WO2024067295A1 - Modified red blood cell and use thereof for delivering medicament - Google Patents

Abstract

Provided is a modified red blood cell. An active agent is conjugated to an extracellular portion of a membrane protein of a red blood cell by means of a linker, wherein the linker comprises a G-containing small peptide and a maleimidoalkyl chain (C2-8). The maleimidoalkyl chain (C2-8) 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 sorting enzyme recognition motif by means of a sorting enzyme-mediated reaction. A covalently modified RBC and use thereof for delivering a medicament and a probe are also provided.

Description

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

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 technique

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

At present, there are many mature and feasible methods for developing drug delivery carriers by modifying red blood cells, which can be mainly divided into: genetically modified red blood cell carriers, non-genetically modified red blood cell carriers and red blood cell membrane modification, such as installing proteins by direct encapsulation, non-covalent linkage of exogenous peptides, or by fusing proteins with RBC surface protein-specific antibodies. However, such modified red blood cells have limitations in in vivo applications. For example, encapsulation can damage the cell membrane, thereby affecting the in vivo survival rate of engineered cells. In addition, the non-covalent connection between polymer particles and red blood cells is easily dissociated, and the payload will be quickly degraded in vivo.

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 cuts between threonine and glycine to form a covalent acyl-enzyme intermediate between the enzyme and the substrate protein. This intermediate is disintegrated by nucleophilic attack of oligoglycine of a peptide or protein, wherein the peptide or protein typically has three consecutive oligoglycine residues (3×glycine, G ₃ ) at the N-terminus. Previous studies have genetically overexpressed the membrane protein KELL with an LPXTG motif at the C-terminus on RBCs, which can be connected to the N-terminus of a protein/peptide modified with 3×glycine or G _(n≥3) by using wtSrtA. These drug-carrying RBCs have shown efficacy in treating diseases in animal models. However, this requires engineering of hematopoietic stem cells or progenitor cells (HSPCs) and the steps of differentiating these cells into mature RBCs, which greatly limits its application.

In addition, how to increase the effective load of red blood cells without affecting their integrity and function, thereby increasing the concentration of drugs at the target site and enhancing the effect of drugs on disease treatment, is still a problem that needs to be solved urgently. The present application solves this problem to a certain extent by providing an improved RBC delivery system.

SUMMARY OF THE INVENTION

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

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

In one embodiment, the present invention provides a modified erythrocyte, wherein a linker comprising a maleimido alkyl chain ( _{C 2-8} ) is covalently bound to a free thiol group on the surface of the erythrocyte membrane. In a specific embodiment, the linker is covalently bound to a free thiol group on the surface of the erythrocyte membrane through 6-maleimidocaproic acid or 4-maleimidobutyric acid contained therein, thereby obtaining an erythrocyte carrying the linker.

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

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 is composed of an amino acid sequence K (GGG), wherein the glycine (G) in the brackets is conjugated with the side chain ε-amino group of the adjacent lysine (K) to form a branch chain, 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 COCH ₂ CH ₂ -PEG ₆ -NH.

In a preferred embodiment, the G-containing small peptide has a structure selected from the following: 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), or K[(COCH ₂ CH ₂ -PEG ₆ -NH)-GGG]-GGG-K[(COCH ₂ CH ₂ -PEG ₆ -NH)-GGG]-GGG-K[(COCH ₂ CH ₂ -PEG ₆ -NH)-GGG]-NH ₂ (SEQ ID NO: 16), wherein the glycine (oligoglycine) in the brackets is conjugated with 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 COCH ₂ CH ₂ -PEG ₆ -NH. In one embodiment, the above 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, a plurality of identical or different active agents are conjugated to a linker comprising a plurality of branching units through the above reaction.

In one embodiment, the linker comprises a G-containing small peptide and a maleimido alkyl chain (C _2-8 ), and is connected to the membrane protein of the erythrocyte through its maleimido alkyl chain (C _2-8 ), 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 consists 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 small peptide and 6-maleimido hexanoic acid. In a specific embodiment, PEGn is contained between the G-containing small peptide and 6-maleimido hexanoic acid, wherein n=1-20. In a specific embodiment, multiple identical or different active agents are conjugated to erythrocytes at the same time through the linker. Preferably, at least two active agents are conjugated to erythrocytes at the same time. In one embodiment, the above-mentioned active agents are the same active agents.

