CN112569360A

CN112569360A - Anti-tumor medicine composition based on blocking PD-1/PD-L1 and application thereof

Info

Publication number: CN112569360A
Application number: CN201910952044.7A
Authority: CN
Inventors: 胡卓伟; 花芳; 尚爽; 陈菲; 杨雨薇
Original assignee: Institute of Materia Medica of CAMS
Current assignee: Institute of Materia Medica of CAMS
Priority date: 2019-09-30
Filing date: 2019-09-30
Publication date: 2021-03-30

Abstract

The invention provides an anti-tumor pharmaceutical composition for blocking and reducing the expression of TRIB3 based on immune check points and application thereof, and clarifies a biological mechanism of the anti-tumor pharmaceutical composition in a tumor treatment process. The immune checkpoint-based anti-tumor drug combination comprises an immune checkpoint inhibitor and a small molecule compound which can reduce the expression of TRIB 3. The small molecule can activate autophagy by reducing expression of oncogenic protein TRIB3 to recruit more killer tumor T cells and increase their killing activity. The synergistic effect of the inhibitor and the PD-L1/PD-1 inhibitor is generated, and more killer T cells are recruited and the activity killing property of the killer T cells is enhanced compared with the single use, so that the tumor growth is effectively inhibited. Therefore, the pharmaceutical composition has better clinical application prospect.

Description

Anti-tumor medicine composition based on blocking PD-1/PD-L1 and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to an anti-tumor medicine composition based on blocking of PD-1/PD-L1 immune check points and application thereof.

Background

Cancer has been the first enemy of human health in our country and even globally. In recent years, the incidence and mortality of malignant tumors in China tend to increase year by year due to the reasons of environmental pollution, fast life pace, aging population and the like. According to the report of 'the current situation and the trend of Chinese tumors' in 2018, the prevalence rate of cancer in China is on a medium level internationally. On average, 1 million people diagnosed cancer per day and 7 people per minute. Tumors such as lung cancer, liver cancer, colorectal cancer, gastric cancer and the like directly threaten the life safety of people in China. Despite the increasing progress of medical treatment, the fatality rate of malignant tumors still remains high, and the search for new treatment is still a major scientific problem to be solved urgently.

The most common previous tumor treatment regimens include surgical resection, chemotherapy, and radiation therapy. However, the serious attacks of chemotherapy and radiotherapy on the immune system of the patient and the subsequent recurrence and metastasis of the tumor make the patient ultimately unable to benefit from the treatment. Immunotherapy of tumors is an emerging therapeutic approach in recent years, and is a novel therapeutic concept proposed by tumor immunologists after recognizing the key role played by immune cells in the tumor microenvironment. The therapy is to control and eliminate tumor by restarting and maintaining normal anti-tumor immune response of the body. In recent years, the therapy has been applied to the treatment of various tumors, and is listed as the first ten scientific breakthroughs in 2013 by the Science journal.

Among the immunotherapies, relevant therapies directed to the immune checkpoint PD-1/PD-L1 are of interest. PD-1(programmed death 1) is mainly expressed by exhausted or inhibitory lymphocytes, is an important immunosuppressive molecule and is a member of the CD28 superfamily. While PD-L1(programmed death ligand 1), the receptor for PD-1, is expressed predominantly by inflammatory epithelial cells or activated stromal cells and is an induced product downstream of the T-cell effector molecule IFN-g. The combination of PD-1 and PD-L1 can inhibit the activation of killer T lymphocytes and cause the immune escape of tumors. Blocking the interaction of PD-1/PD-L1 restores the functional effects of tumor-specific T cells. However, it has been found that not all patients are sensitive to this therapy. The research shows that the infiltrated lymphocytes are less observed in tumor tissue sections of patients with cancer species such as pancreatic cancer, prostatic cancer, ovarian cancer and the like, and the patients are called cold tumors by scientists; in other cancer types, such as lung, liver, bladder, etc., a number of recruited killer lymphocytes are observed in the tumor tissue, and are known by scientists as "hot tumors". Since binding of PD-1/PD-L1 is only possible in "hot tumors", only such tumor patients would benefit from anti-PD-1/PD-L1 therapy. Furthermore, even with "hot tumors," only a fraction of patients are sensitive to this therapy. These indicate that the application of pure anti-PD-1/PD-L1 therapy is very limited. Therefore, a novel PD-1/PD-L1 combined treatment method is sought, so that the method has very important scientific significance and social significance for overcoming the defects and limitations of the treatment method.

