Anucleate cell-derived vaccines

Abstract

The present invention provides 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. 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, wherein 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.

Description

ANUCLEATE CELL-DERIVED VACCINES CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 62 / 797,185, filed on January 25, 2019, U.S. Provisional Application No. 62 / 797,187, filed on January 25, 2019, U.S. Provisional Application No. 62 / 933,301, filed on November 8, 2019, and U.S. Provisional Application No. 62 / 933,302, filed on November 8, 2019, the entire contents of each of which are incorporated herein by reference. FIELD OF THE INVENTION

[0002] The present disclosure relates generally to methods for stimulating an immune response or methods of treating cancer, infectious diseases or viral-associated disease by delivering an anucleate cell-derived vesicle to an individual, wherein the anucleate cell-derived vesicles are loaded with an antigen and / or adjuvant. In some embodiments, the antigen and / or adjuvant is delivered to an anucleate cell by passing a cell suspension through a cell-deforming constriction. BACKGROUND

[0003] The complexity of the immune system and immune response to foreign matter makes challenging the development of efficacious approaches for triggering an in vivo antigen-specific immune response. In addition to the continued development of agents, such as small molecules and polypeptide- and / or nucleotide-based vaccines, capable of triggering antigen-specific immune responses, carrier strategies for nse with such agents are in need of further development to optimize delivery and immune response. Carriers known in the art, including polymer-based carriers, particle carriers, liposomes, and cell-based vesicles, such as those derived from red blood cells, still face challenges limiting their use for triggering an in vivo antigen-specific immune response. For example, use of red blood cells as a carrier is difficult due to challenges associated with manipulation of red blood cells to associate antigenic material given that red blood cells are irregularly shaped (biconcave), anucleate, and transcriptionally inactive. As a result, standard transfection techniques do not work. To overcome these challenges, methods of using red blood cells as a carrier for triggering an immune response have focused on conjugating materials to the surface of erythrocytes. See, e.g., Lorentz et al., Sci. Adv, 1:e15001122015; Grimm et al., Sci Rep, 5, 2015; and Kontos ez al., Proc Natl Acad Sci USA, 110, 2013. Initial work using surface conjugation has shown promising results with model antigens and mouse models of Type 1 diabetes but has some significant drawbacks including: (a) the need for chemically modified antigens for attachment; (b) the limited surface area for loading; and (c) immunogenicity.

[0004] References that describe methods of using microfluidic constrictions to deliver compounds to cells include W02013059343, W02015023982, W02016070136, W02016077761, and WO / 2017 / 192785.

[0005] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety. BRIEF SUMMARY OF THE INVENTION

[0006] In some aspects, the invention provides methods for delivering an antigen into an anucleate cell-derived vesicle, the method comprising: a) passing a cell suspension comprising an input (e.g., parent) anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell-derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle. In some embodiments, the input anucleate cell further comprises an adjuvant.

[0007] In some aspects, the invention provides methods for delivering an adjuvant into an anucleate cell-derived vesicle, the method comprising: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the adjuvant to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell-derived vesicle with the adjuvant for a sufficient time to allow the adjuvant to enter the anucleate cell-derived vesicle. In some embodiments, input anucleate cell further comprises an antigen.

[0008] In some aspects, the invention provides methods for delivering an antigen and an adjuvant into an anucleate cell-derived vesicle, the method comprising: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen and the adjuvant to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell-derived vesicle with the antigen and the adjuvant for a sufficient time to allow the antigen and the adjuvant to enter the anucleate cell-derived vesicle.

[0009] In some embodiments, the invention provides methods for stimulating an immune response to an antigen in an individual, the method comprising administering to the individual an effective amount of an anucleate cell-derived vesicle comprising an antigen, wherein the anucleate cell-derived vesicle comprising the antigen is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell- deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell-derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle. In some embodiments, the method further comprises administering an adjuvant systemically to the individual. In some embodiments, the adjuvant is administered systemically before, after or at the same time as the anucleate cell derived vesicle. In some embodiments, the input anucleate cell comprises an adjuvant.

[0010] In some aspects, the invention provides methods for stimulating an immune response to an antigen in an individual, the method comprising administering to the individual an effective amount of an anucleate cell-derived vesicle comprising an antigen and an adjuvant, wherein the anucleate cell-derived vesicle comprising the antigen and the adjuvant is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen and the adjuvant to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell-derived vesicle with the antigen and the adjuvant for a sufficient time to allow the antigen and the adjuvant to enter the anucleate cell-derived vesicle. In some embodiments, the method further comprises administering an adjuvant systemically to the individual. In some embodiments, the adjuvant is administered systemically before, after or at the same time as the anucleate cell-derived vesicle. In some aspects, the invention provides methods for treating a disease in an individual, comprising administering to the individual an anucleate cell-derived vesicle comprising a disease-associated antigen, wherein an immune response against the antigen ameliorates conditions of the disease, and wherein the anucleate cell-derived vesicle comprising the disease- associated antigen is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell-derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle.

[0011] In some aspects, the invention provides methods for preventing a disease in an individual, comprising administering to the individual an anucleate cell-derived vesicle comprising a disease-associated antigen, wherein an immune response against the antigen prevents development of the disease, and wherein the anucleate cell-derived vesicle comprising the disease-associated antigen is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell- derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle. In some embodiments, the invention provides methods for vaccinating an individual against an antigen, comprising administering to the individual an anucleate cell- derived vesicle comprising the antigen, wherein the anucleate cell-derived vesicle comprising the antigen is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell-derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle. In some embodiments, the method further comprises administering an adjuvant systemically to the individual. In some embodiments, the adjuvant is administered systemically before, after or at the same time as the anucleate cell derived vesicle. In some embodiments, the input anucleate cell comprises an adjuvant.

[0012] In some aspects, the invention provides methods for treating a disease in an individual, comprising administering to the individual an anucleate cell-derived vesicle comprising a disease-associated antigen and an adjuvant, wherein an immune response against the antigen ameliorates conditions of the disease, and wherein the anucleate cell-derived vesicle comprising the disease-associated antigen and the adjuvant is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell- deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen and an adjuvant to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell-derived vesicle with the antigen and the adjuvant for a sufficient time to allow the antigen and the adjuvant to enter the anucleate cell-derived vesicle. In some aspects, the invention provides methods for preventing a disease in an individual, comprising administering to the individual an anucleate cell-derived vesicle comprising a disease-associated antigen and an adjuvant, wherein an immune response against the antigen prevents development of the disease, and wherein the anucleate cell-derived vesicle comprising a disease-associated antigen and an adjuvant is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen and an adjuvant to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell-derived vesicle with the antigen and the adjuvant for a sufficient time to allow the antigen and the adjuvant to enter the anucleate cell-derived vesicle. In some aspects, the invention provides methods for vaccinating an individual against an antigen, comprising administering to the individual an anucleate cell-derived vesicle comprising the antigen and an adjuvant, wherein the anucleate cell-derived vesicle comprising the antigen and the adjuvant is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen and an adjuvant to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell- derived vesicle with the antigen and the adjuvant for a sufficient time to allow the antigen and the adjuvant to enter the anucleate cell-derived vesicle.

[0013] In some aspects, the invention provides methods for treating a disease in an individual, wherein an immune response against a disease-associated antigen ameliorates conditions of the disease, the method comprising a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen to pass through to form an anucleate cell-derived vesicle; b) incubating the anucleate cell-derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle, thereby generating an anucleate cell-derived vesicle comprising the antigen; and c) administering the anucleate cell-derived vesicle comprising the antigen to the individual. In some aspects, the invention provides methods for preventing a disease in an individual, wherein an immune response against a disease-associated antigen prevents development of the disease, the method comprising a) passing a cell suspension comprising an input anucleate cell through a cell- deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen to pass through to form an anucleate cell-derived vesicle; b) incubating the anucleate cell-derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle, thereby generating an anucleate cell-derived vesicle comprising the antigen; and ¢) administering the anucleate cell-derived vesicle comprising 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 an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen to pass through to form an anucleate cell-derived vesicle; b) incubating the anucleate cell-derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle, thereby generating an anucleate cell-derived vesicle comprising the antigen; and ¢) administering the anucleate cell-derived vesicle comprising the antigen to the individual. In some embodiments, the method further comprises administering an extravesicular adjuvant systemically to the individual. In some embodiments, the extravesicular adjuvant is administered before, after or at the same time as the anucleate cell-derived vesicle. In some embodiments, the input anucleate cell comprises an adjuvant.

[0014] In some aspects, the invention provides methods for treating a disease in an individual, wherein an immune response against a disease-associated antigen ameliorates conditions of the disease, the method comprising a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the disease-associated antigen and an adjuvant to pass through to form an anucleate cell-derived vesicle; b) incubating the anucleate cell-derived vesicle with the antigen and the adjuvant for a sufficient time to allow the antigen and the adjuvant to enter the anucleate cell-derived vesicle, thereby generating an anucleate cell- derived vesicle comprising the antigen and the adjuvant; and ¢) administering the anucleate cell- derived vesicle comprising the antigen and the adjuvant to the individual. In some aspects, the invention provides methods for preventing a disease in an individual, wherein an immune response against a disease-associated antigen prevents development of the disease, the method comprising a) passing a cell suspension comprising an input anucleate cell through a cell- deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen and an adjuvant to pass through to form an anucleate cell-derived vesicle; b) incubating the anucleate cell-derived vesicle with the antigen and the adjuvant for a sufficient time to allow the antigen and the adjuvant to enter the anucleate cell-derived vesicle, thereby generating an anucleate cell-derived vesicle comprising the antigen and the adjuvant; and ¢) administering the anucleate cell-derived vesicle comprising the antigen and the adjuvant to the individual. In some embodiments, the invention provides methods for vaccinating an individual against an antigen, the method comprising, a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen and an adjuvant to pass through to form an anucleate cell-derived vesicle; b) incubating the anucleate cell-derived vesicle with the antigen and the adjuvant for a sufficient time to allow the antigen and the adjuvant to enter the anucleate cell-derived vesicle, thereby generating an anucleate cell-derived vesicle comprising the antigen and the adjuvant; and ¢) administering the anucleate cell-derived vesicle comprising the antigen and the adjuvant to the individual. In some embodiments, the method further comprises administering an extravesicular adjuvant systemically to the individual. In some embodiments, the extravesicular adjuvant is administered before, after or at the same time as the anucleate cell derived vesicle.

[0015] In some embodiments of the above aspects, the disease is cancer, an infectious disease or a viral-associated disease. In some embodiments, the anucleate cell-derived vesicle is autologous to the individual. In some embodiments, the anucleate cell-derived vesicle is allogeneic to the individual. In some embodiments, the anucleate cell-derived vesicle is in a pharmaceutical formulation. In some embodiments, the anucleate cell-derived vesicle is administered systemically. In some embodiments, the anucleate cell-derived vesicle is administered intravenously, intraarterially, subcutaneously, intramuscularly, or intraperitoneally.

[0016] In some embodiments of the above aspects, the anucleate cell-derived vesicle is administered to the individual in combination with a therapeutic agent. In some embodiments, the therapeutic agent is administered before, after or at the same time as the anucleate cell- derived vesicle. 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-a, IFN-y, 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 (VICN1) 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 TGFb. In some embodiments, the anucleate cell-derived vesicle is administered to the individual in combination with a chemotherapy or a radiation therapy. In some embodiments, the anucleate cell-derived vesicle is administered to the individual in combination with one or more agents that improve antigen presentation {e.g., CD40 or Ox40L), improve T cell proliferation, and / or improve tumor microenvironments (e.g., ICOS).

[0017] In some embodiments of the above aspects, the antigen is capable of being processed into an MHC class I-restricted peptide and / or an MHC class Il-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 with a polypeptide. In some embodiments, the modified antigen comprises an antigen fused with a targeting peptide. In some embodiments, the modified antigen comprises an antigen fused with a lipid. In some embodiments, the modified antigen comprises an antigen fused with a carbohydrate. In some embodiments, the modified antigen comprises an antigen fused with a nanoparticle. In some embodiments, a nucleic acid encoding the antigen is delivered to the cell. In some embodiments, a plurality of antigens is delivered to the anucleate cell-derived vesicle.

[0018] In some embodiments of the above aspects, the adjuvant is a CpG ODN, IFN-a, STING agonists, RIG-I agonists, 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 anucleate cell is a red blood cell. In some embodiments, the red blood cell is an erythrocyte. In some embodiments, the red blood cell is a reticulocyte. In some embodiments, the input anucleate cell is a platelet. In some embodiments, the input anucleate cell is a mammalian cell. In some embodiments, the input anucleate cell is a monkey, mouse, dog, cat, horse, rat, sheep, goat, pig, or rabbit cell. In some embodiments, the input anucleate cell is a human cell.

[0020] In some embodiments of the above aspects, the constriction is contained within a microfluidic channel. In some embodiments, the microfluidic channel comprises a plurality of constrictions. In some embodiments, the plurality of constrictions is arranged in series and / or in parallel. In some embodiments, the constriction is between a plurality of micropillars; between a plurality of micropillars configured in an array; or between one or more movable plates. In some embodiments, the constriction is a pore or 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 anucleate cell 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 anucleate cell in suspension. In some embodiments, the constriction has a width of about 0.25 um to about 4 um. In some embodiments, the constriction has a width of about 4 pm, about 3.5 um, about 3 pm, about 2.5 um, about 2 um, about 1.5 um, about 1 pm, about 0.5 um, or about 0.25 pm. In some embodiments, the constriction has a width of about 2.2 pm. In some embodiments, the input anucleate cells are passed through the constriction under a pressure ranging from about 10 psi to about 90 psi. In some embodiments, said cell suspension is contacted with the antigen before, concurrently, or after passing through the constriction.

[0021] In some aspects, the invention provides an anucleate cell-derived vesicle comprising an antigen, wherein the anucleate cell-derived vesicle comprising the antigen is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell-derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle; thereby generating the anucleate cell-derived vesicle comprising the antigen. In some embodiments, the input anucleate cell comprises an adjuvant. In some aspects, the invention provides an anucleate cell-derived vesicle comprising an adjuvant, wherein the anucleate cell-derived vesicle comprising the adjuvant is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the adjuvant to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell-derived vesicle with the adjuvant for a sufficient time to allow the adjuvant to enter the anucleate cell-derived vesicle; thereby generating the anucleate cell-derived vesicle comprising the adjuvant. In some embodiments, the input anucleate cell comprises an antigen. In some aspects, the invention provides an anucleate cell-derived vesicle comprising an antigen and an adjuvant, wherein the anucleate cell-derived vesicle comprising the antigen and the adjuvant is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen and the adjuvant to pass through to form an anucleate cell- derived vesicle; and b) incubating the anucleate cell-derived vesicle with the antigen and the adjuvant for a sufficient time to allow the antigen and the adjuvant to enter the anucleate cell- derived vesicle; thereby generating the anucleate cell-derived vesicle comprising the antigen and the adjuvant. In some embodiments, the anucleate cell-derived vesicle is a red blood cell-derived vesicle or a platelet-derived vesicle. In some embodiments, the red blood cell-derived vesicle is an erythrocyte-derived vesicle, or a reticulocyte-derived vesicle.

[0022] In some embodiments of the above anucleate cell-derived vesicles, the antigen is capable of being 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 with a polypeptide. In some embodiments, the modified antigen comprises an antigen fused with a targeting peptide. In some embodiments, the modified antigen comprises an antigen fused with a lipid. In some embodiments, the modified antigen comprises an antigen fused with a carbohydrate. In some embodiments, the modified antigen comprises an antigen fused with a nanoparticle. In some embodiments, a plurality of antigens is delivered to the anucleate cell- derived vesicle.

[0023] In some embodiments of the above anucleate cell-derived vesicles, the adjuvant is a CpG ODN, IFN-q, STING agonists, RIG-I agonists, 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 anucleate cell-derived vesicles, the input anucleate cell is a red blood cell. In some embodiments, the input anucleate cell is an erythrocyte. In some embodiments, the input anucleate cell is a reticulocyte. In some embodiments, the input anucleate cell is a platelet. In some embodiments, the input anucleate cell is a mammalian cell. In some embodiments, the input anucleate cell is a monkey, mouse, dog, cat, horse, rat, sheep, goat, pig, or rabbit cell. In some embodiments, the input anucleate cell is a human cell.

[0025] In some embodiments of the above anucleate cell-derived vesicles, the half-life of the anucleate cell-derived vesicle following administration to a mammal is decreased compared to a half-life of the input anucleate cell following administration to the mammal. In some embodiments, the hemoglobin content of the anucleate cell-derived vesicle is decreased compared to the hemoglobin content of the input anucleate cell. In some embodiments, ATP production of the anucleate cell-derived vesicle is decreased compared to ATP production of the input anucleate cell. In some embodiments, the anucleate cell-derived vesicle exhibits a spherical morphology. In some embodiments, the input anucleate cell is an erythrocyte and wherein the anucleate cell-derived vesicle has a reduced biconcave shape compared to the input anucleate cell. In some embodiments, the anucleate cell-derived vesicle is a red blood cell ghost. In some embodiments, the anucleate cell-derived vesicles prepared by the process have greater than about 1.5 fold more phosphatidylserine on its surface compared to the input anucleate cell. In some embodiments, a population profile of anucleate cell-derived vesicles prepared by the process exhibits higher average phosphatidylserine levels on the surface compared to the input anucleate cells. In some embodiments, at least 50% of the population profile of anucleate cell- derived vesicles prepared by the process exhibits higher phosphatidylserine levels on the surface compared to the input anucleate cells.

[0026] In some embodiments, the anucleate cell-derived vesicle exhibits enhanced uptake in a tissue or cell compared to the input anucleate cell. In some embodiments, the anucleate cell- derived vesicle exhibits enhanced uptake in phagocytic cells and / or antigen presenting cells compared to the input anucleate cell. In some embodiments, the anucleate cell-derived vesicle is modified to enhance uptake in a tissue or cell compared to an unmodified anucleate cell-derived vesicle. In some embodiments, the anucleate cell-derived vesicle is modified to enhance uptake in phagocytic cells and / or antigen presenting cells compared to an unmodified anucleate cell- derived vesicle. In some embodiments, the phagocytic cells and / or antigen presenting cells comprise one or more of a dendritic cell or macrophage. In some embodiments, the tissue or cell comprises one or more of liver or spleen. In some embodiments, the anucleate cell-derived vesicle comprises CD47 on its surface.

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

[0028] In some embodiments of the above anucleate cell-derived vesicles, the constriction is contained within a microfluidic channel. In some embodiments, the microfluidic channel comprises a plurality of constrictions. In some embodiments, the plurality of constrictions is arranged in series and / or in parallel. In some embodiments, the constriction is between a plurality of micropillars; between a plurality of micropillars configured in an array; or between one or more movable plates. In some embodiments, the constriction is a pore or contained within a pore. In some embodiments, the pore is contained in 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 anucleate cell 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 anucleate cell in suspension. In some embodiments, the constriction has a width of about 0.25 pm to about 4 pm. In some embodiments, the constriction has a width of about 4 pm, 3.5 um, about 3 um, about 2.5 pm, about 2 pm, about 1.5 um, about 1 pm, about 0.5 pm, or about 0.25 um. In some embodiments, the constriction has a width of about 2.2 um. In some embodiments, the input anucleate cells are passed through the constriction under a pressure ranging from about 10 psi to about 90 psi. In some embodiments, said cell suspension is contacted with the antigen before, concurrently, or after passing through the constriction.

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

[0030] In some aspects, the invention provides methods for generating an anucleate cell- derived vesicle comprising an antigen, the method comprising: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen to pass through to form an anucleate cell-derived vesicle; b) incubating the anucleate cell-derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle, thereby generating an anucleate cell-derived vesicle comprising the antigen. In some embodiments, the input anucleate cell comprises an adjuvant.

[0031] In some aspects, the invention provides methods for generating an anucleate cell- derived vesicle comprising an adjuvant, the method comprising: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the adjuvant to pass through to form an anucleate cell-derived vesicle; b) incubating the anucleate cell-derived vesicle with the adjuvant for a sufficient time to allow the adjuvant to enter the anucleate cell-derived vesicle, thereby generating an anucleate cell-derived vesicle comprising the adjuvant. In some embodiments, the input anucleate cell comprises an antigen.

[0032] In some aspects, the invention provides methods for generating an anucleate cell- derived vesicle comprising an antigen and an adjuvant, the method comprising: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen and the adjuvant to pass through to form an anucleate cell-derived vesicle; b) incubating the anucleate cell-derived vesicle with the antigen and the adjuvant for a sufficient time to allow the antigen and the adjuvant to enter the anucleate cell-derived vesicle, thereby generating an anucleate cell-derived vesicle comprising the antigen and the adjuvant.

[0033] In some embodiments of the above method for generating an anucleate cell-derived vesicle, the anucleate cell-derived vesicle is a red blood cell-derived vesicle or a platelet derived vesicle. In some embodiments, the red blood cell-derived vesicle is an erythrocyte-derived vesicle or a reticulocyte-derived vesicle.

[0034] In some embodiments of the above method for generating an anucleate cell-derived vesicle, the antigen is capable of being processed into an MHC class I-restricted peptide and / or an MHC class I-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 with a polypeptide. In some embodiments, the modified antigen comprises an antigen fused with a targeting peptide. In some embodiments, the modified antigen comprises an antigen fused with a lipid. In some embodiments, the modified antigen comprises an antigen fused with a carbohydrate. In some embodiments, the modified antigen comprises an antigen fused with a nanoparticle. In some embodiments, a plurality of antigens is delivered to the anucleate cell-derived vesicle.

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

[0036] In some embodiments of the above method for generating an anucleate cell-derived vesicle, the input anucleate cell is a red blood cell. In some embodiments, the input anucleate cell is an erythrocyte. In some embodiments, the input anucleate cell is a reticulocyte. In some embodiments, the input anucleate cell is a platelet. In some embodiments, the input anucleate cell is a mammalian cell. In some embodiments, the input anucleate cell is a monkey, mouse, dog, cat, horse, rat, sheep, goat, pig, or rabbit cell. In some embodiments, the input anucleate cell is a human cell.

[0037] In some embodiments of the above method for generating an anucleate cell-derived vesicle, the half-life of the anucleate cell-derived vesicle following administration to a mammal is decreased compared to a half-life of the input anucleate cell following administration to the mammal. In some embodiments, the hemoglobin content of the anucleate cell-derived vesicle is decreased compared to the hemoglobin content of the input anucleate cell. In some embodiments, ATP production of the anucleate cell-derived vesicle is decreased compared to ATP production of the input anucleate cell. In some embodiments, the anucleate cell-derived vesicle exhibits a spherical morphology. In some embodiments, the input anucleate cell is an erythrocyte and wherein the anucleate cell-derived vesicle has a reduced biconcave shape compared to the input anucleate cell. In some embodiments, the anucleate cell-derived vesicle is ared blood cell ghost. In some embodiments, the anucleate cell-derived vesicles prepared by the process have greater than about 1.5 fold more phosphatidylserine on its surface compared to the input anucleate cell. In some embodiments, a population profile of anucleate cell-derived vesicles prepared by the process exhibits higher average phosphatidylserine levels on the surface compared to the input anucleate cells. In some embodiments, at least 50% of the population profile of anucleate cell-derived vesicles prepared by the process exhibits higher phosphatidylserine levels on the surface compared to the input anucleate cells. In some embodiments, the anucleate cell-derived vesicle exhibits enhanced uptake in a tissue or cell compared to the input anucleate cell. In some embodiments, the anucleate cell-derived vesicle exhibits enhanced uptake in phagocytic cells and / or antigen presenting cells compared to the input anucleate cell. In some embodiments, the anucleate cell-derived vesicle is modified to enhance uptake in a tissue or cell compared to the input anucleate cell. In some embodiments, the anucleate cell-derived vesicle is modified to enhance uptake in phagocytic cells and / or antigen presenting cells compared to an unmodified anucleate cell-derived vesicle. In some embodiments, the phagocytic cells and / or antigen presenting cells comprise one or more of a dendritic cell or macrophage. In some embodiments, the tissue or cell comprises one or more of liver or spleen. In some embodiments, the anucleate cell-derived vesicle comprises CD47 on its surface.

[0038] In some embodiments of the above method for generating an anucleate cell-derived vesicle, the anucleate cell-derived vesicle is not (a) heat processed, (b) chemically treated, and / or (c) subjected to hypotonic or hypertonic conditions during the preparation of the anucleate cell- derived vesicles. In some embodiments, the osmolarity of the cell suspension is maintained throughout the process. In some embodiments, the osmolarity of the cell suspension is maintained between about 200 mOsm and about 400 mOsm throughout the process.

[0039] In some embodiments of the above method for generating an anucleate cell-derived vesicle, the constriction is contained within a microfluidic channel. In some embodiments, the microfluidic channel comprises a plurality of constrictions. In some embodiments, the plurality of constrictions is arranged in series and / or in parallel. In some embodiments, the constriction is between a plurality of micropillars; between a plurality of micropillars configured in an array; or between one or more movable plates. In some embodiments, the constriction is a pore or 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 anucleate cell 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 anucleate cell in suspension. In some embodiments, the constriction has a width of about 0.25 um to about 4 um. In some embodiments, the constriction has a width of about 4 pm, 3.5 um, about 3 um, about 2.5 um, about 2 pm, about 1.5 pm, about 1 um, about 0.5 pm, or about 0.25 um. In some embodiments, the constriction has a width of about 2.2 pm. In some embodiments, the input anucleate cells are passed through the constriction under a pressure ranging from about 10 psi to about 90 psi. In some embodiments, said cell suspension is contacted with the antigen before, concurrently, or after passing through the constriction.

[0040] The present disclosure provides, in one aspect, an anucleate cell-derived vesicle prepared from a parent anucleate cell, the anucleate cell-derived vesicle having one or more of the following properties: (a) a circulating half-life in a mammal is decreased compared to the parent anucleate cell, (b) decreased hemoglobin levels compared to the parent anucleate cell, (c) spherical morphology, (d) increased surface phosphatidylserine levels compared to the parent anucleate cell, or (e) reduced ATP production compared to the parent anucleate cell.

[0041] In some embodiments, the parent anucleate cell is a mammalian cell. In some embodiments, the parent anucleate cell is human cell. In some embodiments, the parent anucleate 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 the anucleate cell-derived vesicle in a mammal is decreased compared to the parent anucleate cell. In some embodiments, the circulating half-life in the mammal is decreased 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 anucleate cell.

