WO2024017192A1 - 包含血细胞的药物递送系统 - Google Patents

包含血细胞的药物递送系统 Download PDF

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WO2024017192A1
WO2024017192A1 PCT/CN2023/107705 CN2023107705W WO2024017192A1 WO 2024017192 A1 WO2024017192 A1 WO 2024017192A1 CN 2023107705 W CN2023107705 W CN 2023107705W WO 2024017192 A1 WO2024017192 A1 WO 2024017192A1
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drug delivery
delivery system
protein
blood cells
cells
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PCT/CN2023/107705
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English (en)
French (fr)
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高晓飞
黄彦杰
俞慧飞
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西湖生物医药科技(上海)有限公司
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Publication of WO2024017192A1 publication Critical patent/WO2024017192A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/18—Erythrocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/49—Urokinase; Tissue plasminogen activator
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/44—Antibodies bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors

Definitions

  • the present invention provides a drug delivery system comprising blood cells, and a novel method of modifying red blood cell membranes for preparing the same.
  • the present invention also relates to methods of treating diseases using the drug delivery system.
  • Red blood cells are the most common cell type in the human body and have been extensively studied for more than 30 years as an ideal in vivo drug delivery system due to their unique biological properties: (i) wide systemic circulation range; (ii) As a biological material, it has good biocompatibility and long in vivo survival time; (iii) large surface area to volume ratio; (iv) no nucleus, mitochondria and other organelles.
  • RBCs have been developed as drug delivery vehicles through direct encapsulation, non-covalent attachment of exogenous peptides, or installation of proteins through fusion with antibodies specific for RBC surface proteins.
  • modified RBCs have proven to have limitations for in vivo applications. For example, encapsulation can disrupt cell membranes, thereby affecting the in vivo lifespan of engineered cells.
  • the non-covalent attachment of polymer particles to RBCs is easily dissociated and the payload will be quickly degraded in the body.
  • Phospholipids are amphipathic.
  • the hydrophilic end usually contains a negatively charged phosphate group, while the hydrophobic end usually consists of two "tails," which are long fatty acid residues.
  • tails which are long fatty acid residues.
  • phospholipids are driven by hydrophobic interactions, leading to the accumulation of fatty acid tails that minimize interactions with water molecules.
  • the result is usually a phospholipid bilayer: a membrane consisting of two layers of phospholipid molecules oriented in opposite directions, with the heads on both sides exposed to the liquid and the tails pointing toward the membrane. This is a major structural motif in all cell membranes and certain other biological structures.
  • phospholipids In biological membranes, phospholipids often appear in bilayers (such as cell membranes) together 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.
  • the maleimide-mediated method is one of the most commonly used methods in bioconjugation. It is used in a variety of methods due to its extremely fast reaction rate and high selectivity for cysteine residues in proteins.
  • a variety of maleimide heterobifunctional reagents are used to prepare targeted therapeutics and study their biological context in proteins, protein-based microarrays, or protein assemblies in protein immobilization.
  • emerging promising antibody-drug conjugates for targeted drug therapy are composed of three main components: a monoclonal antibody, a cytotoxic drug, and a linker molecule that often contains a maleimide group.
  • the imide group binds drugs and antibodies.
  • Maleimides attached to polyethylene glycol chains are often used as flexible linker molecules to attach proteins to surfaces.
  • the double bond easily reacts with the sulfhydryl group on cysteine to form a stable carbon-sulfur bond.
  • Attaching the other end of the polyethylene chain to a bead or solid support can easily separate the protein from other molecules in solution, but only if those molecules do not have sulfhydryl groups.
  • the present invention discovered for the first time that using cell membrane component phospholipid combined with maleimide and polyethylene glycol as a linker for red blood cells can be used to obtain an efficient and stable red blood cell-based drug delivery system.
  • FIG 1a and Figure 1b show the schematic diagram of method 1
  • FIG. 1 Figure 2a and Figure 2b show the schematic diagram of method 2;
  • Figure 3 shows a linker containing a maleimide group-PEG-phospholipid
  • Figure 4 shows a schematic diagram of thiol-based linker coupling of proteins
  • Figure 5 shows eGFP signal intensity on the surface of labeled and unlabeled red blood cells
  • Figure 6 shows the eGFP signal intensity on the cell surface after natural red blood cells were labeled with different concentrations of eGFP-Cys-MAL-PEG2000-DSPE in vitro;
  • Figure 7 shows the eGFP signal intensity on the surface of Cell Far red-stained red blood cells
  • Figure 8a shows the percentage of Far red-positive erythrocytes in mouse blood over time for control erythrocytes (without eGFP-cys labeling) and eGFP-cys-labeled erythrocytes;
  • Figure 8b shows the number of GFP-positive red blood cells as a percentage of Cell Far Red-positive cells in control red blood cells (not labeled by eGFP-cys) and eGFP-cys-labeled red blood cells over time;
  • Figure 9 shows the anti-PD-1 antibody signal on the surface of red blood cells labeled with anti-PD-1 antibody and without anti-PD-1 antibody labeling
  • Figure 10 shows the signal intensity of PD-1 antibodies.
  • Figure 11 shows the UOX signal intensity of UOX-Cys-Mal-PEG-DSPE labeled red blood cells in 2 recipient mice;
  • Figure 12 shows the percentage of Cell Far red positive red blood cells in the blood over time in two recipient mice (Figure 12a) and the percentage of UOX positive red blood cells as a percentage of Cell Far Red positive cells over time ( Figure 12b) ;
  • Figure 13 shows the signal intensity of eGFP on the surface of different types of blood cells detected by flow cytometry
  • Figure 14 shows the eGFP signal intensity and FAR RED signal intensity of eGFP-Cys-MAL-PEG-DOPE labeled red blood cells and unlabeled red blood cells;
  • Figure 15 shows the % Far red positive red blood cell number ( Figure 15a) and the %UOX positive red blood cell number over time ( Figure 15b) of eGFP-Cys-MAL-PEG-DOPE labeled red blood cells and unlabeled red blood cells;
  • Figure 16 shows the signal intensity of eGFP in red blood cells labeled with Mal-PEG2000-phospholipid (DPPE, DOPE, DSPE or DMPE);
  • Figure 17 shows the signal intensity of eGFP obtained by incubating different concentrations of Mal-PEG2000-phospholipid (DOPE or DSPE) with red blood cells for 10 minutes;
  • DOPE Mal-PEG2000-phospholipid
  • Figure 18 shows a schematic diagram of using Mas-PEG-DSPE as a linker to couple prourokinase and red blood cells;
  • Figure 19 shows the signal intensity of prourokinase on the surface of red blood cells labeled with ProUK-cys and without ProUK-cys labeling
  • Figure 20 shows the percentage of input red blood cells with a ProUK signal in the circulation as a function of time (top graph), and the signal intensities of Far Red and ProUK in blood samples obtained at different blood collection times;
  • Figure 21 shows the Far Red signal and ProUK signal intensity on red blood cells labeled with ProUK-cys and without ProUK-cys labeling
  • 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));
  • Figure 23 shows the planned time of blood collection from the orbit in the ProUK-RBC administration group and the free ProUK administration group
  • Figure 24 shows the % of ProUK content in whole blood over time by ELISA in different dosage groups
  • Figure 25 shows the thrombolysis % of the ProUK-RBC administration group and the free ProUK administration group and the RBC control group over time
  • Figure 26 shows the thrombolysis photos of the ProUK-RBC administration group, the free ProUK administration group and the RBC control group over time
  • Figure 27 shows a schematic diagram of the operation of the tail bleeding time measurement method
  • Figure 28 shows tail bleeding times in different dosing groups at low dose levels
  • Figure 29 shows the 24-hour lethality rate of the ProUK-RBC administration group and the unlabeled RBC administration group
  • FIG. 30 shows the sections of the ProUK-RBC administration group (right) and the unlabeled RBC administration group (left) stained with TTC (2,3,5-triphenyltetrazolium chloride) 6 hours after treatment.
  • Figure 31 shows the percentage of cerebral infarct area in the ProUK-RBC administration group and the unlabeled RBC administration group 6 hours after treatment;
  • Figure 32 shows a schematic diagram of the operation of the deep vein thrombosis model
  • Figure 33 shows photos of thrombosis in one mouse in each of the unlabeled RBC administration group, free ProUK administration group and ProUK-RBC administration group.
  • the term “comprises” or “includes” means the inclusion of the stated element, integer or step, but not the exclusion of any other element, integer or step.
  • the term “comprises” or “includes” is used herein, it also encompasses a combination of the stated elements, integers, or steps unless otherwise indicated.
  • reference is made to an antibody variable region that "comprises” a particular sequence it is also intended to encompass antibody variable regions that consist of that particular sequence.
  • drug delivery system refers to any pharmaceutical preparation or preparation capable of delivering the drug contained therein to a target, including cellular preparations, such as red blood cell-based cellular preparations or other blood cell-based cellular preparations; or blood preparations, For example, preparations prepared from human blood.
  • prourokinase is also called “single-chain urokinase-type plasminogen activator”. It is generally a single-chain polypeptide composed of 411 amino acid residues with a molecular weight of about 49KD. It belongs to the serine protease class and contains 12 pairs of disulfide bonds. It can be divided into four domains according to its structure and function, namely epidermal growth factor domain, Kringle domain, serine protease domain and connecting region. Prourokinase is a relatively inactive precursor that must be converted to urokinase before it can become active in the body. However, it shows selectivity for clots by binding to fibrin prior to activation.
  • the prourokinase of the present invention refers to natural human prourokinase, or a natural variant thereof, which may or may not have a signal peptide sequence (for example, MRALLARLLLCVLVVSDSKG (SEQ ID NO: 14)) or a tag (eg His 6 tag).
  • a signal peptide sequence for example, MRALLARLLLCVLVVSDSKG (SEQ ID NO: 14)
  • a tag eg His 6 tag
  • the prourokinase of the present invention is human prourokinase, for example, it comprises or consists of the amino acid sequence shown in SEQ ID NO: 10 or 13. In some embodiments, the prourokinase of the present invention comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence shown in SEQ ID NO: 10 or 13. %, 97%, 98% or 99% identity of the amino acid sequence, and can be activated in vivo to have urokinase activity.
  • thrombosis-related disease refers to any disease associated with thrombosis, including, but not limited to, ischemic stroke, acute myocardial infarction, and deep vein thrombosis.
  • anti-tumor effect refers to a biological effect that can be demonstrated by a variety of means, including but not limited to, for example, reduction in tumor volume, reduction in tumor cell number, reduction in tumor cell proliferation, or reduction in tumor cell survival.
  • tumor and cancer are used interchangeably herein to encompass both solid tumors and hematological tumors.
  • cancer refers to or describes a physiological disorder in mammals typically characterized by unregulated cell growth.
  • cancers suitable for treatment by the antibodies of the invention include lung cancer, pancreatic cancer, breast cancer, gastrointestinal tumors such as colon cancer, colorectal cancer, or rectal cancer, and the like, including metastatic forms of those cancers .
  • neoplastic refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer and tumor as used herein are not mutually exclusive.
  • Red blood cells or “RBCs” are the most common type of blood cells and are the primary carriers of oxygen in vertebrates through the bloodstream and through the circulatory system to body tissues.
  • the cytoplasm of red blood cells is rich in hemoglobin, an iron-containing biomolecule that binds oxygen and gives the cells and blood their red color.
  • Cell membranes are composed of proteins and lipids, a structure that provides properties necessary for physiological cell function, such as deformability and stability, while allowing passage through the circulatory system, particularly the capillary network.
  • mature red blood cells are flexible, oval, biconcave discs. They lack a nucleus and most organelles to have maximum space for hemoglobin; they can be thought of as bags for hemoglobin, with the plasma membrane as the bag.
  • An adult produces approximately 2.4 million new red blood cells every second. Approximately 84% of the cells in the human body are 20–30 trillion red blood cells. Nearly half of the blood volume (40% to 45%) is red blood cells.
  • “Adult natural red blood cells” refers to mature natural red blood cells directly isolated from the blood of animals, especially humans (eg, adults or children).
  • Blood preparations or "blood products” as used herein can be used interchangeably and refer to products prepared from (human) blood and used for medical purposes.
  • Blood preparations include whole blood preparations, blood component preparations, plasma preparations or leukoreduced blood preparations.
  • an effective amount refers to that amount or dosage of the drug delivery system or composition or formulation or preparation or combination or combination product of the invention, which after administration to the patient in single or multiple doses, in a patient in need of treatment or prophylaxis produce the desired effect.
  • a “therapeutically effective amount” means an amount effective to achieve the desired therapeutic result, at the required doses and for the required period of time.
  • a therapeutically effective amount is also an amount in which any toxic or detrimental effects of the drug delivery system or composition or formulation or preparation or combination or combination product are outweighed by the therapeutically beneficial effects.
  • a “therapeutically effective amount” preferably inhibits a measurable parameter (eg, thrombus volume, or, for example, tumor volume) by at least about 30%, even more preferably at least about 40%, 45%, 50%, 55%, relative to an untreated subject , 60%, 65%, 70%, 75%, 80%, 85%, 90% or even 100%.
  • prophylactically effective amount means an amount effective to achieve the desired prophylactic result, at the required dosage and for the required period of time. Generally, the prophylactically effective amount will be less than the therapeutically effective amount because the prophylactic dose is administered in the subject before or at an earlier stage of the disease.
  • “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.
  • pharmaceutically acceptable excipient refers to diluents, adjuvants (such as Freund's adjuvant (complete and incomplete)), excipients, carriers or stabilizers, etc., which are administered with the active substance.
  • composition refers to a composition that is in a form that allows the active ingredients contained therein to be biologically active and does not contain additional ingredients that would be unacceptable toxicity to the subject to whom the composition is administered. ingredients.
  • drug combination refers to non-fixed combination products or fixed combination products, including but not limited to pharmaceutical kits and pharmaceutical compositions.
  • non-fixed combination means that the active ingredients (e.g., (i) the drug delivery system of the invention and (ii) other therapeutic agents) are administered in separate entities simultaneously, without specific time limits or at the same or different time intervals, are administered sequentially to the patient, wherein such administration provides prophylactically or therapeutically effective levels of two or more active ingredients in the patient.
  • the drug delivery systems of the invention and other therapeutic agents used in pharmaceutical combinations are administered at levels that do not exceed those used individually.
  • fixed combination means that two or more active ingredients are administered simultaneously to a patient in the form of a single entity.
  • the dosages and/or time intervals of two or more active ingredients are preferably selected so that the combined use of the parts produces an effect in treating the disease or condition that is greater than that achieved by either ingredient alone.
  • Each component may be in a separate formulation form, and the formulation forms may be the same or different.
  • combination therapy refers to the administration of two or more therapeutic agents or treatment modalities (eg, radiation therapy or surgery, such as stent surgery or bypass surgery) to treat a disease described herein.
  • Such administration involves co-administration of the therapeutic agents in a substantially simultaneous manner, for example, in a single capsule having a fixed ratio of the active ingredients.
  • such administration involves co-administration of the individual active ingredients in multiple or separate containers (eg tablets, capsules, powders and liquids). Powders and/or liquids can be reconstituted or diluted to the desired dosage prior to administration.
  • such administration includes the administration of each type of therapeutic agent or treatment modality at approximately the same time or at different times in a sequential manner. In either case, the treatment regimen will provide a beneficial effect of the combination therapy in treating the disorder or condition described herein.
  • the term "therapeutic agent” encompasses any active agent that exerts a therapeutic effect, such as any substance that is 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 immune agonists); also for example any substance effective in preventing or treating thrombosis-related diseases or metabolism-related diseases, such as drugs for anti-thrombosis or thrombolysis, such as urokinase, Tissue plasminogen activator, streptokinase, etc.
  • the above-mentioned therapeutic agents also encompass precursors of active substances that exert a therapeutic effect, which are subsequently converted in the body into active substances, for example prodrugs, such as prourokinase.
  • cytotoxic agent refers to a substance that inhibits or prevents cellular function and/or causes cell death or destruction.
  • Cyhemotherapeutic agents include chemical compounds useful in treating cancer or immune system diseases.
  • small molecule drugs refers to low molecular weight compounds capable of modulating biological processes.
  • Small molecules are defined as molecules with a molecular weight of less than 10 kD, usually less than 2 kD and preferably less than 1 kD. 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.
  • immune modulators As therapeutic agents, small molecules can be more permeable to cells, less susceptible to degradation, and less likely to elicit an immune response than larger molecules.
  • the term "immunomodulator” as used herein refers to natural or synthetic agents or drugs that inhibit or modulate immune responses.
  • the immune response can be a humoral response or a cellular response.
  • Immunomodulators include immunosuppressants or immune agonists.
  • immune modulators of the invention include immune checkpoint inhibitors or immune checkpoint agonists.
  • treating means slowing, interrupting, retarding, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.
  • prevention includes the inhibition of the onset or progression of a disease or condition or symptoms of a particular disease or condition.
  • subjects with a family history of cardiovascular disease are candidates for a preventive regimen.
  • prophylaxis generally refers to administration of drugs to prevent recurrence of a blood clot (e.g., venous thrombosis) after initial treatment has resolved.
  • tissue or cell sample refers to a collection of cells or fluid obtained from a patient or subject.
  • the source of the tissue or cell sample may be solid tissue, like from fresh, frozen and/or preserved organ or tissue samples or biopsy or aspiration samples; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid) ), peritoneal fluid (ascites), or interstitial fluid; cells from a subject at any time during pregnancy or development.
  • Tissue samples may contain compounds that are not naturally associated with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.
  • the present invention provides a drug delivery system comprising modified blood cells coupled to proteins or peptides through linkers.
  • the linker is a linker containing a maleimide group and a phospholipid.
  • the linker is a maleimide group-phospholipid.
  • phospholipid refers to a class of lipids whose molecules have a hydrophilic "head” containing a phosphate group, and two hydrophobic "tails” derived from fatty acids, linked by glycerol molecules.
  • the phosphate group can be modified with simple organic molecules such as choline, ethanolamine, or serine.
  • the phospholipids can be PC series, such as DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC, and DEPC; PG series, such as DMPG, DPPG, DSPG, and POPG; PA series, such as DMPA, DPPA, DSPA etc.; PE series, such as 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); and PS series, such as DOPS.
  • DSPE 1,2-dist
  • the phospholipid can be a PE series, such as 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).
  • DSPE 1,2-distearoyl-sn-glyceryl-3-phosphatidylethanolamine
  • DEPE 1,2-diacryloyl-sn-glyceryl- 3-Phosphatidylethanolamine
  • DOPE 1,2-dioleoyl-sn-glyceryl-3-phosphatidylethanol
  • the maleimide group in the linker and the phospholipid are also connected through PEG.
  • the linker is maleimide-PEG-phospholipid.
  • the PEG has a molecular weight of, for example, 400-5000, such as 2000-5000, such as PEG2000, PEG2500, PEG3000, PEG3500, PEG4000, PEG4500 or PEG5000.
  • the PEG linker may have a free carboxyl group at the carboxyl terminus and a free amino group at the amino terminus in order to form a peptide bond with the maleimide group and the phospholipid group, respectively.
  • the PEG used to link the maleimide group to the phospholipid may comprise the structure NH2 - PEG-COOH, such as NH2-( CH2CH2O ) n -COOH.
