Modified red blood cells and use thereof for delivery of drugs

Abstract

A modified red blood cell is provided in which an active agent is conjugated to an extracellular portion of a red cell membrane protein via a linker wherein the linker comprises a G-containing small peptide and a maleimidoalkyl chain (C2-8) conjugated to the membrane protein of the red blood cell wherein the linker comprises a G-containing small peptide and a maleimidoalkyl chain (C2-8), and the G-containing small peptide is conjugated with an active agent containing a sortase recognition motif through a sortase-mediated reaction. Also provided are covalently modified RBCs and their use for delivery of drugs and probes.

Description

Modified red blood cells and their use for drug delivery Technical Field

[0001] The present invention relates generally to modified red blood cells (RBCs), and more particularly to covalently modified RBCs and their use for the delivery of drugs and probes. Background Art

[0002] Recent developments in drug delivery systems for prolonged drug retention in the treatment of a variety of human diseases have attracted considerable attention. However, many systems still face various challenges and limitations, such as poor stability, unwanted toxicity, and immune responses. Red blood cells (RBCs), the most common cell type in the human body, have been extensively studied for over 30 years as ideal in vivo drug delivery systems due to their unique biological properties, including: (i) a wide range of in vivo circulation with a long survival time; (ii) high biosafety, low immunogenicity, and good biocompatibility as biomaterials; (iii) a large surface area to volume ratio; and (iv) the absence of a nucleus, mitochondria, and other organelles.

[0003] Currently, several mature and feasible approaches have been developed to modify red blood cells (RBCs) for drug delivery. These approaches can be broadly categorized into genetically modified RBC carriers, non-genetically modified RBC carriers, and RBC membrane modifications. These approaches involve direct encapsulation, non-covalent attachment of exogenous peptides, or protein attachment via fusion with RBC surface protein-specific antibodies. However, these modified RBCs have limitations for in vivo application. For example, encapsulation can disrupt the cell membrane, thereby compromising the in vivo survival of the engineered cells. Furthermore, the non-covalent attachment of polymer particles to RBCs is easily dissociated, resulting in rapid degradation of the payload in vivo.

[0004] Bacterial sortases are transpeptidases that can modify proteins in a covalent and site-specific manner. Wild-type sortase A (wtSrtA) from Staphylococcus aureus recognizes the LPXTG motif and cleaves between threonine and glycine to form a covalent acyl-enzyme intermediate between the enzyme and the substrate protein. This intermediate is disassembled by nucleophilic attack by oligoglycine of a peptide or protein, which typically has three consecutive oligoglycine residues (3×glycine, G3) at the N-terminus. Previous studies have genetically overexpressed the membrane protein KELL with the LPXTG motif at its C-terminus on RBCs, which can be linked to 3×glycine or G3 by using wtSrtA. (n≥3)These drug-carrying RBCs have demonstrated therapeutic efficacy in animal models. However, this requires engineering hematopoietic stem or progenitor cells (HSPCs) and differentiating them into mature RBCs, significantly limiting their application.

[0005] In addition, how to increase the effective load of RBCs without affecting their integrity and function, thereby increasing the concentration of drugs at the target site and enhancing the drug's effect on disease treatment, remains an urgent problem that needs to be solved. This application solves this problem to a certain extent by providing an improved RBC delivery system.

[0006] SUMMARY OF THE INVENTION

[0007] In a first aspect, the present invention provides a modified red blood cell (RBC).

[0008] In one embodiment, the present invention provides a modified red blood cell, wherein the red blood cell is treated with a reducing agent so that the disulfide bonds in the extracellular domain of at least one endogenous membrane protein of the red blood cell (e.g., at an internal site of the extracellular domain) are reduced to have free sulfhydryl groups.

[0009] In one embodiment, the present invention provides a modified red blood cell comprising a maleimidoalkyl chain (C 2- 8) is covalently bound to the free sulfhydryl groups on the surface of the erythrocyte membrane. In a specific embodiment, the linker is covalently bound to the free sulfhydryl groups on the surface of the erythrocyte membrane through the 6-maleimidocaproic acid or 4-maleimidobutyric acid contained therein, thereby obtaining an erythrocyte carrying the linker.

[0010] In one embodiment, the linker comprises a small peptide containing oligoglycine (also referred to herein as a "G-containing small peptide").

[0011] In one embodiment, the G-containing small peptide contained in the linker is a linear small peptide or a branched small peptide. In a specific embodiment, the branched small peptide comprises 2 or more branching units, wherein one or more active agents are each coupled to the corresponding branching unit. In a specific embodiment, the branching units have the same structure. In a specific embodiment, the branching unit consists of the amino acid sequence K (GGG), wherein the glycine (G) in the brackets is conjugated to the side chain ε-amino group of the adjacent lysine (K) to form a branch, and the lysine forms a peptide bond with other amino acids through its α-amino group to form the main chain of the "G-containing small peptide". In a specific embodiment, an extension chain can be added between K and G in the branch unit K (GGG), such as COCH2CH2-PEG6-NH.

[0012] In a preferred embodiment, the G-containing small peptide has a structure selected from the group consisting of GGGSK (SEQ ID NO: 11), K(GGG)-GGG-K(GGG) (SEQ ID NO: 12), K(GGG)-GGG-K(GGG)-GGG-K(GGG) (SEQ ID NO: 13), K(GGG)-GGG-K(GGG)-GGG-K(GGG)-GGG-K(GGG) (SEQ ID NO: 14), or K(GGG)-GGG-K(GGG)-GGG-K(GGG)-GGG-K(GGG)-GGG-K(GGG) (SEQ ID NO: 15). NO: 15), or K[(COCH2CH2-PEG6-NH)-GGG]-GGG-K[(COCH2CH2-PEG6-NH)-GGG]-GGG-K[(COCH2CH2-PEG6-NH)-GGG]-NH2 (SEQ ID NO: 16), wherein the glycine (oligoglycine) in the brackets is conjugated to the ε-amino group of the lysine side chain to form a branched chain. In a specific embodiment, an extension chain can be added between K and G in the branching unit K(GGG), such as COCH2CH2-PEG6-NH. In one embodiment, the above-mentioned oligoglycine reacts with an active agent containing a sortase recognition motif under the mediation of a sortase to be conjugated together. In a specific embodiment, multiple identical or different active agents are conjugated to a linker comprising multiple branching units through the above reaction.

[0013] In one embodiment, the linker comprises a small peptide containing G and a maleimido alkyl chain (C 2-8 ), and through its maleimido alkyl chain (C 2-8 ) is connected to the membrane protein of the red blood cell and is connected to the active agent containing the sortase recognition motif through a sortase-mediated reaction by means of its G-containing small peptide. In a specific embodiment, the linker is composed of a G-containing small peptide and a maleimido alkyl chain (C 2-8 ). In a preferred embodiment, the linker consists of a G-containing peptide and 6-maleimidocaproic acid. In a specific embodiment, PEGn is located between the G-containing peptide and 6-maleimidocaproic acid, where n = 1-20. In a specific embodiment, the linker allows for simultaneous conjugation of multiple, identical or different, active agents to erythrocytes. Preferably, at least two active agents are simultaneously conjugated to erythrocytes. In one embodiment, the active agents are the same active agent.

[0014] In a specific embodiment, the linker has a structure as shown in Table 1 or Table 3.

[0015] In one embodiment, red blood cells may be conjugated to multiple such linker molecules on their membrane surface.

[0016] In one embodiment, the erythrocytes have not been genetically engineered to express a protein comprising a sortase recognition motif or a nucleophilic receptor sequence, and preferably the erythrocytes are native erythrocytes, such as native human erythrocytes.

[0017] In one embodiment, the present invention provides red blood cells conjugated with an active agent, wherein the active agent is conjugated to the red blood cells carrying the linker described above.

[0018] In one embodiment, the active agent is modified to comprise a recognition motif for sortase and is attached to a linker on the red blood cell via a sortase-mediated reaction, and preferably via sortase-mediated glycine conjugation and / or sortase-mediated lysine side chain epsilon-amino conjugation.

