CN113501882B - Preparation method and application of interfering peptide for regulating PD-L1 protein - Google Patents

Abstract

The invention discloses a preparation method and application of an interfering peptide for regulating and controlling PD-L1 protein, firstly, exploring and synthesizing an interfering peptide for relieving PD-L1 immune suppression function; secondly, at the level of cell experiments, verifying the effect of the interfering peptide on inhibiting the interaction between PD-L1 and TRAF 2; finally, in the mature mouse in situ tumor model, the interfering peptide is used in combination with the tumor immunotherapy antibody to prove the therapeutic effect of the interfering peptide in tumor treatment.

Description

Preparation method and application of an interfering peptide that regulates PD-L1 protein

Technical field

The invention belongs to the technical field of drug preparation, and specifically relates to a preparation method and application of an interfering peptide that regulates PD-L1 protein.

Background technique

Cancer, a disease caused by abnormal cell proliferation, is seriously affecting the health and quality of life of modern people. Therefore, the prevention, screening and treatment of cancer have become hot and difficult research topics in the biomedical field. In cancer treatment, immunotherapy has been shown to be effective in some cancer types. Cancer immunotherapy is a cancer treatment method targeting immune checkpoints. Immune checkpoints refer to receptor-ligand combinations that regulate T cell activity. Cancer cells can efficiently activate immunosuppressive signaling pathways through immune checkpoints, thereby inhibiting the activation of T cells in the body, ultimately completing immune evasion and further proliferating and migrating. Immune checkpoints mainly include CTLA-4 (cytotoxic T-lymphocyte antigen-4) and PD-1 (programmed cell deathprotein 1) expressed on the surface of T cells, as well as PD-L1 (PD-L1) expressed on the surface of many cells, including some cancer cells. 1ligand).

PD-L1 is a member of the B7 superfamily. It is a type I single-pass transmembrane protein containing four N-linked polysaccharides, consisting of an extracellular region, a transmembrane region, and a cytoplasmic region. The extracellular region contains an IgV domain and an IgC domain. PD-1 is also a type I transmembrane protein that can interact with the IgV extracellular domain of PD-L1. PD-L1 is a ligand for the inhibitory receptor PDCD1/PD-1. It can interact with PD-1, thereby inhibiting the killing function of cytotoxic T cells (CTLs), and can play a negative regulatory role in the immune response. At the same time Plays an important role in immune tolerance.

Although there are already some drugs targeting the PD-1/PD-L1 immune checkpoint on the market, some of these treatment options are not effective for patients, and some patients even develop tumor resistance. This makes it necessary to further develop treatments that can be used in combination with drugs targeting PD-1/PD-L1. At the same time, small molecule drugs generally have a long development cycle and are difficult to meet the needs of cancer patients for better treatment options.

Therefore, there is an urgent need to explore more macromolecule drug treatments that can be used in combination with drugs targeting PD-1/PD-L1.

Contents of the invention

The purpose of the present invention is to provide a preparation method and application of an interfering peptide that regulates PD-L1 protein.

The technical solution of the present invention is: a method for preparing an interfering peptide that regulates PD-L1 protein, which method includes the following steps:

(a) Myc-PD-L1 plasmid and Flag-TRAF2 full-length and corresponding truncated plasmids were co-transfected in HEK293T cells, and the interaction surface between TRAF2 and PD-L1 was confirmed through co-immunoprecipitation and immunoblotting experiments;

(b) Design targeted interference peptides for the interaction surface between TRAF2 and PD-L1;

(c) Fusion of an HIV-TAT sequence at the N-terminus of the targeted interfering peptide;

(d) Design the targeted interfering peptide segment as a D-amino acid retroisomer to obtain the final interfering peptides TRAF2-DRI-1 and TRAF2-DRI-2.

Further, in step (b), the interaction surface between TRAF2 and PD-L1 is amino acids 317-335 and 491-508 of TRAF2.

Further, in step (b), the two amino acid sequences of the targeting interference peptide are DQDKIEALSNKVQQLERSI and YVRDDAIFIKAIVDLTGL respectively.

Further, in step (c), the HIV-TAT sequence is YGRKKRRQRRR.

Further, in step (d), the amino acid sequences of the final interfering peptides TRAF2-DRI-1 and TRAF2-DRI-2 are ISRELQQVKNSLAEIKDQDPPRRRQRRKKRG and LGTLDVIAKIFIADDRVYPPRRRQRRKKRG respectively, and the amino acid type is D type.