[0043] In some embodiments, the parent anucleate cell is a human cell and wherein the circulating half-life of the anucleate cell-derived vesicle is 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, about 120 days.

[0044] In some embodiments, the parent anucleate cell is a red blood cell, wherein the hemoglobin levels in the anucleate cell-derived vesicle are decreased compared to the parent anucleate cell. In some embodiments, the hemoglobin levels in the anucleate cell-derived vesicle are decreased 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 anucleate cell. In some embodiments, the hemoglobin levels in the anucleate cell-derived vesicle are about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, or about 50% the level of hemoglobin in the parent anucleate cell.

[0045] In some embodiments, the parent anucleate cell is an erythrocyte and wherein the anucleate cell-derived vesicle is spherical in morphology. In some embodiments, the parent anucleate cell is an erythrocyte and wherein the anucleate cell-derived vesicle has a reduced biconcave shape compared to the parent anucleate cell.

[0046] In some embodiments, the parent anucleate cell is a red blood cell or an erythrocyte and wherein the anucleate cell-derived vesicle is a red blood cell ghost (RBC ghost).

[0047] In some embodiments, the anucleate cell-derived vesicle has increased surface phosphatidylserine levels compared to the parent anucleate cell. In some embodiments, the anucleate cell-derived vesicles prepared by the process has greater than about 1.5 fold more phosphatidylserine on its surface compared to the parent anucleate cell. In some embodiments, the anucleate cell-derived vesicle has 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 its surface compared to the parent anucleate cell.

[0048] In some embodiments, the anucleate cell-derived vesicle has reduced ATP production compared to the parent anucleate cell. In some embodiments, the anucleate cell-derived vesicle produces ATP at less than about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, or about 50% the level of ATP produced by the parent anucleate cell. In some embodiments, the anucleate cell-derived vesicle does not produce ATP.

[0049] In some embodiments, the anucleate cell-derived vesicle is modified to enhance uptake in a tissue or cell compared to the parent anucleate cell. In some embodiments, the anucleate cell-derived vesicle is modified to enhance uptake in liver or spleen or by a phagocytic cell or an antigen-presenting cell compared to the uptake of the parent anucleate cell.

[0050] In some embodiments, the anucleate cell-derived vesicle comprises CD47 on its surface.

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

[0052] In some embodiments, the anucleate cell-derived vesicle was prepared by a process comprising: passing a suspension comprising the input parent anucleate cells through a cell deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input parent anucleate cell in the suspension, thereby causing perturbations of the anucleate cell large enough for a payload to pass through; thereby producing an anucleate cell-derived vesicle.

[0053] In some embodiments, the anucleate cell-derived vesicle comprises a payload. In some embodiments, the payload is a polypeptide, a nucleic acid, a lipid, a carbohydrate, a small molecule, a complex, a nanoparticle.

[0054] In some embodiments, the anucleate cell-derived vesicle was prepared by a process comprising: (a) passing a cell suspension comprising the input parent anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input parent anucleate cell in the suspension, thereby causing perturbations of the input parent anucleate cell large enough for the payload to pass through to form an anucleate cell- derived vesicle; and (b) incubating the anucleate cell-derived vesicle with the payload for a sufficient time to allow the payload to enter the anucleate cell-derived vesicle; thereby producing an anucleate cell-derived vesicle comprising the payload.

[0055] In some embodiments, the anucleate cell-derived vesicle comprises an antigen. In some embodiments, the anucleate cell-derived vesicle comprises adjuvant. In some embodiments, the anucleate cell-derived vesicle comprises an antigen and / or a tolerogenic factor.

[0056] In some embodiments, the anucleate cell-derived vesicle was prepared by a process comprising: (a) passing a cell suspension comprising the input parent anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input parent anucleate cell in the suspension, thereby causing perturbations of the input parent anucleate cell large enough for the antigen to pass through to form an anucleate cell- derived vesicle; and (b) incubating the anucleate cell-derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle; thereby producing an anucleate cell-derived vesicle comprising an antigen.

[0057] In some embodiments, the anucleate cell-derived vesicle was prepared by a process comprising: (a) passing a cell suspension comprising the input parent anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input parent anucleate cell in the suspension, thereby causing perturbations of the input parent anucleate cell large enough for the adjuvant to pass through to form an anucleate cell- derived vesicle; and (b) incubating the anucleate cell-derived vesicle with the adjuvant for a sufficient time to allow the adjuvant to enter the anucleate cell-derived vesicle; thereby producing an anucleate cell-derived vesicle comprising an adjuvant.

[0058] In some embodiments, the anucleate cell-derived vesicle comprises an antigen and an adjuvant, wherein the anucleate cell-derived vesicle was prepared by a process comprising: (a) passing a cell suspension comprising the input parent anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input parent anucleate cell in the suspension, thereby causing perturbations of the input parent anucleate cell large enough for the antigen and the adjuvant to pass through to form an anucleate cell-derived vesicle; and (b) incubating the anucleate cell-derived vesicle with the antigen and adjuvant for a sufficient time to allow the antigen and the adjuvant to enter the anucleate cell-derived vesicle; thereby producing an anucleate cell-derived vesicle comprising an antigen and an adjuvant.

[0059] In some embodiments, the anucleate cell-derived vesicle comprises an antigen and a tolerogenic factor, wherein the anucleate cell-derived vesicle was prepared by a process comprising: (a) passing a cell suspension comprising the input parent anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input parent anucleate cell in the suspension, thereby causing perturbations of the input parent anucleate cell large enough for the antigen and the tolerogenic factor to pass through to form an anucleate cell-derived vesicle; and (b) incubating the anucleate cell-derived vesicle with the antigen and the tolerogenic factor for a sufficient time to allow the antigen and the tolerogenic factor to enter the anucleate cell-derived vesicle; thereby producing an anucleate cell-derived vesicle comprising an antigen and an tolerogenic factor.

[0060] In some embodiments, the constriction is contained within a microfluidic channel. In some embodiments, the microfluidic channel comprises a plurality of constrictions. In some embodiments, the plurality of constrictions are arranged in series and / or in parallel. In some embodiments, the constriction is between a plurality of micropillars, between a plurality of micropillars configured in an array, or between one or more movable plates. In some embodiments, the constriction is a pore or 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 um to about 4 um. In some embodiments, the constriction has a width of about 4 pm, 3.5 um, about 3 um, about 2.5 um, about 2 um, about 1.5 pm , about 1 pm, about 0.5 um, or about 0.25 pm. In some embodiments, the constriction has a width of about 2.2 pm. In some embodiments, the input parent anucleate cells are passed through the constriction under a pressure ranging from about 10 psi to about 150 psi. In some embodiments, the cell suspension is contacted with the payload before, concurrently, or after passing through the constriction.

[0061] In some embodiments, the antigen is capable of being processed into an MHC class I- restricted peptide and / or an MHC class Il-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 transplant 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 the cell.

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

[0064] In some embodiments, a plurality of antigens is delivered to the anucleate cell.

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

[0066] The present disclosure provides, in another aspect, a composition comprising a plurality of anucleate cell-derived vesicles according to the description herein.

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

[0068] The present disclosure provides, in another aspect, a composition comprising a plurality of anucleate cell-derived vesicles prepared from a population of a parent anucleate cell, the composition having one or more of the following properties: (a) greater than about 20% of the anucleate cell-derived vesicles in the composition have a circulating half-life in a mammal that is decreased compared to the average of the population of the parent anucleate cell, (b) greater than 20% of the anucleate cell-derived vesicles in the composition have decreased hemoglobin levels compared to the average of the population of the parent anucleate cell, (c) greater than 20% of the anucleate cell-derived vesicles in the composition have spherical morphology, (d) greater than 20% of the anucleate cell-derived vesicles in the composition are RBC ghosts, (€) greater than 20% of the anucleate cell-derived vesicles in the composition vesicles in the composition have higher levels of phosphatidylserine compared to the average of the population of the parent anucleate cell, or (f) greater than 20% of the anucleate cell-derived vesicles in the composition have reduced ATP production compared to the average of the population of the parent anucleate cell.

[0069] In some embodiments, the parent anucleate cell used to prepare the composition is a mammalian cell. In some embodiments, the parent anucleate cell used to prepare the composition is a human cell. In some embodiments, the parent anucleate cell used to prepare the composition is a red blood cell or a platelet. In some embodiments, the red blood cell is an erythrocyte or a reticulocyte.

[0070] In some embodiments, the circulating half-life of 20% of the anucleate cell-derived vesicles in the composition in a mammal is decreased compared to the parent anucleate cell or the average of the population of the parent anucleate cell. In some embodiments, the circulating half-life of 20% of the anucleate cell-derived vesicles in the composition in the mammal is decreased by more than about 50%, about 60%, about 70%, about 80% or about 90% compared to the parent anucleate cell or the average of the population of the parent anucleate cell. In some embodiments, the parent anucleate cell used to prepare the composition is a human cell and wherein the circulating half-life of 20% of the anucleate 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, about 10 days.

[0071] In some embodiments, the parent anucleate cell used to prepare the composition is a red blood cell and wherein the hemoglobin levels of 20% of the anucleate cell-derived vesicles in the composition are decreased compared to the parent anucleate cell or the average of the population of the parent anucleate cell.

[0072] In some embodiments, the hemoglobin levels of 20% of the anucleate cell-derived vesicles in the composition of the anucleate cell-derived vesicle are decreased 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 anucleate cell or the average of the population of the parent anucleate cell. In some embodiments, the hemoglobin levels of 20% of the anucleate cell-derived vesicles in the composition are about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, or about 50% the level of hemoglobin in the parent anucleate cell or the average of the population of the parent anucleate cell.

[0073] In some embodiments, the parent anucleate cell used to prepare the composition is an erythrocyte and wherein greater than 20% of the anucleate cell-derived vesicles in the composition are spherical in morphology. In some embodiments, the parent anucleate cell used to prepare the composition is an erythrocyte and wherein greater than 20% of the anucleate cell- derived vesicles in the composition have a reduced biconcave shape compared to the parent anucleate cell.

[0074] In some embodiments, the parent anucleate cell used to prepare the composition is a red blood cell or an erythrocyte and wherein greater than 20% of the anucleate cell-derived vesicles in the composition are red blood cell ghosts.

[0075] In some embodiments, the anucleate cell-derived vesicles in the composition comprise surface phosphatidylserine. In some embodiments, 20% of the anucleate cell-derived vesicles in the composition comprise increased surface phosphatidylserine levels compared to the parent anucleate cells or the average of the population of the parent anucleate cell. In some embodiments, 20% of the anucleate cell-derived vesicles in the composition 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% higher surface phosphatidylserine levels compared to a composition comprising a plurality of parent anucleate cells.

[0076] In some embodiments, 20% of the anucleate cell-derived vesicles in the composition have reduced ATP production compared to the parent anucleate cell or the average of the population of the parent anucleate cell. In some embodiments, 20% of the anucleate 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% the level of ATP produced by the parent anucleate cell or the average of the population of the parent anucleate cell. In some embodiments, the anucleate cell-derived vesicle in the composition does not produce ATP.

[0077] In some embodiments, the parent anucleate cell used to prepare the composition was not (a) heat processed, (b) chemically treated, and / or (¢) subjected to hypotonic or hypertonic conditions during the preparation of the compositions. In some embodiments, osmolarity was maintained during preparation of the anucleate cell-derived vesicles from the parent anucleate cell. In some embodiments, the osmolarity was maintained between about 200 mOsm and about 600 mOsm. In some embodiments, the osmolarity was maintained between about 200 mOsm and about 400 mOsm.

[0078] In some embodiments, the anucleate cell-derived vesicles of the composition were prepared by a process comprising: passing a suspension comprising the input parent anucleate cells through a cell deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input parent anucleate cells in the suspension, thereby causing perturbations of the anucleate cells large enough for a payload to pass through; thereby producing the anucleate cell-derived vesicles.

[0079] In some embodiments, the anucleate 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, a nanoparticle.

[0080] In some embodiments, the anucleate cell-derived vesicles of the composition were prepared by a process comprising: (a) passing a cell suspension comprising the input parent anucleate cells through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input parent anucleate cells in the suspension, thereby causing perturbations of the input parent anucleate cells large enough for the payload to pass through to form an anucleate cell-derived vesicles; and (b) incubating the anucleate cell-derived vesicles with the payload for a sufficient time to allow the payload to enter the anucleate cell-derived vesicles; thereby producing an anucleate cell-derived vesicles comprising the payload.

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

[0082] In some embodiments, the anucleate cell-derived vesicles of the composition were prepared by a process comprising: (a) passing a cell suspension comprising the input parent anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input parent anucleate cell in the suspension, thereby causing perturbations of the input parent anucleate cell large enough for the antigen to pass through to form an anucleate cell-derived vesicle; and (b) incubating the anucleate cell-derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle; thereby producing an anucleate cell-derived vesicle comprising an antigen.

[0083] In some embodiments, the anucleate cell-derived vesicles of the composition were prepared by a process comprising: (a) passing a cell suspension comprising the input parent anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input parent anucleate cell in the suspension, thereby causing perturbations of the input parent anucleate cell large enough for the adjuvant to pass through to form an anucleate cell-derived vesicle; and (b) incubating the anucleate cell-derived vesicle with the adjuvant for a sufficient time to allow the adjuvant to enter the anucleate cell-derived vesicle; thereby producing an anucleate cell-derived vesicle comprising an adjuvant.

[0084] In some embodiments, the anucleate cell-derived vesicles of the composition comprises an antigen and an adjuvant, wherein the anucleate cell-derived vesicles of the composition were prepared by a process comprising: (a) passing a cell suspension comprising the input parent anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input parent anucleate cell in the suspension, thereby causing perturbations of the input parent anucleate cell large enough for the antigen and the adjuvant to pass through to form an anucleate cell-derived vesicle; and (b) incubating the anucleate cell-derived vesicle with the antigen and the adjuvant for a sufficient time to allow the antigen and the adjuvant to enter the anucleate cell-derived vesicle; thereby producing an anucleate cell-derived vesicle comprising an antigen and / or an adjuvant.

[0085] In some embodiments, the anucleate cell-derived vesicle of the composition comprises an antigen and a tolerogenic factor, wherein the anucleate cell-derived vesicles of the composition were prepared by a process comprising: (a) passing a cell suspension comprising the input parent anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input parent anucleate cell in the suspension, thereby causing perturbations of the input parent anucleate cell large enough for the antigen and the tolerogenic factor to pass through to form an anucleate cell-derived vesicle; and (b) incubating the anucleate cell-derived vesicle with the antigen and the tolerogenic factor for a sufficient time to allow the antigen and the tolerogenic factor to enter the anucleate cell-derived vesicle; thereby producing an anucleate cell-derived vesicle comprising an antigen and / or an tolerogenic factor.

[0086] In some embodiments, the constriction used to prepare the composition is contained within a microfluidic channel. In some embodiments, the microfluidic channel used to prepare the composition comprises a plurality of constrictions. In some embodiments, the plurality of constrictions are arranged in series and / or in parallel. In some embodiments, the constriction used to prepare the composition is between a plurality of micropillars, between a plurality of micropillars configured in an array, or between one or more movable plates. In some embodiments, the constriction used to prepare the composition is a pore or contained within a pore. In some embodiments, the pore used to prepare the composition is contained in a surface. In some embodiments, the surface used to prepare the composition is a filter. In some embodiments, the surface used to prepare the composition is a membrane. In some embodiments, the constriction size used to prepare 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 to prepare the composition has a width of about 0.25 pm to about 4 pm. In some embodiments, the constriction used to prepare the composition has a width of about 4 pm, 3.5 um, about 3 um, about 2.5 pm, about 2 pm, about 1.5 um, about 1 um, about 0.5 um, or about 0.25 um. In some embodiments, the constriction used to prepare the composition has a width of about 2.2 pm. In some embodiments, the input parent anucleate cells used to prepare the composition are passed through the constriction under a pressure ranging from about 10 psi to about 150 psi. In some embodiments, the cell suspension used to prepare the composition is contacted with the antigen before, concurrently, or after passing through the constriction.

[0087] In some embodiments, the antigen of the composition is capable of being 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 transplant 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.

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

[0089] In some embodiments, the anucleate cell-derived vesicle of the composition comprises a plurality of antigens, wherein the plurality of antigens is delivered to the anucleate cell.

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

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

[0092] The present disclosure provides, in another aspect, a method of making a composition comprising a plurality of anucleate cell-derived vesicles prepared from parent anucleate cells, the composition having one or more of the following properties: (a) greater than 20% of the anucleate cell-derived vesicles in the composition have a circulating half-life in a mammal that is decreased compared to the parent anucleate cell, (b) greater than 20% of the anucleate cell- derived vesicles in the composition have decreased hemoglobin levels compared to the parent anucleate cell, (c) greater than 20% of the anucleate cell-derived vesicles in the composition have spherical morphology, (d) greater than 20% of the anucleate cell-derived vesicles in the composition are RBC ghosts, (e) greater than 20% of the anucleate cell-derived vesicles in the composition have higher levels of phosphatidylserine, or (f) greater than 20% of the anucleate cell-derived vesicles in the composition have reduced ATP production compared to the parent anucleate cell; the method comprising passing a cell suspension comprising the parent anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the parent anucleate cell in the suspension, thereby causing perturbations of the parent anucleate cell large enough for a payload to pass through to form an anucleate cell- derived vesicle, thereby producing an anucleate cell-derived vesicle.

[0093] In some embodiments, the constriction used in the methods of making described herein is contained within a microfluidic channel. In some embodiments, the microfluidic channel used in the methods of making described herein comprises a plurality of constrictions. In some embodiments, the plurality of constrictions used in the methods of making described herein are arranged in series and / or in parallel. In some embodiments, the constriction used in the methods of making described herein is between a plurality of micropillars, between a plurality of micropillars configured in an array, or between one or more movable plates. In some embodiments, the constriction used in the methods of making described herein is a pore or contained within a pore. In some embodiments, the pore used in the methods of making described herein is contained in a surface. In some embodiments, the surface used in the methods of making described herein is a filter. In some embodiments, the surface used in the methods of making described herein is a membrane. In some embodiments, the constriction size used in the methods of making 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 methods of making described herein has a width of about 0.25 um to about 4 um. In some embodiments, the constriction used in the methods of making described herein has a width of about 4 pm, 3.5 um, about 3 pum, about 2.5 pm, about 2 pm, about 1.5 um, about 1 pm, about 0.5 pm, or about 0.25 pm. In some embodiments, the constriction used in the methods of making described herein has a width of about 2.2 pm. In some embodiments, the input parent anucleate cells used in the methods of making described herein are passed through the constriction under a pressure ranging from about 10 psi to about 150 psi. In some embodiments, the cell suspension used in the methods of making described herein is contacted with a payload before, concurrently, or after passing through the constriction such that the payload enters the cell.

[0094] In some embodiments, the payload used in the methods of making 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 a tolerogenic factor.

[0095] The present disclosure provides, in another aspect, a method for treating a disease or disorder in an individual in need thereof, the method comprising administering a anucleate cell- derived vesicle described herein. The present disclosure provides, in another aspect, 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 anucleate cell-derived vesicles used in the methods for treating described herein comprise a therapeutic payload. In some embodiments, the individual has cancer and wherein the payload comprises an antigen. In some embodiments, the individual has cancer and wherein 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 viral-associated disease and wherein the payload comprises an antigen. In some embodiments, the individual has an infectious disease or a viral- associated disease and wherein 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 wherein the payload comprises an antigen. In some embodiments, the individual has an autoimmune disease and wherein the payload comprises an antigen and / or a tolerogenic factor.

[0096] The present disclosure provides, in another aspect, a method for preventing a disease or disorder in an individual in need thereof, the method comprising administering a anucleate cell-derived vesicle described herein. The present disclosure provides, in another aspect, 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 anucleate cell-derived vesicles used in the methods for preventing described herein comprise an antigen. In some embodiments, the individual has cancer and wherein 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 wherein the payload comprises an antigen. In some embodiments, the individual has an infectious disease and wherein 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 DESCRIPTION OF THE DRAWINGS

[0097] FIG. 1A shows the percentage of antigen-specific T cells as measured by tetramer staining for each condition. FIG. 1B shows the percentage of IFN-y positive cells as measured by intracellular cytokine staining (ICS) for each condition after re-stimulation with OVA epitope SIINFEKL (circular dots). Stimulation with anti-CD28 alone (without SIINFEKL) is used as a negative control (square dots), while unspecific stimulation by PMA / Ionomycin is used as a positive control (triangular dots). FIG. 1C shows the amounts of IFN-y in each cell as measured by the mean fluorescence intensity (MFI) of each cell in ICS for each condition. FIG. 1D shows the percentage of IL-2 positive cells as measured by intracellular cytokine staining (ICS) for each condition after re-stimulation with OVA epitope SIINFEKL (circular dots). Stimulation with anti-CD28 alone (without SIINFEKL) is used as a negative control (square dots), while unspecific stimulation by PMA / Ionomycin is used as a positive control (triangular dots). FIG. 1E shows the amounts of IL-2 in each cell as measured by the mean fluorescence intensity (MFI) of each cell in ICS for each condition.

[0098] FIG. 2A shows the percentage of antigen-specific T cells as measured by tetramer staining for each condition. FIG. 2B shows the percentage of IFN-y positive cells as measured by intracellular cytokine staining (ICS) for each condition after re-stimulation with OVA epitope SIINFEKL (circular dots). FIG. 2C shows the percentage of IL-2 positive cells as measured by intracellular cytokine staining (ICS) for each condition after re-stimulation with OVA epitope SIINFEKL (circular dots). For both FIGs. 2B and 2C, Stimulation with anti-CD28 alone (without SINFEKL) is used as a negative control (square dots), while unspecific stimulation by PMA / Ionomycin is used as a positive control (triangular dots).

[0099] FIG. 3A shows the percentage of antigen-specific T cells as measured by tetramer staining for each condition. FIG. 3B shows the percentage of IFN-y positive cells as measured by intracellular cytokine staining (ICS) for each condition after re-stimulation with OVA epitope SIINFEKL (circular dots). FIG. 3C shows the percentage of IL-2 positive cells as measured by intracellular cytokine staining (ICS) for each condition after re-stimulation with OVA epitope SIINFEKL (circular dots). For both FIGs. 3B and 3C, Stimulation with anti-CD28 alone (without SIINFEKL) is used as a negative control (square dots), while unspecific stimulation by PMA / Ionomycin is used as a positive control (triangular dots).

[0100] FIG. 4 shows lactate levels of red blood cellderived vesicles that has been processed by constriction mediated delivery (SQZ) versus the unprocessed input red blood cells .

[0101] FIG. 5A shows the images from brightfield microscopy, fluorescent microscopy for CellTrace Violet staining (CT) as well as fluorescent microscopy for FITC labeled Dextran (D- FITC), for untreated RBCs (Untrtd), RBCs incubated with D-FITC (No SQZ), as well as RBC- derived vesicles with D-FITC loaded using SQZ (SQZ). FIG. 5B shows the levels of phosphatidylserine staining for untreated RBCs (Untrt), RBCs incubated with D-FITC (No SQZ), as well as RBC-derived vesicles with D-FITC loaded using SQZ (SQZ).

[0102] FIG. 6A shows the representative schematics of an experiment to determine the circulating half-life of anucleate cell-derived vesicles generated by SQZ-processing. FIG. 6B shows the circulating levels of the separately labeled RBCs and SQZ-loaded RBC-derived vesicles over time. FIG. 6C shows the forward and side scatter in the flow plot of the mixture of RBCs and SQZ-loaded RBC-derived vesicles that were injected into mice.

[0103] FIG. 7A shows the appearance of cell pellet and supernatant after centrifugation of untreated RBCs (NC), and RBC-derived vesicles that were SQZ-processed at pressure of 10psi and 12 psi, respectively. FIG. 7B shows the loss of hemoglobin (hemolysis) as measured by HemoCue® system for untreated RBCs (NC), RBC-derived vesicles that were SQZ-processed at pressure of 10psi and 12 psi, and RBCs diluted in water (Lysis Control).

[0104] FIGs. 8A and 8B show the loss of hemoglobin (hemolysis) as quantified by liquid chromatography / mass spectrometry of 2 hemoglobin peptide, respectively, in RBCs incubated with B9-23 (Endo Control) and RBC-derived vesicles that were SQZ-loaded with B9-23 (SQZ).

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

[0106] FIG. 10 shows the in vivo persistence of unprocessed murine RBCs and SQZ- processed murine RBC vesicles in recipient mice.

[0107] FIG. 11A shows the organs involved in internalization of SQZ-processed RBC-derived vesicles. FIG. 11B shows the cell types within liver and spleen that are involved in internalization of SQZ-processed RBC-derived vesicles.

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

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

[0110] FIG. 14 shows the endogenous CD8+ T cell response upon ex vivo E7 re-stimulation for mice induced by RBC-derived vesicles SQZ-loaded with (i) Poly I:C only, (ii) E7 only, or (iii) E7 and Poly L.C.

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

[0112] FIGs. 16A and 16B show the effect of prophylactic administration of RBC-derived vesicles SQZ-loaded with E7 and Poly I:C on the tumor growth and survival respectively in a murine model receiving E7-positive tumor.

[0113] FIGs. 17A and 17B show the effect of therapeutic administration of RBC-derived vesicles SQZ-loaded with E7 and Poly I:C at different dosages on the tumor growth and survival respectively in a murine model carrying E7-positive tumor.

[0114] FIGs. 18A and 18B show the effect of therapeutic administration of RBC-derived vesicles SQZ-loaded with E7 and Poly I.C with different dosing regimens on the tumor growth and survival respectively in a murine model carrying E7-positive tumor.

[0115] FIGs. 19A-19D show the antigen-specific immune response induced by RBC-derived vesicles SQZ-loaded with E7 and Poly I:C, specifically the recruitment of CD8+ T cells into an E7 positive tumor (FIG. 19A), the percentage of CD8+ T cells within the tumor that is specific to E7 (FIG. 19B), the ratio of E7-specific CD8+ T cells versus regulatory T cells in the tumor (FIG. 19C), and correlation of E7-specific CD8+ T cells versus tumor weight (FIG. 19D), when a murine model carrying a E7-positive tumor was administered with RBC-derived vesicles SQZ- loaded with E7 and Poly L:C.