[0019] In one embodiment, the sortase is sortase A (SrtA), e.g., Staphylococcus aureus transpeptidase A, e.g., Staphylococcus aureus transpeptidase A variant (mgSrtA). For example, mgSrtA comprises, consists essentially of, or consists of an amino acid sequence having at least 60% identity to the amino acid sequence shown in SEQ ID NO: 3.

[0020] In one embodiment, the sortase recognition motif comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of LPXTG, LPXAG, LPXSG, LPXLG, LPXVG, LGXTG, LAXTG, LSXTG, NPXTG, MPXTG, IPXTG, SPXTG, VPXTG, YPXRG, LPXTS, and LPXTA, wherein X is any amino acid. It is understood that after the active agent is linked to the linker, the last residue (e.g., the fifth residue from the N-terminus to the C-terminus) of the sortase recognition motif is replaced by the amino acid to which the linker is attached, resulting in a structure consisting of the first four amino acid residues of the sortase recognition motif and the linker, as described elsewhere herein. In a specific embodiment, the sortase recognition motif provided herein is LPETG. In one embodiment, the sortase recognition motif can be modified to improve its recognition efficiency. Preferably, LPETG is modified to improve its affinity for the sortase, for example, by adding a G to the C-terminus of the recognition sequence, such as the modified sequence LPETGG.

[0021] In one embodiment, the active agent comprises a binding agent, therapeutic agent or detection agent, including, for example, a protein, an antibody or a functional antibody fragment thereof, an antigen, such as a tumor antigen, an MHC-peptide complex, a drug, such as a small molecule drug (e.g., an anti-tumor agent, such as a chemotherapeutic agent), an enzyme (e.g., a functional metabolic enzyme, such as UOX, or a therapeutic enzyme), a hormone, a cytokine, a growth factor, an antimicrobial agent, a probe, a ligand, a receptor, an immune tolerance-inducing peptide, a targeting moiety, a prodrug, or any combination thereof.

[0022] In one embodiment, after the transamidation reaction of sortase, the red blood cells have a conjugated structure of "linker-LPXT-active agent" on their membrane surface.

[0023] In one embodiment, the active agent can be linked to the sortase recognition motif via a flexible peptide segment. In a specific embodiment, the active agent is a peptide molecule, and preferably the flexible peptide segment fuses the sortase recognition motif to the C-terminus of the active agent peptide molecule. In a specific embodiment, the flexible peptide segment has (GS) n In a specific embodiment, (GS) n For example, (GS) 2， (GS) 3， (GS)4.

[0024] In some embodiments, the red blood cells conjugated with the active agent have the following structures: UOX-LPET-G1-RBC, UOX-LPET-G2-RBC, UOX-LPET-G3-RBC, UOX-LPET-G4-RBC, UOX-LPET-G5-RBC, UOX-LPET-G6-RBC, anti-PD1 mAb-LPET-G3-RBC, anti-PD1 mAb-LPET-G1-RBC, or anti-PD1 mAb-1-LPET-GAASK-RBC.

[0025] In the second aspect, the present application provides a linker molecule, which is composed of a G-containing small peptide and a maleimide alkyl chain (C 2-8 )composition.

[0026] In one embodiment, the maleimido alkyl chain (C 2-8 ) are 6-maleimidocaproic acid and 4-maleimidobutyric acid.

[0027] In one embodiment, the G-containing peptide is a linear or branched peptide. In a specific embodiment, the branched peptide comprises two or more branching units, wherein one or more active agents are each coupled to the corresponding branching unit. In a specific embodiment, the branching units have the same structure. In a specific embodiment, the branching unit consists of the amino acid sequence K(GGG), wherein the glycine in the brackets is conjugated to the side chain ε-amino group of lysine to form a branch, and lysine forms a peptide bond with other amino acids through its α-amino group to form the main chain of the G-containing peptide.

[0028] In a preferred embodiment, the G-containing small peptide has a structure selected from the following: GGGSK, K(GGG)-GGG-K(GGG), K(GGG)-GGG-K(GGG)-GGG-K(GGG), K(GGG)-GGG-K(GGG)-GGG-K(GGG)-GGG-K(GGG) or K(GGG)-GGG-K(GGG)-GGG-K(GGG)-GGG-K(GGG)-GGG-K(GGG), wherein the glycine in the brackets is conjugated to the ε-amino group of the lysine side chain to form a branch.

[0029] In a preferred embodiment, the small peptide is conjugated together through the reaction of oligoglycine in the branch chain with an active agent containing a sortase recognition motif under the mediation of sortase. In a specific embodiment, multiple identical or different active agents are conjugated to a linker comprising multiple branching units through the above reaction.

[0030] In one embodiment, the linker is linked to the membrane protein of the erythrocyte via its maleimido alkyl chain (C2-8), and is linked to an active agent containing a sortase recognition motif via a sortase-mediated reaction via its G-containing small peptide. In a specific embodiment, the linker allows multiple identical or different active agents to be conjugated to the erythrocyte simultaneously. Preferably, at least two active agents are conjugated to the erythrocyte simultaneously. In one embodiment, the active agents are the same active agent.

[0031] In a specific embodiment, the linker has the structure shown in Table 1.

[0032] In a specific embodiment, the present invention provides a use of the linker in modifying red blood cells, wherein the red blood cells and the active agent are coupled together via the linker.

[0033] In a third aspect, the present application provides a method for preparing the red blood cells described in the first aspect, comprising:

[0034] 1) treating erythrocytes so that the linker molecule of the second aspect is attached to the extracellular domain of an endogenous membrane protein of the erythrocytes; and / or,

[0035] 2) treating the active agent such that the active agent comprises a sortase recognition motif; and / or,

[0036] 3) In the presence of sortase, contacting the erythrocytes obtained in step 1) with the active agent obtained in step 2) under conditions suitable for the reaction of the sortase, so that the sortase conjugates the active agent to the endogenous membrane protein of the erythrocyte via a linker.

[0037] In one embodiment, red blood cells are treated with a reducing agent such that disulfide bonds in the extracellular domain of at least one endogenous membrane protein of the red blood cell (eg, at an internal site of the extracellular domain) are reduced to have free sulfhydryl groups.

[0038] In a specific embodiment, the linker molecule is linked to the free sulfhydryl group on the extracellular domain of the endogenous membrane protein of red blood cells through the 6-maleimidocaproic acid contained in the linker molecule.

[0039] In a specific embodiment, the active agent is linked to the G-containing peptide in the linker molecule through the sortase recognition motif LPXTG, and after a transamidation reaction of the sortase, an active agent-LPXT-linker structure is formed.

[0040] In a specific embodiment, multiple active agents are conjugated to the linker via a linker molecule having a branching unit. In a more specific embodiment, multiple active agents are conjugated to the red blood cell via a linker molecule having a branching unit.

[0041] In one embodiment, the present invention provides a modified erythrocyte obtained by the method of the third aspect, wherein the membrane surface of the erythrocyte is conjugated with an active agent via a linker.

[0042] In a fourth aspect, the present invention provides a composition comprising the red blood cells described in the first aspect.

[0043] In one embodiment, the composition is a pharmaceutical composition, optionally comprising a pharmaceutically acceptable carrier that is compatible with red blood cells.

[0044] In a fifth aspect, the present invention provides a method for diagnosing, treating or preventing a disease in a subject in need thereof, comprising administering to the subject the red blood cells or composition as described in the present application.

[0045] In one embodiment, the disease is selected from the group consisting of tumors or cancers, metabolic diseases, bacterial infections, viral infections, autoimmune diseases, and inflammatory diseases.

[0046] In a sixth aspect, the present invention provides a method of delivering an active agent to a subject in need thereof, comprising administering to the subject a red blood cell or a composition as described in the present disclosure.

[0047] In a seventh aspect, the present invention provides a method for increasing the plasma half-life of an active agent, comprising:

[0048] 1) treating the active agent so that it contains a sortase recognition motif;

[0049] 2) providing red blood cells carrying a linker, or optionally, for red blood cells not carrying a linker, treating the red blood cells as described above, such that the linker molecule of the second aspect is attached to the extracellular domain of an endogenous membrane protein of the red blood cells; and

[0050] 3) conjugating the active agent obtained in step 1) and the red blood cells obtained in step 2) in the presence of a sortase under suitable conditions, wherein the conditions are suitable for the sortase to conjugate the sortase substrate to at least one endogenous non-engineered membrane protein of the red blood cells through a sortase-mediated reaction, preferably through sortase-mediated glycine conjugation and / or sortase-mediated lysine side chain ε-amino conjugation.

