Polypeptides derived from FC

Abstract

The present disclosure relates to polypeptides comprising a transmembrane domain and an FcRn binding site (e.g., a modified Fc domain), as well as nanovesicles (e.g., extracellular vesicles (EVs) and hybridosomes) comprising such polypeptides. The polypeptides can facilitate the isolation and purification of nanovesicles comprising such polypeptides. The polypeptides and nanovesicles can be used for therapeutic and / or diagnostic applications. Also provided are nucleic acids and expression vectors encoding such polypeptides, as well as cells expressing the polypeptides. Additionally provided are methods for producing nanovesicles comprising such polypeptides and methods for purifying these nanovesicles. Compositions comprising such polypeptides or nanovesicles and their uses are also described.

Description

[Technical field]

[0001] (Priority) This application claims the benefit of priority to U.S. Patent No. 63 / 174,855, filed April 14, 2021, which is incorporated by reference in its entirety.

[0002] (Reference to electronically submitted sequence listing) This application incorporates by reference the Sequence Listing submitted herewith as a text file entitled "14497-004-228_Sequence_Listing.txt", which was created on April 11, 2022, and is 176,537 bytes in size.

[0003] (1. Field) The present disclosure relates to polypeptides comprising a transmembrane domain and an FcRn binding site (e.g., a modified Fc domain), as well as nanovesicles (e.g., extracellular vesicles (EVs) and hybridosomes) comprising such polypeptides. The polypeptides can facilitate the isolation and purification of nanovesicles comprising such polypeptides. The polypeptides and nanovesicles can be used for therapeutic and / or diagnostic applications. Also provided are nucleic acids and expression vectors encoding such polypeptides, as well as cells expressing the polypeptides. Additionally provided are methods for producing nanovesicles comprising such polypeptides and methods for purifying these nanovesicles. Compositions comprising such polypeptides or nanovesicles and their uses are also described. [Background technology]

[0004] (2. Background of the Disclosure) Despite major breakthroughs in identifying new promising drug candidates, the translation of these findings to the clinic is often hindered by challenges in delivering effective drug dosages to the site of disease. Recently discovered cell-to-cell communication pathways may provide the missing puzzle piece for more precise drug delivery. It has become clear that almost every cell in our body can establish connections with neighboring and distant cells by releasing small "balloons" called extracellular vesicles (EVs). The discovery that these EVs, especially exosomes, are functional shuttles for signaling molecules has led to the determination that genetically they could be ideal nanoscale candidates for drug delivery systems for modern pharmaceuticals. However, this idea is coupled with several challenges, including those regarding the preparation and isolation of EVs and the extension of their half-life in the circulation. Therefore, appropriate methods and compositions involved in the generation, isolation and purification of EVs are needed to better enable therapeutic use and other applications of EV-based technologies.

[0005] Thus, there is a need for improved methods for the preparation of membrane vesicles that are suitable for industrial constraints and allow the generation of therapeutic quality vesicle preparations. To that end, International Patent Application Publication WO2019 / 081474 discloses a chromatographic technique for capturing EVs engineered to contain Fc-binding polypeptides by using the Fc domain of an antibody that binds to a chromatographic matrix and inducing elution of the captured EVs by lowering the pH to less than 8, preferably less than 6. However, significant improvements over said methods are needed, especially as the EV therapeutic field progresses towards clinical application and impact of EV-based therapy.

[0006] Citation of any reference herein shall not be construed as an admission that such is prior art to the present disclosure. Summary of the Invention

[0007] (3. Disclosure Overview) In one aspect, provided herein is a polypeptide comprising: a. a transmembrane domain; and bi capable of specifically binding to the Fc binding site of FcRn; and ii. a modified Fc domain of an immunoglobulin that lacks the ability to form homodimers.

[0008] In certain embodiments, the equilibrium dissociation constant of the modified Fc domain binding to FcRn at pH 6.5 is at most 10 -4 In certain embodiments, the equilibrium dissociation constant of the modified Fc domain binding to FcRn at pH 7.4 is at least 10 -4 It has a value of M.

[0009] In certain embodiments, the modified Fc domain is capable of specifically binding to an amino acid sequence between positions 135 and 158 of human FcRn (SEQ ID NO: 7) and / or mouse FcRn (SEQ ID NO: 8). [ka] wherein each of X1, X2, X3, X4, X5, X6, X7 and X8 is any amino acid.

[0010] In certain embodiments, the polypeptide does not substantially bind to C1q, FcγRI, FcγRII or FcγRIII.

[0011] In certain embodiments, the complement dependent cytotoxicity (CDC) activity of the modified Fc domain, the antibody dependent cell-mediated cytotoxicity (ADCC) activity of the modified Fc domain, the antibody dependent cell-mediated phagocytosis (ADCP) activity of the modified Fc domain, and / or the antibody dependent intracellular neutralization (ADIN) activity of the modified Fc domain is reduced by at least 10%, 20%, 30%, 40% or 50% compared to the unmodified Fc domain.

[0012] In certain embodiments, the complement dependent cytotoxicity (CDC) activity of the modified Fc domain, the antibody dependent cell-mediated cytotoxicity (ADCC) activity of the modified Fc domain, the antibody dependent cell-mediated phagocytosis (ADCP) activity of the modified Fc domain, and / or the antibody dependent intracellular neutralization (ADIN) activity of the modified Fc domain is reduced by at least 1.5, 2, 3, 4, or 5 fold compared to the unmodified Fc domain.

[0013] In certain embodiments, the FcRn-binding polypeptide comprises, from N-terminus to C-terminus: a. a modified CH2 domain that has been modified to reduce effector function relative to the unmodified CH2 domain; b. a modified CH3 domain that has been modified to lack homodimerization relative to the unmodified CH3 domain; c. a linker sequence; and d. a transmembrane domain.

[0014] In certain embodiments, the FcRn-binding polypeptide comprises, from the C-terminus to the N-terminus: a. a modified CH3 domain modified to lack homodimerization relative to the unmodified CH3 domain; b. a modified CH2 domain modified to reduce effector function relative to the unmodified CH2 domain; c. a linker sequence; and d. a transmembrane domain.

[0015] In various embodiments, the transmembrane domain is a multi-spanning transmembrane domain.

[0016] In specific embodiments, the polypeptide is an scFv, (scFv)2, Fab, Fab', F(ab')2, F(abl)2, Fv, dAb, Fd fragment, diabody, F(ab)2, F(ab'), F(ab')3, Fd, Fv, disulfide linked Fv, dAb, sdAb, nanobody, CDR, di-scFv, bi-scFv, tascFv (tandem scFv), avibody (e.g., diabody, triabody, tetrabody), T, or the like. -Further comprising a targeting domain selected from the group consisting of a cell engager (BiTE), a V-NAR domain, an Fcab, an IgGACH2, a DVD-Ig, a probody, an intrabody, a DARPin, a sentinelin, an affibody, an affilin, an affitin, an anticalin, an avimer, a finomer, a Kunitz domain peptide, a monobody, an adnectin, a tribody, and a nanophytin.

[0017] In another aspect, provided herein is a nucleic acid encoding a polypeptide described herein.

[0018] In another aspect, provided herein is an expression vector comprising a nucleic acid described herein.

[0019] In another aspect, provided herein is a cell comprising a nucleic acid described herein or an expression vector described herein.

[0020] In another aspect, provided herein are extracellular vesicles comprising the polypeptides described herein.

[0021] In another aspect, provided herein is a hybridosome comprising a polypeptide described herein.

[0022] In another aspect, provided herein is a method for purifying extracellular vesicles (EVs), comprising: a. providing EVs, the EVs associated with a first binding partner, the first binding partner being capable of binding to the Fc binding site of FcRn in a pH-dependent manner; and b. contacting the EVs associated with the first binding partner with a second binding partner at a first pH, the second binding partner comprising the Fc binding site of FcRn and associated with a solid matrix; and c. eluting the EVs associated with the first binding partner from the solid matrix at a second pH. In certain embodiments, the method comprises a washing step at a first pH. In certain embodiments, the first pH is less than 6.5. In certain embodiments, the second pH is greater than 7.4.

[0023] In another aspect, provided herein is a method for purifying extracellular vesicles (EVs), comprising: a. providing EVs, the EVs associated with a first binding partner, the first binding partner being capable of binding to the Fc binding site of FcRn in a pH-dependent manner, and comprising or consisting of a polypeptide as described herein; and b. contacting the EVs associated with the first binding partner with a second binding partner at a first pH, the second binding partner comprising the Fc binding site of FcRn and associated with a solid matrix; and c. eluting the EVs associated with the first binding partner from the solid matrix at a second pH. In certain embodiments, the method comprises a washing step at a first pH. In certain embodiments, the first pH is less than 6.5. In certain embodiments, the second pH is greater than 7.4.

[0024] The present disclosure aims to meet existing needs in the art, such as to provide a means for isolation / separation of extracellular vesicles that can be performed at conditions (e.g., pH values) closer to physiological conditions, and to allow for a longer half-life of EVs in circulation, significantly enhancing the therapeutic potential of EVs for therapeutic drug delivery.

[0025] Provided herein are FcRn-binding polypeptides (often referred to herein as FcRn-binding agents) that contain a transmembrane domain.

[0026] In one aspect, provided herein is a system for purification of nanovesicles of interest (e.g., EVs), comprising a neonatal Fc receptor (FcRn) binding agent and a mammalian FcRn, wherein the FcRn binding agent and the FcRn bind to each other with high affinity under a first set of conditions and with lower affinity under a second set of conditions. 3.1 Illustrative Implementations 1. A polypeptide comprising: a. a transmembrane domain; and bi capable of specifically binding to the Fc binding site of FcRn; and ii. lacks the ability to form homodimers Engineered Fc domains of immunoglobulins The polypeptide comprising: 2. The equilibrium dissociation constant of the engineered Fc domain binding to FcRn at pH 6.5 is at most 10 -4 2. The polypeptide of item 1, having a value of M. 3. The equilibrium dissociation constant of the engineered Fc domain binding to FcRn at pH 7.4 is at least 10 -4 3. The polypeptide of claim 1 or 2, having a value of M. 4. The polypeptide according to any one of items 1 to 3, wherein the modified Fc domain is capable of specifically binding to an amino acid sequence between positions 135 to 158 of human FcRn (sequence number 7) and / or mouse FcRn (sequence number 8). 5. The modified Fc domain has the amino acid sequence [ka] wherein each of X1, X2, X3, X4, X5, X6, X7 and X8 is any amino acid. 6. The polypeptide of any one of paragraphs 1 to 5, wherein the polypeptide does not substantially bind to C1q, FcγRI, FcγRII, or FcγRIII. 7. a. Complement-dependent cytotoxicity (CDC) activity of modified Fc domains; b. antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the modified Fc domain; c. antibody-dependent cell-mediated phagocytosis (ADCP) activity of the modified Fc domain; and / or d. Antibody-dependent intracellular neutralization (ADIN) activity of modified Fc domains The polypeptide according to any one of items 1 to 6, wherein the amount of the modified Fc domain is reduced by at least 10%, 20%, 30%, 40% or 50% compared to an unmodified Fc domain. 8. a. Complement-dependent cytotoxicity (CDC) activity of the modified Fc domain; b. antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the modified Fc domain; c. antibody-dependent cell-mediated phagocytosis (ADCP) activity of the modified Fc domain; and / or d. Antibody-dependent intracellular neutralization (ADIN) activity of modified Fc domains The polypeptide according to any one of paragraphs 1 to 7, wherein the polypeptide has at least a 1.5, 2, 3, 4, or 5-fold reduction compared to an unmodified Fc domain. 9. An FcRn-binding polypeptide comprising, from N-terminus to C-terminus: a. a modified CH2 domain that has been modified to have reduced effector function relative to the unmodified CH2 domain; b. a modified CH3 domain that has been modified to lack homodimerization relative to the unmodified CH3 domain; c. a linker sequence; and d. Transmembrane domain The polypeptide according to any one of items 1 to 8, comprising: 10. An FcRn-binding polypeptide comprising, from C-terminus to N-terminus: a. a modified CH3 domain that has been modified to lack homodimerization relative to the unmodified CH3 domain; b. a modified CH2 domain that has been modified to have reduced effector function relative to the unmodified CH2 domain; c. a linker sequence; and d. Transmembrane domain The polypeptide according to any one of items 1 to 9, comprising: 11. The polypeptide of any one of paragraphs 1 to 10, wherein the transmembrane domain is a multi-spanning transmembrane domain. 12. 12. The polypeptide of any one of paragraphs 1-11, further comprising a targeting domain selected from the group consisting of: scFv, (scFv)2, Fab, Fab', F(ab')2, F(abl)2, Fv, dAb, Fd fragment, diabody, F(ab)2, F(ab'), F(ab')3, Fd, Fv, disulfide-linked Fv, dAb, sdAb, nanobody, CDR, di-scFv, bi-scFv, tascFv (tandem scFv), avibody (e.g., diabody, triabody, tetrabody), T-cell engager (BiTE), V-NAR domain, Fcab, IgGACH2, DVD-Ig, probody, intrabody, darpin, centilin, affibody, affilin, affitin, anticalin, avimer, finomer, Kunitz domain peptide, monobody, adnectin, tribody, and nanophytin. 13. A nucleic acid encoding the polypeptide of any one of items 1 to 12. 14. An expression vector comprising the nucleic acid described in item 13. 15. A cell comprising the nucleic acid according to item 13 or the expression vector according to item 14. 16. An extracellular vesicle comprising a polypeptide described in any one of items 1 to 12. 17. A hybridosome comprising the polypeptide described in any one of items 1 to 12. 18. A method for purifying extracellular vesicles (EVs), comprising: a. providing an EV, the EV associated with a first binding partner, the first binding partner capable of binding to the Fc binding site of FcRn in a pH-dependent manner; and b. contacting the EVs associated with the first binding partner with a second binding partner at a first pH, the second binding partner comprising the Fc binding site of FcRn and associated with a solid matrix; and c. Eluting the EVs associated with the first binding partner from the solid matrix at a second pH. The method comprising: 19. The method of claim 18, wherein the method includes a washing step at a first pH. 20. The method of claim 18 or 19, wherein the first pH is less than 6.5. 21. The method of any one of claims 18 to 20, wherein the second pH is greater than 7.4. 22. A method for purifying EVs, comprising: a. providing an EV, said EV associating with a first binding partner, said first binding partner being capable of binding to the Fc binding site of FcRn in a pH-dependent manner, said first binding partner comprising or consisting of a polypeptide according to any one of claims 1 to 12; and b. contacting the EVs associated with the first binding partner with a second binding partner at a first pH, the second binding partner comprising the Fc binding site of FcRn and associated with a solid matrix; and c. Eluting the EVs associated with the first binding partner from the solid matrix at a second pH. The method comprising: 23. The method of claim 22, wherein the method includes a washing step at a first pH. 24. The method of claim 22 or 23, wherein the first pH is less than 6.5. 25. The method of any one of claims 22 to 24, wherein the second pH is greater than 7.4. [Brief description of the drawings]

[0027] (4. BRIEF DESCRIPTION OF THE DRAWINGS) [Figure 1] FIG. 1 is a schematic diagram of a nanovesicle comprising an FcRn-binding polypeptide containing a type 1 transmembrane domain.

[0028] [Diagram 2] Figure 2 shows examples of the location of the modified Fc (CH2 and monomeric CH3) relative to the transmembrane helices (TMH) of different transmembrane scaffolds, including the T1, T2, and PT scaffolds. In the case of the PT scaffold, the FcRn binding site can be located at the N-terminus (PTa), C-terminus (PTb), or extracellular loop (PTc).

[0029] [Diagram 3] FIG. 3. Exemplary structures of FcRn-binding polypeptides comprising monomeric Fc fused to a scaffold protein derived from the extracellular domain of an Eph receptor.

[0030] [Figure 4] Figure 4. Western blot showing EVs engineered and purified from conditioned media.

[0031] [Diagram 5] 5A-5D. Flow cytometry histograms of different cell lines stained with fluorescent anti-human Fc domain antibodies as described in Example 2.

[0032] [Figure 6] Figure 6. Nanoparticle tracking analysis (NTA) measurements of EVs incubated at different pH levels for 20 min.

[0033] [Figure 7] Figure 7. Anti-FcRn Western blot showing purification of scFcRn.

[0034] [Figure 8] Figure 8. Anti-EphA4 Western blot showing detection of EphA4 fusion protein in concentrated conditioned medium expressed from constructs loaded onto an scFcRn column. The first lane is the load, the second lane is a sample of the flow-through, and the third lane is a sample of the elution fraction.

[0035] [Figure 9]Figures 9A and 9B. Anti-EphA4 Western blot showing detection of EphA4 fusion protein in concentrated conditioned medium expressed from constructs loaded onto an scFcRn column at different pH. In Figure 9A, the conditioned medium was not acidified, and in Figure 9B, the conditioned medium was acidified as described in Example 7. The first lane is the elution sample, the second lane is the flow-through sample, and the third lane is the conditioned medium sample.

[0036] [Figure 10] Figures 10A and 10B show the binding curves of human FcRn binding immunoassays with EVs expressing modified Fc domains (Figure 10A), native EVs (Figure 10A), human IgG1 (Figure 10B) and mouse IgG1 (Figure 10B).

[0037] [Figure 11] Figure 11. DNA vector copy numbers per μl of mouse plasma 3, 6, 21, and 24 days after IV administration of EVs containing a scaffold protein displaying a modified Fc domain compared to IV administration of the LNP formulation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] (5. Detailed Description) Provided herein are polypeptides comprising a transmembrane domain and an FcRn binding site (e.g., a modified Fc domain of an immunoglobulin). In certain embodiments, the FcRn binding site (e.g., a modified Fc domain of an immunoglobulin) can specifically bind to the Fc binding site of FcRn and lacks the ability to form homodimers. Various aspects and embodiments of the polypeptides are described in Section 5.2.

[0039] Also provided are nucleic acids encoding the polypeptides described herein, expression vectors comprising the nucleic acids described herein, and cells comprising the nucleic acids or expression vectors described herein, all of which are further described in Section 5.3.

[0040] Further provided are nanovesicles (e.g., EVs and hybridosomes) comprising the polypeptides described herein. Nanovesicles (e.g., EVs and hybridosomes) are further described in Section 5.4.

[0041] Methods for producing nanovesicles (e.g., EVs or hybridosomes) are provided and are further described in Section 5.4. Methods for purifying nanovesicles (e.g., EVs or hybridosomes) are also provided and are further described in Section 5.5.

[0042] Compositions and kits comprising the polypeptides, nanovesicles (e.g., EVs or hybridosomes), nucleic acids, expression vectors, or cells described herein are provided and are further described in Section 5.6.

[0043] Therapeutic and diagnostic uses of the polypeptides, nanovesicles (e.g., EVs or hybridosomes), compositions, or kits described herein are provided and are further described in Section 5.7.

[0044] It is an object of the present disclosure to overcome the problems associated with the isolation and purification of nanovesicles (e.g., EVs). Furthermore, the present disclosure aims to meet other existing needs in the art, such as developing a generally applicable affinity purification strategy for purifying nanovesicles (e.g., EVs) with high yield and high specificity. In particular, previously known methods for purifying exosomes are not ideal for the large-scale production and scale-up required for commercial production of EV therapeutics. The present disclosure enables the purification of engineered EVs with high affinity at a much larger scale than would be achievable with previously known methods.

[0045] The inventors have developed methods and compositions that allow for specific interactions between engineered nanovesicles (e.g., EVs) and FcRn receptors that may be present intracellularly or as binders for affinity chromatography. In particular, it has been demonstrated that some nanovesicles (e.g., EVs) have poor colloidal stability at low pH, contain pH-labile components, and are not suitable for the low pH elution typically employed in fc binding-based affinity chromatography, but efficient affinity chromatography requires fc binders whose binding specificity can be tuned in the pH range of about 5-8.

[0046] Disclosed herein are nanovesicles (e.g., EVs) comprising FcRn-binding agents that bind to FcRn with high affinity and specificity. Advantageously, some of the FcRn-binding agents described herein have one or more improved or desired pharmacokinetic properties, such as circulating half-life. Without wishing to be bound by theory, it is believed that nanovesicles (e.g., EVs) can have a range of circulating half-lives in humans, which may affect, for example, interactions with serum and cellular components, interactions with FcRn, receptor-mediated endocytosis, drug dosage, and anti-drug antibody generation. Nucleic acid molecules encoding fusion polypeptides, expression vectors, host cells, compositions (e.g., pharmaceutical compositions), kits, containers, and methods of making FcRn-binding nanovesicles (e.g., EVs) are also provided. The polypeptides (e.g., antibody molecules or fusion proteins) and pharmaceutical compositions disclosed herein can be used (alone or in combination with other drugs or treatment modalities) to treat, prevent, and / or diagnose disorders and conditions, e.g., disorders and conditions associated with a target molecule (e.g., protein) or cell, e.g., a disorder or condition described herein.

[0047] Without wishing to be bound by theory, in some embodiments, the engineering of Fc for FcRn binding or half-life extension as disclosed herein is believed to be done in the context of various effector functions mediated by Fc. For example, structural information can be used to investigate the interaction of Fc with FcRn at neutral and acidic pH. This structural information can be used to identify different structures for improving FcRn binding at acidic pH. Combinations of Fc mutations can be evaluated for binding to FcRn and other Fc receptors. For example, Fc variants that have extended half-life and retain, or in some cases, reduced effector functions such as ADCC and CDC can be identified. With the increasing interest in employing nanovesicles (e.g., EVs) as therapeutics for the prevention and treatment of different diseases, there is an increasing need to develop nanovesicles containing FcRn-binding polypeptides with long half-life, for example, for the treatment or prevention of chronic diseases.

[0048] Furthermore, all FcRn-binding polypeptides and proteins identified herein can be freely combined into fusion proteins using conventional strategies for fusing polypeptides. As a non-limiting example, all FcRn-binding polypeptides described herein can be freely combined with one or more EV polypeptides in any combination. FcRn-binding polypeptides can also be combined with each other to generate fusion proteins containing two or more FcRn-binding polypeptides. Furthermore, any and all features (e.g., any and all members of the Markush group described herein) can be freely combined with any and all other features (e.g., any and all members of any other Markush group described herein), e.g., any EV containing an FcRn-binding polypeptide can be purified and / or isolated using any FcRn domain-containing polypeptide. Furthermore, where the teachings herein refer to a singular nanovesicle (e.g., EV) (and / or an EV comprising an FcRn-binding polypeptide) and / or to nanovesicles (e.g., EVs) as separate, naturally occurring nanoparticle-like vesicles, it should be understood that all such teachings are equally relevant and applicable to a plurality of nanovesicles (e.g., EVs) and populations of nanovesicles (e.g., EVs).

[0049] (5.1 Definition) As used herein, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. So, for example, reference to a "polypeptide" can include two or more such molecules, and the like.

[0050] As used herein, the terms "about" and "approximately," when used to modify a quantity specified in a numerical value or range, indicate a reasonable deviation from the numerical value that is known to one of ordinary skill in the art, e.g., ±20%, ±10%, or ±5%, within the intended meaning of the recited value.

[0051] The terms "genetically modified" and "genetically engineered" EVs indicate that the EVs are derived from genetically modified / engineered cells that contain recombinant or exogenous protein products that are incorporated into the nanovesicles (e.g., EVs) typically produced by those cells. The term "modified EVs" indicates that the vesicles have been modified using either genetic or chemical approaches, e.g., via genetic engineering of the EV-producing cells, or, e.g., via chemical conjugation to attach moieties to the exosome surface.

[0052] A "binding domain" is a peptide region, such as a fragment of a polypeptide derived from an immunoglobulin (e.g., an antibody), that specifically binds to one or more specific binding partners. When multiple binding partners are present, the partners share sufficient binding determinants to detectably bind to the binding domain. Preferably, a binding domain is a contiguous sequence of amino acids.

[0053] The term "FcRn" refers to the neonatal Fc receptor. FcRn functions to recycle IgG from the lysosomal degradation pathway, resulting in reduced clearance and increased half-life. FcRn is a heterodimeric protein consisting of two polypeptides, the 50 kDa class I major histocompatibility complex-like protein (a-FcRn) and the 15 kDa β2 microglobulin (β2ι). FcRn binds with high affinity to the CH2-CH3 portion of the Fc domain of IgG. FcRn interacts with the Fc region of antibodies and promotes their recycling via rescue from normal lysosomal degradation. This process is a pH-dependent process that occurs within endosomes at acidic pH (e.g., pH less than 6.5) but not under physiological pH conditions of the bloodstream (e.g., non-acidic pH). An acidic pH is a pH less than about 7.0, e.g., about pH 6.5, about pH 6.0, about pH 5.5, about pH 5.0. An elevated non-acidic pH is a pH of about 7 or higher, for example, about pH 7.4, about pH 7.6, about pH 7.8, about pH 8.0, about pH 8.5, or about pH 9.0. FcRn then promotes recycling of the FcRn-binding polypeptide to the cell surface, followed by release into the bloodstream when the FcRn-FcRn-binding polypeptide complex is exposed to the extracellular neutral pH environment.

[0054] As used herein, "FcRn binding site" refers to the region of an Fc polypeptide that binds to FcRn.

[0055] As used herein, "Fc binding site" refers to the region of an FcRn polypeptide that binds to the Fc domain of an immunoglobulin.

[0056] The term "specifically binds" refers to a molecule (e.g., a Fab, scFv, or modified Fc polypeptide (or target-binding portion thereof) that binds to an epitope or target in a sample with greater affinity, greater avidity, and / or longer duration for that epitope or target than it would bind to another epitope or non-target compound (e.g., a structurally distinct antigen). In some embodiments, a Fab, scFv, or modified Fc polypeptide (or target-binding portion thereof) that specifically binds to an epitope or target is a Fab that specifically binds to an epitope or target with greater affinity, greater avidity, and / or longer duration than it would bind to another epitope or non-target compound (e.g., a structurally distinct antigen). A Fab, scFv, or modified Fc polypeptide (or target-binding portion thereof) that binds to an epitope or target with at least 5-fold greater affinity than the epitope or non-target compound, e.g., at least 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 100-fold, 1000-fold, 10,000-fold, or more affinity. As used herein, the terms "specific binding," "specifically binds," or "is specific" for a particular epitope or target refers to, for example, an affinity of at least 10-fold greater than the epitope or target to which it binds. -4 M or less, e.g., 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 This can be represented by a molecule with an equilibrium dissociation constant KD of M. One of skill in the art will recognize that a Fab or scFv that specifically binds to a target from one species may also specifically bind to an ortholog of that target.

[0057] As used herein, the terms "CH3 domain" and "CH2 domain" refer to immunoglobulin constant region domain polypeptides. For purposes of this application, CH2 and CH3 domain polypeptides may be numbered according to the IMGT (ImMunoGeneTics) numbering scheme, where CH2 domain numbering is 1-110 and CH3 domain numbering is 1-107 according to the IMGT Scientific chart numbering (IMGT website). CH2 and CH3 domains are part of the Fc region of an immunoglobulin. Alternatively, CH2 and CH3 domain polypeptides may be numbered according to the EU numbering scheme, where CH2 domain numbering spans residues 231-340 and CH3 domain numbering spans residues 341-447 according to the EU numbering scheme. The Fc region refers to the segment of amino acids from about 231 to about 447 when numbered according to the EU numbering scheme. "EU numbering scheme" refers to the EU numbering rules for antibody constant regions as described in Kabat et al., Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, 5th ed., 1991, each of which is incorporated herein by reference in its entirety.

