Fusion protein Ag85b-EPDL1 capable of automatically generating PD-L1 antibody and application of fusion protein Ag85b-EPDL1

Abstract

The invention provides a fusion protein Ag85b-EPDL1 capable of automatically generating a PD-L1 antibody and application of the fusion protein Ag85b-EPDL1, and belongs to the technical field of medicines. The fusion protein Ag85b-EPDL1 is obtained by fusing a protein Ag85b of mycobacterium tuberculosis (BCG) which has been used clinically with a PD-L1 extracellular domain, the fusion protein Ag85b-EPDL1 automatically generates a PD-L1 antibody by adopting a subcutaneous immune method, and the combination of PD-1 on the surface of a T cell and PD-L1 on the surface of a tumor cell can be blocked, so that the depletion of the T cell is reversed, the function of the in-vivo T cell is recovered, and the tumor cell activity is improved. According to the present invention, the tumor killing effect of the T cells can be recovered so as to achieve the anti-tumor effect, and the self-generated antibody can reduce the side effects such as high price, frequent drug use, short antibody maintenance time, cardiotoxicity and the like caused by the monoclonal antibody;

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a fusion protein Ag85b-EPDL1 that autogenously produces PD-L1 antibody and its application. Background Art

[0002] In the complex process of tumorigenesis and disease progression, T cell exhaustion significantly affects the efficacy of traditional cancer therapies. After tumorigenesis, the metastatic spread of tumor cells to surrounding tissues and the vascular system becomes the main cause of cancer-related death. The immune surveillance of tumor cells by immune cells (including tumor-specific CD8+ T cells) in the TME plays a crucial role in controlling metastasis.

[0003] Studies have confirmed that T cell exhaustion exists in cancer patients. T cell exhaustion refers to a progressive state in which T cells are functionally impaired due to continuous antigen stimulation, characterized by increased expression of immunosuppressive receptors, but weakened effector functions, reduced self-renewal ability, etc. General characteristics of T cell exhaustion include upregulated expression of immune checkpoints such as PD1, CTLA-4, TIM-3, and LAG-3. These immune checkpoints bind to their ligands, inhibiting T cell activation and immune responses. T cell exhaustion is one of the main causes of cancer immune escape. T cell exhaustion creates an environment that supports tumor development and metastatic spread. Impaired effector functions and a reduced number of CD8+ T cells in the TME lead to uncontrolled tumor growth and accelerated metastatic progression, reducing the patient's survival period.

[0004] Immune checkpoint inhibitors (ICIs) targeting programmed cell death 1 (PD-1) or its ligand 1 (PD-L1) have achieved great clinical success in anti-tumor treatment. For example, Nivolumab and Pembrolizumab targeting PD-1, and Atezolizumab and Durvalumab targeting PD-L1, have shown significant progress and remarkable efficacy in different cancer types. These drugs achieve therapeutic effects by disrupting the PD-1 / PD-L1 interaction, releasing T cells from inhibition, and restoring their anti-tumor activity. However, PD-1 / PD-L1 inhibitors can induce a wide range of immune-related adverse events (irAEs), among which cardiotoxicity is the most fatal adverse reaction. ICI-related cardiotoxicity events occur in different forms, including myocarditis, cardiomyopathy, and myocardial fibrosis. Multiple mechanisms of irAEs have been proposed, including cardiotoxicity, release of pro-inflammatory cytokines, and direct cytotoxic activity of T cells against non-tumor cells. Immune checkpoint inhibitors can induce damage to the intestinal mucosal barrier and intestinal barrier dysfunction in mice, leading to impaired cardiac function and further affecting the treatment effect of tumors.

[0005] Therefore, there is a need for a therapy that can replace monoclonal antibodies to inhibit tumor growth without causing the side effects of monoclonal antibodies, such as cardiotoxicity, colon damage, etc., and can maintain the PD-L1 antibody level for a long time. Summary of the Invention

[0006] Aiming at the defects in the prior art, the purpose of the present invention is to provide a fusion protein Ag85b-EPDL1, which can maintain the PD-L1 antibody level in the body for a long time, thereby facilitating tumor treatment, and at the same time can reduce the toxic and side effects of tumor treatment.

[0007] The purpose of the present invention is achieved by the following technical solutions:

[0008] The present invention provides a fusion protein Ag85b-EPDL1, and the amino acid sequence of the fusion protein Ag85b-EPDL1 is shown in SEQ ID NO.1.

[0009] The present invention provides a drug, comprising the fusion protein Ag85b-EPDL1 described in the above technical solution.

[0010] Preferably, the drug comprises a pharmaceutically acceptable carrier.

[0011] The present invention provides the application of the fusion protein Ag85b-EPDL1 described in the above technical solution or the drug described in the above technical solution in the preparation of PD-L1 antibodies.

[0012] The present invention provides the application of the fusion protein Ag85b-EPDL1 described in the above technical solution or the drug described in the above technical solution in the preparation of drugs for preventing and / or treating diseases related to T cell exhaustion.

