PD-L1 single-chain antibody, PD-L1 targeting bionic exosome and application of PD-L1 single-chain antibody and PD-L1 targeting bionic exosome

Abstract

The invention provides a PD-L1 single-chain antibody. The PD-L1 single-chain antibody comprises a heavy chain variable region, a light chain variable region and a connecting peptide for connecting the heavy chain variable region and the light chain variable region. The invention further provides a PD-L1 targeting bionic exosome, the PD-L1 targeting bionic exosome comprises a bionic exosome and the PD-L1 single-chain antibody, the bionic exosome is formed by fusing a cancer cell-derived exosome and a liposome, and the PD-L1 single-chain antibody is coupled to the bionic exosome. The invention also provides an application of the PD-L1 targeting bionic exosome. The PD-L1 single-chain antibody and the PD-L1 targeting bionic exosome provided by the invention have obvious affinity activity on PD-L1 protein and have targeting specificity, so that the PD-L1 single-chain antibody and the PD-L1 targeting bionic exosome have good targeting and cancer treatment prospects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and specifically relates to a PD-L1 single-chain antibody and a PD-L1 targeting biomimetic exosome modified by the same. BACKGROUND

[0002] Ceritinib belongs to the second generation of tyrosine kinase inhibitor (TKI) drugs, and can inhibit the ALK signaling pathway of non-small cell lung cancer as a first-line drug for ALK-positive non-small cell lung cancer, thereby exerting a therapeutic effect. However, there are few reports on ceritinib in relation to pancreatic cancer. The only study shows that ceritinib and gemcitabine combined through the ALK signaling pathway to inhibit the proliferation, migration and invasion of pancreatic cancer. However, due to the poor water solubility of ceritinib, easy metabolism by CYP3A enzyme and P-gp-mediated efflux, the bioavailability of ceritinib is low. Based on these problems, it is necessary to construct a new tumor cell-targeted drug delivery system to achieve the efficient delivery of ceritinib to tumor parenchymal cells and the anti-cancer effect.

[0003] Based on the natural homing ability, high affinity, good stability and low immunogenicity of exosomes, and the long circulation and passive targeting characteristics of liposomes, the exosomes secreted by human pancreatic cancer cells have been successfully constructed as drug carriers by the method of self-assembly after membrane fusion with liposomes. For example, patent application CN116064394A discloses a hybrid exosome of tumor cell exosome and liposome, a preparation method thereof and an anti-tumor use thereof. The biomimetic exosome disclosed in the patent application has a certain targeting property, but the targeting property is weak, which is not enough to solve the problems of targeted therapy and immune escape of tumors.

[0004] PD-L1 is an important immunosuppressive molecule closely related to the immune escape of tumor. The high expression of PD-L1 on the surface of tumor cells, combined with PD-1 on cytotoxic T lymphocytes, activates the PD-1 / PD-L1 signal system, inhibits the activity of cytotoxic T lymphocytes, and activates the immune escape of tumor cells. Because PD-L1 is expressed in up to 90% of pancreatic cancer patients, the PD-L1 on the surface of pancreatic cancer cells can be selected as a target, and the PD-L1 single-chain fragment variable (scFv) can be used as a targeting molecule. Recombinant antibodies (whole / fragments) are increasingly valued in the field of targeted drug delivery due to their high specificity, ability to modify structure at the genetic level, large-scale expression in prokaryotic / eukaryotic cells, and other advantages. Single-chain antibodies are the most widely used and studied antibody form, which is composed of heavy and light chains of variable regions of antibody fragments, connected by a 10-25 amino acid polypeptide linker. Compared with whole antibodies or Fab, single-chain antibodies have the advantages of small molecular weight, long blood half-life, weak immunogenicity, and large-scale expression and purification through prokaryotic expression systems, and have good application prospects in targeted therapy.

[0005] In recent years, single-chain antibody modified liposomes have attracted attention in the treatment of leukemia, brain cancer, colorectal cancer, gastric cancer, head and neck squamous cell carcinoma, and other tumors. However, there are few studies on PD-L1 scFv modified nanodrug delivery systems. Some studies have used cell membranes expressing PD-L1 scFv to coat polydopamine modified gold nanoparticles to achieve the combination of immunotherapy and photothermal therapy (Xiao Y, Zhu T, Zeng Q, et al. Functionalized biomimetic nanoparticles combining programmed death-1 / programmed death-ligand 1 blockadewith photothermal ablation for enhanced colorectal cancer immunotherapy), but PD-L1 scFv is not combined with nanoparticles by chemical coupling. SUMMARY

[0006] In view of the problems in the prior art, the inventors screened a fully humanized monoclonal antibody against PD-L1 using phage display technology, thereby constructing a PD-L1 single-chain antibody (PD-L1 scFv). Furthermore, the inventors coupled the PD-L1 single-chain antibody to drug-loaded biomimetic exosomes through a chemical bond, combined the biological homing property of the biomimetic exosomes with the immune recognition ability of the PD-L1 scFv, and thereby prepared a novel PD-L1-targeted ceritinib biomimetic exosome. The PD-L1-targeted ceritinib biomimetic exosome can realize the synergistic effect of targeted ceritinib therapy and blocked PD-L1 immunotherapy, thereby providing a new technical platform for the combined treatment of chemotherapeutic drugs and antibody immunotherapy for solid tumors such as pancreatic cancer.

[0007] To achieve the above object, the present application provides the following technical solutions: In a first aspect, the present application provides a PD-L1 single-chain antibody, which comprises a heavy chain variable region (VH), a light chain variable region (VL), and a linker connecting the heavy chain variable region and the light chain variable region; wherein: The heavy chain variable region comprises: a VH CDR1 having an amino acid sequence of SEQ ID NO: 1, or a sequence obtained by substitution, deletion, or addition of one or several, e.g., 2, 3, 4, or 5 amino acids in SEQ ID NO: 1; a VH CDR2 having an amino acid sequence of SEQ ID NO: 2, or a sequence obtained by substitution, deletion, or addition of one or several, e.g., 2, 3, 4, or 5 amino acids in SEQ ID NO: 2; a VH CDR3 having an amino acid sequence of SEQ ID NO: 3, or a sequence obtained by substitution, deletion, or addition of one or several, e.g., 2, 3, 4, or 5 amino acids in SEQ ID NO: 3; and / or The light chain variable region comprises: a VL CDR1 having an amino acid sequence of SEQ ID NO: 4, or a sequence obtained by substitution, deletion, or addition of one or several, e.g., 2, 3, 4, or 5 amino acids in SEQ ID NO: 4; a VL CDR2 having an amino acid sequence of SEQ ID NO: 5, or a sequence obtained by substitution, deletion, or addition of one or several, e.g., 2, 3, 4, or 5 amino acids in SEQ ID NO: 5; VL CDR3, the amino acid sequence of which is SEQ ID NO: 6, or a sequence obtained by substitution, deletion, or addition of one or a few amino acids, for example, 2, 3, 4, or 5, in SEQ ID NO: 6.

[0008] Specifically, SEQ ID NOs: 1 to 6 are as follows: SEQ ID NO: 1: SYAIS; SEQ ID NO: 2: RIIPILGIANYAQKFQG; SEQ ID NO: 3: PYGSGSSLYAFDI; SEQ ID NO: 4: SGSSSNIGNNYVS; SEQ ID NO: 5: DNNKRPS; SEQ ID NO: 6: GTWDSSLSAVV.

[0009] In the present application, the complementarity determining regions of the heavy chain variable region and the light chain variable region are defined using the Kabat numbering system.

[0010] According to a preferred embodiment of the present application, the amino acid sequence of the heavy chain variable region comprises SEQ ID NO: 7 or is as set forth in SEQ ID NO: 7.

[0011] SEQ ID NO: 7: QVQLQQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCASPYGSGSSLYAFDIWGQGTMVTVSS.

[0012] According to another preferred embodiment of the present application, the amino acid sequence of the light chain variable region comprises SEQ ID NO: 8 or is as set forth in SEQ ID NO: 8.

[0013] SEQ ID NO: 8: HVILTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAVVFGGGTKVTVL.

[0014] According to some embodiments of the present application, the amino acid sequence of the connecting peptide consists of 1 to 5 amino acid sequences as shown in SEQ ID NO: 9; preferably, the amino acid sequence of the connecting peptide consists of 4 amino acid sequences as shown in SEQ ID NO: 9.

[0015] SEQ ID NO: 9: GGGGS.

[0016] According to a particularly preferred embodiment of the present application, the amino acid sequence of the PD-L1 single-chain antibody comprises or is as shown in SEQ ID NO: 10.

[0017] SEQ ID NO: 10: MQVQLQQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCASPYGSGSSLYAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSHVILTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAVVFGGGTKVTVLHHHHHH.

[0018] In a second aspect, the present application provides a PD-L1 targeting biomimetic exosome, comprising a biomimetic exosome formed by fusing an exosome derived from a cancer cell with a liposome and a PD-L1 single-chain antibody according to the present application, wherein the PD-L1 single-chain antibody is coupled to the biomimetic exosome.

