A pd-l1 targeting biomimetic exosome, and a pharmaceutical composition comprising the same and application thereof

Abstract

The present application provides a PD-L1 targeting biomimetic exosome, which comprises a biomimetic exosome formed by fusion of a fibroblast-derived exosome and a liposome loaded with a Pin1 inhibitor and a PD-L1 single-chain antibody, wherein the PD-L1 single-chain antibody is coupled to the biomimetic exosome. The present application also provides a pharmaceutical composition comprising the PD-L1 targeting biomimetic exosome and use of the PD-L1 targeting biomimetic exosome. The present application realizes the purposes of simultaneously eliminating tumor fibroblasts and tumor cells and improving the pancreatic cancer tumor microenvironment by combination of the PD-L1 targeting biomimetic exosome and a PD-L1 targeting biomimetic exosome loaded with ceritinib.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine. Specifically, the present application relates to a PD-L1 targeted biomimetic exosome, a pharmaceutical composition comprising the same and applications thereof. BACKGROUND

[0002] Pancreatic cancer is different from many other types of cancer in that the tumor tissue has less tumor cell content, and the dense interstitial tumor can account for 90% of the tumor volume. Cancer-associated fibroblasts (CAFs) are the main cell population in the interstitial tumor of pancreatic tumor, which forms a physical barrier between blood vessels and tumor cells. Conventional drug delivery methods will cause the drug to be blocked in the stroma, which seriously hinders the penetration of therapeutic drugs into the deep layer of tumor tissue, thereby reducing the treatment effect of pancreatic cancer. Therefore, effectively down-regulating CAFs to destroy this physical barrier will help to enhance the penetration and accumulation of therapeutic drugs, thereby improving the treatment effect. Current tumor treatment strategies targeting CAFs mainly include killing CAFs or interfering with the function of CAFs. However, clinical studies have found that the removal of CAFs by Hedgehog inhibitors can lead to increased metastasis of pancreatic cancer cells and significantly shorten the survival of patients. Further studies have found that CAFs have high heterogeneity, and there are different subtypes such as "tumor suppression" and "tumor promotion". Although eliminating "tumor promotion" CAFs can reduce tissue tension and promote the penetration of therapeutic drugs into tumor tissue to achieve the purpose of inhibiting tumors, it also exists the risk of misjudging the tumor suppressive "tumor suppression" CAFs, and the removal of CAFs can lead to a more loose and disordered tumor structure, thereby promoting the invasion of cancer cells into the surrounding tissue and spreading to distant sites, and increasing the risk of tumor metastasis. Therefore, it is not feasible to blindly eliminate CAFs, and targeting specific cancer-promoting pathways of CAFs may be a better treatment option.

[0003] The study found that the peptide-based prolyl cis-trans isomerase Pin1 was highly expressed in 71.5% of pancreatic cancer patients and in 51.9% of CAFs. Pin1 plays an important role in regulating the pancreatic cancer tumor microenvironment (TME). It can promote the activation of quiescent fibroblasts to CAFs by regulating the TGF-β signaling pathway, and activated CAFs secrete a large amount of extracellular matrix (ECM) components (such as collagen, fibronectin, IL-6, etc.), leading to fibrosis of the tumor microenvironment, further promoting the proliferation and invasion of tumor cells. Moreover, Pin1 regulates the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), leading to ECM deposition, increased fibrosis and hardening of the tumor microenvironment, forming a physical barrier that further limits drug delivery and immune cell infiltration. In addition, Pin1 also promotes the differentiation of regulatory T cells (Tregs) and other immunosuppressive cells and inhibits the function of effector immune cells, leading to tumor immune escape, thereby weakening the efficacy of immunotherapy. Therefore, Pin1 inhibitors can be selected to improve the pancreatic cancer TME. Sulfopin is a high-specificity Pin1 inhibitor discovered based on covalent binding drug screening, which can inhibit the proliferation of CAFs and the secretion of various cytokines by CAFs, and can increase the expression of PD-L1 on the surface of tumor cells, so Sulfopin is selected as an anti-tumor fibrosis drug. However, Sulfopin has problems such as low cell activity, non-specific distribution, and toxic side effects, so it is necessary to construct a targeted Sulfopin delivery system to selectively inhibit fibroblasts in pancreatic cancer and enhance tumor immunity, in order to overcome the treatment bottleneck of pancreatic cancer TME "stromal densification".

[0004] Anaplastic lymphoma kinase (ALK) is a tyrosine protein kinase closely related to malignant tumors. It can be activated by fusion with other genes and regulate the growth, differentiation and migration of tumors through a series of downstream cell signaling pathways. Studies have shown that blocking ALK signaling by TAE684 or crizotinib can inhibit the growth and proliferation of pancreatic tumors and induce apoptosis. Ceritinib, as a second-generation ALK tyrosine kinase inhibitor, has obvious antitumor activity against ALK-positive cancer. It also has the activity of an inhibitor of IGF-1R, and functions against CAFs and epithelial-mesenchymal transition (EMT) in the tumor microenvironment. In addition, ceritinib combined with gemcitabine can significantly inhibit the growth of pancreatic cancer. A phase I clinical trial of ceritinib combined with chemotherapy for patients with metastatic pancreatic ductal adenocarcinoma (PDAC) is currently underway (NCT02227940). Therefore, ceritinib has a good application prospect as an antitumor drug for the treatment of PDAC. However, due to the low solubility of ceritinib, CYP3A-mediated metabolism and P-glycoprotein (P-gp)-mediated efflux, its efficacy has been limited. In order to overcome these limitations, a highly efficient targeted drug delivery system needs to be developed to improve the therapeutic effect of ceritinib.

[0005] PD-L1 is an important immune checkpoint related to tumor immune escape. Since PD-L1 is highly expressed in up to 90% of pancreatic cancer patients, the currently approved PD-L1 antibody drugs such as atezolizumab and durvalumab have been shown to have good clinical efficacy. Moreover, CAFs not only directly hinder the antitumor function of cytotoxic T cells through the PD-L1 / PD1 pathway, but also upregulate the expression of PD-L1 in tumor cells, promoting immune evasion. PD-L1 can be used as a potential target for the treatment of pancreatic cancer. SUMMARY

[0006] To solve the above problems in the prior art, the inventors propose: for the PD-L1 target, a PD-L1 single-chain fragment variable (scFv) is used as a targeting antibody to modify a biomimetic exosome, which can not only be used as a targeting molecule to specifically and efficiently deliver a drug preparation to a pancreatic cancer tissue with high expression of PD-L1, but also act on an immunosuppressive TME to overcome the influence of increased PD-L1 expression caused by Sulfopin or CAFs; further, the biomimetic exosome is combined with a Pin1 inhibitor Sulfopin, an ALK inhibitor Ceritinib, and a drug for regulating the TGF-β signaling pathway and targeting CAFs in a tumor microenvironment to inhibit the synthesis and deposition of ECM, so as to improve and balance the condition of TME heterogeneity and interstitial fibrosis, and destroy various cellular components and physical barriers that prevent drug penetration and uptake, thereby achieving the effect of treating pancreatic cancer.

[0007] Therefore, based on the complex characteristics and molecular characteristics in the pancreatic cancer TME described above, the present application constructs two PD-L1 targeting biomimetic exosome systems, one of which uses a biomimetic exosome obtained by fusing a fibroblast-derived exosome with a liposome to realize its homing to the pancreatic cancer tumor microenvironment, and the other of which uses a biomimetic exosome obtained by fusing a pancreatic cancer cell-derived exosome with a liposome to realize its homing to the pancreatic cancer tumor cell, for sequentially delivering Sulfopin (Sul) and Ceritinib (Cer) to remodel the highly fibrotic tumor microenvironment of pancreatic cancer and achieve the multiple effects of synergistic triple combination drug therapy.

[0008] To achieve the above object, the present application provides the following technical solutions:

[0009] In a first aspect, the present application provides a PD-L1 targeting biomimetic exosome, which comprises a biomimetic exosome formed by fusing a fibroblast-derived exosome with a liposome loaded with a Pin1 inhibitor and a PD-L1 single-chain antibody, wherein the PD-L1 single-chain antibody is coupled to the biomimetic exosome.

[0010] According to some embodiments of the present application, the PD-L1 single-chain antibody 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:

[0011] The heavy chain variable region comprises:

[0012] 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, for example 2, 3, 4, or 5, amino acids in SEQ ID NO: 1;

[0013] a VH CDR2 having the 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;

[0014] a VH CDR3 having the 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;

[0015] and / or

[0016] the light chain variable region comprises:

[0017] a VL CDR1 having the 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;

[0018] a VL CDR2 having the 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;

[0019] a VL CDR3 having the amino acid sequence of SEQ ID NO: 6, 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: 6.

