CN117563006A

CN117563006A - Application of POU2F1 activator in preparation of PD-L1PD-1 monoclonal antibody tumor immunotherapy medicament

Info

Publication number: CN117563006A
Application number: CN202311501391.0A
Authority: CN
Inventors: 张明; 高嫦娥; 税铁军; 杨润祥; 陈楠; 杨凯云; 王常安; 杨芳; 周洁; 杨加鹏; 杨银菊; 鲍明亮; 王建逵; 郭刚; 张莹; 杨欣; 沈媛; 杨从波; 董雯; 安以均
Original assignee: Third Affiliated Hospital of Kunming Medical University
Current assignee: Third Affiliated Hospital of Kunming Medical University
Priority date: 2023-11-13
Filing date: 2023-11-13
Publication date: 2024-02-20

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of a POU2F1 activator in preparation of a PD-L1PD-1 monoclonal antibody tumor immunotherapy medicament, wherein the invention provides application of the POU2F1 activator in preparation of the PD-L1/PD-1 monoclonal antibody tumor immunotherapy medicament, and the POU2F1 activator is used for preparing the PD-L1/PD-1 monoclonal antibody tumor immunotherapy medicament by taking a mechanism of targeting up-regulating CRK as a target site detection reagent. The invention discovers that the up-regulation of POU2F1 can directly regulate the transcription level of PD-L1 in tumors by up-regulating the downstream transcription factor CRK, thereby enhancing the curative effect of PD-1 monoclonal antibody blocking treatment on tumors. The mechanism directly discusses the existing problem of the action of the PD-1 blocker, and develops a new mode of combined medication for providing a new development direction of PD-L1/PD-1 monoclonal antibody immunodetection or immunotherapy auxiliary drugs.

Description

Application of POU2F1 activator in preparation of PD-L1PD-1 monoclonal antibody tumor immunotherapy medicament

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of a POU2F1 activator in preparation of a PD-L1PD-1 monoclonal antibody immunity treatment drug.

Background

Colorectal cancer (CRC) is one of the most common malignant tumors of the digestive tract. Investigation shows that the male is the fourth (10.46%), the female is the third (9.17%), the mortality rate male is the fifth (7.44%), the female is the fourth (9.09%), the male is the rising trend in the new cases of colorectal cancer, the new cases of colorectal cancer are 38.8 ten thousand every year, and the death cases are 18.73 ten thousand.

In recent years, research has found that the onset of colorectal cancer is closely related to the immune escape of tumors, but the exact mechanism is not clear. Studies show that after the signal channel of the programmed death molecule 1 (PD-1)/PD-1 ligand (PD-1 ligand, PD-L1) is activated, an immunosuppressive tumor microenvironment can be formed, so that tumor cells can evade the immune monitoring and killing of organisms, and the PD-1/PD-L1 is blocked, the signal channel can reverse the tumor immunosuppressive microenvironment, and the endogenous anti-tumor immune effect is enhanced. At present, aiming at the targeting blocking of PD-L1, remarkable curative effect advantages are obtained in early clinical tests of various tumors, and the prospect is quite considerable, so that the mechanism of a PD1/PD-L1 signal path is clear, the curative effect of immune checkpoint inhibitors and the optimal combined drug scheme are researched, and proper people are selected to be indistinct. The current PD-1 inhibitors applied to colorectal cancer clinical trials include nivolumab (nivolumab) and pembrolizumab (pembrolizumab), and there have been a number of clinical trials to evaluate the objective remission rate of PD-1/PD-L1 inhibitors to colorectal cancer patients, and although some PD-1/PD-L1 inhibitors are approved for immunotherapy of colorectal cancer, there is still an immune escape phenomenon in the immunotherapy of colorectal cancer, leading to tumor recurrence or metastasis.

POU2F1, also known as OCT1, is one of the members of the POU domain transcription factor family. As transcription factors, POU2F1 has similar DNA binding sequences as other members of the POU family, and can activate or inhibit various transcription factors such as immunoglobulin genes, interleukin-related genes and the like in B cells. More and more studies indicate that POU2F1 plays a crucial role in the cellular malignant transformation process, but its role in tumor immunity is not yet clear.