[0058] As used herein, the term "scaffold protein" refers to a polypeptide that can be used to anchor FcRn-binding polypeptides to nanovesicles. In some embodiments, the scaffold protein is a polypeptide that does not naturally occur in nanovesicles (e.g., EVs). In some embodiments, the scaffold protein comprises a synthetic polypeptide. In some embodiments, the scaffold protein comprises a modified protein, and the corresponding unmodified protein naturally occurs in nanovesicles, e.g., exosomes. In some embodiments, the scaffold protein comprises a protein that naturally occurs in EVs, or a fragment thereof, e.g., a fragment of an EV protein, and the protein is expressed at a level higher than the naturally occurring level.

[0059] In some embodiments, the scaffold protein comprises a fusion protein comprising (i) a naturally occurring EV protein or a fragment thereof and (ii) a heterologous polypeptide (e.g., an FcRn binding polypeptide, an antigen binding domain, or any combination thereof).

[0060] As used herein, the term "scaffold protein" of the present disclosure, or grammatical variations thereof, refers to a

[0061] (i) a polypeptide (naturally expressed, chemically or enzymatically synthesized, or recombinantly produced) that comprises at least one FcRn binding site and further comprises a transmembrane domain that spans the membrane of the nanovesicle, e.g., the exosome;

[0062] (ii) any functional fragment of (i);

[0063] (iii) any functional variant of (i) or (ii);

[0064] (iv) any derivative of any of (i) to (iii);

[0065] (v)(i) any peptide corresponding to a domain derived from a protein capable of spanning the membrane of a nanovesicle (e.g., an exosome), or a molecule containing such a polypeptide, or a combination thereof;

[0066] (vi) an FcRn-binding polypeptide described herein;

[0067] (vii) any of molecules (i)-(vi) comprising at least one unnatural amino acid; or

[0068] (viii) Any combination of (i) to (vii) It may be

[0069] This makes it suitable for use as a scaffold to target (attach) FcRn binding sites to surface nanovesicles, e.g., exosomes.

[0070] As used herein, the term "surface-modified" refers to nanovesicles that contain scaffold proteins to which molecules of interest (e.g., proteins) are attached. The scaffold proteins can be altered by chemical, physical, or biological methods, or by being produced from cells that have been modified by chemical, physical, or biological methods. In particular, the scaffold proteins can be altered by genetic engineering, such that cells previously modified by genetic engineering produce such modified scaffold proteins.

[0071] A "fused" polypeptide sequence is linked via a peptide bond between two subject polypeptide sequences.

[0072] As used herein, the term "domain" refers to a unit (e.g., a segment) of a polypeptide that can fold independently into a stable tertiary structure. Generally, domains are responsible for separate functional properties of a protein and can often be added, removed, or transferred to other proteins without losing the function of the remainder of the protein and / or the domain. Several separate domains can be combined in different combinations to form a multi-domain polypeptide. Traditionally, the length of a polypeptide that spans a domain has been worked out by using atomic coordinates from an experimentally determined three-dimensional structure of the protein. More recently, proteins that lack an experimentally determined three-dimensional (3D) structure have been assigned domains by computational methods based on sequence homology. Because many proteins do not have a separated structure, sequence-based approaches have received much more attention. Sequence-based approaches include template-based, homology modeling-based and machine learning-based techniques, depending on whether the prediction methods utilize 3D structures or homologous sequences, as reviewed in Wang, Yan et al., Computational and structural biotechnology journal Vol. 19, pp. 1145-1153. Feb. 2, 2021. Some computationally predicted domains are cataloged in publicly available databases (e.g., the Pfam database in J. Mistry, S. et al., Nucleic Acids Research (2020), 2021, or the NCBI Conserved Domain Database (CDD) https: / / www.ncbi.nlm.nih.gov / Structure / cdd / cdd.shtml).

[0073] The term "interdomain linker" refers to a segment of a polypeptide that connects two adjacent domains together. Interdomain linkers facilitate domain movement and provide flexibility to modulate the interdomain arrangement, as described in Bhaskara RM, et al., J Biomol Struct Dyn. 2013 Dec; 31(12):1467-80. Interdomain linkers modulate the interactions through the length, conformation, intermolecular interactions, and local structure of adjacent domains, thereby affecting the overall arrangement between domains. The above databases based on predicted structural domains (Pfam database or NCBI conserved domain database) provide generalizations of domains and may provide only approximations of domain boundaries (e.g., to distinguish residues that are within a domain or that are interdomain linkers). Thus, the domain sequences described herein (e.g., sequences in Tables 2-20) may include polypeptide sequences that include the corresponding domains as well as interdomain linkers. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues at the N-terminus or C-terminus of a cataloged domain sequence can be an interdomain linker. One of skill in the art can determine the segments of the polypeptide chain that correspond to the domain and the interdomain linker, where the transition from the domain (i.e., at the domain boundary) to the interdomain linker occurs.

[0074] The term "fusion polypeptide" refers to an FcRn-binding polypeptide or an amino acid sequence derived from a polypeptide operably linked to at least a second polypeptide or an amino acid sequence derived from at least a second polypeptide. The individualized elements of the fusion protein can be linked in any of a variety of ways, including, for example, direct binding, use of an intermediate or spacer peptide, use of a linker region, use of a hinge region, or use of both a linker region and a hinge region. In some embodiments, the linker region is within the sequence of the hinge region, or alternatively, the hinge region may be within the sequence of the linker region. Preferably, the linker region is a peptide sequence. For example, the linker peptide comprises in the range of 0-40 amino acids, for example, 0-35 amino acids, 0-30 amino acids, 0-25 amino acids, or 0-20 amino acids. Preferably, the hinge region is a peptide sequence. For example, the hinge peptide comprises in the range of 0-75 amino acids, for example, 0-70 amino acids, 0-65 amino acids, or 0-62 amino acids.

[0075] With respect to a CH3 or CH2 domain, the terms "wild-type," "native," and "naturally occurring" are used herein to refer to a domain having a sequence that occurs in nature.

[0076] As used herein, with respect to a mutant polypeptide or mutant polynucleotide, the term "mutant" is used interchangeably with "variant". In certain embodiments, with respect to a reference sequence of a given wild-type CH3 or CH2 domain (e.g., Fc domain) of IgG or a wild-type scaffold protein reference sequence, the variant can include a naturally occurring allelic variant. A "non-naturally occurring" variant refers to a variant or mutant domain that does not exist in a cell in nature and is generated, for example, by genetic modification of a parent Fc domain polynucleotide using genetic engineering or mutagenesis techniques to introduce appropriate modifications into the nucleic acid sequence encoding the polypeptide, or by protein / peptide synthesis. A "variant" includes any sequence that contains at least one amino acid mutation with respect to the wild type. Mutations can include substitutions, insertions, and deletions (e.g., truncations) of one or more amino acids in another protein, as well as frameshifts or rearrangements. Similarly, with respect to a polynucleotide, the term "variant" refers to a polynucleotide that differs in base sequence from a particular parent polynucleotide. The identity of the parent polypeptide or polynucleotide will be clear from the context. A variant can contain one or more specific substitutions, insertions, and / or deletions relative to the parent sequence and have a percent sequence identity.

[0077] The term "amino acid substitution" refers to the replacement of at least one existing amino acid residue with another different amino acid residue (substitute amino acid residue). The substitute amino acid residue is a "naturally occurring amino acid residue" and may be selected from the group consisting of alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gin, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

[0078] The term "amino acid insertion" refers to the incorporation of at least one amino acid residue at a given position in the amino acid sequence. In one embodiment, the insertion will be of one or two amino acid residues. The inserted amino acid residue(s) can be any naturally occurring or non-natural amino acid residue. The term "amino acid deletion" refers to the removal of at least one amino acid residue at a given position in the amino acid sequence.

[0079] The term "unnatural amino acid residue" refers to an amino acid residue that can be covalently linked to an adjacent amino acid residue in a polypeptide chain other than the naturally occurring amino acid residues listed above. Examples of unnatural amino acid residues include norleucine, ornithine, norvaline, homoserine. Further examples are described in Ellman et al., Meth. Enzym. 202 (1991) 301-336. Exemplary methods for the synthesis of unnatural amino acid residues are reported, for example, in Noren et al., Science 244 (1989) 182 and Ellman et al., supra.

[0080] With respect to a reference polypeptide sequence, "percent (%) amino acid sequence identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0081] The terms "extracellular vesicles", "EVs" or "exosomes" are used interchangeably herein and are understood to refer to any type of vesicle that can be obtained from cells of any form, such as microvesicles (e.g., any vesicle shed from the plasma membrane of a cell), exosomes (e.g., any vesicle derived from the endosome, lysosome and / or endo-lysosomal pathway), apoptotic bodies, ARMM (arrestin domain-containing protein 1-mediated microvesicles), fusosomes, microparticles and cell-derived vesicular structures. In general, extracellular vesicles have a hydrodynamic diameter ranging from 20 nm to 1000 nm and can contain a variety of macromolecular carriers within the interior space and / or across the membrane presented on the outer surface of the extracellular vesicle. The carriers can include nucleic acids, proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. By way of example, and without limitation, extracellular vesicles include apoptotic bodies, cell fragments, vesicles derived from cells by direct or indirect manipulation (e.g., by continuous extrusion, sonication, or treatment with alkaline solutions), vesiculated organelles, and vesicles produced by living cells (e.g., by direct cell membrane budding or fusion of late endosomes with the cell membrane). Extracellular vesicles may be derived from living or dead organisms, explanted tissues or organs, and / or cultured cells. In preferred embodiments, EVs according to the present disclosure are exosomes, microvesicles (MVs), or any other type of vesicles secreted from the endosomal, endolysosomal, and / or lysosomal pathways, or from the cell membrane of a parent cell. Furthermore, when the teachings herein refer to EVs as singular EVs and / or separate natural nanoparticle-like vesicles, it is to be understood that all such teachings are equally relevant and applicable to multiple EVs and populations of EVs.

[0082] It will be apparent to those skilled in the art that when describing medical and scientific uses and applications of nanovesicles (e.g., EVs), the present disclosure generally relates to a plurality of nanovesicles (e.g., EVs), i.e., a population of nanovesicles (e.g., EVs), which may include thousands, millions, billions, or even trillions of nanovesicles (e.g., EVs). As can be seen from the experimental section below, nanovesicles (e.g., EVs) can be as high as 10 per unit volume (e.g., per ml). 5 , 10 8 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 18 , 10 25 , 10 30 The nanovesicles (e.g., EVs) may be present in concentrations of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3500, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 10000, 15000, 20000, 25000, 35000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 150000, 100000, 150000, 250000, 350000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000, 15 ...

[0083] The term "nanovesicles" refers to lipid nanovesicles derived from source cells (i.e., extracellular vesicles) or synthetic lipid nanoparticles, and natural / synthetic hybrids (such as hybridosomes). Nanovesicles typically contain lipids or fatty acids as well as polypeptides, and may further contain payloads, targeting moieties, or other molecules. Furthermore, when teachings herein refer to a singular nanovesicle, it is understood that all such teachings are equally relevant and applicable to a plurality of nanovesicles and populations of nanovesicles. When describing medical and scientific uses and applications of nanovesicles, it will be apparent to those skilled in the art that the present disclosure typically relates to a plurality of nanovesicles, i.e., populations of nanovesicles that may contain thousands, millions, billions, or even trillions of nanovesicles. As can be seen from the experimental section below, nanovesicles can be as small as 10 nanovesicles per unit volume (e.g., per ml). 5 , 10 8 , 10 10 , 10 11 , 1012 , 10 13 , 10 14 , 10 15 , 10 18 , 10 25 , 10 30 The nanovesicles may be present in a concentration of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 100, 200, 35, 40, 50, 60, 70, 80, 90, 100, 150, 100, 150, 200, 250, 350, 400, 450, 500, 550, 600, 700, 800, 900, 1000, 1500, 1000, 1500, 2500, 3500, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 1500

[0084] Furthermore, the nanovesicles (eg, EVs) of the present disclosure may also contain additional payloads in addition to the FcRn-binding polypeptides that may be bound to the nanovesicle surface.

[0085] The term "source cell" or "EV source cell" or "parent cell" or "cell source" or "EV-producing cell" or any other similar term may be understood to relate to any type of mammalian cell that can generate nanovesicles (e.g., EVs) under appropriate conditions, e.g., in suspension or adherent culture or any other type of culture system. Source cells according to the present disclosure may also include cells that generate nanovesicles (e.g., EVs) in vivo. Source cells according to the present disclosure may be selected from a wide range of cells and cell lines that may grow in suspension or adherent culture or are adapted for suspension growth. In general, nanovesicles (e.g., EVs) may be derived from essentially any cell source, whether primary cell source or immortalized cell line. EV source cells may be any embryonic, fetal, and adult somatic stem cell type, including induced pluripotent stem cells (iPSCs) and other stem cells derived by any method. The source cells may be either allogeneic, autologous, or even xenogeneic in nature to the patient being treated, i.e., the cells may be from the patient himself, or from an unrelated donor, a matched donor, or a mismatched donor. In certain circumstances, allogeneic cells may be preferred from a medical perspective, as they may provide immunomodulatory effects that are unlikely to be obtained from autologous cells of a patient suffering from a particular indication. For example, in the context of treating inflammatory or degenerative diseases, allogeneic MSCs or AEs may be highly beneficial as a source of EV-generating cells due to their nanovesicles (e.g., EVs) and in particular the inherent immunomodulation of their nanovesicles (e.g., EVs). Cell lines of particular interest include human umbilical endothelial cells (HUVEC), human embryonic kidney (HEK) cells such as HEK293 cells, HEK293T cells, serum-free HEK293 cells, suspension HEK293 cells, endothelial cell lines such as microvascular or lymphatic endothelial cells, erythrocytes, erythroid progenitor cells, chondrocytes, MSCs of different origins, amniotic cells, amniotic epithelial (AE) cells, any cells obtained via amniocentesis or from the placenta, airway or alveolar epithelial cells, fibroblasts, endothelial cells, epithelial cells, etc.

[0086] The term "buffer substance" refers to a substance that, when in a solution, is able to buffer changes in the pH value of the solution, for example due to the addition or release of acidic or basic substances.

[0087] As used herein, the terms "isolate", "isolated", and "isolating" or "purify", "purified", and "purifying" as well as "extracted" and "extracting" are used interchangeably and refer to the state of a preparation (e.g., multiple known or unknown amounts and / or concentrations) of desired FcRn-binding nanovesicles (e.g., EVs) that have undergone one or more purification processes, such as selection or enrichment, of the desired FcRn-binding EV preparation. In some embodiments, isolation or purification as used herein is a process of removing or partially removing nanovesicles comprising FcRn-binding polypeptides from a produced cell-containing sample (e.g., fractionation). In some embodiments, the isolated nanovesicles comprising an FcRn-binding polypeptide composition have no detectable undesirable activity or have a level or amount of undesirable activity below an acceptable level or amount. In other embodiments, the isolated exosome composition has an amount and / or concentration of desired nanovesicles comprising FcRn-binding polypeptides at or above an acceptable amount and / or concentration. In other embodiments, the isolated nanovesicles comprising the FcRn-binding polypeptide composition are enriched compared to the starting material (e.g., the cell preparation from which the composition is derived). This enrichment can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, or more than 99.9999% compared to the starting material. In some embodiments, the isolated nanovesicles comprising the FcRn-binding polypeptide preparation are substantially free of residual biological products. In some embodiments, the isolated nanovesicles comprising the FcRn-binding polypeptide preparation are 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% free of any contaminating biological material. Residual biological products may include non-biological substances (including chemicals) or undesirable nucleic acids, proteins, lipids, or metabolic products. Substantially free of residual biological products can also mean that the nanovesicles comprising the FcRn-binding polypeptide composition do not contain detectable producing cells, and only the nanovesicles comprising the FcRn-binding polypeptide are detectable.

[0088] The terms "polynucleotide" and "nucleic acid" refer interchangeably to a chain of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase. Polynucleotides can include modified nucleotides, such as methylated nucleotides and their analogs. Examples of polynucleotides contemplated herein include single- and double-stranded DNA, single- and double-stranded RNA, and hybrid molecules having a mixture of single and double strands.

[0089] Typically, as in the case of fusion proteins, the two components typically included in a fusion protein (i.e., the FcRn-binding polypeptide containing a transmembrane domain and the scaffold protein) may be directly linked contiguously in the fusion protein or may be linked and / or attached to each other using various linkers. Any of the peptide linkers may comprise a length of at least 5 residues, at least 10 residues, at least 15 residues, at least 20 residues, at least 25 residues, at least 30 residues or more. In other embodiments, the linker comprises a length of 2-4 residues, 2-4 residues, 2-6 residues, 2-8 residues, 2-10 residues, 2-12 residues, 2-14 residues, 2-16 residues, 2-18 residues, 2-20 residues, 2-22 residues, 2-24 residues, 2-26 residues, 2-28 residues, or 2-30 residues. In some embodiments, the first linker comprises a flexible linker. In some embodiments, the first linker comprises a glycine-serine linker, i.e., a linker consisting primarily or entirely of a stretch of glycine and serine residues. In some embodiments, the first linker comprises a (G4S)n linker (GGGGS)n (SEQ ID NO:1), where "n" indicates the number of repeats of the motif and is an integer from 1 to 10. In some embodiments, the first linker comprises a G4S(GGGGS; SEQ ID NO:2) linker, a (G4S)2(GGGGS GGGGS; SEQ ID NO:3) linker, a (G4S)3(GGGGS GGGGS GGGGS; SEQ ID NO:4) linker, or a (G4S)2-G4 (SEQ ID NO:5) linker.

[0090] As used herein, "single-chain Fv" or "scFv" refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. Methods for producing scFvs are well known in the art. For a review of methods for producing scFvs, see Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 1, Rosenburg and Moore (eds.). Springer-Verlag, New York, pp. 269-315 (1994).

[0091] Wherever an aspect or embodiment is described herein in terms of "comprising", it is understood that analogous aspects are also provided that are otherwise described in terms of "consisting of" and / or "consisting essentially of".

[0092] 5.2 Disclosed Polypeptides In particular, provided herein are certain FcRn-binding polypeptides comprising (i) at least one FcRn-binding site and (ii) a transmembrane (TM) domain. In certain embodiments, the transmembrane domain is a multi-spanning transmembrane domain.

[0093] In one aspect, an FcRn-binding polypeptide for use with the methods and compositions provided herein shall be understood to refer to a polypeptide that comprises an FcRn-binding site that is capable of binding to FcRn with high affinity at a pH below physiological pH and is anchored in the membrane by at least one transmembrane domain or a fragment thereof.

[0094] In certain embodiments, the FcRn-binding polypeptide comprises a transmembrane domain (e.g., a scaffold protein) and an FcRn-binding site (e.g., a modified Fc domain of an immunoglobulin) that can specifically bind to the Fc-binding site of a neonatal Fc receptor and lacks the ability to form homodimers. In certain embodiments, the equilibrium dissociation constant of the FcRn-binding site (e.g., a modified Fc domain of an immunoglobulin) that binds to FcRn at pH 6.5 is at most 10 -4 In certain embodiments, the equilibrium dissociation constant of an FcRn binding site (e.g., a modified Fc domain of an immunoglobulin) that binds to FcRn at pH 7.4 is at least 10 -4 In certain embodiments, the FcRn binding site (e.g., a modified Fc domain of an immunoglobulin) has the amino acid sequence [Table 1] wherein each of X1, X2, X3, X4, X5, X6, X7, and X8 is any amino acid. In certain embodiments, the FcRn-binding site (e.g., a modified Fc domain of an immunoglobulin) can specifically bind to an amino acid sequence between positions 135 and 158 of human FcRn (SEQ ID NO: 7) and / or mouse FcRn (SEQ ID NO: 8). [Table 2]

[0095] In certain embodiments, the FcRn-binding polypeptide of the present disclosure comprises an FcRn-binding site (e.g., Fc domain) derived from the C-terminal region of an immunoglobulin heavy chain polypeptide. In some embodiments, the Fc polypeptide can comprise two linked Ig-like fold structural domains (e.g., CH2 and CH3 domains), and at acidic pH, FcRn can bind to amino residues in both CH2 and CH3 structural domains (FcRn-binding site). In some embodiments, the FcRn polypeptide can comprise an Ig-like fold structural domain (e.g., CH2 of the Fc domain), and at acidic pH, FcRn can bind to amino residues in the CH2 structural domain (e.g., FcRn-binding site). In some embodiments, the FcRn polypeptide can comprise an Ig-like fold structural domain (e.g., CH3 domain of the Fc polypeptide), and at acidic pH, FcRn can bind to amino residues in the CH3 structural domain (FcRn-binding site).

[0096] In some embodiments, the FcRn-binding polypeptide of the present disclosure can comprise one or more FcRn-binding sites from various mammalian species (e.g., human) and various immunoglobulin subtypes, such as IgG (as non-limiting examples, IgG, lgG1, lgG2, lgG3, lgG4, lgG2a, lgG2d, and / or lgG2c). In some embodiments, the FcRn-binding site is or comprises a human Fc structural domain, such as a human IgG Fc structural domain comprising an amino acid sequence derived from a human IgG Fc polypeptide sequence. For example, in some embodiments, the FcRn-binding site is or comprises a human Fc structural domain comprising an amino acid sequence derived from a human IgG1 Fc polypeptide sequence (see SEQ ID NO: 9 for the amino acid sequence of wild-type human IgG1 Fc). In other embodiments, the FcRn-binding site is or comprises a human Fc structural domain comprising an amino acid sequence derived from a human IgG2 Fc polypeptide (see SEQ ID NO: 10 for the amino acid sequence of wild-type human IgG2 Fc). In other embodiments, the FcRn-binding site is or comprises a human Fc structural domain comprising an amino acid sequence derived from a human IgG3 Fc polypeptide (see SEQ ID NO: 11 for the amino acid sequence of wild-type human IgG3 Fc). In other embodiments, the FcRn-binding site is or comprises a human Fc structural domain comprising an amino acid sequence derived from a human IgG4 Fc polypeptide (see SEQ ID NO: 12 for the amino acid sequence of wild-type human IgG4 Fc).

[0097] In some embodiments, the CH2 domain of the FcRn-binding polypeptide can be easily obtained from any suitable antibody. Optionally, the CH2 domain is of human origin. The CH2 domain may or may not be linked to a hinge of a linker amino acid sequence (e.g., at its N-terminus). In one embodiment, the CH2 domain is a naturally occurring human CH2 domain of IgG1, 2, 4 or 4 subtype. In one embodiment, the CH2 domain is a fragment of the CH2 domain (e.g., at least 10, 20, 30, 40 or 50 amino acids in length). In one embodiment, the CH2 domain, when present in a polypeptide described herein, will retain binding to FcRn, particularly human FcRn.

[0098] In one embodiment, the FcRn-binding polypeptide described herein may comprise an Fc domain, the Fc domain exhibiting a three-dimensional structure that can be superimposed on the Fc structure of a wild-type Fc domain of an antibody (e.g., IgG). In certain embodiments, the polypeptide described herein comprises an Fc domain, the Fc domain exhibiting a three-dimensional structure, the portion between the equivalent Cα positions of the beta chains of which can be superimposed on the wild-type Fc domain of an antibody (e.g., IgG) with a root mean square deviation (RMSD) of at most 1, 2, 4, 4, 5, 6, 7, 8, 9, 10 or 15 Å. For example, the structure of the Fc domain can be superimposed on the Fc domains of IgG1, IgG2, and IgG4 subtypes, as described in Tam SH, et al., Antibodies (Basel). 2017;6(3):12. Methods for comparing two biological structures by calculating the RMSD of the superimposed structures are well known in the art (as described in Xu, Y., Xu, D. and Liang, J., 2007. Computational methods for protein structure prediction and modeling. Springer).

[0099] In another aspect, a polypeptide for use with the methods and compositions provided herein shall be understood to relate to an FcRn-binding polypeptide having at least one mutation, e.g., a substitution, deletion or insertion, compared to a wild-type immunoglobulin heavy chain Fc polypeptide sequence, but retaining the overall Ig fold or structure of a native Fc domain.

[0100] In some embodiments, the FcRn-binding polypeptide of the present disclosure comprises a modified Fc domain of an immunoglobulin having the ability to bind to the Fc-binding site of FcRn. The modified Fc domain can be at least 50% homologous to any sequence of the Fc portion of any IgG antibody. In some embodiments, the FcRn-binding polypeptide can be at least 60% homologous to any sequence of the Fc portion of any IgG antibody. In certain embodiments, the modified Fc domain can be at least 70% homologous to any sequence of the Fc portion of any IgG antibody. In certain embodiments, the modified Fc domain can be at least 80% homologous to any sequence of the Fc portion of any IgG antibody. In certain embodiments, the modified Fc domain can be at least 90% homologous to any sequence of the Fc portion of any IgG antibody.

[0101] In some embodiments, the modified Fc domain may be at least 50% homologous to any of SEQ ID NOs: 13-34. In certain embodiments, the FcRn-binding portion may be at least 60% homologous to any of SEQ ID NOs: 13-34. In certain embodiments, the modified Fc domain may be at least 70% homologous to any of SEQ ID NOs: 13-34. In certain embodiments, the modified Fc domain may be at least 80% homologous to any of SEQ ID NOs: 13-34. In certain embodiments, the modified Fc domain may be at least 90% homologous to any of SEQ ID NOs: 13-34. In certain embodiments, the modified Fc domain may be at least 95% or at least 98% homologous to any of SEQ ID NOs: 13-34.

[0102] In some embodiments, the FcRn-binding polypeptide comprises a native FcRn-binding site (e.g., Fc domain). In some embodiments, the FcRn-binding comprises a modification that alters FcRn-binding. In some embodiments, the modified Fc domain of the present disclosure is mutated or modified to further enhance FcRn-binding. In these embodiments, the mutated or modified Fc polypeptide may comprise the following mutations: M252Y, S254T, T256E, L309N, T250Q, M428L, N434S, N434A, T307A, E380A, using the EU numbering system. In some embodiments, the mutated or modified Fc polypeptide comprises one or more mutations selected from the group consisting of M252Y, S256T, T256E, M428L, M428V, N434S, and combinations thereof. CH2 and CH3 domain modifications for enhanced FcRn binding are presented in US Ser. No. 16 / 845,894, which is incorporated herein by reference.

[0103] In various embodiments, the FcRn-binding polypeptide of the present disclosure has increased binding affinity to the Fc-binding site of FcRn at acidic pH and decreased binding affinity to the Fc-binding site of FcRn at near neutral pH. In a preferred embodiment, the FcRn-binding polypeptide of the present disclosure has increased propensity to form a complex with FcRn at acidic pH (e.g., pH 6.5) as opposed to neutral pH (e.g., pH 7.4).

