Novel drug delivery system based on engineered red blood cell

Abstract

The present disclosure relates to a novel drug delivery system, which comprises a lipid particle and a modified red blood cell. The present disclosure also relates to preparation of the drug delivery system and therapeutic use thereof.

Description

A novel drug delivery system based on engineered red blood cells

[0001] Cross-reference to related applications

[0002] This application is based on and claims priority to PCT patent application No. PCT / CN2024 / 114536, filed on August 26, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to a novel drug delivery system, its preparation and application, the drug delivery system comprising lipid particles and modified red blood cells. Background Technology

[0004] In recent years, with the rapid development of lipid nanoparticles (LNPs) as a delivery system, mRNA drugs have made significant progress in the fields of medicine and therapy. Biotechnology companies (such as Moderna and BioNTech) have demonstrated the great potential of mRNA-LNP drugs by developing two COVID-19 mRNA vaccines. mRNA-LNPs continue to show therapeutic potential in a range of applications, including viral vaccines, protein replacement therapies, and cancer immunotherapy.

[0005] However, mRNA delivery systems still face challenges in targeted delivery (Hou et al., 2021; Rohner et al., 2022). To achieve therapeutic effects, mRNA molecules must reach specific target cells and produce sufficient target proteins (protein expression levels need to be increased 50-1000 times compared to vaccine drugs). Existing delivery methods (lipid nanoparticle-based delivery systems) are severely limited in terms of tissue bioavailability, efficiency of delivery to target tissues, and endosome escape. Although targeted delivery of nucleic acid drugs is currently achieved by modifying LNP formulations or mounting antibodies on LNPs, the actual preparation processes are very complex and cannot truly achieve tissue / organ targeting (Cheng et al., 2020). For example, antibody-LNP conjugations mainly accumulate in the liver and cannot effectively reach immune organs, while adding cationic compounds (such as DOTAP) to LNPs can introduce safety risks (Cheng et al., 2020; Rurik et al., 2022).

[0006] Furthermore, mRNA-LNP drugs can easily trigger the innate immune response of the body, leading to adverse reactions or side effects, which is also a potential toxicity problem (Vlatkovic, 2021). When mRNA-LNP is extended to non-vaccine disease treatment, higher doses and longer administration cycles are required, which will increase the risk of mRNA-LNP.

[0007] Therefore, in order to realize the better clinical application prospect of mRNA drugs, it is necessary to improve the in vivo protein expression level of mRNA drugs while avoiding causing toxicity. Therefore, it is necessary to overcome the biological barriers in the process of drug delivery (such as nuclease in plasma and recognition and removal by mononuclear phagocyte system), improve the endosome escape efficiency (the endosome escape efficiency of existing drugs is less than 1%), and further improve the targeting delivery efficiency (Gilleron et al., 2013). In addition, existing mRNA-LNP drugs based on different delivery methods (such as subcutaneous injection, intramuscular injection, intravenous injection) will enter different solid tissue organs, such as lymph nodes, muscle tissue and liver. However, it is still very difficult to enter other solid tissues. For example, in order to mobilize the immune system, the number of lymph node immune cells is small and dispersed, so there are still difficulties in fully activating a stronger immune response, which limits its application in tumor immunity (Hou et al., 2021). Therefore, it is necessary to optimize the delivery method of mRNA drugs so that they can be targeted to deliver to tissues with rich immune cells and effectively expressed in the targeted tissues.

[0008] Therefore, there is a need in the art to develop new delivery systems that can deliver nucleic acids, such as mRNA, to tissues with rich immune cells and allow them to be successfully expressed in the targeted tissues or cells. SUMMARY

[0009] The present application develops a new nucleic acid drug delivery system, which utilizes the unique biological characteristics of red blood cells (natural targeting to peripheral immune organs, high biocompatibility, etc.) to target the delivery of nucleic acid drugs to the spleen.

[0010] Red blood cells are naturally enriched in the spleen and cleared by phagocytes in the spleen, and are considered a good transport carrier for targeting the spleen, which can achieve targeted delivery of immune drugs and effectively activate immunity (Buffet et al., 2011). The spleen, as one of the largest lymphoid organs in the human body, is rich in a large number of immune cells and is an important reservoir of immune cells. The spleen is divided into red pulp and white pulp, which contain a large number of antigen-presenting cells and lymphocytes, respectively, and play an important role in infection, tumor immunity, and autoimmune diseases (Bronte and Pittet, 2013; Lewis et al., 2019). Red blood cells are naturally enriched in the spleen and cleared by phagocytes in the spleen, and are considered a good transport carrier for targeting the spleen, which can achieve targeted delivery of immune drugs and effectively activate immunity (Buffet et al., 2011). Red blood cells have the advantage of spleen enrichment and have close interaction with immune cells in the spleen.

[0011] Although some red blood cell products carrying drugs such as small molecules or polypeptides, especially antibodies, have entered clinical research in recent years, the mechanism of action of some of these products is to enter the spleen and activate its immune cells (McArdel et al., 2021). However, unlike RNA, the function of these delivered small molecules or polypeptides is limited to the function of the delivered drug itself, and cannot achieve diversified gene expression in immune cells in the spleen, thereby achieving targeted modification of immune cells in the spleen. Moreover, the existing red blood cell delivery method is mainly non-covalent coupling (Ukidve et al., 2020; Zhao et al., 2021). Moreover, the coupling of red blood cells with LNP in previous studies is generally non-covalent coupling. Moreover, even for such small molecules or proteins that can directly function in target cells when transported to the spleen, the existing red blood cell delivery method still has two major problems (1) low drug coupling efficiency, i.e., low drug loading on the surface of red blood cells; (2) high risk of drug off-targeting, i.e., lipid nanoparticles fall off before reaching target organs and target cells with red blood cells, resulting in low delivery efficiency.

[0012] Therefore, the present application first develops such a red blood cell delivery system, which applies a specific covalent coupling process, which not only successfully delivers nucleic acid drugs to the spleen, but also successfully expresses them in the spleen, thereby achieving efficient and safe targeted delivery of nucleic acid drugs, and thereby expanding the application of nucleic acid drugs in various diseases.

[0013] In summary, the present application covalently links nucleic acid drugs, such as mRNA drugs (mRNA-LNP), to red blood cells, and uses the natural distribution of red blood cells to efficiently deliver nucleic acid drugs to antigen-presenting cells (such as macrophages, dendritic cells) in the spleen. Red blood cells efficiently deliver the nucleic acid drugs they carry to cells through phagocytosis, and depending on the target of the nucleic acid drugs, such as mRNA drugs, carried, immune activation or immune suppression effects can be achieved.

[0014] In addition, the red blood cell delivery system of the present application can bypass the endosome-lysosome pathway, efficiently enter myeloid cells through phagocytosis of red blood cells, and release mRNA in the cytoplasm, improving protein expression efficiency.

[0015] In some embodiments, without wishing to be limited by theory, the nucleic acid drug delivery system of the present application has at least one or more of the following technical effects: (1) red blood cells themselves have no nucleus, greatly reducing the safety risk compared to other cell therapies; (2) red blood cells have high biocompatibility, long in vivo circulation time, can effectively protect the nucleic acid drugs carried from being cleared, and enhance the efficiency of targeted delivery; (3) red blood cells carrying mRNA drugs can bypass the endosome-lysosome pathway and release mRNA in the cytoplasm, thereby improving protein expression efficiency; and / or (4) red blood cells naturally target the spleen, a natural immune organ (containing nearly 1 / 3 of the immune cells in the human body), and interact with immune cells in the spleen, which can fully exert the great potential of the mRNA drugs carried in immune regulation. In addition, in the present application, mRNA-LNP is covalently coupled to the surface of red blood cell membranes through enzyme-catalyzed reaction or chemical reaction, so that a higher drug load of mRNA-LNP can be coupled to the surface of red blood cells (about 10 times higher than prior art), and the coupled drugs are more stable, are metabolized with the clearance of red blood cells, and avoid non-specific off-target effects.

[0016] In some embodiments, the present application successfully achieves one or more of the following aspects:

[0017] 1. The first covalent coupling system of red blood cells and mRNA-LNP drugs (mRNA-LNP-RBC) is constructed, and through screening and testing of various coupling methods, the optimal mRNA-LNP-RBC engineering connection method is determined. This includes improvement and optimization of LNP formula, and optimization of reaction conditions, etc.

[0018] 2. In vitro characterization of mRNA-LNP-RBC is completed, including identification of its phenotype and quantitative analysis of the mRNA carried.

[0019] 3. Demonstrated the advantage of mRNA-LNP-RBC over mRNA-LNP in intracellular lysosomal escape, such as bypassing the endosome-lysosome pathway, efficiently entering myeloid cells through phagocytosis of red blood cells, and releasing mRNA in the cytoplasm, improving protein expression efficiency.

[0020] 4. Demonstrated the advantage of mRNA-LNP-RBC over mRNA-LNP drugs in gene delivery and expression in the spleen organ.

[0021] 5. Demonstrated the advantage of mRNA-LNP-RBC over mRNA-LNP drugs in gene delivery and expression in the spleen CD11b+ myeloid cells, a group of immune cells. BRIEF DESCRIPTION OF DRAWINGS

[0022] The following drawings illustrate preferred embodiments of the application. For the purpose of illustrating the application, there is shown in the drawings a preferred embodiment of the application. It should be understood, however, that the application is not limited to the precise embodiments shown in the drawings.

[0023] Figure 1 shows three process coupling paths of mRNA-LNP-RBC.

[0024] Figure 2 shows mRNA-LNP-RBC drug loading detection (where the mRNA used is Luciferase).

[0025] Figure 3 shows the results of in vitro characterization of mRNA-LNP-RBC (where the mRNA used in this result is Luciferase). A, flow detection results. B, confocal imaging results.

[0026] Figure 4 shows in vitro mRNA-LNP-RBC gene delivery efficiency studies (where the mRNA used in this result is Luciferase or GFP).

[0027] Figure 5 shows in vitro mRNA-LNP-RBC gene delivery pathway studies (where the mRNA used in this result is Luciferase).

[0028] Figure 6 shows Luc-LNP-RBC-1, Luc-LNP-RBC-2, Luc-LNP-RBC-3 tissue distribution and expression in vivo.

[0029] Figure 7 shows Luc-LNP and Luc-LNP-RBC-1 tissue distribution and expression in vivo.

[0030] Figure 8 shows GFP-LNP and GFP-LNP-RBC-1 distribution and expression in the spleen. A, GFP positive rate in different cell populations in the spleen. B, proportion of GFP+ positive cells. C, distribution of GFP+ positive cells.

[0031] Figure 9 shows the Luc-mRNA-LNP-RBC-1 in vivo pharmacokinetic study. A. The percentage of Luc-mRNA-LNP-RBC-1 in peripheral blood. B. The content of Luc-mRNA-LNP on Luc-mRNA-LNP-RBC-1.

[0032] Definitions

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0034] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Nucleic acids are written left to right in 5' to 3' orientation, unless otherwise indicated; amino acid sequences are written left to right in amino to carboxyl orientation, unless otherwise indicated. It is to be understood that this application is not limited to the particular methodology, protocols, and reagents described, as these can vary.

[0035] As used herein, the term "and / or" means any one of the items, or any two or more of the items.

[0036] As used herein, the term "comprising" or "including," or "having" means including, but not limited to, the stated elements, integers or steps. In this connection, the term "comprising" or "including" or "having" also means that it is possible to comprise, or have, additional elements, integers or steps other than those recited.

[0037] The terms "patient," "individual," and "subject" refer to any mammal that can benefit from the treatment or compositions disclosed herein. Thus, the methods and compositions disclosed herein can have medical and / or veterinary applications. In preferred forms, the mammal is a human.

[0038] As used herein, a "lipid particle" is a composition comprising one or more lipids and one or more therapeutic and / or prophylactic agents. Lipid particles are typically micrometer-sized, or smaller, in size, which can also be larger than micrometer-sized, and can include a lipid bilayer. Lipid particles encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes, preferably are lipid nanoparticles. Lipid nanoparticles are nanometer-sized (1-1000 nm) sized, electron-dense core, spherical or polyhedral particles, which can entrap a negatively charged nucleic acid in the core portion of the particle by mutual attraction of positive and negative charges due to the presence of ionizable lipids. For example, the diameter of a lipid nanoparticle is about 50-500 nm, such as about 50-350 nm. Therapeutic or prophylactic agents, such as nucleic acids, can be encapsulated in the lipid portion of the lipid nanoparticle or in an aqueous space enclosed by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesired effects such as an adverse immune response induced by mechanisms of the host organism or cell.

[0039] "Lipid nanoparticle (LNP)" refers to a particle that contains multiple (i.e., more than one) lipid molecules that are physically associated with one another through intermolecular forces. Lipid nanoparticles can be, for example, microspheres (including unilamellar and multilamellar vesicles, such as liposomes), dispersed phase in an emulsion, micelles in a suspension, or an internal phase.

[0040] The term "antibody fragment" includes a portion of an intact antibody. In preferred embodiments, the antibody fragment is an antigen binding fragment.

[0041] "Antigen binding fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; dAb (domain antibody); linear antibodies; single-chain antibody (e.g., scFv); single domain antibodies such as VHH; diabodies or fragments thereof; or a camelid antibody.

[0042] The term "antigen" refers to a molecule that elicits an immune response. This immune response can involve either antibody production, or the activation of specific immunecells, or both. The skilled artisan will understand that any macromolecule, including essentially any protein or peptide, can serve as an antigen. Furthermore, an antigen can be derived from recombinant or genomic DNA.

[0043] A "complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is hypervariable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contacts"). CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of a heavy chain and light chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. CDRs located within the variable domain of an antibody heavy chain are referred to as HCDR1, HCDR2, and HCDR3, while CDRs located within the variable domain of an antibody light chain are referred to as LCDR1, LCDR2, and LCDR3. The precise amino acid sequence boundaries of each CDR in a given light chain variable region or heavy chain variable region amino acid sequence can be determined using any of a number of well-known antibody CDR assignment systems, or combinations thereof, including, for example: Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (on the World Wide Web at imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering with a large number of crystal structures (North et al., "A New Clustering of Antibody CDR Loop Conformations", Journal of Molecular Biology, 406, 228-256 (2011)).

[0044] The term "drug loading" refers to the amount of drug loaded per unit weight or unit volume or per individual red blood cell. In the present application, the drug loading of red blood cells is typically measured in μg / mL as a dosage unit.

[0045] The term "nucleic acid drug" refers to a drug or active agent comprising a nucleic acid that can be used for prophylaxis, therapy or diagnosis. Thus, "nucleic acid drug" as described herein encompasses drugs for therapy or prophylaxis, and also diagnostic agents for diagnosis.

[0046] The term "treatment" refers to clinical intervention designed to alter the natural course of the disease in the individual being treated. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.

[0047] The term "prevention" includes inhibition of the occurrence or development of a disease or condition or a symptom of a particular disease or condition. In some embodiments, a subject with a family history of cancer is a candidate for a prophylactic regimen. Typically, in the context of cancer, the term "prevention" refers to administration of a drug prior to the onset of signs or symptoms of cancer, particularly in a subject at risk for cancer.

[0048] The term "effective amount" refers to the amount or dose of the modified red blood cells or compositions of the present application, which, when administered to a patient as a single dose or as multiple doses, produces the intended effects in the patient being treated or prevented. An effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the consideration of numerous factors, such as species of mammal; body mass, age and general health condition; the particular disease involved; the extent or severity of the disease; the patient's response; the particular drug involved; the mode of administration; the bioavailability characteristics of the preparation administered; the dosing regimen to be followed; and any concomitant therapy being administered.

[0049] The term "therapeutically effective amount" refers to the amount of the modified red blood cells or compositions of the present application that is effective, at dosages and for periods of time necessary to achieve the desired therapeutic result. The therapeutically effective amount of the modified red blood cells or compositions of the present application can vary according to factors such as the disease state, age, sex, and weight of the individual. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., uric acid levels, tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 50%, 60%, or 70%, and still more preferably by at least about 80% or 90%, relative to an untreated subject.

[0050] The term "prophylactically effective amount" refers to the amount of the modified red blood cells or compositions of the present application that is effective, at dosages and for periods of time necessary to prevent the occurrence or development of the desired prophylactic result. Typically, a prophylactically effective amount will be less than a therapeutically effective amount since a prophylactic dose is used before symptoms of the disease are present, or as soon as possible after appearance of the first signs of the disease.

