Extracellular vesicles and their use for antibody delivery

Abstract

The present invention provides a composition for delivering an antibody or its antigen-binding portion to the target central nervous system. [Solution] Disclosed herein is a method for delivering polypeptides, such as antibodies or their antigen-binding moieties, to the central nervous system of a target by administering polypeptides (e.g., antibodies or their antigen-binding moieties) bound to the surface of extracellular vesicles (EVs) derived from nerve cells, such as neural progenitor cells or neural stem cells, across the blood-brain barrier. Conjugates containing nerve EVs bound to polypeptides such as antibodies or their antigen-binding moieties, and methods for using them are also provided. Disclosed herein is also a method for delivering polypeptides, such as antibodies or their antigen-binding moieties, by administering polypeptides (e.g., antibodies or their antigen-binding moieties) loaded into the lumen of extracellular vesicles (EVs) derived from nerve cells, across the blood-brain barrier.

Description

Related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 930,178, filed on 4 November 2019. The entire contents of the aforementioned priority application are incorporated herein by reference. [Technical Field]

[0002] This application includes an electronically submitted sequence list in ASCII format, which is incorporated herein by reference in its entirety. An ASCII copy thereof, created on October 29, 2020, has the filename A106525_1040WO_SL.txt and a size of 86,402 bytes. [Background technology]

[0003] Recent failures of antibody-based drugs, including solanezumab and aducanumab, for the treatment of Alzheimer's disease, have raised many questions about the future of therapeutic antibody therapy in the CNS, including the use of intravenous (IV) administration for effective bioavailability in the CNS. In mice, antibody concentrations are measurable in the brain at approximately 0.1% of the infused dose when administered IV, and are detectable up to 72 hours later, albeit at extremely low levels (Non-Patent Literature 1). Similarly, antibodies delivered IV have been reported to be at CSF levels of 0.1% of those found in serum in human clinical trials, indicating that IgG does not readily cross the human blood-brain barrier (BBB) ​​(Non-Patent Literature 2).

[0004] Bispecific antibodies have been generated in which one variable region binds to a BBB transcytosis protein, and the other variable region binds to a specific target of interest. Transferrin receptors, in particular, have been shown to be potent transporters across the BBB, but their use in human trials has been hindered by safety concerns (Non-Patent Literature 3). Other potentially bispecific targets, including CD98hc (Non-Patent Literature 4) and FC5 (Non-Patent Literature 5), which cross the BBB, have been investigated in recent years, but have yet to be clinically proven. Adeno-associated viruses (AAVs) have also shown promise for both their ability to cross the BBB (Non-Patent Literature 6) and their ability to deliver exogenous genes capable of encoding secretory antibodies in the brain (Non-Patent Literature 7). However, AAVs are prone to high clearance rates, particularly upon re-administration, and the potential decline of neurons or other CNS cells due to the excessive energy expended in producing these antibodies. Therefore, there is a need in the art for further methods of delivering therapeutic proteins, such as antibodies, to the central nervous system. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Banks et al., Peptides, 2002. 23(12): p. 2223-6 [Non-Patent Document 2] Rubenstein et al., Blood. 2003. 101(2): p. 466-8 [Non-Patent Document 3] Couch et al., Sci Transl Med, 2013. 5(183): p. 183ra57, 1-12 [Non-Patent Document 4] Zuchero et al., Neuron, 2016. 89(1): p. 70-82 [Non-Patent Document 5] Muruganandam et al., 2002. 16(2): p. 240-242 [Non-Patent Document 6] Deverman et al., Nat Biotechnol, 2016. 34(2): p. 204-9 [Non-Patent Document 7] Ryan et al., 2010. 18(8): p. 1471-1481 [Overview of the project] [Means for solving the problem]

[0006] To improve the delivery of therapeutic proteins, such as antibodies, to the target brain and central nervous system, the Specified provides a method for using nerve extracellular vesicles (EVs) as carriers for therapeutic proteins. In one embodiment, the Specified provides a method for delivering an antibody or its antigen-binding moiety to the target central nervous system, the method comprising administering a conjugate comprising the antibody or its antigen-binding moiety and an extracellular vesicle derived from a nerve cell to the target, wherein the antibody or its antigen-binding moiety is bound to the surface of the extracellular vesicle and the associated protein by a clicklinker.

[0007] In one embodiment, this specification provides a method for delivering a polypeptide to a target central nervous system, the method comprising the step of administering a conjugate to the target comprising the polypeptide and an extracellular vesicle derived from a nerve cell, wherein the polypeptide is conjugated to the surface of the extracellular vesicle and associated proteins by a clicklinker. In the exemplary embodiment, the polypeptide is an exogenous polypeptide. In other exemplary embodiments, the polypeptide is a therapeutic polypeptide.

[0008] In some embodiments, the conjugate is administered intravenously.

[0009] In some embodiments, the conjugate is administered intranasally.

[0010] In some embodiments, the conjugate is delivered to the brain of the subject. In some embodiments, the conjugate is delivered across the blood-brain barrier of the subject.

[0011] In some embodiments, the nerve cells are neural progenitor cells. In some such embodiments, the neural progenitor cells are derived from human pluripotent cells. In certain embodiments, the human pluripotent cells are human embryonic stem cells. In other embodiments, the human pluripotent cells are induced pluripotent stem cells.

[0012] In some embodiments, the antibody or antigen-binding portion thereof is IgG.

[0013] In some embodiments, the antibody or antigen-binding portion thereof is an antibody fragment selected from the group consisting of Fab, F(ab’)2, scFv, tandem scFv, diabody, minibody, and single domain antibody.

[0014] In some embodiments, the antibody or antigen-binding portion thereof is a humanized antibody or antigen-binding portion thereof.

[0015] In some embodiments, the antibody or antigen-binding portion thereof is a fully human antibody or antigen-binding portion thereof.

[0016] In some embodiments, the click linker is formed from the reaction between an azide click reactant and an alkyne click reactant.

[0017] In some embodiments, the click linker is formed from the reaction between an azide and dibenzocyclooctyne (DBCO).

[0018] In some embodiments, the click linker is formed from the reaction between a tetrazine and a trans-cyclooctene.

[0019] In some embodiments, the click linker is formed from the reaction between a tetrazine and a norbornene.

[0020] In some embodiments, the antibody is delivered to the target brain.

[0021] In some embodiments, the antibody is delivered to the target central nervous system.

[0022] In some embodiments, the EV further comprises exogenous nucleic acids and / or exogenous proteins. In certain embodiments, the EV comprises siRNA and / or antisense nucleic acids.

[0023] In some embodiments, the Specified herein provides compositions comprising an antibody-EV (Ab-EV) conjugate, the conjugate comprising an antibody or its antigen-binding moiety and an extracellular vesicle (EV) derived from a nerve cell, wherein the antibody or its antigen-binding moiety is bound to the surface of the EV by a clicklinker.

[0024] In some embodiments, the nerve cells are neural progenitor cells. In some such embodiments, the neural progenitor cells are derived from human pluripotent cells. In certain embodiments, the human pluripotent cells are human embryonic stem cells. In other embodiments, the human pluripotent cells are induced pluripotent stem cells.

[0025] In some embodiments, the clicklinker is formed from one or more of the following reactions: between azide and dibenzocyclooctin, between tetrazine and transcyclooctene, between tetrazine and norbornene, between azide and alkyne, between strain-enhancing azide and alkyne, between strain-enhancing azide and nitrone, between alkene and azide, between alkene and tetrazine, and / or between alkene and tetrazole.

[0026] In some embodiments, the antibody or its antigen-binding portion is IgG.

[0027] In some embodiments, the antibody or its antigen-binding portion is an antibody fragment selected from the group consisting of Fab, F(ab')2, scFv, tandem scFv, diabody, minibody, and single-domain antibodies.

[0028] In some embodiments, the antibody or its antigen-binding portion is a humanized antibody or its antigen-binding portion.

[0029] In some embodiments, the antibody or its antigen-binding moiety is one or more of solanezumab, aducanumab, nivolumab, bevacizumab, ocrelizumab, natalizumab, dinutuximab, gantenerumab, lecanemab, or ubrituximab.

[0030] In some embodiments, this specification provides a method for loading an antibody or its antigen-binding moiety into the lumen of an extracellular vesicle (EV), the method comprising: i) treating the EV with a saponin to make the membrane of the EV permeable; ii) sonicating the treated EV; and iii) loading an antibody or its antigen-binding moiety into the lumen of the EV by attaching it to the EV.

[0031] In some embodiments, EV is treated with approximately 0.05% to 0.3% saponin.

[0032] In some embodiments, the method for loading an antibody or its antigen-binding portion into the lumen of an extracellular vesicle (EV) includes the step of incubating the EV for a sufficient period of time to load at least 10% of the antibody into the EV at the time of loading the antibody or its antigen-binding portion.

[0033] In some embodiments, the Specified provides a method for delivering an antibody or its antigen-binding moiety to a target central nervous system (CNS), the method comprising administering an extracellular vesicle (EV) containing the antibody or its antigen-binding moiety in the lumen of the EV, the EV being derived from a nerve cell.

[0034] In some embodiments, the nerve cells are neural progenitor cells. In some such embodiments, the neural progenitor cells are derived from human pluripotent cells. In certain embodiments, the human pluripotent cells are human embryonic stem cells. In other embodiments, the human pluripotent cells are induced pluripotent stem cells.

[0035] In some embodiments, the antibody or its antigen-binding portion is IgG.

[0036] In some embodiments, the antibody or its antigen-binding portion is an antibody fragment selected from the group consisting of Fab, F(ab')2, scFv, tandem scFv, diabody, minibody, and single-domain antibodies.

[0037] In some embodiments, the antibody or its antigen-binding portion is a humanized antibody or its antigen-binding portion.

[0038] In some embodiments, the antibody or its antigen-binding portion is a fully human antibody or its antigen-binding portion.

[0039] In some embodiments, the antibody is delivered to the target brain.

[0040] In some embodiments, the antibody is delivered to the target spinal cord.

[0041] In some embodiments, the EV further comprises exogenous nucleic acids and / or exogenous proteins. In some embodiments, the EV comprises exogenous siRNA and / or antisense nucleic acids. In some embodiments, the EV further comprises small molecules. [Brief explanation of the drawing]

[0042] [Figure 1]This is a schematic diagram illustrating an exemplary method for binding an antibody (Ab) to an extracellular vesicle (EV) using a copper-free click chemistry reaction. An azide group was added to the unreactive antibody. Separately, the extracellular vesicles (EVs) were modified with a DIBO group by SDP coupling, which attacks the amine group. Next, the antibody was bound to the EVs during mixing using a copper-free click chemistry reaction. [Figure 2A-2B] Images of mouse brain sections obtained from mice administered with either unbound antibodies (Figure 2A) or antibodies bound to extracellular vesicles derived from neural progenitor cells (Figure 2B) are provided. Nuclei (blue) and antibody (white) signals are shown. [Figure 3A-B] This shows the loading efficiency of antibodies (Figure 3A) or luciferase proteins (Figure 3B) into the lumen of extracellular vesicles (AB126) derived from neural progenitor cells under various loading conditions. [Modes for carrying out the invention]

[0043] A.Definition To make the present invention easier to understand, certain terms are defined first. Furthermore, it should be noted that whenever a value or range of a parameter is stated, the intermediate values ​​and intermediate ranges between the stated values ​​are also part of the present invention.

[0044] The terms "about" or "approximately" usually mean within 5%, or more preferably within 1%, of a given value or range.

[0045] In this specification, the term “antibody” is used in its broadest sense and is not limited to these, but encompasses a variety of structures that bind to a target antigen, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), nanobodies, monobodies, antibody mimetics, and antibody fragments exhibiting desired antigen-binding activity.

[0046] In some embodiments, the antibody comprises an immunoglobulin molecule containing four polypeptide chains interconnected by disulfide bonds, namely two heavy (H) chains and two light (L) chains, and its polymer (e.g., IgM). Each heavy chain (HC) contains a heavy chain variable region (or domain) (hereinafter abbreviated as HCVR or VH) and a heavy chain constant region (or domain). The heavy chain constant region contains three domains, namely CH1, CH2, and CH3. Each light chain (LC) contains a light chain variable region (hereinafter abbreviated as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain (CL1). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which contain more conserved regions called framework regions (FRs). VH and VL each consist of three CDRs and four FRs, which are arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), a class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or a subclass.

[0047] As used herein, the term “CDR” or “complementarity-determining region” refers to non-adjacent antigen-binding sites found within the variable regions of both heavy-chain and light-chain polypeptides. These specific regions have been described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991), by Chothia et al., J. Mol. Biol. 196:901-917 (1987), and by MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where the definition includes overlaps or subsets of amino acid residues when compared to one another.

[0048] The term “Fc domain” is used to define the C-terminal region of an immunoglobulin heavy chain, which can be produced by papain digestion of an intact antibody. The Fc domain may be a native sequence Fc domain or a variant Fc domain. The Fc domain of an immunoglobulin generally contains two constant domains, namely a CH2 domain and a CH3 domain, and optionally a CH4 domain. Modifying antibody effector function by substituting amino acid residues in the Fc portion is well known in the art (Winter, et al., U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821). The Fc domain of an antibody mediates several important effector functions, such as cytokine induction, ADCC, phagocytosis, complement-dependent cell-mediated cytotoxicity (CDC), and half-life / clearance rate of antibodies and antibody-antigen complexes. In certain embodiments, the effector function of an Fc-domain-containing binding protein is modified by altering (e.g., deleting, inserting, or substituting) at least one amino acid residue within the Fc domain of that binding protein.

[0049] As used herein, “intact” or “full-length” antibody refers to an antibody comprising four polypeptide chains, namely two heavy (H) chains and two light (L) chains. In one embodiment, the intact antibody is an intact IgG antibody.

[0050] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies constituting that population are identical in sequence and bind to the same epitope, with the exception of possible variant antibodies, such as those containing naturally occurring mutations or variant antibodies that may arise during the production of a monoclonal antibody preparation (such variants are generally present in trace amounts). In contrast to polyclonal antibody preparations, which typically contain multiple different antibodies against multiple different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed to a single determinant at one antigen. Thus, the modifier “monoclonal” indicates a characteristic of the antibody that it is obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be produced by a variety of techniques, including, but are not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and these methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0051] As used herein, the term "human antibody" refers to an antibody having a variable region in which both the framework region and the CDR region are derived from a human germline immunoglobulin sequence. Furthermore, if the antibody contains a constant region, that constant region is also derived from a human germline immunoglobulin sequence. The human antibodies of the present invention may contain amino acid residues not encoded by a human germline immunoglobulin sequence (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as mouse, is grafted onto a human framework sequence.

[0052] The term "humanized antibody" is intended to refer to an antibody in which a germline-derived CDR sequence from a single non-human mammalian species, such as a mouse, is grafted onto a human framework sequence. Further modifications of the framework region may be performed within the human framework sequence and / or the non-human CDR sequence. For example, the "humanized form" of an antibody that is a non-human antibody refers to an antibody that has been humanized.

[0053] The term "chimeric antibody" is intended to refer to an antibody in which the variable region sequence originates from one species and the constant region sequence originates from another species, for example, an antibody in which the variable region sequence originates from a mouse antibody and the constant region sequence originates from a human antibody.

[0054] An antibody "antibody fragment," "antigen-binding fragment," or "antigen-binding portion" refers to a molecule other than the intact antibody that contains a part of the intact antibody and binds to the antibody to which the intact antibody binds. Examples of antibody fragments, though not limited to them, include Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0055] A "multispecific antigen-binding polypeptide" or "multispecific antibody" targets two or more antigens or epitopes. A "bispecific," "dual-specific," or "bifunctional" antigen-binding polypeptide or antibody is a hybrid molecule possessing two different antigen-binding sites. Bispecific antigen-binding polypeptides and antibodies are examples of, but are not limited to, multispecific antigen-binding polypeptides or antibodies, and can be produced in a variety of ways, including hybridoma fusion or fab' fragment or semi-antibody linkage. See, for example, Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79:315-321, Kostelny et al., 1992, J. Immunol. 148:1547-1553, and Brinkmann and Kontermann. 2017. MABS. 9(2):182-212. The two binding sites of a bispecific antigen-binding polypeptide or antibody will, for example, bind to two different epitopes, which may be on the same protein target or different protein targets.