[0104] In some embodiments, the equilibrium dissociation constant of an FcRn-binding polypeptide that binds to FcRn at an acidic pH is at least 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 or 10 -9M. In some embodiments, the equilibrium dissociation constant of the FcRn-binding polypeptide that binds to FcRn at pH 6.5 is equal to the equilibrium dissociation constant of the modified Fc domain that binds to FcRn at pH 6.5. In some embodiments, the equilibrium dissociation constant of the FcRn-binding polypeptide that binds to FcRn at pH 6.5 is increased by at least 5%, 10%, 20%, 30%, 40%, 50% or 60% compared to the equilibrium dissociation constant of the modified Fc domain fragment that binds to FcRn.

[0105] In some embodiments, the equilibrium dissociation constant of an FcRn-binding polypeptide that binds to FcRn at neutral pH is 10 -5 , 10 -4 , 10 -3 , 10 -2 or 10 -1 In some embodiments, the equilibrium dissociation constant of the FcRn-binding polypeptide that binds to FcRn at neutral pH is equal to the equilibrium dissociation constant of the modified Fc domain that binds to FcRn at neutral pH. In some embodiments, the equilibrium dissociation constant of the FcRn-binding polypeptide that binds to FcRn at pH 6.5 is increased by at least 20%, 30%, 40%, 50% or 60% compared to the equilibrium dissociation constant of the modified Fc domain fragment that binds to FcRn.

[0106] In some embodiments, the three-dimensional structure of the modified Fc domain of the FcRn-binding polypeptide that binds to FcRn at acidic pH has a binding interface that spans a larger surface area (e.g., more than 1000 Å) than the structure at physiological pH. In certain embodiments, the buried surface area at the interface between the modified Fc domain chain and the polypeptide chain of FcRn can be larger than the buried area at the interface between Fc and other proteins (e.g., protein A, protein G, or rheumatoid factor) that bind to the CH2-CH3 interdomain region of Fc. Methods for calculating the buried surface area between are well known in the art (as described in Xu, Y., Xu, D., and Liang, J., 2007. Computational methods for protein structure prediction and modeling, Springer).

[0107] In one embodiment of all aspects described herein, the FcRn-binding polypeptide has a binding affinity for an FcRn selected from human FcRn, cynomolgus monkey FcRn, mouse FcRn, rat FcRn, sheep FcRn, dog FcRn, and rabbit FcRn at an acidic pH. In some embodiments, the FcRn-binding polypeptide has increased binding affinity for mouse FcRn compared to human FcRn.

[0108] Methods for analyzing the binding affinity and binding kinetics of the FcRn-binding polypeptides and Fc domains of FcRn-binding polypeptides disclosed herein are known in the art. These methods include, but are not limited to, solid-phase binding assays (e.g., ELISA assays), immunoprecipitation, surface plasmon resonance (e.g., Biacore™), kinetic exclusion assays (e.g., KinExA®), flow cytometry, fluorescence-activated cell sorting (FACS), biolayer interferometry (e.g., Octet® (ForteBio)), and Western blot analysis. In some embodiments, ELISA is used to determine binding affinity. Methods for performing ELISA assays are known in the art. In some embodiments, surface plasmon resonance (SPR) is used to determine binding affinity and / or binding kinetics. In some embodiments, kinetic exclusion assays are used to determine binding affinity and / or binding kinetics. In some embodiments, biolayer interference assays are used to determine binding affinity and / or binding kinetics.

[0109] 5.2.1 Modification of CH2 and CH3 domain homodimers In various embodiments, the FcRn-binding polypeptides described herein are engineered to not dimerize with another Fc domain (e.g., not form homodimers with another modified Fc domain or not form heterodimers with endogenous Fc domain). In one embodiment, the Fc domain contains modifications that disrupt heterodimerization, for example, by electrostatic engineering of contact residues in the naturally charged CH3-CH3 interface, or modifications of hydrophobic patches (e.g., not dimerize through interactions with another CH3 domain, referred to as monomeric CH3). In certain embodiments, specifically herein, the CH3 domain of the Fc domain contains one or more amino acid modifications (e.g., amino acid substitutions) to disrupt the CH3 dimerization interface. In such embodiments, the CH3 domain modifications will prevent aggregation of the protein scaffold caused by exposure of hydrophobic residues when the CH2-CH3 domain is in monomeric form. At the same time, CH3 domain modifications useful in the present disclosure will also not interfere with the ability of the Fc derived polypeptide to bind to a neonatal Fc receptor (FcRn), e.g., human FcRn.

[0110] In one aspect, the FcRn-binding polypeptide described herein may comprise a monomeric CH3 domain, which exhibits a three-dimensional structure that can be superimposed on the CH3 structure of a wild-type CH3 domain of an antibody (e.g., IgG). In a particular embodiment, the polypeptide described herein comprises a monomeric CH3 domain, which exhibits a three-dimensional structure, the portion between the equivalent Cα positions of the beta chains of which can be superimposed on the wild-type CH3 domain of an antibody (e.g., IgG) with a root mean square deviation (RMSD) of at most 1, 2, 4, 4, 5, 6, 7, 8, 9, 10 or 15 Å. For example, the structure of the monomeric CH3 domain can be superimposed on the CH3 domain of IgG1, IgG2, and IgG4 subtypes, as described in Tam SH, et al., Antibodies (Basel). 2017;6(3):12.

[0111] Monomeric modified Fc domains that can be used to prevent homodimer formation have been described in various publications. For example, see US Patent Publication No. 2006 / 0074225, WO2006 / 031994, WO2011 / 063348, and Ying et al. (2012) J. Biol. Chem. 287(23):19399-19407, the disclosures of each of which are incorporated herein by reference. To prevent homodimer formation, one or more residues that make up the CH3-CH3 interface are replaced with charged amino acids, making the interaction electrostatically unfavorable. For example, WO2011 / 063348 provides replacing positively charged amino acids in the interface, such as lysine, arginine, or histidine, with different amino acids (e.g., negatively charged amino acids such as aspartic acid or glutamic acid), and / or replacing negatively charged amino acids in the interface with different (e.g., positively charged) amino acids. In one embodiment, the CH3 domain described herein comprises amino acid alterations (e.g., substitutions) at 1, 2, 3, 4, 5, 6, 7 or 8 positions of R355, D356, E357, K370, K392, D399, K409 and K439 (according to EU numbering).In certain embodiments, two or more charged residues in the interface are changed to opposite charges.Exemplary CH3 domains include those that comprise K392D and K409D mutations, and D399K and D356K mutations.

[0112] A further strategy to maintain the monomeric Fc domain includes replacing one or more large hydrophobic residues that constitute the CH3-CH3 interface with small polar amino acids. Taking human IgG as an example, the large hydrophobic residues of the CH3-CH3 interface include Y349, L351, L368, L398, V397, F405, and Y407 of the Fc domain. The low polar amino acid residues include asparagine, cysteine, glutamine, serine, and threonine. Thus, in one embodiment, the CH3 domain described herein includes amino acid modifications (e.g., substitutions) at 1, 2, 3, 4, 5, 6, 7, or 8 positions at R355, D356, E357, K370, K392, D399, K409, and K439. In the study described in WO2011 / 063348, two of the positively charged Lys residues located close to the CH3 domain interface were mutated to Asp. Then, in the background of these two Lys to Asp mutations, threonine scanning mutagenesis was performed on the structurally conserved large hydrophobic residues. The Fc domains containing K392D and K409D mutations, together with various substitutions with threonine, were analyzed for monomer formation. Exemplary monomeric Fc domains include those with K392D, K409D and Y349T substitutions, and those with K392D, K409D and F405T substitutions.

[0113] In Ying et al. (2012) J. Biol. Chem. 287(23):19399-19407, amino acid substitutions were made in the CH3 domain at residues L351, T366, L368, P395, F405, T407 and K409. Different combinations of mutations led to disruption of the CH3 dimerization interface without causing protein aggregation. In one embodiment, the CH3 domain described herein comprises amino acid modifications (e.g., substitutions) at 1, 2, 3, 4, 5, 6 or 7 positions at L351, T366, L368, P395, F405, T407 and / or K409. In one embodiment, the CH3 domain described herein comprises amino acid modifications L351Y, T366Y, L368A, P395R, F405R, T407M and K409A. In one embodiment, the CH3 domain comprises the amino acid modifications L351S, T366R, L368H, P395K, F405E, T407K and K409A. In one embodiment, the CH3 domain described herein comprises the amino acid modifications L351K, T366S, P395V, F405R, T407A and K409Y.

[0114] In various embodiments, the modified Fc domains of the present disclosure exhibit reduced dimerization compared to wild-type Fc molecules. Thus, embodiments of the present disclosure include compositions comprising a population of FcRn-binding polypeptides as described herein, wherein the amount of Fc domain-Fc domain homodimers exhibited by said FcRn-binding polypeptides is less than 10%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, less than 95%, less than 97%, or less than 99% of the population. Dimerization can be measured by several techniques known in the art. Preferred methods for measuring homodimerization of modified Fc domains include size exclusion chromatography (SEC), analytical ultracentrifugation (AUC), dynamic light scattering (DLS), and native PAGE.

[0115] 5.2.2 Additional Fc Domain Modifications In some embodiments, the FcRn-binding polypeptide of the present disclosure contains one or more additional modifications. Non-limiting examples of other mutations that can be introduced into the modified Fc domain include, for example, mutations to increase serum stability and / or extend half-life, mutations to modulate effector functions, mutations to affect glycosylation, and / or mutations to reduce immunogenicity in humans.

[0116] In some embodiments, the FcRn-binding polypeptides described herein comprise modifications that reduce effector function, i.e., reduce the ability to induce a specific biological function upon binding to an Fc receptor (other than FcRn) expressed on or within an effector cell that mediates effector function. Examples of Fc receptor effector functions include, but are not limited to, Clq binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. In some embodiments, the modified Fc domains present in the FcRn-binding polypeptides described herein may comprise further modifications that modulate effector function.

[0117] In some embodiments, the complement dependent cytotoxicity (CDC) activity of the modified Fc domain; the antibody dependent cell-mediated cytotoxicity (ADCC) activity of the modified Fc domain; the antibody dependent cell-mediated phagocytosis (ADCP) activity of the modified Fc domain; and / or the antibody dependent intracellular neutralization (ADIN) activity of the modified Fc domain is reduced by at least 10%, 20%, 30%, 40%, or 50% compared to the unmodified Fc domain.

[0118] In some embodiments, the complement dependent cytotoxicity (CDC) activity of the modified Fc domain; the antibody dependent cell-mediated cytotoxicity (ADCC) activity of the modified Fc domain; the antibody dependent cell-mediated phagocytosis (ADCP) activity of the modified Fc domain; and / or the antibody dependent intracellular neutralization (ADIN) activity of the modified Fc domain is reduced by at least 1.5, 2, 3, 4, or 5 fold compared to the unmodified Fc domain.

[0119] In some embodiments, the FcRn-binding polypeptide comprises, from the N-terminus to the C-terminus: (a) a modified CH2 domain that has been modified to reduce effector function relative to the unmodified CH2 domain; (b) a modified CH3 domain that has been modified to lack homodimerization relative to the unmodified CH3 domain; (c) a linker sequence; and (d) a transmembrane domain.

[0120] In some embodiments, the FcRn-binding polypeptide comprises, from the C-terminus to the N-terminus: (a) a modified CH3 domain that has been modified to lack homodimerization relative to the unmodified CH3 domain; (b) a modified CH2 domain that has been modified to reduce effector function relative to the unmodified CH2 domain; (c) a linker sequence; and (d) a transmembrane domain.

[0121] In some embodiments, the FcRn-binding polypeptides described herein may comprise modifications that reduce or eliminate effector function. Exemplary modifications include CH2 domain modifications that reduce effector function, including, but not limited to, substitutions in the CH2 domain, designated modCH2, for example, at positions 234 and 235 according to the EU numbering scheme. For example, in some embodiments, the modified Fc domain may comprise alanine residues at positions 234 and 235. Thus, the FcRn-binding polypeptide may have L234A and L235A substitutions.

[0122] In one embodiment, the CH2 domain, when present in an FcRn-binding polypeptide described herein, reduces or eliminates binding to Fcγ receptors, in particular FcγRIIIA (CD16). An FcRn-binding polypeptide comprising a CH2 domain that cannot bind to CD16 will not be able to activate or mediate ADCC by cells (e.g., NK cells, T cells) that do not express the effector cell antigen of interest (e.g., NKp46, CD3, etc.).

[0123] In one embodiment, the CH2 domain, when present in an FcRn-binding polypeptide described herein, will reduce or substantially abolish antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), FcR-mediated cell activation (e.g., cytokine release via FcR cross-linking), and / or FcR-mediated platelet activation / depletion.

[0124] In one embodiment, the CH2 domain, when present in an FcRn-binding polypeptide described herein, substantially loses binding to an activating Fcγ receptor, such as FcγRIIIA (CD16), FcγRIIA (CD32A) or CD64, or an inhibitory Fc receptor, such as FcγRIIB (CD32B). In one embodiment, the CH2 domain, when present in an FcRn-binding polypeptide described herein, further substantially loses binding to the first component of complement (C1q).

[0125] For example, substitutions at residues 233-236 in the CH2 domain of human IgG1 or IgG2 and at residues 327, 330 and 331 of IgG4 have been shown to greatly reduce binding to Fcγ receptors and thus ADCC and CDC. Furthermore, Idusogie et al. (2000) J Immunol. 164(8):4178-84 demonstrated that alanine substitutions at different positions, including K322, significantly reduced complement activation.

[0126] Additional CH2 domain modifications or mutations that modulate effector function include, but are not limited to, a mutation at position 329 substituting proline with glycine or arginine, or with an amino acid residue large enough to disrupt the Fc / Fcy receptor interface formed between proline 329 of Fc and tryptophan residues Trp87 and Trp110 of FcyRIII.

[0127] Additional exemplary substitutions in the CH2 domain include S228P, E233P, L235E, N297A, N297D, and P331S according to the EU numbering scheme. Multiple substitutions may also be present, such as L234A and L235A in the human IgGl Fc region; L234A, L235A, and P329G in the human IgGl Fc region; S228P and L235E in the human IgG4 Fc region; L234A and G237A in the human IgGl Fc region; L234A, L235A, and G237A in the human IgGl Fc region; V234A and G237A in the human IgG2 Fc region; L235A, G237A, and E318A in the human IgG4 Fc region; and S228P and L236E in the human IgG4 Fc region according to the EU numbering scheme. In some embodiments, one FcRn-binding polypeptide may have one or more amino acid substitutions that modulate ADCC, for example substitutions at positions 298, 333, and / or 334 according to the EU numbering scheme.

[0128] In one embodiment, the FcRn-binding polypeptide has reduced binding to human Fcy receptors (e.g., CD16, CD32A, CD32B and / or CD64), e.g., compared to a full-length wild-type human lgG1 Fc domain. In one embodiment, the polypeptide has reduced (e.g., partially or completely lost) antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), Fc receptor-mediated cell activation / depletion (e.g., cytokine release via FcR cross-linking), and / or Fc receptor-mediated platelet activation / depletion, as mediated by immune effector cells, e.g., compared to an FcRn-binding polypeptide having a wild-type Fc domain of the human lgG1 isotype.

[0129] In one embodiment, the CH2 domain that retains binding to the FcRn receptor but has reduced binding to the Fcγ receptor lacks or has modified N-linked glycosylation, for example at residue N297 according to the EU numbering scheme. For example, the FcRn-binding polypeptide can be expressed in a cell line that naturally has high enzymatic activity for fucosyl addition to N-acetylglucosamine that does not result in glycosylation at N297. In another embodiment, the CH2 domain can have one or more substitutions that result in the absence of the canonical Asn-X-Ser / Thr N-linked glycosylation motif at residues 297-299, which can also reduce binding to the Fcγ receptor. Thus, the CH2 domain can have substitutions at N297 and / or adjacent residues (e.g., 298, 299).

[0130] In one embodiment, the FcRn binding polypeptide comprises a CH2 domain derived from an lgG2 Fc mutant, which exhibits reduced FcγR binding ability but has preserved FcRn binding. In a particular embodiment, the FcRn binding polypeptide comprises mutations V234A, G237A, P238S according to the EU numbering system. In another embodiment, the FcRn binding polypeptide comprises mutations V234A, G237A, H268Q or H268A, V309L, A330S, P331S according to the EU numbering system. In a particular embodiment, the FcRn binding polypeptide comprises a CH2 domain derived from an lgG2 Fc, which comprises mutations V234A, G237A, P238S, H268A, V309L, A330S, P331S, and optionally P233S according to the EU numbering system.

[0131] In one embodiment, the FcRn-binding polypeptide comprises at least one amino acid modification in the CH2 domain of the Fc domain (e.g., possesses 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications), optionally further in combination with one or more amino acid modifications in other domains (e.g., the CH3 domain). Any combination of Fc domain modifications can be made. In one embodiment, the FcRn-binding polypeptide of the present disclosure with reduced binding to human Fcγ receptor comprises at least one amino acid modification (e.g., possesses 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) relative to the wild-type CH2 domain within amino acid residues 237-340 (EU numbering), such that the FcRn fusion polypeptide comprising such a CH2 domain has reduced affinity for the human Fcγ receptor of interest relative to a comparable polypeptide comprising the wild-type CH2 domain.

[0132] In one aspect, the FcRn-binding polypeptide described herein may comprise a CH2 domain (e.g., a modified CH2 domain), which exhibits a three-dimensional structure that can be superimposed on the CH2 structure of a wild-type CH2 domain of an antibody (e.g., an IgG). In certain embodiments, the polypeptide described herein comprises a modified CH2 domain, which exhibits a three-dimensional structure, the portion between the equivalent Cα positions of the beta chains of which can be superimposed on the wild-type CH2 domain of an antibody (e.g., an IgG) with a root mean square deviation (RMSD) of at most 1, 2, 4, 4, 5, 6, 7, 8, 9, 10 or 15 Å. For example, the structure of the modified CH2 domain can be superimposed on the CH2 domain of IgG1, IgG2, and IgG4 subtypes, as described in Tam SH, et al., Antibodies (Basel). 2017;6(3):12.

[0133] In some embodiments, the FcRn-binding polypeptide comprising the CH3 domain described herein may comprise a modification that reduces the activation of tripartite motif-containing protein 21 (TRIM21).Exemplary CH3 domain mutations that reduce the activation of TRIM21 include, but are not limited to, a substitution in the CH3 domain at position 433 according to the EU numbering scheme.For example, in some embodiments, the CH3 domain can comprise an alanine residue at position 433 according to the EU numbering scheme.

[0134] In certain aspects, provided herein are FcRn-binding polypeptides immobilized on a signal neutral protein scaffold in nanovesicles (e.g., EVs and hybridosomes) for attachment of molecules of interest.

[0135] In further aspects, the FcRn binding site of the Fc polypeptide useful in the present disclosure may include internal amino acids near the interdomain interface (e.g., hinge region) between the CH2 and CH3 domains, but not the C-terminus or N-terminus of the CH2 and CH3 domains (e.g., fc polypeptide). Thus, in certain embodiments, both ends of the structural domain that includes the FcRn binding site are not relevant to the FcRn binding function and can be modified (e.g., linked to a heterologous protein) without significantly altering the FcRn binding function of the polypeptide. Polypeptides that include domains (distal to the N-terminus or C-terminus) with internal FcRn binding sites accessible for structural complementation (e.g., FcRn binding) may provide design advantages when tethered to either the N-terminus or C-terminus of scaffold proteins that include different types of transmembrane domains (types 1, II, and PT), as described in section ). Further provided herein are polypeptides comprising an internal FcRn binding site with accessible N- and C-termini that can be fused to a scaffold protein and optionally a heterologous protein for additional functions such as cell type specific targeting, receptor decoy, or purification. In some embodiments, fusing the FcRn binding site to a protein scaffold that protrudes from the membrane allows FcRn to access the FcRn binding site. In certain embodiments, this results in a long protrusion of the FcRn binding site from the membrane (e.g., when fused to an ephrin receptor scaffold protein), which is flexible to bend and / or reconfigure while maintaining stability at the same time. Stable membrane anchoring can streamline the configuration of the resulting fusion protein in that the molecule of interest can be directed to the outer surface of a nanovesicle (e.g., EV or hybridosome) or cell.

[0136] In some embodiments, the FcRn binding polypeptides described herein do not substantially bind to C1q, FcγRI, FcγRII or FcγRIII.

[0137] Table 1. Examples of wild-type IgG1 Fc domains and engineered Fc domains [Table 3] TIFF2024514156000007.tif245170TIFF2024514156000008.tif247170TIFF2024514156000009.tif172170

[0138] 5.2.3 Structure of FcRn-binding Polypeptides In one embodiment, the FcRn-binding polypeptide comprises an FcRn-binding site linked to a scaffold protein (e.g., a scaffold protein) comprising a transmembrane domain. In some embodiments, the FcRn-binding site comprises a modified Fc domain of an immunoglobulin having the ability to bind to the Fc-binding site of FcRn.

[0139] (a) A transmembrane domain (e.g., a scaffold protein containing a transmembrane domain) In various embodiments, the FcRn-binding polypeptide of the present disclosure comprises a transmembrane domain of a membrane-associated protein or a transmembrane protein that comprises one or more transmembrane regions that are embedded in and cross the cell membrane at least once. Such a transmembrane region or a functional fragment thereof can be used as a membrane anchor for the FcRn-binding polypeptide. The transmembrane domain useful in the FcRn-binding polypeptide of the present disclosure can be derived from a transmembrane protein that is associated with any of various membranes of a cell, including, but not limited to, the cell membrane, the endoplasmic reticulum membrane, the Golgi complex membrane, the lysosomal membrane, the nuclear membrane, and the mitochondrial membrane. Examples of transmembrane proteins that associate with any of these different types of membranes are routinely found in proteomic datasets of EV samples (e.g., www.exocarta.org), and in some cases, when the endocytosis signal of a transmembrane protein such as CD63 is mutated to relocalize it from the endosomal membrane to the plasma membrane, the resulting secreted EVs contain higher amounts of the altered transmembrane protein than the endosome-targeted version of the same protein (see Fordjour et al., bioRxiv; 2019).

[0140] There are four general classes or types of transmembrane proteins (Types I-IV, see Nelson and Cox, Principles of Biochemistry (2008)). Type I transmembrane proteins have an N-terminal region that targets the lumen of the endoplasmic reticulum (ER) and a C-terminal region that is directed to the cytoplasm. Type II transmembrane proteins have an N-terminal region that targets a cytoplasmic domain and a C-terminal region that is directed to the ER lumen. PT type transmembrane proteins are "multiple-spanning" or polytopic transmembrane proteins that have two or more segments of the translated protein that span the cell membrane.

[0141] In various embodiments, the transmembrane domain in the FcRn-binding polypeptide of the present disclosure comprises all or a part of the transmembrane region of a transmembrane protein that normally crosses the membrane of a cell with which the transmembrane protein is normally associated. The transmembrane domain of the FcRn-binding polypeptide of the present disclosure may comprise not only the transmembrane region of the transmembrane protein, but also additional amino acids of the transmembrane protein located in adjacent regions either upstream (N-terminus) and / or downstream (C-terminus) of the transmembrane region or the membrane-embedded region of the transmembrane protein. For example, in certain embodiments, the entire transmembrane region of the transmembrane protein will be used. In additional embodiments, the entire transmembrane region of the transmembrane protein and all or a part of any upstream or downstream region of the membrane-embedded portion may be used as the transmembrane domain of the FcRn-binding polypeptide according to the present disclosure. Additional amino acids located either upstream (N-terminus) and / or downstream (C-terminus) from the membrane-embedded portion of a transmembrane protein that may be part of the transmembrane anchor of an FcRn-binding polypeptide of the present disclosure may have a size range including, but not limited to, 1-10 amino acids, 1-20 amino acids, 1-30 amino acids, 1-40 amino acids, 1-50 amino acids, 1-60 amino acids, 1-70 amino acids, 1-80 amino acids, 1-90 amino acids, 1-100 amino acids, 1-200 amino acids, 1-300 amino acids, 1-400 amino acids, 1-500 amino acids, 1-600 amino acids, 1-700 amino acids, 1-800 amino acids, and 1-900 amino acids. In some embodiments, the fragment transmembrane domain lacks at least 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, or 800 amino acids from the N-terminus of the native protein. In some embodiments, the fragment transmembrane domain lacks at least 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, or 800 amino acids from the C-terminus of the native protein. In some embodiments, the sequence encodes a fragment of a transmembrane domain lacking at least 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, or 800 amino acids from both the N-terminus and the C-terminus of the native protein.In some embodiments, the sequence encodes a fragment of a transmembrane domain protein that lacks one or more functional or structural domains of the native protein.

[0142] The FcRn-binding polypeptides comprising a transmembrane domain as described herein may also comprise the entire cytoplasmic region attached to the transmembrane region of a transmembrane protein, or a truncation of one or more amino acids of the cytoplasmic region, for example, to eliminate undesired signaling functions of the cytoplasmic tail. For example, the presence of a kinase domain in the C-terminal part of the cytoplasmic region of a transmembrane protein may serve as a signaling domain. Thus, when all or part of the membrane-embedded (transmembrane) region and adjacent cytoplasmic C-terminal region of a kinase transmembrane protein are used as the transmembrane domain of the fusion protein of the present disclosure, any known functional kinase signal can be eliminated or destroyed, so that the fusion protein comprising the transmembrane region and any adjacent cytoplasmic region does not activate the host cell.

[0143] Tables 2-4 below provide a list of some non-limiting examples of scaffold proteins containing single-spanning (Table 2) and multiple-spanning (Tables 3 and 4) transmembrane domains, along with Uniprot database entries. The transmembrane domain that may be used in the FcRn-binding polypeptide of the present disclosure may use a portion of the transmembrane region sequence sufficient to anchor the FcRn-binding polypeptide to the nanovesicle. Other portions of the transmembrane protein, including segments of adjacent regions upstream or downstream of the transmembrane region, may be included in the FcRn-binding polypeptide, so long as their inclusion does not enhance, or at least significantly reduce, the presentation of the FcRn-binding polypeptide on the surface of the nanovesicle. Fusing a polypeptide containing an FcRn-binding site to an accessible end of the surface of a scaffold protein containing a transmembrane domain can create a structure that is flexible to bending and / or reorganization, while at the same time being stable. Furthermore, in some embodiments, the ectodomain of the scaffold protein may provide a long protrusion for reaching, such that the ectodomain of the scaffold protein protrudes from the membrane. In some embodiments, the FcRn-binding polypeptide is selected from the group consisting of SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ [ka] (e.g., the transmembrane domain of membrane-bound IgG).