  • the phospholipid is linked to a maleimide at the amino group of its hydrophilic "head” containing a phosphate group, for example, with or without PEG.
  • the hydrophobic "tail” of the phospholipid is incorporated into the cell membrane of the blood cells and thereby serves to connect the blood cells.
  • the amino group of the hydrophilic "head” containing the phosphate group of the phospholipid is linked to the maleimide via PEG or directly, while its hydrophobic "tail” is incorporated into the cell membrane of the blood cell to Connect blood cells.
  • the phospholipid is DSPE.
  • the linker has a chemical formula as shown in Figure 3.
  • Blood cells suitable for use in the drug delivery system of the invention can be any cell derived from the blood of an animal, including, for example, red blood cells, lymphocytes, T cells, B cells, monocytes, NK cells, dendritic cells or megakaryocytes.
  • the animal is a mammal, such as a human.
  • the blood cells are cells from the immune system, such as lymphocytes (eg, T cells or NK cells), or dendritic cells.
  • the cells are native cells.
  • the cells are adult cells. In some embodiments, the cells are native adult cells.
  • the blood cells used to prepare the present invention are blood cells isolated directly from blood, such as human (eg, adult or pediatric) blood.
  • the cells are red blood cells, such as human red blood cells, such as native human red blood cells, such as native adult red blood cells, such as mature red blood cells isolated from human (eg, adult or pediatric) blood.
  • human red blood cells such as native human red blood cells, such as native adult red blood cells, such as mature red blood cells isolated from human (eg, adult or pediatric) blood.
  • the blood cells are autologous blood cells, ie, blood cells obtained from the blood of the subject to be treated.
  • the blood cells are allogeneic isolated blood cells, ie, blood cells isolated from an individual different from the subject to be treated (eg, a healthy individual).
  • proteins or polypeptides suitable for use in the drug delivery systems of the invention include proteins or peptides for labeling, or proteins or peptides for prevention or treatment.
  • protein or peptide for labeling refers to any protein or peptide that is detectable for use in the detection of a reagent to which it is conjugated or conjugated (e.g., blood cells), which may itself be detectable (e.g., fluorescent protein label ) or, in the case of enzymatic labeling, can catalyze a detectable chemical change in a substrate compound or composition.
  • Proteins or peptides used as markers 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, sheep IgG, rabbit IgG, alkaline phosphatase, acid phosphatase, glucose oxidase or beta-galactosidase.
  • fluorescent proteins e.g., green fluorescent protein, such as eGFP
  • Protein or peptide for prevention or treatment refers to any protein that can be used as a drug to prevent or treat disease, including, for example, cytokines or growth factors or their receptors, hormones, enzymes, antibodies, etc. .
  • the protein is an enzyme, such as a functional metabolic enzyme or a therapeutic enzyme, such as an enzyme that functions in metabolism or other physiological processes in mammals.
  • the protein is an enzyme that functions in carbohydrate metabolism, amino acid metabolism, organic acid metabolism, porphyrin metabolism, purine or pyrimidine metabolism, and/or lysosomal storage.
  • proteins contained in drug delivery systems suitable for use in the present invention are functional metabolic enzymes.
  • Exemplary enzymes include urate oxidase, such as from Aspergillus flavus.
  • the urate oxidase 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., His tag), e.g., at its N-terminus or at its C terminus.
  • the urate oxidase comprises or consists of the amino acid sequence shown in SEQ ID NO: 5 or 6.
  • the urate oxidase comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher of the amino acid sequence shown in SEQ ID NO: 5 or 6 The identity of the amino acid sequence.
  • the protein suitable for inclusion in the drug delivery system of the present invention is a protease, such as a serine protease, such as prourokinase, such as human prourokinase.
  • the human prourokinase comprises a signal peptide (e.g., comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 14) and/or a tag (e.g., a His tag), e.g., at its N-terminus or at its C terminus.
  • the prourokinase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 10 or 13.
  • the prourokinase comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher with the amino acid sequence set forth in SEQ ID NO: 10 or 13 The identity of the amino acid sequence.
  • the prourokinase is encoded by the nucleotide sequence set forth in SEQ ID NO: 11.
  • the prourokinase protein has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher similarity to the nucleotide sequence set forth in SEQ ID NO: 11 Identity is encoded by the nucleotide sequence.
  • the peptides suitable for inclusion in the drug delivery systems of the present invention are labeled peptides or various functional peptides.
  • proteins suitable for inclusion in drug delivery systems of the present invention are antibodies or antigen-binding fragments thereof.
  • antibodies suitable for use in the invention are antibodies in the form of IgG1 or in the form of IgG2 or in the form of IgG3 or in the form of IgG4.
  • antibodies suitable for use in the invention are monoclonal antibodies.
  • antibodies suitable for use in the invention are humanized antibodies.
  • antibodies suitable for use in the invention are human antibodies.
  • antibodies suitable for use in the invention are chimeric antibodies. In one embodiment, antibodies suitable for use in the invention are full-length antibodies.
  • antigen-binding fragments suitable for use in the antibodies of the invention are selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single chain antibodies (eg scFv), (Fab')2, single chain antibodies Domain antibodies such as VHH, dAb (domain antibody) or linear antibodies or half-antibodies.
  • Exemplary antibodies include antibodies to immune checkpoint molecules, such as antibodies that specifically bind PD-1, PD-L1, or PD-L2, such as PD-1 antibodies.
  • 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 PA Brolizumab, camrelizumab, tislelizumab, toripalimab, sintilimab or nivolumab or antigen-binding fragments thereof (see for example CN108473977B, CN104250302B, CN105531288B , CN105026428B, WO2008156712A1 or WO2006121168A1).
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises pembrolizumab, camrelizumab, tislelizumab, toripalimab, sintilizumab
  • One or more CDRs of anti- or nivolumab or its antigen-binding fragment preferably 3 CDRs, namely HCDR1, HCDR2H and HCDR3; or LCDR1, LCDR2 and LCDR3, more preferably 6 CDRs, namely HCDR1, HCDR2, HCDR3 , LCDR1, LCDR2 and LCDR3
  • the anti-PD-1 antibodies or antigen-binding fragments thereof of the invention comprise pembrolizumab, camrelizumab, tislelizumab, toripalimab, cinti
  • the anti-PD-1 antibodies or antigen-binding fragments thereof of the invention comprise pembrolizumab, camrelizumab, tislelizumab, toripalimab, cinti Heavy chain variable region and light chain variable region of lizumab or nivolumab.
  • the anti-PD-1 antibodies or antigen-binding fragments thereof of the invention comprise pembrolizumab, camrelizumab, tislelizumab, toripalimab, cinti Heavy and light chains of lizumab or nivolumab.
  • 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 such a protein.
  • proteins or peptides suitable for inclusion in drug delivery systems of the present invention are proteins or peptides that can serve as antigens.
  • Antigens may be naturally occurring or synthetic, such as those naturally produced and/or genetically encoded by pathogens, infected cells, tumor cells (eg, tumors or cancer cells), viruses, bacteria, fungi, or parasites.
  • the antigen is an autoantigen or a graft-associated antigen.
  • the antigen is, for example, a viral capsid, envelope or coat, or a surface protein of a bacterial, fungal, protozoan or parasitic cell.
  • the antigen is any antigen known in the art for use in vaccines.
  • the antigen is a tumor antigen (TA).
  • proteins suitable for inclusion in drug delivery systems of the present invention are "prodrugs," ie, proteins that are metabolized or otherwise converted to a biologically, pharmaceutically, or therapeutically active form upon administration in vivo.
  • Proteins or peptides used in drug delivery systems are processed to have exposed or free thiol groups for Michael addition reaction with maleimide to form stable carbon-sulfur bonds, for example, the protein or peptide contains half Cystine serves to provide free thiol groups.
  • the protein or peptide is a protein or peptide obtained after treatment (e.g., co-incubation) with a thiol reducing agent (e.g., TCEP), which contains a half-molecule containing a free thiol group. Cystine residue.
  • a thiol reducing agent e.g., TCEP
  • the protein or peptide is one obtained by inserting one or more (eg, 1-10) cysteines, which contains a free thiol group. Cysteine residues.
  • 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.
  • 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.
  • the cysteine is inserted into the C-terminus or N-terminus of the protein or peptide, eg, before the N-terminal most amino acid or after the C-terminal most amino acid.
  • the protein or peptide further comprises a signal peptide, for example, it comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14.
  • a green fluorescent protein comprising an inserted cysteine comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1 or 2.
  • the green fluorescent protein comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher with the amino acid sequence set forth in SEQ ID NO: 1 or 2 An amino acid sequence of identity that has a cysteine at the very C terminus.
  • a uricase containing an inserted cysteine comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3 or 7.
  • the urate oxidase comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher of the amino acid sequence set forth in SEQ ID NO: 3 or 7
  • the urate oxidase is encoded by the nucleotide sequence shown in SEQ ID NO:4.
  • the urate oxidase is composed of at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher with the nucleotide sequence set forth in SEQ ID NO:4 is encoded by a nucleotide sequence of identity that has a cysteine at its very C terminus.
  • the prourokinase comprising an inserted cysteine comprises or consists of the amino acid sequence set forth in SEQ ID NO: 8 or 12.
  • the prourokinase comprises SEQ ID NO: The amino acid sequence shown in 8 or 12 has an identity of at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher, which has a cysteine at the most C-terminus .
  • the prourokinase is encoded by the nucleotide sequence set forth in SEQ ID NO:9.
  • the prourokinase protein has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher similarity to the nucleotide sequence set forth in SEQ ID NO:9
  • the identity is encoded by a nucleotide sequence that has a cysteine at its very C terminus.
  • the invention relates to a method of obtaining the drug delivery system of the invention, comprising:
  • the invention relates to a method of obtaining the drug delivery system of the invention, comprising:
  • step (3) incubating the protein obtained in step (3) with blood cells, and then incorporating the phospholipids therein into the cell membrane of the cells;
  • maleimide suitable for connection with phospholipids (or a maleimide coupled to blood cells through phospholipids) and Conditions for Michael addition reaction of thiol groups exposed on proteins or peptides, e.g., optimal temperature, pH, reaction time, concentration.
  • the invention provides a pharmaceutical composition or formulation comprising a drug delivery system of the invention.
  • compositions or preparations may also optionally contain suitable pharmaceutical auxiliaries, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art.
  • the invention provides a blood formulation, such as a human blood formulation, comprising a drug delivery system of the invention.
  • the human blood preparation of the present invention contains a drug delivery system of 0.5-1000 ⁇ g/mL, such as 1-500 ⁇ g/mL, 1-50 ⁇ g/mL, 1-10 ⁇ g/mL, 0.5 ⁇ g/mL-5 ⁇ g/mL , 0.5 ⁇ g/mL-3 ⁇ g/mL or 1 ⁇ g/mL-3 ⁇ g/mL, drug delivery system, such as 0.5 ⁇ g/mL, 0.6 ⁇ g/mL, 0.7 ⁇ g/mL, 0.8 ⁇ g/mL, 0.9 ⁇ g/mL, 1 ⁇ g/mL, 1.5 ⁇ g/mL, 2 ⁇ g/mL, 2.5 ⁇ g/mL, 3 ⁇ g/mL, 4 ⁇ g/mL, 5 ⁇ g/mL, 6 ⁇ g/mL, 7 ⁇ g/mL, 8 ⁇ g/mL, 9 ⁇ g/
  • the drug delivery system comprised in the blood preparation is a drug delivery system comprising red blood cells.
  • the blood preparations of the present invention are leukoreduced blood preparations, ie, blood preparations in which leukocytes have been filtered.
  • the blood preparations of the present invention are human blood leukoreduced blood preparations.
  • the blood preparations of the invention may be allogeneic blood preparations, such as allogeneic human blood preparations.
  • the blood cells eg, red blood cells
  • the blood preparation are from healthy subjects.
  • blood preparations of the present invention may be autologous blood preparations, such as autologous human blood preparations.
  • the blood cells eg, red blood cells
  • the blood preparation are from the subject to be treated.
  • a blood preparation or composition of the present invention may comprise a drug delivery system and unmodified blood cells (eg, red blood cells).
  • unmodified blood cells eg, red blood cells
  • at least a selected percentage of the blood cells (eg, red blood cells) in the composition are modified, ie, coupled to a protein or peptide, to obtain the drug delivery system of the invention.
  • at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96% , 97%, 98%, 99% or more of the cells are the drug delivery system of the present invention.
  • a blood formulation or composition of the invention may comprise two or more drug delivery systems of the invention.
  • the compositions comprise a drug delivery system of the invention.
  • the compositions comprise an effective amount of a drug delivery system of the invention, e.g., up to about 10 14 cells, e.g., 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 modified blood cells (drug delivery system).
  • the number of modified blood cells can range between any two of the aforementioned numbers.
  • 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.
  • the composition administered to the subject includes up to about 10 modified blood cells (drug delivery system), such as about 10 3 , 10 4 , 10 5 , 10 6 , 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 in between.
  • the invention also provides a combination product (eg, a pharmaceutical combination product) comprising a drug delivery system of the invention, together with one or more other therapeutic agents.
  • a combination product eg, a pharmaceutical combination product
  • the combination products of the invention can be used in the treatment methods of the invention.
  • the present invention also provides a complete pharmaceutical kit containing the combination product, for example, the complete pharmaceutical kit contains in the same package:
  • the other therapeutic agent is selected from any substance that is effective in the disease to be treated by the pharmaceutical system, such as any substance that is effective in metabolism-related diseases, thrombosis-related diseases, or cancer, including, for example, chemotherapeutic agents, Cytotoxic agents, other antibodies, vaccines, small molecule drugs or immunomodulators (e.g. immunosuppressants or immune agonists; drugs for antithrombosis or thrombolysis, e.g. urokinase, tissue plasminogen activator, Streptokinase, etc.
  • chemotherapeutic agents e.g. immunosuppressants or immune agonists
  • drugs for antithrombosis or thrombolysis e.g. urokinase, tissue plasminogen activator, Streptokinase, etc.
  • the invention relates to a method of preventing or treating a disease, such as cancer, in a subject, comprising administering to said subject an effective amount of a drug delivery system described herein.
  • the drug delivery system includes an antibody for preventing or treating tumors (eg, a PD-1 antibody).
  • Cancer can be early, intermediate or late or metastatic.
  • the cancer can be a solid tumor or a hematological tumor.
  • the cancer is a gastrointestinal tumor, such as colon or colorectal cancer; or lung, pancreatic, or breast cancer.
  • 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.
  • the tumor or cancer is a cancer characterized by having elevated protein levels and/or nucleic acid levels (e.g., elevated expression) of PD-1, PD-L1, and/or PD-L2 (e.g., associated with compared with healthy individuals or healthy tissues of the same individual), for example, the cancer has increased protein levels and/or nucleic acid levels of PD-1, PD-L1 and/or PD-L2 in tumor cells (e.g., increased expression High) (e.g. compared to healthy cells from a healthy individual or to healthy cells from healthy tissue of the same individual).
  • elevated protein levels and/or nucleic acid levels e.g., elevated expression
  • PD-1, PD-L1, and/or PD-L2 e.g., associated with compared with healthy individuals or healthy tissues of the same individual
  • the cancer has increased protein levels and/or nucleic acid levels of PD-1, PD-L1 and/or PD-L2 in tumor cells (e.g., increased expression High) (
  • the present invention relates to a method of antithrombotic or thrombolytic or preventing or treating a thrombosis-related disease, said method comprising administering to said subject an effective amount of a drug delivery system described herein.
  • the drug delivery system comprises a protein or peptide, such as prourokinase, that is antithrombotic or thrombolytic or prevents or treats thrombosis-related diseases.
  • thrombosis-related diseases such as ischemic stroke, acute myocardial infarction, and deep vein thrombosis
  • thromboprophylaxis can help reduce morbidity and mortality.
  • the currently used thrombolytic drugs plasminogen activator often require a large amount of injection in a short period of time due to its short half-life in the body, and because it can cause bleeding, tissue remodeling and neurotoxicity after flowing out of the blood vessel. and other serious side effects, its clinical application is greatly limited, and it is not suitable for preventing thrombosis.
  • various modifications have been made to plasminogen activators to prolong the residence time of the drugs in blood vessels, but these modifications are not suitable for preventing thrombosis.
  • Red blood cells have well-defined biological functions, safety, and long circulating half-lives.
  • red blood cells are restricted in blood vessels, which can effectively extend the half-life of thromboprevention or treatment drugs in the body, thereby achieving the function of treating and/or preventing thrombosis with a very small dose.
  • the drug is fixed on the cells, side effects such as tissue remodeling and neurotoxicity caused by drug leakage from blood vessels can be avoided.
  • red blood cells are an important component of blood clots, using red blood cells to carry anti-thrombotic drugs can break down blood clots from the inside at the beginning of their formation. This method greatly reduces the dosage and can effectively avoid existing serious side effects. Therefore, using red blood cells to carry proteins or peptides (eg, prourokinase) used to prevent or treat thrombosis-related diseases can be effectively used for the treatment and/or prevention of thrombosis.
  • proteins or peptides eg, prourokinase
  • 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.
  • stroke such as ischemic stroke, myocardial infarction (eg, acute myocardial infarction), and venous thrombosis, such as deep vein thrombosis.
  • Metabolism-related diseases refer to diseases associated with metabolic abnormalities in mammals, often characterized by a lack of functional enzymes or excessive uptake of metabolites. Therefore, deposition of metabolites in the circulation and tissues causes tissue damage. Metabolism-related diseases include, for example, diseases related to defects in carbohydrate metabolism, amino acid metabolism, organic acid metabolism, purine or pyrimidine metabolism.
  • the present invention considers the application of blood cells carrying functional metabolic enzymes for the treatment of metabolism-related diseases.
  • 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 for gout.
  • metabolism-related diseases include, for example, chronic hyperuricemia, such as gout, such as refractory gout (gout that is resistant to other conventional therapies).
  • the drug delivery systems of the present invention (and compositions, pharmaceutical compositions, formulations, combinations, etc., such as blood preparations) comprising the same may be administered by any suitable method, preferably infusion, such as parenteral infusion.
  • Parenteral infusion includes intravenous or intraarterial administration.
  • the drug delivery system of the invention is administered by intravenous or arterial infusion.
  • the appropriate dosage of the drug delivery system of the 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, and the severity of the disease and course, whether administration is for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the judgment of the attending physician.
  • the antibody is suitably administered to the patient in a single treatment or over a series of treatments.
  • a 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.
  • dose or total cell number may be expressed as cells/kg.
  • the dose may be about 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 cells/kg.
  • the course of treatment lasts from about 1 week to 12 months or longer, such as 1, 2, 3 or 4 weeks or 2, 3, 4, 5 or 6 months.
  • subjects can be treated approximately every 2-4 weeks.
  • the number of cells, dosage and/or dosing interval may be selected based on various factors, such as the subject's weight and/or blood volume, the condition being treated, the subject's response, etc.
  • the exact number of cells required may vary from subject to subject, depending on, for example, the subject's species, age, weight, sex, and general condition, the severity of the disease or condition, the specific cells, and the agent to which the cells are conjugated. Factors such as identity and activity, mode of administration, concurrent therapies.
  • the drug delivery systems of the present invention are administered in conjunction with one or more therapeutic modalities or other therapeutic agents.
