Immune checkpoint inhibitor as well as preparation method and application thereof

Abstract

The invention relates to an immune checkpoint inhibitor as well as a preparation method and application thereof. The immune checkpoint inhibitor is a compound composed of a PD-L1 inhibitory peptide, a TLR9 agonist and an immunogenic cell death inducer. The immune checkpoint inhibitor can effectively stimulate the innate immune system and enhance the anti-tumor immune response, and experiments prove that the immune checkpoint inhibitor can be used as a new lung cancer immunotherapy drug with a prospect and has a good market application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of tumor treatment, and particularly relates to an immune checkpoint inhibitor and a preparation method and application thereof. Background Art

[0002] Immunogenic cell death (ICD) is a unique form of cell death that can activate anti-tumor immune responses. In recent years, research on ICD in the field of lung cancer has made significant progress. Traditional chemotherapy drugs (such as doxorubicin, cisplatin, etc.) and radiotherapy have been found to induce ICD in lung cancer cells, thereby activating the body's immune system and inhibiting tumor growth and metastasis. The characteristics of ICD in lung cancer cells include the release of inflammasomes, the exposure of calreticulin (CRT) on the cell surface, and the release of high-mobility group box protein (HMGB1). These characteristics are closely related to the host's anti-tumor immune response. For example, CRT exposure can promote the maturation and antigen presentation of dendritic cells (DCs), thereby activating the cytotoxic response of CD8+T cells. Inflammasomes and HMGB1 can act as danger signals to stimulate the activation and antigen presentation function of DC cells.

[0003] In order to improve the application potential of ICD in the treatment of lung cancer, researchers have adopted a variety of strategies. One of them is to enhance the anti-tumor immune response by combining adjuvants with immunotherapy drugs (such as immune checkpoint inhibitors). This combined treatment strategy has shown good efficacy and feasibility in clinical trials. In addition, the application of nanotechnology and drug carriers has greatly improved the tumor targeting and bioavailability of drugs that induce ICD, and reduced the toxic side effects of drugs. However, there is still a lack of relevant research on the treatment of lung cancer. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide an immune checkpoint inhibitor and its preparation method and application. The inhibitor can effectively stimulate the innate immune system and enhance the anti-tumor immune response. Experimental verification shows that it can be used as a promising new drug for lung cancer immunotherapy and has good market application prospects.

[0005] The present invention provides an immune checkpoint inhibitor, which is a complex composed of a PD-L1 inhibitory peptide, a TLR9 agonist and an immunogenic cell death inducer.

[0006] Preferably, the sequence of the PD-L1 inhibitory peptide is CNYSKPTDRQYHF.

[0007] Preferably, the TLR9 agonist is cytosine CPG.

[0008] Preferably, the immunogenic cell death inducer is doxorubicin DOX.

[0009] Preferably, the immune checkpoint inhibitor is delivered via ferritin HFn.

[0010] Most preferably, the immune checkpoint inhibitor is a CPG / DOX@PD-L1-HFn complex.

[0011] The present invention also provides a method for preparing an immune checkpoint inhibitor, comprising the following steps:

[0012] The chimeric protein pHFn(+) was designed using the PD-L1 inhibitory peptide, MMP2 cleavage site and positive charge in the ferritin cavity; DOX was then loaded into the DNA double strand of CpG to obtain a CpG / DOX complex; CpG / DOX was encapsulated into the chimeric protein pHFn(+) by the acid assembly method to obtain a CpG / DOX@PD-L1-HFn complex.

[0013] The present invention also provides an application of an immune checkpoint inhibitor in the preparation of a lung cancer immunotherapy drug.

[0014] Depend on Figure 1 As shown, after the pHFn(+)@CpG / DOX complex enters the blood system, it is degraded by matrix metalloproteinases (MMPs) to release the PD-L1 blocker, and the complex is decomposed into the PD-L1 blocker and HFn(+)@CpG / DOX. Among them, HFn(+)@CpG / DOX is recognized by lung cancer cells through specific binding to transferrin receptor 1 (TfR1). After recognition, HFn(+)@CpG / DOX enters lung cancer cells through lysosomal internalization. HFn(+)@CpG / DOX entering the cell will release double-stranded DNA containing CPG oligonucleotide sequence and doxorubicin (DOX). The former acts as an immunomodulator to exert immunomodulatory effects, while the latter induces immunogenic death of lung cancer cells. The immunomodulatory effect of the double-stranded DNA of the CPG oligonucleotide sequence will synergistically block the immune checkpoints with the PD-L1 blocker released outside the tumor cells in the early stage, thereby exerting combined immunotherapy to kill lung cancer cells.

[0015] Beneficial Effects

[0016] The immune checkpoint inhibitor of the present invention can effectively stimulate the innate immune system and enhance the anti-tumor immune response. Experimental verification shows that it can be used as a promising new drug for lung cancer immunotherapy and has good market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the principle of the immune checkpoint inhibitor of the present invention.

[0018] Figure 2AI is a characterization of the immune checkpoint inhibitors of the present invention.

[0019] Figure 3 AC is the in vivo distribution test of the immune checkpoint inhibitors of the present invention.