[0116] FIGs. 20A-20C show the ghost formation, the efficiency of payload delivery, and the surface phosphatidylserine levels respectively when human RBC-derived vesicles were generated by SQZ-processing in the presence of E7-SLP (payload).

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

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

[0119] FIGs. 23A-23C show the efficiency of payload delivery, the ghost formation, and the surface phosphatidylserine levels in ghost and non-ghost populations, respectively, when murine RBC-derived vesicles were generated by SQZ-processing.

[0120] FIG. 24A shows the representative schematics of an experiment to determine if in vivo antigen-dependent tolerance to a viral capsid is induced by anucleate cell-derived vesicles with SQZ-loaded antigen. FIG. 24B shows the percentage of IFN-y positive cells as measured by intracellular cytokine staining (ICS) in splenocytes of naive mice, mice treated with RBC incubated with SNYNKSVNV (Peptide), or mice treated with SNYNKSVNV-loaded RBC- derived vesicles (SQZ). FIG. 24C shows the luciferase levels in serum for mice in Peptide group and SQZ group over the course of 43 days.

[0121] FIG. 25A shows the representative schematics of an experiment to determine if in vivo antigen-dependent tolerance to an antibody is induced by anucleate cell-derived vesicles with SQZ-loaded antigen. FIG. 25B shows the levels of circulating rat IgG2b in serum for 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. FIG. 25C shows the levels of circulating rat IgG2b in serum for mice in control, free rat IgG2b, and SQZ group on Day 76.

[0122] FIG. 26A shows the representative schematics of an experiment to determine if in vivo antigen-dependent tolerance to B9-23 is induced by anucleate cell-derived vesicles with SQZ- loaded antigen. FIG. 26B shows the percentage of IFN-y or IL-2 positive cells as measured by intracellular cytokine staining (ICS) after re-stimulation with AAV-NL virus in splenocytes of control mice, mice treated with HEL-loaded RBC-derived vesicles (SQZ HEL), or mice treated Ins B9-23-loaded RBC-derived vesicles (SQZ FAM). FIG. 26C shows the representative schematics of an experiment to determine if in vive antigen-dependent tolerance to 1040-p31 is induced by anucleate cell-derived vesicles with SQZ-loaded antigen. FIG. 26D shows the levels of serum blood glucose measured in control mice and mice treated with 1040-31-loaded RBC- derived vesicles (SQZ). FIG. 26E shows the disease onset for control mice and SQZ mice, as determined from serum blood glucose measurements. DETAILED DESCRIPTION

[0123] The present application provides anucleate cells, including anucleate cell-derived vesicles (such as those prepared from an input anucleate cell), and compositions thereof, wherein the anucleate cells and / or anucleate cell-derived vesicles are loaded and / or admixed with one or more of an antigen, adjuvant, or therapeutic agent. The present application also provides methods of generating anucleate cell-derived vesicles via constriction-mediated delivery (SQZ) described herein and methods of use thereof. The present application further provides methods of stimulating an immune response and of treating and / or preventing diseases in individuals using anucleate cell-derived vesicles generated via constriction-mediated delivery (SQZ) described herein.

[0124] The disclosure of the present application is based, at least in part, on the finding that input anucleate cells can be processed by constriction-mediated delivery (SQZ) to generate anucleate cell-derived vesicles. The disclosure of the present application is also based, at least in part, on the finding that anucleate cell-derived vesicles with antigen(s) and / or adjuvant(s) (whether or not encapsulated within the anucleate cell-derived vesicle) can induce an in vivo antigen-specific immune response.

[0125] The invention provides methods for delivering an antigen and / or an adjuvant into an anucleate cell-derived vesicle, the method comprising: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen and / or adjuvant to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell- derived vesicle with the antigen and / or the adjuvant for a sufficient time to allow the antigen and / or adjuvant to enter the anucleate cell-derived vesicle.

[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 an anucleate cell-derived vesicle comprising an antigen, wherein the anucleate cell-derived vesicle comprising the antigen is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell- deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen to pass through to form an anucleate cell-derived vesicle; and b) incubating the perturbed input anucleate cell with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle. In some embodiments, an adjuvant is also delivered to the anucleate cell-derived vesicle. In other embodiments, an adjuvant is administered systemically to the individual in combination with the anucleate cell-derived vesicle comprising the antigen.

[0127] In certain aspects, the invention provides an anucleate cell-derived vesicle comprising an antigen and / or an adjuvant, wherein the anucleate cell-derived vesicle comprising the antigen and / or adjuvant is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen and / or adjuvant to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell- derived vesicle with the antigen and / or adjuvant for a sufficient time to allow the antigen and / or adjuvant to enter the anucleate cell-derived vesicle; thereby generating the anucleate cell-derived vesicle comprising the antigen and / or adjuvant.

[0128] In certain aspects, the invention provides methods for generating an anucleate cell derived vesicle comprising an antigen and / or an adjuvant, the method comprising: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen and / or adjuvant to pass through to form an anucleate cell-derived vesicle; b) incubating the anucleate cell-derived vesicle with the antigen and / or adjuvant for a sufficient time to allow the antigen and / or adjuvant to enter the anucleate cell-derived vesicle, thereby generating an anucleate cell-derived vesicle comprising the antigen and / or adjuvant.

[0129] In some aspects, the present application provides anucleate cell-derived vesicles (such as those prepared from a parent anucleate cell), and compositions thereof, wherein the anucleate cell-derived vesicles are loaded with a payload, such as any one or more of an antigen, adjuvant, or tolerogenic factor. The present application also provides methods of making compositions of anucleate cell-derived vesicles described herein and methods of use thereof.

[0130] The disclosure of the present application is also based, at least in part, on the finding that compositions comprising anucleate cell-derived vesicles comprising a payload, such as an antigen(s) and / or adjuvant(s), can induce an in vive antigen-specific immune response. The disclosure of the present application is also based, at least in part, on the finding that higher doses of compositions comprising anucleate cell-derived vesicles loaded with an antigen(s) and / or an adjuvant(s) can induce a greater in vivo antigen-specific immune response. Furthermore, the disclosure of the present application is based, at least in part, on the finding that the in vivo antigen-specific immune response can be modulated based on: the adjuvant of the composition; the amount of payload, such as an antigen, encapsulated in an anucleate cell- derived vesicle; and / or the dosing strategy used for administration of the composition comprising anucleate cell-derived vesicles. The disclosure of the present application is also based, at least in part, on the finding that a composition comprising a plurality of anucleate cell- derived vesicles can be actively tuned to generate anucleate cell-derived vesicles, such as a population of anucleate cell-derived vesicles, within the composition having one or more select properties. Generation of a composition of anucleate cell-derived vesicles having desired amounts and / or properties of the anucleate cell-derived vesicle therein is achieved, e.g., by adjusting one or more of the preparation parameters when the anucleate cell-derived vesicles are prepared from parent anucleate cells.

[0131] Thus, in some aspects, provided herein are anucleate cell-derived vesicles prepared from a parent anucleate cell, the anucleate cell-derived vesicles having one or more of the following properties: (a) a circulating half-life in a mammal is decreased compared to the parent anucleate cell, (b) decreased hemoglobin levels compared to the parent anucleate cell, (c) spherical morphology, (d) increased surface phosphatidylserine levels compared to the parent anucleate cell, or (e) reduced ATP production compared to the parent anucleate cell.

[0132] In another aspect, provided herein are compositions comprising a plurality of any anucleate cell-derived vesicles described herein. In some embodiments, the composition has one or more of the following properties: (a) greater than 20% of the anucleate cell-derived vesicles in the composition have a circulating half-life in a mammal that is decreased compared to the parent anucleate cell, (b) greater than 20% of the anucleate cell-derived vesicles in the composition have decreased hemoglobin levels compared to the parent anucleate cell, (c) greater than 20% of the anucleate cell-derived vesicles in the composition have spherical morphology, (d) greater than 20% of the anucleate cell-derived vesicles in the composition are RBC ghosts, (e) greater than 20% of the anucleate cell-derived vesicles in the composition have higher levels of phosphatidylserine, or (f) greater than 20% of the anucleate cell-derived vesicles in the composition have reduced ATP production compared to the parent anucleate cell.

[0133] In another aspect, provided herein are compositions comprising a plurality of any anucleate cells admixed with an adjuvant, as described herein.

[0134] In another aspect, provided herein are methods of making a composition disclosed herein, e.g., a method of making a composition comprising a plurality of anucleate cell-derived vesicles prepared from parent anucleate cells, the composition having one or more of the following properties: (a) greater than 20% of the anucleate cell-derived vesicles in the composition have a circulating half-life in a mammal that is decreased compared to the parent anucleate cell, (b) greater than 20% of the anucleate cell-derived vesicles in the composition have decreased hemoglobin levels compared to the parent anucleate cell, (c) greater than 20% of the anucleate cell-derived vesicles in the composition have spherical morphology, (d) greater than 20% of the anucleate cell-derived vesicles in the composition are RBC ghosts, (e) greater than 20% of the anucleate cell-derived vesicles in the composition have higher levels of phosphatidylserine, or (f) greater than 20% of the anucleate cell-derived vesicles in the composition have reduced ATP production compared to the parent anucleate cell; the method comprising passing a cell suspension comprising the parent anucleate cell through a cell- deforming constriction, wherein a diameter of the constriction is a function of a diameter of the parent anucleate cell in the suspension, thereby causing perturbations of the parent anucleate cell large enough for a payload to pass through to form an anucleate cell-derived vesicle, thereby producing an anucleate cell-derived vesicle.

[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 an individual in need thereof, the method comprising administering any of the anucleate cell-derived vesicles described herein. In some embodiments, the method for use is a method for preventing a disease or disorder an individual in need thereof, the method comprising administering any of the anucleate cell-derived vesicles described herein. Definitions

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

[0137] As used herein, the singular form “a”, “an”, and “the” includes plural references unless indicated otherwise.

[0138] The terms “comprising,” “having,” “containing,” and “including,” and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. 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 thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of’ or “consisting of.”

[0139] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of that range 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 the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.

[0141] As used herein, “anucleate cell” refers to a cell lacking a nucleus. Such cells can include, but are not limited to, platelets, red blood cells (RBCs) such as erythrocytes and reticulocytes. Reticulocytes are immature (e.g., not yet biconcave) red blood cells, typically comprising about 1% of the red blood cells in the human body. Reticulocytes are also anucleate. In certain embodiments, the systems and methods described herein are used the treatment and / or processing of enriched (e.g., comprising a greater percentage of the total cellular population than would be found in nature), purified, or isolated (e.g., from their natural environment, in substantially pure or homogeneous form) populations of anucleate cells (e.g., RBCs, reticulocytes, and / or platelets). In certain embodiments, the systems and methods described herein are used for the treatment and / or processing of whole blood containing RBCs (e.g., erythrocytes or reticulocytes), platelets as well as other blood cells. Purification or enrichment of these cell types is accomplished 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 comprising liquid enclosed by a lipid bilayer. In some examples, the lipid bilayer is sourced from naturally existing lipid composition. In some examples, the lipid bilayer can be sourced from a cellular membrane. In some examples, vesicles can be derived from various kinds of entities, such as cells. In such examples, a vesicle can retain molecules (such as intracellular proteins or membrane components) from the originating entity. For example, a vesicle derived from a red blood cell may contain any number of intracellular proteins that were in the red blood cell and / or membrane components of the red blood cell. In some examples, a vesicle can contain any number of molecules intracellularly in addition to the desired payload.

[0143] As used herein “payload” refers to the material that is being delivered into, such as loaded in, the anucleate cell-derived vesicle (e.g., an RBC-derived vesicle). “Payload,” “cargo,” “delivery material,” and “compound” are used interchangeably herein. In some embodiments, a payload may refer to a protein, a small molecule, a nucleic acid (e.g., RNA and / or DNA), a lipid, a carbohydrate, a macromolecule, a vitamin, a polymer, fluorescent dyes and fluorophores, carbon nanotubes, quantum dots, nanoparticles, and steroids. In some embodiments, the payload may refer to a protein or small molecule drug. In some embodiments, the payload may comprise one or more compounds.

[0144] The term “pore” as used herein refers to an opening, including without limitation, a hole, tear, cavity, aperture, break, gap, or perforation within a material. In some examples, (where indicated) the term refers to a pore within a surface of the present disclosure. In other examples, (where indicated) a 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. The term includes a pliable sheet-like structure that acts as a boundary or lining. In some examples, the 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 through the pores. In some examples the term refers to a surface or membrane containing pores.

[0147] The term “heterologous” as it relates to nucleic acid sequences such as coding sequences and control sequences, denotes sequences that are not normally joined together, and / or are not normally associated with a particular cell. Thus, a “heterologous” region of a nucleic acid construct or a vector is a segment of nucleic acid within or attached to another nucleic acid molecule that is not found in association with the other molecule in nature. For example, a heterologous region of a nucleic acid construct could include a coding sequence flanked by sequences not found in association with the coding sequence in nature. Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., synthetic sequences having codons different from the native gene). Similarly, a cell transformed with a construct which is not normally present in the cell would be considered heterologous for purposes of this invention. Allelic variation or naturally occurring mutational events do not give rise to heterologous DNA, as used herein.

[0148] The term “heterologous” as it relates to amino acid sequences such as peptide sequences and polypeptide sequences, denotes sequences that are not normally joined together, and / or are not normally associated with a particular cell. Thus, a “heterologous” region of a peptide sequence is a segment of amino acids within or attached to another amino acid molecule that is not found in association with the other molecule in nature. For example, a heterologous region of a peptide construct could include the amino acid sequence of the peptide flanked by 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 where the peptide sequence itself is not found in nature (e.g., synthetic sequences having amino acids different as coded from the native gene). Similarly, a cell transformed with a vector that expresses an amino acid construct which is not normally present in the cell would be considered heterologous for purposes of this invention. Allelic variation or naturally occurring mutational events do not give rise to heterologous peptides, as used herein.

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

[0150] The term “homologous” as used herein refers to a molecule which is derived from the same organism. In some examples the term refers to a nucleic acid or protein which is normally found or expressed within the given organism.

[0151] As used herein, “treatment” or “treating” is an approach for obtaining beneficial or desired results, including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing or improving the quality of life, increasing weight gain, and / or prolonging survival. Also encompassed by “treatment” is a reduction of pathological consequence of cancer (such as, for example, tumor volume). The methods of the invention contemplate any one or more of these aspects of treatment.

[0152] As used herein, the term “modulate” may refer to the act of changing, altering, varying, or otherwise modifying the presence, or an activity of, a particular target. For example, modulating an immune response may refer to any act leading to changing, altering, varying, or otherwise modifying an immune response. In other examples, modulating the expression of a nucleic acid may include, but not limited to a change in the transcription of a nucleic acid, a change in mRNA abundance (e.g., increasing mRNA transcription), a corresponding change in degradation of mRNA, a change in mRNA translation, and so forth.

[0153] As used herein, the term “inhibit” may refer to the act of blocking, reducing, eliminating, or otherwise antagonizing the presence, or an activity of, a particular target. Inhibition may refer to partial inhibition or complete inhibition. For example, inhibiting an immune response may refer to any act leading to a blockade, reduction, elimination, or any other antagonism of an immune response. In other examples, inhibition of the expression of a nucleic acid may include, but not limited to reduction in the transcription of a nucleic acid, reduction of mRNA abundance (e.g., silencing mRNA transcription), degradation of mRNA, inhibition of mRNA translation, gene editing and so forth. In other examples, inhibition of the expression of a protein may include, but not be limited to, reduction in the transcription of a nucleic acid encoding the protein, reduction in the stability of mRNA encoding the protein, inhibition of translation of the protein, reduction in stability of the protein, and so forth.

[0154] As used herein, the term “suppress” may refer to the act of decreasing, reducing, prohibiting, limiting, lessening, or otherwise diminishing the presence, or an activity of, a particular target. Suppression may refer to partial suppression or complete suppression. For example, suppressing an immune response may refer to any act leading to decreasing, reducing, prohibiting, limiting, lessening, or otherwise diminishing an immune response. In other examples, suppression of the expression of a nucleic acid may include, but not limited to reduction in the transcription of a nucleic acid, reduction of mRNA abundance (e.g., silencing mRNA transcription), degradation of mRNA, inhibition of mRNA translation, and so forth. In other examples, suppression of the expression of a protein may include, but not be limited to, reduction in the transcription of a nucleic acid encoding the protein, reduction in the stability of mRNA encoding the protein, inhibition of translation of the protein, reduction in stability of the protein, and so forth.

[0155] As used herein, the term “enhance” may refer to the act of improving, boosting, heightening, or otherwise increasing the presence, or an activity of, a particular target. For example, enhancing an immune response may refer to any act leading to improving, boosting, heightening, or otherwise increasing an immune response. In other examples, enhancing the expression of a nucleic acid may include, but not limited to increase in the transcription of a nucleic acid, increase in mRNA abundance (e.g., increasing mRNA transcription), decrease in degradation of mRNA, increase in mRNA translation, and so forth. In other examples, enhancing the expression of a protein may include, but not be limited to, increase in the transcription of a nucleic acid encoding the protein, increase in the stability of mRNA encoding the protein, increase in translation of the protein, increase in the stability of the protein, and so forth.

[0156] As used herein, the term “induce” may refer to the act of initiating, prompting, stimulating, establishing, or otherwise producing a result. For example, inducing an immune response may refer to any act leading to initiating, prompting, stimulating, establishing, or otherwise producing a desired immune response. In other examples, inducing the expression of a nucleic acid may include, but not limited to initiation of the transcription of a nucleic acid, initiation of mRNA translation, and so forth. In other examples, inducing the expression of a protein may include, but not be limited to, increase in the transcription of a nucleic acid encoding the protein, increase in the stability of mRNA encoding the protein, increase in translation of the protein, increase in the stability of the protein, and so forth.

[0157] The term “polynucleotide” or “nucleic acid” as used herein refers to a polymeric form of nucleotides of any length, including ribonucleotides and deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of the polynucleotide can comprise sugars and phosphate groups (as may typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups. The backbone of the polynucleotide can comprise repeating units, such as N-(2-aminoethyl)- glycine, linked by peptide bonds (i.e., peptide nucleic acid). Alternatively, the backbone of the polynucleotide can comprise a polymer of synthetic subunits such as phosphoramidates and thus can be an oligodeoxynucleoside phosphoramidate (P-NH2) or a mixed phosphoramidate- phosphodiester oligomer. In addition, a double-stranded polynucleotide can be obtained from the single stranded polynucleotide product of chemical synthesis either by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using a DNA polymerase with an appropriate primer.

[0158] The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Therefore as used herein, polypeptide includes short peptides. Such polymers of amino acid residues may 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 the definition. The terms also include post-translational modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of the present invention, a “polypeptide” refers to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.

[0159] As used herein, the term “adjuvant” refers to a substance which modulates and / or engenders an immune response. Generally, the adjuvant is administered in conjunction with an antigen to effect enhancement of an immune response to the antigen as compared to antigen alone. Various adjuvants are described herein.

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

[0161] As used herein, by “pharmaceutically acceptable” or “pharmacologically compatible” is meant a material that is not biologically or otherwise undesirable, e.g., the material may 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. Pharmaceutically acceptable carriers or excipients have preferably met the required standards of toxicological and manufacturing testing and / or are included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

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

[0163] As used herein, “microfluidic systems” refers to systems in which low volumes (e.g., m\L, nL, pL, fL) of fluids are processed to achieve the discrete treatment of small volumes of liquids. Certain implementations described herein include multiplexing, automation, and high throughput screening. The fluids (e.g., a buffer, a solution, a payload-containing solution, or a cell suspension) can be moved, mixed, separated, or otherwise processed. In certain embodiments described herein, microfluidic systems are used to apply mechanical constriction to a cell suspended in a buffer, inducing perturbations in the cell (e.g., holes) that allow a payload or compound to enter the cytosol of the cell.

[0164] As used herein, a “constriction” may refer to a portion of a microfluidic channel defined by an entrance portion, a centerpoint, and an exit portion, wherein the centerpoint is defined by a width, a length, and a depth. In other examples, a constriction may refer to a pore or may be a portion of a pore. The pore may be contained on a surface (e.g., a filter and / or membrane).

[0165] As used herein, “width of constriction” refers to the width of the microfluidic channel at the centerpoint. In some embodiments, the constriction has a width of less than about 6um. For example, in some embodiments the constriction may be less than about any of 0.6 um, 0.7 um, 0.8 pm, 0.9 pm, 1.0 pm, 1.5 pm, or 2 um. In some embodiments, the constriction has a width of less than about 4 um. In certain aspects of the invention, the constriction has a width between about 0.5 um and about 4 pm. In further embodiments, the constriction has a width between about 3 pm and about 4 pm. In further embodiments, the constriction has a width between about 2 um and about 4 pm. In further aspects, the constriction has a width of about 3.9 pum or less. In further aspects, the constriction has a width of about 3.9 um or less.In further aspects, the constriction has a width of about 2.2 um. In certain embodiments, the constriction is configured such that a single cell passes through the constriction at a time.

[0166] As used herein “length of constriction” refers to the length of the microfluidic channel at the centerpoint. In certain aspects of the invention, the length of the constriction is about 30 um or less. In some embodiments, the length of the constriction is between about 10 pm and about 30 um. In certain embodiments, the length of the constriction is between about 10 pm and about 20 pm. For example, the length of the constriction may be about any of 11 pm, 12 pm, 13 pum, 14 pm, 15 pm, 20 pm, or 25 pm including all integers, decimals, and fractions between about 10 pm and about 30 um. The length of the constriction can vary to increase the length of time a cell is under constriction (e.g., greater lengths result in longer constrictions times at a given flow rate). The length of the constriction can vary to decrease the length of time a cell is under constriction (e.g., shorter lengths result in shorter constriction times at a given flow rate).

[0167] As used herein, “depth of constriction” refers to the depth of the microfluidic channel at the centerpoint. The depth of constriction can be adjusted to provide a tighter constriction and thereby enhance delivery, similar to adjustments of the constriction width. In some embodiments, the depth of the constriction is between about 1 pm and about 1 mm, including all integers, decimals, and fractions between about 1 pm and about 1 mm. In some embodiments, the depth is about 20 pum. In some embodiments the depth is uniform throughout the channel. In certain embodiments, the depth is decreased at the point of constriction to result in a greater constriction of the cell. In some embodiments, the depth is increased at the point of constriction to result in a lesser constriction of the cell. In some embodiment, the depth of the constriction is greater than the width of the constriction. In certain embodiments, the depth of constriction is less than the width of the constriction. In some embodiments, the depth of constriction and the width of the constriction are equal.

[0168] In some embodiments, the dimensions of the microfluidic device are denoted by length of constriction, width of constriction, and number of constrictions in series. For example, a microfluidic device with a constriction length of 30 um, a width of 5 um, and 5 constrictions in series is represented herein as 30 x 5 x 5 (L x W x # of constrictions).

[0169] In some embodiments, the microfluidic system comprises at least one microfluidic channel comprising at least one constriction. In some embodiments, the microfluidic system comprises multiple microfluidic channels each comprising at least one constriction. For example, the microfluidic system may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 500, 1000, 10,000, 20,000 or greater microfluidic channels, including all integers from 10 to 50, 50 to 100, 100 to 500, 500, 1000, 10000 to 20,000, and the like. In certain aspects, the multiple microfluidic channels each comprising one constriction are arranged in parallel. In certain aspects, the multiple microfluidic channels each comprising one constriction are arranged linearly in series. In certain aspects of the invention, the microfluidic system comprises one microfluidic channel comprising multiple constrictions. For example, one microfluidic channel may comprise 2, 3, 4, 5, 10, 20, or greater constrictions. In some embodiments, the microfluidic system comprises multiple microfluidic channels comprising multiple constrictions. In some aspects of the invention, the multiple microfluidic channels comprising multiple constrictions are arranged in parallel. In some aspects of the invention, the multiple microfluidic channels comprising multiple constrictions are arranged linearly in series.

[0170] The entrance portion may comprise a “constriction angle” that can vary to increase or decrease how quickly the diameter of the channel decreases towards the centerpoint of the constriction. The constriction angle can vary to minimize clogging of the microfluidic system while cells are passing therethrough. For example, the constriction angle may be between 1 and 140 degrees. In certain embodiments, the constriction angle may be between 1 and 90 degrees. The exit portion may also comprise an angle to reduce the likelihood of turbulence / eddies that can result in non-laminar flow. For example, the angle of the exit portion may be between 1 and 140 degrees. In certain embodiments, the angle of the exit portion may be between 1 and 90 degrees.

[0171] The cross-section of the microfluidic channel, the entrance portion, the centerpoint, and the exit portion may vary. Non-limiting examples of various cross-sections include circular, elliptical, an elongated slit, square, hexagonal, or triangular cross-sections.

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

[0173] Cells are moved (e.g., pushed) through the constriction by application of pressure. In some embodiments, said pressure is applied by a cell driver. As used herein, a cell driver is a device or component that applies a pressure or force to the buffer or solution in order to drive a cell through a constriction. In some embodiments, a pressure can be applied by a cell driver at the inlet. In some embodiments, a vacuum pressure can be applied by a cell driver at the outlet. In certain embodiments, the cell driver is adapted to supply a pressure 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 a 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, human muscle, gravity, a microfluidic pumps, and a syringe. Modifications to the pressure applied by the cell driver also affect the velocity at which the cells pass through the microfluidic channel (e.g., increases in the amount of pressure will result in increased cell velocities). When a cell (e.g., an anucleate cell) passes through the constriction, its membrane is perturbed causing temporary disruptions in the membrane and resulting in the uptake of the payload that is present in the surrounding medium. As used herein, these temporary disruptions are referred to as “perturbations.” Perturbations created by the methods described herein are breaches in a cell that allow material from outside the cell to move into the cell. Non-limiting examples of perturbations include a hole, a tear, a cavity, an aperture, a pore, a break, a gap, or a perforation. The perturbations (e.g., pores or holes) created by the methods described herein are not formed as a result of assembly of protein subunits to form a multimeric pore structure such as that created by complement or bacterial hemolysins. Methods for stimulating an immune response to an antigen in an individual Methods for delivering an antigen into an anucleate cell-derived vesicle

[0174] In certain aspects, there is provided a method for delivering an antigen into an anucleate cell-derived vesicle, the method comprising passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen to pass through to form an anucleate cell-derived vesicle; and incubating the anucleate cell-derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle. In some embodiments, the anucleate cell-derived vesicle further comprises an adjuvant. In some embodiments, the input anucleate cell further comprises an adjuvant.