The second technical solution of the present invention is: the application of an interfering peptide that regulates PD-L1 protein in the preparation of tumor drugs.

The third technical solution of the present invention is: the application of an interfering peptide that regulates PD-L1 protein in tumor treatment.

The present invention provides a preparation method and application of an interfering peptide that regulates PD-L1 protein. Its advantages are:

1. The interfering peptides TRAF2-DRI-1 and TRAF2-DRI-2, which target the TRAF2 protein, can block the interaction between PD-L1 and TRAF2 and significantly inhibit the tumor immune evasion function of PD-L1. When used in combination with tumor immune antibody drugs , can effectively limit the growth of tumors and provide new ideas for tumor immunotherapy programs;

2. The interfering peptides TRAF2-DRI-1 and TRAF2-DRI-2, which target the TRAF2 protein, contain the HIV-TAT sequence, which facilitates the peptides to directly pass through the cell membrane and enter the cytoplasm to play a role. They do not require any carriers and can avoid carrier-induced toxic side effects;

3. D-type amino acids degrade more slowly in animals than natural L-type amino acids. DRI-modified interfering peptides have been proven to have good tolerance and therapeutic effects in previous clinical trials. The target TRAF2 involved in the present invention The protein-interfering peptides TRAF2-DRI-1 and TRAF2-DRI-2 are feasible for clinical trials;

4. The interfering peptides TRAF2-DRI-1 and TRAF2-DRI-2 targeting the TRAF2 protein can be obtained directly through existing mature peptide synthesis technology, with controllable quality, high purity and great drug potential.

Description of drawings

Figure 1 is a schematic diagram of using co-immunoprecipitation method to confirm the interaction surface between TRAF2 and PD-L1 by overexpressing the full length and corresponding truncations of PD-L1 and TRAF2 in HEK293T cells;

Figure 2 shows the tertiary structure diagram of the TRAF2 protein hexamer and the protein sequence diagrams of the interfering peptides TRAF2-DRI-1 and TRAF2-DRI-2;

Figure 3 is a schematic diagram showing the molecular weight detection of synthesized interfering peptides TRAF2-DRI-1 and TRAF2-DRI-2 using mass spectrometry (MS);

Figure 4 is a schematic diagram for detecting the purity of the synthesized interfering peptides TRAF2-DRI-1 and TRAF2-DRI-2 using high performance liquid chromatography (HPLC);

Figure 5 is a diagram using co-immunoprecipitation method to verify the inhibitory effect of interfering peptides TRAF2-DRI-1 and TRAF2-DRI-2 on the interaction between TRAF2 and PD-L1;

Figure 6 shows the use of proximity ligation technology (Proximity Ligation Assay, PLA) to verify the inhibitory effect of interfering peptides TRAF2-DRI-1 and TRAF2-DRI-2 on the co-localization of TRAF2 and PD-L1;

Figure 7 shows the promotion effect of combined immunotherapy of interfering peptides DRI-1 and DRI-2 with PD1 monoclonal antibody on tumor treatment by measuring tumor growth on BALB/c mice inoculated with 4T1 tumor cell line in the mammary pad. schematic diagram.

Detailed ways

Since the protein-protein interaction surface is generally large, macromolecular drugs (such as interfering peptides, etc.) may be better able to block protein-protein interactions than small molecule compounds. According to the basic functions of PD-L1 protein and the mechanism of suppressing immune inspection, the preparation method of an interfering peptide that regulates PD-L1 protein according to the present invention reveals the interaction between PD-L1 and TRAF2, and clarifies the interaction between PD-L1 and TRAF2. The interaction surface between L1 and TRAF2, and based on this interaction surface, two interfering peptide drugs (TRAF2-DRI-1 and TRAF2-DRI-2) were designed and synthesized. These two interfering peptides can destroy the hydrophobic interaction, Release PD-L1's inhibition of immune recognition of immune cells, and combine interfering peptide drugs with tumor immune antibody drugs to achieve the effect of inhibiting tumor occurrence, development, and tumor growth.