[0020] In particular, SEQ ID NOs: 1 to 6 are as follows:

[0021] SEQ ID NO: 1 : SYAIS;

[0022] SEQ ID NO: 2: RIIPILGIANYAQKFQG;

[0023] SEQ ID NO: 3: PYGSGSSLYAFDI;

[0024] SEQ ID NO: 4: SGSSSNIGNNYVS;

[0025] SEQ ID NO: 5: DNNKRPS;

[0026] SEQ ID NO: 6: GTWDSSLSAVV.

[0027] 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.

[0028] According to a preferred embodiment of the 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.

[0029] SEQ ID NO: 7:

[0030] QVQLQQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCASPYGSGSSLYAFDIWGQGTMVTVSS.

[0031] According to another preferred embodiment of the 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.

[0032] SEQ ID NO: 8:

[0033] HVILTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAVVFGGGTKVTVL.

[0034] According to some embodiments of the application, the amino acid sequence of the connecting peptide consists of 1 to 5 amino acid sequences as set forth in SEQ ID NO: 9; preferably, the amino acid sequence of the connecting peptide consists of 4 amino acid sequences as set forth in SEQ ID NO: 9.

[0035] SEQ ID NO: 9: GGGGS.

[0036] According to a particularly preferred embodiment of the application, the amino acid sequence of the PD-L1 single-chain antibody comprises SEQ ID NO: 10 or is as set forth in SEQ ID NO: 10.

[0037] SEQ ID NO: 10:

[0038] MQVQLQQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCASPYGSGSSLYAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSHVILTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAVVFGGGTKVTVLHHHHHH.

[0039] According to some embodiments of the application, the fibroblast cells are human fetal lung fibroblasts or human pancreatic cancer-associated fibroblasts. Preferably, the human fetal lung fibroblasts are human fetal lung fibroblasts MRC-5.

[0040] According to some embodiments of the application, the Pinl inhibitor is selected from one or more of Sulfopin, juglone, epigallocatechin gallate (EGCG), KPT-6566 and tretinoin.

[0041] Sulfopin has the chemical name 2-Chloro-N-(2,2-dimethylpropyl)-N-(tetrahydro-l,l-dioxido-3- thienyl)-acetamide. Sulfopin has the CAS number 2451481-08-4, a molecular weight of 281.80 and a molecular formula of C

[0042] 3-thienyl)-acetamide). Sulfopin has the CAS number 2451481-08-4, a molecular weight of 281.80 and a molecular formula of C 11 H 20 CIN03S, having the following structure:

[0043] .

[0044] KPT-6566 has the chemical name 2-[[4-[[[4-(tert-butyl)phenyl]sulfonyl]imino]-l-oxo-l,4- dihydro-2-naphthalenyl]sulfanyl]acetic acid. It has the CAS number 881487-77-0, a molecular weight of 443.54 and a molecular formula of C 22 H 21 N05S2, having the following structure:

[0045] .

[0046] EGCG is chemically named as Epigallocatechin gallate. Its CAS number is 989-51-5, the molecular weight is 458.372, the molecular formula is C 22 H 18 O 11 The structural formula is as follows:

[0047]

[0048] The CAS number of tretinoin is 302-79-4, the molecular weight is 300.435, the molecular formula is C 20 H 28 O2, and the structural formula is as follows:

[0049]

[0050] According to some embodiments of the present application, the particle size of the PD-L1 targeting biomimetic exosome is 70 nm to 120 nm, the Zeta potential is -10 mV to -30 mV, the encapsulation efficiency is 20% to 30%, the drug loading capacity is 1.0% to 2.0%, and the coupling rate of the PD-L1 single-chain antibody is 50% to 55%;

[0051] Preferably, the average particle size of the PD-L1 targeting biomimetic exosome is about 90 nm, the Zeta potential is about -20 mV, the encapsulation efficiency is about 28%, the drug loading capacity is about 1.4%, and the coupling rate of the PD-L1 single-chain antibody is about 51%;

[0052] 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.

[0053] The preparation method of the PD-L1 targeting biomimetic exosome of the present application can refer to the related schemes in the prior art, for example, using freeze-thaw method or membrane extrusion method.

[0054] As an example, the PD-L1 targeting biomimetic exosome of the present application can be prepared by a method comprising the following steps:

[0055] (1) Fibroblast-derived exosomes are fused with liposomes by freeze-thaw method or membrane extrusion method to form biomimetic exosomes;

[0056] (2) The biomimetic exosomes are added into a glutaraldehyde aqueous solution to obtain an aldehyde-modified biomimetic exosome solution, and then the PD-L1 single-chain antibody is added into the aldehyde-modified biomimetic exosome solution and incubated overnight, so that the PD-L1 single-chain antibody is covalently coupled to the surface of the biomimetic exosomes.

[0057] The fibroblast-derived exosomes can be isolated and extracted from the fibroblast culture supernatant by using the ExoQuick reagent method combined with ultrafiltration. The liposomes can be prepared by using the thin film hydration method, and the blank liposomes are prepared by using the raw material of DSPE-PEG2000-NH2. The drug can be loaded into the blank liposomes by using the ammonium sulfate gradient method. In addition, the liposomes can also be prepared by using the reverse phase evaporation method, the solvent injection method, the pH gradient method, the calcium acetate gradient method, and the like. 2000 The blank liposomes are prepared by using the raw material of DSPE-PEG2000-NH2 through the thin film hydration method. Then, the drug can be loaded into the blank liposomes by using the ammonium sulfate gradient method. In addition, the liposomes can also be prepared by using the reverse phase evaporation method, the solvent injection method, the pH gradient method, the calcium acetate gradient method, and the like.

[0058] In a second aspect, the present application provides a pharmaceutical composition for treating cancer, comprising a first PD-L1 targeting biomimetic exosome and optionally a pharmaceutically acceptable adjuvant; wherein the first PD-L1 targeting biomimetic exosome is the PD-L1 targeting biomimetic exosome according to the present application.

[0059] Preferably, the cancer is a pan-solid tumor expressing PD-L1.

[0060] 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.

[0061] Further preferably, the cancer is pancreatic cancer.

[0062] According to some embodiments of the present application, the pharmaceutical composition further comprises a second PD-L1 targeting biomimetic exosome; wherein the second PD-L1 targeting biomimetic exosome comprises a biomimetic exosome formed by fusing a cancer cell-derived exosome with a liposome loaded with an anti-cancer drug 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.

[0063] According to some embodiments of the present application, the cancer cell is a cancer cell highly expressing PD-L1.

[0064] Preferably, the cancer cell is a pancreatic cancer cell highly expressing PD-L1.

[0065] More preferably, the cancer cell is selected from any one of BxPC-3 cells, CFPAC-1 cells, HuP-T3 cells and SU86.86 cells.

[0066] Most preferably, the cancer cell is a HuP-T3 cell.

[0067] According to some embodiments of the present application, the anticancer drug is selected from one or more of gemcitabine, irinotecan, bosutinib, AZ5104, dasatinib and sorafenib.

[0068] Preferably, the anticancer drug is dasatinib and / or sorafenib.

[0069] More preferably, the anticancer drug is sorafenib.

[0070] 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%.

[0071] 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%.

[0072] 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.

[0073] The preparation method of the second PD-L1 targeting biomimetic exosome of the present application is similar to that of the first PD-L1 targeting biomimetic exosome, except that the cell source of the exosome and the loaded drug are adjusted, and thus the present application is not described herein.

[0074] In a third aspect, the present application provides use of the PD-L1 targeting biomimetic exosome or the pharmaceutical composition according to the present application in the preparation of a medicament for treating cancer.

[0075] According to some embodiments of the present application, the cancer is a pan-solid tumor expressing PD-L1.

[0076] 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.

[0077] More preferably, the cancer is pancreatic cancer.

[0078] Compared with the prior art, the technical scheme of the present application has the following beneficial effects:

[0079] The present application utilizes the exosomes secreted by human embryonic lung fibroblasts MRC-5, and after membrane fusion with liposomes loaded with pin1 inhibitor Sulfopin, self-assembly, and then PD-L1 single-chain antibody screened by covalent coupling phage peptide library display technology, a PD-L1 targeted Sulfopin biomimetic exosome (PDL1-Sul-HyE M ) is successfully constructed. In the treatment of patients, the PD-L1 targeted Sulfopin biomimetic exosome is administered first to kill tumor fibroblasts and eliminate the therapeutic obstacles of tumor microenvironment, and then the PD-L1 targeted Ceritinib biomimetic exosome (PDL1-Cer-HyE H ) constructed by the exosomes secreted by human pancreatic cancer HuP-T3 cells is used to eliminate tumor cells. Through this sequential therapy, the purposes of eliminating tumor fibroblasts and tumor cells at the same time and improving the pancreatic cancer tumor microenvironment are achieved.