[0019] According to some embodiments of the present application, the cancer cell is a PD-L1 highly-expressing cancer cell.

[0020] Preferably, the cancer cell is a PD-L1 highly-expressing pancreatic cancer cell.

[0021] More preferably, the cancer cell is selected from any one of BxPC-3 cell, CFPAC-1 cell, HuP-T3 cell and SU86.86 cell.

[0022] Most preferably, the cancer cell is a HuP-T3 cell.

[0023] According to some embodiments of the present application, the liposome is loaded with a drug for treating cancer.

[0024] Preferably, the drug for treating cancer is selected from one or more of gemcitabine, irinotecan, bosutinib, AZ5104, dasatinib and sorafenib.

[0025] More preferably, the drug for treating cancer is dasatinib and / or sorafenib.

[0026] Most preferably, the drug for treating cancer is sorafenib.

[0027] According to some embodiments of the present application, the PD-L1 targeting biomimetic exosome has a particle size of 80 nm to 92 nm, a Zeta potential of -16 mV to -12.5 mV, an encapsulation efficiency of 70% to 85%, a drug loading of 3% to 3.5%, and a coupling rate of the PD-L1 single-chain antibody of 60% to 62%.

[0028] Preferably, the PD-L1 targeting biomimetic exosome has an average particle size of about 87 nm, a Zeta potential of about -14 mV, an encapsulation efficiency of about 79%, a drug loading of about 3%, and a coupling rate of the PD-L1 single-chain antibody of about 60%.

[0029] According to some embodiments of the present application, the PD-L1 targeting biomimetic exosome has at least one of the specific marker proteins CD9, CD81 and PDCD6IP.

[0030] It should be noted that in the present application, the terms "PD-L1 targeting biomimetic exosome" and "PD-L1 single-chain antibody modified biomimetic exosome" have the same meaning and can be used interchangeably.

[0031] The preparation method of the PD-L1 targeting biomimetic exosome of the present application can refer to the related schemes in the prior art, which is not limited by the present application.

[0032] As an example, the PD-L1 targeting biomimetic exosome of the present application can be prepared by a method comprising the following steps: (1) fusing cancer cell-derived exosomes and liposomes by freeze-thaw method or membrane extrusion method to form biomimetic exosomes; (2) adding the biomimetic exosomes into a glutaraldehyde aqueous solution to obtain an aldehyde-modified biomimetic exosome solution, then adding the PD-L1 single-chain antibody into the aldehyde-modified biomimetic exosome solution and incubating overnight, so as to covalently couple the PD-L1 single-chain antibody to the surface of the biomimetic exosomes.

[0033] Among them, the cancer cell-derived exosomes can be separated and extracted from the cancer cell culture supernatant by ExoQuick reagent method combined with ultrafiltration method. The liposomes can be prepared by a method comprising egg phospholipid, cholesterol and DSPE-PEG 2000The raw material of -NH2 is used to prepare blank liposomes by a thin film hydration method. Then the drug can be loaded into the blank liposomes by an ammonium sulfate gradient method.

[0034] In a third aspect, the present application provides a pharmaceutical composition for treating cancer, comprising the PD-L1 targeting biomimetic exosome according to the present application and optionally a pharmaceutically acceptable adjuvant.

[0035] Preferably, the cancer is a pan-solid tumor of PD-L1 expression.

[0036] More preferably, the cancer is selected from one or more of pancreatic cancer, non-small cell lung cancer, bladder cancer, head and neck cancer, gastric cancer, breast cancer, colorectal cancer, kidney cancer, melanoma and liver cancer.

[0037] Further preferably, the cancer is pancreatic cancer.

[0038] In a fourth aspect, the present application provides use of the PD-L1 targeting biomimetic exosome according to the present application in the preparation of a medicament for treating cancer.

[0039] Preferably, the cancer is a pan-solid tumor of PD-L1 expression.

[0040] More preferably, the cancer is selected from one or more of pancreatic cancer, non-small cell lung cancer, bladder cancer, head and neck cancer, gastric cancer, breast cancer, colorectal cancer, kidney cancer, melanoma and liver cancer.

[0041] Further preferably, the cancer is pancreatic cancer.

[0042] Compared with the prior art, the technical scheme of the present application has the following beneficial effects: The PD-L1 single-chain antibody and the PD-L1 targeting biomimetic exosome provided by the present application have obvious affinity activity to PD-L1 protein and have targeting specificity.

[0043] It has been verified through experiments that the PD-L1 single-chain antibody provided by the present application has obvious affinity activity to PD-L1 protein in a concentration range of 0.001 μM to 1 μM, and its affinity activity is concentration-dependent. The results of ELISA test and cell fluorescence imaging analysis both show that the PD-L1 single-chain antibody and the PD-L1 targeting biomimetic exosome provided by the present application have strong affinity to PD-L1 highly-expressed pancreatic cancer HuP-T3 cells, but have no obvious affinity to PD-L1 lowly-expressed pancreatic cancer Panc0403 cells, thereby verifying the targeting specificity thereof.

[0044] In addition, in vitro experiments also prove that the PD-L1 targeting ceritinib biomimetic exosome of the application can specifically bind to the PD-L1 on the surface of pancreatic cancer cells, significantly improves the uptake of the drug loaded by the pancreatic cancer cells with high expression of PD-L1, and blocks the specific binding of PD-L1 and PD-1 to restore T cell activity and enhance anti-tumor immunity. The PD-L1 targeting ceritinib biomimetic exosome of the application shows good activity in inhibiting the proliferation of tumor cells, indicating that it has good targeting and cancer treatment prospects. BRIEF DESCRIPTION OF DRAWINGS

[0045] Hereinafter, the embodiments of the application will be described in detail with reference to the accompanying drawings, in which: Figure 1 The results of detecting the binding activity of AH06280, AH06281 and AH06283 antibodies to PD-L1 by SPR in Example 1 of the application are shown.

[0046] Figure 2 The results of detecting the affinity activity of AH06280, AH06281 and AH06283 antibodies to PD-L1 by ELISA in Example 1 of the application are shown.

[0047] Figure 3 The results of detecting the purity of AH06280 PD-L1 scFv by SDS-PAGE in Example 1 of the application are shown.

[0048] Figure 4 The results of detecting the affinity activity of AH06280 PD-L1 single-chain antibody to PD-L1 by ELISA in Example 1 of the application are shown.

[0049] Figure 5 The results of detecting the expression level of PD-L1 in different pancreatic cancer cell lines by immunoblotting in Example 2 of the application are shown.

[0050] Figure 6 The in vitro killing activity of different drugs on pancreatic cancer cells in Example 2 of the application is shown.

[0051] Figure 7 The encapsulation rate of different drugs in Example 2 of the application is shown.

[0052] Figure 8 The transmission electron microscope photos of different ceritinib preparations derived from HuP-T3 cells in Example 3 of the application are shown.

[0053] Figure 9 The results of detecting the expression of exosome marker proteins in the PD-L1 targeting ceritinib biomimetic exosome by Western blotting in Example 3 of the application are shown.

[0054] Figure 10 The results of detecting the affinity activity of PD-L1 targeting sorafenib biomimetic exosomes to PD-L1 receptor coated antigen by ELISA in Example 4 of the present application are shown.

[0055] Figure 11 The results of detecting the affinity activity of PD-L1 targeting sorafenib biomimetic exosomes to different pancreatic cancer cells by ELISA in Example 4 of the present application are shown. Among them, A shows the affinity activity of PD-L1 targeting sorafenib biomimetic exosomes to HuP-T3 cells; B shows the affinity activity of PD-L1 targeting sorafenib biomimetic exosomes to Panc0403 cells.

[0056] Figure 12 The results of PD-L1 targeting sorafenib biomimetic exosomes uptake in different pancreatic cancer cells in Example 4 of the present application are shown. Among them, A is the fluorescence map of PD-L1 targeting sorafenib biomimetic exosomes uptake in HuP-T3 cells; B is the quantitative results of PD-L1 targeting sorafenib biomimetic exosomes uptake in HuP-T3 cells; C is the fluorescence map of PD-L1 targeting sorafenib biomimetic exosomes uptake in Panc0403 cells; D is the quantitative results of PD-L1 targeting sorafenib biomimetic exosomes uptake in Panc0403 cells.

[0057] Figure 13 The killing activity of PD-L1 targeting sorafenib biomimetic exosomes to different pancreatic cancer cells in Example 5 of the present application is shown. Among them, A shows the killing activity of PD-L1 targeting sorafenib biomimetic exosomes to HuP-T3 cells; B shows the killing activity of PD-L1 targeting sorafenib biomimetic exosomes to Panc0403 cells.

[0058] Figure 14 The results of different sorafenib preparations inducing HuP-T3 cell apoptosis in Example 5 of the present application are shown.

[0059] Figure 15 The in vitro killing activity comparison of sorafenib and dasatinib to pancreatic cancer HuP-T3 cells and Panc0403 cells in Example 2 of the present application is shown. DETAILED DESCRIPTION

[0060] The present application will be further described in detail below in conjunction with specific embodiments, and the examples given are only to illustrate the present application, but not to limit the scope of the present application.

