PD-L1 targeting bionic exosome, pharmaceutical composition containing PD-L1 targeting bionic exosome and application of PD-L1 targeting bionic exosome

Abstract

The invention provides a PD-L1 targeting bionic exosome, the PD-L1 targeting bionic exosome comprises a bionic exosome and a PD-L1 single-chain antibody, the bionic exosome is formed by fusing an exosome derived from fibroblast and a liposome loaded with a Pin1 inhibitor, and the PD-L1 single-chain antibody is coupled to the bionic exosome. The invention also provides a pharmaceutical composition containing the PD-L1 targeting bionic exosome, and an application of the PD-L1 targeting bionic exosome. According to the present invention, the PD-L1 targeting bionic exosome and the ceritinib-loaded PD-L1 targeting bionic exosome are subjected to drug combination so as to achieve the purposes of simultaneous removal of tumor fibroblasts and tumor cells and improvement of the pancreatic cancer tumor microenvironment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine. Specifically, the present invention relates to a PD-L1-targeted biomimetic exosome, a pharmaceutical composition containing the same, and applications thereof. Background Art

[0002] Pancreatic cancer is different from many other types of cancers. In its tumor tissue, the content of tumor cells is relatively low, while the dense tumor stroma can account for 90% of the tumor volume. Cancer-associated fibroblasts (CAFs) are the main cell population in the pancreatic tumor stroma. They form a physical barrier between blood vessels and tumor cells. Conventional drug delivery methods will cause drugs to be blocked in the stroma, seriously hindering the deep penetration of therapeutic drugs into the tumor tissue, thereby reducing the therapeutic effect of pancreatic cancer. Therefore, effectively downregulating CAFs to destroy this physical barrier will help enhance the penetration and accumulation of therapeutic drugs, thereby improving the therapeutic effect. Currently, the tumor treatment strategies targeting the regulation of CAFs mainly include killing CAFs or interfering with the functions of CAFs, etc. However, clinical studies have found that the elimination of CAFs by Hedgehog inhibitors can lead to an increase in the metastasis of pancreatic cancer cells and a significant shortening of the patient's survival period. Further research has found that CAFs have a high degree of heterogeneity and there are different subtypes such as "tumor-suppressing" and "tumor-promoting". Although the elimination of "tumor-promoting" CAFs will reduce tissue tension and promote the penetration of therapeutic drugs into the tumor tissue to achieve the purpose of inhibiting tumors, there is also the risk of accidentally injuring the tumor-suppressing "tumor-suppressing" CAFs. Moreover, the removal of CAFs may lead to a looser and more disordered tumor structure, thereby promoting the invasion of cancer cells into the surrounding tissues and spreading to distant sites, increasing the risk of tumor metastasis. Therefore, simply eliminating CAFs is not feasible, and targeting specific pro-tumor pathways of CAFs may be a better treatment option.

[0003] Research findings show that peptidyl-prolyl cis-trans isomerase Pin1 is highly expressed in 71.5% of pancreatic cancer patients and in 51.9% of patients' cancer-associated fibroblasts (CAFs). Pin1 plays an important role in regulating the tumor microenvironment (TME) of pancreatic cancer. It can promote the activation of quiescent fibroblasts into CAFs by regulating the TGF-β signaling pathway, and the activated CAFs secrete a large amount of extracellular matrix (ECM) components (such as collagen, fibronectin, IL-6, etc.), leading to the fibrosis of the tumor microenvironment and further promoting the proliferation and invasion of tumor cells. Moreover, Pin1 regulates the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), resulting in ECM deposition, increasing the fibrosis and sclerosis of the tumor microenvironment, forming a physical barrier, and further restricting drug delivery and immune cell infiltration. In addition, Pin1 also promotes the differentiation of immunosuppressive cells such as regulatory T cells (Tregs) and inhibits the function of effector immune cells, leading to tumor immune escape and thus weakening the efficacy of immunotherapy. Therefore, Pin1 inhibitors can be selected to improve the TME of pancreatic cancer. Among them, Sulfopin is a highly specific 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. Therefore, Sulfopin is selected as an anti-tumor fibrosis drug. However, Sulfopin has problems such as low cell activity, non-specific distribution, and side effects. Therefore, a targeted modified Sulfopin delivery system needs to be constructed to achieve selective inhibition of fibroblasts in pancreatic cancer, enhance tumor immunity, and overcome the treatment bottleneck of "dense matrix" in the TME of pancreatic cancer.

[0004] Anaplastic Lymphoma kinase (ALK), a tyrosine protein kinase closely related to malignant tumors, can be activated by fusing with other genes and regulate tumor growth, differentiation, and migration through a series of downstream cell signaling pathways. Research has shown that blocking ALK signaling with TAE684 or crizotinib can inhibit the growth, cell proliferation, and induce apoptosis of pancreatic tumors. Ceritinib, as a second-generation ALK tyrosine kinase inhibitor, has significant anti-tumor activity against ALK-positive cancers and also has inhibitor activity targeting IGF-1R, functioning against cancer-associated fibroblasts (CAFs) and epithelial-mesenchymal transition (EMT) in the tumor microenvironment. In addition, combination therapy with ceritinib and gemcitabine can also significantly inhibit the growth of pancreatic cancer, and a phase I clinical trial of ceritinib combined with chemotherapy drugs for the treatment of patients with metastatic pancreatic ductal adenocarcinoma (PDAC) is underway (NCT02227940). Therefore, choosing ceritinib as an anti-tumor drug for the treatment of PDAC has good application prospects. However, due to the low solubility of ceritinib, CYP3A-mediated metabolism, and P-glycoprotein (P-gp)-mediated efflux, its efficacy is limited. To overcome these limitations, an 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, currently approved PD-L1 antibody drugs such as atezolizumab and durvalumab have been proven to have good clinical efficacy. Moreover, CAFs not only directly hinder the anti-tumor function of cytotoxic T cells through the PD-L1 / PD1 pathway but also upregulate the expression of PD-L1 in tumor cells, promoting immune escape. PD-L1 can be a potential target for the treatment of pancreatic cancer. Summary of the Invention

[0006] In view of the problems existing in the above-mentioned prior art, the inventors propose that for the PD-L1 target, a biomimetic exosome modified with a PD-L1 single-chain antibody (scFv) as a targeting antibody can not only specifically and efficiently deliver a pharmaceutical preparation to pancreatic cancer tissues with high PD-L1 expression as a targeting molecule, but also act on the immunosuppressive tumor microenvironment (TME) to overcome the increased PD-L1 expression caused by Sulfopin or cancer-associated fibroblasts (CAFs). Furthermore, it is combined with Sulfopin, a Pin1 inhibitor that regulates the TGF-β signaling pathway, targets CAFs in the tumor microenvironment, and inhibits the synthesis and deposition of the extracellular matrix (ECM), and ceritinib (Ceritinib), an ALK inhibitor, in a triple combination. By affecting the ECM, CAFs, and epithelial-mesenchymal transition (EMT), it can improve and balance the highly heterogeneous and stromal fibrotic TME, and disrupt various cellular components and physical barriers that prevent drug penetration and uptake, thereby jointly exerting the efficacy of treating pancreatic cancer.

[0007] Therefore, based on the above-mentioned complex characteristics and molecular features in the pancreatic cancer TME, the present invention constructs two PD-L1-targeted biomimetic exosome systems. One uses a biomimetic exosome formed by fusing fibroblast-derived exosomes with liposomes to achieve its homing to the pancreatic cancer tumor microenvironment, and the other uses a biomimetic exosome formed by fusing pancreatic cancer cell-derived exosomes with liposomes to achieve its homing to pancreatic cancer tumor cells, for sequentially delivering Sulfopin (Sul) and ceritinib (Cer) to reshape the highly fibrotic tumor microenvironment of pancreatic cancer and achieve the multiple effects of synergistic enhancement of the triple combination therapy.