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

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

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 natural erythrocytes, such as natural human erythrocytes.

In one embodiment, the present invention provides erythrocytes conjugated with an active agent, wherein the active agent is conjugated to the erythrocytes carrying the linker described above.

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

In one embodiment, the sortase is sortase A (SrtA), such as Staphylococcus aureus transpeptidase A, such as 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.

In one embodiment, the sortase recognition motif comprises or 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 connected to the linker, the last (e.g., the 5th from the N-terminal to the C-terminal) residue of the sortase recognition motif is replaced by the amino acid to which the connection occurs, and a structure of the first 4 amino acid residues of the sortase recognition motif-linker is formed, 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 efficiency of recognition, preferably, LPETG is modified to improve its affinity with the sortase, for example, G is added to the C-terminal of the recognition sequence, for example, the modified sequence is LPETGG.

In one embodiment, the active agent comprises a binding agent, a therapeutic agent or a 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.

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.

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 a sequence of (GS) _n , n=1-10. In a specific embodiment, (GS) _n is, for example, (GS) _2, (GS) _3, (GS) ₄ .

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.

In a second aspect, the present application provides a linker molecule, which is composed of a G-containing small peptide and a maleimido alkyl chain (C _2-8 ).

In one embodiment, the maleimido alkyl chain (C _2-8 ) is 6-maleimidocaproic acid, 4-maleimidobutyric acid.

In one embodiment, the G-containing small peptide is a linear or 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 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 small peptide.

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.

In a preferred embodiment, the small peptide is conjugated together by reacting the 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.

In one embodiment, the linker is connected to the membrane protein of the erythrocyte through its maleimido alkyl chain (C2-8), 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, multiple identical or different active agents are conjugated to the erythrocyte at the same time through the linker. Preferably, at least two active agents are conjugated to the erythrocyte at the same time. In one embodiment, the above-mentioned active agents are the same active agent.

In a specific embodiment, the linker has a structure as shown in Table 1.

In a specific embodiment, there is provided use of the linker described in this aspect in modifying erythrocytes, wherein the erythrocytes and an active agent are coupled together via the linker.

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

1) treating erythrocytes so that the linker molecule of the second aspect is linked to the extracellular domain of the endogenous membrane protein of the erythrocytes; 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 via a linker.

In one embodiment, the erythrocytes are treated with a reducing agent so that disulfide bonds in the extracellular domain (eg, at an internal site of the extracellular domain) of at least one endogenous membrane protein of the erythrocyte are reduced to have free sulfhydryl groups.

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

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

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 cells via a linker molecule having a branching unit.

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.

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

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

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 red blood cells or the composition as described in the present application.

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 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.

In a seventh aspect, the present invention provides a method for 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 erythrocytes carrying a linker, or optionally, for erythrocytes not carrying a linker, treating the erythrocytes as described above, such that the linker molecule of the second aspect is attached to the extracellular domain of the endogenous membrane protein of the erythrocyte; and

3) conjugating the active agent obtained in step 1) with the erythrocytes 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 erythrocyte through a sortase-mediated reaction, preferably through sortase-mediated glycine conjugation and/or sortase-mediated lysine side chain ε-amino conjugation.

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.

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 disorder, condition or disease, or in the preparation of a medicament for delivering an active agent.

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.

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

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

Figure 2 shows the effect of different linkers on the drug loading capacity of red blood cells

Figure 3 Flow cytometry was used to detect the effect of G1 linker or G3 linker on the coupling efficiency on the surface of natural red blood cells. Control group: unlabeled red blood cells; experimental group: red blood cells labeled with eGFP-LPET-G1, red blood cells labeled with eGFP-LPET-G3. The histogram shows the eGPF signal on the surface of red blood cells after incubation with the corresponding molecules.

DETAILED DESCRIPTION OF THE INVENTION

definition

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.

In this disclosure, unless otherwise indicated, the 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 implement 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.

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

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 other elements or features not listed or mentioned.

