Anucleate cell-derived vaccines

Abstract

To provide methods for stimulating an immune response to an antigen comprising administering, to an individual, an anucleate cell-derived vesicle comprising an antigen and / or an adjuvant.SOLUTION: In some embodiments, the anucleate cell-derived vesicle comprising the antigen and / or adjuvant is generated by passing a cell suspension containing an input anucleate cell through a constriction, where the constriction deforms the input anucleate cell thereby causing a perturbation of the cell to form an anucleate cell-derived vesicle such that an antigen and / or an adjuvant enters the anucleate cell-derived vesicle. In some embodiments, the anucleate cell-derived vesicle comprising the antigen and / or adjuvant is delivered to an individual, and the antigen is delivered to and processed in an immunogenic environment to treat a disease, prevent a disease, and / or vaccinate an individual against an antigen.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 797,185, filed January 25, 2019, U.S. Provisional Patent Application No. 62 / 797,187, filed January 25, 2019, U.S. Provisional Patent Application No. 62 / 933,301, filed November 8, 2019, and U.S. Provisional Patent Application No. 62 / 933,302, filed November 8, 2019, the entire contents of each of which are incorporated herein by reference.

[0002] The present disclosure generally relates to methods for stimulating an immune response or treating cancer, infectious disease, or virus-related disease by delivering anuclear cell-derived vesicles to an individual, wherein the anuclear cell-derived vesicles are loaded with an antigen and / or an adjuvant. In some embodiments, the antigen and / or adjuvant are delivered to the anuclear cells by passing a cell suspension through a cell-deforming constriction. [Background technology]

[0003] The complexity of the immune system and immune responses to foreign substances makes it difficult to develop effective approaches for eliciting in vivo antigen-specific immune responses. In addition to the continued development of drugs such as small molecules and polypeptide- and / or nucleotide-based vaccines that can elicit antigen-specific immune responses, carrier strategies for use with such drugs require further development to optimize delivery and immune responses. Carriers known in the art, including polymer-based carriers, particulate carriers, liposomes, and cell-based vesicles such as vesicles derived from red blood cells, still face challenges that limit their use for eliciting in vivo antigen-specific immune responses. For example, given that red blood cells are irregularly shaped (biconcave), anucleate, and transcriptionally inactive, the use of red blood cells as carriers is difficult due to the challenges associated with manipulating red blood cells to associate antigenic materials. As a result, standard transfection techniques are ineffective. To overcome these challenges, methods for using red blood cells as carriers to elicit immune responses have focused on conjugating materials to the red blood cell surface. For example, Lorentz et al., Sci. Adv, l:el5001122015; Grimm et al., Sci Rep, 5, 2015; and Kontos See, e.g., Wang et al., Proc Natl Acad Sci USA, 110, 2013. Initial studies using surface conjugation have shown promising results with model antigens and mouse models of type 1 diabetes, but have several significant weaknesses, including (a) the need for chemically modified antigens for attachment; (b) limited surface area for loading; and (c) immunogenicity.

[0004] References describing methods of using microfluidic constrictions to deliver compounds to cells include WO2013059343, WO2015023982, WO2016070136, WO2016077761 and WO / 2017 / 192785. All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Lorentz et al., Sci. Adv, l:el5001122015 [Non-patent document 2] Grimm et al., Sci Rep, 5, 2015 [Non-patent document 3] Kontos et al., Proc Natl Acad Sci USA, 110, 2013 Summary of the Invention [Means for solving the problem]

[0006] In some aspects, the present invention provides a method for delivering an antigen into anuclear cell-derived vesicles, the method comprising: a) passing a cell suspension containing input (e.g., parent) anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigen and forming anuclear cell-derived vesicles; and b) incubating the anuclear cell-derived vesicles with the antigen for a time sufficient to allow entry of the antigen into the anuclear cell-derived vesicles. In some embodiments, the input anuclear cells further comprise an adjuvant.

[0007] In some aspects, the present invention provides a method for delivering an adjuvant into anuclear cell-derived vesicles, the method comprising: a) passing a cell suspension containing input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the adjuvant and form anuclear cell-derived vesicles; and b) incubating the anuclear cell-derived vesicles with an adjuvant for a time sufficient to allow entry of the adjuvant into the anuclear cell-derived vesicles. In some embodiments, the input anuclear cells further comprise an antigen.

[0008] In some aspects, the present invention provides a method for delivering antigens and adjuvants into anuclear cell-derived vesicles, the method comprising the steps of: a) passing a cell suspension containing input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigen and adjuvant, to form anuclear cell-derived vesicles; and b) incubating the anuclear cell-derived vesicles with the antigen and adjuvant for a time sufficient to allow entry of the antigen and adjuvant into the anuclear cell-derived vesicles.

[0009] In some embodiments, the present invention provides a method for stimulating an immune response to an antigen in an individual, the method comprising administering to the individual an effective amount of anuclear cell-derived vesicles containing an antigen, wherein the anuclear cell-derived vesicles containing the antigen are prepared by a process comprising: a) passing a cell suspension containing input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigen and form anuclear cell-derived vesicles; and b) incubating the anuclear cell-derived vesicles with the antigen for a time sufficient to allow the antigen to enter the anuclear cell-derived vesicles. In some embodiments, the method further comprises systemically administering an adjuvant to the individual. In some embodiments, the adjuvant is administered systemically before, after, or simultaneously with the anuclear cell-derived vesicles. In some embodiments, the input anuclear cells comprise an adjuvant.

[0010] In some aspects, the present invention provides a method for stimulating an immune response to an antigen in an individual, the method comprising administering to the individual an effective amount of anuclear cell-derived vesicles comprising an antigen and an adjuvant, wherein the anuclear cell-derived vesicles comprising the antigen and adjuvant are prepared by a process comprising: (a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow the antigen and adjuvant to pass through, thereby forming anuclear cell-derived vesicles; and (b) incubating the anuclear cell-derived vesicles with the antigen and adjuvant for a time sufficient to allow the antigen and adjuvant to enter the anuclear cell-derived vesicles. In some embodiments, the method further comprises systemically administering an adjuvant to the individual. In some embodiments, the adjuvant is administered systemically before, after, or simultaneously with the anuclear cell-derived vesicles. In some aspects, the invention provides methods for treating a disease in an individual, the method comprising administering to the individual anuclear cell-derived vesicles comprising a disease-associated antigen, wherein an immune response to the antigen ameliorates the disease state, and the anuclear cell-derived vesicles comprising the disease-associated antigen are prepared by a process comprising: a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, wherein the diameter of the constriction is a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigen and form anuclear cell-derived vesicles; and b) incubating the anuclear cell-derived vesicles with the antigen for a time sufficient to allow entry of the antigen into the anuclear cell-derived vesicles.

[0011] In some aspects, the invention provides methods for preventing disease in an individual, the method comprising administering to the individual anuclear cell-derived vesicles comprising a disease-associated antigen, wherein an immune response to the antigen prevents the onset of the disease, and the anuclear cell-derived vesicles comprising the disease-associated antigen are prepared by a process comprising: a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, wherein the diameter of the constriction is a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigen and form anuclear cell-derived vesicles; and b) incubating the anuclear cell-derived vesicles with the antigen for a time sufficient to allow entry of the antigen into the anuclear cell-derived vesicles. In some embodiments, the present invention provides a method for vaccinating an individual against an antigen, the method comprising administering to the individual anuclear cell-derived vesicles comprising the antigen, wherein the anuclear cell-derived vesicles comprising the antigen are prepared by a process comprising: a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigen and form anuclear cell-derived vesicles; and b) incubating the anuclear cell-derived vesicles with the antigen for a time sufficient to allow the antigen to enter the anuclear cell-derived vesicles. In some embodiments, the method further comprises systemically administering an adjuvant to the individual. In some embodiments, the adjuvant is administered systemically before, after, or simultaneously with the anuclear cell-derived vesicles. In some embodiments, the input anuclear cells comprise an adjuvant.

[0012] In some aspects, the invention provides methods for treating a disease in an individual, the method comprising administering to the individual anuclear cell-derived vesicles comprising a disease-associated antigen and an adjuvant, wherein an immune response to the antigen ameliorates the disease state, and the anuclear cell-derived vesicles comprising the disease-associated antigen and an adjuvant are prepared by a process comprising: a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow the antigen and adjuvant to pass through, thereby forming anuclear cell-derived vesicles; and b) incubating the anuclear cell-derived vesicles with the antigen and adjuvant for a time sufficient to allow the antigen and adjuvant to enter the anuclear cell-derived vesicles. In some aspects, the invention provides methods for preventing disease in an individual, the method comprising administering to the individual anuclear cell-derived vesicles comprising a disease-associated antigen and an adjuvant, wherein an immune response to the antigen prevents the onset of the disease, and the anuclear cell-derived vesicles comprising the disease-associated antigen and an adjuvant are prepared by a process comprising: a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in suspension, thereby causing disruption of the input anuclear cells large enough to allow the antigen and adjuvant to pass through, thereby forming anuclear cell-derived vesicles; and b) incubating the anuclear cell-derived vesicles with the antigen and adjuvant for a time sufficient to allow the antigen and adjuvant to enter the anuclear cell-derived vesicles.In some aspects, the present invention provides a method for vaccinating an individual against an antigen, the method comprising administering to the individual anuclear cell-derived vesicles comprising an antigen and an adjuvant, wherein the anuclear cell-derived vesicles comprising the antigen and adjuvant are prepared by a process comprising the steps of: a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow the antigen and adjuvant to pass through, thereby forming anuclear cell-derived vesicles; and b) incubating the anuclear cell-derived vesicles with the antigen and adjuvant for a time sufficient to allow the antigen and adjuvant to enter the anuclear cell-derived vesicles.

[0013] In some aspects, the invention provides methods for treating a disease in an individual, wherein an immune response to a disease-associated antigen ameliorates the disease state, the method comprising: a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigen and form anuclear cell-derived vesicles; b) incubating the anuclear cell-derived vesicles with the antigen for a time sufficient to allow entry of the antigen into the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles containing the antigen; and c) administering the anuclear cell-derived vesicles containing the antigen to the individual. In some aspects, the invention provides methods for preventing disease in an individual, wherein an immune response to a disease-associated antigen prevents the onset of the disease, the method comprising: a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigen and form anuclear cell-derived vesicles; b) incubating the anuclear cell-derived vesicles with the antigen for a time sufficient to allow entry of the antigen into the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles containing the antigen; and c) administering the anuclear cell-derived vesicles containing the antigen to the individual. In some aspects, the invention provides methods for vaccinating an individual against an antigen, the method comprising: a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigen and form anuclear cell-derived vesicles; b) incubating the anuclear cell-derived vesicles with the antigen for a time sufficient to allow entry of the antigen into the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles containing the antigen; and c) administering the anuclear cell-derived vesicles containing the antigen to the individual.In some embodiments, the method further comprises systemically administering an extravesicular adjuvant to the individual. In some embodiments, the extravesicular adjuvant is administered before, after, or simultaneously with the anuclear cell-derived vesicles. In some embodiments, the input anuclear cells comprise an adjuvant.

[0014] In some aspects, the present invention provides methods for treating a disease in an individual, wherein an immune response to a disease-associated antigen ameliorates the disease state, the method comprising the steps of: a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow the disease-associated antigen and adjuvant to pass through, to form anuclear cell-derived vesicles; b) incubating the anuclear cell-derived vesicles with the antigen and adjuvant for a time sufficient to allow the antigen and adjuvant to enter the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles comprising the antigen and adjuvant; and c) administering the anuclear cell-derived vesicles comprising the antigen and adjuvant to the individual. In some aspects, the present invention provides methods for preventing disease in an individual, wherein an immune response to a disease-associated antigen prevents the onset of the disease, the method comprising: a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigen and adjuvant and forming anuclear cell-derived vesicles; b) incubating the anuclear cell-derived vesicles with the antigen and adjuvant for a time sufficient to allow the antigen and adjuvant to enter the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles comprising the antigen and adjuvant; and c) administering the anuclear cell-derived vesicles comprising the antigen and adjuvant to the individual.In some embodiments, the present invention provides a method for vaccinating an individual against an antigen, the method comprising: (a) passing a cell suspension containing input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigen and adjuvant and form anuclear cell-derived vesicles; (b) incubating the anuclear cell-derived vesicles with the antigen and adjuvant for a time sufficient to allow the antigen and adjuvant to enter the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles containing the antigen and adjuvant; and (c) administering the anuclear cell-derived vesicles containing the antigen and adjuvant to the individual. In some embodiments, the method further comprises systemically administering an extravesicular adjuvant to the individual. In some embodiments, the extravesicular adjuvant is administered before, after, or simultaneously with the anuclear cell-derived vesicles.

[0015] In some embodiments of the above aspects, the disease is cancer, an infectious disease, or a virus-related disease. In some embodiments, the anuclear cell-derived vesicles are autologous to the individual. In some embodiments, the anuclear cell-derived vesicles are allogeneic to the individual. In some embodiments, the anuclear cell-derived vesicles are present in a pharmaceutical formulation. In some embodiments, the anuclear cell-derived vesicles are administered systemically. In some embodiments, the anuclear cell-derived vesicles are administered intravenously, intraarterially, subcutaneously, intramuscularly, or intraperitoneally.

[0016] In some embodiments of the above aspects, the anuclear cell-derived vesicles are administered to an individual in combination with a therapeutic agent. In some embodiments, the therapeutic agent is administered before, after, or simultaneously with the anuclear cell-derived vesicles. In some embodiments, the therapeutic agent is an immune checkpoint inhibitor and / or a cytokine. In some embodiments, the cytokine is one or more of IFN-α, IFN-γ, IL-2, IL-10, or IL-15. In some embodiments, the immune checkpoint inhibitor is targeted to any one of PD-1, PD-L1, CTLA-4, TIM-3, LAG3, TIGIT, VISTA, TIM1, B7-H4 (VTCN1), and BTLA. In some embodiments, the therapeutic agent is a bispecific agent; for example, a bispecific agent comprising a cytokine component and a targeting component. In some embodiments, the bispecific agent comprises a targeting component and a trap for a molecule such as TGFβ. In some embodiments, the anuclear cell-derived vesicles are administered to an individual in combination with chemotherapy or radiation therapy. In some embodiments, anuclear cell-derived vesicles are administered to an individual in combination with one or more agents that improve antigen presentation (e.g., CD40 or Ox40L), improve T cell proliferation, and / or improve the tumor microenvironment (e.g., ICOS).

[0017] In some embodiments of the above aspects, the antigen may be processed into an MHC class I-restricted peptide and / or an MHC class II-restricted peptide. In some embodiments, the antigen is a CD-1-restricted antigen. In some embodiments, the antigen is a disease-associated antigen. In some embodiments, the antigen is a tumor antigen. In some embodiments, the antigen is derived from a lysate. In some embodiments, the lysate is a tumor lysate. In some embodiments, the antigen is a viral antigen, a bacterial antigen, or a fungal antigen. In some embodiments, the antigen is a microorganism. In some embodiments, the antigen is a polypeptide. In some embodiments, the antigen is a lipid antigen. In some embodiments, the antigen is a carbohydrate antigen. In some embodiments, a nucleic acid encoding the antigen is delivered to a cell. In some embodiments, the antigen is a modified antigen. In some embodiments, the modified antigen comprises an antigen fused to a polypeptide. In some embodiments, the modified antigen comprises an antigen fused to a targeting peptide. In some embodiments, the modified antigen comprises an antigen fused to a lipid. In some embodiments, the modified antigen comprises an antigen fused to a carbohydrate. In some embodiments, the modified antigen comprises an antigen fused to a nanoparticle. In some embodiments, a nucleic acid encoding the antigen is delivered to the cell. In some embodiments, multiple antigens are delivered in anuclear cell-derived vesicles.

[0018] In some embodiments of the above aspects, the adjuvant is CpG ODN, IFN-α, a STING agonist, a RIG-I agonist, poly I:C, polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose (HILTONOL®), imiquimod, resiquimod, and / or lipopolysaccharide (LPS). In some embodiments, the adjuvant is low molecular weight poly I:C.

[0019] In some embodiments of the above aspects, the input anucleated cells are red blood cells. In some embodiments, the red blood cells are red blood cells. In some embodiments, the red blood cells are reticulocytes. In some embodiments, the input anucleated cells are platelets. In some embodiments, the input anucleated cells are mammalian cells. In some embodiments, the input anucleated cells are monkey, mouse, dog, cat, horse, rat, sheep, goat, pig, or rabbit cells. In some embodiments, the input anucleated cells are human cells.

[0020] In some embodiments of the above aspects, the constriction is contained within a microfluidic channel. In some embodiments, the microfluidic channel includes multiple constrictions. In some embodiments, multiple constrictions are arranged in series and / or parallel. In some embodiments, the constriction is between multiple micropillars; between multiple micropillars arranged in an array; or between one or more movable plates. In some embodiments, the constriction is a pore or is contained within a pore. In some embodiments, the pore is contained in a surface. In some embodiments, the surface is a filter. In some embodiments, the surface is a membrane. In some embodiments, the constriction size is a function of the diameter of the input anuclear cells in suspension. In some embodiments, the constriction size is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% of the diameter of the input anuclear cells in suspension. In some embodiments, the constriction has a width of about 0.25 μm to about 4 μm. In some embodiments, the constriction has a width of about 4 μm, about 3.5 μm, about 3 μm, about 2.5 μm, about 2 μm, about 1.5 μm, about 1 μm, about 0.5 μm, or about 0.25 μm. In some embodiments, the constriction has a width of about 2.2 μm. In some embodiments, the input anuclear cells are passed through the constriction under pressure ranging from about 10 psi to about 90 psi. In some embodiments, the cell suspension is contacted with an antigen prior to, simultaneously with, or after passing through the constriction.

[0021] In some aspects, the present invention provides anuclear cell-derived vesicles comprising an antigen, prepared by a process comprising: (a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigen and form anuclear cell-derived vesicles; and (b) incubating the anuclear cell-derived vesicles with the antigen for a time sufficient to allow entry of the antigen into the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles comprising the antigen. In some embodiments, the input anuclear cells comprise an adjuvant. In some aspects, the present invention provides anuclear cell-derived vesicles comprising an adjuvant, prepared by a process comprising: a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the adjuvant and form anuclear cell-derived vesicles; and b) incubating the anuclear cell-derived vesicles with the adjuvant for a time sufficient to allow entry of the adjuvant into the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles comprising the adjuvant. In some embodiments, the input anuclear cells comprise an antigen. In some aspects, the present invention provides anuclear cell-derived vesicles comprising an antigen and an adjuvant, prepared by a process comprising: a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow the antigen and adjuvant to pass through, to form anuclear cell-derived vesicles; and b) incubating the anuclear cell-derived vesicles with the antigen and adjuvant for a time sufficient to allow the antigen and adjuvant to enter the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles comprising the antigen and adjuvant.In some embodiments, the anucleated cell-derived vesicles are red blood cell-derived vesicles or platelet-derived vesicles. In some embodiments, the red blood cell-derived vesicles are red blood cell-derived vesicles or reticulocyte-derived vesicles.

[0022] In some embodiments of the anucleate-derived vesicles described above, the antigen may be processed into an MHC class I-restricted peptide and / or an MHC class II-restricted peptide. In some embodiments, the antigen is a CD-1-restricted antigen. In some embodiments, the antigen is a disease-associated antigen. In some embodiments, the antigen is a tumor antigen. In some embodiments, the antigen is derived from a lysate. In some embodiments, the lysate is a tumor lysate. In some embodiments, the antigen is a viral antigen, a bacterial antigen, or a fungal antigen. In some embodiments, the antigen is a microorganism. In some embodiments, the antigen is a polypeptide. In some embodiments, the antigen is a lipid antigen. In some embodiments, the antigen is a carbohydrate antigen. In some embodiments, a nucleic acid encoding the antigen is delivered to a cell. In some embodiments, the antigen is a modified antigen. In some embodiments, the modified antigen comprises an antigen fused to a polypeptide. In some embodiments, the modified antigen comprises an antigen fused to a targeting peptide. In some embodiments, the modified antigen comprises an antigen fused to a lipid. In some embodiments, the modified antigen comprises an antigen fused to a carbohydrate. In some embodiments, the modified antigen comprises an antigen fused to a nanoparticle, hi some embodiments, multiple antigens are delivered in anuclear cell-derived vesicles.

[0023] In some embodiments of the anucleate cell-derived vesicles described above, the adjuvant is CpG ODN, IFN-α, a STING agonist, a RIG-I agonist, poly I:C, polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose (HILTONOL®), imiquimod, resiquimod, and / or LPS. In some embodiments, the adjuvant is low molecular weight poly I:C.

[0024] In some embodiments of the above-described anucleated cell-derived vesicles, the input anucleated cells are red blood cells. In some embodiments, the input anucleated cells are red blood cells. In some embodiments, the input anucleated cells are reticulocytes. In some embodiments, the input anucleated cells are platelets. In some embodiments, the input anucleated cells are mammalian cells. In some embodiments, the input anucleated cells are monkey, mouse, dog, cat, horse, rat, sheep, goat, pig, or rabbit cells. In some embodiments, the input anucleated cells are human cells.

[0025] In some embodiments of the above-described anuclear cell-derived vesicles, the half-life of the anuclear cell-derived vesicles after administration to a mammal is reduced compared to the half-life of the input anuclear cells after administration to a mammal. In some embodiments, the hemoglobin content of the anuclear cell-derived vesicles is reduced compared to the hemoglobin content of the input anuclear cells. In some embodiments, the ATP production of the anuclear cell-derived vesicles is reduced compared to the ATP production of the input anuclear cells. In some embodiments, the anuclear cell-derived vesicles exhibit a spherical morphology. In some embodiments, the input anuclear cells are red blood cells, and the anuclear cell-derived vesicles have a reduced biconcave shape compared to the input anuclear cells. In some embodiments, the anuclear cell-derived vesicles are red blood cell ghosts. In some embodiments, the anuclear cell-derived vesicles prepared by the process have about 1.5 times more phosphatidylserine on their surface compared to the input anuclear cells. In some embodiments, the population profile of vesicles derived from anucleated cells prepared by the process exhibits a higher average surface phosphatidylserine level compared to the input anucleated cells, hi some embodiments, at least 50% of the population profile of vesicles derived from anucleated cells prepared by the process exhibits a higher surface phosphatidylserine level compared to the input anucleated cells.

[0026] In some embodiments, vesicles derived from anuclear cells exhibit enhanced uptake in tissues or cells compared to input anuclear cells. In some embodiments, vesicles derived from anuclear cells exhibit enhanced uptake in phagocytes and / or antigen-presenting cells compared to input anuclear cells. In some embodiments, vesicles derived from anuclear cells have been modified to enhance uptake in tissues or cells compared to vesicles derived from unmodified anuclear cells. In some embodiments, vesicles derived from anuclear cells have been modified to enhance uptake in phagocytes and / or antigen-presenting cells compared to vesicles derived from unmodified anuclear cells. In some embodiments, the phagocytes and / or antigen-presenting cells comprise one or more of dendritic cells or macrophages. In some embodiments, the tissue or cells comprise one or more of liver or spleen. In some embodiments, the vesicles derived from anuclear cells comprise CD47 on their surface.

[0027] In some embodiments, the anucleated cell-derived vesicles are not (a) heat-treated, (b) chemically treated, and / or (c) subjected to hypotonic or hypertonic conditions during preparation of the anucleated cell-derived vesicles. In some embodiments, the osmolality of the cell suspension is maintained throughout the process. In some embodiments, the osmolality of the cell suspension is maintained between 200 mOsm and 400 mOsm throughout the process.

[0028] In some embodiments of the above-described anucleated cell-derived vesicles, the constriction is contained within a microfluidic channel. In some embodiments, the microfluidic channel includes multiple constrictions. In some embodiments, the multiple constrictions are arranged in series and / or parallel. In some embodiments, the constriction is between multiple microposts; between multiple microposts configured in an array; or between one or more movable plates. In some embodiments, the constriction is a pore or is contained within a pore. In some embodiments, the pore is contained on a surface. In some embodiments, the surface is a membrane. In some embodiments, the constriction size is a function of the diameter of the input anucleated cells in suspension. In some embodiments, the constriction size is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% of the diameter of the input anucleated cells in suspension. In some embodiments, the constriction has a width of about 0.25 μm to about 4 μm. In some embodiments, the constriction has a width of about 4 μm, 3.5 μm, about 3 μm, about 2.5 μm, about 2 μm, about 1.5 μm, about 1 μm, about 0.5 μm, or about 0.25 μm. In some embodiments, the constriction has a width of about 2.2 μm. In some embodiments, the input anuclear cells are passed through the constriction under pressure ranging from about 10 psi to about 90 psi. In some embodiments, the cell suspension is contacted with an antigen prior to, simultaneously with, or after passing through the constriction.

[0029] In some aspects, the present invention provides a composition comprising a plurality of anuclear cell-derived vesicles as described herein. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[0030] In some aspects, the present invention provides a method for generating anuclear cell-derived vesicles containing an antigen, the method comprising: a) passing a cell suspension containing input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigen and forming anuclear cell-derived vesicles; and b) incubating the anuclear cell-derived vesicles with the antigen for a time sufficient to allow entry of the antigen into the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles containing the antigen. In some embodiments, the input anuclear cells comprise an adjuvant.

[0031] In some aspects, the present invention provides a method for generating anuclear cell-derived vesicles containing an adjuvant, the method comprising: a) passing a cell suspension containing input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the adjuvant and form anuclear cell-derived vesicles; and b) incubating the anuclear cell-derived vesicles with an adjuvant for a time sufficient to allow entry of the adjuvant into the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles containing the adjuvant. In some embodiments, the input anuclear cells contain an antigen.

[0032] In some aspects, the present invention provides a method for generating anuclear cell-derived vesicles comprising an antigen and an adjuvant, the method comprising: a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigen and adjuvant and form anuclear cell-derived vesicles; and b) incubating the anuclear cell-derived vesicles with the antigen and adjuvant for a time sufficient to allow the antigen and adjuvant to enter the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles comprising the antigen and adjuvant.

[0033] In some embodiments of the above-described methods for generating anuclear cell-derived vesicles, the anuclear cell-derived vesicles are red blood cell-derived vesicles or platelet-derived vesicles. In some embodiments, the red blood cell-derived vesicles are red blood cell-derived vesicles or reticulocyte-derived vesicles.

[0034] In some embodiments of the above-described methods for generating anucleate-derived vesicles, the antigen may be processed into an MHC class I-restricted peptide and / or an MHC class II-restricted peptide. In some embodiments, the antigen is a CD-1-restricted antigen. In some embodiments, the antigen is a disease-associated antigen. In some embodiments, the antigen is a tumor antigen. In some embodiments, the antigen is derived from a lysate. In some embodiments, the lysate is a tumor lysate. In some embodiments, the antigen is a viral antigen, a bacterial antigen, or a fungal antigen. In some embodiments, the antigen is a microorganism. In some embodiments, the antigen is a polypeptide. In some embodiments, the antigen is a lipid antigen. In some embodiments, the antigen is a carbohydrate antigen. In some embodiments, a nucleic acid encoding the antigen is delivered to the cell. In some embodiments, the antigen is a modified antigen. In some embodiments, the modified antigen comprises an antigen fused to a polypeptide. In some embodiments, the modified antigen comprises an antigen fused to a targeting peptide. In some embodiments, the modified antigen comprises an antigen fused to a lipid. In some embodiments, the modified antigen comprises an antigen fused to a carbohydrate. In some embodiments, the modified antigen comprises an antigen fused to a nanoparticle, hi some embodiments, multiple antigens are delivered in anuclear cell-derived vesicles.

[0035] In some embodiments of the above-described methods for generating anucleate-derived vesicles, the adjuvant is CpG ODN, IFN-α, a STING agonist, a RIG-I agonist, poly I:C, polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose (HILTONOL®), imiquimod, resiquimod, and / or LPS. In some embodiments, the adjuvant is low molecular weight poly I:C.