CRK adaptor proteins consist of two spliced isoforms comprising SH2 and SH3 modular domains that play a key role in signal transduction downstream of cell adhesion molecules through tyrosine kinase-protein interactions. Analysis of TCGA colorectal cancer sequencing data shows that POU2F1 expression is positively correlated with CRK expression, bioinformatics shows that POU2F1 can be combined with a CRK promoter, and existing researches show that CRK is one of molecular regulation mechanisms for promoting PD-1/PD-L1. CRK has been reported to be overexpressed in a variety of tumors including ovarian cancer, glioblastoma multiforme, lung cancer, breast cancer, colorectal cancer, and osteosarcoma; its expression level is generally correlated with tumor grade and inversely correlated with overall patient survival. The specific mechanism of its action in the immune response is still unclear.

Disclosure of Invention

The invention aims at the application of the POU2F1 activator in the preparation of PD-L1PD-1 monoclonal antibody tumor immunotherapy medicaments, provides a new research and development direction of PD-L1/PD-1 monoclonal antibody tumor immunodetection or immunotherapy auxiliary medicaments, and develops a new combined medication mode.

The invention aims at realizing the following technical scheme:

the invention provides an application of a POU2F1 activator in preparing PD-L1/PD-1 monoclonal antibody tumor immunotherapy medicaments;

furthermore, the application is to prepare PD-L1/PD-1 monoclonal antibody tumor immunotherapy medicaments by using a mechanism of targeted up-regulation of CRK by using the POU2F1 activator as a target site detection reagent.

The invention also provides application of the POU2F1 activator and the PD-L1/PD-1 monoclonal antibody medicine in preparing tumor immunity auxiliary therapeutic medicines.

Further, the tumor comprises colorectal cancer.

The invention also provides an application of the CRK serving as a target site detection reagent combined with the POU2F1 activator in preparing a PD-L1/PD-1 monoclonal antibody tumor immunotherapy medicament.

Further, the tumor comprises colorectal cancer.

The invention also provides application of the CRK serving as a target spot detection reagent in preparation of PD-L1/PD-1 monoclonal antibody tumor immune adjuvant therapy drugs.

Further, the tumor comprises colorectal cancer.

Compared with the prior art, the invention has obvious technical progress. The beneficial effects of the invention are as follows: the up-regulation of POU2F1 can directly regulate the transcription level of PD-L1 in tumors by up-regulating the downstream transcription factor CRK, thereby enhancing the curative effect of PD-1 monoclonal antibody blocking treatment on tumors. The mechanism directly discusses the existing problem of the action of the PD-1 blocker, and develops a new mode of combined medication for providing a new development direction of PD-L1/PD-1 monoclonal antibody immunodetection or immunotherapy auxiliary drugs.

Drawings

FIG. 1 is a graph of experimental data of a mouse model of example 1, wherein FIG. 1A is a schematic diagram of the experimental flow of the mouse, FIG. 1B is a graph of the change in volume of a tumor body of a subcutaneous transplanted tumor of the mouse measured in real time, the tumor body volume of the mouse is measured every three days from the beginning of the intervention, and a volume curve is drawn, the mouse is taken out of the body on the day of the sacrifice to carry out in vitro measurement and a histogram is drawn, the data are expressed by mean ± standard error, the result statistical analysis adopts single factor analysis of variance for overall group comparison, and Dunnett-t test is adopted for further two group comparison, NS, P >0.05; * P < 0.01; * P < 0.001. FIG. 1C is a statistical plot of in vitro measurements of tumor volumes of subcutaneous transplants of mice on the day of sacrifice, data expressed as mean ± standard error, results statistical analysis using one-way anova for overall group comparison and Dunnett-t test for further group comparison, NS, P >0.05; * P < 0.01; * P < 0.001. Fig. 1D is a graph of the change in weight of mice measured in real time, from the beginning of the intervention, the general state of the mice was observed and the weight of the mice was measured every three days, and the weight curves were plotted, the data were expressed as mean ± standard error, and the results were statistically analyzed for overall group-to-group comparison using one-way analysis of variance, and further for two-group comparison using Dunnett-t test. FIG. 1E is a graph of Kaplan-Meier survival of mice, with comparison of group differences using the Gehan-Breslow-Wilcoxon test, with NS, P >0.05; * P is less than 0.05; * P < 0.01.