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

[0009] In a first aspect, the present invention provides a PD-L1-targeted biomimetic exosome, which comprises 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.

[0010] According to some embodiments of the present invention, 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] VH CDR1, whose amino acid sequence is SEQ ID NO: 1, or a sequence obtained by substitution, deletion, or addition of one or several, such as 2, 3, 4, or 5 amino acids in SEQ ID NO: 1;

[0013] The VH CDR2, whose amino acid sequence is SEQ ID NO: 2, or a sequence obtained by substitution, deletion or addition of one or several, such as 2, 3, 4 or 5 amino acids in SEQ ID NO: 2;

[0014] The VH CDR3, whose amino acid sequence is SEQ ID NO: 3, or a sequence obtained by substitution, deletion or addition of one or several, such as 2, 3, 4 or 5 amino acids in SEQ ID NO: 3;

[0015] and / or

[0016] The light chain variable region contains:

[0017] The VL CDR1, whose amino acid sequence is SEQ ID NO: 4, or a sequence obtained by substitution, deletion or addition of one or several, such as 2, 3, 4 or 5 amino acids in SEQ ID NO: 4;

[0018] The VL CDR2, whose amino acid sequence is SEQ ID NO: 5, or a sequence obtained by substitution, deletion or addition of one or several amino acids, such as 2, 3, 4 or 5 in SEQ ID NO: 5;

[0019] The VL CDR3, whose amino acid sequence is SEQ ID NO: 6, or a sequence obtained by substitution, deletion or addition of one or several amino acids, such as 2, 3, 4 or 5 in SEQ ID NO: 6.

[0020] Specifically, SEQ ID NO: 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 invention, 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 present invention, the amino acid sequence of the heavy-chain variable region comprises SEQ ID NO: 7 or is as shown in SEQ ID NO: 7.

[0029] SEQ ID NO: 7:

[0030] QVQLQQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCASPYGSGSSLYAFDIWGQGTMVTVSS.

[0031] According to another preferred embodiment of the present invention, the amino acid sequence of the light-chain variable region comprises SEQ ID NO: 8 or is as shown in SEQ ID NO: 8.

[0032] SEQ ID NO: 8:

[0033] HVILTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAVVFGGGTKVTVL.

[0034] According to some embodiments of the present invention, 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.

[0035] SEQ ID NO: 9: GGGGS.

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

[0037] SEQ ID NO: 10:

[0038] MQVQLQQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCASPYGSGSSLYAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSHVILTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAVVFGGGTKVTVLHHHHHH。

[0039] According to some embodiments of the present invention, the fibroblast is a human embryonic lung fibroblast or a human pancreatic cancer-associated fibroblast. Preferably, the human embryonic lung fibroblast is the human embryonic lung fibroblast MRC-5.

[0040] According to some embodiments of the present invention, the Pin1 inhibitor is selected from one or more of Sulfopin, juglone, epigallocatechin gallate (EGCG), KPT-6566, and retinoic acid.

[0041] The chemical name of Sulfopin is 2-Chloro-N-(2,2-dimethylpropyl)-N-(tetrahydro-1,1-dioxido-

[0042] 3-thienyl)-acetamide. The CAS number of Sulfopin is 2451481-08-4, the molecular weight is 281.80, and the molecular formula is C 11 H 20 ClNO3S, and the structural formula is as follows:

[0043] 。

[0044] The chemical name of KPT-6566 is 2-[[4-[[[4-(tert-butyl)phenyl]sulfonyl]imino]-1-oxo-1,4-dihydro-2-naphthalenyl]thio]acetic acid. Its CAS number is 881487-77-0, the molecular weight is 443.54, and the molecular formula is C 22 H 21 NO5S2, and the structural formula is as follows:

[0045] 。

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

[0047]

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

[0049]

[0050] According to some embodiments of the present invention, the particle size of the PD-L1 targeting biomimetic exosomes 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 is 1.0% to 2.0%, and the conjugation rate of the PD-L1 single-chain antibody is 50% to 55%;

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

[0052] According to some embodiments of the present invention, the PD-L1 targeting biomimetic exosomes have at least one of the specific marker proteins CD9, CD81, and PDCD6IP.

[0053] The preparation method of the PD-L1 targeting biomimetic exosomes of the present invention can refer to the relevant schemes in the prior art, such as the freeze-thaw method or the membrane extrusion method.

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

[0055] (1) Making the exosomes derived from fibroblasts fuse with liposomes to form biomimetic exosomes by the freeze-thaw method or the membrane extrusion method;

[0056] (2) Adding the biomimetic exosomes into an aqueous glutaraldehyde solution, reacting to obtain an aldehyde-group modified biomimetic exosome solution, and then adding the PD-L1 single-chain antibody to the aldehyde-group modified biomimetic exosome solution and incubating overnight, so that the PD-L1 single-chain antibody is covalently conjugated to the surface of the biomimetic exosomes.

[0057] Among them, fibroblast-derived exosomes can be isolated and extracted from fibroblast culture supernatant by using ExoQuick reagent method in combination with ultrafiltration method. Liposomes can be prepared from raw materials including lecithin, cholesterol and DSPE-PEG with a molar ratio of (50-70):(15-40):(2-5) 2000 -NH2 through the thin film hydration method to obtain blank liposomes. Then, drugs can also be loaded into the blank liposomes by the ammonium sulfate gradient method. In addition, the liposomes can also be prepared by conventional methods such as reverse phase evaporation method, solvent injection method, pH gradient method, calcium acetate gradient method, etc.

[0058] In a second aspect, the present invention provides a pharmaceutical composition for treating cancer, which comprises a first PD-L1-targeted biomimetic exosome and an optional pharmaceutically acceptable excipient; wherein, the first PD-L1-targeted biomimetic exosome is the PD-L1-targeted biomimetic exosome according to the present invention.

[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, renal cancer, melanoma and liver cancer;

[0061] Even more preferably, the cancer is pancreatic cancer.

[0062] According to some embodiments of the present invention, the pharmaceutical composition further comprises a second PD-L1-targeted biomimetic exosome; wherein, the second PD-L1-targeted biomimetic exosome comprises a biomimetic exosome formed by fusing exosomes derived from cancer cells with liposomes loaded with anti-cancer drugs and the PD-L1 single-chain antibody according to the present invention, wherein the PD-L1 single-chain antibody is conjugated to the biomimetic exosome.

[0063] According to some embodiments of the present invention, the cancer cells are cancer cells with high expression of PD-L1.

[0064] Preferably, the cancer cells are pancreatic cancer cells with high expression of PD-L1.

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

[0066] Most preferably, the cancer cells are HuP-T3 cells.

[0067] According to some embodiments of the present invention, the anti-cancer drug is selected from one or more of gemcitabine, irinotecan, bosutinib, AZ5104, dasatinib, and ceritinib.

[0068] Preferably, the anti-cancer drug is dasatinib and / or ceritinib.

[0069] More preferably, the anti-cancer drug is ceritinib.

[0070] According to some embodiments of the present invention, the PD-L1 targeted 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 the PD-L1 single-chain antibody of 60% to 62%.

[0071] Preferably, the PD-L1 targeted 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 the PD-L1 single-chain antibody of about 60%.

[0072] According to some embodiments of the present invention, the PD-L1 targeted biomimetic exosomes have at least one of the specific marker proteins CD9, CD81, and PDCD6IP.

[0073] The preparation method of the second PD-L1 targeted biomimetic exosomes of the present invention is similar to that of the first PD-L1 targeted biomimetic exosomes, except that the cell source of the exosomes and the loaded drug are adjusted. Therefore, the present invention will not be elaborated herein.

[0074] In a third aspect, the present invention provides the use of the PD-L1 targeted biomimetic exosomes according to the present invention or the pharmaceutical composition according to the present invention in the preparation of a drug for treating cancer.