[0036] In some embodiments of the above-described methods for generating vesicles derived from anucleated cells, the input anucleated cells are red blood cells. In some embodiments, the input anucleated cells are red blood cells. In some embodiments, the input anucleated cells are reticulocytes. In some embodiments, the input anucleated cells are platelets. In some embodiments, the input anucleated cells are mammalian cells. In some embodiments, the input anucleated cells are monkey, mouse, dog, cat, horse, rat, sheep, goat, pig, or rabbit cells. In some embodiments, the input anucleated cells are human cells.

[0037] In some embodiments of the above-described method for producing anuclear cell-derived vesicles, the half-life of the anuclear cell-derived vesicles after administration to a mammal is reduced compared to the half-life of the input anuclear cells after administration to a mammal. In some embodiments, the hemoglobin content of the anuclear cell-derived vesicles is reduced compared to the hemoglobin content of the input anuclear cells. In some embodiments, the ATP production of the anuclear cell-derived vesicles is reduced compared to the ATP production of the input anuclear cells. In some embodiments, the anuclear cell-derived vesicles exhibit a spherical morphology. In some embodiments, the input anuclear cells are red blood cells, and the anuclear cell-derived vesicles have a reduced biconcave shape compared to the input anuclear cells. In some embodiments, the anuclear cell-derived vesicles are red blood cell ghosts. In some embodiments, the anuclear cell-derived vesicles prepared by the process have about 1.5-fold more phosphatidylserine on their surface compared to the input anuclear cells. In some embodiments, the population profile of vesicles derived from anuclear cells prepared by the process exhibits a higher average surface phosphatidylserine level compared to the input anuclear cells. In some embodiments, at least 50% of the population profile of vesicles derived from anuclear cells prepared by the process exhibits a higher surface phosphatidylserine level compared to the input anuclear cells. In some embodiments, vesicles derived from anuclear cells exhibit enhanced uptake in tissues or cells compared to the input anuclear cells. In some embodiments, vesicles derived from anuclear cells exhibit enhanced uptake in phagocytes and / or antigen-presenting cells compared to the input anuclear cells. In some embodiments, vesicles derived from anuclear cells have been modified to enhance uptake in tissues or cells compared to the input anuclear cells. In some embodiments, vesicles derived from anuclear cells have been modified to enhance uptake in phagocytes and / or antigen-presenting cells compared to vesicles derived from unmodified anuclear cells. In some embodiments, the phagocytes and / or antigen-presenting cells comprise one or more of dendritic cells or macrophages. In some embodiments, the tissue or cells comprise one or more of the liver or spleen.In some embodiments, the anuclear cell-derived vesicles comprise CD47 on their surface.

[0038] In some embodiments of the above-described methods for producing anuclear cell-derived vesicles, during the preparation of the anuclear cell-derived vesicles, the anuclear cell-derived vesicles are not (a) heat-treated, (b) chemically treated, and / or (c) subjected to hypotonic or hypertonic conditions. In some embodiments, the osmolality of the cell suspension is maintained throughout the process. In some embodiments, the osmolality of the cell suspension is maintained between about 200 mOsm and about 400 mOsm throughout the process.

[0039] In some embodiments of the above-described methods for generating anucleated cell-derived vesicles, the constriction is contained within a microfluidic channel. In some embodiments, the microfluidic channel includes multiple constrictions. In some embodiments, multiple constrictions are arranged in series and / or parallel. In some embodiments, the constriction is between multiple microposts; between multiple microposts configured in an array; or between one or more movable plates. In some embodiments, the constriction is a pore or is contained within a pore. In some embodiments, the pore is contained in a surface. In some embodiments, the surface is a filter. In some embodiments, the surface is a membrane. In some embodiments, the constriction size is a function of the diameter of the input anucleated cells in suspension. In some embodiments, the constriction size is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% of the diameter of the input anucleated cells in suspension. In some embodiments, the constriction has a width of about 0.25 μm to about 4 μm. In some embodiments, the constriction has a width of about 4 μm, 3.5 μm, about 3 μm, about 2.5 μm, about 2 μm, about 1.5 μm, about 1 μm, about 0.5 μm, or about 0.25 μm. In some embodiments, the constriction has a width of about 2.2 μm. In some embodiments, the input anuclear cells are passed through the constriction under pressure ranging from about 10 psi to about 90 psi. In some embodiments, the cell suspension is contacted with an antigen prior to, simultaneously with, or after passing through the constriction.

[0040] In one aspect, the present disclosure provides anuclear cell-derived vesicles prepared from parent anuclear cells, the anuclear cell-derived vesicles having one or more of the following characteristics: (a) a reduced circulating half-life in a mammal compared to the parent anuclear cells, (b) a reduced hemoglobin level compared to the parent anuclear cells, (c) a spherical morphology, (d) an increased surface phosphatidylserine level compared to the parent anuclear cells, or (e) reduced ATP production compared to the parent anuclear cells.

[0041] In some embodiments, the parent anucleated cell is a mammalian cell. In some embodiments, the parent anucleated cell is a human cell. In some embodiments, the parent anucleated cell is a red blood cell or a platelet. In some embodiments, the red blood cell is an erythrocyte or a reticulocyte.

[0042] In some embodiments, the circulating half-life of vesicles derived from anucleated cells in a mammal is reduced compared to the parent anucleated cells, ie, the circulating half-life in a mammal is reduced by more than about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% compared to the parent anucleated cells.

[0043] In some embodiments, the parent anucleated cells are human cells, and the vesicles derived from the anucleated cells have a circulation half-life of less than about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 6 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 10 days, about 25 days, about 50 days, about 75 days, about 100 days, or about 120 days.

[0044] In some embodiments, the parent anucleated cells are red blood cells, and the hemoglobin level in the anucleated cell-derived vesicles is reduced compared to the parent anucleated cells. In some embodiments, the hemoglobin level in the anucleated cell-derived vesicles is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 99%, or about 100% compared to the parent anucleated cells. In some embodiments, the hemoglobin level in the anucleated cell-derived vesicles is about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, or about 50% of the hemoglobin level in the parent anucleated cells.

[0045] In some embodiments, the parent anucleated cells are red blood cells and the anucleated cell-derived vesicles have a spherical morphology. In some embodiments, the parent anucleated cells are red blood cells and the anucleated cell-derived vesicles have a reduced biconcave shape compared to the parent anucleated cells.

[0046] In some embodiments, the parent anucleated cells are red blood cells or red blood cells, and the anucleated cell-derived vesicles are red blood cell ghosts (RBC ghosts).

[0047] In some embodiments, the vesicles derived from anuclear cells have increased surface phosphatidylserine levels compared to the parent anuclear cells. In some embodiments, the vesicles derived from anuclear cells prepared by the process have about 1.5 times more phosphatidylserine on their surface compared to the parent anuclear cells. In some embodiments, the vesicles derived from anuclear cells have about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 99%, about 100%, or more than about 100% more phosphatidylserine on their surface compared to the parent anuclear cells.

[0048] In some embodiments, vesicles derived from anuclear cells have reduced ATP production compared to the parent anuclear cells. In some embodiments, vesicles derived from anuclear cells produce ATP at about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, or about 50% of the level of ATP produced by the parent anuclear cells. In some embodiments, vesicles derived from anuclear cells do not produce ATP.

[0049] In some embodiments, the anuclear cell-derived vesicles are modified to enhance uptake in tissues or cells compared to the parent anuclear cell, hi some embodiments, the anuclear cell-derived vesicles are modified to enhance uptake in the liver or spleen or by phagocytes or antigen-presenting cells compared to uptake by the parent anuclear cell.

[0050] In some embodiments, the anuclear cell-derived vesicles comprise CD47 on their surface.

[0051] In some embodiments, the parent anucleated cells were not (a) heat-treated, (b) chemically treated, and / or (c) subjected to hypotonic or hypertonic conditions during preparation of the anucleated cell-derived vesicles. In some embodiments, osmolality was maintained during preparation of the anucleated cell-derived vesicles from the parent anucleated cells. In some embodiments, osmolality was maintained between about 200 mOsm and about 600 mOsm. In some embodiments, osmolality was maintained between about 200 mOsm and about 400 mOsm.

[0052] In some embodiments, the anucleated cell-derived vesicles are prepared by a process comprising passing a suspension containing input parent anucleated cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input parent anucleated cells in the suspension, thereby causing disruption of the anucleated cells large enough to allow passage of a payload, thereby generating anucleated cell-derived vesicles.

[0053] In some embodiments, the anuclear cell-derived vesicles comprise a payload, which in some embodiments is a polypeptide, a nucleic acid, a lipid, a carbohydrate, a small molecule, a complex, or a nanoparticle.

[0054] In some embodiments, the anuclear cell-derived vesicles are prepared by a process comprising: (a) passing a cell suspension containing input parent anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input parent anuclear cells in the suspension, thereby causing disruption of the input parent anuclear cells large enough to allow passage of a payload and forming anuclear cell-derived vesicles; and (b) incubating the anuclear cell-derived vesicles with the payload for a time sufficient to allow entry of the payload into the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles containing the payload.

[0055] In some embodiments, the anuclear cell-derived vesicles comprise an antigen. In some embodiments, the anuclear cell-derived vesicles comprise an adjuvant. In some embodiments, the anuclear cell-derived vesicles comprise an antigen and / or a tolerogenic factor.

[0056] In some embodiments, the anuclear cell-derived vesicles are prepared by a process comprising: (a) passing a cell suspension containing input parent anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input parent anuclear cells in the suspension, thereby causing disruption of the input parent anuclear cells large enough to allow passage of the antigen and forming anuclear cell-derived vesicles; and (b) incubating the anuclear cell-derived vesicles with the antigen for a time sufficient to allow entry of the antigen into the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles containing the antigen.

[0057] In some embodiments, the anuclear cell-derived vesicles are prepared by a process comprising: (a) passing a cell suspension containing input parent anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input parent anuclear cells in the suspension, thereby causing disruption of the input parent anuclear cells large enough to allow passage of the adjuvant and form anuclear cell-derived vesicles; and (b) incubating the anuclear cell-derived vesicles with an adjuvant for a time sufficient to allow entry of the adjuvant into the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles containing the adjuvant.

[0058] In some embodiments, the anuclear cell-derived vesicles comprise an antigen and an adjuvant, and the anuclear cell-derived vesicles are prepared by a process comprising: (a) passing a cell suspension comprising input parent anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input parent anuclear cells in the suspension, thereby causing disruption of the input parent anuclear cells large enough to allow the antigen and adjuvant to pass through, to form anuclear cell-derived vesicles; and (b) incubating the anuclear cell-derived vesicles with the antigen and adjuvant for a time sufficient to allow the antigen and adjuvant to enter the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles comprising the antigen and adjuvant.

[0059] In some embodiments, the anuclear cell-derived vesicles comprise antigens and tolerogenic factors, and the anuclear cell-derived vesicles are prepared by a process comprising: (a) passing a cell suspension comprising input parent anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input parent anuclear cells in the suspension, thereby causing disruption of the input parent anuclear cells large enough to allow passage of the antigens and tolerogenic factors to form anuclear cell-derived vesicles; and (b) incubating the anuclear cell-derived vesicles with the antigens and tolerogenic factors for a time sufficient to allow entry of the antigens and tolerogenic factors into the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles comprising the antigens and tolerogenic factors.

[0060] In some embodiments, the constriction is contained within a microfluidic channel. In some embodiments, the microfluidic channel includes multiple constrictions. In some embodiments, multiple constrictions are arranged in series and / or parallel. In some embodiments, the constriction is between multiple microposts; between multiple microposts configured in an array; or between one or more movable plates. In some embodiments, the constriction is a pore or is contained within a pore. In some embodiments, the pore is contained in a surface. In some embodiments, the surface is a filter. In some embodiments, the surface is a membrane. In some embodiments, the constriction size is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% of the cell diameter. In some embodiments, the constriction has a width of about 0.25 μm to about 4 μm. In some embodiments, the constriction has a width of about 4 μm, 3.5 μm, about 3 μm, about 2.5 μm, about 2 μm, about 1.5 μm, about 1 μm, about 0.5 μm, or about 0.25 μm. In some embodiments, the constriction has a width of about 2.2 μm. In some embodiments, the input parent anucleated cells are passed through the constriction under pressure ranging from about 10 psi to about 150 psi. In some embodiments, the cell suspension is contacted with a payload prior to, simultaneously with, or after passing through the constriction.

[0061] In some embodiments, the antigen may be processed into an MHC class I-restricted peptide and / or an MHC class II-restricted peptide.

[0062] In some embodiments, the antigen is a disease-associated antigen. In some embodiments, the antigen is a tumor antigen. In some embodiments, the antigen is derived from a lysate. In some embodiments, the antigen is derived from a graft lysate. In some embodiments, the lysate is a tumor lysate. In some embodiments, the antigen is a viral antigen, a bacterial antigen, or a fungal antigen. In some embodiments, the viral antigen is a virus, a viral particle, or a viral capsid. In some embodiments, the antigen is a microorganism. In some embodiments, the antigen is a polypeptide. In some embodiments, the antigen is a lipid antigen. In some embodiments, the antigen is a carbohydrate antigen. In some embodiments, a nucleic acid encoding the antigen is delivered to a cell.

[0063] In some embodiments, the antigen is a modified antigen. In some embodiments, the modified antigen comprises an antigen fused to a polypeptide. In some embodiments, the modified antigen comprises an antigen fused to a targeting peptide. In some embodiments, the modified antigen comprises an antigen fused to a lipid. In some embodiments, the modified antigen comprises an antigen fused to a carbohydrate. In some embodiments, the modified antigen comprises an antigen fused to a nanoparticle.

[0064] In some embodiments, multiple antigens are delivered to anucleated cells.

[0065] In some embodiments, the adjuvant is CpG ODN, IFN-α, a STING agonist, a RIG-I agonist, poly I:C, imiquimod, resiquimod, and / or lipopolysaccharide (LPS).

[0066] In another aspect, the present disclosure provides a composition comprising a plurality of anuclear cell-derived vesicles as described herein.

[0067] In another aspect, the present disclosure provides a composition comprising a plurality of anuclear cell-derived vesicles prepared from parent anuclear cells, the composition having one or more of the following characteristics: (a) more than about 20% of the anuclear cell-derived vesicles in the composition have a reduced circulatory half-life in a mammal compared to the parent anuclear cells; (b) more than 20% of the anuclear cell-derived vesicles in the composition have a reduced hemoglobin level compared to the parent anuclear cells; (c) more than 20% of the anuclear cell-derived vesicles in the composition have a spherical morphology; (d) more than 20% of the anuclear cell-derived vesicles in the composition are RBC ghosts; (e) more than 20% of the anuclear cell-derived vesicles in the composition have a higher level of phosphatidylserine compared to the parent anuclear cell population; or (f) more than 20% of the anuclear cell-derived vesicles in the composition have reduced ATP production compared to the parent anuclear cells.

[0068] In another aspect, the present disclosure provides a composition comprising a plurality of anuclear cell-derived vesicles prepared from a population of parent anuclear cells, the composition having one or more of the following characteristics: (a) more than about 20% of the anuclear cell-derived vesicles in the composition have a reduced mammalian circulatory half-life compared to the average for the population of parent anuclear cells; (b) more than 20% of the anuclear cell-derived vesicles in the composition have a reduced hemoglobin level compared to the average for the population of parent anuclear cells; (c) more than 20% of the anuclear cell-derived vesicles in the composition have a spherical morphology; (d) more than 20% of the anuclear cell-derived vesicles in the composition are RBC ghosts; (e) more than 20% of the anuclear cell-derived vesicles in the composition have a higher level of phosphatidylserine compared to the average for the population of parent anuclear cells; or (f) more than 20% of the anuclear cell-derived vesicles in the composition have reduced ATP production compared to the average for the population of parent anuclear cells.

[0069] In some embodiments, the parent anucleated cells used in preparing the composition are mammalian cells. In some embodiments, the parent anucleated cells used in preparing the composition are human cells. In some embodiments, the parent anucleated cells used in preparing the composition are red blood cells or platelets. In some embodiments, the red blood cells are erythrocytes or reticulocytes.

[0070] In some embodiments, the circulating half-life of 20% of the anuclear cell-derived vesicles in the composition in a mammal is reduced compared to the parent anuclear cells or the average of a population of parent anuclear cells. In some embodiments, the circulating half-life of 20% of the anuclear cell-derived vesicles in the composition in a mammal is reduced by more than about 50%, about 60%, about 70%, about 80%, or about 90% compared to the parent anuclear cells or the average of a population of parent anuclear cells. In some embodiments, the parent anuclear cells used to prepare the composition are human cells, and the circulating half-life of 20% of the anuclear cell-derived vesicles in the composition is less than about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 6 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, or about 10 days.

[0071] In some embodiments, the parent anucleated cells used to prepare the composition are red blood cells, and 20% of the anucleated cell-derived vesicles in the composition have a reduced hemoglobin level compared to the parent anucleated cells or the average of a population of parent anucleated cells.

[0072] In some embodiments, the hemoglobin level of 20% of the anuclear cell-derived vesicles in the composition is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% compared to the parent anuclear cells or the average of the parent anuclear cells in the population. In some embodiments, the hemoglobin level of 20% of the anuclear cell-derived vesicles in the composition is about 1%, 5%, 10%, 20%, 30%, 40%, or 50% of the hemoglobin level of the parent anuclear cells or the average of the parent anuclear cells in the population.

[0073] In some embodiments, the parent anucleated cells used to prepare the composition are red blood cells, and more than 20% of the anucleated cell-derived vesicles in the composition have a spherical shape. In some embodiments, the parent anucleated cells used to prepare the composition are red blood cells, and more than 20% of the anucleated cell-derived vesicles in the composition have a reduced biconcave shape compared to the parent anucleated cells.

[0074] In some embodiments, the parent anucleated cells used to prepare the composition are red blood cells or erythrocytes, and more than 20% of the anucleated cell-derived vesicles in the composition are red blood cell ghosts.

[0075] In some embodiments, the anuclear cell-derived vesicles in the composition comprise surface phosphatidylserine. In some embodiments, 20% of the anuclear cell-derived vesicles in the composition comprise increased surface phosphatidylserine levels compared to the parent anuclear cells or the average of a population of parent anuclear cells. In some embodiments, 20% of the anuclear cell-derived vesicles in the composition have surface phosphatidylserine levels that are about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 99%, about 100%, or more than about 100% higher than a composition comprising a plurality of parent anuclear cells.

[0076] In some embodiments, 20% of the anuclear cell-derived vesicles in the composition have reduced ATP production compared to the parent anuclear cells or the average of a population of parent anuclear cells. In some embodiments, 20% of the anuclear cell-derived vesicles in the composition produce ATP at less than about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, or about 50% of the level of ATP produced by the parent anuclear cells or the average of a population of parent anuclear cells. In some embodiments, the anuclear cell-derived vesicles in the composition do not produce ATP.

[0077] In some embodiments, the parent anucleated cells used in the preparation of the composition were not (a) heat-treated, (b) chemically treated, and / or (c) subjected to hypotonic or hypertonic conditions during the preparation of the composition. In some embodiments, osmolality was maintained during the preparation of anucleated cell-derived vesicles from the parent anucleated cells. In some embodiments, osmolality was maintained between about 200 mOsm and about 600 mOsm. In some embodiments, osmolality was maintained between about 200 mOsm and about 400 mOsm.

[0078] In some embodiments, the anucleated cell-derived vesicles of the composition are prepared by a process comprising passing a suspension comprising input parent anucleated cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input parent anucleated cells in the suspension, thereby causing disruption of the anucleated cells large enough to allow passage of a payload, thereby generating anucleated cell-derived vesicles.

[0079] In some embodiments, the anuclear cell-derived vesicles of the composition comprise a payload. In some embodiments, the payload is a therapeutic payload. In some embodiments, the payload is a polypeptide, a nucleic acid, a lipid, a carbohydrate, a small molecule, a complex, or a nanoparticle.

[0080] In some embodiments, the anuclear cell-derived vesicles of the composition are prepared by a process comprising: (a) passing a cell suspension comprising input parent anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input parent anuclear cells in the suspension, thereby causing disruption of the input parent anuclear cells large enough to allow passage of a payload and forming anuclear cell-derived vesicles; and (b) incubating the anuclear cell-derived vesicles with the payload for a time sufficient to allow the payload to enter the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles containing the payload.

[0081] In some embodiments, the anuclear cell-derived vesicles of the composition comprise an antigen. In some embodiments, the anuclear cell-derived vesicles of the composition comprise an adjuvant. In some embodiments, the anuclear cell-derived vesicles of the composition comprise an antigen and a tolerogenic factor.

[0082] In some embodiments, the anuclear cell-derived vesicles of the composition are prepared by a process comprising: (a) passing a cell suspension comprising input parent anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input parent anuclear cells in the suspension, thereby causing disruption of the input parent anuclear cells large enough to allow passage of the antigen and forming anuclear cell-derived vesicles; and (b) incubating the anuclear cell-derived vesicles with the antigen for a time sufficient to allow entry of the antigen into the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles containing the antigen.

[0083] In some embodiments, the anuclear cell-derived vesicles of the composition are prepared by a process comprising: (a) passing a cell suspension comprising input parent anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input parent anuclear cells in the suspension, thereby causing disruption of the input parent anuclear cells large enough to allow passage of the adjuvant and forming anuclear cell-derived vesicles; and (b) incubating the anuclear cell-derived vesicles with an adjuvant for a time sufficient to allow entry of the adjuvant into the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles containing the adjuvant.

[0084] In some embodiments, the anuclear cell-derived vesicles of the composition comprise an antigen and an adjuvant, and the anuclear cell-derived vesicles of the composition were prepared by a process comprising the steps of: (a) passing a cell suspension comprising input parent anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input parent anuclear cells in the suspension, thereby causing disruption of the input parent anuclear cells large enough to allow passage of the antigen and adjuvant and forming anuclear cell-derived vesicles; and (b) incubating the anuclear cell-derived vesicles with the antigen and adjuvant for a time sufficient to allow entry of the antigen and adjuvant into the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles comprising the antigen and / or adjuvant.

[0085] In some embodiments, the anuclear cell-derived vesicles of the composition comprise an antigen and a tolerogenic factor, and the anuclear cell-derived vesicles of the composition are prepared by a process comprising: (a) passing a cell suspension comprising input parent anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input parent anuclear cells in the suspension, thereby causing disruption of the input parent anuclear cells large enough to allow passage of the antigen and tolerogenic factor to form anuclear cell-derived vesicles; and (b) incubating the anuclear cell-derived vesicles with the antigen and tolerogenic factor for a time sufficient to allow entry of the antigen and tolerogenic factor into the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles comprising the antigen and / or tolerogenic factor.

[0086] In some embodiments, the constriction used in the preparation of the composition is contained within a microfluidic channel. In some embodiments, the microfluidic channel used in the preparation of the composition contains multiple constrictions. In some embodiments, the multiple constrictions are arranged in series and / or parallel. In some embodiments, the constriction used in the preparation of the composition is present between multiple microposts, between multiple microposts arranged in an array, or between one or more movable plates. In some embodiments, the constriction used in the preparation of the composition is a pore or is contained within a pore. In some embodiments, the pore used in the preparation of the composition is contained within a surface. In some embodiments, the surface used in the preparation of the composition is a filter. In some embodiments, the surface used in the preparation of the composition is a membrane. In some embodiments, the size of the constriction used in the preparation of the composition is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% of the cell diameter. In some embodiments, the constriction used in the preparation of the composition has a width of about 0.25 μm to about 4 μm. In some embodiments, the constriction used in the preparation of the composition has a width of about 4 μm, 3.5 μm, about 3 μm, about 2.5 μm, about 2 μm, about 1.5 μm, about 1 μm, about 0.5 μm, or about 0.25 μm. In some embodiments, the constriction used in the preparation of the composition has a width of about 2.2 μm. In some embodiments, the input parent anucleated cells used in the preparation of the composition are passed through the constriction under pressure ranging from about 10 psi to about 150 psi. In some embodiments, the cell suspension used in the preparation of the composition is contacted with an antigen prior to, simultaneously with, or after passing through the constriction.

[0087] In some embodiments, the antigen of the composition may be processed into an MHC class I-restricted peptide and / or an MHC class II-restricted peptide. In some embodiments, the antigen is a disease-associated antigen. In some embodiments, the antigen is a tumor antigen. In some embodiments, the antigen is derived from a lysate. In some embodiments, the lysate is a graft lysate. In some embodiments, the lysate is a tumor lysate. In some embodiments, the antigen is a viral antigen, a bacterial antigen, or a fungal antigen. In some embodiments, the antigen is a microorganism. In some embodiments, the antigen is a polypeptide. In some embodiments, the antigen is a lipid antigen. In some embodiments, the antigen is a carbohydrate antigen. In some embodiments, a nucleic acid encoding the antigen is delivered to a cell.

[0088] In some embodiments, the antigen of the composition is a modified antigen. In some embodiments, the modified antigen comprises an antigen fused to a polypeptide. In some embodiments, the modified antigen comprises an antigen fused to a targeting peptide. In some embodiments, the modified antigen comprises an antigen fused to a lipid. In some embodiments, the modified antigen comprises an antigen fused to a carbohydrate. In some embodiments, the modified antigen comprises an antigen fused to a nanoparticle.

[0089] In some embodiments, the anuclear cell-derived vesicles of the composition comprise multiple antigens, and the multiple antigens are delivered to the anuclear cell.

[0090] In some embodiments, the adjuvant of the composition is CpG ODN, IFN-α, a STING agonist, a RIG-I agonist, poly I:C, imiquimod, resiquimod, and / or LPS.

[0091] In some embodiments, the composition is a pharmaceutical composition.

[0092] In another aspect, the present disclosure provides a method for making a composition comprising a plurality of anuclear cell-derived vesicles prepared from parent anuclear cells, wherein the composition has the following properties: (a) more than 20% of the anuclear cell-derived vesicles in the composition have a reduced circulating half-life in a mammal compared to the parent anuclear cells; (b) more than 20% of the anuclear cell-derived vesicles in the composition have a reduced hemoglobin level compared to the parent anuclear cells; (c) more than 20% of the anuclear cell-derived vesicles in the composition have a spherical morphology; (d) more than 20% of the anuclear cell-derived vesicles in the composition are RBC ghosts; (e) more than 20% of the anuclear cell-derived vesicles in the composition are RBC ghosts; and (f) more than 20% of the anuclear cell-derived vesicles in the composition have one or more of the following: (i) more than 20% of the anuclear cell-derived vesicles in the composition have higher levels of phosphatidylserine; or (ii) more than 20% of the anuclear cell-derived vesicles in the composition have reduced ATP production compared to the parent anuclear cells, the method comprising passing a cell suspension containing the parent anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the parent anuclear cells in the suspension, thereby causing disruption of the parent anuclear cells large enough to allow passage of a payload to form anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles.

[0093] In some embodiments, the constriction used in the fabrication methods described herein is contained within a microfluidic channel. In some embodiments, the microfluidic channel used in the fabrication methods described herein contains multiple constrictions. In some embodiments, the multiple constrictions used in the fabrication methods described herein are arranged in series and / or parallel. In some embodiments, the constriction used in the fabrication methods described herein is between multiple microposts, between multiple microposts configured in an array, or between one or more movable plates. In some embodiments, the constriction used in the fabrication methods described herein is a pore or is contained within a pore. In some embodiments, the pore used in the fabrication methods described herein is contained in a surface. In some embodiments, the surface used in the fabrication methods described herein is a filter. In some embodiments, the surface used in the fabrication methods described herein is a membrane. In some embodiments, the constriction size used in the fabrication methods described herein is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% of the cell diameter. In some embodiments, the constriction used in the production methods described herein has a width of about 0.25 μm to about 4 μm. In some embodiments, the constriction used in the production methods described herein has a width of about 4 μm, 3.5 μm, about 3 μm, about 2.5 μm, about 2 μm, about 1.5 μm, about 1 μm, about 0.5 μm, or about 0.25 μm. In some embodiments, the constriction used in the production methods described herein has a width of about 2.2 μm. In some embodiments, the input parent anucleated cells used in the production methods described herein are passed through the constriction under pressure ranging from about 10 psi to about 150 psi. In some embodiments, the cell suspension used in the production methods described herein is contacted with a payload before, simultaneously with, or after passing through the constriction, such that the payload enters the cells.