[0051] The term "pharmaceutical composition" refers to a composition that is in a form suitable for its intended use, and that contains an active ingredient in an effective amount to achieve its intended purpose, and that does not contain additional ingredients that are unacceptable with respect to toxicity to the subject to which the composition will be administered. The term "sortase acceptor motif" refers to a motif comprising an oligoglycine that serves as an amine nucleophile attack intermediate with the sortase recognition motif and ultimately covalently couples to the recognition motif. In one embodiment, the sortase acceptor motif is located at the N-terminus or C-terminus of a polypeptide.

[0052] The term "sortase recognition motif" refers to a polypeptide that is cleaved by a sortase molecule and is capable of forming a covalent bond with the sortase after cleavage. In one embodiment, the sortase recognition motif comprises LPXTG / A, where X is any amino acid. In one embodiment, the sortase cleavage occurs between T and G / A.

[0053] The term "linker" refers to a bifunctional or multifunctional molecule that can link (conjugate) two molecules or entities together, typically having two reactive functionalities. Linkers used in conjugates can be broadly classified as non-cleavable or cleavable. They can also be classified as straight linkers and branched linkers depending on whether the linker is branched or not. Branched linkers can comprise branched units, each of which can be coupled to at least one drug or molecule.

[0054] The term "red blood cell" or "RBC" is the most common blood cell type and the main carrier of oxygen in the blood stream through the circulatory system to the body tissues in vertebrates. The cytoplasm of red blood cells is rich in hemoglobin, an iron-containing biological molecule that can bind oxygen and give the cell and blood its red color. The cell membrane is composed of proteins and lipids, a structure that provides the properties necessary for physiological cell functions, such as deformability and stability, while allowing passage through the circulatory system, particularly the capillary network. In humans, mature red blood cells are flexible, oval biconcave disks. They lack a cell nucleus and most organelles to have maximum space for hemoglobin; they can be viewed as bags of hemoglobin with a plasma membrane. About 84% of the cells in the human body are 20-30 trillion red blood cells. Almost half the volume of blood (40 to 45%) is red blood cells.

[0055] The term "adult native red blood cell" refers to a mature native red blood cell obtained directly from the blood of an animal, particularly a human (e.g., an adult or a child).

[0056] The "blood preparation" or "blood product" described herein can be used interchangeably, which refers to a product prepared from (human) blood as a raw material for use in medical products. Blood preparations include whole blood preparations, component blood preparations, plasma preparations, or leukoreduced blood preparations.

[0057] The term "therapeutic agent" as described herein encompasses any substance effective in preventing or treating a tumor, e.g., a cancer, including chemotherapeutic agents, cytotoxic agents, other antibodies, vaccines, small molecule drugs, or immunomodulatory agents (e.g., immunosuppressive or immunostimulatory agents).

[0058] The term "immunomodulatory agent" as used herein refers to a natural or synthetic agent or drug that suppresses or modulates an immune response, e.g., an immunomodulatory protein. The immune response can be a humoral response or a cellular response. Immunomodulatory agents include immunosuppressive or immunostimulatory agents. In some embodiments, the immunomodulatory agents of the application include immune checkpoint inhibitors or immune checkpoint agonists.

[0059] The term "cytotoxic agent" as used herein refers to an agent that inhibits or prevents the function of cells and / or causes cell death or destruction.

[0060] "Chemotherapeutic agents" include chemical compounds useful in the treatment of cancer or immune system disorders.

[0061] The term "small molecule drug" refers to a low molecular weight compound capable of modulating a biological process. A "small molecule" is defined as a molecule having a molecular weight of less than 10 kD, typically less than 2 kD, and preferably less than 1 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimetics. As therapeutic agents, small molecules can penetrate cells more than macromolecules, be less susceptible to degradation, and be less likely to elicit an immune response.

[0062] The terms "tumor" and "cancer" are used interchangeably herein to encompass both solid tumors and hematological tumors.

[0063] The term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth.

[0064] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when referred to herein.

[0065] The term "pharmaceutically acceptable excipient" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, carrier, or stabilizer, etc., that is administered to an active agent.

[0066] The term "pharmaceutical composition" refers to a composition that is in a form suitable for administration to a subject in need of treatment, and that allows the active ingredients contained therein to exist in an effective and biologically active form.

[0067] The terms "pharmaceutical combination" or "combination product" are used interchangeably to mean either a non-fixed combination or a fixed combination comprising, but not limited to, a kit, a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) the lipid particle-erythrocyte conjugate of the application, and (ii) the other therapeutic agent) are presented in a separate entity and are administered simultaneously, with or without

[0068] The term "combination therapy" refers to the administration of two or more therapeutic agents, or treatment modalities (e.g., radiotherapy or surgery), to treat a disease described herein. Such administration includes coadministration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients. Alternatively, such administration

[0069] As used herein, "treat" means to reduce, interrupt, retard, alleviate, stop, decrease, or reverse the progression or severity of an existing symptom, disorder, condition, or disease.

[0070] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector associated with a host cell into which it has been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0071] A "subject / patient / individual sample" refers to a collection of cells or fluid obtained from a patient or subject. The source of the tissue or cell sample can be a solid tissue, such as from a fresh, frozen and / or preserved organ or tissue sample or biopsy sample or a puncture sample; blood or any blood component; a bodily fluid, such as cerebrospinal fluid, amniotic fluid (amniotic water), peritoneal fluid (ascites), or interstitial fluid; a cell from a subject at any time of gestation or development. The tissue sample can contain compounds that are not naturally admixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.

[0072] DETAILED DESCRIPTION

[0073] The present invention relates to a novel lipid particle-erythrocyte conjugate comprising a lipid particle and a modified erythrocyte, wherein the lipid particle is covalently conjugated to the membrane surface of the modified erythrocyte, optionally via a linker.

[0074] In some embodiments, one or more lipid particles are conjugated to an erythrocyte. In some embodiments, 10-10,000 lipid particles are comprised on each erythrocyte.

[0075] I. Lipid particle

[0076] In some embodiments, the lipid particle is a lipid nanoparticle (LNP).

[0077] In some embodiments, the size of the lipid nanoparticle is about 1 to about 2,500 nm, for example in one embodiment about 50 to about 600 nm, for example in one sub-embodiment about 50 to about 400 nm, for example in one sub-embodiment about 50 to about 250 nm, preferably in one sub-embodiment about 50 to about 150 nm.

[0078] In one embodiment, the LNP comprises one or more lipids selected from the group consisting of an ionizable lipid, a helper lipid, a sterol, and a PEG lipid.

[0079] In one embodiment, ionizable lipids suitable for use in the lipid particles of the application include, but are not limited to, 3-(didodecylamino)-N1,N1,4-tridodecyl-1- piperazin-ethylamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4- tridodecyl-1,4-piperazinediethylamine (KL22), 14,25-ditridecyl-15,18,21,24- tetraaza-triacontane (KL25), 1,2-dilinoleyl-oxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA) (abbreviated MC3), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]- dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)- octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA), (2R)-2-({8-[-cholest-5- en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1- amine (Octyl-CLinDMA(2R)), (2S)-2-({8-[-cholest-5-en-3-yloxy]octyl}oxy)-N,N- dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2S)), Bis((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino} 9-heptadecyloctanoate (SM102), and / or combinations thereof.

[0080] In one embodiment, the ionizable lipid is selected from MC3 or SM102.

[0081] In one embodiment, the helper lipid suitable for use in the lipid particles of the application includes, but is not limited to, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-di-undecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-dibehenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-dibehenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyloleyl phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, and / or combinations thereof.

[0082] In one embodiment, the helper lipid is selected from DSPC.

[0083] In one embodiment, the steroid suitable for use in the lipid particles of the application includes, but is not limited to, cholesterol, coprostanol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, and / or combinations thereof.

[0084] In one embodiment, the steroid is selected from cholesterol.

[0085] In one embodiment, the PEG lipids suitable for use in the lipid particles of the application include, but are not limited to, 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (DSPE-PEG), PEG-distearyl glycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleoyl, PEG-distearoyl, PEG-diacryloyl glycine amide (PEG-DAG), PEG-dipalmitoyl phosphatidyl ethanolamine (PEG-DPPE), PEG-l,2-dimyristyl oxyl propyl-3-amine (PEG-c-DMA), and / or combinations thereof.

[0086] In one embodiment, the PEG lipid is selected from DMG-PEG, preferably DMG-PEG2000.

[0087] II. Modified Red Blood Cells

[0088] The lipid particles in the present application can form conjugates with any suitable cell or membrane structure.

[0089] The membrane structure can be any biological membrane structure, preferably selected from the group consisting of red blood cell membrane, macrophage membrane, stem cell membrane, neutrophil membrane, hematopoietic stem cell membrane, red blood cell progenitor membrane, platelet membrane, reticulocyte, mature red blood cell membrane.

[0090] In humans, red blood cells (RBCs) are the most abundant blood cells in the circulatory system. Unlike other blood cells, red blood cells lack a nucleus and are flexible, allowing them to change shape to fit into blood vessels in the body, and primarily serve the function of oxygen supply in the body. Key protein markers on the surface of red blood cells allow them to circulate in the body for long periods without being cleared by macrophages, thus having a long half-life, which makes them excellent candidates for drug carriers. Mature red blood cells, which are anucleate, do not contain any genetic material, and thus have good safety compared to other genetic and cell therapies. In some embodiments, the cells suitable for the present application are red blood cells (e.g., mature red blood cells). Unless otherwise specified or otherwise clear from the context, when the present application refers to red blood cells, it generally refers to mature red blood cells.

[0091] In some embodiments, the cells suitable for the present application are red blood cells are obtainable from a eukaryotic organism, such as a mammal, such as a primate, e.g., chimpanzee or human; bovine; canine; feline; rodent, e.g., guinea pig, rat, mouse; lagomorph; or avian; reptilian; or fish, preferably from a human. In certain embodiments, the red blood cells are human red blood cells, e.g., human native red blood cells.

[0092] In some embodiments, the RBCs of the application are natural adult red blood cells. In some embodiments, the RBCs are mature red blood cells isolated from human (e.g. adult or pediatric) blood, or are mature red blood cells obtained from differentiation of stem cells (e.g. pluripotent stem cells such as induced pluripotent stem cells).

[0093] In some embodiments, the application 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 application contemplates the use of immunocompatible red blood cells, which have the same blood type (e.g. at least with respect to the ABO blood group system, and in some embodiments, the D blood group system) or can be a compatible blood type as the individual to whom the cells will be administered. In some embodiments, the RBCs are mature red blood cells having the same blood type as the subject to whom they will be administered.

[0094] In some embodiments, the red blood cells suitable for use in the application are modified red blood cells. For example, the application provides methods of engineering adult natural red blood cells to enable the natural red blood cells to be used to efficiently carry therapeutic drugs.

[0095] In some embodiments, the red blood cells suitable for use in the application are red blood cells which have been modified to comprise a nucleophilic group or an electrophilic group.

[0096] In some embodiments, the red blood cells suitable for use in the application are red blood cells which have been treated with a reducing agent, for example a thiol reducing agent.

[0097] In some embodiments, the RBCs are mature red blood cells (e.g. natural adult red blood cells) obtained by treatment with a thiol reducing agent, which comprise cysteine residues comprising thiol groups. In some embodiments, the thiol reducing agent is tris(2-carboxyethyl)phosphine (TCEP).

[0098] In some embodiments, the red blood cells suitable for use in the application are red blood cells which have been treated with a reducing agent, for example a thiol reducing agent, obtained by a method comprising:

[0099] (i) isolating and concentrating red blood cells from (human) whole blood, optionally by leukodepletion; or obtaining red blood cells from stem cells, for example pluripotent stem cells such as induced pluripotent stem cells;

[0100] (ii) treating the red blood cells with a reducing agent, for example a thiol reducing agent (e.g. TCEP), to surface chemically modify the red blood cells;

[0101] (iii) collecting and concentrating the modified red blood cells.

[0102] In some embodiments, the surface chemical modification in (ii) comprises the following steps:

[0103] mixing a thiol-based reducing agent with the concentrated red blood cells, wherein the concentration of the reducing agent is between 0.1 mM and 50 mM, such as 0.5 mM and 10.0 mM, 0.5 mM and 5.0 mM; preferably about 5.0 mM.

[0104] III. Lipid particle-red blood cell conjugates

[0105] III-1. Directly covalently coupled lipid particle-red blood cell conjugates and methods of making the same

[0106] In some embodiments, one or more lipid particles are directly covalently coupled to a red blood cell to obtain a lipid particle-red blood cell conjugate. In some embodiments, the lipid particle-red blood cell conjugate has the following structure: Lipid particle-Red blood cell.

[0107] In some embodiments, the red blood cell comprises a nucleophilic group and the lipid particle comprises an electrophilic group, wherein the nucleophilic group reacts with the electrophilic group to form a covalent bond.

[0108] In some embodiments, the thiol group of a cysteine residue on a red blood cell membrane protein can form a chemical bond with a reactive functional group of a lipid particle to make a lipid particle-red blood cell conjugate.

[0109] Nucleophilic groups on red blood cell membrane proteins include, but are not limited to, (i) N-terminal amino groups, (ii) side chain amino groups, such as the amino group of lysine, (iii) side chain thiol groups, such as the thiol group of cysteine, and (iv) sugar hydroxyl or amino groups (in the case of glycosylation), such as the side chain amino group. Amino, thiol, and hydroxyl groups are nucleophilic and can react with electrophilic groups on a lipid particle selected from the group consisting of (i) active esters, such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides, such as haloacetamides; and (iii) aldehyde, ketone, carboxyl, and maleimide groups, to form a covalent bond.

[0110] In some embodiments, the electrophilic group on the lipid particle is a maleimide and the nucleophilic group on the red blood cell membrane protein is a side chain thiol group, such as the thiol group after reduction of cysteine (e.g., the thiol group of free cysteine, or the thiol group exposed after reduction of a disulfide bond).

[0111] In another embodiment, the lipid containing an electrophilic group is covalently coupled to a membrane protein on a red blood cell, e.g., to a membrane protein on a red blood cell bearing a nucleophilic group, e.g., a thiol group, preferably the red blood cell is a mature red blood cell, e.g., an adult native red blood cell. In another embodiment, the lipid containing a maleimide is linked to a thiol group on a red blood cell, preferably the red blood cell is a mature red blood cell, e.g., an adult native red blood cell.

[0112] In some embodiments, the RBC is a mature red blood cell (e.g., a native adult red blood cell derived from a stem cell, e.g., a pluripotent stem cell, e.g., an iPSC) obtained after treatment with a reducing agent, e.g., a thiol-based reducing agent, as described above, which comprises a cysteine residue containing a thiol group.

[0113] In some embodiments, the lipid particle, e.g., LNP, comprises a lipid containing a maleimide group (MAL), and optionally one or more other lipids suitable for use in a lipid particle. In some embodiments, the amount of the lipid containing a maleimide group in the lipid particle, e.g., LNP, is 0.1-10% (e.g., 0.1-5% or 0.1-1% or 0.5-1%, e.g., about 0.75%).

[0114] In one embodiment, the lipid containing a maleimide group suitable for use in a lipid particle of the application includes, but is not limited to, an ionizable lipid containing a maleimide group, a helper lipid containing a maleimide group, a sterol containing a maleimide group, and / or a PEG lipid containing a maleimide group.

[0115] In one embodiment, the lipid containing a maleimide group suitable for use in a lipid particle of the application is a PEG lipid containing a maleimide group.

[0116] In one embodiment, the lipid containing a maleimide group suitable for use in a lipid particle of the application includes a DSPE-PEG lipid containing a maleimide group, preferably DSPE-PEG(2000) containing a maleimide group, e.g., DSPE-PEG(2000)-MAL (DSPE-PEG2000-Maleimide).

[0117] In one embodiment, the lipid particle, e.g., LNP, of the application comprises DSPE-PEG(2000)-MAL, and optionally one or more other lipids suitable for use in a lipid particle. In some embodiments, the amount of the DSPE-PEG(2000)-MAL in the lipid particle, e.g., LNP, is 0.1-10% (e.g., 0.1-5% or 0.1-1% or 0.5-1%, e.g., about 0.75%).

[0118] In one embodiment, the DSPE-PEG(2000)-MAL of the application has the molecular structure shown below:

[0119] In some embodiments, the lipids of the lipid particles, e.g., LNP, of the application are selected from MC3 or SM102; Cholesterol; DSPC; DSPE-PEG2000-Maleimide; DMG-PEG2000.