[0144] Table 2. Examples of bitopic transmembrane proteins that can be used as scaffold proteins [Table 4] TIFF2024514156000012.tif249170TIFF2024514156000013.tif249170TIFF2024514156000014.tif203170

[0145] Table 3. Examples of polytopic transmembrane proteins that can use scaffold proteins with accessible N- or C-termini [Table 5]

[0146] Table 4. Examples of polytopic transmembrane proteins that can contain transmembrane domains using scaffold proteins [Table 6] TIFF2024514156000017.tif246170TIFF2024514156000018.tif200170

[0147] Further non-limiting examples of other scaffold proteins containing transmembrane domains that can be used with the present disclosure include aminopeptidase N (CD13); neprilysin, also known as membrane metalloendopeptidase (MME); ectonucleotide pyrophosphatase / phosphodiesterase family member 1 (ENPP1); neuropilin-1 (NRP1); PDGFR, GPI-anchored protein, lactadherin, LAMP2, and LAMP2B.

[0148] In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a transmembrane domain of a wild-type ephrin receptor (e.g., an ephrin receptor TM domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 35-48). In some embodiments, the transmembrane domain of the polypeptide is a transmembrane domain of a wild-type ephrin receptor (e.g., an ephrin receptor TM domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 35-48).

[0149] In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the transmembrane domain of a wild-type FPRP (e.g., an FPRP TM domain comprising the amino acid sequence of SEQ ID NO: 49). In some embodiments, the transmembrane domain of the polypeptide is the transmembrane domain of a wild-type FPRP (e.g., an FPRP TM domain comprising the amino acid sequence of SEQ ID NO: 49).

[0150] Table 5. Non-exclusive examples of TM domains [Table 7]

[0151] In some embodiments, the transmembrane domain comprises the amino acid sequence of a wild-type ephrin receptor transmembrane domain (e.g., an ephrin receptor transmembrane domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 35-48) excluding one amino acid mutation, two amino acid mutations, three amino acid mutations, four amino acid mutations, five amino acid mutations, six amino acid mutations, seven amino acid mutations, or eight or more amino acid mutations. The mutation(s) can be substitution(s), insertion(s), deletion(s), or any combination thereof.

[0152] In some embodiments, the transmembrane domain comprises the amino acid sequence of a wild-type FPRP transmembrane domain (e.g., an FPRP transmembrane domain comprising the amino acid sequence of SEQ ID NO: 50) except for one amino acid mutation, two amino acid mutations, three amino acid mutations, four amino acid mutations, five amino acid mutations, six amino acid mutations, seven amino acid mutations, or eight or more amino acid mutations. The mutation(s) may be substitution(s), insertion(s), deletion(s), or any combination thereof.

[0153] In some embodiments, the FcRn binding polypeptide comprises a transmembrane domain homodomain dimerization motif that increases the interaction between two or more polypeptides in the transmembrane domain. In certain embodiments, the transmembrane domain homodomain dimerization motif is a transmembrane leucine zipper motif. In certain embodiments, the transmembrane domain homodimerization motif is a transmembrane glycine zipper motif. Methods for modifying and assaying transmembrane domain dimerization are known in the art, see, for example, Bocharov et al., J Biol Chem. 2008 Oct 24;283(43):29385-95.

[0154] In some embodiments, the transmembrane domain comprises the amino acid sequence of a wild-type ephrin receptor transmembrane domain (e.g., an ephrin receptor transmembrane domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 35-48), and its length is 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids or more at the N-terminus and / or C-terminus of SEQ ID NOs: 35-48.

[0155] In some embodiments, the transmembrane domain comprises the amino acid sequence of a wild-type FPRP transmembrane domain (e.g., an FPRP transmembrane domain comprising the amino acid sequence of SEQ ID NO:50), and its length is 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids or more at the N-terminus and / or C-terminus of SEQ ID NO:50.

[0156] (b) Selection of transmembrane scaffold proteins Several factors can be considered when selecting a scaffold protein for use as a transmembrane domain in the FcRn-binding polypeptide of the present disclosure, including an awareness of which particular transmembrane protein type (I, II, or polytopic type) is being considered for use as a source of the transmembrane domain, an awareness of the natural intracellular location of the transmembrane protein, and an awareness of whether the FcRn-binding polypeptide and the transmembrane domain in the fusion protein according to the present disclosure may affect each other's function in the overall process described herein for the presentation of the FcRn-binding polypeptide on the surface of nanovesicles.

[0157] As mentioned above, the four types of scaffold proteins can be distinguished from one another by the relative orientation of their N- and C-termini with respect to the cytoplasm and endoplasmic reticulum or nanovesicle (e.g., EV) lumen, and by whether the transmembrane region of the protein passes through the nanovesicle (e.g., EV) membrane only once (a "single-spanning" transmembrane region) or contains two or more transmembrane regions such that the protein as a whole passes through the membrane more than once (a multiply-spanning transmembrane region).

[0158] Knowing that the transmembrane region is derived from a particular type of transmembrane protein suggests a preferred orientation and location of the transmembrane domain relative to the FcRn-binding site in the FcRn-binding polypeptide of the present disclosure. This is particularly important for type I and type II transmembrane proteins, which have fixed orientation and location of the N-terminus and C-terminus relative to the cytoplasm and nanovesicle (e.g., EV) lumen on either side of the transmembrane region. For example, when a transmembrane region from a type I transmembrane protein is used as a scaffold protein (called a T1 scaffold) for the FcRn-binding polypeptide of the present disclosure, the FcRn-binding site is at the N-terminus of the transmembrane domain (as depicted in FIG. 2). Thus, the most common configuration of the FcRn-binding polypeptide of the present disclosure having a transmembrane domain from type I is a linear structure from N-terminus to C-terminus exemplified as follows: (1) (FcRn binding site)-L-(T1 scaffold) where L represents a direct peptide bond or a linker sequence of one or more amino acid residues connecting the two domains. See FIG. 1 for a schematic of a nanovesicle comprising an FcRn-binding polypeptide containing a type I transmembrane domain.

[0159] In addition, FcRn-binding polypeptides that contain a type I-derived transmembrane domain preferably contain an N-terminal signal sequence (e.g., a signal peptide), which is characteristic of type I transmembrane proteins that directs the N-terminus of the fusion protein through the ER membrane and into the ER lumen.

[0160] In specific embodiments, the FcRn-binding polypeptide is linked to a T1 scaffold protein comprising the ectodomain and transmembrane domain from EphA1. In some embodiments, the FcRn-binding polypeptide comprises an amino acid sequence identical or similar to the entire ectodomain and transmembrane domain region of wild-type EphA1 or a fragment thereof, and has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the entire ectodomain and transmembrane domain region of wild-type EphA1, and said polypeptide exhibits reduced or no binding to ephrin compared to the parent Eph receptor. In some embodiments, the polypeptide comprises an amino acid sequence identical or similar to SEQ ID NO: 50 or a fragment thereof, having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 50, and the polypeptide exhibits reduced or no binding to an ephrin compared to a parent Eph receptor. In some embodiments, a portion of the EphA1-derived polypeptide is fused to one or more heterologous proteins.

[0161] In specific embodiments, the FcRn-binding polypeptide is linked to a T1 scaffold protein comprising the ectodomain and transmembrane domain from EphA2. In some embodiments, the FcRn-binding polypeptide comprises an amino acid sequence identical or similar to the entire ectodomain and transmembrane domain region of wild-type EphA2 or a fragment thereof, and has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the entire ectodomain and transmembrane domain region of wild-type EphA2, and said polypeptide exhibits reduced or no binding to ephrin compared to the parent Eph receptor. In some embodiments, the polypeptide comprises an amino acid sequence identical or similar to SEQ ID NO:51 or a fragment thereof, having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:51, and the polypeptide exhibits reduced or no binding to an ephrin compared to the parent Eph receptor. In some embodiments, a portion of the EphA2-derived polypeptide is fused to one or more heterologous proteins.

[0162] In a specific embodiment, the FcRn-binding polypeptide is linked to a T1 scaffold protein comprising the ectodomain and transmembrane domain from EphA3. In some embodiments, the FcRn-binding polypeptide comprises an amino acid sequence identical or similar to the entire ectodomain and transmembrane domain region of wild-type EphA3 or a fragment thereof, and has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the entire ectodomain and transmembrane domain region of wild-type EphA3, and said polypeptide exhibits reduced or no binding to ephrin compared to the parent Eph receptor. In some embodiments, the polypeptide comprises an amino acid sequence identical or similar to SEQ ID NO: 52 or a fragment thereof, having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 52, and the polypeptide exhibits reduced or no binding to an ephrin compared to a parent Eph receptor. In some embodiments, a portion of the EphA3-derived polypeptide is fused to one or more heterologous proteins.

[0163] In a specific embodiment, the FcRn-binding polypeptide is linked to a T1 scaffold protein comprising the ectodomain and transmembrane domain from EphA4. In some embodiments, the FcRn-binding polypeptide comprises an amino acid sequence identical or similar to the entire ectodomain and transmembrane domain region of wild-type EphA4 or a fragment thereof, and has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of the entire ectodomain and transmembrane domain region of wild-type EphA4, and said polypeptide exhibits reduced or no binding to ephrin compared to the parent Eph receptor. In some embodiments, the polypeptide comprises an amino acid sequence identical or similar to SEQ ID NO: 53 or a fragment thereof, having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 53, and the polypeptide exhibits reduced or no binding to an ephrin compared to the parent Eph receptor. In some embodiments, a portion of the EphA4-derived polypeptide is fused to one or more heterologous proteins.

[0164] In specific embodiments, the FcRn-binding polypeptide is linked to a T1 scaffold protein comprising the ectodomain and transmembrane domain from EphA5. In some embodiments, the FcRn-binding polypeptide comprises an amino acid sequence identical or similar to the entire ectodomain and transmembrane domain region of wild-type EphA5 or a fragment thereof, and has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of the entire ectodomain and transmembrane domain region of wild-type EphA5, and said polypeptide exhibits reduced or no binding to ephrin compared to the parent Eph receptor. In some embodiments, the polypeptide comprises an amino acid sequence identical or similar to SEQ ID NO: 54 or a fragment thereof, having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 54, and the polypeptide exhibits reduced or no binding to an ephrin compared to the parent Eph receptor. In some embodiments, a portion of the EphA5-derived polypeptide is fused to one or more heterologous proteins.

[0165] In a specific embodiment, the FcRn-binding polypeptide is linked to a T1 scaffold protein comprising the ectodomain and transmembrane domain from EphA6. In some embodiments, the FcRn-binding polypeptide comprises an amino acid sequence identical or similar to the entire ectodomain and transmembrane domain region of wild-type EphA6 or a fragment thereof, and has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of the entire ectodomain and transmembrane domain region of wild-type EphA6, and said polypeptide exhibits reduced or no binding to ephrin compared to the parent Eph receptor. In some embodiments, the polypeptide comprises an amino acid sequence identical or similar to SEQ ID NO: 55 or a fragment thereof, having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 55, and the polypeptide exhibits reduced or no binding to an ephrin compared to the parent Eph receptor. In some embodiments, a portion of the EphA6-derived polypeptide is fused to one or more heterologous proteins.

[0166] In specific embodiments, the FcRn-binding polypeptide is linked to a T1 scaffold protein comprising the ectodomain and transmembrane domain from EphA7. In some embodiments, the FcRn-binding polypeptide comprises an amino acid sequence identical or similar to the entire ectodomain and transmembrane domain region of wild-type EphA7 or a fragment thereof, and has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the entire ectodomain and transmembrane domain region of wild-type EphA7, and said polypeptide exhibits reduced or no binding to ephrin compared to the parent Eph receptor. In some embodiments, the polypeptide comprises an amino acid sequence identical or similar to SEQ ID NO: 56 or a fragment thereof, having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 56, and the polypeptide exhibits reduced or no binding to an ephrin compared to a parent Eph receptor. In some embodiments, a portion of the EphA7-derived polypeptide is fused to one or more heterologous proteins.

[0167] In specific embodiments, the FcRn-binding polypeptide is linked to a T1 scaffold protein comprising the ectodomain and transmembrane domain from EphA8. In some embodiments, the FcRn-binding polypeptide comprises an amino acid sequence identical or similar to the entire ectodomain and transmembrane domain region of wild-type EphA8 or a fragment thereof, and has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the entire ectodomain and transmembrane domain region of wild-type EphA8, and said polypeptide exhibits reduced or no binding to ephrin compared to the parent Eph receptor. In some embodiments, the polypeptide comprises an amino acid sequence identical or similar to SEQ ID NO: 57 or a fragment thereof, having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 57, and the polypeptide exhibits reduced or no binding to an ephrin compared to the parent Eph receptor. In some embodiments, a portion of the EphA8-derived polypeptide is fused to one or more heterologous proteins.

[0168] In specific embodiments, the FcRn-binding polypeptide is linked to a T1 scaffold protein comprising the ectodomain and transmembrane domain from EphA10. In some embodiments, the FcRn-binding polypeptide comprises an amino acid sequence identical or similar to the entire ectodomain and transmembrane domain region of wild-type EphA10 or a fragment thereof, and has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the entire ectodomain and transmembrane domain region of wild-type EphA10, and said polypeptide exhibits reduced or no binding to ephrin compared to the parent Eph receptor. In some embodiments, the polypeptide comprises an amino acid sequence identical or similar to SEQ ID NO: 58 or a fragment thereof, having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 58, and the polypeptide exhibits reduced or no binding to an ephrin compared to the parent Eph receptor. In some embodiments, a portion of the EphA10-derived polypeptide is fused to one or more heterologous proteins.

[0169] In specific embodiments, the FcRn-binding polypeptide is linked to a T1 scaffold protein comprising the ectodomain and transmembrane domain from EphB1. In some embodiments, the FcRn-binding polypeptide comprises an amino acid sequence identical or similar to the entire ectodomain and transmembrane domain region of wild-type EphB1 or a fragment thereof, and has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the entire ectodomain and transmembrane domain region of wild-type EphB1, and said polypeptide exhibits reduced or no binding to ephrin compared to the parent Eph receptor. In some embodiments, the polypeptide comprises an amino acid sequence identical or similar to SEQ ID NO: 59 or a fragment thereof, having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 59, and the polypeptide exhibits reduced or no binding to an ephrin compared to a parent Eph receptor. In some embodiments, a portion of the EphB1-derived polypeptide is fused to one or more heterologous proteins.

[0170] In specific embodiments, the FcRn-binding polypeptide is linked to a T1 scaffold protein comprising the ectodomain and transmembrane domain from EphB2. In some embodiments, the FcRn-binding polypeptide comprises an amino acid sequence identical or similar to the entire ectodomain and transmembrane domain region of wild-type EphB2 or a fragment thereof, and has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of the entire ectodomain and transmembrane domain region of wild-type EphB2, and said polypeptide exhibits reduced or no binding to ephrin compared to the parent Eph receptor. In some embodiments, the polypeptide comprises an amino acid sequence identical or similar to SEQ ID NO: 60 or a fragment thereof, having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 60, and the polypeptide exhibits reduced or no binding to an ephrin compared to a parent Eph receptor. In some embodiments, a portion of the EphB2-derived polypeptide is fused to one or more heterologous proteins.

[0171] In a specific embodiment, the FcRn-binding polypeptide is linked to a T1 scaffold protein comprising the ectodomain and transmembrane domain from EphB3. In some embodiments, the FcRn-binding polypeptide comprises an amino acid sequence identical or similar to the entire ectodomain and transmembrane domain region of wild-type EphB3 or a fragment thereof, and has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of the entire ectodomain and transmembrane domain region of wild-type EphB3, and said polypeptide exhibits reduced or no binding to ephrin compared to the parent Eph receptor. In some embodiments, the polypeptide comprises an amino acid sequence identical or similar to SEQ ID NO:61 or a fragment thereof, having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:61, and the polypeptide exhibits reduced or no binding to an ephrin compared to a parent Eph receptor. In some embodiments, a portion of the EphB3-derived polypeptide is fused to one or more heterologous proteins.

[0172] In a specific embodiment, the FcRn-binding polypeptide is linked to a T1 scaffold protein comprising the ectodomain and transmembrane domain from EphB4. In some embodiments, the FcRn-binding polypeptide comprises an amino acid sequence identical or similar to the entire ectodomain and transmembrane domain region of wild-type EphB4 or a fragment thereof, and has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of the entire ectodomain and transmembrane domain region of wild-type EphB4, and said polypeptide exhibits reduced or no binding to ephrin compared to the parent Eph receptor. In some embodiments, the polypeptide comprises an amino acid sequence identical or similar to SEQ ID NO:62 or a fragment thereof, having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:62, and the polypeptide exhibits reduced or no binding to an ephrin compared to a parent Eph receptor. In some embodiments, a portion of the EphB4-derived polypeptide is fused to one or more heterologous proteins.

[0173] In a specific embodiment, the FcRn-binding polypeptide is linked to a T1 scaffold protein comprising the ectodomain and transmembrane domain from EphB6. In some embodiments, the FcRn-binding polypeptide comprises an amino acid sequence identical or similar to the entire ectodomain and transmembrane domain region of wild-type EphB6 or a fragment thereof, and has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of the entire ectodomain and transmembrane domain region of wild-type EphB6, and said polypeptide exhibits reduced or no binding to ephrin compared to the parent Eph receptor. In some embodiments, the polypeptide comprises an amino acid sequence identical or similar to SEQ ID NO:63 or a fragment thereof, having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:63, and the polypeptide exhibits reduced or no binding to an ephrin compared to a parent Eph receptor. In some embodiments, a portion of the EphB6-derived polypeptide is fused to one or more heterologous proteins.

[0174] Table 6. Scaffold proteins containing ectodomains and transmembrane domains from exemplary Eph receptors [Table 8] TIFF2024514156000021.tif249170TIFF2024514156000022.tif245170TIFF2024514156000023.tif186170

[0175] When the transmembrane region of a type II transmembrane protein is employed as a scaffold protein (referred to as a T2 scaffold), the FcRn binding site preferably comprises a domain arrangement in which the type II-derived transmembrane domain is N-terminal to the FcRn binding site. For example, an FcRn fusion protein may comprise a domain arrangement in which, from the N-terminus to the C-terminus (as depicted in FIG. 2), a scaffold protein or a fragment thereof comprising a type II-derived transmembrane domain is linked to a CH2 domain (of the modified Fc domain) which is in turn linked to a monomeric CH3 domain (of the modified Fc domain).

[0176] Thus, the most common configuration of the FcRn-binding polypeptides of the present disclosure that contain a type II derived transmembrane domain is a linear structure from N-terminus to C-terminus depicted as follows: (2) (T2 scaffold)-L-(FcRn binding site) where L represents a direct peptide bond or a linker sequence of one or more amino acid residues connecting the two domains.

[0177] As a non-limiting example, an FcRn-binding polypeptide is constructed by fusing a modified Fc polypeptide to the C-terminus of a transmembrane protein scaffold (T2 scaffold) derived from polytopic type II of AT1B3 (Uniprot P54709) that shares at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with AT1B3 according to SEQ ID NO: 64 or a functional fragment thereof. [Table 9]

[0178] In some embodiments, when a transmembrane region from a polytopic transmembrane protein is employed as a scaffold protein (referred to as a PT scaffold), the position of the transmembrane domain relative to the FcRn binding site varies depending on how many membrane-spanning regions of the transmembrane region are selected and what the orientation (as depicted in FIG. 2) of the membrane-spanning region(s) selected is from N-terminus to C-terminus with respect to the cytoplasmic and ER sides of the cell membrane. Exemplary PT scaffolds with non-cytoplasmic ends are listed in Table 3. It will be clear to one skilled in the art which end of a PT scaffold or a fragment thereof is non-cytoplasmic.

[0179] Thus, possible linear configurations for the FcRn-binding polypeptides of the present disclosure utilizing transmembrane domains derived from scaffold proteins containing polytopic transmembrane domains (PT scaffolds) can be exemplified as follows and may include the use of multiple transmembrane domains: (3) (FcRn binding site)-L-(PT scaffold), and / or (4) (PT scaffold)-L-(FcRn binding site), L in each formula represents a direct peptide bond or a linker of one or more amino acid residues connecting the two domains.

[0180] In some embodiments, the PT scaffold protein comprising polytopic transmembrane domain is positioned at the C-terminal domain relative to the FcRn binding site, similar to the arrangement for using the scaffold protein comprising transmembrane domain from type I, while in other embodiments, the PT scaffold protein comprising polytopic transmembrane domain is positioned at the N-terminal domain relative to the FcRn binding site.Unlike the scaffold protein comprising transmembrane domain from type I, the scaffold protein comprising transmembrane PT may not require an N-terminal signal sequence to guide the N-terminus of the PT scaffold into the ER membrane and into the ER lumen.However, in the case of the FcRn binding polypeptide protein comprising transmembrane domain from PT, an N-terminal signal sequence may still be required to achieve the desired position of the FcRn binding polypeptide on the surface of nanovesicle.

[0181] As a non-limiting example, an FcRn-binding polypeptide is constructed by fusing a modified Fc polypeptide to the C-terminus of a polytopic transmembrane domain derived from a protein scaffold of Zip2 (containing a modification at position H63A that reduces metal transport relative to the wild-type sequence) that shares at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with Zip2 according to SEQ ID NO: 65 or a functional fragment thereof.

[0182] As a further non-limiting example, an FcRn-binding polypeptide is constructed by fusing a modified Fc polypeptide to the N-terminus of a PT scaffold of Zip2 (containing a modification at position H63A that reduces metal transport relative to the wild-type sequence) that shares at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with Zip2 according to SEQ ID NO: 66 or a functional fragment thereof.

[0183] As a further non-limiting example, an FcRn-binding polypeptide is constructed by fusing a modified Fc polypeptide to both the N-terminus and C-terminus of a PT scaffold of Zip2 (containing a modification at position H63A that reduces metal transport relative to the wild-type sequence), both termini of which are located on the surface of the nanovesicle and share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with Zip2 according to SEQ ID NO: 67 or a functional fragment thereof. [Table 10]

[0184] In certain embodiments, in the case of a polytopic scaffold protein transmembrane region that includes both N-terminus and C-terminus toward the cytoplasm, the FcRn binding site can be located on the extracellular loop between two adjacent membrane-spanning fragments of the transmembrane domain.Exemplary PT scaffolds with cytoplasmic termini are listed in Table 4.It will be clear to those skilled in the art that the loop from the PT scaffold transmembrane domain is extracellular as opposed to cytoplasmic.

[0185] Thus, possible linear configurations for FcRn-binding polypeptides of the present disclosure utilizing transmembrane domains derived from scaffold proteins containing polytopic transmembrane domains (PT scaffolds) (as depicted in FIG. 2 ) may be exemplified as follows and may include the use of multiple transmembrane domains: (6):(TMH1)-(CL1)-(TMH2)-(L1)-(FcRn binding site)-(L2)-(TMH3)-(CL2) Each L in the formula represents a direct peptide bond or a linker of one or more amino acid residues connecting two domains, TMH represents a "transmembrane helix" and CL represents a "cytoplasmic loop."

[0186] (c) Standard Assays for Determining Polypeptides Useful as Scaffold Proteins In addition to the specific features of the present disclosure elucidated in the examples below, it is clear that proteins comprising a transmembrane region can be employed in assays to determine whether a particular scaffold protein is useful as an FcRn-binding polypeptide according to the present disclosure. In such scaffold protein assays, recombinant nucleic acid molecules encoding the amino acid sequence of a fusion protein comprising an FcRn-binding site fused in-frame with a candidate transmembrane domain are generated by standard methods (e.g., nucleic acid synthesis, recombinant DNA technology, and / or polymerase chain reaction (PCR)). Candidate scaffold proteins typically comprise a portion of a membrane protein that resides in or passes through a cellular or intracellular membrane according to the characteristics of the transmembrane domain described herein. Thus, as a non-limiting example, to test or evaluate any candidate polypeptide as a scaffold protein, a nucleic acid encoding a candidate transmembrane domain is linked in-frame to a nucleic acid encoding a common portion of FcRn-binding polypeptides comprising an FcRn-binding site. The resulting recombinant nucleic acid encoding the candidate FcRn-binding polypeptide fusion protein can then be inserted into an expression vector. Cells of a mammalian cell line, such as HEK293 cells used in the examples below, can be transfected with the expression vector. The transfected cells can then be isolated and grown in culture under conditions that allow expression of the protein encoded on the expression vector. Samples of the medium or nanovesicles isolated therefrom can be assayed for the amount of FcRn-binding polypeptide immobilized on the nanovesicles (e.g., EVs) (e.g., using an enzyme-linked immunosorbent assay (ELISA)), flow cytometry (e.g., using a fluorescent anti-Fc domain antibody), or functional binding to FcRn at acidic pH (e.g., using Lumit™ FcRn competition assay). An increase in the level of FcRn-binding polypeptide in the medium of transfected cells compared to the level of FcRn-binding polypeptide in the medium of untransfected control cells indicates that the scaffold protein, and thus the candidate FcRn-binding polypeptide, is useful as a scaffold according to the present disclosure.Preferably, the level of FcRn-binding polypeptide present in nanovesicles secreted into the medium of a culture of cells expressing the fusion protein is at least 1.5-fold higher than that in the medium of control cells. Enhancing the level of FcRn-binding polypeptide secreted from nanovesicles (e.g., exosomes) is also a therapeutically and commercially important property, and an increase of 1.5-fold or more can provide a significant reduction in production costs and a significant increase in the availability of therapeutically and commercially important nanovesicles.

[0187] In some embodiments, an FcRn-binding polypeptide comprising an FcRn-binding site and a scaffold protein may also contain an additional polypeptide domain or sequence. Such additional polypeptide domain may perform various functions, for example, such domain may (i) contribute to increasing the surface concentration of the FcRn-binding polypeptide, (ii) cause the clustering of the scaffold protein, thereby increasing the binding activity of the FcRn-binding polypeptide, (iii) function as a linker that optimizes the interaction between the scaffold protein and the FcRn-binding site, and / or (iv) improve the fixation in the nanovesicle membrane, as well as various other functions.

[0188] (5.2.4 Functional part) In a further aspect, the FcRn-binding polypeptide provided herein, in addition to being able to conditionally bind to FcRn, can also comprise one or more functional moieties (e.g., a fusion moiety, preferably a targeting domain that can target nanovesicles (e.g., EVs or hybridosomes) comprising the polypeptide to a specific organ, tissue, or cell type). In a preferred embodiment, the one or more functional moieties are proteins (e.g., peptides or polypeptides). In a preferred embodiment, the one or more functional moieties are fused in frame to the remainder of the polypeptide. In a particular embodiment, the one or more functional moieties are covalently fused to the remainder of the polypeptide via a linker.

[0189] Such one or more functional moieties can be at the N-terminus or C-terminus of the remainder of the polypeptide (e.g., fused at the N-terminus and / or C-terminus) or located between different domains of the remainder of the polypeptide. In certain embodiments, one or more functional moieties are presented towards the external space of the nanovesicle. In some embodiments, one or more functional moieties are at the N-terminus of the transmembrane domain of the scaffold protein (e.g., fused at the N-terminus). In some embodiments, one or more functional moieties are at the N-terminus of the modified Fc domain (e.g., fused at the N-terminus). In some embodiments, one or more functional moieties are at the C-terminus of the modified Fc domain (e.g., fused at the N-terminus). In some embodiments, one or more functional moieties are at the C-terminus of the transmembrane domain of the scaffold protein (e.g., fused at the C-terminus). In some embodiments, one or more functional moieties are at the N-terminus of the transmembrane domain of the scaffold protein (e.g., fused at the N-terminus). In certain embodiments, one or more functional moieties are presented towards the lumen of the nanovesicle. In some embodiments, the one or more functional moieties are C-terminal to (e.g., fused at) the transmembrane domain of the scaffold protein. In some embodiments, the one or more functional moieties are C-terminal to (e.g., fused at) the C-terminus of) the modified Fc domain.