[0075] According to some embodiments of the present invention, 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 solution of the present invention has the following beneficial effects:

[0079] The present invention utilizes exosomes secreted by human embryonic lung fibroblasts MRC-5, which self-assemble after membrane fusion with liposomes loaded with the pin1 inhibitor Sulfopin, and then are covalently coupled with a PD-L1 single-chain antibody screened by phage peptide library display technology, successfully constructing a PD-L1-targeted Sulfopin biomimetic exosome (PDL1-Sul-HyE M ). When treating patients, the PD-L1-targeted Sulfopin biomimetic exosome is administered first to kill tumor fibroblasts and remove the treatment obstacles in the tumor microenvironment, and then a PD-L1-targeted ceritinib biomimetic exosome (PDL1-Cer-HyE H ) constructed by using exosomes secreted by human pancreatic cancer HuP-T3 cells is used to eliminate tumor cells. Through this sequential therapy, the purpose of simultaneously eliminating tumor fibroblasts and tumor cells and improving the tumor microenvironment of pancreatic cancer is achieved.

[0080] Verified by experiments, both the PD-L1-targeted ceritinib biomimetic exosome and the PD-L1-targeted Sulfopin biomimetic exosome of the present invention can specifically recognize the PD-L1 receptor on the cell surface and achieve active targeting of cells with high expression of PD-L1 through receptor-mediated endocytosis. When these two PD-L1-targeted biomimetic exosomes are used in combination, the strongest inhibitory effect is shown on both HuP-T3 cells and MRC-5 cells, indicating that combined administration can achieve the purpose of simultaneously eliminating tumor cells and tumor fibroblasts. In addition, the present invention proves through a scratch repair experiment that the combined administration of these two PD-L1-targeted biomimetic exosomes almost completely inhibits the scratch repair ability of HuP-T3 cells. Moreover, the present invention also proves through a Transwell migration experiment that the combined administration of these two PD-L1-targeted biomimetic exosomes almost completely inhibits the migration ability of HuP-T3 cells. Description of the Drawings

[0081] Hereinafter, the embodiments of the present invention will be described in detail in conjunction with the drawings, where:

[0082] Figure 1 Shows the results of detecting the binding activity of antibodies AH06280, AH06281, and AH06283 to PD-L1 by SPR in Example 1 of the present invention.

[0083] Figure 2 Shows the results of detecting the affinity activity of antibodies AH06280, AH06281, and AH06283 to PD-L1 by ELISA in Example 1 of the present invention.

[0084] Figure 3 Shows the results of detecting the purity of AH06280 PD-L1 scFv by SDS-PAGE in Example 1 of the present invention.

[0085] Figure 4 Shows the results of detecting the affinity activity of the AH06280 PD-L1 single-chain antibody against PD-L1 by ELISA in Example 1 of the present invention.

[0086] Figure 5 Shows the transmission electron microscope photos of different preparations in Example 3 of the present invention. Among them, A shows the transmission electron microscope photos of different ceritinib preparations derived from HuP-T3 cells; B shows the transmission electron microscope photos of different Sulfopin preparations derived from MRC-5 cells.

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

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

[0089] Figure 8 Shows 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 present invention. 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 Shows 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 present invention. Among them, A shows the images of the effects of all ceritinib-containing preparations and Sulfopin-containing preparations on the scratch repair of HuP-T3 cells at 0 h, 24 h, and 48 h; B shows the quantitative results of the inhibition of the scratch repair of HuP-T3 cells by all ceritinib-containing preparations and Sulfopin-containing preparations at 24 h; C shows the quantitative results of the inhibition of the scratch repair of HuP-T3 cells by all ceritinib-containing preparations and Sulfopin-containing preparations at 48 h.

[0091] Figure 10Shows the effects of co-culture and various ceritinib and Sulfopin preparations on the migration ability of HuP-T3 cells in Example 7 of the present invention. Among them, A shows the effect of co-culturing HuP-T3 cells with MRC-5 cells on the migration ability of HuP-T3 cells, **** P < 0.001; B shows the effects of PD-L1-targeted ceritinib biomimetic exosomes and PD-L1-targeted Sulfopin biomimetic exosomes and their combination on the migration ability of HuP-T3 cells in the co-culture system. a: p < 0.05 indicates a significant difference compared with the control group; b: p < 0.05 indicates a significant difference compared with free ceritinib or Sulfopin; c: p < 0.05 indicates a significant difference compared with ceritinib or Sulfopin liposomes; d: p < 0.05 indicates a significant difference compared with ceritinib or Sulfopin biomimetic exosomes; e: p < 0.05 indicates a significant difference compared with ceritinib or Sulfopin-targeted biomimetic exosomes. Detailed implementation methods

[0092] The present invention will be further described in detail below in combination with the specific implementation methods. The given examples are only for clarifying the present invention and do not limit the scope of the present invention.

[0093] The experimental methods and conditions used in the following examples are all conventional methods and conditions unless otherwise specified. The reagents used in the following examples are all commercially available products unless otherwise specified.

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

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

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

[0097] (1) Materials

[0098] PD-L1.Fc (Cat#: Z03371, lot#: B60051712crip, 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: 4×10 10 , Genscript);

[0103] Host bacterium: Escherichia coli TG1 (E.coli TG1);

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

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

[0106] 2×YT: 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 plate (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: 0.1M TEA, trimethylamine (for phage display screening);

[0113] 0.1M 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 HCl (Genscript);

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

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

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

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

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

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

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

[0130] 125 - ml Erlenmeyer 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] S series 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) Method

[0139] (2.1) Biopanning of phage display library

[0140] Human naïve phage display library (kappa and lambda, size: 4×10 10 ) The stock solution was precipitated with PEG / NaCl and then resuspended in PBS for screening. The phage library was screened for human PD-L1. Fc used a modified standard procedure developed by GenScript. Biopanning was performed with solid-phase screening in the first and second rounds and cell screening in the third round. Specifically, human IgG1 (as negative screening to reduce Fc binders) and human PD-L1 were coated on the microplate. The Fc concentration was 50 μg / ml, coating buffer, overnight at 4°C. Human naïve phage display library phage particles (2×10 12Dilute with blocking buffer (pfu / pool). Add the library into wells coated with human IgG1, and incubate with gentle shaking for 1 hour to absorb Fc conjugates. To completely reduce Fc-binding phages, add 10 μg of human Fc as a soluble competitor into the depleted phage pool, and incubate with gentle shaking for 1 hour to compete for binding with Fc-binding phages first. Then transfer the phage particles into wells coated with human PD-L1 Fc, and incubate with shaking at room temperature for 1 hour. After incubation, decant the unbound / nonspecifically bound phages, wash 10 - 15 times with wash buffer and 5 times with PBS. When rinsing the cells, collect approximately 2×10 7 CHO-PD-L1 cells, wash with PBS, then block with 1 mL of 3% (w / v) milk in PBS (MPBS) and rotate at RT for 45 minutes. Meanwhile, 2×10 12 phage particles extracted from the recovered library stock are also blocked with MPBS, and add 1×10 7 washed CHO-K1 cells into each pool, and rotate at RT for 45 minutes. Centrifuge the phage / cell suspension at 500 g for 3 minutes. Add the purified phages into PD-L1 CHO cells, rotate and incubate at RT for 1 hour, centrifuge at 500 g for 3 minutes, and wash 8 times with 1% BSA-PBS wash buffer. Elute the bound phages (wells or cells) with TEA and neutralize with 0.1 M Tris-HCl (pH 7.4). Infect 10 ml of exponentially growing Escherichia coli TG1 with the phage eluate at 37°C. Amplify and rescue the phages with M13K07 helper phage according to the standard procedure, and prepare phage particles for subsequent biological screening. The phages amplified in the first round are used as the phage input for the second round of biological screening. In the third round, plate the infected TG1 cells on LB-Amp+ plates. Verify the binding activity by monoclonal phage enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS).