[0120] In some embodiments, the lipids of the lipid particles, e.g., LNP, of the application consist of the following 5: MC3 or SM102; Cholesterol; DSPC; DSPE-PEG2000-Maleimide; and DMG-PEG2000. In some embodiments, the lipids of the lipid particles, e.g., LNP, of the application consist of the following 5: MC3 or SM102; Cholesterol; DSPC; DSPE-PEG2000-Maleimide; and DMG-PEG2000, wherein the MC3 or SM102 molar ratio is 40-60%, Cholesterol = 30-45%, DSPC = 5-15%, DSPE-PEG2000-Maleimide = 0.1-10% (e.g., 0.1%-5% or 0.1%-1% or 0.5%-1%, e.g., about 0.75%), DMG-PEG2000 = 0.1-10% (e.g., 0.1%-5% or 0.1%-1% or 0.5%-1%); e.g., MC3 or SM102 molar ratio is about 50%, Cholesterol = about 38.5%, DSPC = about 10%, DSPE-PEG2000-Maleimide = about 0.75%, DMG-PEG2000 = about 0.75%; e.g., MC3 or SM102 molar ratio is 50%, Cholesterol = 38.5%, DSPC = 10%, DSPE-PEG2000-Maleimide = 0.75%, DMG-PEG2000 = 0.75%.

[0121] In some embodiments, the lipid particle-erythrocyte conjugate has the structure: LNP-DSPE-PEG(2000)-MAL-S-RBC, wherein

[0122] LNP-DSPE-PEG(2000)-MAL refers to any LNP as defined herein comprising DSPE-PEG(2000)-MAL, wherein optionally the amount of DSPE-PEG(2000)-MAL in the LNP is 0.1-10% (e.g., 0.1%-5% or 0.1%-1% or 0.5%-1%, e.g., about 0.75%), optionally the LNP can further comprise one or more additional lipids as defined herein (e.g., the lipids described herein as suitable for use in lipid particles);

[0123] RBC refers to any red blood cell as defined herein, for example, a reductant-treated red blood cell, for example, a red blood cell treated with a thiol reductant;

[0124] -S- refers to a group containing S formed from the reaction of a maleimide group with a thiol group of an RBC, wherein the S atom is derived from the thiol group of the RBC;

[0125] Optionally, multiple lipid particles can be coupled to an RBC, wherein the lipid particles can be the same or different.

[0126] In some embodiments, the present application provides a method of preparing a directly covalently coupled lipid particle-red blood cell conjugate, comprising mixing a lipid particle containing a maleimide group and a reductant-treated red blood cell such that the two are covalently coupled via the maleimide group and a nucleophilic group on a membrane protein of the red blood cell, for example, comprising

[0127] (1) loading a lipid containing a maleimide group into a lipid particle;

[0128] (2) performing a surface chemical modification of adult native red blood cells to expose a nucleophilic group on a membrane protein of the red blood cell; and collecting the modified red blood cells and concentrating;

[0129] (3) mixing the lipid particle of (1) and the red blood cells obtained in (2) such that the two are covalently coupled via the maleimide group and the nucleophilic group on a membrane protein of the red blood cell; in some embodiments, the ratio between the lipid particle and the red blood cell is 0.1-1000 μg lipid particle per 1 mL red blood cells;

[0130] (4) collecting the lipid particle-red blood cell conjugate obtained in (3).

[0131] In one embodiment, the nucleophilic group on a membrane protein of the red blood cell is a side chain thiol group.

[0132] In one embodiment, the side chain thiol group is a thiol group after reduction of a cysteine.

[0133] In one embodiment, the red blood cell is chemically modified using a thiol reductant.

[0134] In one embodiment, the thiol reductant is TCEP.

[0135] In some embodiments, the final concentration of TCEP is between 0.1 mM and 10 mM, for example, 0.5 mM and 10.0 mM, 0.5 mM and 5.0 mM, preferably about 2.5 mM.

[0136] Lipid particle-erythrocyte conjugates covalently coupled via linkers and methods of making the same

[0137] In some embodiments, one or more lipid particles are covalently coupled to an erythrocyte via a linker. In some embodiments, the lipid particle-erythrocyte conjugate has the following structure: lipid particle-linker-erythrocyte.

[0138] In some embodiments, a linker can be used to link one or more lipid particles to an erythrocyte (e.g., to a membrane protein of an erythrocyte), forming a lipid particle-erythrocyte conjugate. In some embodiments, one or more lipid particles linked to one or more linkers can be linked to an erythrocyte. In some embodiments, the linker is a bivalent linker. In some embodiments, the lipid particle-erythrocyte conjugate can be made using a linker having a reactive functional group for covalent linkage to both the erythrocyte and the lipid particle. In some embodiments, the linker reacts with a nucleophilic group of the lipid particle, and with a nucleophilic group of a membrane protein of the erythrocyte, respectively, to form covalent bonds. For example, in some embodiments, an amino group on a lysine on the membrane protein of the erythrocyte can form a chemical bond with a reactive functional group of the linker, which in turn is coupled to a thiol group on the lipid particle to make the lipid particle-erythrocyte conjugate.

[0139] Nucleophilic groups on the membrane protein of the erythrocyte include, but are not limited to, (i) N-terminal amino groups, (ii) side chain amino groups, such as the amino group of lysine, (iii) side chain thiol groups, such as the thiol group of cysteine, and (iv) sugar hydroxyl or amino groups (in the case of glycosylation), such as the side chain amino group. Amino, thiol, and hydroxyl groups are nucleophilic and can react with electrophilic groups on linker moieties and linkers including (i) active esters, such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides, such as haloacetamides; and (iii) aldehyde, ketone, carboxyl, and maleimide groups, to form covalent bonds.

[0140] In some embodiments, the nucleophilic group of the erythrocyte is an N-terminal amino group or a side chain amino group (e.g., the amino group of lysine), and / or the nucleophilic group on the lipid particle is a side chain thiol group (e.g., the thiol group of a free cysteine, or a thiol group exposed upon reduction of a disulfide bond).

[0141] In some embodiments, the lipid particle, e.g., LNP, comprises a thiol group (SH)-containing lipid, and optionally one or more other lipids suitable for use in a lipid particle. In some embodiments, the amount of the thiol group (SH)-containing lipid in the lipid particle is 0.1-10% (e.g., 0.1-5% or 0.1-1% or 0.5-1%, e.g., about 0.75%). In another embodiment, the thiol group (SH)-containing lipid suitable for use in the lipid particles of the application includes, but is not limited to, one or more of the following: an ionizable thiol group (SH)-containing lipid, a helper thiol group (SH)-containing lipid, a steroid thiol group (SH)-containing lipid, and / or a PEG thiol group (SH)-containing lipid.

[0142] In another embodiment, the thiol group (SH)-containing lipid suitable for use in the lipid particles of the application is a PEG thiol group (SH)-containing lipid, preferably a DSPE-PEG thiol group (SH)-containing lipid, most preferably DSPE-PEG(2000) thiol group (SH)-containing lipid, e.g., DSPE-PEG(2000)-SH.

[0143] In another embodiment, the lipid particle, e.g., LNP, of the application comprises DSPE-PEG(2000)-SH, and optionally one or more other lipids suitable for use in a lipid particle. In some embodiments, the amount of the DSPE-PEG(2000)-SH in the lipid particle, e.g., LNP, is 0.1-10% (e.g., 0.1-5% or 0.1-1% or 0.5-1%, e.g., about 0.75%).

[0144] In one embodiment, the DSPE-PEG(2000)-SH of the present disclosure has the molecular structure shown below:

[0145] In another embodiment, the lipids of the lipid particle, e.g., LNP, of the application are selected from MC3 or SM102; cholesterol; DSPC; DSPE-PEG2000-SH; DMG-PEG2000.

[0146] In another embodiment, the lipids of the lipid particle, e.g., LNP, of the application are selected from MC3 or SM102; cholesterol; DSPC; DSPE-PEG2000-SH; DMG-PEG2000.

[0147] In another embodiment, the lipids of the present liposomal particles, e.g., LNP, are composed of the following 5: MC3 or SM102; cholesterol; DSPC; DSPE-PEG2000-SH; and DMG-PEG2000, wherein the MC3 or SM102 molar ratio is 40-60%, cholesterol = 30-45%, DSPC = 5-15%, DSPE-PEG2000-SH = 0.1-10% (e.g., 0.1%-5% or 0.1%-1% or 0.5%-1%, e.g., about 0.75%), DMG-PEG2000 = 0.1-10% (e.g., 0.1%-5% or 0.1%-1% or 0.5%-1%); e.g., MC3 or SM102 molar ratio is about 50%, cholesterol = about 38.5%, DSPC = about 10%, DSPE-PEG2000-SH = about 0.75%, DMG-PEG2000 = about 0.75%; e.g., MC3 or SM102 molar ratio is 50%, cholesterol = 38.5%, DSPC = 10%, DSPE-PEG2000-SH = 0.75%, DMG-PEG2000 = 0.75%.

[0148] In one embodiment, the linker has a functional group that is capable of reacting with a thiol group present on the liposomal particle to form a covalent linkage to the liposomal particle. Non-limiting exemplary such reactive functional groups include maleimide, haloacetamide, alpha-haloacetyl, active ester such as succinimidyl ester, 4-nitrophenyl ester, pentafluorophenyl ester, tetrafluorophenyl ester, anhydride, acid chloride, sulfonyl chloride, isocyanate, and isothiocyanate, preferably maleimide (MAL).

[0149] In some embodiments, the linker has a functional group that can form a covalent linkage to the red blood cell by reacting with an amino group (-NH2) on lysine in the red blood cell. Non-limiting exemplary such reactive functional groups include, but are not limited to, active esters, e.g., NHS ester, HOBt ester, haloformate, and acid halide, preferably N-hydroxysuccinimidyl ester (NHS ester).

[0150] The linker can comprise one or more linker components. Exemplary linker components include 6-maleimidocaproyl ("MC"), maleimidopropionyl ("MP"), p-aminobenzyloxy carbonyl ("PAB"), NHS esters such as N-succinimidyl 4-(2-pyridylthio) pentanoate ("SPP") and 4-(N-maleimidomethyl)cyclohexane-l-carboxylate ("MCC"). The linker can also comprise an amino acid, e.g., one or more glycines. Various linker components are known in the art.

[0151] In some embodiments, the linker can be attached to the lipid particle by reaction with the thiol group of a free cysteine residue of the lipid particle, and to the red blood cell by reaction with the amino group (-NH2) on lysine in the red blood cell membrane protein.

[0152] Exemplary linkers include, but are not limited to:

[0153] bis-maleimide-trioxyethyleneglycol (BMPEO), N-(beta-maleimidopropyloxy)-N- hydroxysuccinimide ester (BMPS), N-(epsilon-maleimidocaproyloxy) succinimide ester (EMCS), N-[gamma-maleimidobutyryloxy] succinimide ester (GMBS), 1,6-hexanediol-bis- vinylsulfone (HBVS), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate- (6-aminohexanoate) (LC-SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 4-(4-N-maleimidophenyl) butyric acid hydrazide (MPBH), 3-(bromoacetamido) propionic acid succinimidyl ester (SBAP), iodoacetic acid succinimidyl ester (SIA), (4-iodoacetyl) aminobenzoic acid succinimidyl ester (SIAB), N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), N-succinimidyl-4-(2- pyridylthio) pentanoate (SPP), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1- carboxylate (SMCC), NHS-(PEG)n-MAL (where n = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or above, e.g., NHS-PEG2-MAL, NHS-PEG3-MAL, NHS-PEG6-MAL, NHS-PEG8-MAL, NHS-PEG24-MAL), succinimidyl 4-(p-maleimidophenyl) butyrate (SMPB), succinimidyl 6-[(beta- maleimidopropionamido) hexanoate] (SMPH), imidothiolane (IT), sulfo-EMCS, sulfo- GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and (4- vinylsulfone) benzoic acid succinimidyl ester (SVSB), and include bis-maleimide reagents (such as dithiobis-maleimidoethane (DTME), 1,4-bismaleimidobutane (BMB), 1,4- bis-maleimidyl-2,3-dihydroxybutane (BMDB), bis-maleimido-hexane (BMH), bis- maleimidoethane (BMOE), BM(PEG)2, and BM(PEG)3), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis-(p- azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)- ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).In some embodiments, the bismaleimide reagent allows for the thiol group of a cysteine in an antibody to be linked to a thiol-containing drug moiety, linker, or linker-drug intermediate. Other functional groups that react with thiol groups include, but are not limited to, iodoacetamide, bromoacetamide, vinylpyridine, disulfide, pyridyl disulfide, isocyanate, and isothiocyanate.

[0154] In some embodiments, the linker is 4-(N-maleimidomethyl)cyclohexane-1-carboxylate succinimidyl ester or a water-soluble analog thereof such as Sulfo-SMCC (Sulfo-SMCC), or SMCC-GGG, or succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate-(6-aminohexanoate) (LC-SMCC), or NHS-(PEG)n-MAL NHS-(PEG)n-MAL (where n = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more), such as NHS-PEG2-MAL or NHS-PEG 24 -MAL.

[0155] In some embodiments, the linker is succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate-(6-aminohexanoate) (LC-SMCC), 4-(N-maleimidomethyl)cyclohexane-1-carboxylate succinimidyl ester (SMCC), or Sulfo-SMCC, preferably Sulfo-SMCC.

[0156] In some embodiments, the lipid particle-erythrocyte conjugate has the structure: LNP-DSPE-PEG(2000)-S-SMCC-RBC, wherein

[0157] LNP-DSPE-PEG(2000) refers to any LNP as defined herein comprising DSPE-PEG(2000)-SH, wherein optionally the amount of DSPE-PEG(2000)-SH in the LNP is 0.1-10% (e.g. 0.1-5% or 0.1-1% or 0.5-1%, e.g. about 0.75%), optionally the LNP can further comprise one or more other lipids as defined herein (e.g. lipids suitable for use in a lipid particle as described herein);

[0158] RBC refers to any erythrocyte as defined herein;

[0159] SMCC refers to SMCC or a derivative thereof, such as LC-SMCC, SMCC, or Sulfo-SMCC,

[0160] - S- refers to a group comprising S formed by the reaction of a thiol group (SH) in the LNP with the maleimide group of the linker SMCC, wherein the S atom is derived from the thiol group of the lipid DSPE-PEG(2000)-SH in the LNP;

[0161] Optionally, multiple lipid particles can be coupled to the RBC, wherein the lipid particles can be the same or different.

[0162] In some embodiments, the present application also provides a method of preparing a lipid particle-erythrocyte conjugate covalently coupled via a linker, comprising mixing an erythrocyte linked to a linker and a lipid particle containing a thiol group (SH) such that the two are covalently coupled via the linker, for example, which comprises

[0163] (1) reacting a nucleophilic group on a membrane protein of an erythrocyte (e.g., NH2 on lysine) with a bivalent linker reagent to form an erythrocyte linked to a linker via a covalent bond, in some embodiments, the linker reagent is, for example, SMCC or sulfo-SMCC at a final concentration between 0.1 mM and 10 mM, for example, 0.5 mM and 10.0 mM, 0.5 mM and 5.0 mM, preferably about 0.5 mM; preferably the erythrocytes are collected and concentrated;

[0164] (2) pre-loading a lipid containing a thiol group (SH) into a lipid particle;

[0165] (3) mixing the erythrocyte linked to a linker in (1) and the lipid particle obtained in (2) such that the two are covalently coupled via the linker; in some embodiments, the ratio between the lipid particle and the erythrocyte is 0.1-1000 pg of lipid particle per 1 mL of erythrocyte;

[0166] (4) collecting the lipid particle-erythrocyte conjugate obtained in (3).

[0167] In another embodiment, the lipid containing a thiol group (SH) is selected from one or more of the following: an ionizable lipid containing a thiol group, a helper lipid containing a thiol group, a steroid containing a thiol group, and / or a PEG lipid containing a thiol group.

[0168] In another embodiment, the lipid containing a thiol group is a PEG lipid containing a thiol group, preferably a DSPE-PEG lipid containing a thiol group, most preferably a DSPE-PEG(2000) containing a thiol group, such as DSPE-PEG(2000)-SH.

[0169] In another embodiment, the thiol-containing lipid is coupled to a membrane protein on a red blood cell via a linker, for example to a membrane protein on a red blood cell bearing lysines, preferably the red blood cell is a mature red blood cell, for example an adult native red blood cell. In another embodiment, the thiol-containing lipid is linked to an amino group on a red blood cell via a linker, preferably the red blood cell is a mature red blood cell, for example an adult native red blood cell.

[0170] In another embodiment, the linker has a functional group capable of reacting with the thiol group on the thiol-containing lipid to form a covalent linkage to the LNP, and it has a functional group capable of reacting with a nucleophilic group on a red blood cell membrane protein to link to the red blood cell.