[0056] The term "antibody mimetic" or "antibody imitation" refers to a molecule that is not structurally related to an antibody but can specifically bind to an antigen. Examples of antibody mimetic molecules, though not limited to them, include adonectin (i.e., fibronectin-binding molecules), afrin, afimer, afitin, alpha-body, afibody, DARPin, antikalin, avimer, finomer, Kunitz domain peptide, monobody, nanoCLAMP, nanobody, unibody, verbabody, aptamer, and peptide molecules. All of these mimic conventional antibody binding but utilize binding structures that are generated and function through different mechanisms. The term "autologous EV" is used to describe a population of EVs obtained from cells derived from a single subject or patient to whom EVs are administered.

[0057] As used herein, the term “central nervous system” or “CNS” refers to all structures within the dura mater. Such structures include, but are not limited to, the cells and tissues of the brain and spinal cord. The CNS also includes the cerebrospinal fluid that fills the ventricles and central canal of the spinal cord.

[0058] As used herein, the terms “effective dose” or “therapeutic effective dose” mean an amount of a drug, such as an amount of a composition containing exosomes (EVs), that is sufficient to reduce or improve the severity and / or duration of a disorder or one or more of its symptoms, prevent the progression of the disorder, cause regression of the disorder, prevent the recurrence, onset, sign, or progression of one or more symptoms associated with the disorder, or enhance or improve the preventive or therapeutic effect of another therapy.

[0059] The term "embryonic stem cell" preferably refers to pluripotent cells isolated from blastocyst-stage embryos of primates, including humans.

[0060] In this specification, the terms “extracellular vesicles” and “EVs” are used to refer to vesicles approximately 10 nm to 10 μm in size, surrounded by a lipid bilayer, such as a portion of the plasma membrane. EVs may contain fluids, macromolecules, solutes, and metabolites from cells. The term “EVs” also includes lipid vesicles engineered to contain bioactive molecules found in cell-derived EVs, such as neural EVs. These terms encompass both exosomes and ectosomes. EVs can be obtained from suitable biological sources using a combination of several isolation techniques, such as centrifugation, filtration, and ultracentrifugation. Exosomes are released during the exocytosis of multivesicular bodies (MVBs). Ectosomes are vesicles that aggregate at the plasma membrane and are released from the plasma membrane. In some examples, the size of EVs is approximately 20 nm to 10 μm, 20 nm to 1 μm, 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 100 nm, 30 nm to 160 nm, or 80 nm to 160 nm. In some embodiments, EVs are exosomes with a size of approximately 20 to 150 nm. EVs can be isolated from any suitable biological sample of mammalian origin, including, but not limited to, whole blood, serum, plasma, breast milk, cerebrospinal fluid, amniotic fluid, ascites, or bone marrow. In some embodiments, EVs can be isolated from cultured mammalian cells (e.g., immature dendritic cells (wild-type or immortalized), induced and uninduced pluripotent stem cells, fibroblasts, platelets, immune cells, reticulocytes, tumor cells, mesenchymal stem cells, satellite cells, hematopoietic stem cells, pancreatic stem cells, white and beige preadipocytes, etc.). In the exemplary embodiment, EVs can be isolated from nerve cells (e.g., neural progenitor cells, neural stem cells, glial cells, astrocytes, neurons, etc.).

[0061] The term "human pluripotent stem cells" (with "human embryonic stem cells (hESCs)" and "human induced pluripotent stem cells (hiPSCs)" being subsets thereof) is characterized by its ability, under appropriate conditions, to produce offspring of multiple different cell types, particularly neural stem cells and progenitor cells, neural crest cells, mesenchymal stem cells (MSCs), and associated proliferative and non-proliferative neurons, derived from preembryonic, embryonic, fetal tissue, or adult stem cells (in the case of human induced pluripotent stem cells) at any point after fertilization. The term encompasses both established lineages of various types of stem cells and pluripotent cells derived from primary tissues, as described herein.

[0062] As used herein, “pharmaceutically acceptable” refers to materials such as carriers, excipients, or diluents that do not neutralize the biological activity or properties of a compound, and are relatively low in toxicity. That is, the material can be administered to an individual without causing undesirable biological effects or harmful interactions with any component of the composition in which the material is contained. In some examples, a “pharmaceutically acceptable” carrier or excipient refers to a compound suitable for use in contact with human and animal tissues with a reasonable benefit / risk ratio, without excessive toxicity, irritation, allergic reactions, immunogenicity, or complications.

[0063] As used herein, “linker” means a divalent chemical moiety comprising a chain of atoms that covalently bonds an antibody to an extracellular matrix (EV) to form an antibody-EV conjugate. Known methods of conjugating either peptides or macromolecules can be used in the context of this disclosure. Generally, covalent bonding of an antibody to an EV requires the linker to have two reactive functional groups, i.e., to be divalent in terms of reactivity. Divalent linker reaction products useful for conjugating two or more functional or biologically active moieties are known, and such conjugation methods are described, for example, in Hermanson, GT (1996) Bioconjugate Techniques, Academic Press: New York, pp. 234-242. The disclosure of that document relates to linkers suitable for covalent bonding and is incorporated herein by reference. In some embodiments, the compositions and methods described herein utilize a “click linker,” which is generated from a reaction between two complementary click functional groups.

[0064] When the term "linker" is used to describe the binding site of a bond, it refers to a bond between the linker and the extracellular membrane binding site, and / or between the linker and the site-directed modified polypeptide, where one or both of the reactive ends are absent (converted to a chemical moiety) or incomplete (e.g., only the carbonyl part of a carboxylic acid remains). Accordingly, as used herein, linkers include, but are not limited to, linkers containing a chemical moiety formed by a coupling reaction between a reactive functional group in the linker and a nucleophilic group or other reactive substituent in the antibody, and linkers containing a chemical moiety formed by a coupling reaction between a reactive functional group in the linker and a nucleophilic group in the extracellular matrix (EV).

[0065] Examples of chemical moieties formed by these coupling reactions include reactions between chemically reactive functional groups, such as nucleophile / electrophile pairs (e.g., thiol / haloalkyl pairs, amine / carbonyl pairs, or thiol / α,β-unsaturated carbonyl pairs), and diene / dienophile pairs (e.g., particularly azide / alkyne pairs, or diene / α,β-unsaturated carbonyl pairs). Examples of coupling reactions between reactive functional groups to form chemical moieties include, but are not limited to, alkylation of thiols, alkylation of hydroxyls, alkylation of amines, condensation of amines or hydroxylamines, hydrazine formation, amidation, esterification, disulfide formation, cycloaddition (e.g., particularly [4+2]Diels-Alder cycloaddition, [3+2]Hüsgen cycloaddition), aromatic substitution of nucleophiles, aromatic substitution of electrophiles, and other reaction modes known in the art or described herein. A suitable linker may contain electrophilic functional groups for reaction with nucleophilic functional groups of antibodies, extracellular viable (EVs), or both.

[0066] The term “neuron” refers to a cell associated with one or more nerves, which are fibrous bundles composed of neurons. Neurons typically originate from neural progenitor cells (NPCs). In some embodiments, nerves can be obtained in vitro from neural progenitor cells or pluripotent stem cells. In the exemplary embodiments, the nerves referred to herein are human nerve cells. In some embodiments, nerves can be neurons, glial cells, astrocytes, oligodendrocytes, microglia, Schwann cells, or glial cells. In other embodiments, nerves can be neural progenitor cells or neural stem cells. The terms “neuron" and “neural stem cells” refer to pluripotent cells that have the ability to differentiate into a limited repertoire of multiple nerve and glial cell types. In some embodiments, neural progenitor cells can be obtained in vitro from pluripotent stem cells, for example, induced pluripotent stem cells (iPS cells) or embryonic stem cells (ES cells). In the exemplary embodiments, the neural progenitor cells referred to herein are human neural progenitor cells. In some embodiments, the neural progenitor cells may be untransformed. In some embodiments, the neural progenitor cells are proliferative. In some embodiments, the neural progenitor cells maintain their phenotype without differentiation.

[0067] The term "neuronal EV" is used to refer to cell-derived EVs, such as those derived from nerve cells, e.g., neural progenitor cells. The term also refers to vesicles that have been manipulated to have substantially the same biological activity as cell-derived EVs by containing a sufficient number of bioactive molecules.

[0068] As used herein, “neurovascular repair” refers to the recovery that occurs after neurovascular injury to the brain. Neurovascular injury refers to damage to the major blood vessels that supply the brain, brainstem, and upper spinal cord, including the vertebral cortex, basilar artery, and carotid artery. These vessels are located both extracranially and intracranially, and the injury may occur in one or both of these locations. In one example, neurovascular repair occurs when there is increased neurogenesis and angiogenesis, and / or mobilization of glial cells to remove bleeding and create an environment for healing. Astrocytes, pericytes, matrix proteases, and vascular smooth muscle cells also play important roles in neurovascular repair.

[0069] As used herein, the term “sample” refers to a specimen taken from an object (e.g., cells (e.g., nerve cells), tissue, blood, blood components (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, pancreatic juice, chorionic villi samples, and bone marrow).

[0070] As used herein, the term “subject” refers to any organism that is the target of administration or treatment. “Subject” can be an organism, such as a mammal (e.g., human, non-human mammal, non-human primate, primate, laboratory animal, mouse, rat, hamster, cat, or dog). In one embodiment, the subject is a human subject. The term “patient” refers to a human subject under the care of a clinician, such as an internist. The subject may be male or female.

[0071] The terms “to treat” and “treatment” refer to the medical management of a subject with the aim of reducing, improving, stabilizing (i.e., preventing worsening), preventing, or curing a disease, pathological condition, or disorder. This term includes active treatment (treatment aimed at reducing the disease, pathological condition, or disorder), causal treatment (treatment directed at the cause of the associated disease, pathological condition, or disorder), palliative treatment (treatment designed to relieve symptoms), preventive treatment (treatment aimed at minimizing, or partially or completely inhibiting, the onset of the associated disease, pathological condition, or disorder), and supportive treatment (treatment used to complement another therapy). Treatment includes, whether detectable or undetectable, a reduction in the degree of the disease or condition, prevention of the progression of the disease or condition, a delay or slowing of the progression of the disease or condition, improvement or relief of the disease or condition, and remission (partial or complete). To “improve” or “alleviate” a disease or condition means that the severity and / or undesirable clinical symptoms of the disease, disability, or condition are reduced, and / or the progression is slowed or prolonged, compared to the degree or time course of the condition without treatment. Treatment does not require complete improvement of the symptoms or disease, but includes embodiments that reduce the symptoms and / or underlying risk factors. “Treatment” can also mean an extension of survival compared to the survival expected without treatment. Those who require treatment include those who already have the condition or disability, those who are prone to developing the condition or disability, or those who should be prevented from developing the condition or disability. The term “prevent” does not require 100% elimination of the possibility of the event. Rather, this term indicates that the likelihood of the event occurring is reduced in the presence of the compound or method.

[0072] The present invention may utilize conventional cell culture methods, chemical synthesis methods, and other biological and pharmaceutical techniques that are within the scope of the skills in the art. Such techniques are either publicly known or, if not, are well described in the literature. Standard techniques for cell proliferation, standard techniques for cell isolation, and, where applicable, standard techniques for cloning, DNA isolation, amplification, and purification for enzymatic reactions involving DNA ligases, DNA polymerases, restriction endonucleases, etc., as well as various isolation techniques, are known and commonly used by those skilled in the art.A number of standard techniques are described in Sambrook et al., 1989 Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, New York, Maniatis et al., 1982 Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, New York, Wu (Ed.) 1993 Meth. Enzymol. 218, Part I, Wu (Ed.) 1979 Meth. Enzymol. 68, Wu et al., (Eds.) 1983 Meth. Enzymol. 100 and 101, Grossman and Moldave (Eds.) 1980 Meth. Enzymol. 65, Miller (Ed.) 1972 Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, Old and Primrose, 1981 Principles of Gene Manipulation, University of California Press, Berkeley, Schleif and Wensink, 1982 Practical Methods in Molecular Biology, Glover (Ed.) 1985 DNA Cloning Vol. I and II, IRL Press, Oxford, UK, Hames and Higgins (Eds.) 1985 Nucleic Acid Hybridization, IRL Press, Oxford, UK; and Setlow and Hollaender 1979 Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum Press, New York.Where abbreviations and nomenclature are used, they are considered standard in the art and are commonly used in professional journals, such as those cited herein.

[0073] Where a range of values ​​is provided, unless it is not evident from the context that it is not (for example, in the case of a group containing many carbon atoms, in which case the number of carbon atoms in each range is provided), it is understood that each intermediate value between the upper and lower limits of that range, up to 1 / 10 of the unit of the lower limit, and any other mentioned value or intermediate value within the range mentioned are included in the invention. The upper and lower limits of these smaller ranges, which may be independently included within these smaller ranges, are also included in the invention, subject to any particularly excluded limits within the range mentioned. If the range mentioned includes one or both of these limit values, the range excluding one or both of these included limit values ​​is also included in the invention.

[0074] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the present invention, but preferred methods and materials are described herein.

[0075] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context makes it clear otherwise.

[0076] B. Delivery of extracellular vesicles (EVs) and therapeutic proteins The present invention relates to a method for delivering a therapeutic polypeptide, such as an antibody, to the central nervous system of a target, for example, by administering a polypeptide conjugated to the surface of nerve extracellular vesicles (EVs), such as nerve cell-derived exosomes and / or microvesicles, for example, across the blood-brain barrier. Further provided are methods for using polypeptide-EV conjugates, such as antibody (Ab)-EV conjugates, in various applications related to the treatment of neurological disorders and injuries, including, but not limited to, Alzheimer's disease, Huntington's disease, Parkinson's disease, dementia, and cancers of the nervous system, such as glioblastoma or cancer that has metastasized to the central nervous system. Furthermore, conjugates comprising nerve EVs coupled to an antibody or its antigen-binding fragment, and methods for using the same are provided herein.

[0077] The present invention also relates to a method for delivering therapeutic polypeptides, such as antibodies, to a target central nervous system, for example, across the blood-brain barrier, by administering the polypeptides loaded into the lumen of nerve extracellular vesicles (EVs), such as nerve cell-derived exosomes and / or microvesicles. Further provided are, but are not limited to, methods for using lumen-loaded EVs in various applications related to the treatment of neurological disorders and injuries, including Alzheimer's disease, Huntington's disease, Parkinson's disease, dementia, and cancers of the nervous system, such as glioblastoma or cancer that has metastasized to the central nervous system.

[0078] Extracellular vesicles (EVs) are a hybrid group of small structures surrounded by a lipid bilayer, such as a portion of the cellular plasma membrane. EVs can be approximately 10 nm to 10 μm in diameter, most commonly 25 to 500 nm. EVs can be broadly divided into two types: exosomes and ectosomes. Exosomes can be formed by cells through inward budding of the endosomal membrane during the maturation of the multivesicle body (MVB). The exosome can then be released into the extracellular space through fusion of the MVB with the cell surface. Exosomes are typically 25 to 500 nm in diameter, and in some embodiments may be approximately 25 to 250 nm, 50 to 150 nm, or 50 to 200 nm. Ectosomes, also known as microvesicles, can be formed by cells through budding of the plasma membrane. Ectosome size can vary from approximately 10 nm to 10 μm, and in some embodiments it may be approximately 10 to 1000 nm, or approximately 50 to 500 nm.

[0079] EVs suitable for use in the conjugates and methods of the present invention may be derived from any suitable source. For example, EVs may be derived from nerve cells, such as neural stem cells, neural progenitor cells, or differentiated nerve cells such as neurons, glial cells, or astrocytes. EVs suitable for use in the methods described herein may also be produced by synthesis. In exemplary embodiments, the EVs used in the antibody conjugates described herein are derived from neural progenitor cells or neural stem cells.

[0080] EVs are involved in intercellular communication, enabling the movement of substances from EVs to cells through fusion with the cell membrane, or, but not limited to, through uptake into cells via caveolin-independent and caveolin-dependent mechanisms, clathrin-independent and clathrin-dependent mechanisms, macrophages, and / or phagocytosis. EVs have been reported to be involved in numerous physiological processes, including immunomodulation, angiogenesis, endothelial cell migration associated with tumor growth, or damage reduction in ischemia-reperfusion injury. Many of these functions are mediated by proteins, nucleic acids, or lipids contained within or on the surface of vesicles.