The terms "patient," "individual," and "subject" refer to any mammal to which the treatment or composition disclosed herein can be applied. Thus, the methods and compositions disclosed herein can have medical and/or veterinary applications. In a preferred form, the mammal is a human.

As used herein, the term "sequence identity" refers to the number of completely matching nucleotides or amino acids after appropriate alignment using a standard algorithm, with respect to the degree of identity of the sequences over a comparison window. Thus, the "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions at which the same nucleic acid base (e.g., A, T, C, G) appears in the two sequences to produce the number of matching positions, dividing the number of matching positions by The total number of positions in the comparison window (i.e., the window size) is then multiplied by 100 to generate the percent sequence identity. For example, "sequence identity" may be understood to mean the "percent match" calculated by the DNASIS computer program (version 2.5 for Windows; available from Hitachi Software Engineering, Inc., South San Francisco, California, USA).

In the context of polypeptides, the term "mutation" refers to replacing at least one amino acid residue in the 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.

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.

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.

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 blood tumors such as leukemia and lymphoma.

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 usually measured in μg/mL as a dosage unit.

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 relieving the disease state, and alleviating or improving prognosis.

The term "prevention" includes inhibition of the occurrence or development of a disease or disorder or symptoms of a particular disease or disorder. In some embodiments, subjects with a family history of cancer are candidates for preventive regimens. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug before the signs or symptoms of cancer occur, particularly in a subject at risk for cancer.

The term "effective amount" refers to such an amount or dosage of the modified red blood cells or compositions of the present invention, which produces the desired effect in a patient in need of treatment or prevention after being administered to the patient in a single or multiple doses. The effective amount can be easily determined by the attending physician who is a person skilled in the art by considering a variety of factors such as the species of the mammal; body 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.

The term "therapeutically effective amount" refers to an amount that effectively achieves 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 suppresses measurable parameters (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%.

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, since a prophylactic dose is used in a subject before or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

The term "pharmaceutical composition" refers to a composition that is in a form that permits 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.

Red blood cells (RBC)

In the human body, red blood cells are the largest blood cells in the circulatory system. Unlike other blood cells, red blood cells lack a nucleus and are flexible. They can change their shape to adapt to human blood vessels and are mainly responsible for the oxygen supply function in the body. Key protein markers on the surface of red blood cells allow them to circulate in the body for a long time without being cleared by macrophages, thus having a long half-life. This property makes them an excellent candidate for drug carriers. Mature red blood cells without nuclei do not contain any genetic material, so they have good safety compared to other gene and cell therapies.

In one aspect, the present invention provides red blood cells conjugated with an active agent, wherein the active agent is connected to the extracellular domain of at least one endogenous membrane protein of RBC through 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 the free sulfhydryl or amino group of the membrane protein through the maleimide portion 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 sulfhydryls in preparation for connection with the maleimide portion of the linker. In another specific embodiment, the endogenous membrane protein of the modified red blood cells is connected to a linker comprising a maleimide portion and a G-containing small 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.

Unless otherwise specified or it is clear from the context that otherwise means, when the 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.

In some embodiments, the RBC is not genetically engineered. In some embodiments, the invention provides red blood cells having an active agent conjugated thereto by a sortase-mediated reaction. In some embodiments, the conjugated active agent can be one or more active agents described herein. In some embodiments, the active agent can be conjugated to the red blood cell via a linker listed in Table 1.

After the active agent is connected to the linker, the last (e.g., the 5th from the N-terminus to the C-terminus) residue of the sortase recognition motif is replaced by the amino acid to which the connection occurs, thereby forming a structure of the first 4 amino acid residues of the sortase recognition motif-linker, that is, the active agent after connection comprises the first 4 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.

In some embodiments, the present invention contemplates the use of autologous red blood cells isolated from an individual, which are 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 may be a compatible blood type as the individual to whom the cells are to be administered.