In the preparation method of an interfering peptide for regulating PD-L1 protein according to the present invention, the interfering peptide is first designed to target amino acids 317-335 and 491-508 of TRAF2. The sequences of these two amino acids are DQDKIEALSNKVQQLERSI and YVRDDAIFIKAIVDLTGL respectively. Natural amino acids are L-form;

Then, in order to promote the uptake of TRAF2-DRI-1 and TRAF2-DRI-2 by cells, the N-terminal of the interfering peptide was fused with an HIV-TAT sequence. This sequence is a hydrophilic sequence with the amino acid sequence YGRKKRRQRRR, which is beneficial to cells. Absorb peptides without consuming energy;

Next, in order to improve the stability of the peptides in cell and animal experiments, the peptides were designed as D-amino acid retroisomers to obtain the final amino acid sequences of the interfering peptides TRAF2-DRI-1 and TRAF2-DRI-2. They are ISRELQQVKNSLAEIKDQDPPRRRQRRKKRG and LGTLDVIAKIFIADDRVYPPRRRQRRKKRG respectively;

Finally, D-type amino acids were used as raw materials to synthesize the interfering peptides TRAF2-DRI-1 and TRAF2-DRI-2, with purity above 95%.

The Myc-PD-L1 plasmid and Flag-TRAF2 full-length and corresponding truncated plasmids were co-transfected into HEK293T cells, and the interaction surface between TRAF2 and PD-L1 was confirmed through co-immunoprecipitation and immunoblotting experiments;

Design targeted interference peptides for the interaction surface, namely amino acids 317-335 and 491-508 of TRAF2;

HEK293T cells were co-transfected with Myc-PD-L1 plasmid and Flag-TRAF2 full length. After 24 hours, interfering peptides TRAF2-DRI-1 and TRAF2-DRI-2 were added. Co-immunoprecipitation and immunoblotting experiments were performed to verify the pairing of interfering peptides. Inhibitory effect of the interaction between PD-L1 and TRAF2;

Myc-PD-L1 and Flag-TRAF2 full-length plasmids were co-transfected into HeLa cells. After 24 hours, the interfering peptides TRAF2-DRI-1 and TRAF2-DRI-2 were added. Proximity ligation reaction experiments were performed to verify that the interfering peptides were effective against PD-L1. Inhibitory effect of co-localization with TRAF2;

In BALB/c female mice, breast cancer cells 4T1 were injected into the mammary pad to establish an orthotopic tumor model. Three days later, anti-PD1 antibodies or a combination of interfering peptides and anti-PD1 antibodies were used to verify the role of interfering peptides in tumor immunotherapy.

The administration method of interfering peptide is intraperitoneal injection, and the administration frequency is once every 2 days.

The dosage of interfering peptide and anti-PD1 antibody is 100ug each.

In order to make the above objects, features and advantages of the present invention more obvious and understandable, the technical solutions of the present invention will be further described below in conjunction with the embodiments. However, the present invention is not limited to the listed embodiments, but also includes any other known changes within the scope of the rights claimed by the present invention.

Reference herein to "one embodiment" or "an embodiment" refers to a particular feature, structure, or characteristic that may be included in at least one implementation of the invention. "In one embodiment" appearing in different places in this specification does not all refer to the same embodiment, nor is it a separate or selective embodiment that is mutually exclusive with other embodiments.

Example 1

Design of interfering peptide drugs TRAF2-DRI-1 and TRAF2-DRI-2.

1. Experimental materials

TRAF2 full-length and truncation protein expression plasmids Flag-TRAF2 FL, Flag-TRAF21-491, Flag-TRAF2 1-365, Flag-TRAF2 291-508, Flag-TRAF2317-508 and Flag-TRAF2 336-508 , Myc protein expression plasmid Myc-PD-L1, polyethylenimine PEI, fetal bovine serum, DMEM medium, penicillin/streptomycin solution (Gibco), HEK 293T cell line (ATCC source), Flag beads (Sigma), Flag antibody, Myc antibody and related secondary antibodies (CST Company).

2. Experimental methods

HEK 293T cells were spread into a 10cm cell culture dish. When the cell density reached 70%, Myc-PD-L1 plasmid, Flag-TRAF2 full length and the corresponding truncated body were co-transfected into the 10cm cell culture dish. After 24 hours Collect cells, use Flag beads to enrich Flag-TRAF2 protein, and finally detect the interaction between PD-L1 and TRAF2 through denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blot.