[0171] In another embodiment, the nucleophilic group on a red blood cell membrane protein includes, but is not limited to, (i) an N-terminal amino group, (ii) a side chain amino group, for example the amino group of lysine, (iii) a side chain thiol group, for example the thiol group of cysteine, and (iv) a sugar hydroxyl or amino group (in the case of glycosylation), for example a side chain amino group. Amino, thiol, and hydroxyl groups are nucleophilic and are capable of reacting with electrophilic groups on linker moieties and linker reagents including (i) active esters, for example NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides, for example haloacetamides; and (iii) aldehyde, ketone, carboxyl, and maleimide groups.

[0172] In another embodiment, the nucleophilic group on a red blood cell membrane protein is the -NH2of a lysine side chain, and the linker has a functional group capable of reacting with the -NH2of a lysine side chain on a red blood cell membrane protein to link to the red blood cell.

[0173] In another embodiment, the linker is succinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate-(6-aminohexanoate) (LC-SMCC), 4-(N-maleimidomethyl)cyclohexane-l-carboxylic acid succinimidyl ester (SMCC), or sulfo-SMCC, preferably sulfo-SMCC.

[0174] In one embodiment, the final concentration of the linker, for example SMCC or sulfo-SMCC, is between 0.1 mM and 10 mM, for example 0.5 mM and 10.0 mM, 0.5 mM and 5.0 mM, preferably about 0.5 mM.

[0175] In one embodiment, the nucleophilic group on a red blood cell membrane protein is an N-terminal amine group or a side chain amine group.

[0176] III-3. Lipid particle-red blood cell conjugates covalently coupled via a sortase recognition motif and methods of making the same

[0177] In some embodiments, the red blood cells of the application are reacted with a sortase recognition motif in a lipid particle to form a covalent conjugate. In some embodiments, the lipid particle-red blood cell conjugate has the structure: lipid particle-linker-red blood cell.

[0178] In some embodiments, the RBCs are mature red blood cells (e.g., native adult red blood cells) obtained after treatment with a reducing agent, e.g., a thiol-based reducing agent, as described above, which contain cysteine residues with thiol groups, e.g., with free sulfhydryl groups, resulting from reduction of disulfide bonds in, e.g., the extracellular domains of at least one endogenous membrane protein (e.g., at internal sites in the extracellular domain).

[0179] In some embodiments, the lipid particle, e.g., LNP, is pre-loaded or contains a sortase recognition motif-containing lipid, and optionally one or more other lipids suitable for use in a lipid particle. In some embodiments, the sortase recognition motif-containing lipid is present in the lipid particle in an amount of 0.1-10% (e.g., 0.1-5% or 0.1-1% or 0.5-1%, e.g., about 0.75%).

[0180] In yet another embodiment, the sortase recognition motif-containing lipids suitable for use in the lipid particles of the application include, but are not limited to, one or more of: a sortase recognition motif-containing ionizable lipid, a sortase recognition motif-containing helper lipid, a sortase recognition motif-containing sterol, and / or a sortase recognition motif-containing PEG lipid. In yet another embodiment, the sortase recognition motif-containing lipid suitable for use in the lipid particles of the application is a sortase recognition motif-containing PEG lipid, preferably a sortase recognition motif-containing DSPE-PEG, most preferably a sortase recognition motif-containing DSPE-PEG(2000).

[0181] The term "sortase" also known as transamidase refers to an enzyme having transamidase activity. Transamidases generally catalyze the formation of a peptide bond (amide bond) between an acyl donor and a nucleophilic acyl acceptor. Sortases recognize substrates comprising a sortase recognition motif, such as the amino acid sequence LPXTG. Sortases cleave the recognition motif between the residues threonine and glycine. Molecules recognized by sortases (i.e. comprising a sortase recognition motif) are sometimes referred to herein as "sortase substrates". It has been shown that a triglycine and even a diglycine motif on the N-terminus is sufficient to support the SrtA reaction (Clancy, K.W. et al., Peptide science 94 (2010) 385-396). Suitable sortases will be apparent to the skilled person and include, but are not limited to, sortase A, sortase B, sortase C and sortase D. The amino acid sequences of sortases and nucleotide sequences encoding them are known to the skilled person. In a particular embodiment, the sortase 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, resulting in a sortase A-substrate thioester intermediate; then, this thioester acyl-enzyme intermediate is resolved by nucleophilic attack of the amino group of a second substrate containing oligoglycine, resulting in a covalently linked conjugate molecule and regeneration of sortase A.

[0182] For enzymatic conjugation, a soluble truncated sortase A lacking the transmembrane region can be used, such as truncated SrtA comprising amino acid residues 60-206 for S. aureus. Sortase A-mediated reactions result in the linkage of molecules containing a sortase recognition sequence (sortase motif) to molecules containing a sortase acceptor sequence (e.g. one or more N-terminal glycine residues).

[0183] In some embodiments, the present application contemplates the use of variants of naturally occurring sortases. A great deal of structural information is available regarding sortases, e.g., sortase A, including NMR or crystal structures of SrtA alone or in combination with sortase recognition sequences (see, e.g., Zong Y et al. J. Biol Chem. 2004, 279, 31383-31389). The active site and substrate binding pocket of S. aureus SrtA have been determined. One of ordinary skill in the art can produce functional variants by, e.g., deletions or substitutions that do not disrupt or significantly alter the active site or substrate binding pocket of a sortase. In some embodiments, directed evolution of SrtA can be performed by utilizing the FRET (fluorescence resonance energy transfer)-based selection assay described by Chen et al. Sci. Rep. 2016, 6(1), 31899. In some embodiments, functional variants of S. aureus SrtA can be those described in CN10619105A and CN109797194A. In some embodiments, S. aureus SrtA variants can be truncation variants, e.g., (compared to wild-type S. aureus SrtA) removal of 25-60 (e.g., 30, 35, 40, 45, 50, 55, 59, or 60) amino acids from the N-terminus.

[0184] In some embodiments, a functional variant of S. aureus SrtA useful in the present application can be a S. aureus SrtA variant comprising one or more of D124G, Y187L, E189R, and F200L mutations at the amino acid positions of D124, Y187, E189, and F200, and optionally further comprising one or more of P94S / R, D160N, D165A, K190E, and K196T mutations. In some embodiments, the above-mentioned mutated amino acid positions are numbered according to the numbering of wild-type S. aureus SrtA. 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 present application. 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 (e.g., 30, 35, 40, 45, 50, 55, 59, or 60) amino acids removed from the N-terminus.

[0185] In some embodiments, the sortase variant can 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.

[0186] In one particular embodiment, the sortase is a S. aureus transpeptidase A variant (mgSrtA). In some embodiments, the sortase comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence having at least 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the nucleic acid encoding the sortase comprises, or consists of, the nucleotide sequence set forth in SEQ ID NO: 2, or an amino acid sequence having at least 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence set forth in SEQ ID NO: 2.

[0187] In yet another embodiment, the sortase recognition motif comprises or consists of LPXTG. Common recognition motifs are, for example, LPKTG, LPATG, LPNTG. In some embodiments, LPETG is used. However, motifs falling outside of this consensus sequence can also be recognized. For example, in some embodiments, the 4thposition of the motif comprises an "A", "S", "L", or "V" instead of a "T", such as LPXAG, LPXSG, LPXLG, or LPXVG, for example LPNAG or LPESG, LPELG, or LPEVG. In some embodiments, the 5thposition of the motif comprises an "A" instead of a "G", such as LPXTA, for example LPNTA. In some embodiments, the 2ndposition of the motif comprises a "G" or "A" instead of a "P", such as LGXTG or LAXTG, for example LGATG or LAETG. In some embodiments, the 1stposition of the motif comprises an "I" or "M" instead of an "L", such as MPXTG or IPXTG, for example MPKTG, IPKTG, IPNTG, or IPETG. Pishesha et al. 2018 describe various recognition motifs for sortase A.

[0188] 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, for example in certain embodiments an amino acid selected from D, E, A, N, Q, K, or R. In a particular embodiment, the sortase recognition motif provided herein is LPETG. In one embodiment, the sortase recognition motif can be modified to increase its efficiency of recognition, preferably LPETG is modified to increase its affinity for sortase, for example by adding a G at the C-terminus of the recognition sequence, for example the modified sequence is LPETGG.

[0189] In one embodiment, the lipid particle, e.g., LNP, of the application further comprises DSPE-PEG(2000) comprising a sortase recognition motif, and the sortase recognition motif is LPETG. In yet another embodiment, the sortase recognition motif can be modified to increase its affinity; preferentially, the modification is the addition of a G at the C-terminus of the sortase recognition motif, for example LPETGG.

[0190] In yet another embodiment, the sortase recognition motif-containing lipid suitable for use in the lipid particles, e.g., LNP, of the application comprises or consists of DSPE-PEG(2000)-LPETG.

[0191] In yet another embodiment, the lipid particles, e.g., LNP, of the application comprise DSPE-PEG(2000)-LPETG, and optionally one or more other lipids suitable for use in lipid particles. In some embodiments, the DSPE-PEG(2000)-LPETG is present in the lipid particles, e.g., LNP, at a level of 0.1-10% (e.g., 0.1-5% or 0.1-1% or 0.5-1%, e.g., about 0.75%).

[0192] In one embodiment, the DSPE-PEG(2000)-LPETG of the application has the molecular structure shown below:

[0193] In yet another embodiment, the lipids of the lipid particles, e.g., LNP, of the application are selected from MC3 or SM102; cholesterol; DSPC; DSPE-PEG2000-LPETG; DMG-PEG2000.

[0194] In yet another embodiment, the lipids of the lipid particles, e.g., LNP, of the application are selected from MC3 or SM102; cholesterol; DSPC; DSPE-PEG2000-LPETG; DMG-PEG2000.

[0195] In yet another embodiment, the lipids of the lipid particles, e.g., LNP, of the application are selected from MC3 or SM102; cholesterol; DSPC; DSPE-PEG2000-LPETG; DMG-PEG2000.

[0196] In yet another embodiment, the sortase recognition motif is conjugated to the extracellular part of the membrane protein of the red blood cell via a linker. In one embodiment, the linker comprises a G peptide and a maleimidoalkyl chain (C2-8), preferably the maleimidoalkyl chain (C2-8) is conjugated to the membrane protein of the red blood cell and / or the G peptide is conjugated to the lipid containing the sortase recognition motif via a sortase mediated reaction.

[0197] In yet another embodiment, the G peptide is a linear G peptide or a branched G peptide. In yet another embodiment, the G peptide is a branched G peptide comprising two or more branching units, wherein one or more lipidic particles are coupled to one or more branching units. In a particular embodiment, the branching units have the same structure.

[0198] In yet another embodiment, the branching units consist of the amino acid sequence K(GGG), wherein the glycines in brackets are conjugated to the epsilon-amino group of the lysine side chain to form a branch, and the lysine forms a peptide bond via its alpha amino group to other amino acids to constitute the backbone of the "G peptide". Optionally, a spacer can be added between K and G in the branching unit K(GGG), such as COCH2CH2-PEG6-NH.

[0199] In yet another embodiment, the G peptide has the 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, wherein the glycines in brackets are conjugated to the epsilon-amino group of the lysine side chain to form a branch. In yet another embodiment, the G peptide has the structure K(GGG)-GGG-K(GGG)-GGG-K(GGG).

[0200] In yet another embodiment, the maleimidoalkyl chain (C 2-8 ) is 6-maleimidocaproic acid or 4-maleimidobutyric acid, preferably 6-maleimidocaproic acid.

[0201] In yet another embodiment, (PEG)n is contained between the G-small peptide and the 6-maleimidocaproic acid, wherein n = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more.

[0202] In a preferred embodiment, the small peptides are conjugated together by the oligoglycine in the branch, which reacts with the lipid particle containing the sortase recognition motif under the mediation of sortase. In a specific embodiment, multiple identical or different lipid particles are conjugated on the linker containing multiple branching units by the reaction described above. In one embodiment, the linker is connected to the membrane protein of the red blood cell through its maleimidocaproyl chain (C2-8), and is connected to the lipid particle containing the sortase recognition motif through the sortase-mediated reaction of its G-small peptide. In a specific embodiment, multiple identical or different lipid particles are simultaneously conjugated on the red blood cell through the linker. Preferably, at least two lipid particles are simultaneously conjugated on the red blood cell. In one embodiment, the lipid particles conjugated to the red blood cell are identical.

[0203] wherein, when there is only one oligoglycine (e.g., GGG) in the linker that can react with the lipid particle containing the sortase recognition sequence, the G-small peptide is referred to as a G1 small peptide; when there are two oligoglycines in the linker that can react with the lipid particle containing the sortase recognition sequence, the G-small peptide is referred to as a G2 small peptide; when there are three oligoglycines in the linker that can react with the lipid particle containing the sortase recognition sequence, the G-small peptide is referred to as a G3 small peptide; and so on, to obtain a G4 small peptide, a G5 small peptide, respectively. Meanwhile, the linker containing the corresponding small peptide can also be referred to as G1, G2, G3, G4, G5. G6 is a G-small peptide with a branched chain (by PEG10) and containing three linkers. For those containing two or more oligoglycines that can react with the lipid particle containing the sortase recognition sequence, respectively, they constitute the branching units of the G-small peptide, and the linker obtained therefrom is a branched linker.

[0204] In a preferred embodiment, the linker has the structure shown below (G3):

[0205] In one embodiment, the sortase recognition motif is conjugated on the extracellular portion of the membrane protein of the red blood cell through the G3 structure described above.

[0206] In yet another embodiment, a plurality of lipids comprising a sortase recognition motif are conjugated to a membrane protein of a red blood cell via a branched linker.

[0207] In yet another embodiment, the DSPE-PEG(2000) is modified to comprise a sortase recognition motif.

[0208] In yet another embodiment, the DSPE-PEG(2000) can be linked to a sortase recognition motif via a flexible peptide segment (GS)n, where n = 1-10.

[0209] In some embodiments, the lipid particle-red blood cell conjugate has the structure: LNP-DSPE-PEG(2000)-LPETG-linker-RBC, preferably LNP-DSPE-PEG(2000)-LPETG-(G3)-RBC, where

[0210] LNP-DSPE-PEG(2000)-LPETG refers to any LNP as defined herein comprising DSPE-PEG(2000)-LPETG, wherein the amount of DSPE-PEG(2000)-LPETG in the LNP is 0.1-10% (e.g. 0.1-5% or 0.1-1% or 0.5-1%, e.g. about 0.75%), optionally the LNP can further comprise one or more other lipids as defined herein (e.g. lipids suitable for use in a lipid particle as described herein);

[0211] RBC refers to any red blood cell as defined herein, e.g. a reductant-treated red blood cell, e.g. a red blood cell treated with a thiol reductant;

[0212] The linker is any linker as defined herein comprising a G small peptide and a maleimidoalkyl chain (C2-8) (e.g. 6-maleimidocaproic acid or 4-maleimidobutyric acid), preferably a linker having the structure (G3) as above;

[0213] wherein the DSPE-PEG(2000)-LPETG is conjugated to the red blood cell membrane (e.g. the extracellular domain of a membrane protein) via a linker.

[0214] In some embodiments, a plurality of lipid particles can be covalently attached to the RBC, wherein the lipid particles can be the same or different, e.g. a plurality of lipid particles are conjugated to the linker via a linker molecule having a branching unit, and / or a plurality of lipid particles are conjugated to the red blood cell via a linker molecule having a branching unit. In some embodiments, a plurality of lipid particles attached thereto can be conjugated to the red blood cell via a plurality of linker molecules having a branching unit.

[0215] The present application also provides a method of preparing a lipid particle-erythrocyte conjugate covalently coupled via a sortase recognition motif, comprising mixing a erythrocyte linked to a linker and a lipid particle containing a sortase recognition motif in the presence of a sortase under conditions suitable for the sortase to react, such that the sortase conjugates the lipid particle to the erythrocyte via the linker, for example comprising:

[0216] (1) treating the erythrocyte with a reducing agent, such that the linker molecule is attached to an extracellular domain of an endogenous membrane protein of the erythrocyte; and collecting the erythrocyte and concentrating;

[0217] (2) treating the lipid particle, such that the lipid particle contains a sortase recognition motif;

[0218] (3) contacting the erythrocyte obtained in step 1) with the lipid particle obtained in step 2) in the presence of a sortase under conditions suitable for the sortase to react, such that the sortase conjugates the LNP to the endogenous membrane protein of the erythrocyte via the second linker; in some embodiments, the ratio between the lipid particle and the erythrocyte is 0.1-1000 pg of the lipid particle per 1 mL of the erythrocyte.

