Drug delivery system comprising blood cells

Abstract

The present invention provides a drug delivery system comprising blood cells, and a novel modified erythrocyte membrane method for preparing the same. The invention also relates to a method for treating diseases by using the drug delivery system.

Description

Drug delivery system involving blood cells

[0001] The present invention provides a drug delivery system comprising blood cells and a novel method for modifying erythrocyte membranes for preparing the drug delivery system. The present invention also relates to a method for treating a disease using the drug delivery system.

[0002] Background of the Invention

[0003] Recent advances in drug delivery systems for prolonged drug retention to treat a variety of human diseases have attracted considerable attention. However, many systems remain subject to numerous challenges and limitations, such as poor stability, unwanted toxicity, and immune responses. Red blood cells (RBCs), the most common cell type in the human body, have been extensively studied for over 30 years as ideal in vivo drug delivery systems due to their unique biological properties: (i) extensive systemic circulation; (ii) good biocompatibility as a biomaterial with long in vivo survival; (iii) a large surface-to-volume ratio; and (iv) the absence of a nucleus, mitochondria, and other organelles.

[0004] RBCs have been developed as drug delivery vehicles by attaching proteins through direct encapsulation, non-covalent attachment of exogenous peptides, or fusion with antibodies specific for RBC surface proteins. However, these modified RBCs have demonstrated limitations in in vivo applications. For example, encapsulation can disrupt the cell membrane, thereby impacting the in vivo lifespan of the engineered cells. Furthermore, the non-covalent attachment of polymer particles to RBCs is easily dissociated, resulting in rapid degradation of the payload in vivo.

[0005] Therefore, there is still a need in the art for a stable and efficient modification method that can encapsulate drugs into RBCs to obtain a stable and efficient drug delivery system.

[0006] Summary of the Invention

[0007] Phospholipids are amphiphilic. The hydrophilic end typically contains a negatively charged phosphate group, while the hydrophobic end typically consists of two "tails," which are long fatty acid residues. In aqueous solution, phospholipids are driven by hydrophobic interactions, resulting in the accumulation of the fatty acid tails, thereby minimizing interactions with water molecules. The result is usually a phospholipid bilayer: a membrane composed of two layers of phospholipid molecules oriented in opposite directions, with the heads on both sides exposed to the liquid and the tails pointing towards the membrane. This is a major structural motif in all cell membranes and certain other biological structures. In biological membranes, phospholipids are often found in bilayers (such as cell membranes) along with other molecules (such as proteins, glycolipids, and sterols). A lipid bilayer occurs when the hydrophobic tails align with each other, forming a membrane with the hydrophilic heads on the side facing the water.

[0008] Maleimide-mediated methods are among the most commonly used in bioconjugation. Due to their extremely rapid reaction speed and high selectivity for cysteine ​​residues in proteins, they are used in a variety of approaches. A variety of maleimide heterobifunctional reagents are used to prepare targeted therapeutics and study the biological context of proteins, protein-based microarrays, or protein assemblies for protein immobilization. For example, antibody-drug conjugates, an emerging and promising class of targeted therapeutics, consist of three main components: a monoclonal antibody, a cytotoxic drug, and a linker molecule that typically contains a maleimide group. The imide group binds to both the drug and the antibody.

[0009] Maleimide attached to a polyethylene glycol chain is commonly used as a flexible linker molecule to attach proteins to surfaces. The double bond readily reacts with the sulfhydryl group on cysteine ​​to form a stable carbon-sulfur bond. Attaching the other end of the polyethylene chain to beads or solid supports can easily separate proteins from other molecules in solution, provided these molecules lack sulfhydryl groups.

[0010] The present invention first discovered that using cell membrane component phospholipids combined with maleimide and polyethylene glycol as red blood cell linkers can be used to obtain an efficient and stable red blood cell-based drug delivery system. Description of the drawings:

[0011] Figures 1a and 1b show schematic diagrams of method 1;

[0012] Figures 2a and 2b show schematic diagrams of method 2;

[0013] Figure 3 shows a linker comprising a maleimide group-PEG-phospholipid;

[0014] Figure 4 shows a schematic diagram of linker-based conjugation of thiols to proteins;

[0015] Figure 5 shows the eGFP signal intensity on the surface of labeled and unlabeled erythrocytes;

[0016] Figure 6 shows the eGFP signal intensity on the cell surface of natural erythrocytes after being labeled with different concentrations of eGFP-Cys-MAL-PEG2000-DSPE in vitro;

[0017] Figure 7 shows the eGFP signal intensity on the surface of erythrocytes stained with Cell Far red;

[0018] FIG8 a shows the percentage of Far red positive erythrocytes in mouse blood over time for control erythrocytes (not labeled with eGFP-cys) and erythrocytes labeled with eGFP-cys;

[0019] FIG8 b shows the percentage of GFP-positive erythrocytes to Cell Far Red-positive cells over time in control erythrocytes (not labeled with eGFP-cys) and erythrocytes labeled with eGFP-cys;

[0020] FIG9 shows anti-PD-1 antibody signals on the surface of red blood cells labeled with and without anti-PD-1 antibody;

[0021] FIG10 shows the signal intensity of PD-1 antibodies.

[0022] FIG11 shows the UOX signal intensity of UOX-Cys-Mal-PEG-DSPE labeled erythrocytes in two recipient mice;

[0023] FIG12 shows the percentage of Cell Far Red-positive erythrocytes in the blood over time ( FIG12 a ) and the percentage of UOX-positive erythrocytes to Cell Far Red-positive cells over time ( FIG12 b ) in two recipient mice;

[0024] FIG13 shows the signal intensity of eGFP on the surface of different types of blood cells detected by flow cytometry;

[0025] FIG14 shows the eGFP signal intensity and FAR RED signal intensity of eGFP-Cys-MAL-PEG-DOPE labeled erythrocytes and unlabeled erythrocytes;

[0026] FIG15 shows the % Far red positive erythrocyte count over time ( FIG15 a ) and the % UOX positive erythrocyte count over time ( FIG15 b ) of eGFP-Cys-MAL-PEG-DOPE labeled erythrocytes and unlabeled erythrocytes;

[0027] FIG16 shows the signal intensity of eGFP in erythrocytes labeled with Mal-PEG2000-phospholipid (DPPE, DOPE, DSPE or DMPE);

[0028] FIG17 shows the signal intensity of eGFP obtained by incubating erythrocytes with different concentrations of Mal-PEG2000-phospholipid (DOPE or DSPE) for 10 minutes;

[0029] Figure 18 shows a schematic diagram of coupling pro-urokinase and red blood cells using Mas-PEG-DSPE as a linker;

[0030] FIG19 shows the signal intensity of pro-urokinase on the surface of erythrocytes labeled with ProUK-cys and without ProUK-cys;

[0031] FIG20 shows the percentage of circulating red blood cells with ProUK signals that changes over time (upper graph), and the signal intensities of Far Red and ProUK in blood samples obtained at different bleeding times;

[0032] Figure 21 shows the Far Red signal and ProUK signal intensities on erythrocytes labeled with and without ProUK-cys;

[0033] Figure 22 shows the lysis of nascent hemostatic clots and hemostatic clots at different doses of free ProUK (A) and ProUK-RBC (B); and the corresponding thrombolysis % histograms (ProUK (C) and ProUK-RBC (D));

[0034] FIG23 shows the planned orbital blood collection times for the ProUK-RBC administration group and the free ProUK administration group;

[0035] Figure 24 shows the percentage of ProUK content in whole blood over time by ELISA in different dosing groups;

[0036] FIG25 shows the thrombolysis percentage over time in the ProUK-RBC administration group, the free ProUK administration group, and the RBC control group;

[0037] FIG26 shows thrombolysis photos over time in the ProUK-RBC administration group, the free ProUK administration group, and the RBC control group;

[0038] Figure 27 shows a schematic diagram of the operation of the tail bleeding time assay;

[0039] Figure 28 shows the tail bleeding time in different dosing groups at low dose levels;

[0040] FIG29 shows the 24-hour lethality of the ProUK-RBC administration group and the unlabeled RBC administration group;

[0041] FIG30 shows photographs of sections of the ProUK-RBC-administered group (right) and the unlabeled RBC-administered group (left) stained with TTC (2,3,5-triphenyltetrazolium chloride) 6 hours after treatment;

[0042] FIG31 shows the percentage of cerebral infarction area in the ProUK-RBC administration group and the unlabeled RBC administration group 6 hours after treatment;

[0043] FIG32 shows a schematic diagram of the operation of the deep vein thrombosis model;

[0044] FIG33 shows photographs of thrombus formation in one mouse each of the unlabeled RBC-administered group, the free ProUK-administered group, and the ProUK-RBC-administered group.

[0045] Detailed description of the invention:

[0046] I. Definition

[0047] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodology, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0048] To interpret this specification, the following definitions will apply, and wherever appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0049] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0050] As used herein, the term "and / or" means any one of the alternatives or two or more of the alternatives.

[0051] As used herein, the terms "comprising" or "including" are intended to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise indicated, the context of consisting of the stated elements, integers, or steps is also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0052] The "drug delivery system" described herein refers to any drug preparation or product that can deliver the drug it contains to a target, including cell preparations, such as red blood cell-based cell preparations or cell preparations based on other blood cells; or blood preparations, such as preparations prepared using human blood as raw material.

[0053] The term "prourokinase," also known as "single-chain urokinase-type plasminogen activator," is generally a single-chain polypeptide consisting of 411 amino acid residues with a molecular weight of approximately 49 kDa. It belongs to the serine protease class and has 12 disulfide bonds within the molecule. Its structure and function are divided into four domains: the epidermal growth factor domain, the Kringle domain, the serine protease domain, and the linker region. Prourokinase is a relatively inactive precursor that must be converted to urokinase before it can be active in vivo. However, it exhibits clot selectivity by binding to fibrin prior to activation. Compared to other plasminogen activators, it has the advantage of being inactive in plasma and therefore not consuming circulating inhibitors. The amino acid sequence of human prourokinase can be found at Uniprot P00749 (https: / / www.uniprot.org / uniprot / P00749). In one embodiment, the pro-urokinase of the present invention refers to natural human pro-urokinase, or a natural variant thereof, which may or may not have a signal peptide sequence (e.g., MRALLARLLLCVLVVSDSKG (SEQ ID NO: 14)) or a tag (e.g., a His6 tag). In one embodiment, the pro-urokinase of the present invention is human pro-urokinase, for example, it comprises or consists of the amino acid sequence shown in SEQ ID NO: 10 or 13. In some embodiments, the pro-urokinase of the present invention comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 10 or 13, and can be activated in vivo to have urokinase activity.

[0054] The term "thrombosis-related disease" refers to any disease associated with thrombosis, including but not limited to ischemic stroke, acute myocardial infarction, and deep vein thrombosis.

[0055] The term "anti-tumor effect" refers to a biological effect that can be demonstrated by various means, including but not limited to, for example, a reduction in tumor volume, a reduction in tumor cell number, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

[0056] The terms "tumor" and "cancer" are used interchangeably herein to encompass both solid tumors and hematological tumors.

[0057] The term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. In certain embodiments, cancers suitable for treatment by the antibodies of the present invention include lung cancer, pancreatic cancer, breast cancer, gastrointestinal tumors such as colon cancer, colorectal cancer or rectal cancer, etc., including metastatic forms of those cancers.

[0058] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" as used herein are not mutually exclusive.

[0059] Red blood cells, or RBCs, are the most common type of blood cell and the primary carrier of oxygen through the bloodstream and circulatory system in vertebrates to body tissues. The cytoplasm of RBCs is rich in hemoglobin, an iron-containing biomolecule that binds oxygen and gives cells and blood their red color. The cell membrane, composed of proteins and lipids, provides properties essential for physiological cellular function, such as deformability and stability, while also allowing passage through the circulatory system, particularly the capillary network. In humans, mature RBCs are flexible, oval, biconcave discs. They lack a nucleus and most organelles to maximize the space for hemoglobin; they can be considered bags for hemoglobin, with the plasma membrane serving as a sack. Approximately 2.4 million new RBCs are produced in adults every second. RBCs make up approximately 84% of the cells in the human body, numbering 20–30 trillion. Nearly half of the blood volume (40% to 45%) is RBCs.