[0094] In some embodiments, the payload used in the methods described herein is a therapeutic payload. In some embodiments, the payload is a polypeptide, a nucleic acid, a lipid, a carbohydrate, a small molecule, a complex, or a nanoparticle. In some embodiments, the payload is an antigen and / or an adjuvant. In some embodiments, the payload is an antigen and / or an immune tolerogenic factor.

[0095] In another aspect, the present disclosure provides a method for treating a disease or disorder in an individual in need thereof, the method comprising administering an anuclear cell-derived vesicle described herein. In another aspect, the present disclosure provides a method for treating a disease or disorder in an individual in need thereof, the method comprising administering a composition described herein. In some embodiments, the anuclear cell-derived vesicle used in the treatment methods described herein comprises a therapeutic payload. In some embodiments, the individual has cancer and the payload comprises an antigen. In some embodiments, the individual has cancer and the payload comprises an antigen and an adjuvant. In some embodiments, the antigen is a tumor antigen. In some embodiments, the individual has an infectious disease or a virus-related disease and the payload comprises an antigen. In some embodiments, the individual has an infectious disease or a virus-related disease and the payload comprises an antigen and an adjuvant. In some embodiments, the antigen is a viral antigen, a bacterial antigen, or a fungal antigen. In some embodiments, the individual has an autoimmune disease and the payload comprises an antigen. In some embodiments, the individual has an autoimmune disease and the payload comprises an antigen and / or a tolerogenic factor.

[0096] In another aspect, the present disclosure provides a method for preventing a disease or disorder in an individual in need thereof, the method comprising administering an anuclear cell-derived vesicle described herein. In another aspect, the present disclosure provides a method for preventing a disease or disorder in an individual in need thereof, the method comprising administering a composition described herein. In some embodiments, the anuclear cell-derived vesicle used in the prevention methods described herein comprises an antigen. In some embodiments, the individual has cancer and the payload comprises an antigen and an adjuvant. In some embodiments, the disease or disorder is cancer and the antigen is a tumor antigen. In some embodiments, the individual has an infectious disease and the payload comprises an antigen. In some embodiments, the individual has an infectious disease and the payload comprises an antigen and an adjuvant. In some embodiments, the antigen is a viral antigen, a bacterial antigen, or a fungal antigen. [Brief explanation of the drawings]

[0097] [Figure 1-1]Figure 1A shows the percentage of antigen-specific T cells measured by tetramer staining for each condition. Figure 1B shows the percentage of IFN-γ-positive cells measured by intracellular cytokine staining (ICS) for each condition after restimulation with SIINFEKL, an OVA epitope (circular dots). Stimulation with anti-CD28 alone (without SIINFEKL) was used as a negative control (square dots), while nonspecific stimulation with PMA / ionomycin was used as a positive control (triangle dots). Figure 1C shows the amount of IFN-γ in each cell measured by its mean fluorescence intensity (MFI) in ICS for each condition. Figure 1D shows the percentage of IL-2-positive cells measured by intracellular cytokine staining (ICS) for each condition after restimulation with SIINFEKL, an OVA epitope (circular dots). Stimulation with anti-CD28 alone (without SIINFEKL) was used as a negative control (square dots), while nonspecific stimulation with PMA / ionomycin was used as a positive control (triangle dots). Figure 1E shows the amount of IL-2 in each cell, as measured by the mean fluorescence intensity (MFI) of each cell, in ICS for each condition. [Figure 1-2] Same as above. [Figure 1-3] Same as above.

[0098] [Figure 2-1] Figure 2A shows the percentage of antigen-specific T cells measured by tetramer staining for each condition. Figure 2B shows the percentage of IFN-γ-positive cells measured by intracellular cytokine staining (ICS) for each condition after restimulation with the OVA epitope SIINFEKL (circle dots). Figure 2C shows the percentage of IL-2-positive cells measured by intracellular cytokine staining (ICS) for each condition after restimulation with the OVA epitope SIINFEKL (circle dots). In both Figures 2B and 2C, stimulation with anti-CD28 alone (without SIINFEKL) is used as a negative control (square dots), while nonspecific stimulation with PMA / ionomycin is used as a positive control (triangle dots). [Figure 2-2] Same as above.

[0099] [Figure 3-1] Figure 3A shows the percentage of antigen-specific T cells measured by tetramer staining for each condition. Figure 3B shows the percentage of IFN-γ-positive cells measured by intracellular cytokine staining (ICS) for each condition after restimulation with the OVA epitope SIINFEKL (circle dots). Figure 3C shows the percentage of IL-2-positive cells measured by intracellular cytokine staining (ICS) for each condition after restimulation with the OVA epitope SIINFEKL (circle dots). In both Figures 3B and 3C, stimulation with anti-CD28 alone (without SIINFEKL) is used as a negative control (square dots), while nonspecific stimulation with PMA / ionomycin is used as a positive control (triangle dots). [Figure 3-2] Same as above.

[0100] [Figure 4] FIG. 4 shows the lactate levels of red blood cell-derived vesicles (SQZ) processed by constriction-mediated delivery relative to unprocessed input red blood cells.

[0101] [Figure 5] Figure 5A shows images from brightfield, CellTrace Violet staining (CT), and D-FITC fluorescence microscopy of untreated RBCs (Untrtd), RBCs incubated with FITC-labeled dextran (D-FITC) (without SQZ), and RBC-derived vesicles containing D-FITC loaded with SQZ (SQZ). Figure 5B shows the levels of phosphatidylserine staining for untreated RBCs (Untrtd), RBCs incubated with D-FITC (without SQZ), and RBC-derived vesicles containing D-FITC loaded with SQZ (SQZ).

[0102] [Figure 6-1] Figure 6A shows a representative schematic of an experiment to determine the circulating half-life of anucleated cell-derived vesicles generated by SQZ treatment. Figure 6B shows the circulating levels of separately labeled RBCs and SQZ-loaded RBC-derived vesicles over time. Figure 6C shows forward and side scatter in a flow plot of a mixture of RBCs and SQZ-loaded RBC-derived vesicles injected into a mouse. [Figure 6-2] Same as above.

[0103] [Figure 7] Figure 7A shows the appearance of the cell pellet and supernatant after centrifugation of vesicles derived from untreated RBCs (NC) and RBCs treated with SQZ at pressures of 10 and 12 psi, respectively. Figure 7B shows the hemoglobin loss (hemolysis) measured with the HemoCue® System for untreated RBCs (NC), vesicles derived from RBCs treated with SQZ at pressures of 10 and 12 psi, and RBCs diluted in water (lysis control).

[0104] [Figure 8] Figures 8A and 8B show hemoglobin loss (hemolysis) quantified by liquid chromatography / mass spectrometry of two hemoglobin peptides in RBCs incubated with B9-23 (Endo control) and RBC-derived vesicles loaded with B9-23 and SQZ (SQZ), respectively.

[0105] [Figure 9] FIG. 9 shows the percentage of ghost formation in SQZ-mediated induction of RBC-derived vesicles under various constriction widths and driving pressures during SQZ treatment.

[0106] [Figure 10] FIG. 10 shows the in vivo persistence of untreated mouse RBCs and SQZ-treated mouse RBC vesicles in recipient mice.

[0107] [Figure 11] Figure 11A shows the organs involved in the internalization of vesicles derived from SQZ-treated RBCs, and Figure 11B shows the cell types in the liver and spleen involved in the internalization of vesicles derived from SQZ-treated RBCs.

[0108] [Figure 12] Figure 12A shows the expansion of OVA-specific CD4+ T cell proliferation induced by RBC-derived vesicles loaded with OVA and poly I:C in SQZ, and Figure 12B shows the expansion of OVA-specific CD8+ T cell proliferation induced by RBC-derived vesicles loaded with OVA and poly I:C in SQZ.

[0109] [Figure 13] Figure 13 shows endogenous CD8+ T cell responses upon ex vivo re-simulation with SIINFEKL for mice induced with (i) Poly I:C only, (ii) OVA only, or (iii) OVA and Poly I:C SQZ-loaded RBC-derived vesicles.

[0110] [Figure 14] Figure 14 shows endogenous CD8+ T cell responses upon ex vivo restimulation with E7 for mice induced with (i) poly I:C only, (ii) E7 only, or (iii) E7 and poly I:C SQZ-loaded RBC-derived vesicles.

[0111] [Figure 15] FIG. 15 shows quantification of E7-specific CD8+ T cells for mice treated with various priming and boosting dosing regimens of E7 and poly I:C SQZ-loaded RBC-derived vesicles.

[0112] [Figure 16]Figures 16A and 16B show the effect of prophylactic administration of E7 and poly I:C SQZ-loaded RBC-derived vesicles on tumor growth and survival, respectively, in a mouse model bearing E7-positive tumors.

[0113] [Figure 17] Figures 17A and 17B show the effect of therapeutic administration of RBC-derived vesicles SQZ-loaded with E7 and poly I:C at various dosages on tumor growth and survival, respectively, in a mouse model bearing E7-positive tumors.

[0114] [Figure 18] Figures 18A and 18B show the effect of therapeutic administration of E7 and poly I:C SQZ-loaded RBC-derived vesicles at various administration regimens on tumor growth and survival, respectively, in a mouse model bearing E7-positive tumors.

[0115] [Figure 19-1] Figures 19A-19D show the antigen-specific immune response induced by RBC-derived vesicles SQZ-loaded with E7 and poly I:C when administered to a mouse model bearing E7-positive tumors, specifically, the recruitment of CD8+ T cells to E7-positive tumors (Figure 19A), the percentage of intratumoral CD8+ T cells that are specific for E7 (Figure 19B), the ratio of E7-specific CD8+ T cells to regulatory T cells in the tumor (Figure 19C), and the correlation of E7-specific CD8+ T cells with tumor weight (Figure 19D). [Figure 19-2] Same as above.

[0116] [Figure 20-1] Figures 20A-20C show ghost formation, payload delivery efficiency, and surface phosphatidylserine levels, respectively, when human RBC-derived vesicles were generated by SQZ treatment in the presence of E7-SLP (payload). [Figure 20-2] Same as above.

[0117] [Figure 21] FIG. 21 shows the internalization of human RBC-derived vesicles by human monocyte-derived dendritic cells at 37° C. and 0° C.

[0118] [Figure 22] FIG. 22 shows the production and secretion of IFN-γ by CMV antigen-specific CD8+ T cells when co-cultured with CMV antigen-loaded human RBC-derived vesicles and exogenous adjuvant.

[0119] [Figure 23-1] Figures 23A-23C show the efficiency of payload delivery, ghost formation, and surface phosphatidylserine levels in ghost and non-ghost populations, respectively, when mouse RBC-derived vesicles were generated by SQZ treatment. [Figure 23-2] Same as above.

[0120] [Figure 24] Figure 24A shows a representative schematic of an experiment to determine whether antigen-dependent tolerance to viral capsids in vivo is induced by anuclear cell-derived vesicles containing SQZ-loaded antigen. Figure 24B shows the percentage of IFN-γ-positive cells measured by intracellular cytokine staining (ICS) in splenocytes from naive mice, mice treated with RBCs incubated with SNYNKSVNV (peptide), or mice treated with RBC-derived vesicles loaded with SNYNKSVNV (SQZ). Figure 24C shows luciferase levels in serum for mice in the peptide and SQZ groups over a 43-day period.

[0121] [Figure 25]Figure 25A shows a representative schematic of an experiment to determine whether antigen-dependent tolerance to antibodies in vivo is induced by anuclear cell-derived vesicles containing SQZ-loaded antigen. Figure 25B shows the levels of circulating rat IgG2b in the serum of control mice, mice injected with free rat IgG2b, and mice injected with RBC-derived vesicles SQZ-loaded with rat IgG2b (SQZ) on day 20, as determined by ELISA. Figure 25C shows the levels of circulating rat IgG2b in the serum of mice in the control, free rat IgG2b, and SQZ groups on day 76.

[0122] [Figure 26-1] Figure 26A shows a representative schematic diagram of an experiment to determine whether in vivo antigen-dependent tolerance to B9-23 is induced by SQZ-loaded antigen-containing anuclear cell-derived vesicles. Figure 26B shows the percentage of IFN-γ or IL-2-positive cells measured by intracellular cytokine staining (ICS) in splenocytes from control mice, mice treated with HEL-loaded RBC-derived vesicles (SQZ HEL), or mice treated with Ins B9-23-loaded RBC-derived vesicles (SQZ FAM) after restimulation with AAV-NL virus. Figure 26C shows a representative schematic diagram of an experiment to determine whether in vivo antigen-dependent tolerance to 1040-p31 is induced by SQZ-loaded antigen-containing anuclear cell-derived vesicles. Figure 26D shows the serum blood glucose levels measured in control mice and mice treated with 1040-31-loaded RBC-derived vesicles (SQZ). FIG. 26E shows the onset of disease for control and SQZ mice as determined from serum blood glucose measurements. [Figure 26-2] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0123] The present application provides anuclear cells, including anuclear cell-derived vesicles (such as vesicles prepared from input anuclear cells), and compositions thereof, in which the anuclear cells and / or anuclear cell-derived vesicles are loaded with and / or mixed with one or more of an antigen, an adjuvant, or a therapeutic agent. The present application also provides methods for producing anuclear cell-derived vesicles by constriction-mediated delivery (SQZ) as described herein, and methods of using the same. The present application further provides methods for stimulating an immune response in an individual and treating and / or preventing disease using anuclear cell-derived vesicles produced by constriction-mediated delivery (SQZ) as described herein.

[0124] The present disclosure is based, at least in part, on the discovery that input anuclear cells can be processed by constriction-mediated delivery (SQZ) to generate anuclear cell-derived vesicles. The present disclosure is also based, at least in part, on the discovery that anuclear cell-derived vesicles bearing antigen(s) and / or adjuvant(s) (whether encapsulated within the anuclear cell-derived vesicles) can induce an antigen-specific immune response in vivo.

[0125] The present invention provides a method for delivering antigens and / or adjuvants into anuclear cell-derived vesicles, the method comprising the steps of: a) passing a cell suspension containing input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigen and / or adjuvant, to form anuclear cell-derived vesicles; and b) incubating the anuclear cell-derived vesicles with the antigen and / or adjuvant for a time sufficient to allow entry of the antigen and / or adjuvant into the anuclear cell-derived vesicles.

[0126] Certain aspects of the present disclosure relate to methods for stimulating an immune response to an antigen in an individual, the method comprising administering to the individual an effective amount of anuclear cell-derived vesicles comprising an antigen, wherein the anuclear cell-derived vesicles comprising the antigen are prepared by a process comprising: (a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigen and forming anuclear cell-derived vesicles; and (b) incubating the disrupted input anuclear cells with the antigen for a time sufficient to allow entry of the antigen into the anuclear cell-derived vesicles. In some embodiments, an adjuvant is also delivered in the anuclear cell-derived vesicles. In other embodiments, the adjuvant is administered systemically to the individual in combination with the anuclear cell-derived vesicles comprising the antigen.

[0127] In certain aspects, the present invention provides anuclear cell-derived vesicles comprising antigens and / or adjuvants, prepared by a process comprising the steps of: a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigens and / or adjuvants and form anuclear cell-derived vesicles; and b) incubating the anuclear cell-derived vesicles with the antigens and / or adjuvants for a time sufficient to allow entry of the antigens and / or adjuvants into the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles comprising the antigens and / or adjuvants.

[0128] In certain aspects, the present invention provides a method for generating anuclear cell-derived vesicles containing antigen and / or adjuvant, comprising the steps of: a) passing a cell suspension containing input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigen and / or adjuvant and forming anuclear cell-derived vesicles; and b) incubating the anuclear cell-derived vesicles with the antigen and / or adjuvant for a time sufficient to allow entry of the antigen and / or adjuvant into the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles containing the antigen and / or adjuvant.

[0129] In some aspects, the present application provides anuclear cell-derived vesicles (e.g., vesicles prepared from parent anuclear cells) and compositions thereof, in which the anuclear cell-derived vesicles are loaded with a payload, such as any one or more of an antigen, an adjuvant, or a tolerogenic factor. The present application also provides methods of making and using the anuclear cell-derived vesicle compositions described herein.

[0130] The present disclosure is also based, at least in part, on the discovery that compositions comprising anuclear cell-derived vesicles containing a payload, such as antigen(s) and / or adjuvant(s), can induce an in vivo antigen-specific immune response. The present disclosure is also based, at least in part, on the discovery that higher doses of compositions comprising anuclear cell-derived vesicles loaded with antigen(s) and / or adjuvant(s) can induce greater in vivo antigen-specific immune responses. Furthermore, the present disclosure is based, at least in part, on the discovery that in vivo antigen-specific immune responses can be modulated based on the adjuvant of the composition; the amount of payload, such as antigen, encapsulated in the anuclear cell-derived vesicles; and / or the dosing strategy used to administer the composition comprising the anuclear cell-derived vesicles. The present disclosure is also based, at least in part, on the discovery that compositions comprising a plurality of anuclear cell-derived vesicles can be actively tailored to generate anuclear cell-derived vesicles, such as a population of anuclear cell-derived vesicles, within the composition with one or more selected properties. Producing a composition of anuclear cell-derived vesicles having a desired amount and / or characteristics of anuclear cell-derived vesicles therein can be achieved, for example, by adjusting one or more of the preparation parameters when the anuclear cell-derived vesicles are prepared from the parent anuclear cells.

[0131] Thus, in some aspects, provided herein are anuclear cell-derived vesicles prepared from parent anuclear cells, which have one or more of the following characteristics: (a) a reduced circulating half-life in mammals compared to the parent anuclear cells, (b) a reduced hemoglobin level compared to the parent anuclear cells, (c) a spherical morphology, (d) an increased surface phosphatidylserine level compared to the parent anuclear cells, or (e) reduced ATP production compared to the parent anuclear cells.

[0132] In another aspect, the present invention provides a composition comprising a plurality of vesicles derived from any of the anuclear cells described herein.In some embodiments, the composition has one or more of the following characteristics: (a) more than 20% of the anuclear cell-derived vesicles in the composition have a reduced circulatory half-life in mammals compared with parent anuclear cells; (b) more than 20% of the anuclear cell-derived vesicles in the composition have a reduced hemoglobin level compared with parent anuclear cells; (c) more than 20% of the anuclear cell-derived vesicles in the composition have a spherical morphology; (d) more than 20% of the anuclear cell-derived vesicles in the composition are RBC ghosts; (e) more than 20% of the anuclear cell-derived vesicles in the composition have a higher level of phosphatidylserine; or (f) more than 20% of the anuclear cell-derived vesicles in the composition have a reduced ATP production compared with parent anuclear cells.

[0133] In another aspect, provided herein is a composition comprising a plurality of any anuclear cell as described herein in admixture with an adjuvant.

[0134] In another aspect, a method of making a composition disclosed herein, e.g., a method for making a composition comprising a plurality of anuclear cell-derived vesicles prepared from parent anuclear cells, wherein the composition has any of the following properties: (a) more than 20% of the anuclear cell-derived vesicles in the composition have a reduced circulating half-life in a mammal compared to the parent anuclear cells, (b) more than 20% of the anuclear cell-derived vesicles in the composition have a reduced hemoglobin level compared to the parent anuclear cells, (c) more than 20% of the anuclear cell-derived vesicles in the composition have a spherical morphology, (d) more than 20% of the anuclear cell-derived vesicles in the composition are RBC ghosts, (e Provided herein are methods for producing anuclear cell-derived vesicles, the methods comprising: (a) more than 20% of the anuclear cell-derived vesicles in the composition have a higher level of phosphatidylserine; or (b) more than 20% of the anuclear cell-derived vesicles in the composition have reduced ATP production compared to the parent anuclear cells; and the methods comprise passing a cell suspension comprising the parent anuclear cells through a constriction that deforms the cells, wherein the diameter of the constriction is a function of the diameter of the parent anuclear cells in the suspension, thereby causing disruption of the parent anuclear cells large enough to allow passage of a payload to form anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles.

[0135] In another aspect, provided herein are methods for using any of the compositions described herein. In some embodiments, the method for use is a method for treating a disease or disorder in an individual in need thereof, comprising administering any of the anuclear cell-derived vesicles described herein. In some embodiments, the method for use is a method for preventing a disease or disorder in an individual in need thereof, comprising administering any of the anuclear cell-derived vesicles described herein. definition

[0136] For purposes of interpreting this specification, the following definitions shall apply, and whenever appropriate, terms used in the singular shall include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the set forth definition shall control.

[0137] As used herein, the singular forms "a," "an," and "the" include plural references unless otherwise indicated.

[0138] The terms "comprising," "having," "containing," and "including," and other similar forms and their grammatical equivalents, as used herein, are intended to be equivalent in meaning and open-ended in that the item(s) preceding any one of these words are not meant to be an inclusive list of such item(s) or to be limited only to the listed item(s). For example, an article "comprising" components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C, but also one or more other components. As such, it is intended and understood that "comprises" and similar forms, and their grammatical equivalents, include disclosure of "consisting essentially of" or "consisting of" embodiments.

[0139] Where a range of values ​​is presented, it is understood that each intervening value between the upper and lower limit of that range, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0140] The term "about," as used herein, refers to a normal error range for each value, which is readily known to those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments that are directed to the value or parameter itself. For example, a description that refers to "about X" includes a description of "X."

[0141] As used herein, "anuclear cells" refer to cells lacking a nucleus. Such cells include, but are not limited to, red blood cells (RBCs), such as platelets, red blood cells, and reticulocytes. Reticulocytes are immature (e.g., not yet biconcave) red blood cells that typically constitute approximately 1% of the red blood cells in the human body. Reticulocytes are also anuclear. In certain embodiments, the systems and methods described herein are used to treat and / or process enriched (e.g., constituting a greater percentage of the total cell population than can be found in nature), purified, or isolated (e.g., in substantially pure or homogeneous form from its natural environment) populations of anuclear cells (e.g., RBCs, reticulocytes, and / or platelets). In certain embodiments, the systems and methods described herein are used to treat and / or process whole blood containing RBCs (e.g., red blood cells or reticulocytes), platelets, and other blood cells. Purification or enrichment of these cell types is achieved using known methods such as density gradient systems (e.g., Ficoll-Hypaque), fluorescence activated cell sorting (FACS), magnetic cell sorting, or in vitro differentiation of erythroblasts and erythroid precursors.

[0142] The term "vesicle," as used herein, refers to a structure containing a liquid enclosed by a lipid bilayer. In some examples, the lipid bilayer is derived from a naturally occurring lipid composition. In some examples, the lipid bilayer can be derived from a cell membrane. In some examples, vesicles can be derived from various types of entities, such as cells. In such examples, vesicles can retain molecules (such as intracellular proteins or membrane components) from the entity of origin. For example, vesicles derived from red blood cells can contain any number of intracellular proteins that were present in the red blood cells and / or membrane components of the red blood cells. In some examples, vesicles can contain any number of intracellular molecules in addition to the desired payload.

[0143] As used herein, "payload" refers to the material being delivered, such as loaded into anuclear cell-derived vesicles (e.g., RBC-derived vesicles). "Payload," "cargo," "delivery material," and "compound" are used interchangeably herein. In some embodiments, payload can refer to proteins, small molecules, nucleic acids (e.g., RNA and / or DNA), lipids, carbohydrates, macromolecules, vitamins, polymers, fluorescent dyes and fluorophores, carbon nanotubes, quantum dots, nanoparticles, and steroids. In some embodiments, payload can refer to proteins or small molecule drugs. In some embodiments, payload can include one or more compounds.

[0144] The term "pore," as used herein, refers to an opening, including, without limitation, a hole, crevice, cavity, opening, crevice, gap, or perforation in a material. In some instances (where indicated), the term refers to a pore in a surface of the present disclosure. In other instances (where indicated), the pore can refer to a pore in a cell membrane.

[0145] The term "membrane," as used herein, refers to a selective barrier or sheet containing pores. This term includes flexible sheet-like structures that act as boundaries or backings. In some instances, this term refers to a surface or filter containing pores. This term is distinct from the term "cell membrane."

[0146] The term "filter," as used herein, refers to a porous article that allows selective passage of pores. In some instances, the term refers to a surface or membrane that contains pores.

[0147] The term "heterologous," when referring to nucleic acid sequences, such as coding and regulatory sequences, refers to sequences that are not normally linked and / or not normally associated with a particular cell. Thus, a "heterologous" region of a nucleic acid construct or vector is a segment of nucleic acid present within or attached to another nucleic acid molecule that is not found in association with that other molecule in nature. For example, a heterologous region of a nucleic acid construct can include a coding sequence adjacent to sequences not found in association with the coding sequence in nature. Another example of a heterologous coding sequence is a construct in which the coding sequence itself is not found in nature (e.g., a synthetic sequence with codons different from the native gene). Similarly, a cell transformed with a construct that is not normally present in the cell would be considered heterologous for purposes of the present invention. Allelic variants or naturally occurring mutational events do not give rise to heterologous DNA as used herein.

[0148] The term "heterologous," when referring to amino acid sequences, such as peptide and polypeptide sequences, refers to sequences that are not normally spliced ​​and / or not normally associated with a particular cell. Thus, a "heterologous" region of a peptide sequence is a segment of amino acids present within or attached to another amino acid molecule that is not found in association with that other molecule in nature. For example, a heterologous region of a peptide construct can include the amino acid sequence of the peptide adjacent to sequences not found in association with the amino acid sequence of the peptide in nature. Another example of a heterologous peptide sequence is a construct in which the peptide sequence itself is not found in nature (e.g., a synthetic sequence that, when encoded, has amino acids different from those in the native gene). Similarly, a cell transformed with a vector expressing an amino acid construct that is not normally present in the cell would be considered heterologous for purposes of the present invention. Allelic variants or naturally occurring mutational events do not result in a heterologous peptide as used herein.

[0149] The term "exogenous," when used with reference to an agent such as an antigen or adjuvant in relation to a cell, refers to an agent delivered from the extracellular space (i.e., from outside the cell). The cell may or may not already have the agent present, and may or may not produce the agent after the exogenous agent is delivered.

[0150] The term "homologous," as used herein, refers to molecules that are derived from the same organism. In some instances, the term refers to nucleic acids or proteins that are normally found or expressed in a given organism.

[0151] As used herein, "treatment" or "treating" is an approach to obtaining beneficial or desired results, including clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviation of one or more symptoms attributable to the disease, reduction in the extent of the disease, stabilization of the disease (e.g., preventing or slowing the progression of the disease), preventing or slowing the spread of the disease (e.g., metastasis), preventing or slowing the recurrence of the disease, slowing or slowing the progression of the disease, remission of the disease (partial or total), reducing the dose of one or more other medications required to treat the disease, slowing the progression of the disease, increasing or improving quality of life, increasing weight gain, and / or prolonging survival. By "treatment," reduction of the pathological consequences of cancer (e.g., tumor volume, etc.) is also encompassed. The methods of the present invention contemplate any one or more of these aspects of treatment.

[0152] As used herein, the term "modulate" can refer to the act of changing, altering, varying, or otherwise modifying the presence or activity of a particular target. For example, modulating an immune response can refer to any act that results in a change, alteration, variation, or otherwise modification of the immune response. In another example, modulating the expression of a nucleic acid can include, but is not limited to, changes in the transcription of the nucleic acid, changes in mRNA abundance (e.g., increased mRNA transcription), corresponding changes in mRNA degradation, changes in mRNA translation, etc.