[0219] In one embodiment, the erythrocyte is treated with a reducing agent, such that a disulfide bond in an extracellular domain of at least one endogenous membrane protein of the erythrocyte (e.g., on an internal site of the extracellular domain) is reduced to have a free thiol. In a specific embodiment, the linker molecule is attached to the free thiol on the extracellular domain of the erythrocyte endogenous membrane protein via the 6-maleimidocaproic acid contained therein. In a specific embodiment, the lipid particle is attached to the G-containing peptide in the linker molecule via the sortase recognition motif LPXTG, and after the transamidation reaction of the sortase, a structure of lipid particle-LPXT-linker is formed. In a specific embodiment, multiple lipid particles are conjugated to the linker via the linker molecule having a branching unit. In a more specific embodiment, multiple lipid particles are conjugated to the erythrocyte via the linker molecule having a branching unit.

[0220] In some embodiments, the method of treating the erythrocyte, the linker, and the sortase can be found in WO2024 / 067295, which is incorporated herein by reference.

[0221] IV. Payload

[0222] In some embodiments, the lipid particle contains one or more payloads. In some embodiments, the payload is a nucleic acid. In some embodiments, the payload is a nucleic acid, such as DNA and / or RNA.

[0223] In some embodiments, the lipid particles, e.g., LNP, of the application comprise one or more selected from the group consisting of miRNA, siRNA, dsDNA, or mRNA.

[0224] In some embodiments, the mass ratio of lipid to nucleic acid in the lipid particles, e.g., LNP, of the application can be any suitable ratio, e.g., 5: 1 to 50: 1.

[0225] In some embodiments, the mass ratio of lipid to nucleic acid in the lipid particles, e.g., LNP, of the application is 10: 1 to 30: 1.

[0226] In some embodiments, the mass ratio of lipid to nucleic acid in the lipid particles, e.g., LNP, of the application is about 5: 1, 10: 1, 15: 1, 20: 1, 25: 1, or 30: 1.

[0227] In some embodiments, the encapsulation efficiency of the nucleic acid in the lipid particles, e.g., LNP, is greater than 50%, e.g., the encapsulation efficiency can be about 55%, 60%, 65%, 70%, 75%, 79%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc.

[0228] In some embodiments, the lipid particle-erythrocyte conjugate has 10-10,000 nucleic acid molecules per RBC, e.g., about 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or more nucleic acid molecules per RBC, most preferably 9-270 nucleic acid molecules per RBC.

[0229] In one embodiment, the one or more payloads comprise or are an RNA selected from the group consisting of messenger RNA (mRNA), short interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), RNA interference (RNAi) molecule, microRNA (miRNA), antisense RNA, ribozyme, Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), guide RNA, circular RNA (circular RNA), tRNA, and / or combinations thereof.

[0230] In one embodiment, the payload is an mRNA, preferably the mRNA encodes a therapeutic protein. In one embodiment, the RNA comprises a 5'-UTR, an mRNA of a gene of interest, and a 3'-UTR and Poly A linked in sequence. In one embodiment, the RNA is a modified RNA.

[0231] In one embodiment, the one or more payloads comprise or are DNA, for example circular DNA such as a plasmid or linear DNA such as antisense DNA.

[0232] In some embodiments, the lipid particle-erythrocyte conjugate has greater than about 0.01 pg mRNA / mL RBC, for example has about 0.01-300 pg mRNA / mL RBC, 0.05-200 pg mRNA / mL RBC, 0.05-100 pg mRNA / mL RBC, 0.05-50 pg mRNA / mL RBC, or 0.08-10 pg mRNA / mL RBC, most preferably has 0.1-3 pg mRNA / mL RBC.

[0233] In some embodiments, the nucleic acid molecule encapsulated in the lipid particle expresses a therapeutic protein.

[0234] In some embodiments, the nucleic acid molecule encapsulated in the lipid particle can express a therapeutic protein in splenic immune cells. In some embodiments, the therapeutic protein comprises some protein effective to treat a disease, for example a receptor, a ligand, an antibody or antigen binding fragment thereof, or a chimeric antigen receptor. In some embodiments, the protein can be an immunomodulator, for example a protein useful to treat a tumor, for example a protein useful to treat an autoimmune disease.

[0235] In some embodiments, the therapeutic protein is a chimeric antigen receptor. In one embodiment, the chimeric antigen receptor comprises an extracellular binding region that specifically binds an antigen, for example wherein the antigen is a tumor associated antigen.

[0236] V. Pharmaceutical compositions or combinations

[0237] In one aspect, the present application provides a pharmaceutical composition or formulation comprising a lipid particle conjugate. These compositions or formulations can also optionally comprise suitable pharmaceutical adjuvants, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, as known in the art.

[0238] In one aspect, the present application provides a blood preparation, e.g. a human blood preparation, comprising a lipid particle conjugate of the present application. In some embodiments, the human blood preparation of the present application comprises 10-1000 pg / mL of a lipid particle conjugate, e.g. 10-500 pg / mL of a lipid particle conjugate, e.g. at or above 50 pg / mL, 60 pg / mL, 70 pg / mL, 80 pg / mL, 90 pg / mL, 100 pg / mL, 150 pg / mL, 200 pg / mL, 250 pg / mL, 300 pg / mL, 350 pg / mL, 400 pg / mL, 450 pg / mL, or 500 pg / mL, or within any range of said values.

[0239] In some embodiments, the blood preparation of the present application is a leukoreduced blood preparation, i.e. a blood preparation from which leukocytes have been filtered out. In some embodiments, the blood preparation of the present application is a human blood leukoreduced blood preparation.

[0240] In some embodiments, the blood preparation of the present application can be an allogeneic blood preparation, e.g. an allogeneic human blood preparation. In some embodiments, the red blood cells in the blood preparation are from a healthy subject. In some embodiments, the red blood cells in said blood preparation can be mature red blood cells differentiated from stem cells, e.g. obtained by culturing stem cells to differentiate into mature red blood cells.

[0241] In some embodiments, the blood preparation of the present application can be an autologous blood preparation, e.g. an autologous human blood preparation. In some embodiments, the red blood cells in the blood preparation are from the subject to be treated.

[0242] In one aspect, the present application also provides a combination product, e.g. a pharmaceutical combination product, comprising a lipid particle conjugate of the present application, and one or more other therapeutic agents. The combination product of the present application can be used in the therapeutic methods of the present application.

[0243] The present application also provides a kit-of-parts comprising said combination product, e.g. said kit-of-parts comprises within the same package:

[0244] - a first container containing a lipid particle conjugate of the present application or a pharmaceutical composition or formulation comprising the same;

[0245] - a second container comprising one or more other therapeutic agents or a pharmaceutical composition or formulation comprising the same.

[0246] In some embodiments, said therapeutic agent is selected from any agent effective in cancer, including a chemotherapeutic agent, another antibody, a cytotoxic agent, a vaccine, a small molecule drug, or an immunomodulator (e.g. an immunosuppressant or an immunostimulant), preferably the therapeutic agent is selected from a tumor vaccine, an immune checkpoint inhibitor antibody, or an immune stimulant antibody.

[0247] VII. Uses and Therapies

[0248] The present application also provides a method of treating a disease, activating the immune system, or engineering immune cells such as myeloid cells, e.g., CD11b+myeloid cells, in the spleen, comprising administering to a subject a lipid particle-erythrocyte conjugate of the present application, or a pharmaceutical or human blood preparation comprising the same.

[0249] In some embodiments, the disease would benefit from immune system activation, or from engineering of CD11b+myeloid cells.

[0250] In some embodiments, engineering of CD11b+myeloid cells refers to the erythrocytes introducing the nucleic acid drug contained in the lipid particle into CD11b+myeloid cells, e.g., mRNA therein is expressed in CD11b+myeloid cells.

[0251] In some embodiments, the disease is a tumor or an immune system disease.

[0252] In some embodiments, the tumor is a cancer. In some embodiments, the tumor is a tumor antigen (TA) or tumor associated antigen (TAA) positive tumor. In one embodiment, the TA positive tumor refers to having TA expression or having TA nucleic acid in the tumor tissue or tumor cells (e.g., cancer tissue or cancer cells) of the individual. In one embodiment, the TA or TAA positive tumor refers to having TAA expression or having TAA nucleic acid in the tumor tissue or tumor cells (e.g., cancer tissue or cancer cells) of the individual, e.g., as compared to adjacent normal tissue or normal cells (e.g., normal cells in the tissue) of the individual or the same tissue or cells therein of a healthy individual, or as compared, the protein level (e.g., expression) of the TAA is elevated, or the nucleic acid level of the TAA is elevated; or as compared, the activity of the TAA is elevated.

[0253] In some embodiments, the disease is an immune system related disease, e.g., an autoimmune disease.

[0254] In some embodiments, the lipid particle-erythrocyte conjugate of the present application can be used to engineer immune cells such as CD11b+myeloid cells in the spleen, e.g., by administering the conjugate to the individual. In some embodiments, the immune cells, e.g., myeloid cells such as CD11b+myeloid cells, in the spleen comprising the lipid particle-erythrocyte conjugate of the present application migrate to a lesion, e.g., a tumor lesion, are capable of specifically recognizing and phagocytizing tumor cells (e.g., TAA positive tumor cells) targeted by the nucleic acid contained in the lipid particle, achieving tumor killing effect.

[0255] In some embodiments, the immune cells, e.g., myeloid cells such as CD11b+myeloid cells, in the spleen comprising the lipid particle-erythrocyte conjugate of the application are also capable of activating immune cells, e.g., T cells, at a lesion, e.g., a tumor lesion, to effect tumor killing.

[0256] In some embodiments, the lipid particle-erythrocyte conjugate of the application is capable of stimulating the immune system of a host, e.g., enhancing the immune response of cells. "Stimulating the immune system" can include any one or more of an overall increase in immune function, an increase in T cell function, an increase in B cell function, a restoration of lymphocyte function, an increase in IL-2 receptor expression, an increase in T cell responsiveness, an increase in T cell activity or natural killer cell activity or lymphokine-activated killer (LAK) cell activity, an increase in T cell or natural killer cell survival, an increase in expression of cell-killing effector proteins, and the like. The lipid particle-erythrocyte conjugate of the application (as well as compositions, pharmaceutical compositions, formulations, combination products, etc. comprising the same, e.g., blood formulations) can be administered by any suitable method, preferably by infusion or injection, e.g., parenteral infusion. Parenteral infusion includes intravenous or intraarterial administration. Preferably, the lipid particle-erythrocyte conjugate of the application is administered by intravenous or intraarterial infusion.

[0257] For the prevention or treatment of disease, the appropriate dosage of the lipid particle-erythrocyte conjugate of the application (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the lipid particle-erythrocyte conjugate, the severity and course of the disease, whether the administration is for therapeutic or prophylactic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The lipid particle-erythrocyte conjugate is suitably administered to the patient at one time or over a series of treatments.

[0258] In some embodiments, the lipid particle-erythrocyte conjugate of the application, or a pharmaceutical or formulation comprising the same, is administered in combination with one or more modalities of therapy or other therapeutic agents. In some embodiments, the modality of therapy is radiation therapy or surgical treatment. In some embodiments, the therapeutic agent is selected from any agent effective in cancer, including chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, small molecule drugs, or immunomodulatory agents (e.g., immunosuppressive or immunostimulatory agents, e.g., immunomodulatory proteins). Preferably, the therapeutic agent is selected from a tumor vaccine, an immune checkpoint inhibitor antibody, or an immune stimulatory antibody.

[0259] In other aspects, the application provides the use of the lipid particle-erythrocyte conjugate of the application, or a pharmaceutical or human blood formulation comprising the same, in the manufacture or preparation of a medicament for the uses described herein, e.g., for the prevention or treatment of a relevant disease or condition mentioned herein.

[0260] In other aspects, the present application provides a lipid particle-erythrocyte conjugate of the present application, or a pharmaceutical or human blood preparation comprising the same, for use in a use described herein, e.g., for therapy, e.g., for treating a relevant disease or condition mentioned herein.

[0261] In some embodiments, the erythrocytes in the lipid particle-erythrocyte conjugate are from a subject to be treated or a healthy subject. Examples:

[0262] Example 1, Process study of mRNA-LNP-RBC

[0263] 1.1 mRNA-LNP-RBC process path selection and formulation screening

[0264] Purpose of the experiment: to evaluate the feasibility of mRNA-LNP conjugation to erythrocytes through different process paths (chemical conjugation or enzyme catalysis).

[0265] Experimental method:

[0266] mRNA in vitro transcription purification

[0267] In vitro amplification

[0268] In a clean 1.5 mL centrifuge tube, add a certain amount of DNA template (the amino acid sequence and nucleic acid sequence of each protein are shown in the sequence listing), T7 reaction solution, ATP, GTP, CTP, N1-Me-pseudoUTP, cap analog and T7 polymerase according to the requirements of the kit, mix uniformly and react at 37°C for 2 hours.

[0269] Purification and identification

[0270] After the reaction, add an appropriate amount of DNase I and mix uniformly, then continue to react at 37°C for 15 minutes. Mix the reaction system after the reaction with an appropriate amount of enzyme-free water and 7.5M LiCl solution, then freeze at -20°C for 30 minutes. After 30 minutes, take out the reaction system and centrifuge at 15000 rpm in a 4°C centrifuge for 15 minutes to obtain mRNA precipitate. After discarding the supernatant, rinse the precipitate with 500 mL ice ethanol, then centrifuge at 15000 rpm in a 4°C centrifuge for 10 minutes to obtain mRNA precipitate again. After discarding the supernatant, dry at room temperature, then add an appropriate amount of TE solution to dissolve the precipitate to obtain mRNA product. Measure the concentration of the mRNA product using a ultramicro UV-visible spectrophotometer (DS-11+, Denovix).

[0271] The mRNA product of the previous step can be further purified using NanoGeldT20 filler according to the manufacturer's requirements to obtain a higher purity mRNA product to meet the requirements of in vivo experiments. The final product obtained is mRNA containing CapG(OMe)AG and N1-Me-pseudoUTP modification and polyA100.

[0272] The reagents used in the preparation of Path 1-3 are shown in Table 1.

[0273] Table 1

[0274] Path 1: Preparation of mRNA-LNP-RBC-1:

[0275] (a) Preparation and purification of mRNA-LNP

[0276] Different amounts of lipid powder were weighed and dissolved in anhydrous ethanol at room temperature by shaking, and the mass concentration of the lipids was as shown in Table 2.

[0277] Table 2

[0278] Preparation of mRNA-LNP

[0279] In a clean centrifuge tube, the five lipids were mixed according to the specific molar ratio of the formula (MC3 or SM102 = 50%, Cholesterol = 38.5%, DSPC = 10%, DSPE-PEG2000-Maleimide = 0.75%, DMG-PEG2000 = 0.75%) and diluted with anhydrous ethanol to the specified concentration. A certain amount of purified mRNA was taken in a clean centrifuge tube, diluted with 10 mM, pH = 4 citric acid-sodium citrate buffer, so that the final volume of the aqueous mRNA solution was 3 times the volume of the anhydrous ethanol phase containing lipids (V mRNA :V lipid = 3:1). The ethanol phase and the aqueous solution were transferred to two clean syringes, and the syringes were placed in a microfluidic device (Nanovolume Technology NWDPSII120 model; corresponding positions, sample tube and waste tube were installed on the microfluidic device; the flow rate was adjusted to 4 mL / min, the sample volume was 0.4-12 mL, the front waste volume was not less than 0.15 mL, the program was started after the rear, and the preparation process of the sample (the volume ratio of the mixture of the aqueous phase and the ethanol phase was 3:1) was monitored. When the program was completed, the sample tube was removed, and the solvent in the prepared mRNA-LNP was 25% ethanol citric acid-sodium citrate buffer.

[0280] Purification and identification of mRNA-LNP

[0281] The mRNA-LNP solution prepared in the previous step was quickly transferred to a clean centrifuge tube, and 100 times the volume of PBS buffer (pH = 7.4) was added, and then the solution was transferred to a 100 kDa ultrafiltration tube and centrifuged at 2000xg at room temperature. When the volume of the solution was reduced to the original volume, 100 times the PBS buffer was added again, and the ultrafiltration was performed to the required volume. At this time, the solvent in the purified mRNA-LNP was PBS buffer with pH = 7.4. An appropriate amount of mRNA-LNP solution was taken for particle size and particle size distribution index (PDI) detection (Zetasizer Lab, Malvern) and mRNA concentration and encapsulation efficiency test (SpectraMax iD5, Molecular Devices). The results are shown in Table 3.