[0080] Through experiments, it is verified that the PD-L1 targeted Ceritinib biomimetic exosome and the PD-L1 targeted Sulfopin biomimetic exosome of the present application can specifically recognize the PD-L1 receptor on the cell surface, and realize active targeting of high expression PD-L1 cells through receptor-mediated endocytosis. When the two kinds of PD-L1 targeted biomimetic exosomes are used in combination, the strongest inhibitory effect on HuP-T3 cells and MRC-5 cells is shown, which indicates that the combined administration can achieve the purpose of eliminating tumor cells and tumor fibroblasts at the same time. In addition, the present application proves through the scratch repair experiment that the combined administration of the two kinds of PD-L1 targeted biomimetic exosomes almost completely inhibits the scratch repair ability of HuP-T3 cells. And the present application also proves through the Transwell migration experiment that the combined administration of the two kinds of PD-L1 targeted biomimetic exosomes almost completely inhibits the migration ability of HuP-T3 cells. BRIEF DESCRIPTION OF DRAWINGS

[0081] Hereinafter, embodiments of the present application will be described in detail with reference to the accompanying drawings, in which:

[0082] 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 present application are shown.

[0083] 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 present application are shown.

[0084] Figure 3 The results of detecting the purity of AH06280 PD-L1 scFv by SDS-PAGE in Example 1 of the present application are shown.

[0085] Figure 4 The results of detecting the affinity activity of the AH06280 PD-L1 single-chain antibody to PD-L1 by ELISA in Example 1 of the application are shown.

[0086] Figure 5 The transmission electron microscope photos of different formulations in Example 3 of the application are shown. Among them, A shows the transmission electron microscope photos of different Sulfopin formulations derived from HuP-T3 cells; B shows the transmission electron microscope photos of different Sulfopin formulations derived from MRC-5 cells.

[0087] Figure 6 The results of detecting the expression of exosome marker proteins in the PD-L1 targeted biomimetic exosomes derived from HuP-T3 and MRC-5 cells by Western blotting in Example 3 of the application are shown.

[0088] Figure 7 The uptake of the PD-L1 targeted biomimetic exosomes in HuP-T3 cells and MRC-5 cells in Example 4 of the application is shown. Among them, A shows the fluorescence map of the uptake of the PD-L1 targeted biomimetic exosomes in HuP-T3 cells; B shows the quantitative results of the uptake of the PD-L1 targeted biomimetic exosomes in HuP-T3 cells; C shows the fluorescence map of the uptake of the PD-L1 targeted biomimetic exosomes in MRC-5 cells; D shows the quantitative results of the uptake of the PD-L1 targeted biomimetic exosomes in MRC-5 cells.

[0089] Figure 8 The inhibitory effects of different PD-L1 targeted biomimetic exosomes on pancreatic cancer HuP-T3 cells and fibroblast MRC-5 cells in Example 5 of the application are shown. Among them, A shows the inhibitory effect on pancreatic cancer HuP-T3 cells; B shows the inhibitory effect on fibroblast MRC-5 cells.

[0090] Figure 9 The effects of different PD-L1 targeted biomimetic exosomes on the scratch repair ability of pancreatic cancer HuP-T3 cells in Example 6 of the application are shown. Among them, A shows the images of the effects of all formulations containing Sulfopin and formulations containing Sulfopin on the scratch repair of HuP-T3 cells at 0h, 24h and 48h; B shows the quantitative results of the inhibition of all formulations containing Sulfopin and formulations containing Sulfopin on the scratch repair of HuP-T3 cells at 24h; C shows the quantitative results of the inhibition of all formulations containing Sulfopin and formulations containing Sulfopin on the scratch repair of HuP-T3 cells at 48h.

[0091] Figure 10Figure 7 shows the effect of co-culture and various ceritinib and sulfopin formulations on the migration ability of HuP-T3 cells in Example 7 of the present application. Among them, A shows the effect of co-culture of HuP-T3 cells and MRC-5 cells on the migration ability of HuP-T3 cells, **** Figure 7 shows the effect of co-culture and various ceritinib and sulfopin formulations on the migration ability of HuP-T3 cells in Example 7 of the present application. Among them, A shows the effect of co-culture of HuP-T3 cells and MRC-5 cells on the migration ability of HuP-T3 cells, DETAILED DESCRIPTION

[0092] The present application will be further described in detail below with reference to the specific embodiments, and the examples given are only to illustrate the present application, but not to limit the scope of the present application.

[0093] 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.

[0094] Example 1: Screening and preparation of PD-L1 scFv

[0095] 1.1 Screening of PD-L1 fully humanized monoclonal antibody using phage display technology

[0096] 1.1.1 Screening of PD-L1 fully humanized monoclonal antibody

[0097] (1) Materials

[0098] PD-L1.Fc (Cat#: Z03371, lot#: B60051712 crip, Genscript);

[0099] CHO-PD-L1 cells (Genscript);

[0100] CHO-K1 cells (Genscript);

[0101] PD-L1.His (Cat#: Z03425, Genscript);

[0102] Human naïve phage display library (kappa / lambda, size: 4 x 10 10 , Genscript);

[0103] Host bacteria: E. coli TG1;

[0104] M13KO7 helper phage (NEB, Cat. No.: N0315S);

[0105] Coating solution: 0.05 M NaHCO3, pH 9.6;

[0106] 2xYT: 16 g tryptone, 10 g yeast extract and 5 g NaCl dissolved in 1 L ddH2O;

[0107] PBS: 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.4;

[0108] ELISA microtiter plates (Corning, Cat. No.: 9018);

[0109] Blocking solution (MPBS): PBS buffer, pH 7.4, containing 5% skim milk;

[0110] Washing buffer (PBST): PBS buffer, pH 7.4, containing 0.05% Tween20;

[0111] Washing buffer: 1% BSA-PBS (for cell screening);

[0112] Elution buffer: 0.1 M TEA, trimethylamine (for phage display screening);

[0113] 0.1 M Tris-HCl (pH 7.4);

[0114] HRP conjugated Anti-M13 monoclonal antibody (SINO BIOLOGICAL, Cat#: 11973-MM05T-H);

[0115] Anti-fd Bacteriophage-Biotin (B2661, Sigma-Aldrich);

[0116] SA iFluor 647 prepared by Genscript;

[0117] pFASEBA expression vector prepared by GenScript;

[0118] 1 M Isopropyl-D-thiogalactoside (IPTG) (VWR, Cat#: 0487-100G);

[0119] Peroxidase AffiniPure Goat Anti-Human IgG F(ab')2 fragment specific (Jackson, Cat#: 109-035-097);

[0120] Goat anti Human IgG F(ab')2 Fragment specific [FITC] (Jackson, Cat#: 109-096-006);

[0121] Tetramethylbenzidine (TMB, GenScript);

[0122] 1M HC1 (GenScript);

[0123] pcDNA3.4 expression vector and HEK293-6F cells (prepared by GenScript);

[0124] 37°C CO2 incubator (Thermo Scientific, Model. 3951);

[0125] Biosafety cabinet (Thermo Scientific, Model. 1384);

[0126] Orbital shaker (Thermo Scientific, Model. 416);

[0127] Polyethyleneimine (Polysciences, Cat. No. 23966);

[0128] FreeStyle 293 medium (lifetechnologies, Cat. No. 12338-018);

[0129] TN1 (Organotechnie, Cat. No. 19553);

[0130] 125-ml shake flask (Corning, Cat. No. 430421);

[0131] Protein-A resin (GenScript, Cat. No. L00210);

[0132] Binding buffer: 0.15 M NaCl, 20 mM Na2HPO4, pH 7.0;

[0133] Elution buffer: 0.1 M Glycine-HCl, pH 3.2;

[0134] Neutralization buffer: 1 M Tris-HCl, pH 9.0;

[0135] Biacore T200, GR18010468 (GE Healthcare);

[0136] Series S Sensor Chip Protein A (GE Healthcare, Cat. No. 29-1275-55, Lot. No. 10272787);

[0137] HBS-EP + : 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% P20, pH 7.4 (GE Healthcare, Lot. No. BCBX0007).

[0138] (2) Methods

[0139] (2.1) Bio-panning of phage display library

[0140] Human naïve phage display library (kappa and lambda, size: 4 x 1011 10 Stock was precipitated with PEG / NaCl and resuspended in PBS for panning. The phage library was panned against human PD-L1. Fc was coated using a modified standard procedure developed by GenScript. Bio-panning was performed as solid phase panning in round 1 and round 2, and as cell panning in round 3. Specifically, human IgGl (as negative selection to reduce Fc binders) and human PD-L1 were coated on microtiter plates. Fc concentration was 50 μg / ml, coating buffer, 4°C overnight. Human naïve phage display library phage particles (2 x 1011 12pfu / pool) were diluted in blocking buffer. The library was added to human IgGl coated wells and incubated for 1 hour with slow shaking to allow Fc binder absorption. To completely reduce Fc binding phage, 10 μg of human Fc was added to the depleted phage pool as a soluble competitor and incubated for 1 hour with slow shaking to first compete for binding with Fc binding phage. The phage particles were then transferred to human PD-L1. The Fc coated wells were incubated for 1 hour at room temperature with shaking. After incubation, unbound / non-specifically bound phage was decanted and washed 10-15 times with wash buffer and 5 times with PBS. At the time of cell wash, approximately 2 x 10 7 CHO-PD-L1 cells were collected, washed with PBS, and then blocked with 1 mL of 3% (w / v) milk in PBS (MPBS) for 45 minutes at RT. At the same time, 2 x 10 12 phage particles were also blocked with MPBS and 1 x 10 7 washed CHO-K1 cells were added to each well and rotated for 45 minutes at RT. The phage / cell suspension was centrifuged at 500 g for 3 minutes. The purified phage was added to the PD-L1 CHO cells and incubated for 1 hour at RT, centrifuged at 500 g for 3 minutes, and washed 8 times with 1% BSA-PBS wash buffer. Bound phage (wells or cells) was 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. The phage was amplified and rescued using M13K07 helper phage according to standard procedures, and phage particles were prepared for subsequent biopanning. The first round of amplified phage was used as input phage for the second round of biopanning. The third round of infected TG1 cells were plated on LB-Amp+ plates. Monoclonal phage ELISA and flow cytometry (FACS) were used to verify binding activity.