3. Experimental results

Please refer to Figure 1. Figure 1 is a schematic diagram of using co-immunoprecipitation method to confirm the interaction surface between TRAF2 and PD-L1 by overexpressing the full length and corresponding truncated form of PD-L1 and TRAF2 in HEK293T cells. As shown in Figure 1, under overexpression, Myc-PD-L1 has strong interactions with Flag-TRAF2 FL, Flag-TRAF2 291-508 and Flag-TRAF2 317-508 respectively. However, Flag-TRAF2 1-491, Flag-TRAF2 1-365 and Flag-TRAF2336-508 did not interact with Myc-PD-L1. Based on the domain structure of TRAF2, we drew a schematic diagram of the interaction between the full length of TRAF2 and the corresponding truncated form and PD-L1 (Figure 1). Based on the above results, we drew a preliminary conclusion: amino acids 491-508 and 317-335 of TRAF2 are indispensable for the interaction between PD-L1 and TRAF2. Therefore, we designed interfering peptides targeting the amino acids in these two regions to inhibit the interaction between PD-L1 and TRAF2. Example 2 Synthesis and detection of interfering peptide drugs TRAF2-DRI-1 and TRAF2-DRI-2.

Please refer to Figure 2, which shows the tertiary structure diagram of the TRAF2 protein hexamer and the protein sequence diagrams of the interfering peptides TRAF2-DRI-1 and TRAF2-DRI-2. As shown in Figure 2, the amino acid sequences of the interfering peptide drugs TRAF2-DRI-1 and TRAF2-DRI-2 designed by the present invention are ISRELQQVKNSLAEIKDQDPPRRRQRRKKRG and LGTLDVIAKIFIADDRVYPPRRRQRRKKRG respectively, using D-type amino acids as raw materials, synthesized by Gill Biochemical (Shanghai) Co., Ltd. .

Please refer to Figure 3. Figure 3 is a schematic diagram of the molecular weight detection of the synthesized interfering peptides TRAF2-DRI-1 and TRAF2-DRI-2 using mass spectrometry (MS). As shown in Figure 3, the synthesized interfering peptide drugs TRAF2-DRI-1 and TRAF2-DRI-2 were identified with molecular weights of 3595.29 and 3787.38 respectively by the Agilent-6125B liquid mass spectrometry system (Agilent Technologies). HPLC uses an Inertsil ODS-SP liquid chromatography column (Shimadzu, 4.6 mm × 250 mm) as the stationary phase, and mobile phase A (100% acetonitrile, 0.1% trifluoroacetic acid) and mobile phase B (100% ultrapure water, 0.1 % trifluoroacetic acid) for gradient elution, please refer to Figure 4. Figure 4 is a schematic diagram of the purity detection of the synthesized interfering peptides TRAF2-DRI-1 and TRAF2-DRI-2 using high performance liquid chromatography (HPLC). . Table 1 is the purity table of the synthetic interfering peptide drug TRAF2-DRI-1, and Table 2 is the purity table of the synthetic interfering peptide drug TRAF2-DRI-2. As shown in Figure 4, Tables 1 and 2, the purity of TRAF2-DRI-1 and TRAF2-DRI-2 was determined to be greater than 95% and 97%, respectively.

keep time content(%) Peak area Peak height 14.922 95.2 8061310 1171498 15.144 3.3 276977 46636

Table 1

keep time content(%) Peak area Peak height 11.803 97.0 4882820 342686 12.227 1.1 53814 7806

Table 2

Example 3

The use of interfering peptide drugs TRAF2-DRI-1 and TRAF2-DRI-2 can significantly inhibit the interaction between PD-L1 and TRAF2.

1. Experimental materials

Protein expression plasmids Flag-TRAF2 FL and Myc-PD-L1, interfering peptide drugs TRAF2-DRI-1 and TRAF2-DRI-2 prepared according to Example 2, polyethylenimine PEI, fetal bovine serum, DMEM medium, Penicillin/streptomycin solution (Gibco), HEK 293T cell line (ATCC source), Flag beads (Sigma), Flag antibody, Myc antibody and related secondary antibodies (CST Company).

2. Experimental methods

HEK293T cells were spread in a 10cm dish. When the cell density reached 70%, Myc-PD-L1 and Flag-TRAF2 FL plasmids were co-transfected in the 10cm dish. After 12 hours, 100 μM TRAF2-DRI-1 and TRAF2-DRI-2 were used respectively or Cells were co-treated and collected after 24 h. Next, Flag beads were used to enrich Flag-TRAF2, and finally the interaction between PD-L1 and TRAF2 was detected by denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blot.