[0020] Figure 4 AE is an in vivo efficacy test of the immune checkpoint inhibitors of the present invention.

[0021] Figure 5 AD is the safety assessment of the immune checkpoint inhibitors of the present invention.

[0022] Figure 6 AF is a test for the immune microenvironment regulation effect of the immune checkpoint inhibitor of the present invention. DETAILED DESCRIPTION

[0023] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0024] Example 1

[0025] 1. Preparation and identification of pHFn(+)@CpG / DOX

[0026] In this study, a chimeric protein pHFn(+) was designed using the PD-L1 inhibitory peptide (CNYSKPTDRQYHF, CF13), the MMP2 cleavage site, and the positive charge in the ferritin cavity. A His×6 tag was introduced at the N-terminus to facilitate downstream protein purification. The target protein was expressed in Escherichia coli and purified by Q ion exchange column and Ni 2+ The pHFn(+) protein was purified by affinity chromatography and then identified by SDS-PAGE and Western Blot analysis ( Figure 2 A). Reverse phase HPLC further confirmed that the purity of pHFn(+) exceeded 90%. To further verify the responsiveness of MMP2 in nanoparticles, pHFn(+) was verified in real time by MMP2 enzymatic cleavage reaction. Subsequently, SEC-HPLC was used to monitor the enzymatic cleavage products ( Figure 2 B). Subsequently, DOX was loaded into the DNA double strand of CpG. Since DOX would produce fluorescence quenching effect after being embedded in the GC base pair of DNA, the fluorescence intensity of DOX in the solution was monitored under gradient concentration and fixed DOX concentration, and the DOX loading capacity of CpG was calculated. Figure 2As shown in Figure C, when the molar ratio of DOX to PG was 40:1, fluorescence stability was achieved, indicating that each PG molecule was able to encapsulate 40 DOX molecules to form a CpG / DOX complex. After incubation at a ratio of 40:1 for 48 h, the encapsulation efficiency of DOX reached a maximum of 87.0 ± 3.3% ( Figure 2 D). Therefore, incubation of CpG and DOX at a ratio of 40:1 for 48 hours is the optimal condition for preparing CpG / DOX.

[0027] Next, CpG / DOX was encapsulated into pHFn(+) nanoparticles by the acid assembly method. SEC-HPLC further identified that pHFn(+)@CpG / DOX eluted at 5.87 min, with absorption peaks at 280 nm (protein indicator), 260 nm (nucleic acid indicator), and 485 nm (DOX indicator), confirming that pHFn(+) nanoparticles successfully encapsulated CpG / DOX( Figure 2 E). pHFn(+)@CpG / DOX was then characterized using TEM and DLS ( Figure 2 F and G). TEM results describe pHFn(+)@CpG / DOX as uniform spherical particles. DLS results show that the particle size of pHFn(+)@CpG / DOX is 20.5 nm. In addition, Zeta potential results show that the surface potential of pHFn(+)@CpG / DOX is lower than that of HCD, which means that it may have better dispersion stability in solution due to lower aggregation tendency ( Figure 2 H). Finally, pHFn(+)@CpG / DOX was incubated with PBS buffer at two different pH values ​​(pH 7.4 and pH 5.0) at 37 °C for 0, 2, 5, 12, 24, 48, and 72 h. The DOX fluorescence intensity was evaluated using an enzyme labeling instrument. Figure 2 As shown in Figure 1, DOX was significantly released at pH 5.0 (lysosomal acidic pH), but only a small amount was released at pH 7.4 (neutral pH of blood), indicating the stability of DOX loaded with pHFn(+)@CpG / DOX in the blood and its large release when entering the lysosomes of tumor cells.

[0028] 2. In vivo antitumor activity of pHFn(+)@CpG / DOX

[0029] LLC cells were subcutaneously injected into the right forelimb of female BALB / c nude mice to establish a tumor-bearing mouse model. 3 Cy5-labeled pHFn(+) was injected intravenously at 1 hr and in vivo imaging was performed at 0.5, 1, 2, 3, 6, 12, 24, and 48 h after injection. Figure 3As shown in A, free Cy5 did not show significant tumor targeting ability. In contrast, pHFn(+) showed excellent tumor targeting ability, as demonstrated by imaging of the heart, liver, spleen, lung, kidney, and tumors ( Figure 3 B) and fluorescence quantification ( Figure 3 C) confirms this.

[0030] Continue with LLC tumor-bearing mouse model, when the tumor volume reaches 100mm 3 Mice were randomly divided into groups and intravenously injected with PBS, HFn(+), HFn(+)@CpG, HFn(+)@CpG / DOX, or pHFn(+)@CpG / DOX every three days for a total of 5 doses ( Figure 4 A) Tumor volume results Figure 4 As shown in B, the HFn(+)@CpG / DOX group and the pHFn(+)@CpG / DOX group had significant tumor inhibition effects, and the latter showed better therapeutic effects. Figure 4 C It can be seen that except for the DOX group and the GD group, there was no significant change in the body weight of mice in each group. Subsequent survival analysis showed that the survival rate of the HFn(+)@CpG / DOX group was 60%, and the survival rate of the pHFn(+)@CpG / DOX group was 100% ( Figure 4 D) After the experiment, the mice were euthanized and the tumor tissues were stained with H&E and TUNEL. Figure 4 As shown in E, compared with other groups, the tumor tissue necrosis in the pHFn(+)@CpG / DOX group was more severe.