[0013] Preferably, the diseases related to T cell exhaustion include any one or more of viral persistent infection diseases, bacterial persistent infections, and autoimmune diseases; the diseases include any one or more of acquired immunodeficiency syndrome, hepatitis C, and hepatitis B.

[0014] The present invention provides the application of the fusion protein Ag85b-EPDL1 described in the above technical solution or the drug described in the above technical solution in the preparation of anti-tumor drugs.

[0015] Preferably, the tumors include any one or more of melanoma, breast cancer, colon cancer, chronic myeloid leukemia, ovarian cancer, non-small cell cancer, Hodgkin lymphoma, and chronic lymphocytic leukemia.

[0016] The present invention provides a pharmaceutical composition, comprising the fusion protein Ag85b-EPDL1 described in the above technical solution and a drug active ingredient other than the fusion protein Ag85b-EPDL1.

[0017] Preferably, the pharmaceutically active ingredient includes anti-tumor drugs.

[0018] Advantages of the present invention:

[0019] The present invention provides a fusion protein Ag85b-EPDL1, and the amino acid sequence of the fusion protein Ag85b-EPDL1 is shown as SEQ ID NO.1. The present invention first fuses the protein Ag85b of Mycobacterium tuberculosis (BCG) that has been used clinically with the extracellular domain of PD-L1 to obtain the fusion protein Ag85b-EPDL1. Subcutaneous immunization with the fusion protein Ag85b-EPDL1 can enable the body to autogenously produce PD-L1 antibodies, thereby achieving an anti-tumor effect. Moreover, autogenous antibody production can reduce the side effects brought by monoclonal antibodies, such as high cost, frequent medication, short antibody maintenance time, cardiotoxicity, etc. The results of the examples show that the fusion protein Ag85b-EPDL1 provided by the present invention can maintain the PD-L1 antibody level in the body for a long time, and the PD-L1 antibody level in the body can be maintained for up to 21 days; it can significantly inhibit the growth of tumor cells and can also reduce the toxic and side effects of monoclonal antibodies on the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0021] Figure 1 It is a diagram of the expression identification result of the fusion protein Ag85b-EPDL1;

[0022] Figure 2 It is a diagram of the optimization and solubility analysis results of the fusion protein Ag85b-EPDL1;

[0023] Figure 3 It is a diagram of the SDS-PAGE result of the affinity purification of the fusion protein Ag85b-EPDL1;

[0024] Figure 4 It is a diagram of the SDS-PAGE result of the renaturation of the fusion protein Ag85b-EPDL1;

[0025] Figure 5 It is a structural diagram of the fusion protein Ag85b-EPDL1;

[0026] Figure 6 It is a purification verification diagram of the fusion protein Ag85b-EPDL1;

[0027] Figure 7 Graph of the in vivo maintenance time of the PD-L1 antibody after injection of the fusion protein Ag85b-EPDL1 in Example 2;

[0028] Figure 8 Graph of the in vivo maintenance time of the PD-L1 antibody after injection of the anti-PD-L1 monoclonal antibody in Example 2;

[0029] Figure 9 Graph of the antibody production levels in the sera of immunized mice at different dilution multiples in Example 3;

[0030] Figure 10 Graph of the results of the PD-L1 / PD-1 blockade assay in Example 4;

[0031] Figure 11 Graph of the results of the effects of different doses of the fusion protein Ag85b-EPDL1 on the tumor size of tumor-bearing mice at different time periods in Example 5;

[0032] Figure 12 Graph of the results of the effects of different doses of the fusion protein Ag85b-EPDL1 on the tumor weight of tumor-bearing mice in Example 5;

[0033] Figure 13 Graph of the HE staining results of the brains of the control group mice and the mice treated with 10 μg of the fusion protein Ag85b-EPDL1 in Example 5;

[0034] Figure 14 Graph of the HE staining results of the hearts of the control group mice and the mice treated with 10 μg of the fusion protein Ag85b-EPDL1 in Example 5;

[0035] Figure 15 Graph of the HE staining results of the kidneys of the control group mice and the mice treated with 10 μg of the fusion protein Ag85b-EPDL1 in Example 5;

[0036] Figure 16 Graph of the HE staining results of the lungs of the control group mice and the mice treated with 10 μg of the fusion protein Ag85b-EPDL1 in Example 5;

[0037] Figure 17 Graph of the HE staining results of the spleens of the control group mice and the mice treated with 10 μg of the fusion protein Ag85b-EPDL1 in Example 5;

[0038] Figure 18 Graph of the HE staining results of the livers of the control group mice and the mice treated with 10 μg of the fusion protein Ag85b-EPDL1 in Example 5;

[0039] Figure 19HE staining results of tumors of mice in the control group of Example 5 and mice treated with 10 μg of the fusion protein Ag85b-EPDL1;

[0040] Figure 20 Results of continuous monitoring of tumor growth and survival period of the control group in Example 6;

[0041] Figure 21 Results of continuous monitoring of tumor growth and survival period of the monoclonal antibody group in Example 6;

[0042] Figure 22 Results of continuous monitoring of tumor growth and survival period of the fusion protein group in Example 6;

[0043] Figure 23 Results of detection of LDH and AST in the sera of mice in each experimental group of Example 6. Detailed implementation manners

[0044] The present invention provides a fusion protein Ag85b-EPDL1, and the amino acid sequence of the fusion protein Ag85b-EPDL1 is as shown in SEQ ID NO.1. The nucleotide sequence encoding the Ag85b-EPDL1 fusion protein of the present invention is as shown in SEQ ID NO.2.