[0141] (2.2) Monoclonal phage ELISA screening

[0142] Culture single colonies in 96-deep well plates and incubate overnight at 30°C with M13KO7 helper phage. Meanwhile, coat 2 96-well ELISA microtiter plates with 1 μg / ml of the target antigen (using human IgG1 as a control), place them in coating buffer, and incubate overnight at 4°C. Block the plates with PBS plus 5% non-fat milk powder. After washing, take 50 μl of the phage supernatant from each deep well culture and incubate at room temperature for 2 hours. After washing 3 times with wash buffer, add HRP-conjugated anti-M13 monoclonal antibody and incubate at 4°C for 45 minutes. Wash the plates 6 more times, add the substrate solution into the wells for reaction. Measure the absorbance at 450 nm using a spectrophotometer. Select phage conjugates that specifically recognize the antigen for FACS verification.

[0143] (2.3)Phage FACS Screening and DNA Sequencing

[0144] The binding of the phage supernatant of the antigen binder to CHO-PD-L1 cells (control CHO-K1 cells) was detected by flow cytometry using a standard procedure developed by GenScript. The cells were washed in PBS and then seeded at a density of 1×10 5 cells / well (100 μl / well), and 50 μl of the phage supernatant sample 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 μg / ml) was added at 100 μl / well and incubated at 4 °C for 40 min. SA iFluor 647 (1 μg / ml) was added at 100 μl / 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. The antigen and cell binder were subjected to DNA sequencing using a standard procedure developed by GenScript.

[0145] (2.4)Construction of the FASEBA Format Library

[0146] The output plasmid of the third round was amplified and digested with SifⅠ and NotⅠ, and the digested FASEBA vector was used to construct the FASEBA library. High-throughput FASEBA antigen protein screening was performed on the obtained FASEBA library.

[0147] (2.5)FASEBA Format ELISA Screening

[0148] Single Fab colonies were expressed in 96-well plates. The expression and binding activities of the crude proteins secreted by Escherichia coli into the medium to BSA and antigen proteins were detected by ELISA. Specifically, single colonies were cultured in 96-well plates and induced overnight at 30 °C with IPTG. Meanwhile, 96-well ELISA microtiter plates were coated with 1 μg / ml of the target antigen (using human IgG1 as a control) overnight at 4 °C in coating buffer. The culture dishes were blocked with PBS containing 5% skim milk powder. After rinsing, 50 μl of the phage supernatant of each overnight culture was added to the plate and incubated at room temperature for 2 hours. After washing 3 times with the washing buffer, HRP-conjugated anti-human IgG F(ab’)2 monoclonal antibody was added to the plate and incubated at room temperature for 45 min, and then washed 6 times. The substrate solution was added to the wells for reaction. The absorbance was measured at 450 nm using a spectrophotometer. Phage binders that specifically recognize the antigen were selected for FACS verification.

[0149] (2.6)FASEBA Format FACS Screening and DNA Sequencing

[0150] The binding of the phage supernatant of the antigen-binding body to CHO-PD-L1 cells (control CHO-K1 cells) was detected by flow cytometry using a standard procedure developed by GenScript. The cells were washed with PBS and seeded at a density of 1×10 5 cells / well (100 µl / well), and 50 µl of the phage supernatant sample to be tested was added to each well. The cells were incubated at 4 °C for 40 min. The cells were washed twice with cold PBS. After two washes, the cells were loaded and then the fluorescence was detected. Flow cytometry was used to collect data. The antigen and cell conjugates were subjected to DNA sequencing using a standard procedure developed by GenScript.

[0151] (2.7) Construction and production of IgG

[0152] The DNA sequence encoding 3 leader sequences was inserted into pcDNA3.4 to construct an expression plasmid for full-length IgG. HEK293F cells were co-transfected with the heavy-chain and light-chain expression plasmids. The recombinant IgG secreted into the 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 with a BiacoreT200 to confirm binding and determine the affinity.

[0153] (2.8) Affinity determination of purified IgG

[0154] The affinity of the purified antibody for the antigen was determined separately using a surface plasmon resonance (SPR) biosensor, BiacoreT200. 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 evaluation software of the BiacoreT200. 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 conjugates, human IgG1 was eluted in each round, and the Fc conjugates were competitively bound to human Fc protein before binding to PD-L1. Fc was used to obtain cell conjugates. In the 3rd round, the cells were rinsed with CHO-PD-L1 and eluted with CHO-K1. The results are summarized in Table 1.

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

[0158]

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

[0160] (3.2) A total of 222 antigens and cell conjugates were co-identified by ELISA and FACS. DNA sequencing was performed on 95 of these samples, and finally monoclonal antibodies against 8 Complementarity determining regions (CDRs) were obtained, as shown in Table 2.

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

[0162]

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

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

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

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

[0184] 1.1.3 ELISA analysis of the binding activities of three PD-L1 antibodies to PD-L1

[0185] Receptor-coated ELISA was used to detect the affinity activities of three PD-L1 antibodies, namely AH06280, AH06281, and AH06283, against the PD-L1 protein. A 1 μg / ml PD-L1 protein solution (Genscript) was dispensed at 100 μl / well onto an ELISA plate and coated overnight at 4°C. After the coated plate was washed three times with PBS, it was blocked overnight at 4°C with a 1% BSA / PBS solution at 200 μl / well. It was then washed three times with PBST buffer (PBS containing 0.05% Tween-20). Eight serially diluted solutions of AH06280, AH06281, and AH06283 with concentrations of 0.0000128, 0.000064, 0.00032, 0.0016, 0.008, 0.04, 0.2, and 1 μM were added, with three parallel wells for each concentration, and incubated overnight at 4°C. After washing three times with PBST, HRP-streptavidin (1:2000) was added for dilution at 50 μl / well and incubated at 37°C for 90 min. The plate was washed five times with PBST, 100 μl of TMB substrate solution was added to each well, and the reaction was carried out in the dark at room temperature for 2 minutes. Subsequently, 100 μl of 2 mol / L sulfuric acid was added to terminate the reaction, and the absorbance value at 450 nm was immediately measured on a microplate reader. The results of the receptor-coated ELISA experiment are as shown in Figure 2 As shown. It can be seen from the figure that the AH06280 PD-L1 antibody has significantly stronger affinity for the PD-L1 protein than AH06281 and AH06283, and its affinity activity shows concentration dependence 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.

[0186] 1.2 Preparation of PD-L1 scFv antibody

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

[0188]

[0189] First, a prokaryotic expression system for AH06280 PD-L1 scFv was constructed. To facilitate the purification of the single-chain antibody, a 6×His tag was linked to the C-terminus. Then, through the expression of the target protein (including bacterial culture, IPTG induction, and cell disruption for crude extraction to obtain a crude protein extract) and the purification of the target protein (including Ni column affinity purification and size exclusion chromatography), the target protein AH06280 PD-L1 scFv was obtained.

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

[0191] 1) Transform Escherichia coli expression host Bl21(DE3) with the plasmid containing AH06280 PD-L1 scFv;

[0192] 2) Pick a single colony from the transformation plate and inoculate it into 5 ml of LB liquid medium containing antibiotics (ampicillin concentration: 100 μg / ml), and shake it overnight at 37°C and 200 rpm;

[0193] 3) Transfer the above bacterial liquid at a ratio of 1:100 to 100 ml of LB liquid medium containing antibiotics (ampicillin concentration: 100 μg / ml), and culture it at 37°C until OD600 = 1.0;

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

[0195] 5) Collect the above induced culture solution, and collect the bacterial cells by centrifugation at 7000 rpm for 10 min;

[0196] 6) Resuspend the bacterial cells in a buffer (PBS, 0.1 mM EDTA, pH: 7.4);

[0197] 7) Resuspend the initially separated bacterial cells in a cell buffer (1:30), disrupt the cells by sonication, and collect the supernatant after cell disruption by centrifugation at 7000 rpm for 10 min. This is the initial sample solution of the protein AH06280 PD-L1 scFv to be purified, and it is stored at 4°C for later use.