TRIB3 is one of the pseudo-kinase protein family members, and researches prove that the protein is highly expressed in various tumors such as colorectal cancer, lung cancer, liver cancer, breast cancer, gastric cancer and the like, and can promote the development process of the tumors by inhibiting the activation of autophagy flow. Activation of autophagic flux promotes activation of the immune system by degrading various oncogenic proteins to raise more antigens on the cell surface. However, whether the targeted reduction of the expression of TRIB3 can sensitize the responsiveness of a tumor patient to PD-1/PD-L1 therapy is not reported, and it is unclear which small molecule compound is selected to be used as an effective drug for inhibiting the expression of TRIB3 protein.

Disclosure of Invention

The invention aims to solve the defects of the prior art and provide an anti-tumor combination drug based on immune checkpoint blockade and application thereof.

The technical scheme adopted by the invention is as follows:

an anti-tumor pharmaceutical composition based on immune checkpoint blockade, which is characterized by comprising an immune checkpoint inhibitor and a small molecule compound for reducing the expression of TRIB3, wherein the immune checkpoint inhibitor is a programmed death receptor PD-1 or a ligand PD-L1 inhibitor thereof. Preferably, the apoptosis receptor PD-1 inhibitor is Nivolumab (addivo), Pembrolizumab (palboclizumab) or teripril. Preferably, the apoptosis receptor ligand PD-L1 inhibitor is Atezolizumab (astuzumab), Durvalumab (doxoruzumab) or Avelumab (avilamumab).

Preferably, the small molecule compound for reducing the expression level of the TRIB3 protein is P300 acetylase inhibitor C646.

The invention also comprises the application of the antitumor drug composition based on immune checkpoint blockade in the preparation of drugs for treating and preventing cancers or drugs for improving the response effect of tumor patients on immune checkpoint treatment. Further, the tumor in the application is a solid tumor. Further, the solid tumor comprises rectal cancer, liver cancer, pancreatic cancer or lung cancer.

The beneficial technical effects are as follows:

compared with the prior art, the invention has the following advantages:

1. the drug is an anti-tumor combined drug composition based on immune checkpoint inhibition and inhibition of oncogenic protein TRIB3 protein expression, and has wide application in improving the response immune checkpoint treatment effect of tumor patients and establishing clinical anti-tumor treatment strategies.

2. The P300 inhibitor C646 provided by the invention can effectively increase the recruitment and killing activity of killer lymphocytes and effectively enhance the responsiveness of cold tumors to PD-1/PD-L1 medicaments.

For a better understanding and practice, the invention is described in detail below with reference to the accompanying drawings.

Drawings

FIG. 1 protein levels of TRIB3 and acetylated TRIB3 after addition of C646 to colon cancer HCT-8 cells;

FIG. 2 in-tumor CD8 after different drug treatments in MC38 subcutaneous tumor formation model⁺The proportion of T cells;

FIG. 3 intratumoral CD8 after different drug treatments in the MC38 subcutaneous tumor model⁺Killing activity of T cells;

FIG. 4 tumor weights after different drug treatments in the MC38 subcutaneous tumor model;

FIG. 5. growth curves after different drug treatments in the MC38 subcutaneous tumor model;

FIG. 6 in-tumor CD8 after different drug treatments in Hepa1-6 subcutaneous tumor formation model⁺The proportion of T cells;

FIG. 7 intratumoral CD8 after different drug treatments in the Hepa1-6 subcutaneous tumor model⁺Killing activity of T cells;

FIG. 8 tumor weights after different drug treatments in the Hepa1-6 subcutaneous tumor formation model;