[0153] As used herein, the term "inhibit" can refer to the act of blocking, reducing, eliminating, or otherwise antagonizing the presence or activity of a particular target. Inhibition can refer to partial or complete inhibition. For example, inhibition of an immune response can refer to any act that blocks, reduces, eliminates, or otherwise antagonizes an immune response. In other examples, inhibition of nucleic acid expression can include, but is not limited to, reducing nucleic acid transcription, reducing mRNA abundance (e.g., silencing mRNA transcription), mRNA degradation, inhibiting mRNA translation, gene editing, etc. In other examples, inhibition of protein expression can include, but is not limited to, reducing transcription of a nucleic acid encoding the protein, reducing the stability of an mRNA encoding the protein, inhibiting protein translation, reducing protein stability, etc.

[0154] As used herein, the term "inhibit" can refer to the act of decreasing, reducing, inhibiting, limiting, lowering, or otherwise diminishing the presence or activity of a particular target. Inhibition can refer to partial or complete inhibition. For example, inhibition of an immune response can refer to any act that results in a decrease, reduction, inhibition, limiting, lowering, or otherwise diminishing of the immune response. In another example, inhibition of nucleic acid expression can include, but is not limited to, reducing nucleic acid transcription, reducing mRNA abundance (e.g., silencing mRNA transcription), mRNA degradation, inhibiting mRNA translation, and the like. In another example, inhibition of protein expression can include, but is not limited to, reducing transcription of a nucleic acid encoding the protein, reducing the stability of an mRNA encoding the protein, inhibiting protein translation, reducing protein stability, and the like.

[0155] As used herein, the term "enhance" can refer to the act of improving, boosting, enhancing, or otherwise increasing the presence or activity of a particular target. For example, enhancing an immune response can refer to any act that results in improving, boosting, enhancing, or otherwise increasing an immune response. In another example, enhancing expression of a nucleic acid can include, but is not limited to, increasing transcription of the nucleic acid, increasing mRNA abundance (e.g., increasing mRNA transcription), decreasing mRNA degradation, increasing mRNA translation, etc. In another example, enhancing expression of a protein can include, but is not limited to, increasing transcription of a nucleic acid encoding the protein, increasing the stability of an mRNA encoding the protein, increasing protein translation, increasing protein stability, etc.

[0156] As used herein, the term "induce" can refer to the act of initiating, promoting, stimulating, establishing, or otherwise producing a result. For example, inducing an immune response can refer to any act that results in the initiation, promotion, stimulation, establishment, or otherwise producing a desired immune response. In another example, inducing expression of a nucleic acid can include, but is not limited to, initiating transcription of a nucleic acid, initiating mRNA translation, etc. In another example, inducing expression of a protein can include, but is not limited to, increasing transcription of a nucleic acid encoding the protein, increasing the stability of an mRNA encoding the protein, increasing translation of the protein, increasing protein stability, etc.

[0157] The terms "polynucleotide" or "nucleic acid," as used herein, refer to polymeric forms of nucleotides of any length, including ribonucleotides and deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of a polynucleotide can contain sugars and phosphate groups (as typically found in RNA or DNA), or modified or substituted sugar or phosphate groups. The backbone of a polynucleotide can contain repeating units, such as N-(2-aminoethyl)-glycine, linked by peptide bonds (i.e., peptide nucleic acid). Alternatively, the backbone of a polynucleotide can contain polymers of synthetic subunits, such as phosphoramidates, and thus can be oligodeoxynucleoside phosphoramidates (P-NH2) or mixed phosphoramidate-phosphodiester oligomers. Additionally, double-stranded polynucleotides can be obtained from chemically synthesized single-stranded polynucleotide products by either synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using DNA polymerase and appropriate primers.

[0158] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Thus, as used herein, polypeptide includes short peptides. Such polymers of amino acid residues can contain natural or non-natural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by this definition. These terms also include post-translational modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and others. Furthermore, for purposes of the present invention, "polypeptide" refers to proteins containing modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, so long as the protein maintains the desired activity. These modifications may be deliberate, such as by site-directed mutagenesis, or may be accidental, such as by mutations of hosts producing the protein or errors due to PCR amplification.

[0159] As used herein, the term "adjuvant" refers to a substance that modulates and / or generates an immune response. Generally, an adjuvant is administered in conjunction with an antigen to result in an enhanced immune response to the antigen compared to the antigen alone. Various adjuvants are described herein.

[0160] The terms "CpG oligodeoxynucleotide" and "CpG ODN" refer to a DNA molecule containing a dinucleotide of cytosine and guanine separated by a phosphate (also referred to herein as a "CpG" dinucleotide or "CpG"). ODN contains at least one unmethylated CpG dinucleotide. That is, the cytosine in the CpG dinucleotide is not methylated (i.e., not 5-methylcytosine). CpG ODN can have a partial or complete phosphorothioate (PS) backbone.

[0161] As used herein, "pharmaceutically acceptable" or "pharmacologically compatible" means a material that is not biologically or otherwise undesirable, e.g., the material can be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. A pharmaceutically acceptable carrier or excipient preferably meets the required standards of toxicological and manufacturing testing and / or is included in the Inactive Ingredient Guide prepared by the US Food and Drug Administration.

[0162] For any of the structural and functional characteristics described herein, methods for determining these characteristics are known in the art.

[0163] As used herein, a "microfluidic system" refers to a system in which small volumes (e.g., m\L, nL, pL, fL) of fluid are processed to achieve discrete treatment of small volumes of liquid. Certain implementations described herein include multiplexing, automation, and high-throughput screening. Fluids (e.g., buffers, solutions, payload-containing solutions, or cell suspensions) can be moved, mixed, separated, or otherwise processed. In certain embodiments described herein, a microfluidic system is used to apply a mechanical constriction to cells suspended in a buffer to induce a perturbation in the cells (e.g., a pore) that allows a payload or compound to enter the cytosol of the cells.

[0164] As used herein, a "constriction" can refer to a portion of a microfluidic channel defined by an entry portion, a center point, and an exit portion, with the center point defined by a width, length, and depth. In other examples, a constriction can refer to a pore or be part of a pore. A pore can be contained in a surface (e.g., a filter and / or membrane).

[0165] As used herein, "width of a constriction" refers to the width of a microfluidic channel at its center point. In some embodiments, the constriction has a width of less than about 6 μm. For example, in some embodiments, the constriction can be less than about 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1.0 μm, 1.5 μm, or 2 μm. In some embodiments, the constriction has a width of less than about 4 μm. In certain aspects of the invention, the constriction has a width between about 0.5 μm and about 4 μm. In further embodiments, the constriction has a width between about 3 μm and about 4 μm. In further embodiments, the constriction has a width between about 2 μm and about 4 μm. In further aspects, the constriction has a width of about 3.9 μm or less. In further aspects, the constriction has a width of about 3.9 μm or less. In further aspects, the constriction has a width of about 2.2 μm. In certain embodiments, the constriction is configured to allow a single cell to pass through the constriction at a time.

[0166] As used herein, "constriction length" refers to the length of a microfluidic channel at its center point. In certain aspects of the invention, the constriction length is about 30 μm or less. In some embodiments, the constriction length is between about 10 μm and about 30 μm. In certain embodiments, the constriction length is between about 10 μm and about 20 μm. For example, the constriction length can be any of about 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 20 μm, or 25 μm, including all integers, decimals, and fractions between about 10 μm and about 30 μm. The constriction length can be varied to increase the length of time a cell resides within the constriction (e.g., a longer length results in a longer constriction time at a given flow rate). The constriction length can be varied to decrease the length of time a cell resides within the constriction (e.g., a shorter length results in a shorter constriction time at a given flow rate).

[0167] As used herein, "constriction depth" refers to the depth of the microfluidic channel at the center point. Similar to adjusting the constriction width, the constriction depth can be adjusted to create a tighter constriction, thereby enhancing delivery. In some embodiments, the constriction depth is between about 1 μm and about 1 mm, including all integers, decimals, and fractions between about 1 μm and about 1 mm. In some embodiments, the depth is about 20 μm. In some embodiments, the depth is uniform throughout the channel. In certain embodiments, the depth decreases at the point of the constriction, resulting in greater constriction of the cells. In some embodiments, the depth increases at the point of the constriction, resulting in less constriction of the cells. In some embodiments, the constriction depth is greater than the constriction width. In certain embodiments, the constriction depth is less than the constriction width. In some embodiments, the constriction depth and the constriction width are equal.

[0168] In some embodiments, the dimensions of a microfluidic device are designated by the length of the constriction, the width of the constriction, and the number of constrictions in series. For example, a microfluidic device having a constriction length of 30 μm, a width of 5 μm, and five constrictions in series is designated herein as 30×5×5 (L×W×number of constrictions).

[0169] In some embodiments, a microfluidic system includes at least one microfluidic channel containing at least one constriction. In some embodiments, a microfluidic system includes multiple microfluidic channels, each containing at least one constriction. For example, a microfluidic system can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 500, 1000, 10,000, 20,000, or more microfluidic channels, including 10-50, 50-100, 100-500, 500, 1000, 10,000-20,000, or any other integer. In certain aspects, multiple microfluidic channels, each containing a single constriction, are arranged in parallel. In certain aspects, multiple microfluidic channels, each containing a single constriction, are arranged linearly in series. In certain aspects of the invention, a microfluidic system includes a single microfluidic channel containing multiple constrictions. For example, a single microfluidic channel may contain 2, 3, 4, 5, 10, 20 or more constrictions. In some embodiments, a microfluidic system comprises multiple microfluidic channels containing multiple constrictions. In some aspects of the invention, multiple microfluidic channels containing multiple constrictions are arranged in parallel. In some aspects of the invention, multiple microfluidic channels containing multiple constrictions are arranged in linear series.

[0170] The inlet section can include a "constriction angle," which can be varied to increase or decrease the rate at which the channel diameter decreases toward the center point of the constriction. The constriction angle can be varied to minimize clogging of the microfluidic system while allowing cells to pass therethrough. For example, the constriction angle can be between 1 and 140 degrees. In certain embodiments, the constriction angle can be between 1 and 90 degrees. The outlet section can also include an angle that reduces the likelihood of turbulence / vortices that can result in non-laminar flow. For example, the angle of the outlet section can be between 1 and 140 degrees. In certain embodiments, the angle of the outlet section can be between 1 and 90 degrees.

[0171] The cross-sections of the microfluidic channel, inlet section, center point, and outlet section can vary. Non-limiting examples of various cross-sections include circular, oval, elongated slit, square, hexagonal, or triangular cross-sections.

[0172] The velocity of non-nuclear cells (e.g., RBCs) through the microfluidic channels described herein can also be varied to control the delivery of a delivery material to the cells. For example, adjusting the velocity of a cell through a microfluidic channel can vary the amount of time a deforming force is applied to the cell and can vary the rapidity with which the deforming force is applied to the cell. In some embodiments, adjusting the velocity of a cell through a microfluidic channel can vary the amount of time a pressure is applied to the cell and can vary the rapidity with which the pressure is applied to the cell. In some embodiments, cells can be moved through the microfluidic system at a speed of at least 0.1 mm / sec. In further embodiments, cells can be moved through the microfluidic system at a speed between 0.1 mm / sec and 5 m / sec, including all integers and decimals therein. In still further embodiments, cells can be moved through the microfluidic system at a speed between 10 mm / sec and 500 mm / sec, including all integers and decimals therein. In some embodiments, cells can be moved through the system at a speed greater than 5 m / sec.

[0173] Cells are moved (e.g., pushed) through the constriction by applying pressure. In some embodiments, the pressure is applied by a cell driver. As used herein, a cell driver is a device or component that applies pressure or force to a buffer or solution to drive cells through the constriction. In some embodiments, pressure can be applied by the cell driver at the inlet. In some embodiments, vacuum pressure can be applied by the cell driver at the outlet. In certain embodiments, the cell driver is adapted to provide a pressure of about 10 to about 150 psi, such as about 10 to about 90 psi. In further embodiments, the cell driver is adapted to apply a pressure of 120 psi. In certain embodiments, the cell driver is selected from the group consisting of a pressure pump, a gas cylinder, a compressor, a vacuum pump, a syringe pump, a peristaltic pump, a pipette, a piston, a capillary actor, a human heart, a human muscle, gravity, a microfluidic pump, and a syringe. Modifications to the pressure applied by the cell driver also affect the speed at which cells pass through the microfluidic channel (e.g., an increase in the amount of pressure will result in an increase in cell speed). When a cell (e.g., an anuclear cell) passes through a constriction, its membrane is perturbed, causing a temporary disruption in the membrane and resulting in the uptake of a payload present in the surrounding medium. As used herein, such a temporary disruption is referred to as a "perturbation." Perturbations produced by the methods described herein are holes in the cell that allow materials from outside the cell to move into the cell. Non-limiting examples of perturbations include holes, clefts, cavities, openings, pores, clefts, gaps, or perforations. Perturbations (e.g., pores or pores) produced by the methods described herein are not formed as a result of the assembly of protein subunits to form multimeric pore structures, such as those produced by complement or bacterial hemolysin. Methods for stimulating an immune response to an antigen in an individual Method for delivering antigens into anucleate cell-derived vesicles

[0174] In certain aspects, a method for delivering an antigen into anuclear cell-derived vesicles is provided, comprising: passing a cell suspension containing input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigen and form anuclear cell-derived vesicles; and incubating the anuclear cell-derived vesicles with the antigen for a time sufficient to allow entry of the antigen into the anuclear cell-derived vesicles. In some embodiments, the anuclear cell-derived vesicles further comprise an adjuvant. In some embodiments, the input anuclear cells further comprise an adjuvant.

[0175] In certain aspects, a method for delivering an adjuvant into anuclear cell-derived vesicles is provided, comprising: passing a cell suspension containing input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the adjuvant and form anuclear cell-derived vesicles; and incubating the anuclear cell-derived vesicles with the adjuvant for a time sufficient to allow entry of the adjuvant into the anuclear cell-derived vesicles. In some embodiments, the anuclear cell-derived vesicles further comprise an antigen. In some embodiments, the input anuclear cells further comprise an antigen.

[0176] In certain aspects, a method for delivering antigens and adjuvants into anuclear cell-derived vesicles is provided, comprising the steps of passing a cell suspension containing input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigen and adjuvant and forming anuclear cell-derived vesicles; and incubating the anuclear cell-derived vesicles with the antigen and adjuvant for a time sufficient to allow entry of the antigen and adjuvant into the anuclear cell-derived vesicles. Methods for stimulating an immune response

[0177] In certain aspects, a method for stimulating an immune response to an antigen in an individual is provided, the method comprising administering to the individual an effective amount of anuclear cell-derived vesicles comprising an antigen, wherein the anuclear cell-derived vesicles comprising the antigen are prepared by a process comprising: a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigen and form anuclear cell-derived vesicles; and b) incubating the anuclear cell-derived vesicles with the antigen for a time sufficient to allow the antigen to enter the anuclear cell-derived vesicles. In some embodiments, the method further comprises systemically administering an adjuvant to the individual. In some embodiments, the systemic adjuvant is administered before, after, or simultaneously with the anuclear cell-derived vesicles. In some embodiments, the input anuclear cells comprise an adjuvant. In some embodiments, the systemic adjuvant is an extracellular adjuvant. In some embodiments, the systemic adjuvant is an extravesicular adjuvant.In some embodiments, the method of stimulating an immune response to an antigen in an individual enhances an existing immune response to the antigen.

[0178] In certain aspects, a method for stimulating an immune response to an antigen in an individual is provided, the method comprising administering to the individual an effective amount of anuclear cell-derived vesicles comprising an antigen and an adjuvant, wherein the anuclear cell-derived vesicles comprising the antigen and adjuvant are prepared by a process comprising: a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow the antigen and adjuvant to pass through, thereby forming anuclear cell-derived vesicles; and b) incubating the anuclear cell-derived vesicles with the antigen for a time sufficient to allow the antigen and adjuvant to enter the anuclear cell-derived vesicles. In some embodiments, the method further comprises systemically administering an adjuvant to the individual. In some embodiments, the systemic adjuvant is administered before, after, or simultaneously with the anuclear cell-derived vesicles. In some embodiments, the input anuclear cells comprise an adjuvant. In some embodiments, the systemic adjuvant is an extracellular adjuvant. In some embodiments, the systemic adjuvant is an extravesicular adjuvant. In some embodiments, the method of stimulating a pre-existing immune response to an antigen in an individual enhances the immune response to the antigen. Methods for treating or preventing disease in an individual

[0179] In certain aspects, methods are provided for treating a disease in an individual, the method comprising administering to the individual anuclear cell-derived vesicles comprising a disease-associated antigen, wherein an immune response to the antigen ameliorates the disease state, and the anuclear cell-derived vesicles comprising the disease-associated antigen are prepared by a process comprising: a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigen and form anuclear cell-derived vesicles; and b) incubating the anuclear cell-derived vesicles with the antigen for a time sufficient to allow entry of the antigen into the anuclear cell-derived vesicles.

[0180] In certain aspects, methods are provided for preventing disease in an individual, the method comprising administering to the individual anuclear cell-derived vesicles comprising a disease-associated antigen, wherein an immune response to the antigen prevents the onset of the disease, and the anuclear cell-derived vesicles comprising the disease-associated antigen are prepared by a process comprising: a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, wherein the diameter of the constriction is a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigen and form anuclear cell-derived vesicles; and b) incubating the anuclear cell-derived vesicles with the antigen for a time sufficient to allow entry of the antigen into the anuclear cell-derived vesicles.

[0181] In certain aspects, methods are provided for vaccinating an individual against an antigen, the method comprising administering to the individual anuclear cell-derived vesicles comprising the antigen, wherein the anuclear cell-derived vesicles comprising the antigen are prepared by a process comprising: a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigen and form anuclear cell-derived vesicles; and b) incubating the anuclear cell-derived vesicles with the antigen for a time sufficient to allow entry of the antigen into the anuclear cell-derived vesicles.

[0182] In some embodiments of any of the methods described herein, the method further comprises systemically administering an adjuvant to the individual. In some embodiments, the systemic adjuvant is administered before, after, or simultaneously with the anuclear cell-derived vesicles. In some embodiments, the input anuclear cells comprise an adjuvant. In some embodiments, the systemic adjuvant is an extracellular adjuvant. In some embodiments, the systemic adjuvant is an extravesicular adjuvant.

[0183] In another aspect, a method is provided for treating a disease in an individual, the method comprising administering to the individual anuclear cell-derived vesicles comprising a disease-associated antigen and an adjuvant, wherein an immune response to the antigen ameliorates the disease state, and the anuclear cell-derived vesicles comprising the disease-associated antigen and an adjuvant are prepared by a process comprising: a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in suspension, thereby causing disruption of the input anuclear cells large enough to allow the antigen and adjuvant to pass through, thereby forming anuclear cell-derived vesicles; and b) incubating the anuclear cell-derived vesicles with the antigen and adjuvant for a time sufficient to allow the antigen and adjuvant to enter the anuclear cell-derived vesicles.

[0184] In certain aspects, methods are provided for preventing disease in an individual, the method comprising administering to the individual anuclear cell-derived vesicles comprising a disease-associated antigen and an adjuvant, wherein an immune response to the antigen prevents the onset of the disease, and the anuclear cell-derived vesicles comprising the disease-associated antigen and an adjuvant are prepared by a process comprising: a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow the antigen and adjuvant to pass through, thereby forming anuclear cell-derived vesicles; and b) incubating the anuclear cell-derived vesicles with the antigen and adjuvant for a time sufficient to allow the antigen and adjuvant to enter the anuclear cell-derived vesicles.

[0185] In certain aspects, a method is provided for vaccinating an individual against an antigen, the method comprising administering to the individual anuclear cell-derived vesicles comprising an antigen and an adjuvant, wherein the anuclear cell-derived vesicles comprising the antigen and adjuvant are prepared by a process comprising: a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow the antigen and adjuvant to pass through, thereby forming anuclear cell-derived vesicles; and b) incubating the anuclear cell-derived vesicles with the antigen and adjuvant for a time sufficient to allow the antigen and adjuvant to enter the anuclear cell-derived vesicles.

[0186] In certain aspects, methods are provided for treating a disease in an individual, wherein an immune response to a disease-associated antigen ameliorates the disease state, the method comprising: a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigen and form anuclear cell-derived vesicles; b) incubating the anuclear cell-derived vesicles with the antigen for a time sufficient to allow entry of the antigen into the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles containing the antigen; and c) administering the anuclear cell-derived vesicles containing the antigen to the individual.

[0187] In certain aspects, methods are provided for preventing disease in an individual, wherein an immune response to a disease-associated antigen prevents the onset of the disease, the method comprising: a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigen and form anuclear cell-derived vesicles; b) incubating the anuclear cell-derived vesicles with the antigen for a time sufficient to allow entry of the antigen into the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles containing the antigen; and c) administering the anuclear cell-derived vesicles to the individual.

[0188] In certain aspects, methods are provided for vaccinating an individual against an antigen, the methods comprising: a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigen and form anuclear cell-derived vesicles; b) incubating the anuclear cell-derived vesicles with the antigen for a time sufficient to allow entry of the antigen into the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles containing the antigen; and c) administering the anuclear cell-derived vesicles containing the antigen to the individual.

[0189] In some embodiments, the method further comprises systemically administering an adjuvant to the individual. In some embodiments, the systemic adjuvant is administered before, after, or simultaneously with the anuclear cell-derived vesicles. In some embodiments, the input anuclear cells comprise an adjuvant. In some embodiments, the systemic adjuvant is an extracellular adjuvant. In some embodiments, the systemic adjuvant is an extravesicular adjuvant.

[0190] In another aspect, a method is provided for treating a disease in an individual, wherein an immune response to a disease-associated antigen ameliorates the disease state, the method comprising: a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow the disease-associated antigen and adjuvant to pass through, to form anuclear cell-derived vesicles; b) incubating the anuclear cell-derived vesicles with the antigen and adjuvant for a time sufficient to allow the antigen and adjuvant to enter the anuclear cell-derived vesicles, thereby producing anuclear cell-derived vesicles comprising the antigen and adjuvant; and c) administering the anuclear cell-derived vesicles comprising the antigen and adjuvant to the individual.

[0191] In some aspects, a method for preventing disease in an individual is provided, wherein an immune response to a disease-associated antigen prevents the onset of the disease, the method comprising: a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigen and adjuvant and forming anuclear cell-derived vesicles; b) incubating the anuclear cell-derived vesicles with the antigen and adjuvant for a time sufficient to allow the antigen and adjuvant to enter the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles comprising the antigen and adjuvant; and c) administering the anuclear cell-derived vesicles comprising the antigen and adjuvant to the individual.

[0192] In certain aspects, a method for vaccinating an individual against an antigen is provided, comprising the steps of: a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigen and adjuvant and forming anuclear cell-derived vesicles; b) incubating the anuclear cell-derived vesicles with the antigen and adjuvant for a time sufficient to allow the antigen and adjuvant to enter the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles comprising the antigen and adjuvant; and c) administering the anuclear cell-derived vesicles comprising the antigen and adjuvant to the individual.

[0193] In some embodiments, the method further comprises systemically administering an adjuvant to the individual. In some embodiments, the systemic adjuvant is administered before, after, or simultaneously with the anuclear cell-derived vesicles. In some embodiments, the input anuclear cells comprise an adjuvant. In some embodiments, the systemic adjuvant is an extracellular adjuvant. In some embodiments, the systemic adjuvant is an extravesicular adjuvant.

[0194] In some embodiments of any of the methods described herein, the disease is cancer, an infectious disease, or a virus-related disease. In some embodiments, the cancer includes one or more of head and neck, cervical, uterine, rectal, penile, ovarian, testicular, bone, soft tissue, skin (melanoma), stomach, intestinal, colon, prostate, breast, esophageal, liver, lung, pancreatic, brain, or blood cancer. In some embodiments, the infectious disease or virus-related disease is associated with one or more of HPV, EBV, HIV, HBV, RSV, or KSHV. In some embodiments, the disease-associated antigen is an HPV antigen or an HPV-associated antigen. In some embodiments, the HPV antigen is an HPV-16 or HPV-18 antigen. In some embodiments, the HPV antigen is an HPV E6 antigen or HPV E7 antigen. In some embodiments, the HPV-associated disease is an HPV-associated cancer. In some embodiments, the HPV-related cancer is cervical cancer, anal cancer, oropharyngeal cancer, vaginal cancer, vulvar cancer, penile cancer, skin cancer, or head and neck cancer. In some embodiments, the HPV-related disease is an HPV-related infectious disease. In some embodiments, the HPV-related disease can include common warts, plantar warts, flat warts, anogenital warts, anal lesions, epidermal dysplasia, focal epithelial hyperplasia, oral papilloma, verrucous cyst, laryngeal papilloma, squamous intraepithelial lesion (SIL), cervical intraepithelial neoplasia (CIN), vulvar intraepithelial neoplasia (VIN), and vaginal intraepithelial neoplasia (VAIN). In certain embodiments, the disease-related antigen is an EBV antigen or an EBV-associated antigen. In some embodiments, the EBV antigen or EBV-associated antigen is one or more of EBNA-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA-LP, LMP-1, LMP-2A, LMP-2B, or EBER. In some embodiments, the virus-associated disease is an EBV-associated disease. In some embodiments, the EBV-associated disease is multiple sclerosis (MS). In some embodiments, the disease-associated antigen is a human CMV (HCMV) antigen or an HCMV-associated antigen. In some embodiments, the antigen is derived from any of the strains Merlin, Toledo, Davis, Esp, Kerr, Smith, TB40E, TB40F, AD169, or Towne HCMV.In some embodiments, the HCMV antigen or HCMV-associated antigen is derived from one or more of pUL48, pUL47, pUL32, pUL82, pUL83, and pUL99, pUL69, pUL25, pUL56, pUL94, pUL97, pUL144, or pUL128. In some embodiments, the virus-associated disease is an HCMV-associated disease. In other embodiments, the disease-associated antigen is an HIV antigen or an HIV-associated antigen. In some embodiments, the virus-associated disease is an HIV-associated disease. Opportunistic infections are infections that occur more frequently and are more severe in individuals with weakened immune systems, including HIV patients. In some embodiments, the HIV-associated disease is an opportunistic infection, which can include, but is not limited to, candidiasis of the bronchus, trachea, esophagus, or lungs; invasive cervical cancer; coccidioidomycosis; cryptococcosis; chronic intestinal cryptosporidiosis, cytomegalovirus disease; HIV-associated encephalopathy; HSV-associated chronic ulcers or bronchitis, pneumonitis, or esophagitis; histoplasmosis; chronic intestinal isosporosis; Kaposi's sarcoma; lymphoma; tuberculosis; Mycobacterium avium complex (MAC); Pneumocystis carinii pneumonia (PCP); recurrent pneumonia; progressive multifocal leukoencephalopathy; recurrent Salmonella septicemia; cerebral toxoplasmosis; and HIV-associated wasting syndrome.

[0195] In some embodiments, methods are provided for treating an individual by introducing into the individual anuclear cell-derived vesicles containing antigens and / or adjuvants, the anuclear cell-derived vesicles being generated by passing input anuclear cells through a constriction to form anuclear cell-derived vesicles such that the antigens and / or adjuvants enter the anuclear cell-derived vesicles. In some embodiments, the input anuclear cells are autologous cells. For example, the input anuclear cells are isolated from an individual (e.g., a patient) and processed according to the disclosed methods, and the resulting anuclear cell-derived vesicles are introduced back into the same individual. Thus, in some embodiments, the anuclear cell-derived vesicles are autologous to the individual. In other embodiments, the input anuclear cells are allogeneic cells. For example, the anuclear cells are isolated from a different individual (e.g., a donor) and processed according to the disclosed methods, and the resulting anuclear cell-derived vesicles are introduced into the first individual (e.g., a patient). In some embodiments, the anuclear cell-derived vesicles are allogeneic to the individual. In some embodiments, a pool of input anucleated cells from multiple individuals is processed according to the disclosed methods, and the pool of anucleated cell-derived vesicles is introduced into a first individual (e.g., a patient). In some embodiments, the input anucleated cells are isolated from an individual, processed according to the disclosed methods, and the anucleated cell-derived vesicles are introduced into a different individual. In some embodiments, a population of input anucleated cells is isolated from an individual (patient) or a different individual, passed through a constriction to achieve delivery of antigen and / or adjuvant, and then the population of anucleated cell-derived vesicles is re-infused into the patient to augment the therapeutic response.