[0060] “Adult natural red blood cells” refer to mature natural red blood cells directly isolated from the blood of animals, especially humans (eg, adults or children).

[0061] As used herein, "blood preparations" or "blood products" are used interchangeably and refer to products prepared from (human) blood for medical use. Blood preparations include whole blood preparations, blood component preparations, plasma preparations, or leukoreduced blood preparations.

[0062] The term "effective amount" refers to the amount or dosage of the drug delivery system or composition or formulation or preparation or combination or combination product of the present invention, which produces the desired effect in a patient in need of treatment or prevention after single or multiple doses of administration to the patient.

[0063] A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the drug delivery system or composition or formulation or article or combination or combination product are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., thrombus volume, or, for example, tumor volume) by at least about 30%, even more preferably by at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100%, relative to an untreated subject.

[0064] A "prophylactically effective amount" refers to an amount effective to achieve the desired preventive result, at the required dosage and for the required period of time. Typically, a prophylactic amount will be less than a therapeutically effective amount because a prophylactic dose is used in a subject prior to or at an earlier stage of disease.

[0065] "Individual" or "subject" includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0066] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, which is administered together with the active substance.

[0067] The term "pharmaceutical composition" refers to a composition that is in form permitting the active ingredient contained therein to be effective for biological activity, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0068] The term "drug combination" refers to a non-fixed combination product or a fixed combination product, including but not limited to a medicine box, a pharmaceutical composition. The term "non-fixed combination" means that active ingredients (e.g., (i) drug delivery system of the present invention and (ii) other therapeutic agents) are applied to the patient simultaneously, without specific time restrictions or at the same or different time intervals, in sequence, with separate entities, wherein such application provides two or more active ingredients of a preventive or therapeutically effective level in the patient's body. In some embodiments, the drug delivery system of the present invention and other therapeutic agents used in the drug combination are applied at a level not exceeding that when they are used alone. The term "fixed combination" means that two or more active ingredients are applied to the patient simultaneously in the form of a single entity. Preferably, the dosage and / or time interval of two or more active ingredients are selected so that the combined use of each part can produce an effect greater than that achieved by using any one component alone when treating a disease or illness. Each component can be in a separate formulation, and its formulation can be the same or different.

[0069] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiotherapy or surgery, such as stent surgery or bypass surgery) to treat diseases described herein. This administration includes co-administering these therapeutic agents in a substantially simultaneous manner, such as in a single capsule with a fixed ratio of active ingredients. Alternatively, this administration includes co-administration of each active ingredient in a variety of or separate containers (e.g., tablets, capsules, powders, and liquids). The powder and / or liquid can be reconstituted or diluted to the desired dose before administration. In addition, this administration also includes using each type of therapeutic agent or treatment modality in a sequential manner at approximately the same time or at different times. In either case, the treatment regimen will provide the beneficial effects of combination therapy in treating disorders or conditions described herein.

[0070] The term "therapeutic agent" as used herein encompasses any active agent that exerts a therapeutic effect, such as any substance effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytotoxic agents, other antibodies, vaccines, small molecule drugs, or immunomodulators (such as immunosuppressants or immunoagonists); and also encompasses any substance effective in preventing or treating thrombosis-related diseases or metabolic-related diseases, such as drugs for anti-thrombosis or thrombolysis, such as urokinase, tissue-type plasminogen activator, streptokinase, etc. The above-mentioned therapeutic agents also encompass precursors of active substances that exert therapeutic effects, which are subsequently converted into active substances in vivo, such as prodrugs, such as prourokinase.

[0071] The term "cytotoxic agent" as used in the present invention refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction. "Chemotherapeutic agent" includes chemical compounds useful in the treatment of cancer or immune system diseases. The term "small molecule drug" refers to a low molecular weight compound that can regulate biological processes. "Small molecule" is defined as a molecule with a molecular weight of less than 10kD, usually less than 2kD and preferably less than 1kD. Small molecules include but are not limited to inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics and antibody mimetics. As therapeutic agents, small molecules can be more cell-permeable, less susceptible to degradation and less prone to eliciting an immune response than macromolecules. The term "immunomodulator" used herein refers to a natural or synthetic active agent or drug that inhibits or regulates an immune response. The immune response can be a humoral response or a cellular response. Immunomodulators include immunosuppressants or immune agonists. In some embodiments, the immunomodulators of the present invention include immune checkpoint inhibitors or immune checkpoint agonists.

[0072] As used herein, "treat," ...

[0073] As used herein, "prevention" includes inhibition of the onset or development of a disease or condition or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cardiovascular disease are candidates for a preventive regimen. In the context of thrombosis-related diseases, prevention generally refers to administration after initial treatment of a thrombosis (e.g., venous thrombosis) has been alleviated to prevent recurrence of the thrombosis.

[0074] "Subject / patient / individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of a tissue or cell sample can be solid tissue, such as an organ or tissue sample or a biopsy sample or a puncture sample from a fresh, frozen and / or preserved organ; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time during the subject's pregnancy or development. Tissue samples may contain compounds that are not naturally contaminated with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0075] II. Drug Delivery Systems

[0076] The present invention provides a drug delivery system comprising modified blood cells coupled to a protein or peptide via a linker.

[0077] (I) Connector

[0078] In some embodiments, the linker is a linker containing a maleimide group and a phospholipid.

[0079] In some embodiments, the linker is a maleimide group-phospholipid. The term "phospholipid" as used herein refers to a class of lipids whose molecules have a hydrophilic "head" containing a phosphate group and two hydrophobic "tails" derived from fatty acids, connected by a glycerol molecule. The phosphate group can be modified with simple organic molecules such as choline, ethanolamine or serine.

[0080] In some embodiments, the phospholipid can be a PC series, such as DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC and DEPC; a PG series, such as DMPG, DPPG, DSPG and POPG; a PA series, such as DMPA, DPPA, DSPA, etc.; a PE series, such as 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE), 1,2-diacryloyl-sn-glycero-3-phosphatidylethanolamine (DEPE), 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphatidylethanolamine (DMPE) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (DPPE); and a PS series, such as DOPS.

[0081] In a preferred embodiment, the phospholipid can be a PE series, such as 1,2-distearoyl-sn-glyceryl-3-phosphatidylethanolamine (DSPE), 1,2-diacryl-sn-glyceryl-3-phosphatidylethanolamine (DEPE), 1,2-dioleoyl-sn-glyceryl-3-phosphatidylethanolamine (DOPE), 1,2-dimyristoyl-sn-glyceryl-3-phosphatidylethanolamine (DMPE) and 1,2-dipalmitoyl-sn-glyceryl-3-phosphatidylethanolamine (DPPE). In a preferred embodiment, the phospholipid is DSPE or DOPE.

[0082] In some embodiments, the maleimide group and the phospholipid in the linker are further connected by PEG. In some embodiments, the linker is a maleimide group-PEG-phospholipid. In some embodiments, the molecular weight of PEG is, for example, 400-5000, for example, 2000-5000, such as PEG2000, PEG2500, PEG3000, PEG3500, PEG4000, PEG4500 or PEG5000. In some embodiments, in order to form a peptide bond with the maleimide group and the phospholipid group respectively for connection, the PEG linker can have a free carboxyl group at the carboxyl terminal and a free amino group at the amino terminal. In some embodiments, the PEG for connecting the maleimide group and the phospholipid can include the structure of NH2-PEG-COOH, such as NH2-(CH2CH2O) n -COOH.

[0083] In some embodiments, the amino group of the phospholipid containing the hydrophilic "head" of the phospholipid is linked to the maleimide, for example, through or without PEG. In some embodiments, the hydrophobic "tail" of the phospholipid is incorporated into the cell membrane of the blood cell, thereby being used to connect the blood cell. In some embodiments, the amino group of the hydrophilic "head" of the phospholipid containing the phosphate group is linked to the maleimide through PEG or directly, while its hydrophobic "tail" is incorporated into the cell membrane of the blood cell to connect the blood cell.

[0084] In some embodiments, the phospholipid is DSPE. In some specific embodiments, the linker has the chemical formula shown in FIG3 .

[0085] (II) Blood cells

[0086] Blood cells suitable for the drug delivery system of the present invention may be any cells derived from the blood of an animal, including, for example, erythrocytes, lymphocytes, T cells, B cells, monocytes, NK cells, dendritic cells, or megakaryocytes.

[0087] In some embodiments, the animal is a mammal, such as a human.

[0088] In some embodiments, the blood cells are cells from the immune system, such as lymphocytes (e.g., T cells or NK cells), or dendritic cells. In some embodiments, the cells are natural cells. In some embodiments, the cells are adult cells. In some embodiments, the cells are natural adult cells.

[0089] In some embodiments, the blood cells used to prepare the present invention are blood cells isolated directly from blood, such as human (eg, adult or child) blood.

[0090] In some embodiments, the cells are red blood cells, eg, human red blood cells, eg, natural human red blood cells, eg, natural adult red blood cells, eg, mature red blood cells isolated from human (eg, adult or child) blood.

[0091] In some embodiments, the blood cells are autologous isolated blood cells, i.e., blood cells obtained from the blood of the subject to be treated. In some embodiments, the blood cells are allogeneic isolated blood cells, i.e., blood cells isolated from an individual different from the subject to be treated (e.g., a healthy individual).

[0092] (III) Protein or peptide

[0093] In some embodiments, proteins or polypeptides suitable for use in the drug delivery system of the present invention include proteins or peptides for labeling, or proteins or peptides for prevention or treatment.

[0094] As used herein, "protein or peptide for labeling" refers to any protein or peptide that is used for detection of a reagent (e.g., blood cell) to which it is conjugated or coupled, and which can itself be detectable (e.g., fluorescent protein labeling) or, in the case of an enzymatic labeling, can catalyze a chemical change in a detectable substrate compound or composition. Proteins or peptides used as labels include, for example, fluorescent proteins (e.g., green fluorescent protein, such as eGFP), horseradish peroxidase, bovine serum albumin, hemocyanin, chicken ovalbumin, antibodies, bovine IgG, mouse IgG, goat IgG, rabbit IgG, alkaline phosphatase, acid phosphatase, glucose oxidase, or β-galactosidase.

[0095] The "protein or peptide for prevention or treatment" described herein refers to any protein that can be used as a drug to prevent or treat a disease, including, for example, cytokines or growth factors or their receptors, hormones, enzymes, antibodies, etc.

[0096] In some embodiments, the protein is an enzyme, such as a functional metabolic enzyme or a therapeutic enzyme, such as an enzyme that plays a role in metabolism or other physiological processes in mammals. In some embodiments, the protein is an enzyme that plays a role in carbohydrate metabolism, amino acid metabolism, organic acid metabolism, porphyrin metabolism, purine or pyrimidine metabolism and / or lysosomal storage.

[0097] In some embodiments, the protein contained in the drug delivery system suitable for use in the present invention is a functional metabolic enzyme. Exemplary enzymes include uricase, such as the uricase from Aspergillus flavus. In some embodiments, the uricase comprises a signal peptide (e.g., comprising or consisting of the amino acid sequence shown in SEQ ID NO: 14) and / or a tag (e.g., a His tag), such as at its N-terminus or at its C-terminus. In some embodiments, the uricase comprises or consists of the amino acid sequence shown in SEQ ID NO: 5 or 6. In some embodiments, the uricase comprises an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to the amino acid sequence shown in SEQ ID NO: 5 or 6.

[0098] In some embodiments, the protein contained in the drug delivery system suitable for use in the present invention is a protease, such as a serine protease, such as prourokinase, such as human prourokinase. In some embodiments, the human prourokinase comprises a signal peptide (e.g., comprising or consisting of the amino acid sequence shown in SEQ ID NO: 14) and / or a tag (e.g., a His tag), for example, at its N-terminus or at its C-terminus. In some embodiments, the prourokinase comprises or consists of the amino acid sequence shown in SEQ ID NO: 10 or 13. In some embodiments, the prourokinase comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to the amino acid sequence shown in SEQ ID NO: 10 or 13. In some embodiments, the prourokinase is encoded by the nucleotide sequence shown in SEQ ID NO: 11. In some embodiments, the pro-urokinase is encoded by a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the nucleotide sequence shown in SEQ ID NO:11.