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

[0199] 1) Filter the initial sample solution of the protein to be purified through a 0.45 μm filter membrane and wait for loading;

[0200] 2) Prepare a pre-packed Ni ion affinity purification chromatography column, connect the peristaltic pump, chromatography column, and protein UV detector for later use;

[0201] 3) Set an appropriate column flow rate according to the pre-packed column specifications, such as 4 ml / min, and equilibrate the column with the loading buffer (PBS, 100 mM NaCl, pH: 7.4) for about 10 column volumes, and observe the stability of the UV detector reading:

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

[0203] 5) After the UV detector is stable, elute the protein with elution buffers with different imidazole concentration gradients, namely 10 mM, 50 mM, 200 mM, and 500 mM imidazole (elution buffer stock solution: PBS, 100 mM NaCl, 500 mM imidazole, pH: 7.4), and observe the changes in the values of the UV detector to collect each fraction of the eluted protein:

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

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

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

[0207] Perform protein electrophoresis analysis on the obtained target protein AH06280 PD-L1 scFv, and use a BCA kit to determine the concentration. Its purity is greater than 90%, and the results are as Figure 3As shown below. Receptor-coated ELISA was used to detect the affinity activity of AH06280 PD-L1 antibody and its PD-L1 single-chain antibody against PD-L1 protein. A 1 μg / ml PD-L1 protein solution was dispensed at 100 μl / well onto an ELISA plate and coated overnight at 4°C. After the coated plate was washed 3 times with PBS, it was blocked overnight at 4°C with a 1% BSA / PBS solution at 200 μl / well. It was washed 3 times with PBST buffer (PBS containing 0.05% Tween-20). Eight gradient-diluted PD-L1 antibody and PD-L1 single-chain antibody solutions with concentrations of 0.0000128, 0.000064, 0.00032, 0.0016, 0.008, 0.04, 0.2, and 1 μM were added. Three parallel wells were set for each concentration and incubated overnight at 4°C. After washing 3 times with PBST, HRP-streptavidin (1:2000) was added and diluted at 50 μl / well, and incubated at 37°C for 90 min. The plate was washed 5 times with PBST, 100 μl of TMB substrate solution was added to each well, and the reaction was carried out in the dark at room temperature for 2 minutes. Subsequently, 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 measured on an enzyme-linked immunosorbent assay (ELISA) reader. The experimental results of receptor-coated ELISA are as Figure 4 shown. It can be seen from the figure that both the AH06280 PD-L1 antibody and its single-chain antibody have obvious affinity activity for PD-L1 protein, and their affinity activity is concentration-dependent within a certain range.

[0208] Example 2: Preparation of PD-L1-targeted ceritinib and Sulfopin biomimetic exosomes

[0209] 2.1 Preparation of ceritinib liposomes and Sulfopin liposomes

[0210] Phosphatidylcholine, cholesterol, DSPE-PEG 2000 and DSPE-PEG 2000-NH2 (A.V.T. 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 under reduced pressure at 4°C. Then, 250 mM ammonium sulfate solution was added for hydration. First, it was ultrasonically treated at room temperature in a water bath for 5 min, and then further ultrasonically treated in an ultrasonic cell disruptor for 12 min (power 200 W, 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. After extrusion, the liposome suspension was dialyzed in a phosphate buffer solution (PBS) through a dialysis bag (cut-off molecular weight 12,000 - 14,000 Da) for 12 h, twice in total, to obtain blank liposomes. Using the ammonium sulfate gradient method, ceritinib was mixed with blank liposomes (ceritinib:phospholipid = 1:20, w / w). After shaking in a water bath at 40°C for 20 min, the unencapsulated ceritinib (purchased from MCE) was removed by dialysis in PBS solution, and ceritinib liposomes (Cer-Lipo) could be prepared and stored at 4°C for later use.

[0211] The preparation method of Sulfopin liposomes (Sul-Lipo) is the same as that of ceritinib liposomes (Cer-Lipo), with the only difference being that Sulfopin (purchased from Shanghai Tauto Biochemical Technology Co., Ltd.) is used to replace ceritinib in the above method.

[0212] 2.2 Extraction of pancreatic cancer cell 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 Lamboid Trading 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 Medicine, Chinese Academy of Medical Sciences) were cultured in MEM medium (Beijing Lamboid Trading Co., Ltd.) containing 10% fetal bovine serum. All cells were cultured in an incubator at 37°C with 5% CO2.

[0214] The ExoQuick reagent method combined with ultrafiltration was used to separate and extract exosomes secreted by HuP-T3 cells into the supernatant culture medium. HuP-T3 cells were cultured in a 150 mm culture dish until 60% confluence, and then switched to RPMI 1640 medium (purchased from Beijing Lanbolide Trading Co., Ltd.) containing 5% exosome-free serum (purchased from VivaCell). After continued culture for 48 h, the cell culture supernatant was collected and centrifuged at 2000×g for 10 min at 4°C. The obtained supernatant was then centrifuged at 12000×g for 30 min at 4°C. The supernatant was taken and filtered through a 30 kDa ultrafiltration membrane to intercept and concentrate exosomes. The obtained concentrated solution was added with ExoQuick precipitant (purchased from SBI (System Biosciences) Co., Ltd.) at a ratio of ExoQuick precipitant: supernatant = 1:5 (v / v), gently mixed evenly, and then placed vertically at 4°C for more than 12 h. Centrifuged at 1500×g for 30 min at 4°C, and the precipitate at the bottom was pancreatic cancer cell exosomes. The exosome precipitate was suspended with an appropriate amount of PBS, and the protein concentration of exosomes was determined by the 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 to replace 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. Among them, 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 mixture of exosomes and liposomes was sonicated in a ultrasonic cell disruptor for 6 min (power 200 W, working time 30 s, intermittent time 30 s), and successively extruded through polycarbonate membranes with pore sizes of 400 nm and 200 nm to obtain ceritinib biomimetic exosomes (Cer-BE H ), prepared by the membrane extrusion method, and Sulfopin biomimetic exosomes (Sul-BE M ).

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

[0219] Add 20 μL of 25% glutaraldehyde aqueous solution to 1 ml of ceritinib biomimetic exosomes, react at room temperature for 2 h, and then dialyze to remove the excess glutaraldehyde. Then add 80 μL of the PD-L1 scFv antibody prepared in Example 1 (the concentration of PD-L1 scFv is 0.052 mg / ml), incubate overnight at 4 °C, and remove the uncoupled PD-L1 scFv antibody by dialysis in PBS solution, thereby obtaining 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 ), and the only difference is that Sulfopin biomimetic exosomes are used to replace 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] The thin film hydration method is used to prepare coumarin 6-labeled liposomes. Dissolve lecithin, cholesterol, DSPE-PEG 2000 and DSPE-PEG 2000 -NH2 (59:39:4:0.5, molar ratio) and coumarin 6 (coumarin 6: lipid material = 1: 200, w / w) in chloroform, and form a lipid film by rotary evaporation at 40 °C. Add phosphate buffer PBS for hydration, ultrasonicate at room temperature for 5 min, and then further ultrasonicate in an ultrasonic cell disruptor for 6.8 min (working time is 10 s, intermittent time is 10 s, and the protection temperature is 35 °C). The hydrated liposome suspension is successively extruded through polycarbonate membranes with pore sizes of 400 nm and 200 nm three times, and the free coumarin 6 that is not encapsulated into liposomes is removed by passing through a Sephadex G-50 dextran gel column, thus obtaining coumarin 6 liposomes.