FIG. 2 shows the results of immunofluorescence staining of paraffin sections of tumor tissue samples of patients with colorectal cancer treated with PD-1 in example 2 to label the expression levels of tumor POU2F1 and PD-L1, comparing the progression-free survival time (FIG. 2A, FIG. 2B) and the total survival time (FIG. 2C, FIG. 2D) of the patients according to the level of gene expression, and plotting Kaplan-Meier survival curves using

The Gehan-Breslow-Wilcoxon test performs group-to-group differential comparisons.

FIG. 3 is a graph of experimental data of in vitro study on the POU2F1-CRK-PD-L1 axis in example 3, wherein FIG. 3A and FIG. 3D are respectively the results of fluorescence quantitative PCR detection of POU2F1, CRK and PD-L1 expression of the no-load group after the SW620 gene knockout of the colon cancer cell line POU2F1 or CRK, and the p value is less than or equal to 0.05 after correction, which indicates a statistical difference; FIGS. 3B and 3E show the results of western blot detection of POU2F1, CRK and PD-L1 expression after in vitro gene knockout of the colon cancer cell line with POU2F1 or CRK; fig. 3C and 3F are statistical results of corresponding gray scale analysis, data are expressed as mean ± standard error, the results are compared between the whole sets using single factor analysis of variance and further compared between the two sets using Dunnett-t test, P < 0.01; * P < 0.001.

FIG. 4 is experimental data of direct regulatory mechanism study of POU2F1 on CRK in example 3, wherein FIG. 4A represents western blot detection results of POU2F1 and CRK expression after gene overexpression of POU2F1 on colon cancer cell lines in vitro; FIG. 4B is a graph showing statistical results of gray scale analysis, data represented by mean ± standard error, and results obtained by comparing two groups using unpaired t-test, P < 0.01; * P < 0.001; FIG. 4C constructs a vector comprising a POU2F1 mutant CRK promoter sequence and a vector comprising a wild type PD-L1 promoter sequence as control groups, and transfecting a colon cancer cell line with a POU2F1 multiple expression vector to induce upregulation of POU2F1, respectively, and setting an empty load and a vehicle as control groups, and detecting CRK luciferase activity change by a luciferase reporter gene detection method. The data are expressed by mean ± standard error, the result adopts single factor analysis of variance to carry out overall group comparison, and Dunnett-t test is adopted to further carry out two group comparison, ns P is more than 0.05, P is less than 0.01, P is less than 0.001; FIG. 4D is a mutation map of the predicted CRK binding site of the PD-L1 promoter region. FIG. 4E shows that CRK binds significantly to POU2F1 by ChIP detection using anti-flag antibody and CRK primer for SYBR RT-PCR in colon cancer cell line.

FIG. 5 is a graph of Kaplan-Meier survival, which is obtained by performing immunofluorescence staining on paraffin sections of tumor tissue samples of patients with colon cancer treated with PD-1 in example 4 to mark the expression levels of tumor CRK and PD-L1, comparing the progression-free survival time of the patients (FIG. 5A and FIG. 5B) with the total survival time (FIG. 5C and FIG. 5D) according to the level of gene expression, and performing group difference comparison by using Gehan-Breslow-Wilcoxon test.

Detailed Description

Wherein "/" in PD-L1/PD-1 throughout represents the meaning of "and" or ".

Example 1

1. In vivo study of proliferation of knockout adenosine receptor POU2F1 in combination with PD-1mab against mouse engraftment tumor (colon cancer) set forth in group 1.1: scramble+igg2a (control group), shpou2f1+igg2a (POU 2F1 knockdown group), scramble+pd-1mAb (PD-1 mAb treatment group), shpou2f1+pd-1mAb (co-intervention group).