[0045] In the present invention, the tertiary structure of PD-L1 consists of an extracellular domain (ECD), a transmembrane domain and an intracellular region. Since the ECD is responsible for binding to PD-1, the extracellular domain (ECD) is selected as the immunogen, and the ECD is fused with the mycobacterium tuberculosis protein Ag85b to design the fusion protein Ag85b-EPDL1.

[0046] The present invention fuses the protein Ag85b of mycobacterium tuberculosis (BCG) that has been used clinically with the extracellular domain of PD-L1 to design the fusion protein Ag85b-EPDL1. The fusion protein Ag85b-EPDL1 adopts a subcutaneous immunization method to autogenously produce PD-L1 antibodies, which can block the binding of PD-1 on the surface of T cells to PD-L1 on the surface of tumor cells, thereby reversing T cell exhaustion, restoring the function of T cells in the body, restoring the killing of tumors by T cells, and further realizing the anti-tumor effect. Moreover, autogenous antibody production can reduce the side effects brought by monoclonal antibodies, such as high price, frequent medication, short antibody maintenance time, cardiotoxicity, etc.

[0047] In the present invention, the fusion protein Ag85b-EPDL1 can be subcutaneously injected at multiple points. According to the curve graph of the antibody maintenance level in the body, subcutaneous immunization is carried out once every three days to maintain a high level of PD-L1 antibodies in the body to restore the function of T cells in the body by reversing T cell exhaustion and play a therapeutic role.

[0048] The present invention provides a drug, which comprises the fusion protein Ag85b-EPDL1 described in the above technical solution. In the present invention, the drug may comprise a pharmaceutically acceptable carrier.

[0049] The present invention provides the use of the fusion protein Ag85b-EPDL1 described in the above technical solution or the drug described in the above technical solution in the preparation of a PD-L1 antibody. In the present invention, when the fusion protein Ag85b-EPDL1 is injected into an organism, it can enable the organism to produce a PD-L1 antibody, and the PD-L1 antibody can be maintained in the body for a relatively long time, up to 21 days.

[0050] The present invention provides the use of the fusion protein Ag85b-EPDL1 described in the above technical solution or the drug described in the above technical solution in the preparation of a drug for preventing and / or treating diseases related to T cell exhaustion. The fusion protein Ag85b-EPDL1 of the present invention can block the binding of PD-1 on the surface of T cells to PD-L1 on the surface of tumor cells by generating a PD-L1 antibody, reverse T cell exhaustion, and restore the function of T cells in the body. In the present invention, the diseases related to T cell exhaustion include tumors. In the present invention, the diseases related to T cell exhaustion include any one or more of viral persistent infection diseases, bacterial persistent infections, and autoimmune diseases; the diseases include any one or more of acquired immunodeficiency syndrome, hepatitis C, and hepatitis B.

[0051] The present invention provides the use of the fusion protein Ag85b-EPDL1 described in the above technical solution or the drug described in the above technical solution in the preparation of an anti-tumor drug. The anti-tumor in the present invention includes preventing and / or treating tumors. In the present invention, the tumors include any one or more of melanoma, breast cancer, colon cancer, chronic myeloid leukemia, ovarian cancer, non-small cell carcinoma, Hodgkin lymphoma, and chronic lymphocytic leukemia. The results of the examples of the present invention show that the fusion protein Ag85b-EPDL1 can significantly inhibit the growth of melanoma and breast cancer tumors, and reduce the toxic and side effects on the body and the mortality rate of mice.

[0052] The present invention provides a pharmaceutical composition, which comprises the fusion protein Ag85b-EPDL1 described in the above technical solution and a drug active ingredient other than the fusion protein Ag85b-EPDL1. In the present invention, the drug active ingredient includes an anti-tumor drug. The fusion protein Ag85b-EPDL1 provided by the present invention can be used in combination with other anti-tumor drugs to enhance the anti-tumor effect.

[0053] In order to further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

[0054] Example 1

[0055] 1. Fusion protein design

[0056] The tertiary structure of PD-L1 consists of an extracellular domain (ECD), a transmembrane domain, and an intracellular region. Since the ECD is responsible for binding to PD-1, the extracellular domain ECD was selected as the immunogen. The ECD was fused with the Mycobacterium tuberculosis protein Ag85b, and the fusion protein was named Ag85b-EPDL1.