FIG. 9 growth curves after different drug treatments in a Hepa1-6 subcutaneous tumorigenesis model;

FIG. 10 intratumoral CD8 after different drug treatments in the tumor formation model under LLC⁺The proportion of T cells;

FIG. 11 intratumoral CD8 after different drug treatments in the tumor formation model under LLC⁺Killing activity of T cells;

FIG. 12 tumor weights after different drug treatments in the tumor formation model under LLC;

FIG. 13 growth curves after treatment with different drugs in a tumor model under LLC;

FIG. 14 intratumoral CD8 after different drug treatments in Panc02 subcutaneous tumor model⁺The proportion of T cells;

FIG. 15 intratumoral CD8 after different drug treatments in Panc02 subcutaneous tumor model⁺Killing activity of T cells;

FIG. 16 tumor weights after different drug treatments in Panc02 subcutaneous tumor model;

figure 17 growth curves after different drug treatments in Panc02 subcutaneous tumor model.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

The PBS described in the examples refers to phosphate buffer at a concentration of 0.1M and a pH of 7.2.

The room temperature described in the examples is room temperature conventional in the art, preferably 25 ℃.

The experimental results are expressed by mean value plus or minus standard error, and the significant difference is considered to be existed by comparing that p is less than 0.05 and p is less than 0.01 through parameter or nonparametric variance test.

Experimental example 1: the co-immunoprecipitation method proves that C646 can reduce the acetylation and protein expression of tumor cells TRIB3

The co-immunoprecipitation reagents were as follows:

lysate a: 0.6057g Tris base, 1.7532g NaCl, 0.1017g MgCl 2.6H2O, 0.0742g EDTA, 10mL glycerol, 10mL 10% NP40, adding deionized water to 150mL, adjusting the pH value to 7.6 with HCl, diluting to 191mL, mixing well, filtering with 0.45 μm filter membrane, and storing at 4 ℃.

Lysate B: 200. mu.L of 2 M.beta. -phosphoglycerol, 4mL of 2.5M NaF, 2mL of 8mM NaVO3, 2mL of 100mM PMSF, 200. mu.L of 1M DTT, and 200. mu.L each of Leu, Pep, and Apr at 1mg/mL, in a total volume of 9 mL. The mother liquor was stored at-20 ℃. Before use, mother liquor of each component in the solution B is unfrozen, and is respectively added into the solution A according to the composition proportion and uniformly mixed. Protein A/G Plus-Agarose is available from Santa cruz, USA. The specific operation steps are as follows:

colon cancer cells HCT-8 were divided into control and treatment groups, respectively, and the treatment group was treated with 5. mu. m C646 for 24 hours, while the control cells were treated with control solvent for 24 hours. 6 hours before harvesting cells, the deacetylase inhibitors TSA (5 μm) and NAM (10 μm) were added to the two groups of cells, respectively. After 24 hours of C646 treatment, cells were scraped to a 1.5ml centrifuge tube, 500. mu.l of the above COIP lysate was added, ice-cooled for 30min, and centrifuged at 12000rpm at 4 ℃ for 30 min. Each group of proteins was sampled at 200. mu.g, and left as cell lysate Input as a control. Mu.g of TRIB3 antibody or the same Normal IgG as the TRIB3 antibody species was added to the remaining Protein, and at the same time, 15. mu.L of Protein A/G Plus-Agarose was added and the mixture was thoroughly resuspended and slowly vortexed at 4 ℃ overnight. Centrifugation was carried out at 3000rpm for 5min at 4 ℃ and the supernatant was carefully aspirated, leaving only a small amount of supernatant and failing to aspirate the Agarose. Adding 0.5mL of co-immunoprecipitation washing solution, mixing, standing in ice bath for 1min, centrifuging at 4 ℃ and 3000rpm for 30sec, and carefully removing the supernatant by suction. Washing was repeated 5 times, and left for 5min before the final centrifugation. Carefully remove the supernatant by aspiration, add 20-30. mu.l of 2 XSDS gel loading buffer, mix well, denature for 3min at 95 ℃, transfer to ice bath rapidly and cool. Centrifuging at 12000rpm for 2min at room temperature to obtain supernatant as precipitated protein sample, and performing SDS-polyacrylamide gel electrophoresis on part or all of the protein sample. The Western results show that C646 significantly reduced the protein expression level of TRIB3 in the HCT-8 cell line and reduced the acetylation level of TRIB3 (fig. 1, table 1).