[0175] In certain aspects, there is provided a method for delivering an adjuvant into an anucleate cell-derived vesicle, the method comprising passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the adjuvant to pass through to form an anucleate cell-derived vesicle; and incubating the anucleate cell-derived vesicle with the adjuvant for a sufficient time to allow the adjuvant to enter the anucleate cell-derived vesicle. In some embodiments, the anucleate cell-derived vesicle further comprises an antigen. In some embodiments, the input anucleate cell further comprises an antigen.

[0176] In certain aspects, there is provided a method for delivering an antigen and an adjuvant into an anucleate cell-derived vesicle, the method comprising passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen and the adjuvant to pass through to form an anucleate cell-derived vesicle; and incubating the anucleate cell-derived vesicle with the antigen and the adjuvant for a sufficient time to allow the antigen and the adjuvant to enter the anucleate cell-derived vesicle. Methods for stimulating an immune response

[0177] In certain aspects, there is provided a method for stimulating an immune response to an antigen in an individual, the method comprising administering to the individual an effective amount of an anucleate cell-derived vesicle comprising an antigen, wherein the anucleate cell- derived vesicle comprising the antigen is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell-derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle. In some embodiments, the method further comprises administering an adjuvant systemically to the individual. In some embodiments, the systemic adjuvant is administered before, after or at the same time as the anucleate cell-derived vesicle. In some embodiments, the input anucleate cell comprises an adjuvant. In some embodiments, the systemic adjuvant is an extracellular adjuvant. In some embodiment, the systemic adjuvant is an extravesicular adjuvant. In some embodiments, the method of stimulating an immune response to the antigen in the individual enhances a pre-existing immune response to the antigen.

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

[0179] In certain aspects, there is provided a method for treating a disease in an individual, comprising administering to the individual an anucleate cell-derived vesicle comprising a disease-associated antigen, wherein an immune response against the antigen ameliorates conditions of the disease, and wherein the anucleate cell-derived vesicle comprising the disease- associated antigen is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell-derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle.

[0180] In certain aspects, there is provided a method for preventing a disease in an individual, comprising administering to the individual an anucleate cell-derived vesicle comprising a disease-associated antigen, wherein an immune response against the antigen prevents development of the disease, and wherein the anucleate cell-derived vesicle comprising the disease-associated antigen is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell- derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle.

[0181] In certain aspects, there is provided a method for vaccinating an individual against an antigen, comprising administering to the individual an anucleate cell-derived vesicle comprising the antigen, wherein the anucleate cell-derived vesicle comprising the antigen is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell-derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle.

[0182] In some embodiments according to any of the methods described herein, the method further comprises administering an adjuvant systemically to the individual. In some embodiments, the systemic adjuvant is administered before, after or at the same time as the anucleate cell-derived vesicle. In some embodiments, the input anucleate cell comprises an adjuvant. In some embodiments, the systemic adjuvant is an extracellular adjuvant. In some embodiment, the systemic adjuvant is an extravesicular adjuvant.

[0183] In other aspects, there is provided a method for treating a disease in an individual, comprising administering to the individual an anucleate cell-derived vesicle comprising a disease-associated antigen and an adjuvant, wherein an immune response against the antigen ameliorates conditions of the disease, and wherein the anucleate cell-derived vesicle comprising the disease-associated antigen and the adjuvant is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen and an adjuvant to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell-derived vesicle with the antigen and the adjuvant for a sufficient time to allow the antigen and the adjuvant to enter the anucleate cell-derived vesicle.

[0184] In certain aspects, there is provided a method for preventing a disease in an individual, comprising administering to the individual an anucleate cell-derived vesicle comprising a disease-associated antigen and an adjuvant, wherein an immune response against the antigen prevents development of the disease, and wherein the anucleate cell-derived vesicle comprising the disease-associated antigen and the adjuvant is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen and an adjuvant to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell-derived vesicle with the antigen and the adjuvant for a sufficient time to allow the antigen and the adjuvant to enter the anucleate cell-derived vesicle.

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

[0186] In certain aspects, there is provided a method for treating a disease in an individual, wherein an immune response against a disease-associated antigen ameliorates conditions of the disease, the method comprising: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen to pass through to form an anucleate cell-derived vesicle; b) incubating the anucleate cell-derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle, thereby generating an anucleate cell-derived vesicle comprising the antigen; and ¢) administering the anucleate cell-derived vesicle comprising the antigen to the individual.

[0187] In certain aspects, there is provided a method for preventing a disease in an individual, wherein an immune response against a disease-associated antigen prevents development of the disease, the method comprising: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen to pass through to form an anucleate cell-derived vesicle; b) incubating the anucleate cell-derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle, thereby generating an anucleate cell-derived vesicle comprising the antigen; and ¢) administering the anucleate cell-derived vesicle to the individual.

[0188] In certain aspects, there is provided a method for vaccinating an individual against an antigen, the method comprising: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen to pass through to form an anucleate cell-derived vesicle; b) incubating the anucleate cell-derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle, thereby generating an anucleate cell-derived vesicle comprising the antigen; and c) administering the anucleate cell-derived vesicle comprising the antigen to the individual.

[0189] In some embodiments, the method further comprises administering an adjuvant systemically to the individual. In some embodiments, the systemic adjuvant is administered before, after or at the same time as the anucleate cell-derived vesicle. In some embodiments, the input anucleate cell comprises an adjuvant. In some embodiments, the systemic adjuvant is an extracellular adjuvant. In some embodiment, the systemic adjuvant is an extravesicular adjuvant.

[0190] In other aspects, there is provided a method for treating a disease in an individual, wherein an immune response against a disease-associated antigen ameliorates conditions of the disease, the method comprising: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the disease-associated antigen and an adjuvant to pass through to form an anucleate cell-derived vesicle; b) incubating the anucleate cell-derived vesicle with the antigen and the adjuvant for a sufficient time to allow the antigen and the adjuvant to enter the anucleate cell-derived vesicle, thereby generating an anucleate cell-derived vesicle comprising the antigen and the adjuvant; and ¢) administering the anucleate cell-derived vesicle comprising the antigen and the adjuvant to the individual.

[0191] In some aspects, there is provided a method for preventing a disease in an individual, wherein an immune response against a disease-associated antigen prevents development of the disease, the method comprising: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen and an adjuvant to pass through to form an anucleate cell-derived vesicle; b) incubating the anucleate cell-derived vesicle with the antigen and the adjuvant for a sufficient time to allow the antigen and the adjuvant to enter the anucleate cell- derived vesicle, thereby generating an anucleate cell-derived vesicle comprising the antigen and the adjuvant; and ¢) administering the anucleate cell-derived vesicle comprising the antigen and the adjuvant to the individual.

[0192] In certain aspects, there is provided a method for vaccinating an individual against an antigen, the method comprising: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen and an adjuvant to pass through to form an anucleate cell-derived vesicle; b) incubating the anucleate cell-derived vesicle with the antigen and the adjuvant for a sufficient time to allow the antigen and the adjuvant to enter the anucleate cell- derived vesicle, thereby generating an anucleate cell-derived vesicle comprising the antigen and the adjuvant; and ¢) administering the anucleate cell-derived vesicle comprising the antigen and the adjuvant to the individual.

[0193] In some embodiments, the method further comprises administering an adjuvant systemically to the individual. In some embodiments, the systemic adjuvant is administered before, after or at the same time as the anucleate cell-derived vesicle. In some embodiments, the input anucleate cell comprises an adjuvant. In some embodiments, the systemic adjuvant is an extracellular adjuvant. In some embodiment, the systemic adjuvant is an extravesicular adjuvant.

[0194] In some embodiments according to any of the methods described herein, the disease is cancer, an infectious disease or a viral-associated disease. In some embodiment, the cancer comprises one or more of head and neck, cervical, uterine, rectal, penile, ovarian, testicular, bone, soft tissue, skin (melanoma), gastric, intestinal, colon, prostate, breast, esophageal, liver, lung, pancreatic, brain, or blood cancers. In some embodiments, the infectious disease or the viral-associated 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 an HPV-18 antigen. In some embodiments, the HPV antigen is an HPV E6 antigen or an HPV E7 antigen. In some embodiments, the HPV-associated disease is an HPV-associated cancer. In some embodiments, the HPV-associated 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-associated disease is an HPV-associated infectious disease. In some embodiments, the HPV-associated diseases can include common warts, plantar warts, flat warts, anogenital warts, anal lesions, epidermodysplasia, focal epithelial hyperplasia, mouth papillomas, verrucous cysts, laryngeal papillomatosis, squamous intraepithelial lesions (SILs), cervical intraepithelial neoplasia (CIN), vulvar intraepithelial neoplasia (VIN) and vaginal intraepithelial neoplasia (VAIN). In certain embodiments, the disease-associated 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 viral 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 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, pULA7, pUL32, pUL82, pULS83, and pUL99, pUL69, pUL25, pULS56, pULS4, pULS7, pUL144 or pUL128. In some embodiments, the viral 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 viral-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 people with HIV. In some embodiments, the HIV-associated disease are opportunistic infections, which may include but are not limited to: candidiasis of bronchi, trachea, esophagus, or lungs; invasive cervical cancer; coccidioidomycosis; cryptococcosis; chronic intestinal cryptosporidiosis, Cytomegalovirus diseases; HIV-related encephalopathy; HSV-related chronic ulcers or bronchitis, pneumonitis, or esophagitis; histoplasmosis; chronic intestinal isosporiasis; Kaposi's sarcoma; lymphoma; tuberculosis; Mycobacterium avium complex (MAC); Pneumocystis carinii pneumonia (PCP); recurrent pneumonia; progressive multifocal leukoencephalopathy; recurrent Salmonella septicemia; Toxoplasmosis of brain; and wasting syndrome due to HIV.

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

[0196] In some aspects, the invention provides methods of treating a patient by introducing an anucleate cell-derived vesicle comprising an antigen and / or an adjuvant, generated by passing an input anucleate cell through a constriction to form an anucleate cell-derived vesicle such that the antigen and / or adjuvant enters the anucleate cell-derived vesicle, to the individual. In some embodiments, the treatment comprises multiple (such as any of 2, 3, 4, 5, 6, or more) steps of introducing such anucleate cell-derived vesicles to the individual. For example, in some embodiments, there is provided a method of treating an individual by administering an anucleate cell-derived vesicle comprising an antigen and / or an adjuvant, generated by passing an input anucleate cell through a constriction to form an anucleate cell-derived vesicle such that the antigen and / or adjuvant enters the anucleate cell-derived vesicle, to the individual 2, 3, 4, 5, 6, or more times. In some embodiments, the duration of time between any two consecutive administrations of the cell is at least about 1 day (such at least about any of 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3weeks, 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).

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

[0198] In some embodiments, an input anucleate cell is isolated from a universal blood donor (e.g. an O- blood donor) and then stored and / or frozen for later constriction-mediated delivery. In some embodiments, an antigen is isolated from an individual and delivered to an input anucleate cell isolated from a universal donor. In some embodiments, an input anucleate cell is isolated from a blood donor and then stored and / or frozen for later constriction mediated delivery (SQZ). In some embodiments, an antigen is isolated from an individual and delivered to an input anucleate cell isolated from a blood donor. In some embodiments, an anucleate cell- derived vesicle comprising an antigen and / or an adjuvant is generated by constriction-mediated delivery described above. In some embodiments, the anucleate cell-derived vesicle comprising the antigen and / or the adjuvant is introduced into an individual. In some embodiments, the anucleate cell-derived vesicle comprising the antigen and / or the adjuvant is stored and / or frozen (e.g., for later treatments). In some embodiments, the individual has a matched blood type to the blood donor. In some embodiments, an anucleate cell-derived vesicle comprising an antigen and / or an adjuvant is generated by constriction-mediated delivery described above. In some embodiments, the anucleate cell-derived vesicle comprising the antigen and / or the adjuvant is introduced into an individual. In some embodiments, the individual has a matched blood type to the blood donor. In some embodiments, the individual has a mismatched blood type to the blood donor.

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

[0200] In some embodiments, the method of prevention comprises multiple (such as any of 2, 3,4, 5, 6, or more) steps of administering the anucleate cell-derived vesicles as described herein to the individual. For example, in some embodiments, there is provided a method of vaccinating an individual against an antigen by administering an anucleate cell-derived vesicle comprising an antigen and / or an adjuvant, generated by passing an input anucleate cell through a constriction to form an anucleate cell-derived vesicle such that the antigen and / or adjuvant enters the anucleate cell-derived vesicle, to the individual 2, 3, 4, 5, 6, or more times. In some embodiments, the duration of time between any two consecutive administrations of the cell is at least about 1 day (such at least about any of 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3weeks, 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, provided are methods of vaccinating an individual against an antigen by introducing an anucleate cell-derived vesicle comprising the antigen and / or an adjuvant, generated by passing an input anucleate cell through a constriction to form an anucleate cell-derived vesicle such that the antigen and / or adjuvant enters the anucleate cell- derived vesicle, to the individual. In some embodiments, the input anucleate cell is an autologous cell. For example, the input anucleate cell is isolated from an individual (e.g., a patient), processed according to the methods disclosed, and the resulting anucleate cell-derived vesicle is introduced back into the same individual. Therefore, in some embodiments, the anucleate cell-derived vesicle is autologous to the individual. In other embodiments, the input anucleate cell is an allogeneic cell. For example, the anucleate cell is isolated from a different individual (e.g., the donor), processed according to the methods disclosed, and the resulting anucleate cell-derived vesicle is introduced into the first individual (e.g., the patient). In some embodiments, the anucleate cell-derived vesicle is allogeneic to the individual. In some embodiments, a pool of input anucleate cells from multiple individuals is processed according to the methods disclosed, and a pool of anucleate cell-derived vesicles is introduced into the first individual (e.g., the patient). In some embodiments, the input anucleate cell is isolated from an individual, processed according to the disclosed methods, and the anucleate cell-derived vesicle is introduced into a different individual. In some embodiments, a population of input anucleate cells is isolated from an individual (the patient) or a different individual, passed through the constriction to achieve delivery of an antigen and / or an adjuvant, and then a population of anucleate cell-derived vesicles is re-infused into the patient to induce a prophylactic response.

[0202] In some embodiments, the vaccination comprises multiple (such as any of 2, 3,4, 5, 6, or more) steps of administering the anucleate cell-derived vesicles as described herein to the individual. For example, in some embodiments, there is provided a method of vaccinating an individual against an antigen by administering an anucleate cell-derived vesicle comprising an antigen and / or an adjuvant, generated by passing an input anucleate cell through a constriction to form an anucleate cell-derived vesicle such that the antigen and / or adjuvant enters the anucleate cell-derived vesicle, to the individual 2, 3, 4, 5, 6, or more times. In some embodiments, the duration of time between any two consecutive administrations of the cell is at least about 1 day (such at least about any of 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3weeks, 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).

[0203] Any of the methods described above are carried out in vitro, ex vivo, or in vivo. For in vivo applications, the device may be implanted in a vascular lumen, e.g., an in-line stent in an artery or vein. In some embodiments, the methods are 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 could be employed as a means of enhancing and / or stimulating an immune response in an individual. In some embodiments, the method can be implemented in a typical hospital laboratory with a minimally trained technician. In some embodiments, a patient operated treatment system can be used. In some embodiments, the method is implemented using an in- line blood treatment system, in which blood is directly diverted from a patient, passed through the constriction, resulting in formation of vesicles derived from anucleate cells in the blood and delivery of antigen and / or adjuvant to the anucleate cell-derived vesicles in blood, and directly transfused back into the patient after treatment.

[0204] In some embodiments according to any of the methods described herein, the anucleate cell-derived vesicle is in a pharmaceutical formulation. In some embodiments, the anucleate cell-derived vesicle is administered systemically. In some embodiments, the anucleate cell- derived vesicle is administered intravenously, intraarterially, subcutaneously, intramuscularly, or intraperitoneally. In certain embodiments, the anucleate cell-derived vesicle is administered to the individual in combination with a therapeutic agent. In some embodiments, the therapeutic agent is administered before, after or at the same time as the anucleate cell-derived vesicle.

[0205] In some embodiments, the therapeutic agent is an immune checkpoint inhibitor, and / or a cytokine. In some embodiments, the therapeutic agent comprises one or more of: IFN-a, IFN- v, IL-2 (any of its natural or modified forms), IL-10, or IL-15. In some embodiments, the therapeutic agent is one or more forms of immunotherapy. Immunotherapy includes but is not limited to: monoclonal antibodies, immune checkpoint inhibitors, cytokines, vaccines to treat cancers, and adoptive cell transfer. In some embodiments according to any of the methods described herein, the method further comprises administration of 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 anucleate cell-derived vesicle is administered to the individual in combination with a chemotherapy or a radiation therapy. In some embodiments, the anucleate cell-derived vesicle is administered to the individual in combination with one or more agents that improve antigen presentation, improve T cell proliferation, and / or improve tumor microenvironments. Anucleate cells

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

[0207] In some embodiments, the anucleate cell is a red blood cell. Red blood cells (RBCs) are flexible and oval biconcave discs with cytoplasm rich in the oxygen-carrier biomolecule hemoglobin. RBCs serve as the primary means for oxygen delivery and carbon dioxide removal throughout the human body. RBCs can stay in circulation for up to 120 days, after which they are removed from the body via clearance in the liver and spleen. In some embodiments, the anucleate cell is a precursor to RBCs. In some embodiments, the anucleate cell is a reticulocyte. Reticulocytes are anucleate immature (not yet biconcave) red blood cells and typically comprise 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 anucleate cell is an erythrocyte. In some embodiments, the anucleate cell is a platelet. Platelets, also called thrombocytes, are a component of blood whose function involves blood clotting. Platelets are biconvex discoid (lens-shaped) structures 2-3 um in diameter.

[0208] In some embodiments according to any of the methods described herein, presentation of antigen in an immunogenic environment enhances an immune response to the antigen and / or stimulates an immune response to the antigen. Antigens derived from apoptotic bodies, such as anucleate cell-derived vesicles, which can be cleared in the immunogenic environment of the liver and spleen, may stimulate and / or enhance an immune response to the antigens via activation of T cells. In some embodiments, the immune response is antigen-specific. Anucleate cell-derived vesicles, such as red blood cell-derived vesicles have a limited life span and are unable to self-repair, causing eryptosis, a process analogous to apoptosis, that leads to subsequent removal of the anucleate cell-derived vesicles from the bloodstream. In some embodiments, the antigen may be released upon eryptosis of the anucleate cell-derived vesicles within the immunogenic environment, where it is subsequently engulfed, processed, and presented by an antigen-presenting cell. In some embodiments, the anucleate cell-derived vesicle comprising the antigen is phagocytosed by an antigen-presenting cell, and the antigen is subsequently processed and presented by the antigen-presenting cell. In some embodiments, the anucleate cell-derived vesicle comprising the antigen is phagocytosed by a resident macrophage, and the antigen is subsequently processed and presented by the resident macrophage.

[0209] In some embodiments, the antigen contained in the anucleate cell-derived vesicle is subsequently 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 an immunogenic environment. In some embodiments, the immune response is antigen-specific.

[0210] In some embodiments, the anucleate cell-derived vesicle comprises an adjuvant. In some embodiments, the adjuvant generates or promotes an immunogenic environment, wherein presentation of an antigen in said immunogenic environment stimulates an immune response to the antigen. In some embodiments, the immune stimulation is multi-specific, including stimulation of an immune response to a plurality of antigens.

[0211] In some embodiments according to any of the methods described herein, the method comprises passing a cell suspension comprising an input anucleate cell through a constriction, wherein said constriction deforms the input anucleate cell thereby causing a perturbation of the input anucleate cell to form an anucleate cell-derived vesicle such that an antigen and / or an adjuvant enter the anucleate cell-derived vesicle. In some embodiments, the antigen is presented in an immunogenic environment. In some embodiments, the adjuvant generates or promotes an immunogenic environment, wherein presentation of the antigen in the immunogenic environment stimulates an immune response to the antigen. In some embodiments, the antigen is processed in an immunogenic environment. In some embodiments, the immune stimulation is antigen-specific. In some embodiments, the immune stimulation is multi-specific, including stimulation of an immune response to a plurality of antigens.

[0212] In certain embodiments according to any of the methods described herein, the method comprises passing a first cell suspension comprising a first input anucleate cell through a constriction, wherein said constriction deforms the cell thereby causing a perturbation of the first input anucleate cell such that an antigen enters a vesicle derived from perturbing the first input anucleate cell, passing a second cell suspension comprising a second input anucleate cell through a constriction, wherein said constriction deforms the second input anucleate cell thereby causing a perturbation of the second input anucleate cell such that an adjuvant enters a vesicle derived from perturbing the second input anucleate cell, and introducing a vesicle derived from the first input anucleate cell and a vesicle derived from the second input anucleate cell into the individual, thereby stimulating an immune response to the antigen. Therefore, in some embodiments, the vesicle derived from the first input anucleate cell comprises an antigen and the vesicle derived from the second input anucleate cell comprises an adjuvant. In some embodiments, the antigen is presented in an immunogenic environment. In some embodiments, the adjuvant generates or promotes an immunogenic environment, wherein presentation of the antigen in the immunogenic environment stimulates an immune response to the antigen. In some embodiments, the antigen is processed in an immunogenic environment. In some embodiments, the vesicle derived from the first input anucleate cell and the vesicle derived from the second input anucleate cell are introduced simultaneously. In some embodiments, the vesicle derived from the first input anucleate cell and the vesicle derived from the second input anucleate cell are introduced sequentially. In some embodiments, the vesicle derived from the first input anucleate cell is introduced to the individual before introduction of the vesicle derived from the second input anucleate cell. In some embodiments, the vesicle derived from the first input anucleate cell is introduced to the individual more than any of about 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 introduction of the vesicle derived from the second input anucleate cell. In some embodiments, the vesicle derived from the first input anucleate cell is introduced to the individual more than any of about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days before introduction of the vesicle derived from the second input anucleate cell. In some embodiments, the vesicle derived from the second input anucleate cell is introduced to the individual before introduction of the vesicle derived from the first input anucleate cell. In some embodiments, the vesicle derived from the second input anucleate cell is introduced to the individual more than any of about 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 introduction of the vesicle derived from the first input anucleate cell. In some embodiments, the vesicle derived from the second input anucleate cell is introduced to the individual more than any of about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days before introduction of the vesicle derived from the first input anucleate cell. In some embodiments, the immune stimulation is antigen-specific. In some embodiments, the immune stimulation is multi-specific, including stimulation of an immune response to a plurality of antigens.

[0213] In some embodiments, the stimulated and / or enhanced immune response comprises an increased T cell response. For example, an increased T cell response may 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 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 inflammatory cytokines selected from interlenkin-1 (IL-1), IL-2, IL-12, and IL- 18, tumor necrosis factor (TNF), interferon gamma (IFN-v), 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-a and transforming growth factor-beta (TGF-B). In some embodiments, the stimulated and / or enhanced immune response comprises a change in T cell phenotype. For example, the T cell state 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 a T cell, which otherwise may 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. Anucleate Cell-Derived Vesicles

[0214] In some aspects, the present application provides anucleate cell-derived vesicles prepared from a parent anucleate cell, the anucleate cell-derived vesicle having one or more of the following properties: (a) a circulating half-life in a mammal that is decreased compared to the parent anucleate cell, (b) decreased hemoglobin levels compared to the parent anucleate cell, (c) spherical morphology, (d) increased surface phosphatidylserine levels compared to the parent anucleate cell, or (e) reduced ATP production compared to the parent anucleate cell.

[0215] In certain aspects, there is provided an anucleate cell-derived vesicle comprising an antigen, wherein the anucleate cell-derived vesicle comprising the antigen is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell-derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle; thereby generating the anucleate cell-derived vesicle comprising the antigen. In some embodiments, the input anucleate cell comprises an adjuvant.

[0216] In certain aspects, there is provided an anucleate cell-derived vesicle comprising an adjuvant, wherein the anucleate cell-derived vesicle comprising the adjuvant is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the adjuvant to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell-derived vesicle with the adjuvant for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle; thereby generating the anucleate cell-derived vesicle comprising the adjuvant. In some embodiments, the input anucleate cell comprises an antigen.

[0217] In certain aspects, there is provided an anucleate cell-derived vesicle comprising an antigen and an adjuvant, wherein the anucleate cell-derived vesicle comprising the antigen and the adjuvant is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen and the adjuvant to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell- derived vesicle with the antigen and the adjuvant for a sufficient time to allow the antigen and the adjuvant to enter the anucleate cell-derived vesicle; thereby generating the anucleate cell- derived vesicle comprising the antigen and the adjuvant.

[0218] In some embodiments, the anucleate cell-derived vesicle is a red blood cell-derived vesicle or a platelet-derived vesicle. In some embodiments, the anucleate cell-derived vesicle is an erythrocyte-derived vesicle or a reticulocyte-derived vesicle

[0219] In some embodiments according to any of the anucleate cell-derived vesicles described herein, the input or parent anucleate cell is a mammalian cell. Anucleate cells lack a nucleus. In some embodiments, the input anucleate cell is a monkey, mouse, dog, cat, horse, rat, sheep, goat, pig, or rabbit cell. In some embodiments, the input anucleate cell is a human cell. In some embodiments, the input anucleate cell is a non-mammalian cell. In some embodiments, the input anucleate cell is a chicken, frog, insect, fish, or nematode cell. In some embodiments, the input anucleate cell is an erythrocyte. In some embodiments, the input anucleate cell is a red blood cell. In some embodiments, the input anucleate cell is a precursor to red blood cells. In some embodiments, the input anucleate cell is a reticulocyte. In some embodiments, the input anucleate cell is a platelet.