[0141] (2.2) Monoclonal phage ELISA screening

[0142] Individual colonies were grown in 96 deep well plates and M13KO7 helper phage was saved overnight at 30°C. At the same time, two 96-well ELISA microplates were coated with 1 μg / ml of target antigen (human IgGl as control) in coating buffer and incubated overnight at 4°C. The plates were blocked with PBS plus 5% non-fat milk. After washing, 50 μl of phage supernatant from each deep well culture was added to each well and incubated for 2 hours at room temperature. After washing 3 times with wash buffer, HRP-conjugated anti-M13 monoclonal antibody was added and incubated for 45 minutes at 4°C. The plates were washed 6 more times and substrate solution was added to the wells for reaction. Absorbance was measured at 450 nm using a spectrophotometer. Phage binders that specifically recognized the antigen were selected for FACS verification.

[0143] (2.3) Phage FACS screening and DNA sequencing

[0144] The antigen binding phage supernatant was tested for binding to CHO-PD-L1 cells (control CHO-K1 cells) using standard procedures developed by GenScript. Cells were washed in PBS and then seeded at a density of 1 x 10 5 cells / 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. Cells were washed twice with cold PBS. Anti-fd phage biotin (3 μg / ml) was added at 100 μΐ / well and incubated at 4°C for 40 min. SA iFluor 647 (1 μg / ml) was added at 100 μΐ / well, washed twice and incubated at 4°C for 30 min. After two washes, cells were loaded and then fluorescence was detected. Flow cytometry data was collected. DNA sequencing of antigen and cell binders was performed using standard procedures developed by GenScript.

[0145] (2.4) Construction of FASEBA format library

[0146] The output plasmid from the 3rd round was amplified and digested by Sif I and Not I, 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.

[0147] (2.5) FASEBA format ELISA screening

[0148] Individual Fab colonies were expressed in 96-well plates. The expression and binding activity of crude protein secreted by E. coli into the culture medium to BSA and antigen protein were detected by ELISA. Specifically, individual colonies were cultured in 96-well plates and induced with IPTG at 30°C overnight. Meanwhile, 96-well ELISA microplates were coated with 1 μg / ml of target antigen (human IgG1 as control) in coating buffer at 4°C overnight. The plates were blocked with PBS containing 5% skim milk. After washing, 50 μΐ of phage supernatant from each overnight culture was added to the plates and incubated at room temperature for 2 hours, washed 3 times with washing buffer, 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 6 times, and substrate solution was added to the wells for reaction. The absorbance was measured at 450 nm using a spectrophotometer. Phage binders that specifically recognized the antigen were selected for FACS verification.

[0149] (2.6) FASEBA format FACS screening and DNA sequencing

[0150] 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. Cells were washed with PBS and seeded at a density of 1 x 10 5 cells / well (100 μΐ / well) and 50 μΐ of phage supernatant was added to each well and incubated at 4°C for 40 min. Cells were washed twice with cold PBS. After the second wash, cells were loaded and then fluorescence was detected. Flow cytometry was used to collect data. DNA sequencing was performed on antigen and cell binders using standard procedures developed by GenScript.

[0151] (2.7) Construction and production of IgG

[0152] 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 and light chain expression plasmids. Recombinant IgG secreted into the culture medium was purified using protein A affinity chromatography according to GenScript SOP. The concentration and purity of 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.

[0153] (2.8) Affinity determination of purified IgG

[0154] The affinity of purified antibodies to antigen binding was determined using a surface plasmon resonance (SPR) biosensor, Biacore T200. Antibodies were immobilized on a sensor chip by capture method. Antigen was used as analyte. Dissociation (kd) and association (ka) rate constant data were calculated using Biacore T200 evaluation software. The equilibrium dissociation constant (KD) was calculated from the ratio of kd / ka.

[0155] (3) Results

[0156] (3.1) To reduce the enrichment of Fc binders, human IgG1 was panned in each round and Fc binders were competed with human Fc protein before binding to PD-L1. Fc was used to obtain cell binders and in the 3rd round, cells were washed with CHO-PD-L1 and cells were panned with CHO-K1. The results are summarized in Table 1.

[0157] Table 1: Results of 3 rounds of screening using naïve human phage display library

[0158]

[0159] "Pan" in Table 1 refers to washing away unbound phage and retaining specifically bound phage.

[0160] (3.2) 222 antigens and cell binders were identified by ELISA and FACS, 95 samples were sequenced by DNA, and finally 8 monoclonal antibodies of complementarity determining regions (CDRs) were obtained, as shown in Table 2.

[0161] Table 2: ELISA values and amino acid frequencies of 8 CDR unique clones

[0162]

[0163] (3.3) Three high-frequency clones (AH06280, AH06281, AH06283) in phage and FASEBA libraries were selected to construct complete IgG1, and the purity and yield of the antibodies were shown in Table 3.

[0164] Table 3: Purity and yield of purified AH06280, AH06281, and AH06283 antibodies

[0165] (3.4) The CDR amino acid sequences of the PD-L1 antibodies obtained from the AH06280, AH06281, and AH06283 monoclonal antibodies are shown in SEQ ID NO: 1 to SEQ ID NO: 6, respectively. The format of the heavy chain is:

[0166]

[0167] AH06280:

[0168] Heavy chain:

[0169]

[0170] Light chain:

[0171]

[0172] AH06281:

[0173] Heavy chain:

[0174]

[0175] Light chain:

[0176]

[0177] AH06283:

[0178] Heavy chain:

[0179]

[0180] Light chain:

[0181]

[0182] 1.1.2 SPR analysis of the binding activity of three PD-L1 antibodies to PD-L1

[0183] The method is the same as step (2.8) above. Specifically, the affinity of purified PD-L1 antibodies (AH06280, AH06281, and AH06283) for binding to the antigen was determined using a surface plasmon resonance (SPR) biosensor, Biacore T200. The antibodies were immobilized on the sensor chip using a capture method, with the antigen as the analyte, to obtain dissociation (kd) and binding (ka) rate constant data. The equilibrium dissociation constant (KD) was calculated from the ratio of kd / ka. The affinity of the screened AH06280, AH06281, and AH06283 antibodies was determined using SPR. The results are as follows: Figure 1 As shown, the affinity constants (KD values) of the three antibodies AH06280, AH06281, and AH06283 for PD-L1 are 7.27 × 10⁻⁶. -10 M, 2.69 × 10 -9 M, 9.31×10 -9 M.

[0184] 1.1.3 ELISA analysis of the affinity activity of three PD-L1 antibodies for PD-L1

[0185] Receptor-coated ELISA was used to detect the affinity activity of three PD-L1 antibodies AH06280, AH06281, AH06283 to PD-L1 protein. 1 μg / ml of PD-L1 protein solution (Genscript) was coated on the ELISA plate at 100 μl / well, 4°C overnight; the coated plate was washed 3 times with PBS, then 1% BSA / PBS solution was added at 200 μl / well, 4°C overnight; PBST buffer (PBS containing 0.05% Tween-20) was used to wash 3 times; 8 gradient dilutions of AH06280, AH06281, AH06283 solution 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, 4°C overnight; PBST was washed 3 times, then HRP-streptavidin (1:2000) was added at 50 μl / well, 37°C incubated for 90 min; the plate was washed 5 times with PBST, 100 μl TMB substrate solution was added to each well, and the reaction was carried out at room temperature for 2 min, then 100 μl 2 mol / L sulfuric acid was added to terminate the reaction, and the absorbance value at 450 nm was immediately determined on the microplate reader. The results of receptor-coated ELISA experiment are shown in Figure 2 From the figure, it can be seen that the affinity of AH06280 PD-L1 antibody to PD-L1 protein is significantly stronger than that of AH06281 and AH06283, and its affinity activity is concentration-dependent within a certain range, therefore AH06280 clone with the highest affinity activity is selected as the subsequent PD-L1 scFv single-chain antibody sequence.