Sortase

The term "sortase", also known as transamidase, refers to an enzyme having transamidase activity. Transamidase enzymes generally catalyze the formation of a peptide bond (amide bond) between an acyl donor and a nucleophilic acyl acceptor. Sortase enzymes recognize substrates comprising a sortase recognition motif, such as the amino acid sequence LPXTG. Sortase enzymes cleave the recognition motif between residues threonine and glycine. Molecules recognized by sortase enzymes (i.e., comprising a sortase recognition motif) are sometimes referred to herein as "sortase substrates". It has been shown that triglycine and even diglycine motifs on the N-terminus are sufficient to support the SrtA reaction (Clancy, K.W. et al., Peptide science 94 (2010) 385-396). Suitable sortase enzymes 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 sortase enzymes and the nucleotide sequences encoding them are known to those skilled in the art. In a specific embodiment, the sortase enzyme is Staphylococcus aureus sortase A. In the reaction, first, sortase A 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 to produce a sortase A-substrate thioester intermediate; then, the thioester acyl-enzyme intermediate is decomposed by nucleophilic attack of the amino group of a second substrate containing oligoglycine to produce a covalently linked conjugate molecule and regenerate sortase A.

For enzymatic conjugation, a soluble truncated sortase A lacking the transmembrane region can be used, such as, for S. 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).

In some embodiments, SrtA recognizes the motif LPXTG, wherein common recognition motifs include, for example, LPKTG, LPATG, LPNTG. In some embodiments, LPETG is used. However, motifs falling outside the consensus sequence can also be recognized. For example, in some embodiments, the 4th position of the motif comprises "A", "S", "L" or "V" instead of "T", such as LPXAG, LPXSG, LPXLG or LPXVG, such as LPNAG or LPESG, LPELG or LPEVG. In some embodiments, the 5th position of the motif comprises "A" instead of "G", such as LPXTA, such as LPNTA. In some embodiments, the 2nd position of the motif comprises "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 comprises "I" or "M" instead of "L", such as MPXTG or IPXTG, such as MPKTG, IPKTG, IPNTG or IPETG. Pishesha et al. 2018 describe various recognition motifs for sortase A.

In some embodiments, the sortase recognition sequence is LPXTG, wherein 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, wherein X can be any amino acid, such as 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 efficiency of recognition, preferably, LPETG is modified to improve its affinity with the sortase, such as adding G at the C-terminus of the recognition sequence, such as the modified sequence is LPETGG.

In some embodiments, the present invention contemplates the use of naturally occurring variants of sortases. A large amount of structural information is available for sortases such as sortase A, including NMR or crystal structures of SrtA alone or in combination with a sortase recognition sequence (see, e.g., 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, deletions or substitutions that do not destroy or significantly change the active site or substrate binding pocket of the sortase. In some embodiments, directed evolution of SrtA can be performed by utilizing a FRET (fluorescence resonance energy transfer)-based 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, for example, 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).

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 in D124G, Y187L, E189R and F200L at amino acid positions D124, Y187, E189 and F200, and optionally further comprising one or more mutations in P94S/R, D160N, D165A, K190E and K196T. In some embodiments, the above-mentioned mutant amino acid positions are numbered according to the numbering of wild-type S. aureus SrtA.

In a specific embodiment, the sortase is a Staphylococcus aureus transpeptidase A variant (mgSrtA) comprising, consisting essentially of, or consisting of the amino acid sequence: The sequence has 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) identity with the amino acid sequence set forth in SEQ ID NO:3.

In some embodiments, a sortase A variant having higher transamidase activity than naturally occurring sortase A can be used. In some embodiments, the activity of the sortase A variant is at least about 10, 15, 20, 40, 60, 80, 100, 120, 140, 160, 180, or 200 times that of wild-type S. aureus sortase. In some embodiments, such a sortase variant is used in the compositions or methods of the 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.

In some embodiments, the sortase variants may also contain 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.

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

Connectors

The term "linker" refers to a bifunctional or multifunctional molecule that can connect (conjugate) two molecules or entities together, usually with two reactive functions. The linkers used in conjugates can be roughly divided into non-cleavable or cleavable. They can also be divided into straight linkers and branched linkers according to whether the linker is branched. Branched linkers can contain branching units, each of which can be coupled to at least one drug or molecule.

The linker mentioned in the present application comprises two parts, namely, a "maleimido alkyl chain (C _2-8 ) part" and a "G-containing small peptide" part. In one embodiment, the "G-containing small peptide" part is a branched small peptide comprising 2 or more branch units. In one embodiment, the branch unit can be conjugated to the same or different molecules. In the present 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 erythrocytes.