1.2 experimental procedure, see fig. 1A:

about 6 days before the experiment: 5.10.6.6.6C 57BL/6 mice were subcutaneously injected with SW620 colon cancer shPOU2F1 knockdown cell lines at the right dorsal wing ⁵ 20 mice each were treated with the same number of Scramble cell lines (control).

Day 0 of the experiment: the body weight and tumor volume of the mice were recorded as the tumor volume grew to about 100mm ³ At the beginning, mice inoculated with shPOU2F1 knockdown cell strain and Scramble cell strain were respectively subjected to PD-1mAb injection for 100 ug/mouse/3 days, intraperitoneal injection, and isotype IgG control groups of 10 mice each were set.

Experiment 1, 4, 7, 10 days: the body weight and tumor volume of the mice were weighed every three days, volume = pi/6 x length x width ² And (5) calculating a formula. Day 12 of the experiment: half of the mice were sacrificed, tumor photographs were taken and tissue samples were saved for subsequent data analysis.

Lifetime observation: the remaining mice were observed for duration of survival and the time to death was recorded.

1.3 experimental results: as shown in fig. 1B and 1C, there was no significant difference in tumor volume in the POU2F1 knockout group compared to the control group, the PD-1mab group had significantly reduced tumor volume, while the combined intervention group was significantly smaller than the individual intervention group, with statistical differences. As shown in fig. 1D, there was no significant statistical difference in mouse body weight between the four groups. The medicine has no obvious toxic and side effects on the general condition of mice. As shown in fig. 1E, there was no significant difference in survival time between the POU2F1 knockout group and the PD-1mab group compared to the control group, while the combined intervention group was significantly longer than the control group and the independent intervention group, with statistical differences. The results indicate that the gene knockout adenosine receptor POU2F1 can promote the curative effect of PD-1 monoclonal antibody anti-tumor (colon cancer) immunotherapy.

Example 2

1. Retrospective clinical observation shows that the experimental results of the correlation analysis of the expression levels of the tumor PD-L1 and the POU2F1 in the patients with colon cancer treated by the PD-1 monoclonal antibody (Nawu monoclonal antibody) are shown in the table 1, the expression levels of the tumor PD-L1 and the POU2F1 in the patients with colon cancer treated by the PD-1 monoclonal antibody are obviously positively correlated, and the statistical difference exists, and the P is less than 0.05.

TABLE 1

Note that: table 1 is a study of tumor PD-L1 and POU2F1 expression levels by immunofluorescent staining of paraffin sections of tumor tissue samples from patients treated with PD-1mab, and the number of patients in each category is summarized in table 1, based on which the significance of the correlation was assessed using Fisher's exact test (bilateral test).

2. Retrospective clinical observation the experimental results of the correlation between the tumor PD-L1 and POU2F1 expression and the prognosis of the patient (progression free survival time and total survival time) in the patients with colon cancer treated with PD-1mab (nivolumab) are shown in fig. 2, and compared with the tumor PD-L1 high expression and POU2F1 high expression, the PD-L1 low expression and POU2F1 low expression show better prognosis in the patients with colon cancer treated with PD-1mab, with P < 0.05; * P < 0.01; * P < 0.001.

Example 3

In vitro study of 1POU2F1-CRK Axis modulation of tumor PD-L1 expression levels

1.1 experimental method:

the culture establishment procedure for colorectal cancer cell lines SW620 and HCT116 was as follows: fresh colon cancer tissues which are clinically resected are taken, cut into 1 multiplied by 1cm by using a sterile instrument, placed in a culture dish, added with a proper amount of DMEM/F12 culture medium containing 10% FPS, placed in a constant temperature incubator at 37 ℃, periodically changed in time, and subjected to passage collection after cells grow full, and then subjected to subsequent experiments.

The following operations and analyses were performed in the cells by means of exogenous overexpression of PTRF by lentiviral vectors:

(1) Respectively extracting RNA of the cells, and carrying out RT-PCR analysis to detect the expression change of the mRNA level PD-L1;

the RNA extraction operation steps are as follows:

1) The cell culture medium was removed, washed three times with sterile PBS, added with Trizol extraction reagent, left at room temperature for 5min, and cells were repeatedly aspirated with a pipette and transferred to an enzyme-free EP tube.