[0051] In one embodiment, the method further comprises administering the active agent conjugated to the red blood cells to the subject, eg, directly into the circulatory system, eg, intravenously.

[0052] In an eighth aspect, the present invention provides the use of red blood cells or compositions as described herein in the preparation of a medicament for treating or preventing a disease, or in the preparation of a diagnostic agent for diagnosing a condition, illness or disease, or in the preparation of a medicament for delivering an active agent.

[0053] In one embodiment, the disease is selected from a tumor or cancer, a metabolic disease, a bacterial infection, a viral infection, an autoimmune disease, and an inflammatory disease. In some embodiments, the medicament is a vaccine.

[0054] In one embodiment, the present invention provides red blood cells or compositions of the present disclosure for use in diagnosing, treating or preventing a disease in a subject in need thereof. In some embodiments, the disease is selected from a tumor or cancer, a metabolic disease, a bacterial infection, a viral infection, an autoimmune disease and an inflammatory disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] FIG1 shows the effect of using red blood cells containing different linkers on the plasma urate concentration in mice.

[0056] Figure 2 shows the effect of different linkers on red blood cell drug loading

[0057] Figure 3. Flow cytometry analysis of the effects of G1 or G3 linkers on conjugation efficiency on native erythrocyte surfaces. Control group: unlabeled erythrocytes; experimental groups: erythrocytes labeled with eGFP-LPET-G1 or eGFP-LPET-G3. Histograms show eGPF signals on the erythrocyte surface after incubation with the corresponding molecules.

[0058] Detailed Description of the Invention

[0059] definition

[0060] In order to promote an understanding of the principles of the invention, reference will now be made to the embodiments shown in the accompanying drawings and described in detail. However, it should be understood that this is not intended to limit the scope of the invention.

[0061] In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Any methods and materials similar or equivalent to those described herein can be used to practice the present invention, but preferred methods and materials are described herein. Therefore, the terms defined herein are more fully described by the specification as a whole.

[0062] As used herein, the singular expressions "a," "an," and "the" encompass plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written from left to right in 5' to 3' orientation; amino acid sequences are written from left to right in amino to carboxyl orientation. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary depending on the specific context used by those skilled in the art.

[0063] Unless the context requires otherwise, the terms "comprises," "comprising," and "containing" or similar terms are intended to mean a non-exclusive inclusion, whereby a list of elements or features includes not only those elements mentioned or listed, but may include additional elements or features that are not listed or mentioned.

[0064] The terms "patient," "individual," and "subject" refer to any mammal for which the treatments or compositions disclosed herein can be used. Thus, the methods and compositions disclosed herein may have medical and / or veterinary applications. In a preferred form, the mammal is a human.

[0065] As used herein, term " sequence identity " refers to, with regard to the same degree of sequence on comparison window, after using standard algorithm to carry out suitable comparison, the nucleotide of complete matching or amino acid number.Therefore, " sequence identity percentage ratio " is calculated in the following manner: on comparison window, compare the sequence of two optimal comparisons, determine the quantity of the position that identical nucleic acid base (for example, A, T, C, G) occurs in two sequences to produce the quantity of matching position, the quantity of matching position is divided by the position sum (i.e. window size) in comparison window, and result is multiplied by 100 to produce sequence identity percentage ratio.For example, " sequence identity " can be understood as representing by DNASIS computer program (Windows version 2.5; Can obtain from Hitachi Software Engineering Co., Ltd. in South San Francisco, California, USA) " matching percentage ratio " calculated.

[0066] In the context of polypeptides, the term "mutation" refers to the replacement of at least one amino acid residue in a parent amino acid sequence with a different amino acid residue. The one or more replacement residues can be "naturally occurring amino acid residues" or "non-naturally occurring amino acid residues." Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, aib, and other amino acid residue analogs.

[0067] In the context of polypeptides, the term "position" refers to the position of an amino acid residue in the amino acid sequence of a polypeptide. In any case, the positions are numbered sequentially, with the first amino acid residue being numbered 1.

[0068] The terms "individual" or "subject" are used interchangeably and refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, 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 particular, the individual is a human.

[0069] The terms "cancer" and "cancerous" refer to the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, solid tumors such as lung cancer, lymphoma, breast cancer, liver cancer, bladder cancer, skin cancer, melanoma, colon cancer, rectal cancer, ovarian cancer, cervical cancer, prostate cancer, pancreatic adenocarcinoma, esophageal cancer, head and neck squamous cell carcinoma, thyroid cancer, glioblastoma, glioma, and hematological tumors such as leukemia and lymphoma.

[0070] The term "drug loading" refers to the amount of drug loaded per unit weight or per unit volume or per single red blood cell. In this application, the drug loading of red blood cells is generally measured in μg / mL as a dosage unit.

[0071] The term "treatment" refers to clinical intervention intended to alter the natural course of a disease in the individual being treated. Desired therapeutic effects include, but are not limited to, preventing the appearance or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis.

[0072] The term "prevent" includes the inhibition of the development or progression of a disease or condition or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for a preventative regimen. Generally, in the context of cancer, the term "prevent" refers to the administration of a drug before the development of signs or symptoms of cancer, particularly in a subject at risk for cancer.

[0073] The term "effective amount" refers to an amount or dosage of the modified red blood cells or compositions of the present invention that produces the desired effect in a patient in need of treatment or prevention after administration to the patient in a single or multiple doses. The effective amount can be readily determined by the attending physician, who is skilled in the art, by considering a variety of factors such as the species of the mammal; weight, age, and general health; the specific disease involved; the extent or severity of the disease; the response of the individual patient; the specific antibody administered; the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; and the use of any concomitant therapy.

[0074] The term "therapeutically effective amount" refers to an amount that is effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. The therapeutically effective amount of the modified erythrocytes or compositions of the present invention can vary according to a variety of factors such as disease state, age, sex, and weight of the individual. Relative to untreated subjects, a "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., uric acid content, tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60% or 70%, and still more preferably at least about 80% or 90%.

[0075] The term "prophylactically effective amount" refers to an amount effective to achieve the desired preventive result at the required dosage and for the required period of time. Typically, a prophylactic effective amount will be less than a therapeutically effective amount because a prophylactic dose is used in a subject prior to or at an earlier stage of disease.

[0076] The term "pharmaceutical composition" refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0077] Red blood cells (RBCs)

[0078] In the human body, red blood cells (RBCs) are the most numerous blood cells in the circulation. Unlike other blood cells, RBCs lack a nucleus and are flexible, allowing them to change shape to fit into human blood vessels. Their primary function in the body is to supply oxygen. Key protein markers on the surface of RBCs allow them to circulate in the body for extended periods without being cleared by macrophages, resulting in a long half-life. This property makes them excellent candidates for drug carriers. Mature RBCs without nuclei do not contain any genetic material, and therefore have a good safety profile compared to other gene and cell therapies.

[0079] In one aspect, the present invention provides red blood cells conjugated with an active agent, wherein the active agent is linked to the extracellular domain of at least one endogenous membrane protein of the RBC via a linker. In some embodiments, the red blood cells are modified so that their membrane proteins are covalently linked to the linker. In a specific embodiment, the linker is covalently linked to a free sulfhydryl or amino group of the membrane protein via a maleimide moiety contained therein. In a specific embodiment, the red blood cells are modified with a reducing agent so that the disulfide bonds in some endogenous membrane proteins on the surface of the red blood cells are reduced to free sulfhydryl groups in preparation for connection to the maleimide moiety of the linker. In another specific embodiment, the endogenous membrane protein of the modified red blood cells is linked to a linker comprising a maleimide moiety and a G-containing peptide. In another specific embodiment, under the action of a sortase, the red blood cells carrying the linker are contacted with an active agent containing a sortase recognition motif and a conjugation reaction occurs, thereby obtaining red blood cells carrying the active agent.

[0080] Unless otherwise specified or clear from the context, when this disclosure refers to red blood cells, it generally refers to mature red blood cells.In certain embodiments, the RBC is a human RBC, such as a human naive RBC.