In certain preferred embodiments, the branching unit is an amino acid sequence K (GGG), wherein a 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 a G-containing small peptide.

In certain preferred embodiments, the linker is covalently bound to the free thiol groups on the surface of the erythrocyte membrane through its "maleimido alkyl chain (C _2-8 ) part", thereby being conjugated to the erythrocyte membrane. In a specific embodiment, the "maleimido alkyl chain (C _2-8 ) part" is 6-maleimido hexanoic acid or 4-maleimido butyric acid.

UOX

The term "urate oxidase (Uox)" refers to an enzyme that oxidizes slightly soluble uric acid into more soluble allantoin. Urate oxidase exists in many species, but higher animals such as humans and apes lack biologically active urate oxidase because the urate oxidase gene has mutated in animals, so uric acid exists as the end product of purine metabolism in humans and some other primates.

Gout is the most common inflammatory arthritis in adults, especially in adult men, with a global prevalence of 1% to 4%. Gout occurs when monosodium urate crystals (MSU) are deposited in tissues, causing inflammation and severe pain of gout attacks. The biological precursor of 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.

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.

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.

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

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

The recognition motif LPETG of the sortase was coupled to the UOX protein from Aspergillus flavus using a flexible peptide (GS) _3. In order to improve the efficiency, the recognition motif LPETG can be modified. For example, in the present application, the affinity is increased by adding G to the C-terminus of the recognition motif. Therefore, in this embodiment, a fusion protein of UOX and LPETGG was constructed, and the obtained fusion protein was named UOX-LPETGG, and its amino acid sequence is shown in SEQ ID NO: 1:

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

The coding sequence of UOX-LPETG was synthesized by GenScript, and the sequence was divided into 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 of the fusion protein. After the accuracy of the sequence was confirmed by sequencing, the complete coding nucleic acid was constructed into a suitable expression vector and then transformed into Escherichia coli BL21 (DE3, Tiangen) for protein expression.

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

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 UOX-LPETGG protein was collected by centrifugation at 10,000rpm 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 pH8.8 containing 0-1M NaCl. The components corresponding to the elution peak were analyzed by SDS-PAGE, and the purest components were pooled and merged. The combined eluate was diluted with buffer (20mM Tris8.0) and then loaded onto a Diamond MixA column (Borgron (Shanghai) Biotechnology Co., Ltd.) and eluted with a linear gradient solution of 20mM Tris pH 8.0 containing 0-1M NaCl. The components corresponding to the elution peak were analyzed by SDS-PAGE, and the purest components were pooled. After adding an equal volume of buffer (40mM Tris pH7.5, 2M (NH4)2SO4), the eluted sample was loaded onto a UniHR Phenyl-80L column (Suzhou Na Micro Technology Co., Ltd.), washed with 60% gradient buffer B (20mM Tris7.5), and then eluted with 100% buffer B (20mM Tris7.5). The elution concentration was detected using an Amicon Ultra-15 centrifugal filter device (Millipore). The concentrated eluate was loaded onto an EzLoad 16/60 Chromdex 200pg (Borgron (Shanghai) Biotechnology Co., Ltd.) pre-equilibrated with PBS, and then the target protein peak was collected.

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

Generally, the active agent can be conjugated to the membrane surface of erythrocytes using a general straight linker, but 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 the drug loading capacity of erythrocytes.

1. Screening of linkers containing different numbers of G-containing peptides

In the present application, in order to increase the drug loading of red blood cells, a linker containing a linear and a linear G-containing peptide is 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 called 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 called 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 called a G3 peptide; and so on, G4 peptide and G5 peptide are obtained respectively. At the same time, the linker containing the corresponding peptide can also be called G1, G2, G3, G4, G5. As shown in Table 1, for 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 obtained is a branch linker.

A. Preparation of linkers containing different numbers of G-containing peptides

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.