[0220] In some embodiments, presentation of antigen in an immunogenic environment enhances an immune response to the antigen or induces an immune response to the antigen. Antigens derived from eryptotic bodies, such as anucleate cell-derived vesicles, which can be cleared in the immunogenic environment of the liver and spleen, may stimulate and / or enhance an immune response to the antigens via activation of T cells. In some embodiments, the immune response is antigen-specific. Anucleate cell-derived vesicles, such as RBC-derived vesicles have a limited life span and are unable to self-repair, causing eryptosis, a process analogous to apoptosis, that leads to removal of the anucleate cell-derived vesicle from the bloodstream. In some embodiments, the antigen may be released upon eryptosis of the anucleate cell-derived vesicles within the immunogenic environment, where it is subsequently engulfed, processed, and presented by an antigen-presenting cell. In some embodiments, the anucleate cell-derived vesicle containing the antigen is phagocytosed by an antigen-presenting cell, such as a macrophage, and the antigen is subsequently processed and presented by the antigen-presenting cell. In some embodiments, the antigen-presenting cell is a resident macrophage.

[0221] In some embodiments, the input or parent anucleate cell is a red blood cell. In some embodiments, the input or parent anucleate cell is a platelet. In some embodiments, the red blood cell is an erythrocyte. In some embodiments, the red blood cell is a reticulocyte.

[0222] In some embodiments, the circulating half-life of an anucleate cell-derived vesicle in a mammal is decreased compared to an input or parent anucleate cell. Methods for measuring the half-life of a cell, such as an anucleate cell, e.g., red blood cell, or an anucleate cell-derived vesicle are known in the art. See, e.g., Franco, R. S., Transfus Med Hemother, 39, 2012. For example, in some embodiments, the method for measuring the half-life of an anucleate cell or an anucleate cell-derived vesicle comprises a cohort labeling technique or a random labeling technique. In some embodiments, the method for measuring the half-life of an anucleate cell or an anucleate cell-derived vesicle comprises labeling, reinfusing the cell or vesicle, and measuring the disappearance upon reinfusion. In some embodiments, the method for measuring the half-life of an anucleate cell or an anucleate cell-derived vesicle encompassed in the present application comprises measuring the half-life of an appropriate reference control(s), such as a control comprising an input or parent anucleate cell or a population of input or parent anucleate cells.

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

[0224] In some embodiments, the circulating half-life of the anucleate cell-derived vesicle 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, or 10 days. In some embodiments, the circulating half-life of the anucleate cell-derived vesicle is about any of 0.5 minute, 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 anucleate cell is a human cell and wherein the circulating half-life of the anucleate cell-derived vesicle 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, or 10 days. In some embodiments, the input or parent anucleate cell is a human cell and wherein the circulating half-life of the anucleate cell-derived vesicle is less than about any of 0.5 minute, 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 anucleate cell is a red blood cell, wherein the hemoglobin level in the anucleate cell-derived vesicle is decreased compared to the input or parent anucleate cell. Methods of measuring the hemoglobin level of a cell, such as an anucleate cell, e.g., red blood cell, or an anucleate cell-derived vesicle, e.g., a red blood cell-derived vesicle, is known in the art. See, e.g., Chaudhary, R., J Blood Med, 8, 2017. For example, in some embodiments, the method comprises measuring a metabolic precursor or product to determine the turnover of hemoglobin. In some embodiments, the method comprises measuring one or more hemoglobin-derived (Hb) peptides. In some embodiments, the method for measuring the hemoglobin level of an anucleate cell or an anucleate cell-derived vesicle encompassed in the present application comprises measuring the levels of hemoglobin of an appropriate reference control(s), such as a control comprising an input or parent anucleate cell or a population of input or parent anucleate cell.

[0227] In some embodiments, the anucleate cell is characterized by loss, such as a reduction in the level, of an intracellular component compared to a parent anucleate cell.

[0228] In some embodiments, the hemoglobin level in the anucleate cell-derived vesicle is decreased by at least about any of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% 60%, 65%, 10%, 75%. 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.9%, , as compared to the input or parent anucleate cell. In some embodiments, the hemoglobin level in the anucleate cell-derived vesicle is decreased by about 50% to about 99.9%, such as any of about 70% to about 99.9%, about 85% to about 99.9%, or about 95% to about 99.9%, as compared to the input or parent anucleate cell. In some embodiments, the hemoglobin level in the anucleate cell- derived vesicle is decreased by about any of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.9%, as compared to the input or parent anucleate cell. In some embodiments, the anucleate cell-derived vesicle is devoid of hemoglobin. In some embodiments, the hemoglobin level in the anucleate cell-derived vesicle is about any of 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 anucleate cell.

[0229] In some embodiments, the level of one or more hemoglobin (Hb) peptides in the anucleate cell-derive vesicle is decreased by at least about any of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% 60%, 65%, 10%, 15%, 80%, 85%, 90%, 95%, 96%. 97%, 98%, 99%, 99.9%, or 100%, as compared to the input or parent anucleate cell. In some embodiments, the level of one or more Hb peptides in the anucleate cell-derived vesicle is decreased by about 50% to about 99.9%, such as any of about 70% to about 99.9%, about 85% to about 99.9%, or about 95% to about 99.9%, as compared to the input anucleate cell. In some embodiments, the level of one or more Hb peptides in the anucleate cell-derived vesicle is decreased by about any of 50%, 55%, 60%, 65%, 10%, 75%, 80%, 85%, 90%, 95%, 96%, 91%, 98%, 99%, or 99.9%, as compared to the input or parent anucleate cell.

[0230] In some embodiments, the input or parent anucleate cell is an erythrocyte and wherein the morphology of the anucleate cell-derived vesicle is modulated from that of the input or parent anucleate cell. Morphology concerns the classification of, e.g., the shape, structure, geometry, intensity, form, smoothness, roughness, circularity, volume, surface area, and / or size of a cell or a cell-derived vesicle. Methods for determining (such as measuring) morphology are known in the art. See, e.g., 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 comprises high-content imaging. For example, the morphology of the cell can be assessed by staining with Hoechst dye followed by automated high-content image analysis. In other examples, the morphology can be determined through a shift in the forward and side scatter plots from flow cytometry. In some embodiments, the input or parent anucleate cell is an erythrocyte and wherein the anucleate cell- derived vesicle is spherical in morphology. In some embodiments, the input or parent anucleate cell is an erythrocyte and wherein the anucleate cell-derived vesicle has a reduced biconcave shape compared to the input or parent anucleate cell. In some embodiments, the method for measuring morphology of an anucleate cell or an anucleate cell-derived vesicle encompassed in the present application comprises measuring the morphology of an appropriate reference control(s), such as a control comprising an input or parent anucleate cell or a population of input or parent anucleate cell.

[0231] In some embodiments, the input or parent anucleate cell is an erythrocyte and wherein the anucleate cell-derived vesicle has a reduced biconcave shape, such as reduced by more than about any of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 710%, 75%, 80%, 85%, 90% 95%,96%, 97%, 98%, 99%, or 99.9% as compared to the input or parent anucleate cell.

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

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

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

[0235] In some embodiments, the smallest dimension, such as diameter, of an anucleate cell- derived vesicle in suspension is about 5 pm to about 7.25 um, such as any of about 6 um to about 7 pm, or about 6.25 um to about 6.75 pm. In some embodiments, the smallest dimension, such as diameter, of an anucleate cell-derived vesicle in suspension is at least about 5 um, such as at least about any of 5.25 um, 5.5 pm, 5.75 um, 6 um, 6.25 pm, 6.5 pum, 6.75 pm, 7 pm, or 7.25 pm. In some embodiments, the largest dimension, such as diameter, of an anucleate cell- derived vesicle in suspension is about 5 pm to about 7.25 um, such as any of about 6 um to about 7 pm, or about 6.25 um to about 6.75 um. In some embodiments, the largest dimension, such as diameter, of an anucleate cell-derived vesicle in suspension is no greater than about 7.25 um, such as no greater than about any of 7 um, 6.75 pm, 6.5 ym, 6.25 pm, 6 pm, 5.75 um, 5.5 um, 5.25 um, or 5 um.

[0236] In some embodiments, the anucleate cell-derived vesicle exhibits one or more of the following properties: (a) a circulating half-life in a mammal is decreased compared to the parent anucleate cell, (b) decreased hemoglobin levels compared to the parent anucleate cell, (c) spherical morphology, (d) increased surface phosphatidylserine levels compared to the parent anucleate cell, or (e) reduced ATP production compared to the parent anucleate cell.

[0237] In some embodiments, the input or parent anucleate cell is a red blood cell or an erythrocyte and the anucleate cell-derived vesicle is a red blood cell ghost (RBC ghost).

[0238] In some embodiments, the anucleate cell is characterized by acquisition, such as an increase in the level, of a property compared to an input or parent anucleate cell.

[0239] In some embodiments, the anucleate cell-derived vesicle has increased surface phosphatidylserine levels compared to the input or parent anucleate cell. Phosphatidylserine exposure on the outer cell membrane is a hallmark of apoptosis and is recognized by receptors on phagocytes in a manner that promotes engulfment. Methods of measuring the phosphatidylserine level (such as surface phosphatidylserine level) of a cell, such as an anucleate cell, e.g., red blood cell, or an anucleate cell-derived vesicle are known in the art. See, e.g., Morita, S., er al., J Lipid Res, 53, 2012; Kay, J. G. et al., Sensors (Basel), 11, 2011; and Fabisiak JP et al., Methods Mol Biol, 1105, 2014. In some embodiments, the method for measuring the phosphatidylserine level of an anucleate cell or an anucleate cell-derived vesicle encompassed in the present application comprises measuring the phosphatidylserine level of an appropriate reference control(s), such as a control comprising an input or parent anucleate cell or a population of input or parent anucleate cell.

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

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

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

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

[0244] In some embodiments, the anucleate cell-derived vesicle has about any of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% 60%, 65%, 10%, 15%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, or 200% or more phosphatidylserine on its surface as compared to the input or parent anucleate cell. In some embodiments, the anucleate cell-derived vesicle has about 50% to about 200% more, such as any of about 50% to about 100%, about 100% to about 200%, or about 75% to about 125% more phosphatidylserine on its surface, as compared to the input or parent anucleate cell.

[0245] In some embodiments, the anucleate cell-derived vesicle has about any of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% 60%, 65%, 10%, 15%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, or 200% or more phosphatidylserine on its surface per unit volume as compared to the input or parent anucleate cell. In some embodiments, the anucleate cell- derived vesicle has about 50% to about 200% more, such as any of about 50% to about 100%, about 100% to about 200%, or about 75% to about 125% more phosphatidylserine on its surface per unit volume, as compared to the input or parent anucleate cell.

[0246] In some embodiments, the anucleate cell-derived vesicle has about any of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%. 55% 60%, 65%, 10%, 15%, 80%. 85%, 90%, 95%. 100%, 125%, 150%, 175%, or 200% or more phosphatidylserine on its surface per unit surface area as compared to the input or parent anucleate cell. In some embodiments, the anucleate cell- derived vesicle has about 50% to about 200% more, such as any of about 50% to about 100%, about 100% to about 200%, or about 75% to about 125% more phosphatidylserine on its surface per unit surface area, as compared to the input or parent anucleate cell.

[0247] In some embodiments, the anucleate cell-derived vesicle has about any of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% 60%, 65%, 70%, 15%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, or 200% or more phosphatidylserine on its surface per unit of membrane phospholipid as compared to the input or parent anucleate cell. In some embodiments, the anucleate cell-derived vesicle has about 50% to about 200% more, such as any of about 50% to about 100%, about 100% to about 200%, or about 75% to about 125% more phosphatidylserine on its surface per unit of membrane phospholipid, as compared to the input or parent anucleate cell.

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

[0249] In some embodiments according to any of the anucleate cell-derived vesicles described herein, a population profile of anucleate cell-derived vesicles prepared by the process exhibits higher average phosphatidylserine levels on the surface compared to the input or parent anucleate cells. In some embodiments, a population profile of anucleate cell-derived vesicles prepared by the process exhibits about any of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% 60%, 65%, 10%, 15%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, or 200% higher average phosphatidylserine levels on the surface compared to the input or parent anucleate cells. In some embodiments, a population profile of anucleate cell-derived vesicles prepared by the process exhibits 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 higher average phosphatidylserine levels on the surface compared to the input or parent anucleate cells.

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

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

[0252] In some embodiments, the anucleate cell-derived vesicle has reduced ATP production compared to the input or parent anucleate cell. . In some embodiments, the anucleate cell- derived vesicle has reduced ATP production, or levels of intracellular ATP, over time. Methods of measuring the ATP (such as reduced ATP production, or levels of intracellular ATP, over time) of a cell, such as an anucleate cell, e.g., red blood cell, or an anucleate cell-derived vesicle are known in the art. See, e.g., Morciano, G. et al., Nat Protoc, 12, 2017. In some embodiments, the ATP production is measured via a surrogate or a marker, such as lactate production. In some embodiments, the method for measuring ATP production of an anucleate cell or an anucleate cell-derived vesicle encompassed in the present application comprises measuring the ATP production of an appropriate reference control(s), such as a control comprising an input or parent anucleate cell or a population of input or parent anucleate cell. In some embodiments, the method for measuring ATP production, which allow for comparisons of ATP production between a sample and a control, comprises measuring ATP production of the sample and the control under similar conditions. In some embodiments, the method for measuring ATP production or intracellular ATP levels of an anucleate cell-derived vesicle encompassed in the present application comprises measuring the ATP production or intracellular ATP level of anucleate cell-derived vesicles of a population of the anucleate cell-derived vesicles at a first time and a second time, wherein the first time is before the second time, and comparing the results from the first time and the second time.

[0253] In some embodiments, the anucleate cell-derived vesicle produces 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 anucleate cell. In some embodiments, the anucleate cell-derived vesicle does not produce ATP.

[0254] In some embodiments, ATP production is determined by a lactate assay. In some embodiments, to measure the metabolic activity, such as ATP production, of the input anucleate cells versus anucleate cell-derived vesicles, the level of glycolysis can be indirectly measured over time by monitoring the level of lactate production using a fluorescent enzymatic assay. For example, the input anucleate cells are resuspended in citrate-phosphate-dextrose with adenine (dCPDA-1) buffer at 1 billion cells / mL and model antigen and / or adjuvant (at 20 pg / mL) is delivered at room temperature via SQZ (2.2 um constriction width at 50 psi) to generate anucleate cell-derived vesicles. The anucleate cell-derived vesicles, as well as the unprocessed input anucleate cells are then incubated at 37°C for the indicated time points and supernatant is collected. To quantify the levels of lactate produced by the input anucleate cells versus anucleate cell-derived vesicles, the Lactate-Glo assay (Promega) can be used employed to assay supernatant from the respective time points. Briefly, the supernatants are subjected to inactivation and neutralization steps, prior to the addition of the fluorescent lactate detection reagent. Fluorescence is normalized to a blank control and the absolute lactate levels in the supernatant are determined using a lactate standard curve (0.1-10 uM). In some embodiments, the absolute lactate level is about 0 uM to about 200 uM, such as any of about 0.01 uM to about 10 uM, about 0.01 uM to about

[0255] In some aspects, the present application provides anucleate cell-derived vesicles prepared from a parent anucleate cell, the anucleate cell-derived vesicle having any one or more of the following properties, as further described herein, of: (a) a circulating half-life in a mammal that is decreased compared to the parent anucleate cell, (b) decreased hemoglobin levels compared to the parent anucleate cell, (c) spherical morphology, (d) increased surface phosphatidylserine levels compared to the parent anucleate cell, or (e) reduced ATP production compared to the parent anucleate cell.

[0256] In some aspects, the present application provides anucleate cell-derived vesicles prepared from a parent anucleate cell, the anucleate cell-derived vesicle having any two or more of the following properties, as further described herein, of: (a) a circulating half-life in a mammal that is decreased compared to the parent anucleate cell, (b) decreased hemoglobin levels compared to the parent anucleate cell, (c) spherical morphology, (d) increased surface phosphatidylserine levels compared to the parent anucleate cell, or (e) reduced ATP production compared to the parent anucleate cell.

[0257] In some aspects, the present application provides anucleate cell-derived vesicles prepared from a parent anucleate cell, the anucleate cell-derived vesicle having any three or more of the following properties, as further described herein, of: (a) a circulating half-life in a mammal that is decreased compared to the parent anucleate cell, (b) decreased hemoglobin levels compared to the parent anucleate cell, (c) spherical morphology, (d) increased surface phosphatidylserine levels compared to the parent anucleate cell, or (e) reduced ATP production compared to the parent anucleate cell.

[0258] In some aspects, the present application provides anucleate cell-derived vesicles prepared from a parent anucleate cell, the anucleate cell-derived vesicle having any four or more of the following properties, as further described herein, of: (a) a circulating half-life in a mammal that is decreased compared to the parent anucleate cell, (b) decreased hemoglobin levels compared to the parent anucleate cell, (c) spherical morphology, (d) increased surface phosphatidylserine levels compared to the parent anucleate cell, or (e) reduced ATP production compared to the parent anucleate cell.

[0259] In some aspects, the present application provides anucleate cell-derived vesicles prepared from a parent anucleate cell, the anucleate cell-derived vesicle having the following properties, as further described herein, of: (a) a circulating half-life in a mammal that is decreased compared to the parent anucleate cell, (b) decreased hemoglobin levels compared to the parent anucleate cell, (c) spherical morphology, (d) increased surface phosphatidylserine levels compared to the parent anucleate cell, or (e) reduced ATP production compared to the parent anucleate cell.

[0260] In some embodiments, the anucleate cell-derived vesicle is modified to enhance uptake, such as increase uptake, in a tissue or cell compared to the uptake of the parent anucleate cell. In some embodiments, the anucleate cell-derived vesicle is modified to enhance uptake, such as increase uptake, in liver and / or spleen compared to the uptake of the parent anucleate cell in the respective tissue. In some embodiments, the anucleate cell-derived vesicle is modified to enhance uptake, such as increase uptake, in a phagocytic cell or an antigen-presenting cell, such as a macrophage or a dendritic cell, compared to the uptake of the parent anucleate cell in the respective phagocytic cell. 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 macrophage. In some embodiments, the anucleate cell-derived vesicle is cleared by a phagocytic cell and / or an antigen-presenting cell in the liver and / or spleen, thereby leading to antigen presentation including via CD8+ and CD4+ T cell responses.

[0261] In some embodiments according to any of the anucleate cell-derived vesicles described herein, the anucleate cell-derived vesicle exhibits enhanced uptake in a tissue or cell compared to the input or parent anucleate cell. In some embodiments, the modified anucleate cell-derived vesicle exhibits a rate of uptake in tissue or cell that is enhanced by 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 the input or parent anucleate cell. In some embodiments, the anucleate cell-derived vesicle exhibits enhanced uptake in phagocytic cells and / or antigen presenting cells compared to the input or parent anucleate cell. In some embodiments, phagocytic cells and / or antigen presenting cells comprise macrophages and / or dendritic cells. In some embodiments, the anucleate cell-derived vesicle exhibits enhanced uptake in liver, spleen or macrophages compared to the input or parent anucleate cell. In some embodiments, the anucleate cell-derived vesicle exhibits enhanced uptake in liver and / or spleen or by a phagocytic cell and / or an antigen- presenting cell compared to the uptake of the input or parent anucleate cell. In some embodiments, the anucleate cell-derived vesicle is not cleared in the lungs. In some embodiments, the anucleate cell-derived vesicle is cleared by macrophages in the liver and / or spleen, thereby leading to antigen presentation including via CD8+ and CD4+ T cell responses.

[0262] In some embodiments according to any of the anucleate cell-derived vesicles described herein, the anucleate cell-derived vesicle is modified to enhance uptake in a tissue or cell compared to an unmodified anucleate cell-derived vesicle. In some embodiments, the modified anucleate cell-derived vesicle exhibits a rate of uptake in tissue or cell that is enhanced by 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 an unmodified anucleate cell-derived vesicle. In some embodiments, the anucleate cell-derived vesicle is modified to enhance uptake in phagocytic cells and / or antigen presenting cells compared to an unmodified anucleate cell-derived vesicle. In some embodiments, phagocytic cells and / or antigen presenting cells comprise macrophages and / or dendritic cells. In some embodiments, the anucleate cell-derived vesicle is modified to enhance uptake in liver, spleen or macrophages compared to an unmodified anucleate cell- derived vesicle. In some embodiments, the anucleate cell-derived vesicle is modified to enhance uptake in liver and / or spleen or by a phagocytic cell and / or an antigen-presenting cell compared to the uptake of the input or parent anucleate cell. In some embodiments, the anucleate cell- derived vesicle is not cleared in the lungs. In some embodiments, the anucleate cell-derived vesicle is cleared by macrophages in the liver and / or spleen, thereby leading to antigen presentation including via CD8+ and CD4+ T cell responses.

[0263] In some embodiments, the anucleate cell-derived vesicle comprises CD47 on its surface.

[0264] In some embodiments, the anucleate cell-derived vesicle is not (a) heat processed, (b) chemically treated, and / or (c) subjected to hypotonic or hypertonic conditions during the preparation of the anucleate cell-derived vesicles. In certain embodiments, the anucleate cell- derived vesicle is not heat-treated or heat-shocked. In certain embodiments, the anucleate cell- derived vesicle is not treated by chemicals such as bis(sulfosuccinimidyl)suberate or other cross- linking agents. In certain embodiments, the anucleate cell-derived vesicle is not further modified to express or contain ionophores or other ion transporters. In certain embodiments, the anucleate cell-derived vesicle is not associated with antibodies such as anti-TER119.

[0265] In some embodiments according to any of the anucleate cell-derived vesicles described herein, the osmolarity of the cell suspension is maintained throughout the process. In further embodiments, the osmolarity of the cell suspension is maintained between about 200 mOsm and about 400 mOsm throughout the process. In some embodiments, the osmolarity of the cell suspension is maintained between about 200 mOsm and about 600 mOsm throughout the process. In further embodiments, the osmolarity of the cell suspension is maintained between about 200 mOsm and about 800 mOsm throughout the process. In some embodiments, the osmolarity of the cell suspension is maintained between any one of: 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 osmolarity was maintained during preparation of the anucleate cell-derived vesicle from the input or parent anucleate cell. In some embodiments, the osmolarity was maintained between about 200 mOsm and about 600 mOsm, such as between any of about 200 mOsm and about 300 mOsm, about 200 mOsm and about 400 mOsm, about 200 mOsm and about 500 mOsm, about 300 mOsm and about 500 mOsm or about 350 mOsm and about 450 mOsm. In some embodiments, the osmolarity was maintained between about 200 mOsm and about 400 mOsm.

[0266] In some embodiments, cell suspension is contacted with the antigen before, concurrently, and / or after passing through the constriction.

[0267] In some embodiments according to any of the anucleate cell-derived vesicles described herein, there is provided a composition comprising a plurality of anucleate cell-derived vesicle. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient. Antigens and Adjuvants

[0268] In some embodiments according to any of methods or anucleate 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 being infected by a pathogen, such as a bacterium or a virus. In some embodiments, the lysate is derived from a biopsy of an individual bearing tumors (i.e. tumor biopsy lysates). Thus, in some embodiments, the lysate is a tumor lysate. In some embodiments, the antigen is derived from a transplant 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, according to any of the methods or anucleate cell-derived vesicles described herein, the anucleate cell-derived vesicle comprises an antigen comprising an immunogenic epitope. In some embodiments, the antigen is a disease-associated antigen. In some embodiments, the antigen is derived from peptides or mRNA isolated from a diseased cell. In some embodiments, the antigen is derived from a protein 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, viral antigen, bacterial antigen, or fungal antigen. In some embodiments, the antigen comprises an immunogenic epitope fused to heterologous peptide sequences. In some embodiments, the antigen comprises a plurality of immunogenic epitopes. In some embodiments, some of the plurality of immunogenic epitopes are derived from the same source. For example, in some embodiments, some of the plurality of immunogenic epitope are derived from the same viral antigen. In some embodiments, all of the plurality of immunogenic epitopes are derived from the same source. In some embodiments, none of the plurality of immunogenic epitopes are derived from the same source. In some embodiments, the anucleate cell-derived vesicle comprises a plurality of different antigens. . In some embodiments, a plurality of antigens, such as any of 2, 3,4, 5, 6, 7, 8, 9, or 10 different types of antigens, are delivered to the anucleate cell.

[0270] In some embodiments according to any of the methods or anucleate 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 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, antigens are derived from foreign sources, such as bacteria, fungi, viruses, or allergens. In some embodiments, the antigen is a modified antigen. For example, antigens may be fused with therapeutic agents or targeting peptides. In some embodiments, the modified antigen comprises an antigen fused with a polypeptide. In some embodiments, the modified antigen comprises an antigen fused with a targeting peptide. In some embodiments, the modified antigen comprises an antigen fused with a lipid. In some embodiments, the modified antigen comprises an antigen fused with a carbohydrate. In some embodiments, the modified antigen comprises an antigen fused with a nanoparticle. In some embodiments, a plurality of antigens is delivered to the anucleate cell.

[0271] In some embodiments, according to any of the methods or anucleate cell-derived vesicles described herein, the anucleate cell-derived vesicle comprises 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 polypeptides are non-natural. In some embodiments, the immunogenic peptide epitope fused to the N-terminal flanking polypeptide and / or the C-terminal flanking polypeptide is synthetic. In some embodiments, the N-terminal and / or C-terminal flanking polypeptides are derived from an immunogenic synthetic long peptide (SLP). In some embodiments, the N-terminal and / or C- terminal flanking polypeptides are derived from a disease-associated immunogenic SLP.

[0272] In some embodiments, according to any of the methods or anucleate cell-derived vesicle described herein, the anucleate cell-derived vesicle comprises an antigen, wherein the antigen is capable of being processed into an MHC class I-restricted peptide and / or an MHC class II-restricted peptide. In some embodiments, the antigen is capable of being processed into an MHC class I-restricted peptide. In some embodiments, the antigen is capable of being processed into an MHC class II-restricted peptide. In some embodiments, the antigen comprises a plurality of immunogenic epitopes, and is capable of being processed into an MHC class I- restricted peptide and an MHC class Il-restricted peptide. In some embodiments, the anucleate cell-derived vesicle comprising the antigen is taken up by an antigen presenting cell, and the antigen is processed into one or more MHC class I-restricted peptide and / or one or more MHC class II-restricted peptide by the antigen presenting cell. 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 a plurality of immunogenic epitopes, and is capable of being processed into a plurality of CD-1 restricted antigens . In some embodiments, the anucleate cell-derived vesicle comprising the antigen is taken up by an antigen presenting cell, and the antigen is processed into one or more CD-1 restricted antigens by the antigen presenting cell. In some embodiments, the antigen comprises a plurality of immunogenic epitopes, and is capable of being processed into one or more of (a) a MHC class I-restricted peptide; (b) an MHC class II-restricted peptide; or (c) a CD-1 restricted antigen. In some embodiments, some of the plurality of immunogenic epitopes are derived from the same source. In some embodiments, all of the plurality of immunogenic epitopes are derived from the same source. In some embodiments, none of the plurality of immunogenic epitopes are derived from the same source.