[0057] The amino acid sequence of Ag85b-EPDL1 is shown in SEQ ID NO.1, specifically:

[0058] MTDVSRKIRAWGRRLMIGTAAAVVLPGLVGLAGGAATAGAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGNNSPAVYLLDGLRAQDDYNGWDINTPAFEWYYQSGLSIVMPVGGQSSFYSDWYSPACGKAGCQTYKWETFLTSELPQWLSANRAVKPTGSAAIGLSMAGSSAMILAAYHPQQFIYAGSLSALLDPSQGMGPSLIGLAMGDAGGYKAADMWGPSSDPAWERNDPTQQIPKLVANNTRLWVYCGNGTPNELGGANIPAEFLENFVRSSNLKFQDAYNAAGGHNAVFNFPPNGTHSWEYWGAQLNAMKGDLQSSLGAGGGGGSGGGGSGGGGSFTITAPKDLYVVEYGSNVTMECRFPVERELDLLALVVYWEKEDEQVIQFVAGEEDLKPQHSNFRGRASLPKDQLLKGNAALQITDVKLQDAGVYCCIISYGGADYKRITLKVNAPYRKINQRISVDPATSEHELICQAEGYPEAEVIWTNSDHQPVSGKRSVTTSRTEGMLLNVTSSLRVNATANDVFYCTFWRSQPGQNHTAELIIPELPATHPPQNRTHLEHHHHHH*.

[0059] 2. Fusion protein preparation

[0060] The preparation and purification of the fusion protein Ag85b-EPDL1 was entrusted to Tianjin Mederma Biotechnology Co., Ltd. The relevant steps are as follows:

[0061] 1) Preparation of recombinant strains

[0062] The nucleotide sequence encoding the Ag85b-EPDL1 fusion protein is shown in SEQ ID NO.2, specifically:

[0063]

[0064] Cloning vector and restriction sites: pET-22b(+) NdeI-XhoI. A recombinant vector was prepared using the cloning vector and restriction sites, and after obtaining the recombinant vector, the recombinant vector was introduced into competent cells to obtain recombinant strains.

[0065] 2) Protein expression and purification of recombinant strains

[0066] (1) Activation of recombinant strains. After transferring the recombinant strains, they were cultured overnight at 37 °C to obtain the transferred recombinant strains.

[0067] (2) Expression identification, optimization and solubility analysis

[0068] (i) The transferred recombinant strains were propagated in 5 tubes of LB medium and cultured at 37 °C until the OD of the bacteria 600 was 0.6 - 0.8. The expression of the fusion protein was induced by adding IPTG to a final concentration of 0.5 mM, and after culturing at 37 °C for 4 h, the bacteria were centrifuged, collected, and prepared for SDS-PAGE and Western Blot analysis. The results were as Figure 1 shown. Figure 1 In lane M of [reference] is Protein Marker; lane 1 is the uninduced sample; lanes 2 - 6 are the induced samples.

[0069] (ii) The transferred recombinant strains were propagated in LB medium and cultured at 37 °C until the OD of the bacteria 600 was 0.6 - 0.8. Four tubes of the culture were taken:

[0070] IPTG was added to a final concentration of 0.2 mM in 2 tubes of the culture. One tube was cultured at 37 °C at 220 rpm for 4 h; one tube was cultured at 15 °C at 220 rpm for 16 h.

[0071] IPTG was added to a final concentration of 1 mM in 2 tubes of the culture. One tube was cultured at 37 °C at 220 rpm for 4 h; one tube was cultured at 15 °C at 220 rpm for 16 h.

[0072] The expression of the fusion protein was induced, and samples were prepared for SDS-PAGE analysis under each condition. The bacterial liquid of each condition was centrifuged to collect the bacteria, which were lysed (Tris-NaCl buffer), and samples were prepared from the supernatant and precipitate respectively for SDS-PAGE analysis.

[0073] The SDS-PAGE electrophoresis results were as Figure 2 shown. Among them, Figure 2 the information of each lane in [reference] is shown in Table 1.

[0074] Table 1 Explanation of the information in the SDS-PAGE electrophoresis result diagram

[0075] Lane M: Protein Marker Lane 6: Precipitation sample after induction with 1.0 mM IPTG at 37°C Lane 1: Sample after induction with 0.2 mM IPTG at 15°C Lane 7: Supernatant sample after induction with 1.0 mM IPTG at 37°C Lane 2: Sample after induction with 1.0 mM IPTG at 15°C Lane 8: Precipitation sample after induction with 0.2 mM IPTG at 37°C Lane 3: Sample after induction with 0.2 mM IPTG at 37°C Lane 9: Supernatant sample after induction with 0.2 mM IPTG at 37°C Lane 4: Sample after induction with 1.0 mM IPTG at 37°C Lane 10: Precipitation sample after induction with 1.0 mM IPTG at 15°C Lane 5: Uninduced sample Lane 11: Supernatant sample after induction with 1.0 mM IPTG at 15°C - Lane 12: Precipitation sample after induction with 0.2 mM IPTG at 15°C - Lane 13: Supernatant sample after induction with 0.2 mM IPTG at 15°C

[0076] As described above, the target protein was expressed as detected by SDS-PAGE and Western Blot. Solubility analysis: The target protein was insoluble as detected by SDS-PAGE.