[0081] In some embodiments, the RBCs are not genetically engineered. In some embodiments, the present invention provides red blood cells having an active agent conjugated thereto via a sortase-mediated reaction. In some embodiments, the conjugated active agent can be one or more of the active agents described herein. In some embodiments, the active agent can be conjugated to the red blood cells via a linker listed in Table 1.

[0082] After the active agent is linked to the linker, the last (e.g., the fifth from the N-terminus to the C-terminus) residue of the sortase recognition motif is replaced by the linked amino acid to form a structure of the first four amino acid residues of the sortase recognition motif-linker, that is, the active agent after connection comprises the first four amino acid residues of the sortase recognition motif, which is, for example, selected from LPXT, LPXA, LPXS, LPXL, LPXV, LGXT, LAXT, LSXT, NPXT, MPXT, IPXT, SPXT, VPXT, YPXR, LPXT and LPXT; X represents any amino acid.

[0083] In some embodiments, the present invention contemplates the use of autologous red blood cells isolated from an individual, modified in vitro, and then administered to the individual. In some embodiments, the present invention contemplates the use of immunocompatible red blood cells that have the same blood type (e.g., at least with respect to the ABO blood group system, and in some embodiments, with respect to the D blood group system) or a compatible blood type as the individual to whom the cells will be administered.

[0084] Sortase

[0085] The term "sortase," also known as transamidase, refers to an enzyme with transamidase activity. Transaminases typically catalyze the formation of a peptide bond (amide bond) between an acyl donor and a nucleophilic acyl acceptor. Sorting enzymes recognize substrates comprising a sortase recognition motif, such as the amino acid sequence LPXTG. Sorting enzymes cleave the recognition motif between residues threonine and glycine. Molecules recognized by sortases (i.e., comprising a sortase recognition motif) are sometimes referred to herein as "sortase substrates." Triglycine and even diglycine motifs on the N-terminus have been shown to be sufficient to support the SrtA reaction (Clancy, KW et al., Peptide Science 94 (2010) 385-396). Suitable sortases will be apparent to those skilled in the art, including but not limited to sortase A, sortase B, sortase C, and sortase D. The amino acid sequences of sortases and the nucleotide sequences encoding them are known to those skilled in the art. In a specific embodiment, the sortase is Staphylococcus aureus sortase A. In the reaction, sortase A first recognizes a substrate containing the LPXTG amino acid sequence motif and cleaves the amide bond between Thr and Gly with the help of the active site Cys, producing a sortase A-substrate thioester intermediate; then, this thioester acyl-enzyme intermediate is decomposed by nucleophilic attack of the amino group of a second substrate containing oligoglycine, producing a covalently linked conjugate molecule and regenerating sortase A.

[0086] For enzymatic conjugation, a soluble truncated sortase A lacking the transmembrane region can be used, such as, for Staphylococcus aureus, a truncated SrtA comprising amino acid residues 60 to 206. The sortase A-mediated reaction results in the attachment of molecules containing a sortase recognition sequence (sortase motif) to molecules containing a sortase receptor sequence (e.g., one or more N-terminal glycine residues).

[0087] In some embodiments, SrtA recognizes the motif LPXTG, where common recognition motifs include, for example, LPKTG, LPATG, and LPNTG. In some embodiments, LPETG is used. However, motifs that fall outside this consensus sequence can also be recognized. For example, in some embodiments, the 4th position of the motif contains "A," "S," "L," or "V" instead of "T," such as LPXAG, LPXSG, LPLXLG, or LPXVG, such as LPNAG or LPESG, LPELG, or LPEVG. In some embodiments, the 5th position of the motif contains "A" instead of "G," such as LPXTA, such as LPNTA. In some embodiments, the 2nd position of the motif contains "G" or "A" instead of "P," such as LGXTG or LAXTG, such as LGATG or LAETG. In some embodiments, the 1st position of the motif contains "I" or "M" instead of "L," such as MPXTG or IPXTG, such as MPKTG, IPKTG, IPNTG, or IPETG. Pishesha et al. 2018 described various recognition motifs for sortase A.

[0088] In some embodiments, the sortase recognition sequence is LPXTG, where X is a standard or non-standard amino acid. In some embodiments, X is selected from D, E, A, N, Q, K, or R. In some embodiments, the recognition sequence is selected from LPXTG, LPXAG, LPXSG, LPXLG, LPXVG, LGXTG, LAXTG, LSXTG, NPXTG, MPXTG, IPXTG, SPXTG, VPXTG, YPXRG, LPXTS, and LPXTA, where X can be any amino acid, for example, an amino acid selected from D, E, A, N, Q, K, or R in certain embodiments. In a specific embodiment, the sortase recognition motif provided herein is LPETG. In one embodiment, the sortase recognition motif can be modified to improve its recognition efficiency. Preferably, LPETG is modified to improve its affinity for the sortase, for example, by adding a G to the C-terminus of the recognition sequence, for example, the modified sequence is LPETGG.

[0089] In some embodiments, the present invention contemplates the use of naturally occurring variants of sortases. Regarding sortases such as sortase A, a large amount of structural information is available, including NMR or crystal structures of SrtA alone or in combination with a sortase recognition sequence (see, for example, Zong Y et al. J. Biol Chem. 2004, 279, 31383-31389). The active site and substrate binding pocket of Staphylococcus aureus SrtA have been determined. One of ordinary skill in the art can generate functional variants by, for example, not destroying or significantly changing the active site or substrate binding pocket of the sortase. In some embodiments, the directed evolution of SrtA can be performed by utilizing the FRET (fluorescence resonance energy transfer) selection assay described by Chen et al. Sci. Rep. 2016, 6 (1), 31899. In some embodiments, the functional variants of Staphylococcus aureus SrtA can be those described in CN10619105A and CN109797194A. In some embodiments, the S. aureus SrtA variant can be a truncated variant, eg, with 25-60 (eg, 30, 35, 40, 45, 50, 55, 59, or 60) amino acids removed from the N-terminus (compared to wild-type S. aureus SrtA).

[0090] In some embodiments, the functional variant of S. aureus SrtA useful in the present invention can be a S. aureus SrtA variant comprising one or more mutations at amino acid positions D124, Y187, E189, and F200, including D124G, Y187L, E189R, and F200L, and optionally further comprising one or more mutations at amino acid positions P94S / R, D160N, D165A, K190E, and K196T. In some embodiments, the mutant amino acid positions are numbered according to the numbering of wild-type S. aureus SrtA.

[0091] In a specific embodiment, the sortase is a Staphylococcus aureus transpeptidase A variant (mgSrtA) comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) identical to the amino acid sequence of SEQ ID NO:3.

[0092] In some embodiments, a sortase A variant having higher transamidase activity than naturally occurring sortase A can be used. In some embodiments, the sortase A variant has an activity that is at least about 10, 15, 20, 40, 60, 80, 100, 120, 140, 160, 180, or 200 times greater than that of wild-type S. aureus sortase. In some embodiments, such a sortase variant is used in the compositions or methods of the present invention. In some embodiments, the sortase variant comprises any one or more of the following substitutions relative to wild-type S. aureus SrtA: P94S / R, E105K, E108A, E108Q, D124G, D160N, D165A, Y187L, E189R, K190E, K196T, and F200L mutations. In some embodiments, the SrtA variant can have 25-60 (eg, 30, 35, 40, 45, 50, 55, 59, or 60) amino acids removed from the N-terminus.

[0093] In some embodiments, the sortase variants may further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative amino acid mutations. Conservative amino acid mutations that do not significantly affect protein activity are well known in the art.

[0094] Covalent conjugates comprising two entities that are not covalently associated in vivo can be obtained in vitro by using sortases, in particular sortase A.

[0095] connector

[0096] The term "linker" refers to a bifunctional or multifunctional molecule that can connect (conjugate) two molecules or entities together, typically having two reactive functions. Linkers used in conjugates can be broadly classified as non-cleavable or cleavable. They can also be divided into straight linkers and branched linkers based on whether the linker is branched. Branched linkers can contain branching units, each of which can be coupled to at least one drug or molecule.