[0081] EVs can have a cargo in their lumen, which can be embedded in or attached to a lipid bilayer. This cargo may include proteins, lipids, and / or nucleic acids, such as mRNA or miRNA. When EVs are produced by cells, the composition of the cargo is highly cell-type dependent. For example, EVs derived from astrocytes, neural progenitor cells, and mesenchymal stem cells have been shown to contain different complements of protein cargo (see, for example, U.S. Patent Application Publication 2018 / 0327714, the entirety of which is incorporated herein by reference). EVs from these different cell types also contain nucleic acid molecules of different properties, including mRNA and / or miRNA. In embodiments where EVs are obtained from cells, the EV contains endogenous cargo that reflects the contents of the EV produced by the cell from which this EV originates. In some embodiments, EVs obtained from cells may also contain exogenous cargo. Exogenous cargo comprises proteins, nucleic acids, small molecules, or lipids and is introduced into EVs by manipulating the vesicles following their release into the extracellular space. In other embodiments, EVs obtained from cells may contain exogenous cargo, which is packaged into the EV by the presence of recombinant nucleic acid within the cell from which the EV originates. For example, an EV containing a recombinant protein can be obtained from a cell containing the recombinant nucleic acid encoding this protein. In embodiments in which EVs are produced by synthesis, a suitable cargo for inclusion within the vesicle can be selected. For example, in one embodiment, a synthetic EV may contain one or more proteins, lipids, or nucleic acids present within an EV derived from a nerve cell, such as a neural progenitor cell, neuron, glial cell, or astrocyte.

[0082] Cell-derived extracellular molecules (EVs), such as exosomes, contain a diverse range of biological molecules reflecting their cellular origin. In one embodiment, the present invention provides a population of EVs derived from neurons, such as neural progenitor cells. Such neuronal EVs contain a diverse protein environment derived from neurons, including cytokines and growth factors, as well as coding and non-coding RNA molecules. The cargo contained in neuronal EVs can influence neuronal and vascular function by providing neuroprotection; reducing inflammation; immunomodulation by acting on T cells, macrophages, and microglia; reducing oxidative stress; improving vascular integrity; influencing metabolic activity; and inducing neurogenesis, cell migration, remyelination, and differentiation to induce neurogenesis. While not theoretically bound, the combination of native proteins expressed on the surface of EVs derived from neurons (e.g., neural progenitor cells, neural stem cells) is thought to enable a greater degree of vesicle-mediated localization of the CNS and crossing of the blood-brain barrier compared to EVs from other sources. The ability to target the CNS and / or cross the blood-brain barrier is not hindered by surface labeling EVs with antibodies, as shown herein. Furthermore, EV-antibody conjugates containing EVs derived from neuronal cells (e.g., neural progenitor cells, neural stem cells) can transport functionally intact antibodies and their antigen-binding fragments across the blood-brain barrier for delivery to targets within the brain and central nervous system.

[0083] In some embodiments, nerve cell-derived extracellular proteins (EVs), such as exosomes, may contain membrane proteins, including, but not limited to, CD63, CD81, and CD133. Accordingly, in one embodiment, an EV suitable for use in the conjugates and methods described herein, such as an exosome, contains one or more of the cell surface proteins CD63, CD81, and CD133 (i.e., one or more, two or more, or all three). Furthermore, EVs, such as exosomes, may contain one or more of the following miRNAs (i.e., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or all twelve) of the following: hsa-miR-135a-2, hsa-miR-124-1, hsa-miR-124-2, hsa-miR-124-3, hsa-miR-489, hsa-miR-9-3, hsa-miR-9-2, hsa-miR-9-1, hsa-miR-219b, hsa-miR-219a-2, hsa-miR-363, and / or hsa-miR-20b. In one embodiment, a population of isolated EVs is enriched with respect to one or more of the aforementioned miRNAs. Concentration can be measured either as an absolute amount or as a relative amount, such as when compared to unmodified exosomes derived from non-neuronal cells, for example, mesenchymal stem cells.

[0084] (i) Production of EVs EVs suitable for use in the conjugates and methods disclosed herein can be produced by various cell types or by synthesis. In one embodiment, EVs may be derived from nerve cells such as neural progenitor cells, neurons, astrocytes, oligodendrocytes, microglia, Schwann cells, or glial cells. In some embodiments, EVs may be produced by transformed cell lines. In other embodiments, EVs may be produced by untransformed cell lines. In some embodiments, EVs may be produced by engineered cell lines expressing exogenous polypeptides and / or nucleic acids, such as recombinant cell lines. In other embodiments, polypeptide-EV conjugates may be produced using EVs from other sources, for example, EVs generated from cell types including, but not limited to, platelets, reticulocytes, immune cells, intestinal epithelial cells, tumor cells, HELA cells, mesenchymal stem cells, human embryonic kidney cells (HEK cells), and primary cells of any kind. In other embodiments, EVs may be isolated from bodily fluids such as milk or colostrum.

[0085] In one embodiment, the EV may contain exosomes or consist essentially of exosomes. The exosomes may originate from any of the cell types described above. For example, nerve exosomes may be suitable for use in the conjugates and methods disclosed herein. Neural exosomes may originate from nerve cells, including, but are not limited to, neural progenitor cells, neurons, astrocytes, oligodendrocytes, microglia, Schwann cells, or glial cells. In another embodiment, the exosomes are produced by synthesis.

[0086] In another embodiment, the EV may include ectosomes, also known as microvesicles, or may consist essentially of ectosomes. The microvesicles may be derived from any of the cell types described above. For example, a microvesicle suitable for use in the conjugates and methods disclosed herein may be a neuron microvesicle. Neuron microvesicles may be derived from nerve cells, including, but are not limited to, neural progenitor cells, neurons, astrocytes, oligodendrocytes, microglia, Schwann cells, or glial cells. In another embodiment, the microvesicles are produced by synthesis.

[0087] In some examples, the extracellular viable cells (EVs) of this disclosure can be obtained by culturing cells, such as nerve cells, for a sufficient amount of time for the cells to produce EVs. In some embodiments, the cells used to produce EVs can be obtained from pluripotent stem cells, for example, human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs).

[0088] In some embodiments, extracellular embryos (EVs) are isolated from nerve cells differentiated from pluripotent stem cells, such as neural progenitor cells. Pluripotent stem cells may express one or more of stage-specific embryonic antigens (SSEAs) 3 and 4, and markers detectable using antibodies against Tra-1-60 and Tra-1-81 (Thomson et al., Science 282:1145, 1998). In vitro differentiation of pluripotent stem cells results in loss of expression of SSEA-4, Tra-1-60, and Tra-1-81 (if present), and increased expression of SSEA-1. Undifferentiated pluripotent stem cells typically possess alkaline phosphatase activity, which can be detected by fixing the cells with 4% paraformaldehyde and then developing them using Vector Red as a substrate, as described by the manufacturer (Vector Laboratories, Burlingame, California). Undifferentiated pluripotent stem cells also typically express Oct-4 and TERT, which can be detected by antibody or RT-PCR. In one embodiment, EVs for use in the conjugate described herein can be produced by neural progenitor cells derived from human ES cells. In another embodiment, EVs for use in the conjugate described herein can be produced by neural progenitor cells derived from human iPS cells.

[0089] The types of pluripotent stem cells that may be used include established lines of pluripotent stem cells derived from tissues formed after fertilization, such as proembryonic tissue (e.g., blastocyst), embryonic tissue, or fetal tissue, typically collected at any point during pregnancy, but not necessarily around 10-12 weeks of gestation. Examples of non-exclusive examples include established ethical lines of human embryonic stem cells or human embryonic germ cells, such as human embryonic stem cell lines WA01, WA07, and WA09 (WiCell). The compositions of this disclosure may also be used during the initial establishment or stabilization phase of such cells, in which case the source cells are primary pluripotent cells directly collected from the source tissue. Cells collected from a pluripotent stem cell population already cultured in the absence of feeder cells are also preferred. Furthermore, mutant human embryonic stem cell lines such as BG01v (ViaCyte, San Diego, California), as well as normal human embryonic stem cell lines such as WA01, WA07, WA09 (WiCell, Madison, Wisconsin), and BG01, BG02 (ViaCyte, San Diego, California), are also suitable.

[0090] Human embryonic stem cells (hESCs) can be prepared by methods described in the art, such as those described by Thomson et al. (U.S. Patent No. 5,843,780; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133 ff., 1998; Proc. Natl. Acad. Sci. USA 92:7844, 1995), or they may be commercially available.

[0091] iPSCs are created by dedifferentiating adult somatic cells and returning them to a pluripotent state. iPSCs can also be generated by any suitable method, including, but not limited to, the artificial expression of four genes (c-myc, Klf4, SOX2, OCT4), or similar methods.

[0092] A method for producing human neural progenitor (hNP) cells from human embryonic stem cells (ESCs) is described, for example, in U.S. Patent No. 7,531,354, which is incorporated herein by reference in its entirety. Human neural progenitor cells (hNPs) are known to express markers associated with the earliest pluripotent neural stem cells, including nestin, Musashi-1, SOX1, SOX2, and SOX3. In one example, hNPs express SOX1 (SOX1+). In another example, hNPs express SOX2 (SOX2+). In several other examples, hNPs express SOX3 (SOX3+). In some specific examples, hNPs express at least one of nestin, Musashi-1, SOX1, SOX2, and SOX3. In other examples, hNPs express two or more of nestin, Musashi-1, SOX1, SOX2, and SOX3. In yet another example, the hNP expresses three or more of nestin, Musashi-1, SOX1, SOX2, and SOX3. In some examples, the hNP expresses at least one of nestin, Musashi-1, SOX1, SOX2, and SOX3, but not OCT4. In some other examples, the hNP expresses at least two of nestin, Musashi-1, SOX1, SOX2, and SOX3, but not OCT4. In yet another example, the hNP expresses at least three of nestin, Musashi-1, SOX1, SOX2, and SOX3, but not OCT4. In one specific example, the hNP expresses SOX1, SOX2, and SOX3, but not OCT4. Neural progenitor cells can be cultured with or without feeder cells. In some cases, neural progenitor cells produced according to the method presented in U.S. Patent No. 7,531,354 do not contain feeder cells or embryoid bodies.

[0093] In some cases, the extracellular viable cells (EVs) of this disclosure can be obtained by culturing differentiated nerve cells, such as glial cells, directly or indirectly derived from pluripotent stem cells, in cell culture medium under conditions for EV production for a sufficient time to produce EVs, and then isolating the EVs from the culture medium. Examples of glial cell types include oligodendrocytes, astrocytes, ependymal cells, Schwann cells, microglia, and satellite cells. In one example, the differentiated nerve cells (e.g., glial cells) include astrocytes. Examples of differentiated nerve cells that can be used include hN2® nerve cells (ArunA Biomedical Inc.), NeuroNet® neurons, and AstroPro® astrocytes (ArunA Biomedical Inc.).

[0094] Extracellular vesicles (EVs) can be isolated from cell culture media or tissue culture supernatant. EVs produced from cells can be recovered from the culture medium by any suitable method. Typically, a population of isolated EVs can be prepared from cell cultures or tissue supernatant by centrifugation, size exclusion columns, microfluidic devices, polymer precipitation, filtration, or a combination of these methods. For example, EVs can be prepared as described in U.S. Patent Application Publication No. 20140356382, which is incorporated herein by reference in its entirety. For example, EVs can be prepared by fractional centrifugation, i.e., slow (<2,000 g) centrifugation to pelletize relatively large particles, followed by high (>100,000 g) centrifugation to pelletize the EVs, size filtration using a suitable filter (e.g., a 0.22 μm filter), gradient ultracentrifugation (e.g., using a sucrose gradient), or a combination of these methods.

[0095] In some embodiments, the EV-producing NP cells and / or nerve cells disclosed herein are cultured for approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or for as long as 1, 2, 3, 4, 5, 6, 7, or 8 weeks, or for as long as 1, 2, 3, 4, 5, or 6 months, depending on the cells and their EV-producing capacity. EV-producing cells can be cultured and grown in suitable media under conditions readily determined by those skilled in the art. Cell culture conditions may vary depending on the cell type. The examples presented hereafter illustrate suitable media and conditions. For example, CMRL 1066 medium (Invitrogen) containing exosome-deficient fetal bovine serum (e.g., 10%) and optionally supplemented with glutamine or a glutamine-containing mixture and antibiotics may be used. In some embodiments, cells can be grown attached to a surface; for example, cells can be grown as a monolayer or multilayer on a surface (without feeder cells) and can be grown until 30, 40, 50, 60, 70, 80, 90, 95, or 100% confluent. In other embodiments, cells can be grown as cell aggregates or on microbeads in suspension culture.

[0096] Cell growth media are known in the art and include, in addition to minimally essential media, one or more optional components such as growth factors, ascorbic acid, glucose, non-essential amino acids, salts (including trace elements), glutamine, insulin (if indicated and not excluded), activin A, transferrin, β-mercaptoethanol, and other chemicals known in the art and described elsewhere in this specification. Preferred media are low-protein, serum-free growth media that support nerve cells. The growth factor used may be fibroblast growth factor 2 (FGF2), which may be alone or preferably in combination with leukemia suppressor (LIF). Depending on the NPs or nerve cells to be grown in the growth medium, the inclusion of LIF is preferred but may not be necessary. Further media include basal cell media, which may contain serum, for example, about 0.1% to 20% (preferably about 2% to 10%) of fetal bovine serum, or, in the case of synthetic media, which do not contain fetal bovine serum and KSR, but optionally contain bovine serum albumin (about 1% to 5%, preferably about 2%). In some examples, the media are synthetic, serum-free, and have a low protein content. In other examples, the media are ArunA media and supplements, which allow nerve cultures to maintain a stable karyotype over multiple passages without the need for feeder cells, making them an excellent choice for a variety of research applications, including early-stage drug discovery. The components of the growth media depend on the type of nerve cells being grown, and all of them are known in the art. In one example, the AB2® Neural Cell Culture Media Kit is used, which contains AB2® Basal Neural Medium and ANS® Neural Medium Supplement. In a specific example, the media and supplements described above are specially modified for versatility to meet the requirements of nerve cell culture. AB2® Basal Neural Medium and ANS® Neural Medium Supplement can be used as a base for special media to direct the differentiation of hNP1® strains towards various neural phenotypes.Each lot of culture media and supplements is pre-tested for use by undergoing tests for cell proliferation, sterility, pH, osmotic pressure, and endotoxin levels.

[0097] Other chemical substances that may be optionally added to the culture medium include, depending on the cell type being grown in the medium, a number of components, particularly nicotinamide; members of the TGF-β family including TGF-β1, 2, and 3; activin A; Nodal; bone morphogenetic proteins (BMP2-7); serum albumin; members of the fibroblast growth factor (FGF) family; platelet-derived growth factors-AA and -BB; platelet-rich plasma; insulin growth factor (IGF-I, II, LR-IGF); growth and differentiation factors (GDF-5, -6, -8, -10, 11); glucagon-like peptides-I and II (GLP-I and II); GLP-1 and GLP-2 mimetodes; exendin-4; parathyroid hormone; insulin; progesterone; aprotinin; hydrocortisone; ethanolamine; epidermal growth factor (EGF); gastrin I and II; Examples include copper chelating agents such as triethylenepentamine; forskolin; sodium butyrate; beta-cerulin; ITS; Noggin; neurite growth factor; Nodal; valproic acid; trichostatin A; sodium butyrate; hepatocyte growth factor (HGF); sphingosine-1; VEGF; MG132 (EMD, California); N2 and B27 supplements (Gibco, California); steroid alkaloids such as cyclopamine (EMD, CA); keratinocyte growth factor (KGF); Dickkopf protein family; bovine pituitary extract; islet neogenesis-associated protein (INGAP); Indian hedgehog; sonic hedgehog; proteasome inhibitors; Notch pathway inhibitors; sonic hedgehog inhibitors; heregulin; or one or more of these combinations. If any of these ingredients are included, each ingredient is included in an effective amount.