[0190] Exemplary functional moieties include, but are not limited to, targeting domains such as affinity tags and purification domains. A functional moiety can be a large polypeptide or peptide. In some embodiments, an FcRn-binding polypeptide comprises an FcRn-binding site and an optional targeting moiety, each of which can be modified independently.

[0191] In some embodiments, the targeting domain is preferably located on the surface of the nanovesicle. The targeting domain serves to guide the nanovesicle towards a specific organ, tissue, or cell, and is preferably organ, tissue, or cell specific. One or more targeting domains may be fused to the remainder of the FcRn binding polypeptide. The presence of more than one targeting domain may increase the specificity for the target organ, tissue, or cell. In some embodiments, the targeting domain is or comprises one or more antigen binding molecules. In some embodiments, the targeting domain specifically targets an antigen expressed on cancer cells, metastatic cells, dendritic cells, stem cells, or immune cells. Exemplary antigens expressed on tumor cells include, but are not limited to, BAGE, BCMA, CEA, CD19, CD20, CD33, CD123, CEA, FAP, HER2, LMP1, LMP2, MAGE, Mart1 / MelanA, NY-ESO, PSA, PSMA, RAGE, and survivin.

[0192] In some embodiments, the targeting domain is located in the lumen of the nanovesicle. The targeting domain serves to attach cytoplasmic components (e.g., proteins, protein complexes, viruses) to the scaffold prior to invagination and vesicle formation. The presence of more than one targeting domain can increase the loading efficiency of cytoplasmic components into the lumen of the nanovesicle during biosynthesis. In some embodiments, the targeting domain is or comprises one or more antigen binding molecules. In some embodiments, the targeting domain specifically targets an antigen expressed on adeno-associated virus.

[0193] In certain embodiments, the targeting domain is selected from the group consisting of scFv, (scFv)2, Fab, Fab', F(ab')2, Fv, dAb, Fd fragment, diabody, F(ab')3, disulfide-linked Fv, sdAb (VHH or nanobody), CDR, di-scFv, bi-scFv, tascFv (tandem scFv), triabody, tetrabody, V-NAR domain, Fcab, IgGACH2, DVD-Ig, probody, darpin, centirin, affibody, affilin, affitin, anticalin, avimer, finomer, Kunitz domain peptide, monobody (or adnectin), tribody, and nanophytin. In certain embodiments, the targeting domain is selected from the group consisting of scFv, (scFv)2, Fab, Fab', F(ab')2, F(abl)2, Fv, dAb, Fd fragment, diabody, F(ab)2, F(ab'), F(ab')3, Fd, Fv, disulfide-linked Fv, dAb, sdAb, nanobody, CDR, di-scFv, bi-scFv, tascFv (tandem scFv), avibody (e.g., diabody, triabody, tetrabody), T-cell engager (BiTE), V-NAR domain, Fcab, IgGACH2, DVD-Ig, probody, intrabody, darpin, centirin, affibody, affilin, affitin, anticalin, avimer, finomer, Kunitz domain peptide, monobody, adnectin, tribody, and nanophytin.

[0194] In certain embodiments, the targeting domain specifically binds to a marker. In a specific embodiment, the marker is a tumor-associated antigen. In a specific embodiment, the tumor-associated antigen is human epidermal growth factor receptor 2 (HER2), CD20, CD33, B-cell maturation antigen (BCMA), prostate-specific membrane antigen (PSMA), DLL3, ganglioside GD2 (GD2), CD 123, anoctamin-1 (anol), mesothelin, carbonic anhydrase IX (CAIX), tumor-associated calcium signaling factor 2 (TROP2), carcinoembryonic antigen (CEA), claudin-18.2, receptor tyrosine kinase-like orphan receptor 1 (ROR1), trophoblast glycoprotein (5T4), glycoprotein nonmetastatic melanoma protein B (GPNMB), folate receptor alpha (FR-alpha), pregnancy-associated plasma protein A (PAPP-A), CD37, epithelial cell adhesion molecule (EpCAM), CD2, CD19, CD30, CD38, CD40, CD52, CD70, CD79b, fms-like tyrosine kinase 3 (FLT3), glypican 3 (GPC3), B7 homolog 6 (B7H6), CC chemokine receptor type 4 (CCR4), C-X-C motif chemokine receptor 4 (CXCR4), receptor tyrosine kinase-like orphan receptor 2 (ROR2), CD133, HLA class I histocompatibility antigen, alpha chain E (HLA-E), epidermal growth factor receptor (EGFR / ERBB-1), insulin-like growth factor 1 receptor (IGF1R), and human epidermal growth factor receptor 3.

[0195] In some embodiments, a method for targeting nanovesicles to specific organs, tissues or cells is provided, the method comprising fusing a targeting domain to a portion of an FcRn-binding polypeptide of the present disclosure and expressing the polypeptide within the nanovesicle.

[0196] The antigen-binding molecule serving as the targeting domain may be monospecific, bispecific or multispecific, i.e., it may target one or more epitopes of the same or different targets. The more specificity displayed on the nanovesicle, the more specific the targeting. In some embodiments, the antigen-binding molecule is (i) a full-length antibody molecule (e.g., IgG, IgM, IgA, IgM or IgE); (ii) antibody fragments, such as CDR, Dab, Fab, Fab', F(ab')2, Fd fragment, Fv fragment, disulfide-linked Fv, scFab, nanobody, minimal recognition unit, VHH or V-NAR domains; iii) non-antibody scaffolds, such as affibodies, afftin molecules, affitins, adnectins, anticalins, avimers, centrins, lipocalin muteins, darpins, finomers, knottins, Kunitz-type domains, nanophytins, tetranectins or transbodies; iv) fusion polypeptides comprising one or more antibody domains, e.g., bi-scFv, aBITE, diabody, di-scFv, probody, tascFv (tandem scFv), triabody, tribody, tetrabody, IgGACH2, DVD-Ig, MATCH, minibody, scFv, Fv-Fc, bispecific F(ab')2, F(ab')3, monovalent IgG; v) Soluble T cell receptor (sTCR); vi) peptides, e.g., natural peptides, recombinant peptides, synthetic peptides; and vii) a viral protein, such as a receptor binding domain of a viral spike protein (e.g., a coronavirus) or influenza hemagglutinin (HA), Nipah virus protein F, measles virus F protein, Tupaia paramyxovirus F protein, Paramyxovirus F protein, Hendra virus F protein, Henipavirus F protein, Morbillivirus F protein, Respirovirus F protein, Sendai virus F protein, Rubulavirus F protein, or Abulavirus F protein, or a fragment thereof, respectively; is selected from the group consisting of:

[0197] In further embodiments, the transmembrane region is derived from a particular type of transmembrane protein, suggesting a preferred orientation and location of the transmembrane domain relative to the targeting moiety in the FcRn-binding polypeptides of the present disclosure.

[0198] Therefore, the most common configuration of the FcRn-binding polypeptide of the present disclosure having a type I derived transmembrane domain is a linear structure from N-terminus to C-terminus exemplified as follows: (targeting domain)-L-(FcRn binding site)-L-(T1 scaffold) where each L represents a direct peptide bond or a linker of one or more amino acid residues connecting the two domains.

[0199] In contrast, in embodiments in which the transmembrane region of a type II transmembrane protein is employed as the transmembrane domain, the arrangement of the domains results in a configuration in which the targeting moiety is fused to the FcRn-binding polypeptide of the present disclosure, resulting in a linear structure from N-terminus to C-terminus depicted as follows: (T2 scaffold) -L-(FcRn binding site) -L-(targeting moiety) where each L represents a direct peptide bond or a linker of one or more amino acid residues connecting the two domains.

[0200] In some embodiments, the FcRn-binding polypeptide comprises a targeting moiety that is a bispecific modified Fc domain (e.g., an Fc domain further modified to promote transferrin receptor binding). In some embodiments, the bispecific modified Fc domain comprises a monomeric CH3 domain modified to exhibit higher binding affinity to transferrin receptor compared to the unmodified CH3 domain, and retains the ability to bind to the Fc-binding site of FcRn. In some embodiments, the modified Fc domain that specifically binds to transferrin receptor comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 substitutions in a set of amino acid positions including 380, 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 according to EU numbering. In some embodiments, the modified Fc domain that specifically binds to transferrin receptor is at least 80%, 90%, or 95% similar to SEQ ID NO: 34.

[0201] 5.3 Methods for Producing Nucleic Acids, Expression Vectors, Cells, and Polypeptides Also provided are nucleic acids encoding the polypeptides described herein (e.g., as described in Section 5.2), vectors (e.g., expression vectors) that contain the nucleic acids described herein, and cells (e.g., host cells) that contain the nucleic acids or expression vectors described herein.

[0202] The FcRn-binding polypeptides of the present disclosure can be produced using any number of expression systems, including prokaryotic and eukaryotic expression systems. In some embodiments, the expression system is a mammalian cell expression system, such as the HEK293T system. Many such systems are widely available from commercial sources. In some embodiments, the polynucleotides encoding the polypeptides (particularly the FcRn-binding polypeptides) can be expressed using a single vector, for example, in a bicistronic expression unit or under the control of different promoters. In other embodiments, the polynucleotides encoding the polypeptides (particularly the FcRn-binding polypeptides) can be expressed using separate vectors.

[0203] The polynucleotide may be present in a variety of different forms and / or in different vectors. For example, the polynucleotide may be essentially linear, circular, and / or may have any secondary and / or tertiary and / or higher order structure. Furthermore, the present disclosure also relates to vectors comprising the polynucleotide, such as vectors such as plasmids, any circular or linear DNA polynucleotides, minicircles, viruses (such as adenoviruses, adeno-associated viruses, lentiviruses, retroviruses, etc.), mRNA, and / or modified mRNA.

[0204] In some aspects, the present disclosure provides isolated nucleic acids comprising a nucleic acid sequence encoding any of the polypeptides described herein (particularly, FcRn-binding polypeptides); vectors comprising such nucleic acids; and host cells into which the nucleic acids are introduced, used to replicate the nucleic acids and / or express the polypeptides (particularly, FcRn-binding polypeptides).

[0205] In some embodiments, the polynucleotide (e.g., the isolated polynucleotide) comprises a nucleotide sequence encoding a polypeptide (particularly an FcRn-binding polypeptide) as disclosed herein (e.g., as described above). In some embodiments, the polynucleotide described herein is operably linked to a heterologous nucleic acid, e.g., a heterologous promoter.

[0206] Suitable vectors containing a polynucleotide encoding the polypeptides (especially FcRn-binding polypeptides) or fragments thereof of the present disclosure include cloning vectors and expression vectors. The cloning vector selected may vary depending on the cells intended for use, but useful cloning vectors are generally capable of self-replication, may possess a single target for a specific restriction endonuclease, and / or may carry a gene for a marker that can be used in selecting clones containing the vector. Examples include plasmids and bacterial viruses, such as pUCl8, pUCl9, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColEl, pCRl, RP4, phage DNA, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial vendors such as BioRad, Strategene, and Invitrogen.

[0207] An expression vector is generally a replicable polynucleotide construct that contains the nucleic acid of the present disclosure. Expression vectors can replicate in cells as episomes or as an integrated part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors including adenoviruses, adeno-associated viruses, lentiviruses, retroviruses, and any other vectors. Usually, the coding sequence of a polypeptide is operably linked to a suitable control sequence that can affect the expression of DNA in a suitable host. Such control sequences may include a promoter that affects transcription, an optional operator sequence that controls transcription, a sequence that codes for a suitable ribosome binding site on mRNA, an enhancer, and / or a sequence that controls the termination of transcription and translation.

[0208] Suitable cells for cloning or expressing the polynucleotides or vectors described herein include prokaryotic or eukaryotic cells. In some embodiments, the cells are prokaryotic. In some embodiments, the cells are eukaryotic, such as Chinese hamster ovary (CHO) cells or lymphoid cells. In some embodiments, the cells are human cells, such as human embryonic kidney (HEK) cells. In some embodiments, the cells are human cells, such as human embryonic kidney (HEK) cells. In some embodiments, the cells are non-tumor cell lines derived from human amniotic cells.

[0209] Transfection is the process of introducing nucleic acid into a cell by non-viral methods. Transduction is the process of introducing foreign DNA into another cell via a viral vector. Common transfection methods include calcium phosphate, cationic polymers (such as PEI), magnetic beads, electroporation, and commercially available lipid-based reagents such as Lipofectamine and Fugene. Transduction is primarily used to describe the introduction of recombinant viral vector particles into a target cell, while "infection" refers to the natural infection of humans or animals with wild-type viruses.

[0210] In addition to the above-mentioned standard methods of nucleic acid delivery, the nucleic acids provided herein can be targeted to specific sites within the genome of a cell. Such methods include, but are not limited to, CRISPR-Cas9, TALEN, meganucleases designed against the genomic sequence of interest in the host cell, and other techniques for precise editing of the genome, Cre-lox site-specific recombination; zinc finger-mediated integration; and homologous recombination. The nucleic acid may contain a transposon comprising a nucleic acid encoding a polypeptide of the present disclosure. In some embodiments, the nucleic acid may further comprise a nucleic acid sequence encoding a transposase enzyme. In other embodiments, a system is provided having two nucleic acids, a first plasmid containing a transposon comprising a nucleic acid encoding a polypeptide of the present disclosure, and a second plasmid containing a nucleic acid sequence encoding a transposase enzyme. Both the first and second nucleic acids can be delivered simultaneously to the host cell. Cells expressing the polypeptides described herein (particularly FcRn-binding polypeptides) can also be produced by using a combination of gene insertion (using a transposon) and gene editing (using a nuclease). Exemplary transposons include, but are not limited to, piggyBac and Sleeping Beauty Transposon Systems (SBTS); while exemplary nucleases include, but are not limited to, CRISPR / Cas systems, Transcription Activator-Like Effector Nucleases (TALENs), and Zinc Finger Nucleases (ZFNs).

[0211] The genetically modified cells can contain exogenous sequences by transient or stable transformation. The exogenous sequences can be transformed as a plasmid. The exogenous sequences can be stably integrated into the genome sequence of the cells at a target site or random site. In some embodiments, a stable cell line is generated for the production of nanovesicles (e.g., EVs and hybridosomes) comprising the polypeptides disclosed herein (particularly, FcRn-binding polypeptides). Preferably, the stable cell line is generated when the cells are stably transfected with constructs encoding the polypeptides disclosed herein (particularly, FcRn-binding polypeptides). This advantageously results in consistent production of nanovesicles (e.g., EVs and hybridosomes) of uniform quality and yield.

[0212] Exogenous sequences encoding fragments of the polypeptides described herein (particularly those containing FcRn binding sites) can be inserted into the genomic sequence of the producer cell, located upstream (5'-end) or downstream (3'-end) of the endogenous sequence.Various methods known in the art can be used to introduce exogenous sequences into the producer cell.For example, cells modified using various gene editing methods (e.g., using homologous recombination, transposon-mediated systems, loxP-Cre systems, CRISPR / Cas9 or TALEN) are within the scope of the present disclosure.

[0213] The exogenous nucleic acid sequence may comprise a sequence encoding a polypeptide (particularly an FcRn-binding polypeptide) or a fragment or variant thereof disclosed herein. Extra copies of a sequence encoding a polypeptide (particularly an FcRn-binding polypeptide) may be introduced to generate nanovesicles described herein (e.g., nanovesicles with a higher density of FcRn-binding polypeptides or nanovesicles expressing multiple different FcRn-binding polypeptides on the surface of the nanovesicle). Exogenous sequences encoding a polypeptide (particularly an FcRn-binding polypeptide), variants or fragments thereof may be introduced to generate lumen-engineered and / or surface-modified nanovesicles (EVs or hybridosomes) and, optionally, nanovesicles containing modified or fragmented polypeptides (particularly an FcRn-binding polypeptide).

[0214] In some embodiments, cells can be modified, e.g., transfected, with one or more polypeptides (particularly one or more FcRn-binding polypeptides comprising different scaffold proteins) comprising an exogenous fusion moiety (e.g., a targeting moiety or purification domain) described herein.

[0215] In another aspect, a method for producing a polypeptide (particularly an FcRn-binding polypeptide) described herein is provided. In some embodiments, the method comprises culturing a host cell described herein (e.g., a cell comprising a nucleic acid or expression vector described herein) under conditions suitable for expression of the polypeptide (particularly an FcRn-binding polypeptide). In some embodiments, the polypeptide (particularly an FcRn-binding polypeptide) is then recovered from the host cell (or host cell medium). In some embodiments, the polypeptide (particularly an FcRn-binding polypeptide) is purified, for example, by affinity chromatography.

[0216] 5.4 Nanovesicles (e.g., extracellular vesicles and hybridosomes) and methods for producing nanovesicles Also provided herein are nanovesicles (e.g., extracellular vesicles and hybridosomes) comprising the polypeptides described herein (e.g., as described in Section 5.2). Another aspect of the present disclosure relates to the production and use of surface-engineered nanovesicles. Nanovesicles comprising the polypeptides described herein (e.g., FcRn-binding polypeptides) provide an important advancement and lead to new nanovesicle compositions and methods of making the same. Previously, overexpression of exogenous proteins relied on stochastic or random placement of exogenous proteins on nanovesicles to generate surface-engineered nanovesicles. As a result, the density of the protein of interest on the nanovesicles was low and unpredictable.

[0217] Thus, in one embodiment, there is provided a nanovesicle comprising at least an FcRn binding site, said FcRn binding site being (i) Binds to FcRn at acidic pH (ii) lacks the ability to form homodimers; and (ii) comprises a transmembrane domain; Nanovesicles are provided.

[0218] Nanovesicles of the present disclosure can be natural (i.e., generated from a source cell through secretion from the endosomal, endolysomal and / or lysosomal pathways or from the cell membrane of a source cell) or synthetic nanovesicles. Exemplary nanovesicles include, but are not limited to, extracellular vesicles ("EVs"), microvesicles (MVs), exosomes, apoptotic bodies, ARMMs, fusosomes, microparticles, and cell-derived vesicle structures include membrane particles, membrane vesicles, exosome-like vesicles, ectosome-like vesicles, ectosomes or exo vesicles or hybridosomes.

[0219] In one embodiment, the FcRn-binding polypeptide can be present on a hybridosome, i.e., a hybrid biocompatible carrier comprising structural and biologically active elements resulting from an EV comprising the FcRn-binding polypeptide and a lipid nanoparticle comprising a regulatable membrane fusogenic moiety, as described in WO2015110957. In some embodiments, the isolated hybridosome comprising the FcRn-binding polypeptide of the present disclosure further comprises a therapeutic molecule.

[0220] The present disclosure further provides a method for producing and / or purifying nanovesicles (e.g., EVs and hybridosomes) comprising at least one polypeptide (particularly at least one FcRn-binding polypeptide) as described above. The method may generally comprise the steps of: (i) introducing a nucleic acid encoding the above-mentioned polypeptide (particularly an FcRn-binding polypeptide) into an EV-producing cell; (ii) allowing the EV-producing cell to produce EVs comprising the polypeptide (particularly an FcRn-binding polypeptide), e.g. culturing the cell under suitable conditions. As a result of the presence of a transmembrane domain, the polypeptide (particularly an FcRn-binding polypeptide) is efficiently transported to the membrane of the cell and an FcRn-binding site is presented within or on the surface of the EV. Then, in step (iii), the EVs may be purified from the culture medium. Such a method may optionally comprise the step of (iv) chemically modifying the purified EVs to produce synthetic nanovesicles, e.g., hybridosomes.

[0221] In one embodiment, there is provided a method for producing nanovesicles that are surface-modified with one or more FcRn binding sites, comprising the steps of: (i) providing a nucleic acid or expression vector encoding a polypeptide as described above (in particular an FcRn-binding polypeptide) comprising one or more FcRn-binding sites; (ii) introducing the nucleic acid or expression vector into an EV-producing cell (e.g., a mesenchymal stem cell); (iii) culturing the cells under appropriate conditions to produce EVs (e.g., exosomes); and (iv) purifying EVs (e.g., exosomes) containing the polypeptides (e.g., FcRn-binding polypeptides) so produced from the cell culture. The method includes:

[0222] The method may optionally include (v) chemically modifying the EVs to generate synthetic nanovesicles, such as hybridosomes, for example, produced by contacting the EVs with in vitro generated second vesicles, thereby binding the second vesicles to the EVs, the second vesicles comprising a membrane and a fusogenic, ionizable, cationic lipid (e.g., at a molar concentration of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, and preferably at least 30% of the total lipids of the second vesicles), and optionally a therapeutic agent.

[0223] In one embodiment, a method of generating EVs comprises: a. transfecting cells with a nucleic acid described herein or an expression vector described herein; b. culturing the cells under conditions suitable for the generation of EVs; and c. collecting EVs secreted from the cells.

[0224] In one embodiment, a method of generating a hybridosome comprises contacting a first EV with a second EV, thereby associating the first EV with the second EV and generating a hybridosome, wherein said first EV is generated in vitro, and wherein the first EV comprises (i) a membrane, and (ii) a fusogenic, ionizable, cationic lipid (e.g., at a molar concentration of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, and preferably at least 30% of the total lipids of the first EV), and wherein said second EV is generated by a method of generating EVs described herein.

[0225] Nanovesicles (e.g., EVs and hybridosomes) comprising the polypeptides of the present disclosure (particularly FcRn-binding polypeptides) can be generated from any type of mammalian cell that can generate nanovesicles (e.g., EVs) under appropriate conditions, for example, in suspension or adherent culture or any other type of culture system. Source cells according to the present disclosure can also include cells that can generate nanovesicles (e.g., EVs) in vivo. Source cells can be selected from a wide range of cells and cell lines that grow in suspension or adherent culture or can be adapted to suspension growth. In general, nanovesicles (e.g., EVs and hybridosomes) can be derived from essentially any cell source, whether primary cell source or immortalized cell line. Source cells can be either allogeneic, autologous, or even xenogeneic in nature to the patient to be treated, i.e., the cells can be from the patient himself or from an unrelated donor, a matched donor or a mismatched donor. In certain circumstances, allogeneic cells may be preferable from a medical standpoint, as they can provide immunomodulatory effects that are unlikely to be obtained from autologous cells for patients suffering from certain indications. For example, in the context of the treatment of inflammatory or degenerative diseases, allogeneic MSCs or amniotic epithelium (AE) may be highly beneficial as a source of nanovesicle (e.g., EVs or hybridosomes)-generating cells, due to the inherent immunomodulatory properties of their EVs. Cell lines of particular interest include, but are not limited to, anionic fluid derived cells, induced pluripotent cells, human umbilical cord endothelial cells (HUVEC), human embryonic kidney (HEK) cells, e.g., HEK293 cells, HEK293T cells, serum-free HEK293 cells, suspension HEK293 cells, endothelial cell lines, e.g., microvascular or lymphatic endothelial cells, erythrocytes, erythroid progenitor cells, chondrocytes, MSCs of different origins, amniotic cells, AE cells, any cells obtained via amniocentesis or from the placenta, airway or alveolar epithelial cells, fibroblasts, endothelial cells, and epithelial cells.

[0226] As described above, source cells can be genetically modified to contain one or more exogenous sequences (e.g., encoding one or more fusion proteins) to produce nanovesicles as described herein. Preferably, the exogenous sequences encoding the polypeptides described herein (particularly FcRn-binding polypeptides) are stably integrated into the genomic sequence of the producer cells at targeted or random sites. In some embodiments, stable cell lines are generated for the production of nanovesicles (e.g., EVs) comprising the polypeptides disclosed herein (particularly FcRn-binding polypeptides). This advantageously results in consistent production of nanovesicles (e.g., EVs) of uniform quality and yield.

[0227] In some embodiments, nanovesicles comprising the polypeptides of the present disclosure (particularly, FcRn-binding polypeptides) can be produced from cells transformed with a sequence encoding a full-length scaffold protein fused to an FcRn-binding site (particularly, an FcRn-binding polypeptide) disclosed herein, which may further comprise one or more heterologous proteins (e.g., targeting domains) as described above. Any of the polypeptides described herein (particularly, an FcRn-binding polypeptides) can be expressed from a plasmid, an exogenous sequence inserted into a genome, or other exogenous nucleic acid, such as synthetic messenger RNA (mRNA).

[0228] In one aspect, the present disclosure provides EVs comprising two or more interacting FcRn-binding polypeptides (e.g., scaffold proteins) produced from a cell of the present disclosure. In some embodiments, the surface density or concentration of the polypeptides (e.g., scaffold proteins) on the EVs described herein is increased by dimerization or oligomerization (excluding CH3-CH3 dimerization).

[0229] In some embodiments, the source cells disclosed herein are further modified to include additional exogenous sequences. For example, additional exogenous sequences can be introduced to regulate endogenous gene expression or generate nanovesicles that include a particular polypeptide as a payload. In some embodiments, the source cells are modified to include two exogenous sequences, one encoding a polypeptide described herein (particularly an FcRn-binding polypeptide) or a variant or fragment thereof, and the other encoding a payload. In some embodiments, the source cells are modified to include two exogenous sequences, one encoding a polypeptide described herein (particularly an FcRn-binding polypeptide) or a variant or fragment thereof, and the other encoding a polypeptide described herein (particularly an FcRn-binding polypeptide) including any targeting moiety. In certain embodiments, the source cells can be further modified to include additional exogenous sequences that impart additional functionality to the nanovesicles (e.g., a payload, a targeting moiety, or a purification domain). In some embodiments, the source cell is modified to contain two exogenous sequences, one encoding a polypeptide disclosed herein (particularly an FcRn binding polypeptide) or a variant or fragment thereof, and the other encoding a protein that confers additional functionality to the nanovesicle, hi some embodiments, the source cell is further modified to contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more additional exogenous sequences.

[0230] Thus, the present disclosure further relates to the production and use of EVs comprising at least one FcRn-binding polypeptide, said FcRn-binding polypeptide comprising a transmembrane domain and a modified Fc domain of an immunoglobulin that (i) is capable of specifically binding to the Fc-binding site of FcRn; and (i) lacks the ability to form homodimers. In one embodiment, when the FcRn-binding polypeptide is expressed on a nanovesicle (e.g., an EV), the transmembrane domain or a fragment thereof to which the modified Fc domain is covalently linked (e.g., fused) provides anchoring of the modified Fc domain to the EV membrane, such that the modified Fc domain of the FcRn-binding polypeptide protrudes into the extracellular environment and subsequently allows specific and conditional binding to the Fc-binding site of FcRn.