  • the therapeutic modalities or other therapeutic agents are used to treat tumors, such as cancer.
  • the treatment modality is radiation therapy or surgery.
  • the therapeutic agent is selected from any agent effective in cancer, including chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, small molecule drugs, or immune modulators (eg, immunosuppressives or immune agonists).
  • the treatment modality or agent is used to prevent or treat thrombosis-related disorders.
  • the treatment modality is, for example, a stent or bypass.
  • the therapeutic agent is, for example, an antithrombotic or thrombolytic drug, such as urokinase, tissue plasminogen activator, streptokinase, and the like.
  • the treatment modality or agent is used to treat a metabolism-related disease.
  • the present invention also provides the use of the drug delivery system of the present invention in the preparation of a medicament or formulation for use in the aforementioned methods (eg, for treatment).
  • the blood cells in the drug delivery systems of the invention are from the subject to be treated.
  • the methods of the present invention for treating a disease in a subject include:
  • the blood cells in the drug delivery systems of the invention can be from other subjects, such as healthy subjects.
  • the drug delivery systems of the present invention can be administered as a drug or formulation to a subject to be treated.
  • 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). CaCl 2 and FeCl 3 were purchased from Sionpharm Chemical Reagent Co., Ltd (China). All other chemicals and solvents were purchased from Sionpharm Chemical Reagent Co., Ltd (China).
  • Unlabeled red blood cells or washed red blood cells refer to red blood cells separated from the peripheral blood of healthy individuals by density gradient centrifugation, and the separated red blood cells are washed three times with PBS to remove other components (such as plasma or other blood cells).
  • 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 Westlake University Animal Care and Use Committee (IACUC protocol #19-012-GXF).
  • eGFP was used to test the binding efficiency of the reaction.
  • eGFP is fused to a cysteine at the C terminus and purified (eGFP-Cys).
  • the purity of the synthesized Mal-PEG2000-DSPE linker (synthesized by Xi'an Ruixi Biotechnology) exceeds 99%.
  • the eGFP-Cys cDNA was cloned into pET vector and transformed into E. coli BL21 (DE3) cells for protein expression. Transformed cells were cultured at 37°C until OD 600 reached 0.6-0.8, then 500 ⁇ M IPTG was added. Cells were incubated with IPTG for 4 hours at 37°C, harvested by centrifugation and lysed with pre-chilled lysis buffer (20mM Tris-HCl, pH 7.8, 500mM NaCl). Lysates were sonicated on ice (5 sec on, 5 sec off, 60 cycles, 25% power, Branson Sonifier 550 Ultrasonic Cell Disrupter).
  • Mal-PEG2000-DSPE connector was purchased from Xi'an Ruixi Biotechnology. Its chemical formula is shown in Figure 3, and its purity exceeds 99%.
  • Mal-PEG-phospholipids such as Mal-PEG-DSPE
  • phospholipids such as DSPE
  • Maleimide can undergo a Michael addition reaction with the sulfhydryl groups on the exposed cysteine on the protein to form a stable covalent bond, thereby obtaining Attach the target protein to the cell membrane of red blood cells.
  • Red blood cells were isolated from peripheral blood of C57BL/6J mice by density gradient centrifugation. The isolated red blood cells were washed three times with PBS. Dissolve Mal-PEG2000-DSPE in phosphate buffer at 37°C to a final concentration of 100 ⁇ M. Then 1 ⁇ 10 9 red blood cells were pretreated with 50 ⁇ M MAL-PEG2000-DSPE at 37 °C for 30 min. The pretreated red blood cells were then washed three times with PBS. Preconditioned red blood cells should be used immediately or not stored for long periods of time.
  • the above-mentioned pretreated red blood cells and eGFP-cys were mixed in PBS buffer, where the concentration of red blood cells was 1 ⁇ 10 9 /mL and the concentrations of eGFP-cys were 5, 25, 50 and 100 ⁇ M.
  • the total volume of the reaction mixture was 200 ⁇ L, and the reaction was carried out by rotating at 10 rpm for 1 hour. Stop the reaction, wash with PBS, remove the remaining reaction reagents, obtain labeled red blood cells, and use Beckman Coulter CytoFLEX LX to analyze the labeling efficiency of red blood cells (ie, the percentage of red blood cells coupled with proteins to total red blood cells). 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%.
  • Maleimide groups can undergo Michael addition reactions with sulfhydryl groups on exposed cysteines, but not unexposed cysteines.
  • Maleimides attached to polyethylene glycol chains are often used as flexible linker molecules to attach proteins to surfaces. We use this principle to modify proteins so that the target protein has a linker that can be inserted into the cell membrane ( Figure 2).
  • proteins with exposed cysteines e.g., the cysteine at the C-terminus
  • Michael addition reactions with maleimide groups thereby binding the protein and DSPE into a stable covalent bond.
  • the DSPE end can fuse with the red blood cell membrane to form a stable connection, and the target protein is loaded onto the cell membrane.
  • red blood cells Mix the above red blood cells and eGFP-cys-Mal-PEG2000-DSPE in PBS buffer, the total volume is 200 ⁇ L, the final concentration of red blood cells is 1 ⁇ 10 9 /mL, and the eGFP-cys-Mal-PEG2000-DSPE is 5 and 25 respectively. ,50,100 ⁇ M.
  • the reaction mixture was rotated at 10 rpm and incubated for 1 hour to obtain red blood cell-protein conjugates. Stop the reaction, wash with PBS, remove the remaining reaction reagents, obtain labeled red blood cells, and use Beckman Coulter CytoFLEX LX to analyze the labeling efficiency of red blood cells. (i.e., the percentage of protein-coupled red blood cells relative to total red blood cells), in which unlabeled red blood cells were used as a control group.
  • the labeling efficiency of eGFP was characterized on RBC membranes. After the reaction, the (eGFP-cys-Mal-PEG-DSPE) prepared in the above 1.3.2 and the unlabeled red blood cells (the amount of red blood cells are 1 ⁇ 10 9 /mL respectively) were passed through the flow cytometer. Detect the labeling effect of eGFP. As shown in Figure 5, the histogram shows the eGFP signal on the cell surface.
  • eGFP-cys-Mal-PEG-DSPE-labeled mouse red blood cells prepared in Example 1.3 and the unlabeled mouse red blood cells.
  • the labeled red blood cells (unlabeled) were stained and labeled with the fluorescent dye Cell Trace Far Red dye, and then infused into the mice through the tail vein.
  • Each group had three C57BL/6J mice aged 6-8 weeks.
  • the infusion volume was 1 ⁇ 10 9 cells/each.
  • anti-PD-1 antibodies Prepared by Merck Sharp & Dohme Corp.
  • Cysteine-exposed antibodies were obtained by pretreating 10 ⁇ M anti-PD-1 antibody with 0.2 mM TCEP for 1 h at room temperature. After pretreatment, excess TCEP was removed by dialysis and ultrafiltration to collect the pretreated protein. Pretreated proteins are to be used immediately or not stored for long periods of time.
  • anti-PD-1 antibody-labeled RBCs were prepared as described in Example 1.3, except that the antibody concentration was 5 ⁇ M, and anti-PD-1 antibody was used instead of eGFP-cys.
  • the control was to incubate 5 ⁇ M anti-PD-1 antibody with red blood cells not coupled to Mal-PEG-DSPE as described in Example 1.3. Labeling efficiency was measured 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 antibody; Anti-PD-1-Ma-PEG -DSPE represents the result of 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, where blue: the signal intensity of the anti-PD-1 antibody after applying red blood cells not coupled to Mal-PEG-DSPE and incubated with the anti-PD-1 antibody;, red: applying Signal intensity of anti-PD-1 antibody after incubation of red blood cells coupled with Mal-PEG-DSPE and anti-PD-1 antibody. The results show that this method effectively labels antibodies to the cell surface.
  • 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 with additional cysteine residues in the C-terminal His 6 linker. All constructs were verified by sequencing and then transformed in E. coli BL21(DE3) for protein expression.
  • a single transformed colony was inoculated into 10 ml Luria-Bertani (LB) medium supplemented with ampicillin (100 ⁇ g/ml) and cultured overnight at 37°C with shaking at 220 rpm. Transfer this 10 ml culture to 1 L of fresh LB medium and grow the culture at 37°C with shaking at 220 rpm until OD 600 reaches 0.6. The temperature was then lowered to 20°C and 1 mM IPTG was added for induction.
  • LB Luria-Bertani
  • Cells were harvested 20 h after induction by centrifugation at 8,000 rpm for 10 min at 4°C.
  • the cell pellet was resuspended in low-salt lysis buffer (50mM Tris 7.5, 50mM NaCl) and lysed with sonication.
  • the supernatant collected after centrifugation at 10,000 rpm for 1 h was loaded onto a 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 absorbance at 280 nm and conductivity became stable, and then eluted using a linear gradient of 0-1 M NaCl in 20 mM Tris 7.5. Fractions corresponding to the elution peaks were analyzed by SDS-PAGE and the highest purity fractions were enriched. To avoid cysteine oxidation, 2 mM TCEP was added to the combined fractions and samples were concentrated using an Amicon Ultra-15 centrifugal filter unit (Millipore, Darmstadt, Germany). The concentrated protein was loaded into EzLoad 16/60 Chromdex 200pg (Bestchrom, Shanghai, China) pre-equilibrated with PBS, and the target protein peak was collected.
  • the cell pellet was resuspended in lysis buffer (50mM Tris 7.5, 200mM NaCl, 5mM imidazole) and lysed with sonication.
  • the tagged protein UOX-His 6 -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.
  • UOX-labeled RBCs were prepared by method 1 as described in Example 1.3, in which UOX was used instead of eGFP to obtain UOX-Cys-Mal-PEG-DSPE-labeled RBCs.
  • PBMC peripheral blood mononuclear cells
  • the purpose of this step is to use the lymphocyte separation solution to achieve density gradient centrifugation of cellular components and separate PBMC from different cells such as red blood cells and platelets to ensure subsequent enrichment of T cells.
  • Cell separation The antigens on the cell surface are combined with the corresponding biotin-labeled antibodies (purchased from Biolegend), and the biotin is combined with streptavidin-labeled magnetic beads Streptavidin Particles Plus–DM (BD Company, Cat. No.: 557812). Isolation of cells with specific surface markers by magnetic beads, where,
  • eGFP-labeled cells were prepared as described in Example 1, wherein T cells, B cells, monocytes, NK cells or megakaryocytes were used instead of red blood cells used in this example, and flow cytometry was applied to detect the labeling efficiency.
  • T cells, B cells, monocytes, NK cells or megakaryocytes were used instead of red blood cells used in this example, and flow cytometry was applied to detect the labeling efficiency.
  • flow cytometry was applied to detect the labeling efficiency.
  • this modification method is also suitable for these cell types and showed good labeling efficiency (Figure 13).
  • Example 1.5 Prepare eGFP-Cys-MAL-PEG-DOPE (Xi'an Ruixi Biosynthesis, as shown in Figure 3, where DSPE is replaced by DOPE) labeled red blood cells as described in Example 1.3, only using DOPE instead of DSPE.
  • the standard operating procedure for evaluating the in vivo lifespan of these surface-modified red blood cells was applied in Example 1.5, in which blood was collected on days 1, 3, 4, and 15, and the blood collected on day 15 was used for flow cytometric analysis ( Figure 14, Control: unlabeled red blood cells).
  • eGFP-Cys-MAL-PEG-DOPE-labeled red blood cells (eGFP-Cys-MAL-PEG-DOPE-RBC) not only showed the same characteristics as those of the control group (mice transfused with unlabeled red blood cells) Lifetime, also shows a signal lasting 15 days in the cycle ( Figures 14 and 15).
  • Example 7 Comparison of optimal labeling efficiency concentrations of DSPE, DPPE, DMPE, and DOPE
  • Mal-PEG2000-phospholipid (DPPE, DOPE, DSPE or DMPE) labeled RBCs were prepared as described in Example 1.3 (where Mal-PEG2000-DPPE, Mal-PEG2000-DOPE, Mal-PEG2000-DSPE and Mal-PEG2000-DMPE are all from Xi'an Ruixi Biotechnology), the difference is that the concentrations of Mal-PEG2000-phospholipids in the labeling reaction system are 200 ⁇ g/mL, 80 ⁇ g/mL, 20 ⁇ g/mL and 0 ⁇ g/mL (control group), and the eGFP-cys concentration is 5 ⁇ M/mL . After the labeling reaction, the average fluorescence intensity on the cells was detected by flow cytometry.
  • DPPE and DMPE are also effective for labeling cells.
  • the labeling efficiency of these two phospholipids is the best.
  • DSPE and DOPE showed the best labeling efficiency at a concentration of 200 ⁇ g/mL.
  • the absorbance of the cell supernatant at 410 nM was also detected by ELISA. As shown in Table 1, after incubation with high concentration (200 ⁇ g/mL) of Mal-PEG2000-DPPE, the absorbance of the supernatant increased due to RBC hemolysis. Likewise, RBC hemolysis occurred after incubation with 200 ⁇ g/mL Mal-PEG2000-DMPE. However, high concentrations of Mal-PEG2000-DOPE and Mal-PEG2000-DSPE will not cause red blood cell damage.
  • Red blood cells were isolated from peripheral blood of C57BL/6J mice by density gradient centrifugation. The isolated red blood cells were washed three times with PBS. Dissolve Mal-PEG2000-DSPE in phosphate buffer at 37°C to a final concentration of 100 ⁇ M. Then 1 ⁇ 10 RBCs were pretreated with 50 ⁇ M MAL-PEG2000-DSPE at 37 °C for 30 min. The pretreated red blood cells were then washed three times with PBS. Preconditioned red blood cells should be used immediately or not stored for long periods of time.
  • ProUK-Cys cDNA (the nucleic acid sequence is shown in SEQ ID NO: 9) was cloned into the pcDNA 3.4 vector, and the plasmid was transfected into HEK293F cells for protein expression. After transfection, 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 min at 4°C and filtered through a 0.45 ⁇ M filter. The filtered supernatant was loaded into The designed chromatography system was connected to a HisTrap FF 1mL column (GE Healthcare).
  • ProUK-Cys protein was thus obtained.
  • the amino acid sequence of ProUK-Cys is shown in SEQ ID NO:8 below.
  • the nucleic acid sequence of ProUK-Cys is shown in the following SEQ ID NO:9.
  • Example 8 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 in Example 1. The details are as follows: 10 9 each of unlabeled red blood cell RBCs (control RBCs) and ProUK-RBCs obtained in Example 8.2.3 were stained with Cell Trace Far Red dye. Cell Trace Far Red-stained RBC-ProUK and control RBC were infused into the tail vein of normal recipient C57BL/6J mice (Experimental Animal Center of Westlake University, 6-8 weeks old, 3 mice in each group).
  • Blood was collected from the cheeks of mice at 1, 2, 6, 24, 48, 72, 144, 192, 240, 336, 408, 504, and 576 hours after blood transfusion.
  • the blood sample was incubated with FITC-coupled anti-urokinase antibody (Yiqiao Shenzhou Company, 1 ⁇ L/sample) at 37°C for 15 minutes to detect the ProUK signal, washed three times and then analyzed by flow cytometry. Targeted at 0, 8, and 17 and 24-day flow cytometry results are shown.
  • 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 rates of RBCs in the experimental group and the control group.
  • the engineered RBCs showed good stability in vivo, and the ProUK signal could always be detected on the surface of these RBCs.
  • ProUK-RBCs exhibit good stability in vivo, and ProUK signals can always be detected on the surface of these RBCs.
  • the biological function of ProUK-RBC prepared in Example 8 was further evaluated.
  • RBCs play a very important role, participating in platelet-driven clot and thrombus contraction, resulting in the formation of tightly packed polyhedral RBC arrays that include a nearly impermeable barrier important for hemostasis and wound healing.
  • Whole blood was obtained from C57BL/6J mice and plasma was separated by centrifugation. Washed red blood cells were also obtained from whole blood.
  • the concentrations of ProUK-RBC and unlabeled RBC are both 1 ⁇ 10 7 cells/ ⁇ l ProUK-RBC group:
  • ProUK-RBC 100 ⁇ L of ProUK-RBC prepared in Example 8 at 2.4 ⁇ g/mL
  • Free ProUK+RBC group Add 50 mg/mL ProUK-cys stock solution (the volume change during the dilution process is negligible) to 100 ⁇ l unlabeled RBC to obtain final concentrations of 0, 4, 8, 16, 32, and 64 ⁇ g/mL ProUK. -cys solution.
  • the thrombotic clot was covered with different amounts of ProUK-RBC and ProUK+ unlabeled RBCs as described above at 20°C.
  • nascent clots Nascent clot group
  • different amounts of ProUK-RBC and ProUK + unlabeled RBCs were added to the above Hemostatic clot group before adding CaCl and thrombin.
  • CaCl2 and thrombin was added directly to mouse plasma, followed immediately by CaCl2 and thrombin for clot formation, which lasted 20 min.
  • ProUK carried on RBC is more stable, and with the help of RBC as a carrier, it can better activate the function of plasminogen activator.
  • RBCs are an important component of thrombus. During the process of thrombosis, as long as a small part of ProUK-RBC enters the thrombus, its fibrinolytic activity can be activated to break down the thrombus from the inside. However, due to their tighter structure, ProUK-RBC cannot penetrate the hemostatic clot and can only dissolve thrombosis from the surface. Therefore, ProUK-RBC is more effective in targeting nascent clots.
  • Example 10 ProUK-RBC extends half-life and reduces drug dosage
  • ProUK-RBC can effectively reduce the dose of ProUK.
  • C57/BL6J mice (Experimental Animal Center of West Lake University, 6-8 weeks old, 3 mice in each group, a total of three groups) were injected into the tail vein with equal amounts of free ProUK (i.e. 0.2ug ProUK-cys and 200ul 1 ⁇ 10 7 /ul RBC mixture), ProUK-RBC (i.e. ProUK-RBC 200ul 1 ⁇ 10 7 /ul prepared in Example 8) and unlabeled RBC (i.e. 200ul 1 ⁇ 10 7 /ul unlabeled RBC mixed), blood was collected from the orbit according to the planned time.
  • the ProUK-RBCs group collected blood at 1, 5, 15, 30, 60, and 120 minutes after administration.
  • the RBC and free ProUK groups collected blood at 1, 3, 5, and 120 minutes after administration. Blood was collected at 10, 30, and 60 minutes ( Figure 23).
  • Thrombosis experiments were performed by adding CaCl 2 and thrombin (final concentrations of 20 mM and 0.2 units/ml, respectively) to the mouse whole blood taken out above, and incubated at 37°C for 2 hours. Gently invert the sample to resuspend, use a pipette to aspirate the clot-free red blood cell suspension, add red blood cell lysis solution for 10 min, centrifuge (5 min, 1200 ⁇ g), and measure the amount of released hemoglobin spectrophotometrically at 405 nm. Thrombolysis was standardized in each group by comparing the highest absorbance value (no thrombosis) and the lowest absorbance value (no added thrombolytic component, thrombosis).
  • Example 11 ProUK-RBC improves thrombolytic efficacy in vivo
  • ProUK-RBC showed good thrombolytic ability in in vitro and ex vivo experiments.