[0061] The experimental methods and conditions used in the following examples are conventional methods and conditions unless otherwise specified. The reagents used in the following examples are commercially available unless otherwise specified.

[0062] Example 1: Screening and preparation of PD-L1scFv 1.1 Screening of fully humanized monoclonal antibodies against PD-L1 using phage display technology 1.1.1 Screening of fully humanized monoclonal antibodies against PD-L1 (1) Materials PD-L1.Fc (Cat#: Z03371, lot#: B60051712crip, Genscript); CHO-PD-L1 cells (Genscript); CHO-K1 cells (Genscript); PD-L1.His(Cat#:Z03425,Genscript); Human naïve phage display library (kappa / lambda, size: 4×10⁻⁶) 10 (Genscript) Host bacteria: Escherichia coli TG1 ( E. coli TG1); M13KO7 helper phage (NEB, Cat. No.: N0315S); Coating solution: 0.05 M NaHCO3, pH 9.6; 2×YT: 16 g tryptone, 10 g yeast extract and 5 g NaCl dissolved in 1 L ddH2O; PBS: 137mM NaCl, 2.7mM KCl, 4.3mM Na2HPO4, 1.4mM KH2PO4, pH 7.4; ELISA microtiter plate (Corning, Cat. No.: 9018); Blocking buffer (MPBS): PBS buffer, pH 7.4, containing 5% skim milk; Washing buffer (PBST): PBS buffer, pH 7.4, containing 0.05% Tween 20; Washing buffer: 1% BSA-PBS (for cell screening); Elution buffer: 0.1M TEA, trimethylamine (for phage display screening); 0.1M Tris-HCl (pH 7.4); HRP conjugated Anti-M13 monoclonal antibody (SINO BIOLOGICAL, Cat#: 11973-MM05T-H); Anti-fd Bacteriophage-Biotin (B2661, Sigma-Aldrich); SA iFluor 647 prepared by Genscript; pFASEBA expression vector prepared by GenScript; 1 M Isopropyl-D-thiogalactoside (IPTG) (VWR, Cat#: 0487-100G); Peroxidase AffiniPure Goat Anti-Human IgG F(ab')2 fragment specific (Jackson, Cat#: 109-035-097); Goat anti Human IgG F(ab')2 Fragment specific [FITC] (Jackson, Cat#: 109-096-006); Tetramethylbenzidine (TMB, GenScript); 1M HCl (GenScript); pcDNA3.4 expression vector and HEK293-6F cells (prepared by GenScript); 37°C CO2 incubator (Thermo Scientific, Model. 3951); Biosafety cabinet (Thermo Scientific, Model. 1384); Orbital shaker (Thermo Scientific, Model. 416); Polyethylenimine (Polysciences, Cat. No. 23966); FreeStyle 293 medium (lifetechnologies, Cat. No. 12338-018); TN1 (Organotechnie, Cat. No. 19553); 125-ml shake flask (Corning, Cat. No. 430421); Protein-A resin (GenScript, Cat. No. L00210); Binding buffer: 0.15 M NaCl, 20 mM Na2HPO4, pH 7.0; Elution buffer: 0.1 M Glycine-HCl, pH 3.2; Neutralization buffer: 1 M Tris-HCl, pH 9.0; Biacore T200, GR18010468 (GE Healthcare); Series S Sensor Chip Protein A (GE Healthcare, Cat. No. 29-1275-55, Lot. No. 10272787); HBS-EP + : 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% P20, pH 7.4 (GE Healthcare, Lot. No. BCBX0007).

[0063] (2) Methods (2.1) Bio-panning of phage display library Human naïve phage display library (kappa and lambda, size: 4 x 10 10 ) stocks were precipitated with PEG / NaCl and resuspended in PBS for panning. The phage library was panned against human PD-L1. Fc was used with a modified standard procedure developed by GenScript. Bio-panning was performed with solid phase panning in the first and second round and cell panning in the third round. Specifically, human IgG1 (as negative panning to reduce Fc binders) and human PD-L1 were coated on microtiter plates. The Fc concentration was 50 μg / ml in coating buffer, 4°C overnight. The human naïve phage display library phage particles (2 x 10 12 pfu / pool) were diluted in blocking buffer. The library was added to the human IgG1 coated wells and incubated for 1 hour with slow shaking to absorb Fc binders. To completely reduce Fc binding phage, 10 μg human Fc was added to the depleted phage pool as a soluble competitor and incubated for 1 hour with slow shaking to first compete with Fc binding phage. Then the phage particles were transferred to the human PD-L1. The Fc coated wells were incubated for 1 hour with shaking at room temperature. After incubation, unbound / non-specific bound phage was decanted and washed 10-15 times with washing buffer and 5 times with PBS. Upon cell washing, about 2 x 10 7CHO-PD-L1 cells, washed with PBS, then blocked with 1 mL of 3% (w / v) milk in PBS (MPBS) for 45 minutes at RT. Meanwhile, 2 x 10 12 Phage particles were also blocked with MPBS, 1 x 10 7 washed CHO-K1 cells per well were added and incubated at RT for 45 minutes. The phage / cell suspension was centrifuged at 500 g for 3 minutes. Purified phage was added to the PD-L1 CHO cells and incubated at RT for 1 hour, centrifuged at 500 g for 3 minutes, and washed 8 times with 1% BSA-PBS wash buffer. Bound phage (wells or cells) were eluted with TEA and neutralized with 0.1 M Tris-HCl (pH 7.4). The phage eluate was used to infect 10 mL of exponentially growing E. coli TG1 at 37°C. Phage were amplified and rescued using M13K07 helper phage according to standard procedures, and phage particles were prepared for subsequent biopanning. Phage from the first round of amplification were used as input for the second round of biopanning. Infected TG1 cells from the third round were plated on LB-Amp+ plates. Monoclonal phage ELISA and flow cytometry (FACS) were used to verify binding activity.

[0064] (2.2) Monoclonal phage ELISA screening Individual colonies were grown in 96-deep well plates with M13KO7 helper phage at 30°C overnight. Meanwhile, two 96-well ELISA microplates were coated with 1 μg / mL of target antigen (human IgGl as control) in coating buffer at 4°C overnight. The plates were blocked with PBS plus 5% nonfat dry milk. After washing, 50 μL of phage supernatant from each deep well culture was added to each well and incubated at room temperature for 2 hours. After washing three times with wash buffer, HRP-conjugated anti-M13 monoclonal antibody was added and incubated at 4°C for 45 minutes. The plates were washed six more times, and substrate solution was added to the wells and allowed to react. Absorbance was measured at 450 nm using a spectrophotometer. Phage binders that specifically recognized the antigen were selected for FACS verification.

[0065] (2.3) Phage FACS screening and DNA sequencing Flow cytometry was used to detect the binding of phage supernatant to CHO-PD-L1 cells (control CHO-K1 cells) using standard procedures developed by GenScript. Cells were washed in PBS, then blocked with 1 x 10 5Cells were seeded at a density of 1 cell / well (100 mΐ / well) and 50 mΐ of the phage supernatant to be tested was added to each well, and incubated at 4°C for 40 min. The cells were washed twice with cold PBS. Anti-fd phage biotin (3 pg / ml) was added at 100 mΐ / well, and incubated at 4°C for 40 min. SA iFluor 647 (1 pg / ml) was added at 100 mΐ / well, washed twice, and incubated at 4°C for 30 min. After two washes, the cells were loaded and then the fluorescence was detected. Flow cytometry was used to collect data. DNA sequencing of the antigen and cell binders was performed using standard procedures developed by GenScript.

[0066] (2.4) Construction of FASEBA format library The output plasmid from the 3rd round was amplified and digested, and the digested FASEBA vector was used to construct the FASEBA library. The obtained FASEBA library was subjected to high-throughput FASEBA antigen protein screening. Sfil and Notl The obtained FASEBA library was subjected to high-throughput FASEBA antigen protein screening.

[0067] (2.5) FASEBA format ELISA screening Individual Fab colonies were expressed in 96-well plates. The expression and binding activity of BSA and antigen protein of crude protein secreted by E. coli into the culture medium were detected by ELISA. Specifically, individual colonies were cultured in 96-well plates, and induced with IPTG at 30°C overnight. At the same time, 96-well ELISA microplates were coated with 1 pg / ml target antigen (human IgG1 as control) in coating buffer at 4°C overnight. The culture dishes were blocked with PBS added with 5% skimmed milk powder. After washing, 50 mΐ of phage supernatant from each overnight culture was added to the plates, and incubated at room temperature for 2 hours, washed with washing buffer for 3 times, and then HRP-conjugated anti-human IgG F(ab’)2 monoclonal antibody was added to the plates, and incubated at room temperature for 45 minutes, washed for 6 times, and then substrate solution was added to the wells for reaction. The absorbance was measured at 450 nm by spectrophotometer. Phage binders that specifically recognized the antigen were selected for FACS verification.