[0098] In some cases, suitable media may be prepared from components such as Dulbecco's Modified Eagle Medium (DMEM), Gibco #11965-092; Knockout Dulbecco's Modified Eagle Medium (KO DMEM), Gibco #10829-018; Ham F12 / 50% DMEM basal medium; 200 mM L-glutamine, Gibco #15039-027; Non-essential amino acid solution, Gibco 11140-050; β-mercaptoethanol, Sigma #M7522; Human recombinant basic fibroblast growth factor (bFGF), Gibco #13256-029. Other suitable reactants include Neurobasal (Gibco), BrainPhys (Stem Cell Technologies), and / or NeuroDiff (Stem Cell Technologies).

[0099] Cell media are commercially available and can be supplemented with commercially available components, including those that do not contain synthetic heterologous components, such as those available from Invitrogen Corp. (GIBCO), Cell Applications, Inc., Biological Industries, Beth HaEmek (Israel), and Calbiochem. Those skilled in the art can easily modify the cell media to produce one or more of the target cells according to the present invention.

[0100] The EV-producing cells of this disclosure may be cultured on a layer of feeder cells that support the cells in various ways. Methods for culturing cells on a layer of feeder cells are known in the art. The cells can be grown on a cell support or matrix as an adhesive monolayer or as cell aggregates in suspension. In some examples, the use of a cell support may be preferred depending on the cells used to produce EVs. When a cell support is used, it preferably contains at least one substrate protein. Examples of substrate proteins include extracellular matrix proteins, such as laminin, tenascin, thrombospondin, and mixtures thereof, which exhibit proliferation-promoting activity and contain domains homologous to epidermal growth factor (EGF). Other substrate proteins that may be used include, for example, collagen, fibronectin, vitronectin, polylysine, polyornithine, and mixtures thereof. Furthermore, other materials such as methylcellulose of gels and other gels containing one or more of these embryonic stem cell differentiation proteins in effective concentrations may also be used. Examples of differentiated proteins or materials containing these differentiated proteins include, for example, recombinant laminin, BD Cell-Tak™ cell and tissue adhesive, BD™ FIBROGEN human recombinant collagen I, BD™ FIBROGEN human recombinant collagen III, BD Matrigel™ basement membrane matrix, BD Matrigel™ high-concentration (HC) basement membrane matrix, BD™ PuraMatrix™ peptide hydrogel, collagen I, high-concentration (HC) collagen I, collagen II (bovine), collagen III, collagen IV, collagen V, and collagen VI.

[0101] Alternatively, these cells can be cultured in a culture system that does not contain feeder cells, or in a culture system that does not contain feeder cells but still supports cell proliferation for EV production. Cell proliferation in feeder-free culture can be supported by using a culture medium that has been prepared by pre-culturing with another cell type. Alternatively, the proliferation of EV-producing cells in feeder-free culture without differentiation can be supported by using a chemically synthesized culture medium. These methods are known in the art. In certain embodiments of the present invention, cells are grown in a culture medium that does not contain feeder cells.

[0102] EVs can be harvested at various time intervals (for example, approximately 1, 2, 4, 6, 8 days, or 3, 6, 9, 12 days, or longer intervals, depending on the rate of EV production). Exemplary EV yields, as described elsewhere in this specification, during a period of approximately 24 hours to 7 days of culture of proliferating and non-proliferating nerve cells, are at least approximately 1 ng of EV per 1 million cells, at least approximately 10 ng of EV per 1 million cells, at least approximately 50 ng of EV per 1 million cells, at least approximately 100 ng of EV per 1 million cells, at least approximately 500 ng of EV per 1 million cells, at least approximately 750 ng of EV per 1 million cells, and less than approximately 1 million ng of EV per 1 million cells. The EV could be approximately 800 ng, at least approximately 900 ng per 1 million cells, at least approximately 1.0 μg per 1 million cells, at least approximately 1.5 μg per 1 million cells, at least approximately 2.0 μg per 1 million cells, at least approximately 2.5 μg per 1 million cells, at least, for example, approximately 3.0 μg per 1 million cells, at least approximately 5.0 μg per 1 million cells, and at least approximately 10.0 μg per 1 million cells.

[0103] In many cases, extracellular viable cells (EVs) are collected and recovered by ultracentrifugation, fractional centrifugation, or a combination thereof, and the pelletized EVs are recovered and optionally washed with a suitable medium. For example, EV preparations can be prepared from cell cultures or tissue supernatants by centrifugation, filtration, or a combination thereof. In some embodiments, EVs can be prepared by fractional centrifugation, i.e., centrifugation at a low speed (<20,000 g) to pelletize relatively large particles, followed by centrifugation at a high speed (>100,000 g) to pelletize the EVs, size filtration using a suitable filter (e.g., a 0.22 μm filter), gradient ultracentrifugation (e.g., using a sucrose gradient), or a combination thereof. EVs can be purified by fractional centrifugation, micro and ultrafiltration, polymer precipitation, microfluidic separation, immunocapture, and size exclusion chromatography. The above-described methods and / or related methods for the isolation and purification of EVs are described in Thery, et al., Current Protocols in Cell Biology, (2006) 3.221-3.22.29, copyright 2006 by John Wiley & Sons, Inc., Sokolova, et al., Colloids and Surfaces B: Biointerfaces, 2011, 87, 146-150, Wiklander, et al., Journal of Extracellular Vesicles, 2015, 4, 26316, pp. 1-13, and Boing, et al., Journal of Extracellular Vesicles, 2014, 3, 23430, pp. 1-11. Other isolation methods, such as electric field radio frequency and acoustics, may also be developed.

[0104] Methods for producing synthetic vesicles, such as synthetic exosomes, are known in the art. Such methods can be used to produce synthetic vesicles suitable for use in the compositions and methods provided herein. Furthermore, EVs from which the contents, i.e., the lipid bilayer, have been removed or eliminated may also be used to produce artificial EVs.

[0105] (ii) Polypeptide-EV conjugate In one embodiment, the present invention provides a composition comprising a polypeptide-EV conjugate containing an extracellular vesicle derived from nerve cells (e.g., neural progenitor cells or neural stem cells), wherein the polypeptide is conjugated to the surface of the vesicle using click chemistry. In some embodiments, the polypeptide is an antibody or its antigen-binding moiety (referred herein to as an “antibody-EV (Ab-EV) conjugate”). The polypeptide-EV conjugate (e.g., Ab-EV conjugate) may contain an extracellular vesicle derived from nerve cells (e.g., neural progenitor cells or neural stem cells), the extracellular vesicle being conjugated to a polypeptide (e.g., an antibody or its antigen-binding moiety) on its surface. The polypeptide (e.g., antibody) can be conjugated to the surface of the vesicle using click chemistry. In such embodiments, the polypeptide (e.g., antibody) can be coupled to the vesicle by a linker generated from a reaction between two complementary click chemistry functional groups. Further features of the polypeptide-EV conjugate are provided below.

[0106] To prepare polypeptide-EV (e.g., Ab-EV) conjugates provided herein, an antibody or its antigen-binding moiety can be conjugated to EV using a variety of conjugation methods.

[0107] In some embodiments, the polypeptide coupled to the EV is an antibody or its antigen-binding moiety. The antibody can be, for example, a monoclonal antibody, a polyclonal antibody, a multispecific antibody (e.g., a bispecific antibody), a nanobody, a monobody, or an antibody fragment exhibiting desired antigen-binding activity. The antibody or its antigen-binding moiety can take the form of a full-length (intact) antibody, a bispecific antibody, a bivariable domain antibody, a multi-chain or single-chain antibody, and / or its antigen-binding fragment. The antigen-binding fragment includes, but is not limited to, Fab, Fab', (Fab')2, Fv, scFv (single-chain Fv), a surrobody (including alternative light chain constructs), a single-domain antibody, a camelized antibody, and the like. The antibody or its antigen-binding moiety can be, or derived from, any isotype including, for example, IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3 or IgG4), or IgM. In some embodiments, the antibody or its antigen-binding portion is a humanized antibody or its antigen-binding portion. In other embodiments, the antibody or its antigen-binding portion is a fully human antibody or its antigen-binding portion.

[0108] In some embodiments, antibody mimetics are bound to EVs provided herein. Examples of antibody mimetics, but not limited to, adonectin (i.e., fibronectin-type binding molecules), afrin, affimer, afitin, alpha-body, affi-body, DARPin, antikalin, avimer, finomer, Kunitz domain peptide, mono-body, nanoCLAMP, nano-body, uni-body, verba-body, aptamer, and peptide molecules, all of which use binding structures that mimic conventional antibody binding but are generated from different mechanisms and function by different mechanisms.

[0109] Polypeptides (e.g., antibodies or their antigen-binding portions) can be conjugated to the surface of extracellular viable cells (EVs) using "click chemistry" (see, for example, Kolb, HC, Finn, MG, Sharpless, KB Angewandte Chemie, International Edition 2001, 40, 2004-2021; Kolb, HC, Sharpless, KB Drug Discovery Today 2003, 8, 1128-1137; the disclosures in these documents are incorporated herein by reference in their entirety). Polypeptides can be ligated to the EV surface using any suitable click reaction. Click chemistry reactions are advantageous because they are usually rapid, modular, efficient, often produce no toxic waste, can be carried out using water as a solvent, and can be stereospecifically configured.

[0110] As used herein, the term “click functional group” is used interchangeably with the term “click chemistry reactant” or “click reactant” to refer to a reactant that can react rapidly and selectively with the corresponding click reactant (e.g., via a cyclic addition reaction, “click”) under mild conditions in aqueous solution. Mild conditions may include low reactant concentrations, neutral pH, aqueous solution, and ambient temperature. Examples of click functional groups include azides, alkenes, alkynes, dibenzocyclooctin (DBCO), transcyclooctene, nitrones, nitrile imines, nitrile oxides, isonitriles, tetrazoles, and tetrazine groups. Examples of click reactions, but not limited to, include Cu-azide-alkynes, strain-promoting azide-alkynes, Staudinger ligation, tetrazine ligation, photoinducible tetrazole-alkenes, thiol-enes, NHS esters, epoxides, isocyanates, and aldehyde-aminooxys. In some embodiments, the linker that couples the antibody or its antigen-binding portion to the EV is generated from a reaction between two complementary click functional groups ("click linker").

[0111] In one embodiment, the polypeptide (e.g., an antibody or its antigen-binding moiety) or EV in the polypeptide-EV conjugate herein may contain a click functional group, which is a click-functionalized unsaturated group. Examples include molecules containing parental dipoles such as alkenes and alkynes, and associated heteroatom functional groups, such as carbonyls and nitriles. Other click functional groups that can be used as reactants for the click-functionalized unsaturated group are dienes such as tetrazine and tetrazole.

[0112] In another embodiment, the polypeptide (e.g., an antibody or its antigen-binding moiety) or EV in the polypeptide-EV conjugates herein may include a click-functional dipolar group. A click-functional dipolar group is understood to be a compound comprising one or more heteroatoms and having at least one mesomeri structure representing a charged dipole. Examples of click-functional dipolar groups are linear 1,3-dipolar groups, such as azides, oxidized nitriles, diazoalkanes, nitrile imines, and nitrones.

[0113] In certain embodiments, EV-Ab conjugates are prepared by reacting an antibody or its antigen-binding moiety containing a first click functional group with an EV containing a second click functional group. The first and second click functional groups are known to undergo click chemistry reactions. Table 1 shows some, not limited to, pairs of complementary functional groups that may be used to enable the binding of an antibody or its antigen-binding moiety to an EV.

[0114] Table 1. Examples of complementary click functional group pairs for Ab-EV conjugate formation. JPEG2026082945000001.jpg79168

[0115] Other exemplary pairs of click reaction products known to those skilled in the art include, but are not limited to, azide and dibenzocyclooctin (DBCO; also known as DIBO), tetrazine and transcyclooctene, and tetrazine and norbornene.

[0116] In one exemplary embodiment, an azide-labeled extracellular matrix (EV) can be coupled to a DBCO-labeled antibody or its antigen-binding moiety. In another embodiment, a DBCO-labeled EV can be coupled to an azide-labeled antibody or its antigen-binding moiety. In such embodiments, the antibody or its antigen-binding moiety is bound to the EV via a clicklinker formed by the reaction of the azide and DBCO.

[0117] In another exemplary embodiment, a tetrazine-labeled EV can be coupled to a transcyclooctene-labeled antibody or its antigen-binding moiety. In such embodiments, the antibody or its antigen-binding moiety is bound to the EV via a clicklinker formed by the reaction of tetrazine and transcyclooctene.

[0118] In another exemplary embodiment, a tetrazine-labeled EV can be coupled to a norbornene-labeled antibody or its antigen-binding moiety. In such embodiments, the antibody or its antigen-binding moiety is bound to the EV via a clicklinker formed by the reaction of tetrazine and norbornene.

[0119] Exemplary reactions provided herein that can be used to conjugate antibodies to EVs include copper-catalyzed reactions of azide and alkyne (Hüsgen 1,3-bipolar cycloaddition), reactions of diene and dienophile (Diels-Alder), strain-enhanced azide-alkyne cycloaddition, strain-enhanced alkyne-nitrone cycloaddition, reactions of strained alkenes with azide, tetrazine, or tetrazole, [3+2] cycloaddition of alkenes with azide, reverse electron-demand Diels-Alder reactions of alkenes with tetrazine, or photoreactions of alkenes with tetrazole. In some embodiments, the reactions may be carried out in an aqueous environment. In certain embodiments, the reaction is copper-catalyzed or ruthenium-catalyzed (for example, to initiate the reaction between azide and alkyne, for example, in azide-alkyne cycloaddition reactions). Alternatively, the reaction may be a non-copper reaction (for example, strain-enhanced azide-alkyne cycloaddition, strain-enhanced alkyne-nitrone cycloaddition).

[0120] Methods for incorporating click functional groups into proteins and extracellular vesicles are described, for example, in Smyth et al. "Surface functionalization of exosomes using click chemistry." Bioconjugate chemistry 25.10 (2014): 1777-1784, Wang et al. "Integrating protein engineering and bioorthogonal click conjugation for extracellular vesicle modulation and intracellular delivery." PLoS One 10.11 (2015): e0141860, and Lee et al. "Facile metabolic glycan labeling strategy for exosome tracking." Biochimica et Biophysica Acta (BBA)-General Subjects 1862.5 (2018): 1091-1100, each of which is incorporated herein by reference in its entirety.

[0121] The number of proteins, such as antibodies, that bind to EVs can be altered by changing the degree of click binding of EVs. The term "degree of click binding" can be used interchangeably with "degree of substitution" or "DS," and refers to the average number of click reactants per EV. The degree of click substitution can be altered by changing the number of molar equivalents of click reactants relative to the concentration of EVs in the click binding reaction. For example, a click bond reaction may include about 1 to about 5000 molar equivalents of click reactants, such as about 1, about 5, about 10, about 20, about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 1200, about 1500, about 1800, about 2000, about 2200, about 2500, about 2800, about 3000, about 3200, about 3500, about 4000, about 4200, about 4500, or about 5000 molar equivalents of click reactants. The intermediate range of the listed values ​​is also intended to be part of the present invention. For example, the click bond reaction may contain approximately 1 to 50, 10 to 100, 150 to 250, 200 to 500, 400 to 800, 700 to 1000, 1200 to 1600, 1500 to 2000, 1800 to 3500, or 3200 to 5000 molar equivalents of the click reaction product.

[0122] In some embodiments, the EV bonded to the click reactant has a degree of click substitution of about 0.01% to about 100%, for example, about 0.01% to about 0.5%, about 0.1% to about 5%, about 1% to about 10%, about 5% to about 15%, about 10% to about 20%, about 15% to about 25%, about 20% to about 35%, about 30% to about 40%, about 35% to about 50%, about 40% to about 60%, about 50% to about 75%, about 70% to about 90%, or about 85% to about 100%. For example, the EV of the present invention, which is bound to the click reaction product, may be about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

[0123] Using the methods provided herein, any antibody or its antigen-binding moiety can be conjugated to an extracellular matrix (EV) derived from nerve cells. In some embodiments, the antibody is a chimeric antibody or its antigen-binding moiety. In other embodiments, the antibody is a humanized antibody or its antigen-binding moiety. In other embodiments, the antibody is a fully human antibody or its antigen-binding moiety. In exemplary embodiments, the antibody or its antigen-binding fragment specifically binds to a target protein expressed in the brain or central nervous system. For example, in some embodiments, the antibody or its antigen-binding moiety can specifically bind to amyloid-beta polypeptide (Aβ), such as human amyloid-beta polypeptide associated with Alzheimer's disease.