[0223] Prepare the biomimetic exosomes of coumarin 6 liposomes and HuP-T3 cell exosomes and the corresponding PD-L1-targeted biomimetic exosomes. The preparation method is the same as that of the above-mentioned ceritinib biomimetic exosomes and their PD-L1-targeted biomimetic exosomes, and the only difference is that coumarin 6 liposomes are used to replace ceritinib liposomes.

[0224] Prepare biomimetic exosomes of coumarin 6 liposomes and MRC-5 cell-derived exosomes, and corresponding PD-L1-targeted biomimetic exosomes. The preparation method is the same as that of the above-mentioned Sulfopin biomimetic exosomes and their PD-L1-targeted biomimetic exosomes, with the only difference being that coumarin 6 liposomes are used to replace Sulfopin liposomes.

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

[0226] 3.1 Determination of particle size and Zeta potential

[0227] Use a nanoparticle size and zeta potential analyzer Zetasizer Nano ZSP to measure the particle size, polydispersity index (PDI), and Zeta potential of exosomes derived from HuP-T3 cells, blank liposomes, ceritinib liposomes, ceritinib biomimetic exosomes, and PD-L1-targeted ceritinib biomimetic exosomes. 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 their Zeta potential is between -10 mV and -30 mV, indicating that the prepared ceritinib nanosuspensions have a relatively uniform particle size distribution and meet the particle size requirements for passing through the intercellular spaces of tumor neovessel wall cells during blood circulation. At the same time, the negative charge on the surface of the nanosuspensions can play a role in stabilizing the preparation due to charge repulsion.

[0228] Table 4 Particle size, PDI, and Zeta potential of different ceritinib nanosuspensions

[0229]

[0230] At the same time, use a nanoparticle size and zeta potential analyzer Zetasizer Nano ZSP to also measure the particle size, polydispersity index (PDI), and Zeta potential of exosomes derived from MRC-5 cells, blank liposomes, Sulfopin liposomes, Sulfopin biomimetic exosomes, and PD-L1-targeted Sulfopin biomimetic exosomes. The results are shown in Table 5. The average particle size of Sulfopin liposomes, Sulfopin biomimetic exosomes, and PD-L1-targeted Sulfopin biomimetic exosomes is between 70 nm and 100 nm, and their Zeta potential is between -18 mV and -23 mV, indicating that the prepared Sulfopin nanosuspensions have a relatively uniform particle size distribution. At the same time, the negative charge on the surface of the nanosuspensions can play a role in stabilizing the preparation due to charge repulsion.

[0231] Table 5 Particle size, PDI, and Zeta potential of different Sulfopin nanosuspensions

[0232]

[0233] 3.2 Morphological Observation of Bionic Exosomes by Transmission Electron Microscopy

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

[0235] 3.3 Determination of Encapsulation Efficiency

[0236] To detect the drug content in liposomes, bionic exosomes and PD-L1-targeted bionic exosomes, these nanometer preparations loaded with ceritinib or Sulfopin were disrupted 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 ceritinib. Detection conditions: Poroshell 120 EC-C18 chromatographic column (2.1×50 mm, 1.9 µm), gradient elution was carried out with acetonitrile-0.01% formic acid aqueous solution as the mobile phase (5% acetonitrile at the initial 0 minute, rapidly increased to 95% acetonitrile from 0 to 0.5 min, maintained at 95% acetonitrile from 0.5 to 5 min, rapidly decreased to 5% acetonitrile from 5 to 5.01 min), flow rate 1.0 mL / min, column temperature 45 °C, detection wavelength 214 nm, injection volume 0.8 μL.

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

[0238] The calculation formula for the encapsulation efficiency (EE) of the drug (i.e., ceritinib or Sulfopin) is: EE (%) = mass of the drug encapsulated in the nanocarrier / total mass of the added drug × 100%.

[0239] The encapsulation efficiency and drug loading results of ceritinib and Sulfopin in each preparation are shown in Table 6. After calculation, the encapsulation efficiencies of ceritinib liposomes, ceritinib biomimetic exosomes, and PD-L1-targeted ceritinib biomimetic exosomes are 86.66%, 82.19%, and 79.11% respectively, indicating that liposomes, biomimetic exosomes, and PD-L1-targeted biomimetic exosomes have achieved good encapsulation effects on ceritinib. The encapsulation efficiencies of Sulfopin liposomes, Sulfopin biomimetic exosomes, and PD-L1-targeted Sulfopin biomimetic exosomes are 32.13%, 29.27%, and 27.71% respectively.

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

[0241]

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

[0243] The BCA method was used to determine the amount of PD-L1 scFv conjugated to the biomimetic exosomes. Take 0.5 ml of the prepared suspension of PD-L1 scFv-modified biomimetic exosomes, with the added mass of PD-L1 scFv being 0.0152 mg, and dialyze it in 2 ml of PBS solution at 4 °C for 48 h. The BCA kit (Shanghai Beyotime Biotechnology Co., Ltd.) was used to determine the content of PD-L1 scFv in the dialysis solution. According to the formula: coupling rate (%) = (1 - mass of PD-L1 scFv measured in the dialysis solution / added mass of PD-L1 scFv in the biomimetic exosomes) × 100%, the coupling rate of PD-L1 scFv on PD-L1-targeted ceritinib biomimetic exosomes was calculated to be 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 conjugated to the biomimetic exosomes.

[0244] 3.5 Identification of exosome-specific marker proteins by Western blotting

[0245] The prepared PD-L1-targeted biomimetic exosomes from different cell sources were identified for exosome-specific marker proteins at the molecular level by Western blotting. Specifically, a BCA protein concentration assay kit (Shanghai Beyotime Biotechnology Co., Ltd.) was used to measure the protein concentrations of cell lysates, exosomes, biomimetic exosomes, and targeted biomimetic exosomes. After adjusting to the same concentration according to the measurement results, a certain amount of 5× protein loading buffer was added, and the protein was denatured at 100 °C for 10 min. An SDS-PAGE gel was prepared, and the prepared protein samples were loaded. After electrophoresis at 80 V through the stacking gel, the voltage was switched to 120 V until the bromophenol blue reached the bottom edge of the gel. Transfer was carried out at 300 mA for 1 h. After transfer, the membrane was blocked in 5% milk for 2 h. After cutting the corresponding bands, they were incubated with anti-CD9, anti-CD81, and anti-PDCD6IP primary antibodies (Proteintech) overnight at 4 °C. After washing the membrane, the secondary antibody (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) was incubated at room temperature for 2 h, and after washing the membrane, it was exposed. The results are as Figure 6 shown. Western blotting confirmed that the PD-L1-targeted biomimetic exosomes derived from HuP-T3 cells and the PD-L1-targeted biomimetic exosomes derived from MRC-5 cells both retained the expression of exosome-specific marker proteins such as CD9, CD81, and PDCD6IP, indicating the successful preparation of the targeted biomimetic exosomes.