3. Experimental results

Please refer to Figure 5. Figure 5 is a diagram using co-immunoprecipitation method to verify the inhibitory effect of interfering peptides TRAF2-DRI-1 and TRAF2-DRI-2 on the interaction between TRAF2 and PD-L1. As shown in Figure 5, Myc-PD-L1 has a strong interaction with Flag-TRAF2 FL without interfering peptide treatment. After treatment with TRAF2-DRI-1 or TRAF2-DRI-2, the interaction between PD-L1 and TRAF2 was significantly inhibited. And after treating cells with TRAF2-DRI-1 and TRAF2-DRI-2 at the same time, the interaction between PD-L1 and TRAF2 almost disappeared. Based on the above results, the interfering peptides TRAF2-DRI-1 and TRAF2-DRI-2 can significantly inhibit the interaction between PD-L1 and TRAF2.

Example 4

The use of interfering peptide drugs TRAF2-DRI-1 and TRAF2-DRI-2 can significantly inhibit the co-localization of PD-L1 and TRAF2.

1. Experimental materials

Protein expression plasmids Flag-TRAF2 FL and Myc-PD-L1 were prepared according to the interfering peptide drugs TRAF2-DRI-1 and TRAF2-DRI-2 prepared in Example 2, In Situ/> Probe Anti-Human PLUS Kit (Sigma),/> In Situ/> Probe Anti-Human MINUS Kit (Sigma),/> In Situ Detection Reagents Red kit (Sigma), polyethylenimine PEI, fetal calf serum, DMEM medium, penicillin/streptomycin solution (Gibco), HEK 293T cell line (ATCC source), Flag antibody and Myc antibody ( CST Company), Zeiss LSM880 ultra-high resolution inverted confocal microscope.

2. Experimental methods

Spread HeLa cells into a 24-well plate, and put cells into the slide at the same time. When the cell density reaches 30%, Myc-PD-L1 and Flag-TRAF2 FL plasmids were co-transfected into 2 wells. After 12 hours, 100 μM TRAF2-DRI- 1 and TRAF2-DRI-2 were collected together to process cells, and cells were collected after 24 h. Next, refer to the experimental steps for proximity ligation reaction on Sigma's official website, and use the corresponding proximity ligation reaction probe kit and proximity ligation reaction detection kit to perform immunofluorescence experiments. Finally, a Zeiss LSM880 ultra-high-resolution inverted confocal microscope was used to photograph the experimental results under 579nm excitation light.

3. Experimental results

Please refer to Figure 6. Figure 6 is a diagram using Proximity Ligation Assay (PLA) to verify the inhibitory effect of interfering peptides TRAF2-DRI-1 and TRAF2-DRI-2 on the co-localization of TRAF2 and PD-L1. As shown in Figure 6, in the absence of interfering peptide stimulation, Myc-PD-L1 and Flag-TRAF2FL have strong PLA red signals in HeLa cells (see bright spots in the figure), which indicates that PD-L1 and Flag-TRAF2FL have strong PLA red signals in HeLa cells. There is significant co-localization of TRAF2. After treating cells with TRAF2-DRI-1 and TRAF2-DRI-2 simultaneously, the co-localization of PD-L1 and TRAF2 was significantly inhibited. The above results show that the interfering peptides TRAF2-DRI-1 and TRAF2-DRI-2 can significantly inhibit the co-localization of PD-L1 and TRAF2.

Example 5

The combination of the interfering peptide drug TRAF2-DRI and PD1 antibody can significantly inhibit the proliferation of orthotopic breast cancer in mice.

1. Experimental materials

Breast cancer cell line 4T1, 6-8 weeks old BALB/c female mice (Shanghai Leske Company), interfering peptide drugs TRAF2-DRI-1 and TRAF2-DRI-2 prepared in Example 2, Matrigel (BD), anti -mPD-1antibody(BE0146,Bio XCell,West Lebanon,NH),IgG(BE0089,Bio Sterile surgical scissors and surgical forceps, medical suture needles and threads, surgical warming machine, vernier calipers.