[0124] In one exemplary embodiment, the antibody-EV conjugate provided herein may include solanezumab, an anti-amyloid-beta antibody, or its antigen-binding moiety. Solanezumab is a humanized IgG1 monoclonal antibody that targets the Mid domain of Aβ. Solanezumab (also known as 10D5 and m266) is described, for example, in U.S. Patent Nos. 7,320,790, 7,195,761, 8,105,597, 8,591,894, and 8,623,365, each of which is incorporated herein by reference in its entirety.

[0125] In one embodiment, the antibody-EV conjugate described herein comprises an anti-amyloid-β antibody or its antigen-binding moiety, which includes variable heavy chain and / or light chain regions of solanezumab. In one embodiment, the anti-amyloid-β antibody comprises a heavy chain variable region of solanezumab including VH CDR1, VH CDR2, and VH CDR3, and / or a light chain variable region of solanezumab including VL CDR1, VL CDR2, and VL CDR3.

[0126] Table 2 shows the heavy and light chain amino acid sequences of solanezumab. The CDR regions (CDR1, CDR2, and CDR3) of solanezumab are highlighted in bold. The heavy and light chain variable regions of solanezumab are underlined.

[0127] Table 2: Amino acid sequence of solanezumab TIFF2026082945000002.tif210164

[0128] In one embodiment, the antibody-EV conjugate includes one, two, or three heavy chain CDR regions listed in Table 2, or sequences having at least 95%, 96%, 97%, 98%, or 99% identity thereto, and / or one, two, or three light chain CDR regions listed in Table 2, or sequences having at least 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, the antibody or its antigen-binding moiety includes VH CDR1 of SEQ ID NO: 3, VH CDR2 of SEQ ID NO: 4, and VH CDR3 of SEQ ID NO: 5, and / or VL CDR1 of SEQ ID NO: 8, VL CDR2 of SEQ ID NO: 9, and VL CDR3 of SEQ ID NO: 10.

[0129] In one embodiment, the antibody-EV conjugate has a heavy chain variable region (HCVR) having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 2, and / or a light chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 7.

[0130] In one embodiment, the antibody-EV conjugate has a heavy chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 1, and / or a light chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 6.

[0131] In another exemplary embodiment, the antibody-EV conjugate provided herein may include aducanumab, an anti-amyloid-beta antibody, or its antigen-binding moiety. Aducanumab is a fully human IgG1 monoclonal antibody that recognizes the conformational epitope of the aggregated form of Aβ. Aducanumab (also known as BIIB037 and BART) is described, for example, in U.S. Patent Application Publication No. 20150315267, U.S. Patent Application Publication No. 20180333487, International Publication No. 2017211827, and International Publication No. 2019040612, each of which is incorporated herein by reference in its entirety.

[0132] In one embodiment, the antibody-EV conjugate described herein comprises an anti-amyloid-β antibody or its antigen-binding moiety, which includes a variable heavy chain and / or light chain region of aducanumab. In one embodiment, the anti-amyloid-β antibody comprises a heavy chain variable region of aducanumab including VH CDR1, VH CDR2, and VH CDR3, and / or a light chain variable region of aducanumab including VL CDR1, VL CDR2, and VL CDR3.

[0133] Table 3 shows the heavy and light chain amino acid sequences of aducanumab. The CDR regions (CDR1, CDR2, and CDR3) of aducanumab are highlighted in bold. The heavy and light chain variable regions of aducanumab are underlined.

[0134] Table 3: Amino acid sequence of aducanumab TIFF2026082945000003.tif205169

[0135] In one embodiment, the antibody-EV conjugate includes one, two, or three heavy chain CDR regions listed in Table 3, or sequences having at least 95%, 96%, 97%, 98%, or 99% identity thereto, and / or one, two, or three light chain CDR regions listed in Table 3, or sequences having at least 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, the antibody or its antigen-binding moiety includes VH CDR1 of SEQ ID NO: 13, VH CDR2 of SEQ ID NO: 14, and VH CDR3 of SEQ ID NO: 15, and / or VL CDR1 of SEQ ID NO: 18, VL CDR2 of SEQ ID NO: 19, and VL CDR3 of SEQ ID NO: 20.

[0136] In one embodiment, the antibody-EV conjugate has a heavy chain variable region (HCVR) having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 12, and / or a light chain variable region (LCVR) having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 17.

[0137] In one embodiment, the antibody-EV conjugate has a heavy chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 11, and / or a light chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 16.

[0138] In another embodiment, the antibody or its antigen-binding fragment comprises a heavy chain variable region containing an amino acid sequence having at least 95% identity to the anti-amyloid-beta antibody provided herein, for example, at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the anti-amyloid-beta antibody provided herein. In a particular embodiment, the antibody comprises a modified heavy chain (HC) variable region containing the HC variable domain of the anti-amyloid-beta antibody or its variant, the variant differs from the anti-amyloid-beta antibody in that (i) there is a substitution, addition, or deletion of 1, 2, 3, 4, or 5 amino acids; (ii) there is a substitution, addition, or deletion of up to 5, 4, 3, 2, or 1 amino acid; and (iii) there is a substitution, addition, or deletion of 1-5, 1-3, 1-2, 2-5, or 3-5 amino acids. Unlike anti-amyloid-beta antibodies, and / or (iv) having an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to that of anti-amyloid-beta antibodies, and in any of (i) to (iv), the amino acid substitutions may be conserved or non-conserved, and the modified heavy chain variable region can have enhanced biological activity against the heavy chain variable region of anti-amyloid-beta antibodies while maintaining the amyloid-beta binding specificity of the antibody.

[0139] In another exemplary embodiment, the antibody-EV conjugate provided herein may comprise an antibody or its antigen-binding moiety that specifically binds to the human programmed cell death 1 (PD1) protein. An antibody-EV conjugate comprising an antibody or its antigen-binding moiety that specifically binds to PD1 can be used to treat diseases or disorders involving PD1, such as cancers including but not limited to glioblastoma multiforme. In one exemplary embodiment, the antibody-EV conjugate provided herein may comprise nivolumab, an anti-PD1 antibody, or its antigen-binding moiety. Nivolumab is a human IgG4 monoclonal antibody that targets PD1. Nivolumab is described, for example, in U.S. Patent No. 8,008,449, U.S. Patent No. 8,168,179, and U.S. Patent No. 9,387,247, each of which is incorporated herein by reference in its entirety.

[0140] In one embodiment, the antibody-EV conjugate described herein comprises an anti-PD1 antibody or its antigen-binding moiety, which includes variable heavy chain and / or light chain regions of nivolumab. In one embodiment, the anti-PD1 antibody comprises a heavy chain variable region of nivolumab including VH CDR1, VH CDR2, and VH CDR3, and / or a light chain variable region of nivolumab including VL CDR1, VL CDR2, and VL CDR3. The heavy chain and light chain amino acid sequences of nivolumab are shown in Table 4.

[0141] Table 4: Amino acid sequence of nivolumab TIFF2026082945000004.tif202163

[0142] In one embodiment, the antibody-EV conjugate includes one, two, or three heavy chain CDR regions listed in Table 4, or sequences having at least 95%, 96%, 97%, 98%, or 99% identity thereto, and / or one, two, or three light chain CDR regions listed in Table 4, or sequences having at least 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, the antibody or its antigen-binding moiety includes VH CDR1 of SEQ ID NO: 23, VH CDR2 of SEQ ID NO: 24, and VH CDR3 of SEQ ID NO: 25, and / or VL CDR1 of SEQ ID NO: 28, VL CDR2 of SEQ ID NO: 29, and VL CDR3 of SEQ ID NO: 30.

[0143] In one embodiment, the antibody-EV conjugate comprises a heavy chain variable region (HCVR) having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 22, and / or a light chain variable region (LCVR) having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 27.

[0144] In one embodiment, the antibody-EV conjugate comprises a heavy chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 21, and / or a light chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 26.

[0145] In another exemplary embodiment, the antibody-EV conjugate provided herein may comprise an antibody or its antigen-binding moiety that specifically binds to the human vascular endothelial growth factor (VEGF) protein associated with cancer. An antibody-EV conjugate comprising an antibody or its antigen-binding moiety that specifically binds to VEGF can be used to treat diseases or disorders involving VEGF, such as cancers including but not limited to glioblastoma multiforme. In one exemplary embodiment, the antibody-EV conjugate provided herein may comprise bevacizumab, an anti-VEGF antibody, or its antigen-binding moiety. Bevacizumab is a recombinant humanized monoclonal antibody that targets VEGF. Bevacizumab is described, for example, in U.S. Patent No. 6,632,926, U.S. Patent No. 7,169,901, and U.S. Patent No. 7,575,893, each of which is incorporated herein by reference in its entirety.

[0146] In one embodiment, the antibody-EV conjugate described herein comprises an anti-VEGF antibody or its antigen-binding moiety containing variable heavy chain and / or light chain regions of bevacizumab. In one embodiment, the anti-VEGF antibody comprises a heavy chain variable region of bevacizumab containing VH CDR1, VH CDR2, and VH CDR3, and / or a light chain variable region of bevacizumab containing VL CDR1, VL CDR2, and VL CDR3. The heavy chain and light chain amino acid sequences of bevacizumab are shown in Table 5.

[0147] Table 5: Amino acid sequence of bevacizumab TIFF2026082945000005.tif209164

[0148] In one embodiment, the antibody-EV conjugate includes one, two, or three heavy chain CDR regions listed in Table 5, or sequences having at least 95%, 96%, 97%, 98%, or 99% identity thereto, and / or one, two, or three light chain CDR regions listed in Table 5, or sequences having at least 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, the antibody or its antigen-binding moiety includes VH CDR1 of SEQ ID NO: 33, VH CDR2 of SEQ ID NO: 34, and VH CDR3 of SEQ ID NO: 35, and / or VL CDR1 of SEQ ID NO: 38, VL CDR2 of SEQ ID NO: 39, and VL CDR3 of SEQ ID NO: 40.

[0149] In one embodiment, the antibody-EV conjugate comprises a heavy chain variable region (HCVR) having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 32, and / or a light chain variable region (LCVR) having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 37.

[0150] In one embodiment, the antibody-EV conjugate comprises a heavy chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 31, and / or a light chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 36.

[0151] In another exemplary embodiment, the antibody-EV conjugate provided herein may comprise an antibody or its antigen-binding moiety that specifically binds to the human B lymphocyte antigen CD20 (CD20) protein. An antibody-EV conjugate comprising an antibody or its antigen-binding moiety that specifically binds to CD20 can be used to treat diseases or disorders involving CD20, such as multiple sclerosis (MS), including relapsing MS, primary progressive MS, and secondary progressive MS. In one exemplary embodiment, the antibody-EV conjugate provided herein may include ocrelizumab, an anti-CD20 antibody, or its antigen-binding moiety. Ocrelizumab is a humanized IgG1 monoclonal antibody that targets CD20. Ocrelizumab is described, for example, in U.S. Patent No. 5,500,362, U.S. Patent No. 5,677,180, and U.S. Patent No. 7,799,900, each of which is incorporated herein by reference in its entirety.

[0152] In one embodiment, the antibody-EV conjugate described herein comprises an anti-CD20 antibody or its antigen-binding moiety, which includes variable heavy chain and / or light chain regions of ocrelizumab. In one embodiment, the anti-CD20 antibody comprises a heavy chain variable region of ocrelizumab including VH CDR1, VH CDR2, and VH CDR3, and / or a light chain variable region of ocrelizumab including VL CDR1, VL CDR2, and VL CDR3. The heavy chain and light chain amino acid sequences of ocrelizumab are shown in Table 6.

[0153] Table 6: Amino acid sequence of ocrelizumab TIFF2026082945000006.tif237164

[0154] In one embodiment, the antibody-EV conjugate includes one, two, or three heavy chain CDR regions listed in Table 6, or sequences having at least 95%, 96%, 97%, 98%, or 99% identity thereto, and / or one, two, or three light chain CDR regions listed in Table 6, or sequences having at least 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, the antibody or its antigen-binding moiety includes VH CDR1 of SEQ ID NO: 43, VH CDR2 of SEQ ID NO: 44, and VH CDR3 of SEQ ID NO: 45, and / or VL CDR1 of SEQ ID NO: 48, VL CDR2 of SEQ ID NO: 49, and VL CDR3 of SEQ ID NO: 50.

[0155] In one embodiment, the antibody-EV conjugate comprises a heavy chain variable region (HCVR) having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 42, and / or a light chain variable region (LCVR) having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 47.

[0156] In one embodiment, the antibody-EV conjugate comprises a heavy chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 41, and / or a light chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 46.

[0157] In another exemplary embodiment, the antibody-EV conjugate provided herein may comprise an antibody or its antigen-binding moiety that specifically binds to human α-4 integrin protein. An antibody-EV conjugate comprising an antibody or its antigen-binding moiety that specifically binds to α-4 integrin can be used to treat diseases or disorders involving α-4 integrin, such as multiple sclerosis, including relapsing-remitting multiple sclerosis. In one exemplary embodiment, the antibody-EV conjugate provided herein may include natalizumab, an anti-α-4 integrin antibody, or its antigen-binding moiety. Natalizumab is a humanized monoclonal antibody that targets α-4 integrin. Natalizumab is described, for example, in U.S. Patent No. 7,157,086, U.S. Patent No. 6,602,503, and U.S. Patent No. 8,124,350, each of which is incorporated herein by reference in its entirety.

[0158] In one embodiment, the antibody-EV conjugate described herein comprises an anti-α-4 integrin antibody or its antigen-binding moiety containing variable heavy chain and / or light chain regions of natalizumab. In one embodiment, the anti-α-4 integrin antibody comprises a heavy chain variable region of natalizumab containing VH CDR1, VH CDR2, and VH CDR3, and / or a light chain variable region of natalizumab containing VL CDR1, VL CDR2, and VL CDR3. The heavy chain and light chain amino acid sequences of natalizumab are shown in Table 7.

[0159] Table 7: Amino acid sequence of natalizumab TIFF2026082945000007.tif128165

[0160] In one embodiment, the antibody-EV conjugate includes one, two, or three heavy chain CDR regions listed in Table 7, or sequences having at least 95%, 96%, 97%, 98%, or 99% identity thereto, and / or one, two, or three light chain CDR regions listed in Table 7, or sequences having at least 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, the antibody or its antigen-binding moiety includes VH CDR1 of SEQ ID NO: 52, VH CDR2 of SEQ ID NO: 53, and VH CDR3 of SEQ ID NO: 54, and / or VL CDR1 of SEQ ID NO: 56, VL CDR2 of SEQ ID NO: 57, and VL CDR3 of SEQ ID NO: 58.

[0161] In one embodiment, the antibody-EV conjugate comprises a heavy chain variable region (HCVR) having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 51, and / or a light chain variable region (LCVR) having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 55.

[0162] In another exemplary embodiment, the antibody-EV conjugate provided herein may comprise an antibody or its antigen-binding moiety that specifically binds to the human GD2 ganglioside (GD2) protein. An antibody-EV conjugate comprising an antibody or its antigen-binding moiety that specifically binds to GD2 can be used to treat diseases or disorders involving GD2, such as neuroblastoma. In one exemplary embodiment, the antibody-EV conjugate provided herein may include dinutuximab, an anti-GD2 antibody, or its antigen-binding moiety. Dinutuximab is a chimeric human / mouse monoclonal antibody that targets GD2. Dinutuximab is described, for example, in U.S. Patent No. 9,777,068, U.S. Patent No. 10,294,305, and U.S. Patent No. 9,840,566, each of which is incorporated herein by reference in its entirety.

[0163] In one embodiment, the antibody-EV conjugate described herein comprises an anti-GD2 antibody or its antigen-binding moiety containing variable heavy chain and / or light chain regions of dinutuximab. In one embodiment, the anti-GD2 antibody comprises a heavy chain variable region of dinutuximab containing VH CDR1, VH CDR2, and VH CDR3, and / or a light chain variable region of dinutuximab containing VL CDR1, VL CDR2, and VL CDR3. The heavy chain and light chain amino acid sequences of dinutuximab are shown in Table 8.