[0097] The linker mentioned in this application comprises two parts, namely "maleimido alkyl chain (C 2-8 ) portion and a "G-containing small peptide" portion. In one embodiment, the "G-containing small peptide" portion is a branched small peptide comprising two or more branch units. In one embodiment, the branch units can be conjugated to the same or different molecules. In this application, the terms "branch unit" and "branching unit" can be used interchangeably. In one embodiment, the linker can be conjugated to multiple active molecules by virtue of its branching unit. In a specific embodiment, multiple linkers can be conjugated to the membrane surface of red blood cells.

[0098] In certain preferred embodiments, the branching unit is the amino acid sequence K(GGG), wherein the glycine residue adjacent to lysine is conjugated to the side chain ε-amino group of lysine, so that oligoglycine forms a branch chain, and lysine forms a peptide bond with other amino acids through its α-amino group to form the main chain of the G-containing small peptide.

[0099] In certain preferred embodiments, the linker is formed by its "maleimidoalkyl chain (C 2-8 ) portion" is covalently bound to the free sulfhydryl groups on the surface of the erythrocyte membrane, thereby being conjugated to the erythrocyte membrane. In a specific embodiment, the "maleimido alkyl chain (C 2-8 ) portion" are 6-maleimidocaproic acid and 4-maleimidocaproic acid.

[0100] UOX

[0101] The term "urate oxidase (Uox)" refers to the enzyme that oxidizes sparingly soluble uric acid into the more soluble allantoin. Urate oxidase is present in many species, but higher animals such as humans and apes lack biologically active urate oxidase. This is because the urate oxidase gene has mutated in these animals, resulting in uric acid being present as the end product of purine metabolism in humans and some other primates.

[0102] Gout is the most common inflammatory arthritis in adults, especially men, with a global prevalence of 1% to 4%. Gout occurs when monosodium urate crystals (MSU) deposit in tissues, causing inflammation and severe pain of gout attacks. The biological precursor to gout is elevated serum uric acid (UA) levels (i.e., hyperuricemia). When conventional urate-lowering drugs cannot be used, uricase is undoubtedly a valuable treatment option for chronic tophaceous gout.

[0103] Available recombinant UOX (rasburicase, pegloticase) drugs are effective urate-lowering agents for gout. However, current therapies have several limitations. First, UOX is significantly immunogenic and may cause severe allergic reactions. Second, these therapeutic enzymes may be inactivated or cleared in vivo due to their short half-life, limited bioavailability, and / or interactions with plasma proteins.

[0104] The following examples are intended to illustrate the present invention only and therefore should not be construed as limiting the present invention in any way.

[0105] Example 1. Preparation and purification of UOX-LPETGG fusion protein

[0106] 1. Construction of UOX fusion protein molecules with sortase recognition motifs

[0107] The sortase recognition motif LPETG was coupled to the UOX protein from Aspergillus flavus using a flexible peptide (GS) 3. To improve efficiency, the recognition motif LPETG can be modified. For example, in this application, affinity was increased by adding a G to the C-terminus of the recognition motif. Therefore, in this example, a fusion protein of UOX and LPETGG was constructed and named UOX-LPETGG. The amino acid sequence of the fusion protein is shown in SEQ ID NO: 1:

[0108] The nucleotide sequence encoding the fusion protein UOX-LPETGG is shown in SEQ ID NO: 2:

[0109] The UOX-LPETG coding sequence was synthesized by GenScript and consists of three parts: the nucleic acid sequence encoding the UOX protein, the nucleic acid sequence encoding (GS)3, and the nucleic acid sequence encoding the C-terminal LPETGG portion of the fusion protein. After sequence accuracy was confirmed by sequencing, the complete coding nucleic acid was constructed into a suitable expression vector and transformed into Escherichia coli BL21 (DE3, Tiangen) for protein expression.

[0110] A single transformed colony was inoculated into 10 ml of Luria-Bertani (LB) medium containing ampicillin (100 μg / ml, Beyotime) and cultured at 37°C with shaking at 220 rpm. The next day, 10 ml of the culture was transferred to 1 L of fresh LB medium and cultured at 37°C with shaking at 220 rpm until the OD600 reached 0.6. The culture temperature was lowered to 20°C, and 1 mM IPTG (Sigma) was added for induction.

[0111] After induction, the cell pellet was collected by centrifugation, resuspended in low-salt lysis buffer (50mM Tris 8.8, 50mM NaCl), and then sonicated. The supernatant containing the UOX-LPETGG protein was collected by centrifugation at 10,000 rpm for 1 hour and loaded onto a Q Sepharose FF column (Cytiva, Marlborough, USA) pre-equilibrated with QA buffer (20mM Tris 8.8). The column was washed with QA buffer until the absorbance was 280nm and the conductivity was stable, and then eluted with a linear gradient solution of 20mM Tris pH 8.8 containing 0-1M NaCl. The fractions corresponding to the elution peak were analyzed by SDS-PAGE, and the purest fractions were pooled and combined. The combined eluate was diluted with buffer (20 mM Tris 8.0) and then loaded onto a Diamond MixA column (Borgron (Shanghai) Biotechnology Co., Ltd.). Elution was performed using a linear gradient of 20 mM Tris pH 8.0 containing 0-1 M NaCl. Fractions corresponding to the elution peak were analyzed by SDS-PAGE, and the purest fractions were pooled. After adding an equal volume of buffer (40 mM Tris pH 7.5, 2 M (NH4)2SO4), the eluted sample was loaded onto a UniHR Phenyl-80L column (Suzhou Nanomicro Technology Co., Ltd.), washed with a 60% gradient of buffer B (20 mM Tris 7.5), and then eluted with 100% buffer B (20 mM Tris 7.5). The elution concentration was determined using an Amicon Ultra-15 centrifugal filter device (Millipore). The concentrated eluate was loaded onto an EzLoad 16 / 60 Chromdex 200 pg (Bioglone (Shanghai) Biotechnology Co., Ltd.) pre-equilibrated with PBS, and the target protein peak was collected.

[0112] Example 2. Preparation of red blood cells conjugated with UOX-LPETG using different linkers

[0113] Typically, active agents can be conjugated to the membrane surface of erythrocytes using conventional linear linkers. However, the drug loading capacity of erythrocytes obtained in this way sometimes cannot meet clinical needs. Therefore, this example studies the effect of different linkers on erythrocyte drug loading capacity.

[0114] 1. Screening of linkers containing different numbers of G-containing peptides

[0115] In the present application, in order to increase the drug loading capacity of red blood cells, a linker comprising a linear and a linear G-containing peptide was prepared. When the linker contains only one oligoglycine (e.g., GGG) that can react with an active agent containing a sortase recognition sequence, the G-containing peptide is referred to as a G1 peptide; when the linker contains two oligoglycines that can react with an active agent containing a sortase recognition sequence, the G-containing peptide is referred to as a G2 peptide; when the linker contains three oligoglycines that can react with an active agent containing a sortase recognition sequence, the G-containing peptide is referred to as a G3 peptide; and so on, G4 peptide and G5 peptide are obtained respectively. At the same time, linkers containing corresponding peptides can also be referred to as G1, G2, G3, G4, and G5. As shown in Table 1, when containing two or more oligoglycines that can react with active agents containing sortase recognition sequences, they respectively constitute branch units containing G peptides, and the linker thus obtained is a branched linker.

[0116] A. Preparation of linkers containing different numbers of G-containing peptides

[0117] Zhongke Yaguang was commissioned to prepare and synthesize the various linkers described in Table 1, which contain G1, G2, G3, G4, and G5 small peptides respectively.

[0118] B. Modification of red blood cells with different linkers

[0119] Red blood cells were isolated from the peripheral blood of Wistar rats (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) using density gradient centrifugation. The isolated red blood cells were rinsed three times with PBS and then pretreated with 5mM tris(2-carboxyethyl)phosphine (TCEP, Sigma) at 30°C for 1 hour. TCEP treatment reduces the extracellular disulfide bonds in the red blood cell membrane proteins, resulting in free sulfhydryl groups on the red blood cell membrane surface. The pretreated red blood cells were washed three times with PBS and reacted with the linkers containing the G1-G5 small peptides described in Table 1. Specifically, each linker was dissolved in PBS solution and mixed with the red blood cells pretreated with TCEP. The final reaction concentration of the linker was 0.625mM. The reaction was carried out at 30°C for 15 minutes to obtain red blood cells carrying different linkers. The red blood cells carrying different linkers were washed three times with PBS to obtain linker-modified red blood cells, which were named Gn-RBC.