[0186] 1.2 Preparation of PD-L1 scFv antibody

[0187] The sequence of AH06280 PD-L1 scFv (VH-G4Sx3-VL-His6) is as follows: the VH of AH06280 (underlined sequence in SEQ ID NO: 10) at the N-terminus, the 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 the 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:

[0188]

[0189] Firstly, the prokaryotic expression system of AH06280 PD-L1 scFv was constructed, and 6xHis tag was connected at the C-terminal for the convenience of purification of the single-chain antibody. Then, the target protein was expressed (including bacterial culture, IPTG induction, and crude extraction to obtain the crude protein solution), and the target protein was purified (including Ni column affinity purification and molecular sieve chromatography), to obtain the target protein AH06280 PD-L1 scFv.

[0190] The expression method of the target protein AH06280 PD-L1 scFv is as follows:

[0191] 1) The E. coli expression host Bl21 (DE3) was transformed with the plasmid containing AH06280 PD-L1 scFv;

[0192] 2) A single colony was inoculated in 5 ml of LB liquid medium containing antibiotics (amoxicillin concentration: 100 μg / ml) from the transformation plate, and incubated at 37°C, 200 rpm overnight;

[0193] 3) The above bacterial solution was transferred to 100 ml of LB liquid medium containing antibiotics (amoxicillin concentration: 100 μg / ml) at a ratio of 1:100, and incubated at 37°C until OD 600 = 1.0;

[0194] 4) IPTG was added to a final concentration of 0.5 mM, and induced at 37°C, 200 rpm for 4 h;

[0195] 5) The induced culture solution was collected, and the bacterial cells were collected by centrifugation at 7000 rpm for 10 min;

[0196] 6) The bacterial cells were resuspended in buffer (PBS, 0.1 mM EDTA, pH: 7.4);

[0197] 7) The primary separated bacterial cells were resuspended in bacterial buffer (1:30), and the bacteria were broken by ultrasonic, and the broken bacterial supernatant was collected by centrifugation at 7000 rpm for 10 min, which was the initial sample solution of AH06280 PD-L1 scFv to be purified, and was stored at 4°C for standby.

[0198] The purification method of the target protein AH06280 PD-L1 scFv is as follows:

[0199] 1) The initial sample solution to be purified was filtered through a 0.45 μm filter membrane for loading;

[0200] 2) A pre-packed Ni ion affinity purification chromatography column was prepared, and a peristaltic pump, a chromatography column, and a protein UV detector were connected for standby;

[0201] 3) According to the pre-packed column specification, set the appropriate column flow rate, such as 4 ml / min, equilibrate the column with the sample buffer (PBS, 100 mM NaCl, pH: 7.4) for about 10 column volumes, and observe the stable reading of the ultraviolet detector:

[0202] 4) The filtered protein sample is loaded at a flow rate of 4 ml / min, and after loading, the column is washed with the sample buffer for about 10 column volumes;

[0203] 5) After the ultraviolet 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 the value of the ultraviolet detector is observed to collect the eluted protein in each part:

[0204] 6) Take 15 μl of each part of the eluted protein for electrophoresis analysis to determine the part where the target protein is located and preliminarily evaluate the purity of the purified target protein:

[0205] 7) Pour the eluted protein containing the target protein into a dialysis bag and dialyze the protein against protein dialysis solution (PBS, pH 7.4) for more than 4 hours for 3 times to remove imidazole and other substances:

[0206] 8) After dialysis, centrifuge the protein solution and filter it through a 0.2 μm filter membrane to remove bacteria, which is the final product.

[0207] 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 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 dilutions of PD-L1 antibody and PD-L1 single-chain antibody solution 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) dilution 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 in the dark, then added 100 μl of 2 mol / L sulfuric acid to each well to terminate the reaction, and immediately measured the absorbance value at 450 nm on an enzyme label instrument. The results of receptor-coated ELISA experiment are shown in Figure 4 As can be seen from the figure, 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.

[0208] Example 2: Preparation of PD-L1-targeted sunitinib and sulfopin biomimetic exosomes

[0209] 2.1 Preparation of sunitinib liposomes and sulfopin liposomes

[0210] Egg phosphatidylcholine, cholesterol, DSPE-PEG 2000 , and DSPE-PEG 2000-NH2 (Aivivo Shanghai Pharmaceutical Technology Co., Ltd.) 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 4°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 successively 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. Ceritinib was mixed with the blank liposomes (ceritinib: phospholipid = 1:20, w / w) by the ammonium sulfate gradient method. After shaking in a 40°C water bath for 20 min, the ceritinib that was not loaded into the liposomes was removed by dialysis in a PBS solution (purchased from MCE Company), to obtain ceritinib liposomes (Cer-Lipo), which were stored at 4°C for later use.

[0211] The preparation method of Sulfopin liposomes (Sul-Lipo) was the same as that of ceritinib liposomes (Cer-Lipo), except that Sulfopin (purchased from Shanghai Taosusheng Biotechnology Co., Ltd.) was used instead of ceritinib in the above method.

[0212] 2.2 Extraction of pancreatic cancer exosomes and fibroblast exosomes

[0213] Human pancreatic cancer cells HuP-T3 (a gift from Dr. Wang Liewei's laboratory at Mayo Clinic) were cultured in RPMI 1640 medium (Beijing Lambolide Trade Co., Ltd.) containing 10% fetal bovine serum (PAN, Germany). Human embryonic lung fibroblasts MRC-5 (purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were cultured in MEM medium (Beijing Lambolide Trade Co., Ltd.) containing 10% fetal bovine serum. All cells were cultured in a 37°C incubator containing 5% CO2.

[0214] ExoQuick reagent method combined with ultrafiltration method was used to isolate and extract exosomes secreted into the supernatant of HuP-T3 cell culture. HuP-T3 cells were cultured in a 150 mm culture dish to 60% confluence, and then replaced with RPMI 1640 medium containing 5% exosome-free serum (purchased from VivaCell company) and continued to culture for 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 by 30 kDa ultrafiltration membrane. The obtained concentrated solution was added with ExoQuick precipitant (purchased from SBI (System Biosciences) company) at a ratio of ExoQuick precipitant: supernatant = 1:5 (v / v), and then gently mixed uniformly and placed vertically at 4°C for 12 h or more. Centrifugation was performed at 1500 x g for 30 min at 4°C, and the precipitate at the bottom was pancreatic cancer cell exosomes. The exosome precipitate was suspended with appropriate PBS, and the protein concentration of the exosomes was determined by Bicinchoninic Acid Assay (BCA) method. The extracted exosomes were stored at -80°C for later use.

[0215] The preparation method of fibroblast exosomes was the same as that of pancreatic cancer cell exosomes, except that MRC-5 cells were used instead of HuP-T3 cells in the above method.

[0216] 2.3 Preparation of ceritinib biomimetic exosomes and sulfopin biomimetic exosomes

[0217] Exosomes with a concentration of 15 mg / mL and drug-loaded liposomes with a phospholipid concentration of 56000 μmol / L were mixed at a volume ratio of 9:50, in which HuP-T3 cell exosomes were membrane-fused with ceritinib liposomes to prepare ceritinib biomimetic exosomes (Cer-BE H ), and MRC-5 cell exosomes were membrane-fused with sulfopin liposomes to prepare sulfopin biomimetic exosomes (Sul-BE M ). The exosome and liposome mixture was ultrasonicated in an ultrasonic cell crusher for 6 min (power 200 W, working time 30 s, and intermittent time 30 s), and then sequentially extruded through polycarbonate membranes with pore sizes of 400 nm and 200 nm to obtain ceritinib biomimetic exosomes (Cer-BE H ) and sulfopin biomimetic exosomes (Sul-BE M ) prepared by membrane extrusion.

[0218] 2.4 Preparation of PD-L1-targeted ceritinib biomimetic exosomes and PD-L1-targeted sulfopin biomimetic exosomes

[0219] In 1 ml of ceritinib biomimetic exosomes, 20 μL of 25% glutaraldehyde aqueous solution was added, and after reaction at room temperature for 2 h, the excess glutaraldehyde was removed by dialysis. Then 80 μL of PD-L1 scFv antibody prepared in Example 1 (the concentration of PD-L1 scFv was 0.052 mg / ml) was added, and it was left overnight at 4°C. The uncoupled PD-L1 scFv antibody was removed by dialysis in a PBS solution, thereby preparing PD-L1 targeted ceritinib biomimetic exosomes (PD-L1@Cer-BE H ).

[0220] The preparation method of PD-L1 targeted sulfopin biomimetic exosomes (PD-L1@Sul-BE M ) is the same as that of PD-L1 targeted ceritinib biomimetic exosomes (PD-L1@Cer-BE H ), except that sulfopin biomimetic exosomes are used instead of ceritinib biomimetic exosomes in the above method.