[0282] Table 3 Detection results of mRNA-LNP under process path 1

[0283] (b) Preparation and purification of mRNA-LNP-RBC-1

[0284] TCEP-treated concentrated red blood cells (RBC)

[0285] An appropriate amount of EDTA anticoagulant whole blood (mouse or human) was taken in a centrifuge tube, 10 times the volume of PBS buffer (pH = 7.4) was added, mixed evenly, and centrifuged at 900xg at room temperature for 4 minutes. After removing the supernatant, 10 times the volume of PBS buffer was added and mixed, and centrifuged at 900xg at room temperature for 4 minutes, and the supernatant was discarded. Take 1 part of 0.5M TCEP stock solution, thaw it, dilute it with PBS buffer to 5mM to get 2X TCEP PBS solution, add equal volume of 2X TCEP PBS solution to the concentrated red blood cells obtained in the previous step to make the final concentration of TCEP 2.5mM, and place the centrifuge tube in a rotary mixer at 37°C for 1 hour. After the reaction, 10 times the volume of PBS buffer was added and mixed, and centrifuged at 900xg at room temperature for 4 minutes, and the supernatant was discarded. Repeat the above washing step once. Obtain TCEP-treated concentrated red blood cells.

[0286] Preparation of mRNA-LNP-RBC-1

[0287] Take the appropriate amount of the TCEP treated concentrated red blood cells obtained in the previous step into a centrifuge tube, add the corresponding volume of PBS buffer and purified mRNA-LNP as described in step (2) (a) above, so that the ratio of mRNA-LNP to concentrated red blood cells is 100 μg mRNA-LNP per 1 mL of concentrated RBC, and the concentration of RBC in the system is 2.5E9 / mL. Place the mixed centrifuge tube in a rotary shaker at 37°C for 1 hour. After the reaction, add 10 times the volume of PBS buffer and mix, centrifuge at 900xg for 4 minutes at room temperature, and discard the supernatant; repeat the above washing step once to obtain the coupled mRNA-LNP-RBC-1, i.e. mRNA-LNP-Mal.

[0288] Path 2: Preparation of mRNA-LNP-RBC-2

[0289] (a) Preparation and purification of mRNA-LNP

[0290] Weigh different amounts of lipid powder, and dissolve the lipids in anhydrous ethanol at room temperature by shaking, so that the mass concentration of the lipids is as shown in the table below:

[0291] Preparation of mRNA-LNP

[0292] In a clean centrifuge tube, mix the five lipids according to the specific molar ratio (MC3 or SM102 = 50%, cholesterol = 38.5%, DSPC = 10%, DSPE-PEG2000-SH = 0.75%, DMG-PEG2000 = 0.75%) according to the formula, and then dilute with anhydrous ethanol to the specified concentration. Take a certain amount of purified mRNA in a clean centrifuge tube, dilute with 10 mM, pH = 4 citric acid-sodium citrate buffer, so that the final volume of the aqueous mRNA solution is 3 times the volume of the anhydrous ethanol phase containing lipids (V mRNA :V lipid = 3:1). Transfer the ethanol phase and the aqueous solution to two clean syringes, place the syringes in the corresponding positions on the microfluidic device, load the sample tube and waste tube on the microfluidic device, adjust the corresponding parameters (flow rate, sample volume, waste volume, etc.) of the program, start the program, monitor the preparation process of the sample (the volume ratio of the aqueous phase to the ethanol phase is 3:1), and when the program is completed, remove the sample tube. At this time, the solvent in the prepared mRNA-LNP is 25% ethanol citric acid-sodium citrate buffer.

[0293] Purification and identification of mRNA-LNP

[0294] The mRNA-LNP solution prepared in the previous step was quickly transferred to a clean centrifuge tube, and 100 times the volume of PBS buffer (pH = 7.4) was added, and then the solution was transferred to a 100 kDa ultrafiltration tube and centrifuged at 2000xg at room temperature. When the volume of the solution was reduced to the original volume, 100 times the PBS buffer was added again, and the ultrafiltration was performed to the required volume. At this time, the solvent in the purified mRNA-LNP was PBS buffer with pH = 7.4. An appropriate amount of mRNA-LNP solution was taken for particle size and particle size distribution index (PDI) detection (Zetasizer Lab, Malvern) and mRNA concentration and encapsulation rate test (SpectraMax iD5, Molecular Devices). The results are shown in Table 4.

[0295] Table 4 Detection results of mRNA-LNP under process path 2

[0296] (b) Preparation and purification of mRNA-LNP-RBC-2

[0297] SMCC-treated concentrated red blood cells

[0298] An appropriate amount of EDTA anticoagulant whole blood (mouse or human) was taken in a centrifuge tube, 10 times the volume of PBS buffer (pH = 7.4) was added, mixed evenly, and centrifuged at 900xg at room temperature for 4 minutes. After removing the supernatant, 10 times the volume of PBS buffer was added and mixed, and then centrifuged at 900xg at room temperature for 4 minutes, and the supernatant was discarded. Take 2 mg of sulfo-SMCC powder, dissolve it in 1.8 mL of ultrapure water, add 0.2 mL of 10x PBS buffer, mix evenly to get 1 mg / mL (2.29 mM) SMCC PBS solution, and add an appropriate volume of SMCC PBS solution to the concentrated red blood cells obtained in the previous step to make the final concentration of SMCC 0.5 mM. The centrifuge tube was placed in a rotary mixer at 37°C for 1 hour. After the reaction, 10 times the volume of PBS buffer was added and mixed, and then centrifuged at 900xg at room temperature for 4 minutes, and the supernatant was discarded. Repeat the above washing step once to obtain SMCC-treated concentrated red blood cells.

[0299] Preparation of mRNA-LNP-RBC-2

[0300] Take the appropriate amount of SMCC treated concentrated red blood cells obtained in the above step into a centrifuge tube, add the corresponding volume of PBS buffer and purified mRNA-LNP with the same characteristics as in step (3) (a) above, so that the ratio of mRNA-LNP to concentrated red blood cells is 100 pg mRNA-LNP per 1 mL of concentrated RBC, and the concentration of RBC in the system is 2.5E9 / mL. Place the mixed centrifuge tube in a rotary shaker at 37°C for 1 hour. After the reaction, add 10 times the volume of PBS buffer and mix, centrifuge at 900xg for 4 minutes at room temperature, and discard the supernatant; repeat the above washing step once to obtain the coupled mRNA-LNP-RBC-2, i.e. mRNA-LNP-SH.

[0301] Path 3: Preparation of mRNA-LNP-RBC-3

[0302] (a) Preparation and purification of mRNA-LNP

[0303] Weigh different amounts of lipid powder, and dissolve them in anhydrous ethanol at room temperature by shaking, so that the mass concentration of the lipids is as shown in the table below:

[0304] Preparation of mRNA-LNP

[0305] In a clean centrifuge tube, mix the five lipids according to the specific molar ratio (MC3 or SM102 = 50%, cholesterol = 38.5%, DSPC = 10%, DSPE-PEG2000-LPETG = 0.75%, DMG-PEG2000 = 0.75%) according to the formula, and then dilute with anhydrous ethanol to the specified concentration. Take a certain amount of purified mRNA in a clean centrifuge tube, dilute it with 10 mM, pH = 4 citric acid-sodium citrate buffer, so that the final volume of the aqueous mRNA solution is 3 times the volume of the anhydrous ethanol phase containing lipids (V mRNA :V lipid = 3:1). Transfer the ethanol phase and the aqueous solution to two clean syringes, place the syringes in the corresponding positions on the microfluidic device, and install the sample tube and waste tube on the microfluidic device; adjust the corresponding parameters (flow rate, sample volume, waste volume, etc.) of the program, start the program, monitor the preparation process of the sample (the volume ratio of the aqueous phase to the ethanol phase is 3:1), and when the program is completed, remove the sample tube. At this time, the solvent in the prepared mRNA-LNP is 25% ethanol citric acid-sodium citrate buffer.

[0306] Purification and identification of mRNA-LNP

[0307] The mRNA-LNP solution prepared in the previous step was quickly transferred to a clean centrifuge tube, and 100 times the volume of PBS buffer (pH = 7.4) was added, and then the solution was transferred to a 100 kDa ultrafiltration tube and centrifuged at 2000xg at room temperature. When the volume of the solution was reduced to the original volume, 100 times the PBS buffer was added again, and the ultrafiltration was performed to the required volume. At this time, the solvent in the purified mRNA-LNP was PBS buffer with pH = 7.4. An appropriate amount of mRNA-LNP solution was taken for particle size and particle size distribution index (PDI) detection (Zetasizer Lab, Malvern) and mRNA concentration and encapsulation rate test (SpectraMax iD5, Molecular Devices). The results are shown in Table 5.

[0308] Table 5 Detection results of mRNA-LNP under process path 3

[0309] (b) Preparation and purification of mRNA-LNP-RBC-3

[0310] TCEP-treated concentrated red blood cells

[0311] An appropriate amount of EDTA anticoagulant whole blood (mouse or human) was taken in a centrifuge tube, 10 times the volume of PBS buffer (pH = 7.4) was added, mixed evenly, and centrifuged at 900xg at room temperature for 4 minutes. After removing the supernatant, 10 times the volume of PBS buffer was added and mixed, and centrifuged at 900xg at room temperature for 4 minutes, and the supernatant was discarded. Take 1 part of 0.5M TCEP stock solution, thawed, and dilute with PBS buffer to 5mM to obtain 2X TCEP PBS solution. Add an equal volume of 2X TCEP PBS solution to the concentrated red blood cells obtained in the previous step to make the final concentration of TCEP 2.5mM, and place the centrifuge tube in a rotary mixer at 37°C for 1 hour. After the reaction, 10 times the volume of PBS buffer was added and mixed, and centrifuged at 900xg at room temperature for 4 minutes, and the supernatant was discarded. Repeat the above washing step once. TCEP-treated concentrated red blood cells were obtained.

[0312] G small peptide-coupled concentrated red blood cells

[0313] An appropriate amount of TCEP-treated concentrated red blood cells obtained in the previous step was taken in a centrifuge tube, an equal volume of PBS buffer and an appropriate amount of 10mg / mL (6.90mM) G small peptide solution were added to make the final concentration of G small peptide 0.3125mM, and the centrifuge tube was placed in a rotary mixer at 37°C for 15 minutes. After the reaction, 10 times the volume of PBS buffer was added and mixed, and centrifuged at 900xg at room temperature for 4 minutes, and the supernatant was discarded. Repeat the above washing step once. G small peptide-coupled concentrated red blood cells were obtained.

[0314] Preparation of mRNA-LNP-RBC-3

[0315] Take an appropriate amount of G peptide-coupled concentrated red blood cells obtained in the previous step and add the corresponding volume of PBS buffer, mRNA-LNP purified and characterized as in step (3) (a) above, and mgSortase transpeptidase (SEQ ID No: 1) into a centrifuge tube. The ratio of mRNA-LNP to concentrated red blood cells is 100 pg mRNA-LNP per 1 mL of concentrated RBC, and the concentration of mgSortase transpeptidase used is 10 pM. The concentration of RBC in the system is 2.5E9 / mL. Place the mixed centrifuge tube in a rotary shaker at 37°C for 1 hour. After the reaction, add 10 times the volume of PBS buffer and mix. Centrifuge at 900xg for 4 minutes at room temperature, and discard the supernatant. Repeat the above washing step once to obtain the coupled mRNA-LNP-RBC-3, i.e., mRNA-LNP-LPETG.

[0316] 1.2 Preparation of mRNA-LNP without linker

[0317] Weigh an appropriate amount of different lipid powders and dissolve them in anhydrous ethanol at room temperature by shaking. The mass concentration of the lipids is as shown in the following table:

[0318] Preparation of mRNA-LNP

[0319] In a clean centrifuge tube, mix the four lipids according to the specific molar ratio of the formula (MC3 = 50%, cholesterol = 38.5%, DSPC = 10%, DMG-PEG2000 = 1.5%) and dilute with anhydrous ethanol to the specified concentration. Take a certain amount of purified mRNA in a clean centrifuge tube and dilute it with 10 mM, pH = 4 citric acid-sodium citrate buffer. The final volume of the mRNA-containing aqueous solution is 3 times the volume of the anhydrous ethanol phase containing lipids (V mRNA :V lipid = 3:1). Transfer the ethanol phase and the aqueous solution to two clean syringes, respectively. Place the syringes in the corresponding positions on the microfluidic device, load the sample tube and waste tube on the microfluidic device, adjust the corresponding parameters (flow rate, sample volume, waste volume, etc.) of the program, and start the program. Monitor the preparation process of the sample (the volume ratio of the aqueous phase to the ethanol phase is 3:1). When the program is complete, remove the sample tube. At this time, the solvent in the prepared mRNA-LNP is 25% ethanol citric acid-sodium citrate buffer.

[0320] Table 6 Detection results of mRNA-LNP without linker

[0321] Example 2, in vitro characterization of mRNA-LNP-RBC

[0322] Objective: To study the distribution of mRNA-LNP on the surface of red blood cells in mRNA-LNP-RBC-1.

[0323] Method: Luc-mRNA (mRNA encoding Luciferase, Luciferase amino acid sequence: SEQ ID NO: 3; nucleotide sequence: SEQ ID NO: 4) was labeled with the same HiSynthT7 Co-transcription RNA synthesis kit and replaced 20% N1-Me-pseudo UTP with Cy5-UTP (APExBIO, B8333) to obtain fluorescent Luc-mRNA, i.e. Cy5-Luc-mRNA. Cy5-Luc-mRNA was mixed with the corresponding proportion of lipid formula microfluidic to obtain Cy5-Luc-mRNA-LNP (preparation method see process path 1 described in Example 1); red blood cells were labeled with CFSE dye (ThermoFisher, C34554) to obtain fluorescently labeled red blood cells.

[0324] CFSE+Cy5-Luc-mRNA-LNP-RBC-1 was prepared with CFSE+RBCs and Cy5-Luc-mRNA (preparation method see process path 1 described in Example 1). The labeling efficiency was evaluated by flow detection (Beckman Coulter CytoFLEX) method, and CFSE+Cy5-Luc-mRNA-LNP-RBC-1 was imaged by Zessis980 confocal microscope.

[0325] Results: As shown in Figure 3, the flow detection results showed that 99.96% of red blood cells were coupled with Cy5-Luc-mRNA-LNP, and the confocal imaging results further showed that all red blood cells in the field of view were coupled with Cy5-Luc-mRNA-LNP, and Cy5-Luc-mRNA was uniformly distributed on the surface of red blood cells. This result suggests that mRNA-LNP can be successfully and efficiently coupled to the surface of red blood cells.

[0326] Example 3, mRNA-LNP-RBC gene delivery efficiency study

[0327] 3.1 mRNA-LNP-RBC gene delivery / expression ratio

[0328] Experimental purpose: Unlike mRNA-LNP entering cells by endocytosis, mRNA-LNP-RBC enters cells by phagocytosis, so the difference between the two methods may lead to different mRNA escape efficiencies. This example evaluates the mRNA-LNP-RBC gene delivery efficiency and compares it with the traditional cationic liposome transfection method.

[0329] Experimental method: Bone marrow-derived macrophages (BMDM) were isolated from the bone marrow of C57BL / 6 mice (Vital River) and cultured for 7 days under the induction of M-CSF cytokine (Peprotech, 315-02), and then induced with LPS (MCE, HY-D1056) for 8 hours. After induction, Luc-LNP or GFP-LNP (preparation method see Example 1.2, 500 ng / well, wherein the amino acid sequence of GFP is SEQ ID NO: 5; the nucleotide sequence is SEQ ID NO: 6) or Luc-LNP-RBC-1 (Luciferase mRNA-LNP-RBC-1) or GFP-LNP-RBC-1 (GFP mRNA-LNP-RBC-1) prepared according to the method of process path 1 of Example 1 (10 μL / well, equivalent to 50 ng / well of mRNA content) were added to the BMDM cells. After 24 hours, the delivery of mRNA and the production of proteins were detected by flow cytometry method, and the escape efficiency of the delivered mRNA was evaluated according to the amount of mRNA entering and the efficiency of protein expression.

[0330] Experimental results: As shown in Figure 4, compared with mRNA-LNP-RBC-1, mRNA-LNP delivered more mRNA, but the expression of the target protein was much lower than mRNA-LNP-RBC-1, i.e. mRNA-LNP-RBC-1 can achieve more efficient protein expression with lower mRNA amount, and it is verified in 2 target points. The results show that mRNA-LNP-RBC-1 has higher lysosome escape compared with mRNA-LNP gene delivery method, achieving more efficient mRNA expression.

