutic target in the screening of a medicament for the treatment of castration-resistant prostate cancer.

3. Use of an antibody targeting IL-1 β for the preparation of a pharmaceutical composition for the treatment of castration-resistant prostate cancer or for the treatment of an anti-tumor.

4. The use according to claim 3, wherein the pharmaceutical composition comprises an antibody targeting PD-1.

5. The use according to claim 3, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or adjuvant.

6. Use of an IL-1 β -targeting antibody in combination with a PD-1-targeting antibody for the preparation of a pharmaceutical composition for the treatment of castration-resistant prostate cancer or an anti-tumor.

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