[0099] In some embodiments, the peptide contained in the drug delivery system suitable for use in the present invention is a marker peptide or various functional peptides.

[0100] In some embodiments, the protein contained in the drug delivery system suitable for use in the present invention is an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody suitable for use in the present invention is an antibody in the form of IgG1, an antibody in the form of IgG2, an antibody in the form of IgG3, or an antibody in the form of IgG4. In some embodiments, the antibody suitable for use in the present invention is a monoclonal antibody. In some embodiments, the antibody suitable for use in the present invention is a humanized antibody. In some embodiments, the antibody suitable for use in the present invention is a human antibody. In some embodiments, the antibody suitable for use in the present invention is a chimeric antibody. In one embodiment, the antibody suitable for use in the present invention is a full-length antibody. In one embodiment, the antigen-binding fragment of the antibody suitable for use in the present invention is selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, single-domain antibody such as VHH, dAb (domain antibody) or linear antibody or half antibody.

[0101] Exemplary antibodies include antibodies to immune checkpoint molecules, such as antibodies that specifically bind to PD-1, PD-L1, or PD-L2, such as PD-1 antibodies. In one embodiment of the present invention, the antibody or antigen-binding fragment thereof against PD-1 is an anti-PD-1 antibody or antigen-binding fragment thereof disclosed in CN108473977B, CN104250302B, CN105531288B, CN105026428B, WO2008156712A1 or WO2006121168A1, such as pembrolizumab, carrelizumab, tislelizumab, toripalimab, sintilimab or nivolumab or an antigen-binding fragment thereof (see, for example, CN108473977B, CN104250302B, CN105531288B, CN105026428B, WO2008156712A1 or WO2006121168A1). In one embodiment, the anti-PD-1 antibody or its antigen-binding fragment comprises one or more CDRs (preferably 3 CDRs, i.e., HCDR1, HCDR2H, and HCDR3; or LCDR1, LCDR2, and LCDR3, more preferably 6 CDRs, i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) of pembrolizumab, carrelizumab, tislelizumab, toripalimab, sintilimab, or nivolumab, or an antigen-binding fragment thereof, or comprises the VH and / or VL of pembrolizumab, carrelizumab, tislelizumab, toripalimab, sintilimab, or nivolumab, or an antigen-binding fragment thereof, or comprises the heavy chain and / or light chain of said antibody. In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments thereof of the present invention comprise heavy chain variable region CDR1, CDR2, and CDR3, and light chain variable region CDR1, CDR2, and CDR3, from pembrolizumab, camrelizumab, tislelizumab, toripalizumab, sintilimab, or nivolumab. In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments thereof of the present invention comprise heavy chain variable region and light chain variable region from pembrolizumab, camrelizumab, tislelizumab, toripalizumab, sintilimab, or nivolumab. In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments thereof of the present invention comprise heavy and light chains from pembrolizumab, camrelizumab, tislelizumab, toripalizumab, sintilimab, or nivolumab.

[0102] In some embodiments, the protein contained in the drug delivery system suitable for use in the present invention is a cytokine or growth factor or its receptor, or a functional fragment or domain of the protein.

[0103] In some embodiments, the protein or peptide contained in the drug delivery system suitable for the present invention is a protein or peptide that can be used as an antigen. Antigen can be naturally occurring or synthetic, such as antigens naturally produced and / or genetically encoded by pathogens, infected cells, tumor cells (such as tumors or cancer cells), viruses, bacteria, fungi or parasites. In some embodiments, the antigen is an autoantigen or a transplant-associated antigen. In some embodiments, the antigen is, for example, a viral capsid, envelope or shell, or a surface protein of a bacteria, fungi, protozoa or parasite cell. In some embodiments, the antigen is any antigen used in vaccines known in the art. In some embodiments, the antigen is a tumor antigen (TA).

[0104] In some embodiments, proteins included in drug delivery systems suitable for use in the present invention are "prodrugs," ie, proteins that are metabolized or otherwise converted to a biologically, pharmaceutically, or therapeutically active form after administration in vivo.

[0105] The protein or peptide used in the drug delivery system is treated to have an exposed or free thiol group for Michael addition reaction with maleimide to form a stable carbon-sulfur bond. For example, the protein or peptide contains cysteine ​​to provide a free thiol group.

[0106] In some embodiments, the protein or peptide (eg, antibody) is obtained by treating (eg, co-incubating with) a thiol reducing agent (eg, TCEP), and comprises a cysteine ​​residue containing a free thiol group.

[0107] In some embodiments, the protein or peptide (e.g., enzyme) is a protein or peptide obtained by inserting one or more (e.g., 1-10) cysteines, which contain cysteine ​​residues containing free thiol groups. The cysteine ​​can be inserted at any position of the protein or peptide, as long as its insertion does not affect the function of the protein or peptide. Preferably, the cysteine ​​is inserted at a position corresponding to the surface of the three-dimensional structure of the protein or peptide that does not affect the structure and function of the protein or peptide. In some embodiments, the cysteine ​​is inserted into the C-terminus or N-terminus of the protein or peptide, for example, before the most N-terminal amino acid or after the most C-terminal amino acid.

[0108] In some embodiments, the protein or peptide further comprises a tag suitable for purification, such as (His)n (eg, n=1, 2, 3, 4, 5, 6), which is located at the C-terminus or N-terminus of the protein or peptide.

[0109] In some embodiments, the protein or peptide further comprises a signal peptide, for example, comprising or consisting of the amino acid sequence shown in SEQ ID NO: 14.

[0110] In some embodiments, the green fluorescent protein comprising an inserted cysteine ​​comprises, or consists of, the amino acid sequence of SEQ ID NO: 1 or 2. In some embodiments, the green fluorescent protein comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 1 or 2, which has a cysteine ​​at the extreme C-terminus.

[0111] In some embodiments, the uricase comprising an inserted cysteine ​​comprises or consists of the amino acid sequence of SEQ ID NO:3 or 7. In some embodiments, the uricase comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:3 or 7, which has a cysteine ​​at its C-terminus. In some embodiments, the uricase is encoded by the nucleotide sequence of SEQ ID NO:4. In some embodiments, the uricase is encoded by a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the nucleotide sequence of SEQ ID NO:4, which has a cysteine ​​at its C-terminus.

[0112] In some embodiments, the pro-urokinase comprising an inserted cysteine ​​comprises or consists of the amino acid sequence set forth in SEQ ID NO: 8 or 12. In some embodiments, the pro-urokinase comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 8 or 12, which has a cysteine ​​at its extreme C-terminus. In some embodiments, the pro-urokinase is encoded by the nucleotide sequence set forth in SEQ ID NO: 9. In some embodiments, the pro-urokinase is encoded by a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the nucleotide sequence set forth in SEQ ID NO: 9, which has a cysteine ​​at its extreme C-terminus.

[0113] III. Methods of Drug Delivery Systems

[0114] In one embodiment, the present invention relates to a method for obtaining the drug delivery system of the present invention, comprising:

[0115] (1) providing a linker comprising maleimide and phospholipid;

[0116] (2) providing a protein or peptide comprising a free thiol group;

[0117] (3) incubating the linker described in (1) with blood cells, thereby incorporating the phospholipids therein into the cell membrane of the cells;

[0118] (4) incubating the cells prepared in step (3) with the thiol groups of the protein in step (2) under conditions suitable for a Michael addition reaction to occur; and

[0119] (5) Obtain a drug delivery system comprising proteins and blood cells coupled via a linker.

[0120] In one embodiment, the present invention relates to a method for obtaining the drug delivery system of the present invention, comprising:

[0121] (1) providing a linker comprising maleimide and phospholipid;

[0122] (2) providing a protein or peptide comprising a free thiol group;

[0123] (3) incubating the linker in (1) and the thiol group of the protein in (2) under conditions suitable for Michael addition reaction to obtain a protein coupled to a phospholipid;

[0124] (4) incubating the protein obtained in step (3) with blood cells, thereby incorporating the phospholipids therein into the cell membrane of the cells; and

[0125] (5) Obtain a drug delivery system comprising proteins and blood cells coupled via a linker.

[0126] It should be understood that those skilled in the art are able to select conditions suitable for the Michael addition reaction between maleimide linked to phospholipids (or maleimide coupled to blood cells via phospholipids) and thiol groups exposed on proteins or peptides, for example, optimal temperature, pH, reaction time, and concentration, based on the properties of the maleimide, thiol group, etc.

[0127] It will also be appreciated that one of ordinary skill in the art will be able to select appropriate conditions (e.g., physiological conditions, such as optimal temperature (e.g., 37° C.), pH, reaction time, concentration) to incubate a phospholipid-containing linker (or a protein or peptide coupled to a phospholipid-containing linker) to incorporate the phospholipid into the cell membrane of the cell.

[0128] IV. Pharmaceutical Compositions or Formulations

[0129] In one aspect, the present invention provides a pharmaceutical composition or formulation comprising the drug delivery system of the present invention. These compositions or formulations may also optionally comprise suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, as known in the art.

[0130] In one aspect, the present invention provides a blood preparation, such as a human blood preparation, comprising a drug delivery system of the present invention. In some embodiments, the human blood preparation of the present invention comprises a drug delivery system of 0.5-1000 μg / mL, such as 1-500 μg / mL, 1-50 μg / mL, 1-10 μg / mL, 0.5 μg / mL-5 μg / mL, 0.5 μg / mL-3 μg / mL or 1 μg / mL-3 μg / mL, Drug delivery systems, such as 0.5μg / mL, 0.6μg / mL, 0.7μg / mL, 0.8μg / mL, 0.9μg / mL, 1μg / mL, 1.5μg / mL, 2μg / mL, 2.5μg / mL, 3μg / mL, 4μg / mL, 5μg / mL, 6μg / mL, 7μg / mL, 8μg / mL, 9μg / mL, 10μg / mL, 15μg / mL, 20μg / mL, 25μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, 45 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, 350 μg / mL, 400 μg / mL, 450 μg / mL, or 500 μg / mL or more, or within any range of recited values.

[0131] In some embodiments, the drug delivery system included in the blood preparation is a drug delivery system comprising red blood cells. Thus, in some embodiments, the blood preparation of the present invention is a leukoreduced blood preparation, i.e., a blood preparation in which white blood cells have been filtered out. In some embodiments, the blood preparation of the present invention is a leukoreduced human blood preparation.

[0132] In some embodiments, the blood preparation of the present invention can be an allogeneic blood preparation, such as an allogeneic human blood preparation.In some embodiments, the blood cells (such as red blood cells) in the blood preparation are from healthy subjects.

[0133] In some embodiments, the blood preparation of the present invention can be an autologous blood preparation, such as an autologous human blood preparation.In some embodiments, the blood cells (eg, red blood cells) in the blood preparation are from the subject to be treated.

[0134] In some embodiments, blood preparation of the present invention or compositions can include drug delivery system and unmodified blood cell (such as erythrocyte).In some embodiments, in compositions, the blood cell (such as erythrocyte) of at least selected percentage is modified, i.e., obtains drug delivery system of the present invention by coupling with protein or peptide.For example, in some embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more cell is drug delivery system of the present invention.In some embodiments, blood preparation of the present invention or compositions can include two or more drug delivery systems of the present invention.

[0135] In some embodiments, the composition comprises a drug delivery system of the present invention. In some embodiments, the composition comprises an effective amount of a drug delivery system of the present invention, for example, up to about 10 14 cells, for example, about 10, 10 2 , 10 3 , 10 4 , 10 5 , 5×10 5 , 10 6 , 5×10 6 , 10 7 , 5×10 7 , 10 8 , 5×10 8 , 10 9 , 5×10 9 , 10 10 , 5×10 10 , 10 11 , 5×10 11 , 10 12 , 5×10 12 , 10 13 , 5×10 13 , 10 14 or 5×10 14 In some embodiments, the number of modified blood cells can be within a range between any two of the aforementioned numbers.