2) One fifth of the Trizol volume of chloroform was added to the EP tube, turned upside down, manually shaken for 30s, centrifuged at 4℃for 15min at 12000 rpm.

3) After centrifugation, the EP tube was carefully removed from the centrifuge, the liquid level in the tube was divided into three layers, the upper aqueous phase was RNA, the lower organic phase was protein and DNA, and the RNA of the upper aqueous phase was carefully aspirated into another enzyme-free EP tube.

4) Isopropanol with the same volume as that of the aqueous phase RNA is added into the EP tube, and the mixture is turned over and mixed uniformly, and the mixture is kept stand at room temperature for 10min.

5) The EP tube was placed in a 4℃centrifuge and centrifuged at 12000rpm for 15min, the EP tube was removed, pellet RNA was visible at the bottom of the centrifuge tube, and the supernatant was carefully discarded.

6) 1mL of 75% ethanol was added to the EP tube, turned upside down, and RNA was vortexed to precipitate for 30s. The EP tube was centrifuged at 12000rpm for 15 minutes in a centrifuge at 4 ℃.

7) The supernatant from the EP tube was decanted, 1mL of absolute ethanol was added to the EP tube, the mixture was inverted upside down, and RNA pellet was vortexed and centrifuged at 12000rpm for 15 minutes at 30s at 4 ℃.

8) The supernatant from the EP tube was discarded and the EP tube was placed in an ultra clean bench and air dried for 10-20 minutes.

9) The RNA pellet was dissolved by pipetting 20. Mu.L of DEPC water using a pipette.

The fluorescent quantitative PCR experiment steps are as follows:

1) Primers were designed at NCBI using Primer-BLAST and aligned in the database, after which the company was found to synthesize primers.

2) A fluorescent quantitative PCR experiment was performed in a qPCR octant according to a total volume of 20. Mu.L of the formulated mixture of 10. Mu.L of the 2 XSYBR Green qPCR mixture, 1. Mu.L of the gene upstream primer (10. Mu.M), 1. Mu.L of the gene downstream primer (10. Mu.M), 2. Mu.L of the template DNA/cDNA, 6. Mu.L of nuclease-free DEPC water, and a real-time fluorescent quantitative PCR system.

3) Three repetitions are respectively arranged for each sample, the experimental result is obtained through a delta CT calculation formula according to the melting curve and CT value of the PCR experiment, and the GAPDH is used as an internal reference to compare the relative expression level of the target genes among the groups.

(2) Protein of cell is extracted respectively for Western Blot analysis to detect the expression change of protein level PD-L1

The method comprises the following specific steps of extracting total cell proteins:

1) Before extracting the protein, preparing cell protein lysate, generally using high-efficiency RIPA lysate, adding PMSF to make its final concentration be 10mM, mixing, and placing on ice for use.

2) The medium was removed and the cells were washed three times with sterile PBS and the ppa lysate containing PMSF was added to the petri dish.

3) The dish was shaken to allow the lysate to infiltrate the cells, which were then placed on ice for 30 minutes to further lyse the cells.

4) The cell lysate mixture was pipetted into a clean 1.5mL EP tube using a pipette and centrifuged at 12000rpm for 15 minutes in a centrifuge at 4 ℃.

5) After centrifugation, the cell lysis supernatant was transferred to a new EP tube using a pipette, BCA protein concentration was detected, and total cell protein was quantified.