[0246] Example 4: Targeting analysis of PD-L1-targeted biomimetic exosomes derived from HuP-T3 and MRC-5 cells

[0247] Since ceritinib and Sulfopin have no fluorescence, coumarin 6 was used as a fluorescent probe to prepare coumarin 6-labeled liposomes, biomimetic exosomes, and PD-L1-targeted biomimetic exosomes for studying the accumulation and distribution of biomimetic exosomes derived from HuP-T3 cells and their corresponding PD-L1-targeted biomimetic exosomes in HuP-T3 cells, and for studying the accumulation and distribution of biomimetic exosomes derived from MRC-5 cells and their corresponding PD-L1-targeted biomimetic exosomes in MRC-5 cells. Specifically, 2×10 4HuP-T3 cells or MRC-5 cells were respectively seeded into 24-well plates and incubated in an incubator at 37 °C and 5% CO2 for 24 h, with 3 replicates in each group. For HuP-T3 cells, coumarin 6, coumarin 6 liposomes, HuP-T3 cell-derived biomimetic exosomes and their corresponding PD-L1-targeted biomimetic exosomes (final concentration of coumarin was 1.2 μg / mL) were added respectively. For MRC-5 cells, coumarin 6, coumarin 6 liposomes, MRC-5 cell-derived biomimetic exosomes and their corresponding PD-L1-targeted biomimetic exosomes (final concentration of coumarin was 1.2 μg / mL) were added respectively. Incubation was continued for 0, 5, 15, 30, 45 and 60 min. Thereafter, the cells were washed 3 times with ice-cold PBS and fixed with 2% paraformaldehyde at room temperature for 30 min. Then the nuclei were stained with 10 μg / mL DAPI (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) for 30 min and washed 3 times with PBS. Finally, image analysis was performed using a Cytation 1 multimode microplate reader. Blue fluorescence represents the cell nuclei and green fluorescence represents coumarin 6. The cell uptake results are shown in Figure 7 as follows.

[0248] As can be seen Figure 7 from Figure 7 A and Figure 7 B, in HuP-T3 cells with high PD-L1 expression, compared with free coumarin 6 and coumarin 6 liposomes, the coumarin 6 biomimetic exosomes derived from autologous cells had significantly higher intracellular uptake, indicating that homologous exosomes contribute to improving the degree of cell uptake; compared with non-targeted coumarin 6 biomimetic exosomes, the uptake of PD-L1-targeted coumarin 6 biomimetic exosomes by HuP-T3 cells increased significantly. Similarly, as can be seen Figure 7 from Figure 7 C and Figure 7 D, the fibroblast MRC-5 had a higher uptake degree of coumarin 6 biomimetic exosomes derived from autologous cells than free coumarin 6 and coumarin 6 liposomes, and the uptake of PD-L1-targeted coumarin 6 biomimetic exosomes was significantly stronger than that of non-targeted coumarin 6 biomimetic exosomes. The results show that PD-L1-targeted biomimetic exosomes can specifically recognize the PD-L1 receptor on the cell surface and achieve active targeting of cells with high PD-L1 expression through receptor-mediated endocytosis.

[0249] Example 5: Combined inhibition of the in vitro activities of two targeted ceritinib and Sulfopin biomimetic exosomes against 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. Cells in the logarithmic growth phase were seeded into 96-well cell culture plates at a density of 5×10 3 cells / well and cultured in an incubator at 37°C and 5% CO2 for 24 h; different ceritinib preparations were added to HuP-T3 cells, and the concentration gradients of ceritinib were 0.1953125, 0.390625, 0.78125, 1.5625, 3.125, 6.25, 12.5, and 25 μM; different Sulfopin preparations were added to MRC-5 cells, and the concentration gradients of Sulfopin were 1.5625, 3.125, 6.25, 12.5, 25, 50, 100, and 200 μM. Medium was used as the blank, and untreated cells were used as the control. Three replicate wells were set up in each group and cultured in an incubator at 37°C and 5% CO2 for 72 h; 10 μL of MTS solution (purchased from Promega) was added to each well and incubated at 37°C for an additional 2 h; the absorbance value (OD) at 490 nm was read using a microplate reader. The cell survival rate was calculated according to the following formula: Survival rate = (OD of drug-treated wells - OD of blank wells) / (OD of control wells - OD of blank wells) × 100%.

[0251] The results are as Figure 8 shown. Compared with ceritinib, ceritinib liposomes, and ceritinib biomimetic exosomes, the killing activity of PD-L1-targeted ceritinib biomimetic exosomes against HuP-T3 cells was significantly enhanced due to receptor-mediated endocytosis ( Figure 8 A); similarly, in MRC-5 cells, compared with Sulfopin, Sulfopin liposomes, and Sulfopin biomimetic exosomes, PD-L1-targeted Sulfopin biomimetic exosomes also showed a stronger inhibitory effect ( Figure 8 B). Moreover, in each drug 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 combination drug administration can achieve the purpose of simultaneously eliminating tumor cells and tumor fibroblasts.

[0252] Meanwhile, the combination index (CI) of drugs was used to evaluate the synergistic effect of the combined use of two targeted biomimetic exosomes. The calculation formula is: CI = (C A,x / IC x,A )+(C B,x / IC x,B )where C A,x and C B,x are the doses of drugs A and B required to reach the specific X-effect concentration (such as 50% effect concentration) at the combined dose, while IC x,A and IC x,B are the doses of single drugs A and B required to reach the same effect concentration respectively.

[0253] In this example, drug A is the PD-L1-targeted ceritinib biomimetic exosome, drug B is the PD-L1-targeted Sulfopin biomimetic exosome, and the X concentration is selected as the drug concentration at the IC 50 value, which is the concentration required to inhibit cell proliferation by 50%. CI less than 1 indicates that the combined use of the two targeted biomimetic exosomes can produce a synergistic effect.

[0254] The drug concentration value at 50% inhibitory effect was calculated using graphpad prism 9 software. For HuP-T3 cells, calculated by graphpad prism 9 software: 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, calculated by graphpad prism 9 software: 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 two PD-L1-targeted biomimetic exosomes for HuP-T3 cells and MRC-5 cells are 0.787 and 0.698 respectively, indicating that the combined use of the two targeted biomimetic exosomes can produce a synergistic effect on both HuP-T3 cells and MRC-5 cells.

[0255] Example 6: Effects of two 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 effects of various ceritinib and Sulfopin preparations on the scratch repair ability of HuP-T3 cells. HuP-T3 cells in the logarithmic growth phase were collected and seeded in 24-well plates at a density of 1.2×10 5 cells / well, and 1 mL of cell suspension was added to each well. The plates were placed in an incubator at 37 °C and 5% CO2 until the cell confluence reached over 85%. A 200 μL pipette tip was used to draw a vertical line in the central part of the culture plate. The cells were washed 3 times with PBS, and 1 mL of fresh RPMI 1640 medium containing 10% fetal bovine serum (PAN, Germany) (purchased from Beijing Lamboid Trading Co., Ltd.) was added again. Then, PBS (as the 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, and the combination of PD-L1 targeted ceritinib biomimetic exosome + PD-L1 targeted Sulfopin biomimetic exosome (drug concentration: 1.0 μM) were added to each well respectively. Immediately after adding the drugs, the Cytation1 cell imaging microplate detection system was used to take pictures at 0 h, three positions were taken for each well, and the coordinates of each position were recorded. Then, pictures were taken at the same positions at 24 h and 48 h respectively. The scratch healing rate was calculated: cell healing rate (%) = [(scratch width at 0 h - scratch width at 24 h or 48 h) / scratch width at 0 h] × 100%, and the differences in scratch healing among groups were compared.

[0257] The results are as Figure 9 shown. The ability of various ceritinib preparations to inhibit 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 them, the PD-L1 targeted ceritinib biomimetic exosome or the PD-L1 targeted Sulfopin biomimetic exosome had the strongest inhibitory effect, and the combined administration of these two PD-L1 targeted biomimetic exosomes almost completely inhibited the scratch repair ability of HuP-T3 cells.