[0136] As used herein, the term "effective amount" refers to an amount sufficient to achieve a biological response or effect of interest, for example, reducing one or more symptoms or manifestations of a disease or disorder or modulating an immune response. In some embodiments, the composition administered to a subject comprises up to about 10 14 modified blood cells (drug delivery system), for example about 10 3 , 10 4 , 10 5 , 106 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 or 10 14 modified blood cells (drug delivery system), or any number or range therebetween.

[0137] In one aspect, the present invention also provides a combination product (eg, a pharmaceutical combination product) comprising the drug delivery system of the present invention and one or more other therapeutic agents. The combination product of the present invention can be used in the treatment method of the present invention.

[0138] The present invention also provides a kit comprising the combination product, for example, the kit comprises in the same package:

[0139] - a first container containing the drug delivery system of the present invention or a pharmaceutical composition or formulation comprising the same;

[0140] - A second container comprising one or more additional therapeutic agents or a pharmaceutical composition or formulation comprising the same.

[0141] In some embodiments, the other therapeutic agent is selected from any substance that is effective in the disease to be treated by the drug system, such as any substance that is effective in metabolic-related diseases, thrombosis-related diseases or cancer, including, for example, chemotherapeutic agents, cytotoxic agents, other antibodies, vaccines, small molecule drugs or immunomodulators (such as immunosuppressants or immunoagonists; drugs for anti-thrombosis or thrombolysis, such as urokinase, tissue plasminogen activator, streptokinase, etc.

[0142] V. Preventive or therapeutic methods or uses

[0143] (I) Indications

[0144] Tumor or cancer

[0145] In one aspect, the present invention relates to a method for preventing or treating a subject disease, such as cancer, comprising administering to the subject an effective amount of a drug delivery system as described herein. In some specific embodiments, the drug delivery system comprises an antibody for preventing or treating a tumor (e.g., a PD-1 antibody).

[0146] The cancer can be in the early, middle, or late stages or be metastatic. In some embodiments, the cancer can be a solid tumor or a hematological tumor. In some embodiments, the cancer is a gastrointestinal tumor, such as colon cancer or colorectal cancer; or a lung cancer, pancreatic cancer, or breast cancer. In some embodiments, the tumor is a tumor or cancer that is resistant or insensitive to a known drug, such as a known PD-1 inhibitor, such as an anti-PD-1 antibody, such as a refractory tumor or cancer.

[0147] In some embodiments, the tumor or cancer is a cancer characterized as having elevated protein levels and / or nucleic acid levels (e.g., elevated expression) of PD-1, PD-L1, and / or PD-L2 (e.g., compared to a healthy individual or healthy tissue from the same individual), for example, tumor cells of the cancer have elevated protein levels and / or nucleic acid levels (e.g., elevated expression) of PD-1, PD-L1, and / or PD-L2 (e.g., compared to healthy cells from a healthy individual or healthy cells from healthy tissue from the same individual).

[0148] Thrombosis-related diseases

[0149] In one aspect, the present invention relates to a method for anti-thrombosis or thrombolysis or prevention or treatment of thrombosis-related diseases, the method comprising administering to the subject an effective amount of a drug delivery system as described herein. In some specific embodiments, the drug delivery system comprises a protein or peptide for anti-thrombosis or thrombolysis or prevention or treatment of thrombosis-related diseases, such as pro-urokinase.

[0150] Thrombosis-related diseases, such as ischemic stroke, acute myocardial infarction, and deep vein thrombosis, remain the leading cause of death and disability worldwide. Thrombosis prevention can help reduce morbidity and mortality. Currently used thrombolytic drugs, plasminogen activators, often have a short half-life in the body, requiring large injections within a short period of time when a stroke occurs. Furthermore, they can cause serious side effects such as bleeding after bleeding out of the blood vessels, tissue remodeling, and neurotoxicity. Consequently, their clinical application is greatly limited, making them unsuitable for thrombosis prevention. To overcome the clinical limitations of these thrombolytic drugs, various modifications have been made to plasminogen activators to prolong their residence time in the blood vessels, but these modifications are not suitable for thrombosis prevention.

[0151] Erythrocytes have clear biological functions, safety, and a long circulation half-life. As natural drug carriers, erythrocytes are confined to blood vessels and can effectively prolong the half-life of thrombosis prevention or treatment drugs in the body, thereby achieving the function of treating and / or preventing thrombosis with extremely small doses. At the same time, because the drugs are fixed to the cells, side effects such as tissue remodeling and neurotoxicity caused by drug leakage from blood vessels can also be avoided. In addition, since erythrocytes are an important component of thrombi, using erythrocytes to carry antithrombotic drugs can decompose them from the inside at the beginning of thrombosis. This method greatly reduces the dosage and can effectively avoid existing serious side effects. Therefore, the use of erythrocytes to carry proteins or peptides (such as prourokinase) used to prevent or treat thrombosis-related diseases can be effectively used for the treatment and / or prevention of thrombosis.

[0152] In some embodiments, thrombosis-related diseases include, for example, stroke, such as ischemic stroke, myocardial infarction (eg, acute myocardial infarction), and venous thrombosis, such as deep vein thrombosis.

[0153] Metabolic-related diseases

[0154] Metabolic diseases are diseases associated with metabolic abnormalities in mammals, typically characterized by a lack of functional enzymes or excessive uptake of metabolites. Consequently, the accumulation of metabolites in the circulation and tissues causes tissue damage. Examples of metabolic diseases include those associated with defects in carbohydrate metabolism, amino acid metabolism, organic acid metabolism, or purine or pyrimidine metabolism.

[0155] Due to the widespread distribution of blood cells, such as RBCs, in the human body, the present invention contemplates the use of blood cells carrying functional metabolic enzymes for the treatment of metabolic-related diseases. For example, blood cells, such as red blood cells, containing functional metabolic enzymes will take up metabolites in the patient's plasma. Exemplary enzymes include urate oxidase, which is used for gout.

[0156] In some embodiments, the metabolic-related disease includes, for example, chronic hyperuricemia, such as gout, such as refractory gout (gout that is resistant to other conventional therapies).

[0157] (II) Methods of treatment or use

[0158] The drug delivery system of the present invention (and compositions, pharmaceutical compositions, preparations, combination products, etc. comprising the same, such as blood preparations) can be administered by any suitable method, preferably by infusion, such as parenteral infusion. Parenteral infusion includes intravenous or intra-arterial administration. Preferably, the drug delivery system of the present invention is administered by intravenous or arterial infusion.

[0159] For the prevention or treatment of disease, the appropriate dosage of the drug delivery system of the present invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and progression of the disease, whether it is administered for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient as a single treatment or over a series of treatments.

[0160] In some embodiments, the subject receives a single dose of cells during treatment, or receives multiple doses of cells, such as 2 to 5, 10, 20 or more doses. In some embodiments, the dose or total number of cells can be expressed as cells / kg. For example, a dose can be about 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 Cells / kg. In some embodiments, the course of treatment lasts from about 1 week to 12 months or longer, for example, 1, 2, 3 or 4 weeks or 2, 3, 4, 5 or 6 months. In some embodiments, the subject can be treated once every 2-4 weeks. It will be understood by those of ordinary skill in the art that the number of cells, dosage and / or dosing interval can be selected based on various factors, such as the subject's body weight and / or blood volume, the condition being treated, the subject's response, etc. The exact number of cells required can vary from subject to subject, depending on factors such as the subject's species, age, weight, sex and general condition, the severity of the disease or condition, specific cells, the identity and activity of the agent conjugated to the cells, the mode of administration, concurrent therapy, etc.

[0161] In some embodiments, the drug delivery systems of the present invention, or drugs or formulations comprising the same, are administered in combination with one or more treatment modalities or other therapeutic agents.

[0162] In some embodiments, the treatment modality or other therapeutic agent is used to treat a tumor, such as cancer.

[0163] In some embodiments, the treatment modality is radiotherapy or surgery.In some embodiments, the therapeutic agent is selected from any substance effective in cancer, including chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, small molecule drugs or immune modulation (such as immunosuppressants or immune agonists).

[0164] In some embodiments, the treatment modality or therapeutic agent is used to prevent or treat a thrombosis-related disease.

[0165] In some embodiments, the treatment modality is, for example, a stent or a bypass. In some embodiments, the therapeutic agent is, for example, an antithrombotic or thrombolytic drug, such as urokinase, tissue plasminogen activator, streptokinase, and the like.

[0166] In some embodiments, the treatment modality or therapeutic agent is used to treat a metabolic-related disease.

[0167] In another aspect, the present invention also provides use of the drug delivery system of the present invention in preparing a medicament or formulation for use in the aforementioned method (eg, for treatment).

[0168] In some embodiments, the blood cells in the drug delivery system of the present invention are from the subject to be treated. In some embodiments, the method of treating a disease in a subject of the present invention comprises:

[0169] (1) Collecting blood from the subject to be treated;

[0170] (2) preparing the drug delivery system of the present invention, for example, by the method of the present invention;

[0171] (3) Infusing a therapeutically effective amount of the drug delivery system into the subject.

[0172] In some embodiments, the blood cells in the drug delivery system of the present invention can be from other subjects, such as healthy subjects. In some embodiments, the drug delivery system of the present invention can be administered as a drug or formulation to a subject to be treated.

[0173] Any documents cited herein, including patents, patent applications, and publications, are hereby incorporated by reference in their entirety.

[0174] Any or all of the features discussed above and throughout this application may be combined in various embodiments of the present invention. The following examples further illustrate the present invention, however, it should be understood that the examples are described in an illustrative and non-limiting manner and are not intended to and should not limit the scope of the present invention in any way, and that various modifications may be made by those skilled in the art. Example:

[0175] Material

[0176] ProUK was purchased from Tasly Biopharma (China). ProUK-cys was expressed and purified using HEK293F cells. Thrombin was purchased from Sigma-Aldrich (USA). Anti-urokinase / uPA-HRP (Clone #05) and anti-urokinase / uPA (Clone #1A2G4G11) were purchased from Sino Biological Inc (China). MAL-PEG2000-DSPE was purchased from Xi'an Ruixi Biological Technology Co., Ltd (China). CaCl2 and FeCl3 were purchased from Sionpharm Chemical Reagent Co., Ltd (China). All other chemicals and solvents were purchased from Sionpharm Chemical Reagent Co., Ltd (China).

[0177] Unlabeled erythrocytes or washed erythrocytes refer to erythrocytes separated from peripheral blood of healthy individuals by density gradient centrifugation, and the separated erythrocytes are washed three times with PBS to remove other components (such as plasma or other blood cells).

[0178] experimental animals

[0179] C57BL / 6J mice or SD rats were purchased from the Institutional Animal Care and Use Committee of Westlake University. Animals were handled in accordance with institutional guidelines, and all animal studies were approved by the Institutional Animal Care and Use Committee of Westlake University (IACUC protocol #19-012-GXF).

[0180] Statistical analysis

[0181] In all graphs and tables, data are presented as mean ± standard deviation. One-way analysis of variance was used to analyze data involving multiple treatment groups. All statistical analyses were performed using GraphPad Prism version 8.4 for Windows. Differences between groups were considered significant at a P value of < 0.05.

[0182] Example

[0183] Example 1 Preparation of eGFP-labeled erythrocytes using DSPE

[0184] eGFP was used to test the binding efficiency of the reaction. eGFP was expressed and purified by fusion with a cysteine ​​at the C-terminus (eGFP-Cys). The synthetic Mal-PEG2000-DSPE linker (synthesized by Xi'an Ruixi Biotechnology) had a purity exceeding 99%.