  • Tail bleeding time assay is the most widely used method to assess hemostasis in mice. Specific steps are as follows:
  • Figure 28 shows the anti-thrombotic ability in three groups at low dose levels, of which 13 mice in the RBC group, 10 mice in the free ProUK group, and 13 mice in the ProUK-RBC group met the hemostatic time requirements.
  • This experiment can evaluate the thrombolytic ability of ProUK-RBC on newly formed clots. The results showed no significant difference between the free ProUK group and the RBC group, which was due to the low dose of free ProUK. However, at such a low dose, the bleeding time of the ProUK-RBC group was longer than that of the RBC group and the free ProUK group ( Figure 28). The ProUK-RBC group still showed good anti-thrombotic ability, which strongly proved the effectiveness of ProUK-RBC.
  • MCAO cerebral thrombosis in animals.
  • the traditional thread embolization method is not suitable for prevention models, so we used microthrombus particle injection to construct a cerebral thrombosis model.
  • Micro-thrombus particles cannot directly block blood vessels, but when these particles enter the body, they will form larger thrombus, block blood vessels, and cause stroke. Specific steps are as follows:
  • ProUK-RBC (1 ⁇ 10 7 / ⁇ l) and unlabeled RBC (1 ⁇ 10 7 / ⁇ l) were injected into the tail veins of the two groups of rats respectively, with the volume being 200 ⁇ L/rat.
  • microthrombosis solution 100 ⁇ L; 1.4 ⁇ 10 particles
  • the catheter was withdrawn, and the ligatures were removed from the carotid artery and PPA (obstructing downstream vessels, resulting in >80% Blood flow ceases and results in extensive ipsilateral cerebral infarction). Survival rates were calculated after 24 hours.
  • FeCl 3 can induce vascular endothelial cell damage and simulate endogenous thrombosis.
  • ProUK-RBC ProUK-RBC 200ul 1 ⁇ 10 7 cells/ul
  • free ProUK with 0.2ug ProUK-cys and 200ul 1 ⁇ 10 7 cells/ul unlabeled RBC mix
  • RBC 200ul 1 ⁇ 10 7 /ul unlabeled RBC
  • mice were anesthetized with intraperitoneal (IP) injection of tribromoethanol.
  • IP intraperitoneal

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Abstract

本发明提供了一种包含血细胞的药物递送系统，以及用于制备其的新的修饰红细胞膜的方法。本发明还涉及应用所述药物递送系统对疾病进行治疗的方法。

Description

包含血细胞的药物递送系统
本发明提供了一种包含血细胞的药物递送系统，以及用于制备其的新的修饰红细胞膜的方法。本发明还涉及应用所述药物递送系统对疾病进行治疗的方法。
发明背景
用于延长治疗多种人类疾病的药物保留时间的药物递送系统的最新进展引起了广泛关注。然而，许多系统仍然受到多种挑战和限制，例如稳定性差、不需要的毒性和免疫反应。红细胞(RBC)是人体中最常见的细胞类型，由于具有以下的独特的生物学特性，作为理想的体内药物递送系统已被广泛研究了30多年：(i)广泛的全身循环范围；(ii)作为生物材料具有良好的生物相容性，具有长的体内存活时间；(iii)大的表面积体积比；(iv)没有细胞核、线粒体和其他细胞器。
通过直接封装、外源肽的非共价连接或通过与RBC表面蛋白特异性抗体融合来安装蛋白质，RBC已被开发为药物递送载体。已经证明，这种修饰的RBC在体内应用方面存在局限性。例如，封装会破坏细胞膜，从而影响工程化细胞的体内寿命。此外，聚合物颗粒与RBC的非共价连接很容易解离，有效载荷将在体内很快降解。
因此，本领域中仍然需要稳定高效的能将药物封装到RBC中的修饰方法，以获得稳定且高效的药物递送系统。
发明内容
磷脂是两亲性的。亲水端通常包含带负电的磷酸基团，而疏水端通常由两个“尾巴”组成，它们是长脂肪酸残基。在水溶液中，磷脂由疏水相互作用驱动，导致脂肪酸尾的积累，从而最小化与水分子的相互作用。结果通常是磷脂双层：由两层相反方向的磷脂分子组成的膜，两侧的头部都暴露在液体中，而尾部则指向膜。这是所有细胞膜和某些其他生物结构的主要结构基序。在生物膜中，磷脂常与其他分子(如蛋白质、糖脂和甾醇)一起出现在双层(如细胞膜)中。当疏水尾部相互对齐，在面向水的一侧形成亲水头部的膜时，就会出现脂双层。
马来酰亚胺介导的方法是生物偶联中最常用的方法之一。由于反应速度极快，对蛋白质中半胱氨酸残基的选择性极高，因此用于多种方法。多种马来酰亚胺异双功能试剂用于制备靶向治疗剂，并研究其蛋白质、基于蛋白质的微阵列或蛋白质固定化中蛋白质组装体的生物学背景。例如，新兴的有前途的靶向药物治疗抗体-药物偶联物由三个主要成分组成：单克隆抗体、细胞毒性药物和通常含有马来酰亚胺基团的连接分子。酰亚胺基团与药物和抗体结合。
连接到聚乙二醇链的马来酰亚胺通常用作柔性连接分子以将蛋白质附着到表面。双键容易与半胱氨酸上的巯基反应形成稳定的碳硫键。将聚乙烯链的另一端连接到珠子或固体支持物上可以很容易地将蛋白质与溶液中的其他分子分开，但前提是这些分子没有巯基。
本发明首次发现，使用细胞膜成分磷脂结合马来酰亚胺和聚乙二醇作为红细胞的接头，能够用于获得高效稳定的基于红细胞的药物递送系统。
附图说明：
图1a和图1b显示了方法1的示意图；
图2a和图2b显示了方法2的示意图；
图3显示了包含马来酰亚胺基团-PEG-磷脂的接头；
图4显示了蛋白质的硫醇基于接头偶联的示意图；
图5显示了标记和未标记的红细胞表面上的eGFP信号强度；
图6显示了天然红细胞在体外被不同浓度eGFP-Cys-MAL-PEG2000-DSPE标记后细胞表面上的eGFP信号强度；
图7显示了Cell Far red染色的红细胞表面上的eGFP信号强度；
图8a显示了对照红细胞(未经eGFP-cys标记)和经eGFP-cys标记的红细胞在小鼠血液中随时间的Far red阳性红细胞数的百分比；
图8b显示了对照红细胞(未经eGFP-cys标记)和经eGFP-cys标记的红细胞中随时间的GFP阳性红细胞数占Cell Far Red阳性细胞的百分比；
图9显示了经抗PD-1抗体标记和未经抗PD-1抗体标记的红细胞表面上的抗PD-1抗体信号；
图10显示了PD-1抗体的信号强度。
图11显示了UOX-Cys-Mal-PEG-DSPE标记的红细胞在2只受体小鼠中的UOX信号强度；
图12显示了两只受体小鼠中随时间的在血液中的Cell Far red阳性红细胞数的百分比(图12a)和随时间的UOX阳性红细胞数占Cell Far Red阳性细胞的百分比(图12b)；
图13显示了应用流式细胞术检测的在不同类型的血细胞表面的eGFP的信号强度；
图14显示了eGFP-Cys-MAL-PEG-DOPE标记的红细胞和未标记的红细胞的eGFP信号强度和FAR RED信号强度；
图15显示了eGFP-Cys-MAL-PEG-DOPE标记的红细胞和未标记的红细胞随时间的％Far red阳性红细胞数(图15a)和随时间的％UOX阳性红细胞数(图15b)；
图16显示了Mal-PEG2000-磷脂(DPPE、DOPE、DSPE或DMPE)标记的红细胞中eGFP的信号强度；
图17显示了不同浓度的Mal-PEG2000-磷脂(DOPE或DSPE)与红细胞孵育10分钟获得eGFP的信号强度；
图18显示了应用Mas-PEG-DSPE作为接头偶联尿激酶原和红细胞的示意图；
图19显示了经ProUK-cys标记和未经ProUK-cys标记的红细胞表面上的尿激酶原的信号强度；
图20显示了循环中输入的具有ProUK的信号的红细胞随时间变化的百分比(上图)，和在不同取血时间获得的血样中Far Red和ProUK的信号强度；
图21显示了经ProUK-cys标记和未经ProUK-cys标记的红细胞上的Far Red信号和ProUK信号强度；
图22显示了不同剂量的游离ProUK(A)和ProUK-RBC(B)在不同剂量下对新生止血凝块和止血凝块的溶解；以及相应的溶栓％柱状图(ProUK(C)和ProUK-RBC(D))；
图23显示了ProUK-RBC给药组和游离ProUK给药组计划从眼眶采血的时间；
图24显示了不同给药组中ELISA对全血中随时间ProUK含量所占％；
图25显示了ProUK-RBC给药组和游离ProUK给药组和RBC对照组随时间的溶栓％；
图26显示了ProUK-RBC给药组和游离ProUK给药组和RBC对照组随时间的溶栓照片；
图27显示了尾部出血时间测定法的操作示意图；
图28显示了低剂量水平下不同给药组中尾部出血时间；
图29显示了ProUK-RBC给药组和未标记RBC给药组的24小时致死率；
图30显示了ProUK-RBC给药组(右)和未标记RBC给药组(左)在处理后6小时使用TTC(2，3，5—氯化三苯基四氮唑)染色后进行切片的照片；
图31显示了ProUK-RBC给药组和未标记RBC给药组在处理后6小时的脑梗塞面积百分比；
图32显示了深静脉血栓形成模型的操作示意图；
图33显示了未标记RBC给药组、游离的ProUK给药组和ProUK-RBC给药组中各一只小鼠血栓形成的照片。
发明详述：
I.定义
在下文详细描述本发明前，应理解本发明不限于本文中描述的特定方法学、方案和试剂，因为这些可以变化。还应理解本文中使用的术语仅为了描述具体实施方案，而并不意图限制本发明的范围，其仅会由所附权利要求书限制。除非另外定义，本文中使用的所有技术和科学术语与本发明所属领域中普通技术人员通常的理解具有相同的含义。
为了解释本说明书，将使用以下定义，并且只要适当，以单数形式使用的术语也可以包括复数，并且反之亦然。要理解，本文所用的术语仅是为了描述具体的实施方案，并且不意欲是限制性的。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
如本文所用，术语“和/或”意指可选项中的任一项或可选项的两项或多项。
如本文所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。
本文所述的“药物递送系统”是指能够将其所包含的药物输送至靶标的任何药物制剂或制品，包括细胞制剂，例如基于红细胞的细胞制剂或基于其他血细胞的细胞制剂；或血液制剂，例如以人血为原料制备的制剂。
术语“尿激酶原”也称作“单链尿激酶型纤溶酶原激活剂”，一般是由411个氨基酸残基组成的单链多肽，分子量为49KD左右，属于丝氨酸蛋白酶类，分子内有12对二硫键。其按结构功能可分为四个结构域，即表皮生长因子结构域、Kringle结构域、丝氨酸蛋白酶结构域和连接区。尿激酶原是一种相对无活性的前体，必须先转化为尿激酶，然后才能在体内发挥活性。然而，它通过在激活前与纤维蛋白结合来显示对凝块的选择性。与其他纤溶酶原激活剂相比，它的优势在于它在血浆中无活性，因此不消耗循环抑制剂。人尿激酶原的氨基酸序列登录号可以参见Uniprot P00749(https://www.uniprot.org/uniprot/P00749)。在一个实施方案中，本发明所述的尿激酶原是指天然人尿激酶原，或其天然变体，其可以具有或不具有信号肽序列(例如MRALLARLLLCVLVVSDSKG(SEQ ID NO:14))或标签(例如His6标签)。在一个实施方案中，本发明的尿激酶原是人尿激酶原，例如其包含SEQ ID NO:10或13所示的氨基酸序列或由其组成。在一些实施方案中，本发明的尿激酶原包含与SEQ ID NO:10或13所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，且能够在体内被激活具有尿激酶活性。
术语“血栓形成相关疾病”是指与血栓形成相关的任何疾病，包括但不限于缺血性中风、急性心肌梗塞和深静脉血栓。
术语“抗肿瘤作用”指可以通过多种手段展示的生物学效果，包括但不限于例如，肿瘤体积减少、肿瘤细胞数目减少、肿瘤细胞增殖减少或肿瘤细胞存活减少。
术语“肿瘤”和“癌症”在本文中互换地使用，涵盖实体瘤和血液肿瘤。
术语“癌症”指向或描述哺乳动物中特征通常为细胞生长不受调节的生理疾患。在某些实施方案中，适合于通过本发明的抗体来治疗的癌症包括肺癌、胰腺癌、乳腺癌、胃肠道肿瘤例如结肠癌、结直肠癌或直肠癌等，包括那些癌症的转移性形式。
术语“肿瘤”指所有赘生性(neoplastic)细胞生长和增殖，无论是恶性的还是良性的，及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”和“肿瘤”在本文中提到时并不互相排斥。
“红细胞”或“RBC”是最常见的血细胞种类，也是脊椎动物向身体组织经血流通过循环系统输送氧气的主要载体。红细胞的细胞质富含血红蛋白，血红蛋白是一种含铁的生物分子，可以结合氧气，使细胞和血液呈红色。细胞膜由蛋白质和脂质组成，这种结构提供了生理细胞功能所必需的特性，例如可变形性和稳定性，同时可以穿过循环系统，特别是毛细血管网络。在人类中，成熟的红细胞是柔韧的椭圆形双凹圆盘。它们缺乏细胞核和大多数细胞器，以具有容纳血红蛋白的最大空间；它们可以被视为血红蛋白的袋子，以质膜为袋子。成人每秒大约产生240万个新红细胞。人体中大约84％的细胞是20–30万亿个红细胞。近一半的血液体积(40％到45％)是红细胞。
“成体天然红细胞”是指从动物，特别是人(例如成人或儿童)的血液中直接分离获得的成熟天然红细胞。
本文所述的“血液制剂”或“血液制品”可以互换使用，是指由(人类)血液为原料制备，用在医疗用的制品。血液制剂包括全血制剂、成分血制剂、血浆制剂或减白血液制剂。
术语“有效量”指本发明的药物递送系统或组合物或制剂或制品或组合或组合产品的这样的量或剂量，其以单一或多次剂量施用患者后，在需要治疗或预防的患者中产生预期效果。
“治疗有效量”指以需要的剂量并持续需要的时间段，有效实现所需治疗结果的量。治疗有效量也是这样的一个量，其中药物递送系统或组合物或制剂或制品或组合或组合产品的任何有毒或有害作用不及治疗有益作用。相对于未治疗的对象，“治疗有效量”优选地抑制可度量参数(例如血栓量，或例如肿瘤体积)至少约30％、甚至更优选地至少约40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％甚至100％。
“预防有效量”指以需要的剂量并持续需要的时间段，有效实现所需预防结果的量。通常，由于预防性剂量在对象中在疾病较早阶段之前或在疾病较早阶段使用，故预防有效量将小于治疗有效量。
“个体”或“受试者”包括哺乳动物。哺乳动物包括但不限于，家养动物(例如，牛，羊，猫，狗和马)，灵长类动物(例如，人和非人灵长类动物如猴)，兔，以及啮齿类动物(例如，小鼠和大鼠)。在一些实施方案中，个体或受试者是人。
术语“药用辅料”指与活性物质一起施用的稀释剂、佐剂(例如弗氏佐剂(完全和不完全的))、赋形剂、载体或稳定剂等。
术语“药物组合物”指这样的组合物，其以允许包含在其中的活性成分以生物学活性有效的形式存在，并且不包含对施用所述组合物的受试者具有不可接受的毒性的另外的成分。
术语“药物组合”是指非固定组合产品或固定组合产品，包括但不限于药盒、药物组合物。术语“非固定组合”意指活性成分(例如，(i)本发明的药物递送系统以及(ii)其他治疗剂)以分开的实体被同时、无特定时间限制或以相同或不同的时间间隔、依次地施用于患者，其中这类施用在患者体内提供预防或治疗有效水平的两种或更多种活性成分。在一些实施方案中，药物组合中使用的本发明的药物递送系统和其他治疗剂以不超过它们单独使用时的水平施用。术语“固定组合”意指两种或更多种活性成分以单个实体的形式被同时施用于患者。优选对两种或更多种活性成分的剂量和/或时间间隔进行选择，从而使各部分的联合使用能够在治疗疾病或病症时产生大于单独使用任何一种成分所能达到的效果。各成分可以各自呈单独的制剂形式，其制剂形式可以相同也可以不同。