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

[0274] In some embodiments, according to any of the methods or any of the anucleate cell- derived vesicles described herein, the anucleate cell-derived vesicle comprises an adjuvant. In some embodiments, the adjuvant is a CpG oligodeoxynucleotide (ODN), IFN-a, STING agonists, RIG-I agonists, 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 a CpG ODN. In some embodiments, the adjuvant is low molecular weight poly I:C. In some embodiments, the CpG ODN is no greater than about 50 (such as no greater than about any of 45, 40, 35, 30, 25, 20, or fewer) nucleotides 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 the nucleotide sequences as disclosed in US provisional application US 62 / 641,987. In some embodiments, the anucleate cell-derived vesicle comprises a plurality of different CpG ODN. For example, in some embodiments, the anucleate cell-derived vesicle comprises a plurality of different CpG ODN? selected from among Class A, Class B, and Class C CpG ODN. ). In some embodiments, a plurality of adjuvants, such as any of 2, 3, 4, 5, 6, 7, 8, 9, or 10 different types of adjuvants, is delivered to the anucleate cell.

[0275] In some embodiments, the anucleate cell-derived vesicle comprises an antigen and / or an adjuvant. In some embodiments, the anucleate cell-derived vesicle comprises the antigen at a concentration between about 1 pM and about 10 mM. In some embodiments, the anucleate cell- derived vesicle comprises the adjuvant at a concentration between about 1 pM and about 10 mM. In some embodiments, the anucleate cell-derived vesicle comprises the antigen at a concentration between about 0.1 uM and about 10 mM. In some embodiments, the anucleate cell-derived vesicle comprises the adjuvant at a concentration between about 0.1 pM and about 10 mM. For example, in some embodiments, the concentration of adjuvant in the anucleate cell- derived vesicle is any of less than about 1 pM, about 10 pM, about 100 pM, about 1 nM, about 10 nM, about 100 nM, about 1 uM, about 10 uM, about 100 uM, about 1 mM or about 10 mM. In some embodiments, the concentration of adjuvant in the anucleate cell-derived vesicle is greater than about 10 mM. In some embodiments, the concentration of antigen in the anucleate cell-derived vesicle is any of less than about 1 pM, about 10 pM, about 100 pM, about 1 nM, about 10 nM, about 100 nM, about 1 uM, about 10 uM, about 100 uM, about 1 mM or about 10 mM. In some embodiments, the concentration of antigen in the anucleate cell-derived vesicle is greater than about 10 mM. In some embodiments, the concentration of antigen in the anucleate cell-derived vesicle is any of 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 pM, between about 1 pM and about 10 uM, between about 10 uM and about 100 pM, between about 100 uM and about 1 mM, or between 1 mM and about 10 mM. In some embodiments, the concentration of adjuvant in the anucleate cell-derived vesicle is any of 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 pM, between about 1 uM and about 10 pM, between about 10 uM and about 100 uM, between about 100 uM 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 anucleate cell- derived vesicle is any of between about 10000:1 to about 1:10000. For example, in some embodiments, the molar ratio of adjuvant to antigen in the anucleate cell-derived vesicle is about any of 10000:1, about 1000:1, about 100:1, about 10:1, about 1:1, about 1:10, about 1:100, about 1:1000, or about 1:10000. In some embodiments, the anucleate cell-derived vesicle comprises a complex comprising: a) the antigen, b) the adjuvant, and / or c) the antigen and the adjuvant.

[0277] In some embodiments, according to any of the methods or anucleate cell-derived vesicles described herein, the anucleate cell-derived vesicle further comprises an additional agent that enhances the function of the anucleate cell-derived vesicle as compared to a corresponding anucleate cell-derived vesicle that does not comprise the additional agent. In some embodiments, the additional agent is a stabilizing agent or a co-factor. 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, according to any of the methods or anucleate cell-derived vesicles described herein, the anucleate cell-derived vesicle further comprises one or more therapeutic agents. Other payloads

[0279] In some embodiments, the payload is a tolerogenic factor. In 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, 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 an uricase (including semi-synthetic forms, e.g., Pegloticase) glucocerebrosidase (e.g., Imiglucerase, velaglucerase alfa, -glucosidase), tissue non-specific alkaline phosphatase (TNSALP) (e.g., Asfotase alfa), lysosomal acid lipase (e.g., Sebelipase alfa), alpha-glucosidase (e.g., alglucosidase alfa), a-L-iduronidase (e.g., Iaronidase), Iduronate sulfatase (e.g., Idursulfase), heparan sulfate, keratin sulfate, chondroitin 6-sulfate (e.g., elosulfase alfa), N- acetylgalactosamine-4-sulfatase (e.g., galsulfase), B-glucuronidase, hyaluronidase, a- galactosidase A (e.g., agalsidase beta), phenylalanine hydroxylase, medium-chain acyl-CoA dehydrogenase, gliadin, acetylcholine receptor and receptor-associated proteins, thyroid stimulating hormone receptor (TSHR), desmoglein 1 and 3, aquaporin 4, GADD63, insulin, pro- insulin, and pre-pro-insulin.

[0281] In some embodiments, the anucleate cell-derived vesicle was prepared by a process comprising: (a) passing a cell suspension comprising the input parent anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input parent anucleate cell in the suspension, thereby causing perturbations of the input parent anucleate cell large enough for the payload to pass through to form an anucleate cell- derived vesicle; and (b) incubating the anucleate cell-derived vesicle with the payload for a sufficient time to allow the payload to enter the anucleate cell-derived vesicle; thereby producing an anucleate cell-derived vesicle comprising the payload.

[0282] In some embodiments, the anucleate cell-derived vesicle comprises an antigen and a tolerogenic factor. In some embodiments, the tolerogenic factor enhances suppression of an immune response to an antigen and / or enhances the induction of tolerance to an antigen. In some embodiments, the tolerogenic factor may promote tolerogenic presentation of the antigen by an antigen-presenting cell. In some embodiments, the tolerogenic factor comprises a polypeptide. In some embodiments, the polypeptide is IL-4, IL-10, IL-13, IL-35, IFN-a, or TGF-p. 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 to knock down expression of inflammatory genes. In some embodiments, the tolerogenic factor is a DNA sequence that binds NF-«B and prevents NF-kB activation and downstream signaling. In some embodiments, the tolerogenic factor is a small molecule.

[0283] In some embodiments, the tolerogenic factor modulates expression and / or activity of an immunomodulatory agent (such as an immunostimulatory agent (e.g., a costimulatory molecule), an immunosuppressive agent, or an inflammatory or anti-inflammatory molecule). In some embodiments, the tolerogenic factor inhibits expression and / or activity of an immunostimulatory agent (e.g., a costimulatory molecule), enhances expression and / or activity of an immunosuppressive molecule, inhibits expression and / or activity of an inflammatory molecule, and / or enhances expression and / or activity of an anti-inflammatory molecule. In some embodiments, the tolerogenic factor inhibits the activity of a costimulatory molecule. Interaction between costimulatory molecules and their ligands is important to sustain and integrate TCR signaling to stimulate optimal T cell proliferation and differentiation. In some embodiments, the tolerogenic factor decreases expression of a costimulatory molecule. Exemplary costimulatory molecules expressed on antigen-presenting cells include, without limitation, CD40, CD80, CD86, CD54, CD83, CD79, 0x40 or ICOS Ligand. In some embodiments, the costimulatory molecule is CD80 or CD86. In some embodiments, the tolerogenic factor inhibits the expression of a nucleic acid that expresses or modulates expression of the costimulatory molecule. In some embodiments, the tolerogenic factor deletes a nucleic acid that expresses or modulates expression of the costimulatory molecule. In some embodiments, deletion of the nucleic acid that expresses or modulates expression of the costimulatory molecule is achieved via gene editing. In some embodiments, the tolerogenic factor inhibits the costimulatory molecule. In some embodiments, the tolerogenic factor is a siRNA that inhibits the costimulatory molecule. In some embodiments, the tolerogenic factor increases the activity of a transcriptional regulator that suppresses expression of the costimulatory molecule. In some embodiments, the tolerogenic factor increases the activity of a protein inhibitor that suppresses expression of the costimulatory molecule. In some embodiments, the tolerogenic factor comprises nucleic acid encoding a suppressor of the costimulatory molecule. In some embodiments, the tolerogenic factor degrades the costimulatory molecule. In some embodiments, the tolerogenic factor labels the costimulatory molecule for destruction. For example, the tolerogenic factor may enhance ubiquitination of the costimulatory molecule, thereby targeting it for destruction.

[0284] In some embodiments, the tolerogenic factor enhances the expression and / or activity of an immunosuppressive molecule. In some embodiments, the immunosuppressive molecule is a co-inhibitory molecule, a transcriptional regulator, or an immunosuppressive molecule. Co- inhibitory molecules negatively regulate the activation of lymphocytes. Exemplary co-inhibitory molecules include, without limitation, PD-L1 , PD-L2, HVEM, B7-H3, TRAIL, immunoglobulin-like transcripts (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 the co-inhibitory molecule. In some embodiments, the tolerogenic factor increases expression of a co-inhibitory molecule. In some embodiments, the tolerogenic factor encodes the co-inhibitory molecule. In some embodiments, the tolerogenic factor increases the activity of the co-inhibitory molecule. In some embodiments, the tolerogenic factor increases the activity of a transcriptional regulator that may enhance expression of the co-inhibitory molecule. In some embodiments, the tolerogenic factor increases the activity of a polypeptide that increases expression of the co-inhibitory molecule. In some embodiments, the tolerogenic factor comprises nucleic acid encoding an enhancer of the 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 expression and / or activity of an immunosuppressive molecule. Exemplary immunosuppressive molecules include, without limitation, 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-B (TGF-B), IFN-q, 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 the immunosuppressive molecule. In some embodiments, the tolerogenic factor increases the activity of the immunosuppressive molecule. In some embodiments, the tolerogenic factor increases the activity of a transcriptional regulator that enhances expression of the immunosuppressive molecule. In some embodiments, the tolerogenic factor increases the activity of a polypeptide that enhances expression of the immunosuppressive molecule. In some embodiments, the tolerogenic factor comprises nucleic acid encoding an enhancer of the immunosuppressive molecule. In some embodiments, the tolerogenic factor inhibits a negative regulator of an immunosuppressive molecule.

[0286] In some embodiments, the tolerogenic factor inhibits expression and / or activity of an inflammatory molecule. In some embodiments, the inflammatory molecule is an inflammatory transcription factor. In some embodiments, the tolerogenic factor inhibits the inflammatory transcription factor. In some embodiments, the tolerogenic factor decreases expression of an inflammatory transcription factor. In some embodiments, the inflammatory transcription factor is NF-KB, an interferon regulatory factor (IRF), or a molecule associated with the JAK-STAT signaling pathway. The NF-kB pathway is a prototypical proinflammatory signaling pathway that mediates the expression of proinflammatory genes including cytokines, chemokines, and adhesion molecules. Interferon regulatory factors (IRFs) constitute a family of transcription factors that can regulate the expression of proinflammatory genes. The JAK-STAT signaling pathway transmits 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 a cell surface receptor, Janus kinases (JAKs), and Signal Transducer and Activator of Transcription (STAT) proteins. Exemplary JAK-STAT molecules include, without limitation, JAKI, JAK2, JAK 3, Tyk2, STATI STAT2, STAT3, STAT4, STATS (STATSA and STATSB), and STATS. In some embodiments, the tolerogenic factor enhances expression of a suppressor of cytokine signaling (SOCS) protein. SOCS proteins may inhibit signaling through the JAK-STAT pathway. In some embodiments, the tolerogenic factor inhibits the expression of a nucleic acid encoding the inflammatory transcription factor. In some embodiments, the tolerogenic factor deletes a nucleic acid encoding the inflammatory transcription factor. In some embodiments, the tolerogenic factor increases the activity of a transcriptional regulator that suppresses expression of the inflammatory transcription factor. In some embodiments, the tolerogenic factor increases the activity of a protein inhibitor that suppresses expression of the inflammatory transcription factor. In some embodiments, the tolerogenic factor comprises nucleic acid encoding a suppressor of the inflammatory transcription factor.

[0287] In some embodiments, the tolerogenic factor enhances 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 the anti-inflammatory transcription factor. In some embodiments, the tolerogenic factor increases expression of an anti-inflammatory transcription factor. In some embodiments, the tolerogenic factor enhances expression of nucleic acid encoding the anti-inflammatory transcription factor. In some embodiments, the tolerogenic factor decreases the activity of a transcriptional regulator that suppresses expression of the anti-inflammatory transcription factor. In some embodiments, the tolerogenic factor decreases the activity of a protein inhibitor that suppresses expression of the anti-inflammatory transcription factor. In some embodiments, the tolerogenic factor comprises nucleic acid encoding an enhancer of the 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, IncRNA, tRNA, gRNA, and shRNA. In some embodiments, the nucleic acid is homologous to a nucleic acid in the cell. In some embodiments, the nucleic acid is heterologous to a 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, IncRNA, tRNA, or shRNA. For example, the tolerogenic factor can include siRNA to knock down expression of inflammatory genes. In some embodiments, the tolerogenic factor is a DNA sequence that binds NF-kB and prevents NF-kB 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, antibody, fusion protein, antigen, synthetic protein, reporter marker, or selectable marker. In some embodiments, the protein is a gene-editing protein or nuclease such as a zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), mega nuclease, CRE recombinase, transposase, RNA-guided endonuclease (e.g., CAS9 enzyme), DNA-guided endonuclease, or integrase enzyme. In some embodiments, the fusion proteins can include, without limitation, chimeric protein drags such as antibody drug conjugates or recombinant fusion proteins such as proteins tagged with GST or streptavidin. In some embodiments, the compound is a transcription factor. Exemplary transcription factors include, without limitation, Oct4, Sox2, c-Myc, Kif-4, T-bet, GATA3, FoxP3, and RORyt. In some embodiments, the polypeptide is IL-4, IL-10, IL-13, IL-35, IFN-a, or TGFp. 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. These complexes contain sequence-specific DNA-binding domains in combination with nonspecific DNA cleavage nucleases. These complexes enable targeted genome editing, including adding, disrupting, or changing the sequence of a specific gene. In some embodiments, a disabled Cas9 (dCas9) is used to block or induce transcription of a target gene. In some embodiments, the tolerogenic factor contains a Cas9 protein and a guide RNA and donor DNA. In some embodiments, the tolerogenic factor includes a nucleic acid encoding for a Cas9 protein and a guide RNA or donor DNA. In some embodiments, the gene editing complex targets 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 an inflammatory molecule. Exemplary small molecules include, without limitation, pharmaceutical agents, metabolites, or radionuclides. In some embodiments, the pharmaceutical agent is a therapeutic drug and / or 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 noncovalently) to a therapeutic molecule. In some embodiments, the nanoparticle contains a nucleic acid, such as mRNA or cDNA.

[0292] In some embodiments, the anucleate cell-derived vesicle comprises a cytokine. In some embodiments, the anucleate cell-derived vesicle comprises an agent for modulating genetic material, such as DNA. In some embodiments, the anucleate cell-derived vesicle comprises a gene editing component, such as a CRISPR component. In some embodiments, the anucleate cell-derived vesicle comprises an agent for modulating RNA, such as decreasing the presence of an RNA species. In some embodiments, the anucleate cell-derived vesicle comprises a siRNA. Methods for Generating Anucleate Cell-Derived Vesicles

[0293] In certain aspects, there is provided a method for generating an anucleate cell-derived vesicle comprising an antigen, the method comprising: a) passing a cell suspension comprising an input (e.g., parent) anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen to pass through to form an anucleate cell-derived vesicle; b) incubating the anucleate cell-derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle, thereby generating an anucleate cell-derived vesicle comprising the antigen. In some embodiments, the input anucleate cell comprises an adjuvant.

[0294] In certain aspects, there is provided a method for generating an anucleate cell-derived vesicle comprising an adjuvant, the method comprising: a) passing a cell suspension comprising an input (e.g., parent) anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the adjuvant to pass through to form an anucleate cell-derived vesicle; b) incubating the anucleate cell-derived vesicle with the adjuvant for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle, thereby generating an anucleate cell-derived vesicle comprising the adjuvant. In some embodiments, the input anucleate cell comprises an adjuvant.

[0295] In certain aspect, there is provided a method for generating an anucleate cell-derived vesicle comprising an antigen and an adjuvant, the method comprising: a) passing a cell suspension comprising an input (e.g., parent) anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen and the adjuvant to pass through to form an anucleate cell-derived vesicle; b) incubating the anucleate cell-derived vesicle with the antigen and the adjuvant for a sufficient time to allow the antigen and the adjuvant to enter the anucleate cell-derived vesicle, thereby generating an anucleate cell-derived vesicle comprising the antigen and the adjuvant.

[0296] In some embodiments, the anucleate cell-derived vesicle is a red blood cell-derived vesicle, or a platelet-derived vesicle. In some embodiments, the anucleate cell-derived vesicle is an erythrocyte-derived vesicle or a reticulocyte-derived vesicle.

[0297] In some embodiments according to any of the methods described herein, the input (e.g., parent) anucleate cell is a mammalian cell. In some embodiments, the input anucleate cell is a monkey, mouse, dog, cat, horse, rat, sheep, goat, pig, or rabbit cell. In some embodiments, the input anucleate cell is a human cell. In some embodiments, the input anucleate cell is a non- mammalian cell. In some embodiments, the input anucleate cell is a chicken, frog, insect, fish, or nematode cell. In some embodiments, the input anucleate cell is an erythrocyte. In some embodiments, the input anucleate cell is a red blood cell. In some embodiments, the input anucleate cell is a precursor to RBCs. In some embodiments, the input anucleate cell is a reticulocyte. In some embodiments, the input anucleate cell is a platelet.

[0298] In some embodiments, presentation of antigen in an immunogenic environment enhances an immune response to the antigen or induces an immune response to the antigen. Antigens derived from eryptotic bodies, such as anucleate cell-derived vesicles, which can be cleared in the immunogenic environment of the liver and spleen, may stimulate or enhance an immune response to the antigens via activation of T cells. In some embodiments, the immune response is antigen-specific. Anucleate cell-derived vesicles, such as RBC-derived vesicles have a limited life-span and are unable to self-repair, causing eryptosis, a process analogous to apoptosis, that leads to removal of the anucleate cell-derived vesicles from the bloodstream. In some embodiments, the antigen may be released upon eryptosis of the anucleate cell-derived vesicles within the immunogenic environment, where it is subsequently engulfed, processed, and presented by an antigen-presenting cell. In some embodiments, the anucleate cell-derived vesicle containing the antigen is phagocytosed by an antigen-presenting cell, such as a macrophage, and the antigen is subsequently processed and presented by the antigen presenting cell. In some embodiments, the antigen presenting cell is a resident macrophage.

[0299] In some embodiments, the circulating half-life of an anucleate cell-derived vesicle in a mammal is decreased compared to an input (e.g., parent) anucleate cell. Methods for measuring the half-life of a cell, such as an anucleate cell, e.g., red blood cell, or an anucleate cell-derived vesicle are known in the art. See, e.g., Franco, R. S., Transfus Med Hemother, 39, 2012. For example, in some embodiments, the method for measuring the half-life of an anucleate cell or an anucleate cell-derived vesicle comprises a cohort labeling technique or a random labeling technique. In some embodiments, the method for measuring the half-life of an anucleate cell or an anucleate cell-derived vesicle comprises labeling, reinfusing the cell or vesicle, and measuring the disappearance upon reinfusion. In some embodiments, the method for measuring the half-life of an anucleate cell or an anucleate cell-derived vesicle encompassed in the present application comprises measuring the half-life of an appropriate reference control(s), such as a control comprising an input anucleate cell or a population of input anucleate cells.

[0300] In some embodiments, the circulating half-life in the mammal is decreased by more than about 50%, such as more than about any of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, as compared to the input (e.g., parent) anucleate cell. In some embodiments, the circulating half-life in the mammal is decreased by about 50% to about 99.9%, such as any of about 70% to about 99.9%, about 85% to about 99.9%, or about 95% to about 99.9%, as compared to the input anucleate cell. In some embodiments, the circulating half-life in the mammal is decreased by about any of 50%, 55%, 60%, 65%, 70%, 15%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.9%, as compared to the input anucleate cell.

[0301] In some embodiments, the circulating half-life of the anucleate cell-derived vesicle 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 circulating half-life of the anucleate cell-derived vesicle is about any of 0.5 minute, 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) anucleate cell is a human cell and wherein the circulating half-life of the anucleate cell-derived vesicle 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 day, 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, or 100 days s. In some embodiments, the input anucleate cell is a human cell and wherein the circulating half-life of the anucleate cell-derived vesicle is about any of 0.5 minute, 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) anucleate cell is a red blood cell, wherein the hemoglobin level in the anucleate cell-derived vesicle is decreased compared to the input anucleate cell. Methods of measuring the hemoglobin level of a cell, such as an anucleate cell, e.g., red blood cell, or an anucleate cell-derived vesicle is known in the art. See, e.g., Chaudhary, R., J Blood Med, 8, 2017. For example, in some embodiments, the method comprises measuring a metabolic precursor or product to determine the turnover of hemoglobin. In some embodiments, the method for measuring the hemoglobin level of an anucleate cell or an anucleate cell-derived vesicle encompassed in the present application comprises measuring the hemoglobin levels of an appropriate reference control(s), such as a control comprising an input anucleate cell or a population of input anucleate cell.

[0304] In some embodiments, the hemoglobin level in the anucleate cell-derived vesicle is decreased by at least about any of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% 60%, 65%, 10%, 15%, 80%, 85%, 90%, 95%, 96%, 91%, 98%, 99%, 99.9%, or 100%, as compared to the input (e.g., parent) anucleate cell. In some embodiments, the hemoglobin level in the anucleate cell-derived vesicle is decreased by about 50% to about 99.9%, such as any of about 70% to about 99.9%, about 85% to about 99.9%, or about 95% to about 99.9%, as compared to the input anucleate cell. In some embodiments, the hemoglobin level in the anucleate cell-derived vesicle is decreased by about any of 50%, 55%, 60%, 65%, 70%, 75%. 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.9%, as compared to the input anucleate cell.

[0305] In some embodiments, the hemoglobin level in the anucleate cell-derived vesicle is about any of 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) anucleate cell.

[0306] In some embodiments, the input (e.g., parent) anucleate cell is an erythrocyte and wherein the morphology of the anucleate cell-derived vesicle is modulated from that of the input anucleate cell. Morphology concerns the classification of, e.g., the shape, structure, geometry, intensity, form, smoothness, roughness, circularity, volume, surface area and / or size of a cell or a cell-derived vesicle. Methods for determining (such as measuring) morphology are known in the art. See, e.g., Boutros er 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 comprises high-content imaging. For example, the morphology of the cell can be assessed by staining with Hoechst dye followed by automated high-content image analysis. In other examples, the morphology can be determined through a shift in the forward and side scatter plots from flow cytometry. In some embodiments, the input anucleate cell is an erythrocyte and wherein the anucleate cell-derived vesicle is spherical in morphology. In some embodiments, the input anucleate cell is an erythrocyte and wherein the anucleate cell-derived vesicle has a reduced biconcave shape compared to the input anucleate cell. In some embodiments, the method for measuring morphology of an anucleate cell or an anucleate cell-derived vesicle encompassed in the present application comprises measuring the morphology of an appropriate reference control(s), such as a control comprising an input anucleate cell or a population of input anucleate cell.

[0307] In some embodiments, the input (e.g., parent) anucleate cell is an erythrocyte and wherein the anucleate cell-derived vesicle has a reduced biconcave shape, such as reduced by more than about any of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 10%, 715%, 80%. 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.9%, as compared to the input anucleate cell.

[0308] In some embodiments, the input (e.g., parent) anucleate cell is a red blood cell or an erythrocyte and wherein the anucleate cell-derived vesicle is a red blood cell ghost (RBC ghost).

[0309] In some embodiments, the half-life of the anucleate cell-derived vesicle can be further modified. In some embodiments, the half-life of the anucleate cell-derived vesicle is increased by the further modification. For example, the anucleate cell-derived vesicle may be modified to increase the time the anucleate cell-derived vesicle circulates in the blood stream before clearance in the liver and spleen. In some embodiments, the half-life of the anucleate cell- derived vesicle is further decreased by the modification. For example, the anucleate cell-derived vesicle may be modified to decrease the time the anucleate cell circulates in the blood stream before clearance in the spleen. In some embodiments, an altered ratio of phospholipids on the surface of the anucleate cell-derived vesicle decreases the half-life of the anucleate cell-derived vesicle. In some embodiments, an increased ratio of phosphatidylserine to other phospholipids on the surface of the anucleate cell-derived vesicle decreases the half-life of the anucleate cell- derived vesicle. For example, the presence of phosphatidylserine on the surface of the anucleate cell-derived vesicle can be further increased to decrease the half-life of the anucleate cell, such as by using any method known in the art for increasing surface phosphatidylserine (see Hamidi etal., J. Control. Release, 2007, 118(2): 145-60). In some embodiments, the anucleate cell- derived vesicle is incubated with lipids or phospholipids prior to delivery to an individual. In some embodiments, the anucleate cell-derived vesicle is treated by chemicals such as bis(sulfosuccinimidyl)suberate or other cross-linking agents, prior to delivery to an individual. In other embodiments, the surface phosphatidylserine of the anucleate cell-derived vesicle can be decreased to increase the half-life of the anucleate cell-derived vesicle. In some embodiments, the anucleate cell-derived vesicle is treated with flippase prior to delivery to an individual. Generally, flippases are enzymes that transport phospholipids from the external leaflet to the cytosolic leaflet in the plasma membrane. In some embodiments, the anucleate cell-derived vesicle is treated with an enzyme that cleaves phosphatidylserines, prior to delivery to an individual. A non-limiting example of an enzyme that cleaves phosphatidylserine is phosphatidylserine carboxylase.