[0221] 2.5 Preparation of coumarin 6 labeled liposomes, biomimetic exosomes and PD-L1 targeted biomimetic exosomes

[0222] Thin film hydration method was used to prepare coumarin 6 labeled liposomes. Egg phospholipid, cholesterol, DSPE-PEG 2000 and DSPE-PEG 2000 -NH2 (59:39:4:0.5, molar ratio) and coumarin 6 (coumarin 6:lipid = 1:200, w / w) were dissolved in chloroform, and a lipid film was formed by rotary evaporation at 40°C. Phosphate buffer PBS was added for hydration, and after ultrasonic treatment at room temperature for 5 min, further ultrasonic treatment was carried out in an ultrasonic cell crusher for 6.8 min (working time 10 s, intermittent time 10 s, protection temperature 35°C). The hydrated liposome suspension was successively extruded through polycarbonate membranes with pore sizes of 400 nm and 200 nm for 3 times, and free coumarin 6 not packaged into liposomes was removed by Sephadex G-50 dextran gel column, thereby obtaining coumarin 6 liposomes.

[0223] Coumarin 6 liposomes, biomimetic exosomes with HuP-T3 cell exosomes and corresponding PD-L1 targeted biomimetic exosomes were prepared by the same method as that for the preparation of ceritinib biomimetic exosomes and their PD-L1 targeted biomimetic exosomes described above, except that coumarin 6 liposomes were used instead of ceritinib liposomes.

[0224] The coumarin 6 liposomes and MRC-5 cell exosomes were prepared into biomimetic exosomes and corresponding PD-L1 targeting biomimetic exosomes, and the preparation method was the same as that of the above-mentioned Sulfopin biomimetic exosomes and PD-L1 targeting biomimetic exosomes, and the only difference was that the Sulfopin liposomes were replaced by coumarin 6 liposomes.

[0225] Example 3: Characterization of PD-L1 targeting biomimetic exosomes

[0226] 3.1 Determination of particle size and zeta potential

[0227] The particle size, polydispersity index (PDI) and zeta potential of the exosomes derived from HuP-T3 cells, blank liposomes, sorafenib liposomes, sorafenib biomimetic exosomes and PD-L1 targeting sorafenib biomimetic exosomes were determined by nanoparticle size potential instrument Zetasizer Nano ZSP, and the results are shown in Table 4. The average particle size of sorafenib liposomes, sorafenib biomimetic exosomes and PD-L1 targeting sorafenib biomimetic exosomes was between 70 nm and 90 nm, and the zeta potential was between -10 mV and -30 mV, indicating that the prepared sorafenib nanofomulation had a relatively uniform particle size distribution, and met the particle size requirement of passing through the intercellular space of tumor neovascular wall during blood circulation. At the same time, the negative charge on the surface of the nanofomulation can play a stabilizing effect due to charge repulsion.

[0228] Table 4 Particle size, PDI and zeta potential of different sorafenib nanofomulations

[0229]

[0230] At the same time, the particle size, polydispersity index (PDI) and zeta potential of the exosomes derived from MRC-5 cells, blank liposomes, Sulfopin liposomes, Sulfopin biomimetic exosomes and PD-L1 targeting Sulfopin biomimetic exosomes were also determined by nanoparticle size potential instrument Zetasizer Nano ZSP, and the results are shown in Table 5. The average particle size of Sulfopin liposomes, Sulfopin biomimetic exosomes and PD-L1 targeting Sulfopin biomimetic exosomes was between 70 nm and 100 nm, and the zeta potential was between -18 mV and -23 mV, indicating that the prepared Sulfopin nanofomulation had a relatively uniform particle size distribution, and the negative charge on the surface of the nanofomulation could play a stabilizing effect due to charge repulsion.

[0231] Table 5 Particle size, PDI and zeta potential of different Sulfopin nanofomulations

[0232]

[0233] 3.2 Transmission electron microscopy for morphological observation of the biomimetic exosomes

[0234] The size and shape of the above preparations were observed by transmission electron microscopy. Figure 5 Transmission electron microscopy results of different crizotinib formulations derived from HuP-T3 cells and different Sulfopin formulations derived from MRC-5 cells. As can be seen from the figure, these preparations are spherical, with uniform particle size, approximately between 70 nm and 120 nm.

[0235] 3.3 Determination of encapsulation efficiency

[0236] To detect the content of drugs in liposomes, biomimetic exosomes and PD-L1 targeted biomimetic exosomes, these nanocarriers loaded with crizotinib or Sulfopin were destroyed with acetonitrile-water (50:50, v / v), then centrifuged, and the supernatant was taken for detection. UPLC (Agilent 1290+6120 MSD chromatographic system) was used to detect the content of crizotinib, and the detection conditions were as follows: Poroshell 120 EC-C18 chromatographic column (2.1 x 50 mm, 1.9 pm), gradient elution with acetonitrile-0.01% formic acid aqueous solution as mobile phase (initial 0 min 5% acetonitrile, 0-0.5 min rapidly increased to 95% acetonitrile, 0.5-5 min maintained 95% acetonitrile, 5-5.01 min rapidly decreased to 5% acetonitrile), flow rate 1.0 mL / min, column temperature 45°C, detection wavelength 214 nm, injection volume 0.8 pL.

[0237] HPLC (Shimadzu Essentia LC-16 chromatographic system) was used to detect the content of Sulfopin, and the detection conditions were as follows: Phenomenex Luna C18 chromatographic column (250 x 4.6 mm, 5 pm), mobile phase acetonitrile-0.01% formic acid water (80:20, v:v), flow rate 1.0 mL / min, detection wavelength 214 nm, injection volume 20 pL.

[0238] The formula for calculating the drug (i.e. crizotinib or Sulfopin) encapsulation efficiency (EE) is: EE (%) = mass of encapsulated drug in nanocarriers / total mass of added drug x 100%.

[0239] The encapsulation efficiency and drug loading of sorafenib and Sulfopin in each preparation are shown in Table 6. It is calculated that the encapsulation efficiency of sorafenib liposomes, sorafenib biomimetic exosomes and PD-L1 targeted sorafenib biomimetic exosomes is 86.66%, 82.19% and 79.11% respectively, indicating that the liposomes, biomimetic exosomes and PD-L1 targeted biomimetic exosomes achieve good loading effect on sorafenib. The encapsulation efficiency of Sulfopin liposomes, Sulfopin biomimetic exosomes and PD-L1 targeted Sulfopin biomimetic exosomes is 32.13%, 29.27% and 27.71% respectively.

[0240] Table 6 Encapsulation efficiency of different sorafenib / Sulfopin nano-preparations

[0241]

[0242] 3.4 Determination of the coupling rate of PD-L1 scFv

[0243] The amount of PD-L1 scFv coupled to the biomimetic exosomes was determined by BCA method. 0.5 ml of prepared PD-L1 scFv modified biomimetic exosome suspension was taken, in which the added mass of PD-L1 scFv was 0.0152 mg, and was dialyzed in 2 ml of PBS solution at 4°C for 48 h. The content of PD-L1 scFv in the dialysate was determined by BCA kit (Shanghai Biyun Tian Biotechnology Co., Ltd.), and the coupling rate (%) = (1 - the mass of PD-L1 scFv measured in the dialysate / the added mass of PD-L1 scFv in the biomimetic exosomes) x 100% was calculated according to the formula. The coupling rate of PD-L1 scFv on PD-L1 targeted sorafenib biomimetic exosomes was 58.27±3.86%, and the coupling rate of PD-L1 scFv on PD-L1 targeted Sulfopin biomimetic exosomes was 51.26±2.89%, indicating that the PD-L1 scFv targeting molecule has been successfully coupled to the biomimetic exosomes.

[0244] 3.5 Identification of exosome specific marker protein by Western blotting method

[0245] The prepared PD-L1 targeting biomimetic exosomes of different cell sources were identified at the molecular level by Western blotting method for exosome-specific marker proteins. Specifically, the protein concentrations of cell lysates, exosomes, biomimetic exosomes and targeting biomimetic exosomes were determined by 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 the prepared protein sample, 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 1h, block in 5% milk for 2h after transfer, cut the corresponding bands and incubate CD9, CD81 and PDCD6IP primary antibodies (Proteintech Co., Ltd.) at 4℃ overnight, wash the membrane and incubate the secondary antibody (Beijing Zhongshanjinqiao Biotechnology Co., Ltd.) at room temperature for 2h, and then wash the membrane and expose. The results are shown in Figure 6 Figure 6, Western blotting confirmed that the HuP-T3 cell-derived PD-L1 targeting biomimetic exosomes and the MRC-5 cell-derived PD-L1 targeting biomimetic exosomes both retained the expression of exosome-specific marker proteins CD9, CD81 and PDCD6IP, indicating the successful preparation of the targeting biomimetic exosomes.