[0185] 1.1 Expression and purification of recombinant proteins in Escherichia coli

[0186] The eGFP-Cys cDNA was cloned into the pET vector and transformed into E. coli BL21 (DE3) cells for protein expression. The transformed cells were cultured at 37°C until the OD 600 The pH value was 0.6-0.8, and then 500 μM IPTG was added. The cells were cultured with IPTG at 37°C for 4 hours, harvested by centrifugation and lysed with pre-cooled lysis buffer (20 mM Tris-HCl, pH 7.8, 500 mM NaCl). The lysate was sonicated on ice (5 seconds on, 5 seconds off, 60 cycles, 25% power, Branson Sonifier 550 ultrasonic cell disruptor). All supernatants were centrifuged at 14,000 x g for 40 minutes at 4°C and filtered through a 0.45 μM filter. The filtered supernatant was loaded onto the same The protein was eluted on a HisTrap FF 1 mL column (GE Healthcare) connected to a designed chromatography system. The protein was eluted with an elution buffer containing 20 mM Tris-HCl, pH 7.8, 500 mM NaCl, and 300 mM imidazole. All eluted fractions were analyzed on an SDS-PAGE gel. The eGFP-Cys protein was obtained. The amino acid sequence of eGFP-Cys is shown below in SEQ ID NO: 1.

[0187] 1.2 Obtaining the connector

[0188] The Mal-PEG2000-DSPE linker was purchased from Xi'an Ruixi Biological, and its chemical formula is shown in FIG3 . The purity is over 99%.

[0189] 1.3 Red blood cell labeling: Method 1

[0190] As shown in Figure 1, Mal-PEG-phospholipid (e.g., Mal-PEG-DSPE) can be first linked to red blood cells via phospholipid (e.g., DSPE) to obtain pre-modified red blood cells with maleimide groups on the cell membrane surface. Then, a pre-treated protein containing exposed thiol groups is incubated with the pre-modified red blood cells. Maleimide can react with the thiol groups on the exposed cysteine ​​on the protein through Michael addition to form a stable covalent bond, thereby connecting the target protein to the cell membrane of the red blood cell. The specific experimental steps are as follows:

[0191] 1.3.1 Red blood cell pretreatment

[0192] Red blood cells were isolated from the peripheral blood of C57BL / 6J mice by density gradient centrifugation. The isolated red blood cells were washed three times with PBS. Mal-PEG2000-DSPE was dissolved in phosphate buffer at 37°C to a final concentration of 100 μM. 1×10 cells were then pretreated with 50 μM Mal-PEG2000-DSPE at 37°C. 9 The pretreated red blood cells were then washed three times with PBS. The pretreated red blood cells were used immediately or not stored long-term.

[0193] 1.3.2 MAL-PEG2000-DSPE-mediated labeling of erythrocytes

[0194] The pretreated erythrocytes were mixed with eGFP-cys in PBS buffer, where the concentration of erythrocytes was 1×10 9 The total volume of the reaction mixture was 200 μL, and the reaction was carried out by rotating at 10 rpm for 1 hour. The reaction was stopped, washed with PBS, and the remaining reaction reagents were removed to obtain labeled red blood cells. The labeling efficiency of red blood cells (i.e., the percentage of red blood cells coupled to the protein in the total red blood cells) was analyzed using Beckman Coulter CytoFLEX LX. No eGFP signal was detected in cells not labeled by this method, while the eGFP positivity rate of cells labeled by this method was 100%.

[0195] 1.4 Red blood cell labeling: Method 2 (Figure 2)

[0196] Maleimide groups can undergo Michael addition reactions with sulfhydryl groups on exposed cysteines, but not on unexposed cysteines. Maleimides attached to polyethylene glycol chains are commonly used as flexible linker molecules to attach proteins to surfaces. We exploit this principle to modify proteins with linkers that can insert into cell membranes (Figure 2).

[0197] As shown in Figure 2, a protein with an exposed cysteine ​​(e.g., a C-terminal cysteine) undergoes a Michael addition reaction with a maleimide group, thereby linking the protein and DSPE in a stable covalent bond. The DSPE end can then fuse with the red blood cell membrane to form a stable connection, allowing the target protein to be loaded onto the cell membrane. The specific experimental steps are as follows:

[0198] 1.4.1 Protein pretreatment

[0199] The linker Mal-PEG2000-DSPE and eGFP-cys protein were added to PBS to a final volume of 1 mL. The final concentrations of Mal-PEG2000-DSPE and eGFP-cys were 2 mM and 1 mM, respectively. This means the two substrates were mixed in a 1:2 ratio of eGFP-cys:MAL-PEG2000-DSPE for the reaction (Figure 4). The reaction buffer was rotated and incubated at 10 rpm for 1 hour. After the reaction, the eGFP-CysMal-PEG2000-DSPE product was collected by protein purification, dialysis, and ultrafiltration to remove excess linker.

[0200] 1.4.2 eGFP-Cys-MAL-PEG2000-DSPE-mediated labeling of erythrocytes

[0201] The above red blood cells and eGFP-cys-Mal-PEG2000-DSPE were mixed in PBS buffer to a total volume of 200 μL, with a final concentration of 1×10 9 The concentrations of eGFP-cys-Mal-PEG2000-DSPE were 5, 25, 50, and 100 μM, respectively. The reaction mixture was rotated and incubated at 10 rpm for 1 hour to obtain the erythrocyte-protein conjugate. The reaction was stopped, washed with PBS, and the remaining reaction reagents were removed. The labeled erythrocytes were then analyzed using a Beckman Coulter CytoFLEX LX to determine the erythrocyte labeling efficiency (i.e., the percentage of protein-conjugated erythrocytes relative to the total erythrocyte count). Unlabeled erythrocytes served as a control.

[0202] 1.5 Characterization of labeling efficacy

[0203] The labeling efficiency of eGFP was characterized on the RBC membrane. After the reaction, the eGFP-cys-Mal-PEG-DSPE prepared in 1.3.2 and the unlabeled red blood cells (red blood cells were 1×10 9 / mL) were used to detect the labeling effect of eGFP by flow cytometry. As shown in Figure 5, the histogram shows the eGFP signal on the cell surface. The results showed that 100% of natural red blood cells were labeled with eGFP-Cys-MAL-PEG2000-DSPE in vitro (Figure 5), and the signal intensity was dose-dependent (see Figure 6, where red: unlabeled; blue: labeled with 5μM eGFP-cys-Mal-PEG-DSPE; orange: labeled with 25μM eGFP-cys-Mal-PEG-DSPE; light green: labeled with 50μM eGFP-cys-Mal-PEG-DSPE; dark green: labeled with 100μM eGFP-cys-Mal-PEG-DSPE). As shown in Figures 5 and 6, after incubation with eGFP-Cys-MAL-PEG-DSPE, the eGFP signal on the surface of red blood cells was significantly higher than that of unlabeled red blood cells.

[0204] 1.6 In vivo lifespan of eGFP-Cys-MAL-PEG-DSPE-labeled erythrocytes

[0205] To evaluate the lifespan of these surface-modified erythrocytes in vivo, we then stained the eGFP-cys-Mal-PEG-DSPE-labeled mouse erythrocytes (eGFP-cys-mal-PEG-DSPE-RBCs) and unlabeled erythrocytes (unlabeled) prepared in Example 1.3 with the fluorescent dye Cell Trace Far Red dye and infused them into mice through the tail vein. Each group consisted of three 6-8 week old C57BL / 6J mice, and the infusion volume was 1×10 9 cells / each.

[0206] Mice were bled by cheek sampling on days 1, 3, 4, 7, 8, 11, 15, 18, 22, 25, 29, and 34 after infusion.

[0207] Blood samples from day 29 were analyzed by flow cytometry. Far Red-positive cells were selected for analysis of the percentage of red blood cells expressing eGFP signals. The results are shown in Figure 7 . Figure 7 shows that in the labeled cell group (eGFP-cys-mal-PEG-DSPE-RBC), all Far Red-positive cells were eGFP-positive, indicating that the cells still carried the eGFP protein.

[0208] Blood samples on days 1, 3, 4, 7, 8, 11, 15, 18, 22, 25, 29, and 34 were analyzed by flow cytometry. Far red positive cells were selected for analysis of the percentage of red blood cells with eGFP signals. Far red positive cells represent the percentage of transfused red blood cells in the blood circulation. We found that eGFP-Cys-MAL-PEG-DSPE-labeled red blood cells (eGFP-cys-mal-PEG-DSPE-RBC) not only showed the same lifespan as the red blood cells of the control group (mice transfused with RBCs lacking eGFP-Cys-MAL-PEG-DSPE labeling, control RBC), but also showed an eGFP signal that lasted for 34 days in the cycle (see Figure 8). No obvious hemolysis was observed.

[0209] Example 2. Preparation of RBCs labeled with anti-PD-1 antibodies using DSPE

[0210] To further confirm that this method can be applied to labeling of different types of proteins, we used anti-PD-1 antibody ( manufactured by Merck Sharp & Dohme Corp.) was used for labeling.

[0211] 10 μM anti-PD-1 antibody was pretreated with 0.2 mM TCEP at room temperature for 1 hour to expose the cysteine ​​residues of the antibody. Following pretreatment, the pretreated protein was collected by dialysis and ultrafiltration to remove excess TCEP. The pretreated protein was used immediately or not stored long-term.

[0212] Then, RBCs labeled with anti-PD-1 antibodies were prepared as described in Example 1.3, except that the antibody concentration was 5 μM, in which anti-PD-1 antibodies were used instead of eGFP-cys. As a control, 5 μM anti-PD-1 antibodies were incubated with red blood cells not coupled to Mal-PEG-DSPE as described in Example 1.3. The labeling efficiency was detected by flow cytometry. The histogram shows the PD-1 antibody signal on the cell surface (Figure 9, where Unlabeled represents the result after incubation of red blood cells not coupled to Mal-PEG-DSPE with anti-PD-1 antibodies; Anti-PD-1-Ma-PEG-DSPE represents the result after incubation of red blood cells coupled to Mal-PEG-DSPE with anti-PD-1 antibodies). Figure 10 shows the signal intensity of the PD-1 antibody. Blue: Signal intensity of the anti-PD-1 antibody after incubation with erythrocytes not conjugated with Mal-PEG-DSPE; Red: Signal intensity of the anti-PD-1 antibody after incubation with erythrocytes conjugated with Mal-PEG-DSPE. This result demonstrates that this method effectively labels the antibody on the cell surface.

[0213] Example 3. Preparation of erythrocytes labeled with urate oxidase (UOX) using DSPE

[0214] 3.1 Recombinant protein expression and purification in E. coli

[0215] The coding sequence of UOX-Cys (Aspergillus flavus uricase) (amino acid sequence: SEQ ID NO: 3; nucleic acid sequence: SEQ ID NO: 4) was codon-optimized for expression in E. coli and synthesized by GenScript. Subclones were generated by standard PCR procedures and inserted into the pET-30a (GenScript) vector, which has an additional cysteine ​​residue at the C-terminal His6 linker. All constructs were verified by sequencing and then transformed into E. coli BL21 (DE3) for protein expression.

[0216] A single transformed colony was inoculated into 10 ml of Luria-Bertani (LB) medium supplemented with ampicillin (100 μg / ml) and cultured overnight at 37°C with shaking at 220 rpm. The 10 ml culture was transferred to 1 L of fresh LB medium and cultured at 37°C with shaking at 220 rpm until the OD 600 The temperature was then lowered to 20°C and 1 mM IPTG was added for induction.

[0217] Cells were harvested 20 h after induction by centrifugation at 8,000 rpm for 10 minutes at 4°C. For proteins without a His6 tag, the cell pellet was resuspended in low salt lysis buffer (50 mM Tris 7.5, 50 mM NaCl) and lysed by sonication. The supernatant collected after centrifugation at 10,000 rpm for 1 hour was loaded onto an SP Sepharose FF column (Cytiva, Marlborough, USA) pre-equilibrated with SPA buffer (20 mM Tris 7.5). The column was washed with SPA buffer until the absorbance and conductivity at 280 nm became stable and then eluted using a linear gradient of 0-1 M NaCl in 20 mM Tris 7.5. The fractions corresponding to the elution peak were analyzed by SDS-PAGE, and the purest fractions were enriched. To avoid cysteine ​​oxidation, 2 mM TCEP was added to the combined fractions, and the samples were concentrated using Amicon Ultra-15 centrifugal filter devices (Millipore, Darmstadt, Germany). The concentrated protein was loaded onto an EzLoad 16 / 60 Chromdex 200 pg (Bestchrom, Shanghai, China) pre-equilibrated with PBS, and the target protein peak was collected. For proteins with a His6 tag, the cell pellet was resuspended in lysis buffer (50 mM Tris 7.5, 200 mM NaCl, 5 mM imidazole) and lysed by sonication. The tagged protein UOX-His6-Cys was purified on a Ni Sepharose 6FF affinity column (Cytiva) and an anion exchange column, followed by size exclusion chromatography. All proteins were stored at -80°C.