[0164] Table 8: Amino acid sequence of dinutuximab TIFF2026082945000008.tif246164

[0165] In one embodiment, the antibody-EV conjugate includes one, two, or three heavy chain CDR regions listed in Table 8, or sequences having at least 95%, 96%, 97%, 98%, or 99% identity thereto, and / or one, two, or three light chain CDR regions listed in Table 8, or sequences having at least 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, the antibody or its antigen-binding moiety includes VH CDR1 of SEQ ID NO: 61, VH CDR2 of SEQ ID NO: 62, and VH CDR3 of SEQ ID NO: 63, and / or VL CDR1 of SEQ ID NO: 66, VL CDR2 of SEQ ID NO: 67, and VL CDR3 of SEQ ID NO: 68.

[0166] In one embodiment, the antibody-EV conjugate comprises a heavy chain variable region (HCVR) having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 60, and / or a light chain variable region (LCVR) having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 65.

[0167] In one embodiment, the antibody-EV conjugate comprises a heavy chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 59, and / or a light chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 64.

[0168] In another exemplary embodiment, the antibody-EV conjugate provided herein may comprise an antibody or its antigen-binding moiety that specifically binds to the human amyloid-beta protein associated with Alzheimer's disease. An antibody-EV conjugate comprising an antibody or its antigen-binding moiety that specifically binds to amyloid-beta can be used to treat amyloid-beta-related diseases or disorders, such as Alzheimer's disease. In one exemplary embodiment, the antibody-EV conjugate provided herein may comprise gantenerumab, an anti-amyloid-beta antibody, or its antigen-binding moiety. Gantenerumab is a human IgG1 monoclonal antibody that targets amyloid-beta. Gantenerumab is described, for example, in U.S. Patent Nos. 7,794,719, 8,216,577, and 8,329,886, each of which is incorporated herein by reference in its entirety.

[0169] In one embodiment, the antibody-EV conjugate described herein comprises an anti-amyloid-β antibody or its antigen-binding moiety, which includes variable heavy chain and / or light chain regions of gantenerumab. In one embodiment, the anti-amyloid-β antibody comprises a heavy chain variable region of gantenerumab including VH CDR1, VH CDR2, and VH CDR3, and / or a light chain variable region of gantenerumab including VL CDR1, VL CDR2, and VL CDR3. The heavy chain and light chain amino acid sequences of gantenerumab are shown in Table 9.

[0170] Table 9: Amino acid sequence of gantenerumab TIFF2026082945000009.tif246165TIFF2026082945000010.tif29164

[0171] In one embodiment, the antibody-EV conjugate includes one, two, or three heavy chain CDR regions listed in Table 9, or sequences having at least 95%, 96%, 97%, 98%, or 99% identity thereto, and / or one, two, or three light chain CDR regions listed in Table 9, or sequences having at least 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, the antibody or its antigen-binding moiety includes VH CDR1 of SEQ ID NO: 71, VH CDR2 of SEQ ID NO: 72, and VH CDR3 of SEQ ID NO: 73, and / or VL CDR1 of SEQ ID NO: 76, VL CDR2 of SEQ ID NO: 77, and VL CDR3 of SEQ ID NO: 78.

[0172] In one embodiment, the antibody-EV conjugate comprises a heavy chain variable region (HCVR) having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 70, and / or a light chain variable region (LCVR) having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 75.

[0173] In one embodiment, the antibody-EV conjugate comprises a heavy chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 69, and / or a light chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 74.

[0174] In another exemplary embodiment, the antibody-EV conjugate provided herein may include lecanemab, an anti-amyloid-beta antibody, or its antigen-binding moiety. Lecanemab is a humanized mouse monoclonal antibody that targets amyloid-beta. Lecanemab is described, for example, in U.S. Patents 8,106,164, 8,999,936, and 9,573,994, each of which is incorporated herein by reference in its entirety.

[0175] In one embodiment, the antibody-EV conjugate described herein comprises an anti-amyloid-β antibody or its antigen-binding moiety, which includes variable heavy chain and / or light chain regions of lecanemab. In one embodiment, the anti-amyloid-β antibody comprises a heavy chain variable region of lecanemab including VH CDR1, VH CDR2, and VH CDR3, and / or a light chain variable region of lecanemab including VL CDR1, VL CDR2, and VL CDR3. The heavy chain and light chain amino acid sequences of lecanemab are shown in Table 10.

[0176] Table 10: Amino acid sequence of lecanemab TIFF2026082945000011.tif164164

[0177] In one embodiment, the antibody-EV conjugate comprises a heavy chain variable region (HCVR) having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 80, and / or a light chain variable region (LCVR) having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 82.

[0178] In one embodiment, the antibody-EV conjugate comprises a heavy chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 79, and / or a light chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 81.

[0179] In another exemplary embodiment, the antibody-EV conjugate provided herein may comprise an antibody or its antigen-binding moiety that specifically binds to the human B lymphocyte antigen CD20 (CD20) protein. As described above, an antibody-EV conjugate comprising an antibody or its antigen-binding moiety that specifically binds to CD20 can be used to treat diseases or disorders involving CD20, such as multiple sclerosis, for example, relapsing-remitting multiple sclerosis. In one exemplary embodiment, the antibody-EV conjugate provided herein may comprise ublituximab, an anti-CD20 antibody, or its antigen-binding moiety. Ublituximab is a chimeric human / mouse monoclonal antibody that targets CD20. Ublituximab is described, for example, in U.S. Patent No. 9,234,045, U.S. Patent No. 9,694,071, and U.S. Patent No. 9,873,745, each of which is incorporated herein by reference in its entirety.

[0180] In one embodiment, the antibody-EV conjugate described herein comprises an anti-CD20 antibody or its antigen-binding moiety containing variable heavy chain and / or light chain regions of ublituximab. In one embodiment, the anti-CD20 antibody comprises heavy chain variable regions of ublituximab including VH CDR1, VH CDR2, and VH CDR3, and / or light chain variable regions of ublituximab including VL CDR1, VL CDR2, and VL CDR3. The heavy chain and light chain amino acid sequences of ublituximab are shown in Table 11.

[0181] Table 11: Amino acid sequence of ublituximab TIFF2026082945000012.tif241164

[0182] In one embodiment, the antibody-EV conjugate includes one, two, or three heavy chain CDR regions listed in Table 11, or sequences having at least 95%, 96%, 97%, 98%, or 99% identity thereto, and / or one, two, or three light chain CDR regions listed in Table 11, or sequences having at least 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, the antibody or its antigen-binding moiety includes VH CDR1 of SEQ ID NO: 85, VH CDR2 of SEQ ID NO: 86, and VH CDR3 of SEQ ID NO: 87, and / or VL CDR1 of SEQ ID NO: 90, VL CDR2 of SEQ ID NO: 91, and VL CDR3 of SEQ ID NO: 92.

[0183] In one embodiment, the antibody-EV conjugate comprises a heavy chain variable region (HCVR) having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 84, and / or a light chain variable region (LCVR) having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 89.

[0184] In one embodiment, the antibody-EV conjugate comprises a heavy chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 83, and / or a light chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 88.

[0185] In one embodiment, the antibody-EV conjugate described herein contains human immunoglobulin. Immunoglobulin is a sterile solution prepared from human plasma containing human antibodies. Immunoglobulin products are marketed under the trade names GAMUNEX C®, GAMMAKED®, HIZENTRA®, PRIVIGEN®, and GAMMAGARD®. Antibody-EV conjugates containing immunoglobulin, but not limited to these, can be used to treat disorders including chronic inflammatory demyelinating polyneuropathy (CIDP) and multifocal motor neuropathy.

[0186] In some embodiments, the antibody-labeled EVs provided herein may further include, but are not limited to, small molecules, nucleic acids (e.g., short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), and double-stranded RNA (dsRNA)), proteins, and / or peptides.

[0187] In some cases, extracellular molecules (EVs) are isolated from cells, then the desired molecule is loaded into the EVs, and finally the EVs are administered to the subject. In other cases, the desired molecule can be loaded into the EVs by simultaneously incubating cells that produce EVs with one or more therapeutic agents. In several other cases, the desired nucleic acid, protein, or peptide can be loaded into the EVs by overexpression of the desired molecule in the cells used for EV production, so that the desired molecule is loaded into the EVs during production. In yet another case, the desired molecule can be loaded into the EVs by overexpression of a carrier molecule (e.g., a protein) that facilitates the transport of the desired molecule into exosomes. For example, a carrier peptide from bovine leukemia virus protein can be expressed as a fusion protein with the polypeptide of interest to facilitate the loading of the polypeptide of interest into exosomes. This method is described, for example, in U.S. Patent No. 9,546,371, the entire contents of which are incorporated herein by reference. In other embodiments, the carrier molecule is expressed together with an inhibitory nucleic acid in the cells used for EV production, thereby loading the inhibitory nucleic acid into the EVs.

[0188] In the above example, the desired molecule may be exogenous or endogenous. An exogenous molecule refers to a molecule that is added to an EV from an external source, such as one that is not natural to the EV, in vitro, in vivo, or ex vivo. An endogenous molecule refers to a molecule that is naturally present in or related to the EV in vitro, in vivo, or ex vivo. When an exogenous molecule is added to an EV from an external source, for example by overexpression or post-production insertion, it may be of the same type as molecules naturally present in the EV (such as nucleic acids or proteins).

[0189] Before or after surface labeling of the EV with an antibody or its antigen-binding moiety, a desired cargo molecule can be loaded into the EV. The desired molecules described herein can be introduced into the EV by a number of different techniques, including incubation, sonication, electroporation, or the use of transfection reagents such as membrane permeabilizing agents (e.g., polyols, surfactants, sugars). The electroporation conditions may vary depending on the charge and size of the desired molecule. Typical voltages are 20 V / cm to 1,000 V / cm, e.g., 20 V / cm to 100 V / cm, and capacitances are typically 25 μF to 250 μF, e.g., 25 μF to 125 μF. Voltages of 150 mV to 250 mV, particularly 200 mV, are preferred for loading antibodies into the EV. Alternatively, the desired molecule (e.g., exogenous protein and / or peptide) may be loaded into the EV using a transfection reagent. Although EVs are small in size, conventional transfection agents can be used to transfect EVs with the desired molecule (e.g., exogenous proteins and / or peptides). In one example, the therapeutic protein or peptide may be incorporated into the EV by transforming or transfecting a host cell with a nucleic acid construct expressing the therapeutic protein or peptide of interest, so that the therapeutic protein or peptide is incorporated into the EV when the EV is produced from the cell. In another example, an inhibitory nucleic acid construct may be incorporated into the EV by transforming or transfecting a host cell with or without a carrier / adapter molecule of the inhibitory nucleic acid construct, so that the inhibitory nucleic acid construct is incorporated into the EV.

[0190] (iii) EVs in which antibodies have been loaded into the EV lumen In one embodiment, the present invention provides a composition comprising an EV in which a polypeptide (e.g., an antibody or an antigen-binding fragment thereof) is loaded into the lumen of the EV. An EV suitable for this purpose may contain extracellular vesicles derived from nerve cells (e.g., neural progenitor cells or neural stem cells) for loading the antibody or its antigen-binding portion. Any of the antibodies described herein can be used for loading into the EV lumen in addition to, or instead of, loading onto the EV surface by, for example, click chemistry as described above.

[0191] Polypeptides (e.g., antibodies or their antigen-binding portions) can be loaded into the EV lumen using various methods.

[0192] In some embodiments, antibodies are loaded into vesicles using a surfactant suitable for membrane permeabilization, such as a saponin. In some embodiments, antibodies are loaded into vesicles by sonication. In some embodiments, antibodies are loaded into vesicles by electroporation. In some embodiments, antibodies are loaded into vesicles by incubation (e.g., at room temperature) with shaking (e.g., 100 rpm to 1500 rpm). In some embodiments, antibodies are loaded into vesicles using a combination of the above methods (e.g., saponin permeabilization and sonication, electroporation and sonication, saponin permeabilization and electroporation, etc.). Proteins, such as antibodies, can be added to the vesicles before, during, or after permeabilization, sonication, and / or electroporation. In some embodiments, the vesicles are permeabilized using a surfactant, sonication, and / or electroporation in the presence of the protein to be loaded into the lumen, such as an antibody. In other embodiments, the vesicles are permeabilized using a surfactant, sonication, and / or electroporation, and then incubated with the protein to be loaded, such as an antibody. In some embodiments, the protein to be loaded (e.g., antibody) is attached to the vesicle at a concentration of about 25 to about 2000 μg / mL. In some embodiments, the protein to be loaded (e.g., antibody) is attached to the vesicle at a concentration of about 50 to about 1500 μg / mL. In some embodiments, the protein to be loaded (e.g., antibody) is attached to the vesicle at a concentration of about 100 to about 1000 μg / mL. In some embodiments, the protein to be loaded (e.g., antibody) is attached to the vesicle at concentrations of approximately 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 600 μg / mL, 700 μg / mL, 800 μg / mL, 900 μg / mL, 1,000 μg / mL, 1,500 μg / mL, or 2,000 μg / mL.

[0193] In some embodiments, antibodies are loaded into the lumen of vesicles using a surfactant suitable for membrane permeabilization. Examples of such surfactants include saponins, Tween-20, and other agents known in the art to be suitable for permeabilization of extracellular vesicles. In some embodiments, the EV membrane is permeabilized using about 0.01%, about 0.03%, about 0.05%, about 0.07%, about 0.09%, about 0.1%, about 0.12%, about 0.14%, about 0.16%, about 0.18%, about 0.2%, about 0.22%, about 0.24%, about 0.26%, about 0.28%, or about 0.3% (w / v) of a surfactant (e.g., saponin). In some embodiments, antibodies are loaded into the lumen of vesicles by permeabilization of the EV membrane with saponin. For example, the membrane can be permeated by incubating an EV preparation with, for example, 0.01% to 5% (w / v) of saponin before antibody addition. In some embodiments, before antibody addition, for example, about 0.01%, about 0.03%, about 0.05%, about 0.07%, about 0.09%, about 0.1%, about 0.12%, about 0.14%, about 0.16%, about 0.18%, about 0.2%, about 0.22%, about 0.24%, about 0.26%, about 0.28%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1% The above membrane can be permeated by incubating the EV preparation with 0.0%, approximately 1.2%, approximately 1.4%, approximately 1.6%, approximately 1.8%, approximately 2.0%, approximately 2.2%, approximately 2.4%, approximately 2.8%, approximately 3.0%, approximately 3.2%, approximately 3.4%, approximately 3.6%, approximately 3.8%, approximately 4.0%, approximately 4.2%, approximately 4.4%, approximately 4.6%, approximately 4.8%, or approximately 5.0% (w / v) of saponin. In some embodiments, EV can be incubated with saponin for 1 to 20 minutes before antibody addition. In some embodiments, EV can be incubated with saponin for 1 to 10 minutes before antibody addition. In some embodiments, EV can be incubated with saponin for 1 to 5 minutes before antibody addition.The antibody can then be added to the permeabilized EV and incubated for 1 to 30 minutes (e.g., 1 to 20 minutes, 1 to 15 minutes, 1 to 10 minutes, or 1 to 5 minutes) while optionally gently mixing or shaking (e.g., 100 rpm, 200 rpm, 300 rpm, 400 rpm, 500 rpm, 600 rpm, 700 rpm, 800 rpm, 900 rpm, 1000 rpm, 1100 rpm, 1200 rpm, 1300 rpm, 1400 rpm, 1500 rpm).

[0194] In some embodiments, antibodies are loaded into the lumen of vesicles by sonication. Those skilled in the art will understand that sonication can be performed with various combinations of amplitude, pulse duration, and cycles, as illustrated herein in Table 12. For example, the amplitude of the ultrasonic treatment can be set to any amplitude from 10% to 100% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%), and this can be combined with various pulse times (e.g., 4 seconds on / 2 seconds off, 4 seconds on / 4 seconds off, 4 seconds on / 8 seconds off, 2 seconds on / 2 seconds off, 2 seconds on / 4 seconds off, 2 seconds on / 8 seconds off, 8 seconds on / 2 seconds off, 8 seconds on / 4 seconds off, 8 seconds on / 8 seconds off) and the number of cycles (e.g., 2 to 36 cycles, e.g., 2 cycles, 4 cycles, 6 cycles, 8 cycles, 10 cycles, 12 cycles, 14 cycles, 16 cycles, 18 cycles, 20 cycles, 22 cycles, 24 cycles, 26 cycles, 28 cycles, 30 cycles, 32 cycles, 34 cycles, or 36 cycles). In some embodiments, the amplitude of the ultrasonic treatment can be set to any amplitude from 10% to 100% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) over a certain duration (e.g., 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, or longer). In some embodiments, antibodies are loaded into the lumen of vesicles by electroporation using known methods, for example, by varying the voltage (e.g., 100V, 150V, 200V, 250V, 300V, 350V, 400V, 450V, 500V, 550V, 600V, 650V, 700V, 750V, 800V, 850V, 900V, 950V, 1kV, or higher) and the duration and number of pulses (e.g., 1 pulse, 2 pulses, 3 pulses, 4 pulses, 5 pulses, 6 pulses, 7 pulses, 8 pulses, 9 pulses, 10 pulses, 11 pulses, 12 pulses, 13 pulses, 14 pulses, 15 pulses, 16 pulses, 17 pulses, 18 pulses, 19 pulses, 20 pulses, or higher).