术语“组合疗法”是指施用两种或更多种治疗剂或治疗方式(例如放射疗法或手术，例如支架手术或搭桥手术)以治疗本文所述疾病。这种施用包括以基本上同时的方式共同施用这些治疗剂，例如以具有固定比例的活性成分的单一胶囊。或者，这种施用包括对于各个活性成分在多种或在分开的容器(例如片剂、胶囊、粉末和液体)中的共同施用。粉末和/或液体可以在施用前重构或稀释至所需剂量。此外，这种施用还包括以大致相同的时间或在不同的时间以顺序的方式使用每种类型的治疗剂或治疗方式。在任一情况下，治疗方案将提供组合疗法在治疗本文所述的病症或病状中的有益作用。
本文所述的术语“治疗剂”涵盖行使治疗作用的任何活性剂，例如在预防或治疗肿瘤，例如癌症中有效的任何物质，包括化疗剂、细胞毒性剂、其它抗体、疫苗、小分子药物或免疫调节剂(例如免疫抑制剂或免疫激动剂)；还例如在预防或治疗血栓形成相关疾病或代谢相关疾病中有效的任何物质，例如用于抗血栓形成或溶栓的药物，例如尿激酶、组织型纤溶酶原激活剂、链激酶等。上述治疗剂还涵盖行使治疗作用的活性物质的前体，其随后在体内被转化为活性物质，例如前药，如尿激酶原。
术语“细胞毒性剂”用在本发明中指抑制或防止细胞功能和/或引起细胞死亡或破坏的物质。“化疗剂”包括在治疗癌症或免疫系统疾病中有用的化学化合物。术语“小分子药物”是指低分子量的能够调节生物过程的化合物。“小分子”被定义为分子量小于10kD、通常小于2kD和优选小于1kD的分子。小分子包括但不限于无机分子、有机分子、含无机组分的有机分子、含放射性原子的分子、合成分子、肽模拟物和抗体模拟物。作为治疗剂，小分子可以比大分子更能透过细胞、对降解更不易感和更不易于引发免疫应答。本文使用的术语“免疫调节剂”指抑制或调节免疫应答的天然或合成活性剂或者药物。免疫应答可以是体液应答或细胞应答。免疫调节剂包含免疫抑制剂或免疫激动剂。在一些实施方案中，本发明的免疫调节剂包括免疫检查点抑制剂或免疫检查点激动剂。
用于本文时，“治疗”指减缓、中断、阻滞、缓解、停止、降低、或逆转已存在的症状、病症、病况或疾病的进展或严重性。
用于本文时，“预防”包括对疾病或病症或特定疾病或病症的症状的发生或发展的抑制。在一些实施方式中，具有心血管家族病史的受试者是预防性方案的候选。在血栓形成相关疾病的背景下，预防一般是指在血栓(例如静脉血栓)初步治疗得到缓解后给药，用于预防血栓复发。
“受试者/患者/个体样品”指从患者或受试者得到的细胞或流体的集合。组织或细胞样品的来源可以是实体组织，像来自新鲜的、冷冻的和/或保存的器官或组织样品或活检样品或穿刺样品；血液或任何血液组分；体液，诸如脑脊液、羊膜液(羊水)、腹膜液(腹水)、或间隙液；来自受试者的妊娠或发育任何时间的细胞。组织样品可能包含在自然界中天然不与组织混杂的化合物，诸如防腐剂、抗凝剂、缓冲剂、固定剂、营养物、抗生素、等等。
II.药物递送系统
本发明提供了一种药物递送系统，其包含修饰的血细胞，所述修饰的血细胞通过接头与蛋白质或肽偶联。
(I)接头
在一些实施方案中，所述接头是含有马来酰亚胺基团和磷脂的接头。
在一些实施方案中，所述接头为马来酰亚胺基团-磷脂。本文所用的术语“磷脂”，是指一类脂质，其分子具有包含磷酸基团的亲水性“头部”，和两个衍生自脂肪酸的疏水性“尾部”，由甘油分子连接。磷酸基团可以用简单的有机分子如胆碱、乙醇胺或丝氨酸修饰。
在一些实施方案中，磷脂可以是PC系列，例如DDPC、DLPC、DMPC、DPPC、DSPC、DOPC、POPC和DEPC；PG系列，例如DMPG、DPPG、DSPG和POPG；PA系列，例如DMPA、DPPA、DSPA等；PE系列，例如1，2-二硬脂酰-sn-甘油基-3-磷脂酰乙醇胺(DSPE)、1，2-二丙烯酰-sn-甘油基-3-磷脂酰乙醇胺(DEPE)、1，2-二油酰-sn-甘油基-3-磷脂酰乙醇胺(DOPE)、1，2-二肉豆蔻酰-sn-甘油基-3-磷脂酰乙醇胺(DMPE)和1，2-二棕榈酰-sn-甘油基-3-磷脂酰乙醇胺(DPPE)；以及PS系列，例如DOPS。
在优选的实施方案中，磷脂可以是PE系列，例如1，2-二硬脂酰-sn-甘油基-3-磷脂酰乙醇胺(DSPE)、1，2-二丙烯酰-sn-甘油基-3-磷脂酰乙醇胺(DEPE)、1，2-二油酰-sn-甘油基-3-磷脂酰乙醇胺(DOPE)、1，2-二肉豆蔻酰-sn-甘油基-3-磷脂酰乙醇胺(DMPE)和1，2-二棕榈酰-sn-甘油基-3-磷脂酰乙醇胺(DPPE)。在优选的实施方案中，磷脂是DSPE或DOPE。
在一些实施方案中，所述接头中的马来酰亚胺基团和磷脂之间还通过PEG连接。在一些实施方案中，所述接头是马来酰亚胺基团-PEG-磷脂。在一些实施方案中，PEG的分子量为例如400-5000，例如2000-5000，例如PEG2000、PEG2500、PEG3000、PEG3500、PEG4000、PEG4500或PEG5000。在一些实施方案中，为了分别与马来酰亚胺基团和磷脂基团形成肽键进行连接，PEG接头可以在羧基末端具有游离羧基，且在氨基末端具有游离氨基。在一些实施方式中，用于连接马来酰亚胺基团和磷脂的PEG可以包含NH2-PEG-COOH的结构，例如NH2-(CH2CH2O)n-COOH。
在一些实施方案中，磷脂在其包含磷酸基团的亲水性“头部”的氨基处与马来酰亚胺连接，例如通过或不通过PEG。在一些实施方案中，磷脂的疏水性“尾部”掺入到血细胞的细胞膜中，从而用于连接血细胞。在一些实施方案中，磷脂的包含磷酸基团的亲水性“头部”的氨基处与马来酰亚胺通过PEG或直接连接，同时其疏水性“尾部”掺入到血细胞的细胞膜中以连接血细胞。
在一些实施方案中磷脂为DSPE。在一些具体的实施方案中，所述接头具有如图3所示的化学式。
(II)血细胞
适用于本发明的药物递送系统的血细胞可以是来源于动物的血液的任何细胞，包括例如红细胞、淋巴细胞、T细胞、B细胞、单核细胞、NK细胞、树突细胞或巨核细胞。
在一些实施方案中，所述动物是哺乳动物，例如人。
在一些实施方案中，所述血细胞是来自免疫系统的细胞，例如淋巴细胞(例如T细胞或NK细胞)，或树突细胞。在一些实施方案中，所述细胞是天然细胞。在一些实施方案中，所述细胞是成体细胞。在一些实施方案中，所述细胞是天然成体细胞。
在一些实施方案中，用于制备本发明的血细胞是直接从血液(例如人(例如成人或儿童)血液)中分离的血细胞。
在一些是实施方案中，所述细胞是红细胞，例如人红细胞，例如天然人红细胞，例如天然成体红细胞，例如从人(例如成人或儿童)血液中分离的成熟红细胞。
在一些实施方案中，所述血细胞是自体分离的血细胞，即从待治疗的受试者的血液获得的血细胞。在一些实施方案中，所述血细胞是异体分离的血细胞，即从与待治疗受试者不同的个体(例如健康个体)分离获得的血细胞。
(III)蛋白质或肽
在一些实施方案中，适用于本发明的药物递送系统的蛋白质或多肽包括用于标记的蛋白质或肽，或用于预防或治疗的蛋白质或肽。
本文所述的“用于标记的蛋白质或肽”是指用于其所缀合或偶联的试剂(例如血细胞)检测的任何蛋白质或肽，其本身可以是可检测的(例如，荧光蛋白标记)或在酶促标记的情况下可以催化可检测的底物化合物或组合物的化学改变。用作标记的蛋白质或肽包括例如荧光蛋白(例如绿色荧光蛋白，如eGFP)、辣根过氧化物酶、牛血清白蛋白、血蓝蛋白、鸡卵白蛋白、抗体、牛IgG、鼠IgG、羊IgG、兔IgG、碱性磷酸酶、酸性磷酸酶、葡萄糖氧化酶或β-半乳糖苷酶。
本文所述的“用于预防或治疗的蛋白质或肽”是指可以用作药物来预防或治疗疾病的任何蛋白质，包括例如细胞因子或生长因子或其受体、激素类、酶类、抗体等。
在一些实施方案中，蛋白质是酶类，例如功能性代谢酶或治疗性酶，例如在哺乳动物中的代谢或其他生理过程中起作用的酶。在一些实施方案中，蛋白质是在碳水化合物代谢、氨基酸代谢、有机酸代谢、卟啉代谢、嘌呤或嘧啶代谢和/或溶酶体贮积中起作用的酶。
在一些实施方案中，适用于本发明的药物递送系统中所含的蛋白质是功能性代谢酶。示例性的酶包括尿酸氧化酶，例如来自黄曲霉的尿酸氧化酶。在一些实施方案中，所述尿酸氧化酶包含信号肽(例如包含SEQ ID NO:14所示的氨基酸序列或由其组成)和/或标签(例如His标签)，例如在其N末端或在其C末端。在一些实施方案中，所述尿酸氧化酶包含SEQ ID NO:5或6所示的氨基酸序列，或由所述氨基酸序列组成。在一些实施方案中，所述尿酸氧化酶包含与SEQ ID NO:5或6所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％、99％或更高的同一性的氨基酸序列。
在一些实施方案中，适用于本发明的药物递送系统中所含的蛋白质是蛋白酶，例如丝氨酸蛋白酶，例如尿激酶原，如人尿激酶原。在一些实施方案中，所述人尿激酶原包含信号肽(例如包含SEQ ID NO:14所示的氨基酸序列或由其组成)和/或标签(例如His标签)，例如在其N末端或在其C末端。在一些实施方案中，所述尿激酶原包含SEQ ID NO:10或13所示的氨基酸序列，或由所述氨基酸序列组成。在一些实施方案中，所述尿激酶原包含与SEQ ID NO:10或13所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％、99％或更高的同一性的氨基酸序列。在一些实施方案中，所述尿激酶原由SEQ ID NO:11所示的核苷酸序列所编码。在一些实施方案中，所述尿激酶原由与SEQ ID NO:11所示的核苷酸序列具有至少85％、90％、95％、96％、97％、98％、99％或更高的同一性的核苷酸序列所编码。
在一些实施方案中，适用于本发明的药物递送系统中所含的肽是标记肽或各种功能肽。
在一些实施方案中，适用于本发明的药物递送系统中所含的蛋白质是抗体或其抗原结合片段。在一些实施方案中，适用于本发明的抗体是IgG1形式的抗体或IgG2形式的抗体或IgG3形式的抗体或IgG4形式的抗体。在一些实施方案中，适用于本发明的抗体是单克隆抗体。在一些实施方案中，适用于本发明的抗体是人源化的抗体。在一些实施方案中，适用于本发明的抗体是人抗体。在一些实施方案中，适用于本发明的抗体是嵌合抗体。在一个实施方案中，适用于本发明的抗体是全长抗体。在一个实施方案中，适用于本发明的抗体的抗原结合片段选自以下的抗体片段：Fab、Fab’、Fab’-SH、Fv、单链抗体(例如scFv)、(Fab’)2、单结构域抗体例如VHH、dAb(domain antibody)或线性抗体或半抗体。
示例性抗体包括免疫检查点分子的抗体，例如特异性结合PD-1、PD-L1或PD-L2的抗体，例如PD-1抗体。在本发明的一个实施方案中，所述针对PD-1的抗体或其抗原结合片段为CN108473977B、CN104250302B、CN105531288B、CN105026428B、WO2008156712A1或WO2006121168A1中公开的抗PD-1抗体或其抗原结合片段，例如帕博利珠单抗、卡瑞利珠单抗、替雷利珠单抗、特瑞普利单抗、信迪利单抗或纳武利尤单抗或其抗原结合片段(参见例如CN108473977B、CN104250302B、CN105531288B、CN105026428B、WO2008156712A1或WO2006121168A1)。在一个实施方案中，所述抗PD-1抗体或其抗原结合片段包含帕博利珠单抗、卡瑞利珠单抗、替雷利珠单抗、特瑞普利单抗、信迪利单抗或纳武利尤单抗或其抗原结合片段的一个或多个CDR(优选3个CDR，即HCDR1、HCDR2H和HCDR3；或LCDR1、LCDR2和LCDR3，更优选6个CDR，即HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3)，或包含帕博利珠单抗、卡瑞利珠单抗、替雷利珠单抗、特瑞普利单抗、信迪利单抗或纳武利尤单抗或其抗原结合片段的VH和/或VL，或包含所述抗体的重链和/或轻链。在一些实施方案中，本发明的抗PD-1抗体或其抗原结合片段包含来自帕博利珠单抗、卡瑞利珠单抗、替雷利珠单抗、特瑞普利单抗、信迪利单抗或纳武利尤单抗的重链可变区CDR1、CDR2和CDR3，以及轻链可变区CDR1、CDR2和CDR3。在一些实施方案中，本发明的抗PD-1抗体或其抗原结合片段包含来自帕博利珠单抗、卡瑞利珠单抗、替雷利珠单抗、特瑞普利单抗、信迪利单抗或纳武利尤单抗的重链可变区和轻链可变区。在一些实施方案中，本发明的抗PD-1抗体或其抗原结合片段包含来自帕博利珠单抗、卡瑞利珠单抗、替雷利珠单抗、特瑞普利单抗、信迪利单抗或纳武利尤单抗的重链和轻链。
在一些实施方案中，适用于本发明的药物递送系统中所含的蛋白质是细胞因子或生长因子或其受体，或所述蛋白的功能性片段或结构域。
在一些实施方案中，适用于本发明的药物递送系统中所含的蛋白质或肽是可以用作抗原的蛋白质或肽。抗原可以是天然存在的或合成的，例如由病原体、感染的细胞、肿瘤细胞(例如肿瘤或癌细胞)、病毒、细菌、真菌或寄生虫天然产生和/或遗传编码的抗原。在一些实施方案中，抗原是自身抗原或移植物相关抗原。在一些实施方案中，抗原是例如病毒衣壳、包膜或外壳，或细菌、真菌、原生动物或寄生虫细胞的表面蛋白。在一些实施方案中，抗原是本领域已知的疫苗中使用的任何抗原。在一些实施方案中，抗原是肿瘤抗原(TA)。
在一些实施方中，适用于本发明的药物递送系统中所含的蛋白质为“前药”，即在体内施用后代谢或以其它方式转化成生物学、药学或治疗活性形式的蛋白质。
用于药物递送系统的蛋白质或肽经处理具有暴露的或游离的硫醇基，用于与马来酰亚胺发生迈克尔加成反应形成稳定的碳硫键，例如，所述蛋白质或肽包含半胱氨酸用于提供游离的硫醇基。
在一些实施方案中，所述蛋白质或肽(例如抗体)是通过硫醇类还原剂(例如TCEP)处理后(例如与其共孵育)所获得的蛋白质或肽，其包含含有游离硫醇基的半胱氨酸残基。
在一些实施方案中，所述蛋白质或肽(例如酶类)是经过插入1个或多个(例如1-10个)半胱氨酸来获得的蛋白质或肽，其包含含有游离硫醇基的半胱氨酸残基。所述半胱氨酸可以在蛋白质或肽的任何位置插入，只要其插入不影响蛋白质或肽的功能即可。优选地，所述半胱氨酸被插入到不影响蛋白质或肽结构和功能的对应于蛋白质或肽三维结构表面的位置上。在一些实施方案中，所述半胱氨酸被插入到蛋白质或肽的C末端或N末端，例如插入在最N末端氨基酸前面或插入在最C末端氨基酸后面。
在一些实施方案中，所述蛋白质或肽还包含适用于纯化的标签，例如(His)n(例如n＝1、2、3、4、5、6)，所述标签例如位于蛋白质或肽的C末端或N末端。
在一些实施方案中，所述蛋白质或肽还包含信号肽，例如其包含SEQ ID NO:14所示的氨基酸序列或由所述序列组成。
在一些实施方案中，包含插入的半胱氨酸的绿色荧光蛋白包含SEQ ID NO:1或2所示的氨基酸序列，或由所述氨基酸序列组成。在一些实施方案中，所述绿色荧光蛋白包含与SEQ ID NO:1或2所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％、99％或更高的同一性的氨基酸序列，其在最C末端具有半胱氨酸。
在一些实施方案中，包含插入的半胱氨酸的尿酸氧化酶包含SEQ ID NO:3或7所示的氨基酸序列，或由所述氨基酸序列组成。在一些实施方案中，所述尿酸氧化酶包含与SEQ ID NO:3或7所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％、99％或更高的同一性的氨基酸序列，其在最C末端具有半胱氨酸。在一些实施方案中，所述尿酸氧化酶由SEQ ID NO:4所示的核苷酸序列所编码。在一些实施方案中，所述尿酸氧化酶由与SEQ ID NO:4所示的核苷酸序列具有至少85％、90％、95％、96％、97％、98％、99％或更高的同一性的核苷酸序列所编码，其在其最C末端具有半胱氨酸。
在一些实施方案中，包含插入的半胱氨酸的尿激酶原包含SEQ ID NO:8或12所示的氨基酸序列，或由所述氨基酸序列组成。在一些实施方案中，所述尿激酶原包含与SEQ ID NO: 8或12所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％、99％或更高的同一性的氨基酸序列，其在最C末端具有半胱氨酸。在一些实施方案中，所述尿激酶原由SEQ ID NO:9所示的核苷酸序列所编码。在一些实施方案中，所述尿激酶原由与SEQ ID NO:9所示的核苷酸序列具有至少85％、90％、95％、96％、97％、98％、99％或更高的同一性的核苷酸序列所编码，其在其最C末端具有半胱氨酸。
III.药物递送系统的方法
在一个实施方案中，本发明涉及一种获得本发明的药物递送系统的方法，其包括：
(1)提供包含马来酰亚胺和磷脂的接头；
(2)提供一种蛋白质或肽，其包含游离的硫醇基；
(3)使(1)中所述接头与血细胞一起孵育，进而将其中的磷脂掺入所述细胞的细胞膜中；
(4)将步骤(3)中制备的细胞与(2)中的蛋白质的硫醇基在适合于发生迈克尔加成反应的条件下一起孵育；和
(5)获得包含通过接头偶联的蛋白质和血细胞作为药物递送系统。
在一个实施方案中，本发明涉及一种获得本发明的药物递送系统的方法，其包括：
(1)提供包含马来酰亚胺和磷脂的接头；
(2)提供一种蛋白质或肽，其包含游离的硫醇基；
(3)使(1)中的接头与(2)中的蛋白质的硫醇基在适合于发生迈克尔加成反应的条件下一起孵育，获得与磷脂偶联的蛋白质；
(4)将步骤(3)中获得的蛋白质与血细胞一起孵育，进而将其中的磷脂掺入所述细胞的细胞膜中；和
(5)获得包含通过接头偶联的蛋白质和血细胞作为药物递送系统。
应了解，所属领域的技术人员能够根据马来酰亚胺、硫醇基等的性质选择适合于与磷脂连接的马来酰亚胺(或通过磷脂与血细胞偶联的马来酰亚胺)与暴露于蛋白质或肽上的硫醇基进行迈克尔加成反应的条件，例如，最佳温度、pH、反应时间、浓度。
还应理解，本领域普通技术人员能够选择合适的条件(例如，生理条件，例如最佳温度(例如，37℃)、pH、反应时间、浓度)来孵育包含磷脂的接头(或与包含磷脂的接头偶联的蛋白质或肽)从而将磷脂掺入细胞的细胞膜中。
IV.药物组合物或制剂
在一方面，本发明提供了包含本发明的药物递送系统的药物组合物或制剂。这些组合物或制剂还可以任选地包含合适的药用辅料，如本领域中已知的药用载体、药用赋形剂，包括缓冲剂。