[0218] 3.2 Red blood cell labeling

[0219] UOX-labeled RBCs were prepared by method 1 as described in Example 1.3, wherein UOX was used instead of eGFP to obtain UOX-Cys-Mal-PEG-DSPE-labeled red blood cells.

[0220] In vivo lifespan of UOX-His6-Cys-Mal-PEG-DSPE labeled RBCs

[0221] We used UOX-His6-Cys-Mal-PEG-DSPE labeled mouse erythrocytes (dose: 1×10 9 The 1×10 9Cells / each, denoted as UOX1 and UOX2, respectively), these red blood cells were simultaneously stained and labeled with the fluorescent dye Cell Far Red. The percentage of Cell Far Red and UOX-Cys-Mal-PEG-DSPE-positive RBCs in vivo was regularly analyzed using the same method as in Example 1.5, but blood was collected and analyzed on days 1, 3, 6, 10, 12, 17, and 20, with the blood sample collected on day 20 also being analyzed by flow cytometry ( FIG11 ). We found that UOX-Cys-Mal-PEG-DSPE-labeled red blood cells showed the same lifespan in the two recipient mice and exhibited a persistent signal for more than 20 days during the cycle (see FIG11 and 12 ).

[0222] Example 4. Preparation of other mammalian cells labeled with eGFP using DSPE

[0223] We further validated the approach for other blood cells. We tested whether the approach worked on T cells, B cells, monocytes, NK cells, and megakaryocytes.

[0224] PBMCs were isolated from human peripheral blood of healthy volunteers. Whole blood was diluted 1:1 with phosphate buffered saline, and peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation at 1200 x g for 15 minutes using lymphocyte separation solution (Lymphoprep™, STEMCELL Technologies) and lymphocyte separation tubes.

[0225] The purpose of this step is to achieve density gradient centrifugation of cell components using lymphocyte separation solution to separate PBMCs from different cells such as red blood cells and platelets to ensure subsequent enrichment of T cells.

[0226] Cell separation: Cell surface antigens are bound to corresponding biotin-labeled antibodies (purchased from Biolegend), and biotin is bound to streptavidin-labeled magnetic beads Streptavidin Particles Plus-DM (BD, Cat. No. 557812). Cells with specific surface markers are separated by magnetic beads, where:

[0227] Then, eGFP-labeled cells were prepared as described in Example 1, except that T cells, B cells, monocytes, NK cells, or megakaryocytes were used instead of the red blood cells used in that example, and the labeling efficiency was examined by flow cytometry. The results showed that this modified method was also applicable to these cell types and exhibited good labeling efficiency ( FIG. 13 ).

[0228] Example 5. In vivo lifespan of eGFP-Cys-MAL-PEG-DOPE labeled erythrocytes

[0229] As described in Example 1.3, eGFP-Cys-MAL-PEG-DOPE (Xi'an Ruixi Biosynthesis, as shown in Figure 3, where DSPE is replaced by DOPE) labeled red blood cells were prepared, except that DOPE was used instead of DSPE. The standard operating procedure for evaluating the in vivo lifespan of these surface-modified red blood cells in Example 1.5 was applied, where blood was collected on days 1, 3, 4, and 15, and flow cytometric analysis was performed on the blood collected on day 15 (Figure 14, Control: unlabeled red blood cells). We found that eGFP-Cys-MAL-PEG-DOPE labeled red blood cells (eGFP-Cys-MAL-PEG-DOPE-RBC) not only showed the same lifespan as the red blood cells of the control group (mice transfused with unlabeled red blood cells), but also showed a signal that lasted for 15 days in the cycle (Figures 14 and 15).

[0230] Example 6. Comparison of labeling efficiency of DSPE and DOPE

[0231] Erythrocyte labeling efficiency was measured by flow cytometry. Mal-PEG2000-DOPE and Mal-PEG2000-DSPE-labeled RBCs were prepared as described in Example 1.3. The differences in the labeling reaction system were that the concentration of eGFP-cys was 5 μM / mL, and the concentrations of DSPE or DOPE ranged from 2 μg / mL to 512 μg / mL, respectively. Mal-PEG2000-DOPE and Mal-PEG2000-DSPE were incubated with RBCs for 10, 30, and 60 minutes, respectively. The MFI was determined by flow cytometry.

[0232] As shown in FIG17 , under the same incubation time or the same concentration of eGFP, the eGFP signal on the surface of mature RBCs was stronger using DOPE, indicating that DOPE has better efficiency than DSPE in modifying RBCs.

[0233] Example 7. Comparison of the optimal labeling efficiency concentrations of DSPE, DPPE, DMPE, and DOPE

[0234] Mal-PEG2000-phospholipid (DPPE, DOPE, DSPE, or DMPE)-labeled RBCs were prepared as described in Example 1.3 (Mal-PEG2000-DPPE, Mal-PEG2000-DOPE, Mal-PEG2000-DSPE, and Mal-PEG2000-DMPE were all from Xi'an Ruixi Biotechnology). The differences were that the concentrations of Mal-PEG2000-phospholipid in the labeling reaction system were 200 μg / mL, 80 μg / mL, 20 μg / mL, and 0 μg / mL (control group), respectively, and the eGFP-cys concentration was 5 μM / mL. After the labeling reaction, the mean fluorescence intensity on the cells was measured by flow cytometry.

[0235] As shown in Figure 16, DPPE and DMPE were also effective for labeling cells. The labeling efficiency of these two phospholipids was the highest when the concentration of eGFP-Cys-Mal-PEG2000-phospholipid was 80 μg / mL. DSPE and DOPE showed the highest labeling efficiency at a concentration of 200 μg / mL.

[0236] The absorbance of the cell supernatant at 410 nM was also measured by ELISA. As shown in Table 1, after incubation with a high concentration (200 μg / mL) of Mal-PEG2000-DPPE, the absorbance of the supernatant increased due to RBC hemolysis. Similarly, after incubation with 200 μg / mL Mal-PEG2000-DMPE, RBC hemolysis occurred. However, high concentrations of Mal-PEG2000-DOPE and Mal-PEG2000-DSPE did not cause red blood cell damage.

[0237] Table 1. Absorbance of supernatant at 410 nm

[0238] Example 8 Preparation of Prourokinase Activator-Red Blood Cell Conjugate

[0239] In this experiment, we first modified ProUK by adding a cysteine ​​residue to the C-terminus of the protein to provide a free thiol group. Next, we used DSPE, a cell membrane component analog that can fuse with the RBC membrane. The maleimide group can undergo a Michael addition reaction with the free thiol group on ProUK. We used Mal-PEG-DSPE to couple cells to proteins. Mal-PEG-DSPE and RBCs were incubated at 37°C to fuse with the cell membrane. The unfused Mal-PEG-DSPE was washed away, and ProUK containing a free thiol group at the C-terminus was added to react the protein with the pretreated RBCs (Figure 18) (See also Figure 1 for this experiment). The entire reaction process was carried out under physiological buffer conditions to avoid damage to the RBCs.

[0240] The specific steps are as follows:

[0241] 8.1. RBC Pretreatment

[0242] Red blood cells were isolated from the peripheral blood of C57BL / 6J mice by density gradient centrifugation. The isolated red blood cells were washed three times with PBS. Mal-PEG2000-DSPE was dissolved in phosphate buffer at 37°C to a final concentration of 100 μM. 1×10 cells were then pretreated with 50 μM Mal-PEG2000-DSPE at 37°C. 9 The pretreated RBCs were then washed three times with PBS. The pretreated RBCs were used immediately or not stored long-term.

[0243] 8.2. MAL-PEG2000-DSPE-mediated RBC labeling

[0244] 8.2.2 ProUK pre-processing

[0245] 8.2.2.1 C-terminal cysteine ​​modification of ProUK

[0246] ProUK-Cys cDNA (nucleic acid sequence as shown in SEQ ID NO: 9) was cloned into pcDNA 3.4 vector and the plasmid was transfected into HEK293F cells for protein expression. The transfected HEK293F cells were cultured at 37°C for 5 days and the supernatant was collected for purification. All supernatants were centrifuged at 14,000 x g for 40 minutes at 4°C and filtered through a 0.45 μM filter. The filtered supernatant was loaded onto the same The protein was eluted on a HisTrap FF 1 mL column (GE Healthcare) connected to a designed chromatography system. The protein was eluted with an elution buffer containing 20 mM Tris-HCl, pH 7.8, 500 mM NaCl, and 300 mM imidazole. All eluted fractions were analyzed on an SDS-PAGE gel. The ProUK-Cys protein was obtained. The amino acid sequence of ProUK-Cys is shown below in SEQ ID NO: 8. The nucleic acid sequence of ProUK-Cys is shown below in SEQ ID NO: 9.

[0247] 8.2.3. Conjugation of erythrocytes to ProUK-cys

[0248] The above RBCs and ProUK-cys were mixed in PBS buffer with a total volume of 200 μL and a final concentration of 1×10 RBCs. 9 / mL, and the ProUK-cys concentration was 10μM. The reaction mixture was rotated and incubated at a speed of 10 rpm for 1 hour to react. The reaction was stopped, washed with PBS, and the remaining reaction reagents were removed to obtain the red blood cell-ProUK conjugate ProUK-RBC. The red blood cell labeling efficiency (i.e., the percentage of red blood cells coupled with the protein in the total red blood cells) was analyzed using Beckman Coulter CytoFLEX LX, where red blood cells not labeled with ProUK (unlabeled red blood cells) were used as a control group. The results showed that this method can effectively carry ProUK protein to the RBC surface (Figure 19, the red blood cells without Pro-UK labeling have almost 0 ProUK signals, while the red blood cells with ProUK signals in ProUK-RBC prepared by the above method are 100%).

[0249] 8.2.4 Lifespan of the ProUK-RBC Protein Conjugate in Vivo

[0250] In order to evaluate the in vivo lifespan of the surface-modified RBCs obtained in 8.2.3, the in vivo lifespan of Pro-UK-RBCs was evaluated as described in 1.5 of Example 1. Specifically, 10 unlabeled RBCs (control RBCs) and 10 ProUK-RBCs obtained in Example 8.2.3 were each 9 Cell Trace Far Red dye was used to stain the RBC-ProUK and control RBCs. Cell Trace Far Red dyed RBC-ProUK and control RBCs were infused into the tail vein of normal recipient C57BL / 6J mice (Experimental Animal Center of Westlake University, 6-8 weeks old, 3 mice per group).

[0251] The mice were bled from the cheek at 1, 2, 6, 24, 48, 72, 144, 192, 240, 336, 408, 504, and 576 hours after transfusion. The blood samples were incubated with FITC-conjugated anti-urokinase antibody (Sino Biological, 1 μL / sample) at 37°C for 15 minutes to detect ProUK signal, washed three times, and analyzed by flow cytometry. The flow cytometry results on days 0, 8, 17, and 24 are shown in the figure. Cell Trace Far Red positive cells were selected for analysis of the percentage of RBCs with ProUK signal. Cell Trace Far Red positive cells represent the percentage of transfused RBCs in the circulation. The results are shown in Figure 20. There was no statistically significant difference in the in vivo survival rate of RBCs between the experimental and control groups.

[0252] Mice were bled from the cheek 24 hours after transfusion. Blood samples were incubated with FITC-conjugated anti-urokinase antibody at 37°C for 15 minutes to detect ProUK signal, washed three times, and then analyzed using a Merck Amnis ImageStream Mark II. The results are shown in Figure 21, showing that ProUK-RBCs are morphologically identical to unlabeled red blood cells (RBCs), demonstrating that the Mal-PEG-DSPE labeling procedure does not significantly damage RBCs, leading to their premature removal.