6) The Western blot Western immunoblotting experiment comprises the following specific steps:

SDS-PAGE gel was prepared and placed in an electrophoresis tank with the gel plate clamped by the device. And adding electrophoresis buffer solution, adding the sample into the corresponding hole, covering a cover of the electrophoresis tank, connecting an electrophoresis apparatus, and setting the constant pressure to be 80-100 kv. About 90-120 minutes, when bromophenol blue in the loading buffer solution runs out of the bottom of the gel plate, electrophoresis is stopped, and transfer is performed. The PVDF film was cut into rectangular films of 5X 8cm in size and activated by soaking in anhydrous methanol. Preparing a film transfer buffer solution, placing the film transfer clamp into a tray, carefully taking off the glue on the glue plate, placing the glue on a sponge cushion and filter paper of the film transfer clamp, covering the activated PVDF film, covering the glue and the PVDF film by using another layer of filter paper and the sponge cushion, and finally clamping the clamp. The film transfer groove is placed on ice, the film transfer buffer solution is poured into the film transfer groove, the film transfer clamp is placed at the corresponding position of the film transfer groove according to the positive electrode and the negative electrode, the cover of the film transfer groove is covered, the electrophoresis apparatus is connected and electrified, the voltage is set to 80kv, and the time is 90 minutes. Bovine serum albumin blocking buffer (BSA blocking solution) was prepared, the PVDF membrane was removed by opening the clamp, placed in a blocking cuvette containing 5% BSA blocking solution and slowly shaken on a desiccation shaker, and incubated at room temperature for 1-2 hours for blocking. Preparing primary antibody working solution according to the antibody use concentration of the antibody specification, taking out the sealing solution from the sealing small box, recovering, directly adding the prepared primary antibody working solution, placing on a shaking table at 4 ℃, and slowly shaking overnight. And (3) on a rotating day, recovering the primary antibody working solution, adding a PBST buffer solution into the PVDF membrane, placing the PVDF membrane on a decoloring shaking table at room temperature for rapid shaking, cleaning the PVDF membrane for 8-10 minutes, and cleaning three times by using the PBST. Preparing secondary antibody working solution of corresponding species, pouring out PBST buffer solution after the PVDF membrane is cleaned for the last time, adding the secondary antibody working solution again, slowly shaking at room temperature on a shaking table, and incubating for 1-2 hours. And (3) carrying out Westernblot PVDF film exposure on the prepared chemiluminescent liquid.

(3) The specific operation steps of the cell gene intervention are as follows:

POU2F1 was overexpressed in SW620 cells. SW620 cells were transfected with POU2F1 over-expression lentiviral vector constructed using GV348 vector. SW620 cells were transfected with CRK siRNA to knock down CRK. Transfection was performed when the cell density reached 70-90% confluence, and manipulations were performed in a biosafety cabinet. Serum-free medium was added to 1.5mL of sterile EP, and Lipofectamine3000 reagent was added thereto for thorough mixing. A serum-free medium was added to another 1.5mL of sterile EP, siRNA was added to make a premix, and then the corresponding P3000 reagent was added and thoroughly mixed. The diluted siRNA was then added in a 1:1 ratio to a 1.5mL EP tube diluted with Lipofectamine3000 reagent and incubated for 5-10 minutes at room temperature. siRNA liposome complexes were added to the corresponding cell culture dishes, cells were incubated in a carbon dioxide incubator at 37℃for 2-4 days, and then corresponding analytical experiments were performed on transfected cells.

1.2 experimental results:

referring to fig. 3A, through qPCR analysis, it was found that in colon cancer cell line SW620, gene knockdown POU2F1 vs empty group, POU2F1, CRK and PD-L1 gene expression were significantly down-regulated, with statistical differences; as shown in fig. 3B and 3c, western bolt detects gene knockdown tumor POU2F1, compared with the control group, the expression levels of CRK and PD-L1 can be significantly reduced, and statistical differences exist; referring to FIG. 3D, through qPCR analysis, it is found that in colon cancer cell line SW620, CRK gene expression is significantly down-regulated compared with that of empty vector, and there is a statistical difference between CRK and PD-L1 gene expression; as shown in fig. 3E and 3f, western bolt detection gene knockdown tumor CRK can significantly inhibit the expression level of PD-L1 compared with the control group, and has statistical differences; 2POU2F1 regulates the transcription level of CRK in combination with the CRK promoter.