[0120] 1×10 9 / mL Gn-mal-RBC was conjugated to the UOX-LPETGG obtained in Example 1 via a sortase (mg SrtA) reaction. In the conjugation reaction, the concentration of mg SrtA was 10 μM and the concentration of UOX-LPETGG was 25 μM. The conjugated red blood cell product was named UOX-LPET-Gn-RBC and stored at 2-8°C.

[0121] The amino acid sequence of mg SrtA is shown in SEQ ID NO: 3:

[0122] The nucleotide sequence of mg SrtA is shown in SEQ ID NO: 4:

[0123] C. Effects of different linkers on red blood cell drug loading

[0124] A sandwich ELISA was used to examine the conjugation of UOX protein to erythrocyte membranes. Specifically, the wells of a PVC microtiter plate were coated with anti-UOX antibody-1 (purchased from Hangzhou Huaan Biotechnology Co., Ltd.) at a concentration of 0.5 μg / mL in ELISA coating buffer (pH 9.6, purchased from Beijing Solebao Technology Co., Ltd.) overnight at 4°C. The coating buffer was removed, and the wells were rinsed twice with 200 μL of PBS. Remaining protein-binding sites in the coated wells were blocked by adding 200 μL of blocking buffer (5% skim milk / PBS) to each well at 37°C for 1 hour. The wells were then washed twice with 200 μL of PBS. UOX-LPET-Gn-RBCs were lysed with RIPA buffer (R&D) at 4°C for 10 minutes, and 100 μL of lysis solution was added to each well. Each plate included a positive control (in duplicate) and a blank control, which were incubated at 37°C for 1 hour. The solution was removed, and the wells were washed twice with 200 μL of PBS. Add 100 μL of diluted anti-UOX antibody-2 solution (1 μg / mL, HRP-conjugated, purchased from Hangzhou Huaan Biotechnology Co., Ltd.) to each well and incubate at 37°C for 1 hour. Wash four times with 200 μL of PBS. Add TMB solution (purchased from Beijing Solebao Technology Co., Ltd.) to each well and incubate for 10-15 minutes. Then, add an equal volume of stop solution (purchased from Beijing Solebao Technology Co., Ltd.) and measure the optical density at 450 nm.

[0125] The results are shown in Table 2. As the number of G-containing peptides contained in the linker increases, the drug loading capacity of the engineered erythrocytes obtained after the reaction with erythrocytes increases significantly. The UOX content in the erythrocytes carrying UOX conjugated by the G2 linker is 2.5 times that of the erythrocytes carrying UOX conjugated by the G1 linker, and the UOX content in the erythrocytes carrying UOX conjugated by the G3, G4 or G5 linkers is more than 3 times that of the erythrocytes carrying UOX conjugated by the G1 linker.

[0126] However, the study found that further increasing the number of G-containing peptides contained in the linker did not significantly increase the obtained red blood cell drug loading capacity, and the difficulty of synthesizing the linker was further increased.

[0127] As described above, the use of G2, G3, G4 or G5 linkers to modify erythrocytes results in higher drug loading efficiency of the modified erythrocytes than that of the G1 linker.

[0128] Table 2. Effects of different linkers on drug loading capacity of red blood cells

[0129] D. Effects of different linkers on red blood cell activity

[0130] We transfused UOX-LPET-G1-RBCs, UOX-LPET-G3-RBCs, UOX-LPET-G4-RBCs, UOX-LPET-G5-RBCs, and control RBCs (unmodified mouse peripheral blood red blood cells) into NPSG mice (Shanghai Jihui Laboratory Animal Breeding Co., Ltd.), with two mice per group, for a total of 10 mice. The RBC transfusion dose was 4e10 RBCs / kg. Plasma urate levels were measured 7 days after transfusion using a urate detection kit (Abcam). The experimental results are shown in Figure 1, which show that compared with the control RBC infusion group, the plasma urate of mice was reduced to a certain extent after 7 days of infusion of the red blood cell preparation modified with the G peptide linker prepared in this application. Among them, the urate level in the plasma of mice treated with UOX-LPET-G3-RBC, UOX-LPET-G4-RBC, and UOX-LPET-G5-RBC was significantly lower than the urate level in the plasma of mice treated with UOX-LPET-G1-RBC, indicating that the red blood cells modified with G3, G4 or G5 linkers all make the in vivo efficacy (ability to reduce plasma urate) of the modified red blood cells higher than that of the red blood cells modified with G1 linkers.

[0131] 2. Screening of connectors containing branch units of different lengths

[0132] To investigate whether branch units of different lengths in the branched connector would affect the drug loading capacity of red blood cells, the following experiments were performed.

[0133] A. Preparation of joints

[0134] Zhongke Yaguang was commissioned to prepare the linker described in Synthesis Table 3 and named it G6.

[0135] B. Modification of red blood cells with different linkers

[0136] Red blood cells were separated from the peripheral blood of C57 / B6 mice (Shanghai Jihui Experimental Animal Breeding Co., Ltd.) by density gradient centrifugation. The separated red blood cells were washed 3 times with PBS and then pretreated with 2.5mM TCEP (sigma) at 30°C for 1 hour. The pretreated red blood cells were washed 3 times with PBS, and the treated red blood cells were reacted with a connector comprising G1 and G3 described in Table 1 and a connector comprising G6 in Table 3. Specifically, each connector (G1, G3, G6) was dissolved in PBS solution and mixed with the red blood cells pretreated with TCEP, and the final reaction concentration of the connector was 0.625mM. At 30°C, the reaction was carried out for 15 minutes to obtain red blood cells carrying different connectors. The red blood cells carrying different connectors were washed three times with PBS to obtain connector-modified red blood cells, and the modified red blood cells were named Gn-RBC based on the name of the connector, where n is an integer of 1-5.

[0137] Then, 1×10 9 / mL Gn-RBC was conjugated with UOX-LPETGG obtained in Example 1. In the conjugation reaction, the concentration of mg SrtA was 10 μM and the UOX-LPETGG substrate was 25 μM. The final red blood cell product after conjugation was named UOX-LPET-Gn-RBC and stored at 2-8°C.

[0138] Table 3. Structure of G6 (6-mal-PEG10-(PEG6-GGG)3)

[0139] C. Effect of different linker lengths on red blood cell drug loading

[0140] A sandwich ELISA was used to examine the conjugation of UOX protein to erythrocyte membranes. Specifically, the wells of a PVC microtiter plate were coated with anti-UOX antibody-1 (purchased from Hangzhou Huaan Biotechnology Co., Ltd.) at a concentration of 0.5 μg / mL in ELISA coating buffer (pH 9.6, purchased from Beijing Solebao Technology Co., Ltd.) overnight at 4°C. The coating buffer was removed, and the wells were rinsed twice with 200 μL of PBS. Free protein-binding sites in the wells were blocked with 200 μL of blocking buffer (5% skim milk / PBS) at 37°C for 1 hour, and then washed twice with 200 μL of PBS. UOX-LPET-Gn-RBCs were lysed with RIPA buffer (R&D) at 4°C for 10 minutes, and 100 μL of lysis solution was added to each well of the plate. Each plate included a positive control (in duplicate) and a blank control, which were incubated at 37°C for 1 hour. The solution was removed, and the wells were washed twice with 200 μL of PBS. Add 100 μL of diluted anti-UOX antibody-2 solution (1 μg / mL, HRP-conjugated, purchased from Hangzhou Huaan Biotechnology Co., Ltd.) to each well and incubate at 37°C for 1 hour. Wash four times with 200 μL of PBS. Add TMB solution (purchased from Beijing Solebao Technology Co., Ltd.) to each well and incubate for 10-15 minutes. Then, add an equal volume of stop solution (purchased from Beijing Solebao Technology Co., Ltd.) and measure the optical density at 450 nm.

[0141] The results are shown in Figure 2. The conjugation effect of UOX and erythrocytes obtained using linker G3 (Mal-(GGG)3 in Figure 2) is approximately 2.3 times that of the conjugation effect of UOX and erythrocytes obtained using linker G1 (Mal-SKGGG in Figure 2). Further increasing the length of the linker (G6, Mal-PEG10-(GGG)3 in Figure 2) does not significantly increase the conjugation of UOX and erythrocytes.