[0253] It can also be seen from Figures 20 and 21 that ProUK levels remained constant on Cell Trace Far Red positive cells for more than 24 days.

[0254] Therefore, the engineered RBCs exhibited good stability in vivo, and ProUK signals were consistently detectable on the surface of these RBCs.

[0255] Example 9 ProUK-RBC improves thrombolytic efficacy in vitro

[0256] As shown in Example 8, ProUK-RBCs exhibited good stability in vivo, and ProUK signals were consistently detected on the surface of these RBCs. In this example, the biological functions of the ProUK-RBCs prepared in Example 8 were further evaluated.

[0257] During thrombosis, RBCs play a crucial role, participating in platelet-driven clot and thrombus retraction, leading to the formation of tightly packed polyhedral RBC arrays that comprise a nearly impermeable barrier important for hemostasis and wound healing.

[0258] After quantifying the ProUK content on RBCs by ELISA, we used mixtures of washed RBCs / free ProUK at varying ratios, washed RBCs / ProUK-RBCs, and an equal amount of washed RBCs (control). These thrombolytic components were added to thrombi formed from washed mouse RBCs, mouse plasma, CaCl2, and thrombin. Thrombolytic activity was assessed by comparing the number of free RBCs that did not form thrombi at 37°C. The specific steps were as follows:

[0259] Whole blood was obtained from C57BL / 6J mice, and plasma was separated by centrifugation. Washed red blood cells were also obtained from the whole blood.

[0260] Hemostatic clot group:

[0261] A 100 μL volume of CaCl2 and thrombin (20 mM and 0.2 units / ml final concentration, respectively) was added to 50 μL of mouse plasma and washed RBCs to form a clot for 20 minutes. After clot formation, different amounts of ProUK-RBCs and ProUK+unlabeled RBCs were added as follows.

[0262] The specific experimental design is as follows: the concentration of ProUK-RBC and unlabeled RBC is 1×10 7 Cells / μl ProUK-RBC group:

[0263] 2.4 μg / mL: ProUK-RBC (100 μL of 2.4 μg / mL ProUK-RBC prepared in Example 8);

[0264] 1.2 μg / mL: 50 μL ProUK-RBC and 50 μL unlabeled RBC;

[0265] 0.6 μg / mL: 25 μL ProUK-RBC and 75 μL unlabeled RBC;

[0266] 0.3 μg / mL: 12.5 μL ProUK-RBC and 87.5 μL unlabeled RBC;

[0267] 0.15 μg / mL: 6.25 μL ProUK-RBCs and 93.75 μL unlabeled RBCs.

[0268] 0μg / mL: 100μl unlabeled red blood cells

[0269] Free ProUK+RBC group: 50 mg / mL ProUK-cys stock solution (volume change during dilution was negligible) was added to 100 μl of unlabeled RBC to obtain ProUK-cys solutions with final concentrations of 0, 4, 8, 16, 32, and 64 μg / mL.

[0270] To simulate the lysis of hemostatic clots (Hemostatic clot group), thrombus clots were covered with different amounts of ProUK-RBCs and ProUK+unlabeled RBCs at 20°C.

[0271] Nascent clot group:

[0272] To dissolve nascent clots (Nascent clot group), different amounts of ProUK-RBCs and ProUK+unlabeled RBCs (including unlabeled RBCs containing 0 μg / mL ProUK-cys) from the above Hemostatic clot group were added directly to mouse plasma before the addition of CaCl2 and thrombin, and then CaCl2 and thrombin were immediately added for clot formation, which lasted for 20 minutes.

[0273] The two groups of clots were incubated separately at 37°C for 2 hours. The samples were then gently inverted to resuspend, and the clot-free RBC suspension was aspirated with a pipette. RBC lysis buffer (Solarbio Biotech) was added for 10 minutes, followed by centrifugation (5 minutes, 1200 × g), and the amount of released hemoglobin was determined spectrophotometrically at 405 nm. Thrombolysis was standardized for each group by comparing the highest absorbance value (no thrombus formation) with the lowest absorbance value (0 μg / mL, no thrombolytic agent added, thrombus formation). The results showed that free ProUK required approximately 30 μg / mL to effectively prevent thrombus formation, with no significant difference in thrombolytic performance between hemostatic and nascent clots (Figures 22A and C). In the ProUK-RBC group, only 0.15-0.3 μg / mL of ProUK was required to effectively prevent thrombus formation, and the thrombolytic effect on nascent clots was superior to that on hemostatic clots (Figures 22B and D). The dose of free ProUK that completely lysed thrombus was approximately 100 times that of ProUK-RBC, and the ability of ProUK-RBC to lyse nascent clots (-50%) was 10 times higher than that of hemostatic clots (-5%).

[0274] Therefore, ProUK loaded onto RBCs is more stable, and with the help of RBCs as a carrier, it can better activate the function of plasminogen activators. Furthermore, RBCs are an important component of blood clots. During thrombosis, even a small number of ProUK-RBCs can enter the clot and activate its fibrinolytic activity, breaking down the clot from within. However, due to their more compact structure, ProUK-RBCs cannot penetrate the hemostatic clot and can only dissolve clots from the surface. Therefore, ProUK-RBCs are more effective in targeting nascent clots.

[0275] Example 10 ProUK-RBC prolongs half-life and reduces drug dosage

[0276] The above studies have shown that ProUK-RBC can effectively reduce the dose of ProUK. We used 10ug / kg (the effective dose of free ProUK verified in clinical practice is 1mg / kg) of free ProUK or ProUK-RBC to verify the in vitro antithrombotic function of the ProUK-RBC of the present invention.

[0277] C57 / BL6J mice (Animal Center of Westlake University, 6-8 weeks old, 3 mice per group, totaling three groups) were injected with equal amounts of free ProUK (i.e., 0.2 μg ProUK-cys and 200 μl 1×10 7 / ul RBC mixture), ProUK-RBC (i.e., ProUK-RBC prepared in Example 8 200ul 1×10 7 / ul) and unlabeled RBC (i.e. 200ul 1×10 7 Blood was collected from the eye sockets at the planned time. The ProUK-RBCs group had blood collected 1, 5, 15, 30, 60, and 120 minutes after administration, and the RBC and free ProUK group had blood collected 1, 3, 5, 10, 30, and 60 minutes after administration ( FIG23 ).

[0278] After quantifying the ProUK concentration in whole blood by ELISA using Anti-Urokinase / uPA-HRP (Clone #05) and Anti-Urokinase / uPA (Clone #1A2G4G11) (both purchased from Sino Biological), we found that the free ProUK group was almost undetectable after about 5 minutes, while the ProUK-RBC group still retained about 90% in the blood at 30 minutes ( Figure 24 ).

[0279] We further evaluated the in vitro thrombolytic ability of ProUK-RBCs by analyzing the amount of thrombus and the amount of free red blood cells to analyze the antithrombotic capacity.

[0280] Thrombosis was performed by adding CaCl2 and thrombin (20 mM and 0.2 units / ml final concentrations, respectively) to whole blood from the mice removed as described above and incubating at 37°C for 2 hours. The sample was gently inverted to resuspend, and the clot-free red blood cell suspension was aspirated with a pipette. Red blood cell lysis buffer was added for 10 minutes, and the sample was centrifuged (5 minutes, 1200 × g). The amount of released hemoglobin was measured spectrophotometrically at 405 nm. Thrombolysis was standardized for each group by comparing the highest absorbance value (no thrombus formation) to the lowest absorbance value (thrombus formation without the addition of thrombolytic agent).

[0281] The results showed that the ProUK group was unable to effectively dissolve thromboses at the initial time point, while the ProUK-RBC group still had approximately 75% thrombolytic activity after 2 hours (Figures 25 and 26). Due to the low initial dose, the ProUK group had a short plasma half-life and poor thrombolytic effect. However, we found that the ProUK-RBC group, even at this low concentration, prolonged the half-life of ProUK and demonstrated effective thrombolytic activity.

[0282] Example 11 ProUK-RBC improves thrombolytic efficacy in vivo

[0283] 11.1 Tail Time Measurement Method

[0284] ProUK-RBCs demonstrated excellent thrombolytic activity in vitro and ex vivo. We next tested the antithrombotic effects of Pro-UK-RBCs prepared in Example 8 in vivo. The tail bleeding time assay is the most widely used method for assessing hemostasis in mice. The specific steps are as follows:

[0285] C57 / BL6J mice were anesthetized with intraperitoneal (IP) injection of tribromoethanol, and the tail hair was cleaned with scissors. Then, ProUK-RBC (ProUK-RBC 200ul 1×10 7 / ul), free ProUK (with 0.2ug ProUK-cys and 200ul 1×10 7 / ul unlabeled RBC) and RBC (200ul 1×10 7 The tail of C57 / BL6J mice (Westlake University, 6-8 weeks old, 18 mice per group) was injected into the tail vein with 100 μl of unlabeled RBCs. The tail was immediately transected 8 mm from the tip using a scalpel. The bleeding tail stump was immersed in physiological saline warmed to 37°C, and the hemostasis time was determined (Figure 27). Bleeding time less than 60 seconds or greater than 900 seconds was related to experimental operation or individual animal differences, and the data were excluded.

[0286] Figure 28 shows the antithrombotic activity of the three groups at low dose levels, with 13 mice in the RBC group, 10 mice in the free ProUK group, and 13 mice in the ProUK-RBC group meeting the hemostasis time requirement. This experiment allows for the evaluation of the thrombolytic activity of ProUK-RBC against nascent clots. The results showed no significant difference between the free ProUK group and the RBC group, due to the low dose of free ProUK. However, at this low dose, the ProUK-RBC group had a longer bleeding time than both the RBC and free ProUK groups ( Figure 28 ). The ProUK-RBC group still demonstrated good antithrombotic activity, strongly demonstrating the effectiveness of ProUK-RBC.

[0287] 11.2 Evaluation of Thromboprophylaxis in a Rat Model of Cerebral Atherosclerosis Induced by Fibrin Occlusion (MCAO)

[0288] MCAO is a classic model of cerebral thrombosis in animals. Traditional suture embolization methods are not suitable for preventive models, so we used microthrombotic particle injection to create a cerebral thrombosis model. Microthrombotic particles cannot directly block blood vessels, but once these particles enter the body, they will form larger thrombi, blocking blood vessels and causing stroke. The specific steps are as follows:

[0289] Whole blood was collected from SD rats and washed to remove other components to obtain washed red blood cells. The red blood cells were prepared as ProUK-RBCs using the method of Example 8 and used in the experiment 11.2. Fresh plasma was also obtained from SD rats.

[0290] 10 mg / mL fibrinogen, 20 mM CaCl2, and 0.2 U / mL thrombin were added to fresh SD rat plasma (Bdeir, K. et al. Urokinase mediates fibrinolysis in the pulmonary microvasculature. Blood 96, 1820-1826 (2000).). After incubation at room temperature for 1 hour, the clot was incubated at 4°C overnight to promote contraction. The blood clot was processed using a homogenizer to prepare microthrombi with a diameter of 1.5-3.5 μm, which were dissolved in PBS solution to obtain a microthrombosis solution (1.4×10 6 particles / 100 μL).

[0291] Get male SD rats (200-350g, 2 groups, 10 in each group, 6-8 weeks old) and use 2% isoflurane anesthesia animals, with the mask with the mixture of 70% N2O and 30% O2 containing 1.5% isoflurane, keep rectal temperature at 37 ± 0.5 ℃, expose the left common carotid artery, external carotid artery and internal carotid artery through midline incision. Use silk thread to suture the distal end of the internal carotid artery, temporarily ligate the left internal carotid artery, external carotid artery and pterygopalatine artery (PPA). A section of inner diameter vinyl catheter is connected to a syringe, introduced into the inner catheter, and fixed with silk thread. Remove the ligature around the internal carotid artery and advance the catheter into the internal carotid artery cavity.