B. Modification of red blood cells with different linkers

Red blood cells were separated from the peripheral blood of Wistar rats (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) by density gradient centrifugation. The separated red blood cells were rinsed with PBS three times, and then pretreated with 5mM tri(2-carboxyethyl)phosphine (TCEP, Sigma) at 30°C for 1hr. Through the treatment with TCEP, the disulfide bonds located outside the cell in the red blood cell membrane protein were reduced, so that the membrane surface of the red blood cells contained free thiol groups. The pretreated red blood cells were washed with PBS three times, and the linkers containing G1-G5 small peptides described in Table 1 were reacted with the above-treated red blood cells. Specifically, each linker was dissolved with PBS solution and mixed with the red blood cells pretreated with TCEP, and the final reaction concentration of the linker was 0.625mM. At 30°C, the reaction was carried out for 15min to obtain red blood cells carrying different linkers. The red blood cells carrying different linkers were washed with PBS three times to obtain linker-modified red blood cells, which were named Gn-RBC.

1×10 ⁹ /mL Gn-mal-RBC was coupled with UOX-LPETGG obtained in Example 1 by 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.

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

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

C. Effect of different linkers on drug loading in red blood cells

The conjugation effect of UOX protein with erythrocyte membrane was detected by sandwich ELISA. Specifically, the wells of PVC microtiter plates 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 Solebow Technology Co., Ltd.) overnight at 4°C; the coating solution was removed and the wells were rinsed twice with 200 μL PBS; 200 μL blocking buffer (5% skim milk/PBS) was added to each well, and the remaining protein binding sites in the coated wells were blocked at 37°C for 1 hour; and washed twice with 200 μL PBS. UOX-LPET-Gn-RBC was lysed with RIPA buffer (R&D) at 4°C for 10 minutes, and then 100 μL lysis solution was added to each well of the plate. Each plate included a positive control (duplicate) and a blank control, incubated at 37°C for 1 hour. The solution was removed and washed twice with 200 μL 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 4 times with 200 μL PBS. Add TMB solution (purchased from Beijing Solebold Technology Co., Ltd.) to each well, incubate for 10-15 minutes, then add an equal volume of stop solution (purchased from Beijing Solebold Technology Co., Ltd.) and detect the optical density at 450 nm.

The results are shown in Table 2. As the number of G-containing small peptides contained in the linker increases, the drug loading of the engineered erythrocytes obtained after reacting 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.

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, and the difficulty of synthesizing the linker was further increased.

As described above, using G2, G3, G4 or G5 linkers to modify erythrocytes makes the drug loading efficiency of the modified erythrocytes higher than that of the G1 linker.

Table 2. Effects of different linkers on drug loading in red blood cells

D. Effects of different linkers on red blood cell activity

We transfused UOX-LPET-G1-RBC, UOX-LPET-G3-RBC, UOX-LPET-G4-RBC, UOX-LPET-G5-RBC and control RBC (unmodified mouse peripheral blood red blood cells) into NPSG mice (Shanghai Jihui Experimental Animal Breeding Co., Ltd.), 2 mice in each group, a total of 10 mice. The red blood cell transfusion volume was 4e10RBC/kg. The plasma urate level was measured using a urate detection kit (Abcam) 7 days after transfusion. The experimental results are shown in Figure 1, indicating that compared with the control RBC infusion group, after 7 days of infusion of the red blood cell preparation modified with the G small peptide linker prepared in the present application, the plasma urate of mice was reduced to a certain extent. 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 the efficacy of red blood cells modified with G1 linkers.

2. Screening of linkers containing branch units of different lengths

In order to explore whether branch units of different lengths in the branching connector would affect the drug loading capacity of red blood cells, the following experiment was performed.

A. Preparation of joints

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

B. Modification of red blood cells with different linkers

Red blood cells were isolated from the peripheral blood of C57/B6 mice (Shanghai Jihui Experimental Animal Breeding Co., Ltd.) by density gradient centrifugation. The isolated red blood cells were washed three times with PBS and then pre-warmed with 2.5 mM TCEP (Sigma) at 30°C. Treat 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 containing G1 and G3 described in Table 1 and the connector containing G6 in Table 3. Specifically, each connector (G1, G3, G6) was dissolved with a 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, react 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.

Then, 1×10 ⁹ /mL Gn-RBC was conjugated with UOX-LPETGG obtained in Example 1 by sortase (mg SrtA) reaction. 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.