[0231] In one embodiment, nanovesicles (e.g., EVs) comprising at least one FcRn-binding polypeptide of the present disclosure have increased binding affinity to the Fc-binding site of FcRn at acidic pH and decreased binding affinity to the Fc-binding site of FcRn at near neutral pH. In a preferred embodiment, EVs comprising at least one FcRn-binding polypeptide of the present disclosure have increased propensity to form complexes with FcRn at acidic pH as opposed to neutral pH.

[0232] In some embodiments, the equilibrium dissociation constant of a nanovesicle (e.g., EV) comprising at least one FcRn-binding polypeptide of the present disclosure that binds to FcRn in an acidic environment is at least 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 or 10 -9M. In some embodiments, the equilibrium dissociation constant of an EV comprising at least one FcRn-binding polypeptide of the present disclosure that binds to FcRn at an acidic pH (e.g., a pH less than 6.5) is equal to the equilibrium dissociation constant of the modified Fc domain that binds to FcRn at pH 6.5. In some embodiments, the equilibrium dissociation constant of an EV comprising at least one FcRn-binding polypeptide of the present disclosure that binds to FcRn at pH 6.5 is increased by at least 5%, 10%, 20%, 30%, 40%, 50% or 60% compared to the equilibrium dissociation constant of the modified Fc domain fragment that binds to FcRn.

[0233] In some embodiments, the equilibrium dissociation constant of an EV comprising at least one FcRn-binding polypeptide of the present disclosure that binds to FcRn at neutral pH is 10 -5 , 10 -4 , 10 -3 , 10 -2 or 10 -1 In some embodiments, the equilibrium dissociation constant of an EV comprising at least one FcRn-binding polypeptide of the present disclosure that binds to FcRn at neutral pH is equal to the equilibrium dissociation constant of the modified Fc domain that binds to FcRn at neutral pH. In some embodiments, the equilibrium dissociation constant of an EV comprising at least one FcRn-binding polypeptide of the present disclosure that binds to FcRn at pH 6.5 is increased by at least 20%, 30%, 40%, 50% or 60% compared to the equilibrium dissociation constant of the modified Fc domain fragment that binds to FcRn.

[0234] In one embodiment of all aspects described herein, at acidic pH, the EV comprising at least one FcRn-binding polypeptide of the present disclosure has a binding affinity for an FcRn selected from human FcRn, cynomolgus monkey FcRn, mouse FcRn, rat FcRn, sheep FcRn, dog FcRn, and rabbit FcRn. In some embodiments, the FcRn-binding polypeptide has increased binding affinity for mouse FcRn over human FcRn.

[0235] Methods for analyzing binding affinity and binding kinetics for EVs comprising at least one FcRn-binding polypeptide of the present disclosure disclosed herein are known in the art. These methods include, but are not limited to, solid-phase binding assays (e.g., ELISA assays), immunoprecipitation, surface plasmon resonance (e.g., Biacore™ (GE Healthcare, Piscataway, NJ)), kinetic exclusion assays (e.g., KinExA®), flow cytometry, fluorescence-activated cell sorting (FACS), biolayer interferometry (e.g., Octet® (ForteBio, Inc., Menlo Park, CA)), and Western blot analysis. In some embodiments, ELISA is used to determine binding affinity. In some embodiments, surface plasmon resonance (SPR) is used to determine binding affinity and / or binding kinetics. In some embodiments, kinetic exclusion assays are used to determine binding affinity and / or binding kinetics. In some embodiments, biolayer interference assays are used to determine binding affinity and / or binding kinetics.

[0236] In some embodiments, EVs comprising at least one FcRn-binding polypeptide as described herein may comprise modifications in the FcRn-binding polypeptide that reduce or eliminate effector function. Thus, in some embodiments, EVs comprising at least one FcRn-binding polypeptide as described herein comprise modifications that reduce effector function, i.e., have a reduced ability to induce a particular biological function upon binding to an Fc receptor (other than FcRn) expressed on or in an effector cell and mediating effector function. Examples of Fc receptor effector functions include, but are not limited to, Clq binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. In some embodiments, modified Fc domains present in the FcRn-binding polypeptides as described herein may comprise additional modifications that modulate effector function.

[0237] In some embodiments, EVs comprising at least one FcRn-binding polypeptide have a modified Fc domain that lacks the ability to form homodimers to avoid protein misfolding in the producing cells, which can cause cytotoxicity to the protein cells and lead to protein instability and / or aggregation of EVs.

[0238] In another embodiment, an EV comprising at least one FcRn-binding polypeptide of the present disclosure may have the ability to specifically bind to an antigen on a specific cell in addition to binding to FcRn. In some embodiments, an EV comprising at least one FcRn-binding polypeptide comprises an additional targeting domain. In some embodiments, an EV comprises an FcRn-binding polypeptide that can be fused to a targeting domain, such as an antigen-binding fragment (e.g., Fab, Fv, or scFv) that specifically binds to an antigen. In some embodiments, an EV comprising at least one FcRn-binding polypeptide contains an FcRn-binding site and optionally a targeting moiety, each of which can be independently modified. In some embodiments, the modification allows an EV comprising at least one FcRn-binding polypeptide to specifically bind to FcRn at acidic pH. The targeting moiety can be used to target an EV comprising at least one FcRn-binding polypeptide to an antigen on a specific organ, tissue, or cell.

[0239] In some embodiments, EVs comprising at least one FcRn-binding polypeptide described herein exhibit superior characteristics compared to EVs known in the art. For example, FcRn-binding polypeptides or fragments thereof comprising different transmembrane domains are enriched on the EV surface compared to naturally occurring EVs or EVs generated using conventional EV proteins. In some embodiments, EVs comprising FcRn-binding polypeptides described herein can express more (e.g., 2, 3, 4, 5 or more) FcRn-binding sites, such that not many EVs are required. Furthermore, the surface of EVs comprising engineered FcRn-binding polypeptides of the present disclosure can have higher, more specific, or more controlled biological activity (e.g., targeting to specific cells or half-life) compared to naturally occurring EVs or EVs generated using conventional transmembrane domains (e.g., Lamp2b, PTGFRN, CD63, or CD81).

[0240] In further embodiments, the FcRn-binding polypeptide may be present on a hybridosome, a hybrid biocompatible carrier comprising structural and bioactive elements resulting from an EV comprising the FcRn-binding polypeptide and a lipid nanoparticle comprising a regulatable membrane fusogenic moiety, as described in WO2015110957. As a result of the presence of the FcRn-binding polypeptide, the resulting hybridosome can be isolated from unfused lipid nanoparticles by affinity chromatography methods described herein. In some embodiments, the isolated hybridosome comprising the FcRn-binding polypeptide of the present disclosure further comprises a therapeutic agent.

[0241] (5.4.1 Therapeutic molecules) In some embodiments, nanovesicles (e.g., EVs or hybridosomes) comprising the FcRn-binding polypeptides disclosed herein are engineered or modified to deliver one or more (e.g., 2, 3, 4, 5 or more) therapeutic molecules to target cells.

[0242] The therapeutic molecule may be any inorganic or organic compound. The therapeutic molecule may reduce, inhibit, attenuate, shrink, stop, or stabilize the onset or progression of a disease, disorder, or cell proliferation in an animal, such as a mammal or human. Examples of therapeutic molecules that can be introduced into nanovesicles (e.g., EVs or hybridosomes) containing FcRn-binding polypeptides include therapeutic agents, such as nucleic acids (e.g., DNA or mRNA molecules encoding polypeptides such as enzymes, mRNA molecules encoding polypeptides such as antigens, or RNA molecules with regulatory functions such as miRNA, dsDNA, and lncRNA), amino acids (e.g., amino acids that contain detectable moieties or toxins or interfere with translation), polypeptides (e.g., enzymes, enzymes for gene editing, nucleic acid binding proteins, antibodies, intrabodies, single chain variable fragments (scFv), affibodies, bi- and multispecific antibodies or binders, affibodies, darpins, receptors, ligands, or fragments thereof), lipids, carbohydrates, and small molecules (e.g., small molecule drugs and toxins). In certain embodiments, the therapeutic molecule may be a substance used in the diagnosis, treatment, or prevention of disease, or as a component of a drug. In some embodiments, the payload may refer to a compound that facilitates obtaining diagnostic information about a target site in the body of an organism, such as a mammal or human. For example, an imaging agent may be classified as an active agent in the present disclosure, since it is a substance that provides the imaging information required for diagnosis.

[0243] Further non-limiting examples of therapeutic nucleic acids contemplated for use in the present disclosure include siRNA, small or short hairpin RNA (shRNA), guide RNA (gRNA), single guide RNA (sgRNA), clustered regularly interspaced short palindromic repeats RNA (crRNA), transactivated clustered regularly interspaced short palindromic repeats RNA (tracrRNA), immunostimulatory oligonucleotides, plasmids, antisense nucleic acids, and ribozymes. In certain embodiments, the therapeutic nucleic acid can be linear DNA, circular DNA, or DNA, including artificial chromosomes. In some embodiments, the therapeutic DNA is maintained as an episome. In some embodiments, the therapeutic DNA is integrated into the genome. The therapeutic RNA can be chemically modified RNA, for example, can include one or more backbone modifications, sugar modifications, non-standard bases, or caps. Backbone modifications include, for example, phosphorothioate, N3' phosphoramidite, boranophosphate, phosphonoacetate, thioPACE, morpholino phosphoramidite, or PNA. Sugar modifications include, for example, 2'-0-Me, LNA, UNA, and 2'-0-MOE. Non-standard bases include, for example, 5-bromo-U, and 5-iodo-U, 2,6-diaminopurine, C-5 propynylpyrimidine, difluorotoluene, difluorobenzene, dichlorobenzene, 2-thiouridine, pseudouridine, and dihydrouridine. Caps include, for example, ARCA. Additional modifications are discussed, for example, in Deleavey et al., "Designing Chemically Modified Oligonucleotides for Targeted Gene Silencing," Chemistry & Biology, Vol. 19, Issue 8, August 24, 2012, pp. 937-954.

[0244] Non-limiting examples of other suitable therapeutic molecules include pharmacologically active drugs and genetically active molecules, including anti-neoplastic agents, anti-inflammatory agents, hormones or hormone antagonists, ion channel modulators, and neuroactive agents.Examples of suitable payloads of therapeutic agents include those described in The Pharmacological Basis of Therapeutics, Goodman and Gilman, McGraw-Hill, New York, NY, (1996), 9th Edition, Sections: Drugs Acting at Synaptic and Neuroeffector Junctional Sites; Drugs Acting on the Central Nervous System; Autacoids: Drug Therapy of Inflammation; Water, Salts and Ions; Drugs Affecting Renal Function and Electrolyte Metabolism; Cardiovascular Drugs; Drugs Affecting Gastrointestinal Function; Drugs Affecting Uterine Motility; Motility; Chemotherapy of Parasitic Infections; Chemotherapy of Microbial Diseases; Chemotherapy of Neoplastic Diseases; Drugs Used for Immunosuppression; Drugs Acting on Blood-Forming organs; Hormones and Hormone Antagonists; Vitamins, Dermatology; and Toxicology.Suitable payloads further include toxins, and biological and chemical warfare agents, see, for example, Somani, SM (ed.), Chemical Warfare Agents, Academic Press, New York (1992).

[0245] In one aspect, nanovesicles comprising a scaffold protein and a modified Fc domain can confer several desirable properties to the nanovesicles, including increased serum half-life, shorter blood clearance, and improved affinity purification. In some embodiments, the nanovesicles described herein can be modified to increase or decrease their half-life in circulation. In some embodiments, the half-life of the therapeutic agent carrier in nanovesicles comprising a polypeptide described herein in circulation can be altered by modifying the half-life of the nanovesicles. In some cases, the half-life can be increased, for example, by about 1.5-fold to 20-fold for the therapeutic agent payload maintained in nanovesicles comprising a polypeptide described herein, when compared to the half-life measured in a serum-containing solution and the therapeutic agent not contained in the nanovesicles.

[0246] In certain embodiments, the presence or absence of nanovesicles and / or therapeutic molecular payloads in the circulatory system is determined by the presence or absence of a particular polypeptide or fragment thereof, such as a modified Fc domain polypeptide or functional fragment thereof, on the nanovesicle.

[0247] In some embodiments, nanovesicles comprising a polypeptide as described herein can reside in the circulatory system or tissues of a subject for extended periods of time, allowing for more efficient delivery of a therapeutic effect than can be achieved by nanovesicles lacking said polypeptide. The increased half-life is particularly advantageous when compared to current EV-based therapies that do not include a scaffold protein comprising a modified Fc domain.

[0248] An effective amount of a scaffold protein comprising a modified Fc domain includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 60, 80, 100 or more polypeptides per nanovesicle, or an effective amount is an amount that is capable of extending the half-life of a nanovesicle by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 400%, 800%, 1,000%, or 10,000% relative to the half-life exhibited by the nanovesicle without the polypeptide.

[0249] In some embodiments, the nanovesicles (e.g., EVs or hybridosomes) described herein have properties that can be demonstrated by the following methods. In some embodiments, the contents of the nanovesicles can be extracted for study and characterization. In some embodiments, the nanovesicles are isolated and characterized by metrics including, but not limited to, size, shape, morphology, or molecular composition, such as nucleic acids, proteins, metabolites, and lipids, as well as half-life and pharmacodynamics.

[0250] In some embodiments, the methods described herein include measuring the size of the nanovesicles and / or population of nanovesicles contained in the purified fraction. In some embodiments, the size of the nanovesicles is measured as the longest measurable dimension. In general, the longest dimension of a typical nanovesicle is also referred to as its diameter.

[0251] The size of nanovesicles can be measured using various methods known in the art, such as nanoparticle tracking analysis, multi-angle light scattering, single-angle light scattering, size exclusion chromatography, analytical ultracentrifugation, field-flow fractionation, laser diffraction, tunable resistive pulse sensing, or dynamic light scattering.

[0252] In some embodiments, the methods described herein include measuring the density of FcRn-binding polypeptides on the nanovesicle surface. Surface density can be calculated or presented as mass per unit area, number of proteins per area, number of molecules or intensity of molecular signal per nanovesicle, molar amount of protein, etc. Surface density can be experimentally measured using methods known in the art, such as biolayer interferometry (BLI), FACS, Western blotting, fluorescence (e.g., GFP fusion protein) detection, nanoflow cytometry, ELISA, alphaLISA, and / or densitometry by measuring bands on protein gels.

[0253] 5.5 Purification of Nanovesicles Containing FcRn-Binding Polypeptides To use nanovesicles for medical purposes, it is necessary that the nanovesicles are free or almost free of impurities in the culture supernatant, including but not limited to macromolecules such as nucleic acids, contaminating proteins, lipids, carbohydrates, metabolites, small molecules, metals, or combinations thereof. The present disclosure provides a method for purifying nanovesicles comprising FcRn-binding polypeptides from contaminating macromolecules. In some embodiments, the purified nanovesicles comprising FcRn-binding polypeptides are substantially free of contaminating macromolecules.

[0254] In some cases, the isolation, purification, and removal of nanovesicles comprising FcRn-binding polypeptides are performed by column chromatography using a column in which FcRn and a solid support (e.g., a resin) are packed in the column. In some embodiments, a sample containing nanovesicles comprising the FcRn-binding polypeptides of the present disclosure is loaded onto the column, passed through, and bound, optionally a washing buffer is passed through the column, and then an elution buffer is applied to the column to collect an eluate containing nanovesicles comprising the FcRn-binding polypeptides of the present disclosure. These steps can be performed at atmospheric pressure or with the application of additional pressure. In some cases, the isolation, purification, and elution of nanovesicles comprising FcRn-binding polypeptides are performed using a batch process. For example, a sample is added to FcRn attached to a solid support in a container, mixed, and then the solid support is separated, followed by removing the liquid phase, washing, centrifugation, addition of an elution buffer, centrifugation again, and removal of the eluate. In some cases, a hybrid method can be employed. For example, a sample is added to FcRn attached to a solid support in a container, and then the solid support bound to nanovesicles containing an FcRn-binding polypeptide is loaded onto a column, followed by washing and elution on the column.

[0255] Generally speaking, the affinity purification method of nanovesicles comprising the FcRn-binding polypeptide of the present disclosure will result in a pure and highly enriched population of nanovesicles. However, additional separation, purification, and / or refinement steps may be included both upstream and / or downstream of the affinity purification step. Suitable complementary purification steps include size-exclusion liquid chromatography, bead elution liquid chromatography, ion-exchange purification (such as anion exchange), charged membrane separation, and various other purification and / or refinement strategies used in the art.

[0256] In some embodiments, nanovesicles containing FcRn-binding polypeptide samples are isolated or purified with an FcRn-binding agent, and then treated with a different binding agent (e.g., a protein affinity binding agent, an ion exchange resin, or a mixed-mode resin). In some embodiments, two or more columns are used in series, where each of the multiple columns contains a different binding agent specific for a different target protein. In some embodiments, a single column contains multiple binding agents, each of which is specific for a different target protein.

[0257] Also provided herein is a method for separating nanovesicles comprising an FcRn-binding polypeptide from nanovesicles not comprising said polypeptide (e.g., non-surface-modified EVs or lipid nanoparticles). In a specific embodiment, a subpopulation of nanovesicles comprising an FcRn-binding polypeptide is distinguished from other subpopulations by forming a complex with FcRn at acidic pH.

[0258] In one embodiment, the chromatographic material comprising FcRn as a ligand has a stability of at least three cycles in the methods and uses described herein. A cycle is a pH gradient from a first pH value to a second pH value of each method. Thus, in one embodiment, a cycle is a pH gradient from a pH value of about pH 5.5 to a pH value of about pH 8.8.

[0259] Once at least one elution fraction has been collected, the composition of the elution fraction can be analyzed. For example, the concentration of nanovesicles containing FcRn-binding polypeptides, host cell proteins, contaminating proteins, DNA, carbohydrates, or lipids can be measured in each elution fraction. Other properties of the nanovesicles in each elution fraction can also be measured. Properties include average size, average charge density, and other physiological properties related to biodistribution, cellular uptake, half-life, pharmacodynamics, efficacy, dosing, immune response, loading efficiency, stability, or reactivity to other compounds.

[0260] In some embodiments, nanovesicles comprising FcRn-binding polypeptide variants with increased affinity for FcRn (i.e., increased retention time on an FcRn column compared to native nanovesicles as described herein, but still eluting at pH values ​​below pH 7.4) may be predicted to have a longer serum half-life compared to those with reduced affinity for FcRn. Nanovesicles comprising FcRn-binding polypeptide variants with increased affinity for FcRn have applications in mammalian, particularly human, therapeutics, such as for treating chronic diseases or disorders, where a longer half-life of the administered EVs is desired.

[0261] Some embodiments of the present invention relate to the isolation, purification and sub-fractionation of nanovesicles using specific binding interactions (i.e., affinity purification) between FcRn-binding polypeptides (e.g., scaffold proteins of the present disclosure linked to a polypeptide comprising an FcRn-binding site) enriched on the nanovesicle membrane and immobilized binding agents (e.g., FcRn). These methods generally include the steps of (1) applying or loading a sample comprising nanovesicles of the present disclosure onto the immobilized agent, (2) optionally washing away unbound sample components using an appropriate buffer that maintains the binding interaction between the FcRn-binding polypeptide and the binding agent displayed on the nanovesicles, and (3) eluting (dissociating and recovering) the nanovesicles from the immobilized binding agent by changing the buffer conditions to eliminate the binding interaction.

[0262] In some embodiments, the affinity purification method of purifying nanovesicles comprising at least one polypeptide described herein (particularly at least one FcRn-binding polypeptide) has a superior recovery rate compared to other affinity purification of nanovesicles known in the art. For example, nanovesicles comprising at least one polypeptide described herein (particularly at least one FcRn-binding polypeptide) can be eluted from the immobilized binding partner at a milder pH (e.g., pH 7-9) compared to conventional affinity purification methods that require a pH of less than 5, and in some cases less than 3, to elute (e.g., dissociate) the nanovesicles from the immobilized binding partner (e.g., Protein A).

[0263] The use of nanovesicles (e.g., EVs or hybridosomes) for medical purposes further requires that the nanovesicles (e.g., EVs or hybridosomes) are not in aggregated form and exhibit colloidal stability, and highly acidic pH may cause colloidal instability. An important aspect of the present disclosure is to provide a method for purifying nanovesicles (e.g., EVs or hybridosomes) comprising at least one FcRn-binding polypeptide under more physiological conditions, such as physiological pH values.

[0264] Some aspects of the present disclosure relate to the isolation and purification of nanovesicles (e.g., EVs or hybridosomes) comprising FcRn-binding polypeptides using specific binding interactions between a first binding partner (e.g., an FcRn-binding polypeptide present on the nanovesicle membrane) and a second binding partner (e.g., an immobilized FcRn). These methods generally include the steps of: (1) applying or loading a sample containing nanovesicles comprising a first binding partner (e.g., an FcRn-binding polypeptide present on the nanovesicle membrane) onto a matrix containing a second binding partner (e.g., an immobilized FcRn); (2) optionally washing away unbound sample components using an appropriate buffer that maintains the binding interaction between the first binding partner (e.g., an FcRn-binding polypeptide present on the nanovesicle membrane) and the second binding partner (e.g., an immobilized FcRn); and (3) eluting (dissociating and recovering) the nanovesicles comprising the FcRn-binding polypeptide from the immobilized bound FcRn agent by changing the buffer conditions such that the binding interaction between the binding partners is abolished.

[0265] In some embodiments, the second binding partner is FcRn, optionally immobilized on a suitable matrix or chromatographic material. In some embodiments, the second binding partner used in this isolation and purification process is FcRn protein produced in vitro by a cell produced by genetic modification or transfection, or FcRn protein modified and isolated by chemical, physical or other biological methods. Optionally, the FcRn protein is a non-mutated FcRn protein or a mutated FcRn protein, such as a variant or fragment of an FcRn protein. Optionally, the FcRn is a fusion protein. In a specific embodiment, the FcRn is a soluble single-chain FcRn, as produced according to the method of Feng et al. (2011), Protein Expr. Purif. 79:66-71. In one embodiment, the soluble FcRn forms a non-covalent heterodimer with beta-2-microglobulin (B2M).

[0266] In a specific embodiment, provided herein is a method for purifying EVs, comprising: a. providing EVs, the EVs associated with a first binding partner, the first binding partner being capable of binding to the Fc binding site of FcRn in a pH-dependent manner; b. contacting the EVs associated with the first binding partner with a second binding partner at a first pH, the second binding partner comprising the Fc binding site of FcRn and associated with a solid matrix; and c. eluting the EVs associated with the first binding partner from the solid matrix at a second pH. In certain embodiments, the method further comprises a washing step at the first pH. In certain embodiments, the first pH is less than 6.5. In certain embodiments, the second pH is greater than 7.4.

[0267] In a specific embodiment, provided herein is a method for purifying EVs, comprising: a. providing EVs, the EVs associated with a first binding partner, the first binding partner capable of binding to the Fc binding site of FcRn in a pH-dependent manner, and comprising or consisting of a polypeptide described herein; b. contacting the EVs associated with the first binding partner with a second binding partner at a first pH, the second binding partner comprising the Fc binding site of FcRn and associated with a solid matrix; and c. eluting the EVs associated with the first binding partner from the solid matrix at a second pH. In certain embodiments, the method further comprises a washing step at the first pH. In certain embodiments, the first pH is less than 6.5. In certain embodiments, the second pH is greater than 7.4.

[0268] In a specific embodiment, provided herein is a method for purifying hybridosomes, comprising: a. providing hybridosomes, the hybridosomes associated with a first binding partner, the first binding partner being capable of binding to the Fc binding site of FcRn in a pH-dependent manner; b. contacting the hybridosomes associated with the first binding partner with a second binding partner at a first pH, the second binding partner comprising the Fc binding site of FcRn and associated with a solid matrix; and c. eluting the hybridosomes associated with the first binding partner from the solid matrix at a second pH. In certain embodiments, the method further comprises a washing step at a first pH. In certain embodiments, the first pH is less than 6.5. In certain embodiments, the second pH is greater than 7.4.

[0269] In a specific embodiment, provided herein is a method for purifying hybridosomes, comprising: a. providing hybridosomes, the hybridosomes associated with a first binding partner, the first binding partner can bind to the Fc binding site of FcRn in a pH-dependent manner, and comprising or consisting of a polypeptide as described herein; b. contacting the hybridosomes associated with the first binding partner with a second binding partner at a first pH, the second binding partner comprises the Fc binding site of FcRn and is associated with a solid matrix; and c. eluting the hybridosomes associated with the first binding partner from the solid matrix at a second pH. In certain embodiments, the method further comprises a washing step at a first pH. In certain embodiments, the first pH is less than 6.5. In certain embodiments, the second pH is greater than 7.4. In one embodiment of all aspects described herein, the FcRn is selected from human FcRn, cynomolgus monkey FcRn, mouse FcRn, rat FcRn, sheep FcRn, dog FcRn and rabbit FcRn.

[0270] In one embodiment of all aspects described herein, the beta-2-microglobulin is selected from human beta-2-microglobulin, cyno beta-2-microglobulin, mouse beta-2-microglobulin, rat beta-2-microglobulin, sheep beta-2-microglobulin, dog beta-2-microglobulin and rabbit beta-2-microglobulin.

[0271] In one embodiment, the heterodimer is composed of beta-2-microglobulin and soluble FcRn from the same species. In one embodiment, the heterodimer is composed of beta-2-microglobulin and soluble FcRn from different species.

[0272] Therefore, the chromatographic materials comprising a complex of neonatal Fc receptor (FcRn) and beta-2-microglobulin as ligands described herein can be used for the isolation / separation of extracellular vesicles presenting monomeric Fc, thus providing an alternative to conventional Protein A affinity chromatography. In addition, by using the chromatographic materials described herein, separation can be performed under more physiological conditions, such as pH values, compared to conventional Protein A affinity chromatography.

[0273] Methods for preparing soluble and functional FcRn are known in the art. One method involves expressing soluble human FcRn (sFcRn) in mammalian cells as a single-chain soluble fusion protein (see SEQ ID NOs: 70 and 71 for the amino acid sequences of human single-chain FcRn and mouse single-chain FcRn, respectively). Highly hydrophilic beta-2-microglobulin is linked to a hydrophobic heavy chain via a 15 amino acid linker. The single-chain fusion protein format not only improves the expression level of the heavy chain but also simplifies the purification process (Feng et al. (2011), Protein Expr. Purif. 79:66-71). The use of an immobilized non-covalent complex of FcRn and beta-2-microglobulin (b2m) as an affinity chromatography ligand in affinity chromatography of soluble Fc fusion proteins in a positive linear pH gradient is described in WO2013 / 120929. Recombinant FcRn and its variants for the purification of Fc-containing soluble fusion proteins are described in WO 2010 / 048313.