[0292] First, the rats in the two groups were injected with ProUK-RBC (1×10 7 / μl) and unlabeled RBC (1×10 7 After administration, microthrombosis solution (100 μL; 1.4×10 6 The patients were injected with a bolus of 5000 particles, the catheters were removed, and the ligatures (which blocked downstream vessels, resulting in >80% cessation of blood flow and extensive ipsilateral cerebral infarction) were removed from the carotid artery and PPA. Survival was calculated 24 hours later.

[0293] We evaluated prophylactic ProUK-RBC intervention in SD rats by administering ProUK-RBC prior to a lethal dose of microthrombi. ProUK-RBC reduced 24-hour mortality by 40% (Figure 29). Six hours after treatment, we analyzed cerebral infarct size in both groups of animals by sectioning the rat brains with TTC (2,3,5-triphenyltetrazolium chloride) staining to measure ischemic area. ProUK-RBC effectively reduced infarct size (Figures 30 and 31).

[0294] 11.3 Prevention of Intrinsic Thrombosis in a Deep Vein Thrombosis Model

[0295] FeCl3 can induce vascular endothelial cell damage and simulate endogenous thrombosis.

[0296] C57 / BL6J mice were injected with ProUK-RBC (ProUK-RBC 200ul 1×10 7 / ul), free ProUK (with 0.2ug ProUK-cys and 200ul 1×10 7 / ul unlabeled RBC) and RBC (200ul 1×10 7 5 minutes later, femoral vein thrombosis was induced with FeCl3, and the specific steps were as follows:

[0297] Mice were anesthetized with an intraperitoneal (IP) injection of tribromoethanol. For the femoral vein thrombosis model, a midline skin incision was made and the vessel was gently exposed by blunt dissection. Filter paper soaked in 20% FeCl3 was placed on the anterior surface of the vein for 90 seconds.

[0298] Thrombi were observed 3 minutes and 18 hours after the above induction ( FIG. 32 ).

[0299] The results showed that after FeCl3 induction, both the RBC and free ProUK groups developed deep thrombi, while the ProUK-RBC group showed no obvious thrombi. Eighteen hours later, significant thrombi were still present in the RBC and free ProUK groups, while the ProUK-RBC group had mild, but inconspicuous, perivascular thrombi (Figure 33, one mouse per group). ProUK-RBCs also effectively prevented thrombosis in venous thrombosis caused by endothelial damage.

Claims

1. A drug delivery system comprising a modified blood cell coupled to a protein or peptide via a linker, wherein the linker comprises maleimide-PEG-phospholipid, the protein or peptide is treated to have an exposed or free thiol group for Michael addition reaction with maleimide to form a stable carbon-sulfur bond, and the phospholipid is incorporated into the blood cell membrane.

2. the drug delivery system of claim 1, wherein the hemocyte is selected from the hemocyte deriving from animal blood, such as erythrocyte, lymphocyte, T cell, B cell, monocyte, NK cell, dendritic cell or megakaryocyte, preferably, the animal is mammal such as human.

3. The drug delivery system of claim 2, wherein the blood cells are red blood cells, such as natural red blood cells, such as natural adult red blood cells.

4. The drug delivery system of any one of claims 1-3, wherein the PEG molecular weight is 400-5000, such as 2000-5000, such as PEG2000, PEG2500, PEG3000, PEG3500, PEG4000, PEG4500 or PEG5000.

5. the drug delivery system of any one of claim 1-4, wherein phospholipid is PE series phospholipid, for example 1,2-distearoyl-sn-glyceryl-3-phosphatidylethanolamine (DSPE), 1,2-diacryloyl-sn-glyceryl-3-phosphatidylethanolamine (DEPE), 1,2-dioleoyl-sn-glyceryl-3-phosphatidylethanolamine (DOPE), 1,2-dimyristoyl-sn-glyceryl-3-phosphatidylethanolamine (DMPE) and 1,2-dipalmitoyl-sn-glyceryl-3-phosphatidylethanolamine (DPPE), preferably, the phospholipid is DSPE or DOPE.

6. The drug delivery system of any one of claims 1 to 4, wherein the linker is maleimide-PEG-DSPE, for example having the chemical formula shown below:

7. The drug delivery system of any one of claims 1-6, wherein the processing of the protein or peptide comprises (1) treating the protein or peptide with a thiol reducing agent (e.g., TCEP) to make it contain a cysteine ​​residue containing a free thiol group; or (2) by inserting one or more cysteines at a position that does not affect the outcome or function of the protein or peptide, thereby making it contain a cysteine ​​residue containing a free thiol group, for example, by inserting one cysteine ​​at the C-terminus (such as after the most C-terminal amino acid) or the N-terminus (such as before the most N-terminal amino acid) of the protein or peptide. 8 . The drug delivery system according to claim 1 , wherein the protein is a protein or peptide for labeling, and a protein or peptide for prevention or treatment.

9. the drug delivery system of claim 8, wherein the protein or peptide used for labeling are selected from fluorescent protein (for example green fluorescent protein, as eGFP), horseradish peroxidase, bovine serum albumin, hemocyanin, chicken ovalbumin, antibody, ox IgG, mouse IgG, goat IgG, rabbit IgG, alkaline phosphatase, acid phosphatase, glucose oxidase or beta-galactosidase.

10. The drug delivery system of claim 9, wherein the protein or peptide for prevention or treatment is any protein that can be used as a drug to prevent or treat a disease, including, for example, cytokines or growth factors or their receptors, hormones, enzymes, antibodies, etc.

11. The drug delivery system of claim 10, wherein the antibody is an antibody to an immune checkpoint molecule or an antigen-binding fragment thereof, such as an antibody that specifically binds to PD-1, PD-L1 or PD-L2, such as a human antibody, a humanized antibody or a chimeric antibody.

12. The drug delivery system of claim 11, wherein the antibody that specifically binds to PD-1 is selected from pembrolizumab, carrelizumab, tislelizumab, toripalimab, sintilimab or nivolumab or an antigen-binding fragment thereof, or the antibody that specifically binds to PD-1 comprises 3 CDRs of the heavy chain variable region and 3 CDRs of the light chain variable region of an antibody selected from pembrolizumab, carrelizumab, tislelizumab, toripalimab, sintilimab or nivolumab.

13. The drug delivery system according to claim 11 or 12, wherein the thiol groups on the cysteine ​​residues of the antibody are exposed by incubation with a thiol reducing agent (such as TCEP).

14. The drug delivery system according to any one of claims 11 to 13, for use in treating a tumor, such as a solid tumor, such as a solid cancer.

15. The drug delivery system of claim 8, wherein the protein is a protease, such as a serine protease, such as prourokinase, such as human prourokinase, such as the human prourokinase (1) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 10 or 13; (2) comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 10 or 13; (3) encoded by the nucleotide sequence shown in SEQ ID NO: 11; or (4) Encoded by a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identical to the nucleotide sequence shown in SEQ ID NO:

11.

16. The drug delivery system of claim 15, wherein the protein comprises a cysteine ​​residue containing a free thiol group by inserting a cysteine ​​before the most N-terminal amino acid or after the most C-terminal amino acid, for example, the human prourokinase inserts a cysteine ​​at its C-terminus, the prourokinase comprising the inserted cysteine (1) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 8 or 12; (2) comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to the amino acid sequence shown in SEQ ID NO: 8 or 12, which has a cysteine ​​at the extreme C-terminus; (3) encoded by the nucleotide sequence shown in SEQ ID NO: 9; or (4) Encoded by a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identical to the nucleotide sequence shown in SEQ ID NO: 9, which has a cysteine ​​at its C-terminus.

17. The drug delivery system according to claim 15 or 16, which is used for preventing or treating thrombosis-related diseases, for example, stroke such as ischemic stroke, myocardial infarction (eg, acute myocardial infarction), and venous thrombosis such as deep vein thrombosis.

18. The drug delivery system of claim 8, wherein the protein is a functional metabolic enzyme, such as urate oxidase, such as urate oxidase from Aspergillus flavus, such as the urate oxidase (1) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 5 or 6; or (2) An amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence shown in SEQ ID NO: 5 or 6.

19. The drug delivery system of claim 18, wherein the protein comprises a cysteine ​​residue containing a free thiol group by inserting a cysteine ​​before the most N-terminal amino acid or after the most C-terminal amino acid, for example, the uricase has a cysteine ​​inserted at its C-terminus, the uricase comprising the inserted cysteine (1) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3 or 7; (2) comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to the amino acid sequence shown in SEQ ID NO: 3 or 7, which has a cysteine ​​at the extreme C-terminus; (3) encoded by the nucleotide sequence shown in SEQ ID NO: 4; or (4) Encoded by a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identical to the nucleotide sequence shown in SEQ ID NO: 4, which has a cysteine ​​at its C-terminus.

20. The drug delivery system according to claim 18 or 19, which is used for preventing or treating metabolic diseases such as chronic hyperuricemia, such as gout, such as refractory gout (gout resistant to other conventional therapies).

21. A method for preparing the drug delivery system of any one of claims 1-20, comprising (1) providing a linker comprising maleimide and phospholipid; (2) providing a protein or peptide comprising a free thiol group; (3) incubating the linker described in (1) with blood cells, thereby incorporating the phospholipids therein into the cell membrane of the cells; (4) incubating the cells prepared in step (3) with the thiol groups of the protein in step (2) under conditions suitable for a Michael addition reaction; or (3') incubating the linker in (1) and the thiol group of the protein in (2) under conditions suitable for Michael addition reaction to obtain a protein coupled to a phospholipid; (4') incubating the protein obtained in step (3') with blood cells, thereby incorporating the phospholipids therein into the cell membrane of the cells; and (5) Obtain a drug delivery system comprising proteins and blood cells coupled via a linker.

22. A pharmaceutical composition or formulation comprising the drug delivery system according to any one of claims 1 to 20 or the drug delivery system prepared according to claim 21, and pharmaceutically acceptable excipients.

23. The pharmaceutical composition or formulation of claim 22, wherein the formulation is a human blood preparation, such as an autologous or allogeneic human blood preparation.

24. The pharmaceutical composition or formulation of claim 22 or 23, wherein the human blood preparation is a human leukopenic blood preparation.

25. The pharmaceutical composition or formulation of any one of claims 22-24, wherein the human blood preparation comprises a drug delivery system of 1 μg / mL-1000 μg / mL, for example, a drug delivery system of 1-10 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, 45 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, 350 μg / mL, 400 μg / mL, 450 μg / mL, or 500 μg / mL or more.

26. Use of the drug delivery system of any one of claims 11 to 13 in the preparation of a drug or human blood preparation for treating a tumor, such as a cancer, such as a solid tumor, such as a tumor or cancer having elevated protein levels and / or nucleic acid levels (e.g., elevated expression) of PD-1, PD-L1 and / or PD-L2 in a subject, such as a gastrointestinal tumor, such as colon cancer or colorectal cancer; or lung cancer, pancreatic cancer or breast cancer.

27. Use of the drug delivery system of claim 15 or 16 in the preparation of a medicament or a human blood preparation for antithrombotic or thrombolytic treatment in a subject.

28. Use of the drug delivery system of claim 15 or 16 in the preparation of a medicament or a human blood preparation for preventing or treating a thrombosis-related disease in a subject.

29. The use according to claim 28, wherein the thrombosis-related disease is selected from the group consisting of stroke such as ischemic stroke, myocardial infarction (eg acute myocardial infarction) and venous thrombosis such as deep vein thrombosis.

30. The purposes of the drug delivery system of claim 18 or 19 in preparing medicine or human blood preparation, wherein the medicine or human blood preparation are used for treating metabolism-related diseases in a subject, such as chronic hyperuricemia, such as gout, such as refractory gout (gout tolerated to other conventional therapies).

31. The use according to any one of claims 26 to 30, wherein the blood cells in the drug delivery system are from the subject to be treated or a healthy subject.

32. The use according to any one of claims 26 to 31, wherein the medicament or human blood preparation is administered in combination with one or more treatment modalities or other therapeutic agents.

33. The use according to claim 32, wherein the treatment modality is surgical treatment.

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