TABLE 1 inhibition of TRIB3 protein and its acetylation level by C646

Number of repetitions	Protein inhibition rate	Acetylation inhibition rate of TRIB3
			1	95％	95％
2	92％	91％
			3	87％	88％

Experimental example 2: the effect of a combination of C646, based on immune checkpoint inhibition and reducing the expression of TRIB3, on tumor growth was studied by a mouse colon cancer MC38 neoplasia experiment.

Firstly, a stable transfer cell line (MC38T 3) of MC38 stably knocking down TRIB3 is constructed by using Trib3shRNA virus^KD) Then the cell is infected by Trib3K197R virus (MC38T 3)^KD/K197R^OE). K197R is obtained by mutating lysine 197 of TRIB3 to arginine, which inhibits ubiquitination of TRIB3 and maintains stable and high expression of TRIB 3.

The above mouse experiments were divided into 5 groups: isotype control antibody and control solvent treated group (MC38), C646 treated alone group (MC38), immune checkpoint PD-L1 blockingAntibody single treatment group (MC38), C646/PD-L1 blocking antibody combined treatment group (MC38) and C646/PD-L1 blocking antibody combined treatment group (MC38T 3)^K ^D/K197R^OE)。

The mouse product used in the above tumor formation experiment is C57BL/6, and the tumor cell strain used is mouse colon cancer cell strain MC 38. Subcutaneous planting of MC38 or MC38T3^KD/K197R^OEOne week after the cells were administered by intraperitoneal injection of PD-L1 or C646.

The results indicate that more CD8 was recruited by treatment with C646⁺T cells (fig. 2, table 2); the combination of the two drugs can further increase CD8 compared with single drug⁺T cell killing activity (FIG. 3, Table 3), and more importantly, the C646/PD-L1 blocking antibody combination inhibited tumor growth better than the single administration (FIG. 4, FIG. 5, Table 4).

TABLE 2 Intra-tumor CD8 after different drug treatments in MC38 subcutaneous tumor model⁺Proportion of T cells (%)

TABLE 3 IFN-g in tumors after different drug treatment in MC38 subcutaneous tumor model⁺/CD8⁺Proportion of T cells (%)

TABLE 4 tumor weights after different drug treatments in MC38 subcutaneous tumor model

Experimental example 3: the effect of the combination of C646 which is based on the inhibition of immune check points and reduces the expression of TRIB3 on the growth of tumors is researched through a mouse liver cancer Hepa1-6 tumor formation experiment.

Firstly, constructing a stable transgenic cell line (Hepa1-6T 3) of Hepa1-6 stably knocking down TRIB3 by using Trib3shRNA virus^KD) Then the cell is infected by Trib3K197R virus (Hepa1-6T 3)^KD/K197R^OE). K197R is obtained by mutating lysine 197 of TRIB3 to arginine, which inhibits ubiquitination of TRIB3 and maintains stable and high expression of TRIB 3.

The above mouse experiments were divided into 5 groups: isotype control antibody and control solvent treatment group (Hepa 1-6), C646 single treatment group (Hepa 1-6), immune checkpoint PD-L1 blocking antibody single treatment group (Hepa 1-6), C646/PD-L1 blocking antibody combined treatment group (Hepa 1-6) and C646/PD-L1 blocking antibody combined treatment group (Hepa1-6T 3)^KD/K197R^OE)。

The mouse product used in the above tumor formation experiment is C57BL/6, and the tumor cell strain used is mouse colon cancer cell strain Hepa 1-6. Subcutaneously planting Hepa1-6 or Hepa1-6T3^KD/K197R^OEOne week after the cells were administered by intraperitoneal injection of PD-L1 or C646.