[0077] (3) Scale-up expression and purification

[0078] The optimal expression clone strain was cultured in LB medium (1 L) at 37 °C until OD 600 = 0.6 - 0.8, induced at 37 °C for 4 h, and the cells were harvested;

[0079] After harvesting the cells, the cells were centrifuged again to collect the cells, resuspended, and sonicated.

[0080] After sonication, centrifugation was performed. The precipitate was dissolved in denaturing buffer and then centrifuged. The supernatant was purified by Ni column affinity chromatography. The reagents used for Ni column affinity chromatography are as follows:

[0081] Equilibration buffer: "Tris-NaCl-Urea" buffer, pH 8.0, specifically: 0.05 M Tris HCl, 2% SDS, 5 M Urea, 1% bmerceptoethanol (β-mercaptoethanol).

[0082] Washing buffer: "Tris-NaCl-Urea" buffer, pH 8.0 with 50 mM imidazole, specifically: 0.05 M Tris HCl, 2% SDS, 5 M Urea, 1% bmerceptoethanol 50 mM imidazole.

[0083] Elution buffer: "Tris-NaCl-Urea" buffer, pH 8.0 with 500 mM imidazole, specifically: 0.05 M Tris HCl, 2% SDS, 5 M Urea, 1% bmerceptoethanol 500 mM imidazole.

[0084] Each elution fraction was collected for SDS-PAGE electrophoresis analysis, and the analysis results are as Figure 3 shown. Among them, Figure 3 Lane M in is Protein Marker; Lane 1 is the precipitate after sonication; Lane 2 is the supernatant after sonication; Lane 3 is the flow-through; Lane 4 is the washing sample; Lane 5 is the elution sample.

[0085] As detected by SDS-PAGE, the target protein can be obtained by affinity purification.

[0086] (4) Protein refolding

[0087] The eluted sample was refolded and added to Buffer (50 mM Tris, 300 mM NaCl, 10% Glycerol, 2 mM DTT, pH 8.0), filtered and sterilized, and SDS-PAGE was performed to obtain the fusion protein Ag85b-EPDL1. The SDS-PAGE detection results of protein refolding are as Figure 4 shown. Figure 4 In the figure, lane M is Protein Marker; lane S is the refolded sample.

[0088] The structural schematic diagram of the fusion protein Ag85b-EPDL1 is as Figure 5 shown.

[0089] The verification diagram (by SDS-PAGE) of the fusion protein Ag85b-EPDL1 is as Figure 6 shown.

[0090] Example 2

[0091] Detection of the maintenance time of PD-L1 antibody

[0092] C57 mice were randomly divided into 3 experimental groups, namely the fusion protein experimental group, the PD-L1 monoclonal antibody group and the control group, with 5 mice in each group.

[0093] Among them, in the fusion protein experimental group: 50 μg of the fusion protein Ag85b-EPDL1 was subcutaneously injected into C57 mice (C57BL / 6J mice).

[0094] In the PD-L1 monoclonal antibody group: 100 μg of the PD-L1 monoclonal antibody [bioxcell anti-mouse PDL1 (B7-H1) clone number 10F.9G2] was injected into the tail vein of C57 mice.

[0095] In the control group: The mice were injected with PBS of the same volume as the fusion protein Ag85b-EPDL1 Z injection solution in the fusion protein experimental group.

[0096] In the fusion protein experimental group, the PD-L1 monoclonal antibody group and the control group, blood was collected from the submandibular vein before injection, and blood was collected from the submandibular vein on the 4th, 7th, 10th, 14th, 21st, and 28th days after immunization. The collected blood was centrifuged at 6000 rpm for 5 min, and the serum was taken and stored at -20 °C for subsequent antibody level detection.

[0097] Antibody level detection:

[0098] Add 5 μg / mL of recombinant PD-L1 protein [Recombinant Mouse PD-L1 (C-6His) (Cat.No.: CJ88)] to a 96-well plate, 100 μL per well, and incubate overnight at 4°C. The next day, take out the 96-well plate and wash it three times with PBS and Tween 20 (0.1% PBST), 3 minutes each time. After washing, pat it dry and block it with 5% skim milk powder. Place it at 37°C for 40 minutes and then wash it three times with PBST, standing for 3 minutes each time. After washing, pat it dry. Add 100 μL / well of the diluted serum dilutions obtained from different time samplings of mice in each test group in sequence, and set up blank control wells (without adding mouse serum) at the same time. Place it at 37°C for 1 hour. The washing steps are the same as above (wash three times with PBST, standing for 3 minutes each time). After washing, add 100 μL / well of the goat anti-mouse antibody labeled with horseradish peroxidase (HRP) (volume ratio 1:5000) dilution and place it at 37°C for 1 hour. After placement, wash it, and the washing steps are the same as above (wash three times with PBST, standing for 3 minutes each time). After washing, add 100 μL / well of TMB chromogenic solution, incubate at room temperature in the dark for 15 minutes, then add the TMB chromogenic termination solution, and measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader.