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

[0259] The Transwell migration assay was used to investigate the effects of various ceritinib and Sulfopin preparations on the migration ability of HuP-T3 cells. Pancreatic cancer HuP-T3 cells and fibroblast MRC-5 cells were co-cultured. The specific procedure was as follows: (1) HuP-T3 cells (3×10 4 cells / well) in 100 μL of serum-free medium were seeded into the upper chamber, and 600 μL of MRC-5 cells (3×10 4 cells / well) were seeded into the lower chamber. One lower chamber was set as a control well with 600 μL of medium containing 10% FBS. Different media were used for the upper and lower chambers. The RPMI 1640 medium (Beijing Lamboid Trading Co., Ltd.) was used for the upper chamber, and the MEM medium (Beijing Lamboid Trading Co., Ltd.) containing 10% fetal bovine serum (PAN, Germany) was used for the lower chamber. 1 μM of different ceritinib preparations and Sulfopin preparations were added to the lower chamber containing MRC-5 cells, and the chambers were further cultured in an incubator at 37°C and 5% CO2 for 24 h. (2) After 24 h, the medium in the chambers was aspirated, washed 2-3 times with PBS, and the cells that had perforated the chambers were fixed with 600 μL of 4% paraformaldehyde fixative for 30 min. The chambers were washed again 2-3 times with PBS. (3) Stained with 600 μL of crystal violet staining solution in the dark for 30 min. Then, after washing the staining solution in the chambers with PBS buffer, the cells that had not perforated the chambers were removed with a cotton swab. (4) The chambers were placed under an inverted microscope for observation and photography. More than 3 fields of view were photographed for each well.

[0260] The experimental results are as Figure 10 shown. As can be seen from Figure 10 A, after co-culture with fibroblast MRC-5 cells, the number of migrated HuP-T3 cells in the co-culture group was significantly increased compared with the group of HuP-T3 cells cultured alone, indicating that co-culture of pancreatic cancer HuP-T3 cells with fibroblast cells can significantly improve their migration ability. As can be seen from Figure 10 B, various ceritinib preparations inhibited the migration ability of HuP-T3 cells stronger than the corresponding Sulfopin preparations. Compared with the control group, all ceritinib-containing preparations and Sulfopin-containing preparations had a significant ability to inhibit the migration of HuP-T3 cells. Among them, the PD-L1-targeted ceritinib biomimetic exosomes had a stronger inhibitory effect. Moreover, when the two PD-L1-targeted biomimetic exosomes were administered in combination, the effect was the most obvious, almost completely inhibiting the migration ability of HuP-T3 cells.

[0261] The above are only several exemplary embodiments of the present invention, and do not impose any form of limitation on the present invention. Although the present invention is disclosed above in preferred embodiments, it is not intended to limit the present invention. Any equivalent or equivalent implementation obtained by making some changes or modifications using the disclosed technical content by any person skilled in the relevant art without departing from the technical solution of the present invention falls within the scope of the present invention.

Claims

1. A PD-L1-targeted biomimetic exosome, which comprises a biomimetic exosome formed by fusing exosomes derived from fibroblasts with liposomes loaded with Pin1 inhibitors and a PD-L1 single-chain antibody, wherein, The PD-L1 single-chain antibody is conjugated to the biomimetic exosome.

2. The PD-L1 targeted biomimetic exosome according to claim 1, wherein, 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 comprises: VH CDR1, whose amino acid sequence is SEQ ID NO: 1, or a sequence obtained by substitution, deletion, or addition of one or several amino acids in SEQ ID NO: 1; VH CDR2, whose amino acid sequence is SEQ ID NO: 2, or a sequence obtained by substitution, deletion, or addition of one or several amino acids in SEQ ID NO: 2; VH CDR3, whose amino acid sequence is SEQ ID NO: 3, or a sequence obtained by substitution, deletion, or addition of one or several amino acids in SEQ ID NO: 3; and / or The light-chain variable region comprises: VL CDR1, whose amino acid sequence is SEQ ID NO: 4, or a sequence obtained by substitution, deletion, or addition of one or several amino acids in SEQ ID NO: 4; VL CDR2, whose amino acid sequence is SEQ ID NO: 5, or a sequence obtained by substitution, deletion, or addition of one or several amino acids in SEQ ID NO: 5; VL CDR3, whose amino acid sequence is SEQ ID NO: 6, or a sequence obtained by substitution, deletion, or addition of one or several amino acids in SEQ ID NO:

6.

3. The PD-L1-targeted biomimetic exosome according to claim 2, wherein, The amino acid sequence of the heavy-chain variable region comprises SEQ ID NO: 7 or is 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 is as shown in SEQ ID NO:

8.

4. The PD-L1-targeted biomimetic exosome according to claim 2, wherein, The amino acid sequence of the linker peptide consists of 1 to 5 amino acid sequences as shown in SEQ ID NO:

9.

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

9.

6. The PD-L1-targeted biomimetic exosome according to any one of claims 2 to 5, wherein, The amino acid sequence of the PD-L1 single-chain antibody comprises SEQ ID NO: 10 or is as shown in SEQ ID NO:

10.

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

8. The PD-L1-targeted biomimetic exosome according to claim 1, wherein, The particle size of the PD-L1 targeted biomimetic exosomes 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 is 1.0% to 2.0%, and the conjugation rate of the PD-L1 single-chain antibody is 50% to 55%.

9. The PD-L1-targeted biomimetic exosome according to claim 8, wherein, The average particle size of the PD-L1 targeted biomimetic exosomes is about 90 nm, the Zeta potential is about -20 mV, the encapsulation efficiency is about 28%, the drug loading is about 1.4%, and the conjugation rate of the PD-L1 single-chain antibody is about 51%.

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

11. A pharmaceutical composition for treating cancer, which comprises a first PD-L1-targeted biomimetic exosome and an optional pharmaceutically acceptable excipient; wherein, The first PD-L1 targeted biomimetic exosomes are the PD-L1 targeted biomimetic exosomes according to any one of claims 1 to 410.

12. The pharmaceutical composition according to claim 11, wherein, The cancer is a pan-solid tumor with PD-L1 expression.

13. The pharmaceutical composition according to claim 12, wherein, 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, renal cancer, melanoma, and liver cancer.

14. The pharmaceutical composition according to claim 13, wherein, The cancer is pancreatic cancer.

15. The pharmaceutical composition according to claim 11, further comprising a second PD-L1 targeting biomimetic exosome; wherein, The second PD-L1 targeted biomimetic exosomes comprise biomimetic exosomes formed by fusing exosomes derived from cancer cells with liposomes loaded with anticancer drugs and the PD-L1 single-chain antibody according to any one of claims 2 to 6, wherein the PD-L1 single-chain antibody is conjugated to the biomimetic exosomes.

16. The pharmaceutical composition according to claim 15, wherein, The cancer cells are cancer cells with high expression of PD-L1.

17. The pharmaceutical composition according to claim 16, wherein, The cancer cells are pancreatic cancer cells with high expression of 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 are HuP-T3 cells.

20. The pharmaceutical composition according to claim 15, 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 drug is dasatinib and / or ceritinib.

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

23. The pharmaceutical composition according to claim 15, wherein, The particle size of the second PD-L1 targeted biomimetic exosomes is 80 nm to 92 nm, the Zeta potential is -16 mV to -12.5 mV, the encapsulation efficiency is 70% to 85%, the drug loading is 3% to 3.5%, and the conjugation rate of the PD-L1 single-chain antibody is 60% to 62%.

24. The pharmaceutical composition according to claim 23, wherein, The average particle size of the second PD-L1 targeted biomimetic exosomes is about 87 nm, the Zeta potential is about -14 mV, the encapsulation efficiency is about 79%, the drug loading is about 3%, and the conjugation rate of the PD-L1 single-chain antibody is about 60%.

25. The pharmaceutical composition according to claim 15, wherein, The second PD-L1 targeted biomimetic exosomes have at least one of the specific marker proteins CD9, CD81, and PDCD6IP.

26. Use of the PD-L1 targeted biomimetic exosomes according to any one of claims 1 to 10 or the pharmaceutical composition according to any one of claims 11 to 25 in the preparation of a drug for treating cancer.

27. The use according to claim 26, wherein The cancer is a pan-solid tumor with PD-L1 expression.

28. The use according to claim 27, wherein 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, renal cancer, melanoma, and liver cancer.

29. The use according to claim 28, wherein, The cancer is pancreatic cancer.

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