The results indicate that more CD8 was recruited by treatment with C646⁺T cells (fig. 6, table 5); the combination of the two drugs can further increase CD8 compared with single drug⁺Killing activity of T cells (fig. 7, table 6); more importantly, the combination of C646/PD-L1 blocking antibody inhibited tumor growth better than the combination of C646/PD-L1 blocking antibody alone (FIG. 8, FIG. 9, Table 7).

TABLE 5 Intra-tumor CD8 after different drug treatments in Hepa1-6 subcutaneous tumor model⁺Proportion of T cells (%)

TABLE 6 Intra-tumoral IFN-g after treatment with different drugs in the Hepa1-6 subcutaneous tumorigenesis model⁺/CD8⁺Proportion of T cells (%)

TABLE 7 tumor weights after different drug treatments in Hepa1-6 subcutaneous tumor models

Experimental example 4: the effect of a combination of C646, based on immune checkpoint inhibition and reducing the expression of TRIB3, on tumor growth was studied by a mouse lung cancer LLC tumorigenesis experiment.

Firstly, constructing a stable transgenic cell line (LLC T3) of LLC stable knock-down TRIB3 by using Trib3shRNA virus^KD) And then infecting the cells with Trib3K197R virus (LLC T3)^KD/K197R^OE). K197R is obtained by mutating lysine 197 of TRIB3 to arginine, which inhibits ubiquitination of TRIB3 and maintains stable and high expression of TRIB 3.

The above mouse experiments were divided into 5 groups: isotype control antibody and control solvent treatment group (LLC), C646 single treatment group (LLC), immune checkpoint PD-L1 blocking antibody single treatment group (LLC), C646/PD-L1 blocking antibody combined treatment group (LLC), and C646/PD-L1 blocking antibody combined treatment group (LLC T3)^KD/K197R^OE)。

The mouse product used in the above tumor formation experiment is C57BL/6, and the tumor cell strain used is mouse colon cancer cell strain LLC. Subcutaneous implantation of LLC or LLC T3^KD/K197R^OEOne week after the cells were administered by intraperitoneal injection of PD-L1 or C646.

The results indicate that more CD8 was recruited by treatment with C646⁺T cells (fig. 10, table 8); the combination of the two drugs can further increase CD8 compared with single drug⁺Killing activity of T cells (fig. 11, table 9); more importantly, the combination of C646/PD-L1 blocking antibody inhibited tumor growth better than the combination of C646/PD-L1 blocking antibody alone (FIG. 12, FIG. 13, Table 10).

TABLE 8 Intra-tumoral CD8 after different drug treatments in LLC subcutaneous tumorigenesis model⁺Proportion of T cells (%)

TABLE 9 Intratumoral IFN-g after treatment with different drugs in LLC subcutaneous tumorigenesis model⁺CD8⁺Proportion of T cells (%)

TABLE 10 tumor weights after different drug treatments in LLC subcutaneous tumor model

Experimental example 5: effect of combination of checkpoint inhibition by mouse pancreatic cancer Panc02 with C646 that reduces the expression of TRIB3 on tumor growth.

Firstly, constructing a Panc02 stable transgenic cell line (Panc02T 3) for stably knocking down TRIB3 by using Trib3shRNA virus^KD) Then the cell is infected by Trib3K197R virus to construct (Panc02T 3)^KD/K197R^OE). K197R is obtained by mutating lysine 197 of TRIB3 to arginine, which inhibits ubiquitination of TRIB3 and maintains stable and high expression of TRIB 3.

The above mouse experiments were divided into 5 groups: isotype control antibody and control solvent treated group (Panc02), C646 single treated group (Panc02), immune checkpoint PD-L1 blocking antibody single treated group (Panc02), C646/P D-L1 blocking antibody combined treated group (Panc02), and C646/PD-L1 blocking antibody combined treated group (Pan C02T 3)^KD/K197R^OE)。

The mouse product used in the above tumor formation experiment is C57BL/6, and the tumor cell strain used is mouse colon cancer cell strain Panc 02. Planting Panc02 or Panc02T3 under skin^KD/K197R^OEOne week after cells were PD-L1 or C646 by intraperitoneal injection.