在一个方面，本发明提供了包含本发明的药物递送系统的血液制剂，例如人血制剂。在一些实施方案中，本发明的人血制剂包含0.5-1000μg/mL的药物递送系统，例如1-500μg/mL，1-50μg/mL、1-10μg/mL、0.5μg/mL-5μg/mL、0.5μg/mL-3μg/mL或1μg/mL-3μg/mL、 的药物递送系统，例如在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、30μg/mL、35μg/mL、40μg/mL、45μg/mL、50μg/mL、60μg/mL、70μg/mL、80μg/mL、90μg/mL、100μg/mL、150μg/mL、200μg/mL、250μg/mL、300μg/mL、350μg/mL、400μg/mL、450μg/mL、或500μg/mL以上，或在所述数值的任何范围内。
在一些实施方案中，包含在血液制剂中的药物递送系统为包含红细胞的药物递送系统。因此，在一些实施方案中，本发明的血液制剂是减白血液制剂，即滤除了白细胞的血液制剂。在一些实施方案中，本发明的血液制剂是人血减白血液制剂。
在一些实施方案中，本发明的血液制剂可以是异体血液制剂，例如异体人血制剂。在一些实施方案中，血液制剂中的血细胞(例如红细胞)来自健康受试者。
在一些实施方案中，本发明的血液制剂可以是自体血液制剂，例如自体人血制剂。在一些实施方案中，血液制剂中的血细胞(例如红细胞)来自待治疗的受试者。
在一些实施方案中，本发明的血液制剂或组合物可以包含药物递送系统和未被修饰的血细胞(例如红细胞)。在一些实施方案中，组合物中至少选定百分比的血细胞(例如红细胞)被修饰，即与蛋白质或肽偶联获得本发明的药物递送系统。例如，在一些实施方案中，至少5％、10％、15％、20％、25％、30％、40％、50％、60％、70％、80％、90％、95％、96％、97％、98％、99％或更多的细胞为本发明的药物递送系统。在一些实施方案中，本发明的血液制剂或组合物可以包含两种或多种本发明的药物递送系统。
在一些实施方案中，组合物包含本发明的药物递送系统。在一些实施方案中，组合物包含有效量的本发明的药物递送系统，例如，至多约1014个细胞，例如，约10、102、103、104、105、5×105、106、5×106、107、5×107、108、5×108、109、5×109、1010、5×1010、1011、5×1011、1012、5×1012、1013、5×1013、1014或5×1014个修饰的血细胞(药物递送系统)。在一些实施例中，修饰的血细胞的数量可以在前述数量中的任意两个之间的范围内。
如本文所用，术语“有效量”是指足以实现感兴趣的生物学应答或作用的量，例如，减少疾病或病症的一种或多种症状或表现或调节免疫应答。在一些实施方案中，施用于受试者的组合物包含至多约1014个修饰的血细胞(药物递送系统)，例如约103、104、105、106、107、108、109、1010、1011、1012、1013或1014个修饰的血细胞(药物递送系统)，或任何介于其间的数目或范围。
在一方面，本发明还提供了组合产品(例如药物组合产品)，其包含本发明的药物递送系统，以及一种或多种其它治疗剂。本发明的组合产品可用于本发明的治疗方法中。
本发明还提供了包含所述组合产品的成套药盒，例如所述成套药盒在同一包装内包含：
-含有本发明的药物递送系统或包含其的药物组合物或制剂的第一容器；
-包含一种或多种其它治疗剂或包含其的药物组合物或制剂的第二容器。
在一些实施方案中，所述其它治疗剂选自在所述药物系统待治疗疾病中有效的任何物质，例如在代谢相关疾病、血栓形成相关疾病或癌症中有效的任何物质，例如包括化疗剂、细胞毒性剂、其它抗体、疫苗、小分子药物或免疫调节剂(例如免疫抑制剂或免疫激动剂；用于抗血栓形成或溶栓的药物，例如尿激酶、组织型纤溶酶原激活剂、链激酶等。
V.预防或治疗方法或用途
(I)适应症
肿瘤或癌症
在一方面中，本发明涉及预防或治疗受试者疾病，如癌症的方法，所述方法包括向所述受试者施用有效量的本文所述的药物递送系统。在一些具体的实施方案中，所述药物递送系统包含用于预防或治疗肿瘤的抗体(例如PD-1抗体)。
癌症可以处于早期、中期或晚期或是转移性癌。在一些实施方案中，癌症可以是实体肿瘤或血液肿瘤。在一些实施方案中，所述癌症是胃肠道肿瘤，例如结肠癌或结直肠癌；或者是肺癌、胰腺癌或乳腺癌。在一些实施方案中，所述肿瘤是对已知药物，例如已知PD-1抑制剂，例如抗PD-1抗体具有耐受性或不敏感的肿瘤或癌症，例如难治性肿瘤或癌症。
在一些实施方案中，所述肿瘤或癌症是表征为具有升高的PD-1、PD-L1和/或PD-L2的蛋白质水平和/或核酸水平(例如表达升高)的癌症(例如与健康个体或同一个体的健康组织相比)，例如，所述癌症的肿瘤细胞中具有升高的PD-1、PD-L1和/或PD-L2的蛋白质水平和/或核酸水平(例如表达升高)(例如与来自健康个体的健康细胞或与来自同一个体的健康组织的健康细胞相比)。
血栓形成相关疾病
在一个方面，本发明涉及抗血栓形成或溶栓或预防或治疗血栓形成相关疾病的方法，所述方法包括向所述受试者施用有效量的本文所述的药物递送系统。在一些具体的实施方案中，所述药物递送系统包含用于抗血栓形成或溶栓或预防或治疗血栓形成相关疾病的蛋白质或肽，例如尿激酶原。
血栓形成相关疾病，如缺血性中风、急性心肌梗塞和深静脉血栓形成，仍然是全球死亡或残疾的主要原因，血栓预防有助于降低发病率和死亡率。目前使用的溶栓药物纤溶酶原激活剂往往由于其在体内的半衰期短，发生中风时需要在短时间内大量注射，并且由于其可引起如流出血管后的出血、组织重塑和神经毒性等严重的副作用，其临床应用受到很大限制，不适合预防血栓形成。为了克服这类溶栓药物在临床上的局限性，对纤溶酶原激活剂进行了多种修饰，以延长药物在血管中的停留时间，但这些修饰并不适合预防血栓形成。
红细胞具有明确的生物学功能、安全性和长的循环半衰期。作为天然药物载体，红细胞被限制在血管中，可有效延长血栓预防或治疗药物在体内的半衰期，从而达到应用极小剂量就能治疗和/或预防血栓形成的功能。同时，由于药物被固定在细胞上，还可以避免药物从血管渗出引起的如组织重塑和神经毒性等副作用。此外，由于红细胞是血栓的重要组成部分，利用红细胞携带抗血栓形成药物可以在血栓形成之初从内部将其分解。这种方法大大减少了剂量，可以有效避免现有的严重副作用。因此，应用红细胞携带用于预防或治疗血栓形成相关疾病的蛋白质或肽(例如尿激酶原)，能够有效用于血栓形成的治疗和/或预防。
在一些实施方案中，血栓形成相关疾病包括例如中风如缺血性中风、心肌梗塞(例如急性心肌梗塞)和静脉血栓例如深静脉血栓。
代谢相关疾病
代谢相关疾病是指与哺乳动物的代谢异常有关的疾病，其通常的特征在于缺乏功能酶或代谢物的过度摄入。因此，代谢物在循环和组织中的沉积引起组织损伤。代谢相关疾病例如与碳水化合物代谢、氨基酸代谢、有机酸代谢、嘌呤或嘧啶代谢缺陷相关的疾病。
由于血细胞如RBC在人体中的广泛分布，本发明考虑应用携带功能性代谢酶的血细胞用于代谢相关疾病的治疗。例如，包含功能性代谢酶的血细胞例如红细胞将摄取患者血浆中的代谢物。示例性的酶包括用于痛风的尿酸氧化酶。
在一些实施方式中，代谢相关疾病包括例如慢性高尿酸血症，例如痛风，例如难治性的痛风(对其他常规疗法耐受的痛风)。
(II)治疗方法或用途
本发明的药物递送系统(以及包含其的组合物、药物组合物、制剂、组合产品等，例如血液制剂)可以通过任何合适的方法给药，优选输注，例如肠胃外输注。肠胃外输注包括静脉内或动脉内给药。优选地，通过静脉或动脉输注施用本发明的药物递送系统。
为了预防或治疗疾病，本发明的药物递送系统的合适剂量(当单独或与一种或多种其他的治疗剂组合使用时)将取决于待治疗疾病的类型、抗体的类型、疾病的严重性和进程、以预防目的施用还是以治疗目的施用、以前的治疗、患者的临床病史和对所述抗体的应答，和主治医师的判断力。所述抗体以一次治疗或经过一系列治疗合适地施用于患者。
在一些实施方案中，受试者在治疗过程中接受单剂量的细胞，或接受多剂量的细胞，例如2至5、10、20或更多剂量。在一些实施方案中，剂量或总细胞数可以表示为细胞/kg。例如，剂量可以是约103、104、105、106、107、108个细胞/kg。在一些实施方案中，治疗过程持续约1周至12个月或更长时间，例如1、2、3或4周或2、3、4、5或6个月。在一些实施方案中，受试者可以约每2-4周治疗一次。本领域普通技术人员将理解，细胞的数量、剂量和/或给药间隔可以基于各种因素选择，例如受试者的体重和/或血量、所治疗的病症、受试者的反应等。所需细胞的确切数目可因受试者而异，这取决于诸如受试者的物种、年龄、体重、性别和一般状况、疾病或病症的严重性、特定细胞、与细胞缀合的药剂的身份和活性、施用模式、并行疗法等因素。
在一些实施方案中，本发明的药物递送系统，或包含其的药物或制剂，与一种或多种治疗方式或其他治疗剂联合施用。
在一些实施方案中，所述治疗方式或其他治疗剂用于治疗肿瘤，例如癌症。
在一些实施方案中，所述治疗方式是放疗或手术治疗。在一些实施方案中，所述治疗剂选自癌症中有效的任何物质，包括化疗剂、其他抗体、细胞毒性剂、疫苗、小分子药物或免疫调剂(例如免疫抑制剂或免疫激动剂)。
在一些实施方案中，所述治疗方式或治疗剂用于预防或治疗血栓形成相关疾病。
在一些实施方案中，所述治疗方式例如支架或搭桥。在一些实施方案中，所述治疗剂例如用于抗血栓形成或溶栓的药物，例如尿激酶、组织型纤溶酶原激活剂、链激酶等。
在一些实施方案中，所述治疗方式或治疗剂用于治疗代谢相关疾病。
再一方面，本发明也提供本发明药物递送系统在制备用于前述方法(例如用于治疗)的药物或制剂中的用途。
在一些实施方案中，本发明的药物递送系统中的血细胞来自待治疗的受试者。在一些实施方案中，本发明的治疗受试者疾病的方法包括：
(1)从待治疗的受试者采血；
(2)制备本发明的药物递送系统，例如通过本发明所述的方法；
(3)将治疗有效量的药物递送系统输注入受试者。
在一些实施方案中，本发明的药物递送系统中的血细胞可以来自其他受试者，例如健康受试者。在一些实施方案中，本发明的药物递送系统可以作为药物或制剂施用给待治疗的受试者。
本文中所引用的任何文献，包括专利、专利申请和文献，均以其全文引入本文。
本发明上文以及整个本申请中所论述的任何或所有特征可以在本发明的各种实施方案中组合。以下实施例进一步说明本发明，然而，应理解实施例以说明而非限定的方式来描述，不意在且不应当以任何方式限制本发明的保护范围，并且本领域技术人员可以进行多种修改。
实施例：
材料
ProUK购自Tasly Biopharma(China)。ProUK-cys利用HEK293F细胞表达并纯化。凝血酶购自Sigma-Aldrich(USA)。抗尿激酶/uPA-HRP(Clone#05)和抗尿激酶/uPA(Clone#1A2G4G11)购自Sino Biological Inc(China)。MAL-PEG2000-DSPE购自Xi’an ruixi Biological Technology Co.,Ltd(China)。CaCl2和FeCl3购自Sionpharm Chemical Reagent Co.,Ltd(China)。所有其他化学品和溶剂均购自Sionpharm Chemical Reagent Co.,Ltd(China)。
未标记的红细胞或洗涤的红细胞是指通过密度梯度离心法从健康个体外周血中分离的红细胞，所述分离的红细胞用PBS洗涤3次去除其他成分(例如血浆或其他血细胞)。
实验动物
C57BL/6J小鼠或SD大鼠购自西湖大学动物管理和使用委员会(Institutional Animal Care and Use Committee)。动物按照机构指南进行处理，所有动物研究均得到西湖大学动物管理和使用委员会的批准(IACUC协议#19-012-GXF)。
统计分析
在所有图表中，数据表示为平均值±标准差。单因素方差分析用于分析涉及多组处理的数据。所有统计分析均使用适用于Windows的GraphPad Prism 8.4版进行。在P值<0.05时，组间差异被认为是显著的。
实施例
实施例1通过使用DSPE的eGFP标记的红细胞的制备
eGFP用于测试反应的结合效率。eGFP在C末端融合表达一个半胱氨酸并纯化(eGFP-Cys)。合成的Mal-PEG2000-DSPE接头(由西安瑞禧生物合成)纯度超过99％。
1.1重组蛋白在大肠杆菌中的表达和纯化
将eGFP-Cys cDNA克隆到pET载体中并转化到大肠杆菌BL21(DE3)细胞中进行蛋白质表达。转化的细胞在37℃下培养直到OD600达到0.6-0.8，然后加入500μM IPTG。将细胞与IPTG在37℃下培养4小时，通过离心收获并用预冷的裂解缓冲液(20mM Tris-HCl,pH 7.8,500mM NaCl)进行裂解。裂解物在冰上进行超声处理(5秒开，5秒关，60个循环，25％功率，Branson Sonifier 550超声波细胞破碎仪)。所有上清液在4℃下14,000x g离心40分钟后用0.45μM过滤器过滤。将过滤后的上清液上样到与设计色谱系统相连的HisTrap FF 1mL色谱柱(GE Healthcare)上。用含有20mM Tris-HCl、pH7.8、500mM NaCl和300mM咪唑的洗脱缓冲液洗脱蛋白质。所有洗脱的组分在SDS-PAGE凝胶上分析。由此获得eGFP-Cys蛋白质。eGFP-Cys的氨基酸序列如以下SEQ ID NO:1所示。
1.2接头的获得
Mal-PEG2000-DSPE接头购自西安瑞禧生物，其化学式如图3所示，纯度超过99％。
1.3红细胞标记:方法1
如图1所示，可以先将Mal-PEG-磷脂(例如Mal-PEG-DSPE)通过磷脂(例如DSPE)与红细胞连接，获得在细胞膜表面具有马来酰亚胺基团的预修饰的红细胞，然后将预处理含有暴露的巯基的蛋白质与预修饰的红细胞孵育，马来酰亚胺可以与蛋白质上暴露的半胱氨酸上的巯基发生迈克尔加成反应，形成稳定的共价结合，进而获得将靶标蛋白与红细胞的细胞膜相连。具体实验步骤如下：
1.3.1红细胞预处理
通过密度梯度离心法从C57BL/6J小鼠外周血中分离红细胞。将分离的红细胞用PBS洗涤3次。将Mal-PEG2000-DSPE溶解在37℃的磷酸盐缓冲液中至终浓度为100μM。然后用50μM MAL-PEG2000-DSPE在37℃下预处理1×109个红细胞30分钟。然后将预处理的红细胞用PBS洗涤3次。预处理的红细胞立即使用或不可长期储存。
1.3.2 MAL-PEG2000-DSPE介导的红细胞的标记
将上述预处理后的红细胞与eGFP-cys混合在PBS缓冲液中，其中红细胞的浓度为1×109个/mL，eGFP-cys的浓度为5、25、50和100μM。反应混合液总体积为200μL，在10rpm下旋转1小时进行反应。停止反应，用PBS洗涤，去除其余反应试剂，获得标记的红细胞，用Beckman Coulter CytoFLEX LX分析红细胞的标记效率(即偶联了蛋白质的红细胞占总红细胞的百分比)。未通过该方法标记的细胞未检测到eGFP信号，而通过该方法标记的细胞eGFP阳性率为100％。
1.4红细胞标记：方法2(图2)
马来酰亚胺基团可以与暴露的半胱氨酸上的巯基发生迈克尔加成反应，但未暴露的半胱氨酸不会发生这种反应。连接到聚乙二醇链的马来酰亚胺通常用作柔性连接分子以将蛋白质附着到表面。我们利用这一原理对蛋白质进行修饰，使目标蛋白质带有可插入细胞膜的接头(图2)。
如图2所示，具有暴露的半胱氨酸(例如C末端的半胱氨酸)的蛋白质与马来酰亚胺基团发生迈克尔加成反应，从而将蛋白质和DSPE以稳定的共价键形式连接。之后，DSPE端可以与红细胞细胞膜融合形成稳定的连接，进而靶标蛋白被加载到细胞膜上。其具体实验步骤如下：
1.4.1蛋白质预处理
将接头Mal-PEG2000-DSPE和eGFP-cys蛋白分别加入PBS中，终体积为1mL，Mal-PEG2000-DSPE和eGFP-cys蛋白的终浓度分别为2mM和1mM，即这两种反应底物以1:2＝eGFP-cys:MAL-PEG2000-DSPE的比例混合用于反应(图4)。将反应缓冲液在10rpm的速度下旋转孵育1小时。反应结束后，通过蛋白质纯化透析和超滤去除多余的接头收集eGFP-CysMal-PEG2000-DSPE产物。
1.4.2 eGFP-Cys-MAL-PEG2000-DSPE介导的红细胞的标记
将上述红细胞和eGFP-cys-Mal-PEG2000-DSPE在PBS缓冲液中混合，总体积200μL，终浓度红细胞为1×109个/mL，eGFP-cys-Mal-PEG2000-DSPE分别为5、25、50、100μM。将反应混合液以10rpm的速度旋转孵育1小时，获得红细胞-蛋白质偶联物。停止反应，用PBS洗涤，去除其余反应试剂，获得标记的红细胞，用Beckman Coulter CytoFLEX LX分析红细胞的标记效率。(即偶联了蛋白质的红细胞占总红细胞的百分比)，其中应用未标记的红细胞作为对照组。
1.5标记功效表征
eGFP的标记效率在RBC膜上表征。反应结束后，上述1.3.2制备的(eGFP-cys-Mal-PEG-DSPE)和未经eGFP标记(unlabeled)的红细胞(红细胞量分别为1×109个/mL)分别通过流式细胞仪检测eGFP的标记效果。如图5所示，直方图显示细胞表面上的eGFP信号。结果表明，100％的天然红细胞在体外被eGFP-Cys-MAL-PEG2000-DSPE标记(图5)，信号强度呈剂量依赖性(参见图6，其中红色：未标记；蓝色：应用5μM eGFP-cys-Mal-PEG-DSPE标记；橙色：应用25μM eGFP-cys-Mal-PEG-DSPE标记；浅绿色：应用50μM eGFP-cys-Mal-PEG-DSPE标记；深绿色：应用100μM eGFP-cys-Mal-PEG-DSPE标记)。如图5和图6所示，与红细胞与eGFP-Cys-MAL-PEG-DSPE孵育后，红细胞表面的eGFP信号显著高于未标记的红细胞。
1.6 eGFP-Cys-MAL-PEG-DSPE标记的红细胞的体内寿命
为了评估这些表面修饰的红细胞在体内的寿命，我们接下来将实施例1.3制备的eGFP-cys-Mal-PEG-DSPE标记的小鼠红细胞(eGFP-cys-mal-PEG-DSPE-RBC)和未标记的红细胞(unlabeled)分别用荧光染料Cell Trace Far Red染料染色标记后，分别尾静脉输注到小鼠中，每组3只6-8周龄的C57BL/6J小鼠，输注量为1×109个细胞/每只。
在输注后第1、3、4、7、8、11、15、18、22、25、29、34天对小鼠进行脸颊取血。