[0068] (2.6) FASEBA format FACS screening and DNA sequencing Flow cytometry was used to detect the binding of antigen-binding phage supernatant to CHO-PD-L1 cells (control CHO-K1 cells) using standard procedures developed by GenScript. The cells were washed with PBS, and 1 x 10 5Cells were seeded at a density of 1 cell / well (100 μΐ / well) and 50 μΐ of the phage supernatant to be tested was added to each well, and incubated at 4°C for 40 min. The cells were washed twice with cold PBS. After the second wash, the cells were loaded and then the fluorescence was detected. Flow cytometry was used to collect data. DNA sequencing was performed for the antigen and cell binders using standard procedures developed by GenScript.

[0069] (2.7) Construction and production of IgG DNA sequences encoding 3 leader sequences were inserted into pcDNA3.4 to make expression plasmids for full-length IgG. HEK293F cells were co-transfected with heavy chain and light chain expression plasmids. Recombinant IgG secreted into the culture medium was purified by protein A affinity chromatography according to the SOP of GenScript. The concentration and purity of the purified protein were determined by OD 280 and SDS-PAGE, respectively. Surface plasmon resonance (SPR) was used to detect binding confirmation and affinity using Biacore T200.

[0070] (2.8) Affinity determination of purified IgG The affinity of the purified antibody to bind to the antigen was determined individually using a surface plasmon resonance (SPR) biosensor, Biacore T200. The antibody was immobilized on the sensor chip by the capture method. The antigen was used as the analyte. The dissociation (kd) and association (ka) rate constant data were calculated using the Biacore T200 evaluation software. The equilibrium dissociation constant (KD) was calculated from the ratio of kd / ka.

[0071] (3) Results (3.1) To reduce the enrichment of the Fc binder, human IgG1 was panned in each round, and the Fc binder was competitively bound to the human Fc protein before binding to PD-L1. Fc was used to obtain the cell binder, and CHO-PD-L1 was used to flush the cells in the 3rd round and CHO-K1 was used to pan the cells. The results are summarized in Table 1.

[0072] Table 1: Results of 3 rounds of screening using a naïve human phage display library “Panning” in Table 1 refers to washing away unbound phage, and retaining specifically bound phage.

[0073] (3.2) A total of 222 antigen and cell binders were identified by ELISA and FACS, and 95 samples were subjected to DNA sequencing, and finally 8 monoclonal antibodies with complementarity determining regions (CDRs) were obtained, as shown in Table 2.

[0074] Table 2: ELISA values and amino acid frequencies of 8 CDR unique clones (3.3) Three high frequency clones (AH06280, AH06281, AH06283) in the phage and FASEBA library were selected to construct complete IgG1, and the purity and yield of which are shown in Table 3.

[0075] Table 3: Purity and yield of purified AH06280, AH06281, AH06283 antibodies (3.4) The CDR amino acid sequences of the PD-L1 antibodies obtained from the AH06280, AH06281, AH06283 monoclonal are shown in SEQ ID NO: 1 to SEQ ID NO: 6, respectively. Among them, the format of the heavy chain is: AH06280: Heavy chain: Light chain: AH06281: Heavy chain: Light chain: AH06283: Heavy chain: Light chain: 1.1.2 SPR analysis of the binding activity of three PD-L1 antibodies to PD-L1 Methods are the same as step (2.8) described above. Specifically, the surface plasmon resonance (SPR) biosensor Biacore T200 was used to determine the affinity of the purified AH06280, AH06281 and AH06283 three PD-L1 antibodies to the antigen. Through the capture method, the antibody was fixed on the sensor chip, and the antigen was used as the analyte, and the dissociation (kd) and binding (ka) rate constant data were obtained. The equilibrium dissociation constant (KD) was calculated from the ratio of kd / ka. The affinity of the screened AH06280, AH06281, AH06283 antibodies was determined by SPR. The results are shown in Table 4. Figure 1The affinity constants KD of the three antibodies AH06280, AH06281 and AH06283 to PD-L1 were 7.27 x 10 -10 M, 2.69 x 10 -9 M, 9.31 x 10 -9 M.

[0076] 1.1.3 ELISA analysis of the affinity activity of three PD-L1 antibodies to PD-L1 The affinity activity of the three PD-L1 antibodies AH06280, AH06281 and AH06283 to PD-L1 protein was detected by receptor-coated ELISA. 1 μg / ml of PD-L1 protein solution (Genscript) was coated on an ELISA plate at a volume of 100 μl / well at 4°C overnight; the coated plate was washed with PBS for 3 times, and then blocked with 1% BSA / PBS solution at 200 μl / well at 4°C overnight; washed with PBST buffer (PBS containing 0.05% Tween-20) for 3 times; 8 gradient dilutions of AH06280, AH06281 and AH06283 were added, with concentrations of 0.0000128, 0.000064, 0.00032, 0.0016, 0.008, 0.04, 0.2 and 1 μM respectively, 3 parallel holes for each concentration, 4°C incubation overnight; washed with PBST for 3 times, then added HRP-streptavidin (1:2000) diluted solution at 50 μl / well, 37°C incubation for 90 min; washed the plate with PBST for 5 times, added 100 μl TMB substrate solution to each well, reacted at room temperature for 2 min, then added 100 μl 2 mol / L sulfuric acid to terminate the reaction, and immediately measured the absorbance value at 450 nm on the microplate reader. The results of the receptor-coated ELISA experiment are shown in Figure 2 As can be seen from the figure, the affinity of the AH06280 PD-L1 antibody to PD-L1 protein was significantly stronger than that of AH06281 and AH06283, and its affinity activity was concentration-dependent within a certain range, therefore the AH06280 clone with the highest affinity activity was selected as the subsequent PD-L1 scFv single-chain antibody sequence.

[0077] 1.2 Preparation of PD-L1 scFv antibody AH06280 PD-L1 scFv (VH-G4Sx3-VL-His6) sequence is according to the VH of AH06280 (underlined sequence in SEQ ID NO: 10) at the N-terminus, with a connecting peptide (italic sequence in SEQ ID NO: 10) in the middle, followed by the VL of AH06280 (bold sequence in SEQ ID NO: 10), and finally a His6 tag sequence (underlined and italic sequence in SEQ ID NO: 10) at the C-terminus. The complete sequence of AH06280 PD-L1 scFv is as follows: Firstly, the prokaryotic expression system of AH06280 PD-L1 scFv was constructed, and a 6xHis tag was connected at the C-terminus for the convenience of purification of the single-chain antibody. Then, the target protein AH06280 PD-L1 scFv was obtained through expression of the target protein (including bacterial culture, IPTG induction, and crude extraction to obtain protein crude extract) and purification of the target protein (including Ni column affinity purification and molecular sieve chromatography).

[0078] The expression method of the target protein AH06280 PD-L1 scFv is as follows: 1) Transforming E. coli expression host Bl21 (DE3) with a plasmid containing AH06280 PD-L1 scFv; 2) Picking a single colony from the transformation plate and inoculating it into 5 ml of LB liquid medium containing an antibiotic (amoxicillin concentration: 100 μg / ml) and incubating it at 37°C, 200 rpm overnight; 3) Transferring the above bacterial solution into 100 ml of LB liquid medium containing an antibiotic (amoxicillin concentration: 100 μg / ml) at a ratio of 1:100 and incubating it at 37°C until OD 600 = 1.0; 4) Adding IPTG to a final concentration of 0.5 mM and inducing it at 37°C, 200 rpm for 4 h; 5) Collecting the above induced culture solution and centrifuging it at 7000 rpm for 10 min to collect the bacterial cells; 6) Resuspending the bacterial cells in a buffer (PBS, 0.1 mM EDTA, pH: 7.4); 7) Adding the primary separated bacterial cells to the bacterial buffer (1:30) for resuspension, sonicating the bacteria, and centrifuging it at 7000 rpm for 10 min to collect the supernatant of the broken bacteria, which is the initial sample solution of AH06280 PD-L1 scFv to be purified, and stored at 4°C for standby use.

[0079] The purification method of the target protein AH06280 PD-L1 scFv is as follows: 1) The protein sample to be purified is filtered through a 0.45 μm filter membrane before loading; 2) A pre-packed Ni ion affinity chromatography column is prepared, and a peristaltic pump, chromatography column and protein UV detector are connected for use; 3) According to the specifications of the pre-packed column, an appropriate column flow rate is set, such as 4 ml / min, and the column is equilibrated with loading buffer (PBS, 100 mM NaCl, pH: 7.4) for about 10 column volumes, and the UV detector reading is observed to be stable: 4) The filtered protein sample is loaded at a flow rate of 4 ml / min, and after loading, the column is washed with loading buffer for about 10 column volumes; 5) After the UV detector is stable, the protein is eluted with different imidazole concentration gradients, i.e. 10 mM, 50 mM, 200 mM and 500 mM imidazole elution buffer (elution buffer stock solution: PBS, 100 mM NaCl, 500 mM imidazole, pH: 7.4), and the change in UV detector value is observed to collect the eluted protein fractions: 6) Take 15 μl of each fraction of eluted protein for electrophoresis analysis to determine the fraction in which the target protein is located, and to preliminarily assess the purity of the purified target protein: 7) The eluted protein solution containing the target protein is poured into a dialysis bag, and the protein is dialyzed against protein dialysis solution (PBS, pH 7.4) for more than 4 h for 3 times to remove imidazole and other substances: 8) After dialysis, the protein solution is centrifuged and filtered through a 0.2 μm filter membrane to remove bacteria, which is the final product.