As shown in fig. 4A and fig. 4b, the westernbolt detection gene over-expresses tumor POU2F1, compared with the control group, can significantly promote the expression level of CRK, and has statistical difference; in FIG. 4C, the luciferase reporter gene detects whether POU2F1 is combined with the CRK promoter, and compared with a control group, the over-expression of CRK or DPCPX treatment can lead to the remarkable enhancement of the luciferase activity of the tumor PD-L1 promoter, but when the POU2F1 combining site of the CRK promoter is mutated, the effect disappears, which indicates that CRK is directly combined with the PD-L1 promoter, and the result has statistical difference; the predicted mutation map of CRK binding site in the PD-L1 promoter region is shown in FIG. 4D. As shown in fig. 4e, chip detection suggests that CRK significantly binds to PD-L1 and is statistically different from the control. The result shows that POU2F1 is combined with CRK promoter to regulate CRK transcription; the gene knockdown POU2F1 can obviously up-regulate CRK, and the CRK can be used as a transcription factor to directly regulate and control PD-L1 transcription from an mRNA level, so that the target POU2F1-CRK axis can directly regulate and control the expression level of tumor PD-L1.

Example 4

1. Retrospective clinical observation of correlation analysis of tumor PD-L1 and CRK expression levels in PD-1mab (Nawuzumab) treatment colon cancer patients

The experimental results are shown in Table 2, the tumor PD-L1 and CRK expression levels in the patients with colon cancer treated by the PD-1 monoclonal antibody are obviously positively correlated, and have statistical differences, and P is less than 0.05.

TABLE 2

Note that: table 2 is a study of tumor PD-L1 and CRK expression levels by immunofluorescent staining of paraffin sections of tumor tissue samples from patients with colon cancer treated with PD-1mab, and the number of patients in each category is summarized in table 2, based on which data the significance of the correlation of the two was assessed using Fisher's exact test (bilateral test).

2. Retrospective clinical observation of correlation of tumor PD-L1 and CRK expression with patient prognosis (time to progression free survival and total survival) in treatment of colon cancer patients with PD-1mab (nivolumab)

As shown in fig. 5, in the patient with colon cancer treated with PD-1mab, compared with the tumor PD-L1 high expression and CRK high expression, PD-L1 low expression and CRK low expression show better prognosis, and there is a statistical difference, P < 0.05; * P < 0.01; * P < 0.001.

In conclusion, POU2F1 serving as a biomarker can be applied to detection before the PD-L1/PD-1 monoclonal antibody immunotherapy is used; the transcription factor POU2F1 can also be used as a target site detection reagent for preparing PD-L1/PD-1 monoclonal antibody tumor immunotherapy medicaments; the POU2F1 activator and the PD-L1/PD-1 monoclonal antibody can be combined to prepare the tumor immunity auxiliary therapeutic medicine. The CRK can be used as a target detection reagent to be applied to the preparation of PD-L1/PD-1 monoclonal antibody tumor immunity auxiliary treatment medicines or the CRK can be used as a target detection reagent to be combined with the POU2F1 activator to be applied to the preparation of PD-L1/PD-1 monoclonal antibody tumor immunity treatment medicines. The tumor is preferably a solid tumor; further preferred is colon or rectal cancer.

Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.

Claims

Application of POU2F1 activator in preparing PD-L1/PD-1 monoclonal antibody tumor immunotherapy medicine.
2. The use of claim 1, wherein the use is for the preparation of a PD-L1/PD-1 mab immunotherapeutic agent using the mechanism of POU2F1 activator targeted up-regulation of CRK as a target site detection reagent.
3. The use of claim 1, wherein the tumor comprises colorectal cancer.
The application of the POU2F1 activator and the PD-L1/PD-1 monoclonal antibody medicine in preparing tumor immune auxiliary therapeutic medicine.
5. The use of claim 4, wherein the tumor comprises colorectal cancer.
Application of CRK as target site detection reagent combined with POU2F1 activator in preparing PD-L1/PD-1 monoclonal antibody tumor immunotherapy medicine.
7. The use of claim 6, wherein the tumor comprises colorectal cancer.
Application of CRK as target detection reagent in preparing PD-L1/PD-1 monoclonal antibody tumor immune adjuvant therapy medicine.
9. The use of claim 8, wherein the tumor comprises colorectal cancer.