[0099] The in vivo maintenance time of the PD-L1 antibody after injecting the fusion protein Ag85b-EPDL1 and the PD-L1 antibody after injecting the PD-L1 monoclonal antibody is as Figures 7 - 8 shown. Figure 7 is the in vivo maintenance time graph of the PD-L1 antibody after injecting the fusion protein Ag85b-EPDL1; Figure 8 is the in vivo maintenance time graph of the PD-L1 antibody after injecting the PD-L1 monoclonal antibody. Figures 7 - 8 In the [figure], the ordinate is the content of the PD-L1 antibody, the unit is μg / mL, the abscissa is the time after antibody injection, and pre is the detection level of the PD-L1 antibody in the mouse serum before injecting the fusion protein.

[0100] From Figures 7 - 8 it can be seen that the PD-L1 antibody in mice immunized with the fusion protein is maintained in vivo for a long time, while the PD-L1 antibody in mice injected with the PD-L1 monoclonal antibody has returned to the baseline level at 24 h.

[0101] Example 3

[0102] Antibody production levels in the sera of immunized mice at different dilution multiples

[0103] Subcutaneously inject 50 μg of the fusion protein Ag85b-EPDL1 into C57 mice (C57BL / 6J mice). On the fourth day after immunization, take the sera of C57 mice subcutaneously immunized with 50 μg of the fusion protein Ag85b-EPDL1, and dilute the sera 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, and 1000-fold respectively.

[0104] Add 5 μg / mL recombinant PD-L1 protein to a 96-well plate, 100 μL per well, and incubate overnight at 4°C. The next day, take out the 96-well plate and wash it three times with PBS and Tween 20 (0.1% PBST), 3 minutes each time. After washing, pat it dry and block it with 5% skim milk powder. After placing it at 37°C for 40 minutes, wash it 3 times with PBST, let it stand for 3 minutes each time, and pat it dry after washing.

[0105] Add 100 μL / well of the diluted immune mouse serum in sequence.

[0106] At the same time, set up control wells and add non-immune mouse serum (the treatment method of non-immune mice is the same as in Example 2).

[0107] And set up wells adding PD-L1 monoclonal antibody as positive control. Specifically: add 100 μL of 20 μg / mL PD-L1 monoclonal antibody. The PD-L1 monoclonal antibody is the same as in Example 2, which is [bioxcell anti-mouse PDL1 (B7-H1) clone number 10F.9G2].

[0108] Place it at 37°C for 1 h. The washing steps are the same as above. After washing, add 100 μL / well of the dilution of goat anti-mouse antibody labeled with horseradish peroxidase (HRP) (volume ratio 1:5000) and place it at 37°C for 1 h. After placing, wash it. The washing steps are the same as above. Add 100 μL / well of TMB chromogenic solution, incubate at room temperature in the dark for 15 min, then add TMB chromogenic termination solution, and measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader.

[0109] The antibody production levels of immune mouse sera at different dilution multiples are shown in Table 2 and Figure 9 as follows. Figure 9 The anti-PDL1 mAb in [[ ]] is the detection result of the well adding PD-L1 monoclonal antibody. 20x, 50x, 100x, 200x, 500x, 1000x are the detection results of sera at different dilution multiples after immunization with the fusion protein Ag85b-EPDL1. PBS is the detection result of the control well. 20x means dilution by 20 times, and so on.

[0110] Table 2 Antibody production levels of immune mouse sera at different dilution multiples

[0111]

[0112]

[0113] From Table 1 and Figure 9It can be obtained that the antibody level in the serum after immunization with the fusion protein Ag85b-EPDL1 is equivalent to that of the PDL1 antibody in 100 μL of 20 μg / mL PDL1 monoclonal antibody after the serum is diluted 20-fold, indicating the level of antibody production after immunization with the fusion protein.

[0114] Example 4

[0115] PD-L1 / PD-1 Blocking Assay

[0116] (1) Preparation of immunized mice: C57 mice were subcutaneously injected with 50 μg of the fusion protein Ag85b-EPDL1. On the fourth day after immunization, the sera of C57 mice subcutaneously immunized with 50 μg of the fusion protein Ag85b-EPDL1 were taken and diluted 10-fold, 20-fold, 100-fold, and 200-fold, respectively. Then, the 10-fold serum dilution (10x), 20-fold serum dilution (20x), 100-fold serum dilution (100x), and 200-fold serum dilution (200x) were used for subsequent experiments.