[0080] The obtained target protein AH06280 PD-L1 scFv is subjected to protein electrophoresis analysis, and the concentration is determined by BCA kit, and the purity is greater than 90%, and the results are as follows Figure 3The affinity activity of the AH06280 PD-L1 antibody and its PD-L1 single-chain antibody to PD-L1 protein was detected by receptor-coated ELISA. 1 μg / ml of PD-L1 protein solution was coated on an ELISA plate at an amount of 100 μl / well at 4°C overnight; the coated plate was washed with PBS for 3 times, and then blocked with 1% BSA / PBS solution at 200 μl / well at 4°C overnight; washed with PBST buffer (0.05% Tween-20 in PBS) for 3 times; 8 gradient-diluted PD-L1 antibody and PD-L1 single-chain antibody solutions were added, with concentrations of 0.0000128, 0.000064, 0.00032, 0.0016, 0.008, 0.04, 0.2, and 1 μM respectively, 3 parallel wells for each concentration, and incubated at 4°C overnight; washed with PBST for 3 times, and then added with HRP-streptavidin (1:2000) diluted at 50 μl / well, and incubated at 37°C for 90 min; the plate was washed with PBST for 5 times, 100 μl of TMB substrate solution was added to each well, and reacted at room temperature for 2 min in the dark, and then 100 μl of 2 mol / L sulfuric acid was added to each well to terminate the reaction, and the absorbance value at 450 nm was immediately determined on an enzyme-labeled instrument. The results of the receptor-coated ELISA experiment are shown in FIG. 2. Figure 4 As can be seen from the figure, the AH06280 PD-L1 antibody and its single-chain antibody have obvious affinity activity to PD-L1 protein, and the affinity activity is concentration-dependent within a certain range.

[0081] Example 2: Preparation of PD-L1-targeted sunitinib biomimetic exosomes 2.1 Western blotting method for detecting the expression of PD-L1 protein in different pancreatic cancer cell lines Eight human pancreatic cancer cell lines MIAPaCa-2, AsPC-1, BxPC-3, PANC-1, CFPAC-1, Panc0403, HuP-T3, and SU86.86 (originally purchased from the ATCC cell library in the United States) were selected for cell culture. Among them, MIAPaCa-2, PANC-1, CFPAN-1, and Panc0403 cells were cultured in DMEM medium (Beijing Lambolide Trade Co., Ltd.) containing 10% fetal bovine serum (Germany PAN); AsPC-1, HuP-T3, BxPC-3, and SU86.86 cells were cultured in RPMI 1640 medium (Beijing Lambolide Trade Co., Ltd.) containing 10% fetal bovine serum. All cells were cultured in a 37°C incubator containing 5% CO2.

[0082] The eight strains of pancreatic cancer cells in logarithmic growth phase and in good cell state were collected, rinsed with pre-cooled PBS, and then added with RIPA (containing a final concentration of 1 mM PMSF) lysis solution and placed on ice for lysis for 30 min. 4°C, 12000 x g centrifugation for 20 min, collection of supernatant for BCA protein quantification; adjustment to the same concentration, addition of a certain amount of 5 x protein loading buffer, 100°C protein denaturation for 10 min, and then storage at -80°C. The expression level of PD-L1 in these cells was detected by Western, which provided a basis for selecting appropriate cell strains for subsequent research. The specific method of Western was as follows: 10% SDS-PAGE gel was prepared, and the protein samples prepared from the aforementioned different pancreatic cancer cells were loaded, 80V electrophoresis through the concentrated gel, and then 120V electrophoresis to the bromophenol blue to the lower edge of the gel; 300mA transmembrane for 1 hour, after transmembrane, blocking in 5% milk for 2 hours, cutting the corresponding band, and then incubating PD-L1 primary antibody (CST company), 4°C overnight, washing the membrane, incubating secondary antibody (Beijing Zhongsu Jinqiao Biotechnology Co., Ltd.) at room temperature for 2 hours, washing the membrane, and then exposing. The results are shown in Figure 5 Fig. 1, in these cell lines, the PD-L1 expression level of HuP-T3 cells was relatively high, while the PD-L1 expression of Panc0403 cells was almost not expressed. Therefore, the PD-L1 highly expressed HuP-T3 cells and the PD-L1 lowly expressed Panc0403 cells were selected for subsequent experiments.

[0083] 2.2 Comparison of in vitro killing activity of different drugs on pancreatic cancer Ten commonly used antitumor drugs were selected, including gemcitabine, SN-38, irinotecan, PLX4032, paclitaxel, bosutinib, AZ5104, AIM-100, dasatinib and seribantib (purchased from MCE company), and MTS method was used to detect the in vitro antitumor activity of PD-L1 highly expressed pancreatic cancer HuP-T3 cells and PD-L1 lowly expressed pancreatic cancer Panc0403 cells, and the specific steps were as follows: (1) logarithmic growth phase cells were taken at 5 x 10 3The density of individual wells is 90 μΐ, and each well is inoculated in a 96-well cell culture plate. The cells are incubated at 37°C in a 5% CO2saturated humidity incubator for 24 h. (2) 10 μΐ of the test drug diluted with PBS or 10% DMSO PBS solution is added to each well, and a control group and a blank group are set up. Each group has three replicates, and the cells are incubated at 37°C in a 5% CO2incubator for 72 h. (3) 10 μΐ of MTS reagent (purchased from Promega) is added to each well, and the cells are incubated at 37°C for 2 h. (4) The absorbance value is read at 490 nm using a microplate reader, and the cell survival rate is calculated according to the following formula: survival rate % = (OD drug well-OD blank well) / (OD control well-OD blank well) x 100%.

[0084] The results of the in vitro killing activity of different drugs on pancreatic cancer cells are shown in Table 2. Figure 6 Except for SN-38, PLX4032 and paclitaxel, which have poor sensitivity in at least one cell line, the rest of the drugs show good in vitro antitumor activity in the two cells.

[0085] The results of the in vitro killing activity of different drugs on pancreatic cancer HuP-T3 cells and Panc0403 cells are shown in Table 3. Figure 15

[0086] 2.3 Preparation of different drug liposomes and comparison and screening of encapsulation efficiency Based on the above MTS results, gemcitabine, irinotecan, bosutinib, AZ5104, dasatinib and sunitinib were screened for liposome preparation. Gemcitabine, irinotecan and sunitinib were prepared into liposomes using the ammonium sulfate gradient method, while bosutinib, AZ5104 and dasatinib were prepared into liposomes using the thin film hydration method, and the drug encapsulation efficiency was detected. AIM-100 was not obtained due to unsuccessful encapsulation, and the results of the encapsulation efficiency of the rest of the drugs are shown in Table 4. Figure 7 The encapsulation efficiency of gemcitabine, bosutinib, AZ5104 and irinotecan is low, while the encapsulation efficiency of dasatinib and sunitinib is more than 85%.

[0087] For dasatinib and sunitinib, the inventors considered the following: Both are marketed drugs, but sunitinib is a second-generation ALK inhibitor and is a TGF- ​ / SMADs, IGF-1R as the target, which is a first-line treatment drug for ALK-positive solid tumor NSCLC; and dasatinib as an inhibitor that can inhibit a variety of receptor and non-receptor tyrosine kinases, is used for the treatment of adult chronic, accelerated, myeloid or lymphoid chronic myeloid leukemia that is resistant to previous treatment or cannot tolerate, such as leukemia resistant to imatinib. The inventors found through research that, compared with dasatinib, ceritinib has been approved for the treatment of solid tumors, and the drug targets TGF- / SMADs pathway, an important molecular target of tumor microenvironment, which can promote the synthesis, deposition and epithelial-mesenchymal transition (EMT) of CAFs in the tumor microenvironment, while ceritinib itself also has ACK1 non-receptor tyrosine kinase inhibitory activity, and the function of an inhibitor targeting IGF-1R, so it is more beneficial to the tumor microenvironment, and the development of nanodelivery drugs for the treatment of pancreatic cancer and other solid tumors.

[0088] In addition, from Figure 15 The comparison results of the in vitro killing activity of ceritinib and dasatinib shown in the table can be seen that, at low concentrations, such as when the concentration of ceritinib is 1.25×10 -5 mol / L, the survival rate of HuP-T3 and Panc0403 cells is only 0.21% and 0.90%; while when the concentration of dasatinib is 1.0×10 -5 mol / L, the survival rate of HuP-T3 and Panc0403 cells is 36.73% and 68.99% respectively, indicating that the killing activity of ceritinib on pancreatic cancer HuP-T3 and Panc0403 cells at low concentrations is significantly stronger than that of dasatinib, and when the concentration is higher than 1.25×10 -5 mol / L, HuP-T3 and Panc0403 cells can be almost completely eliminated. Therefore, combined with the difference between the cytotoxic activity of these drugs and the encapsulation rate of liposome drug loading, ceritinib with higher killing activity on pancreatic cancer cells and higher encapsulation rate is finally selected as an antitumor drug for the subsequent preparation of PD-L1 targeted biomimetic exosomes and the study of its antitumor effect.