通过流式细胞术分析第29天的血液样品。选择Cell Far red阳性细胞用于分析具有eGFP信号的红细胞百分比。结果见图7。图7显示，标记的细胞组(eGFP-cys-mal-PEG-DSPE-RBC)中，Far red阳性细胞均为eGFP阳性，表明该细胞依然搭载有eGFP蛋白。
通过流式细胞术分析第1、3、4、7、8、11、15、18、22、25、29、34天的血液样品。选择Far red阳性细胞用于分析具有eGFP信号的红细胞百分比。Far red阳性细胞表示血液循环中输注的红细胞的百分比。我们发现，eGFP-Cys-MAL-PEG-DSPE标记的红细胞(eGFP-cys-mal-PEG-DSPE-RBC)不仅显示出与对照组(输注了缺乏eGFP-Cys-MAL-PEG-DSPE标记的RBC的小鼠，control RBC)的红细胞相同的寿命，而且在周期中表现出持续34天的eGFP信号(参见图8)。未观察到明显溶血。
实施例2.通过使用DSPE的抗PD-1抗体标记的RBC的制备
为了进一步确认这种方法可以应用于不同类型蛋白质的标记，我们使用了抗PD-1抗体(由Merck Sharp & Dohme Corp.制备)用于标记。
将10μM抗PD-1抗体在室温下用0.2mM TCEP预处理1小时，获得半胱氨酸暴露的抗体。在预处理之后，通过透析和超滤去除过量的TCEP来收集预处理的蛋白质。预处理的蛋白质立即使用或不可长期保存。
然后，如实施例1.3所述制备抗PD-1抗体标记的RBC，区别在于抗体浓度为5μM，其中用抗PD-1抗体代替eGFP-cys。对照为应用如实施例1.3所述的将5μM的抗PD-1抗体与未与Mal-PEG-DSPE偶联的红细胞进行孵育。通过流式细胞术检测标记效率。直方图显示细胞表面的PD-1抗体信号(图9，其中Unlabeled表示应用未与Mal-PEG-DSPE偶联的红细胞与抗PD-1抗体孵育后的结果；Anti-PD-1-Ma-PEG-DSPE表示与Mal-PEG-DSPE偶联的红细胞与抗PD-1抗体孵育后的结果)。图10显示了PD-1抗体的信号强度，其中蓝色：应用未与Mal-PEG-DSPE偶联的红细胞与抗PD-1抗体孵育后的抗PD-1抗体的信号强度；，红色：应用与Mal-PEG-DSPE偶联的红细胞与抗PD-1抗体孵育后的抗PD-1抗体的信号强度。结果显示这种方法将抗体有效的标记到细胞表面。
实施例3.通过使用DSPE的尿酸氧化酶(UOX)标记的红细胞的制备
3.1大肠杆菌中的重组蛋白表达和纯化
将UOX-Cys(黄曲霉尿酸酶)(氨基酸序列：SEQ ID NO:3；核酸序列SEQ ID NO:4)的编码序列进行密码子优化以在大肠杆菌中表达并由GenScript合成。通过标准PCR程序产生亚克隆，并将亚克隆插入pET-30a(GenScript)载体中，所述pET-30a载体的C末端His6接头带有额外的半胱氨酸残基。所有构建体均通过测序验证，然后在大肠杆菌BL21(DE3)中转化以进行蛋白质表达。
将单个转化菌落接种到补充有氨苄青霉素(100μg/ml)的10ml Luria-Bertani(LB)培养基中，在37℃下以220rpm振摇过夜培养。将该10ml培养物转移到1L新鲜的LB培养基中，并在37℃下以220rpm振荡培养培养物，直到OD600达到0.6。然后将温度降至20℃并添加1mM IPTG用于诱导。
在通过在4℃下以8,000rpm离心10分钟进行诱导后的20h收获细胞。对于没有His6标签的蛋白质，将细胞沉淀重悬在低盐裂解缓冲液(50mM Tris 7.5,50mM NaCl)中并用超声处理裂解。将以10,000rpm离心1h后收集的上清液上样到用SPA缓冲液(20mM Tris 7.5)预平衡的SP Sepharose FF柱(Cytiva,Marlborough,USA)上。用SPA缓冲液洗涤柱直到280nm处的吸光度和电导率变得稳定，然后使用20mM Tris 7.5中的0-1M NaCl线性梯度洗脱。通过SDS-PAGE分析对应于洗脱峰的级分，并富集纯度最高的级分。为避免半胱氨酸氧化，将2mM TCEP添加到组合馏分中，并使用Amicon Ultra-15离心过滤装置(Millipore,Darmstadt,Germany)进行样品浓缩。将浓缩蛋白上样至用PBS预平衡的EzLoad 16/60 Chromdex 200pg(Bestchrom,Shanghai,China)，收集目标蛋白峰。对于带有His6标签的蛋白质，将细胞沉淀重悬在裂解缓冲液(50mM Tris 7.5,200mM NaCl,5mM咪唑)中并用超声处理裂解。在Ni Sepharose 6FF亲和柱(Cytiva)和阴离子交换柱上纯化标记的蛋白质UOX-His6-Cys，然后进行尺寸排阻色谱。所有蛋白质均储存在-80℃。
3.2红细胞标记
UOX标记的RBC通过如实施例1.3中所述的方法1制备，其中用UOX代替eGFP，获得UOX-Cys-Mal-PEG-DSPE标记的红细胞。
UOX-His6-Cys-Mal-PEG-DSPE标记的RBC的体内寿命
我们将UOX-His6-Cys-Mal-PEG-DSPE标记的小鼠红细胞(剂量：1×109个/小鼠)尾静脉输注到2只受体小鼠(6-8周龄的C57BL/6J小鼠，输注量为1×109个细胞/每只，分别用UOX1和UOX2表示)中，这些红细胞同时被荧光染料Cell Far Red染色标记。定期分析体内Cell Far red和UOX-Cys-Mal-PEG-DSPE阳性RBC的百分比，方法同实施例1.5，但是在第1、3、6、10、12、17、20天采血分析，其中第20天采血的样品还应用流式细胞分析(图11)。我们发现，UOX-Cys-Mal-PEG-DSPE标记的红细胞在2只受体小鼠中显示出相同的寿命，并且在周期中表现出超过20天的持续信号(参见图11和12)。
实施例4.通过使用DSPE的eGFP标记的其他哺乳动物细胞的制备
我们进一步验证了该方法是否适用于其他血细胞。我们测试了这种方法是否对T细胞、B细胞、单核细胞、NK细胞和巨核细胞有效。
PBMC从健康志愿者的人外周血中分离。全血用磷酸盐缓冲液1:1稀释，外周血单核细胞(PBMC)通过使用淋巴细胞分离溶液(LymphoprepTM,STEMCELL Technologies)和淋巴细胞分离管以1200x g离心15分钟分离。
本步骤的目的是通过淋巴细胞分离溶液实现细胞成分的密度梯度离心，将PBMC从如红细胞、血小板等不同细胞中分离出来，以保证T细胞的后续富集。
细胞分离：细胞表面的抗原与相应的生物素标记的抗体(购自Biolegend)结合，生物素与链霉亲和素标记的磁珠Streptavidin Particles Plus–DM(BD公司,货号：557812)结合。通过磁珠分离具有特定表面标志物的细胞，其中，
然后，如实施例1所述制备eGFP标记的细胞，其中用T细胞、B细胞、单核细胞、NK细胞或巨核细胞代替该实施例中使用的红细胞，并应用流式细胞术检测标记效率。结果表明，这种修饰方法也适用于这些细胞类型，并显示出良好的标记效率(图13)。
实施例5.eGFP-Cys-MAL-PEG-DOPE标记的红细胞的体内寿命
如实施例1.3所述制备eGFP-Cys-MAL-PEG-DOPE(西安瑞禧生物合成，如图3所示，其中DSPE替换为DOPE)标记的红细胞，仅用DOPE代替DSPE。应用实施例1.5中的评估这些表面修饰的红细胞体内寿命的标准操作过程，其中在第1、3、4、15天取血，应用第15天采血的进行流式细胞分析(图14，Control:未标记的红细胞)。我们发现，eGFP-Cys-MAL-PEG-DOPE标记的红细胞(eGFP-Cys-MAL-PEG-DOPE-RBC)不仅显示出与对照组(输注了未标记的红细胞的小鼠)的红细胞相同的寿命，在周期中也表现出持续15天的信号(图14和15)。
实施例6.DSPE和DOPE的标记效率比较
通过流式细胞术测量红细胞标记效率。如实施例1.3所述制备Mal-PEG2000-DOPE和Mal-PEG2000-DSPE标记的RBC，不同在于标记反应系统中，eGFP-cys的浓度为5μM/mL，DSPE或DOPE分别为2μg/mL-512μg/mL，Mal-PEG2000-DOPE和Mal-PEG2000-DSPE与红细胞的孵育时间分别为10分钟、30分钟和60分钟。MFI是通过流式细胞术检测的。
如图17所示，在相同孵育时间或相同浓度的eGFP下，使用DOPE在成熟RBC表面eGFP信号更强，这表明DOPE在修饰RBC方面比DSPE具有更好的效率。
实施例7.DSPE、DPPE、DMPE、DOPE最佳标记效率浓度的比较
如实施例1.3所述制备Mal-PEG2000-磷脂(DPPE、DOPE、DSPE或DMPE)标记的RBC(其中Mal-PEG2000-DPPE、Mal-PEG2000-DOPE、Mal-PEG2000-DSPE和Mal-PEG2000-DMPE均来自西安瑞禧生物)，区别在于标记反应系统中Mal-PEG2000-磷脂的浓度分别为200μg/mL、80μg/mL、20μg/mL和0μg/mL(对照组)，eGFP-cys浓度为5μM/mL。标记反应后，通过流式细胞术检测细胞上的平均荧光强度。
如图16所示，DPPE和DMPE对于标记细胞也是有效的。当eGFP-Cys-Mal-PEG2000-磷脂浓度为80μg/mL时，这两种磷脂的标记效率最好。DSPE和DOPE在200μg/mL的浓度下显示出最好的标记效率。
还通过ELISA检测了细胞上清液在410nM处的吸光度。如表1所示，在与高浓度(200μg/mL)的Mal-PEG2000-DPPE孵育后，由于RBC溶血，上清液的吸光度增加。同样，在与200μg/mL Mal-PEG2000-DMPE孵育后，发生RBC溶血。而高浓度的Mal-PEG2000-DOPE和Mal-PEG2000-DSPE不会导致红细胞损伤。
表1.上清液在410nm处的吸光度

实施例8尿激酶原激活剂-红细胞偶联物的制备
在本次实验中，我们首先通过在蛋白质的C末端添加半胱氨酸来修饰ProUK，以提供游离的巯基。接下来，我们使用了DSPE，一种可以与RBC膜融合的细胞膜成分类似物。马来酰亚胺基团可以与ProUK上的游离巯基发生迈克尔加成反应。我们使用Mal-PEG-DSPE进行细胞与蛋白质的偶联。Mal-PEG-DSPE和RBC在37℃孵育以与细胞膜融合，未融合的Mal-PEG-DSPE被洗掉，加入C末端含有游离巯基的ProUK，使蛋白质与预处理的RBC反应(图18)(该实验还可参考图1)。整个反应过程在生理缓冲条件下进行，避免对RBC造成损伤。
具体步骤如下：
8.1.RBC预处理
通过密度梯度离心法从C57BL/6J小鼠外周血中分离红细胞。将分离的红细胞用PBS洗涤3次。将Mal-PEG2000-DSPE溶解在37℃的磷酸盐缓冲液中至终浓度为100μM。然后用50μM MAL-PEG2000-DSPE在37℃下预处理1×109个RBC 30分钟。然后将预处理的红细胞用PBS洗涤3次。预处理的红细胞立即使用或不可长期储存。
8.2.MAL-PEG2000-DSPE介导的RBC的标记
8.2.2 ProUK预处理
8.2.2.1 ProUK的C末端半胱氨酸修饰
将ProUK-Cys cDNA(核酸序列如SEQ ID NO:9所示)克隆到pcDNA 3.4载体中，并将该质粒转染至HEK293F细胞中进行蛋白质表达。转染后的HEK293F的细胞在37℃下培养5天，收集上清进行纯化。所有上清液在4℃下14,000x g离心40分钟后用0.45μM过滤器过滤。将过滤后的上清液上样到与设计色谱系统相连的HisTrap FF 1mL色谱柱(GE Healthcare)上。用含有20mM Tris-HCl、pH7.8、500mM NaCl和300mM咪唑的洗脱缓冲液洗脱蛋白质。所有洗脱的级分在SDS-PAGE凝胶上分析。由此获得ProUK-Cys蛋白质。ProUK-Cys的氨基酸序列如以下SEQ ID NO:8所示。ProUK-Cys的核酸序列如以下SEQ ID NO:9所示。
8.2.3.红细胞与ProUK-cys的偶联
将上述RBC和ProUK-cys在PBS缓冲液中混合，总体积200μL，终浓度为RBC 1×109个/mL，ProUK-cys浓度为10μM。将反应混合液以10rpm的速度旋转孵育1小时进行反应。停止反应，用PBS洗涤，去除其余反应试剂，获得红细胞-ProUK偶联物ProUK-RBC。用Beckman Coulter CytoFLEX LX分析红细胞的标记效率(即偶联了蛋白质的红细胞占总红细胞的百分比)，其中未经ProUK标记的红细胞(未标记的红细胞)作为对照组。结果表明该方法可以有效地将ProUK蛋白搭载至RBC表面(图19，未经Pro-UK标记的红细胞中具有ProUK信号的几乎为0，而经上述方法制备的ProUK-RBC中具有ProUK信号的红细胞为100％)。
8.2.4红细胞蛋白质偶联物ProUK-RBC在体内的寿命
为了评估8.2.3中获得的表面修饰的RBC在体内的寿命，如实施例1中1.5所述评估Pro-UK-RBC在体内的寿命。具体如下：将未标记的红细胞RBC(对照RBC)和实施例8.2.3中获得的ProUK-RBC各109个用Cell Trace Far Red染料染色。将Cell Trace Far Red染色的RBC-ProUK和对照RBC尾静脉内输注到正常受体C57BL/6J小鼠(西湖大学实验动物中心，6-8周龄，每组3只)中。
在输血后的第1、2、6、24、48、72、144、192、240、336、408、504、576小时对小鼠进行脸颊取血。将血样与FITC偶联的抗尿激酶抗体(义翘神州公司，1μL/样品)在37℃孵育15分钟以检测ProUK信号，洗涤3次后进行流式细胞仪分析，针对第0、8、17和24天的流式细胞结果给出了图。选择Cell Trace Far Red阳性细胞用于分析具有ProUK信号的RBC百分比。Cell Trace Far Red阳性细胞表示循环中输注的RBC的百分比。结果见图20，实验组和对照组中的RBC体内存活率无任何统计学上的显著差异。
小鼠在输血后24小时进行脸颊取血。将血样与FITC偶联的抗尿激酶抗体在37℃孵育15分钟以检测ProUK信号并洗涤3次，然后通过Merck Amnis ImageStream MarkII进行分析。结果见图21，可见ProUK-RBC在形态方面与未标记的红细胞(RBC)一致(图21)，说明Mal-PEG-DSPE标记程序不会对RBC造成显著损害，从而导致其过早去除。
从图20和图21还可以看出，ProUK水平在Cell Trace Far Red阳性细胞上保持恒定超过24天。
因此，工程化的RBC在体内表现出良好的稳定性，并且在这些RBC的表面上始终可以检测到ProUK信号。
实施例9 ProUK-RBC提高体外溶栓功效
如实施例8所示，ProUK-RBC在体内表现出良好的稳定性，并且在这些RBC的表面上始终可以检测到ProUK信号。本实施例中进一步评估了实施例8中制备的ProUK-RBC的生物学功能。
在血栓形成过程中，RBC发挥着非常重要的作用，参与血小板驱动的凝块和血栓的收缩，从而形成紧密排列的多面体RBC阵列，其中包括对止血和伤口愈合重要的几乎不可渗透的屏障。
通过ELISA对RBC上的ProUK含量进行定量后，我们使用不同比例的洗涤的RBC/游离的ProUK的混合物、洗涤的RBC//ProUK-RBC的混合物和等量的洗涤的RBC(对照)。将这些溶栓成分添加到由洗涤过的小鼠RBC、小鼠血浆、CaCl2和凝血酶形成的血栓中。通过比较在37℃时未形成血栓的游离的RBC的数量来评估溶栓能力。具体步骤如下：
从C57BL/6J小鼠小鼠中获取全血，离心分离血浆。还从全血中获得洗涤的红细胞。
止血凝块(Hemostatic clot)组：
将100μL体积的CaCl2和凝血酶(分别为20mM和0.2单位/ml最终浓度)添加到含有各50μL的小鼠血浆和洗涤的RBC中形成凝块，该过程持续20分钟。形成凝块后添加下述不同量的ProUK-RBC和ProUK+未标记的红细胞。
具体实验量设计如下：其中ProUK-RBC和未标记的RBC的浓度均为1×107细胞/μl ProUK-RBC组:
2.4μg/mL：ProUK-RBC(2.4μg/mL的实施例8制备的ProUK-RBC 100μL)；
1.2μg/mL：50μL ProUK-RBC以及50μL未标记的RBC；
0.6μg/mL：25μL ProUK-RBC以及75μL未标记的RBC；
0.3μg/mL：12.5μL ProUK-RBC以及87.5μL未标记的RBC；
0.15μg/mL：6.25μL ProUK-RBC以及93.75μL未标记的RBC。
0μg/mL:100μl未标记的红细胞
Free ProUK+RBC组：将50mg/mL ProUK-cys原液(稀释过程中体积变化可忽略不计)加入到100μl未标记的RBC，获得0、4、8、16、32、64μg/mL终浓度的ProUK-cys溶液。
为了模拟止血凝块(Hemostatic clot组)的溶解，在20℃下用上述不同量的ProUK-RBC和ProUK+未标记的红细胞覆盖血栓凝块。
新生凝块(Nascent clot)组：
为了溶解新生凝块(Nascent clot组)，在添加CaCl2和凝血酶之前，将上述Hemostatic clot组不同量的ProUK-RBC和ProUK+未标记的红细胞(包括含有0μg/mLProUK-cys的未标记的红细胞)直接添加到小鼠血浆中，然后立即添加CaCl2和凝血酶用于形成凝块，该过程持续20分钟。
将上述两组凝块分别在37℃下孵育2小时。之后轻轻倒置样品重悬，用移液管吸出无凝块的红细胞悬液，加入红细胞裂解液(索莱宝生物)10分钟，离心(5分钟，1200×g)，用分光光度法在405nm处确定释放的血红蛋白的量。通过比较最高吸光度值(无血栓形成)和最低吸光度值(0μg/mL，未添加溶栓成分，血栓形成)对各组的溶栓进行标准化。结果表明，游离的ProUK需要在约30μg/mL时有效预防血栓形成，在止血凝块和新生凝块的溶栓性能方面无显著差异(图22A和C)。在ProUK-RBC组，仅需0.15-0.3μg/mL ProUK即可有效预防血栓形成，新生凝块的溶栓效果优于止血凝块(图22B和D)。游离的ProUK完全溶解血栓的剂量相当于ProUK-RBC剂量的约100倍，ProUK-RBC溶解新生凝块的能力(～50％)比止血凝块高10倍(～5％)。