[0310] In some embodiments, the anucleate cell-derive vesicle exhibits one or more of the following properties: (a) a circulating half-life in a mammal is decreased compared to the parent anucleate cell, (b) decreased hemoglobin levels compared to the parent anucleate cell, (c) spherical morphology, (d) increased surface phosphatidylserine levels compared to the parent anucleate cell, or (e) reduced ATP production compared to the parent anucleate cell.

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

[0312] In some embodiments, according to any of the methods or anucleate cell-derived vesicles described herein, the anucleate cell-derived vesicle further comprises an additional agent that enhances the function of the anucleate cell-derived vesicle as compared to a corresponding anucleate cell-derived vesicle that does not comprise the additional agent. In some embodiments, the additional agent is a stabilizing agent or a co-factor. 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 anucleate cell-derived vesicles further comprise one or more therapeutic agents.

[0313] In some embodiments, the anucleate cell-derived vesicle comprises an antigen and a tolerogenic factor, wherein the anucleate cell-derived vesicle was prepared by a process comprising: (a) passing a cell suspension comprising the input parent anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input parent anucleate cell in the suspension, thereby causing perturbations of the input parent anucleate cell large enough for the antigen and the tolerogenic factor to pass through to form an anucleate cell-derived vesicle; and (b) incubating the anucleate cell-derived vesicle with the antigen and the tolerogenic factor for a sufficient time to allow the antigen and the tolerogenic factor to enter the anucleate cell-derived vesicle; thereby producing an anucleate cell-derived vesicle comprising an antigen and an tolerogenic factor.

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

[0315] In some embodiments, the constriction is a pore or 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 any of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 715%, or 80% of a diameter of the cell, such as the largest diameter of an anucleate cell in suspension. In some embodiments, the constriction size is less than about any of 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10% of a diameter of the cell, such as the largest diameter of an anucleate cell in suspension. In some embodiments, the constriction has a width of about 0.1 um to about 4 pm, such as any of about 1 pm to about 3 pm, about 1.75 pum to about 2.5 pum, or about 2 pm to about 2.5 pm. In some embodiments, the constriction has a width of about any of 4 um, 3.5 pm, 3 um, 2.5 um, 2 um, 1.5 pm, 1 um, 0.5 pm, or about 0.25 um. In some embodiments, the constriction has a width of about any of 1.8 um, 1.9 um, 2 um, 2.1 pm, 2.2 pm, 2.3 um, 2.4 pm, 2.5 um or 2.6 pm. In some embodiments, the constriction has a width of about 2.2 pm.

[0317] In some embodiments, the input parent anucleate cells are passed through the constriction under a pressure ranging from about 10 psi to about 150 psi, such as any of about 30 psi to about 60 psi, about 10 psi to about 40 psi, about 50 psi to about 90 psi. In some embodiments, the input parent anucleate 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 parent anucleate 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 (such as first contacted) with the payload before passing through the constriction. In some embodiments, the cell suspension is contacted (such as first contacted) with the payload concurrently with passing through the constriction. In some embodiments, the cell suspension is contacted (such as first contacted) with the payload after passing through the constriction. In some embodiments, the cell suspension is at least contacted with the payload concurrently with passing through the constriction and after passing through the constriction. In some embodiments, the cell suspension is contacted with the payload before passing through the constriction, concurrently with passing through the constriction, and after passing through the constriction.

[0319] In some embodiments, an anucleate cell-derived vesicle comprising an antigen and an adjuvant as described herein is an activating antigen carrier (AAC).

[0320] In some embodiments, an anucleate cell-derived vesicle comprising an antigen for tolearization as described herein is an tolerizing antigen carrier (TAC). Compositions

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

[0322] In some embodiments, provided is a composition comprising a plurality of anucleate cell-derived vesicles prepared from parent anucleate cells, the composition having any one or more of the following properties: (a) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 710%, 15%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have a circulating half-life in a mammal that is decreased compared to the parent anucleate cell, (b) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 10%, 75%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have decreased hemoglobin levels compared to the parent anucleate cell, (c) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 710%, 75%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have spherical morphology, (d) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 10%, 75%, 80%, 85%, 90% or 95%, of the anucleate cell- derived vesicles in the composition are RBC ghosts, (e) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 10%, 75%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition vesicles in the composition have higher levels of phosphatidylserine, or (f) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 710%, 75%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have reduced ATP production compared to the parent anucleate cell.

[0323] In some embodiments, the composition comprising a plurality of anucleate cell-derived vesicles may be actively tuned to generate a desired profile of anucleate cell-derived vesicles within the composition having one or more select properties, including one or more of: (a) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 10%, 15%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have a circulating half-life in a mammal that is decreased compared to the parent anucleate cell, (b) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 10%, 715%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have decreased hemoglobin levels compared to the parent anucleate cell, (c) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 15%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have spherical morphology, (d) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 710%, 75%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition are RBC ghosts, (e) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 10%, 15%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition vesicles in the composition have higher levels of phosphatidylserine, or (f) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 10%, 15%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have reduced ATP production compared to the parent anucleate cell.

[0324] In some embodiments, the composition comprising a plurality of anucleate cell-derived vesicles having a desired profile of select properties is prepared from parent anucleate cells using the methods of making described herein, including use of microfluidic constrictions, wherein parameters of the methods of making, including constriction dimension, speed of passing a parent anucleate cell through the constriction, constriction architecture (e.g., Weir structure and size), processing time, pressure, and buffer composition, are selected to produce the composition comprising a plurality of anucleate cell-derived vesicles having the desired profile of select properties. In some embodiments, the method of making a composition comprising a plurality of anucleate cell-derived vesicles having a desired profile of select properties comprises selecting a set of parameters, including constriction dimension, speed of passing a parent anucleate cell through the constriction, constriction architecture (e.g., Weir structure and size), processing time, pressure, and buffer composition, to produce the composition. In some embodiments, the method of making a composition comprising a plurality of anucleate cell-derived vesicles having a desired profile of select properties comprises using a set of parameters, including constriction dimension, speed of passing a parent anucleate cell through the constriction, constriction architecture (e.g., Weir structure and size), processing time, pressure, and buffer composition, to produce the composition.

[0325] For example, in some embodiments, the composition comprising a plurality of anucleate cell-derived vesicles having a desired profile of select properties is prepared using a set of parameters comprising a constriction dimension, e.g., about 2.2 pm or about 2.5 um, and a pressure, e.g., about 30 psi or about 50 psi. In some embodiments, the composition comprising a plurality of anucleate cell-derived vesicles having a desired profile of select properties is prepared using a set of parameters comprising a constriction dimension of about 2.2 um and a pressure of about 30 psi. In some embodiments, the composition comprising a plurality of anucleate cell-derived vesicles having a desired profile of select properties is prepared using a set of parameters comprising a constriction dimension of about 2.2 pm and a pressure of about 50 psi. In some embodiments, the composition comprising a plurality of anucleate cell-derived vesicles having a desired profile of select properties is prepared using a set of parameters comprising a constriction dimension of about 2.5 um and a pressure of about 30 psi. In some embodiments, the composition comprising a plurality of anucleate cell-derived vesicles having a desired profile of select properties is prepared using a set of parameters comprising a constriction dimension of about 2.5 pm and a pressure of about 50 psi.

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

[0327] In some embodiments, the parent anucleate cell is a red blood cell. In some embodiments, the parent anucleate cell is a platelet. In some embodiments, the red blood cell is an erythrocyte. In some embodiments, the red blood cell is a reticulocyte.

[0328] In some embodiments, the circulating half-life of at least about 20%, such as at least about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 15%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition in a mammal is decreased compared to the parent anucleate cell. In some embodiments, the circulating half-life of at least about 75%, such as at least about any of 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition in a mammal is decreased compared to the parent anucleate cell. In some embodiments, the circulating half-life of about any of 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 10%, 75%, 80%, 85%, 90% or 95% of the anucleate cell-derived vesicles in the composition in a mammal is decreased compared to the parent anucleate cell. In some embodiments, the circulating half-life of at least about 20%, such as at least about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 710%, 75%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition in the mammal is decreased by more than about any of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 90% compared to the parent anucleate cell. In some embodiments, the parent anucleate cell is a human cell, and the circulating half-life of at least about 20%, such as at least about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 10%, 75%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition 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, or 10 days. In some embodiments, the parent anucleate cell is a human cell, and the circulating half-life of at least about 20%, such as at least about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition is 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 anucleate cell is a red blood cell, and the hemoglobin levels of at least about 20%, such as at least about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 10%, 15%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition are decreased compared to the parent anucleate cell. In some embodiments, the hemoglobin levels of at least about 75%, such as at least about any of 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition in a mammal is decreased compared to the parent anucleate cell. In some embodiments, the hemoglobin levels of 20%, such as at least about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 15%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition of the anucleate cell-derived vesicle are decreased by at least about any of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% 10%, 15%, 80%, 85%, 90%, 95%, 99%, or 100% compared to the parent anucleate cell. In some embodiments, the hemoglobin levels of at least about 20%, such as at least about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition are about any of 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% the level of hemoglobin in the parent anucleate cell.

[0330] In some embodiments, the parent anucleate cell is an erythrocyte, and at least about 20%, such as at least about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have a modulated morphology as compared to the parent anucleate cell. In some embodiments, the parent anucleate cell is an erythrocyte, and at least about 20%, such as at least about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 710%, 75%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition are spherical in morphology. In some embodiments, the parent anucleate cell is an erythrocyte, and at least about 20%, such as at least about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 15%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have a reduced, such as reduced by more than about any of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 10%, 75%, 80%, 85%, 90% or 95%, bioconcave shape compared to the parent anucleate cell.

[0331] In some embodiments, the parent anucleate cell is a red blood cell or an erythrocyte, and at least about 20%, such as at least about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 10%, 15%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition are red blood cell ghosts.

[0332] In some embodiments, at least about 20%, such as at least about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 10%, 75%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition comprise surface phosphatidylserine. In some embodiments, at least about 20%, such as at least about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 10%, 75%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition comprise increased surface phosphatidylserine levels compared to the parent anucleate cells. In some embodiments, at least about 20%, such as at least about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 10%, 715%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have greater than about any of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 10%, 15%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, or 200% higher surface phosphatidylserine levels compared to a composition comprising a plurality of parent anucleate cells.

[0333] In some embodiments, at least about 20%, such as at least about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 15%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have reduced ATP production compared to the parent anucleate cell. In some embodiments, at least about 20%, such as at least about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 710%, 15%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition produce ATP at less than about any of 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% the level of ATP produced by the parent anucleate cell. In some embodiments, ATP production of a sample and a control is measured under similar conditions. In some embodiments, at least about 20%, such as at least about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 10%, 15%, 80%, 85%. 90% or 95%, do not produce ATP.

[0334] In some embodiments, provided is a composition comprising a plurality of anucleate cell-derived vesicles prepared from parent anucleate cells, the composition comprising the following property, as further described herein, of greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 10%, 15%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have a circulating half-life in a mammal that is decreased compared to the parent anucleate cell.

[0335] In some embodiments, provided is a composition comprising a plurality of anucleate cell-derived vesicles prepared from parent anucleate cells, the composition comprising the following property, as further described herein, of greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 10%, 75%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have decreased hemoglobin levels compared to the parent anucleate cell.

[0336] In some embodiments, provided is a composition comprising a plurality of anucleate cell-derived vesicles prepared from parent anucleate cells, the composition comprising the following property, as further described herein, of greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 15%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have spherical morphology.

[0337] In some embodiments, provided is a composition comprising a plurality of anucleate cell-derived vesicles prepared from parent anucleate cells, the composition comprising the following property, as further described herein, of greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 15%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition are RBC ghosts.

[0338] In some embodiments, provided is a composition comprising a plurality of anucleate cell-derived vesicles prepared from parent anucleate cells, the composition comprising the following property, as further described herein, of greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 15%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition vesicles in the composition have higher levels of phosphatidylserine.

[0339] In some embodiments, provided is a composition comprising a plurality of anucleate cell-derived vesicles prepared from parent anucleate cells, the composition comprising the following property, as further described herein, of greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 15%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have reduced ATP production compared to the parent anucleate cell.

[0340] In some embodiments, provided is a composition comprising a plurality of anucleate cell-derived vesicles prepared from parent anucleate cells, the composition comprising any two of the following properties, as further described herein, of (a) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 710%, 75%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have a circulating half-life in a mammal that is decreased compared to the parent anucleate cell, (b) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 10%, 75%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have decreased hemoglobin levels compared to the parent anucleate cell, (c) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 10%, 75%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have spherical morphology, (d) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 710%, 75%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition are RBC ghosts, (e) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 15%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition vesicles in the composition have higher levels of phosphatidylserine, or (f) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 710%, 75%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have reduced ATP production compared to the parent anucleate cell.

[0341] In some embodiments, provided is a composition comprising a plurality of anucleate cell-derived vesicles prepared from parent anucleate cells, the composition comprising any three of the following properties, as further described herein, of (a) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 10%, 75%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have a circulating half-life in a mammal that is decreased compared to the parent anucleate cell, (b) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 10%, 75%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have decreased hemoglobin levels compared to the parent anucleate cell, (c) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 10%, 15%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have spherical morphology, (d) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 15%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition are RBC ghosts, (e) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 10%, 15%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition vesicles in the composition have higher levels of phosphatidylserine, or (f) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 10%, 15%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have reduced ATP production compared to the parent anucleate cell.

[0342] In some embodiments, provided is a composition comprising a plurality of anucleate cell-derived vesicles prepared from parent anucleate cells, the composition comprising any four of the following properties, as further described herein, of (a) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 710%, 75%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have a circulating half-life in a mammal that is decreased compared to the parent anucleate cell, (b) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 10%, 15%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have decreased hemoglobin levels compared to the parent anucleate cell, (c) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 710%, 15%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have spherical morphology, (d) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition are RBC ghosts, (e) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 15%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition vesicles in the composition have higher levels of phosphatidylserine, or (f) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 710%, 15%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have reduced ATP production compared to the parent anucleate cell.

[0343] In some embodiments, provided is a composition comprising a plurality of anucleate cell-derived vesicles prepared from parent anucleate cells, the composition comprising any five of the following properties, as further described herein, of (a) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 710%, 75%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have a circulating half-life in a mammal that is decreased compared to the parent anucleate cell, (b) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 710%, 15%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have decreased hemoglobin levels compared to the parent anucleate cell, (c) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 15%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have spherical morphology, (d) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition are RBC ghosts, (e) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 15%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition vesicles in the composition have higher levels of phosphatidylserine, or (f) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 10%, 15%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have reduced ATP production compared to the parent anucleate cell.

[0344] In some embodiments, provided is a composition comprising a plurality of anucleate cell-derived vesicles prepared from parent anucleate cells, the composition comprising the following properties, as further described herein, of (a) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 10%, 75%, 80%, 85%. 90% or 95%, of the anucleate cell-derived vesicles in the composition have a circulating half-life in a mammal that is decreased compared to the parent anucleate cell, (b) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 710%, 75%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have decreased hemoglobin levels compared to the parent anucleate cell, (c) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 710%, 75%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition have spherical morphology, (d) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 10%, 15%, 80%, 85%, 90% or 95%, of the anucleate cell- derived vesicles in the composition are RBC ghosts, (e) greater than about 20%, such as greater than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 10%, 15%, 80%, 85%, 90% or 95%, of the anucleate cell-derived vesicles in the composition vesicles in the composition hav...

Claims

CLAIMS ‘What is claimed is: : A method for delivering an antigen into an anucleate cell-derived vesicle, the method comprising: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell-derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle. 2 The method of claim 1, wherein the input anucleate cell further comprises an adjuvant.

3. A method for delivering an adjuvant into an anucleate cell-derived vesicle, the method comprising: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the adjuvant to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell-derived vesicle with the adjuvant for a sufficient time to allow the adjuvant to enter the anucleate cell-derived vesicle.

4. The method of claim 3, wherein the input anucleate cell further comprises an antigen.

5. A method for delivering an antigen and an adjuvant into an anucleate cell-derived vesicle, the method comprising: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen and the adjuvant to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell-derived vesicle with the antigen and the adjuvant for a sufficient time to allow the antigen and the adjuvant to enter the anucleate cell-derived vesicle.

6. A method for stimulating an immune response to an antigen in an individual, the method comprising administering to the individual an effective amount of an anucleate cell-derived vesicle comprising an antigen, wherein the anucleate cell-derived vesicle comprising the antigen is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell-derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle.

7. The method of claim 6, wherein the method further comprises administering an adjuvant systemically to the individual.

8. The method of claim 7, wherein the adjuvant is administered systemically before, after or at the same time as the anucleate cell derived vesicle.

9. The method of any one of claims 6-8, wherein the input anucleate cell comprises an adjuvant.

10. A method for stimulating an immune response to an antigen in an individual, the method comprising administering to the individual an effective amount of an anucleate cell-derived vesicle comprising an antigen and an adjuvant, wherein the anucleate cell-derived vesicle comprising the antigen and the adjuvant is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen and the adjuvant to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell-derived vesicle with the antigen and the adjuvant for a sufficient time to allow the antigen and the adjuvant to enter the anucleate cell-derived vesicle.

11. The method of claim 10, wherein the method further comprises administering an adjuvant systemically to the individual.

12. The method of claim 11, wherein the adjuvant is administered systemically before, after or at the same time as the anucleate cell-derived vesicle.

13. A method for treating a disease in an individual, comprising administering to the individual an anucleate cell-derived vesicle comprising a disease-associated antigen, wherein an immune response against the antigen ameliorates conditions of the disease, and wherein the anucleate cell-derived vesicle comprising the disease-associated antigen is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell-derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle.

14. A method for preventing a disease in an individual, comprising administering to the individual an anucleate cell-derived vesicle comprising a disease-associated antigen, wherein an immune response against the antigen prevents development of the disease, and wherein the anucleate cell-derived vesicle comprising the disease-associated antigen is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell-derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle.

15. A method for vaccinating an individual against an antigen, comprising administering to the individual an anucleate cell-derived vesicle comprising the antigen, wherein the anucleate cell-derived vesicle comprising the antigen is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell-derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle.

16. The method of any one of claims 13-15, wherein the method further comprises administering an adjuvant systemically to the individual.

17. The method of claim 16, wherein the adjuvant is administered systemically before, after or at the same time as the anucleate cell derived vesicle.

18. The method of claim 13-17, wherein the input anucleate cell comprises an adjuvant.

19. A method for treating a disease in an individual, comprising administering to the individual an anucleate cell-derived vesicle comprising a disease-associated antigen and an adjuvant, wherein an immune response against the antigen ameliorates conditions of the disease, and wherein the anucleate cell-derived vesicle comprising the disease-associated antigen and the adjuvant is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen and an adjuvant to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell-derived vesicle with the antigen and the adjuvant for a sufficient time to allow the antigen and the adjuvant to enter the anucleate cell-derived vesicle.

20. A method for preventing a disease in an individual, comprising administering to the individual an anucleate cell-derived vesicle comprising a disease-associated antigen and an adjuvant, wherein an immune response against the antigen prevents development of the disease, and wherein the anucleate cell-derived vesicle comprising a disease-associated antigen and an adjuvant is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen and an adjuvant to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell-derived vesicle with the antigen and the adjuvant for a sufficient time to allow the antigen and the adjuvant to enter the anucleate cell-derived vesicle.

21. A method for vaccinating an individual against an antigen, comprising administering to the individual an anucleate cell-derived vesicle comprising the antigen and an adjuvant, wherein the anucleate cell-derived vesicle comprising the antigen and the adjuvant is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen and an adjuvant to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell-derived vesicle with the antigen and the adjuvant for a sufficient time to allow the antigen and the adjuvant to enter the anucleate cell-derived vesicle.

22. A method for treating a disease in an individual, wherein an immune response against a disease-associated antigen ameliorates conditions of the disease, the method comprising a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen to pass through to form an anucleate cell-derived vesicle; b) incubating the anucleate cell-derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle, thereby generating an anucleate cell-derived vesicle comprising the antigen; and ¢) administering the anucleate cell-derived vesicle comprising the antigen to the individual 23. A method for preventing a disease in an individual, wherein an immune response against a disease-associated antigen prevents development of the disease, the method comprising a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen to pass through to form an anucleate cell-derived vesicle; b) incubating the anucleate cell-derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle, thereby generating an anucleate cell-derived vesicle comprising the antigen; and ¢) administering the anucleate cell-derived vesicle comprising the antigen to the individual. 24, A method for vaccinating an individual against an antigen, the method comprising, a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen to pass through to form an anucleate cell-derived vesicle; b) incubating the anucleate cell-derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle, thereby generating an anucleate cell-derived vesicle comprising the antigen; and ¢) administering the anucleate cell-derived vesicle comprising the antigen to the individual.

25. The method of any one of claims 19-24, wherein the method further comprises administering an extravesicular adjuvant systemically to the individual.

26. The method of claim 25, wherein the extravesicular adjuvant is administered before, after or at the same time as the anucleate cell-derived vesicle.

27. The method of claim19-24, wherein the input anucleate cell comprises an adjuvant.

28. A method for treating a disease in an individual, wherein an immune response against a disease-associated antigen ameliorates conditions of the disease, the method comprising a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the disease-associated antigen and an adjuvant to pass through to form an anucleate cell-derived vesicle: b) incubating the anucleate cell-derived vesicle with the antigen and the adjuvant for a sufficient time to allow the antigen and the adjuvant to enter the anucleate cell-derived vesicle, thereby generating an anucleate cell-derived vesicle comprising the antigen and the adjuvant; and ¢) administering the anucleate cell-derived vesicle comprising the antigen and the adjuvant to the individual 29. A method for preventing a disease in an individual, wherein an immune response against a disease-associated antigen prevents development of the disease, the method comprising a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen and an adjuvant to pass through to form an anucleate cell-derived vesicle; b) incubating the anucleate cell-derived vesicle with the antigen and the adjuvant for a sufficient time to allow the antigen and the adjuvant to enter the anucleate cell-derived vesicle, thereby generating an anucleate cell-derived vesicle comprising the antigen and the adjuvant; and ¢) administering the anucleate cell-derived vesicle comprising the antigen and the adjuvant to the individual.

30. A method for vaccinating an individual against an antigen, the method comprising, a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen and an adjuvant to pass through to form an anucleate cell-derived vesicle; b) incubating the anucleate cell-derived vesicle with the antigen and the adjuvant for a sufficient time to allow the antigen and the adjuvant to enter the anucleate cell-derived vesicle, thereby generating an anucleate cell-derived vesicle comprising the antigen and the adjuvant; and ¢) administering the anucleate cell-derived vesicle comprising the antigen and the adjuvant to the individual.

31. The method of any one of claims 28-30, wherein the method further comprises administering an extravesicular adjuvant systemically to the individual.

32. The method of claim 31, wherein the extravesicular adjuvant is administered before, after or at the same time as the anucleate cell derived vesicle.

33. The method of any one of claims 13-32, wherein the disease is cancer, an infectious disease or a viral-associated disease.

34. The method of any one of claims 6-33 wherein the anucleate cell-derived vesicle is autologous to the individual.

35. The method of any one of claims 6-33, wherein the anucleate cell-derived vesicle is allogeneic to the individual.

36. The method of any one of claims 6-35, wherein the anucleate cell-derived vesicle is in a pharmaceutical formulation.

37. The method of any one of claims 6-36, wherein the anucleate cell-derived vesicle is administered systemically.

38. The method of any one of claims 6-37, wherein the anucleate cell-derived vesicle is administered intravenously, intraarterially, subcutaneously, intramuscularly, or intraperitoneally.

39. The method of any one of claims 6-38, wherein the anucleate cell-derived vesicle is administered to the individual in combination with a therapeutic agent.

40. The method of claim 39, wherein the therapeutic agent is administered before, after or at the same time as the anucleate cell-derived vesicle.

41. The method of claim 39 or 40, wherein the therapeutic agent is an immune checkpoint inhibitor and / or a cytokine.

42. The method of claim 41, wherein the cytokine is one or more of IFN-a, IFN-y, IL-2 or 1-15.

43. The method of claim 41, wherein 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. 44, The method of any one of claims 1, 2, or 4-43, wherein the antigen is capable of being processed into an MHC class I-restricted peptide and / or an MHC class I-restricted peptide.

45. The method of any one of claims 1, 2, or 4-43, wherein the antigen is a CD-1 restricted antigen.

46. The method of any one of claims 1, 2, or 4-45, wherein the antigen is a disease- associated antigen.

47. The method of any one of claims 1, 2, or 4-46, wherein the antigen is a tumor antigen.

48. The method of any one of claims 1, 2, or 4-47, wherein the antigen is derived from a lysate.

49. The method of claim 48, wherein the lysate is a tumor lysate.

50. The method of any one of claims 1, 2, or 4-46, wherein the antigen is a viral antigen, a bacterial antigen or a fungal antigen.

51. The method of any one of claims 1, 2, or 4-46, wherein the antigen is a microorganism.

52. The method of any one of claims 1, 2, or 4-50, wherein the antigen is a polypeptide.

53. The method of any one of claims 1, 2, or 4-50, wherein the antigen is a lipid antigen.

54. The method of any one of claims 1, 2, or 4-50, wherein the antigen is a carbohydrate antigen.

53. The method of any one of claims 1, 2, or 4-54, wherein the antigen is a modified antigen.

56. The method of claim 55, wherein the modified antigen comprises an antigen fused with a polypeptide.

57. The method of claim 56, wherein the modified antigen comprises an antigen fused with a targeting peptide.

58. The method of claim 55, wherein the modified antigen comprises an antigen fused with a lipid.

59. The method of claim 55, wherein the modified antigen comprises an antigen fused with a carbohydrate.

60. The method of claim 55, wherein the modified antigen comprises an antigen fused with a nanoparticle.

61. The method of any one of claims 1-60, wherein a plurality of antigens is delivered to the anucleate cell-derived vesicle.

62. The method of any one of claims 2-5, 7-12, 16-21, 25-61 wherein the adjuvant is a CpG ODN, IFN-q, STING agonists, RIG-I agonists, poly I:C, polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose (HILTONOL®), imiquimod, resiquimod, and / or lipopolysaccharide (LPS).