[0089] 2.4 Preparation of ceritinib liposomes Egg phosphatidylcholine, cholesterol, DSPE-PEG 2000 and DSPE-PEG 2000-NH2 (purchased from Avestia Biologics) was dissolved in chloroform and methanol (3:1, v / v) at a molar ratio of 66:18:4:0.5, and a thin film was formed by rotary evaporation at 40°C under reduced pressure. Then, 250 mM ammonium sulfate solution was added for hydration. The mixture was first ultrasonicated in a water bath at room temperature for 5 min, and then further ultrasonicated in an ultrasonic cell crusher for 12 min (power 200 W, working time 10 s, intermittent time 10 s, and protection temperature 35°C). The hydrated liposome suspension was sequentially extruded through polycarbonate membranes with pore sizes of 400 nm and 200 nm. After extrusion, the liposome suspension was dialyzed in a dialysis bag (molecular weight cut-off 12000-14000 Da) in a phosphate buffer solution (PBS) for 12 h, twice, to obtain blank liposomes. Cetrelimab was mixed with the blank liposomes (Cetrelimab: phospholipid = 1:20, w / w) by the ammonium sulfate gradient method. Cetrelimab was loaded into the blank liposomes by shaking in a 40°C water bath for 20 min. The Cetrelimab liposomes (CER-Lipo) were obtained by dialysis in a PBS solution for 24 h, with the dialysis solution being replaced every 12 h. The CER-Lipo was stored at 4°C for later use.

[0090] 2.5 Extraction of pancreatic cancer cell exosomes ExoQuick reagent method combined with ultrafiltration method was used to separate and extract exosomes secreted by HuP-T3 cells into the supernatant. HuP-T3 cells were cultured in a 150 mm culture dish in a 5% CO2, 37°C incubator using RPMI 1640 culture medium containing 10% fetal bovine serum (purchased from Beijing Lambolide Trade Co., Ltd.) to 60% confluence. The medium was replaced with RPMI 1640 medium containing 5% exosome-free serum (purchased from VivaCell Company), and the cells were cultured for another 48 h. The cell culture supernatant was collected and centrifuged at 2000 x g for 10 min at 4°C. The obtained supernatant was centrifuged at 12000 x g for 30 min at 4°C. The supernatant was subjected to exosome interception and concentration using a 30 kDa ultrafiltration membrane. ExoQuick precipitant (purchased from System Biosciences) was added to the concentrated solution at a ratio of 1:5 (v / v). After gentle mixing, the mixture was placed vertically at 4°C for 12 h or more. The precipitate at the bottom was exosomes after centrifugation at 1500 x g for 30 min at 4°C. The exosome precipitate was suspended in an appropriate amount of PBS, and the protein concentration of the exosomes was determined by the Bicinchoninic Acid Assay (BCA) method. The extracted exosomes were stored at -80°C for later use.

[0091] 2.6 Preparation of cetrelimab biomimetic exosomes The exosomes with a concentration of 300 μg / mL and the sorafenib liposomes with a phospholipid concentration of 100 μmol / L were mixed at a volume ratio of 1:1, and then the following two methods were used to prepare the sorafenib biomimetic exosomes: (1) The exosome and liposome mixture was frozen in liquid nitrogen at -196°C for 15 minutes, and then thawed at room temperature until it melted. This freeze-thaw process was repeated 5 times to obtain the sorafenib biomimetic exosomes prepared by freeze-thawing.

[0092] (2) The exosome and liposome mixture was subjected to ultrasonic cell pulverization for 6 min (power 200 W, working time 30 s, and intermittent time 30 s), and then successively extruded through polycarbonate membranes with pore sizes of 400 nm and 200 nm to obtain the sorafenib biomimetic exosomes prepared by membrane extrusion.

[0093] 2.7 Preparation of PD-L1-targeted sorafenib biomimetic exosomes In 1 ml of sorafenib biomimetic exosomes, 20 μL of 25% glutaraldehyde aqueous solution was added, and the reaction was carried out at room temperature for 2 h. After dialysis to remove excess glutaraldehyde, 80 μL of the PD-L1 scFv antibody prepared in Example 1 was added and incubated at 4°C overnight. The uncoupled PD-L1 scFv antibody was removed by dialysis in a PBS solution, thereby obtaining the PD-L1-targeted sorafenib biomimetic exosomes.

[0094] Example 3: Characterization of PD-L1-targeted sorafenib biomimetic exosomes (PDL1-Cer-HyEH) 3.1 Measurement of particle size and zeta potential The particle size, polydispersity index (PDI) and Zeta potential of the HuP-T3 cell-derived exosomes, blank liposomes, ceritinib liposomes, ceritinib biomimetic exosomes and PD-L1 targeted ceritinib biomimetic exosomes were determined by using a HYPERLINK "http: / / www.baidu.com / link?url=ZT0EXrDwdgZFfDDx4mJYGQOYbQ_AiFc39Z6WBzbbXgsZCeKMizyVG99I3XAS8ZHslvyLgwV0tIBKJk0Ec74QPK" \t "_blank" nano particle size potentiometer Zetasizer Nano ZSP, and the results are shown in Table 4. The average particle size of ceritinib liposomes, ceritinib biomimetic exosomes and PD-L1 targeted ceritinib biomimetic exosomes is between 70 nm and 90 nm, and the Zeta potential is between -10 mV and -30 mV. The results in Table 4 show that the particle size distribution of the prepared nano-preparation is relatively uniform, and meets the particle size requirement of passing through the intercellular space of tumor neovascular wall during blood circulation, and the negative charge on the surface of the nano-preparation can play a role in stabilizing the preparation due to charge repulsion.

[0095] Table 4: Particle size, PDI and Zeta potential of different nano-preparations 3.2 Morphological observation of biomimetic exosomes by transmission electron microscopy The size and shape of the above dosage forms were observed by transmission electron microscopy. Figure 8 The transmission electron microscopy results of different ceritinib preparations derived from HuP-T3 cells. As can be seen from the figure, these preparations are spherical, with uniform particle size of about 70 nm to 100 nm, confirming that the biomimetic exosomes have uniform morphological characteristics of exosomes.

[0096] 3.3 Determination of encapsulation efficiency and drug loading In order to detect the content of ceritinib in ceritinib liposomes, ceritinib biomimetic exosomes and PD-L1 targeted ceritinib biomimetic exosomes, these ceritinib-loaded nano-preparations were destroyed with acetonitrile-water (50:50, v / v), then centrifuged, and the supernatant was taken for UPLC detection of ceritinib content. The specific method of UPLC detection is: using Agilent 1290+6120 MSD chromatographic system and Poroshell 120 EC-C18 chromatographic column (2.1x50mm, 1.9µm), gradient elution with acetonitrile-0.01% formic acid aqueous solution as mobile phase, flow rate 1.0 mL / min, column temperature 45℃, detection wavelength 214 nm, injection volume 0.8μL, detection time 5 min.

[0097] The encapsulation efficiency (EE) of the sorafenib was calculated according to the formula: EE (%) = the mass of the encapsulated sorafenib in the nanocarriers / the total mass of the added sorafenib x 100%. The drug loading DL (%) = the mass of the encapsulated sorafenib in the nanocarriers / (the total mass of the added nanocarriers and sorafenib) x 100%. The encapsulation efficiency of the sorafenib liposomes, the sorafenib biomimetic exosomes and the PD-L1 targeted sorafenib biomimetic exosomes was 86.66%, 82.19% and 79.11%, respectively, and the drug loading was 4.37%, 3.75% and 3.16%, respectively, as shown in Table 5. The data in Table 5 show that the liposomes, the biomimetic exosomes and the PD-L1 targeted biomimetic exosomes all achieved good encapsulation of the sorafenib.

[0098] Table 5: Encapsulation efficiency and drug loading of different sorafenib nanofomulations 3.4 Determination of the coupling rate of PD-L1 scFv The amount of the PD-L1 scFv coupled to the sorafenib biomimetic exosomes was determined by the BCA method. 0.5 ml of the prepared PD-L1 scFv modified biomimetic exosome suspension was taken, in which the added mass of the PD-L1 scFv was 0.0152 mg (the concentration of the PD-L1 scFv was 0.38 mg / mL, and the added volume was 40 μL), and the suspension was dialyzed in 2 ml of a PBS solution at 4°C for 48 h. The content of the PD-L1 scFv in the dialysate was determined by using a BCA kit (purchased from Shanghai Biyun Tian Biotechnology Co., Ltd.), and the coupling rate of the PD-L1 scFv was calculated according to the formula: coupling rate (%) = (1 - the mass of the PD-L1 scFv determined in the dialysate / the added mass of the PD-L1 scFv in the biomimetic exosomes) x 100%, which was 58.27 ± 3.86%, indicating that the PD-L1 scFv targeting molecule was successfully coupled to the biomimetic exosomes.