[0274] In one embodiment, the second binding agent comprises a soluble extracellular domain of FcRn (e.g., SEQ ID NO: 68 for human FcRn) with a C-terminal His-Avi tag or C-Tag co-expressed with β2-microglobulin (SEQ ID NO: 69 for human beta-2-microglobulin) in mammalian cells. In some embodiments, the non-covalent or single-chain FcRn complex is biotinylated and loaded onto streptavidin-derivatized sepharose. In some embodiments, the non-covalent FcRn complex containing the C-Tag is loaded onto C-TagXl beads. [Table 11]

[0275] An exemplary affinity chromatography column is characterized in that it comprises a matrix and a matrix-bound chromatography functional group, the matrix-bound chromatography functional group comprising a complex of neonatal Fc receptor (FcRn) and beta-2-microglobulin. A further exemplary affinity chromatography column is characterized in that it comprises a matrix and a matrix-bound chromatography functional group, the matrix-bound chromatography functional group comprising a single-chain fusion protein of soluble neonatal Fc receptor (FcRn) and beta-2-microglobulin.

[0276] In a preferred embodiment, FcRn is attached to a solid phase, for example, to enable chromatographic purification and / or membrane-based purification. Affinity chromatography is generally based on highly selective interactions between immobilized FcRn ligands and structural elements on target biomolecules (e.g., FcRn-binding sites on FcRn-binding polypeptides). In one embodiment, the target biomolecule is an FcRn-binding polypeptide of the present disclosure, and the structural element is an FcRn-binding site (e.g., modified Fc domain). In another embodiment, the target biomolecule is a nanovesicle comprising an FcRn-binding polypeptide of the present disclosure, and the structural element is a modified Fc domain. The high selectivity of affinity chromatography can be provided by multiple molecular interactions (including hydrogen bonds, hydrophobic interactions, ionic interactions, and / or van der Waals interactions) between FcRn immobilized on a suitable matrix (e.g., chromatography matrix) and the modified Fc domain forming part of the FcRn-binding polypeptide of the present disclosure. Suitable second binding partners for affinity-based purification of FcRn-binding polypeptide-containing nanovesicles are soluble heterodimers of the FcRn receptor and any combination of its derivatives, domains or parts.

[0277] In a further embodiment, the chromatographic methods comprising FcRn as the second binding partner described herein can be used for the isolation / separation of nanovesicles presenting FcRn-binding polypeptides, thus providing an alternative to conventional Protein A affinity chromatography. In addition, by using the chromatographic materials described herein, the separation can be performed under more physiological conditions, such as pH values, compared to conventional Protein A affinity chromatography.

[0278] In some embodiments, isolation or purification as used herein refers to the process of removing or partially removing (e.g., removing a fraction) an FcRn-binding polypeptide or nanovesicles comprising said FcRn-binding polypeptide from a sample containing produced cells. In some embodiments, isolation or purification as used herein refers to the process of removing or partially removing (e.g., removing a fraction) an FcRn-binding polypeptide or nanovesicles (e.g., EVs) comprising said FcRn-binding polypeptide after removing produced cells (e.g., after removing cells by centrifugation or depth filtration).

[0279] FcRn can be chemically immobilized or bound to a solid support (e.g., via biotin-streptavidin binding) so that a population of nanovesicles comprising FcRn-binding polypeptides have specific affinity for FcRn and bind at acidic pH.Various forms of solid support can be used, such as porous agarose beads, microtiter plates, magnetic beads, or membranes.In some embodiments, the solid support forms a chromatography column and can be used for affinity chromatography of nanovesicles comprising FcRn-binding polypeptides.

[0280] "Solid support" refers to non-fluid materials, including particles (including microparticles and beads) made from materials such as polymers, metals (paramagnetic, ferromagnetic particles), glass, and ceramics; gel materials such as silica, alumina, and polymer gels; capillaries that can be made of polymers, metals, glass, and / or ceramics; zeolites and other porous materials; electrodes; microtiter plates; solid strips; and cuvettes, tubes, or other spectrometer sample containers. The solid phase component of the assay is distinguished from an inert solid surface in that the solid support contains at least one moiety on its surface that is intended to chemically interact with the molecule. The solid phase can be an immobilized component such as a chip, tube, strip, cuvette, or microtiter plate, or a non-immobilized component such as beads and microparticles. Microparticles can also be used as solid supports in homogeneous assay formats. A variety of microparticles can be used that allow both non-covalent or covalent attachment of FcRn complexes and other substances. In one embodiment, the solid support is composed of POROS™ beads.

[0281] Conjugation of FcRn to a solid support can be achieved by chemical binding via the N-terminus and / or epsilon amino group (lysine), the epsilon amino groups of different lysines, the carboxy, sulfhydryl, hydroxyl and / or phenolic functions of the amino acid backbone of the protein, and / or the sugar alcohol groups of the carbohydrate structure of the protein.

[0282] (5.5.1 Join) Some embodiments of the present disclosure relate to the isolation and purification of nanovesicles comprising FcRn-binding polypeptides using specific binding interactions between a first binding partner (e.g., an FcRn-binding polypeptide present on the membrane of the nanovesicle) and a second binding partner (e.g., an immobilized FcRn).

[0283] In one embodiment, a sample containing the FcRn-binding polypeptide of the present disclosure, or nanovesicles (e.g., EVs or hybridosomes) containing said polypeptide, is adjusted to a first pH (i.e., acidic pH), and then applied to an FcRn affinity column. In one embodiment, a production mixture or crude or partially purified culture supernatant containing the FcRn-binding polypeptide of the present disclosure is adjusted to a first pH (i.e., acidic pH), and then applied to an FcRn affinity column. In one embodiment, the first pH is less than about pH 6.5. In a preferred embodiment, the first pH is less than about pH 6.5 and greater than pH 5.

[0284] In one embodiment, the first pH value is about pH 5 to about pH 6. In one embodiment, the first pH value is about pH 5 or about pH 5.5 or about pH 6. In one embodiment, the first pH value is selected from about pH 3.5, about pH 3.6, about pH 3.7, about pH 3.8, about pH 3.9, about pH 4.0, about pH 4.1, about pH 4.2, about pH 4.3, about pH 4.4, about pH 4.5, about pH 4.6, about pH 4.7, about pH 4.8, about pH 4.9, about pH 5.0, about pH 5.1, about pH 5.2, about pH 5.3, about pH 5.4, about pH 5.5, about pH 5.6, about pH 5.7, about pH 5.8, about pH 5.9, about pH 6.0, about pH 6.1, about pH 6.2, about pH 6.3, and about pH 6.4.

[0285] The methods described herein require specific interactions between FcRn-binding polypeptides and FcRn-purifying ligands. High-throughput screening can be performed to further identify ideal buffer conditions for specific binding, primarily through altering pH, but also optionally altering salt concentration, and / or decreasing polarity with organic modifiers, ethylene glycol, propylene glycol, or urea. The interaction between the FcRn-binding polypeptides of the present disclosure and the binding agent (e.g., FcRn) can also be altered depending on the sample conditions (e.g., amount of sample loaded per volume of chromatography resin, concentration of FcRn-binding polypeptide, concentration of EVs containing FcRn-binding polypeptide, concentration of impurities), loading buffer (e.g., pH, salt concentration, salt identity, polarity), and other physical conditions (e.g., temperature). In addition, the residence time can be adjusted based on the difference in adsorption rate between the impurities and the FcRn-binding polypeptide or nanovesicles containing said polypeptide. Thus, various purification conditions described herein can be tested to identify ideal conditions for the process.

[0286] Similar approaches can be used to improve purity and yield and aid in enriching, depleting, or isolating subpopulations of nanovesicles containing FcRn-binding polypeptides. These properties can be used to maximize loading and apply more stringent elution conditions, as well as to further increase the concentration of exosomes.

[0287] (5.5.2 Elution) In one aspect, in the uses and methods described herein, recovery of the FcRn-binding polypeptide of the present disclosure bound to an FcRn affinity column or nanovesicles (e.g., EVs or hybridosomes) comprising said FcRn-binding polypeptide is primarily by changing the pH of the buffer solution from a first pH value that promotes binding (i.e., a more acidic pH) to a second pH value that does not promote binding between the binding pair as much (i.e., less acidic, more neutral or more alkaline).

[0288] In some embodiments, elution is performed by a positive linear pH gradient, which indicates a pH gradient starting from a first low pH value (i.e., a more acidic pH value) and ending at a second higher pH value (i.e., a less acidic, neutral or alkaline pH value), and is driven by a change in pH, where the pH of the eluent changes continuously as a function of time. An example of a continuous pH gradient is a linear pH gradient, where the change in pH is a linear function of time. In one embodiment, a continuous pH gradient can be established by utilizing two or more buffers of different pH that are mixed together to form an eluent. The ratio of buffers in the eluent, and therefore the pH of the eluent, can be changed continuously as a function of time. The control of the buffer mixing process is usually controlled by a flow controller that is programmed to produce the desired pH gradient. In one embodiment, the positive linear pH gradient starts from a first pH value of about 5.5 and ends at a second pH value of about 8.8.

[0289] In some embodiments, the change in pH is achieved by a positive step pH gradient that starts low (i.e., a more acidic pH value) and ends higher (i.e., a less acidic, neutral or alkaline pH value), and the change in pH is discontinuous with respect to time, forming one or more steps, or time points where the pH undergoes an abrupt change. This can be achieved by simply replacing a first buffer with a second buffer of different pH as the eluent. In a preferred embodiment of the method, the gradient employed is a step pH gradient. In one embodiment, the positive step pH gradient starts from a first pH value of about 5.5 and ends at a second pH value of about 8.8.

[0290] In one embodiment, the second pH value is about pH 7.3 to about pH 9.5. In one embodiment, the second pH value is about pH 8.5 to about pH 9. In one embodiment, the second pH value is about pH 8.8.

[0291] In one embodiment, the second pH value is selected from about pH 7.1, about pH 7.2, about pH 7.3, about pH 7.4, about pH 7.5, about pH 7.6, about pH 7.7, about pH 7.8, about pH 7.9, about pH 8.0, about pH 8.0, about pH 8.1, about pH 8.2, about pH 8.3, about pH 8.4, about pH 8.5, about pH 8.6, about pH 8.7, about pH 8.8, about pH 8.9, about pH 9.0, about pH 9.1, about pH 9.2, about pH 9.3, about pH 9.4, and about pH 9.5.

[0292] In one embodiment, the first pH value is about pH 3.5, about pH 3.6, about pH 3.7, about pH 3.8, about pH 3.9, about pH 4.0, about pH 4.1, about pH 4.2, about pH 4.3, about pH 4.4, about pH 4.5, about pH 4.6, about pH 4.7, about pH 4.8, about pH 4.9, about pH 5.0, about pH 5.1, about pH 5.2, about pH 5.3, about pH 5.4, about pH 5.5, about pH 5.6, about pH 5.7, about pH 5.8, about pH 5.9, about pH 6.0, about pH 6.1, about pH 6.2 , about pH 6.3, or about pH 6.4, and the second pH value is about 7.1, about pH 7.2, about pH 7.3, about pH 7.4, about pH 7.5, about pH 7.6, about pH 7.7, about pH 7.8, about pH 7.9, about pH 8.0, about pH 8.0, about pH 8.1, about pH 8.2, about pH 8.3, about pH 8.4, about pH 8.5, about pH 8.6, about pH 8.7, about pH 8.8, about pH 8.9, about pH 9.0, about pH 9.1, about pH 9.2, about pH 9.3, about pH 9.4, or about pH 9.5.

[0293] In some embodiments, elution of FcRn-binding nanovesicles comprising FcRn-binding polypeptides can alternatively be achieved by changing the salt concentration and / or polarity using organic modifiers, ethylene glycol, propylene glycol, or urea.

[0294] In some embodiments, apart from adjusting the pH range, elution can also be achieved by adjusting salts, organic solvents, small molecules, detergents, zwitterions, amino acids, polymers, temperature, and any combination of the above. Similar elution agents can be used to improve purity, improve yield, and isolate subpopulations of nanovesicles containing FcRn-binding polypeptides.

[0295] In some embodiments, elution can be performed using multiple elution buffers with different properties, such as pH, salt, organic solvent, small molecule, detergent, zwitterion, amino acid, polymer, temperature, and any combination of the above. Multiple elution fractions can be collected, and the nanovesicles comprising FcRn-binding polypeptide collected in each fraction have different properties. For example, the nanovesicles comprising FcRn-binding polypeptide collected in one fraction have higher purity, smaller or larger average size, or preferred composition, etc., than the FcRn-binding nanovesicles in other fractions.

[0296] In principle, any buffer substance can be used in the methods described herein. In one embodiment, a pharma- ceutically acceptable buffer substance such as phosphoric acid or a salt thereof, acetic acid or a salt thereof, citric acid or a salt thereof, morpholine, 2-(N-morpholino)ethanesulfonic acid (MES) or a salt thereof, histidine or a salt thereof, glycine or a salt thereof, tris(hydroxymethyl)aminomethane (TRIS) or a salt thereof, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) or a salt thereof is used.

[0297] A specific embodiment relates to a method of removing unmodified nanovesicles from a sample using a specific binding interaction between an FcRn binding polypeptide and an immobilized FcRn binding agent. In these cases, nanovesicles bound to the binding agent are not eluted from the binding agent, and the fraction not bound to the binding agent can be collected.

[0298] Selective elution of FcRn-binding polypeptides or nanovesicles comprising said polypeptides can be achieved by varying the concentration of monovalent cationic halide salts (e.g., sodium chloride, potassium chloride, sodium bromide, lithium chloride, sodium iodide, potassium bromide, lithium bromide, sodium fluoride, potassium fluoride, lithium fluoride, lithium iodide, sodium acetate, potassium acetate, lithium acetate, and potassium iodide), divalent or trivalent salts (e.g., calcium chloride, magnesium chloride, calcium sulfate, sodium sulfate, magnesium sulfate, chromium trichloride, chromium sulfate, sodium citrate, iron(III) chloride, yttrium(III) chloride, potassium phosphate, potassium sulfate, sodium phosphate, ferrous chloride, calcium citrate, magnesium phosphate, and ferric chloride) in the elution buffer, or combinations thereof. can be achieved by increasing the concentration of a monovalent cation halide salt (e.g., sodium chloride, potassium chloride, sodium bromide, lithium chloride, sodium iodide, potassium bromide, lithium bromide, sodium fluoride, potassium fluoride, lithium fluoride, lithium iodide, sodium acetate, potassium acetate, lithium acetate, and potassium iodide), a divalent or trivalent salt (e.g., calcium chloride, magnesium chloride, calcium sulfate, sodium sulfate, magnesium sulfate, chromium trichloride, chromium sulfate, sodium citrate, ferric chloride, yttrium chloride, potassium phosphate, potassium sulfate, sodium phosphate, ferrous chloride, calcium citrate, magnesium phosphate, and ferric chloride) at a constant pH using an increasing gradient (step or linear).

[0299] In one embodiment the buffer substance is selected from phosphoric acid or a salt thereof, or acetic acid or a salt thereof, or citric acid or a salt thereof, or histidine or a salt thereof.

[0300] In one embodiment, the buffer substance has a concentration of 5 mM to 500 mM. In one embodiment, the buffer substance has a concentration of 10 mM to 300 mM. In one embodiment, the buffer substance has a concentration of 10 mM to 250 mM. In one embodiment, the buffer substance has a concentration of 10 mM to 100 mM. In one embodiment, the buffer substance has a concentration of 15 mM to 50 mM. In one embodiment, the buffer substance has a concentration of about 20 mM.

[0301] In one embodiment, the buffer substance in the first buffer solution and the buffer substance in the second buffer solution are the same buffer substance. In one embodiment, the buffer substance in the first solution and the buffer substance in the second solution are different buffer substances. In one embodiment, the first solution has a pH value of about pH 3.5 to about pH 7.5. In one embodiment, the first solution has a pH value of about pH 5 to about pH 6. In one embodiment, the first solution has a pH value of about pH 5.5.

[0302] In one embodiment, the second solution has a pH value of about pH 7.0 to about pH 9.5. In one embodiment, the second solution has a pH value of about pH 8 to about pH 9. In one embodiment, the second solution has a pH value of about pH 8.2 to about pH 8.8.

[0303] An exemplary first solution includes 20 mM MES and 150 mM NaCl adjusted to pH 5.5. An exemplary second solution includes 20 mM TRIS and 150 mM NaCl adjusted to pH 8.8. An exemplary second solution includes 20 mM HEPES adjusted to pH 8.6. An exemplary second solution includes 20 mM TRIS adjusted to pH 8.2.

[0304] In one embodiment, the buffer solution comprises an additional salt. In one embodiment, the additional salt is selected from sodium chloride, sodium sulfate, potassium chloride, potassium sulfate, sodium citrate, or potassium citrate. In one embodiment, the buffer solution comprises 50 mM to 1000 mM of additional salt. In one embodiment, the buffer solution comprises 50 mM to 750 mM of additional salt. In one embodiment, the buffer solution comprises 50 mM to 500 mM of additional salt. In one embodiment, the buffer solution comprises 50 mM to 750 mM of additional salt. In one embodiment, the buffer solution comprises about 50 mM to about 300 mM of additional salt.

[0305] In one embodiment, the first and / or second solution contains sodium chloride. In one embodiment, the first and / or second solution contains about 50 mM to about 300 mM sodium chloride.

[0306] (5.5.3 Cleaning) In some embodiments, substantial nanovesicle purity can be achieved by flushing out impurities during the column loading step, eluting impurities during a selective excipient wash, and selectively eluting the product during elution while leaving additional impurities bound to the column. The absorbance measured from the column eluate can indicate the purification of the nanovesicles obtained by the method.

[0307] Optionally, the purity of nanovesicles containing FcRn-binding polypeptides can be further improved by washing the sample before elution. In some embodiments, the excipient can be a washing buffer. The excipient can be a solution with a specific pH range, a salt, an organic solvent, a small molecule, a surfactant, a zwitterion, an amino acid, a polymer, and any combination of the above.

[0308] More specifically, the excipient may include arginine, lysine, glycine, histidine, calcium, sodium, lithium, potassium, iodide, magnesium, iron, zinc, manganese, urea, propylene glycol, aluminum, ammonium, guanidinium polyethylene glycol, EDTA, EGTA, surfactants, chloride, sulfate, carboxylate, sialic acid, phosphate, acetate, glycine, borate, formate, perchlorate, bromine, nitrate, dithiothreitol, beta-mercaptoethanol, or tri-n-butyl phosphate.

[0309] Excipients include cetyltrimethylammonium chloride, octoxynol-9, TRITON™ X-100 (i.e., polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether), and TRITON™ CG-110 available from Sigma-Aldrich; sodium dodecyl sulfate; sodium lauryl sulfate; deoxycholic acid; polysorbate 80 (i.e., polyoxyethylene (20) sorbitan monooleate); polysorbate 20 (i.e., polyoxyethylene (20) sorbitan monolaurate); alcohol ethoxylates; alkyl polyethylene glycol ethers; decyl glucoside; octoglucoside; SafeCare; DOW The surfactants may also include surfactants selected from the group consisting of ECOSURF™ EH9, ECOSURF™ EH6, ECOSUR™ EH3, ECOSURF™ SA7, and ECOSURF™ SA9 available from Lubrizol Chemical; LUTENSOL™ M5, LUTENSOL™ XL, LUTENSOL™ XP, and APG™ 325N available from BASF; TOMADOL™ 900 available from AIR PRODUCTS; NATSURF™ 265 available from CRODA; SAFECARE™ 1000 available from Bestchem; TERGITOL™ L64 available from DOW; caprylic acid; CHEMBETAINE™ LEC available from Lubrizol; and Mackol DG.

[0310] In some embodiments, further methods can be applied to improve purification results.For example, the amount of nanovesicles containing FcRn-binding polypeptides that can be loaded per volume of chromatography resin can be improved by adjusting the feed material, for example, by increasing the concentration of FcRn-binding nanovesicles, decreasing the concentration of impurities, changing the pH, decreasing the salt concentration, decreasing the ionic strength, or changing the specific subpopulation of nanovesicles containing FcRn-binding polypeptides.In certain embodiments, due to mass transport limitations and the delayed adsorption and desorption of nanovesicles on the resin, the amount of nanovesicles containing FcRn-binding polypeptides that can be loaded per volume of chromatography resin can be increased by slowing down the flow rate during column loading and adopting a longer column to increase the residence time.

[0311] In other embodiments, the isolated nanovesicles comprising the FcRn-binding polypeptide composition have a desired amount and / or concentration of FcRn-binding nanovesicles (e.g., EVs) at or above an acceptable amount and / or concentration. In other embodiments, the isolated nanovesicles comprising the FcRn-binding polypeptide composition are enriched compared to the starting material (e.g., produced cell conditioned medium) from which the composition is derived. This enrichment can be greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or 99.9999% compared to the starting material.

[0312] In some embodiments, the isolated nanovesicles comprising the FcRn-binding polypeptide preparation are substantially free of residual biological products. In some embodiments, the isolated nanovesicles comprising the FcRn-binding polypeptide preparation are 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% free of any contaminating biological material. Residual biological products may include non-biological materials (including chemicals) or undesired nucleic acids, proteins, lipids, or metabolites. Substantially free of residual biological products may also mean that the nanovesicles comprising the FcRn-binding polypeptide do not contain detectable producing cells, and only the FcRn-binding nanovesicles are detectable.

[0313] In a further embodiment, the chromatography method comprising FcRn as the second binding partner described herein can be used to isolate / enrich the crude mixture of hybridosomes comprising FcRn-binding polypeptide from unfused lipid nanoparticles.In some embodiments, the isolated hybridosomes comprising FcRn-binding polypeptide preparation are substantially free of unfused lipid nanoparticles.In some embodiments, the isolated hybridosomes comprising FcRn-binding polypeptide preparation are 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% free of any unfused lipid nanoparticles.

[0314] 5.6 Compositions and Kits In another aspect, compositions and kits are provided that include the polypeptides, nanovesicles, nucleic acids, expression vectors, and / or cells of the present disclosure (e.g., as described in Sections 5.2-5.4). Such compositions can be, for example, cosmetic compositions, diagnostic compositions, or pharmaceutical compositions.

[0315] In some embodiments, the pharmaceutical composition comprises an FcRn-binding polypeptide described herein (e.g., an FcRn-binding polypeptide comprising a transmembrane protein and a modified Fc domain capable of specifically binding to FcRn and not forming homodimers), and further comprises one or more pharma- ceutically acceptable carriers and / or excipients. Guidance for preparing the formulations can be found in any number of handbooks on pharmaceutical preparations and formulations known to those skilled in the art.

[0316] In certain embodiments, the compositions described herein are useful as drugs. Typically, such drugs comprise a therapeutically effective amount of the compositions provided herein. Thus, each composition can be used to produce a drug useful for treating disorders. Thus, in one embodiment, pharmaceutical compositions and kits are provided that comprise the polypeptides, nanovesicles, nucleic acids, expression vectors, and / or cells of the present disclosure. In some embodiments, pharmaceutical compositions and kits are provided that comprise the nanovesicles of the present disclosure (i.e., nanovesicles that comprise the above-mentioned polypeptides).

[0317] In some embodiments, the pharmaceutical composition comprises the polypeptides, nanovesicles, nucleic acids, expression vectors, and / or cells described herein, and further comprises one or more pharma- ceutically acceptable carriers, excipients, and / or diluents. Guidance for preparing the formulations can be found in any number of handbooks on pharmaceutical preparations and formulations known to those skilled in the art.

[0318] Pharmaceutically acceptable carriers include any solvents, dispersion media, or coatings that are physiologically compatible and preferably do not interfere with or otherwise inhibit the activity of the active agent. A variety of pharma- ceutically acceptable excipients are well known in the art.

[0319] In some embodiments, the medicament acceptable carrier is suitable for intravenous, intramuscular, oral, intraperitoneal, intrathecal, transdermal, topical, or subcutaneous administration.The medicament acceptable carrier can contain one or more physiologically acceptable compound(s), which acts, for example, to stabilize the composition or to increase or decrease the absorption of the active agent(s).The physiologically acceptable compound can include, for example, carbohydrates, such as glucose, sucrose, or dextran, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the active agent, or excipients or other stabilizers and / or buffers.Other medicament acceptable carriers and their formulations are well known in the art.

[0320] The pharmaceutical compositions can be manufactured in a manner known to those skilled in the art, for example, by conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping or lyophilizing processes. The methods and excipients disclosed herein are merely illustrative and in no way limiting.

[0321] For oral administration, the FcRn-binding polypeptides disclosed herein can be formulated by combining with pharma- ceutical acceptable carriers well known in the art. Such carriers allow the compounds to be formulated as tablets, pills, dragees, capsules, emulsions, lipophilic and hydrophilic suspensions, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by the patient to be treated. Pharmaceutical preparations for oral use can be obtained by mixing the compounds with solid excipients, optionally grinding the resulting mixture, and processing the mixture of granules after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients include, for example, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0322] The FcRn-binding polypeptides disclosed herein can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. For injection, the FcRn-binding polypeptides can be formulated into preparations by dissolving, suspending or emulsifying in aqueous or non-aqueous solvents, for example, vegetable oils or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol, optionally with conventional additives such as solubilizers, isotonicity agents, suspending agents, emulsifying agents, stabilizers and preservatives. In some embodiments, the FcRn-binding polypeptides can be formulated in aqueous solutions, preferably in physiologically compatible buffers, for example, Hank's solution, Ringer's solution, or saline buffer. The preparations for injection can be provided in unit dosage form, for example, in ampoules or multi-dose containers, with the addition of preservatives. The compositions can take the form of suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulating agents such as suspending agents, stabilizing agents and / or dispersing agents.

[0323] In some embodiments, the FcRn-binding polypeptides disclosed herein are prepared for delivery in sustained-release, controlled-release, sustained-release, timed-release or delayed-release formulations, such as semipermeable matrices of solid hydrophobic polymers containing active agents. Various types of sustained-release materials have been established and are well known to those skilled in the art. Current sustained-release formulations include film-coated tablets, multiparticulate or pellet systems, matrix technology using hydrophilic or lipophilic materials, and wax-based tablets with pore-forming excipients. Depending on their design, sustained-release delivery systems can release compounds over a period of hours or days, for example, 4, 6, 8, 10, 12, 16, 20, or 24 hours or more. Typically, sustained-release formulations can be prepared using natural or synthetic polymers, such as polymeric vinylpyrrolidone, e.g., polyvinylpyrrolidone (PVP); carboxyvinyl hydrophilic polymers; hydrophobic and / or hydrophilic hydrocolloids, e.g., methylcellulose, ethylcellulose, hydroxypropylcellulose, and hydroxypropylmethylcellulose; and carboxypolymethylene.

[0324] Pharmaceutical compositions for use in in vivo administration are usually sterile. Sterilization can be accomplished according to methods known in the art, for example, by heat sterilization, steam sterilization, sterile filtration, or irradiation.

[0325] The dosage and desired drug concentration of the pharmaceutical composition of the present disclosure may vary depending on the particular application envisaged. The determination of the appropriate dosage or route of administration is well within the skill of the art. Exemplary suitable dosages are also described in Section 5.7 below.

[0326] (5.7 Therapeutic and diagnostic uses) Nanovesicles comprising the polypeptides of the present disclosure (e.g., as described in Section 5.4), as well as nucleic acids and expression vectors encoding such polypeptides (e.g., as described in Section 5.3), cells capable of expressing such polypeptides (e.g., as described in Section 5.3), and compositions and kits comprising the foregoing (e.g., as described in Section 5.6), can be used to treat, monitor, prevent and / or diagnose a number of diseases and disorders (e.g., cancer, inflammation, or inflammation associated with cancer).