因此，RBC上搭载的ProUK更稳定，在RBC作为载体的帮助下，可以更好地激活纤溶酶原激活剂的功能。此外，RBC是血栓的重要组成部分。在血栓形成过程中，只要有一小部分ProUK-RBC进入血栓，就可以激活其纤溶活性，从内部分解血栓。然而，由于其结构更紧密，ProUK-RBC不能进入止血凝块，只能从表面溶栓。因此，ProUK-RBC在靶向新生凝块方面更有效。
实施例10 ProUK-RBC延长半衰期并减少药物剂量
前述研究已经表明，ProUK-RBC可以有效降低ProUK的剂量。我们使用10ug/kg(临床中验证的游离的ProUK有效剂量为1mg/kg)游离的ProUK或ProUK-RBC来验证本发明的ProUK-RBC离体的抗血栓形成功能。
向C57/BL6J小鼠(西湖大学实验动物中心，6-8周龄，每组3只，共计三组)分别尾静脉注射等量的游离的ProUK(即0.2ug ProUK-cys与200ul 1×107个/ul的RBC混合)、ProUK-RBC(即实施例8制备的ProUK-RBC 200ul 1×107个/ul)和未标记的RBC(即200ul 1×107个/ul的未标记的RBC混合)，按计划时间从眼眶采血，其中ProUK-RBCs组分别于给药后1、5、15、30、60、120分钟采血，RBC和游离ProUK组于给药后1、3、5、10、30、60分钟采血(图23)。
通过ELISA，应用Anti-Urokinase/uPA-HRP(Clone#05)和Anti-Urokinase/uPA(Clone#1A2G4G11)(均购买自义翘神州)对全血中的ProUK浓度进行定量后，我们发现游离的ProUK组在约5分钟后几乎检测不到，而ProUK-RBC组在30分钟时仍有约90％保留在血液中(图24)。
我们进一步评估了ProUK-RBC的体外溶栓能力，通过分析血栓量和游离红细胞量来分析抗血栓形成能力。
通过将CaCl2和凝血酶(分别为20mM和0.2单位/ml终浓度)添加到上述取出的小鼠全血内，进行血栓形成实验，37℃下孵育2小时。轻轻倒置样品重悬，用移液管吸出无凝块的红细胞悬液，加入红细胞裂解液10min，离心(5min，1200×g)，在405nm分光光度法测定释放的血红蛋白量。通过比较最高吸光度值(无血栓形成)和最低吸光度值(未添加溶栓成分，血栓形成)对各组的溶栓进行标准化。
结果显示ProUK组在初始时间点不能有效溶栓，而ProUK-RBC组在2小时后仍有约75％的溶栓能力(图25和26)。由于起始剂量过低，ProUK组由于血浆半衰期短，溶栓效果不佳。但是，我们发现ProUK-RBC组即使在如此低的浓度下也能延长ProUK的半衰期，并显示出有效的溶栓能力。
实施例11 ProUK-RBC提高体内溶栓功效
11.1尾部时间测定法
ProUK-RBC在体外和离体实验中显示出良好的溶栓能力。我们接下来在体内测试了实施8制备的Pro-UK-RBC抗血栓形成作用。尾部出血时间测定法是评估小鼠止血的最广泛使用的方法。具体步骤如下：
用腹膜内(IP)注射三溴乙醇对C57/BL6J小鼠进行麻醉，用剪刀清洁尾毛。然后将与先前实验一致的ProUK-RBC(ProUK-RBC 200ul 1×107个/ul)、游离ProUK(具0.2ug ProUK-cys与200ul 1×107个/ul的未标记的RBC混合)和RBC(200ul 1×107个/ul的未标记的RBC)尾静脉注射C57/BL6J小鼠(西湖大学，6-8周龄，每组18只小鼠)。立即使用手术刀在距尖端8mm处横切尾巴。将流血的尾部残端浸入温至37℃的生理盐水中，并确定止血时间(图27)。出血时间小于60秒或大于900秒与实验操作或动物个体差异有关，数据被排除在外。
图28显示了低剂量水平下三组中的抗血栓能力，其中RBC组有13只小鼠，游离ProUK组有10只小鼠，ProUK-RBC组有13只小鼠符合止血时间规定。该实验可以评估ProUK-RBC对新生凝块的溶栓能力。结果显示游离的ProUK组与RBC组之间无显著差异，这是由于游离的ProUK剂量低。但是，在如此低的剂量下，ProUK-RBC组的出血时间长于RBC组和游离的ProUK组(图28)。ProUK-RBC组仍表现出良好的抗血栓形成能力，有力地证明了ProUK-RBC的有效性。
11.2由纤维蛋白栓塞导致脑血栓的大鼠模型(MCAO)来评估血栓预防
MCAO是动物脑血栓形成的经典模型。传统的线栓塞方法不适合预防模型，因此我们采用微血栓颗粒注射构建脑血栓模型。微血栓颗粒不能直接阻塞血管，但当这些颗粒进入体内后，会形成较大的血栓，阻塞血管，引起中风。具体步骤如下：
从SD大鼠中采集全血，通过洗涤去除其他成分获得洗涤的红细胞。将红细胞通过实施例8的方法制备为ProUK-RBC，用于11.2的实验。同时获得SD大鼠的新鲜血浆。
在新鲜SD大鼠血浆中加入10mg/mL纤维蛋白原、20mM CaCl2和0.2U/mL凝血酶(Bdeir,K.et al.Urokinase mediates fibrinolysis in the pulmonary microvasculature.Blood 96,1820-1826(2000).)。在室温下孵育1小时后，将凝块在4℃下孵育过夜以促进收缩。使用均质器处理血凝块以制备直径为1.5-3.5μm的微血栓，溶于PBS溶液中，获得微血栓形成溶液(1.4×106个颗粒/100μL)。
取雄性SD大鼠(200-350g，2组，每组10只，6-8周龄)用2％异氟烷麻醉动物，用具有含有1.5％异氟烷的70％N2O和30％O2的混合物的面罩保持，保持直肠温度在37±0.5℃，通过正中切口暴露左侧颈总动脉、颈外动脉和颈内动脉。用丝线缝合颈内动脉远端，临时结扎左颈内动脉、颈外动脉和翼腭动脉(PPA)。将一段内径乙烯基导管连接到注射器上，引入内导管，并用丝线固定。去除颈内动脉周围的结扎线并将导管推进颈内动脉腔。
首先针对两组大鼠分别尾静脉注射ProUK-RBC(1×107个/μl)和未标记的RBC(1×107个/μl)，量分别为200μL/只。在给药后，将微血栓形成溶液(100μL；1.4×106个颗粒)以推注形式注射，拔出导管，并且从颈动脉和PPA上去除结扎线(阻塞下游血管，导致>80％的血流停止并导致广泛的同侧脑梗塞)。24小时后计算存活率。
我们在致死剂量的微血栓注射之前给SD大鼠的预防性ProUK-RBC注射，评估了SD大鼠的预防性ProUK-RBC干预。就24小时死亡率而言，ProUK-RBC可将死亡率降低40％(图29)。在这种情况下，我们分析了两组动物在处理后6小时的脑梗塞面积，即大鼠脑部使用TTC(2，3，5—氯化三苯基四氮唑)染色后进行切片后，分析脑部缺血面积。ProUK-RBC可以有效减少脑梗塞的面积(图30和31)。
11.3在深静脉血栓形成模型中预防内源性血栓形成
FeCl3可诱导血管内皮细胞损伤，模拟内源性血栓形成。
将C57/BL6J小鼠分别通过尾静脉注射ProUK-RBC(ProUK-RBC 200ul 1×107个/ul)、游离ProUK(具0.2ug ProUK-cys与200ul 1×107个/ul的未标记的RBC混合)和RBC(200ul 1×107个/ul的未标记的RBC)(每组6只小鼠)。5分钟后用FeCl3诱导股静脉血栓，具体步骤如下：
用腹膜内(IP)注射三溴乙醇来麻醉小鼠。对于大腿静脉血栓形成模型，进行中线皮肤切口，并通过钝性解剖轻轻暴露血管。将滤纸浸泡在20％FeCl3中，置于静脉前表面90秒。
上述诱导后3分钟和18小时观察血栓(图32)。
结果表明，FeCl3诱导后，RBC组和游离的ProUK组均出现较深的血栓，而ProUK-RBC组均未出现明显的血栓。18小时后，RBC组和游离的ProUK组仍有明显血栓，ProUK-RBC组血管周围有轻微但不明显的血栓(图33，每组显示1只小鼠)。ProUK-RBC也在内皮损伤引起的静脉血栓形成中有效预防血栓形成。




Claims (33)

  1. 一种药物递送系统，其包含修饰的血细胞，所述修饰的血细胞通过接头与蛋白质或肽偶联，其中所述接头包含马来酰亚胺-PEG-磷脂，所述蛋白质或肽经处理具有暴露的或游离的硫醇基用于与马来酰亚胺发生迈克尔加成反应形成稳定的碳硫键，所述磷脂掺入到血细胞膜中。
  2. 权利要求1的药物递送系统，其中所述血细胞选自来源于动物血液的血细胞，例如红细胞、淋巴细胞、T细胞、B细胞、单核细胞、NK细胞、树突细胞或巨核细胞，优选地，所述动物是哺乳动物例如人。
  3. 权利要求2的药物递送系统，其中所述血细胞是红细胞，例如天然红细胞，如天然成体红细胞。
  4. 权利要求1-3中任一项的药物递送系统，其中PEG分子量为400-5000，例如2000-5000，例如PEG2000、PEG2500、PEG3000、PEG3500、PEG4000、PEG4500或PEG5000。
  5. 权利要求1-4中任一项的药物递送系统，其中磷脂为PE系列磷脂，例如1，2-二硬脂酰-sn-甘油基-3-磷脂酰乙醇胺(DSPE)、1，2-二丙烯酰-sn-甘油基-3-磷脂酰乙醇胺(DEPE)、1，2-二油酰-sn-甘油基-3-磷脂酰乙醇胺(DOPE)、1，2-二肉豆蔻酰-sn-甘油基-3-磷脂酰乙醇胺(DMPE)和1，2-二棕榈酰-sn-甘油基-3-磷脂酰乙醇胺(DPPE)，优选地，所述磷脂是DSPE或DOPE。
  6. 权利要求1-4中任一项的药物递送系统，其中所述接头是马来酰亚胺-PEG-DSPE，例如具有如下所示的化学式：
  7. 权利要求1-6中任一项所述的药物递送系统，其中对所述蛋白质或肽的处理包括
    (1)通过硫醇类还原剂(例如TCEP)处理所述蛋白质或肽，从而使其包含含有游离硫醇基的半胱氨酸残基；或者
    (2)通过在不影响所述蛋白肽或肽的结果或功能的位置上插入1个或多个半胱氨酸，从而使其包含含有游离的硫醇基的半胱氨酸残基，例如，将1个半胱氨酸插入到蛋白质或肽的C末端(如最C末端氨基酸后面)或N末端(例如最N末端氨基酸前面)。
  8. 权利要求1-7中任一项的药物递送系统，其中所述蛋白质是用于标记的蛋白质或肽，和用于预防或治疗的蛋白质或肽。
  9. 权利要求8的药物递送系统，其中所述用于标记的蛋白质或肽选自荧光蛋白(例如绿色荧光蛋白，如eGFP)、辣根过氧化物酶、牛血清白蛋白、血蓝蛋白、鸡卵白蛋白、抗体、牛IgG、鼠IgG、羊IgG、兔IgG、碱性磷酸酶、酸性磷酸酶、葡萄糖氧化酶或β-半乳糖苷酶。
  10. 权利要求9的药物递送系统，其中用于预防或治疗的蛋白质或肽是可以用作药物来预防或治疗疾病的任何蛋白质，包括例如细胞因子或生长因子或其受体、激素类、酶类、抗体等。
  11. 权利要求10的药物递送系统，其中所述抗体是免疫检查点分子的抗体或其抗原结合片段，例如特异性结合PD-1、PD-L1或PD-L2的抗体，例如人抗体、人源化抗体或嵌合抗体。
  12. 权利要求11的药物递送系统，所述特异性结合PD-1的抗体选自帕博利珠单抗、卡瑞利珠单抗、替雷利珠单抗、特瑞普利单抗、信迪利单抗或纳武利尤单抗或其抗原结合片段，或所述特异性结合PD-1的抗体包含选自帕博利珠单抗、卡瑞利珠单抗、替雷利珠单抗、特瑞普利单抗、信迪利单抗或纳武利尤单抗的抗体的重链可变区的3个CDR和轻链可变区的3个CDR。
  13. 权利要求11或12的药物递送系统，其中所述抗体通过与硫醇类还原剂(例如TCEP)共孵育而暴露了半胱氨酸残基上的硫醇基。
  14. 权利要求11-13中任一项的药物递送系统，其用于治疗肿瘤，例如实体肿瘤，例如实体癌症。
  15. 权利要求8的药物递送系统，其中所述蛋白质是蛋白酶，例如丝氨酸蛋白酶，例如尿激酶原，例如人尿激酶原，例如所述人尿激酶原
    (1)包含SEQ ID NO:10或13所示的氨基酸序列，或由所述氨基酸序列组成；
    (2)包含与SEQ ID NO:10或13所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％、99％或更高的同一性的氨基酸序列；
    (3)由SEQ ID NO:11所示的核苷酸序列所编码；或
    (4)由与SEQ ID NO:11所示的核苷酸序列具有至少85％、90％、95％、96％、97％、98％、99％或更高的同一性的核苷酸序列所编码。
  16. 权利要求15的药物递送系统，其中所述蛋白质通过在最N末端氨基酸前面或在最C末端氨基酸后面插入一个半胱氨酸而包含含有游离硫醇基的半胱氨酸残基，例如，所述人尿激酶原在其C末端插入一个半胱氨酸，所述包含插入的半胱氨酸的尿激酶原
    (1)包含SEQ ID NO:8或12所示的氨基酸序列，或由所述氨基酸序列组成；
    (2)包含与SEQ ID NO:8或12所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％、99％或更高的同一性的氨基酸序列，其在最C末端具有半胱氨酸；
    (3)由SEQ ID NO:9所示的核苷酸序列所编码；或
    (4)由与SEQ ID NO:9所示的核苷酸序列具有至少85％、90％、95％、96％、97％、98％、99％或更高的同一性的核苷酸序列所编码，其在其最C末端具有半胱氨酸。
  17. 权利要求15或16的药物递送系统，其用于预防或治疗血栓形成相关疾病，例如中风如缺血性中风、心肌梗塞(例如急性心肌梗塞)和静脉血栓例如深静脉血栓。
  18. 权利要求8的药物递送系统，其中所述蛋白质是功能性代谢酶，例如尿酸氧化酶，例如来自黄曲霉的尿酸氧化酶，例如所述尿酸氧化酶
    (1)包含SEQ ID NO:5或6所示的氨基酸序列，或由所述氨基酸序列组成；或
    (2)包含与SEQ ID NO:5或6所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％、99％或更高的同一性的氨基酸序列。
  19. 权利要求18的药物递送系统，其中所述蛋白质通过在最N末端氨基酸前面或在最C末端氨基酸后面插入一个半胱氨酸而包含含有游离硫醇基的半胱氨酸残基，例如，所述尿酸氧化酶在其C末端插入一个半胱氨酸，所述包含插入的半胱氨酸的尿酸氧化酶
    (1)包含SEQ ID NO:3或7所示的氨基酸序列，或由所述氨基酸序列组成；
    (2)包含与SEQ ID NO:3或7所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％、99％或更高的同一性的氨基酸序列，其在最C末端具有半胱氨酸；
    (3)由SEQ ID NO:4所示的核苷酸序列所编码；或
    (4)由与SEQ ID NO:4所示的核苷酸序列具有至少85％、90％、95％、96％、97％、98％、99％或更高的同一性的核苷酸序列所编码，其在其最C末端具有半胱氨酸。
  20. 权利要求18或19的药物递送系统，其用于预防或治疗代谢疾病例如慢性高尿酸血症，例如痛风，例如难治性的痛风(对其他常规疗法耐受的痛风)。
  21. 制备权利要求1-20中任一项所述的药物递送系统的方法，其包括
    (1)提供包含马来酰亚胺和磷脂的接头；
    (2)提供一种蛋白质或肽，其包含游离的硫醇基；
    (3)使(1)中所述接头与血细胞一起孵育，进而将其中的磷脂掺入所述细胞的细胞膜中；
    (4)将步骤(3)中制备的细胞与(2)中的蛋白质的硫醇基在适合于发生迈克尔加成反应的条件下一起孵育；
    或者
    (3’)使(1)中的接头与(2)中的蛋白质的硫醇基在适合于发生迈克尔加成反应的条件下一起孵育，获得与磷脂偶联的蛋白质；
    (4’)将步骤(3’)中获得的蛋白质与血细胞一起孵育，进而将其中的磷脂掺入所述细胞的细胞膜中；和
    (5)获得包含通过接头偶联的蛋白质和血细胞作为药物递送系统。
  22. 药物组合物或制剂，其包含权利要求1-20中任一项的药物递送系统或权利要求21所制备的药物递送系统，和药用辅料。
  23. 权利要求22的药物组合物或制剂，其中所述制剂是人血制剂，例如自体或异体的人血制剂。
  24. 权利要求22或23的药物组合物或制剂，其中所述人血制剂是人血减白血液制剂。
  25. 权利要求22-24中任一项的药物组合物或制剂，其中所述人血制剂包含1μg/mL-1000μg/mL的药物递送系统，例如在1-10μg/mL、20μg/mL、25μg/mL、30μg/mL、35μg/mL、40μg/mL、45μg/mL、50μg/mL、60μg/mL、70μg/mL、80μg/mL、90μg/mL、100μg/mL、150μg/mL、200μg/mL、250μg/mL、300μg/mL、350μg/mL、400μg/mL、450μg/mL、或500μg/mL以上的药物递送系统。
  26. 权利要求11-13中任一项的药物递送系统在制备药物或人血制剂中的用途，所述药物或人血制剂用于在受试者中治疗肿瘤例如癌症，例如实体肿瘤，例如具有具有升高的PD-1、PD-L1和/或PD-L2的蛋白质水平和/或核酸水平(例如表达升高)的肿瘤或癌症，例如胃肠道肿瘤，例如结肠癌或结直肠癌；或者是肺癌、胰腺癌或乳腺癌。
  27. 权利要求15或16的药物递送系统在制备药物或人血制剂中的用途，所述药物或人血制剂用于在受试者中抗血栓形成或溶栓。
  28. 权利要求15或16的药物递送系统在制备药物或人血制剂中的用途，所述药物或人血制剂用于在受试者中预防或治疗血栓形成相关疾病。
  29. 权利要求28的用途，其中所述血栓形成相关疾病选自中风如缺血性中风、心肌梗塞(例如急性心肌梗塞)和静脉血栓例如深静脉血栓。
  30. 权利要求18或19的药物递送系统在制备药物或人血制剂中的用途，所述药物或人血制剂用于在受试者中治疗代谢相关疾病，例如慢性高尿酸血症，例如痛风，例如难治性的痛风(对其他常规疗法耐受的痛风)。
  31. 权利要求26-30中任一项的用途，其中所述药物递送系统中的血细胞来自待治疗的受试者或健康受试者。
  32. 权利要求26-31中任一项的用途，其中所述药物或人血制剂与一种或多种治疗方式或其他治疗剂联合施用。
  33. 权利要求32的用途，其中所述治疗方式是手术治疗。
PCT/CN2023/107705 2022-07-18 2023-07-17 包含血细胞的药物递送系统 WO2024017192A1 (zh)

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