[0196] In some aspects, the present invention provides methods for treating a patient by introducing into an individual anuclear cell-derived vesicles containing antigens and / or adjuvants, the anuclear cell-derived vesicles being generated by passing input anuclear cells through a constriction to form anuclear cell-derived vesicles such that the antigens and / or adjuvants enter the anuclear cell-derived vesicles. In some embodiments, the treatment comprises multiple steps (e.g., 2, 3, 4, 5, 6, or more) of introducing such anuclear cell-derived vesicles into the individual. For example, in some embodiments, methods are provided for treating an individual by administering to the individual 2, 3, 4, 5, 6, or more times anuclear cell-derived vesicles containing antigens and / or adjuvants, the anuclear cell-derived vesicles being generated by passing input anuclear cells through a constriction to form anuclear cell-derived vesicles such that the antigens and / or adjuvants enter the anuclear cell-derived vesicles. In some embodiments, the duration between any two consecutive administrations of cells is at least about 1 day (such as at least about 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year or more, including any ranges between these values).

[0197] In some embodiments, input anuclear cells are isolated from an individual and processed according to the disclosed methods, and the resulting anuclear cell-derived vesicles containing antigen and / or adjuvant are then infused back into the same individual (e.g., patient). For example, a population of input anuclear cells are isolated from an individual and passed through a constriction to achieve delivery of antigen and / or adjuvant, and then the resulting anuclear cell-derived vesicles are re-infused into the individual to augment a therapeutic immune response. In some embodiments, input anuclear cells are isolated from an individual and processed according to the disclosed methods, and the resulting anuclear cell-derived vesicles are re-infused into the individual. For example, a population of input anuclear cells are isolated from an individual and passed through a constriction to achieve delivery of antigen and / or adjuvant, and then the resulting anuclear cell-derived vesicles are re-infused into the individual to stimulate and / or enhance an immune response in the individual.

[0198] In some embodiments, input anucleated cells are isolated from a universal blood donor (e.g., a type O blood donor) and then stored and / or frozen for later constriction-mediated delivery. In some embodiments, antigens are isolated from an individual and delivered to input anucleated cells isolated from a universal donor. In some embodiments, input anucleated cells are isolated from a blood donor and then stored and / or frozen for later constriction-mediated delivery (SQZ). In some embodiments, antigens are isolated from an individual and delivered to input anucleated cells isolated from a blood donor. In some embodiments, anucleated cell-derived vesicles containing antigens and / or adjuvants are generated by constriction-mediated delivery as described above. In some embodiments, anucleated cell-derived vesicles containing antigens and / or adjuvants are introduced into an individual. In some embodiments, anucleated cell-derived vesicles containing antigens and / or adjuvants are stored and / or frozen (e.g., for later treatment). In some embodiments, the individual has a blood type compatible with the blood donor. In some embodiments, the anuclear cell-derived vesicles containing antigens and / or adjuvants are produced by the constriction-mediated delivery described above. In some embodiments, the anuclear cell-derived vesicles containing antigens and / or adjuvants are introduced into an individual. In some embodiments, the individual has a blood type that is compatible with the blood donor. In some embodiments, the individual has a blood type that is incompatible with the blood donor.

[0199] In some embodiments, methods are provided for preventing disease in an individual by introducing into the individual anuclear cell-derived vesicles containing antigens and / or adjuvants, the anuclear cell-derived vesicles being generated by passing input anuclear cells through a constriction to form anuclear cell-derived vesicles such that the antigens and / or adjuvants enter the anuclear cell-derived vesicles. In some embodiments, the input anuclear cells are autologous cells. For example, the input anuclear cells are isolated from an individual (e.g., a patient) and processed according to the disclosed methods, and the resulting anuclear cell-derived vesicles are introduced back into the same individual. Thus, in some embodiments, the anuclear cell-derived vesicles are autologous to the individual. In other embodiments, the input anuclear cells are allogeneic cells. For example, the anuclear cells are isolated from a different individual (e.g., a donor) and processed according to the disclosed methods, and the resulting anuclear cell-derived vesicles are introduced into the first individual (e.g., a patient). In some embodiments, the anuclear cell-derived vesicles are allogeneic to the individual. In some embodiments, a pool of input anucleated cells from multiple individuals is processed according to the disclosed methods, and the pool of anucleated cell-derived vesicles is introduced into a first individual (e.g., a patient). In some embodiments, the input anucleated cells are isolated from an individual, processed according to the disclosed methods, and the anucleated cell-derived vesicles are introduced into a different individual. In some embodiments, a population of input anucleated cells is isolated from an individual (patient) or a different individual, passed through a constriction to achieve delivery of antigen and / or adjuvant, and then the population of anucleated cell-derived vesicles is re-infused into the patient to augment the prophylactic response.

[0200] In some embodiments, prophylactic methods include multiple steps (e.g., any of 2, 3, 4, 5, 6, or more) of administering to an individual an anuclear cell-derived vesicle described herein. For example, some embodiments provide methods of vaccinating an individual against an antigen by administering to the individual 2, 3, 4, 5, 6, or more times an anuclear cell-derived vesicle containing an antigen and / or adjuvant, the anuclear cell-derived vesicle being generated by passing input anuclear cells through a constriction to form an anuclear cell-derived vesicle, such that the antigen and / or adjuvant enters the anuclear cell-derived vesicle. In some embodiments, the duration between any two consecutive cell administrations is at least about 1 day (e.g., any of at least about 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, or longer, including any ranges between these values).

[0201] In some embodiments, methods are provided for vaccinating an individual against an antigen by introducing into the individual anucleated cell-derived vesicles containing antigens and / or adjuvants, which vesicles are generated by passing input anucleated cells through a constriction to form anucleated cell-derived vesicles, such that the antigens and / or adjuvants enter the anucleated cell-derived vesicles. In some embodiments, the input anucleated cells are autologous cells. For example, the input anucleated cells are isolated from an individual (e.g., a patient) and processed according to the disclosed methods, and the resulting anucleated cell-derived vesicles are introduced back into the same individual. Thus, in some embodiments, the anucleated cell-derived vesicles are autologous to the individual. In other embodiments, the input anucleated cells are allogeneic cells. For example, the anucleated cells are isolated from a different individual (e.g., a donor) and processed according to the disclosed methods, and the resulting anucleated cell-derived vesicles are introduced into the first individual (e.g., a patient). In some embodiments, the anucleated cell-derived vesicles are allogeneic to the individual. In some embodiments, a pool of input anucleated cells from multiple individuals is processed according to the disclosed methods, and the pool of anucleated cell-derived vesicles is introduced into a first individual (e.g., a patient). In some embodiments, the input anucleated cells are isolated from an individual and processed according to the disclosed methods, and the anucleated cell-derived vesicles are introduced into a different individual. In some embodiments, a population of input anucleated cells is isolated from an individual (patient) or a different individual and passed through a constriction to achieve delivery of antigen and / or adjuvant, and then the population of anucleated cell-derived vesicles is re-infused into the patient to induce a prophylactic response.

[0202] In some embodiments, vaccination involves multiple steps (e.g., any of 2, 3, 4, 5, 6, or more) of administering an anuclear cell-derived vesicle described herein to an individual. For example, in some embodiments, a method of vaccinating an individual against an antigen is provided by administering to the individual 2, 3, 4, 5, 6, or more times an anuclear cell-derived vesicle containing an antigen and / or adjuvant, the anuclear cell-derived vesicle being generated by passing input anuclear cells through a constriction to form an anuclear cell-derived vesicle, such that the antigen and / or adjuvant enters the anuclear cell-derived vesicle. In some embodiments, the duration between any two consecutive cell administrations is at least about 1 day (e.g., any of at least about 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, or more, including any ranges between these values).

[0203] Any of the above-described methods can be performed in vitro, ex vivo, or in vivo. For in vivo applications, the device can be implanted into a vascular lumen, e.g., an in-line stent in an artery or vein. In some embodiments, the method is used as part of a bedside system for ex vivo treatment of patient cells and immediate reintroduction of the cells into the patient. Such methods can be used as a means to enhance and / or stimulate an immune response in an individual. In some embodiments, the method can be performed in a typical hospital laboratory by a minimally trained technician. In some embodiments, a patient-operated treatment system can be used. In some embodiments, the method is performed using an in-line blood treatment system in which blood is diverted directly from a patient and passed through a stenosis, resulting in the formation of vesicles derived from anuclear cells in the blood and the delivery of antigens and / or adjuvants to the anuclear cell-derived vesicles in the blood, which are then directly infused back into the patient after treatment.

[0204] In some embodiments of any of the methods described herein, the anuclear cell-derived vesicles are present in a pharmaceutical formulation. In some embodiments, the anuclear cell-derived vesicles are administered systemically. In some embodiments, the anuclear cell-derived vesicles are administered intravenously, intraarterially, subcutaneously, intramuscularly, or intraperitoneally. In certain embodiments, the anuclear cell-derived vesicles are administered to an individual in combination with a therapeutic agent. In some embodiments, the therapeutic agent is administered before, after, or simultaneously with the anuclear cell-derived vesicles.

[0205] In some embodiments, the therapeutic agent is an immune checkpoint inhibitor and / or cytokine. In some embodiments, the therapeutic agent includes one or more of IFN-α, IFN-γ, IL-2 (either its native or modified form), IL-10, or IL-15. In some embodiments, the therapeutic agent is one or more forms of immunotherapy. Immunotherapies include, but are not limited to, monoclonal antibodies, immune checkpoint inhibitors, cytokines, vaccines for treating cancer, and adoptive cell transfer. In some embodiments of any of the methods described herein, the method further comprises administering an immunotherapy. In some embodiments, the method further comprises administering one or more therapeutic agents. In some embodiments, the immune checkpoint inhibitor is targeted to any one of PD-1, PD-L1, CTLA-4, TIM-3, LAG3, TIGIT, VISTA, TIM1, B7-H4 (VTCN1), and BTLA. In some embodiments, the therapeutic agent is a bispecific agent; for example, a bispecific agent comprising a cytokine component and a targeting component. In some embodiments, the anuclear cell-derived vesicles are administered to an individual in combination with chemotherapy or radiation therapy. In some embodiments, the anuclear cell-derived vesicles are administered to an individual in combination with one or more agents that improve antigen presentation, improve T cell proliferation, and / or improve the tumor microenvironment. anucleate cell

[0206] In some embodiments of any of the methods described herein, the input anuclear cell is a mammalian cell. An anuclear cell lacks a nucleus. In some embodiments, the anuclear cell is a monkey, mouse, dog, cat, horse, rat, sheep, goat, pig, or rabbit cell. In some embodiments, the anuclear cell is a human cell. In some embodiments, the anuclear cell is a non-mammalian cell. In some embodiments, the anuclear cell is a chicken, frog, insect, fish, or nematode cell.

[0207] In some embodiments, the anucleated cells are red blood cells. Red blood cells (RBCs) are flexible, oval, biconcave discs whose cytoplasm is rich in the oxygen-carrier biomolecule hemoglobin. RBCs serve as the primary means of oxygen delivery and carbon dioxide removal throughout the human body. RBCs can remain in circulation for up to 120 days before being removed from the body by clearance in the liver and spleen. In some embodiments, the anucleated cells are precursors of RBCs. In some embodiments, the anucleated cells are reticulocytes. Reticulocytes are anucleated immature (not yet biconcave) red blood cells, typically comprising about 1% of the red blood cells in the human body. Mature red blood cells are also referred to as erythrocytes. In some embodiments, the anucleated cells are red blood cells. In some embodiments, the anucleated cells are platelets. Platelets, also called thrombocytes, are components of blood whose function is involved in blood clotting. Platelets are biconvex discoid (lenticular) structures with a diameter of 2-3 μm.

[0208] In some embodiments of any of the methods described herein, presentation of an antigen in an immunogenic environment enhances and / or stimulates an immune response to the antigen. Antigens derived from apoptotic bodies, such as anuclear cell-derived vesicles, which can be eliminated in the immunogenic environment of the liver and spleen, can stimulate and / or enhance an immune response to the antigen by activating T cells. In some embodiments, the immune response is antigen-specific. Anuclear cell-derived vesicles, such as red blood cell-derived vesicles, have a limited lifespan and are unable to self-repair, subsequently undergoing eryptosis, a process similar to apoptosis, which results in the removal of anuclear cell-derived vesicles from the bloodstream. In some embodiments, antigens can be released after eryptosis of anuclear cell-derived vesicles in an immunogenic environment, where they are then engulfed, processed, and presented by antigen-presenting cells. In some embodiments, anuclear cell-derived vesicles containing antigens are phagocytosed by antigen-presenting cells, and the antigens are then processed and presented by the antigen-presenting cells. In some embodiments, anucleated cell-derived vesicles containing antigens are phagocytosed by resident macrophages, and the antigens are then processed and presented by the resident macrophages.

[0209] In some embodiments, the antigen contained in the anucleate-derived vesicles is then presented. In some embodiments, presentation of the antigen in an immunogenic environment stimulates an immune response to the antigen. In some embodiments, the antigen is processed in the immunogenic environment. In some embodiments, the immune response is antigen-specific.

[0210] In some embodiments, the anuclear cell-derived vesicles comprise an adjuvant. In some embodiments, the adjuvant creates or promotes an immunogenic environment, and presentation of the antigen in said immunogenic environment stimulates an immune response to the antigen. In some embodiments, the immune stimulation is multispecific, including stimulation of an immune response to multiple antigens.

[0211] In some embodiments of any of the methods described herein, the method includes passing a cell suspension containing input anuclear cells through a constriction, where the constriction deforms the input anuclear cells, thereby causing disruption of the input anuclear cells such that antigens and / or adjuvants enter the anuclear cell-derived vesicles, forming anuclear cell-derived vesicles. In some embodiments, the antigen is presented in an immunogenic environment. In some embodiments, the adjuvant creates or promotes an immunogenic environment, and presentation of the antigen in the immunogenic environment stimulates an immune response against the antigen. In some embodiments, the antigen is processed in the immunogenic environment. In some embodiments, the immune stimulation is antigen-specific. In some embodiments, the immune stimulation is multispecific, including stimulation of an immune response against multiple antigens.

[0212] In certain embodiments of any of the methods described herein, the method includes passing a first cell suspension comprising first input anuclear cells through a constriction, wherein the constriction deforms the cells, thereby causing perturbation of the first input anuclear cells such that an antigen enters vesicles derived from the perturbation of the first input anuclear cells; passing a second cell suspension comprising second input anuclear cells through a constriction, wherein the constriction deforms the second input anuclear cells, thereby causing perturbation of the second input anuclear cells such that an adjuvant enters vesicles derived from the perturbation of the second input anuclear cells; and introducing the vesicles derived from the first input anuclear cells and the vesicles derived from the second input anuclear cells into an individual, thereby stimulating an immune response to the antigen. Thus, in some embodiments, vesicles derived from the first input anuclear cells comprise an antigen, and vesicles derived from the second input anuclear cells comprise an adjuvant. In some embodiments, the antigen is presented in an immunogenic environment. In some embodiments, the adjuvant creates or promotes an immunogenic environment, and presentation of the antigen in the immunogenic environment stimulates an immune response to the antigen. In some embodiments, the antigen is processed in the immunogenic environment. In some embodiments, vesicles derived from the first input anuclear cells and vesicles derived from the second input anuclear cells are introduced simultaneously. In some embodiments, vesicles derived from the first input anuclear cells and vesicles derived from the second input anuclear cells are introduced sequentially. In some embodiments, vesicles derived from the first input anuclear cells are introduced into an individual before introduction of vesicles derived from the second input anuclear cells. In some embodiments, the vesicles derived from the first input anuclear cells are introduced into the individual about any of 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, or 24 hours before the introduction of the vesicles derived from the second input anuclear cells.In some embodiments, the vesicles derived from the first input anuclear cells are introduced into the individual about 1, 2, 3, 4, 5, 6, or 7 days before the introduction of the vesicles derived from the second input anuclear cells. In some embodiments, the vesicles derived from the second input anuclear cells are introduced into the individual before the introduction of the vesicles derived from the first input anuclear cells. In some embodiments, the vesicles derived from the second input anuclear cells are introduced into the individual about 1, 5, 10, 15, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, or 24 hours before the introduction of the vesicles derived from the first input anuclear cells. In some embodiments, the vesicles derived from the second input anuclear cells are introduced into the individual about 1, 2, 3, 4, 5, 6, or 7 days before the introduction of the vesicles derived from the first input anuclear cells. In some embodiments, the immune stimulation is antigen-specific. In some embodiments, the immune stimulation is multispecific, including stimulating an immune response against multiple antigens.

[0213] In some embodiments, the stimulated and / or enhanced immune response comprises an increased T cell response. For example, the increased T cell response can include, without limitation, increased T cell activation or proliferation, increased T cell survival, or increased cell functionality. In some embodiments, the increased T cell response comprises increased T cell activation. In some embodiments, the increased T cell response comprises increased T cell survival. In some embodiments, the increased T cell response comprises increased T cell proliferation. In some embodiments, the increased T cell response comprises increased T cell functionality. For example, increased T cell functionality can include, without limitation, modulated cytokine secretion, increased T cell migration to sites of inflammation, and increased T cell cytotoxic activity. In some embodiments, the stimulated and / or enhanced immune response comprises increased pro-inflammatory cytokine production and / or secretion and / or decreased anti-inflammatory cytokine production and / or secretion. In some embodiments, the stimulated and / or enhanced immune response comprises increased production and / or secretion of one or more pro-inflammatory cytokines selected from interleukin-1 (IL-1), IL-2, IL-12, and IL-18, tumor necrosis factor (TNF), interferon gamma (IFN-γ), and granulocyte-macrophage colony-stimulating factor (GM-CSF). In some embodiments, the stimulated and / or enhanced immune response comprises decreased production and / or secretion of one or more anti-inflammatory cytokines selected from IL-4, IL-10, IL-13, IL-35, IFN-α, and transforming growth factor-beta (TGF-β). In some embodiments, the stimulated and / or enhanced immune response comprises a change in T cell phenotype. For example, T cell status may change from a regulatory (Treg) or anti-inflammatory phenotype to a pro-inflammatory phenotype. In some embodiments, the stimulated and / or enhanced immune response suppresses non-specific activation of T cells, which may otherwise subsequently lead to cell death. In some embodiments, the stimulated and / or enhanced immune response comprises a suppressed Treg response. In some embodiments, the stimulated and / or enhanced immune response comprises an increased B cell response.In some embodiments, the increased B cell response comprises increased antibody production. Vesicles derived from anucleated cells

[0214] In some aspects, the present application provides anuclear cell-derived vesicles prepared from parent anuclear cells, the anuclear cell-derived vesicles having one or more of the following characteristics: (a) a reduced circulating half-life in mammals compared to the parent anuclear cells, (b) a reduced hemoglobin level compared to the parent anuclear cells, (c) a spherical morphology, (d) an increased surface phosphatidylserine level compared to the parent anuclear cells, or (e) reduced ATP production compared to the parent anuclear cells.

[0215] In certain aspects, anuclear cell-derived vesicles comprising an antigen are provided, the anuclear cell-derived vesicles being prepared by a process comprising: (a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigen and form anuclear cell-derived vesicles; and (b) incubating the anuclear cell-derived vesicles with the antigen for a time sufficient to allow entry of the antigen into the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles comprising the antigen. In some embodiments, the input anuclear cells comprise an adjuvant.

[0216] In certain aspects, anuclear cell-derived vesicles comprising an adjuvant are provided, the anuclear cell-derived vesicles being prepared by a process comprising: (a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the adjuvant and form anuclear cell-derived vesicles; and (b) incubating the anuclear cell-derived vesicles with the adjuvant for a time sufficient to allow antigen to enter the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles comprising the adjuvant. In some embodiments, the input anuclear cells comprise an antigen.

[0217] In certain aspects, there are provided anuclear cell-derived vesicles comprising antigens and adjuvants, prepared by a process comprising: a) passing a cell suspension comprising input anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigen and adjuvant and form anuclear cell-derived vesicles; and b) incubating the anuclear cell-derived vesicles with the antigen and adjuvant for a time sufficient to allow entry of the antigen and adjuvant into the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles comprising the antigen and adjuvant.

[0218] In some embodiments, the non-nucleated cell-derived vesicles are red blood cell-derived vesicles or platelet-derived vesicles. In some embodiments, the non-nucleated cell-derived vesicles are red blood cell-derived vesicles or reticulocyte-derived vesicles.

[0219] In some embodiments of any of the anucleated cell-derived vesicles described herein, the input or parent anucleated cells are mammalian cells. Anucleated cells lack a nucleus. In some embodiments, the input anucleated cells are monkey, mouse, dog, cat, horse, rat, sheep, goat, pig, or rabbit cells. In some embodiments, the input anucleated cells are human cells. In some embodiments, the input anucleated cells are non-mammalian cells. In some embodiments, the input anucleated cells are chicken, frog, insect, fish, or nematode cells. In some embodiments, the input anucleated cells are red blood cells. In some embodiments, the input anucleated cells are erythroid cells. In some embodiments, the input anucleated cells are precursors of red blood cells. In some embodiments, the input anucleated cells are reticulocytes. In some embodiments, the input anucleated cells are platelets.

[0220] In some embodiments, presentation of an antigen in an immunogenic environment enhances or induces an immune response to the antigen. Antigens derived from eryptotic bodies, such as anuclear cell-derived vesicles that can be eliminated in the immunogenic environment of the liver and spleen, can stimulate and / or enhance an immune response to the antigen by activating T cells. In some embodiments, the immune response is antigen-specific. Anuclear cell-derived vesicles, such as RBC-derived vesicles, have a limited lifespan and cannot self-repair, causing eryptosis, a process similar to apoptosis, which leads to the removal of anuclear cell-derived vesicles from the bloodstream. In some embodiments, antigens can be released after eryptosis of anuclear cell-derived vesicles in an immunogenic environment, where they are then engulfed, processed, and presented by antigen-presenting cells. In some embodiments, anuclear cell-derived vesicles containing antigens are phagocytosed by antigen-presenting cells, such as macrophages, and the antigens are then processed and presented by the antigen-presenting cells. In some embodiments, the antigen-presenting cells are resident macrophages.

[0221] In some embodiments, the input or parent anucleated cells are red blood cells. In some embodiments, the input or parent anucleated cells are platelets. In some embodiments, the red blood cells are erythrocytes. In some embodiments, the red blood cells are reticulocytes.

[0222] In some embodiments, the circulating half-life of vesicles derived from anucleated cells in a mammal is reduced compared to the input or parent anucleated cells. Methods for measuring the half-life of anucleated cells, e.g., cells such as red blood cells, or vesicles derived from anucleated cells, are known in the art. See, e.g., Franco, RS, Transfus Med Hemother, 39, 2012. For example, in some embodiments, methods for measuring the half-life of anucleated cells or vesicles derived from anucleated cells include cohort labeling techniques or random labeling techniques. In some embodiments, methods for measuring the half-life of anucleated cells or vesicles derived from anucleated cells include labeling, re-injecting the cells or vesicles, and measuring their disappearance after re-injection. In some embodiments, methods for measuring the half-life of anucleated cells or vesicles derived from anucleated cells encompassed herein include measuring the half-life of appropriate reference control(s), such as input or parent anucleated cells or a control comprising a population of input or parent anucleated cells.

[0223] In some embodiments, the circulating half-life in a mammal is reduced by more than about 50%, such as by more than about any of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.9%, relative to the input or parent anucleated cells. In some embodiments, the circulating half-life in a mammal is reduced by between about 50% and about 99.9%, such as between about 70% and about 99.9%, between about 85% and about 99.9%, or between about 95% and about 99.9%, relative to the input or parent anucleated cells. In some embodiments, the circulating half-life in a mammal is reduced by any of about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.9% compared to the input or parent anucleated cells.

[0224] In some embodiments, the circulation half-life of anuclear cell-derived vesicles is less than about 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, or 10 days. In some embodiments, the circulation half-life of anuclear cell-derived vesicles is less than about 0.5 minutes, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 3 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 10 days, 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, or 100 days.

[0225] In some embodiments, the input or parent anucleated cells are human cells, and the vesicles derived from the anucleated cells have a circulation half-life of less than about 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, or 10 days. In some embodiments, the input or parent anucleated cells are human cells, and the vesicles derived from the anucleated cells have a circulation half-life of less than about 0.5 minutes, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 3 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 10 days, 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, or 100 days.

[0226] In some embodiments, the input or parent anucleated cells are red blood cells, and the hemoglobin level in the anucleated cell-derived vesicles is reduced compared to the input or parent anucleated cells. Methods for measuring the hemoglobin level of anucleated cells, e.g., cells such as red blood cells, or anucleated cell-derived vesicles, e.g., vesicles derived from red blood cells, are known in the art. See, e.g., Chaudhary, R., J Blood Med, 8, 2017. For example, in some embodiments, the method includes measuring metabolic precursors or products to determine hemoglobin turnover. In some embodiments, the method includes measuring one or more hemoglobin-derived (Hb) peptides. In some embodiments, methods for measuring the hemoglobin level of anucleated cells or anucleated cell-derived vesicles encompassed herein include measuring the hemoglobin level of appropriate reference control(s), such as input or parent anucleated cells or a control comprising a population of input or parent anucleated cells.

[0227] In some embodiments, the anucleated cells are characterized by a loss, such as a reduced level, of an intracellular component compared to the parent anucleated cell.

[0228] In some embodiments, the hemoglobin level in the anucleated cell-derived vesicles is reduced by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.9% compared to the input or parent anucleated cells. In some embodiments, the hemoglobin level in the anucleated cell-derived vesicles is reduced by about 50% to about 99.9%, such as about 70% to about 99.9%, about 85% to about 99.9%, or about 95% to about 99.9%, compared to the input or parent anucleated cells. In some embodiments, the hemoglobin level in the anucleated cell-derived vesicles is reduced by about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.9% compared to the input or parent anucleated cells. In some embodiments, the anucleated cell-derived vesicles lack hemoglobin. In some embodiments, the hemoglobin level in the anucleated cell-derived vesicles is about 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, or 50% of the hemoglobin level in the input or parent anucleated cells.

[0229] In some embodiments, the level of one or more hemoglobin (Hb) peptides in vesicles derived from anucleated cells is reduced by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% compared to the input or parent anucleated cells. In some embodiments, the level of one or more Hb peptides in vesicles derived from anucleated cells is reduced by about 50% to about 99.9%, such as about 70% to about 99.9%, about 85% to about 99.9%, or about 95% to about 99.9%, compared to the input anucleated cells. In some embodiments, the level of one or more Hb peptides in vesicles derived from anucleated cells is reduced by about any of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.9% compared to the input or parent anucleated cells.

[0230] In some embodiments, the input or parent anucleated cells are red blood cells, and the morphology of the anucleated cell-derived vesicles is modulated from that of the input or parent anucleated cells. Morphology relates to, for example, the shape, structure, geometry, strength, form, smoothness, roughness, circularity, volume, surface area, and / or size classification of cells or cell-derived vesicles. Methods for determining (e.g., measuring) morphology are known in the art. See, for example, Boutros et al., Cell, 163, 2015; Girasole, M. et al., Biochim Biophys Acta Biomembr, 1768, 2007; and Chen et al., Comput Math Methods Med, 2012. In some embodiments, the method for determining morphology involves high-content imaging. For example, cell morphology can be assessed by staining with Hoechst dye followed by automated high-content image analysis. In other examples, morphology can be determined by shifts in forward and side scatter plots from flow cytometry. In some embodiments, the input or parent anucleated cells are red blood cells, and the anucleated cell-derived vesicles have a spherical morphology. In some embodiments, the input or parent anucleated cells are red blood cells, and the anucleated cell-derived vesicles have a reduced biconcave shape compared to the input or parent anucleated cells. In some embodiments, methods for measuring the morphology of anucleated cells or anucleated cell-derived vesicles encompassed herein include measuring the morphology of appropriate reference control(s), such as input or parent anucleated cells or a control comprising a population of input or parent anucleated cells.