[0099] 3.5 Identification of exosome specific marker proteins by Western blotting The prepared PD-L1 targeting biomimetic exosomes were identified at the molecular level by Western blotting method for exosome specific marker proteins. Specifically, the protein concentrations of cell lysate, exosomes, biomimetic exosomes and targeting biomimetic exosomes were determined by BCA method (BCA protein concentration determination kit, Shanghai Biyun Tian Biotechnology Co., Ltd.), and after adjusting to the same concentration according to the determination results, a certain amount of 5x protein loading buffer was added, and the protein was denatured at 100℃ for 10 min. Prepare SDS-PAGE gel, load these prepared protein samples, electrophorese through the concentrated gel at 80V, and then electrophorese to bromophenol blue to the lower edge of the gel at 120V. Transfer the membrane at 300mA for 1 hour, block in 5% milk for 2 hours after transfer, cut the corresponding bands and incubate CD9, CD81 and PDCD6IP primary antibodies (Proteintech Company) respectively, 4℃ overnight, wash the membrane, incubate the secondary antibody (Beijing Zhongsu Jin Qiao Biotechnology Co., Ltd.) at room temperature for 2 hours, wash the membrane and expose. The results are shown in Figure 9 Western blotting confirmed that the PD-L1 targeting biomimetic exosomes expressed exosome specific marker proteins CD9, CD81 and PDCD6IP on the exosomes, indicating the successful preparation of the targeting biomimetic exosomes.

[0100] Example 4: Targeting analysis of PD-L1 targeting sunitinib biomimetic exosomes 4.1 Receptor-coated ELISA analysis of the specific affinity of PD-L1 targeting sunitinib biomimetic exosomes to PD-L1 antigen PD-L1 protein solution (purchased from Genscript) was coated on the ELISA plate at 1 μg / ml in an amount of 100 μl / well at 4°C overnight; the coated plate was washed with PBS for 3 times, then blocked with 1% BSA / PBS solution at 200 μl / well at 4°C overnight; washed with PBST buffer (0.05% Tween-20 in PBS) for 3 times; 8 gradient dilutions of ceritinib biomimetic exosomes, PD-L1 targeting ceritinib biomimetic exosomes and PD-L1 scFv were added, with concentrations of 0.0000128, 0.000064, 0.00032, 0.0016, 0.008, 0.04, 0.2, 1 μM respectively, 3 parallel holes for each concentration, incubated at 4°C overnight; washed with PBST for 3 times, then added HRP-streptavidin (purchased from Shanghai Biyun Tian Biotechnology Co., Ltd.) (1:2000) diluted at 50 μl / well, incubated at 37°C for 90 min; washed the plate with PBST for 5 times, added 100 μl / well of substrate reaction solution TMB (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.), reacted at room temperature for 2 min in the dark; added 2 M sulfuric acid 100 μl / well to stop the reaction, and immediately measured the absorbance value at 450 nm on the microplate reader. The results of the receptor-coated ELISA experiment are shown in Figure 10 As can be seen from the figure, the affinity of PD-L1 targeting ceritinib biomimetic exosomes and PD-L1 scFv for PD-L1 protein is much stronger than that of ceritinib biomimetic exosomes, and the affinity activity is concentration-dependent within a certain range, indicating that the PD-L1 single-chain antibody and its modified biomimetic exosomes have obvious affinity activity for PD-L1 protein.

[0101] 4.2 Targeting of PD-L1 targeting ceritinib biomimetic exosomes to pancreatic cancer cells with different expression of PD-L1 4.2.1 Cell ELISA analysis and binding specificity with cells 5 × 10 4The amount of cells of pancreatic cancer HuP-T3 and Panc0403 cells were inoculated in a 96-well plate, washed with PBS for 3 times in a 37°C, 5% CO2 incubator overnight; the cells were fixed with 4% paraformaldehyde for 30 min, washed with PBS for 3 times; the cells were blocked with 1% BSA for 2 h; the ceritinib biomimetic exosomes, PD-L1 targeting ceritinib biomimetic exosomes and PD-L1 scFv were diluted in 8 gradients, and the concentrations were 0.0000128, 0.000064, 0.00032, 0.0016, 0.008, 0.04, 0.2 and 1 μM respectively, 50 μl was added to each well, 3 replicate wells were set for each concentration, and the plate was incubated at 4°C overnight; after washing with PBST for 3 times, HRP-streptavidin (1:2000) was added at a concentration of 50 μl / well, and the plate was incubated at 37°C for 90 min; the plate was washed with PBST for 5 times, 100 μl / well of substrate solution TMB was added, and the plate was reacted at room temperature for 2 min; 100 μl / well of 2 M sulfuric acid was added to stop the reaction, and the absorbance value was measured at 450 nm wavelength, and the results are shown in Figure 11 As can be seen from Figure 11 A, compared with the biomimetic exosomes, the PD-L1 single-chain antibody and the modified biomimetic exosomes have strong affinity for the pancreatic cancer HuP-T3 cells with high expression of PD-L1. Figure 11 As can be seen from

[0102] 4.2.2 Fluorescence image analysis of cell uptake capacity Since ceritinib has no fluorescence, coumarin 6-labeled liposomes, biomimetic exosomes and PD-L1 targeting biomimetic exosomes were prepared using coumarin 6 as a fluorescent probe to study the accumulation and distribution of these nano-preparations in cells.

[0103] Thin film hydration method was used to prepare coumarin 6-labeled liposomes. Specifically, egg phospholipid, cholesterol, DSPE-PEG 2000 and DSPE-PEG 2000-NH2 (59:39:4:0.5, molar ratio) and coumarin 6 (coumarin 6:liposome = 1:200, w / w) were dissolved in chloroform and rotary evaporated at 40°C to form a lipid film. The film was hydrated with phosphate-buffered saline (PBS), sonicated at room temperature for 5 min, and then further sonicated in an ultrasonic cell disruptor for 6.8 min (working time 10 s, interval 10 s, incubation temperature 35°C). The hydrated liposome suspension was extruded three times sequentially through polycarbonate membranes with pore sizes of 400 nm and 200 nm. Free coumarin 6 not encapsulated in the liposomes was removed by passing the film through a Sephadex G-50 dextran gel column, yielding coumarin 6 liposomes.

[0104] The preparation of coumarin 6 biomimetic exosomes and PD-L1 targeted coumarin 6 biomimetic exosomes adopted the same preparation method as that of ceritinib biomimetic exosomes and PD-L1 targeted ceritinib biomimetic exosomes in Example 2, the only difference being that coumarin 6 liposomes were used instead of ceritinib liposomes in the method of Example 2.

[0105] Cell fluorescence imaging analysis includes: 2×10 4 HuP-T3 and Panc0403 cells were seeded into 24-well plates and incubated at 37°C with 5% CO2 for 24 h. Then, coumarin 6, coumarin 6 liposomes, coumarin 6 biomimetic exosomes, and PD-L1-targeting biomimetic exosomes (coumarin final concentration 1.2 μg / mL) were added, and incubation continued for 0, 5, 15, 30, 45, and 60 min. Subsequently, the cells were washed three times with ice-cold PBS and fixed with 2% paraformaldehyde at room temperature for 30 min. The cell nuclei were then stained with 10 μg / mL DAPI (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) for 30 min, followed by three washes with PBS. Finally, image analysis was performed using a Cytation 1 multi-mode microplate reader. Blue fluorescence represents the cell nucleus, and green fluorescence represents coumarin 6. Cell uptake results are shown below. Figure 12 As shown.

[0106] from Figure 12 A and Figure 12 B shows that in HuP-T3 cells with high PD-L1 expression, compared with free coumarin 6 and coumarin 6 liposomes, coumarin 6 biomimetic exosomes derived from autologous cells (i.e., coumarin 6 biomimetic exosomes derived from HuP-T3 cells) showed significantly higher intracellular uptake, indicating that homologous exosomes help improve cellular uptake. Compared with non-targeted coumarin 6 biomimetic exosomes, HuP-T3 cells showed a significant increase in the uptake of PD-L1-targeted coumarin 6 biomimetic exosomes. However, from Figure 12 C and Figure 12As can be seen from D, in PD-L1-low-expressing Panc0403 cells, there was no significant difference in the uptake of coumarin 6 liposomes, coumarin 6 biomimetic exosomes derived from HuP-T3 cells, and PD-L1-targeted coumarin 6 biomimetic exosomes by Panc0403 cells compared with free coumarin 6. Figure 12 The results showed that PD-L1-targeting biomimetic exosomes can specifically recognize the PD-L1 receptor on the surface of HuP-T3 cells and achieve active targeting of cells with high PD-L1 expression through receptor-mediated endocytosis.