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

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

The conjugation effect of UOX protein and erythrocyte membrane was detected by sandwich ELISA. Specifically, in ELISA coating buffer (pH 9.6, purchased from Beijing Solebow Technology Co., Ltd.), anti-UOX antibody-1 (purchased from Beijing Solebow Technology Co., Ltd.) at a concentration of 0.5 μg/mL was used. The wells of PVC microtiter plates were coated with RIPA buffer (R&D, Hangzhou Huaan Biotechnology Co., Ltd.) at 4°C overnight; the coating solution was removed and the wells were rinsed twice with 200 μL PBS; free protein binding sites in the wells were blocked with 200 μL blocking buffer (5% skim milk/PBS) at 37°C for 1 hour; and washed twice with 200 μL PBS. UOX-LPET-Gn-RBCs were lysed with RIPA buffer (R&D) at 4°C for 10 min, and then 100 μL of lysis solution was added to each well of the plate. Each plate included a positive control (duplicate) and a blank control and incubated at 37°C for 1 hour. The solution was removed and washed twice with 200 μL PBS. 100 μL of diluted detection anti-UOX antibody-2 solution (1 μg/mL, conjugated with HRP, purchased from Hangzhou Huaan Biotechnology Co., Ltd.) was added to each well and incubated at 37°C for 1 hour. Washed 4 times with 200 μL PBS. TMB solution (purchased from Beijing Solebow Technology Co., Ltd.) was added to each well, incubated for 10-15 min, then an equal volume of stop solution (purchased from Beijing Solebow Technology Co., Ltd.) was added, and the optical density at 450 nm was detected.

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 about 2.3 times that 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.

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

1. Expression and purification of recombinant protein eGFP-LPETG in E. coli

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 gel (GenScript).

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

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

2. _Gn linker labeled red blood cells

Red blood cells were isolated from the peripheral blood of C57/B6 mice (Shanghai Jihui Experimental Animal Breeding Co., Ltd.) by density gradient centrifugation, respectively, using the same method as in Example 2. The pretreated red blood cells were washed 3 times with PBS and modified with the G1 and G3 connectors described in Table 1, respectively. Specifically, each connector was dissolved with a PBS solution and mixed with the red blood cells pretreated with TCEP, and the final reaction concentration of the connector was 0.625 mM. 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 names of the connectors.

Then, 1×10 ⁹ /mL Gn-RBC was conjugated with eGFP-LPETG (SEQ ID NO: 5) by sortase (mg SrtA) reaction. 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 product after conjugation was named eGFP-LPET-Gn-RBC and stored at 2-8°C.

The labeling effect of eGFP on the erythrocyte membrane was monitored by flow cytometry (Cytoflex, Beckman), that is, 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 labeling effect of eGFP-LPET on the erythrocyte membrane obtained by the G ₃ linker (eGFP-LPET-(GGG) ₃ -mal-RBC in Figure 3) was about 15 times that of the eGFP-LPET labeling on the erythrocyte membrane obtained by the G ₁ linker (eGFP-LPETGGGSK-mal-RBC in Figure 3), which shows that the branched linker can more effectively increase the drug loading capacity of erythrocytes.

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

1. Purification of anti-PD1 mAb-LPETGG fusion protein

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

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

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

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

The nucleotide sequences encoding the above heavy chains or light chains were inserted into the expression vector pcDNA3.1, respectively. Each successfully constructed vector was transfected into CHO-S cells using the ExpiCHO ^™ expression system (ThermoFisher) according to the manufacturer's instructions. The transfected cells were cultured in ExpiCHO ^™ expression medium to express the corresponding heavy chain 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.

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.

2. Modification of red blood cells with different linkers

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 containing G1 and G3 described in Table 1, respectively. Specifically, each connector was dissolved with a 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, react 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.

Then, 1×10 ⁹ /mL Gn-RBC was coupled with anti-PD1 mAb-LPETGG by sortase reaction. In the coupling reaction, the concentration of mg SrtA was 10 μM and the anti-PD1 mAb-LPETGG substrate was 25 μM. After coupling, the final red blood cell product after conjugation was named anti-PD1 mAb-LPET-Gn-RBC and stored at 2-8°C.