[0195] In some embodiments, antibodies are loaded into the lumen of vesicles by incubation (e.g., 30 minutes, 1 hour, 2 hours, 3 hours, 400 rpm, 500 rpm, 600 rpm, 700 rpm, 800 rpm, 900 rpm, 1000 rpm, 1100 rpm, 1200 rpm, 1300 rpm, 1400 rpm, 1500 rpm) with or without shaking (e.g., 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or longer), with or without shaking (e.g., 100 rpm, 200 rpm, 300 rpm, 400 rpm, 500 rpm, 600 rpm, 700 rpm, 800 rpm, 900 rpm, 1000 rpm, 1100 rpm, 1200 rpm, 1300 rpm, 1400 rpm, 1500 rpm). Incubation can be carried out at any suitable temperature. In some embodiments, incubation can be carried out at approximately 4 to approximately 37°C. In some embodiments, incubation can be carried out at room temperature, on ice, at about 4°C, or at about 37°C.

[0196] In some embodiments, as illustrated herein, polypeptides (e.g., antibodies) are loaded into vesicles using any combination of the methods described herein for introducing antibodies into the lumen of vesicles. In some embodiments, the polypeptides (e.g., antibodies) are included (added to the vesicles) during permeabilization of the vesicle membrane. In some embodiments, the polypeptides (e.g., antibodies) are included (added to the vesicles) after permeabilization of the vesicle membrane.

[0197] In certain embodiments, the antibody is loaded into the EV by saponin permeabilization of the EV (e.g., using 0.01-5% saponin), accompanied by sonication at 10-100% amplitude for a duration of 5-30 minutes prior to antibody loading into the EV. In certain embodiments, the antibody is loaded into the EV by saponin permeabilization of the EV (0.2% saponin), accompanied by sonication at 20% amplitude for a duration of 10 minutes prior to antibody loading into the EV. In certain embodiments, the antibody is loaded into the EV by saponin permeabilization of the EV (0.2% saponin), accompanied by sonication at 40% amplitude for a duration of 10 minutes prior to antibody loading into the EV. In certain embodiments, the antibody is loaded into the EV by saponin permeabilization of the EV (0.2% saponin), accompanied by sonication at 20% amplitude for a duration of 5 minutes prior to antibody loading into the EV.

[0198] In some embodiments, antibodies are loaded into EVs by a combination of permeabilization with 0.01-5% saponin and shaking. In some embodiments, antibodies are loaded into EVs by a combination of permeabilization with 0.2% saponin and shaking (e.g., 500 rpm). In some embodiments, antibodies are loaded into EVs by a combination of incubation at room temperature and shaking (e.g., 1000 rpm). In some embodiments, antibodies are loaded into EVs by electroporation. In some embodiments, antibodies are loaded into vesicles by sonication with 20% amplitude, 12 cycles, 4 seconds on and 8 seconds off.

[0199] In some embodiments, antibodies are loaded into the EV by sonication over six cycles with a 60% amplitude and a duration of 4 seconds on / 8 seconds off. In some embodiments, antibodies are loaded into the EV by sonication over six cycles with a 60% amplitude, a duration of 2 seconds on / 4 seconds off, and a subsequent 2-minute incubation on ice.

[0200] In some embodiments, a range of polypeptide (e.g., antibody) concentrations, such as 100 μg / ml, about 200 μg / ml, about 300 μg / ml, about 400 μg / ml, about 500 μg / ml, about 600 μg / ml, about 700 μg / ml, about 800 μg / ml, about 900 μg / ml, or about 1,000 μg / ml, can be loaded into the lumen of the EV.

[0201] After loading antibodies into the lumen of extracellular viable cells (EVs), the EVs loaded with antibodies can be separated from the free antibodies and unloaded EVs using various methods known in the art, such as ultrafiltration, ultracentrifugation, size exclusion, ion exchange, and high-performance liquid chromatography (HPLC), including bioaffinity chromatography.

[0202] C. Formulation, delivery, and administration Polypeptide-charged extracellular vehicles (EVs), such as antibody-charged EVs (including antibody-EV conjugates and EVs with antibodies charged in their lumen), provided herein can be formulated within a pharmaceutical composition for delivery to a target (e.g., a pharmaceutical composition containing neuronal Ab-EV conjugates). The pharmaceutical composition may include a therapeutically effective amount of polypeptide-charged EVs (e.g., antibody-charged neuronal EVs) and a pharmaceutically acceptable carrier. For example, a therapeutically effective amount of polypeptide-charged EVs (e.g., Ab-EV conjugates and / or EVs with charged lumen) can be provided in sterile phosphate-buffered saline. Other suitable excipients, vehicles, and carriers are known in the art. They are described, for example, in Remington's Pharmaceutical Sciences, 18th ed. (1990). It will be understood by those skilled in the art that any conventionally used and inactive dosing agent, vehicle, or carrier may be used for the preparation and administration of pharmaceutical compositions containing the polypeptide-charged EVs of this disclosure. Examples of such methods, vehicles, and carriers are described, for example, in Remington's Pharmaceutical Sciences, 4th ed. (1970), the disclosures of which are incorporated herein by reference.

[0203] Formulations containing polypeptide-loaded extracellular viable cells (e.g., antibody-loaded nerve viable cells) of the Disclosure may take the form of liquids, solids, or semi-solids, such as solutions, suspensions, emulsions, sustained-release formulations, lotions, or aerosols, and may optionally take the form of unit dosage forms suitable for easy administration of precise doses. The pharmaceutical composition typically comprises conventional pharmaceutical carriers and / or excipients, and may further contain other drugs, carriers, adjuvants, additives, etc. The weight percentage ratio of Ab-EV conjugate to one or more excipients can be about 20:1 to about 1:60, or about 15:1 to about 1:45, or about 10:1 to about 1:40, or about 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1 to about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, or 1:35, preferably about 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, or 5:1. In some embodiments, the composition of the present disclosure has a total EV amount of about 1 μg to about 1 g or more, for example, about 1 μg to about 100 μg, about 100 μg to about 200 μg, about 200 μg to about 300 μg, about 300 μg to about 400 μg, about 500 μg to about 600 μg, about 700 μg to about 800 μg, about 900 μg to about 1 mg, about 100 μg to about 500 μg, about 1 mg to about 500 mg, about 5 The total amount of extravalent protein (EV) is approximately mg to 500 mg, approximately 10 mg to 500 mg, approximately 25 mg to 500 mg, approximately 50 mg to 350 mg, approximately 75 mg to 450 mg, approximately 50 mg to 450 mg, approximately 75 mg to 325 mg, approximately 100 mg to 650 mg, or approximately 500 mg to 1 g, and may further optionally contain one or more suitable pharmaceutical carriers, additives and / or excipients.

[0204] In various examples, the pharmaceutical compositions described herein (e.g., pharmaceutical compositions including polypeptide-immobilized EVs such as antibody-immobilized neural EVs) can be formulated for delivery to cells and / or targets via any administration route known to those skilled in the art. The administration methods are generally known or obvious to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences (17th Ed., Mack Pub. Co. 1985).

[0205] Compositions comprising polypeptide-immobilized extracellular molecules (e.g., antibody-immobilized nerve extracellular molecules) provided herein can be delivered to a subject by any suitable route, including, but not limited to, injection, infusion, inhalation, intranasal, intraocular, topical delivery, intercannular delivery, or ingestion. Injections include, but are not limited to, intravenous, intracranial, intrathecal, intramuscular, intraarterial, intracerebroventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injections and infusions. In some examples, administration involves intracerebrospinal injection. In other examples, administration involves aerosol inhalation (e.g., by spraying). In other examples, administration may be systemic (e.g., oral, rectal, nasal, sublingual, oral cavity, or parenteral), intra-intestinal (e.g., systemic effect, but delivered via the CNS), or topical (e.g., topical application to the skin, intravitreal injection). In some embodiments, the administration of polypeptide-loaded extracellular molecules (e.g., antibody-loaded nerve extracellular molecules) allows the antibody-loaded extracellular molecules to be delivered to the central nervous system, and in certain embodiments, across the blood-brain barrier. In some embodiments, administration is performed at a site of disease and / or dysfunction (e.g., the brain). In other embodiments, the site of administration is away from the disease and / or dysfunction (e.g., intravenous or intranasal delivery). In one embodiment, the composition containing polypeptide-loaded extracellular molecules is administered parenterally to the subject. In one embodiment, the composition containing polypeptide-loaded extracellular molecules is administered intravenously to the subject. In another embodiment, the composition containing polypeptide-loaded extracellular molecules is administered intranasally to the subject. In one embodiment, the composition containing polypeptide-loaded extracellular molecules is administered intracranially to the subject. In one embodiment, the composition containing polypeptide-loaded extracellular molecules is administered intrathecally to the subject.

[0206] Injectable compositions for parenteral administration (e.g., intravenous, intramuscular, intrathecal, intracerebrospinal fluid, or intranasal) may contain polypeptide-immobilized extracellular molecules (e.g., antibody-immobilized nerve extracellular molecules) and optionally further components in a suitable IV solution such as sterile saline. In other embodiments, the composition is formulated as a suspension in an aqueous emulsion.

[0207] Liquid pharmaceutical compositions can be prepared by dissolving or dispersing an assembly of polypeptide-immobilized extracellular vehicles (EVs) (e.g., antibody-immobilized nerve EVs) and optionally a pharmaceutical adjuvant in a carrier such as saline solution, aqueous dextrose, glycerol, or ethanol to form a solution or suspension.

[0208] Intravenous formulations may include polypeptide-loaded extracellular viable cells (EVs) as described herein (e.g., antibody-loaded nerve EVs), an isotonic medium, and one or more substances that prevent aggregation of the polypeptide-loaded EVs. Exemplary intravenous / intrathecal / cerebrospinal fluid formulations may contain saline (e.g., physiological saline (NS), about 0.91% w / v NaCl, about 300 mOsm / L) and / or 4% dextrose in 0.18% saline, and optionally 1%, 2%, or 3% human serum albumin. Furthermore, polypeptide-loaded EVs may be destroyed to obtain their contents, which may then be used in the compositions according to the present invention.

[0209] For use in oral liquid preparations, the composition may also be prepared as a solution, suspension, emulsion, or syrup, which is supplied in liquid form or in a dry form suitable for hydration in water or saline. Excipients for oral administration include pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, gelatin, sucrose, and magnesium carbonate. If necessary, the composition may also contain small amounts of non-toxic auxiliary substances such as wetting agents, emulsifiers, or buffers.

[0210] For intranasal, intratracheal, or intrapulmonary administration, the composition may be provided as a liquid or aerosol formulation that can be sprayed into the nose, trachea, and / or lungs.

[0211] When a composition is used in the form of a solid preparation for oral administration, this preparation can be made into tablets, granules, powders, capsules, etc. In tablet formulations, the composition is usually formulated with excipients such as sugars or cellulose preparations, binders such as starch paste or methylcellulose, fillers, disintegrants, and other additives commonly used in the manufacture of pharmaceutical preparations.

[0212] The pharmaceutical compositions provided herein (e.g., pharmaceutical compositions containing polypeptide-charged EVs) may be administered to a subject as a single dose, or multiple doses may be administered over a period of time. For example, two, three, four, five, or more doses may be administered to a subject during a single treatment or over a set period of time. In some examples, six, eight, ten, twelve, fifteen, or twenty or more doses may be administered to a subject as a treatment regimen during a single treatment or over a set period of time. In other examples, administration may be given as needed, for example, as long as symptoms related to neurological disorders persist. In some embodiments, repeated administration is required for the remainder of the subject's life. Exemplary dosing schedules include administration of the pharmaceutical composition containing polypeptide-charged EVs once daily, once every two days, once every three days, once a week, once every two weeks, once a month, once every two months, once every three months, once every six months, once every twelve months, or once every six months or more.

[0213] In some embodiments, the present invention provides a pharmaceutical composition comprising a polypeptide-charged EV (e.g., an antibody-charged nerve EV) further comprising one or more additional therapeutic agents.

[0214] In some embodiments, the EV may include one or more inhibitory nucleic acids. For example, in some embodiments, the EV may include one or more inhibitory nucleic acids selected from short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), antisense oligonucleotide (ASO), and double-stranded RNA (dsRNA).

[0215] In some embodiments, EV may include one or more neurotrophic agents. In some embodiments, EV may include leukemia suppressor (LIF), brain-derived neurotrophic factor (BDNF), epidermal growth factor receptor (EGF), basic fibroblast growth factor (bFGF), FGF-6, glial neurotrophic factor (GDNF), granulocyte colony-stimulating factor (GCSF), hepatocyte growth factor (HGF), IFN-γ, insulin-like growth factor-binding protein (IGFBP-2), IGFBP-6, IL-1ra, IL-6, IL-8, monocyte chemotactic protein (MCP-1), mononuclear phagocyte colony-stimulating factor (M-CSF), and neurotrophic factor (NT3). It may contain one or more chemical substances selected from metalloproteinase tissue inhibitors (TIMP-1), TIMP-2, tumor necrosis factor (TNF-β), vascular endothelial growth factor (VEGF), VEGF-D, urokinase-type plasminogen-activating factor receptor (uPAR), bone morphogenesis protein 4 (BMP4), IL-1-α, IL-3, leptin, stem cell factor (SCF), stromal cell-derived factor-1 (SDF-1), platelet-derived growth factor-BB (PDGFBB), transforming growth factor-β (TGFβ-1), and TGFβ-3.

[0216] The pharmaceutical compositions containing polypeptide-immobilized EVs described herein may be administered to a subject as monotherapy (a single agent) or as combination therapy (the subject is administered the pharmaceutical composition containing polypeptide-immobilized EVs in combination with one or more additional agents). The pharmaceutical compositions containing polypeptide-immobilized EVs and one or more additional agents may be administered to the subject simultaneously, sequentially, or temporarily.

[0217] In one embodiment, the Disclosure provides a pharmaceutical composition comprising polypeptide-immobilized EVs as described herein (for example, a pharmaceutical composition comprising antibody-immobilized neuronal EVs) for use in the treatment of subjects suffering from neurological disorders, such as Alzheimer's disease, Huntington's disease, Parkinson's disease, cancer of the nervous system (e.g., glioblastoma or neuroblastoma), neuromyelitis optica, multiple sclerosis, migraine, chronic inflammatory demyelinating polyneuropathy (CIDP), or multifocal motor neuropathy. In another embodiment, the Disclosure provides a pharmaceutical composition comprising polypeptide-charged EVs (e.g., antibody-charged EVs, including antibody-EV conjugates and EVs with antibodies charged in the lumen) described herein, for use in the manufacture of pharmaceuticals for the treatment of subjects suffering from neurological disorders, such as Alzheimer's disease, Huntington's disease, Parkinson's disease, cancers of the nervous system (e.g., glioblastoma or neuroblastoma), neuromyelitis optica, multiple sclerosis, migraine, chronic inflammatory demyelinating polyneuropathy (CIDP), or multifocal motor neuropathy.

[0218] In some examples, the pharmaceutical formulation may contain approximately 50 ng or more of polypeptide-charged extravalents (EVs) per ml of fluid medium. The exemplary pharmaceutical formulation may contain approximately 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, 1.0 μg, 1.5 μg, 2.0 μg, 2.5 μg, 3.0 μg, 5.0 μg, 10.0 μg, 15.0 μg, 20.0 μg, 100 μg, or more of polypeptide-charged EVs per ml of fluid medium.