[0107] Example 5: In vitro anti-pancreatic cancer activity of targeted ceritinib biomimetic exosomes 5.1 In vitro activity against the growth and proliferation of pancreatic cancer cells In HuP-T3 cells with high PD-L1 expression and Panc0403 cells with low PD-L1 expression, the effects of free ceritinib (as a control), ceritinib liposomes, ceritinib biomimetic exosomes, and PD-L1-targeted ceritinib biomimetic exosomes on cell proliferation activity were detected using the MTS method. Logarithmic growth phase cells were harvested at 5 × 10⁻⁶ cells / year. 3 Cells were seeded at a density of [number] cells / well in 96-well cell culture plates and cultured for 24 h at 37°C in a 5% CO2 incubator. A series of ceritinib concentration gradients were then added to HuP-T3 cells. The ceritinib concentration gradients were 0.097, 0.387, 1.547, 3.094, 6.188, 12.375, and 49.5%. M; For Panc0403 cells, the ceritinib concentration gradient was 2.949, 11.797, 47.188, 94.375, 188.75, 377.5, 755 and 1510. M. Using culture medium as a blank, each group was divided into 3 replicates and incubated at 37°C with 5% CO2 for 72 h. 10 μL of MTS solution (purchased from Promega) was added to each well, and incubation continued at 37°C for 2 h. The absorbance (OD) value at 490 nm was read using a microplate reader. The cell growth inhibition rate was calculated using the following formula: Inhibition rate = (OD of drug-treated wells - OD of blank wells) / (OD of control wells - OD of blank wells) × 100%. Results are as follows: Figure 13 As shown, compared to ceritinib, ceritinib liposomes, and ceritinib biomimetic exosomes, PD-L1-targeting ceritinib biomimetic exosomes exhibit significantly enhanced killing activity against HuP-T3 cells due to receptor-mediated endocytosis. Figure 13 A), and due to the lack of receptor-mediated endocytosis, its killing activity against Panc0403 cells showed no significant difference. Figure 13B). These results show that the PD-L1 single-chain antibody modified biomimetic exosomes of the present application have good biological activity on cells with high expression of PD-L1.

[0108] 5.2 In vitro activity of inducing apoptosis of pancreatic cancer cells The flow cytometry detection technology was used to detect the apoptosis of pancreatic cancer HuP-T3 cells by Annexin V-FITC / 7-AAD double staining method, so as to observe the induction activity of various sunitinib dosage forms on the apoptosis of HuP-T3 cells. HuP-T3 cells were inoculated into 6-well cell culture plates at a concentration of 5×10 5 Each of PBS (as a control), sunitinib, sunitinib liposomes, sunitinib biomimetic exosomes and PD-L1 targeted sunitinib biomimetic exosomes (sunitinib concentration of 12 μM) was added and placed in a cell incubator at 37°C with 5% CO2, and incubated for 18 h. The 6-well cell culture plate was taken out of the cell incubator, the supernatant was carefully aspirated, and the cells were collected and suspended in 500 μl of binding buffer (purchased from Beijing Bynovar Biotechnology Co., Ltd.). 5 μl of fluorescently labeled reagent Annexin V-FITC (purchased from Beijing Bynovar Biotechnology Co., Ltd.) and 5 μl of 7-AAD (7-aminoactinomycin D, purchased from Beijing Bynovar Biotechnology Co., Ltd.) were added under light shielding conditions, and the mixture was placed in the dark at room temperature for 15-20 min. The flow cytometry was used to detect the apoptosis rate of HuP-T3 cells. Figure 14 The effects of different sunitinib dosage forms on the apoptosis of HuP-T3 cells are shown in the figure. The results show that the apoptosis rates of HuP-T3 cells treated with free sunitinib, sunitinib liposomes, sunitinib biomimetic exosomes and PD-L1 targeted sunitinib biomimetic exosomes are 59.1%, 6.8%, 10.24% and 88.1% respectively, indicating that PD-L1 targeted sunitinib biomimetic exosomes cause more apoptosis of HuP-T3 cells than free sunitinib, sunitinib liposomes and sunitinib biomimetic exosomes.

[0109] In this embodiment, the PD-L1-scFv modified sunitinib biomimetic exosomes can specifically bind to PD-L1 on the surface of pancreatic cancer cells, significantly improve the uptake of drug-loaded PD-L1 high expression pancreatic cancer cells, and block the specific binding of PD-L1 and PD-1 to restore T cell activity and enhance anti-tumor immunity, showing good activity in inhibiting the proliferation of tumor cells, indicating that the PD-L1-scFv modified sunitinib biomimetic exosomes of the present application have good targeting and cancer treatment prospects.

[0110] The above merely describes several exemplary embodiments of the present application, and does not limit the present application in any form. Although the present application is disclosed with the preferred embodiments as above, it is not intended to limit the present application. Any skilled person in the art can make some changes or modifications to the above disclosed technical contents without departing from the scope of the technical solutions of the present application, and the equivalent or equivalent embodiments obtained by the changes or modifications fall within the scope of the present application.

Claims

1. A PD-L1 single-chain antibody comprising a heavy chain variable region, a light chain variable region, and a linker peptide connecting the heavy chain variable region and the light chain variable region; wherein: The heavy chain variable region includes: VH CDR1, whose amino acid sequence is SEQ ID NO: 1, or the sequence obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO: 1; VH CDR2, whose amino acid sequence is SEQ ID NO: 2, or the sequence obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO: 2; VH CDR3, whose amino acid sequence is SEQ ID NO: 3, or the sequence obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO: 3; and / or The light chain variable region includes: VL CDR1, whose amino acid sequence is SEQ ID NO: 4, or the sequence obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO: 4; VL CDR2, whose amino acid sequence is SEQ ID NO: 5, or the sequence obtained by substituting, deleting or adding one or more amino acids with SEQ ID NO: 5; VL CDR3, whose amino acid sequence is SEQ ID NO: 6, or a sequence obtained by substituting, deleting or adding one or more amino acids with SEQ ID NO:

6.

2. The PD-L1 single-chain antibody according to claim 1, wherein, The amino acid sequence of the heavy chain variable region comprises SEQ ID NO: 7 or as shown in SEQ ID NO: 7; and / or, the amino acid sequence of the light chain variable region comprises SEQ ID NO: 8 or as shown in SEQ ID NO:

8.

3. The PD-L1 single-chain antibody according to claim 1, wherein, The amino acid sequence of the linker peptide consists of 1 to 5 amino acid sequences as shown in SEQ ID NO: 9; preferably, the amino acid sequence of the linker peptide consists of 4 amino acid sequences as shown in SEQ ID NO:

9.

4. The PD-L1 single-chain antibody according to any one of claims 1 to 3, wherein, The amino acid sequence of the PD-L1 single-chain antibody includes SEQ ID NO: 10 or as shown in SEQ ID NO:

10.

5. A PD-L1-targeting biomimetic exosome, comprising a biomimetic exosome formed by fusing exosomes derived from cancer cells with liposomes and a PD-L1 single-chain antibody according to the present invention, wherein, The PD-L1 single-chain antibody is conjugated to the biomimetic exosome.

6. The PD-L1 targeted biomimetic exosome according to claim 5, wherein, The cancer cells are cancer cells that highly express PD-L1; preferably, the cancer cells are pancreatic cancer cells that highly express PD-L1; more preferably, the cancer cells are selected from any one of BxPC-3 cells, CFPAC-1 cells, HuP-T3 cells and SU86.86 cells; most preferably, the cancer cells are HuP-T3 cells. Preferably, the liposomes carry a drug for treating cancer; more preferably, the drug for treating cancer is selected from one or more of gemcitabine, irinotecan, bosutinib, AZ5104, dasatinib, and ceritinib; even more preferably, the drug for treating cancer is dasatinib and / or ceritinib; most preferably, the drug for treating cancer is ceritinib.

7. The PD-L1 targeted biomimetic exosome according to claim 5, wherein, The PD-L1-targeting biomimetic exosomes have a particle size of 80 nm to 92 nm, a zeta potential of -16 mV to -12.5 mV, an encapsulation efficiency of 70% to 85%, a drug loading of 3% to 3.5%, and a conjugation rate of 60% to 62% for the PD-L1 single-chain antibody. Preferably, the PD-L1-targeting biomimetic exosomes have an average particle size of about 87 nm, a zeta potential of about -14 mV, an encapsulation efficiency of about 79%, a drug loading of about 3%, and a conjugation rate of about 60% for the PD-L1 single-chain antibody.

8. The PD-L1 targeted biomimetic exosome according to claim 5, wherein, The PD-L1-targeting biomimetic exosomes contain at least one of the specific marker proteins CD9, CD81, and PDCD6IP.

9. A pharmaceutical composition for treating cancer, comprising PD-L1-targeting biomimetic exosomes according to any one of claims 5 to 8 and optionally pharmaceutically acceptable excipients; Preferably, the cancer is a pan-solid tumor expressing PD-L1; More preferably, the cancer is selected from one or more of pancreatic cancer, non-small cell lung cancer, bladder cancer, head and neck cancer, gastric cancer, breast cancer, colorectal cancer, kidney cancer, melanoma, and liver cancer; More preferably, the cancer is pancreatic cancer.

10. Use of the PD-L1-targeting biomimetic exosomes according to any one of claims 5 to 8 in the preparation of a medicament for treating cancer; Preferably, the cancer is a pan-solid tumor expressing PD-L1; More preferably, the cancer is selected from one or more of pancreatic cancer, non-small cell lung cancer, bladder cancer, head and neck cancer, gastric cancer, breast cancer, colorectal cancer, kidney cancer, melanoma, and liver cancer; More preferably, the cancer is pancreatic cancer.

Images