[0327] Thus, in one aspect, provided herein is a method of delivering a therapeutic or diagnostic agent to a target cell or tissue, the method comprising providing to the target cell or tissue an extracellular vesicle or hybridosome described herein.

[0328] In one aspect, a method for treating a disease or disorder is provided. The method comprises administering a medicamentously effective amount of a composition (i.e., a composition that contains or can express a polypeptide) described herein to a subject in need thereof. In one embodiment, the method comprises administering a medicamentously effective amount of the pharmaceutical composition described above.

[0329] The subject in need of treatment may be a human or a non-human animal. Typically, the subject is a mammal, such as an ape, dog, guinea pig, horse, monkey, mouse, pig, rabbit or rat. In the case of animal models, the animal may be genetically engineered to develop a disorder or exhibit characteristics of a disease.

[0330] In some embodiments, the subject has cancer, an inflammatory disorder, an autoimmune disease, a chronic disease, inflammation, impaired organ function, an infectious disease, a metabolic disease, a degenerative disorder, a genetic disease (e.g., a genetic defect, a recessive genetic disorder, or a dominant genetic disorder), or an injury. In some embodiments, the subject has an infectious disease, and the nanovesicle comprises an antigen for the infectious disease. In some embodiments, the subject has a genetic defect, and the nanovesicle comprises a protein that the subject is defective in, or a nucleic acid (e.g., mRNA) encoding the protein, or a DNA encoding the protein, or a chromosome encoding the protein, or a nucleus comprising a nucleic acid encoding the protein. In some embodiments, the subject has a dominant genetic disorder, and the nanovesicle comprises a nucleic acid inhibitor (e.g., shRNA, siRNA, or miRNA) of a dominant mutant allele. In some embodiments, the subject has a dominant genetic disorder, and / or the nanovesicle comprises a nucleic acid inhibitor (e.g., shRNA, siRNA, or miRNA) of a dominant mutant allele, and / or the nanovesicle also comprises an mRNA encoding a non-mutated allele of a mutant gene that is not targeted by the nucleic acid inhibitor. In some embodiments, the subject is in need of vaccination, hi some embodiments, the subject is in need of regeneration of, for example, a site of injury.

[0331] In some embodiments, the nanovesicles or compositions described herein are administered to a subject at least 1, 2, 3, 4, or 5 times.

[0332] In some embodiments, nanovesicles comprising a polypeptide as described herein, when administered to a subject, e.g., a mouse or a human, target a tissue, e.g., liver, lung, heart, spleen, pancreas, gastrointestinal tract, kidney, testes, ovaries, brain, reproductive organs, central nervous system, peripheral nervous system, skeletal muscle, endothelium, inner ear, or eye. In some embodiments, at least 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the nanovesicles comprising a polypeptide as described herein in the administered composition are present in the target tissue after 24, 48, or 72 hours.

[0333] In some embodiments, the nanovesicles or compositions described above are administered to a subject in a therapeutically effective amount or therapeutically effective doses. Exemplary dosages include daily dose ranges of about 0.01 mg / kg to about 500 mg / kg, or about 0.1 mg / kg to about 200 mg / kg, or about 1 mg / kg to about 100 mg / kg, or about 10 mg / kg to about 50 mg / kg. However, dosages may vary according to several factors, including the selected route of administration, formulation of the composition, patient response, severity of the condition, subject weight, and the judgment of the prescribing physician. Dosages can be increased or decreased over time, depending on the needs of an individual patient. In some embodiments, a patient is given a low dose initially, and then the dose is increased to an effective dosage tolerated by the patient. Determination of an effective amount is well within the capabilities of one of ordinary skill in the art.

[0334] In some embodiments, the nanovesicles or compositions disclosed herein are used to treat cancer.In certain embodiments, the cancer is a primary cancer of the CNS, such as glioma, glioblastoma multiforme, meningioma, astrocytoma, acoustic neuroma, chondroma, oligodendroglioma, medulloblastoma, glioma, schwannoma, neurofibroma, neuroblastoma, or epidural, intramedullary or intradural tumor.In some embodiments, the cancer is a solid tumor, or in other embodiments, the cancer is a non-solid tumor. Solid tumor cancers include tumors of the central nervous system, breast cancer, prostate cancer, skin cancer (including basal cell carcinoma, cell carcinoma, squamous cell carcinoma and melanoma), cervical cancer, uterine cancer, lung cancer, ovarian cancer, testicular cancer, thyroid cancer, astrocytoma, glioma, pancreatic cancer, mesothelioma, gastric cancer, liver cancer, colon cancer, rectal cancer, renal cancer including nephroblastoma, bladder cancer, esophageal cancer, laryngeal cancer, parotid gland cancer, biliary tract cancer, endometrial cancer, adenocarcinoma, small cell carcinoma, neuroblastoma, adrenocortical carcinoma, epithelial carcinoma, desmoid tumor, desmoplastic small round cell tumor, endocrine tumor, Ewing's sarcoma family tumors, germ cell tumor, hepatoblastoma, hepatocellular carcinoma, non-rhabdomyosarcoma soft tissue sarcoma, osteosarcoma, peripheral primitive neuroectodermal tumor, retinoblastoma, and rhabdomyosarcoma. In some embodiments, there is provided a use of nanovesicles as disclosed herein in the manufacture of a medicament for treating cancer.

[0335] In some embodiments, the nanovesicles or compositions disclosed herein can be used to treat autoimmune or inflammatory diseases. Examples of such diseases include, but are not limited to, ankylosing spondylitis, arthritis, osteoarthritis, rheumatoid arthritis, psoriatic arthritis, asthma, scleroderma, stroke, atherosclerosis, Crohn's disease, colitis, ulcerative colitis, dermatitis, diverticulitis, fibrosis, idiopathic pulmonary fibrosis, fibromyalgia, hepatitis, irritable bowel syndrome (IBS), lupus, systemic lupus erythematosus (SLE), nephritis, multiple sclerosis, and ulcerative colitis. In some embodiments, the use of the nanovesicles disclosed herein in the manufacture of a drug for treating autoimmune or inflammatory diseases is provided.

[0336] In some embodiments, the nanovesicles or compositions as disclosed herein may be used to treat cardiovascular diseases, such as coronary artery disease, heart attack, abnormal heart rhythm or arrhythmia, heart failure, valvular heart disease, congenital heart disease, myocardial disease, cardiomyopathy, pericardial disease, aortic disease, Marfan syndrome, vascular disease, or vascular disease.

[0337] The nanovesicles or compositions of the present disclosure can be administered via a variety of different routes of administration, typically depending on the disease being treated and / or the characteristics of the nanovesicles, compositions, and / or therapeutic molecules, e.g., auricular (ear), buccal, conjunctival, dermal, dental, electroosmotic, intracervical, intrasinus, intratracheal, intraintestinal, epidural, extra-amniotic, extracorporeal, hemodialytic, infiltrative, interstitial, intraperitoneal, intra-amniotic, intra-arterial, intra-articular, intrabiliary, intrabronchial, intravesical, intracardiac, intracartilage, intracaudal, intracavernous, intracavitary, intracerebral, intraventricular, intracisternal, intracorneal, intracoronary (dental), intracoronary, intracavernous, intraspondylotic ... cavernosum), intradermal, intradiscal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intraileal, intralesional, intracavity, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intrapericardial, intraperitoneal, intrathoracic, intraprostatic, intrapulmonary, intrasinus, intraspinal, intrasynovial, intratendon, intratesticular, intrathecal, intrathoracic, intratubular, intratumoral, intratympanic, intrauterine, intravascular, intravenous, intravenous, intravenous injection, intravenous drip, intraventricular, intravesical, intravitreal, iontophoresis, lavage, laryngeal, nasal, nasogastric, occlusive dressing The compositions may be administered to a human or animal subject via intrathecal, intramuscular, intravenous, intravenous, intravenous (IV) administration, intramuscular, intravenous ...

[0338] The nanovesicles disclosed herein may be used for in vivo and / or in vitro detection or diagnostic purposes, including quantitative and / or qualitative detection. Similarly, the polypeptides, nucleic acids, expression vectors and / or cells described in the preceding text may be used accordingly, as detailed in this section.

[0339] For diagnostic applications or detection purposes, nanovesicles may contain detectable moieties, such as moieties that are detectable through biological imaging, including radiology or magnetic resonance imaging. In some embodiments, nanovesicles contain reporter proteins or detectable labels. In some embodiments, the nanovesicles disclosed herein are bound to one or more substances that can be recognized by a detection substance. As an example, nanovesicles may be covalently bound to biotin, and biotin can be detected by its ability to bind to streptavidin.

[0340] In certain embodiments, nanovesicles are useful for detecting their presence in a sample, preferably a biological sample from, for example, a human subject.Non-limiting examples of biological samples include blood, biopsy, cerebrospinal fluid, lymphatic fluid, urine, and / or non-blood tissue.In certain embodiments, biological samples include cells or tissues from human patients.

[0341] Thus, in some embodiments, methods are provided that include the steps of: (i) contacting a subject or biological sample with a nanovesicle of the present disclosure that comprises a detectable moiety; (ii) allowing the nanovesicle to interact with the subject or sample; and (iii) detecting the nanovesicle. Such methods can be in vitro or in vivo methods. In some embodiments, such methods are methods of localizing nanovesicles. EXAMPLES

[0342] 6. Working Examples The examples illustrate the methods and compositions disclosed herein. Although a general description is provided above, it will be understood that various other embodiments may be practiced.

[0343] 6.1 Example 1: Generation of Engineered EVs The DNA sequence of a fusion protein containing an FcRn binding site fused to a transmembrane scaffold protein (EphA4) was designed in silico, and encoded a polypeptide with the following structure:

[0344] Fusion protein 1: EphA4 signal peptide-scFv-linker1-modified monomeric Fc-linker2-EphA4 fragment-linker3-EGFP, with the extracellular domain shown in FIG. 3; and

[0345] Fusion protein 2: EphA4 signal peptide-scFv-linker1-EphA4 fragment-linker3-EGFP.

[0346] The DNA sequence was synthesized by a commercial DNA synthesis vendor and cloned into a lentiviral backbone containing an internal ribosome entry site and an antibiotic selection marker. Lentiviruses were generated using standard protocols, transduced into HEK293T cells, then sorted by flow cytometry for GFP expression, and then monoclonal expanded.

[0347] Cell lines were cultured and EVs were isolated from the culture supernatants of stable clones. Specifically, the EV-containing medium was collected and clarified from debris by differential centrifugation. The supernatant was then filtered through 0.22 μm syringe or bottle-top filters and further processed in different purification steps. For large-scale production, high-density cultures were maintained in a stirred bioreactor in perfusion mode, and the collected perfusion supernatant was pre-clarified and filtered by an alternating tangential flow system fitted with a 0.2 μm hollow fiber filter. EVs were isolated and purified from the clarified conditioned medium using different methods, typically a combination of diafiltration / ultrafiltration and tangential flow filtration (TFF), and flow-through-based multimodal chromatography and / or bind-and-elute chromatography steps. The purified EVs were then frozen and stored for downstream analysis. Western blotting was performed on the purified EVs. As shown in Figure 4, equal protein amounts of fusion protein 1 (left lane) and fusion protein 2 (right lane) expressed on EVs were loaded onto a denaturing polyacrylamide gel. Western blotting of EphA4 using an antibody specific for the EphA4 extracellular domain demonstrated the expression of fusion proteins 1 and 2 on engineered EVs.

[0348] 6.2 Example 2: Selection of source cells expressing FcRn-binding polypeptides In addition to the product cell line generated in Example 1, a stable pool of cells expressing an FcRn polypeptide (fusion protein 3) containing from N-terminus to C-terminus: targeting monobody-linker1-modified monomeric Fc- linker2-EphB2 scaffold-linker3-turboluc (EphB2 scaffold contains residues 195-905 of EphB2, lacks the LBD, and contains the following amino acid substitutions relative to wild-type EphB2: L356A I395A S536E A562S, Y822F) was generated using the same lentivirus scaffold. See SEQ ID NO: 73 for the sequence of the complete fusion protein. In contrast to the previous two GFP-tagged cell lines, the FcRn-binding polypeptide expressed by this cell line does not contain a GFP tag, which allows for flow cytometry-assisted cell sorting (FACS). To select high-expressing cell clones, the transduced cells were monoclonally expanded using limiting dilution and antibiotic selection. The different clones were screened for FcRn-binding polypeptide expression levels by flow cytometry using a fluorescent anti-human Fc domain antibody (Invitrogen catalog 12-4998-82). As shown in Figures 5A-5D, the HEK293T control cells and the cell line expressing fusion protein 2 (described in Example 1) (Figures 5A and 5B, respectively) were not stained by the anti-human Fc domain antibody, whereas all cells of the cell line expressing fusion protein 1 (containing modified monomeric Fc, as described in Example 1) and all cells of the selected clones expressing the FcRn-binding polypeptides of this example were successfully stained by the fluorescent anti-human Fc domain antibody (Figures 5C and 5D, respectively).

[0349] 6.3 Example 3: Low pH Elution of Engineered EVs High expressing clones of producer cells generated in Example 2 were cultured in serum-free chemically defined medium and engineered EVs were isolated from the supernatant as described in Example 1. Engineered and isolated EVs were loaded onto a Protein A affinity chromatography column. Flow settings for the column equilibration, sample loading and column washing procedures in place were selected according to the manufacturer's instructions. FcRn-bound EVs bound to Protein A were eluted into the pre-plated pH neutralizing buffer utilizing an elution buffer containing 0.1 M glycine-HCI, pH 3.0. The flow-through and eluate were fractionated into a 96-well plate and sampled for particle counting by dynamic light scattering (DLS). The minimal UV absorbance and particle / ml measurements for both the flow-through and elution fractions compared to the amount of particles loaded onto the column indicate low recovery.

[0350] To examine the low recovery rate, about 1.8x10 12 The particles / ml of purified EV samples were diluted 1:10 in the reduced pH buffer, mixed thoroughly and incubated for 20 min at room temperature. The incubated EV samples were then diluted by a dilution factor into a neutral pH buffer (50 mM Tris, 50 mM NaCl) to achieve particle concentrations in the linear range on a nanoparticle tracking analysis (NTA) instrument (between 1:2500 at higher pH and 1:500 at lower pH, respectively), and the particle concentration of the samples was then measured. As shown in Figure 6, simply incubating the engineered EVs at a pH below 5 and then returning them to pH 7.4 reduced the particle concentration by approximately 90% (approximately 1.8x10 11 From about 1.8x10 10 ), large aggregates were visible in the measurement video. The aggregation was not reversed by diluting the EVs in a high pH buffer. Therefore, EV elution below pH 5 may lead to irreversible aggregation, and a suboptimal very low pH is required for affinity purification methods.

[0351] 6.4 Example 4: Construction of single-chain FcRn expression vector Recombinant human and mouse single-chain FcRn (scFcRn) constructs containing a mouse IgG kappa chain leader sequence as a secretion signal followed by the mature B2M sequence linked via three (GGGGS) (SEQ ID NO: 72) to the mature sequence of FCGRT heavy chain fused to a C-tag were designed in silico, synthesized by a commercial DNA synthesis vendor, and cloned into lentivectors and transient vectors.

[0352] 6.5 Example 5: Generation of recombinant scFcRn Recombinant human scFcRn and mouse scFcRn polypeptides were expressed in HEK293 cells grown in customized chemically defined medium (containing only small molecules), respectively. Expression of scFcRn in concentrated supernatants was detected by Western blot (human: Invitrogen PA5-97738 antibody, mouse R&D Systems AF6775). For large-scale production, stable cell lines expressing recombinant scFcRn were generated using the vectors of Example 4. The cell lines were cultured on hollow fiber cartridges (5 kDa cutoff) or orbital shakers. Clarified supernatants or partially purified and clarified supernatants (concentrated and diafiltered against PBS in a tangential flow device with a 10 kDa hollow fiber unit) were loaded onto a Capture-Select C-TagXL affinity chromatography column. The column was washed with PBS and scFcRn was eluted with either i) 20 mM Tris, 2.0 M MgCl2 pH 7.4, ii) 50 mM acetic acid pH 3.0, or iii) 20 mM Tris, 2 mM "SEPEA" peptide, pH 7.4. For scFcRn eluted with "SEPEA" peptide, the elution fraction was dialyzed or desalted to remove the peptide. The protein content of the purified scFcRn was measured by bicinchoninic acid assay (BCA) and the product was stored at -20°C. The purity of scFcRn was examined by Western blot and SDS-Page. As shown in Figure 7, binding and elution of scFcRn product was confirmed using mouse FcRn-specific antibody for Western blotting of the clarified supernatant (lane 1), flow-through fraction (lane 2) and elution fraction (lane 3). Alternatively, a two-step affinity chromatography was performed to increase the purity and selection of functional protein. In a first step, the clarified crude supernatant or the partially purified and clarified supernatant was adjusted to pH 5.8 with HCl, filtered through a 0.45 μm filter, and loaded onto a commercially available hIgG-Sepharose column pre-equilibrated with MES buffer pH 5.8 as described above. The column was washed with 5 column volumes of MES buffer pH 5.8.Finally, bound protein was eluted from the column with pH 8.0 buffer (50 mM Tris, pH 8.0, 100 mM NaCl). The purified protein was then loaded onto a Capture-Select C-TagXL column, washed with PBS, and optionally eluted with i) 20 mM Tris, 2.0 M MgCl2 pH 7.4, ii) 50 mM acetic acid pH 3.0, or iii) 20 mM Tris, 2 mM "SEPEA" peptide, pH 7.4, as described above.

[0353] 6.6 Example 6: scFcRn Purification of Engineered EVs The recombinant scFcRn protein of Example 5 was loaded onto a C-tagXL column according to the procedure in the instruction manual. The resin was then washed with 25 mM MES pH 5.8, 150 mM NaCl. A stable cell line expressing a polypeptide containing N- to C-terminal targeting monobody-linker-modified monomeric Fc-linker-EphA4 fragment (containing residues 29-590 of EphA4 and an amino acid substitution of F154A relative to wild-type EphA4) was generated, cultured, and the supernatant was collected, clarified, and concentrated. The pH of the collected supernatant was adjusted to pH 5.8, then loaded onto an equilibrated column and further washed with 25 mM MES pH 5.8, 150 mM NaCl. The bound sample was eluted with 50 mM Tris pH 7.4, 150 mM NaCl (counterflow).

[0354] To confirm the presence of transmembrane FcRn-binding polypeptides in the eluted flow-through and elution samples, the elution fractions were pooled, concentrated, and then examined by Western blotting using an anti-EphA4 antibody (ECM Biosciences, Cat. No. EM2801). As shown in FIG. 8, the elution samples showed enhanced EphA4 signal.

[0355] 6.7 Example 7: pH-Dependent Enrichment with scFcRn Approximately 5 mg of scFcRN protein per ml of resin material was covalently coupled to POROS 20 EP resin according to the procedure in the instruction manual. The resin was then washed with 10 column volumes of 0.2M Tris pH 8.2 with 500 mM NaCl, followed by 10 column volumes of 25 mM Tris pH 8.2, according to the instructions in the manufacturer's manual. After a final wash with Tris-NaCl, the column was equilibrated with MES buffer pH 5.8 and was then ready for use. Successful functionalization of the resin was confirmed by loading purified human IgG1.

[0356] The pH dependence of enrichment of engineered EVs containing FcRn-binding polypeptides was determined by loading clarified and diafiltered supernatants of production cells expressing FcRn-binding polypeptides (i.e., fusion protein 1 described in Example 1) containing transmembrane EphA4 scaffold protein onto scFcRn-functionalized POROS EP resin pre-equilibrated with 50 mM Tris pH 7.4, 150 mM NaCl without pre-adjusting the pH. Alternatively, samples of the same batch of supernatant were pH adjusted as described in Example 6 before loading onto scFcRn-functionalized POROS EP resin pre-equilibrated with 25 mM MES pH 5.8. The column was washed and eluted as described in Example 6. In both cases, the flow-through and elution fractions were pooled and concentrated. To confirm the presence of transmembrane FcRn-binding polypeptides in the elution flow-through and elution samples, the flow-through elution fractions were pooled, concentrated, and then examined by Western blotting using an anti-EphA4 antibody (ECM Biosciences, Cat. No. EM2801). The clarified supernatants were not concentrated. As shown in FIG. 9A, for the non-pH-adjusted supernatants, the pooled and concentrated flow-through fractions retained transmembrane EphA4, whereas for the acidified supernatants in FIG. 9B, the pooled and concentrated elution fractions showed enrichment of FcRn-binding polypeptides, including the transmembrane EphA4 scaffold protein.

[0357] 6.8 Example 8: FcRn Binding Immunoassay Lumit™ FcRn binding immunoassay (Promega) was performed with purified EVs as described in Example 2 and native Hek293 EVs. Samples of the EVs and controls human IgG1 and mouse IgG1 were serially diluted and incubated with split FcRn / tracer according to the manufacturer's instructions (tracer and FcRn were diluted 10-fold). Detection reagent was added and luminescence was detected in a plate reader. As shown in Figure 10A, purified EVs as described in Example 1 were able to bind to FcRn, whereas native EVs did not. As shown in Figure 10B, human IgG1 was able to bind to FcRn, whereas mouse IgG1 was not.

[0358] 6.9 Example 9: Blood Clearance Following IV Administration of Modified Fc Hybridosomes EVs (e.g., exosomes) are believed to have a very short half-life and circulation time. To test the blood clearance of hybridosomes containing the EphB2 scaffold described in Example 2, immunocompetent nude SKH1 mice (6-8 weeks old, n=6 / group) were intravenously injected with DNA-loaded lipid nanoparticles or hybridosomes (0.5 mg / kg). The DNA cargo encoded a promoter, a reporter transgene, and BGH poly(A). Lipid nanoparticles were prepared on a Nanoassemblr™ microfluidic system (Precision NanoSystems) according to the manufacturer's instructions. On day 21 after dosing, animals were re-challenged. To monitor blood clearance, 20 microliters of blood was collected from the tail vein on days 3, 6, 21 (before the second dose), and 24, respectively, and processed to plasma. Two microliters of diluted plasma was used in a Taqman qPCR assay to quantify DNA sequences, specifically BGH polyA sequences, by comparing with a standard curve on the same plate. The efficiency of DNA recovery from naive mouse plasma was determined by spiking mouse plasma with DNA vectors. As shown in Figure 11, hybridosomes containing targeting monobody modified Fc domain fused to EphB2 scaffold protein could be detected in mouse plasma on day 6 after administration, while the plasma copy number on the same day in the LNP-treated group was below the detection limit. [Table 12]

Claims

1. A polypeptide comprising: (a) a transmembrane domain; and (b) i. capable of specifically binding to the Fc binding site of the neonatal Fc receptor (FcRn); and ii. lacks the ability to form homodimers Engineered Fc domains of immunoglobulins The polypeptide comprising:

2. The equilibrium dissociation constant of the engineered Fc domain binding to FcRn at pH 6.5 is up to 10 -4 2. The polypeptide of claim 1, having a value of M.

3. The equilibrium dissociation constant of the engineered Fc domain binding to FcRn at pH 7.4 is at least 10 -4 2. The polypeptide of claim 1, having a value of M.

4. The polypeptide of claim 1, wherein the modified Fc domain specifically binds to an amino acid sequence between positions 135-158 of human FcRn (SEQ ID NO: 7) and / or mouse FcRn (SEQ ID NO: 8).

5. The modified Fc domain has the amino acid sequence 【Chemistry 1】 where X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 and X 8 The polypeptide of claim 1, wherein each of is any amino acid.

6. The polypeptide of claim 1, which does not bind to C1q, FcγRI, FcγRII, or FcγRIII.

7. (a) the complement-dependent cytotoxicity (CDC) activity of the modified Fc domain; (b) antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the modified Fc domain; (c) the antibody-dependent cell-mediated phagocytosis (ADCP) activity of the modified Fc domain; and / or (d) Antibody-dependent intracellular neutralization (ADIN) activity of the modified Fc domain The polypeptide of claim 1, wherein the Fc domain is at least 10% reduced compared to an unmodified Fc domain.

8. The FcRn-binding polypeptide has from N-terminus to C-terminus: (a) a modified CH2 domain that has been modified to reduce effector function relative to an unmodified CH2 domain; (b) a modified CH3 domain that has been modified to lack homodimerization function relative to the unmodified CH3 domain; (c) a linker sequence; and (d) Transmembrane domain The polypeptide of claim 1, comprising:

9. The FcRn-binding polypeptide has from the C-terminus to the N-terminus: (a) a modified CH3 domain that has been modified to lack homodimerization function relative to the unmodified CH3 domain; (b) a modified CH2 domain that has been modified to reduce effector function relative to the unmodified CH2 domain; (c) a linker sequence; and (d) Transmembrane domain The polypeptide of claim 1, comprising:

10. The polypeptide of claim 1, wherein the transmembrane domain is a multi-spanning transmembrane domain.

11. scFv, (scFv) 2 , Fab, Fab', F(ab') 2 , F(abl) 2 , Fv, dAb, Fd fragment, diabody, F(ab) 2 , F(ab'), F(ab') 3 2. The polypeptide of claim 1, further comprising a targeting domain selected from the group consisting of: an Fd, an Fv, a disulfide-linked Fv, a dAb, an sdAb, a nanobody, a CDR, a di-scFv, a bi-scFv, a tascFv (tandem scFv), an avibody (e.g., a diabody, a triabody, a tetrabody), a T-cell engager (BiTE), a V-NAR domain, an Fcab, an IgGACH2, a DVD-Ig, a probody, an intrabody, a DARPin, a centrin, an affibody, an affilin, an affitin, an anticalin, an avimer, an finomer, a Kunitz domain peptide, a monobody, an adnectin, a tribody, and a nanophytin.

12. A nucleic acid encoding a polypeptide according to any one of claims 1 to 11.

13. 13. An expression vector comprising the nucleic acid of claim 12.

14. A cell comprising the nucleic acid of claim 12.

15. An extracellular vesicle comprising a polypeptide described in any one of claims 1 to 11.

16. A hybridosome comprising a polypeptide according to any one of claims 1 to 11.

17. 1. A method for purifying extracellular vesicles (EVs), comprising: (a) providing said EVs, wherein said EVs associate with a first binding partner, said first binding partner binding to the Fc binding site of FcRn in a pH-dependent manner and comprising or consisting of a polypeptide according to any one of claims 1 to 11; and (b) contacting the EVs associated with the first binding partner with a second binding partner at a first pH, the second binding partner comprising the Fc binding site of the FcRn and associated with a solid matrix; and (c) eluting the EVs associated with the first binding partner from the solid matrix at a second pH. The method comprising:

18. 20. The method of claim 17, wherein the method includes a wash step at the first pH.

19. 18. The method of claim 17, wherein the first pH is less than 6.

5.

20. 18. The method of claim 17, wherein the second pH is greater than 7.4.

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