[0219] In other embodiments, the pharmaceutical preparation may contain polypeptide-loaded EVs at about 0.1 μg per 1 ml of the medium, polypeptide-loaded EVs at about 0.2 μg per 1 ml of the medium for intravenous administration, polypeptide-loaded EVs at about 0.3 μg per 1 ml of the medium for intravenous administration, polypeptide-loaded EVs at about 0.4 μg per 1 ml of the medium for intravenous administration, polypeptide-loaded EVs at about 0.5 μg per 1 ml of the medium for intravenous administration, polypeptide-loaded EVs at about 0.6 μg per 1 ml of the medium for intravenous administration, polypeptide-loaded EVs at about 0.7 μg per 1 ml of the medium for intravenous administration, polypeptide-loaded EVs at about 0.8 μg per 1 ml of the medium for intravenous administration, polypeptide-loaded EVs at about 0.9 μg per 1 ml of the medium for intravenous administration, polypeptide-loaded EVs at about 1.0 μg per 1 ml of the medium for intravenous administration, polypeptide-loaded EVs at about 1.5 μg per 1 ml of the medium for intravenous administration, polypeptide-loaded EVs at about 2.0 μg per 1 ml of the medium for intravenous administration, polypeptide-loaded EVs at about 2.5 μg per 1 ml of the medium for intravenous administration, polypeptide-loaded EVs at at least for example about 3.0 μg per 1 ml of the medium for intravenous administration, polypeptide-loaded EVs at at least for example about 5.0 μg per 1 ml of the medium for intravenous administration, polypeptide-loaded EVs at about 10.0 μg per 1 ml of the medium for intravenous administration, polypeptide-loaded EVs at 15.0 μg per 1 ml of the medium for intravenous administration, or polypeptide-loaded EVs at about 20.0 μg or more per 1 ml of the medium for intravenous administration.

[0220] In some examples, the administration of the composition is, in a single administration, 1×10 8 or more polypeptide-loaded EVs per kg. In other examples, the administration of the polypeptide-loaded EV composition is 1×10 8 , 1×10 8 to 1×10 9 , 1×10 9 to 1×10 10 , 1×10 10 to 1×10 11 , 1×10 11 to 1×10 12 , 1×10 12or a higher dose of polypeptide-loaded EVs. In some cases, a single dose is administered to the subject multiple times. In other specific cases, multiple doses to the subject include two or more of the following: intravenous, intracerebrospinal, intravenous infusion, and injection.

[0221] In some examples, the pharmaceutical composition is a dosage form comprising at least 1 mg of polypeptide-loaded EV, at least 5 mg of polypeptide-loaded EV, at least 10 mg of polypeptide-loaded EV, at least 20 mg of polypeptide-loaded EV, at least 25 mg of polypeptide-loaded EV, at least 50 mg of polypeptide-loaded EV, at least 60 mg of polypeptide-loaded EV, at least 75 mg of polypeptide-loaded EV, at least 100 mg of polypeptide-loaded EV, at least 150 mg of polypeptide-loaded EV, at least 200 mg of polypeptide-loaded EV, at least 250 mg of polypeptide-loaded EV, at least 300 mg of polypeptide-loaded EV, about 350 mg of polypeptide-loaded EV, about 400 mg of polypeptide-loaded EV, about 500 mg of polypeptide-loaded EV, about 750 mg of polypeptide-loaded EV, or about 1 g (1,000 mg) or more of polypeptide-loaded EV, either alone or in combination with at least one additional drug in a therapeutically effective amount. In some embodiments, the pharmaceutical composition contains approximately 10 mg to approximately 750 mg, approximately 25 mg to approximately 650 mg, or approximately 30 mg to approximately 500 mg, or approximately 35 mg to approximately 450 mg, most often approximately 50 to approximately 500 mg of polypeptide-loaded extravalents (EVs).

[0222] The therapeutically effective dose of a pharmaceutical composition containing polypeptide-immobilized extracellular viable cells (EVs), such as antibody-immobilized EVs (including antibody-EV conjugates and EVs with antibodies loaded in their lumen), is sufficient to treat or improve one or more symptoms of the condition being treated (e.g., neurological disorders, such as Alzheimer's disease, Huntington's disease, or Parkinson's disease, or cancers of the nervous system, such as glioblastoma), while not exceeding a dose that may cause serious side effects. The therapeutically effective dose may depend on numerous factors, such as the characteristics of the condition being treated and the specific individual being treated.

[0223] D. Treatment methods In some embodiments, the present invention provides a method for treating a subject using a composition comprising polypeptide-charged extracellular viable cells (EVs) (e.g., antibody-charged EVs, including antibody-EV conjugates and EVs with antibodies charged in their lumen). Any composition comprising polypeptide-charged EVs or pharmaceutical composition described herein is suitable for use in any of the methods provided herein. In exemplary embodiments, the EVs of the polypeptide-charged EVs are derived from nerve cells, e.g., neural progenitor cells, neurons, or astrocytes. In other exemplary embodiments, the EVs of the polypeptide-charged EVs are produced by synthesis and contain one or more markers characteristic of nerve EVs, e.g., one or more proteins or nucleic acids present in nerve EVs but not present in EVs derived from MSCs.

[0224] In some embodiments, the present invention provides a method for treating (e.g., curing, suppressing, improving associated symptoms, delaying or preventing progression, delaying or preventing onset, or preventing recurrence or relapse) a neurological disorder in a subject, such as Alzheimer's disease, Huntington's disease, Parkinson's disease, cancer of the nervous system (e.g., glioblastoma or neuroblastoma), neuromyelitis optica, multiple sclerosis, migraine, chronic inflammatory demyelinating polyneuropathy (CIDP), or multifocal motor neuropathy, the method comprising the step of administering to the subject a composition comprising polypeptide-charged EV as described herein in an amount sufficient to treat the disease or disorder in the subject. An amount sufficient to treat the disease or disorder is preferably an effective amount, such as a therapeutic effective amount, as provided herein.

[0225] The change in symptoms as a result of treatment can be measured in comparison to any suitable control. For example, the change in symptoms can be measured against the frequency, severity, duration, or number of times the symptoms were experienced by the same patient before the start of treatment. In other embodiments, the change in symptoms can be measured against the frequency, severity, duration, or number of times the symptoms occurred in different subjects or groups of subjects with similar symptoms who were not treated, i.e., not administered the composition containing Ab-EV. In some embodiments, the degree of relief, when determined in comparison to a suitable control, is at least 5%, i.e., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more.

[0226] In some embodiments, the composition containing polypeptide-loaded EVs is administered to the subject as a single dose. In some embodiments, the composition containing Ab-EVs is administered in multiple doses. For example, in some embodiments, the composition may be administered daily, every two days, every three days, every four days, every five days, every six days, every seven days, every two weeks, every three weeks, every four weeks, every eight weeks, or every twelve weeks.

[0227] It will be readily apparent to those skilled in the art that other suitable modifications and alterations of the methods of the present invention described herein can be readily made using suitable equivalent inventions without departing from the scope or embodiments of the present invention disclosed herein. Although the present invention has been described in detail, it will be better understood by referring to the following examples. The examples are for illustrative purposes only and are not intended to limit the invention. [Examples]

[0228] The following examples are for illustrative purposes only and are not intended to limit the scope of the disclosures provided herein.

[0229] Example 1: Preparation of extracellular vesicles of antibody-labeled neural progenitor cells This example illustrates the binding of an antibody (Ab) to extracellular vesicles (EVs) derived from neural progenitor cells using click chemistry.

[0230] A purified population of extracellular vesicles (EVs) was obtained from neural progenitor cells according to the method provided in U.S. Patent Application Publication 2018 / 0327714 (the entire contents of which are incorporated herein by reference). Briefly, human neural progenitor cells were cultured as described in U.S. Patent Application Publication 2018 / 0327714. After 24 hours of medium change, the cell medium was collected from the confluent culture of neural progenitor cells and frozen at -20°C. The medium was thawed overnight at 4°C and filtered through a 0.22 μm filter unit before purification of extracellular vesicles (EVs). Extracellular vesicles were purified from the filtered cell culture medium using tangential flow filtration.

[0231] Goat anti-rabbit IgG antibody labeled with AlexaFluor Plus 647 was selected for proof-of-concept experiments because it was expected to be deficient in reactivity and immunogenicity in a mouse model. The antibody (Ab) was modified to include an azide group using the SITECLICK® antibody azide modification method (see ThermoFisher catalog number S20026). Manufacturer recommendations were followed.

[0232] Separately, the EVs were modified to exhibit sDIBO via SDP ester groups targeting amine groups on the EV surface. sDIBO (CLICK-IT® SDP ester sDIBO alkyne, catalog number C20025) was applied at a molecular weight of 5.95 e per EV. 5 The EV was reacted with the EV in a specific ratio at room temperature for 2 hours. This number was selected to be approximately 10 times the number of Ab molecules to be added in a later step.

[0233] To qualitatively detect a high signal in the liver using a dose escalation study of released, unbound antibodies, approximately 8 × 10⁴ 13 Since it was shown that one injection of Ab was necessary, this was selected as the dose to crosslink to EV. 5.95 e of Ab per EV. 4 Azide-Ab was mixed with sDIBO-EV in a specific ratio. After mixing, the antibody was conjugated to EV by a copper-free click chemistry reaction (Figure 1).

[0234] Example 2: In vivo administration of EV-Ab conjugate This example describes the in vivo administration of extracellular vesicle-antibody (EV-Ab) conjugates in mice. EV-Ab mixture, Ab alone, and PBS (n=1 / condition) were intravenously injected into animals after overnight incubation. The animals were euthanized approximately 2 hours later to preserve the brain. Tissue was fixed, frozen sections were prepared, and background suppression was optionally achieved by treatment with TrueBlack lipofuscin autofluorescence quencher (Biotium). Sections were stained with anti-goat secondary antibody and then imaged.

[0235] Stained and unstained sections were imaged using a Zeiss LSM 710 confocal microscope. To distinguish the signal from the background, images were acquired at multiple wavelengths, which in some cases involved acquiring lambda images with linear unmixing using Zeiss Zen 2012 Blue software. First, randomly selected frames were imaged from PBS-treated animal brain sections to get a feel for the signal-to-noise ratio. After imaging and analyzing untreated brain sections, animals treated with the Ab-EV mixture were imaged as hemispherical slices using an automated tiling Z-stack. After imaging the entire section at multiple wavelengths at low magnification, the section was manually examined using ImageJ software for zooming and scanning. One hemisphere of an Ab-treated animal was similarly scanned and analyzed. After analysis, the Ab-EV hemisphere (Figure 2B) had a different signal pattern than the PBS-treated brain slices or the Ab-only treated brain slices (Figure 2A). Both mice treated with Ab alone and mice treated with PBS exhibited intense punctate fluorescence, which varied in appearance from region to region, indicating autofluorescence. In areas without punctate fluorescence, the signal was completely suppressed by the TrueBlack lipofuscin autofluorescence quencher (Biotium). Ab-EV hemispheres showed similar autofluorescence in the same regions as PBS-treated brain slices and Ab-only treated brain slices. However, in areas suppressed in Ab-only and PBS-treated brains, Ab-EV showed a high-intensity signal accumulation pattern, indicating high-intensity signal accumulation in the cellular shape (Figure 2B), demonstrating delivery of the Ab-EV conjugate to brain tissue.

[0236] Example 3: Insertion of extracellular vesicles into the lumen This example illustrates the loading of polypeptides (e.g., antibodies or luciferase proteins) into the lumen of extracellular vesicles derived from neural progenitor cells by permeabilization treatment of the EV membrane.

[0237] Antibodies were loaded into exosomes (EVs) derived from neural progenitor cells by saponin permeabilization (0.2% w / w), incubation, electroporation, or sonication. Saponin permeabilization provided surfactant-based loading and was combined with shaking (500 rpm). Incubation was performed at room temperature with shaking (1000 rpm). Electroporation was performed using a Neon transfection system at 500 V, 1 ms width, and 12 pulses. Finally, sonication was performed using a Fisherbrand Model 505 sonicator (500 W, 20 kHz) at 20% amplitude, 4 seconds on / 8 seconds off for 12 cycles. After testing these various loading procedures, antibody loading into the exosome lumen was achieved with more than 25,000 antibody molecules per EV. Figure 3A.

[0238] Luciferase protein (62 kDa) was loaded into neural progenitor cell-derived extracellular viable cells (EVs) by saponin permeabilization, incubation, or sonication. Saponin permeabilization provided surfactant-based loading and was combined with sonication before protein addition. Incubation was performed at room temperature with shaking (1000 rpm). Finally, sonication was performed using a Fisherbrand Model 505 sonicator (500 W, 20 kHz). Multiple combinations of amplitude, pulse time, and cycle count were tested (listed in Table 12 below). Since direct probe sonication disrupts the function of luciferase protein, all sonication protocols were performed using a cup-horn accessory. After testing more than 50 loading procedures, a protein loading efficiency of 15% or higher was achieved. Figure 3B shows the loading efficiency for each condition listed in Table 12.

[0239] Table 12: Conditions for testing polypeptides loaded into the lumen of an EV JPEG2026082945000013.jpg250161JPEG2026082945000014.jpg101161

[0240] Reference The references, patents, pending patent applications, and publications cited throughout this application are expressly incorporated by reference in their entirety.

[0241] Equivalent invention Those skilled in the art will recognize, or can verify by commonplace experiment, numerous equivalent inventions of specific embodiments of the invention described herein. Such equivalent inventions are intended to be included in the following claims.

Claims

1. A composition for delivering an antibody or its antigen-binding portion to the target central nervous system (CNS) across the blood-brain barrier, wherein the composition is A composition comprising a conjugate containing the antibody or its antigen-binding portion and an extracellular vesicle (EV) derived from a nerve cell, wherein the antibody or its antigen-binding portion is bound to the surface of the extracellular vesicle by a linker.

2. The composition according to claim 1, wherein the conjugate or EV is administered intravenously or intranasally.

3. The composition according to claim 1, wherein the nerve cells are neural progenitor cells.

4. The composition according to claim 3, wherein the neural progenitor cells are derived from human pluripotent cells.

5. The composition according to claim 4, wherein the human pluripotent cells are human embryonic stem cells or human induced pluripotent stem cells.

6. The antibody or its antigen-binding portion is IgG or an antibody fragment, and the antibody fragment is selected from the group consisting of Fab, F(ab')2, scFv, tandem scFv, diabody, minibody, and single-domain antibody, and / or The composition according to claim 1, wherein the antibody or its antigen-binding portion is a humanized antibody or a fully human antibody or its antigen-binding portion.

7. The composition according to claim 1, wherein the antibody or its antigen-binding portion is delivered to the target brain or spinal cord.

8. The composition according to claim 1, wherein the EV further comprises exogenous nucleic acids and / or exogenous proteins, exogenous siRNA, antisense nucleic acids, and / or low molecules.

9. The composition according to any one of claims 1 to 8, wherein the linker includes a click linker.

10. The composition according to claim 9, wherein the click linker is formed from a reaction between an azide click reaction product and an alkyne click reaction product, between an azide and dibenzocyclooctine (DBCO), between a tetrazine and transcyclooctene, and / or between a tetrazine and norbornene.

11. The composition according to claim 6, wherein the antibody or its antigen-binding portion is one or more of solanezumab, aducanumab, nivolumab, bevacizumab, ocrelizumab, natalizumab, dinutuximab, gantenerumab, lecanemab, or ubrituximab.

12. The composition according to claim 9, wherein the clicklinker is formed from a reaction between azide and dibenzocyclooctin (DBCO), between tetrazine and transcyclooctene, between tetrazine and norbornene, between azide and alkyne, between strain-promoting azide and alkyne, between strain-promoting azide and nitrone, between alkene and azide, between alkene and tetrazine, and / or between alkene and tetrazole.

13. A method for loading an antibody or its antigen-binding portion into the lumen of an extracellular vesicle (EV), the method comprising: i) treating the EV with a saponin to make the membrane of the EV permeable; ii) sonicating the treated EV; and iii) adding the antibody or its antigen-binding portion to the EV to load the antibody or its antigen-binding portion into the lumen of the EV, wherein the combination of the saponin treatment and the sonication treatment improves the efficiency of EV loading compared to saponin treatment or sonication treatment alone.

14. The method according to claim 13, wherein the EV is treated with approximately 0.05% to 0.3% saponin.

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