KR-102960025-B1 - Anucleated cell-derived vaccine

Abstract

The present invention provides a method for stimulating an immune response to an antigen, comprising administering to an individual an enucleated cell-derived vesicle containing an antigen and/or ajuvant. In some embodiments, an enucleated cell-derived vesicle containing an antigen and/or ajuvant is generated by passing a cell suspension containing an input anucleated cell through a press, wherein the press modifies the input anucleated cell and thereby induces perturbation of the cell to allow the antigen and/or ajuvant to enter the enucleated cell-derived vesicle, thereby forming an enucleated cell-derived vesicle. In some embodiments, an enucleated cell-derived vesicle containing an antigen and/or ajuvant is delivered to an individual, and the antigen is delivered to an immunogenic environment and processed therein to treat a disease,/or prevent a disease, or/or vaccinate the individual against the antigen.

Inventors

• 샤레이, 아몬 알.
• 번스타인, 하워드
• 길버트, 조나단 비.
• 무어, 피놀라
• 브릿젠, 데빈
• 카세로, 루크

Assignees

• 에스큐지 바이오테크놀로지스 컴퍼니

Dates

Publication Date

20260507

Application Date

20200124

Priority Date

20190125

Claims (20)

1. A method for delivering an antigen, an ajuvant, or both an antigen and an ajuvant into an anucleated cell-derived vesicle, wherein the method a) Select one or more parameters including pressure and compression width; b) passing a cell suspension containing introduced anucleated cells through a cell-transforming compression section at one or more selected parameters, thereby inducing perturbation of the introduced anucleated cells to form anucleated cell-derived vesicles; c) Contacting an anucleated cell-derived vesicle with an antigen, an ajuvant, or both antigen and ajuvant, such that the antigen, an ajuvant, or both antigen and ajuvant pass through perturbation involving a transient breakthrough in the input anucleated cell and enter the anucleated cell-derived vesicle. Includes, A method in which a larger amount of anucleated cell-derived vesicles are produced from the input anucleated cell as pressure increases, or as the width of the compression area decreases, or as pressure increases and the width of the compression area decreases.
2. A composition for stimulating an immune response to an antigen in an individual requiring stimulation of an immune response to an antigen, comprising an effective amount of an anucleated cell-derived vesicle containing an antigen, wherein the antigen is A cell suspension containing introduced anucleated cells is passed through a cell-transforming compression section, thereby inducing perturbation of the introduced anucleated cells to form anucleated cell-derived vesicles; Allowing the antigen to pass through perturbations involving a transient breakthrough in the introduced anucleated cell and enter the anucleated cell-derived vesicle It was delivered into anucleated cell-derived vesicle by, Anucleated cell-derived vesicles have the following characteristics: (a) Reduced hemoglobin levels compared to the input anucleated cells; (b) Spherical morphology; (c) Increased surface phosphatidylserine levels compared to the input anucleated cells, or (d) Reduced ATP production compared to input anucleated cells A composition representing one or more of the following.
3. A composition in paragraph 2, wherein the composition further comprises an azuvant, wherein the azuvant is administered systemically to an individual.
4. A composition in which the input nucleus-free cells in paragraph 2 further comprise an ajuvant.
5. In paragraph 1, (i) antigen (1) Can be processed into MHC class I-restricted peptides, MHC class II-restricted peptides, or MHC class I-restricted peptides and MHC class II-restricted peptides, or (2) CD-1 restricted antigen, or (3) If it is a disease-associated antigen, or (4) If it is a tumor antigen, or (5) Derived from a dissolved substance, or (6) If it is a viral antigen, (7) If it is a bacterial antigen, (8) If it is a fungal antigen, (9) Microorganisms, or (10) polypeptide, or (11) If it is a lipid antigen, or (12) Carbohydrate antigen, or (13) It is a modified antigen, or (14) A combination of these; (ii) the ajuvant is selected from CpG ODN, IFN-α, STING agonists, RIG-I agonists, poly I:C, polylysine and carboxymethylcellulose-stabilized polyinosinic acid-polycythidyl acid, imiquimod, resiquimod, lipopolysaccharide (LPS), or a combination thereof; or (iii) A method that is both (i) and (ii) above.