[0331] Therefore, mRNA-LNP carried by red blood cells enters cells based on phagocytosis, achieving higher mRNA delivery efficiency.

[0332] 3.2 mRNA-LNP-RBC gene delivery path research

[0333] Experimental purpose: This study compared the differences between mRNA-LNP and red blood cell-mediated mRNA-LNP delivery pathways through fluorescence co-localization experiments to explore whether red blood cells can bypass the traditional endosome-lysosome pathway, reduce endosome entrapment, and thus improve mRNA delivery efficiency and protein expression levels.

[0334] Experimental method: (1) Co-localization experiment: Bone marrow-derived macrophages (BMDM) were obtained from C57BL / 6 mice (Vital River) and cultured for 7 days under the induction of M-CSF (Peprotech, 315-02), then stimulated with LPS (MCE, HY-D1056) for 8 hours. After induction, Cy5-Luc-LNP (fluorescently labeled Luciferase mRNA, 500 ng / well) or Cy5-Luc-LNP-RBC-1 (10 μL / well, mRNA content equivalent to 50 ng / well) prepared by the process path 1 of Example 1 were added to BMDM, respectively, and incubated for 6 hours. Then, immunofluorescence method was used to detect the co-localization of mRNA with early endosome marker EEA1 (Abeam, ab2900, 1:50) and late endosome / lysosome marker LAMP1 (Cell Signaling Technology, 99437S, 1:50). (2) Phagocytosis inhibition experiment: BMDM of the same source were pretreated with 10 μM Cytochalasin D (Thermo Fisher, PHZ1063) for 1 hour to inhibit phagocytosis, then washed and added with Cy5-Luc-LNP (500 ng / well) or Cy5-Luc-LNP-RBC-1 (10 μL / well, equivalent to mRNA 50 ng / well), respectively, and incubated for 6 hours. Flow cytometry was used to detect the uptake of Cy5-mRNA in BMDM.

[0335] Experimental results: Immunofluorescence results showed (Fig. 5A-B) that 83% of LNP-delivered mRNA was co-localized with EEA1 and 50% with LAMP1; while the co-localization rate of red blood cell-delivered mRNA was less than 5%, indicating that it does not depend on the classic endosome-lysosome pathway. The results of the phagocytosis inhibition experiment showed that Cytochalasin D treatment reduced the uptake level of red blood cell-delivered mRNA by 11.1 times, confirming that phagocytosis is the main mechanism of red blood cell-mediated mRNA uptake (Fig. 5C).

[0336] This study proves that red blood cell-mediated mRNA delivery can bypass the endosome-lysosome pathway, efficiently enter myeloid cells through phagocytosis, and release mRNA in the cytoplasm, improving protein expression efficiency.

[0337] Example 4, Tissue distribution and pharmacokinetics study of mRNA-LNP-RBC

[0338] 4.1 In vivo tissue distribution and gene expression of mRNA-LNP-RBC-1 and mRNA-LNP-RBC-2

[0339] Objective of the experiment: To evaluate the distribution and in vivo expression of mRNA-LNP-RBC in mice and compare with LNP. Luciferase (Luc) and GFP (green fluorescent protein) were selected for evaluation in this experiment.

[0340] Experimental method: Luc-LNP (preparation method see Example 1.3, containing mRNA 500 ng / each, wherein the mRNA is the mRNA encoding Luciferase), Luc-LNP-RBC-1 (2e9 / each, containing mRNA 500 ng / each, wherein the mRNA is the mRNA encoding Luciferase), Luc-LNP-RBC-2 (2e9 / each, containing mRNA 500 ng / each) and Luc-LNP-RBC-3 (2e9 / each, containing mRNA 500 ng / each, wherein the mRNA is the mRNA encoding Luciferase) were prepared and injected into C57BL / 6 mice (Vital River) through the tail vein. The expression of Luciferase in various tissues and organs was detected 20 hours after administration; wherein n = 3 in each group. The specific in vivo expression detection method is as follows: a 15 mg / mL aqueous solution of D-luciferin potassium salt (Biyun Tian, ST198) was prepared and injected into the mice intraperitoneally at 200 uL / each. After 10 min, the heart, liver, spleen, lung and kidney were taken out and analyzed by PerkinElmer IVIS Lumina Series III in vivo imaging instrument.

[0341] GFP-LNP (preparation method see Example 1.3, containing mRNA 500 ng / each, wherein the mRNA is the mRNA encoding GFP) and GFP-LNP-RBC-1 (2e9 / each, containing mRNA 500 ng / each, wherein the mRNA is the mRNA encoding GFP) were prepared and injected into C57BL / 6 mice (Vital River) through the tail vein. The expression of GFP in the spleen was detected by flow cytometry (Beckman Coulter CytoFLEX) 20 hours after administration, wherein n = 2 in each group of mice.

[0342] Experimental results: As shown in Figure 6, Luc-LNP-RBC-1, Luc-LNP-RBC-2 and Luc-LNP-RBC-3 can all achieve in vivo gene delivery and expression, and the expression is mainly concentrated in the spleen. As can be seen from the left graph, the distribution of each Luc-LNP carried by red blood cells in various organs, and the right graph can see the distribution ratio of Luc-LNP carried by red blood cells in the liver and spleen.

[0343] As shown in Figure 7, Luc-LNP is consistent with the literature, and after intravenous infusion, it is mainly distributed in the liver, and Luciferase expression is seen in the liver, and the distribution ratio of liver expression is as high as 97%; while Luc-LNP-RBC-1 is mainly distributed in the spleen, and Luciferase expression is seen in the spleen, and the distribution ratio of spleen expression is as high as 90%. This result suggests that Luc-LNP-RBCs can change the in vivo distribution of Luc-LNP, and achieve effective delivery and expression of genes in the spleen.

[0344] As shown in Figure 8, compared with GFP-LNP, GFP-LNP-RBC-1 can more effectively deliver to the spleen, and mainly express GFP protein in CD11b+myeloid cells (CD11b+F4 / 80+cells and CD11b+F4 / 80-cells), and other immune cells (i.e. cells other than CD11b+F4 / 80+cells and CD11b+F4 / 80-cells, such as lymphocytes, etc.) do not express GFP. It can be seen that red blood cells deliver mRNA-LNP to the spleen, especially the myeloid cells of the spleen, by being phagocytosed by CD11b+myeloid cells in the spleen.

[0345] From the above experimental results, it can be seen that the targeting distribution of Luc-LNP-RBC is consistent with the distribution of RBC (JANDL JH et al., Clinical determination of the sites of red cell sequestration in hemolytic anemias. J Clin Invest. 1956 Aug;35(8):842-67. doi: 10.1172 / JCI103338. PMID: 13345887; PMCID: PMC441656; McArdel, S. L., et al. (2021). Anti-tumor effects of RTX-240: an engineered red blood cell expressing 4-1BB ligand and interleukin-15. Cancer immunology, immunotherapy: CII 70, 2701-2719), that is, red blood cells transport the mRNA carried by them to the organs (spleen) and immune cells (CD11b+myeloid cells) they target, resulting in a significant difference in distribution between Luc-LNP-RBC and mRNA-LNP.

[0346] 4.2 In vivo pharmacokinetic study of mRNA-LNP-RBC

[0347] Experimental purpose: to evaluate the pharmacokinetics of Luc-mRNA-LNP-RBC-1 in C57BL / 6 mice

[0348] Experimental method: As described previously, CFSE+Cy5-Luc-mRNA-LNP-RBC-1 was prepared. CFSE+Cy5-Luc-mRNA-LNP-RBC-1 (2e9 / each, where mRNA is mRNA encoding Luciferase) was injected into C57BL / 6 mice (Vantianhua) via the tail vein, and blood was taken at 0.5 hours after administration (Day 0, D0), 24 hours (D1), 48 hours (D2), 72 hours (D3), 96 hours (D4), 120 hours (D5) for flow detection. The number of animals in each group n=3.

[0349] Experimental results: As shown in Figure 9, Luc-mRNA-LNP-RBC-1 was cleared by about 70% in mice in about 5 days, and the Cy5-Luc-mRNA-LNP signal on the surface decreased with the clearance of red blood cells. Since mRNA-LNP drugs are usually completely cleared within 24h (Lei Ci, et al., Biodistribution of Lipid 5, mRNA, and Its Translated Protein Following Intravenous Administration of mRNA-Encapsulated Lipid Nanoparticles in Rats. Drug Metab Dispos. 2023 Jul; 51(7): 813-823; Kimberly J Hassett et al., mRNA vaccine trafficking and resulting protein expression after intramuscular administration. Mol Ther Nucleic Acids. 2023 Nov 24; 35(1): 102083.), therefore, this result suggests that mRNA-LNP-RBC can effectively improve the in vivo stability of mRNA-LNP.

[0350] 4.3 mRNA-LNP-RBC drug loading detection

[0351] Take 10 μL of the concentrated mRNA-LNP-RBC preparation prepared in the above routes 1, 2 and 3, wherein the mRNA in these mRNA-LNP-RBCs is mRNA encoding Luciferase (amino acid sequence: SEQ ID NO: 3; nucleotide sequence: SEQ ID NO: 4). Add an appropriate amount of reference mRNA (mRNA of GFP (SEQ ID NO: 5; SEQ ID NO: 6)) to the cell lysate (QIAGEN RNeasy Plus Mini Kit, 74136), mix well to obtain the lysate after adding the reference: 600 μL of cell lysate and 50 ng of reference mRNA are needed for each 10 μL of concentrated RBC; lyse N samples of 10 μL of concentrated RBC, and (N+1) portions of lysate with mRNA reference are prepared, i.e. (N+1)*600 μL of lysate is added to (N+1)*50 ng of reference mRNA. Centrifuge at 15000x rpm for 3 minutes, and take the supernatant. Perform gDNA removal, mRNA reverse transcription and Real Time PCR reaction (Light Cycler 96, Roche) according to the instructions of the kit.

[0352] Experimental results: The modified mRNA-LNP (including mRNA-LNP-Mal, mRNA-LNP-SH and mRNA-LNP-LPETG) has no obvious abnormality in physicochemical properties compared with unmodified mRNA-LNP, and can successfully perform red blood cell coupling reaction to obtain mRNA-LNP-RBC. The drug loading detection results of mRNA-LNP-RBC (Figure 2) show that the mRNA-LNP loading on the surface of the engineered red blood cells (mRNA-LNP-RBC-1, mRNA-LNP-RBC-2, mRNA-LNP-RBC-3) prepared by the three process routes is 0.1-3 μg mRNA / mL RBCs, i.e. about 9-270 mRNA molecules can be connected to the surface of each red blood cell. The drug loading detection results of mRNA-LNP mixed with RBC without linker are lower than the detection limit (<10 pg / uL), i.e. the mRNA-LNP connected to RBC by adsorption method is very low. This result shows that the covalent coupling method used in this study significantly improves the LNP drug loading on the surface of RBC.

[0353] SEQUENCE LISTING

[0354] REFERENCES

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Claims

1. A lipid particle-erythrocyte conjugate comprising a lipid particle comprising a nucleic acid and an erythrocyte, wherein the lipid particle is covalently conjugated to the membrane surface of the erythrocyte, optionally the covalent conjugation is via a linker.

2. The lipid particle-erythrocyte conjugate of claim 1, wherein the lipid particle is a lipid nanoparticle (LNP).

3. The lipid particle-erythrocyte conjugate of any one of the preceding claims, wherein the nucleic acid comprises DNA and / or RNA.

4. The lipid particle-erythrocyte conjugate of claim 3, wherein the RNA is selected from the group consisting of messenger RNA (mRNA), short interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), RNA interference (RNAi) molecules, microRNA (miRNA), antisense RNA, ribozyme, Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), a guide RNA, a circular RNA, a tRNA, and / or combinations thereof; and / or wherein the DNA is a circular DNA such as a plasmid or a linear DNA such as an antisense DNA.

5. The lipid nanoparticle of any one of the preceding claims, wherein the lipid nanoparticle comprises one or more lipids selected from the group consisting of an ionizable lipid, a helper lipid, a sterol, and a PEG lipid, for example wherein the ionizable lipid is selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phospho-rac-(l'-guanine) (DMG-PEG), 1,2-dimyristoyl-sn-glycero-3-phospho-rac-(l'-cytosine) (DMC-PEG), 1,2-dioleoyl-sn-glycero-3-phospho-rac-(l'-guanine) (DOPG), 1,2-dioleoyl-sn-glycero-3-phospho-rac-(l'-cytosine) (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-rac-(l'-adenine) (DOPA), 1,2-dioleoyl-sn-glycero-3-phospho-rac-(l'-uracil) (DOPU), 1,2-dioleoyl-sn-glycero-3-phospho-rac-(l'-thymine) (DOPTh), 1,2-dioleoyl-sn-glycero-3-phospho-rac-(l'-guanine) (DOPG), 1,2-dioleoyl-sn-glycero-3-phospho-rac-(l'-cytosine) (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-rac-(l'-adenine) (DOPA), 1,2-dioleoyl-sn-glycero-3-phospho-rac-(l'-uracil) (D ​ The ionizable lipid is preferably selected from the group consisting of 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazinethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-triacontane (KL25), 1,2-dilinoleyl-oxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA) (abbreviated MC3), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA), (2R)-2-({8-[-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA (2R)), (2S)-2-({8-[-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA (2S)), Bis((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino} 9-heptadecyloctanoate (SM102) and / or combinations thereof, preferably the ionizable lipid is selected from MC3 or SM102; The helper lipid is preferably selected from the group consisting of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-di-undecanoyl-sn-glycero-phosphocholine (DUPC), 1 -palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1 -oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1 -hexadecyl-sn-glycero-3-phosphocholine (C16 LysoPC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-dibehenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-dibehenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1 -glycerol) sodium salt (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyloleyl phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1 -stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1 -stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, and / or combinations thereof, more preferably the helper lipid is selected from the group consisting of DSPC; The steroid is preferably selected from the group consisting of cholesterol, coprostanol, sitosterol, ergosterol, elaidosterol, stigmasterol, brassicasterol, and / or combinations thereof, preferably the steroid is preferably selected from the group consisting of cholesterol; and / or The PEG lipid is preferably selected from the group consisting of 1,2-dimyristoyl-sn-glycero-methoxypolyethyleneglycol (DMG-PEG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)] (DSPE-PEG), PEG-distearoylglycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleoyl, PEG-distearoyl, PEG-diacrylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidyl ethanolamine (PEG-DPPE), PEG-l,2-dimyristyloxypropyl-3-amine (PEG-c-DMA) and / or combinations thereof, more preferably the PEG lipid is selected from DMG-PEG, such as DMG-PEG2000.

6. The lipoplex-red blood cell conjugate of any one of the preceding claims, wherein the lipoplex comprises one or more lipids comprising a maleimide group, preferably wherein the amount of the lipid in the lipoplex is 0.1-10% (e.g. 0.1-5% or 0.1-1% or 0.5-1%, e.g. about 0.75%).

7. The lipoplex-red blood cell conjugate of claim 6, wherein the lipid comprising a maleimide group is selected from one or more of the following: an ionizable lipid comprising a maleimide group, a helper lipid comprising a maleimide group, a steroid comprising a maleimide group and / or a PEG lipid comprising a maleimide group, preferably the ionizable lipid, helper lipid, steroid or PEG lipid is selected from a lipid as defined in claim 5.

8. The lipoplex-red blood cell conjugate of any one of the preceding claims, wherein the lipid comprising a maleimide group is a PEG lipid comprising a maleimide group, preferably a DSPE-PEG lipid comprising a maleimide group, most preferably a DSPE-PEG(2000) comprising a maleimide group, such as DSPE-PEG(2000)-MAL.

9. The lipoplex-red blood cell conjugate of any one of the preceding claims, wherein the red blood cell is a reductant-treated red blood cell, e.g. a thiol reductant-treated red blood cell, Optionally, the reductant-treated red blood cell is obtained by a method comprising: (1) isolating and concentrating red blood cells from human whole blood, optionally by leukocyte depletion; or obtaining red blood cells from stem cells, e.g. pluripotent stem cells, e.g. induced pluripotent stem cells; (2) treating the red blood cells with a reductant, e.g. a thiol reductant, to chemically modify the surface of the red blood cells; (3) collecting and concentrating the modified red blood cells.