[0117] (2) 5 μg / mL of recombinant PD-L1 protein was added to each well of a 96-well plate at 100 μL per well and incubated overnight at 4°C. The next day, the 96-well plate was taken out and washed three times with PBS and Tween 20 (0.1% PBST), 3 min each time. After washing, it was patted dry and then blocked with 5% skim milk powder and placed at 37°C for 40 min. Then, it was washed three times with PBST, 3 min of standing each time. After washing, it was patted dry. The 96-well plate was divided into 4 experimental groups, 2 control groups, and a PDL1 monoclonal antibody group. The experimental groups were added with the serum dilutions obtained in (1), and the addition amount of the serum dilution was 100 μL / well; in one of the 2 control wells, non-immunized mouse serum was added, and in the other control well, no serum was added as a blank well; in the PDL1 monoclonal antibody group, 20 μg / mL of PDL1 monoclonal antibody was directly added to the wells at 100 μL / well. Subsequently, it was placed at 37°C for 1 h. The washing steps were the same as above. Then, 100 μL of 2.5 μg / mL streptavidin-labeled PD1 was added to each well, and after incubation at 37°C for 1 h, the washing steps were the same as above. 100 μL / well of the dilution of goat anti-mouse antibody labeled with Streptavidin-HRP (Cat.#STN-NH913, AcroBiosystems) (1:500) was added and placed at 37°C for 1 h. The washing steps were the same as above. 100 μL / well of TMB chromogenic solution was added, and after incubation at room temperature in the dark for 15 min, the TMB chromogenic termination solution was added, and then the absorbance was measured at 450 nm with an ELISA reader.

[0118] Relative inhibition rate = (1 - OD value of experimental group ÷ OD value of blank well) × 100.

[0119] The results of the PD-L1 / PD-1 blocking assay are shown in Table 3 and Figure 10 as follows.

[0120] Table 3 Results of PD-L1 / PD-1 Blocking Assay

[0121] Group Relative inhibition rate Relative inhibition rate Relative inhibition rate 10× 75.80148 61.99137 61.52898 20× 61.34402 71.42417 63.81011 100× 39.11837 20.96178 34.5561 200× 27.34279 25.33909 25.09248 PBS 20.31443 20.43773 12.97781 aPD - L1 36.83724 46.23921 49.66091

[0122] As shown in Table 3 and Figure 10 it can be seen that immunization with the fusion protein Ag85b-EPDL1 can block the binding of PD-L1 to PD-1.

[0123] Example 5 Therapeutic Effects of Different Doses of Fusion Protein in C57BL / 6F10 Tumor-Bearing Mice

[0124] B16F10 cells in logarithmic growth phase were trypsinized, centrifuged, resuspended in PBS for cell counting. 750,000 B16F10 cells were subcutaneously implanted into 4- to 5-week-old C57 mice. Among them, 750,000 B16F10 cells were resuspended in 100 μL of PBS, that is, for 4- to 6-week-old C57 mice, each mouse was subcutaneously injected with 7.5×10 5 cells, and the volume of the injected cell suspension was 100 μL.

[0125] On the 5th day after subcutaneous tumor implantation, different doses of the fusion protein Ag85b-EPDL1 or PBS (injection of PBS as a control) were subcutaneously injected for the first time. The injection amounts of the fusion protein Ag85b-EPDL1 were 5 μg, 10 μg, 25 μg, and 50 μg respectively. The second injection was on the 10th day after subcutaneous tumor implantation, and the injection amounts of the second injection were the same as those of each group in the first injection. The tumor growth was measured every two days starting from the 7th day after subcutaneous tumor implantation. On the 18th day after subcutaneous tumor implantation, the mice were euthanized, and the tumors and organs were taken and placed in fixative for subsequent experiments. After taking the tumors, the tumor weights were weighed.

[0126] The therapeutic effects of different doses of the fusion protein Ag85b-EPDL1 on tumor-bearing mice are as Figures 11 - 12 shown. Among them Figure 11 is the result graph of the influence of different doses of the fusion protein Ag85b-EPDL1 on the tumor size of tumor-bearing mice at different time periods; Figure 12 is the result graph of the influence of different doses of the fusion protein Ag85b-EPDL1 on the tumor weight of tumor-bearing mice. 5, 10, 25, and 50 in the figure refer to 5 μg, 10 μg, 25 μg, and 50 μg.

[0127] From Figure 11 and Figure 12 it can be seen that with the increase of the injection dose of the fusion protein Ag85b-EPDL1, the tumor inhibitory effect is stronger.

[0128] The brains, hearts, kidneys, lungs, spleens, livers, and tumor tissues of the control group mice and the mice treated with 10 μg of the fusion protein Ag85b-EPDL1 were subjected to HE staining, and the staining results are as Figures 13 - 19As shown in the figure. Among them Figure 13 is the HE staining result diagram of the brains of control group mice and mice treated with 10 μg of the fusion protein Ag85b-EPDL1. Figure 14 is the HE staining result diagram of the hearts of control group mice and mice treated with 10 μg of the fusion protein Ag85b-EPDL1. Figure 15 is the HE staining result diagram of the kidneys of control group mice and mice treated with 10 μg of the fusion protein Ag85b-EPDL1. Figure 16 is the HE staining result diagram of the lungs of control group mice and mice treated with 10 μg of the fusion protein Ag85b-EPDL1. Figure 17 is the HE staining result diagram of the spleens of control group mice and mice treated with 10 μg of the fusion protein Ag85b-EPDL1. Figure 18 is the HE staining result diagram of the livers of control group mice and mice treated with 10 μg of the fusion protein Ag85b-EPDL1. Figure 19 is the HE staining result diagram of the tumors of control group mice and mice treated with 10 μg of the fusion protein Ag85b-EPDL1.