6. In paragraph 2, the antigen (1) Can be processed into MHC class I-restricted peptides, MHC class II-restricted peptides, or MHC class I-restricted peptides and MHC class II-restricted peptides, or (2) CD-1 restricted antigen, or (3) If it is a disease-associated antigen, or (4) If it is a tumor antigen, or (5) Derived from a dissolved substance, or (6) If it is a viral antigen, (7) If it is a bacterial antigen, (8) If it is a fungal antigen, (9) Microorganisms, or (10) polypeptide, or (11) If it is a lipid antigen, or (12) Carbohydrate antigen, or (13) It is a modified antigen, or (14) A composition that is a combination of these.
7. A composition according to claim 3, wherein the ajuvant is selected from CpG ODN, IFN-α, STING agonist, RIG-I agonist, poly I:C, polylysine and carboxymethylcellulose-stabilized polyinosinic acid-polycythidyl acid, imiquimod, resiquimod, lipopolysaccharide (LPS), or a combination thereof.
8. A composition according to claim 4, wherein the ajuvant is selected from CpG ODN, IFN-α, STING agonist, RIG-I agonist, poly I:C, polylysine and carboxymethylcellulose-stabilized polyinosinic acid-polycythidyl acid, imiquimod, resiquimod, lipopolysaccharide (LPS), or a combination thereof.
9. A method according to claim 1, wherein the injected nucleus-free cells comprise red blood cells, platelets, or both.
10. A composition according to paragraph 2, wherein the input nucleus-free cells comprise red blood cells, platelets, or both.
11. A method according to claim 1, wherein the cell-deformed compression portion includes a pore or is contained within the pore.
12. A composition according to paragraph 2, wherein the cell-deformed compression portion includes a pore or is contained within the pore.
13. In paragraph 1, (i) the cell-deformed compression portion has a width of 0.25 μm to 4 μm; (ii) the introduced anucleated cells are passed through a cell-transforming compression section under a pressure ranging from 10 psi to 90 psi; or (iii) A method that is both (i) and (ii) above.
14. In paragraph 2, (i) the cell-deformed compression portion has a width of 0.25 μm to 4 μm; (ii) the introduced anucleated cells are passed through a cell-transforming compression section under a pressure ranging from 10 psi to 90 psi; or (iii) A composition that is both (i) and (ii) above.
15. A method according to claim 1 in which the cell suspension comes into contact with the antigen, the ajuvant, or both the antigen and the ajuvant, either jointly or after passing through the cell-transformed compression section.
16. A composition according to paragraph 2, wherein the cell suspension comes into contact with the antigen jointly or subsequently before passing through the cell-transformed compression portion.
17. A nucleated cell-derived vesicle comprising an antigen, an ajuvant, or both an antigen and an ajuvant, wherein the antigen, the ajuvant, or both the antigen and the ajuvant are delivered into the nucleated cell-derived vesicle according to the method of claim 1, and Anucleated cell-derived vesicles have the following characteristics: (a) Reduced hemoglobin levels compared to the input anucleated cells; (b) Spherical morphology; (c) Increased surface phosphatidylserine levels compared to the input anucleated cells, or (d) Reduced ATP production compared to input anucleated cells Anucleated cell-derived vesicles representing one or more of the following.
18. In Paragraph 17, (i) antigen (1) Can be processed into MHC class I-restricted peptides, MHC class II-restricted peptides, or MHC class I-restricted peptides and MHC class II-restricted peptides, or (2) CD-1 restricted antigen, or (3) If it is a disease-associated antigen, or (4) If it is a tumor antigen, or (5) Derived from a dissolved substance, or (6) If it is a viral antigen, (7) If it is a bacterial antigen, (8) If it is a fungal antigen, (9) Microorganisms, or (10) polypeptide, or (11) If it is a lipid antigen, or (12) Carbohydrate antigen, or (13) It is a modified antigen, or (14) A combination of these; (ii) the ajuvant is selected from CpG ODN, IFN-α, STING agonists, RIG-I agonists, poly I:C, polylysine and carboxymethylcellulose-stabilized polyinosinic acid-polycythidyl acid, imiquimod, resiquimod, LPS, or a combination thereof; or (iii) Anucleated cell-derived vesicles that are both (i) and (ii) above.
19. In paragraph 17, anucleated cell-derived vesicle in which the input anucleated cell comprises red blood cells, platelets, or both.
20. A composition comprising a plurality of nucleated cell-derived vesicles, comprising a nucleated cell-derived vesicle of any one of claims 17 to 19.