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

The amount of anti-PD1 mAb conjugated to RBC was measured by sandwich ELISA. Briefly, the wells of PVC microtiter plates were coated with a concentration of 0.5 μg/mL of captured 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 solution was removed and the plate was washed twice with 200 μL PBS; 200 μL blocking buffer (5% skim milk/PBS) was added to each well, and the remaining protein binding sites in the coated wells were blocked at 37°C for 1 h; the plate was washed twice with 200 μL PBS; anti-PD1 mAb-LPET-Gn-RBC was lysed with RIPA buffer (R&D) at 4°C for 10 minutes. 100 μL of lysed red blood cell 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. Remove the solution and wash the plate twice with 200 μL PBS; add 100 μL of diluted detection anti-human FC antibody (1 μg/mL, eBioscience) to each well and incubate at 37°C for 1 hour; wash the plate four times with 200 μL PBS; add TMB solution (SURMODICS) to each well and incubate for 10-15 minutes, then add an equal volume of stop solution (purchased from Beijing Solebow Technology Co., Ltd.) and detect the optical density at 450 nm.

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).

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

Claims (32)

1. A modified erythrocyte, wherein an active agent is conjugated to the extracellular part 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 erythrocyte membrane protein, and the G-containing small peptide is conjugated to the active agent containing a sortase recognition motif via a sortase-mediated reaction, for example, the sortase recognition motif is LPXTG, preferably, the sortase recognition motif is LPETG.
2. The red blood cell of 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 maleimido alkyl chain (C _2-8 ) is 6-maleimidocaproic acid or 4-maleimidobutyric acid.
4. The red blood cell 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 according to any one of claims 1 to 4, wherein the G-containing small peptide is a branched small peptide comprising 2 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 an 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 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 such as COCH ₂ CH ₂ -PEG ₆ -NH can be added between K and G in the branching unit K (GGG).
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[(COCH ₂ CH ₂ -PEG ₆ -NH)-GGG]-GGG-K[(COCH 2 CH 2 -PEG 6 -NH)-GGG]-GGG-K[(COCH ₂ CH ₂ -PEG ₆ -NH)-GGG]-GGG-K[(COCH ₂ CH ₂ -PEG ₆ -NH)-GGG]-NH ₂ .
8. The erythrocyte according to any one of claims 1 to 7, wherein the maleimido alkyl 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 1-9, 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 the membrane protein of the erythrocyte via a branched linker.
12. The erythrocyte of any one of claims 1-11, wherein the active agent is modified to contain 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 according to any one of claims 1 to 13, wherein the active agent is a peptide molecule and is linked to the sortase recognition motif via a flexible peptide segment (GS) _n , wherein n=1-10.
15. The red blood cells according to any one of claims 1-14 have 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 consists of a G-containing small peptide and a maleimido alkyl chain (C _2-8 ).
17. The linker molecule of claim 16, wherein the maleimido alkyl chain (C _2-8 ) is 6-maleimidocaproic acid or 4-maleimidobutyric acid.
18. The linker molecule of claim 16 or 17, wherein the G-containing small peptide is a linear small peptide or a branched small 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 2 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 an 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 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 such as COCH ₂ CH ₂ -PEG ₆ -NH can be added between K and G in the branching unit K (GGG).
21. The linker molecule according to any one of claims 16 to 20, 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[(COCH ₂ CH ₂ -PEG ₆ -NH)-GGG]-GGG-K[(COCH 2 CH 2 -PEG 6 -NH)-GGG]-GGG-K[(COCH ₂ CH ₂ -PEG ₆ -NH)-GGG]-GGG-K[(COCH ₂ CH ₂ -PEG ₆ -NH)-GGG]-NH ₂ .
22. The linker molecule according to 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 according to any one of claims 16 to 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 described in any one of claims 16 to 24 is connected to the extracellular domain of the endogenous membrane protein of the erythrocytes; 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 via a linker.
26. A composition comprising the red blood cells according to any one of claims 1 to 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 1-15 or the composition of claim 26.
28. The method of claim 27, wherein the disease is selected from tumors or cancer, 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 for 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) with the erythrocytes 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 erythrocyte 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 condition, a pathology or a 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 cancer, metabolic diseases, bacterial infections, viral infections, autoimmune diseases and inflammatory diseases.