[0246] Example 4: Targeting analysis of PD-L1 targeting biomimetic exosomes of HuP-T3 and MRC-5 cell sources

[0247] Since ceritinib and sulfopin do not have 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 HuP-T3 cell-derived biomimetic exosomes and their corresponding PD-L1 targeting biomimetic exosomes in HuP-T3 cells, and to study the accumulation and distribution of MRC-5 cell-derived biomimetic exosomes and their corresponding PD-L1 targeting biomimetic exosomes in MRC-5 cells. Specifically, 2x10 4HuP-T3 cells or MRC-5 cells were seeded into 24-well plates, respectively, and incubated at 37 °C in a 5% CO2 incubator for 24 h, with 3 replicates in each group. For HuP-T3 cells, coumarin 6, coumarin 6 liposome, coumarin 6 exosome derived from HuP-T3 cells and its corresponding PD-L1 targeted exosome were added, respectively (the final concentration of coumarin was 1.2 μg / mL). For MRC-5 cells, coumarin 6, coumarin 6 liposome, coumarin 6 exosome derived from MRC-5 cells and its corresponding PD-L1 targeted exosome were added, respectively (the final concentration of coumarin was 1.2 μg / mL). Incubation was continued for 0, 5, 15, 30, 45 and 60 min. Thereafter, the cells were washed with ice-cold PBS for 3 times and fixed with 2% paraformaldehyde at room temperature for 30 min. Then the cell nuclei were stained with 10 μg / mL DAPI (purchased from Shanghai Biyun Tian Biotechnology Co., Ltd.) for 30 min, and washed with PBS for 3 times. Finally, image analysis was performed using Cytation 1 multifunctional microplate reader. Blue fluorescence represents the cell nucleus and green fluorescence represents coumarin 6. The results of cell uptake are shown in Figure 7

[0248] From Figure 7 A and Figure 7 B, it can be seen that in the PD-L1 high expression HuP-T3 cells, the coumarin 6 exosome derived from the self cells has a significantly high intracellular uptake compared with free coumarin 6 and coumarin 6 liposome, indicating that the homologous exosome helps to improve the uptake degree of the cells; the uptake amount of the PD-L1 targeted coumarin 6 exosome by the HuP-T3 cells is significantly increased compared with the non-targeted coumarin 6 exosome. Similarly, from Figure 7 C and Figure 7 D, it can be seen that the uptake degree of the coumarin 6 exosome derived from the self cells by the fibroblast MRC-5 cells is higher than that of free coumarin 6 and coumarin 6 liposome, and the uptake of the PD-L1 targeted coumarin 6 exosome is significantly stronger than that of the non-targeted coumarin 6 exosome. The results show that the PD-L1 targeted exosome can specifically recognize the PD-L1 receptor on the cell surface, and realize the active targeting of the high expression PD-L1 cells through receptor-mediated endocytosis.

[0249] Example 5: In vitro activity of two kinds of targeted sorafenib and sulfopin exosomes combined to inhibit pancreatic cancer cells and fibroblasts

[0250] ​In pancreatic cancer HuP-T3 cells, the MTS method was used to detect the effects of ceritinib, ceritinib liposomes, ceritinib biomimetic exosomes, and PD-L1-targeted ceritinib biomimetic exosomes, as well as their combination with targeted Sulfopin biomimetic exosomes, on cell proliferation activity. In fibroblast MRC-5 cells, the MTS method was used to detect the effects of Sulfopin, Sulfopin liposomes, Sulfopin biomimetic exosomes, and PD-L1-targeted Sulfopin biomimetic exosomes, as well as their combination with targeted ceritinib biomimetic exosomes, on cell proliferation activity. Logarithmic growth phase cells were harvested at 5 × 10⁶ cells / year. 3 HuP-T3 cells were seeded at a density of cells / well in 96-well cell culture plates and cultured at 37°C in a 5% CO2 incubator for 24 h. Different ceritinib formulations were added to HuP-T3 cells at concentration gradients of 0.1953125, 0.390625, 0.78125, 1.5625, 3.125, 6.25, 12.5, and 25 μM. Different Sulfopin formulations were added to MRC-5 cells at concentration gradients of 1.5625, 3.125, 6.25, 12.5, 25, 50, 100, and 200 μM. Using culture medium as a blank and untreated cells as a control, each group was divided into three 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 another 2 h. The absorbance (OD) at 490 nm was read using a microplate reader. Cell viability was calculated using the following formula: Viability = (OD of drug-treated wells - OD of blank wells) / (OD of control wells - OD of blank wells) × 100%.

[0251] The results are as follows Figure 8 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 8 A); Similarly, in MRC-5 cells, PD-L1-targeting Sulfopin biomimetic exosomes also showed a stronger inhibitory effect compared to Sulfopin, Sulfopin liposomes, and Sulfopin biomimetic exosomes. Figure 8 B). Moreover, in each treatment group, the combination of PD-L1-targeted ceritinib biomimetic exosomes and PD-L1-targeted Sulfopin biomimetic exosomes showed the strongest inhibitory effect on both HuP-T3 cells and MRC-5 cells, indicating that the combined administration can achieve the goal of simultaneously eliminating tumor cells and tumor fibroblasts.

[0252] Meanwhile, the drug combination index (CI) was used to evaluate the synergistic effect of the combination of two kinds of targeted biomimetic exosomes, and the calculation formula was: CI = (C A,x / IC x,A ) + (C B,x / IC x,B ) wherein C A,x and C B,x are the doses of drugs A and B required to achieve a specific X effect concentration (such as 50% effect concentration) at the combined dose, and IC x,A and IC x,B are the doses of single drugs A and B required to achieve the same effect concentration.

[0253] In this embodiment, the A drug is PD-L1 targeted ceritinib biomimetic exosomes, the B drug is PD-L1 targeted sulfopin biomimetic exosomes, and the X concentration is the drug concentration when the cell proliferation is inhibited by 50%, i.e. the IC 50 value. CI less than 1 indicates that the combination of two kinds of targeted biomimetic exosomes can produce synergistic effect.

[0254] The drug concentration value when the 50% inhibition effect is achieved is calculated by graphpad prism 9 software. For HuP-T3 cells, the graphpad prism 9 software calculates C A,50 = 0.7891 μM, IC 50, A = 2.029 μM, C B,50 = 0.6683 μM, IC 50, B = 1.678 μM, therefore, CI = 0.7891 / 2.029 + 0.6683 / 1.678 = 0.787. For MRC-5 cells, the graphpad prism 9 software calculates C A,50 = 3.431 μM, IC 50, A = 13.92 μM, C B,50 = 27.44 μM, IC 50, B = 56.82 μM. So CI = 3.431 / 13.92 + 27.44 / 56.82 = 0.729. Therefore, the drug combination index CI values of the combination of two kinds of PD-L1 targeted biomimetic exosomes for HuP-T3 cells and MRC-5 cells are 0.787 and 0.698 respectively, which indicates that the combination of two kinds of targeted biomimetic exosomes can produce synergistic effect on HuP-T3 cells and MRC-5 cells.

[0255] Example 6: Effect of two kinds of targeted Ceritinib and Sulfopin biomimetic exosomes on the scratch repair ability of HuP-T3 cells

[0256] The scratch repair experiment was used to study the effect of various Ceritinib and Sulfopin preparations on the scratch repair ability of HuP-T3 cells. The logarithmic growth phase of HuP-T3 cells was collected and inoculated in a 24-well plate at a density of 1.2 x 10 5 Each well was added with 1 mL of cell suspension, which was placed in a 37°C, 5% CO2 incubator until the cell confluence reached more than 85%, and a 200 μL gun head was used to draw a vertical line in the central part of the culture plate. The cells were washed with PBS for 3 times, and 1 mL of fresh RPMI 1640 culture medium containing 10% fetal bovine serum (Germany PAN) was added. Then PBS (as a control group), Ceritinib, Ceritinib liposome, Ceritinib biomimetic exosome, PD-L1 targeted Ceritinib biomimetic exosome, Sulfopin, Sulfopin liposome, Sulfopin biomimetic exosome, PD-L1 targeted Sulfopin biomimetic exosome, PD-L1 targeted Ceritinib biomimetic exosome + PD-L1 targeted Sulfopin biomimetic exosome combination (drug concentration was 1.0 μM) were added to each well, respectively. After the addition of drugs, 0 h photographing was immediately performed using Cytation1 cell imaging microplate detection system, three positions in each well were photographed, and the coordinates of each position were recorded. Then photographing was performed at the same position at 24 h and 48 h. The scratch healing rate was calculated: cell healing rate (%) = (0 h scratch width-24 h or 48 h scratch width) / 0 h scratch width) x 100%, and the differences in scratch healing between groups were compared.

[0257] The results are shown in Figure 9 The inhibitory ability of various Ceritinib preparations on scratch repair was significantly stronger than that of the corresponding Sulfopin preparations. At 24 h and 48 h, compared with the control group, all Ceritinib-containing preparations and Sulfopin-containing preparations had the ability to inhibit the scratch repair of HuP-T3 cells, among which PD-L1 targeted Ceritinib biomimetic exosome or PD-L1 targeted Sulfopin biomimetic exosome had the strongest inhibitory effect, and the combination of the two PD-L1 targeted biomimetic exosomes almost completely inhibited the scratch repair ability of HuP-T3 cells.