63. The method of claim 62, wherein the adjuvant is low molecular weight poly I:C.

64. The method of any one of claims 1-63, wherein the input anucleate cell is a red blood cell.

65. The method of any one of claims 1-63, wherein the red blood cell is an erythrocyte.

66. The method of any one of claims 1-63, wherein the red blood cell is a reticulocyte.

67. The method of any one of claims 1-63, wherein the input anucleate cell is a platelet.

68. The method of any one of claims 1-67, wherein the input anucleate cell is a mammalian cell.

69. The method of any one of claims 1-68, wherein the input anucleate cell is a monkey, mouse, dog, cat, horse, rat, sheep, goat, pig, or rabbit cell.

70. The method of any one of claims 1-68, wherein the input anucleate cell is a human cell.

71. The method of any one of claims 1-70, wherein the constriction is contained within a microfluidic channel.

72. The method of claim 71, wherein the microfluidic channel comprises a plurality of constrictions.

73. The method of claim 72, wherein the plurality of constrictions are arranged in series and / or in parallel.

74. The method of any one of claims 1-73, wherein the constriction is between a plurality of micropillars; between a plurality of micropillars configured in an array; or between one or more movable plates.

75. The method of any one of claims 1-70, wherein the constriction is a pore or contained within a pore.

76. The method of claim 75, wherein the pore is contained in a surface.

77. The method of claim 76, wherein the surface is a filter.

78. The method of claim 76, wherein the surface is a membrane.

79. The method of any one of claims 1-76, wherein the constriction size is a function of the diameter of the input anucleate cell in suspension.

80. The method of any one of claims 1-79, wherein 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 anucleate cell in suspension.

81. The method of any one of claims 1-79, wherein the constriction has a width of about 0.25 um to about 4 pm.

82. The method of any one of claims 1-79, wherein the constriction has a width of about 4 pm, 3.5 um, about 3 um, about 2.5 um, about 2 um, about 1.5 pm, about 1 pm, about 0.5 um, or about 0.25 um.

83. The method of any one of claims 1-79, wherein the constriction has a width of about 2.2 um.

84. The method of any one of claims 1-83, wherein the input anucleate cells are passed through the constriction under a pressure ranging from about 10 psi to about 90 psi.

85. The method of any one of claims 1-84, wherein said cell suspension is contacted with the antigen before, concurrently, or after passing through the constriction.

86. An anucleate cell-derived vesicle comprising an antigen, wherein the anucleate cell- derived vesicle comprising the antigen is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell-derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle; thereby generating the anucleate cell-derived vesicle comprising the antigen.

87. The anucleate cell-derived vesicle of claim 86, wherein the input anucleate cell comprises an adjuvant.

88. An anucleate cell-derived vesicle comprising an adjuvant, wherein the anucleate cell- derived vesicle comprising the adjuvant is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the adjuvant to pass through to form an anucleate cell-derived vesicle; and b) incubating the anucleate cell-derived vesicle with the adjuvant for a sufficient time to allow the adjuvant to enter the anucleate cell-derived vesicle; thereby generating the anucleate cell-derived vesicle comprising the adjuvant.

89. The anucleate cell-derived vesicle of claim 88, wherein the input anucleate cell comprises an antigen.

90. An anucleate cell-derived vesicle comprising an antigen and an adjuvant, wherein the anucleate cell-derived vesicle comprising the antigen and the adjuvant is prepared by a process comprising the steps of: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen and the adjuvant to pass through to form an anucleate cell-derived vesicle: and b) incubating the anucleate cell-derived vesicle with the antigen and the adjuvant for a sufficient time to allow the antigen and the adjuvant to enter the anucleate cell-derived vesicle; thereby generating the anucleate cell-derived vesicle comprising the antigen and the adjuvant.

91. The anucleate cell-derived vesicle of any one of claims 86-90, wherein the anucleate cell-derived vesicle is a red blood cell-derived vesicle or a platelet-derived vesicle.

92. The anucleate cell-derived vesicle of claim 91, wherein the red blood cell-derived vesicle is an erythrocyte-derived vesicle, or a reticulocyte-derived vesicle.

93. The anucleate cell-derived vesicle of any one of claims 86, 87, or 89-92, wherein the antigen is capable of being processed into an MHC class I-restricted peptide and / or an MHC class II-restricted peptide.

94. The anucleate cell-derived vesicle of any one of claims 86, 87, or 89-92, wherein the antigen is a CD-1 restricted antigen.

95. The anucleate cell-derived vesicle of any one of claims 86, 87, or 89-94, wherein the antigen is a disease-associated antigen.

96. The anucleate cell-derived vesicle of any one of claims 86, 87, or 89-95, wherein the antigen is a tumor antigen.

97. The anucleate cell-derived vesicle of any one of claims 86, 87, or 89-96, wherein the antigen is derived from a lysate.

98. The anucleate cell-derived vesicle of claim 97, wherein the lysate is a tumor lysate.

99. The anucleate cell-derived vesicle of any one of claims 86, 87, or 89-95, wherein the antigen is a viral antigen, a bacterial antigen or a fungal antigen.

100. The anucleate cell-derived vesicle of any one of claims 86, 87, or 89-95, wherein the antigen is a microorganism.

101. The anucleate cell-derived vesicle of any one of claims 86, 87, or 89-99, wherein the antigen is a polypeptide.

102. The anucleate cell-derived vesicle of any one of claims 86, 87, or 89-99, wherein the antigen is a lipid antigen.

103. The anucleate cell-derived vesicle of any one of claims 86, 87, or 89-99, wherein the antigen is a carbohydrate antigen.

104. The anucleate cell-derived vesicle of any one of claims 86, 87, or 89-103, wherein the antigen is a modified antigen.

105. The anucleate cell-derived vesicle of claim 104, wherein the modified antigen comprises an antigen fused with a polypeptide.

106. The anucleate cell-derived vesicle of claim 105, wherein the modified antigen comprises an antigen fused with a targeting peptide.

107. The anucleate cell-derived vesicle of claim 104, wherein the modified antigen comprises an antigen fused with a lipid.

108. The anucleate cell-derived vesicle of claim 104, wherein the modified antigen comprises an antigen fused with a carbohydrate.

109. The anucleate cell-derived vesicle of claim 104, wherein the modified antigen comprises an antigen fused with a nanoparticle.

110. The anucleate cell-derived vesicle of any one of claims 86, 87, or 89-109, wherein a plurality of antigens is delivered to the anucleate cell-derived vesicle.

111. The anucleate cell-derived vesicle of any one of claims 87-110 wherein the adjuvant is a CpG ODN, IFN-q, STING agonists, RIG-I agonists, poly I:C, polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose (HILTONOL®), imiquimod, resiquimod and / or LPS.

112. The anucleate cell-derived vesicle of claim 111, wherein the adjuvant is low molecular weight poly I:C.

113. The anucleate cell-derived vesicle of any one of claims 86-112 wherein the input anucleate cell is a red blood cell.

114. The anucleate cell-derived vesicle of any one of claims 86-112, wherein the input anucleate cell is an erythrocyte.

115. The anucleate cell-derived vesicle of any one of claims 86-112, wherein the input anucleate cell is a reticulocyte.

116. The anucleate cell-derived vesicle of any one of claims 86-112, wherein the input anucleate cell is a platelet.

117. The anucleate cell-derived vesicle of any one of claims 86-116 wherein the input anucleate cell is a mammalian cell.

118. The anucleate cell-derived vesicle of any one of claims 86-117, wherein the input anucleate cell is a monkey, mouse, dog, cat, horse, rat, sheep, goat, pig, or rabbit cell.

119. The anucleate cell-derived vesicle of any one of claims 86-117, wherein the input anucleate cell is a human cell.

120. The anucleate cell-derived vesicle of any one of claims 86-119 wherein a half-life of the anucleate cell-derived vesicle following administration to a mammal is decreased compared to a half-life of the input anucleate cell following administration to the mammal.

121. The anucleate cell-derived vesicle of any one of claims 86-115, or 117-120, wherein a hemoglobin content of the anucleate cell-derived vesicle is decreased compared to the hemoglobin content of the input anucleate cell.

122. The anucleate cell-derived vesicle of any one of claims 86-120, wherein ATP production of the anucleate cell-derived vesicle is decreased compared to ATP production of the input anucleate cell.

123. The anucleate cell-derived vesicle of any one of claims 113, 114, 117-122 wherein the anucleate cell-derived vesicle exhibits one or more of the following properties: (a) a circulating half-life in a mammal that is decreased compared to the input anucleate cell; (b) decreased hemoglobin level compared to the input anucleate cell; (c) a spherical morphology; (d) increased surface phosphatidylserine levels compared to the input anucleate cell, (e) reduced ATP production compared to the input anucleate cell.

124. The anucleate cell-derived vesicle of any one of claims 113, 114, 117-122, wherein the input anucleate cell is an erythrocyte and wherein the anucleate cell-derived vesicle has a reduced biconcave shape compared to the input anucleate cell.

125. The anucleate cell-derived vesicle of claim 113, 114, 117-122 wherein the anucleate cell- derived vesicle is a red blood cell ghost.

126. The anucleate cell-derived vesicle of any one of claims 86-125, wherein the anucleate cell-derived vesicles prepared by the process have greater than about 1.5 fold more phosphatidylserine on its surface compared to the input anucleate cell.

127. The anucleate cell-derived vesicle of any one of claims 86-126, wherein a population profile of anucleate cell-derived vesicles prepared by the process exhibits higher average phosphatidylserine levels on the surface compared to the input anucleate cells.

128. The anucleate cell-derived vesicle of any one of claims 86-127, wherein at least 50% of the population profile of anucleate cell-derived vesicles prepared by the process exhibits higher phosphatidylserine levels on the surface compared to the input anucleate cells 129. The anucleate cell-derived vesicle of any one of claims 86-128, wherein the anucleate cell-derived vesicle exhibits enhanced uptake in a tissue or cell compared to the input anucleate cell.

130. The anucleate cell-derived vesicle of claim 129, wherein the anucleate cell-derived vesicle exhibits enhanced uptake in liver and / or spleen or by a phagocytic cell and / or an antigen- presenting cell compared to the uptake of the input anucleate cell.

131. The anucleate cell-derived vesicle of any one of claims 86-130, wherein the anucleate cell-derived vesicle is modified to enhance uptake in a tissue or cell compared to an unmodified anucleate cell-derived vesicle.

132. The anucleate cell-derived vesicle of claim 131, wherein the anucleate cell-derived vesicle is modified to enhance uptake in liver and / or spleen or by a phagocytic cell and / or an antigen-presenting cell compared to the uptake of the input anucleate cell. 133 The anucleate cell-derived vesicle of any one of claims 86-132, wherein the anucleate cell-derived vesicle comprises CD47 on its surface.

134. The anucleate cell-derived vesicle of any one of claims 86-133, wherein the anucleate cell-derived vesicle is not (a) heat processed, (b) chemically treated, and / or (c) subjected to hypotonic or hypertonic conditions during the preparation of the anucleate cell-derived vesicles.

135. The anucleate cell-derived vesicle of any one of claims 86-134, wherein the osmolarity of the cell suspension is maintained throughout the process.

136. The anucleate cell-derived vesicle of claims 86-135, wherein the osmolarity of the cell suspension is maintained between 200 mOsm and 400 mOsm throughout the process.

137. The anucleate cell-derived vesicle of any one of claims 86-136, wherein the constriction is contained within a microfluidic channel.

138. The anucleate cell-derived vesicle of claim 137, wherein the microfluidic channel comprises a plurality of constrictions.

139. The anucleate cell-derived vesicle of claim 138, wherein the plurality of constrictions are arranged in series and / or in parallel.

140. The anucleate cell-derived vesicle of any one of claims 86-139, wherein the constriction is between a plurality of micropillars; between a plurality of micropillars configured in an array; or between one or more movable plates.

141. The anucleate cell-derived vesicle of any one of claims 86-136, wherein the constriction is a pore or contained within a pore.

142. The anucleate cell-derived vesicle of claim 141, wherein the pore is contained in a surface.

143. The anucleate cell-derived vesicle of claim 142, wherein the surface is a filter.

144. The anucleate cell-derived vesicle of claim 142, wherein the surface is a membrane.

145. The anucleate cell-derived vesicle of any one of claims 86-144, wherein the constriction size is a function of the diameter of the input anucleate cell in suspension.

146. The anucleate cell-derived vesicle of any one of claims 86-144, wherein 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 anucleate cell in suspension.

147. The anucleate cell-derived vesicle of any one of claims 86-146, wherein the constriction has a width of about 0.25 um to about 4 um.

148. The anucleate cell-derived vesicle of any one of claims 86-147, wherein the constriction has a width of about 4 um, 3.5 pm, about 3 pm, about 2.5 pm, about 2 um, about 1.5 um, about 1 um, about 0.5 um, or about 0.25 pm.

149. The anucleate cell-derived vesicle of any one of claims 86-147, wherein the constriction has a width of about 2.2 pm.

150. The anucleate cell-derived vesicle of any one of claims 86-149, wherein the input anucleate cells are passed through the constriction under a pressure ranging from about 10 psi to about 90 psi.

151. The anucleate cell-derived vesicle of any one of claims 86-150, wherein said cell suspension is contacted with the antigen before, concurrently, or after passing through the constriction.

152. A composition comprising a plurality of anucleate cell-derived vesicles of any one of claims 86-151.

153. The composition of claim 152, further comprising a pharmaceutically acceptable excipient.

154. A method for generating an anucleate cell-derived vesicle comprising an antigen, the method comprising: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen to pass through to form an anucleate cell-derived vesicle; b) incubating the anucleate cell-derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleate cell-derived vesicle, thereby generating an anucleate cell-derived vesicle comprising the antigen.

155. The method of claim 154, wherein the input anucleate cell comprises an adjuvant.

156. A method for generating an anucleate cell-derived vesicle comprising an adjuvant, the method comprising: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the adjuvant to pass through to form an anucleate cell-derived vesicle; b) incubating the anucleate cell-derived vesicle with the adjuvant for a sufficient time to allow the adjuvant to enter the anucleate cell-derived vesicle, thereby generating an anucleate cell-derived vesicle comprising the adjuvant.

157. The method of claim 156, wherein the input anucleate cell comprises an antigen.

158. A method for generating an anucleate cell-derived vesicle comprising an antigen and an adjuvant, the method comprising: a) passing a cell suspension comprising an input anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the input anucleate cell in the suspension, thereby causing perturbations of the input anucleate cell large enough for the antigen and the adjuvant to pass through to form an anucleate cell-derived vesicle: b) incubating the anucleate cell-derived vesicle with the antigen and the adjuvant for a sufficient time to allow the antigen and the adjuvant to enter the anucleate cell-derived vesicle, thereby generating an anucleate cell-derived vesicle comprising the antigen and the adjuvant.

159. The method of any one of claims 154-158, wherein the anucleate cell-derived vesicle is a red blood cell-derived vesicle or a platelet derived vesicle.

160. The method of claim 159, wherein the red blood cell-derived vesicle is an erythrocyte- derived vesicle or a reticulocyte-derived vesicle.

161. The method of any one of claims 154, 155 or 157-160, wherein the antigen is capable of being processed into an MHC class I-restricted peptide and / or an MHC class II-restricted peptide.

162. The method of any one of claims 154, 155 or 157-160, wherein the antigen is a CD-1 restricted antigen.

163. The method of any one of claims 154, 155 or 157-162, wherein the antigen is a disease- associated antigen.

164. The method of any one of claims 154, 155 or 157-163, wherein the antigen is a tumor antigen.

165. The method of any one of claims 154, 155 or 157-164, wherein the antigen is derived from a lysate.

166. The method of claim 165, wherein the lysate is a tumor lysate.

167. The method of any one of claims 154, 155 or 157-163, wherein the antigen is a viral antigen, a bacterial antigen or a fungal antigen.

168. The method of any one of claims 154, 155 or 157-163, wherein the antigen is a microorganism.

169. The method of any one of claims 154, 155 or 157-167, wherein the antigen is a polypeptide.

170. The method of any one of claims 154, 155 or 157-167, wherein the antigen is a lipid antigen.

171. The method of any one of claims 154, 155 or 157-167, wherein the antigen is a carbohydrate antigen.

172. The method of any one of claims 154, 155 or 157-171, wherein the antigen is a modified antigen.

173. The method of claim 172, wherein the modified antigen comprises an antigen fused with a polypeptide.

174. The method of claim 173, wherein the modified antigen comprises an antigen fused with a targeting peptide.

175. The method of claim 174, wherein the modified antigen comprises an antigen fused with a lipid.

176. The method of claim 175, wherein the modified antigen comprises an antigen fused with a carbohydrate.

177. The method of claim 176, wherein the modified antigen comprises an antigen fused with a nanoparticle.

178. The method of any one of claims 154, 155 or 157-177, wherein a plurality of antigens is delivered to the anucleate cell-derived vesicle.

179. The method of any one of claims 155-178 wherein the adjuvant is a CpG ODN, IFN-q, STING agonists, RIG-I agonists, poly I:C, polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose (HILTONOL®), imiquimod, resiquimod, and / or LPS.

180. The method of claim 179, wherein the adjuvant is a low molecular weight poly I.C.

181. The method of any one of claims 154-180 wherein the input anucleate cell is a red blood cell.

182. The method of any one of claims 154-181, wherein the input anucleate cell is an erythrocyte.

183. The method of any one of claims 154-181, wherein the input anucleate cell is a reticulocyte.

184. The method of any one of claims 154-180, wherein the input anucleate cell is a platelet.

185. The method of any one of claims 154-184, wherein the input anucleate cell is a mammalian cell.

186. The method of any one of claims 154-185, wherein the input anucleate cell is a monkey, mouse, dog, cat, horse, rat, sheep, goat, pig, or rabbit cell.

187. The method of any one of claims 154-185, wherein the input anucleate cell is a human cell.

188. The method of any one of claims 154-187, wherein a half-life of the anucleate cell- derived vesicle following administration to a mammal is decreased compared to a half-life of the input anucleate cell following administration to the mammal.

189. The method of any one of claims 181-183, or 185-188, wherein a hemoglobin content of the anucleate cell-derived vesicle is decreased compared to the hemoglobin content of the input anucleate cell.

190. The method of any one of claims 181-189, wherein ATP production of the anucleate cell- derived vesicle is decreased compared to ATP production of the input anucleate cell.

191. The method of any one of claims 181-182 or 185-190, wherein the anucleate cell-derived vesicle exhibits one or more of the following properties: (a) a circulating half-life in a mammal that is decreased compared to the input anucleate cell; (b) decreased hemoglobin level compared to the input anucleate cell; (c) a spherical morphology; (d) increased surface phosphatidylserine levels compared to the input anucleate cell, (e) reduced ATP production compared to the input anucleate cell.

192. The method of any one of claims 181-182 or 185-191, wherein the input anucleate cell is an erythrocyte and wherein the anucleate cell-derived vesicle has a reduced biconcave shape compared to the input anucleate cell.

193. The method of claim 181-182 or 185-192, wherein the anucleate cell-derived vesicle is a red blood cell ghost.

194. The method of any one of claims 154-193, wherein the anucleate cell-derived vesicles prepared by the process have greater than about 1.5 fold more phosphatidylserine on its surface compared to the input anucleate cell.

195. The anucleate cell-derived vesicle of any one of claims 154-194, wherein a population profile of anucleate cell-derived vesicles prepared by the process exhibits higher average phosphatidylserine levels on the surface compared to the input anucleate cells.

196. The anucleate cell-derived vesicle of any one of claims 154-195, wherein at least 50% of the population profile of anucleate cell-derived vesicles prepared by the process exhibits higher phosphatidylserine levels on the surface compared to the input anucleate cells.

197. The anucleate cell-derived vesicle of any one of claims 154-196, wherein the anucleate cell-derived vesicle exhibits enhanced uptake in a tissue or cell compared to the input anucleate cell.

198. The anucleate cell-derived vesicle of claim 197, wherein the anucleate cell-derived vesicle exhibit enhanced uptake in liver and / or spleen or by a phagocytic cell and / or an antigen- presenting cell compared to the uptake of the input anucleate cell.

199. The anucleate cell-derived vesicle of any one of claims 154-198, wherein the anucleate cell-derived vesicle is modified to enhance uptake in a tissue or cell compared to the input anucleate cell.

200. The anucleate cell-derived vesicle of claim 199, wherein the anucleate cell-derived vesicle is modified to enhance uptake in liver and / or spleen or by a phagocytic cell and / or an antigen-presenting cell compared to the uptake of the input anucleate cell.

201. The anucleate cell-derived vesicle of any one of claims 154-200, wherein the anucleate cell-derived vesicle comprises CD47 on its surface.

202. The method of any one of claims 154-201, wherein the anucleate cell-derived vesicle is not (a) heat processed, (b) chemically treated, and / or (c) subjected to hypotonic or hypertonic conditions during the preparation of the anucleate cell-derived vesicles.

203. The method of any one of claims 154-202, wherein the osmolarity of the cell suspension is maintained throughout the process.

204. The method of claims 154-203, wherein the osmolarity of the cell suspension is maintained between about 200 mOsm and about 400 mOsm throughout the process.

205. The method of any one of claims 154-204, wherein the constriction is contained within a microfluidic channel.

206. The method of claim 205, wherein the microfluidic channel comprises a plurality of constrictions.

207. The method of claim 206, wherein the plurality of constrictions are arranged in series and / or in parallel.

208. The method of any one of claims 154-207, wherein the constriction is between a plurality of micropillars; between a plurality of micropillars configured in an array; or between one or more movable plates.

209. The method of any one of claims 154-208, wherein the constriction is a pore or contained within a pore.

210. The method of claim 209, wherein the pore is contained in a surface.

211. The method of claim 210, wherein the surface is a filter.

212. The method of claim 210, wherein the surface is a membrane.

213. The method of any one of claims 154-212, wherein the constriction size is a function of the diameter of the input anucleate cell in suspension.

214. The method of any one of claims 154-213, wherein 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 anucleate cell in suspension.

215. The method of any one of claims 154-214, wherein the constriction has a width of about 0.25 um to about 4 um.

216. The method of any one of claims 154-215, wherein the constriction has a width of about 4 um, 3.5 pm, about 3 pm, about 2.5 um, about 2 um, about 1.5 pm, about 1 um, about 0.5 pm, or about 0.25 um.

217. The method of any one of claims154-215, wherein the constriction has a width of about 2.2 um.

218. The method of any one of claims 154-217, wherein the input anucleate cells are passed through the constriction under a pressure ranging from about 10 psi to about 90 psi.

219. The method of any one of claims 154-218, wherein said cell suspension is contacted with the antigen before, concurrently, or after passing through the constriction.

220. A composition comprising a population of anucleate cell-derived vesicles prepared by the method of any one of claims 154-219.

221. An anucleate cell-derived vesicle prepared from a parent anucleate cell, the anucleate cell- derived vesicle having one or more of the following properties: (a) a circulating half-life in a mammal is decreased compared to the parent anucleate cell, (b) decreased hemoglobin levels compared to the parent anucleate cell, (c) spherical morphology, (d) increased surface phosphatidylserine levels compared to the parent anucleate cell, or (e) reduced ATP production compared to the parent anucleate cell.

222. A composition comprising a plurality of anucleate cell-derived vesicles prepared from parent anucleate cells, the composition having one or more of the following properties: (a) greater than about 20% of the anucleate cell-derived vesicles in the composition have a circulating half-life in a mammal that is decreased compared to the parent anucleate cell, (b) greater than 20% of the anucleate cell-derived vesicles in the composition have decreased hemoglobin levels compared to the parent anucleate cell, (c) greater than 20% of the anucleate cell-derived vesicles in the composition have spherical morphology, (d) greater than 20% of the anucleate cell-derived vesicles in the composition are RBC ghosts, (e) greater than 20% of the anucleate cell-derived vesicles in the composition vesicles in the composition have higher levels of phosphatidylserine compared to the population of parent anucleate cells, or (f) greater than 20% of the anucleate cell-derived vesicles in the composition have reduced ATP production compared to the parent anucleate cell.

223. A composition comprising a plurality of anucleate cell-derived vesicles prepared from a population of a parent anucleate cell, the composition having one or more of the following properties: (a) greater than about 20% of the anucleate cell-derived vesicles in the composition have a circulating half-life in a mammal that is decreased compared to the average of the population of the parent anucleate cell, (b) greater than 20% of the anucleate cell-derived vesicles in the composition have decreased hemoglobin levels compared to the average of the population of the parent anucleate cell, (c) greater than 20% of the anucleate cell-derived vesicles in the composition have spherical morphology, (d) greater than 20% of the anucleate cell-derived vesicles in the composition are RBC ghosts, (e) greater than 20% of the anucleate cell-derived vesicles in the composition vesicles in the composition have higher levels of phosphatidylserine compared to the average of the population of the parent anucleate cell, or (f) greater than 20% of the anucleate cell-derived vesicles in the composition have reduced ATP production compared to the average of the population of the parent anucleate cell.

224. A method of making a composition comprising a plurality of anucleate cell-derived vesicles prepared from parent anucleate cells, the composition having one or more of the following properties: (a) greater than 20% of the anucleate cell-derived vesicles in the composition have a circulating half-life in a mammal that is decreased compared to the parent anucleate cell, (b) greater than 20% of the anucleate cell-derived vesicles in the composition have decreased hemoglobin levels compared to the parent anucleate cell, (c) greater than 20% of the anucleate cell-derived vesicles in the composition have spherical morphology, (d) greater than 20% of the anucleate cell-derived vesicles in the composition are RBC ghosts, (e) greater than 20% of the anucleate cell-derived vesicles in the composition have higher levels of phosphatidylserine, or (f) greater than 20% of the anucleate cell-derived vesicles in the composition have reduced ATP production compared to the parent anucleate cell; the method comprising passing a cell suspension comprising the parent anucleate cell through a cell-deforming constriction, wherein a diameter of the constriction is a function of a diameter of the parent anucleate cell in the suspension, thereby causing perturbations of the parent anucleate cell large enough for a payload to pass through to form an anucleate cell-derived vesicle, thereby producing an anucleate cell-derived vesicle.

225. A method for treating a disease or disorder in an individual in need thereof, the method comprising administering the anucleate cell-derived vesicle of claim 221.

226. A method for treating a disease or disorder in an individual in need thereof, the method comprising administering the composition of claim 222.

227. A method for preventing a disease or disorder in an individual in need thereof, the method comprising administering the anucleate cell-derived vesicle of claim 221.

228. A method for preventing a disease or disorder in an individual in need thereof, the method comprising administering the composition of claim 222.

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