[0231] In some embodiments, the input or parent anucleated cells are red blood cells, and the vesicles derived from the anucleated cells have a reduced biconcave shape compared to the input or parent anucleated cells, such as a reduction of more than any of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.9%.

[0232] In some embodiments, the anuclear cell-derived vesicles are characterized by a spherical morphology, including a substantially spherical morphology. In some embodiments, the spherical morphology of the anuclear cell-derived vesicles is assessed qualitatively. In some embodiments, the spherical morphology of the anuclear cell-derived vesicles is assessed quantitatively.

[0233] In some embodiments, the anucleated cell-derived vesicles have a reduced surface area to volume ratio compared to the input or parent anucleated cells, such as a reduction of more than any of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0234] In some embodiments, the variation between each diameter measurement of the plurality of diameter measurements of an anuclear cell-derived vesicle is less than about 50%, such as less than any of about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%, and the plurality of diameter measurements includes at least two diameter measurements measuring the diameter at different points of the anuclear cell-derived vesicle.

[0235] In some embodiments, the smallest dimension, such as the diameter, of the anucleated cell-derived vesicles in the suspension is about 5 μm to about 7.25 μm, such as about 6 μm to about 7 μm or about 6.25 μm to about 6.75 μm. In some embodiments, the smallest dimension, such as the diameter, of the anucleated cell-derived vesicles in the suspension is at least about 5 μm, such as at least about 5.25 μm, 5.5 μm, 5.75 μm, 6 μm, 6.25 μm, 6.5 μm, 6.75 μm, 7 μm, or 7.25 μm. In some embodiments, the largest dimension, such as the diameter, of the anucleated cell-derived vesicles in the suspension is about 5 μm to about 7.25 μm, such as about 6 μm to about 7 μm or about 6.25 μm to about 6.75 μm. In some embodiments, the maximum dimension, e.g., diameter, of the anuclear cell-derived vesicles in the suspension is about 7.25 μm or less, such as any of about 7 μm, 6.75 μm, 6.5 μm, 6.25 μm, 6 μm, 5.75 μm, 5.5 μm, 5.25 μm, or 5 μm or less.

[0236] In some embodiments, the anucleated cell-derived vesicles exhibit one or more of the following characteristics: (a) a decreased circulating half-life in mammals compared to the parent anucleated cell, (b) a decreased hemoglobin level compared to the parent anucleated cell, (c) a spherical morphology, (d) an increased surface phosphatidylserine level compared to the parent anucleated cell, or (e) a reduced ATP production compared to the parent anucleated cell.

[0237] In some embodiments, the input or parent anucleated cells are red blood cells or red blood cells, and the anucleated cell-derived vesicles are red blood cell ghosts (RBC ghosts).

[0238] In some embodiments, the anucleated cells are characterized by the acquisition, such as an increased level, of a trait compared to the input or parent anucleated cells.

[0239] In some embodiments, vesicles derived from anucleated cells have increased surface phosphatidylserine levels compared to input or parent anucleated cells. Phosphatidylserine exposure on the cell outer membrane surface is evidence of apoptosis and is recognized by receptors on the surface of phagocytes in a manner that promotes phagocytosis. Methods for measuring phosphatidylserine levels (such as surface phosphatidylserine levels) of anucleated cells, such as cells such as red blood cells, or vesicles derived from anucleated cells are known in the art. For example, Morita, S., et al., J Lipid Res, 53, 2012; Kay, JG et al. al., Sensors (Basel), 11, 2011; and Fabisiak JP et al., Methods Mol Biol, 1105, 2014. In some embodiments, the methods encompassed herein for measuring phosphatidylserine levels in anucleated cells or vesicles derived from anucleated cells include measuring the phosphatidylserine levels of appropriate reference control(s), such as input or parent anucleated cells or a control comprising a population of input or parent anucleated cells.

[0240] In some embodiments, the anuclear cell-derived vesicles prepared by the process have more than about 1.5-fold more phosphatidylserine on their surface compared to the input or parent anuclear cells, such as more than about 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 10-fold, 15-fold, 20-fold, or 25-fold more. In some embodiments, the anuclear cell-derived vesicles have more than about 1.5-fold more phosphatidylserine on their surface compared to the input or parent anuclear cells, such as more than about 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 10-fold, 15-fold, 20-fold, or 25-fold more.

[0241] In some embodiments, the anuclear cell-derived vesicles prepared by the process have more than about 1.5-fold more phosphatidylserine on their surface per unit volume compared to the input or parent anuclear cells, such as more than about any of 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 10-fold, 15-fold, 20-fold, or 25-fold more. In some embodiments, the anuclear cell-derived vesicles have more than about 1.5-fold more phosphatidylserine on their surface per unit volume compared to the input or parent anuclear cells, such as more than about any of 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 10-fold, 15-fold, 20-fold, or 25-fold more.

[0242] In some embodiments, the anuclear cell-derived vesicles prepared by the process have more than about 1.5-fold more phosphatidylserine on their surface per unit surface area compared to the input or parent anuclear cells, such as more than about any of 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 10-fold, 15-fold, 20-fold, or 25-fold more. In some embodiments, the anuclear cell-derived vesicles have more than about 1.5-fold more phosphatidylserine on their surface per unit surface area compared to the input or parent anuclear cells, such as more than about any of 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 10-fold, 15-fold, 20-fold, or 25-fold more.

[0243] In some embodiments, the anucleated cell-derived vesicles prepared by the process have more than about 1.5-fold more phosphatidylserine on their surface per unit of membrane phospholipid compared to the input or parent anucleated cells, such as more than about 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 10-fold, 15-fold, 20-fold, or 25-fold more. In some embodiments, the anucleated cell-derived vesicles have more than about 1.5-fold more phosphatidylserine on their surface per unit of membrane phospholipid compared to the input or parent anucleated cells, such as more than about 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 10-fold, 15-fold, 20-fold, or 25-fold more.

[0244] In some embodiments, vesicles derived from anucleated cells have about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, or 200% or more phosphatidylserine on their surface compared to the input or parent anucleated cells. In some embodiments, vesicles derived from anucleated cells have about 50% to about 200% more phosphatidylserine on their surface compared to the input or parent anucleated cells, such as about 50% to about 100%, about 100% to about 200%, or about 75% to about 125% more.

[0245] In some embodiments, vesicles derived from anucleated cells have about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, or 200% or more phosphatidylserine on their surface per unit volume compared to the input or parent anucleated cells. In some embodiments, vesicles derived from anucleated cells have about 50% to about 200% more phosphatidylserine on their surface per unit volume compared to the input or parent anucleated cells, such as about 50% to about 100%, about 100% to about 200%, or about 75% to about 125% more phosphatidylserine.

[0246] In some embodiments, vesicles derived from anucleated cells have about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, or 200% or more phosphatidylserine on their surface per unit surface area compared to the input or parent anucleated cells. In some embodiments, vesicles derived from anucleated cells have about 50% to about 200% more phosphatidylserine on their surface per unit surface area compared to the input or parent anucleated cells, such as about 50% to about 100%, about 100% to about 200%, or about 75% to about 125% more phosphatidylserine.

[0247] In some embodiments, vesicles derived from anucleated cells have about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, or 200% or more phosphatidylserine on their surface per unit of membrane phospholipid compared to the input or parent anucleated cells. In some embodiments, vesicles derived from anucleated cells have about 50% to about 200% more phosphatidylserine on their surface per unit of membrane phospholipid compared to the input or parent anucleated cells, such as about 50% to about 100%, about 100% to about 200%, or about 75% to about 125% more phosphatidylserine.

[0248] In some embodiments of any of the anuclear cell-derived vesicles described herein, greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99.5% of the total membrane phosphatidylserine is localized in the external membrane leaflet of the anuclear cell-derived vesicles, hi some embodiments, greater than 50% of the total membrane phosphatidylserine is localized in the external membrane leaflet of the anuclear cell-derived vesicles.

[0249] In some embodiments of any of the anuclear cell-derived vesicles described herein, the population profile of the anuclear cell-derived vesicles prepared by the process exhibits a higher average surface phosphatidylserine level compared to the input or parent anuclear cells. In some embodiments, the population profile of the anuclear cell-derived vesicles prepared by the process exhibits a higher average surface phosphatidylserine level compared to the input or parent anuclear cells by about any of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, or 200%. In some embodiments, the population profile of anucleated cell-derived vesicles prepared by the process exhibits a higher average surface phosphatidylserine level by about any of 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, or 100-fold compared to the input or parent anucleated cells.

[0250] In some embodiments of any of the anuclear cell-derived vesicles described herein, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the population profiles of the anuclear cell-derived vesicles prepared by the process exhibit higher surface phosphatidylserine levels compared to the input or parent anuclear cells, or in some embodiments, at least 50% of the population profiles of the anuclear cell-derived vesicles prepared by the process exhibit higher surface phosphatidylserine levels compared to the input or parent anuclear cells.

[0251] In some embodiments, the half-life of anuclear cell-derived vesicles can be further modified. In some embodiments, the half-life of anuclear cell-derived vesicles is increased by further modification. For example, anuclear cell-derived vesicles can be modified to increase the time that anuclear cell-derived vesicles circulate in the bloodstream before clearance in the liver and spleen. In some embodiments, the half-life of anuclear cell-derived vesicles is further decreased by modification. For example, anuclear cell-derived vesicles can be modified to decrease the time that anuclear cell circulates in the bloodstream before clearance in the liver and spleen. In some embodiments, changing the ratio of phospholipids on the surface of anuclear cell-derived vesicles decreases the half-life of anuclear cell-derived vesicles. In some embodiments, increasing the ratio of phosphatidylserine to other phospholipids on the surface of anuclear cell-derived vesicles decreases the half-life of anuclear cell-derived vesicles. The presence of phosphatidylserine on the surface of vesicles derived from anuclear cells can be further increased to decrease the half-life of the vesicles derived from anuclear cells, such as by using any method known in the art for increasing surface phosphatidylserine (see, for example, Hamidi et al., J. Control. Release, 2007, 118(2): 145-60). In some embodiments, vesicles derived from anuclear cells are incubated with lipids or phospholipids prior to delivery to an individual. In some embodiments, vesicles derived from anuclear cells are treated with chemicals such as bis(sulfosuccinimidyl) suberate or other crosslinking agents prior to delivery to an individual. In other embodiments, the surface phosphatidylserine of vesicles derived from anuclear cells can be decreased to increase the half-life of the vesicles derived from anuclear cells. Flippases are enzymes that transport phospholipids from the external surface to the cytosolic surface in the plasma membrane. In some embodiments, vesicles derived from anuclear cells are treated with flippases prior to delivery to an individual. In some embodiments, the anuclear cell-derived vesicles are treated with an enzyme that cleaves phosphatidylserine prior to delivery to an individual. A non-limiting example of an enzyme that cleaves phosphatidylserine is phosphatidylserine carboxylase.

[0252] In some embodiments, vesicles derived from anucleated cells have reduced ATP production compared to input or parent anucleated cells. In some embodiments, vesicles derived from anucleated cells have reduced ATP production or intracellular ATP levels over time. Methods for measuring ATP (such as reduced ATP production or intracellular ATP levels over time) in vesicles derived from anucleated cells, e.g., cells such as red blood cells, or anucleated cells are known in the art. See, e.g., Morciano, G. et al., Nat Protoc, 12, 2017. In some embodiments, ATP production is measured by a surrogate or marker, such as lactate production. In some embodiments, methods for measuring ATP production in anucleated cells or vesicles derived from anucleated cells encompassed herein include measuring ATP production in appropriate reference control(s), such as input or parent anucleated cells or a control comprising a population of input or parent anucleated cells. In some embodiments, methods for measuring ATP production, which allow for comparison of ATP production between the sample and the control, include measuring ATP production in the sample and the control under similar conditions. In some embodiments, a method for measuring ATP production or intracellular ATP levels of vesicles derived from anucleated cells encompassed herein includes measuring ATP production or intracellular ATP levels of vesicles derived from anucleated cells of a population of vesicles derived from anucleated cells at a first time point and a second time point, where the first time point is before the second time point, and comparing the results from the first and second time points.

[0253] In some embodiments, vesicles derived from anucleated cells produce ATP at less than about any of 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the level of ATP produced by the input or parent anucleated cells. In some embodiments, vesicles derived from anucleated cells do not produce ATP.

[0254] In some embodiments, ATP production is determined by lactate assay. In some embodiments, the level of glycolysis can be indirectly measured over time by monitoring the level of lactate production using a fluorescent enzyme assay to measure metabolic activity, such as ATP production, of vesicles derived from input anucleated cells versus non-nucleated cells. For example, input anucleated cells are resuspended at 1 billion cells / mL in citrate-phosphate-dextrose (dCPDA-1) buffer containing adenine, and model antigens and / or adjuvants (20 μg / mL) are delivered at room temperature using an SQZ tube (50 psi, 2.2 μm constriction width) to generate anucleated cell-derived vesicles. The anucleated cell-derived vesicles and untreated input anucleated cells are then incubated at 37°C for the indicated times, and the supernatants are collected. To quantify the level of lactate produced by vesicles derived from input anucleated cells versus non-nucleated cells, the supernatants at each time point can be assayed using the Lactate-Glo assay (Promega). Briefly, the supernatant is subjected to an inactivation and neutralization step prior to the addition of a fluorescent lactate detection reagent. Fluorescence is normalized to a blank control, and absolute lactate levels in the supernatant are determined using a lactate calibration curve (0.1-10 μM). In some embodiments, absolute lactate levels are from about 0 μM to about 200 μM, such as from about 0.01 μM to about 10 μM, or from about 0.01 μM to about 200 μM.

[0255] In some aspects, the present application provides anuclear cell-derived vesicles prepared from parent anuclear cells, which have any one or more of the following characteristics, as further described herein: (a) a reduced circulating half-life in a mammal compared to the parent anuclear cells, (b) a reduced hemoglobin level compared to the parent anuclear cells, (c) a spherical morphology, (d) an increased surface phosphatidylserine level compared to the parent anuclear cells, or (e) reduced ATP production compared to the parent anuclear cells.

[0256] In some aspects, the present application provides anuclear cell-derived vesicles prepared from parent anuclear cells, which have any two or more of the following characteristics, as further described herein: (a) a reduced circulating half-life in mammals compared to the parent anuclear cells, (b) a reduced hemoglobin level compared to the parent anuclear cells, (c) a spherical morphology, (d) an increased surface phosphatidylserine level compared to the parent anuclear cells, or (e) reduced ATP production compared to the parent anuclear cells.

[0257] In some aspects, the present application provides anuclear cell-derived vesicles prepared from parent anuclear cells, which have any three or more of the following properties, as further described herein: (a) a reduced circulating half-life in mammals compared to the parent anuclear cells, (b) a reduced hemoglobin level compared to the parent anuclear cells, (c) a spherical morphology, (d) an increased surface phosphatidylserine level compared to the parent anuclear cells, or (e) reduced ATP production compared to the parent anuclear cells.

[0258] In some aspects, the present application provides anuclear cell-derived vesicles prepared from parent anuclear cells, which have any four or more of the following characteristics, as further described herein: (a) a reduced circulating half-life in mammals compared to the parent anuclear cells, (b) a reduced hemoglobin level compared to the parent anuclear cells, (c) a spherical morphology, (d) an increased surface phosphatidylserine level compared to the parent anuclear cells, or (e) reduced ATP production compared to the parent anuclear cells.

[0259] In some aspects, the present application provides anuclear cell-derived vesicles prepared from parent anuclear cells, which have the following properties, as further described herein: (a) a reduced circulating half-life in a mammal compared to the parent anuclear cells, (b) a reduced hemoglobin level compared to the parent anuclear cells, (c) a spherical morphology, (d) an increased surface phosphatidylserine level compared to the parent anuclear cells, or (e) reduced ATP production compared to the parent anuclear cells.

[0260] In some embodiments, the anucleated cell-derived vesicles are modified to enhance uptake, such as increasing uptake in a tissue or cell compared to uptake of the parent anucleated cell. In some embodiments, the anucleated cell-derived vesicles are modified to enhance uptake, such as increasing uptake in the liver and / or spleen compared to uptake of the parent anucleated cell in the respective tissue. In some embodiments, the anucleated cell-derived vesicles are modified to enhance uptake, such as increasing uptake in phagocytes or antigen-presenting cells, such as macrophages or dendritic cells, compared to uptake of the parent anucleated cell in the respective phagocyte. In some embodiments, the macrophage is an adipose tissue macrophage, monocyte, Kupffer cell, sinus histiocyte, alveolar macrophage, tissue macrophage, microglia, Hofbauer cell, intraglomerular mesangial cell, osteoclast, epitheloid cell, red pulp macrophage, peritoneal macrophage, or LysoMac. In some embodiments, the antigen-presenting cell is a professional antigen-presenting cell. In some embodiments, the antigen-presenting cell is a non-professional antigen-presenting cell. In some embodiments, the antigen-presenting cell is a dendritic cell or a macrophage. In some embodiments, the anucleate-derived vesicles are eliminated by phagocytes and / or antigen-presenting cells in the liver and / or spleen, thereby resulting in antigen presentation, including by CD8+ and CD4+ T cell responses.

[0261] In some embodiments of any of the anuclear cell-derived vesicles described herein, the anuclear cell-derived vesicles exhibit enhanced uptake in tissues or cells compared to the input or parent anuclear cells. In some embodiments, the modified anuclear cell-derived vesicles exhibit enhanced tissue or cellular uptake rates of greater than any one of about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 20-fold, about 30-fold, about 50-fold, about 100-fold, about 200-fold, about 500-fold, or about 1000-fold compared to the input or parent anuclear cells. In some embodiments, the anuclear cell-derived vesicles exhibit enhanced uptake in phagocytes and / or antigen-presenting cells compared to the input or parent anuclear cells. In some embodiments, the phagocytes and / or antigen-presenting cells comprise macrophages and / or dendritic cells. In some embodiments, anuclear cell-derived vesicles exhibit enhanced uptake in the liver, spleen, or macrophages compared to input or parent anuclear cells. In some embodiments, anuclear cell-derived vesicles exhibit enhanced uptake in the liver and / or spleen or by phagocytes and / or antigen-presenting cells compared to input or parent anuclear cells. In some embodiments, anuclear cell-derived vesicles are not eliminated in the lung. In some embodiments, anuclear cell-derived vesicles are eliminated by macrophages in the liver and / or spleen, resulting in antigen presentation, including by CD8+ and CD4+ T cell responses.

[0262] In some embodiments of any of the anuclear cell-derived vesicles described herein, the anuclear cell-derived vesicles are modified to enhance uptake in tissues or cells compared to vesicles derived from unmodified anuclear cells. In some embodiments, the modified anuclear cell-derived vesicles exhibit an enhanced tissue or cellular uptake rate of more than any one of about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 20-fold, about 30-fold, about 50-fold, about 100-fold, about 200-fold, about 500-fold, or about 1000-fold compared to vesicles derived from unmodified anuclear cells. In some embodiments, the anuclear cell-derived vesicles are modified to enhance uptake in phagocytes and / or antigen-presenting cells compared to vesicles derived from unmodified anuclear cells. In some embodiments, the phagocytes and / or antigen-presenting cells include macrophages and / or dendritic cells. In some embodiments, vesicles derived from anuclear cells are modified to enhance uptake in the liver, spleen, or macrophages compared to vesicles derived from unmodified anuclear cells. In some embodiments, vesicles derived from anuclear cells are modified to enhance uptake in the liver and / or spleen or by phagocytes and / or antigen-presenting cells compared to uptake by input or parent anuclear cells. In some embodiments, vesicles derived from anuclear cells are not eliminated in the lung. In some embodiments, vesicles derived from anuclear cells are eliminated by macrophages in the liver and / or spleen, resulting in antigen presentation, including by CD8+ and CD4+ T cell responses.

[0263] In some embodiments, the anuclear cell-derived vesicles comprise CD47 on their surface.

[0264] In some embodiments, the vesicles derived from anuclear cells have not been (a) heat-treated, (b) chemically treated, and / or (c) subjected to hypotonic or hypertonic conditions during preparation of the vesicles derived from anuclear cells. In certain embodiments, the vesicles derived from anuclear cells have not been heat-treated or heat-shocked. In certain embodiments, the vesicles derived from anuclear cells have not been treated with chemicals such as bis(sulfosuccinimidyl) suberate or other crosslinking agents. In certain embodiments, the vesicles derived from anuclear cells have not been further modified to express or contain ionophores or other ion transporters. In certain embodiments, the vesicles derived from anuclear cells do not associate with antibodies such as anti-TER119.

[0265] In some embodiments of any of the anuclear cell-derived vesicles described herein, the osmolality of the cell suspension is maintained throughout the process. In further embodiments, the osmolality of the cell suspension is maintained between about 200 mOsm and about 400 mOsm throughout the process. In some embodiments, the osmolality of the cell suspension is maintained between about 200 mOsm and about 600 mOsm throughout the process. In further embodiments, the osmolality of the cell suspension is maintained between about 200 mOsm and about 800 mOsm throughout the process. In some embodiments, the osmolality of the cell suspension is maintained between about 200 mOsm and about 300 mOsm, about 300 mOsm and about 400 mOsm, about 400 mOsm and about 500 mOsm, about 500 mOsm and about 600 mOsm, about 600 mOsm and about 700 mOsm, about 700 mOsm and about 800 mOsm, about 200 mOsm and about 400 mOsm, about 400 mOsm and about 600 mOsm, or about 600 mOsm and about 800 mOsm. In some embodiments, the osmolality was maintained during preparation of anucleated cell-derived vesicles from input or parent anucleated cells. In some embodiments, the osmolality is maintained between about 200 mOsm and about 600 mOsm, such as between about 200 mOsm and about 300 mOsm, between about 200 mOsm and about 400 mOsm, between about 200 mOsm and about 500 mOsm, between about 300 mOsm and about 500 mOsm, or between about 350 mOsm and about 450 mOsm. In some embodiments, the osmolality is maintained between about 200 mOsm and about 400 mOsm.

[0266] In some embodiments, the cell suspension is contacted with an antigen prior to, concurrently with, and / or after passage through the constriction.

[0267] In some embodiments of any of the anuclear cell-derived vesicles described herein, a composition is provided comprising a plurality of anuclear cell-derived vesicles, in some embodiments, the composition further comprises a pharmaceutically acceptable excipient. Antigens and adjuvants

[0268] In some embodiments of any of the methods or anuclear cell-derived vesicles described herein, the antigen is a disease-associated antigen. In further embodiments, the antigen is a tumor antigen. In some embodiments, the antigen is derived from a lysate. In some embodiments, the lysate is derived from a biopsy of an individual. In some embodiments, the lysate is derived from a biopsy of an individual infected with a pathogen, such as a bacterium or a virus. In some embodiments, the lysate is derived from a biopsy of an individual with a tumor (i.e., a tumor biopsy lysate). Thus, in some embodiments, the lysate is a tumor lysate. In some embodiments, the antigen is derived from a graft lysate. In some embodiments, the lysate is derived from a biopsy of a transplanted organ. In some embodiments, the antigen is a viral antigen, a bacterial antigen, or a fungal antigen. In some embodiments, the antigen is a microorganism.

[0269] In some embodiments of any of the methods or anuclear cell-derived vesicles described herein, the anuclear cell-derived vesicles comprise an antigen comprising an immunogenic epitope. In some embodiments, the antigen is a disease-associated antigen. In some embodiments, the antigen is derived from a peptide or mRNA isolated from a diseased cell. In some embodiments, the antigen is derived from a protein that is ectopically expressed or overexpressed in a diseased cell. In some embodiments, the antigen is derived from a neoantigen, e.g., a cancer-associated neoantigen. In some embodiments, the antigen comprises a neoepitope, e.g., a cancer-associated neoepitope. In some embodiments, the antigen is a non-self antigen. In some embodiments, the antigen is a mutated or otherwise altered self-antigen. In some embodiments, the antigen is a tumor antigen, a viral antigen, a bacterial antigen, or a fungal antigen. In some embodiments, the antigen comprises an immunogenic epitope fused to a heterologous peptide sequence. In some embodiments, the antigen comprises multiple immunogenic epitopes. In some embodiments, some of the multiple immunogenic epitopes are derived from the same source. For example, in some embodiments, some of the multiple immunogenic epitopes are derived from the same viral antigen. In some embodiments, all of the multiple immunogenic epitopes are derived from the same source. In some embodiments, none of the multiple immunogenic epitopes are derived from the same source. In some embodiments, the vesicles derived from anuclear cells contain multiple different antigens. In some embodiments, multiple antigens, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 different types of antigens, are delivered to anuclear cells.

[0270] In some embodiments of any of the methods or anuclear cell-derived vesicles described herein, the antigen is a polypeptide antigen. In some embodiments, the antigen is a non-protein antigen. For example, in some embodiments, the antigen is a lipid antigen. In some embodiments, the antigen is a carbohydrate antigen, such as a polysaccharide. In some embodiments, the antigen is a glycolipid. In some embodiments, a nucleic acid encoding the antigen is delivered to the cell. In some embodiments, the antigen is a whole microorganism, such as an intact bacterium. In some embodiments, the antigen is a disease-associated antigen. In some embodiments, the antigen is derived from an exogenous source, such as a bacterium, a fungus, a virus, or an allergen. In some embodiments, the antigen is a modified antigen. For example, the antigen can be fused to a therapeutic agent or a targeting peptide. In some embodiments, the modified antigen comprises an antigen fused to a polypeptide. In some embodiments, the modified antigen comprises an antigen fused to a targeting peptide. In some embodiments, the modified antigen comprises an antigen fused to a lipid. In some embodiments, the modified antigen comprises an antigen fused to a carbohydrate. In some embodiments, the modified antigen comprises an antigen fused to a nanoparticle. In some embodiments, multiple antigens are delivered to anucleated cells.

[0271] In some embodiments of any of the methods or anuclear cell-derived vesicles described herein, the anuclear cell-derived vesicles comprise an antigen, wherein the antigen comprises an immunogenic epitope. In some embodiments, the antigen is a polypeptide, and the immunogenic epitope is an immunogenic peptide epitope. In some embodiments, the immunogenic peptide epitope is fused to an N-terminal flanking polypeptide and / or a C-terminal flanking polypeptide. In some embodiments, the immunogenic peptide epitope fused to the N-terminal flanking polypeptide and / or the C-terminal flanking polypeptide is a non-naturally occurring sequence. In some embodiments, the N-terminal and / or C-terminal flanking polypeptide is non-natural. In some embodiments, the immunogenic peptide epitope fused to the N-terminal and / or C-terminal flanking polypeptide is synthetic. In some embodiments, the N-terminal and / or C-terminal flanking polypeptide is derived from an immunogenic synthetic long peptide (SLP). In some embodiments, the N-terminal and / or C-terminal flanking polypeptide is derived from a disease-associated immunogenic SLP.