[0142] Example 3 Preparation of eGFP-LPET-labeled red blood cells using a linker

[0143] 1. Expression and Purification of Recombinant Protein eGFP-LPETG in Escherichia coli

[0144] mg SrtA (SEQ ID NO: 3) and eGFP-LPETG cDNA were cloned into the pET vector and transformed into Escherichia coli BL21 (DE3) cells for protein expression. The transformed cells were cultured at 37°C until OD600 reached 0.6, and then 500 μM IPTG (sigma) was added. After culture at 37°C for 4 hours, the cells were collected by centrifugation and lysed with pre-cooled lysis buffer (20 mM Tris-HCl, pH 7.8, 500 mM NaCl). The lysate was sonicated on ice (5 seconds on, 5 seconds off, 60 cycles, 25% power, Branson Sonifier 550 ultrasonic cell disruptor). All supernatants were centrifuged at 4°C 14,000g for 40 minutes and filtered with a 0.45 μM filter. The filtered supernatant was loaded into the same The chromatography system was connected to a HisTrap FF 1 ml column (GE Healthcare). The protein was eluted with an elution buffer containing 20 mM Tris-HCl, pH 7.8, 500 mM NaCl, and 300 mM imidazole. All eluted fractions were analyzed on SDS-PAGE gels (GenScript).

[0145] The amino acid sequence of eGFP-LEPTG is shown in SEQ ID NO: 5:

[0146] The nucleotide sequence of eGFP-LEPTG is shown in SEQ ID NO: 6:

[0147] 2.G n Linker-labeled red blood cells

[0148] Red blood cells were isolated from the peripheral blood of C57 / B6 mice (Shanghai Jihui Experimental Animal Breeding Co., Ltd.) by density gradient centrifugation, using the same method as in Example 2. The pretreated red blood cells were washed three times with PBS and modified with the G1 and G3 linkers described in Table 1, respectively. Specifically, each linker was dissolved in PBS solution and mixed with the red blood cells pretreated with TCEP, and the final reaction concentration of the linker was 0.625 mM. At 30°C, the reaction was carried out for 15 minutes to obtain red blood cells carrying different linkers. The red blood cells carrying different linkers were washed three times with PBS to obtain linker-modified red blood cells, and the modified red blood cells were named Gn-RBC based on the name of the linker.

[0149] Then, 1×10 9 / mL Gn-RBC was conjugated with eGFP-LPETG (SEQ ID NO: 5). In the conjugation reaction, the concentration of mg SrtA (SEQ ID NO: 3) was 10 μM and the eGFP-LEPTG substrate was 25 μM. The final conjugated product was named eGFP-LPET-Gn-RBC and stored at 2-8°C.

[0150] The eGFP labeling effect on the erythrocyte membrane was simultaneously monitored by flow cytometry (Cytoflex, Beckman). The efficiency of eGFP-LPETG coupling to Gn-RBC was characterized by the eGFP (FITC) fluorescence signal on the cell membrane. The results showed that the eGFP-LPET labeling effect on the erythrocyte membrane obtained by the G3 linker (eGFP-LPET-(GGG)3-mal-RBC in Figure 3) was approximately 15 times that of the eGFP-LPET labeling on the erythrocyte membrane obtained by the G1 linker (eGFP-LPETGGGSK-mal-RBC in Figure 3). (See Figure 3) This shows that the branched linker can more effectively increase the drug loading capacity of erythrocytes.

[0151] Example 4 Preparation of anti-PD1 mAb-LPETGG labeled red blood cells using different linkers

[0152] 1. Purification of Anti-PD1 mAb-LPETGG Fusion Protein

[0153] The heavy chain amino acid sequence of anti-PD1 mAb-LPETGG is shown in SEQ ID NO: 7:

[0154] The heavy chain nucleotide sequence of anti-PD1 mAb-LPETGG is shown in SEQ ID NO: 8:

[0155] The light chain amino acid sequence of the anti-PD1 antibody is shown in SEQ ID NO: 9:

[0156] The nucleotide sequence of the light chain of the anti-PD1 antibody is shown in SEQ ID NO: 10:

[0157] The nucleotide sequences encoding the heavy chain or light chain were inserted into the expression vector pcDNA3.1. TM Each successfully constructed vector was transfected into CHO-S cells using the ExpiCHO expression system (ThermoFisher). TMThe cells are cultured in expression medium to express the corresponding heavy or light chain, thereby assembling the corresponding anti-PD1 antibody. Since LPETGG is linked to the C-terminus of the antibody heavy chain, it is called anti-PD1Ab-LPETGG.

[0158] The culture supernatant with anti-PD1 mAb-LPETGG protein was then harvested and purified using protein A affinity chromatography (Cytiva), Q Sepharose FF column (Cytiva), and Bestdex G-25 (Borgron (Shanghai) Biotechnology Co., Ltd.) according to the manufacturer's instructions, and the purified target protein was concentrated and stored at −80°C.

[0159] 2. Modification of red blood cells with different linkers

[0160] Red blood cells were separated from the peripheral blood of C57 / B6 mice (Shanghai Jihui Experimental Animal Breeding Co., Ltd.) by density gradient centrifugation. The separated red blood cells were washed 3 times with PBS. The red blood cells were then pretreated with 2.5mM TCEP (sigma) at 30°C for 1 hour. The pretreated red blood cells were washed 3 times with PBS, and the treated red blood cells were reacted with the connectors G1 and G3 described in Table 1. Specifically, each connector was dissolved with PBS solution and mixed with the red blood cells pretreated with TCEP, and the final reaction concentration of the connector was 0.625mM. At 30°C, the reaction was carried out for 15 minutes to obtain red blood cells carrying different connectors. The red blood cells carrying different connectors were washed three times with PBS to obtain connector-modified red blood cells, and the modified red blood cells were named Gn-RBC based on the name of the connector, where n is an integer from 1 to 5.

[0161] Then, 1×10 9 / mL Gn-RBC was conjugated with anti-PD1 mAb-LPETGG. In the conjugation reaction, the concentration of mg SrtA was 10 μM and the anti-PD1 mAb-LPETGG substrate was 25 μM. After conjugation, the final conjugated RBC product was designated anti-PD1 mAb-LPET-Gn-RBC and stored at 2-8°C.

[0162] 3. Effects of different linkers on red blood cell drug loading

[0163] The amount of anti-PD1 mAb conjugated to RBCs was measured by sandwich ELISA. Briefly, the wells of a PVC microtiter plate were coated with a 0.5 μg / mL concentration of a capture human PD-1 His tag (ACRO) in ELISA coating buffer (pH 9.6, purchased from Beijing Solebao Technology Co., Ltd.) at 4°C overnight. The coating buffer was removed, and the plate was washed twice with 200 μL of PBS. Remaining protein-binding sites in the coated wells were blocked by adding 200 μL of blocking buffer (5% skim milk / PBS) to each well for 1 hour at 37°C. The plate was washed twice with 200 μL of PBS. The anti-PD1 mAb-LPET-Gn-RBCs were lysed with RIPA buffer (R&D) at 4°C for 10 minutes. 100 μL of lysed RBC sample was added to each well and incubated at 37°C for 1 hour. The experiment was performed in duplicate, and each plate included a positive control and a blank control. The solution was removed and the plate was washed twice with 200 μL PBS; 100 μL of diluted detection anti-human FC antibody (1 μg / mL, eBioscience) was added to each well and incubated at 37°C for 1 hour; the plate was washed four times with 200 μL PBS; TMB solution (SURMODICS) was added to each well and incubated for 10-15 minutes, then an equal volume of stop solution (purchased from Beijing Solebold Technology Co., Ltd.) was added, and the optical density was detected at 450 nm.

[0164] The results are shown in Table 4. The conjugation effect of anti-PD1 mAb and red blood cells obtained using linker G3 (anti-PD1 mAb-LPET-G3) is approximately 2.3 times that of the conjugation effect of anti-PD1 mAb and red blood cells obtained using linker G1 (anti-PD1 mAb-LPET-G1).