[0031] To evaluate the potential safety risks of pHFn(+)@CpG / DOX, H&E staining was performed on the heart, liver, spleen, lung, and kidney of each group of mice after treatment. Figure 5 As shown in A, no obvious tissue lesions were found in each group. Further, the liver and kidney function-related indicators of mice in each group were evaluated. Figure 5 BC results showed no significant abnormalities in liver and kidney function. Hematological examination results showed no bone marrow suppression ( Figure 5 D). These results indicate that pHFn(+)@CpG / DOX has good antitumor activity in vivo and has no significant effect on normal organ tissues.

[0032] 3. Regulatory effect of pHFn(+)@CpG / DOX in TME

[0033] To elucidate the role of pHFn(+)@CpG / DOX in inducing ICD in vivo, immunofluorescence staining of mouse tumor tissues was performed to evaluate the expression of CRT and HMGB1. Figure 6As shown in A, the HMGB1 level in the pHFn(+)@CpG / DOX treatment group was significantly decreased compared with the PBS and other treatment groups. In contrast, the CRT expression in the pHFn(+)@CpG / DOX treatment group was significantly increased, exceeding that of the PBS and other treatment groups. This indicates that pHFn(+)@CpG / DOX can effectively induce ICD in tumor tissues. In addition, the activation of DC cells in the draining lymph nodes was evaluated by flow cytometry. Figure 6 As shown in Figure B, in the pHFn(+)@CpG / DOX-treated group, the proportion of CD11c+CD80+CD86+ cells reached 29.4%, which was significantly higher than that in the PBS group (2.5%) and the HFn(+)@CpG / DOX group (16.7%). Subsequently, we detected the infiltration of CD8+T cells (effector T cells) and CD25+T cells (immunosuppressive T cells) in tumor tissues. Figure 6 As shown in C, compared with the PBS treatment group, the infiltration of CD8+T cells in the pHFn(+)@CpG / DOX treatment group increased significantly, while the infiltration of CD25+T cells decreased significantly. This indicates that pHFn(+)@CpG / DOX effectively activates the anti-tumor immune response of T cells in tumor tissues. Further flow cytometry results are shown in Figure 6 As shown in D, the proportion of CD3+CD8+ cells in pHFn(+)@CpG / DOX reached 24.3%, which was significantly higher than that in the PBS group (10.9%) and the HFn(+)@CpG / DOX group (17.6%). At the same time, the secretion of cytokines TGF-β, TNF-α, IL-2, and IFN-γ in tumor tissues was detected. Figure 6 As shown in E, pHFn(+)@CpG / DOX promoted the secretion of TNF-α, IL-2, and IFN-γ, while inhibiting the secretion of TGF-β, indicating that the inflammatory and anti-tumor responses of tumor tissues were significantly enhanced. Figure 6 The ELISPOT results of F showed that pHFn(+)@CpG / DOX could stimulate the production of T cells with multiple tumor antigen specificity. Statistical analysis of immune spots showed that the number of IFN-γ+T cells in the pHFn(+)@CpG / DOX-treated group was 3.36 times that of the PBS group and 1.26 times that of the HFn(+)@CpG / DOX group, indicating that pHFn(+)@CpG / DOX could induce a stronger immune response level.

Claims

1. An immune checkpoint inhibitor, characterized in that: The immune checkpoint inhibitor is a complex composed of a PD-L1 inhibitory peptide, a TLR9 agonist and an immunogenic cell death inducer.

2. The immune checkpoint inhibitor according to claim 1, characterized in that: The sequence of the PD-L1 inhibitory peptide is CNYSKPTDRQYHF.

3. The immune checkpoint inhibitor according to claim 1, characterized in that: The TLR9 agonist is cytosine CPG.

4. The immune checkpoint inhibitor according to claim 1, characterized in that: The immunogenic cell death inducer is doxorubicin DOX.

5. The immune checkpoint inhibitor according to claim 1, characterized in that: The immune checkpoint inhibitors are delivered via ferritin HFn.

6. The immune checkpoint inhibitor according to claim 1, characterized in that: The immune checkpoint inhibitor is a CPG / DOX@PD-L1-HFn complex.

7. A method for preparing the immune checkpoint inhibitor according to any one of claims 1 to 6, comprising the following steps: The chimeric protein pHFn(+) was designed using the PD-L1 inhibitory peptide, MMP2 cleavage site and positive charge in the ferritin cavity; DOX was then loaded into the DNA double strand of CpG to obtain a CpG / DOX complex; CpG / DOX was encapsulated into the chimeric protein pHFn(+) by the acid assembly method to obtain a CpG / DOX@PD-L1-HFn complex.

8. Use of the immune checkpoint inhibitor according to any one of claims 1 to 6 in the preparation of immunotherapeutic drugs for lung cancer.

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