[0272] In some embodiments of any of the methods or anuclear cell-derived vesicles described herein, the anuclear cell-derived vesicles comprise an antigen, and the antigen may be processed into an MHC class I-restricted peptide and / or an MHC class II-restricted peptide. In some embodiments, the antigen may be processed into an MHC class I-restricted peptide. In some embodiments, the antigen may be processed into an MHC class II-restricted peptide. In some embodiments, the antigen comprises multiple immunogenic epitopes and may be processed into an MHC class I-restricted peptide and an MHC class II-restricted peptide. In some embodiments, the anuclear cell-derived vesicles comprising the antigen are taken up by antigen-presenting cells, and the antigen is processed by the antigen-presenting cells into one or more MHC class I-restricted peptides and / or one or more MHC class II-restricted peptides. In some embodiments, the antigen is a CD-1-restricted antigen. In some embodiments, the CD-1-restricted antigen is a lipid antigen. In some embodiments, the antigen comprises multiple immunogenic epitopes and may be processed into multiple CD-1-restricted antigens. In some embodiments, vesicles derived from anuclear cells containing antigens are taken up by antigen-presenting cells, and the antigens are processed by the antigen-presenting cells into one or more CD-1 restricted antigens. In some embodiments, the antigen comprises multiple immunogenic epitopes and can be processed into one or more of: (a) MHC class I restricted peptides; (b) MHC class II restricted peptides; or (c) CD-1 restricted antigens. In some embodiments, some of the multiple immunogenic epitopes are derived from the same source. In some embodiments, all of the multiple immunogenic epitopes are derived from the same source. In some embodiments, none of the multiple immunogenic epitopes are derived from the same source.

[0273] In some embodiments of any of the methods or anuclear cell-derived vesicles described herein, the anuclear cell-derived vesicles comprise a plurality of antigens comprising a plurality of immunogenic epitopes. In some embodiments, following administration of an anuclear cell-derived vesicle comprising a plurality of antigens comprising a plurality of immunogenic epitopes to an individual, none of the plurality of immunogenic epitopes reduces an immune response in the individual against any of the other immunogenic epitopes.

[0274] In some embodiments of any of the methods or anuclear cell-derived vesicles described herein, the anuclear cell-derived vesicles comprise an adjuvant. In some embodiments, the adjuvant is CpG oligodeoxynucleotide (ODN), IFN-α, a STING agonist, a RIG-I agonist, poly I:C (low and / or high molecular weight), polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose (HILTONOL®), imiquimod, resiquimod, and / or lipopolysaccharide (LPS). In some embodiments, the adjuvant is CpG ODN. In some embodiments, the adjuvant is low molecular weight poly I:C. In some embodiments, the CpG ODN is about 50 nucleotides or less (e.g., about 45, 40, 35, 30, 25, 20, or fewer) in length. In some embodiments, the CpG ODN is a class A CpG ODN, a class B CpG ODN, or a class C CpG ODN. In some embodiments, the CpG ODN comprises a nucleotide sequence disclosed in U.S. Provisional Patent Application No. 62 / 641,987. In some embodiments, the anuclear cell-derived vesicles comprise a plurality of different CpG ODNs. For example, in some embodiments, the anuclear cell-derived vesicles comprise a plurality of different CpG ODNs selected from among class A, class B, and class C CpG ODNs. In some embodiments, a plurality of adjuvants, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 different types of adjuvants, are delivered to the anuclear cell.

[0275] In some embodiments, anuclear cell-derived vesicles comprise an antigen and / or an adjuvant. In some embodiments, anuclear cell-derived vesicles comprise an antigen at a concentration of about 1 pM to about 10 mM. In some embodiments, anuclear cell-derived vesicles comprise an adjuvant at a concentration of about 1 pM to about 10 mM. In some embodiments, anuclear cell-derived vesicles comprise an antigen at a concentration of about 0.1 μM to about 10 mM. In some embodiments, anuclear cell-derived vesicles comprise an adjuvant at a concentration of about 0.1 μM to about 10 mM. For example, in some embodiments, the concentration of adjuvant in anuclear cell-derived vesicles is any of about 1 pM, about 10 pM, about 100 pM, about 1 nM, about 10 nM, about 100 nM, about 1 μM, about 10 μM, about 100 μM, about 1 mM, or less than about 10 mM. In some embodiments, the concentration of adjuvant in anuclear cell-derived vesicles is greater than about 10 mM. In some embodiments, the concentration of antigen in anuclear cell-derived vesicles is less than about 1 pM, about 10 pM, about 100 pM, about 1 nM, about 10 nM, about 100 nM, about 1 μM, about 10 μM, about 100 μM, about 1 mM, or about 10 mM. In some embodiments, the concentration of antigen in anuclear cell-derived vesicles is greater than about 10 mM. In some embodiments, the concentration of antigen in the anuclear cell-derived vesicles is between about 1 pM and about 10 pM, between about 10 pM and about 100 pM, between about 100 pM and about 1 nM, between about 1 nM and about 10 nM, between about 10 nM and about 100 nM, between about 100 nM and about 1 μM, between about 1 μM and about 10 μM, between about 10 μM and about 100 μM, between about 100 μM and about 1 mM, or between 1 mM and about 10 mM. In some embodiments, the concentration of adjuvant in the anuclear cell-derived vesicles is between about 1 pM and about 10 pM, between about 10 pM and about 100 pM, between about 100 pM and about 1 nM, between about 1 nM and about 10 nM, between about 10 nM and about 100 nM, between about 100 nM and about 1 μM, between about 1 μM and about 10 μM, between about 10 μM and about 100 μM, between about 100 μM and about 1 mM, or between 1 mM and about 10 mM.

[0276] In some embodiments, the molar ratio of adjuvant to antigen in the anuclear cell-derived vesicles is anywhere between about 10,000:1 and about 1:10,000. For example, in some embodiments, the molar ratio of adjuvant to antigen in the anuclear cell-derived vesicles is any of 10,000:1, about 1,000:1, about 100:1, about 10:1, about 1:1, about 1:10, about 1:100, about 1:1,000, or about 1:10,000. In some embodiments, the anuclear cell-derived vesicles comprise a) an antigen, b) an adjuvant, and / or c) a complex comprising an antigen and an adjuvant.

[0277] In some embodiments of any of the methods or anuclear cell-derived vesicles described herein, the anuclear cell-derived vesicles further comprise an additional agent that enhances the function of the anuclear cell-derived vesicles compared to corresponding anuclear cell-derived vesicles without the additional agent. In some embodiments, the additional agent is a stabilizer or cofactor. In some embodiments, the agent is albumin. In some embodiments, the albumin is mouse, bovine, or human albumin. In some embodiments, the additional agent is a divalent metal cation, glucose, ATP, potassium, glycerol, trehalose, D-sucrose, PEG1500, L-arginine, L-glutamine, or EDTA.

[0278] In some embodiments of any of the methods or anuclear cell-derived vesicles described herein, the anuclear cell-derived vesicles further comprise one or more therapeutic agents. Other payloads

[0279] In some embodiments, the payload is a tolerogenic factor, hi some embodiments, the payload is a polypeptide, a nucleic acid, a lipid, a carbohydrate, a small molecule, a complex (such as a protein-based complex, a nucleic acid complex, a protein-protein complex, a nucleic acid-nucleic acid complex, or a protein-nucleic acid complex), a nanoparticle, a virus, or a viral particle.

[0280] In some embodiments, the payload is selected from the group consisting of uricase (including semi-synthetic forms, e.g., pegloticase), glucocerebrosidase (e.g., imiglucerase, velaglucerase alfa, β-glucosidase), tissue-nonspecific alkaline phosphatase (TNSALP) (e.g., asfotase alfa), lysosomal acid lipase (e.g., sebelipase alfa), alpha-glucosidase (e.g., alglucosidase alfa), α-L-iduronidase (e.g., laronidase), iduronate sulfatase (e.g., idursulfase), heparan sulfate, keratinocyte growth factor receptor 1 (EGFR), and erythrocyte stimulating factor receptor 1 (CRP). and the enzyme is selected from the group consisting of chondroitin sulfate, chondroitin 6-sulfate (e.g., elosulfase alpha), N-acetylgalactosamine-4-sulfatase (e.g., galsulfase), β-glucuronidase, hyaluronidase, α-galactosidase A (e.g., agalsidase beta), phenylalanine hydroxylase, medium-chain acyl-CoA dehydrogenase, gliadin, acetylcholine receptor and receptor-associated protein, thyroid-stimulating hormone receptor (TSHR), desmoglein 1 and 3, aquaporin 4, GADD65, insulin, proinsulin, and preproinsulin.

[0281] In some embodiments, the anuclear cell-derived vesicles are prepared by a process comprising: (a) passing a cell suspension containing input parent anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input parent anuclear cells in the suspension, thereby causing disruption of the input parent anuclear cells large enough to allow passage of a payload and forming anuclear cell-derived vesicles; and (b) incubating the anuclear cell-derived vesicles with the payload for a time sufficient to allow entry of the payload into the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles containing the payload.

[0282] In some embodiments, the anucleate-derived vesicles comprise an antigen and a tolerogenic factor. In some embodiments, the tolerogenic factor enhances suppression of an immune response to the antigen and / or enhances induction of tolerance to the antigen. In some embodiments, the tolerogenic factor can promote tolerogenic presentation of an antigen by antigen-presenting cells. In some embodiments, the tolerogenic factor comprises a polypeptide. In some embodiments, the polypeptide is IL-4, IL-10, IL-13, IL-35, IFN-α, or TGF-β. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the polypeptide is a fragment of a therapeutic polypeptide. In some embodiments, the polypeptide is conjugated to a carbohydrate. In some embodiments, the tolerogenic factor is a nucleic acid. In some embodiments, the nucleic acid can include, without limitation, mRNA, DNA, miRNA, or siRNA. For example, the tolerogenic factor can include siRNA for knocking down the expression of an inflammatory gene. In some embodiments, the tolerogenic agent is a DNA sequence that binds to NF-κB and prevents NF-κB activation and downstream signaling, hi some embodiments, the tolerogenic agent is a small molecule.

[0283] In some embodiments, the tolerogenic factor modulates the expression and / or activity of an immunomodulatory agent, such as an immunostimulatory agent (e.g., a costimulatory molecule), an immunosuppressant, or an inflammatory or anti-inflammatory molecule. In some embodiments, the tolerogenic factor inhibits the expression and / or activity of an immunostimulatory agent (e.g., a costimulatory molecule), enhances the expression and / or activity of an immunosuppressive molecule, inhibits the expression and / or activity of a proinflammatory molecule, and / or enhances the expression and / or activity of an anti-inflammatory molecule. In some embodiments, the tolerogenic factor inhibits the activity of a costimulatory molecule. The interaction between a costimulatory molecule and its ligand is important for the persistence and integration of TCR signaling to stimulate optimal T cell proliferation and differentiation. In some embodiments, the tolerogenic factor reduces the expression of a costimulatory molecule. Exemplary costimulatory molecules expressed on the surface of antigen-presenting cells include, without limitation, CD40, CD80, CD86, CD54, CD83, CD79, Ox40, or ICOS ligand. In some embodiments, the costimulatory molecule is CD80 or CD86. In some embodiments, the tolerogenic factor inhibits expression of a nucleic acid that expresses or modulates the expression of a costimulatory molecule. In some embodiments, the tolerogenic factor deletes a nucleic acid that expresses or modulates the expression of a costimulatory molecule. In some embodiments, deletion of a nucleic acid that expresses or modulates the expression of a costimulatory molecule is achieved by gene editing. In some embodiments, the tolerogenic factor inhibits a costimulatory molecule. In some embodiments, the tolerogenic factor is an siRNA that inhibits a costimulatory molecule. In some embodiments, the tolerogenic factor increases the activity of a transcriptional regulator that suppresses the expression of a costimulatory molecule. In some embodiments, the tolerogenic factor increases the activity of a protein inhibitor that suppresses the expression of a costimulatory molecule. In some embodiments, the tolerogenic factor comprises a nucleic acid encoding an inhibitor of a costimulatory molecule. In some embodiments, the tolerogenic factor degrades a costimulatory molecule. In some embodiments, the tolerogenic factor marks a costimulatory molecule for destruction. For example, tolerogenic factors can enhance the ubiquitination of costimulatory molecules, thereby targeting them for destruction.

[0284] In some embodiments, the tolerogenic factor enhances the expression and / or activity of an immunoinhibitory molecule. In some embodiments, the immunoinhibitory molecule is a co-inhibitory molecule, a transcriptional regulator, or an immunoinhibitory molecule. The co-inhibitory molecule negatively regulates lymphocyte activation. Exemplary co-inhibitory molecules include, but are not limited to, PD-L1, PD-L2, HVEM, B7-H3, TRAIL, immunoglobulin-like transcript (ILT) receptors (ILT2, ILT3, ILT4), FasL, CTLA4, CD39, CD73, and B7-H4. In some embodiments, the co-inhibitory molecule is PD-L1 or PD-L2. In some embodiments, the tolerogenic factor increases the activity of a co-inhibitory molecule. In some embodiments, the tolerogenic factor increases the expression of a co-inhibitory molecule. In some embodiments, the tolerogenic factor encodes a co-inhibitory molecule. In some embodiments, the tolerogenic factor increases the activity of a co-inhibitory molecule. In some embodiments, the tolerogenic factor increases the activity of a transcriptional regulator that can enhance the expression of a co-inhibitory molecule. In some embodiments, the tolerogenic factor increases the activity of a polypeptide that increases the expression of a co-inhibitory molecule. In some embodiments, the tolerogenic factor comprises a nucleic acid encoding an enhancer of a co-inhibitory molecule. In some embodiments, the tolerogenic factor inhibits an inhibitor of a co-inhibitory molecule.

[0285] In some embodiments, the tolerogenic factor increases the expression and / or activity of an immunosuppressive molecule. Exemplary immunosuppressive molecules include, but are not limited to, arginase-1 (ARG1), indoleamine 2,3-dioxygenase (IDO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), nitric oxide (NO), nitric oxide synthase 2 (NOS2), thymic stromal lymphopoietin (TSLP), vascular intestinal peptide (VIP), hepatocyte growth factor (HGF), transforming growth factor-β (ΤGF-β), IFN-α, IL-4, IL-10, IL-13, and IL-35. In some embodiments, the immunosuppressive molecule is NO or IDO. In some embodiments, the tolerogenic factor encodes an immunosuppressive molecule. In some embodiments, the tolerogenic factor increases the activity of an immunosuppressive molecule. In some embodiments, the tolerogenic factor increases the activity of a transcriptional regulator that enhances expression of an immunosuppressive molecule. In some embodiments, the tolerogenic factor increases the activity of a polypeptide that enhances expression of an immunosuppressive molecule. In some embodiments, the tolerogenic factor comprises a nucleic acid encoding an enhancer of an immunosuppressive molecule. In some embodiments, the tolerogenic factor inhibits a negative regulator of an immunosuppressive molecule.

[0286] In some embodiments, the tolerogenic factor inhibits the expression and / or activity of a pro-inflammatory molecule. In some embodiments, the pro-inflammatory molecule is an inflammatory transcription factor. In some embodiments, the tolerogenic factor inhibits an inflammatory transcription factor. In some embodiments, the tolerogenic factor reduces the expression of an inflammatory transcription factor. In some embodiments, the inflammatory transcription factor is a molecule associated with the NF-κB, interferon regulatory factor (IRF), or JAK-STAT signaling pathway. The NF-κB pathway is a prototypic pro-inflammatory signaling pathway that mediates the expression of pro-inflammatory genes, including cytokines, chemokines, and adhesion molecules. Interferon regulatory factors (IRFs) constitute a family of transcription factors that can regulate the expression of pro-inflammatory genes. The JAK-STAT signaling pathway transduces information from extracellular cytokine signals to the nucleus, resulting in DNA transcription and expression of genes involved in immune cell proliferation and differentiation. The JAK-STAT system consists of cell surface receptors, Janus kinases (JAKs), and signal transducers and activators of transcription (STAT) proteins. Exemplary JAK-STAT molecules include, but are not limited to, JAK1, JAK2, JAK3, Tyk2, STAT1, STAT2, STAT3, STAT4, STATS (STAT5A and STAT5B), and STAT6. In some embodiments, the tolerogenic factor enhances expression of a suppressor of cytokine signaling (SOCS) protein. SOCS proteins can inhibit signaling through the JAK-STAT pathway. In some embodiments, the tolerogenic factor inhibits expression of a nucleic acid encoding an inflammatory transcription factor. In some embodiments, the tolerogenic factor deletes a nucleic acid encoding an inflammatory transcription factor. In some embodiments, the tolerogenic factor increases the activity of a transcriptional regulator that suppresses expression of an inflammatory transcription factor. In some embodiments, the tolerogenic factor increases the activity of a protein inhibitor that suppresses expression of an inflammatory transcription factor. In some embodiments, the tolerogenic factor comprises a nucleic acid encoding an suppressor of an inflammatory transcription factor.

[0287] In some embodiments, the tolerogenic factor enhances the expression and / or activity of an anti-inflammatory molecule. In some embodiments, the anti-inflammatory molecule is an anti-inflammatory transcription factor. In some embodiments, the tolerogenic factor enhances an anti-inflammatory transcription factor. In some embodiments, the tolerogenic factor increases the expression of an anti-inflammatory transcription factor. In some embodiments, the tolerogenic factor enhances the expression of a nucleic acid encoding an anti-inflammatory transcription factor. In some embodiments, the tolerogenic factor decreases the activity of a transcriptional regulator that suppresses the expression of an anti-inflammatory transcription factor. In some embodiments, the tolerogenic factor decreases the activity of a protein inhibitor that suppresses the expression of an anti-inflammatory transcription factor. In some embodiments, the tolerogenic factor comprises a nucleic acid encoding an enhancer of an anti-inflammatory transcription factor.

[0288] In some embodiments, the tolerogenic factor comprises a nucleic acid. In some embodiments, the tolerogenic factor is a nucleic acid. Exemplary nucleic acids include, without limitation, recombinant nucleic acids, DNA, recombinant DNA, cDNA, genomic DNA, RNA, siRNA, mRNA, saRNA, miRNA, lncRNA, tRNA, gRNA, and shRNA. In some embodiments, the nucleic acid is homologous to the nucleic acid in the cell. In some embodiments, the nucleic acid is heterologous to the nucleic acid in the cell. In some embodiments, the tolerogenic factor is a plasmid. In some embodiments, the nucleic acid is a therapeutic nucleic acid. In some embodiments, the nucleic acid encodes a therapeutic polypeptide. In some embodiments, the tolerogenic factor comprises a nucleic acid encoding an siRNA, mRNA, miRNA, lncRNA, tRNA, or shRNA. For example, the tolerogenic factor can comprise an siRNA for knocking down expression of an inflammatory gene. In some embodiments, the tolerogenic factor is a DNA sequence that binds to NF-κB and prevents NF-κB activation and downstream signaling.

[0289] In some embodiments, the tolerogenic factor comprises a polypeptide. In some embodiments, the tolerogenic factor is a polypeptide. In some embodiments, the protein or polypeptide is a therapeutic protein, an antibody, a fusion protein, an antigen, a synthetic protein, a reporter marker, or a selectable marker. In some embodiments, the protein is a gene-editing protein or nuclease, such as a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a CRE recombinase, a transposase, an RNA-guided endonuclease (e.g., CAS9 enzyme), a DNA-guided endonuclease, or an integrase enzyme. In some embodiments, the fusion protein can include, without limitation, a chimeric protein drug, such as an antibody-drug conjugate, or a recombinant fusion protein, such as a GST- or streptavidin-tagged protein. In some embodiments, the compound is a transcription factor. Exemplary transcription factors include, but are not limited to, Oct4, Sox2, c-Myc, Klf-4, T-bet, GATA3, FoxP3, and RORγt. In some embodiments, the polypeptide is IL-4, IL-10, IL-13, IL-35, IFN-α, or TGFβ. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the polypeptide is a fragment of a therapeutic polypeptide. In some embodiments, the polypeptide is a peptide nucleic acid (PNA).

[0290] In some embodiments, the tolerogenic factor comprises a protein-nucleic acid complex. In some embodiments, the tolerogenic factor is a protein-nucleic acid complex. In some embodiments, protein-nucleic acid complexes such as clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 are used in genome editing applications. Such complexes contain a sequence-specific DNA-binding domain combined with a non-specific DNA-cleaving nuclease. Such complexes enable targeted genome editing, including the addition, destruction, or alteration of the sequence of a specific gene. In some embodiments, disabled Cas9 (dCas9) is used to block or induce transcription of a target gene. In some embodiments, the tolerogenic factor comprises a Cas9 protein, a guide RNA, and donor DNA. In some embodiments, the tolerogenic factor comprises a nucleic acid encoding the Cas9 protein and a guide RNA or donor DNA. In some embodiments, the gene editing complex targets the expression of a costimulatory molecule (e.g., CD80 and / or CD86).

[0291] In some embodiments, the tolerogenic factor comprises a small molecule. In some embodiments, the tolerogenic factor is a small molecule. In some embodiments, the small molecule inhibits the activity of a costimulatory molecule, enhances the activity of a co-inhibitory molecule, and / or inhibits the activity of a pro-inflammatory molecule. Exemplary small molecules include, without limitation, a pharmaceutical agent, a metabolite, or a radionuclide. In some embodiments, the pharmaceutical agent is a therapeutic agent and / or a cytotoxic agent. In some embodiments, the compound comprises a nanoparticle. Examples of nanoparticles include gold nanoparticles, quantum dots, carbon nanotubes, nanoshells, dendrimers, and liposomes. In some embodiments, the nanoparticle contains or is linked (covalently or non-covalently) to a therapeutic molecule. In some embodiments, the nanoparticle contains a nucleic acid, such as mRNA or cDNA.

[0292] In some embodiments, the vesicles derived from anuclear cells comprise cytokines. In some embodiments, the vesicles derived from anuclear cells comprise agents for modulating genetic material such as DNA. In some embodiments, the vesicles derived from anuclear cells comprise gene editing components such as CRISPR components. In some embodiments, the vesicles derived from anuclear cells comprise agents for modulating RNA, such as reducing the presence of RNA species. In some embodiments, the vesicles derived from anuclear cells comprise siRNA. Methods for generating anucleate cell-derived vesicles

[0293] In certain aspects, methods are provided for generating antigen-containing anuclear cell-derived vesicles, comprising: (a) passing a cell suspension containing input (e.g., parent) anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigen and form anuclear cell-derived vesicles; and (b) incubating the anuclear cell-derived vesicles with the antigen for a time sufficient to allow entry of the antigen into the anuclear cell-derived vesicles, thereby generating antigen-containing anuclear cell-derived vesicles. In some embodiments, the input anuclear cells comprise an adjuvant.

[0294] In certain aspects, a method for generating anuclear cell-derived vesicles containing an adjuvant is provided, comprising: a) passing a cell suspension containing input (e.g., parent) anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the adjuvant and form anuclear cell-derived vesicles; and b) incubating the anuclear cell-derived vesicles with the adjuvant for a time sufficient to allow antigen to enter the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles containing the adjuvant. In some embodiments, the input anuclear cells contain the adjuvant.

[0295] In certain aspects, a method for generating anuclear cell-derived vesicles comprising an antigen and an adjuvant is provided, comprising: a) passing a cell suspension comprising input (e.g., parent) anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input anuclear cells in the suspension, thereby causing disruption of the input anuclear cells large enough to allow passage of the antigen and adjuvant, to form anuclear cell-derived vesicles; and b) incubating the anuclear cell-derived vesicles with the antigen and adjuvant for a time sufficient to allow entry of the antigen and adjuvant into the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles comprising the antigen and adjuvant.

[0296] In some embodiments, the non-nucleated cell-derived vesicles are red blood cell-derived vesicles or platelet-derived vesicles. In some embodiments, the non-nucleated cell-derived vesicles are red blood cell-derived vesicles or reticulocyte-derived vesicles.

[0297] In some embodiments of any of the methods described herein, the input (e.g., parent) anucleated cells are mammalian cells. In some embodiments, the input anucleated cells are monkey, mouse, dog, cat, horse, rat, sheep, goat, pig, or rabbit cells. In some embodiments, the input anucleated cells are human cells. In some embodiments, the input anucleated cells are non-mammalian cells. In some embodiments, the input anucleated cells are chicken, frog, insect, fish, or nematode cells. In some embodiments, the input anucleated cells are red blood cells. In some embodiments, the input anucleated cells are erythroid cells. In some embodiments, the input anucleated cells are precursors of RBCs. In some embodiments, the input anucleated cells are reticulocytes. In some embodiments, the input anucleated cells are platelets.

[0298] In some embodiments, presentation of an antigen in an immunogenic environment enhances or induces an immune response to the antigen. Antigens derived from eryptotic bodies, such as anuclear cell-derived vesicles, which can be eliminated in the immunogenic environment of the liver and spleen, can stimulate or enhance an immune response to the antigen by activating T cells. In some embodiments, the immune response is antigen-specific. Anuclear cell-derived vesicles, such as RBC-derived vesicles, have a limited lifespan and cannot self-repair, causing eryptosis, a process similar to apoptosis, which results in the removal of anuclear cell-derived vesicles from the bloodstream. In some embodiments, antigens can be released after eryptosis of anuclear cell-derived vesicles in an immunogenic environment, where they are then engulfed, processed, and presented by antigen-presenting cells. In some embodiments, anuclear cell-derived vesicles containing antigens are phagocytosed by antigen-presenting cells, such as macrophages, and the antigens are then processed and presented by the antigen-presenting cells. In some embodiments, the antigen-presenting cells are resident macrophages.

[0299] In some embodiments, the circulating half-life of vesicles derived from anuclear cells in a mammal is reduced compared to the input (e.g., parent) anuclear cells. Methods for measuring the half-life of anuclear cells, e.g., cells such as red blood cells, or vesicles derived from anuclear cells, are known in the art. See, e.g., Franco, RS, Transfus Med Hemother, 39, 2012. For example, in some embodiments, methods for measuring the half-life of anuclear cells or vesicles derived from anuclear cells include cohort labeling techniques or random labeling techniques. In some embodiments, methods for measuring the half-life of anuclear cells or vesicles derived from anuclear cells include labeling, reinjecting the cells or vesicles, and measuring their disappearance after reinjection. In some embodiments, methods for measuring the half-life of anuclear cells or vesicles derived from anuclear cells encompassed herein include measuring the half-life of appropriate reference control(s), such as input anuclear cells or a control comprising a population of input anuclear cells.

[0300] In some embodiments, the circulating half-life in a mammal is reduced by more than about 50%, such as by more than about any of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, relative to the input (e.g., parent) anucleated cells. In some embodiments, the circulating half-life in a mammal is reduced by about 50% to about 99.9%, such as by any of about 70% to about 99.9%, about 85% to about 99.9%, or about 95% to about 99.9%, relative to the input anucleated cells. In some embodiments, the circulatory half-life in a mammal is reduced by any of about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.9% compared to the input anuclear cells.

[0301] In some embodiments, the circulation half-life of anuclear cell-derived vesicles is less than about 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 10 days, 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, or 100 days. In some embodiments, the circulation half-life of anuclear cell-derived vesicles is less than about 0.5 minutes, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 3 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 10 days, 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, or 100 days.

[0302] In some embodiments, the input (e.g., parent) anucleated cells are human cells, and the circulation half-life of the anucleated cell-derived vesicles is less than about any of 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 10 days, 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, or 100 days. In some embodiments, the input anucleated cells are human cells, and the circulation half-life of the anucleated cell-derived vesicles is about any of 0.5 minutes, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 3 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 10 days, 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, or 100 days.

[0303] In some embodiments, the input (e.g., parent) anucleated cells are red blood cells, and the hemoglobin level in anucleated cell-derived vesicles is reduced compared to the input anucleated cells. Methods for measuring the hemoglobin level of anucleated cells, such as red blood cells, or anucleated cell-derived vesicles, are known in the art. See, e.g., Chaudhary, R., J Blood Med, 8, 2017. For example, in some embodiments, the method includes measuring metabolic precursors or products to determine hemoglobin turnover. In some embodiments, methods for measuring the hemoglobin level of anucleated cells or anucleated cell-derived vesicles encompassed herein include measuring the hemoglobin level of appropriate reference control(s), such as input anucleated cells or a control comprising a population of input anucleated cells.

[0304] In some embodiments, the hemoglobin level in the anucleated cell-derived vesicles is reduced by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% compared to the input (e.g., parent) anucleated cells. In some embodiments, the hemoglobin level in the anucleated cell-derived vesicles is reduced by about 50% to about 99.9%, such as about 70% to about 99.9%, about 85% to about 99.9%, or about 95% to about 99.9%, compared to the input anucleated cells. In some embodiments, the hemoglobin levels in vesicles derived from anucleated cells are reduced by about any of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.9% compared to the input anucleated cells.