10. The lipoplex-red blood cell conjugate of claim 9, wherein the thiol reductant is TCEP.

11. The lipoplex-red blood cell conjugate of any one of the preceding claims, having the structure: LNP-DSPE-PEG(2000)-MAL-S-RBC, wherein LNP-DSPE-PEG(2000)-MAL means a lipid nanoparticle as defined in claim 8 comprising DSPE-PEG(2000)-MAL, optionally, the amount of DSPE-PEG(2000)-MAL in the LNP is 0.1-10% (e.g. 0.1-5% or 0.1-1% or 0.5-1%, e.g. about 0.75%), optionally the LNP can further comprise one or more other lipids as defined in claim 5; RBC means a red blood cell as defined in claim 9; -S- means a group comprising S formed by reaction of the maleimide group with a thiol group of the red blood cell, wherein the S atom is derived from the thiol group of the RBC.

12. A method of preparing a lipid particle-red blood cell conjugate as defined in any of the preceding claims, comprising mixing the lipid particle comprising a maleimide group and a red blood cell treated with a reducing agent, such that both are covalently coupled via the maleimide group and a nucleophilic group on a membrane protein of the red blood cell, e.g. the method comprises (1) loading the lipid comprising a maleimide group into a lipid particle, (2) performing a surface chemical modification of adult native red blood cells to expose a nucleophilic group on a membrane protein of the red blood cell; and collecting the modified red blood cells and concentrating; (3) mixing the lipid particle of (1) and the red blood cells obtained in (2) (e.g. 0.1-1000 pg lipid particle per 1 ml red blood cells), such that both are covalently coupled via the maleimide group and a nucleophilic group on a membrane protein of the red blood cell; (4) collecting the lipid particle-red blood cell conjugate obtained in (3).

13. The method of claim 12, wherein the nucleophilic group on a membrane protein of the red blood cell is a side chain thiol group.

14. The method of claim 13, wherein the side chain thiol group is a thiol group after reduction of cysteine.

15. The method of any of claims 12-14, wherein the red blood cells are surface chemically modified using a thiol-based reducing agent.

16. The method of claim 15, wherein the thiol-based reducing agent is TCEP.

17. The method of claim 16, wherein the final concentration of TCEP is between 0.1 mM and 10 mM, e.g. 0.5 mM and 10.0 mM, 0.5 mM and 5.0 mM, preferably about 2.5 mM.

18. A lipid particle-red blood cell conjugate as defined in any of claims 1-5, the lipid particle comprising a lipid comprising a thiol group (SH), preferably the amount of the lipid comprising a thiol group (SH) in the lipid particle is 0.1-10% (e.g. 0.1-5% or 0.1-1% or 0.5-1%, e.g. about 0.75%).

19. The lipid particle-erythrocyte conjugate of claim 18, wherein the thiol group (SH)-containing lipid is selected from one or more of the group consisting of a thiol group (SH)-containing ionizable lipid, a thiol group (SH)-containing helper lipid, a thiol group (SH)-containing sterol, and / or a thiol group (SH)-containing PEG lipid, preferably the ionizable lipid, helper lipid, sterol, or PEG lipid is selected from the lipids as defined in claim 5; for example wherein the thiol group (SH)-containing lipid is a thiol group (SH)-containing PEG lipid, preferably a thiol group (SH)-containing DSPE-PEG lipid, most preferably a thiol group (SH)-containing DSPE-PEG(2000), such as DSPE-PEG(2000)-SH.

20. The lipid particle-erythrocyte conjugate of claim 18 or 19, wherein the thiol group (SH)-containing lipid is linked to an amino group on the erythrocyte via a linker, preferably the erythrocyte is a mature erythrocyte, such as an adult native erythrocyte.

21. The lipid particle-erythrocyte conjugate of claim 20, wherein the linker has a functional group capable of reacting with the thiol group (SH) on the thiol group (SH)-containing lipid to form a covalent linkage to the lipid particle, and it has a functional group capable of reacting with a nucleophilic group on a membrane protein of the erythrocyte to link to the erythrocyte.

22. The lipid particle-erythrocyte conjugate of claim 21, wherein the nucleophilic group on the membrane protein of the erythrocyte includes, but is not limited to, (i) an N-terminal amino group, (ii) a side chain amino group, such as the amino group of lysine, (iii) a side chain thiol group, such as the thiol group of cysteine, and (iv) a sugar hydroxyl or amino group (in the case of glycosylation), such as the side chain amino group, and the linker has an electrophilic group: (i) an active ester, such as an NHS ester, an HOBt ester, a haloformate, and an acid halide; (ii) an alkyl and benzyl halide, such as a haloacetamide; and (iii) an aldehyde, a ketone, a carboxyl, and a maleimide group, wherein the nucleophilic group reacts with the electrophilic group to form a covalent linkage.

23. The lipid particle-erythrocyte conjugate of claim 21, wherein the nucleophilic group on the membrane protein of the erythrocyte is the -NH2 of the lysine side chain, and the linker has a functional group capable of reacting with the -NH2 of the lysine side chain on the membrane protein of the erythrocyte to form a covalent linkage to link to the erythrocyte.

24. The lipid particle-erythrocyte conjugate of any one of claims 20-23, wherein the linker is succinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate-(6-aminohexanoate) (LC-SMCC), 4-(N-maleimidomethyl)cyclohexane-l-carboxylic acid succinimidyl ester (SMCC), or sulfo-SMCC, preferably sulfo-SMCC.

25. The lipid particle-erythrocyte conjugate of any one of claims 18-24, having the structure: LNP-DSPE-PEG(2000)-S-SMCC-RBC, wherein LNP-DSPE-PEG(2000) refers to a lipid nanoparticle as defined in claim 19 comprising DSPE-PEG(2000)-SH, wherein optionally the amount of said DSPE-PEG(2000)-SH in the LNP is between 0.1-10% (e.g. 0.1-5% or 0.1-1% or 0.5-1%, e.g. about 0.75%), optionally the LNP can further comprise one or more other lipids as defined in claim 5; RBC refers to a red blood cell; SMCC refers to a linker LC-SMCC, SMCC or sulfo-SMCC as defined in claim 24, -S- refers to a group comprising S formed by reaction of a thiol group (SH) in the LNP with a maleimide group of the linker SMCC, wherein the S atom is derived from the thiol group of the lipid DSPE-PEG(2000)-SH in the LNP; Optionally, multiple lipid particles can be coupled to the RBC, wherein the lipid particles can be the same or different.

26. A method of preparing a lipid particle-red blood cell conjugate as defined in any one of claims 18-25, comprising mixing a red blood cell linked to a linker and a lipid particle comprising a thiol group (SH) such that the two are covalently coupled via the linker, e.g. (1) reacting a nucleophilic group on a red blood cell membrane protein (e.g. NH2 on lysine) with a bivalent linker reagent to form a red blood cell linked to a linker via a covalent bond, preferably the red blood cells are collected and concentrated; (2) pre-loading a lipid comprising a thiol group (SH) into a lipid particle; (3) mixing the red blood cell linked to a linker in (1) and the lipid particle obtained in (2) (e.g. 0.1-1000 pg lipid particle per 1 mL red blood cells) such that the two are covalently coupled via the linker; (4) collecting the lipid particle-red blood cell conjugate obtained in (3).

27. The method of claim 26, wherein the nucleophilic group on a red blood cell membrane protein is an N-terminal amine group or a side chain amine group.

28. The method of claim 26 or 27, wherein the bivalent linker is LC-SMCC, SMCC or sulfo-SMCC, preferably SMCC, e.g. the final concentration of SMCC is between 0.1 mM and 10 mM, e.g. 0.5 mM and 10.0 mM, 0.5 mM and 5.0 mM, preferably about 0.5 mM.

29. The lipid particle-red blood cell conjugate of any one of claims 1-5, wherein the lipid particle is pre-loaded or comprises a lipid comprising a sortase recognition motif.

30. The lipid particle-red blood cell conjugate of claim 29, wherein the lipid comprising a sortase recognition motif is selected from one or more of: an ionizable lipid comprising a sortase recognition motif, a helper lipid comprising a sortase recognition motif, a steroid comprising a sortase recognition motif and / or a PEG lipid comprising a sortase recognition motif.

31. Lipid particle-erythrocyte conjugate according to claim 29 or 30, wherein the lipid comprising a sortase recognition motif is a PEG lipid comprising a sortase recognition motif, preferably DSPE-PEG comprising a sortase recognition motif, most preferably DSPE-PEG(2000) comprising a sortase recognition motif, optionally said DSPE-PEG(2000) is modified to comprise a sortase recognition motif; for example said DSPE-PEG(2000) is linked via a flexible peptide segment (GS) n to a sortase recognition motif, wherein n = 1-10.

32. The lipoplex-erythrocyte conjugate of any one of claims 29-31, wherein the sortase recognition motif comprises or consists of LPXTG, preferably the sortase recognition motif is LPETG.

33. The lipoplex-erythrocyte conjugate of any one of claims 29-32, wherein the sortase recognition motif can be modified to increase its affinity; preferably the modification is the addition of a G at the C-terminus of the sortase recognition motif, such as LPETGG.

34. The lipoplex-erythrocyte conjugate of any one of claims 29-33, the sortase recognition motif containing lipid comprises or consists of DSPE-PEG(2000)-LPETG.

35. Lipid particle-erythrocyte conjugate according to any one of claims 29-34, wherein the sortase recognition motif is conjugated to the extracellular part of a membrane protein of the erythrocyte via a linker, for example the linker comprises a G nane peptide and a maleimidoalkyl chain (C 2-8 ), which maleimidoalkyl chain (C 2-8 ) is conjugated to the membrane protein of the erythrocyte and the G nane peptide is conjugated to the lipid containing the sortase recognition motif via a sortase mediated reaction.

36. The lipoplex-erythrocyte conjugate of any one of claim 35, wherein the G-small peptide is a linear small peptide or a branched small peptide.

37. The lipoplex-erythrocyte conjugate of claim 35 or 36, wherein the G-small peptide is a branched small peptide comprising two or more branching units, wherein one or more lipoplexes are conjugated to one or more branching units.

38. The lipoplex-erythrocyte conjugate of claim 37, the branching unit consists of the amino acid sequence K(GGG), wherein the glycines in parentheses are conjugated to the side chain epsilon-amino group of the lysine to form a branch, and the lysine forms a peptide bond with other amino acids through its alpha amino group to constitute the "backbone" of the "G-small peptide", optionally, a spacer can be added between K and G in the branching unit K(GGG), such as COCH2CH2-PEG6-NH.

39. The lipoplex-erythrocyte conjugate of any one of claims 34-37, the G-small peptide has the 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, preferably the G-small peptide has the structure K(GGG)-GGG-K(GGG)-GGG-K(GGG).

40. Lipid particle-erythrocyte conjugate according to any one of claims 35 to 39, wherein the maleimidylalkyl chain (C 2-8 ) is 6-maleimidocaproic acid or 4-maleimidobutyric acid, preferably 6-maleimidocaproic acid.

41. The lipoplex-erythrocyte conjugate of any one of claims 35-40, wherein there is (PEG)n between the G-small peptide and the 6-maleimidocaproic acid, wherein n = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more.

42. The lipid particle-erythrocyte conjugate of any one of claims 35-41, the linker having the structure (G3) shown below:

43. The lipid particle-erythrocyte conjugate of any one of claims 35-42, wherein the plurality of lipids comprising a sortase recognition motif are conjugated to a membrane protein (e.g., an extracellular domain of a membrane protein) of the erythrocyte via a branched linker.

44. The lipid particle-erythrocyte conjugate of any one of claims 29-43, wherein the erythrocyte is a reductant-treated erythrocyte, e.g., a thiol reductant-treated erythrocyte, such as obtained by a method comprising: (1) isolating and concentrating erythrocytes from human whole blood, optionally by leukodepletion; or obtaining erythrocytes from stem cells, e.g., pluripotent stem cells, e.g., induced pluripotent stem cells; (2) treating the erythrocytes with a thiol reductant to chemically modify the surface of the erythrocytes; (3) collecting and concentrating the modified erythrocytes.

45. The lipid particle-erythrocyte conjugate of claim 44, wherein the thiol reductant is TCEP.

46. The lipid particle-erythrocyte conjugate of any one of claims 29-45, having the structure: LNP-DSPE-PEG(2000)-LPETG-linker-RBC, preferably LNP-DSPE-PEG(2000)-LPETG-(G3)-RBC; wherein LNP-DSPE-PEG(2000)-LPETG refers to a lipid nanoparticle as defined in claim 34 comprising DSPE-PEG(2000)-LPETG, wherein the amount of DSPE-PEG(2000)-LPETG in the LNP is 0.1-10% (e.g., 0.1%-5% or 0.1%-1% or 0.5%-1%, e.g., about 0.75%), optionally the LNP can further comprise one or more additional lipids as defined in claim 5; the erythrocyte is an erythrocyte as defined in claim 44; the linker is a linker as defined in any one of claims 35-42; and wherein the DSPE-PEG(2000)-LPETG is conjugated to an extracellular domain of a membrane protein of the erythrocyte via the linker.

47. A method of making the lipid particle-erythrocyte conjugate of any one of claims 29-46, comprising mixing an erythrocyte linked to a linker and a lipid particle comprising a sortase recognition motif in the presence of a sortase under conditions suitable for a reaction to occur with the sortase to conjugate the lipid particle to the erythrocyte via the linker, e.g., comprising: (1) treating the erythrocyte with a reductant to allow a linker molecule as defined in any one of claims 35-42 to attach to an extracellular domain of an endogenous membrane protein of the erythrocyte; and collecting and concentrating the erythrocyte; and / or, (2) treating the lipid particle to allow the lipid particle to comprise a sortase recognition motif; and / or, (3) contacting the erythrocyte obtained in step 1) with the lipid particle obtained in step 2) in the presence of a sortase under conditions suitable for a reaction to occur with the sortase to conjugate the lipid particle to the endogenous membrane protein of the erythrocyte via the second linker (e.g., 0.1-1000 pg of the lipid particle per 1 mL of the erythrocyte). ​ ​ ​ ​ ​ ​ ​ 48. The method of claim 47, wherein the reducing agent is a thiol-based reducing agent, such as TCEP.

49. A pharmaceutical composition or preparation comprising the lipid particle- red blood cell conjugate of any one of claims 1-11, 18-25, and 29-46, or the lipid particle- red blood cell conjugate prepared by the method of any one of the preceding claims 12-17, 26-28, and 47-48, and a pharmaceutically acceptable excipient.

50. The pharmaceutical composition or preparation of any one of claim 49, wherein the preparation is a human blood preparation, such as an autologous or allogeneic human blood preparation.

51. The pharmaceutical composition or preparation of any one of claims 49 or 50, wherein the human blood preparation is a human blood leukoreduced blood preparation.

52. The pharmaceutical composition or preparation of any one of claims 49-51, wherein the human blood preparation comprises 10-1000 pg / mL of the lipid particle- red blood cell conjugate, such as at or above 50 pg / mL, 60 pg / mL, 70 pg / mL, 80 pg / mL, 90 pg / mL, 100 pg / mL, 150 pg / mL, 200 pg / mL, 250 pg / mL, 300 pg / mL, 350 pg / mL, 400 pg / mL, 450 pg / mL, or 500 pg / mL; or wherein the human blood preparation comprises the lipid particle- red blood cell conjugate having 10-10000 nucleic acid molecules per RBC, such as about 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or more nucleic acid molecules per RBC, most preferably 9-270 nucleic acid molecules per RBC; or 53. Use of the lipid particle- red blood cell conjugate of any one of claims 1-11, 18-25, and 29-46, or the lipid particle- red blood cell conjugate prepared by the method of any one of claims 12-17, 26-28, and 47-48, in the manufacture of a medicament or a human blood preparation for modifying CD11b+myeloid cells in the spleen, preferably for modulating the immune system of a host (e.g., enhancing or attenuating the immune response of cells) or for treating cancer or treating an autoimmune disease in a subject. wherein the human blood preparation comprises lipid particle-erythrocyte conjugates having greater than about 0.01 pg mRNA / 10 10 RBCs, for example having about 0.01-300 pg mRNA / 10 10 RBCs, 0.05-200 pg mRNA / 10 10 RBCs, 0.05-100 pg mRNA / 10 10 RBCs, 0.05-50 pg mRNA / 10 10 RBCs, or 0.08-10 pg mRNA / 10 10 RBCs, most preferably having 0.1-3 pg mRNA / 10 10 RBCs.

54. The use of claim 53, wherein the red blood cells in the lipid particle- red blood cell conjugate are from a subject to be treated or a healthy subject.

55. The use of claim 53 or 54, wherein the medicament or human blood preparation is administered in combination with one or more therapeutic modalities or other therapeutic agents.

56. The use of any one of claims 53-55, wherein the therapeutic modality is radiation therapy or surgical treatment, and / or the therapeutic agent is selected from a chemotherapeutic agent, another antibody, a cytotoxic agent, a vaccine, and the like. ​

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