[0129] It can be seen from Figures 13 - 19 that the fusion protein Ag85b-EPDL1 treatment group can effectively treat tumors without causing side effects on other organs.

[0130] Example 6 Therapeutic effect of the fusion protein Ag85b-EPDL1 in the Balbc orthotopic 4T1 triple-negative breast cancer model

[0131] Take logarithmically growing 4T1 cells, digest them with trypsin, centrifuge, resuspend them with PBS for cell counting, and inoculate 2×10 5 4T1-Luc cells into the left mammary fat pad of 4-6 week-old Balbc mice. The volume of the 2×10 5 4T1-Luc cell suspension is 30 μL, and after injection, suture with surgical thread. After confirming successful model establishment, divide the mice into 3 experimental groups, with 6 mice in each experimental group. They are the model control group, the monoclonal antibody group, and the fusion protein group.

[0132] Among them, in the fusion protein group, on the 5th day after tumor inoculation, 10 μg of the fusion protein Ag85b-EPDL1 was subcutaneously injected into the mice, and the injection was given once every three days.

[0133] The monoclonal antibody group was injected with the PD-L1 monoclonal antibody [bioxcell anti-mouse PDL1 (B7-H1) clone number 10F.9G2]. Starting from the 5th day after tumor inoculation, it was injected via the tail vein once every two days, and the injection volume each time was 100 μg / mouse.

[0134] The model control group was injected with PBS.

[0135] 1. Small animal imaging: First, each Balbc mouse was anesthetized by intraperitoneal injection of 100 μL of 5% chloral hydrate. After anesthesia, 200 μL / mouse of 15 mg / mL D-luciferin potassium salt was injected for small animal imaging, and the tumor growth and survival period were continuously monitored. The monitoring results are as Figures 20 - 22 shown. Among them, Figure 20 is the continuous monitoring result chart of tumor growth and survival period in the control group; Figure 21 is the continuous monitoring result chart of tumor growth and survival period in the monoclonal antibody group; Figure 22 is the continuous monitoring result chart of tumor growth and survival period in the fusion protein group. The × in the figure indicates the death of the mouse.

[0136] From Figures 20 - 22 the results, it shows that the tumor volume was inhibited after treatment with the fusion protein Ag85b-EPDL1, and the survival period was the longest. Due to the toxicity of the monoclonal antibody, the survival period in the monoclonal antibody group was shortened.

[0137] 2. On the 21st day after tumor inoculation, the sera of the surviving mice were taken to detect LDH and AST in the mouse sera. The detection results are shown in Tables 4 - 5 and Figure 23 shown. Among them, anti PDL1 mAb corresponds to the monoclonal antibody group; Ag85B-EPDL1 corresponds to the fusion protein group; PBS corresponds to the model control group.

[0138] Table 4 Detection results of AST in the sera of mice in each test group

[0139]

[0140] Table 5 Detection results of LDH in the sera of mice in each test group

[0141]

[0142] From Tables 4 - 5 and Figure 23 it can be obtained that AST and LDH in the sera of mice in the monoclonal antibody group were significantly higher than those in the fusion protein group and the model control group, while AST and LDH in the sera of mice in the fusion protein group were comparable to those in the model control group. It can be seen that the monoclonal antibody group will have a significant adverse effect on each organ, while the fusion protein will hardly have an adverse effect on each organ.

[0143] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments without creative work based on this embodiment, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A fusion protein Ag85b-EPDL1, characterized in that, The amino acid sequence of the fusion protein Ag85b-EPDL1 is shown in SEQ ID NO.

1.

2. A drug, characterized in that, It includes the fusion protein Ag85b-EPDL1 described in claim 1.

3. The drug according to claim 2, wherein The drug includes a pharmaceutically acceptable carrier.

4. Use of the fusion protein Ag85b-EPDL1 described in claim 1 or the drug described in claim 2 or 3 in the preparation of a PD-L1 antibody.

5. Use of the fusion protein Ag85b-EPDL1 described in claim 1 or the drug described in claim 2 or 3 in the preparation of a drug for preventing and / or treating diseases related to T cell exhaustion.

6. The application according to claim 5, wherein The diseases related to T cell exhaustion include any one or more of viral persistent infection diseases, bacterial persistent infections, and autoimmune diseases; the diseases include any one or more of AIDS, hepatitis C, and hepatitis B.

7. Use of the fusion protein Ag85b-EPDL1 described in claim 1 or the drug described in claim 2 or 3 in the preparation of an anti-tumor drug.

8. The application according to claim 7, wherein The tumors include any one or more of melanoma, breast cancer, colon cancer, chronic myeloid leukemia, ovarian cancer, non-small cell carcinoma, Hodgkin lymphoma, and chronic lymphocytic leukemia.

9. A pharmaceutical composition, characterized in that, It includes the fusion protein Ag85b-EPDL1 described in claim 1 and a drug active ingredient other than the fusion protein Ag85b-EPDL1.

10. The pharmaceutical composition according to claim 9, characterized in that, The drug active ingredient includes an anti-tumor drug.

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