[0305] In some embodiments, the hemoglobin level in vesicles derived from anucleated cells is about 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, or 50% of the hemoglobin level in the input (e.g., parent) anucleated cells.

[0306] In some embodiments, the input (e.g., parent) anucleated cells are red blood cells, and the morphology of the anucleated cell-derived vesicles is modulated from that of the input anucleated cells. Morphology relates to, for example, the shape, structure, geometry, strength, form, smoothness, roughness, circularity, volume, surface area, and / or size classification of cells or cell-derived vesicles. Methods for determining (e.g., measuring) morphology are known in the art. See, for example, Boutros et al., Cell, 163, 2015; Girasole, M. et al., Biochim Biophys Acta Biomembr, 1768, 2007; and Chen et al., Comput Math Methods Med, 2012. In some embodiments, the method for determining morphology involves high-content imaging. For example, cell morphology can be assessed by staining with Hoechst dye followed by automated high-content image analysis. In other examples, morphology can be determined by shifts in forward and side scatter plots from flow cytometry. In some embodiments, the input anuclear cells are red blood cells, and the anuclear cell-derived vesicles have a spherical morphology. In some embodiments, the input anuclear cells are red blood cells, and the anuclear cell-derived vesicles have a reduced biconcave shape compared to the input anuclear cells. In some embodiments, methods for measuring the morphology of anuclear cells or anuclear cell-derived vesicles encompassed herein include measuring the morphology of appropriate reference control(s), such as a control comprising input anuclear cells or a population of input anuclear cells.

[0307] In some embodiments, the input (e.g., parent) anucleated cells are red blood cells, and the anucleated cell-derived vesicles have a reduced biconcave shape compared to the input anucleated cells, such as a reduction of more than any of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.9%.

[0308] In some embodiments, the input (e.g., parent) anucleated cells are red blood cells or red blood cells, and the anucleated cell-derived vesicles are red blood cell ghosts (RBC ghosts).

[0309] In some embodiments, the half-life of anuclear cell-derived vesicles can be further modified. In some embodiments, the half-life of anuclear cell-derived vesicles is increased by further modification. For example, anuclear cell-derived vesicles can be modified to increase the time that anuclear cell-derived vesicles circulate in the bloodstream before clearance in the liver and spleen. In some embodiments, the half-life of anuclear cell-derived vesicles is further decreased by modification. For example, anuclear cell-derived vesicles can be modified to decrease the time that anuclear cell circulates in the bloodstream before clearance in the spleen. In some embodiments, changing the ratio of phospholipids on the surface of anuclear cell-derived vesicles decreases the half-life of anuclear cell-derived vesicles. In some embodiments, increasing the ratio of phosphatidylserine to other phospholipids on the surface of anuclear cell-derived vesicles decreases the half-life of anuclear cell-derived vesicles. For example, any method known in the art for increasing surface phosphatidylserine (Hamidi et al., 2004) can be used. al., J. Control. Release, 2007, 118(2): The presence of phosphatidylserine on the surface of vesicles derived from anuclear cells can be further increased to decrease the half-life of the anuclear cells, such as by using a vesicle-cleaving agent (see, e.g., 145-60). In some embodiments, the anuclear cell-derived vesicles are incubated with a lipid or phospholipid prior to delivery to an individual. In some embodiments, the anuclear cell-derived vesicles are treated with a chemical, such as bis(sulfosuccinimidyl) suberate or other crosslinking agent, prior to delivery to an individual. In other embodiments, the surface phosphatidylserine of vesicles derived from anuclear cells can be reduced to increase the half-life of the anuclear cell-derived vesicles. In some embodiments, the anuclear cell-derived vesicles are treated with a flippase prior to delivery to an individual. Generally, flippases are enzymes that transport phospholipids from the outer leaflet to the cytosolic leaflet of the plasma membrane. In some embodiments, the anuclear cell-derived vesicles are treated with an enzyme that cleaves phosphatidylserine prior to delivery to an individual. A non-limiting example of an enzyme that cleaves phosphatidylserine is phosphatidylserine carboxylase.

[0310] In some embodiments, the anucleated cell-derived vesicles exhibit one or more of the following characteristics: (a) a decreased circulating half-life in mammals compared to the parent anucleated cell, (b) a decreased hemoglobin level compared to the parent anucleated cell, (c) a spherical morphology, (d) an increased surface phosphatidylserine level compared to the parent anucleated cell, or (e) a reduced ATP production compared to the parent anucleated cell.

[0311] In some embodiments of any of the methods described herein, the osmolality of the cell suspension is maintained throughout the process. In further embodiments, the osmolality of the cell suspension is maintained between 200 mOsm and 400 mOsm throughout the process. In some embodiments, the osmolality of the cell suspension is maintained between 200 mOsm and 600 mOsm throughout the process. In further embodiments, the osmolality of the cell suspension is maintained between 200 mOsm and 800 mOsm throughout the process. In some embodiments, the osmolality of the cell suspension is maintained between any one of 200 mOsm and 300 mOsm, 300 mOsm and 400 mOsm, 400 mOsm and 500 mOsm, 500 mOsm and 600 mOsm, 600 mOsm and 700 mOsm, and 700 mOsm and 800 mOsm.

[0312] In some embodiments of any of the methods or anuclear cell-derived vesicles described herein, the anuclear cell-derived vesicles further comprise an additional agent that enhances the function of the anuclear cell-derived vesicles compared to corresponding anuclear cell-derived vesicles without the additional agent. In some embodiments, the additional agent is a stabilizer or cofactor. In some embodiments, the agent is albumin. In some embodiments, the albumin is mouse, bovine, or human albumin. In some embodiments, the additional agent is a divalent metal cation, glucose, ATP, potassium, glycerol, trehalose, D-sucrose, PEG1500, L-arginine, L-glutamine, or EDTA. In some embodiments, the anuclear cell-derived vesicles further comprise one or more therapeutic agents.

[0313] In some embodiments, the anuclear cell-derived vesicles comprise antigens and tolerogenic factors, and the anuclear cell-derived vesicles are prepared by a process comprising: (a) passing a cell suspension comprising input parent anuclear cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input parent anuclear cells in the suspension, thereby causing disruption of the input parent anuclear cells large enough to allow passage of the antigens and tolerogenic factors to form anuclear cell-derived vesicles; and (b) incubating the anuclear cell-derived vesicles with the antigens and tolerogenic factors for a time sufficient to allow entry of the antigens and tolerogenic factors into the anuclear cell-derived vesicles, thereby generating anuclear cell-derived vesicles comprising the antigens and tolerogenic factors.

[0314] In some embodiments, the constriction is contained within a microfluidic channel. In some embodiments, the microfluidic channel comprises multiple constrictions. In some embodiments, the multiple constrictions are arranged in series and / or parallel. In some embodiments, the constriction is between multiple microposts; between multiple microposts arranged in an array; or between one or more movable plates. In some embodiments, the constriction is formed by multiple microposts. In some embodiments, the constriction is formed between multiple microposts arranged in an array. In some embodiments, the constriction is formed by one or more movable plates.

[0315] In some embodiments, the constriction is or is contained within a pore. In some embodiments, the pore is contained in a surface. In some embodiments, the surface is a filter. In some embodiments, the surface is a membrane.

[0316] In some embodiments, the constriction size is about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% of the diameter of the cells, e.g., the maximum diameter of anucleated cells in suspension. In some embodiments, the constriction size is less than about 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10% of the diameter of the cells, e.g., the maximum diameter of anucleated cells in suspension. In some embodiments, the constriction has a width of about 0.1 μm to about 4 μm, e.g., about 1 μm to about 3 μm, about 1.75 μm to about 2.5 μm, or about 2 μm to about 2.5 μm. In some embodiments, the stenosis has a width of about 4 μm, 3.5 μm, 3 μm, 2.5 μm, 2 μm, 1.5 μm, 1 μm, 0.5 μm, or about 0.25 μm. In some embodiments, the stenosis has a width of about 1.8 μm, 1.9 μm, 2 μm, 2.1 μm, 2.2 μm, 2.3 μm, 2.4 μm, 2.5 μm, or 2.6 μm. In some embodiments, the stenosis has a width of about 2.2 μm.

[0317] In some embodiments, the input parental anucleated cells are passed through the constriction under a pressure ranging from about 10 psi to about 150 psi, such as from about 30 psi to about 60 psi, from about 10 psi to about 40 psi, or from about 50 psi to about 90 psi. In some embodiments, the input parental anucleated cells are passed through the constriction under a pressure of at least about any of 5 psi, 10 psi, 15 psi, 20 psi, 25 psi, 30 psi, 35 psi, 40 psi, 45 psi, 50 psi, 55 psi, 60 psi, 65 psi, 70 psi, 75 psi, 80 psi, 85 psi, 90 psi, 95 psi, 100 psi, 105 psi, 110 psi, 115 psi, 120 psi, 125 psi, 130 psi, 135 psi, 140 psi, 145 psi, or 150 psi. In some embodiments, the input parental anucleated cells are passed through the constriction under a pressure of at least about 5 psi and less than about any of 150 psi, 145 psi, 140 psi, 135 psi, 130 psi, 125 psi, 120 psi, 115 psi, 110 psi, 105 psi, 100 psi, 95 psi, 90 psi, 85 psi, 80 psi, 75 psi, 70 psi, 65 psi, 60 psi, 55 psi, 50 psi, 45 psi, 40 psi, 35 psi, 30 psi, 25 psi, 20 psi or 15 psi.

[0318] In some embodiments, the cell suspension is contacted (e.g., first contacted) with the payload before passing through the constriction. In some embodiments, the cell suspension is contacted (e.g., first contacted) with the payload simultaneously with passing through the constriction. In some embodiments, the cell suspension is contacted (e.g., first contacted) with the payload after passing through the constriction. In some embodiments, the cell suspension is contacted with the payload at least simultaneously with and after passing through the constriction. In some embodiments, the cell suspension is contacted with the payload before passing through the constriction, simultaneously with and after passing through the constriction.

[0319] In some embodiments, the anuclear cell-derived vesicles comprising an antigen and an adjuvant described herein are activated antigen carriers (AAC).

[0320] In some embodiments, the anuclear cell-derived vesicles containing antigens for tolearization described herein are tolerizing antigen carriers (TACs). composition

[0321] In some aspects, the present application provides a composition comprising a plurality of any of the anuclear cell-derived vesicles described herein.

[0322] In some embodiments, a composition comprising a plurality of anuclear cell-derived vesicles prepared from parent anuclear cells, wherein the composition has the following characteristics: (a) more than about 20% of the anuclear cell-derived vesicles in the composition, e.g., more than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, have a reduced circulating half-life in a mammal compared to the parent anuclear cells; and (b) more than about 20% of the anuclear cell-derived vesicles in the composition have a reduced circulating half-life in a mammal compared to the parent anuclear cells. %, e.g., more than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the anucleated cell-derived vesicles in the composition have reduced hemoglobin levels compared to the parent anucleated cells; (c) more than about 20%, e.g., more than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the anucleated cell-derived vesicles in the composition have reduced hemoglobin levels compared to the parent anucleated cells. (d) more than about 20% of the anuclear cell-derived vesicles in the composition are RBC ghosts, e.g., more than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%; (e) more than about 20% of the anuclear cell-derived vesicles in the composition are RBC ghosts, e.g., more than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%; %, 85%, 90%, or 95% of the anucleated cell-derived vesicles in the composition have higher levels of phosphatidylserine; or (f) more than about 20%, for example, more than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the anucleated cell-derived vesicles in the composition have reduced ATP production compared to the parent anucleated cells.

[0323] In some embodiments, a composition comprising a plurality of anuclear cell-derived vesicles has: (a) more than about 20% of the anuclear cell-derived vesicles in the composition, e.g., more than any of about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, have a reduced circulating half-life in a mammal compared to the parent anuclear cell; (b) more than about 20% of the anuclear cell-derived vesicles in the composition, e.g., more than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, have a reduced circulating half-life in a mammal compared to the parent anuclear cell; (c) more than about 20%, e.g., more than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the anuclear cell-derived vesicles in the composition have a spherical morphology; (d) more than about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the anuclear cell-derived vesicles in the composition have a spherical morphology; (e) more than about 20% of the vesicles, e.g., more than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, are RBC ghosts; (f) more than about 20% of the anucleated cell-derived vesicles in the composition, e.g., more than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% are RBC ghosts; The composition may be actively tailored to produce a desired profile of anuclear cell-derived vesicles within the composition having one or more selective properties, including one or more of: (f) having dihydrogen stearate; or (g) having more than about 20%, for example, more than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the anuclear cell-derived vesicles in the composition have reduced ATP production compared to the parent anuclear cells.

[0324] In some embodiments, a composition comprising a plurality of anuclear cell-derived vesicles having a desired profile of selection characteristics is prepared from parent anuclear cells using a production method described herein that includes the use of a microfluidic constriction, and production method parameters, including constriction dimensions, speed at which the parent anuclear cells are passed through the constriction, constriction structure (e.g., weir structure and size), processing time, pressure, and buffer composition, are selected to produce a composition comprising a plurality of anuclear cell-derived vesicles having a desired profile of selection characteristics. In some embodiments, a method for producing a composition comprising a plurality of anuclear cell-derived vesicles having a desired profile of selection characteristics comprises selecting a set of parameters, including constriction dimensions, speed at which the parent anuclear cells are passed through the constriction, constriction structure (e.g., weir structure and size), processing time, pressure, and buffer composition, to generate the composition. In some embodiments, a method for producing a composition comprising a plurality of anuclear cell-derived vesicles having a desired profile of selection characteristics comprises generating the composition using a set of parameters, including constriction dimensions, speed at which the parent anuclear cells are passed through the constriction, constriction structure (e.g., weir structure and size), processing time, pressure, and buffer composition.

[0325] For example, in some embodiments, a composition comprising a plurality of anuclear cell-derived vesicles having a desired profile of selection characteristics is prepared using a set of parameters including, for example, a constriction dimension of about 2.2 μm or about 2.5 μm and a pressure of, for example, about 30 psi or about 50 psi. In some embodiments, a composition comprising a plurality of anuclear cell-derived vesicles having a desired profile of selection characteristics is prepared using a set of parameters including, for example, a constriction dimension of about 2.2 μm and a pressure of about 30 psi. In some embodiments, a composition comprising a plurality of anuclear cell-derived vesicles having a desired profile of selection characteristics is prepared using a set of parameters including, for example, a constriction dimension of about 2.2 μm and a pressure of about 50 psi. In some embodiments, a composition comprising a plurality of anuclear cell-derived vesicles having a desired profile of selection characteristics is prepared using a set of parameters including, for example, a constriction dimension of about 2.5 μm and a pressure of about 30 psi. In some embodiments, a composition comprising a plurality of anuclear cell-derived vesicles having a desired profile of selection characteristics is prepared using a set of parameters including, for example, a constriction dimension of about 2.5 μm and a pressure of about 50 psi.

[0326] In some embodiments, the parent anuclear cell is a mammalian cell, including but not limited to, a cell from a human, bovine, equine, feline, canine, rodent, or primate. In some embodiments, the parent anuclear cell is a human cell. In some embodiments, the parent anuclear cell is an anuclear cell from a mammal, including but not limited to, a human, bovine, equine, feline, canine, rodent, or primate.

[0327] In some embodiments, the parent anucleated cells are red blood cells. In some embodiments, the parent anucleated cells are platelets. In some embodiments, the red blood cells are erythrocytes. In some embodiments, the red blood cells are reticulocytes.

[0328] In some embodiments, the circulating half-life of at least about 20%, e.g., at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the anuclear cell-derived vesicles in the mammalian composition is reduced compared to the parent anuclear cell, hi some embodiments, the circulating half-life of at least about 75%, e.g., at least about 80%, 85%, 90%, or 95% of the anuclear cell-derived vesicles in the mammalian composition is reduced compared to the parent anuclear cell. In some embodiments, the circulating half-life of about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the anuclear cell-derived vesicles in the mammalian composition is reduced compared to the parent anuclear cells. In some embodiments, the circulating half-life of at least about 20%, e.g., at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the anuclear cell-derived vesicles in the mammalian composition is reduced by about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more than 90% compared to the parent anuclear cells. In some embodiments, the parent anucleated cells are human cells, and at least about 20%, e.g., at least about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, of the anucleated cell-derived vesicles in the composition have a circulating half-life of less than about any of 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, or 10 days. In some embodiments, the parent anucleated cells are human cells, and at least about 20%, e.g., at least about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, of the anucleated cell-derived vesicles in the composition have a circulating half-life of about any of 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 3 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, or 10 days.

[0329] In some embodiments, the parent anucleated cells are red blood cells, and at least about 20%, e.g., at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the anucleated cell-derived vesicles in the composition have a reduced hemoglobin level compared to the parent anucleated cells. In some embodiments, at least about 75%, e.g., at least about 80%, 85%, 90%, or 95% of the anucleated cell-derived vesicles in the mammalian composition have a reduced hemoglobin level compared to the parent anucleated cells. In some embodiments, 20% of the anucleated cell-derived vesicles in the composition, e.g., at least about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, have hemoglobin levels that are reduced by at least about any of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% compared to the parent anucleated cells. In some embodiments, at least about 20%, e.g., at least about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, of the anuclear cell-derived vesicles in the composition have a hemoglobin level that is about any of 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the level of hemoglobin in the parent anuclear cell.

[0330] In some embodiments, the parent anucleated cells are red blood cells, and at least about 20%, e.g., at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the anucleated cell-derived vesicles in the composition have a modulated morphology compared to the parent anucleated cells. In some embodiments, the parent anucleated cells are red blood cells, and at least about 20%, e.g., at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the anucleated cell-derived vesicles in the composition have a spherical morphology. In some embodiments, the parent anucleated cells are red blood cells, and at least about 20%, e.g., at least about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the anucleated cell-derived vesicles in the composition have a biconcave shape that is reduced compared to the parent anucleated cells, e.g., by more than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0331] In some embodiments, the parent anucleated cells are red blood cells or red blood cells, and at least about 20%, e.g., at least about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the anucleated cell-derived vesicles in the composition are red blood cell ghosts.

[0332] In some embodiments, at least about 20% of the anuclear cell-derived vesicles in the composition, e.g., at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, comprise surface phosphatidylserine. In some embodiments, at least about 20% of the anuclear cell-derived vesicles in the composition, e.g., at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, comprise increased surface phosphatidylserine levels compared to the parent anuclear cells. In some embodiments, at least about 20%, e.g., at least about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the anucleated cell-derived vesicles in the composition have surface phosphatidylserine levels that are greater than or equal to about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, or 200% higher than a composition comprising a plurality of parent anucleated cells.

[0333] In some embodiments, at least about 20%, e.g., at least about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the anuclear cell-derived vesicles in the composition have reduced ATP production compared to the parent anuclear cells. In some embodiments, at least about 20%, e.g., at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the anuclear cell-derived vesicles in the composition produce ATP that is less than about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the level of ATP produced by the parent anuclear cell. In some embodiments, ATP production in the sample and the control is measured under similar conditions. In some embodiments, at least about 20%, e.g., at least about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, do not produce ATP.

[0334] In some embodiments, a composition is provided comprising a plurality of anuclear cell-derived vesicles prepared from parent anuclear cells, the composition comprising the following property, as further described herein: greater than about 20%, e.g., greater than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the anuclear cell-derived vesicles in the composition have a reduced circulating half-life in a mammal compared to the parent anuclear cells.

[0335] In some embodiments, a composition is provided comprising a plurality of anuclear cell-derived vesicles prepared from parent anuclear cells, the composition comprising the following characteristic, as further described herein: greater than about 20%, e.g., greater than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the anuclear cell-derived vesicles in the composition have reduced hemoglobin levels compared to the parent anuclear cells.

[0336] In some embodiments, a composition is provided comprising a plurality of anuclear cell-derived vesicles prepared from parent anuclear cells, wherein the composition has any of the following characteristics: greater than about 20%, e.g., greater than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the anuclear cell-derived vesicles in the composition have a spherical morphology, as further described herein.

[0337] In some embodiments, a composition is provided comprising a plurality of anuclear cell-derived vesicles prepared from parent anuclear cells, wherein the composition has any of the following characteristics: greater than about 20%, e.g., greater than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the anuclear cell-derived vesicles in the composition are RBC ghosts, as further described herein.

[0338] In some embodiments, a composition is provided comprising a plurality of anuclear cell-derived vesicles prepared from parent anuclear cells, wherein the composition has the following characteristics: greater than about 20%, e.g., greater than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the anuclear cell-derived vesicles in the composition have a higher level of phosphatidylserine, as further described herein.

[0339] In some embodiments, a composition is provided comprising a plurality of anuclear cell-derived vesicles prepared from parent anuclear cells, wherein the composition has any of the following characteristics: greater than about 20%, e.g., greater than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the anuclear cell-derived vesicles in the composition have reduced ATP production compared to the parent anuclear cells, as further described herein.

[0340] In some embodiments, a composition comprising a plurality of anuclear cell-derived vesicles prepared from parent anuclear cells, wherein the anuclear cell-derived vesicles in the composition have the following characteristics, as further described herein: (a) more than about 20%, e.g., more than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the anuclear cell-derived vesicles in the composition have a reduced circulating half-life in a mammal compared to the parent anuclear cells; and (b) the anuclear cell-derived vesicles in the composition have a reduced circulating half-life in a mammal compared to the parent anuclear cells. (c) more than about 20%, e.g., more than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, of the anucleated cell-derived vesicles in the composition have reduced hemoglobin levels compared to the parent anucleated cells; (d) more than about 20%, e.g., more than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the anuclear cell-derived vesicles in the composition are RBC ghosts; (e) more than about 20%, e.g., more than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the anuclear cell-derived vesicles in the composition are RBC ghosts; or (f) more than about 20%, e.g., more than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the anucleated cell-derived vesicles in the composition have reduced ATP production compared to the parent anucleated cells.

[0341] In some embodiments, a composition comprising a plurality of anuclear cell-derived vesicles prepared from parent anuclear cells, wherein the anuclear cell-derived vesicles in the composition have the following characteristics, as further described herein: (a) more than about 20%, e.g., more than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the anuclear cell-derived vesicles in the composition have a reduced circulating half-life in a mammal compared to the parent anuclear cells; and (b) the anuclear cell-derived vesicles in the composition have a reduced circulating half-life in a mammal compared to the parent anuclear cells. (c) more than about 20%, e.g., more than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, of the anucleated cell-derived vesicles in the composition have reduced hemoglobin levels compared to the parent anucleated cells; (d) more than about 20%, e.g., more than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the anuclear cell-derived vesicles in the composition are RBC ghosts; (e) more than about 20%, e.g., more than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the anuclear cell-derived vesicles in the composition are RBC ghosts; or (f) more than about 20%, e.g., more than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the anuclear cell-derived vesicles in the composition have reduced ATP production compared to the parent anuclear cells.

[0342] In some embodiments, a composition comprising a plurality of anuclear cell-derived vesicles prepared from parent anuclear cells, wherein the anuclear cell-derived vesicles have the following properties, as further described herein: (a) greater than about 20%, e.g., greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the anuclear cell-derived vesicles in the composition have a reduced circulating half-life in a mammal compared to the parent anuclear cells; (b) the anuclear cell-derived vesicles in the composition have a reduced circulating half-life in a mammal compared to the parent anuclear cells; (c) more than about 20%, e.g., more than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the anucleated cell-derived vesicles in the composition have reduced hemoglobin levels compared to the parent anucleated cells; (d) more than about 20% of the anuclear cell-derived vesicles in the composition are RBC ghosts, e.g., more than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%; (e) more than about 20% of the anuclear cell-derived vesicles in the composition are RBC ghosts, e.g., more than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%; %, 75%, 80%, 85%, 90% or 95% of the anucleated cell-derived vesicles in the composition have higher levels of phosphatidylserine; or (f) more than about 20%, for example, more than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the anucleated cell-derived vesicles in the composition have reduced ATP production compared to the parent anucleated cells.

[0343] In some embodiments, a composition comprising a plurality of anuclear cell-derived vesicles prepared from parent anuclear cells, wherein the anuclear cell-derived vesicles have the following properties, as further described herein: (a) greater than about 20%, e.g., greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the anuclear cell-derived vesicles in the composition have a reduced circulating half-life in a mammal compared to the parent anuclear cells; (b) the anuclear cell-derived vesicles in the composition have a reduced circulating half-life in a mammal compared to the parent anuclear cells; (c) more than about 20%, e.g., more than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the anucleated cell-derived vesicles in the composition have reduced hemoglobin levels compared to the parent anucleated cells; (d) more than about 20% of the anuclear cell-derived vesicles in the composition are RBC ghosts, e.g., more than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%; (e) more than about 20% of the anuclear cell-derived vesicles in the composition are RBC ghosts, e.g., more than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%; %, 75%, 80%, 85%, 90% or 95% of the anucleated cell-derived vesicles in the composition have higher levels of phosphatidylserine; or (f) more than about 20%, for example, more than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the anucleated cell-derived vesicles in the composition have reduced ATP production compared to the parent anucleated cells.

[0344] In some embodiments, a composition comprising a plurality of anuclear cell-derived vesicles prepared from parent anuclear cells, wherein the anuclear cell-derived vesicles have the following properties, as further described herein: (a) greater than about 20%, e.g., greater than any of about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the anuclear cell-derived vesicles in the composition have a reduced circulating half-life in a mammal compared to the parent anuclear cells; and (b) the anuclear cell-derived vesicles in the composition have a reduced circulating half-life in a mammal compared to the parent anuclear cells. (c) more than about 20% of the anuclear cell-derived vesicles in the composition, e.g., more than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, have reduced hemoglobin levels compared to the parent anuclear cells; (d) more than about 20% of the anuclear cell-derived vesicles in the composition are RBC ghosts, e.g., more than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%; (e) more than about 20% of the anuclear cell-derived vesicles in the composition are RBC ghosts, e.g., more than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, , 65%, 70%, 75%, 80%, 85%, 90% or 95% have higher levels of phosphatidylserine, and (f) more than about 20%, for example, more than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the anucleated cell-derived vesicles in the composition have reduced ATP production compared to the parent anucleated cells.

[0345] In some embodiments described herein, one or more properties of a composition comprising a plurality of anuclear cell-derived vesicles are based on a comparison to a parent anuclear cell population from which the anuclear cell-derived vesicles were prepared. In some embodiments, the comparison is based on an average value measured for the parent anuclear cell population. In some embodiments, the comparison is based on a range of values ​​measured for the parent anuclear cell population.In some embodiments, a composition comprising a plurality of anuclear cell-derived vesicles prepared from a population of parent anuclear cells, wherein the anuclear cell-derived vesicles have the following characteristics: (a) more than about 20% of the anuclear cell-derived vesicles in the composition, e.g., more than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, have a reduced circulating half-life in a mammal compared to the average value of the population of parent anuclear cells; and (b) more than about 20% of the anuclear cell-derived vesicles in the composition have a reduced circulating half-life in a mammal compared to the average value of the population of parent anuclear cells. (c) more than 20%, e.g., more than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the anucleated cell-derived vesicles in the composition have reduced hemoglobin levels compared to the mean value of the parent anucleated cell population. (d) more than 20% of the anuclear cell-derived vesicles in the composition are RBC ghosts, e.g., about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the anuclear cell-derived vesicles in the composition are RBC ghosts; (e) more than 20% of the anuclear cell-derived vesicles in the composition are RBC ghosts, e.g., about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the anuclear cell-derived vesicles in the composition are RBC ghosts; or (f) more than 20%, e.g., more than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the anucleated cell-derived vesicles in the composition have one or more of the following characteristics: (i) either more than 95% have higher levels of phosphatidylserine compared to the average of the parent anucleated ...

Claims

[Claim 1] The invention described in this specification.

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