Description

Anucleated cell-derived vaccine Cross-reference regarding related applications The present application claims priority to U.S. Provisional Application No. 62/797,185 filed January 25, 2019, U.S. Provisional Application No. 62/797,187 filed January 25, 2019, U.S. Provisional Application No. 62/933,301 filed November 8, 2019, and U.S. Provisional Application No. 62/933,302 filed November 8, 2019, the full contents of each of which are incorporated herein by reference. Field of invention The present disclosure generally relates to a method for stimulating an immune response by delivering an enucleated cell-derived vesicle to an individual or a method for treating cancer, an infectious disease or a virus-associated disease, wherein the enucleated cell-derived vesicle is loaded with an antigen and/or ajuvant. In some embodiments, the antigen and/or ajuvant is delivered to the enucleated cell by passing a cell suspension through a cell-transforming compression section. The complexity of the immune system and immune responses to foreign substances makes it difficult to develop effective approaches to trigger antigen-specific immune responses in vivo. In addition to the continued development of agents capable of triggering antigen-specific immune responses, such as small molecule and polypeptide- and/or nucleotide-based vaccines, carrier strategies for use with these agents require further development to optimize delivery and immune response. Carriers known in the relevant art, including polymer-based carriers, particle carriers, liposomes, and cell-based vesicles, such as those derived from red blood cells, still face difficulties that limit their use in triggering antigen-specific immune responses in vivo. For example, considering that red blood cells are irregular in shape (dioplex), anucleated, and transcriptionally inactive, the use of red blood cells as carriers is difficult due to the challenges associated with manipulating red blood cells to associate antigenic substances. Consequently, standard transfection techniques do not work. To overcome these difficulties, methods using red blood cells as carriers to trigger an immune response have focused on conjugating substances to the surface of mature red blood cells. For example, see the literature [Lorentz et al ., Sci. Adv , l:el5001122015]; [Grimm et al ., Sci Rep , 5, 2015]; and [Kontos et al ., Proc Natl Acad Sci USA , 110, 2013]. Early work using surface conjugation showed promising results with model antigens of type 1 diabetes and mouse models, but has some significant drawbacks, including (a) the need for chemically modified antigens for attachment; (b) limited surface area for loading; and (c) immunogenicity. References describing a method of using a microfluidic compression unit to deliver a compound to a cell include WO2013059343, WO2015023982, WO2016070136, WO2016077761, and WO/2017/192785. All references cited herein, including patent applications and publications, are incorporated by reference in their entirety. A brief summary of the invention In some aspects, the present invention provides a method for delivering an antigen into an anucleated cell-derived vesicle, comprising: a) passing a cell suspension containing an input (e.g., mother) anucleated cell through a cell-transforming compression section—wherein the diameter of the compression section is a function of the diameter of the input anucleated cell in the suspension, thereby causing perturbation of the input anucleated cell large enough for the antigen to pass through to form an anucleated cell-derived vesicle; and b) incubating the anucleated cell-derived vesicle with the antigen for a sufficient time to allow the antigen to enter the anucleated cell-derived vesicle. In some embodiments, the input anucleated cell further comprises an ajuvant. In some aspects, the present invention provides a method for delivering an ajuvant into an ajuvant-derived vesicle, comprising: a) passing a cell suspension containing an input anucleated cell through a cell-modifying compression section—wherein the diameter of the compression section is a function of the diameter of the input anucleated cell in the suspension, thereby causing perturbation of the input anucleated cell large enough for the ajuvant to pass through to form an anucleated cell-derived vesicle; and b) incubating the ajuvant with the ajuvant for a sufficient time to allow the ajuvant to enter the ajuvant-derived vesicle. In some embodiments, the input anucleated cell further comprises an antigen. In some aspects, the present invention provides a method for delivering an antigen and an ajuvant into an anucleated cell-derived vesicle, comprising: a) passing a cell suspension containing an input anucleated cell through a cell-transforming compression section, wherein the diameter of the compression section is a function of the diameter of the input anucleated cell in the suspension, thereby causing perturbation of the input anucleated cell