[0258] Example 7: Effect of two kinds of targeted Ceritinib and Sulfopin biomimetic exosomes on the migration ability of HuP-T3 cells after co-culture

[0259] Transwell migration assay was used to study the effect of various Sunitinib and Sulfopin formulations on the migration ability of HuP-T3 cells. Pancreatic cancer HuP-T3 cells and fibroblast MRC-5 cells were selected for co-culture. The specific process is as follows: (1) 100 μL of serum-free medium of HuP-T3 cells (3 x 10 4 cells / chamber) were plated in the upper chamber, and 600 μL of MRC-5 cells (3 x 10 4 cells / chamber) were plated in the lower chamber, and one lower chamber was set as a 600 μL medium containing 10% FBS control well. Different media were used in the upper and lower chambers, respectively, in which RPMI 1640 medium (Beijing Lambolide Trade Co., Ltd.) was used in the upper chamber, and MEM medium containing 10% fetal bovine serum (Germany PAN) was used in the lower chamber. 1 μM of different Sunitinib formulations and Sulfopin formulations were added to the lower chamber containing MRC-5 cells, and the chamber was placed in a 37°C, 5% CO2 incubator for further incubation for 24 h. (2) After 24 h, the medium in the chamber was aspirated, and the cells were washed with PBS for 2-3 times, and the cells in the chamber were fixed with 600 μL of 4% paraformaldehyde for 30 min, and the chamber was washed with PBS for 2-3 times again. (3) 600 μL of crystal violet staining solution was used for staining in the dark for 30 min, and then the staining solution in the chamber was washed clean with PBS buffer, and the cells in the chamber that were not perforated were removed with a cotton swab. (4) The chamber was observed and photographed under an inverted microscope, and more than 3 fields of view were photographed for each well.

[0260] The experimental results are shown in Figure 10 A. As can be seen from Figure 10 A, after co-culture with fibroblasts MRC-5, the number of HuP-T3 cell migration in the co-culture group was significantly increased compared with the HuP-T3 cell culture group alone, indicating that co-culture of pancreatic cancer HuP-T3 cells with fibroblasts can significantly improve the migration ability. As can be seen from Figure 10 B, various Sunitinib formulations inhibit the migration ability of HuP-T3 cells more strongly than the corresponding Sulfopin formulations. Compared with the control group, all Sunitinib-containing formulations and Sulfopin-containing formulations have a significant ability to inhibit the migration of HuP-T3 cells, among which the PD-L1 targeted Sunitinib biomimetic exosomes have a stronger inhibitory effect, and when the two PD-L1 targeted biomimetic exosomes are administered in combination, the effect is most obvious, and the migration ability of HuP-T3 cells is almost completely inhibited.

[0261] 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-targeting biomimetic exosome, comprising a biomimetic exosome formed by fusing fibroblast-derived exosomes with liposomes carrying a Pin1 inhibitor and a PD-L1 single-chain antibody, wherein, The PD-L1 single-chain antibody is conjugated to the biomimetic exosome; The PD-L1 single-chain antibody comprises 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; VH CDR2, its amino acid sequence is SEQ ID NO: 2; VH CDR3, its amino acid sequence is SEQ ID NO: 3; and The light chain variable region includes: VL CDR1, whose amino acid sequence is SEQ ID NO: 4; VL CDR2, whose amino acid sequence is SEQ ID NO: 5; VL CDR3, whose amino acid sequence is SEQ ID NO:

6.

2. The PD-L1 targeted biomimetic exosome according to claim 1, wherein, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

8.

3. The PD-L1 targeted biomimetic exosome 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.

4. The PD-L1 targeted biomimetic exosome according to claim 3, wherein, The amino acid sequence of the linker peptide consists of four amino acid sequences as shown in SEQ ID NO:

9.

5. The PD-L1-targeting biomimetic exosome according to any one of claims 1 to 4, wherein, The amino acid sequence of the PD-L1 single-chain antibody is shown in SEQ ID NO:

10.

6. The PD-L1 targeted biomimetic exosome according to claim 1, wherein, The fibroblasts are human embryonic lung fibroblasts or human pancreatic cancer-associated fibroblasts; The Pin1 inhibitor is selected from one or more of 2-chloro-N-(1,1-dioxatetrahydrothiophen-3-yl)-N-neopentylacetamide (Sulfopin), juglone, epigallocatechin gallate (EGCG), 2-[[4-[[[4-(tert-butyl)phenyl]sulfonyl]imino]-1-oxo-1,4-dihydro-2-naphthyl]thio]acetic acid (KPT-6566) and retinoic acid.

7. The PD-L1 targeted biomimetic exosome according to claim 1, wherein, The PD-L1-targeting biomimetic exosomes have a particle size of 70 nm to 120 nm, a zeta potential of -10 mV to -30 mV, an encapsulation efficiency of 20% to 30%, a drug loading of 1.0% to 2.0%, and a conjugation rate of 50% to 55% for the PD-L1 single-chain antibody.

8. The PD-L1 targeted biomimetic exosome according to claim 7, wherein, The PD-L1-targeting biomimetic exosomes have an average particle size of 90 nm, a zeta potential of -20 mV, an encapsulation efficiency of 28%, a drug loading of 1.4%, and a conjugation rate of 51% for the PD-L1 single-chain antibody.

9. The PD-L1 targeted biomimetic exosome according to claim 1, wherein, The PD-L1-targeting biomimetic exosomes contain at least one of the specific marker proteins CD9, CD81, and PDCD6IP.

10. A pharmaceutical composition for treating cancer, comprising a first PD-L1-targeting biomimetic exosome and optional pharmaceutically acceptable excipients; wherein, The first PD-L1-targeting biomimetic exosome is the PD-L1-targeting biomimetic exosome according to any one of claims 1 to 9; The cancer in question is pancreatic cancer.

11. The pharmaceutical composition of claim 10, further comprising a second PD-L1-targeting biomimetic exosome; wherein, The second PD-L1-targeting biomimetic exosome comprises a biomimetic exosome formed by fusing exosomes derived from cancer cells with liposomes loaded with an anticancer drug and a PD-L1 single-chain antibody, wherein the PD-L1 single-chain antibody is coupled to the biomimetic exosome; The PD-L1 single-chain antibody comprises 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; VH CDR2, its amino acid sequence is SEQ ID NO: 2; VH CDR3, its amino acid sequence is SEQ ID NO: 3; and The light chain variable region includes: VL CDR1, whose amino acid sequence is SEQ ID NO: 4; VL CDR2, whose amino acid sequence is SEQ ID NO: 5; VL CDR3, whose amino acid sequence is SEQ ID NO:

6.

12. The pharmaceutical composition according to claim 11, wherein, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

8.

13. The pharmaceutical composition according to claim 11, wherein, The amino acid sequence of the linker peptide consists of 1 to 5 amino acid sequences as shown in SEQ ID NO:

9.

14. The pharmaceutical composition according to claim 13, wherein, The amino acid sequence of the linker peptide consists of four amino acid sequences as shown in SEQ ID NO:

9.

15. The pharmaceutical composition according to any one of claims 11 to 14, wherein, The amino acid sequence of the PD-L1 single-chain antibody is shown in SEQ ID NO:

10.

16. The pharmaceutical composition according to claim 11, wherein, The cancer cells in question are those that highly express PD-L1.

17. The pharmaceutical composition according to claim 16, wherein, The cancer cells were pancreatic cancer cells that highly expressed PD-L1.

18. The pharmaceutical composition according to claim 17, wherein, The cancer cells are selected from any one of BxPC-3 cells, CFPAC-1 cells, HuP-T3 cells, and SU86.86 cells.

19. The pharmaceutical composition according to claim 18, wherein, The cancer cells were HuP-T3 cells.

20. The pharmaceutical composition according to claim 11, wherein, The anticancer drug is selected from one or more of gemcitabine, irinotecan, bosutinib, AZ5104, dasatinib, and ceritinib.

21. The pharmaceutical composition according to claim 20, wherein, The anticancer drugs mentioned are dasatinib and / or ceritinib.

22. The pharmaceutical composition according to claim 21, wherein, The anticancer drug in question is ceritinib.

23. The pharmaceutical composition according to claim 11, wherein, The second 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 conjugation rate of 60% to 62% for the PD-L1 single-chain antibody.

24. The pharmaceutical composition according to claim 23, wherein, The second PD-L1-targeting biomimetic exosome has an average particle size of 87 nm, a zeta potential of -14 mV, an encapsulation efficiency of 79%, a drug loading of 3%, and a conjugation rate of 60% for the PD-L1 single-chain antibody.

25. The pharmaceutical composition according to claim 11, wherein, The second PD-L1-targeting biomimetic exosome has at least one of the specific marker proteins CD9, CD81 and PDCD6IP.

26. Use of the PD-L1-targeting biomimetic exosome according to any one of claims 1 to 9 or the pharmaceutical composition according to any one of claims 10 to 25 in the preparation of a medicament for treating cancer, wherein the cancer is pancreatic cancer.

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