The results indicate that more CD8 was recruited by treatment with C646⁺T cells (fig. 14, table 11); the combination of the two drugs can further increase CD8 compared with single drug⁺Killing activity of T cells (fig. 15, table 12); more importantly, the combination of C646/PD-L1 blocking antibody inhibited tumor growth better than the administration alone (FIG. 16, FIG. 17, Table 13).

TABLE 11 intra-tumor CD8 after different drug treatments in Panc02 subcutaneous tumor model⁺Proportion of T cells (%)

TABLE 12 Intra-tumor IFN-g following different drug treatment in Panc02 subcutaneous tumor model⁺CD8⁺Proportion of T cells (%)

TABLE 13 tumor weights after different drug treatments in Panc02 subcutaneous tumor model

The mouse PD-L1 blocking antibody adopted in the embodiment of the invention is a common PD-L1 inhibitor in the experiment, and due to the interaction of PD-1/PD-L1, the same experiment effect can be obtained by adopting the PD-1 inhibitor to carry out the experiment. Since in mouse experiments PD-1 inhibitors or PD-L1 inhibitors may work synergistically with C646 to better inhibit tumors, the same clinically used PD-1 inhibitors including but not limited to the following drugs Nivolumab (Ulmacevo), embrocolizumab (Pabolizumab), Terepril, or the clinically used PD-L1 inhibitors including but not limited to the following drugs Atezolizumab (Attributizumab), Durvalumab (Durvalumab) or Avelumab (Avenumab) may also work synergistically with C646 or other small molecule drugs that decrease the expression of TRIB3 protein to inhibit tumors.

In conclusion, the anti-tumor combination drug composition based on immune checkpoint inhibition and reduction of the expression of TRIB3 provided by the invention is widely applied to improvement of the treatment effect of tumor patients responding to immune checkpoint and formulation of clinical anti-tumor treatment strategies.

The present invention is not limited to the above-described embodiments, and various modifications and variations of the present invention are intended to be included within the scope of the claims and the equivalent technology of the present invention if they do not depart from the spirit and scope of the present invention.

Claims

1. An anti-tumor pharmaceutical composition based on immune checkpoint blockade, comprising an immune checkpoint inhibitor and a small molecule compound that reduces the expression of TRIB 3.

2. The immune checkpoint blockade based antitumor pharmaceutical composition as claimed in claim 1, wherein the immune checkpoint inhibitor is an inhibitor of programmed death receptor PD-1 or its ligand PD-L1.

3. The immune checkpoint blockade based antitumor pharmaceutical composition as claimed in claim 2, wherein the programmed death receptor PD-1 inhibitor is an inhibitory antibody to PD-1; the apoptosis receptor ligand PD-L1 inhibitor is an inhibitory antibody to PD-L1.

4. The immune checkpoint blockade based antitumor pharmaceutical composition as claimed in claim 3 wherein the apoptosis receptor PD-1 inhibitory antibody is Nivolumab, Pembrolizumab or Terepril; the apoptosis receptor ligand PD-L1 inhibitory antibody is Atezolizumab, Durvalumab or Avelumab.

5. The immune checkpoint blockade based antitumor pharmaceutical composition as claimed in claim 1, wherein the small molecule compound that reduces the expression of TRIB3 comprises the P300 acetylase inhibitor C646 capable of reducing the acetylation level of TRIB3, thereby facilitating the degradation thereof.

6. Use of the immune checkpoint blockade based anti-tumor pharmaceutical composition of claim 1 for the preparation of a medicament for the treatment and/or prevention of cancer or for the preparation of a medicament for increasing the effect of a tumor patient's response to immune checkpoint therapy.

7. The use of claim 6, wherein the tumor is a solid tumor.

8. The use of claim 7, wherein the solid tumor comprises colorectal cancer, liver cancer, pancreatic cancer, or lung cancer.