2. Experimental methods

(1) Use trypsin to digest 4T1 cells in the logarithmic growth phase, centrifuge and wash the cells with PBS to remove residual serum components in the culture medium, and centrifuge again;

(2) Resuspend the cells in appropriate PBS and adjust the density to 2×10 ⁵ 4T1/50 μL, then mix the cell suspension with Matrigel 1:1 and place it in an ice box for later use;

(3) Inject 300 μL avertin intraperitoneally. After the mouse is completely anesthetized, use a shaver to shave the hair on the fourth pair of breast areas of the mouse, and wipe and disinfect with an alcohol cotton ball;

(4) Cut the epidermis to find the fourth pair of breast pads, use tweezers to pick up the breast pad tissue, and inject 100 μL of mixed cell suspension into the breast pads;

(5) After the cell injection is completed, quickly suture the wound, wipe it gently with an alcohol cotton ball, place the mouse on a 37°C surgical warming machine, and wait for its recovery;

(6) Three days after tumor cell inoculation, BALB/c female mice were divided into 4 groups, with 6 mice in each group. The first group was injected with PBS and IgG, the second group was injected with interfering peptide and IgG, the third group was injected with PBS and anti-mPD-1 antibody, and the fourth group was injected with interfering peptide and anti-mPD-1 antibody. All injections were performed intraperitoneally, with IgG and anti-mPD-1 antibodies injected every three days, 100ug each time. The interfering peptide is injected every two days, 100ug per animal;

(7) After starting to inject the antibody, use a vernier caliper to measure the size of the tumor at the mouse breast pad every three days, and calculate the tumor volume.

3. Experimental results

Please refer to Figure 7. Figure 7 shows the effectiveness of combined immunotherapy with interfering peptides DRI-1 and DRI-2 and PD1 monoclonal antibody on BALB/c mice inoculated with the 4T1 tumor cell line in the mammary pad by measuring tumor growth. Schematic diagram of the promotion effect of tumor treatment. As shown in Figure 7, in the mouse orthotopic breast cancer model, the combined use of the interfering peptide drug TRAF2-DRI and the PD1 antibody can significantly inhibit tumor growth compared with the use of the PD1 antibody alone, indicating that the TRAF2-DRI interfering peptide Can play a good role in tumor treatment.

In summary, the present invention provides a preparation method and application of an interfering peptide that regulates PD-L1 protein. The provided interfering peptide drugs TRAF2-DRI-1 and TRAF2-DRI-2 can effectively block PD-L1. The interaction with TRAF2 further inhibits the tumor immune evasion function of PD-L1, thereby achieving the purpose of enhancing the effect of tumor immunotherapy.

It should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solution of the present invention can be carried out. Modifications or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention shall be included in the scope of the claims of the present invention.

Claims (2)

1. A preparation method of an interfering peptide for regulating and controlling PD-L1 protein is characterized by comprising the following steps of

The method comprises the following steps:

(a) Co-transfecting Myc-PD-L1 plasmid and Flag-TRAF2 full length and corresponding truncated plasmid in HEK293T cells, and confirming the interaction surface of TRAF2 and PD-L1 through co-immunoprecipitation and immunoblotting experiments;

(b) Designing a targeting interference peptide segment aiming at the interaction surface of the TRAF2 and the PD-L1, wherein the interaction surface of the TRAF2 and the PD-L1 is 317-335 and 491-508 amino acids of the TRAF2, and the sequences of two sections of amino acids of the targeting interference peptide segment are DQDKIEALSNKVQQLERSI and YVRDDAIFIKAIVDLTGL respectively;

(c) Fusing a segment of HIV-TAT sequence at the N end of the targeting interfering peptide segment, wherein the HIV-TAT sequence is YGRKRRQRRR;

(d) The targeted interfering peptide segment is designed into D-type amino acid retroisomer to obtain final interfering peptides TRAF2-DRI-1 and TRAF2-DRI-2, wherein the amino acid sequences of the final interfering peptides TRAF2-DRI-1 and TRAF2-DRI-2 are ISRELQQVKNSLAEIKDQDPPRRRQRRKKRG and ISRELQQVKNSLAEIKDQDPPRRRQRRKKRG respectively

LGTLDVIAKIFIADDRVYPPRRRQRRKKRG the amino acid type is D-form.

2. The preparation of a medicament for treating breast cancer, wherein the medicament comprises an interfering peptide for regulating and controlling PD-L1 protein according to claim 1

Application in the material.