[0165] Table 4. Effects of different linkers on red blood cell drug loading (anti-PD1 mAb-RBC)

Claims

1. A modified erythrocyte, wherein the active agent is conjugated to the extracellular portion of the erythrocyte membrane protein via a linker, wherein the linker comprises a G-containing small peptide and a maleimido alkyl chain (C 2-8 ), the maleimido alkyl chain (C 2-8 ) is conjugated to the membrane protein of the red blood cell, and the G-containing small peptide is conjugated to an active agent containing a sortase recognition motif through a sortase-mediated reaction, for example, the sortase recognition motif is LPXTG, preferably, the sortase recognition motif is LPETG.

2. The red blood cell according to claim 1, wherein the sortase recognition motif can be modified to increase its affinity; preferably, the modification is to add G to the C-terminus of the sortase recognition motif, such as LPETGG.

3. The erythrocyte according to claim 1 or 2, wherein the maleimidoalkyl chain (C 2-8 ) is 6-maleimidocaproic acid or 4-maleimidobutyric acid.

4. The erythrocyte according to any one of claims 1 to 3, wherein the G-containing small peptide is a linear small peptide or a branched small peptide.

5. The erythrocyte of any one of claims 1-4, wherein the G-containing small peptide is a branched small peptide comprising two or more branching units, wherein one or more active agents are coupled to one or more branching units.

6. The erythrocyte according to any one of claims 1 to 5, wherein the branching unit consists of the amino acid sequence K(GGG), wherein the glycine in the brackets is conjugated to the ε-amino group of the side chain of lysine to form a branch chain, and lysine forms peptide bonds with other amino acids through its α-amino group to constitute the main chain of the "G-containing small peptide", and optionally, an extension chain can be added between K and G in the branching unit K(GGG), such as COCH2CH2-PEG6-NH.

7. The erythrocyte according to any one of claims 1 to 6, wherein the G-containing small peptide has the following structure: GGGSK, K(GGG)-GGG-K(GGG), K(GGG)-GGG-K(GGG)-GGG-K(GGG), K(GGG)-GGG-K(GGG)-GGG-K(GGG)-GGG-K(GGG), K(GGG)-GGG-K(GGG)-GGG-K(GGG)-GGG-K(GGG)-GGG-K(GGG) or K[(COCH2CH2-PEG6-NH)-GGG]-GGG-K[(COCH2CH2-PEG6-NH)-GGG]-GGG-K[(COCH2CH2-PEG6-NH)-GGG]-NH2.

8. The erythrocyte of any one of claims 1 to 7, wherein the maleimidoalkyl chain (C 2-8 ) is 6-maleimidocaproic acid.

9. The erythrocyte according to any one of claims 1 to 8, wherein PEGn is contained between the G-containing small peptide and the 6-maleimidocaproic acid, wherein n=1-20.

10. The red blood cell according to any one of claims 1 to 9, wherein the linker has a structure as shown in Table 1 or Table 3.

11. The erythrocyte of any one of claims 1-10, wherein a plurality of active agents are conjugated to a membrane protein of the erythrocyte via a branched linker.

12. The red blood cell of any one of claims 1-11, wherein the active agent is modified to comprise a recognition motif for a sortase.

13. The red blood cell of any one of claims 1-12, wherein the active agent comprises a binding agent, a therapeutic agent, or a detection agent.

14. The erythrocyte of any one of claims 1 to 13, wherein the active agent is a peptide molecule and is activated by a flexible peptide segment (GS). n Linked to a sortase recognition motif, where n=1-10.

15. The red blood cell of any one of claims 1-14, having the following structure: UOX-LPET-G1-RBC, UOX-LPET-G2-RBC, UOX-LPET-G3-RBC, UOX-LPET-G4-RBC, UOX-LPET-G5-RBC, UOX-LPET-G6-RBC, anti-PD1 mAb-LPET-(GGG)3-RBC, anti-PD1 mAb-LPET-GGGSK-RBC, or anti-PD1 mAb-1-LPET-GAASK-RBC.

16. A linker molecule comprising a G-containing peptide and a maleimido alkyl chain (C 2-8 )composition.

17. The linker molecule of claim 16, wherein the maleimido alkyl chain (C 2-8 ) is 6-maleimidocaproic acid or 4-maleimidobutyric acid. The linker molecule according to claim 16 or 17 , wherein the G-containing peptide is a linear peptide or a branched peptide.

19. The linker molecule of any one of claims 16 to 18, wherein the G-containing small peptide is a branched small peptide comprising two or more branching units, wherein one or more active agents are coupled to one or more branching units.

20. The linker molecule according to any one of claims 16 to 19, wherein the branching unit consists of the amino acid sequence K(GGG), wherein the glycine in the brackets is conjugated to the ε-amino group of the side chain of lysine to form a branch chain, and lysine forms a peptide bond with other amino acids through its α-amino group to constitute the main chain of the "G-containing small peptide", and optionally, an extension chain can be added between K and G in the branching unit K(GGG), such as COCH2CH2-PEG6-NH.

21. The linker molecule of any one of claims 16-20, wherein the G-containing peptide has the following structure: GGGSK, K(GGG)-GGG-K(GGG), K(GGG)-GGG-K(GGG)-GGG-K(GGG), K(GGG)-GGG-K(GGG)-GGG-K(GGG)-GGG-K(GGG), K(GGG)-GGG-K(GGG)-GGG-K(GGG)-GGG-K(GGG) or K[(COCH2CH2-PEG6-NH)-GGG]-GGG-K[(COCH2CH2-PEG6-NH)-GGG]-GGG-K[(COCH2CH2-PEG6-NH)-GGG]-NH2.

22. The linker molecule of any one of claims 16 to 21, wherein the maleimido alkyl chain (C 2-8 ) is 6-maleimidocaproic acid.

23. The linker molecule according to any one of claims 16 to 22, wherein PEGn is contained between the G-containing small peptide and the 6-maleimidocaproic acid, wherein n=1-20.

24. The linker molecule of any one of claims 16-23, wherein the linker has a structure as shown in Table 1 or Table 3.

25. A method for preparing the red blood cells according to any one of claims 1 to 15, comprising: 1) treating erythrocytes with a reducing agent so that the linker molecule according to any one of claims 16 to 24 is linked to the extracellular domain of the endogenous membrane protein of the erythrocyte; and / or, 2) treating the active agent such that the active agent comprises a sortase recognition motif; and / or, 3) In the presence of sortase, contacting the erythrocytes obtained in step 1) with the active agent obtained in step 2) under conditions suitable for the reaction of the sortase, so that the sortase conjugates the active agent to the endogenous membrane protein of the erythrocyte through a linker.

26. A composition comprising the red blood cells of any one of claims 1-15, and optionally a pharmaceutically acceptable carrier.

27. A method for diagnosing, treating or preventing a disease in a subject in need thereof, comprising administering to the subject the red blood cells of any one of claims 1 to 15 or the composition of claim 26.

28. The method of claim 27, wherein the disease is selected from the group consisting of tumors or cancers, metabolic diseases, bacterial infections, viral infections, autoimmune diseases, and inflammatory diseases.

29. A method of delivering an active agent to a subject in need thereof, comprising administering to the subject the red blood cells of any one of claims 1-15 or the composition of claim 26.

30. A method of increasing the plasma half-life of an active agent, comprising: 1) treating the active agent so that it contains a sortase recognition motif; 2) providing a red blood cell carrying the linker according to any one of claims 16 to 24; and 3) conjugating the active agent obtained in step 1) and the red blood cells obtained in step 2) in the presence of a sortase under suitable conditions, wherein the conditions are suitable for the sortase to conjugate the sortase substrate to at least one endogenous non-engineered membrane protein of the red blood cells through a sortase-mediated reaction, preferably through sortase-mediated glycine conjugation and / or sortase-mediated lysine side chain ε-amino conjugation.

31. Use of the red blood cells of any one of claims 1 to 15 or the composition of claim 26 in the preparation of a medicament for treating or preventing a disease, or in the preparation of a diagnostic agent for diagnosing a disorder, condition or disease, or in the preparation of a medicament for delivering an active agent.

32. The use of claim 31, wherein the disease is selected from tumors or cancers, metabolic diseases, bacterial infections, viral infections, autoimmune diseases and inflammatory diseases.

Images